Related Application

This application is a continuation-in-part of U.S. Application Ser. No. 07/386,527 filed 27 July 1989.

Field of he Invention

This invention is in the field of cardiovascular therapeutics and relates to a class of compounds useful in control of hypertension. Of particular interest is a class of compounds which prevent or control hypertension by selective action on the renal sympathetic nervous system.

Background of the Invention

Hypertension has been linked to increased sympathetic nervous system activity stimulated through any of four mechanisms, namely (1) by increased vascular resistance, (2) by increased cardiac rate, stroke volume and output, (3) by vascular muscle defects or (4) by sodium retention and ^' renin release [J. P. Koepke et al, The Kidney ____ Hypertension. B. M. Brenner and J. H. Laragh (Editors), Vol. 1, p. 53 (1987)]. As to this fourth mechanism in particular, stimulation of the renal sympathetic nervous system can affect renal function and maintenance of homeostasis. For example, an increase in efferent renal sympathetic nerve activity may cause increased renal vascular resistance, renin release and sodium retention [A. Zanchetti et al. Handbook of Hypertension, Vol. 8, Ch. 8, pp. 151-172 (1986) ] . Such sympathetically mediated renal vasoconstriction has been identified as an element in the

pathogenesis of early essential hypertension in man. [R. E. Katholi, Amer. J Phvsiol.. 45., F1-F14 (1983) ] .

Proper renal function is essential to maintenance of homeostasis so as to avoid hypertensive conditions. Excretion of sodium is key to maintaining extracellular fluid volume, blood volume and ultimately the effects of these volumes on arterial pressure. Under steady-state conditions, arterial pressure rises to that pressure level which will cause balance between urinary output and water/salt intake. If a perturbation in normal kidney function occurs causing renal sodium and water retention, as with sympathetic stimulation of the kidneys, arterial pressure will increase to a level to maintain sodium output equal to intake. In hypertensive patients, the balance between sodium intake and output is achieved at the expense of an elevated arterial pressure.

During the early stages of genetically spontaneous or desoxycorticosterone acetate-sodium chloride (DOCA-NaCl) induced hypertension in rats, a positive sodium balance has been observed to precede hypertension. Also, surgical sympathectomy of the kidneys has been shown to reverse the positive sodium balance and delay the onset of hypertension [R. E. Katholi, Amer. ____ Physiol.. 245. F1-F14 (1983) ] . Other chronic sodium retaining disorders are linked to heightened sympathetic nervous system stimulation of the kidneys. Congestive heart failure, cirrhosis and nephrosis are characterized by abnormal chronic sodium retention leading to edema and ascites. These studies support the concept that renal selective pharmacological inhibition of heightened sympathetic nervous system activity to the kidneys may be an effective therapeutic treatment for chronic sodium-retaining disorders, such as

hypertension, congestive heart failure, cirrhosis, and nephrosis.

One approach to reduce sympathetic nervous system effects on renal function is to inhibit the synthesis of one or more compounds involved as inter¬ mediates in the "catecholamine cascade", that is, the pathway involved