US009302982B2

(12) United States Patent (10) Patent No.: US 9,302,982 B2 Christian (45) Date of Patent: *Apr. 5, 2016

(54) PHARMACEUTICAL DOPAMINE 6,339,064 B1 1/2002 McDevitt et al. GIYCOCONJUGATECOMPOSITIONS AND 355 R. 393 Eas 51462 METHODS OF THEIR PREPARATION AND 8,352.752 B2 8/2012 Christian ...... 514.42 USE 9,023,818 B2 5/2015 Christian 2006/0189547 A1 8, 2006 Christian (75) Inventor: Samuel T. Christian, Alabaster, AL (US) FOREIGN PATENT DOCUMENTS (73) Assignee: GLYCON LLC, Riverside, AL (US) WO 9728174 8, 1997 WO 9905089 2, 1999 (*) Notice: Subject to any disclaimer, the term of this WO O197244 12/2001 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 255 days. This patent is Subject to a terminal dis- Thomas et al., “Parkinson's Disease” Human Molecular Genetics claimer. (2007) vol. 16 reviss 2, pp. R183-R194.* Rao et al., “Parkinson's Disease:Diagnosis and Treatment” American Family Physician (2006) vol. 74 No. 2 pp. 2046-2054.* (21) Appl. No.: 13/551,131 “Remington: The Science and Practice of Pharmacy, 20' edition”, (22) Filed: Jul. 17, 2012 iamsEdited A by Wii Alfonso M. pp.R. Gennaro,218, publishedity. 2000 by Lippincott PP Will O O Czarnocki et al., "Enantioselective Synthesis of (R)-(-) Laudanosine (65) Prior Publication Data and (R)-(-) Glaucine from E. Acid', E.A. Asym US 2013/0203855A1 Aug. 8, 2013 metry, vol. 7, No. 9, Sep. 1996, pp. 2711-2720. Doherty, “The Synthesis of Glyconyl Peptides”,Journal of Biologi Related U.S. Application Data cal Chemistry, vol. 201(2), May 1953, pp. 857-866. Fernandez et al., “Synthesis and biological studies of glycosyl (63) Continuation-in-part of application No. 1 1/965,444, dopamine deriviatives”. Carbohyd. Res. vol. 327, 2000, pp. 353-365. filed on Dec. 27, 2007, now Pat. No. 8,252,752, which Glinsky et al., “Inhibition of colony formation in agarose of meta is a continuation of application No. 10/625,645, filed static human breast carcinoma and melanoma cells by Synthetic on Jul. 22, 2003, now Pat. No. 7,345,031, application glycoamine analogs”. Clin. Exp. Metastasis, vol. 14(3), May 1996, No. 13/551,131, which is a continuation-in-part of pp. 253-267. application No. 12/913,543, filed on Oct. 27, 2010, Haavik et al., "Tyrosine hydroxylase and Parkinson's disease'. now Pat. No. 9.023.818, which is a continuation of Molecular Neurobiology, vol. 16, No. 3, Jun. 1998, pp. 285-309. application No. 1 1/343 266 filed on Jan. 30, 2006 Jakaset al. “Synthesis and 13C NMR investigation of novel Amadori now abandoned,s which is a continuation of application compoundsopioid peptide, (1-amino leucine—enkephalin'. -1-deoxy-D-fructose J. Chem. derivatives) Soc., Perkin related Trans. to the No. 09/547.501, filed on Apr. 12, 2000, now vol. 2, DOI 10.1039/P29960000789, 1996, pp. 789-794. abandoned. Jiang et al., “Dopaminergic properties and experimental anti Parkinsonian effects . . . . Clin. Neuropharmacol vol. 27, No. 2, (51) Int. Cl. 2004, 63-73. A6 IK3I/7008 (2006.01) Knoerzer et al., “Dopaminergic Benzoaphenanthridines: Resolu A 6LX3L/7032 (2006.01) tion and Pharmacological Evaluation of the Enantiomers of C7H5/04 (2006.01) Dihydrexidine, the Full Efficacy D1 Dopamine Receptor Agonist'. C7H 5/18 (2006.01) Journal of Medicinal Chemistry, vol. 37 No. 15, Jul. 1994, pp. 2453 C07C 235/08 (2006.01) iRoberto et al., “Giycoconjugates of amines: alkylation of pri A6 IK 47/48 (2006.01) marv and second amines Russ. Chem. Bull vol 47. No. 6 iss. A6 IK3I/704 (2006.01) s: Hisplay v w w K-1 - w C07H 17/00 (2006.01) Likhoshersfov et al., “Synthesis of N-chloroacetyi-B- (52) U.S. Cl. glycopyranosylamines, derivatives of monosaccharides and CPC ...... C07C 235/08 (2013.01); A61 K3I/704 lactose.”. Russ. Chern. B1 vol: 45, 1996, pp. 1760-1763. (2013.01); A61K47/48092 (2013.01); C07H Maher et al., “Substrate specificity and kinetic parameters of 5/04 (2013.01); C07H 1700 (2013.01) GLUT3". Biochem. J. vol.315, 1996, pp. 827-831. (58) Field of Classification Search (Continued) CPC. C07H 15/2013; C07H5/06; A61K31/7032: A61K 3.1 F7008 See application file for complete search history. Primary Examiner — Eric Olson (74) Attorney, Agent, or Firm — Kilpatrick Townsend & (56) References Cited Stockton LLP U.S. PATENT DOCUMENTS (57) ABSTRACT 3,929,813 A 12/1975 Higuchi 3,962.447 A 6/1976 Higuchi et al. Hydrophilic transportable N-linked glycosyl dopaminergic 4,032,676 A 6, 1977 Heins et al. prodrug compounds and methods of their use. 4,190,672 A 2, 1980 Fahn 5,380,837 A 1/1995 Nakada et al. 5,639,737 A 6, 1997 Rubin 9 Claims, 22 Drawing Sheets

U.S. Patent Apr. 5, 2016 Sheet 1 of 22 US 9,302,982 B2

360- P=0.6349 340 320 500 280 260 240 220 200 180 160 140 120 P=0.0066 100 80 60 40 20 Z Before(n=2 2 ) AfterNoC ( r 6) AfterMPTP(n=18) FG 1

U.S. Patent Apr. 5, 2016 Sheet 3 of 22 US 9,302,982 B2

O Before IPX-750 IPX-750 St week 700 IPX-750 2nd week ZIPX-750 3rd week NY IPX-750 4th week

as 500 3. , N soto 3.WN NN i 200 WN NZN

% SNN Z 3.3 t SNWN | 2NZN3N t NZN MPTP+NoCI(n=6) MPTP-IPX-750 MPTP-IPX-750 (20mg/kg)(n=6) (80mg/kg)(n=6) Roto-Rod test for different time of IPX-750 treatment FIG.3 U.S. Patent Apr. 5, 2016 US 9,302,982 B2

700 O After MPP After treotment of IPX-750 600 WOsh-Out 1st Week ZZ Wosh-out 2nd week

500

?

500

200

100

<> N N` MPTP+PX-750 MPTP+IPX-750 (20mg/kg)(n=6) (80mg/kg)(n=6)

FIG.4 U.S. Patent Apr. 5, 2016 Sheet 5 of 22 US 9,302,982 B2

6-OHDA+ 6-OHDA+ 6-OHDA 6-OHDA+ Apomorphine 0.9% NgC PX-750 IPX-750 (80mg/kg) (200mg/kg) Acute effects of IPX-750 FIG.5 U.S. Patent Apr. 5, 2016 Sheet 6 of 22 US 9,302,982 B2

is NZNZ W 2 N

N

r" as cry co - to r- in CN " . any led Sapko u000 U.S. Patent Apr. 5, 2016 Sheet 7 of 22 US 9,302,982 B2

is is is 55 E E2 E . . . . . ÉS 33 3 SSS3 -s a N. N. N. N. x:it a is s 33s is a g a set is is is is is ww. SS is a a r

sSS s SSSSSSSSSSSSSSSSSSS SXSS s s XXX RSSSSSSSSSSSSSSSSSSS Xs deRys SS sSSSS SSSSSSSSSSS

N sExx s XXXXXXSSSS s : wa + HE Š& eaw –2aÉs

• 2 - al a &YsS E . &Si; s SES ZZZZZZZZZZZZZZZ N

S. 2 S se S S S S 3 g S S S se c was saw was was views (SPU003S) au PO1-Dog U.S. Patent Apr. 5, 2016 Sheet 8 of 22 US 9,302,982 B2

OThe lost treatment 120 1st week wash-out 2nd week wo sh-out 3rd week WOsh-out OO

80

k (spu008S)ºupou-0308 60 40

O N N```` Hetero HetN Ø>s oC (PX-750 20mg/kg) Rot O-rod T t with IPX-750 (20mg/kg)/0.9% NoC in heterozygous/WT Mice FIG.8 U.S. Patent Apr. 5, 2016 Sheet 9 of 22 US 9,302,982 B2

U.S. Patent Apr. 5, 2016 Sheet 12 of 22 US 9,302,982 B2

|Gipons]fra

U.S. Patent Apr. 5, 2016 Sheet 13 of 22 US 9,302,982 B2

Spl Cenotoxic Compounds. Spl cnd GLUT1 mRNA e.g. eioposide, cisplotin, -radiation decrease after myogenesis Myobicsts-kiyotubes Differentition

Repressor

re cities Sp1 Repressor. FO Cell cycle plO7 RelA of NFy

-27Rip to -J.3%ip Housekeeping - Eoholicer-fi) -ette to -2dio is -33bp to -99bp

Growth Factors e.g. insulin, Serum, PDGF, Oncogene products

IkB Phosphorylation (inhibitory subunit of NFkB)

Doponine-induced Apoptosis Inlommctory Cytokines c.g. IL-1, THF-, a FN- 7 FIG. 1 OA U.S. Patent Apr. 5, 2016 Sheet 14 of 22 US 9,302,982 B2

yPTP Insecticides:

e.g.,

-- Rolenone, 02/03 Agonists

pyrethroids, Alpha-Synuclein chlorpyrifos, +

permethrin Growth foctors

e.g. Scrum, PDCF, l C2/03 Receptor Activotion Oncogene products Kurr1 mRNA Down-Regulation and Blocks Nurri Nuclear Trorislocation

ove CLU RNA f

+16.7Kbp to +1.8Kbp Enhancer-2E2) in intron 2

Guti ni 3 tip at 2.18%ip to 24Kip 80kDo cylosolic protein

tronscribed from 3'UTR of CLUI in recction to hypoxic

C or tumorigenesis, i.e., an

pge \ mRNA stabilizer protein

P53, HPTF Targeted Endonucleose Stabilized Sensitive it rRNA Ubiquinotor WHL Pro PI3K/AKT Gut frMA

Closes, Signaling ProteoSome Clucose Degradation deprivation Hific A inctive Hypoxie: Ferroprotein Sensor Co2t Fe2+ chelotion OH-pyridinones Growth Fociors: e.g. insulin, IGF-1 FIG. 1 OB U.S. Patent Apr. 5, 2016 Sheet 15 of 22 US 9,302,982 B2

jossaJdeu-c)

U.S. Patent Apr. 5, 2016 Sheet 16 of 22 US 9,302,982 B2

(01-6'0E005) uchopp1783 OXd10/pub(01-6(9.3935)

|dS

U.S. Patent Apr. 5, 2016 Sheet 18 of 22 US 9,302,982 B2

U.S. Patent Apr. 5, 2016 Sheet 19 of 22 US 9,302,982 B2

|-5H$

&###

U.S. Patent Apr. 5, 2016 Sheet 20 of 22 US 9,302,982 B2

udsoqd-90

U.S. Patent US 9,302,982 B2

30

U.S. Patent Apr. 5, 2016 Sheet 22 of 22 US 9,302,982 B2

|-

+ [×] US 9,302,982 B2 1. 2 PHARMACEUTICAL DOPAMINE 878); benzenemethanamines (e.g., U.S. Pat. No. 2,599,000; GLYCOCONUGATE COMPOSITIONS AND U.S. Pat. No. 5,190,965), piperidines (e.g., Budipine, U.S. METHODS OF THEIR PREPARATION AND Pat. No. 4,016,280; Biperiden, U.S. Pat. No. 2,789,110; Tri USE hexylphenidyl, U.S. Pat. No. 2.682.543), pyrrolidines (e.g., Procyclidine, U.S. Pat. No. 2,891,890), tropines (e.g., Benz PRIOR RELATED APPLICATIONS tropine, U.S. Pat. No. 2,595.405; Hyoscyamine, Fodor et al. 1961), criptines (e.g., Bromocriptine, U.S. Pat. No. 3,752, The present application is a continuation-in-part of U.S. 814) and ergolines (e.g. Pergolide, U.S. Pat. No. 4,166,182). patent application Ser. No. 1 1/965,444, filed Dec. 27, 2007, Metabolic replacement therapy compounds that are endog which issued as U.S. Pat. No. 8.252,752 on Aug. 28, 2012, 10 enously converted to dopamine, e.g., Levodopa, results in which is a continuation of U.S. patent application Ser. No. stimulation of both D1-like and D2-like dopaminergic fami 10/625,645, filed Jul 22, 2003, which issued as U.S. Pat. No. lies of receptors. While agonists are theoretically superior to 7.345,031 on Mar. 18, 2008. The present application is a Levodopa (i.e., because they should not be dependent on continuation-in-part of U.S. patent application Ser. No. enzymatic conversion), in clinical use they have been shown 12/913,543, filed Oct. 27, 2010, now U.S. Pat. No. 9,023,818 15 to lack the therapeutic potency of Levodopa. Direct acting D2 which is a continuation of U.S. patent application Ser. No. agonists (e.g., bromocriptine, lisuride and pergolide) have 1 1/343,266, filed Jan. 30, 2006, which is abandoned and is a shown limited efficacy in monotherapy and are primarily used continuation of U.S. patent application Ser. No. 09/547,501, as add-on therapy to L-Dopa. Recent identification of novel filed Apr. 12, 2000, which is abandoned. The foregoing pat structural classes of D1-selective isochroman dopamine ago ents and patent applications are incorporated herein by refer nists has led to renewed interest in possible use of D1 selec ence in their entirety. tive agonists in treatments for Parkinson's and other neuro logical diseases. However, any interest in dopaminergic FIELD OF THE INVENTION agonists has recently been tempered by reports that direct neural injections of dopamine may be toxic to certain neurons The invention relates generally to dopaminergic composi 25 (e.g. Rabinovic et al., Luo et al.); possibly by overloading tions and methods of their preparation and use for treating nascent vesicular monoamine transporters (e.g. Reveron et neurological diseases including Parkinson's and related dis al.) and inducing apoptosis (e.g. Hou at al.; Panet et al.: CaSCS. Weingarten et al.) through postulated formation of toxic dopamine oxidative metabolites (e.g., Daily et al.) that might BACKGROUND OF THE INVENTION 30 be transportable by a dopamine transporter (DAT; Xia et al.). L-dopa, Levodopa, Cardiodopa (an inhibitor of dopa Parkinson's disease reportedly affects one person in fifty decarboxylase), Deprenyl (inhibiting dopamine degrading over fifty years of age and one in twenty over seventy. A monoamine oxidase), Sinemet (a controlled release form of degenerative disease of the nervous system described in 1817 Levodopa) and their combinations and derivatives suffer from and characterized by progressive loss of nigrostriatal neu 35 many major disadvantages. Commonly these agents have rons, a shaking palsy with tremor at rest, muscular rigidity poor aqueous solubility and relatively short half-lives. and slowness of movement, the possible etiology, the cell Observed side effects accompanying chronic use include biology, biochemistry and pathophysiology are still areas of motor fluctuation, dysfunctions, peak-dose dyskinesia, intense speculation and ongoing research. Diseases related by requirements for frequent dosing involuntary movements, clinical symptomology, and progressive clinical symptomol 40 psychosis, confusion, visual hallucinations, bradykinesia, ogy in Parkinson's patients, including post-encephalitic Syn rigidity, tremors, gastrointestinal and gentiourinary dyanto dromes, Wilson's disease, Parkinsonism secondary to cere nomia, hypotension and cognitive decline (Hurtig, 1997). brovascular trauma and stroke, dementia, Alzheimer's Often after 3-5 years of treatment patients develop complex disease, Lou Gehrig's disease, psychomotor retardation, cer dose-related unpredictable response fluctuations usually tain schizophreniform behavior, anxiety and depression. The 45 leading to a progressive decrease in therapeutic efficacy and primary biochemical defect in Parkinson's disease is loss of also possible onset of serious side effects such as abnormal nigrostriatal dopamine synthesis. involuntary movements, end-of-dose deterioration and Catecholamines including dopamine, norepinephrine and abrupt near instantaneous on-off changes in patient disability. epinephrine are produced by chromaffin cells in the adrenal Adaptation” by neural tissues to chronic administration is medulla responding as a specialized ganglion to sympathetic 50 complex, and may include down-regulation of dopamine enervation from preganglionic fibers of the splanchic nerve. receptor expression as well as metabolic changes in post However, catecholamines do not cross the blood-brain bar striatal neurons. In certain patients dyskinesia and response rier, hence, the need for synthesis within the CNS. L-Dope, fluctuations would desirably be controlled by continuous the precursor of dopamine, readily crosses the blood-brain intravenous infusion of drug at a constant level, however, barrier but is unstable and rapidly inactivated in blood. 55 because of the low aqueous solubility of Levodopa this is not Levodopa (a precursor of dopamine) and its derivatives are a feasible solution. In addition to these neurologic disadvan used for treatments of Parkinson's disease. Dopamine admin tages, metabolism of oral dope compounds to dopamine in the istered intravenously, while not crossing the blood brain bar stomach and gastrointestinal tract (even in the presence of rier, binds D1-like and D2-like dopamine receptors in the decarboxylase inhibitors) can often lead to unwanted side periphery and is reportedly useful in certain treatments for 60 effects including severe nausea and hypotension. Levodopa peripheral defects Such as congestive heart failure and hyper methyl and ethyl esters given orally suffer many of these same tension (e.g., Kuchel, 1999). problems. Thus, all current therapies suffer from serious side Pharmaceutical compositions for treatments of Parkin effects, bioavailability problems, or both, and there has been sonism include: Levodopa (e.g., U.S. Pat. No. 3.253,023, a long-felt need for improved pharmaceutically active agents U.S. Pat. No. 3,405,159), Carbidopa (e.g., U.S. Pat. No. 65 for metabolic replacement therapy in Parkinson's and related 3,462.536), aminoindans (e.g., U.S. Pat. No. 5,891.923), ben diseases (Hurtig, 1997). There has also been a long-standing Zhydrylamines (e.g., Diphenhydramine, U.S. Pat. No. 2,427, need for improved dopaminergic catechol agonists with US 9,302,982 B2 3 4 improved bioavailability and penetrability of myelinated may be coupled (or uncoupled) to modulatory second mes nerves, i.e., for peripheral use in treatments of e.g. hyperten senger systems, (e.g., down-regulation of DAT accompany sion and congenital heart diseases. ing activation of protein kinase C by phorbolesters), and ionic Molecular cloning studies have identified several genes currents (Melikian et al., 1999; reviewed in Figlewicz, 1999). encoding dopamine receptors. D1-like receptors, (recognized Radiotracer imaging methods have been used to localize DAT pharmacologically by the SCH23390 agonist), activate ade (e.g., within the nucleus accumbens and mid-brain regions) nylate cyclase resulting in increased intracellular cAMP. Two and D1 and D2 receptors (e.g., in nigrostrial pathways) in the gene products have been identified, i.e., D1A and D1B. D1B brains of normal subjects, as well as in patients with Parkin may have been previously identified pharmacologically as D5 son's disease and neuropsychiatric diseases such as Schizo and may be responsible for SCH23390 specific agonist activ 10 phrenia (reviewed in Verhoeff 1999). Structure activity stud ity. D2-like dopamine receptors, (recognized pharmacologi ies of antagonists have Suggested that: (i) the DAT transporter cally by spiperone and Sulpride agonists), appear to be may be sensitive to N-substitution (Choi et al., 2000); (ii) encoded by three genes with multiple possible splice variants N-phenyl-substituted analogues may inhibit transport expressed in different brain regions, i.e., D2S, D2L, D3 and (Prakash et al., 1999: Husbands, et al., 1999); (iii) certain D4. D2-like receptors do not appearadenylate cyclase-linked 15 energetically unfavored boat conformations of rings may and may decrease intercellular cAMP levels, perhaps a result have high affinity for DAT (Prakash at al., 1999); (iv) struc of kinase-mediated phosphorylation. D2-like receptors have tural rearrangement of the DAT protein may occur and be been identified as a potential target for development of anti required for inward transport (Chen et al., 2000); (v) the DAT psychotic agents and treatments for Schizophrenia, i.e., based protein contains an endogenous Zn" binding site (Loland et on antipsychotic effects of chlorpromazine occurring with al., 1999); (vi) DAT transporter function is sensitive to aro resultant drug-induced Parkinson's symptoms and increased matic substitutions (Husbands, et al., 1999); and, (vii) appar risk of tardive dyskinesia. Schizophrenia is (at present) ent ordered kinetics for DAT transporter function is Nabind believed to result from hyperactive dopaminergic transmis ing first, then dopamine and then Cl. sion in the mesolimbic region of the brain. While antipsy Several tissue enzyme systems exist for altering catechola chotic drugs with fewer side-effects have been developed 25 mines, including dopamine. Monoamine oxidases, MAO-A (e.g., haloperidol, fluphenazine, clozapine, olanzapine, ris in neural tissues and MAO-B in other tissues including stom peridone), to date, no consensus antipsychotic dopaminergic ach and intestine, are oxioreductases that deaminate dopam antagonist pharmacologic or receptor profile has emerged ine and other catecholamines with preferential activity mani and approaches under active consideration include: (i) com fest for 2-phenylethylamine and benzylamine. Catechol-O- bination approaches for blockade of D2-like and D1-like 30 methyltransferase is a cytosolic enzyme that catalyzes receptors as well as 5-HT, and C. adrenergic receptors; (ii) addition of a methyl group, usually at the 3 position of a selective approaches for blocking D2 subtypes, e.g., D3 and/ benzyl ring. O-methoxylated derivatives may be further or D4 or D2L/S and D4; and (iii) attempts to develop partial modified by conjugation with glucuronic acid. Non-neuronal agonists to compete with dopamine binding. dopamine transporter uptake mechanisms may also exist, In pharmacologic studies conducted over the past 20 years, 35 e.g., in kidney (Sugamoriet. al., 1999). the results seem to Suggest relatively stringent structural Oral delivery of drugs constitutes special chemical chal requirements for activation of the D1 receptors, particularly lenges, i.e., general simultaneous requirements for intestinal in regard to any nitrogenatoms present in the compound (e.g., penetration, blood borne delivery, blood-brain-barrier pen see Seller et al., 1991; Berger et al., 1989; Brewster et al., etrability and maintenance of functional (receptor binding 1990; Kaiseret al., 1982; Dandridgeet. al., 1984: Brewsteret 40 and/or metabolic) utility. CNS active drugs constitute yet al. 1990; Weinstock et al., 1985; Riggs et al.; Seiler et al., additional special and challenging problems, i.e., low pH 1982: Shah et al., 1996: Knoerzeret al., 1994). In addition, the stability (or protection) and intestinal transport. Intestinal nature of the terminal group (i.e., amino), or presence or intracellular transport mechanisms for amino acids, vitamins length of an n-alkyl chain (Iorio et. al., 1986) may reportedly and Sugars are varied. Glucose transport has recently been influence binding interactions at D1 sites. Based on experi 45 reviewed (Takata et. al., 1997). Transport mechanisms for ence with different pharmacophores, several receptor models glucose include intestinal transport vesicles and Na/glucose have been proposed (Seiler and Markstein, 1989; Petersson co-transporters (SGLTs), i.e., driving active transport of glu et. al., 1990; Brewster et. al., 1990; Knoerzer et al., 1994; cose and galactose across the intestinal brush border by har Snyder et. al., 1995; Minor et. al., 1994). By comparison, nessing Na' gradients across the cell membrane. Net rates of pharmacologic Studies of D2-like receptors suggest some 50 vesicle transport and exocytosis have been estimated to be in what less rigid overall structural requirements, but also the range of 10 thousand to 1 million per second (Wright et. restrictions around any nitrogen atoms (e.g., see McDermed al, 1997). Missense mutations in SGLT1 reportedly result in et al. 1979: Freeman and McDermed, 1982.: Liljefors et al., potentially lethal inability to transport glucose and galactose 1986; van de Waterbeemdet al., 1987). (Martin et. al., 1996). Certain sugar specificity’s, structural The Na/Cl dependent dopamine transporter, DAT1, 55 requirements and capabilities of Na'-dependent glucose granule system mediates calcium-dependent outward transport carriers have been investigated with impure receptor dopamine release into the synaptic cleft and inward energy membrane preparations, and/or mixtures of receptors, with dependent dopamine vesicular re-uptake into the cytoplasm the findings that the glucosyl transporter in human erythro of presynaptic neurons. Loading of biosynthetic dopamine cytes (i.e., GLUT1): (i) seems to require that the ring oxygen into granules is effected by the vesicular monoamine trans 60 atoms at positions C1, C3, C4, and possibly C6, be capable of porter (VMAT2: reviewed in Miller et. al., 1999). DAT may forming hydrogen bonds with the transporter protein, and (ii) also control movements of other monoamines in braintissues. a hydrophobic group at C5 may increase affinity for the Cocaine, amphetamines, phencyclidine and certain anti-de transporter (Barnett et al., 1973). Intestinal glucose trans pressants and uptake inhibitors interfere with dopamine porter mechanisms reportedly prefer: (i) B-anomers to C-ano transport by DAT (e.g., see Jones at al., 1999; Giros et. al., 65 mers; (ii) B-D-glucose to B-D-galactose; and, (iii) 1992). DAT function may be regulated by steroid hormones, B-glucoside>O-glucoside>B-galactoside>O-galactoside. The has second order dependence on Na" (Earles et. al., 1999) and C-anomers of glucose and galactose were reportedly hydro US 9,302,982 B2 5 6 lyzed to their aglycone constituents during a non-Na'-depen Neuraxial delivery of many cyclic and heterocyclic com dent desglucosylation transport (Mizuma et. al., 1992, 1993, pounds is problematic. Objects of the invention provide new 1994). Apparently unrelated studies of antiviral glycosides classes of CNS-active compounds which circumvent prob have reportedly found that: (i) C1 phenyl-substituted glyco lems of low aqueous solubility of dopaminergic compounds sides and para-Substituted butyl-phenyl derivatives may and the varied transport, receptor binding and stability prob inhibit glucose transporters (Arita et al., 1980); (ii) C1 O-acyl lems encountered with dopaminergic drugs, including their glycoside derivatives with alkyl chains or carbonyl groups (as relatively poor blood-brain barrier penetrability. an aglycone Substituent) may act as non-penetrating inhibi tors of glucose transport (Ramaswamy et. al., 1976); and (iii) SUMMARY OF THE INVENTION 10 1-5-anhydroglucitol and 6-deoxyglucose may be transport Hydrophilic transportable N-linked glycosyl dopaminer able (Alvarado et. al., 1960). Thus, like dopaminergic recep gic prodrug compounds, their methods of preparation and tor binding, the art Suggests that special chemical structural uses are disclosed. The compounds are described by the gen requirements may exist for intestinal transport. eral structure of FORMULAI, Unlike intestinal transport, neural glucose transport at the 15 blood brain barrier appears to be mediated: (i) by endothelial A-B-D-E Formula I cells and a sodium-independent facilitative transporter wherein: each of “ ” constitutes a single bond; the 'A'- known as GLUT1 (Kumagaiet. al., 1999); and (ii) at neuronal moiety constitutes a dopaminergic cyclic radical; the 'B'- cells by GLUT3 (Vannucci, S.J. et al., 1998). GLUT1 also a moiety constitutes a “bridging alkyl moiety; the “D'-moiety predominant glucose transporter expressed in human eryth constitutes a nitrogen “linker'; and, the “E”-moiety consti rocytes. Neural tissue is almost entirely dependent on glucose tutes a saccharide as set forth further below, e.g., a mono-, di-, transport for normal metabolic activity because tissue stores tri- or oligosaccharide. Preferred compounds thus configured of glucose are low (relative to demand). Thus, current under are: (i) ligands for a dopaminergic receptor; (ii) transportable standing Suggest that GLUT1/3 competitive agents might in an intact form by an intestinal saccharide transporter sys have undesirable side effects. Specificity of neural GLUT1/3 25 tem; (iii) transportable in an intact form by an endothelial is an area of active current investigation. blood brain barrier saccharide transporter system; (iv) trans In mammals, glucuronidation of drug metabolites is com portable by neural dopamine transporters; and (V) metaboliz mon, e.g., involving the hepatic glucuronostransferase sys able to provide a metabolic replacement therapy. tem and enzyme systems in kidney and intestine. Catechola mine glucuronidation is reportedly an important metabolic 30 BRIEF DESCRIPTION OF THE FIGURES pathway in the rat and dopamine glucuronides we reportedly identified in rat cerebrospinal fluid (Wanget. al., 1983). Many FIG.1. MPTP treatment of mice resulted in neural lesions drugs investigated for dopaminergic agonists and antagonist that significantly reduced coordination and balance as mea properties are reportedly metabolized and/or excreted as glu sured by the time animals were able to remain on a slowly curonides, e.g., SCH23390 (a Schering prototype D1 receptor 35 rotating rod, “Rota-Rod', as depicted graphically in the figure antagonist; Barnett, et. al., 1992), CGS 15873 (a Ciba-Geigy and as set forth further in regard to EXAMPLE 13, below. dopamine agonist; Leal et. al., 1992), Carmoxirole (a Merck Rota-Rod performance times of C57BL/6 mice before and dopamine agonist; Meyer et. al., 1992), Olanzapine (a Lilly after lesioning with MPTP, or as a negative control, NaCl. dopaminergic compound; Mattiuz et al. 1997) and CP-93, FIG. 2. MPTP-lesioned mice (FIG. 1) were treated with 393 (a Pfizer anxiolytic drug candidate; Prakash et al., 1998). 40 COMPOUND #1 twice daily and tested for Rota-Rod perfor Within this general class of cyclic Parkinson's drugs, several mance times at weekly intervals as depicted graphically in the investigators have suggested glucuronidation as one common figure and set forth further in regard to EXAMPLE 13, below. mechanism for targeting removal of phenolic drugs by uri FIG.3. Significant improvements in Rota-rod performance nary and biliary excretion, e.g., Mico et al., 1986 (indolone times were observed in COMPOUND H1-treated MPTP-lei agonists); Gerding et al., 1990 (N-0437, a tetralin agonist); 45 Sioned mice (FIG. 1), i.e., as as depicted graphically in the Wang et al., 1983 (catecholamines); Green et. al., 1996 (hy figure and set forth further in regard to EXAMPLE 13, below. droxylated and carboxylated phenolic compounds); Poo (p<0.05.) chiariet. al., 1986 (Ibopamine); Claustreet. al., 1990 and FIG. 4. Washout experiments to determine how long Alexander et. al., 1984 (dopamine). Shindo et. al., 1973 therapeutic effects remained evident after cessation of COM reportedly studied absorption of L- and D-dopa in vitro in 50 POUND #1 therapy in MPTP-leisioned mice, i.e., as as ligated rat intestinal loops and found active transport and depicted graphically in the figure and set forth further in metabolism to dopamine glucuronides. regard to EXAMPLE 13, below. (p<0.05). The blood brain barrier effectively limits neuraxial deliv FIG. 5. The effects of COMPOUND # 1 were tested in the ery of many pharmaceutically active compounds, including 6-hydroxydopamine (6-OHDA) rat nigrostriatal-leision dopamine. Approaches disclosed for delivering drugs to the 55 model and rotational behavior induced by apomorphine, i.e., brain include lipophilic additions and modifications of hydro as depicted graphically in the figure and set forth further in philic drugs, (e.g., N-methylpyridinium-2-carbaldoxime regard to EXAMPLE 14, below. chloride: 2-PA; U.S. Pat. Nos. 3,929,813 and 3,962,447: FIG. 6. Treatment of 6-OHDA leisioned rats (FIG.5) with Bodor et. al., 1976, 1978 and 1981); linkage of prodrugs to COMPOUND #1 significantly decreased apomorphine-in biologically active compounds, (e.g., phenylethylamine 60 duced rotation in 6-hydroxydopamine-leisioned animals, i.e., coupled to nicotinic acid and modified to form N-methylni as depicted graphically in the figure and set forth further in cotinic acid esters and amides; Bodor et. al., 1981 and 1983; regard to EXAMPLE 14, below. (p<0.05). PCT/US83/00725; U.S. Pat. No. 4,540,564); derivatization to FIG. 7. Nurr1 genetically deficient stem-cell knockout centrally acting amines (e.g., dihydropyridinium quaternary mice were treated with COMPOUND # 1 or Saline and amine derivatives; PCT/US85/00236); and enclosing com 65 observed weekly for RotoRod performance (time) as pounds in cyclodextrin complexes (e.g., Yakshet. al., U.S. Pat. depicted graphically in the figure and set forth further in No. 5,180,716). regard to EXAMPLE 15, below. *p-0.05. US 9,302,982 B2 7 8 FIG. 8. Washout of COMPOUND #1 treatment in Nurr1 vides N-phenyl derivative compounds that bind DAT and do deficient mice as evidenced by returining disability in the not inhibit normal loading of Na" and Cl by DAT, or inhibit Rota-Rod performance time measurements (FIG. 1) of ani normal structural conformational changes in the DAT protein mals after removal of therapy, i.e., as depicted graphically in required for inward transport of dopamine. In other objects the figure and as set forth further in regard to EXAMPLE 15, the invention provides domaminergic agonist and partial ago below. *p-0.05. nist compounds that do not down-regulate transporter func FIG. 9 schematically depicts the disclosed regulatory tion, e.g., by activating a cellular protein kinase. In other mechanisms operative at the Tyrosine Hydroxylase gene by objects, the invention provides compounds that are not modi cis- and trans-acting elements affecting the promoter region fied by monoamine oxidases, catechol-O-methyltransferase of the gene, i.e., as set forth further in regard to EXAMPLE 10 or glucuronidation mechanisms operative in the intestine and 17, below. In FIG. 9-12, positive (+) signs associated with stomach. In still other objects, the invention provides dopam arrows depict that the factor at the beginning of the arrow inergic prodrug NCEs which, unexpectedly, are not metabo increases expression or activity of the factor at the arrow lized or glucuronidated by intestinal transport mechanisms, head; negative (-) signs depict that the indicated factor and instead, are transported intact. In still other objects, the decreases expression or activity; and, all indicated activities 15 invention provides a class of dopaminergic NCES that, unex are as they would occur in wild-type neural cells in the sub pectedly, do not alter intestinal or brain glucose or dopamine Stantia nigra. transport in an adverse manner. In other objects, the invention FIG. 10 schematically depicts the disclosed regulatory provides dopamine NCEs having a high degree of innate mechanisms operative at the GLUT1 (vascular endothelial aqueous solubility, e.g., up to 500 mg/ml. Thus, in other glucose transporter) gene by cis- and trans-acting elements objects the invention provides hydrophilic therapeutic NCE affecting the promoter region of the gene, i.e., as set forth prodrug pharmaceutical compositions which, when adminis further in regard to EXAMPLE 17, below. tered orally are sequentially transportable in an intact form FIG. 11 schematically depicts the disclosed regulatory across the intestine; are also transportable in blood; are also mechanisms operative at the GLUT3 (neural glucose trans transportable across the blood brain barrier; are transportable porter) gene by cis- and trans-acting elements affecting the 25 by DAT transporters; and are capable of binding to dopamine promoter region of the gene, i.e., as set forth further in regard receptors. In other objects, the invention provides pharma to EXAMPLE 17, below. ceutical compositions for metabolic replacement therapy in FIG. 12 schematically depicts the disclosed regulatory subjects with Parkinsonism and related diseases. In yet other mechanisms operative at the DAT (dopamine re-uptake neu objects, because the special aqueous solubility of the instant ral transporter) gene by cis- and trans-acting elements affect 30 NCE prodrug compounds, the invention provides novel phar ing the promoter region of the gene, i.e., as set forth further in maceutical compositions containing relatively high concen regard to EXAMPLE 17, below. trations of active ingredients allowing administration of a FIG. 13 schematically depicts the disclosed regulatory therapeutically effective unit dose in a relatively small vol mechanisms operative at the Nurr gene by cis- and trans ume, i.e., a particular advantage for multi-dose, timed-re acting elements affecting the promoter region of the gene, i.e., 35 lease, Subcutaneous and Intradermal, intranasal, buccal, and as set forth further in regard to EXAMPLE 17, below. trouch pharmaceutical compositions, as well as for pharma FIG. 14 schematically depicts coordinate regulatory ceutical compositions designed to achieve steady-state mechanisms exerted by SP1, SP2, Nurr1 and Nurr2 at the plasma concentrations. In still other objects, the invention gene regulatory regions of the DAT, VMAT2 (the dopamine provides bioavailable dopaminergic NCE prodrugs lacking in vesicular storage transporter), TH, GLUT1, GLUT3, D1A 40 a reactive carboxylic acid, making co-administration of a (Dopaminergic receptor-1A) and membrane transporters decarboxylase or a monoamine oxidase inhibitorunnecessary Adenine nucleotide translocate-2 and beta-galactosidase-sia in a treatment for Parkinson's disease. In other objects, the lyltransferase, i.e., as set forth further in regard to EXAMPLE invention provides dopaminergic NCE prodrug compounds 17, below. that may be activatable by a brain amidase, e.g., glucosamini 45 dase, galactosaminidase and the like. In other objects, the DETAILED DESCRIPTION OF THE PREFERRED invention provides dopaminergic NCE prodrug compounds EMBODIMENT that may be capable of promoting their own transport by upregulating expression and transport rates of intestinal sac Objects of the invention provide improved novel chemical charine transporters in gastrointestinal cells. In other objects, entities (NCEs) for metabolic replacement therapy in Parkin 50 the invention provides dopaminergic prodrug NCE com sons and related disease. Objects of the invention also pro pounds comprising a saccharide-transporter-enhancing func vide dopaminergic NCEs with simultaneously enhanced tionality, (i.e. a dopaminergic moiety), that may compensate hydrophilicity, i.e., facilitating blood-borne transport; for Parkinson's malabsorption, erratic gastrointestinal improved intestinal transport; improved blood-brain-barrier absorption, irregular gastric contractions, and the like. In still endothelial transport; and improved neuronal transport. In 55 other objects, the invention provides dopaminergic NCE pro other objects, the invention provides NCEs that are N-substi drug compounds comprising a dopamine-receptor-enhancing tuted dopaminergic compounds that, unexpectedly, fulfill the functionality (i.e., a Sugar or oligosaccharide moiety) capable structural binding requirements of dopaminergic receptors of upregulating dopaminergic receptor function, i.e., particu despite their N-substitution. In yet other objects, the invention larly useful in advanced Parkinsonism where a limited num provides a NCES comprising dopaminergic (dihydroxy-phe 60 ber of functional nigrostriatal neurons may be available and nyl-) glycosyl-compounds which, unexpectedly, do not possible glutamate-induced dyskinesia is evident. In other inhibit intestinal glucose transporters, but instead, are trans objects, the invention provides stable dopaminergic pharma portable by these proteins. In other objects, the invention ceutical compositions Suitable for transcutaneous delivery, provides N-Substituted compounds unexpectedly transport i.e., not possible with many prior dopa compounds because of able by dopamine transporters (DAT) in the brain. In still 65 their chemical instability. other objects, the invention provides N-phenyl-derivatives Applicants do not believe it has been appreciated, until that do not inhibit DAT. In other objects, the invention pro now, that a single chemical entity can affect glucose trans US 9,302,982 B2 10 porters and dopamine receptors to promote its own transport and receptor binding. Fischer et. al., 1995 reported that Formula II tryptamine, 5-OH-tryptamine and dopamine may elicit about R3 a 3-5 fold increase in glucose transport with about 1.8- and R R4 1.5-fold increases in the amount of cell surface GLUT1 and 1 GLUT4 transporters, respectively. Whitfield et al., 1974 sug gested that catecholamines, including dopamine, might stimulate carrier-mediated transport of 3-O-methylglucose B and galactose in avian erythrocytes. Coffey at al., 1994 Sug- 10 gested that binding of a radiolabeled tropane to a rat striatal wherein, membrane dopamine receptors might be increased in the Ring 1 comprises am optionally Substituted cyclic or het presence of Sucrose, fructose and mannose, but not dextrose erocyclic ring, or an optionally Substituted aromatic ring, or N-methyl-D-glucosamine (Coffey, et al. 1994). These 15 composed of about 4 to about 8 carbonatoms, among which respective reports utilized separate dopaminergic and Sugar are counted “X” and “Y”: preferably, Ring 1 comprises an chemical entities, not a single chemical entity, to achieve their optionally Substituted aryl or heteroaryl ring; and most pref measured results. erably, a substituted aryl ring; wherein, R. R. R. and R. comprise the Subject optional ring Substituents; Embodiments of the invention provide dopaminergic com-o. each of X and Y are optional and when present comprise a pounds having improved hydrophilicity, bioavailability and carbon atom, a halogen atom or a lower alkyl, preferably, a blood brain barrier penetration. In other embodiments, the carbon atom or a lower alkyl chain having 2 carbon atoms, invention provides novel compounds capable of binding to a most preferably a single carbon atom; dopaminergic receptor, a dopamine transporter and a glucose Ro comprises hydrogen; transporter protein. In other embodiments, the invention pro- 25 R. R. or R comprise a group selected from among hydro vides dopaminergic pro-drug compounds that are transport gen, hydroxyl, halogen, halo-lower alkyl, alkoxy, alkoxy able by all of the following: namely, intestinal transporters, lower alkyl, halo-alkoxy, thioamido, amidosulfonyl, alkoxy blood transporters and blood-brain-barrier transporters. In carbonyl, carboxamide, amino-carbonyl, and alkylamine yet other embodiments, the invention provides dopaminergic carbonyl: prodrug compounds comprising glycosyl-pro-drug com- 30 R comprises hydroxyl; and, preferably, both R- and R pounds that are: (i) ligands for a dopaminergic receptor; (ii) comprise hydroxyl and R and R comprise hydrogen. transportable in an intact form by an intestinal saccharide “Bridge', when used in reference to the B-moiety, of FOR transporter system; (iii) transportable in an intact form by an MULAI (Supra), is intended to mean a group according to endothelial blood brainbarrier saccharide transporter system; FORMULA III, below, (as depicted linked through single (iv) transportable by neural dopamine transporters; and (v) 35 bonds to each of the A-moiety and the D-moiety, Supra): metabolizable to provide metabolic replacement therapy. In yet other embodiments, the invention provides novel dopam Formula III inergic agonist and partial agonist compounds finding a vari Rs ety of different potential therapeutic uses in treating periph 40 eral diseases including e.g., congestive heart disease and Y. D hypertension. ofRs' For purposes of organizing the following disclosure, as R6 well as, improved understanding of the scope and breadth of the instant compounds and their constituent structure, 45 wherein, embodiments of the invention are described by the general Z is optional and when present comprises a lower alkyl structure of FORMULAI, optionally substituted with Rs and Rs; preferably, Z is absent or a lower alkyl comprising 1 or 2 carbon atoms; most pref A-B-D-E Formula I erably, Z is absent or a one carbon atom; and, Rs and Rs. 50 (when present) and R and R (when present) are groups wherein: each of “ ” constitutes a single bond; the 'A'- selected from among hydrogen, hydroxyl, alkoxyl, carboxyl, moiety constitutes a dopaminergic cyclic radical; the 'B'- alkoxylcarbonyl, aminocarbonyl, alkylamino-carbonyl and moiety constitutes a “bridging alkyl moiety; the “D'-moiety dialkylamino-carbonyl. constitutes a nitrogen “linker'; and, the “E”-moiety consti “Linker, when used in reference to the D-moiety, FOR tutes a saccharide as set forth further below, e.g., a mono-, di-, 55 MULAI (supra) is intended to mean an optionally R-sub tri- or oligosaccharide. While certain preferred instant com stituted amide or amine linking the B-moiety with the E-moi pounds according to FORMULA I are set forth below as ety, i.e., through each of two single bonds, according to representative examples, i.e., FORMULAS VII and VIII, FORMULA IV, below (depicted linking the B- and E-moi below, whereby certain of the preferred constituents are dis eties of FORMULA 1): namely, closed, before addressing the specifics, the meanings of gen 60 eral terms relating to FORMULAI are provided as follows: namely, Formula IV "Dopaminergic cyclic radical”, as used in reference to the “A-moiety'. FORMULA I (supra), is intended to mean a 65 group according to FORMULA II, below, (as depicted linked through a single bond to the B-moiety, Supra): US 9,302,982 B2 11 12 wherein, N comprises a nitrogen atom of a primary or and, while for purposes of illustration the Fischer and Hay secondary amine or an amide, preferably R, is a hydrogen or worth formulas are set forth in FORMULAS Va, Vb, Vc and methyl, most preferably, R, is hydrogen. Vd depicting the configuration of a glucosyl residue, the Thus, according to the foregoing disclosure, the assem Scope of the invention is not intended to be so limited, as set blage of constituents A-B-D-E (FORMULAI) comprises forth further in the accompanying disclosure, below: compounds having the general structure according to FOR The numbers “1”, “2, “3”, “4”, “5”, “6” and the like MULAV: namely, appearing in FORMULAVIa and VIb, are intended, to refer to particular numbered carbon atoms in the respective differ ent Sugar residues, e.g., C1, C2, Cs, C4, Cs and Co. (i.e., Formula V 10 CHOH). In certain optional embodiments, carbon atoms R3 depleted in FORMULAS VIa, VIb, VIc and VId may be R R4 optional, e.g., in trioses C. C. and C are absent. In aldoses, according to convention, positional numbering of carbon 1 atoms is initiated from the chain terminal aldehyde and in 15 ketoses, from the chain terminal carbon atom nearest the ketone. In alternative embodiments, C., C and C (FORMU R-SS,Rss-Rs LAS VIa, VIb, VIc and VId) are optional; preferably, C is present; most preferably, all of C, C and C are present; ... n. The optional dotted line connecting the Cs oxygenatom to p1Ns. the C carbon atom (FORMULAVIb) is intended to mean an optional ester bond, in the absence of which bond FOR MULAVIb is acyclic as depicted in FORMULAVla: wherein, the constituents of Formula V are as set forth In certain embodiments, the linkage of the E-moiety to the above; the relationship of “Z” with Rs. Rs. RandR may be D-moiety amine or amide (Supra) occurs through a single cis or trans; and, the relationship of “N” with R. R. R. “Z” 25 bond formed between the subject amine or amide nitrogen and “E” may be either cis or trans. and either of the C, C, C, C, Cs or C carbon atoms of the “Saccharide' is intended to mean a mono-, di-, tri- or subject sugar (FORMULASVIa-VId). Preferably, with hexo oligosaccharide made up of n sugar Subunits linked to each syl residues the linkage is between the D-moiety amine or other by glycosidic bonds, which Subunits, when n is greater amide and the C. C. C or Cs carbon, most preferably, the than 1, may be the same or different in respect to: (i) the type 30 linkage is between the D-moiety amine oramide and the C or of constituent Sugar residues (e.g., homo- or heteropoly C. carbon. Preferably, with pentosyl residues (i.e., the C. meric); and, (ii) the localization of axial and equatorial ring residue depicted in FORMULAS VIa-VId is absent), linkage Substituents, i.e., Rs (supra); (iii) the number of carbon is between the D-moiety amine or amide and the C or Cs atoms (i.e., C Supra); and (iv) the ring carbon locations and carbon atom (i.e., as depicted in FORMULAS VIa-VId). orientations of hydroxyl groups. 35 Preferably, with tetraosyl residues (i.e., when both C and C. "Sugar, used interchangeably with monosaccharide, of FORMULAS VIa-VIdare absent), linkage is between the when used in reference to constituents groups of the “B-moi D-moiety amine or amide and the Cs or C carbon atom (i.e., ety” (Supra), is intended to mean a Substituted or unsubsti as depicted in FORMULAS VIa-VId). Preferably, with trio tuted Sugar residue having 3 carbon atoms (trios) 4 carbons syl residues (i.e., when C. C. and C of FORMULAS VIa (tetraose), 5 carbons (pentose), 6 carbons (hexose), 7 carbons 40 VId are absent), linkage is between the D-moiety amine or (heptose), 8 carbons (octose) or 9 carbon atoms (nonose). amide and the Cs or C carbon (i.e., as depicted in FORMU FORMULAVIa, VIb, VIc and VId illustrate the interrelated LAS VIa-VId). Most preferably, the E-moiety is hexosyland straight chain, hemiacetal and acetal forms of a hexose Sugar, linkage is between the D-moiety amine or amide and the C. i.e., depicted in FORMULA VIa and VIb using a modified C or C carbon atoms. Fischer projection formula, and depicted in FORMULASVIc 45 The E-moiety optional substitutions according to FOR and VId using modified Haworth projection formulas: MULAS VIa, VIb, VIc and VId comprise Rs, R. R. R. and

CH-R1 CH-R of - O R8 R10 se R10 R11 CH-R R9 Ro

Formula VIa Formula VIb Formula VIc Formula WId

wherein, R; preferably, when the D-moiety forms a carbon to nitro The bi-directional arrows between FORMULAS VIa, VIb, gen bond with C, then Rs is hydrogen; preferably, when the VIc and VId are Intended to mean that the subject sugar 65 D-moiety forms a carbon to nitrogenbond with C, then R is residues are interconvertible between Straight chain, aldosyl, hydrogen; when the D-moiety forms a carbon-to-nitrogen furanosyland pyranosyl forms at Some equilibrium constant; bond with C, then Rio is hydrogen; when the D-moiety forms US 9,302,982 B2 13 14 a carbon-to-nitrogen bond with Cathen R is hydrogen; most erythro-tetrauloSonic acid, threo-tetrauloSonic acid, ribo preferably, the D-moiety forms a carbon-to-nitrogen bond pentulosonic acid, arabino-pentuloSonic acid, Xylo-pentu with either of C or C. losonic acid, lyxo-pentuloSonic acid, gluco-hexuloSonic acid, Substituents Rs. R. Rio, R and R2 (according to FOR allo-hexuloSonic acid, altro-hexuloSonic acid, manno-hexu MULAS VIa-VId) are selected from hydroxyl, hydrogen, losonic acid, gulo-hexulosonic acid, ido-hexuloSonic acid, methyl, halogen, lower alkyl, halo-lower alkyl, alkoxyl, galacto-heXuloSonic acid, talo-hexulosonic acid and their ketone, carboxyl, amine, amido, N-acetyl, N-methyl, derivatives. Representative aldaric acids include erythraric N-linked lower alkyl, aminocarbonyl, alkylaminocarbonyl, acid, threaric acid, ribaric acid, arabinaric acid, Xylaric acid, dialkylaminocarbonyl, phosphate, Sulfate, and thiol. In cer lyXaric acid, allaric acid, altraric acid, glucaric acid, mannaric tain alternative embodiments, R may be a monosaccharide 10 acid, gularic acid, idaric acid, galactaric acid, talaric acid and or disaccharide, with the proviso that R when present as a their derivatives. Representative of amino Sugar include Substituent in a monosaccharide glucosyl Sugar R2 is not erhtyrosamine, threosamine, ribosamine, arabinosamine, carboxyl, i.e., the instant Sugar is not a C-glucuronic acid; Xylosamine, lyxosamine, allosamine, altrosamine, glu Preferably, two of either Rs. R. R. R. and R are cosamine, N-acetylglucosamine, N-methlglucosamine man hydroxyl; most preferably, Ro and R2 are hydroxyl. 15 nosamine, gulosamine, idosamine, galactosamine, Representative examples of E-moiety Sugar residues talosamine and their derivatives. Representative uronic acids include the following: namely, polyhydroxy Caldehydes include erythroSuronic acid, threoSuronic acid, ribosuronic (e.g. aldoses and ketoaldoses); polyols resulting from e.g., acid, arabinoSuronic acid, Xylosuronic acid, lyxoSuronic acid, reduction of the Caldehyde carbonyl to a hydroxyl (e.g., allosuronic acid, altroSuronic acid, glucuronic acid, manno alditols and ketoses); polyhydroxy acids resulting e.g., from Suronic acid, gulosuronic acid, idoSurnic acid, galactosurnilc oxidation of the C aldehyde and/or the chain terminal acid, taloSuronic acid and their derivatives. Representative hydroxyl (e.g., aldonic, ketoaldonic, aldaric and ketoaldaric); keto-uronic acids include keto-crythroSuronic acid, keto amino-Sugars resulting from replacement of any hydroxyl in threoSuronic acid, keto-ribosuronic acid, keto-arabinosu the chain with an amino (e.g., aldosamines and ketosamines); ronic acid, keto-Xylosuronic acid, keto-lyxoSuronic acid, aldehydro-acids resulting e.g. from oxidation of only the 25 keto-alloSuronic acid, keto-altroSuronic acid, keto-glucu chain terminal hydroxyl in an aldehydro-sugar (e.g., uronic ronic acid, keto-mannoSuronic acid, keto-gulosuronic acid, acids and keto-uronic acids); and their various lactones, i.e., keto-idoSuronic acid, keto-galactosuronic acid, keto-talosur cyclic esters of hydroxy carboxylic acids containing one nic acid and their derivatives. Representative lactones include 1-oxacycloalkan-2-one structure. The Subject Sugars may be erythrolactone, threolactone, ribolactone, arabinolacton, straight chains and/or cyclic 3-, 4-, 5-, 6-, 7-, 8- and 9-mem 30 Xyloslactone, lyxoslactone, allolactone, altrolacone, gluco bered Sugar residues (e.g., hemiacetals and acetals) option lactone, mannolactone, gulolactone, idolactone, galactolac ally substituted and linked with the D-moiety as set forth, tone, talolactone and their derivatives. supra. Representative triosyl residues include the aldoses D Preferably, the subject E-moiety comprises an aldose or and L-glyceraldehyde and derivatives thereof e.g., glyceral ketose pentose or hexose Sugar selected from the group con dehyde and glyceric acid phosphates; the keto-Sugars D- and 35 sisting of D- and L-enantiomers of ribose, glucose, galactose, L-dihydroxyacetone and derivatives thereof. Representative mannose, arabinose, allose, altrose, gulose, idose, talose and tetraosyl residues include the aldoses D- and L-erythrose, their substituted derivatives. Most preferably, the subject threose, Streptose and apiose; the keto-Sugars D- and L-eryth E-moiety comprises an aldose pentosyl or hexosyl Sugar rulose; and derivatives thereof. Representative pentosyl resi selected from ribose, glucose, galactose, glucosamine, galac dues include the D- and L-aldoses ribose, arabinose, Xylose 40 tosamine, N-acetylglucosamine, N-acetylgalactosamine, and lyxose; the D- and L-ketoses ribulose and Xylulose; and, N-acetyl ribosamine, Xylose, mannose and arabinose. derivatives thereof. Representative hexosyl residues include “Di-sacchride', when used in regard to the subject E-moi aldosyl, furanosyl and pyranosyl Sugars e.g., cyclic and acy ety, is intended to mean a polymeric assemblage of 2 Sugar clic D- and L-aldoses such as allose, altrose, glucose, man residues. Representative examples of disaccharides include nose, gulose, idose, galactose, talose, fructose, glucono-1,4- 45 homo-polymeric (e.g., maltose and cellobiose) and het lactone, glucaro-1,4:6.3-dilactone, gluconofuranono-6,3- eropolymeric (e.g., lactose and Sucrose) assemblages of Sug lactone; the ketoses ribo-hexylose, arabino-hexulolose, Xylo ars as set forth Supra. hexylose and lyxo-hexylose; and derivatives thereof. “Tri-saccharide', when used in regard to the subject Representative 7-membered residues (i.e., heptosyl residues) E-moiety, is intended to mean a polymeric assemblage of 3 include e.g., Sedoheptulose and derivatives thereof, and, rep 50 Sugar residues, e.g., as set forth Supra. resentative 9-membered residues (i.e., nanosyl residues) “Oligosaccharide', when used in relation to the subject include N-acetylneuraminic acid and derivatives thereof. E-moiety, is intended to mean a polymeric assemblage of Also representative are, 2-deoxy-ribos, 6-deoxyglucose and about 4 to about 10 constituent homo-monosaccharide Sugars 2-deoxyglucose, Xyloascorbyllactone, digitoxose (2-deoxy (i.e., all the same constituent) or hetero-monosaccharide (i.e., altromethylose), fucose (6-deoxy-galactose), gluoanolac 55 different constituent) Sugars. Each of the Subject constituent tone, galaconolactone, rhamnose (6-deoxy-mannose), fruc Sugars is linked one-to-another in a serial array through a tose (2-keto-arabohexose), aldaric acids, alditols, aldonic series of glycosyl bonds formed between the C and C car acids, ketoaldonic acids, and amino Sugars; with the proviso bon atoms; or alternatively, between the C and C carbon that the E-moiety is not a cyclodextrin. Representative aldi atoms; or alternatively, between the C and C carbonatoms; tols include e.g., erythritol, threitol, ribitol, arabinitol, xylitol, 60 with the proviso that when the E-moiety is according to lyXitol, glucitol, allositol, altrositol, mannositol, gulositol, FORMULAVIa, VIb, VIc or VId and comprises glycosidic idositol, galactositol, talositol and their derivatives. Repre linkage at C-C then Rs and R are hydrogen, when linkage sentative aldonic acids include erythronic acid, threonic acid, is at C-C, then Rs and Ro are hydrogen, and when linkage ribonic acid, arabinonic acid, Xylonic acid, lyXonic acid, glu is at C-C, then Rs and R2 are hydrogen. conic acid, allonic acid, altronic acid, mannonic acid, gulonic 65 Preferably, the subject di-, tri- and oligosaccharide E-moi acid, idonic acid, galactonic acid, tolonic acid and their eties are metabolizable and/or acid hydrolyzable to mono-, derivatives. Representative ketoaldonic acids include di- and tri-saccharides and transportable by saccharide trans US 9,302,982 B2 15 16 porters in mammals; most preferably, when present as an ergic compounds according to FORMULAS VIIIA and oligosaccharide the Subject E-moiety comprises a residue VIIIB, which follow on the next page. selected from the group of metabolizable di- and tri-saccha rides consisting of: (i) homopolymers such as an erythran, a threan, a riban, an arabinan, a Xylan, a lyXan, an allan, an 5 FORMULAVIIIA altran, a glucan (e.g. maltose, isomaltose, cellobiose), a man R12 nan, a gulan, an idan, a galactan, a talan and their substituted R11 derivatives; (ii) heteropolymers such as erythrosides, threo O sides, ribosides, arabinosides, Xylosides, lyxosides, allosides, 2 altrosides, glucosides (e.g., Sucrose; (Glc-B1.4-Frc), galacto 10 sides (e.g., lactose; Gal-B1.4-Glc), mannosides, gulosides, R4 Ro Rio R9 T idosides, talosides and their substituted derivatives. Other 1. N 1. representative oligosaccharides include the following: R3 (CH)n (CH2)m namely, Sucrose glycogen, fucosidolactose, lactulose, lacto 15 bionic acid, amylose, fructose, fructofuranose, Scillabiose, panose, raffinose, amylopectin, hyaluronic acid, chondroitin R2 R Sulfate, heparin, laminarin, lichenin and inulin. Preferably, FORMULAVIIIB the Subject E-moiety, when present as an oligosaccharide, is selected from the group consisting of glucosyland galactosyl R12 homo- and heteropolymers e.g., glucans, galactans, gluco sides and galactosides. The Subject E-moiety is not a cyclo R11 O dextrin or derivative thereof. 2 Thus according to the foregoing disclosure, embodiments of the invention provide a variety of compounds which are 25 R4 Ro R10 Ro within the spirit and bounds of the instant invention. For T example, FORMULAVIIa and VIIb, below, depict an Illus 1NCEN 1. trative E-moiety aldose hexosyl sugar (FORMULA VIIIa) R3 (CH2)n (CH2)m linked at C with A-B-D and Interconvertible with its pyra nose form (FORMULAVIIb). 30 R R

wherein: Ring 1 is the A-moiety (described Supra); Substituents 35

R-Ra, Rs. Rio, RandR areas described (Supra) and Ring 2 is the E-moiety, also as described (Supra); T comprises an optional amine, amide, halogen, thioa mido, oxyamido, ureido, thioureido, thiamido, dithiamido, acetyl, carboxylic acid amide, carboxamide, amino-carbonyl, 40 alkylaminocarbonyl, dialkylaminocrbonyl, preferably amine or amide, most preferably amine; n is an integer selected from within the range of 1 to 4; and, m is an integer selected from within the range of 0 to 4, with 45 the proviso that value of (m+n) is an integer selected from within the range of 1 to 4: R and R are hydroxyl; Ro, RandR are hydrogen; Rs. R. Rio or R are selected the group consisting of 50 hydrogen, hydroxyl, halogen, hydroxyl Substituted lower alkyl, halogen substituted lower alkyl or lower alkyl: Formula VIIa Formula VIIb R is selected from the group consisting of hydrogen, lower alkyl, substituted lower alkyl, hydroxyl, alkoxyl, or wherein the X,Y,Z and R-R are as set forth in regard to halogen, preferably hydrogen, lower alkyl, halogen- or FORMULA I, supra. 55 hydroxyl-substituted lower alkyl, haloalkoxy, hydroxyl, Those of skill in the art will recognize varied synthetic alkoxyl, alkoxycarbonyl, thioalkoxy or halogen, or a routes for assembling test compounds from the constituents monosaccharide, disaccharide, trisaccharide or oligosaccha set forth in the foregoing disclosure. The skill for determining ride. Most preferably, R is hydrogen, hydroxyl or substi the functionality of a test compound according to the inven tuted lower alkyl. tion is also known in the art. For instance, assays for measur 60 In alternative embodiments, compounds according to ing ligand binding to dopaminergic receptors are known as FORMULAVIIIA or VIIIB are provided in which R and R. are assays for determining that a test compound is transport comprise a group selected from the group consisting of able by a saccharide transporter. (Further disclosure of illus hydrogen, hydroxyl, halogen, hydroxyl Substituted lower trative testing methods being provided in the accompanying alkyl, halogen substituted lower alkyl and carboxyl; prefer disclosure below.) 65 ably, R and R comprise hydrogen, hydroxyl or halogen. In other presently preferred embodiments, the invention As used herein, the following additional terms are intended provides N-linked transportable and metabolizable dopamin to have meanings as follows: namely,

US 9,302,982 B2 21 22 glyconolactones followed by reduction to aldoses. Methods to dopamine receptors include assays measuring tyrosine for reducing carbon chain length are also known, e.g., Ruff hydroxylase activity in mouse and rat brain where dopamin degradation involving conversion to a glyconic acid, forma ergic agonist-induced increases in enzyme activity which tion of a calcium salt and oxidation of the salt by hydrogen may be measured using conventional methodology (e.g., peroxide/Fe" or oxidation (e.g., in nitric acid) of an aldose to Kikuchi et al. 1995; Yasuda et al., 1988). Antagonist activity a keto acid with cleavage at the resultant carbonyl to form of a test compound may also be evaluated using assay meth lower carbon numberaldaric dicarboxylic acids. Methods for ods to measure reserpine and gamma-butyrolactone (GBL)- separation of epimers (diastereomers) are also known, e.g., by induced increases in tyrosine hydroxylase activity, i.e., the crystallization of lactone salts with differing physical prop increase inactivity is antagonized by D2 receptor antagonists erties followed by reduction to a single pure aldose or ketose 10 epimer. Methods for converting between epimers are also Such as haloperidol. Post-synaptic D2 receptor antagonist known, e.g., oxidation to a glyconic acid followed by treat activity may be confirmed by increased locomotor activity in ment with pyridine to establish an equilibrium between the reserpine-treated mice, or by evaluating rotation in rats with epimers, separation of the epimers as lactone salts, followed unilateral 6-OH-dopamine lesions (Kikuchit al., 1995). by reduction to the epimeric pure aldose. Methods for sepa 15 Assays for determining that a test compound according to ration of anomeric (i.e., C) diastereomers are also known. FORMULAI is capable of passaging the blood-brain barrier Methods for conversion of aldoses to ketoses, e.g., through are also known. For example, a test compound according to enediols under alkaline conditions, are also appreciated in the the invention may be injected intravenously into test mice and art as are methods for converting aldoses to glyconic mono passage of the blood-brain-barrier may be evaluated by mea carboxylic acids, e.g., by reduction to glycitols and oxidation suring a brain penetration index (BPI), wherein the amount of to glyconic acids. test compound measured per gram of brain tissue is divided According to the instant methods, lactones of Sugars and by the amount of test compound measured per gram of liver their acids are preferred starting materials for synthesis of tissue. For comparison, the BPI value for gamma amino compounds according to FORMULA I, e.g., aldonic and butyric acid (GABA) is about 1.0%. Ex-Vivo assays for deter keto-aldonic acids (Supra). Preferably, the starting materials 25 mining blood brain barrier transport are also known, includ are lactones or aldoses, ketoses, aldonic and keto-aldonic ing e.g., an assay described recently by Duport et al., (1993) monocarboxylic acids, i.e., aldehydo lactones and lactone using in vitro organ culture to measure the likelihood that a acids and their salts. According to instruction provided test compound will passage the blood brain barrier. Briefly, in herein, glucuronic Co acid lactones are not preferred starting the latter assay slices of selected brain regions are overlaid material according to the instant methods, i.e., drug glucu 30 ronides being rapidly metabolized and removed from circu onto an endothelial cell monolayer in vitro and allowed to lation. form tight junctions over the course of about 10 days of Also envisaged as within the scope of the present invention culture. Test compounds are then perfused into the endothe are acid- and alkali-hydrolyzable controlled-release multim lial side of culture and blood brain barrier penetration is ers of the compounds of FORMULAI, wherein a first A-B- 35 detected by measuring the levels of the test compound which D-E molecule is linked through a hydrolyzable cross-linker enter into the organ slice. “R” to a second A-B-D-E molecule, e.g., E-D-B-A-R-A-B- Methods for assessing intestinal transport of a test com D-E. Linkage between the first and the second molecules may pound (i.e., according to FORMULAI) we also known. For be effected at any of the A, B, D or E moieties, e.g., using example, that a compound according to the instant invention methods known in the art. 40 is transportable by an intestinal saccharide transporter may be Methods for determining that a test compound synthesized determined e.g., using the ex situ perfused everted rat Small according to the methods of the invention is dopaminergic, intestine (jejunum) model in a Ussing-type chamber, or alter i.e., capable of binding a dopamine receptor, may be deter natively, using everted intestinal sacs and rings. In the latter mined according to methods known in the art. For example, assays, transport of a test compound according to the inven dopamine receptors and receptor ligand binding assays are 45 tion is determined by applying the test compound to the known in the at including at least assays for D1-like (D1A, everted luminal epithelium and measuring the amount of D1B/D5), and D2-like (D2S, D2L, D3, D4) receptors using compound that reaches the opposite serosal side (e.g., see e.g., brain slices, brain region membrane preparations, iso Mizuma et al., 1994: Diez-Sampedro et al., 1999). For ease of lated neuronal cells, cell lines, synaptosomal membrane measurement, the Subject test compound may be radiolabeled preparations and cells stably transfected with dopamine 50 e.g., with C''. That a sodium-dependent glucose co-trans receptors e.g., C-6 glioma cells stably transfected with rat porter (SGLT1) is specifically involved in transport of a test cDNA encoding D2L or D3; CHO cells stably transfected compound according to the invention may be determined by with cDNA encoding rat D2S, D2L, D3 or D4; and the like. removing sodium ions from the intestinal perfusate solution, The binding interactions between a test compound and a (i.e., the Subject saccharide transporter is Na+ dependent and dopamine receptor binding may be assessed e.g., using radio 55 the rate or amount transported in the absence of Na+ ions labeled test compounds as ligands. Specificity of binding may should decrease), or by adding phlorizin (i.e., an inhibitor of be assessed by competition with known dopamine receptor transport). ligands, e.g., the D1 antagonist SCH23390(R)-(+)-7-chloro Methods for testing compounds to determine that they 8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3- binding to and are transported by glucose co-transporters in benazepine and the D2 antagonist haloperidol. Functionality 60 vitro are also known, e.g., using isolated primary cultures of of a test compound according to the invention as a dopamin rat Villus tip intestinal enterocytes and enterocyte-like cul ergic agonist may, for example, be determined using rat stri tures of HT-29 colon carcinoma cells. That a test compound atal membrane preparations and modulation of second mes according to the invention is transportable may be determined sengers such cAMP, calcium flux or 5'-O-(gamma-Sithio) by adding the compound to the extracellular medium, wash triphosphate (IS-GTPgamma S) binding, e.g., according 65 ing the cells to remove non-specifically associated test com to methods such as those described in Geurts, et. al., 1999 and pound cells (e.g., by centrifugation through isobutyl pthylate others. In vivo tests for assessing binding of test compounds or Ficol), and then measuring the amount of test compound US 9,302,982 B2 23 24 which has entered the cells. Specificity of transport may be “Dosage form' is intended to mean a form of a pharma established, as above, by removing Na+ ions or adding phlo ceutical composition Suitable for administration to a subject rizin. in need thereof. Representative dosage forms include Solids Saccharide transporter, as set forth above, is intended to and liquids, e.g., perenteral and injection Solutions, powders mean a cellular membrane protein capable of binding a sac and granules, emollient creams, syrups and elixirs, nasal and charide and transporting that saccharide from one location to ophthalmic drops, intrabronchial inhalants, timed-release another on/in the cell. Representative examples of saccharide capsules, lozenges, troches, Suppositories, dermal patches, transporters include glucose transporters (e.g., GLUT 1, 2, 3, impregnated bandages and the like. 4 and 5), galactose transporters, mannose transporters, fruc Embodiments of the invention provide pharmaceutical 10 compositions, Supra, containing one or more of the instant tose transporters, arabinose transporters and the like. Those N-linked prodrug compounds in a form Suitable for adminis skilled in the art are cognizant of methods by which test tration to man or domestic animals. Representative examples compounds may be shown capable of binding to a saccharide of forms so Suitable include compositions in which the instant transporter, examples of which are provided below. compound is in Solid and liquid mixtures with optional addi "Brain penetration index’, abbreviated BPI, is intended to 15 tives, stabilizers, carriers, binders, buffers, excipients, emol mean the mathematical ratio calculated as the amount of one lients, disintegrants, lubricating agents, antimicrobial agents or more of the instant compounds in brain tissue per gram of and the like. The instant pharmaceutical compositions are brain tissue, divided by the amount of the compound (or distinct, in that the instant compound comprising the active compounds) in liver tissue per gram liver tissue. The liver ingredient in the Subject compositions has all of the following being chosen as a reference organ because of its intimate properties: namely, (i) it is transportable in an intact form by contact with blood and relative lack of barriers. Measure a saccharide transporter, e.g., as that receptor is expressed in ments of BPI may be made for instance at 5-60 minutes after nature in an intestinal cell or in a red blood cell; (ii) it is administration of a test compound, e.g., by oral, Subcutaneous transportable in an intact form across the blood brain barrier or intravenous routes. The Subject mathematical ratio is com by a saccharide transporter, i.e., as that transporter is monly expressed as a percentage, i.e., by multiplying the ratio 25 expressed in nature in an endothelial cell; (iii) it is transport by 100%. This procedure has the advantage that even for a able by DAT; and, (iv) it is capable of binding to a dopamin sparingly soluble lipophilic drugs, (which tend to remain ergic receptor in a neural cell, i.e., as that receptor is expressed largely at an injection site with slow diffusion into the circu in nature in a neural cell. lation), the amounts of drug in the liver will reflect the actual “Intestinal cell' is intended to mean a columnar epithelial amount which is systemically available and not the initial 30 cell, e.g., a microVillus luminal cell, lining the Small or large dose injected. Certain of the preferred compounds according intestine, or lining the colon. to the instant invention have BPIs in the range of about 2% to "Endothelial cell” is intended to mean a cell lining a blood about 500%, most preferred compounds have a BPI of about vessel, e.g., a capillary cell or a cell of an artery or a vein. 10% to about 200%. “Neural cell' is intended to mean cells of the nervous Methods for determining that a test compound according to 35 system, including neurons, glial cells, Schwann cells and the FORMULA I, i.e., with a drug selected from TABLE A or like. TABLE B, is suitable for use in one or more of the instant “Transportable in an intact form' is intended to mean that methods, (i.e., for treating neurologic dysfunction or for use the subject instant compound is not an inhibitor of GLUT as a CNS-acting drug), are known to those skilled in the art of transporters, and is not substantially chemically altered dur neuropsycopharmacology. For instance, the test compound 40 ing transport, e.g., it is not metabolized or converted to a may be evaluated in behavioral tests in experimental animals; glucuronide during transport, such that when the instant com e.g., to determine whether it exhibits one or more of the pound is transported from one side of a cell to the another side following: namely, a Pergolide-like dopaminergic activity, it remains Substantially unchanged. "Substantially stimulation induced release of dopamine, locomotor activity unchanged’ means that only conservative modifications of in a murine test model, anticonvulsant activity, analgesic 45 certain R. R. Rs. R. R-7, Rs. Ro, R or R2 group Substitu activity, cognition/memory, DAT transportability, activity in ents (Supra) may occur during transport, e.g., removal of a an MPTP-induced model of locomotor impairment. halogen atom and replacement with a hydrogen, conversion “N-linked glycosyl prodrug', when used herein in regard of a hydroxyl to a methoxy (e.g., an —OCH in an acetal or a to a pharmaceutical agent, is intended to mean an 'A'-moiety hemiacetal) and the like. CNS acting prodrug compound according to FORMULAI, 50 In other embodiments, the invention provides pharmaceu linked through an amine or amide according to FORMULAI tical compositions containing one or more of the instant com to a saccharide. pounds in combination with optional stabilizers, carriers, "Pharmaceutical composition', is intended to mean a com binders, buffers, excipients, emollients, disintegrants, lubri position containing one or more N-linked glycosyl CNS cating agents, antimicrobial agents and the like. For oral acting prodrug compounds according to FORMULAI and a 55 administration, the instant pharmaceutical compositions may formulary effective to provide a dosage form suitable for be liquid, Solid or encapsulated. For perenteral administra administration to man or domestic animals. Representative tion, the instant pharmaceutical compositions may be sterile examples of formularies and dosage forms so suitable are liquids or solids may be provided in a form suitable for provided below. reconstitution, e.g., powdered or granulated. “Formulary' is intended to mean an agent added to a phar 60 The instant compounds may be administered alone or in maceutical composition comprising said hydrophilic combination with pharmaceutically acceptable carriers, in N-linked CNS acting prodrug compound. Representative either single or multiple doses. Suitable pharmaceutical car examples of formulary agents include additives, stabilizers, riers may include inert Solid diluents or fillers, sterile aqueous carriers, binders, buffers, excipients, emollient water-in-oil Solutions, and various nontoxic organic solvents. The phar and oil-in-water emulsions, disintegrants, lubricating agents, 65 maceutical compositions formed by combining the instant antimicrobial agents, preservative and the like; as disclosed compound with the pharmaceutically acceptable carrier may further below. then be readily administered in a variety of dosage forms such

US 9,302,982 B2 27 28 magnesium. Other representative pharmaceutically accept which are also evidenced clinically in a patient with Parkin able salts include hydrochloride, hydrobromide, sulfate, son's disease. Representative examples of symptoms evi bisulfate, acetate, oxalate, Valarate, oleate, laurate, borate, denced in patients with Parkinsonism include seizure, loss of benzoate, lactate, phosphate, tosulate, citrate, maleate, fur neuromotor control of muscle movements, tardive dyskine marate. Succinate, tartrate, and the like. sia, Alzheimer's disease, Wilson's disease, post-encephalitic The route of delivery of the instant compounds is deter syndromes, Parkinsonism secondary to trauma and stroke, mined by the disease and the site where treatment is required. dementia, Lou Gehrig's disease, psychomoter retardation, For topical, intrathecal, intramuscular or intra-rectal applica Schizophreniform behavior, anxiety and depression. Clinical tion it may be desirable to apply the instant compounds as a features of Parkinson's related diseases are disclosed in Hur salve, ointment or emollient pharmaceutical composition at 10 the local site, or to place an impregnated bandage or a dermal tig, 1997, incorporated herein by reference in its entirety. timed-release lipid-soluble patch. For intra-rectal applica "Metabolic replacement therapy”, as used herein, is tion, it may be desirable to apply the instant compounds as a intended to mean that the instant compound, when adminis pharmaceutical composition in a Suppository. In other situa tered in the instant pharmaceutical composition, is effective, tions, it may prove desirable to administer the compositions 15 following transport into a neural cell, to satisfy one or meta by intranasal or intrabronchial instillation (e.g., as a pharma bolic requirements of catecholamine synthesis in the neural ceutical composition Suitable for use in a nebulizer), or gas cellofa Subject having a nigrostriatal dopamine insufficiency. trointestinal delivery (e.g., with a capsule, tablet, trouch or Representative examples of compounds so capable include Suppository). In one preferred embodiment, the instant phar derivatives of L-Dope, e.g., Levodopa. maceutical compositions are administered via Suppository The routes and methods for delivery of the instant prepa taking advantage of saccharide transporters in the colon for rations are determined by the particular disease. Disclosure of transport of the instant compound into the blood stream in a therapeutic methods of treating disease appear in Applicants timed-release type manner for metabolic replacement therapy co-pending U.S. patent application Ser. Nos. 10/274,798, in patients with Parkinson's and related diseases. 10/198,798 and 09/547,501, incorporated herein by reference The instant compounds find use in treatment of a variety of 25 in their entirety. pathological central and peripheral nervous system dysfunc In yet other embodiments, the special aqueous solubility of tions, neuromotor conditions and cardiovascular diseases in the instant prodrug compounds provides novel pharmaceuti Subjects in need of treatment. For example, the Subject con cal compositions containing relatively high concentrations of ditions include, but are not limited to, i) toxic dystrophy, (e.g., active ingredients (e.g., up to 500 mg/ml) included in rela chemical or drug-induced secondary dystrophy in the ner 30 tively small Volumes (e.g., up to about 500 mg/ml), allowing Vous system), ii) Vascular impairment e.g. resulting in dam administration of relatively small volumes of therapeutically age to nervous tissues, iii) central nervous system degenera effective unit doses. The latter attribute of the instant com tion or peripheral nerve degeneration, iv) nervous system pounds is a particular advantage in the instant pharmaceutical lesions induced by physical trauma, v) nervous system com compositions, i.e., especially in multi-dose, time-release, plications of illnesses and infections (e.g., viral or bacterial); 35 Subcutaneous and intradermal, buccal, trouch, and Supposi and vi) hereditary nervous system impairment. Representa tory preparations. The Subject attributes may also be espe tive illness, diseases, and conditions having neurologic dys cially useful for achieving steady state plasma levels in a function have been classified and codified (“International subject in need thereof. Where conventional methods of Classification of Diseases, Washington D.C., 1989). Repre administration are ineffective in certain patients, the Subject sentative examples of Subjects in need of treatment may 40 high solubility attributes of the instant compounds make it include humans and domestic animals having e.g., a condi feasible to administer metabolic replacement therapy via an tion of hyper- or hypo-dopaminergic activity, such as may be implantable mini-pump Such as those used for delivery of evident in a patient with Schizophrenia, Parkinson's disease, insulin in patients with Type 1 insulin-dependent diabetes epilepsy, locomotor deficiency, hyperprolactinemia, mellitus. Tourette's syndrome, Huntington's disease, psychosis, 45 Non-limiting illustrative preparations and formulations are chronic psychiatric illness with amotivation, apathy, asocial disclosed in the EXAMPLES, section which follows. ity, psychomotor adverse effects of drugs of abuse (e.g., cocaine, amphetamine, neuroleptics), Subolivopontocerebel Example 1 laratrophy (sCPCA), multiple system atrophy (MSA), bipo lar disorder, chronic alcoholism, cocaine abuse, mood disor 50 Preparation of Dopamine Gluconamide ders, attention deficit disorder, physiologic stress, pesticide exposure (e.g., organochlorine insecticides), juvenile neu Scheme 1 ronal ceroid lipofuscinosis (JNCL), detached personality O syndromes (as e.g. determined using the Karolinska Scales of O Personality questionnaire) and the like. Representative 55 TEA examples of conditions exhibiting hyper-dopaminergic activ HO OH MeOH O -- ity include Schizophrenia, chronic psychiatric illness with Y OH hallucinations and delusions. Also representative are, patients with coronary hypertension, angina, ischemic myocardium HO and the like. In addition, prophylactic methods are envisaged 60 for lowering aortic and pulmonary artery pressure during and OH after coronary bypass Surgery and liver, kidney and heart transplant Surgery. Vasodilation mediated by the instant com pounds is without impairment of oxygen delivery or impair ment of intrinsic neural or hormonal control systems. 65 "Parkinson's related disease', as used herein, is intended to NH2 mean a disease characterized by one or more symptoms US 9,302,982 B2

-continued -continued H H

N O N O

OH OH HO O OH OH OH O HO 10 CH3 OH OH

OH OH CHOH CHOH 15 Dopamine gluconamide (EXAMPLE 1, supra; 0.75 gm, Gluconolactone (1.9 gm, 10.5 mmol) and triethylamine 2.26 mmol) was added to acetone (40 mL) in a 100 mL round bottom flask with stirring. Them, the reaction mixture was (TEA: 1.1 gm, 10.5 mmol) were added to methanol (25 mL) refluxed for 2 hrs., after which time it was allowed to cool to in a 100 mL round bottom flask with stirring. The glucono room temperature (about 22-25C). The resultant white solid lactone was allowed to dissolve. When the solid was dis was removed by filtration and dried in vacuo for 7 hrs. yield ing the isopropylidine protected dopamine gluconamide solved, the solution was stirred for an additional 10 minutes (0.68 gm, 1.83 mmol. 81.0% yield). Melting point of the and then 3-hydroxytyramine (2.0 gm, 10.5 mmol) was added 25 synthesis product was 170° C. slowly, i.e., allowing it to dissolve. The reaction mixture was Example 3 stirred in the dark for about 2 hrs. during which time a white Solid precipitant appeared. The white solid precipitant was Reduction of Isopropylidine Protected Dopamine collected by filtration, washed with methanol (5 mL) and 30 Gluconamide dried in vacuo for 6 hrs. to give dopamine gluconamide (1.69 gm, 5.10 mmol. 48.6% yield). Melting point of the synthesis product was 154-155° C. Predicted: CHN (331.32): C, Scheme 3 35 -50.75%; H, -6.39%; N, -4.23%; analysis results of syn H thetic product: C, 50.65; H, 6.63: N, 4.44. N O

OH 40 O Example 2 OH (1) Borane/THF c-A- O HO (2) MeOH/HCI CH3 OH Protection of Aromatic Dopamine Hydroxyl 45 Residues OH CHOH H

50 N O

Scheme 2 OH HO OH 55 OH HO

OH OH HO CH CH3 OH OH OH 60 CH2OH HO OH Isopropylidene protected dopamine gluconamide (EX OH AMPLE 2. Supra; 0.68 gm, 1.83 mmol) was slowly added to 65 a 1 M Borane solution in THF (25 ml) in a 100 mL round CHOH bottom flask, with stirring. The reaction mixture was refluxed for 2 hrs. and then allowed to cool to room temperature. US 9,302,982 B2 31 32 Excess solvent was removed by rotary evaporation. Methan thick hygroscopic syrup (complicating melting point analy olic HCl was added to the resultant residue and the solution sis). The syrup was dried in vacuo for 6 hrs. to give the refluxed for 2 hrs., after which time solvent was removed by dopamine ribonamine-HCl salt as product (0.20 gm., 0.62 evaporation and the Solid recrystallized using a mixture of mmol, 30.3% yield.) H and 'C-NMR results were consis acetonitrile and ethanol. The recrystallized reduced dopam 5 tent with structure. ine gluconamide product was dried in vacuo for 6 hrs giving the dopamine gluconamine-HCl salt (0.22 gm, 0.62 mmol. Example 7 33.8% recovery). Melting point for the synthesis product was 151-152° C. Predicted CHN (353.80); C, 47.53; H, 6.84; Ready Solution for Administration as a Measured N, 3.96: Analysis result of synthesis product: C, 47.48; H, 10 Dose 6.93: N, 3.88. An illustrative ready solution for administration as a mea Example 4 Sure dose was prepared according to the formulation of TABLE A, below. Preparation of Dopamine Ribonamide 15 TABLE A D-(+)-Ribonic acid gamma-lactone (2.0 gm, 13.5 mmol) was added to methanol (25 mL) in a 100 mL round bottom Component: Amount: flask with stirring until dissolved, and then an additional 5 Compound #1 or #2* 2.5 gm min. 3-Hydroxytyramine (2.6 gm, 13.5 mmol) was added Methyl-p-aminobenzoic acid 0.014 gm slowly, allowing it to dissolve, with stirring, over the course of Propyl-p-aminobenzoic acid 0.020 gm Saccharin sodium 0.050 gm about 10 minutes. Triethylamine (1.4 gm, 13.5 mmol) was Flavoring agent 0.001 gm then added and the reaction mixture refluxed for 4 hr. in the Citric acid 0.200 gm dark, during which time the Solution acquired a slight yellow Sodium citrate 0.320 gm color. Solvents were removed by rotary evaporation using 25 Distilled water USP q.S. to 100 ml anhydrous ethanol as an azeotrope to remove any residual *Compound #1, Dopamine gluconamide (EXAMPLE #1, supra); Compound #2, Dopamine water. The resultant dried product constituted a thick syrup gluconamine, which Solidified upon standing (1 hr.) to give a white Solid. The white solid product was stirred (1 hr.) with acetone (40 mL), again resulting in a white Solid as a product. The result 30 Example 8 ant solid was collected by filtration and dried in vacuo for 6 hrs. yielding dopamine ribonamide (3.83 gm, 12.7 mmol. Powder Composition for Reconstitution Prior to Use 94.1% yield.) H and 'C-NMR results and CHN analyses were consistent with structure. Molting point was 90-91° C. An illustrative powder composition for reconstitution prior Predicted CHN: (301.30): C, 51.82; H, 6.36: N, 4.65; 35 to use was prepared according to the formulation of TABLE Analysis results of synthesis product: C, 51.67; H, 6.40; N. B, below. 4.69. TABLE B Example 5 40 Component: Amount: Preparation of Dopamine Isopropylidine Ribonamide Compound #1 or #2* 2.5 mg Sodium citrate 20.0 mg Aromatic hydroxyl groups in dopamine ribonamide were Sorbitol 2.0 mg protected by synthesizing the isopropylidine compound. Flavoring agent 0.1 mg Distilled water USP for reconstitution: 10.0 ml Dopamine ribonamide (EXAMPLE 4: 1.0 gm, 3.32 mmol) 45 was added to acetone (30 mL) in a 100 mL round bottom flask *Compound #1, Dopamine gluconamide (EXAMPLE #1, supra; Compound #2, Dopamine with stirring. The reaction mixture was refluxed for 5 hrs. and gluconamine, then allowed to cool to room temperature. The resultant white solid was collected by filtration and dried in vacuo for 7 hrs. Example 9 to yield the isopropylidine protected dopamine ribonamide 50 (0.99 g, 2.90 mmol, 87.6% yield). H and 'C-NMR results were consistent with structure. Melting point was found to Tablets for Oral Administration 142-143° C. An illustrative tablet for oral administration was prepared Example 6 55 according to the formulation of TABLE C, below. Reduction of Isopropylidine Protected Dopamide TABLE C Ribonamide Yielding Dopamine Ribonamine Component: Amount: Isopropylidine-protected dopamide ribonamide (EX 60 Compound #1 or #2* 250 mg Starch 17 mg AMPLE 5: 0.70 gm; 2.05 mmol) was added slowly to 1 M Sodium glycolate (starch) 40 mg Borane in THF (25 mL) in a 100 mL round bottom flask with Polyvinal pyrrollidene 7.0 mg stirring. The reaction mixture was refluxed for 2 hr. and Microcrystalline cellulose 45 mg allowed to cool to room temperature. Excess Solvent was Magnesium sterate 2.0 mg 65 removed by rotary evaporation and methanolic HCl was *Compound #1, Dopamine gluconamide (EXAMPLE #1, supra); Compound #2, Dopamine added to the resulting residue. The resuspended residue was gluconamine, refluxed for 2 hr. and solvent was then evaporated yielding a US 9,302,982 B2 33 34 Example 10 cAMP accumulation recorded, Suggesting strongly that the compounds produced according to the instant methods act as Tablet for Sublingual Administration agonists, not antagonists. An illustrative tablet for sublingual administration was 5 Example 12 prepared according to the formulation of TABLE D, below. Dopamine Transporter Binding Activity TABLED To further illustrate biological activity, i.e., transportability Component: Amount: 10 within the brain, Compounds #1 and #2 (Supra) dopamine Compound #1 or #2 250 mg transporter (DAT) binding activity of Compounds #1 and #2 Gum arabic 10 mg was evaluated by measuring their ability to compete uptake of Lactose 90 mg H-labeled dopamine by human DAT-transfected cells over Ammonium glycyrrhiznate 20 mg Sodium saccharin 2 mg the course of a 5 hour incubation period. To obtain differing Flavor 10 mg 15 levels of DAT expression, cells were transiently transfected) Magnesium sterate 7 mg with plasmids containing a DAT cDNA insert (hDAT), or alternatively, control irrelevant clNA insert (Negative Con *Compound #1, Dopamine gluconamide (EXAMPLE #1, supra); Compound #2, Dopamine gluconamine, trol, NC). After 48-72 hrs. culture, dopamine transport was measured in the transiently transfected hDAT-cells by incu bation for 5 hrs. in the presence of H-labeled dopamine Example 11 (Positive Control, PC). Competition of H-dopamine uptake was observed with both Compound #1 and Compound #2. Dopamine Receptor Binding Example 13 To illustrate biological activity, i.e., dopaminergic activity, 25 and putative pharmaceutical utility, dopamine receptor bind Parkinson's Animal Model Studies ing activity of Compounds #1 (EXAMPLE 2, product) and Compound #2 (EXAMPLE 3, product) was tested in vitro Study #1: Anti-Parkinson's Effect in using COS-7 cells transiently transfected with pCD-PS MPTP-Lesioned Mice expression vectors containing human D1, human D5 and 30 human D2 (long) inserts, i.e., according to Materials and Dopaminergic neurotoxin 1.2.3.6-methyl-phenyl-tetrahy Methods disclosed further below. Binding to dopaminergic dropyridine (MPTP) is a chemical contaminant first found in receptors was tested as ability to compete binding of specific contaminated synthetic street heroin. This chemical produces receptor ligands (i.e., H-SCH-23390 for D1; H Parkinsons-like neuropathological changes and clinical fea emonapride for D2), as well as, the ability to trigger intra 35 tures in man, monkey and mice. Like the natural dopamine cellular second messengers, i.e., cAMP. mediator, MPP" is taken up into the dopaminergic nigral Competition binding assays were initiated in duplicate terminal by dopamine transporters (DAT) with resultant cel with 0.5 ml aliquots of membrane preparations from cell lular changes similar to those mitochondrial complex I cultures transfected with cDNA encoding human D1- or defects found in Parkinson's Disease. MPTP-treated mice are D2-receptors. Test compounds (Compounds #1 or #2, Supra) 40 one accepted Parkinson's-like animal models. were added as competitors to achieve a final concentration in The first phase of the project involved testing to measure the assay in the range of 10M to 10' M. As binding ligand, anti-Parkinson’s effects of COMPOUND #1 in an MPTP -SCH-23390 (a D1-selective agonist) or H-Emonapride mouse model of nigral injury. Experimental mice were (a D2-selective agonist) was added to each assay. After 90 lesioned with MPTP (i.e., 40 mg/kg, ip twice, 1 month). minutes incubation at room temperature the assay was termi 45 FIG. 1. MPTP treatment significantly reduced the Rota nated by rapid filtration and membrane bound Hwas deter Rod mean time i.e., by over 65%. Rota-Rod performance mined by Scintillation spectrometry. times of C57BL/6 mice before and after lesioning with Test Compounds #1 and #2 successfully competed H MPTP, or as a negative control, NaCl. SCH-23390 binding to dopamine receptors in cells tran FIG. 2. MPTP-lesioned mice were treated with COM siently expressing receptors, i.e., in a dose-response and uni 50 POUND #1 twice daily (20 mg/kg or 80 mg/kg) and tested at phasic type manner. Under these particular conditions of weekly intervals. The results presented show improved Rota assay, the illustrative test Compounds #1 and #2 showed Rod performance times at the fourth week of COMPOUND selectivity for D5-over D1-receptors, i.e., a property held in #1 treatment, i.e., 8-9 fold better than NaCl-treated controls common with natural dopamine agonist. (p<0.05). Rota-Rod performance of MPTP-lesioned mice Agonist functional activity assays were conducted by 55 after four weeks of treatment with either 20 mg/kg or 80 evaluating ability of test compound to trigger production of mg/kg COMPOUND #1 or, with vehicle control (0.9% second messengers in dopamine receptor transfected COS-7 NaCl). Both treatment groups showed statistically significant cells, i.e., cAMP. Incubation with test compound (or dopam increases (p<0.05) in the Rota-Rod times. (Y-axis: time in ine as a positive control) were conducted at 37°C. (5% CO) seconds). for 15 min. and cAMP accumulation was determined by 60 FIG.3. Significant improvements in Rota-rod performance radioimmunoassay. For comparison, dopamine as a positive times were observed in COMPOUND #1 treated animals control stimulated accumulation of cAMP by about 5-fold in beginning at about the third week of treatment at a dose of 20 D1-transfected cells and about 3-fold in D5-transfectants. In mg/kg, and at about the second week at the higher dose of 80 dopamine receptor transfectants, Compound #2 stimulated mg/kg. Kinetics of onset of anti-Parkinsons therapeutic cAMP accumulation in a dose-response manner to levels near 65 effects on COMPOUND #1 treatment. MPTP-lesioned mice those achieved in dopamine control cultures. Co-incubation treated daily at 20 mg/kg or 80 g/kg COMPOUND #1, or of dopamine with Compound #2 did not reduce the levels of vehicle control (0.9% NaCl). Rota-Rod times were statisti

US 9,302,982 B2 37 38 Rota-Rod time within two weeks of treatment, i.e., a statisti DISCUSSION OF EXAMPLES 13-16 follows a definition cally significant increase in performance when compared of terms and an introductory Background section, below. with pretreatment performance times. *p-0.05. Definition of Terms Used in Example 17: To determine the washout kinetics for COMPOUND #1, Tyrosine hydroxylase (ID is a tetrahydrobiopterin-(BH4; treatment was terminated at the 8” week and RotaRod per 5 6-(R)-erythro-5,6,7,8-tetrahydrobiopterin) requiring, iron formance measurements of mice were made in each of the Successive three weeks. containing monooxygenase enzyme which catalyzes the first FIG. 8. Washout of COMPOUND #1 treatment in Nurr1 and rate-limiting step of catecholamine synthesis, i.e. conver deficient mice. Rota-Rod performance measurements were sion of tyrosine to L-Dopa. TH expression is necessary for performed over three successive weeks following cessation of neurotransmitter specification of all catecholaminergic neu COMPOUND #1 treatment. Rota-Rod performance rons. Nigrostriatal synthesis of dopamine accounts for the degraded significantly within one week and returned to pre majority of dopamine in the central nervous system. Indica treatment values within 2 weeks. *p-0.05. tive of its Importance, TH is regulated by nearly every CONCLUSIONS: The data indicate COMPOUND #1 (20 reported form of regulation including multiple gene-level mg/kg) possesses anti-Parkinson's effects in Nurr1 deficient transcription controls, alternative splicing, mRNA stability, 1CC. 15 DISCUSSION: A discussion of the animal model data is translational control, enzyme stability, feedback inhibition on presented in EXAMPLE 17, below. enzyme activity by end products such as dopamine, allosteric MATERIALS AND METHODS: RotaRod performance modulation of enzyme activity and phosphorylation-depen measurements (time that balance was maintained on the rod) dent activation of the enzyme by various kinase systems. TH were performed (as in Study #1), each week during 8 weeks protein, and the TH gene, are stress responsive. Up-regulation of treatment. After RotaRod testing, COMPOUND #1 (20 of TH/TH activity and expression is triggered during hypoxia mg/kg) was administered twice daily ip; Vehicle control ani and may supply neurotransmitters needed for critical sym mals received NaCl twice daily; WT control animals received pathoadrenal stress-responsive functions such as changing NaCl twice daily. breathing and heart rate and liver function. 25 Dopamine beta-hydroxylase (DBH) catalyzes conversion Example 16 of dopamine (DA) to noradrenaline (NA) and is selectively Measurements of Tyrosine Hydroxylase Levels in expressed in noradrenergic and adrenergic neurons and neu the Nigrostriatal Region of Mice Treated with roendocrine cells. Compound #1 DAT is intended to mean the dopamine (DA) presynaptic 30 monoamine transporter encoded by the DAT gone. DA regu Mice lesioned with MPTP and treated with 20 mg/kg of lates extracellular levels of DA by reuptake of released DA. Compound #1, or NaCl as a negative control, for two months DAT is an important site of action of amphetamine and according to EXAMPLE 13 were sacrificed and brains were prepared for immunocytochemistry with an aim toward cocaine. Hic-5 is an adaptor protein associated with the intra detecting both (i) the numbers of neurons in the nigrostrial 35 cellular domain of DAT and involved in signal transduction region that stain positively for the presence of TH; and, (ii) the mediated by DAT. Hic-5 contains multiple Lin-11, Isl-1 and amount of TH per neuron. Mec-3 (LIM) domains. Hic-3 activation, i.e., phosphoryla The results of these experiments indicated, significantly, tion, occurs during the course of dopamine binding and trans that: port. Hic-5 is a ligand for Nurr-1. GLUT3-LIM protein is (i) the number of neurons staining positively for TH were 40 intended to mean a GLUT3 adaptor protein associated with approximately 20-30% greater, (but not statistically signifi the intracellular domain of GLUT3 and having multiple Lin cant due to the Small numbers of animals per group, i.e., n-6). 11, Isl-1 and/or Mec-3 domains. GLUT3-LIM is activated, in mice treated with Compound #1; and, i.e., phosphorylated, during the course of Compound #1 bind (ii) the amount of TH per neuron was qualitatively much ing and transport. higher in the Compound #1 treated animals. 45 VMAT2 is the vesicular monoamine transporter respon Normal, non-MPTP-lesioned mice treated for two months according to EXAMPLEil 13 with Compound #1 at 20 mg/kg sible for vesicle sequestration of dopamine that has been (daily/ip) showed normal locomotor activity and brain histol transported into the cell via DAT. ogy without any evidence of overt or histologic toxicity. GLUT is intended to mean a family of structurally related, MPTP-lesioned mice treated for two months with Compound membrane-spanning glycoproteins that mediate the facilita #1 at 20 mg/kg (daily/ip) recovered locomotor activity and 50 tive transport of glucose across plasma membranes. Six histologically showed no evidence of any neural toxicity GLUT isoforms have been cloned (to date) with GLUT1 over-and-above that shown by control MPTP-lesioned NaCl widely expressed in endothelial cells of the vasculature treated animals. including neural vasculature; GLUT3 expressed strongly in DISCUSSION: The results obtained in the present the trophoblast and neural cells of the brain and retina; EXAMPLE 16, are discussed in EXAMPLE 17, below. 55 GLUT4 expressed in Somatic cells including muscle and adipose tissue; and GLUT2 expressed primarily in the pan Example 17 creas and liver but also in the intestine and kidney. GLUT2 gene regulation may act in molecular sensor mechanisms Low-Level Delivery of Compounds #1 and #2 determining production of insulin by pancreatic beta-cells Effects Cross-Coupling of Transcriptional 60 and GLUT4 gene regulation may act in hepato-portal Regulation of the Tyrosine Hydroxylase Geneas molecular sensor mechanisms regulating systemic glucose Mediated Through DAT and GLUT Transporters concentrations. GLUT1 is intended to mean the endothelial glucose trans Citations made in this EXAMPLE 17 appear at the end of porter isoform associated with neural endothelial cells. Resi this particular examples section. By convention, genes are 65 dent in the cytosol, these transporters are rapidly mobilized to referred to herein with italices and their protein products in and from the plasma membrane in response to changes in regular text. extracellular glucose concentrations. The GLUT1 gene is US 9,302,982 B2 39 40 stress responsive and hypoxia or hypoglycemia induce leukin-1; TNF-C. is tumor necrosis factor-alpha; IFN-Y is increased levels of GLUT1 mRNA, increased translocation of interferon-gamma; NGF is nerve growth factor; and, FGF is and induces increased. fibroblast growth factor. GLUT3 is intended to mean the neural glucose transporter PACAP is intended to mean pituitary adenylate cyclase isoform associated with neural cells. activating polypeptide. Hypoxia is intended to mean low oxygen tension, e.g., less ERK is intended to mean the extracellular-regulated kinase than 5% O. Hypoxia increases expression of both the involved with c-fos in Ca" calmodulin dependent responses tyrosinehydroxylase (TH) and certan GLUT gene products in to hypoxia; PIK3 is the phosphoinositide-3 kinase cascade. neural cells. The response to hypoxia is a cell-type specific, Nurr1 is Nur-related factor 1 a member of the Nur subfam not generalized, and the gene-level response shows differ 10 ily of orphan nuclear receptors which are ligand activated ences in regulation for different isoforms of GLUT. i.e., nuclear receptors, i.e., Nurr1 is also known as aka NOT, GLUT1 and GLUT3 are activated for increased transcription TINUR, RNR-1, HZF-3, and NR4A2; Nurr2, also known as while GLUT2 is down-regulated. Nurr1a, is an alternatively spliced and frame-shifted carboxy HIF-1 is intended to mean HIF-1C, HIF-1B and/or HIF-2C. 15 truncated form of Nurr which negatively regulates the bind which are hypoxia-inducible transcription factors in Somatic, ing of several members of the Nur subfamily to NBRE sites: (e.g. myoblasts, cardiac muscle cells, fibroblasts, epithelial RXR is the retinoid X receptor activator of Nurr1; and, SRC cells and the like), and neural cells, (e.g. cells of the central is a steroid receptor coactivator of Nurr1 protein. and peripheral nervous system), respectively. HIP-1C. is Background: capable of forming functional regulatory heterodimers with Tyrosine hydroxylase catalyzes the rate-limiting step in aryl hydrocarbon nuclear translocator (ARNT) and is biosynthesis of catecholamines including dopamine, norepi encoded by a gene whose promoter contains a hypoxia nephrine and epinephrine. Regulation of the enzyme and gene response element (HRE). HIF-1 is capable of up-regulating encoding the enzyme represent the central means for control TH, GLUT1 and GLUT3. Activity of HIF-1C protein may ling midbrain synthesis of these important central and sym also be increased in cells exposed to low oxygen tension. 25 pathetic neurotransmitters and adrenomedullary hormones. VHL is intended to mean von Hippel-Lindau protein which Accordingly, tyrosine hydroxylase appears to be modulated interacts with HIF-1C. and decreases HIF-1C. degradation by by nearly every documented form of regulation including e.g. inhibiting ubiquination. all of the following: namely, (i) gene transcriptional regula Pitx3 is intended to mean the pair-like homeodomain tran tion of TH mRNA levels; (ii) alternative mRNA processing/ Scription factor 3 also known as pituitary homeobox 3. 30 splicing that generates four different enzyme isoforms with Pitx3RE is the GGCTT response element for Pitx3. differences in activity; (iii) regulation of mRNA stability; (iv) Pro-OHases is intended to mean prolyl-4-hydroxylases possible translational control; (v) enzyme stability controls: active in hydroxylation of HIF-1 resulting in ubiquitination (vi) enzyme co-factor requirements for tetrahydrobiopterin; and Subsequent proteasome-dependent degradation. (vii) enzyme feedback inhibition of the enzyme protein by NFKB is intended to mean the nuclear transcription factor 35 catecholamine end- and intermediate-products, e.g. dopam kappa B. NFKB is mobilized from the cytoplasm of cells into ine, L-dopa, and NE: (viii) allosteric modulation of enzyme the nucleus in response to cytokines, (e.g., IL-1, TNF-C. activity; and, (ix) phosphorylation-dependent activation of IFN-Y), hypoxia and certain other cellular stress insults. enzyme activity. At the gene level, transcription controls are NFKB increases transcription of stress related genes. exerted as depicted by the inventors in FIG. 9. PKC is protein kinase C. PKA is protein kinase A; PI3K is 40 FIG. 9 schematically depicts regulation of the Tyrosine posphoinositol-3-kinase; PP1 is protein phosphatase 1: PP2A Hydroxylase gene by cis- and trans-acting elements affecting is protein phosphatase 2A; and, MEK 1/2 kinase is mitogen the promoter region of the gene. In FIG. 9-12, positive (+) enhanced kinase 1 or 2. signs associated with arrows depict that the factor at the CREM is intended to mean cAMP-responsive element beginning of the arrow increases expression or activity of the modulator; is also known as CREB, an acronym for cAMP 45 factor at the arrow head; negative (-) signs depict that the response element binding protein; and, CBP, CREB binding indicated factor decreases expression or activity; and, all protein that is a co-activator of CREB. indicated activities are as they would occur in wild-type neu AP-1 is intended to mean activator protein-1 transcription ral cells in the substantia nigra. Unless otherwise indicated, factor, AP-1 is intended to mean a genomic AP-1 binding site all cell-type specific gene regulatory interactions presented in Such as commonly present in a gene promoter region; IRE is 50 FIG. 9-13 are as they occur in human nigrostriatal neural intended to mean an insulin response element in a promoter cells. region of a gene; SRE is a serum-response element in a The MPTP Model of Parkinsonism: promoter region of a gene: TRE is a TPA (phorbol myristate) N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as oxi sensitive response element in a promoter region of a gene; dized by monoamine oxidases in neural cells to MPP" is FSE is a fat specific response element that is present within 55 currently believed to be a potent inhibitor of mitochondrial neural cells at an AP-1 regulatory site that may be responsive electron transport, increasing oxidative stress, and inducing to NGF signaling through the Fos/Jun transcription complex; apoptotic mechanisms responsible for programmed cell death NBRE is the nerve growth factor induced protein I-B binding in the nigrostrial region of mice, monkeys and man. MPTP site response element which is a Nurr1 response element, also chemical-leisioning results in fewer TH-positive nigral neu referred to herein as NRE; GRE is glucocorticoid response 60 rons with decreased expression of pre-synaptic DAT and element; and RAR is the retinoic acid response element. post-synaptic dopaminergic receptors of the D1 and D2 fami VGCC is intended to mean voltage-gated Ca" channels lies and increased expression of the alpha Synuclein gene involved in membrane depolarization changes and Ca" (37); and, with the appearance of behavioral abnormalities influx. well correlated with decreased expression of the DAT, VMAT BDNF is brain-derived neurotrophic factor; GCNF is glial 65 and TH genes (38). MPTP treatment has also been disclosed cell neurotrophic factor, CRH is corticotropin-releasing hor by certain investigators to increase DA turnover in a manner mone; PDGF is platelet-derived growth factor; IL-1 is inter inhibited by pre-treating mice with 17? 3-estradiol, progester

US 9,302,982 B2 43 44 subjects, and this loss of TH-ir neurons was highly correlated that Nurr1 dimeric transcription factors may be activation (r–0.92) with the loss of Nurr1-ir neurons. targets in PKA signaling pathway triggered by peptide hor Nurr1: Regulation by Purine Nucleosides Released in OS. Stress Responses: Multiple Requirements and Other Dopaminergic Diseases: As set forth above, Nurr1 is an orphan member of the 5 In reviewing evidence from recent transgenic knock-out nuclear hormone receptor Superfamily NR4A subgroup along animal model studies, Ells (17) reminds (i) that alterations in with its close relative NOR-1. This subgroup is categorized as dopaminergic neurotransmission have been implicated in a immediate early response genes that are induced through number of human neuropathologies including Parkinson's multiple signal transduction pathways. They have been impli disease, Schizophrenia and attention deficit/hyperactivity dis cated in cell proliferation, differentiation, T-cell apoptosis, 10 order; and, (ii) that there are multiple requirements for devel chondrosarcomas, neurological disorders, inflammation and opment, transmission and maintenance of DA neurons: atherogenesis. However, the mechanism of transcriptional namely, for development the genes engrailed-1 and -2, limX1b activation, coactivator recruitment, and agonist mediated and Nurr1; for neurotransmission tyrosine hydroxylase (TH). activation remains largely obscure. Recent studies of NOR-1 15 vesicular monoamine transporter, dopamine transporter indicate that a variety of 6-MP analogs all efficiently activated (DAT) and the D2 and D3 receptors; and, for neuronal sur NOR-1. These findings lead the inventors to believe that vival alpha-synuclein, glial neurotrophic factor and SuperoX signaling pathways already known to modulate cell prolifera ide dismutase. tion through inhibition of de novo purine and/or nucleic acid Dysregulation in dopaminergic neurotransmission might biosynthesis may also be involved in regulating the activity of 20 play a role in the pathogenesis of schizophrenia, and therefore transcription factors in the NR4A family and Nurr1. genetic components of the dopamine (DA) pathway could Abayratna Wansa and coworkers (13) Suggested that tran conceivable confer a genetic risk. Since NR4A2/Nurr1 is scription factors of the NR4A subgroup may be regulated by essential for the development and maintenance of mesen and mediates the genotoxic stress response, i.e., this subgroup cephalic DA-synthesizing neurons and the Nurr1 gene prod of transcription factors are Suggested to function as sensors to 25 uct forms heterodimer with the retinoid X receptor (RRX), it identify modified purines which could result in genotoxicity. is possible that disturbances in retinoid-signaling could be Ordentlich et al. (14) investigated Nurr1 instead of NOR-1 involved in susceptibility to schizophrenia. However, and found, in similar manner, that Nurr1 appears to be subject Iwayama-Shigeno et al. (18) report that data do not support to regulation by purine nucleosides and may be activated by the notion that haplotype analysis of Schizophrenic polymor 30 phisms in Japanese do not support a role for the NR4A2/ 6-mercaptopurine (14). From these combined findings it now Nurr1 gene plays a major role in determining statistical risk. appears possible to the inventors that altered nucleosides Modulation of the NURR subfamily of nuclear receptors and/or purine nucleosides released in stroke and other geno may also be an important mechanism regulating pathways toxic stress responses may down-regulate expression of associated with inflammatory joint disease. In studies con Nurr1 resulting in-turn in decreased dopamine synthesis. 35 ducted by McEnvoy et al. (19) the signaling mechanisms for Nurr1: Possible Relation by Kinase Pathways: induction of inflammatory mediators in rheumatoid arthritis Studying expression of Nurr1, NGFI-B and NOR-1, Satoh synovial tissue was reported to involve the possible regulation etal. (15) report that all are expressed constitutively in various by the NURR subfamily. Markedly enhanced expression of human neural and non-neural cell lines under the serum NURR1 is observed in synovial tissue of patients with RA containing culture condition and their levels are up-regulated 40 compared with normal Subjects and in primary RA and nor in human neurons by activation of protein kinase A or protein mal synoviocytes and Nurr1 (but not NOR-1 or NUR77) kinase C pathway. mRNA and protein were increased in response to PGE(2), The NGFI-B (Nur77) subfamily of orphan nuclear recep IL-1beta, and TNF-alpha. tors (NRs), which also includes Nurr1 and NOR1, bind the Nurr1: Summary: NurRE regulatory element as either bomo- or heterodimers 45 Recent findings suggest to the inventors that dysregulation formed between subfamily members. These nuclear receptors at the level of Nurr1 has the potential to decrease tyrosine reportedly mediate the activation of pituitary proopiomelano hydroxylase mRNA levels, decreasing levels of dopamine cortin (POMC) gene transcription by the hypothalamic hor synthesis in the substantia nigra—i.e., a Parkinson's hall mone corticotropin-releasing hormone (CRH), a link mark phenotype. Dysregulation in Nurr1 could potentially between neuronal and endocrine components of the hypo- 50 result from any combination of the following: namely, (i) thalamo-pituitary-adrenal axis. CRH effects on POMC tran increased steady-state down-regulation of Nurr1 mediated by Scription do not require de novo protein synthesis, suggesting alpha Synuclein, age-related decreases in Nurr1 or decreased that the signaling mechanisms may be pre-existent in cells. levels of Nurr1 activators (e.g., intracellular levels of purines Elucidating these mechanisms, Maira et al. (16) recently and pyrimidines and the like); (ii) altered Nurr1 protein activ reported that CRH activates Nur factors via the cyclic AMP/ 55 ity resulting in lowered responsiveness to PAKactivity and/or protein kinase A (PKA) pathway. CRH induction of PKA age-related decrease in the activity of one or more Nurr1 apparently rapidly increases nuclear DNA binding activity of co-activator/enhancer (telomeric) proteins like p160/SRC co NGFI-B dimers but not monomers. PKA-activated Nur activators (iii) defects or alterations in Nurr1 dimer formation dimeric factors have enhanced binding to target response with NR4A family member proteins; (iv) mutant altered elements in the POMC gene promoter. Providing additional 60 Nurr1 or Nurr1 co-activator or dimer partner protein; or, (v) mechanistic understanding, Maim et al. (16) reported that genetically altered promoter binding sites in target genes or in p160/SRC coactivators were recruited to the Nur dimers (but the Nurr1 promoter region. not to monomers) and that coactivator recruitment to NurFRE In studies of Nurr1 allelic variation in Parkinson's patients, was enhanced in response to CRH. Thus, in this system PKA Le and coworkers (20) have reportedly identified a homozy activation apparently activates coactivator-induced potentia- 65 gous polymorphism in Intron 6 of the human Nurr1 gene that tion of transcription factor dimmer binding at gene regulatory seems associated at a higher frequency with familial Parkin sites. Taken together, these results suggest to the inventors son's than with spontaneous Parkinson's. US 9,302,982 B2 45 46 At least one recent study suggests an additional possibility: with cAMP. Ca2" flux and possible CREB involvement) may namely, that chronic long-term stimulation at the D2 receptor, up-regulate TH gene expression, e.g. through CRE or AP-1 i.e., in cocaine users, may lead to decreased Nurr1 expression response elements as depicted in FIG.9. However, the results in human brain (21). In other receptor-driven expression sys presented in EXAMPLE 16 also show increased TH protein tems, it is appreciated that compensatory down-regulation of 5 in the nigrostriatal neurons of MPTP-lesioned animals treated transcription can occur in response to chronic long-term with Compound #1. This singular finding indicates that at receptor activation. Thus, it is also possible that chronic pro least some of the important effects of Compound #1 must be duction of inflammatory mediators by microglial cells (mac exerted pre-synaptically, i.e., increasing TH levels and up rophages) in the brain may down-regulate Nurr1 activity. regulating the TH gene—but how? The common perception is Reservoir Delivery: 10 that end-products like dopamine feedback on gene function in Gene transfer studies conducted by others have reported a negative manner. So, either this is a misconception with negative feedback regulation from simultaneous expression respect to Compound #1, or there must be some other expla of the genes for TH; GTP-cyclohydrolase-I, (rate limiting in nation for the findings of increased TH protein in Compound tetrahydrobiopterin -BH1- co-factor synthesis); and, aro #1-treated animals. Theoretically, increased immunorespon matic L-amino decarboxylase (capable of converting the TH 15 sive TH protein levels in treated animals could result from L-Dopa product into dopamine), i.e., the reconstructed cells either: (a) increased protein stability, i.e., decreased turnover; produced dopamine and the synthesis was then apparently (b) increased mRNA stability, i.e., decreased turnover, with down-regulated by the dopamine end-product (29). Negative increased TH protein synthesis; or, (c) from increased mRNA feedback regulation was reportedly overcome in these gene transcription and translation. At present it is believed most therapy studies (29) by also supplying a vesicular monoamine likely that the majority of the increased TH protein results transporter with the aim of sequestering the synthesized from increased TH mRNA transcription and translation. It is L-dopa and dopamine within vesicles. Such findings rein believed less likely that stability of mRNA and protein force the inventors belief in the great importance of Com accounts for the observed changes in the striatum of animals pound #1 binding and transport by DAT and GLUT transport treated with Compound #1. Assuming in argumentum that ers, as well as, the importance of this uptake route in 25 this is the case, then even if Compound #1 is binding and establishing sequestration into vesicles, as discussed further activating D1/D5 it is likely that up-regulation of TH relates below. to some other property of the instant class of compounds. Coordinated DAT/GLUT3 Signaling: Coordinated Therapeutic Mode of Action: DAT and GLUT are transporters, to the inventor's knowl Potential in vim binding sites for Compound #1, i.e., in edge these plasma membrane proteins have not previously 30 addition to D1/D5 receptor sites, may be reasoned as follows: been implicated in signal transduction or gene-level regula namely, tory controls. (i) Dopamine re-uptake transporters, i.e., DAT, mediate Discussion: pre-synaptic re-uptake of dopamine from the synaptic cleft In Vitro and InVivo Studies: and VMAT2 transporters sequester the reuptake-dopamine The results presented in EXAMPLES 13-16, supra, show 35 preventing possible toxicity associated with dopamine oxida that Compound #1 alleviated locomotor symptoms in three tion products. It seems likely to the inventors that DAT different animal models commonly used to evaluate a Parkin reuptake processes are monitored at a gene level and that sons drug candidate: namely, the MPTP-lesioned mouse response elements must exist in the TH regulatory region to model (EXAMPLE 13): 6-hydroxydopamine lesioned rats account for DAT activity. Similarly, DAT associated co-fac (EXAMPLE 1.4); and, the Nurr1 genetic knockout mouse 40 tors such as Hic-5, LIM or other adaptor proteins must serve model (EXAMPLE 15). functions in signal transduction to the TH gene. Thus, is Potential Mechanisms of Action: currently believed highly likely that Compound #1 binding Attempting to explain therapeutic mode of action, binding and translocation at DAT trigger changes effective to up of Compound #1 to neural cells in the striatum may occur regulate the TH gene. Signaling through DAT and regulatory according to one or more of the following mechanisms: 45 affects of DAT transport on TH gene expression have, to the namely, (i) binding as an agonist at D1/D5 receptors post inventor's best knowledge, not been previously disclosed; synaptically to Supply needed dopamine for post-synaptic (ii) It is also believed highly likely that GLUT3 facilitative innervation; (ii) binding as an agonist at D1/D5 receptors transporters on nigral neural cells mediate uptake of Com located on pre-synaptic plasma membrane; (iii) binding as an pound #1. It is believed highly likely that glucose response agonist, at a low level, at a D2/D3 receptor; (iv) transport via 50 elements exist in the TH gene regulatory region and that a pre-synaptic DAT transporters; and, (iv) transport via a pre GLUT3 transport is monitored at a gene level. Again, to the or post-synaptic neural GLUT3 transporter. inventor's best knowledge, signaling through GLUT and The results of in vitro experiments presented in regulatory effects on TH gene expression have not previously EXAMPLES 11 and 12 show that (a) Compound #1 has been disclosed; and, predominantly D1/D5 binding specificity and that receptor 55 (iii) it is believed highly likely that monitoring glucose binding triggers signal transduction with changes in intracel transport through GLUT3 and dopamine reuptake through lular cAMP; (b) Compound #1 is transportable at DAT; and, DAT constitutes a novel important sensor System operative (c) Compound #1 is transportable at GLUT1, i.e., as evi within striatal neural cells for controlling bodily metabolic denced by the locomotor effects disclosed in EXAMPLES responses to stress and trauma. Again, to the inventor's best 13-16 confirming transport via GLUT1 at endothelial cells of 60 knowledge a coordinated DAT/GLUT3 sensor system opera the blood brain barrier. Possible binding at a D2/D3 receptor tive in nigral cells at the level of TN gene regulation has not site in vivo, thus, seems less likely than binding to a D1/D5 been previously disclosed. receptor. Advantageous Properties of the Instant Therapeutic Com D1/D5 Agonist Activity: pounds: Binding at D1/D5 pre- or post-synaptic receptors with 65 Transport of Compound #1 by DAT and GLUT transport associated triggering of a kinase-mediated signal transduc ers insures that uptake and sequestration into VMAT2 and tion cascade (e.g. through PKA, PKC or calmodulin kinase II GLUT3 cytoplasmic vesicles, thereby alleviating potential US 9,302,982 B2 47 48 dopamine intracellular toxicity of the degradation and oxida or activity of the factor at the arrow head; negative (-) tion products of Compound #1, as well as, possible negative signs depict that the indicated factor decreases expres feedback inhibitory activity of dopamine at both the enzyme sion or activity; and, all indicated activities are as they and gene transcription levels. Vesicular storage also allows would occur in wild-type neural cells in the substantia time for processing of pro-drug Compound #1 into glucose nigra. and dopamine, and assures that the processed dopamine is FIG. 10 schematically depicts the disclosed regulatory readily available for synaptic release. Vesicular storage in mechanisms operative at the GLUT (vascular endothe striatal neural cells in the midbrain, acting in combination lial glucose transporter) gene by cis- and trans-acting with gradual release of Compound #1 from GLUT1 receptors elements affecting the promoter region of the gene. on red blood cells and regulated transport of Compound #1 10 FIG. 11 schematically depicts the disclosed regulatory via endothelial cells at the blood brain barrier, are all pres mechanisms operative at the GLUT3 (neural glucose ently believed to be key in achieving the therapeutic efficacy transporter) gene by cis- and trans-acting elements of the instant class of therapeutic agents, i.e., via gradual affecting the promoter region of the gene. sustained release at low levels. FIG. 12 schematically depicts the disclosed regulatory A Novel Drug Target: 15 mechanisms operative at the DAT (dopamine re-uptake In Summary, the finding that TH immunoreactive protein is neural transporter) gene by cis- and trans-acting ele increased in striatal neurons in Compound #1-treated MPTP ments affecting the promoter region of the gene. lesioned mice (EXAMPLE 16) is considered to be highly FIG. 13 schematically depicts the disclosed regulatory significant as it indicates to the inventors a previously un mechanisms operative at the Nurr gene by cis- and trans recognized linkage between TH gene expression and com acting elements affecting the promoter region of the pounds, like Compound #1, that are capable of simulta gene. neously accessing GLUT3 and DAT. Further, the findings FIG. 14 schematically depicts coordinate regulatory indicate highly likely gene regulatory controls exerted on TH mechanisms exerted by SP1, SP2, Nurr1 and Nurr2 at transcription and/or enzyme protein which are responsive to the gene regulatory regions of the DAT, VMAT2 (the GLUT3 and DAT transporter activity. The following addi 25 dopamine vesicular storage transporter), TH, GLUT1, tional reasoning may be posited: namely, GLUT3. D1A (Dopaminergic receptor-1A) and mem 1) Why should there be coupling of signaling at GLUT3, brane transporters Adenine nucleotide translocate-2 and DAT and TH2 Important clues about possible coordinate cou beta-galactosidase-Sialyltransferase. pling of regulation at GLUT3, DAT and TH is evident in 4) To the inventors thinking, a coordinated rapid system for detailed analysis of TH gene regulation: e.g., responsiveness 30 responding to stress, trauma, hypoxia and inflammation of TH gene promoter elements to adrenocorticoid hormones, requires a single effector operative in simultaneously up insulin, glucose levels and hypoxia. Teliologically, the pres regulating the critical dopaminergic gene functions, i.e., the ence of “fight-or-flight glucocorticoid regulatory elements transcription factor Nurr1. A critical system for responding to (GRE) in the TH promoter region along with glucose sensor stress also requires counteracting balances, i.e., Nurr2, as capability Suggests that in the process of responding to a 35 well as a multiplicity of backup components, i.e., the regula “flight' directive from adrenal gland hormones the brain may tory mechanisms set forth in FIG. 9-13. However, such a judge it to be wise to check available bodily reserves of both rapid-response system is undoubtedly not suitable for day-to dopamine and glucose, and if necessary, increase brain glu day balancing and tuning of nigral dopamine products. Thus, cose transport, expression of TH and output of parasympa as set forth schematically in FIG. 14, it is believed most likely thetic and sympathetic mediators; 40 that reciprocal SP1/SP3-mediated balancing controls exerted 2) Assuming in argumentum that the midbrain nigrostrital on DAT, GLUT 3, T, and other dopaminergic components region contains a coordinated sensor for glucose and dopam (FIG. 14) provides basal level balanced +/- regulatory con ine, then what is it and how does it work? Again, the key rate trols to fine-tune ongoing cellular functions, i.e., housekeep limiting enzyme in catecholamine synthesis is tyrosine ing functions. The multiplicity of backup components for hydroxylase. The importance of this enzyme is affirmed by 45 maintaining control of critical catecholamine synthesis are the findings that virtually every form of known regulation is depicted in FIG. 9-13. exerted upon the TH gene. mRNA and protein (as Summa Defects in Existing L-Dopa, Carbidopa and Synemet rized in the Background section above). Even breathing and Treatments: heart rate are regulated by the catecholamine neurotransmit Existing treatments of Parkinson's disease suffer funda ters whose synthesis is catalyzed by TH. Thus, the promoter 50 mental mechanistic flaws in view of the instant disclosed regulatory region of the TH gene and its associated transcrip regulatory interactions, as also supported by certain recent tion factors is believed by the inventors to be the key sensor disclosures from others. For example, Guttman et al. (30) system for balancing dopamine synthesis in response to glu disclosed results of PET studies in patients with early Parkin cose transport (mediated through GLUT3) and dopamine son's disease (PD), wherein binding activity of test com reuptake through DAT. The following details, below, are 55 pounds at dopamine transporters (DAT) was reportedly offered up in answer to the question “how does it work?: reduced after therapeutic administration of L-dopa. Similarly, 3) FIGS. 9-14 disclose the inventors understanding of sig Torstenson et al. (31) disclosed results of L-DOPA influx naling events relating to regulation of the TH (FIG. 9), rates after levodopa infusion as recorded in PET studies in GLUT1 (FIG. 10), GLUT3 (FIG. 11), DAT (FIG. 12) and patients with mild PD or advanced PD. Influx rates were Nurr1 genes (FIG. 13), as well as how coordinate regulation 60 decreased in patients with mild PD and Increased in patients is achieved through the counter-balancing effects of SP1/SP3 with advanced PD, Suggesting to these authors a positive and Nurr1/Nurr2 (FIG. 14). upregulation in patients with advanced disease. In wild-type FIG. 9 schematically depicts regulation of the Tyrosine rats, L-Dopa:carbidopa (50:5 mg/kg) administered three Hydroxylase gene by cis- and trans-acting elements times daily significantly decreased DAT levels (32). Looking affecting the promoter region of the gene. In FIG. 9-12. 65 at DAT transporters expressed Xenopus oocytes and rat stria positive (+) signs associated with arrows depict that the tum, Gulley et al. (33) disclosed down-regulation of DAT factor at the beginning of the arrow increases expression function after repeated brief exposure to dopamine, AMPH or US 9,302,982 B2 49 50 tyramine. Down-regulation of DAT is very counter-produc phenotype and the Survival of Ventral mesencephalic late tive to Successful long-term therapy and may also account for dopaminergic precursor neurons. PANS USA. 1998, the significant fluctuations seen in patient responsiveness, 95:4O13-4O18 with time, to therapy. Defects in Existing D2 Agonist Treatments: 2. Zetterstrom R. H., Solomin L. Jansson L., et al. Dopamine As depicted schematically in FIG.9, D2 receptoragonists, neuron agenesis in Nurr1-deficient mice. Sciences 1997: but not D1 agonists, have been disclosed by others to down 276:248-250 regulate transcription of the tyrosine hydroxylase gene in 3. Castillo S. O. Baffi J. S., Palkovits M. et al. Dopamine vitro, in tissue slices (e.g. 34), in fetal grafts in animals (e.g. biosynthesis is selectively abolished in substantia nigra/ 35) and in patients. Down-regulation may occur as a result of Ventral tegmental area but not in hypothalamic neurons in D2-agonist induced decreased CRE activity (36), i.e., delete mice with targeted disruption of the Nurr1 gene. Mol Cell rious to transcription of the TH gene (FIG.9). Down-regulat Neurosci 1998; 11:36.46 ing synthesis of tyrosine hydroxylase is very counterproduc 4. Iwawaki T., Kohno K. Kobayashi K. Identification of tive to Successful long-term therapy and may also account for potential Nurr1 response element that activates the tyrosine the significant fluctuations seen in patient responsiveness, hydroxylase gene promoter in cultured cells. Biochem with time, to therapy. D1 agonists act in vitro to increase, 15 rather than decrease, CRE activity (36) i.e., A change advan Biophys Res Comm 2000, 274:590-595 tageous to increased TH gene transcription (FIG. 9). 5. Sacchetti P. Mitchell T. R., Grameman J. G., Bannon M. J. At the Gene Level: Nurr1 enhances transcription of the human dopamine As set forth in regard to the FIGURES above, the instant transporter gene through a novel mechanism. J Neurochem invention discloses that agonists binding at presynaptic 2001; 76:1565-1572 D2/D3 receptors down-regulate Nurr1 gene expression (FIG. 6. Le W. D., Zou L. L., Conneely O. M., et al. Selective 13) with resultant decreased positive-transcription regulation agenesis of mesencephalic dopaminergic neurons in Nurr1 at the DAT gene promoter (FIG. 12) and also at the TH gene deficient mice. Exp Neurol 1999; 159: 451–458 (FIG. 9). As a result, despite supplying substrate L-dopa for 7. Le W. D., et al. Reduced Nurr1 expression increases the dopamine synthesis these D2/D3 agonists actions amply any 25 Vulnerability of mesencephalic dopamine neurons to nigrostriatal deficiencies in TH and DAT, i.e., a counter-pro MPTP-induced injury.J. Neurochem. 1999: 73:2218-2221 ductive in a long term therapy designed to correct a defect in 8. Chung S, Sonntag KC, Anderson T. Bjorklund L. M. Park transcription regulation of a TH gene. J. J. Kim D. W. Kang UJ. Isacson O, Kim K S. Genetic At the Protein Level: As schematically depicted in FIG. 12, engineering of mouse embryonic stem cells by Nurr1 dopaminergicagonists at D2/D3 receptors may activate phos 30 enhances differentiation and maturation into dopaminergic phorylation of DAT through the actions of PIK3, PKC and/or neurons. EurJ Neurosci 2002 November; 16(10): 1829-38 MEK 1/2. Phosphorylation of DAT down-regulates dopam 9. Baptist. MJ, O'Farrell C, Daya S, Ahmad R, Miller DW, ine re-uptake by (a) sequestering phosphorylated DAT in Hardy J. Farner M.J. Cookson MR. Coordinate transcrip cytoplasmic vesicules, thereby reducing plasma membrane tional regulation of dopamine synthesis genes by alpha DAT available for re-uptake of dopamine; and, (b) reducing 35 the Vmax of dopamine transport by DAT. Both of these Synuclein in human neuroblastoma cell lines. J Neurochem actions are counter-productive in a long term therapy 2003 May: 85(4):957-68 designed to correct a nigrostriatal dopamine deficiency. 10. Kim KS, Kim CH, Hwang DY, Seo H. Chung S. Hong S Additional Therapeutic Methods of Use: J. Lim L. K. Anderson T. Isacson O. Orphan nuclear recep The Ventral tegmental area (VTA) and substantia nigra 40 tor Nurr1 directly transactivates the promoter activity of pars compacta (SNC) regions of the striatum includes cells the tyrosine hydroxylase gene in a cell-specific manner. J whose synapses impinge in brain areas implicated in locomo Neurochem 2003 May: 85(3):622-34 tion; conditioning, learning and motivation of appetite and 11. Kessler MA, Yang M. Gollomp KL, Jin H, Iacovitti L. feeding behavior; alcohol withdrawal seizures; and, reward. The human tyrosine hydroxylase gene promoter. Brain Res The disclosed association of DAT, GLUT3 and TH (above) 45 Mol Brain Res 2003 Apr. 10; 112(1-2):8-23 indicates additional uses for the compounds of the instant 12. ChuY. Kompoliti K, Cochran EJ, Mufson E.J. Kordower invention in treatments of feeding disorders, e.g., binging J. H. Age-related decreases in Nurr1 immunoreactivity in eating leading to obesity and increasing risk of Type 2 diabe the human substantia nigra. J Comp Neurol 2002 Aug. 26: tes, bulimia nervosa, anorexia and the like. Other uses of the 450(3):203-14 compounds of the instant invention include the following: 50 13. Abayratna Wansa K S. Harris J. M. Yen G, Ordentlich P. namely, (i) reversing DAT blockade by psychoStimulants Muscat G. E. The AF-1 domain of NOR-1/NR4A3 medi Such as cocaine, e.g. in an acute care setting; (ii) treatments ates trans-activation, coactivator recruitment, and activa for alcohol withdrawal symptoms including seizures; (iii) tion by the purine anti-metabolite 6-Mercaptopurine. 2003 treatments for insecticide poisoning related to dysfunctional J Biol Chem April 22; epub ahead of print regulation of TH and DAT; (iv) treatments for sleep apnea 14. Ordentlich P. Yen Y. Zhou S, Heyman RA. Identification and/or narcolepsy as related to dysfunctional regulation of of the anti-neoplastic agent 6-mercaptopurine as an activa TH aid DAT; (v) treatments for schizophrenia, attention defi tor of the orphan nuclear hormone receptor Nurr1, 20033 J cit disorder and hyperactivity syndromes; (vi) treatments for Biol Chem April 22 epub ahead of print panic disorders; ad, (vii) treatments of die rheumatoid syn 15. Satoh J. Kuroda Y. The constitutive and inducible expres ovium, lung and immune systems, wherein the aim of each of 60 sion of Nurr1, a key regulator of dopaminergic neuronal the foregoing treatments is to restore a more normal transcrip differentiation, in human neural and non-neural cell lines. tion regulation of DAT and TH to a patient in need thereof. Neuropathology 2002 December; 22(4):219–32 16. Maira M, Martens C, Batsche E. Grauthier Y. Drouin J. CITATIONS MADE IN EXAMPLE 17 Dimer-specific potentiation of NGF1-B (Nur77) transcrip 65 tional activity by the protein kinase A pathway and AF-1- 1. Saucedo-Cardenas O. Quintana-Hau J D. Le WD), et al. dependent coactivator recruitment. Mol Cell Biol 2003 Nurr1 is essential for the induction of the dopaminergic February; 23(3):763-76 US 9,302,982 B2 51 52 17. Fells, J. B. The control of dopamine neuron development, 33. Gulley, J. M. Doolen, S., Zahniser, N. R. Brief, repeated function and Survival: insights from transgenic mice and exposure to Substrates down-regulates dopamine trans the relevance to human disease. Curr Med Chem 2003 porter function in Xenopus oocytes in vitro and rat dorsal May: 10(10):857-70 striatum in vivo. 2002; J. Neurochem. 83 (2): 400-11. 18. Iwayama-Shigeno Y. Yamada K, Toyota T. Shimizu H, 5 34. Hu G, Jin G Z. (-)-Stepholidine antagonizes the inhibition Hattori E. Yoshitsugu K, Fujisawa T.Yoshida Y. Kobayashi by D2 receptor agonists on synaptosomal tyrosine T, Toru M. Kurumaji A, Detera-Wadleigh S. Yoshikawa T. hydroxylase in rat corpus striatum. Zhongguo Yao Li Xue Distribution of haplotypes derived from three common Bao. 1995 16(4): 376-9. variants of the NR4A2 gene in Japanese patients with 35. Meloni R. Gale, K. Pharmacological evidence for feed schizophrenia. Am J Med Genet 2003 Apr. 1; 118B(1):20-4 10 19. McEvoy A N, Murphy E A, Ponnio T. Conneely O M, back regulation of dopamine metabolism in Solid fetal Bresnihan B, FitzGerald O. Murphy, E. P. Activation of substantia nigra transplants. J Pharmacol Exp Ther. 1990 nuclear orphan receptor NURR1 transcription by 253 (3): 1259-64. NF-kappa B and cyclic adenosine 5'-monophosphate 36. Takeuchi Y., Fukunaga K. Differential regulation of NF response element-binding protein in rheumatoid arthritis 15 kappa B, SRE and CRE by dopamine D1 and D2 receptors synovial tissue. J Immunol 2002 Mar. 15: 168(6):2979-37 intransfected NG108-15 cells.J. Neurochem. 1003 35(3): 20. Xu PY. Liag R, Jankovic J. Hunter C, ZengYX, Ashizawa 729-39. T. Lai D. Le W. D. Association of homozygous 7048G7049 37. Kuhn K, Wellen J, Link N., Maskri L, Lubbert H, Stichel variant in the intron six of Nurr1 gene with Parkinson's CC. The mouse MPTP model: Gene expression changes in disease. Neurology 2002 Mar. 26:58(6):881-4 dopaminergic neurons. Eur. J. Neurosci. 2003, 17(1): 1-12. 21. Bannon M J. Pruetz. B. Manning-Bog A B, Whitty C J, 38. Tillerson J. L., Caudle W. M., Reveron M E, Miler G. W. Michelhaugh S K, Sacchetti P. Granneuman J. G. Mash D Detection of behavioral impairments correlated to neuro C. Schmidt C.J. Decreased expression of the transcription chemical deficits in mice treated with moderate doses of factor NURR1 in dopamine neurons of cocaine abusers. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Exp. Neu Proc Natl AcadSci USA 2002 Apr. 30: 99(9):6382-5 25 rol. 2002, 178(1): 80-90. 22. Saucedo-Cardenas O. Quintana-Hau JD, Le WD, et al. 39. D'Astous M, Morissette M, Tanguay B, Callier S, Di Nurr1 is essential for the induction of the dopaminergic Paolo T. Dehydroepiandrosterone (DHEA) such as 17beta phenotype and the Survival of Ventral mesencephalic late estradiol prevents MPTP-induced dopamine depletion in dopaminergic precursor neurons. PANS USA. 1998: mice. 258 Synapse 2002 47(1): 10-14. 95:4O13-4018 30 23. Zetterstrom R. H., Solomin L. Jansson L., et al. Dopam CITATIONS MADE IN THE BACKGROUND, ine neuron agenesis in Nurr1-deficient mice. Sciences DETAILED DESCRIPTION AND EXAMPLES 1997:276:248-250 1-16 24. Castillo S. O. Baffi J. S. Palkovits M. et al. Dopamine biosynthesis is selectively abolished in substantia nigra/ 35 Alexander, N., Yoneda, S. Vlachakis, N. D. and R. F. Ventral tegmental area but not in hypothalamic neurons in Maronde. 1994. Role of conjugation and red blood cells for mice with targeted disruption of the Nurr1 gene. Mol Cell inactivation of circulating catecholamines. Am. J. Physiol. Neurosci 1998; 11:36-46 247 (1): R203-R207. 25. Iwawaki T., Kohno K. Kobayashi K. Identification of Alvarado, F. and R. K. Crane. 1960. Plorizin as a competitive potential Nurr1 response element that activates the tyrosine 40 inhibitor of the active transport of sugars by hamster small hydroxylase gene promoter in cultured cells. Biochem intestine, in vitro. Biochim. Biophys. Acta 56: 170-172. Biophys Res Comm 2000: 274:590-595 Arita, H. and J. Kawanami. 1980. Studies on uptake of phenyl 26. Sacchetti P. Mitchell T. R., Grameman J. G., Bannon M.J. glycosides as inhibitors of D-glucose uptake by Rhesus Nurr1 enhances transcription of the human dopamine monkey kidney calls. J. Biochem. 88: 1399-1406. transporter gene through a novel mechanism. J Neurochem 45 Barnett, J. E. G., Holman, G. D. and K. A. Munday. 1973. 2001; 76:1565-1572 Structural requirements for binding to the Sugar transport 27. Le W. D. Zou L. L., Conneely O. M., et al. Selective system of the human erythrocyte. Biochem. J. 131: 211 agenesis of mesencephalic dopaminergic neurons in Nurr1 221. deficient mice. Exp Neurol 1999; 159: 451–458 Barnett, A., McQuade, R. D. and C. Tedford. 1992. High 28. Le W. D., et al. Reduced Nurr1 expression increases the 50 lights of D1 dopamine receptor antagonist research. Neu Vulnerability of mesencephalic dopamine neurons to rochem. Int. 20 (Suppl.): 119S-122S. MPTP-induced injury.J. Neurochem. 1999: 73:2218-2221 Bencsics, A., Sershen. H. Baranyi, M., Hashim, A., Lajtha, A. 29. Kang UJ, Lee WY. Chang J. W. Gene therapy for Parkin and E. S. Vizi. 1997, dopamine, as well as norepinephrine, Son's disease: determining the genes necessary for optimal is a link between noradrenergic nerve terminals and sple dopamine replacement in rat models. Hum. Cell. 2001 55 nocytes. Brain Res. 761 (2): 236-243. March;4(1):39-48. Berger, J. G., Chang, W. K., Clader, J. W., Hou, D., Chipkin, 30. Guttman M., Stewart D. Hussey D., Wilson A., Houle S., R. E. and A. T. McPhail. 1989. Synthesis and receptor Kish S. Influence of L-dopa on striatal dopamine trans affinities of some conformationally restricted analogues of porter in early PD. 2001; Neurology 56(11): 1559-64. the dopamine D, selective ligand (5R)-8-chloro-2,3,4,5- 31. Torstenson R. Hartvig P. Langstrom B. Westerberg G., 60 tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol. J. Tedroff J. Differential effects of levodopa on dopaminergic Med. Chem. 32: 1913-1921. function in early and advanced Parkinson's disease. Ann. Brewster, W.K., Nichols, D. E., Riggs, R. M., Mottola, D.M., Neurol. 1997: 41(3): 334-40. Lovenberg., t. W., Lewis, M. H. and R. B. Mailman. 1990, 32. Reveron ME, Savelieva KY. Tillerson JL, McCormack A trans-10,11-dihydroxy-5,6,7,8,12b-hexahydrobenzoa L., Di Monte DA, Miller G. W. L-DOPA does not cause 65 pehnanthridine: A highly potent selective dopamine D full neurotoxicity in VMAT2 heterozygous knockout mice. agonist. J. Med. Chem. 33: 1756-1764. Neurotoxicity 2002 23 (4-5): 611-9. Bodor et al. 1978. J. Pharm. Sci, 67 (5): 685.

US 9,302,982 B2 57 58 intestinal active absorption of monosaccharide conjugates. tinal Na+/glucose cotransporter I mRNA levels is tran Biochim. Biophys. Acts 1200 (2): 117-122. scriptionally regulated. J. Biol. Chem. 273 (16): 95.10 Mizuma, T., Ohta. K. Hayashi, M. and S. Awazu. 1992. 9.516. Intestinal active absorption of Sugar-conjugated com Riggs, M. R. Nichols, D. E. Foreman, M. M., Truex, L. L., pounds by glucose transport system: Implications for 5 Glock, D. and J. D. Kohli. 1987. J. Med. Chem. 30: 1454. improvement of poorly absorbable drugs. Biochem. Phar Schauer, R. 1978. In: Methods in Enzymology, Ed. V. Gins macol. 43: 2037-2039. berg. Academic Press, NY. pp. 64–89. Mizuma, T., Ohta. K. Hayashi, M. and S. Awazu. 1993. Seiler, M. P. and R. Markstein. 1982. Mol. Pharmacol. 22: Comparative study of active absorption by the intestine and 281. disposition of anomers of Sugar-conjugated compounds. 10 Seiler, M. P. and R. Markstein. 1989. J. Mol. Pharmacol. 35: Biochem. Pharmacol. 45(7): 1520-1523. 643. Morgan,T. D., Hopkins, T. L., Kramer, K.J., Roseland, C.R., Seiler, M. P. Hagenbach, A., Wuthrich, H-J. and R. Mark Czapaia, T. H., Tomer, K. B. and Crow, F. W. 1987. N-B- stein. 1991, trans-Hexahydroindolo 4.3-abphenan Alanylnorepinephrine: Biosynthesis in insect cuticle and 15 thridines (“Benzergolines”), the first structural class of possible role in sclerotization. Insect Biochem. 37:255 potent and selective D1 receptor agonists lacking a cat 263. echol group. J. Med. Chem. 34 (1): 303-307. Morgan, M. J. and K. B. Franklin. 1991. Dopamine receptor Shimada, S., Kitayama, S. Lin, C. L., Patel, A., Nanthakumar Subtypes and formalin test analgesia. Pharmacol. Bio E. Gregor, P. Kuhar, M. and G. Uhl. 1991. Cloning and chem. Behav. 40 (2): 317-322. expression of a cocaine-sensitive dopamine transporter Mueller, D.D., Morgan,T. D., Wassenberg, J. D., Hopkins, T. complementary DNA. Science 254 (5031): 576-578. L. and K. J. Kramer. 1993, 1H and 13C NMR of 3-O and Shindo, H., Komai, T. and K. Kawaii. 1973. Metabolism of D 4-O conjugates of dopamine and other catecholamines. and L-isomers of 3.4 dihydroxyphenylalanine (DOPA). V. Bioconjug. Chem. 4(1): 47-53. Mechanism of intestinal absorption of carbon-14 labeled Navarro, H., Arruebo, M. P., Alcalde, A. I. and V. Sorribas. 25 D- and L-dopa in rats. Chem. Pharm. Bull 21 (9): 2031 1993. Effect of erythromycin on D-galactose absorption 2O38. and Sucrase activity in rabbit jejunum. Can. J. Physiol. Shah, J. H. Kline, R. H., Geter-Douglass, B., Izenwasser, S., Pharmacol. 71 (3-4): 191-194. Witkin, J. M. and A. H. Newman. 1996. (+/-)-3-4'-(N.N- Panet H, Barzilai A, Daily D, Melamed E. Offen D. Activation dimethylamino)cinnamylbenzazepine analogs: Novel of nuclear transcription factor kappa B (NF-kappaB) is 30 dopamine D receptor antagonists. J. Med. Chem. 39:3423-3428. essential for dopamine-induced apoptosis in PC12 cells. J Snyder, S. E., Aviles-Garay, F. A., Chakraborti, R., Nichols, Neurochem. 2001 April; 77(2):391-8. D. E., Watts, V.J. and R. B. Mailman. 1995. Synthesis and Petersson, I., Liljefors, T. and K. Bogeso. 1990. Conforma evaluation of 6,7-dihydroxy-2,3,4,8,9,13b-hexahydro-1H tional analysis and structure-activity relationships of selec 35 benzo 6,7cycloheptal 1.2.3ef3 benzazepine, 6,7-dihy tive dopamine D receptor agonists and antagonists of the droxy-2,3,4,8,9,12b-hexahydroanthra-10.4a,4-c.d benzazepine series. J. Med. Chem. 33: 2197-2204. aZepine and 10-(aminomethyl)-9,10-dihydro-1,2- Pokorski, M. and Z. Matysiak. 1998. Fatty acid acylation of dihyroxyanthracene as conformationally restricted dopamine in the carotid body. Med. Hypothesis. 50 (2): analogs of B-phenyldopamine. J. Med. Chem. 38: 2395 131-133. 40 2409. Pocchiari, F., Pataccini, R., Casteinovo, P. Longo, A. and C. Storch, A., Ludolph, A. C. and J. Schwarz 1999. HEK-293 Casagrande. 1986. Ibopamine, an orally active dopamine cells expressing the human dopamine transporter are Sus like drug: Metabolism and pharmacokinetics in rats. Arz ceptible to low concentrations of 1-methyl-4-phenylpuri neim-Forsch. 36 (2A): 334-340. dine acting via impairment of energy metabolism. Neuro Prakash, C., Cui, D., Baxter, J. G., Bright, G. M., Micell, J. 45 chem. Int. 35(5): 393-403. and K. Wilner. 1998. Metabolism and excretion of a new Sugamori, K. S. Lee, F. J., Pristupa, Z. B. and H. B. Niznik, anxiolytic drug candidate, CP-93.393, in healthy male vol 1999. A cognate dopamine transporter-like activity endog unteers. Drug Matab. Dispos. 26 (5): 448-456. enously expressed in a COS-7 kidney derived cell line. Prakash, K. R., Tamiz, A. P., Araldi, G. L., Zhang, M., FEBS Let. 451 (2): 169-174. Johnson, K. M. and A. Kozikowski. 1999. N-phenylalkyl 50 Takata, K., H. Hirano and M. Kasahara. 1997. Transport of Substituted tropate analogs of boat conformation of high glucose across the blood-tissue barriers. Int. Rev. Cytology Selectivity for the dopamine versa serotonin transporter. 172: 1-53. Bioorg. Med. Chem. Lett. 9 (23): 3325-3328. Umegae.Y., H. Nohta and Y. Ohkura. 1988. Anal. Chim. Acta Rabinovic AD, Lewis DA, Hastings T G. Role of oxidative 208: 59. changes in the degeneration of dopamine terminals after 55 Vandenbergh, D.J., Persico, A. M. and G. R. Uhl. 1992. A injection of neurotoxic levels of dopamine. Neuroscience. human dopamine transporter cDNA predicts reduced gly 2000; 101(1):67-76. cosylation, displays a novel repetitive element and pro Ramaswamy, K., Bhattacharyya, B.R. and R. K. Crane. 1976, vides radically-dimorphic TaqI RFLPs. Brain Res. Mol. 1-O-acyl derivatives of glucose as non-penetrating inhibi Brain Res. 15 (1-2): 161-166. tors of glucose transport by hamster Small intestine invitro. 60 Vannucci, S.J., Clark, R. R., Koehler-Stec. E., Li, K., Smith, Biochim. Biophys. Acta 443: 284-287. C. B., Davies, P., Maher, F. and I. A. Simpson. 1998. Reveron ME, Savelieva KY. Tillerson J. L. McCormack AL, Glucose transporter expression in brain: Relationship to Di Monte DA, Miller G. W. L-DOPA does not cause neu cerebral glucose utilization. Dev. Neurosci. 20 (4-5): 369 rotoxicity in VMAT2 heterozygote knockout mice. Neuro 379. toxicology. 2002 October; 23(4-5):611-9. 65 Verhoeff N. P. 1999. Radiotracer imaging of dopaminergic Rhoads, D. B., Rosenbaum, D. H., Unsal, H., Isselbacher, K. transmission in neuropsychiatric disorders. Psychophar J. and L. L. Levitsky. 1991. Circadian periodicity of intes macol. (Berl) 147 (3): 217-249. US 9,302,982 B2 59 60 Wang, P. C., Nguyen, T. B., Kuchel, O. and J. Genest. 1983. 2. The pharmaceutical composition of claim 1, wherein E is Conjugation patterns of endogenous plasma catechola a Sugar residue having a structure: mines in human and rat. J. Lab. Clin. Med. 101 (1): 141 451. Wang, P. C., Kuchel, O., Buu, N. T. and J. Genest. 1983. OH Cathecholamine glucuronidation: An important metabolic pathway for dopamine in the rat. J. Neurochem. 40 (5): OH or 1435-1440. OH OH Weingarten P. Bermak J, Zhou QY. Evidence for non-oxida OH OH tive dopamine cytotoxicity: potent activation of NF-kappa 10 B and lack of protection by anti-oxidants. J Neurochem. OH. 2001 March; 76(6): 1794-804. Weinstock, J., Hieble, J. P. and J. W. Wilson. 1985. Drugs Future 10: 645. OH OH Whitfield, C. F., Rannels, S. R. and H. E. Morgan. 1974. 15 Acceleration of Sugar transport in avian erythrocytes by 3. A pharmaceutical composition comprising catecholamines. J. Biol. Chem. 249 (13): 4181 -4188. an agent selected from the group consisting of an additive, Wright, E. M., Hirsch, J. R. Loo, D.D. and G. A. Zampighi. a stabilizer, a carrier, a binder, a buffer, an excipient, an 1997. Regulation of Na+/glucose cotransporters. J. Exp. emollient, a disintegrant, a lubricating agent, an antimi crobial agent and a preservative and Biol. 200 (2): 287-293. a dopamine ribonamide or a pharmaceutically acceptable Wu, X. and H. H. Gu. 1999. Molecular cloning of the mouse salt thereof. dopamine transporter and pharmacological comparison 4. A pharmaceutical composition comprising with the human homologues. Gene 233 (3):163-170. an agent selected from the group consisting of an additive, Xia X G, Schmidt N, Teismann P. Ferger B, Schulz J. B. a stabilizer, a carrier, a binder, a buffer, an excipient, an Dopamine mediates striatal malonate toxicity vie dopam 25 emollient, a disintegrant, a lubricating agent, an antimi ine transporter-dependent generation of reactive oxygen crobial agent and a preservative and species and D2 but not D1 receptor activation. J Neuro a dopamine amide, wherein the dopamine amide is pro chem. 2001 October; 79(1):63 70. duced by a process comprising: While the preferred embodiment of the invention has been a) reacting a lactone of a Sugar and 3-hydroxytyramine, illustrated and described, it will be appreciated that various 30 and changes can be made therein without departing from the spirit b) collecting the dopamine amide, and scope of the invention. wherein the reaction yields a Sugar residue having a Structure: I claim: 1. A pharmaceutical composition comprising 35 an agent selected from the group consisting of an additive, O OH OH a stabilizer, a carrier, a binder, a buffer, an excipient, an emollient, a disintegrant, a lubricating agent, an antimi OH or crobial agent and a preservative and : a compound having the formula: 40 OH OH OH OH

OH. R3 : R R4 45 OH OH X Yn 5. A process for preparing the pharmaceutical composition R1 R of claim 1, comprising: Rs-23 Rs reacting a lactone of a Sugar and an optionally Substituted phenethylamine, and > R, 50 collecting the phenethylamide. N 6. A process of preparing the pharmaceutical composition E1'NR. of claim 4, comprising reacting a lactone of a Sugar and 3-hydroxytyramine, and wherein: 55 collecting the dopamine amide. Ring 1 is a Substituted aryl ring; 7. A method of ameliorating Parkinson's Disease in a sub X and Y are carbon atoms; ject in need thereof comprising administering to the Subject Ro is hydrogen; the pharmaceutical composition of claim 1. R is hydroxyl; 8. A method for ameliorating Parkinson's Disease in a Subject in need thereof, comprising administering to the Sub N is the nitrogen atom of an amine or an amide; 60 R. R. Rs. Rs, R. R. and R-7 are hydrogen, ject a pharmaceutical composition of claim 3. R is hydroxyl, 9. A method for ameliorating Parkinson's Disease in a Z is a carbon atom, Subject in need thereof, comprising administering to the Sub E is a residue of a straight chain hexose Sugar, ject a pharmaceutical composition of claim 4. or a pharmaceutically acceptable salt thereof. k k k k k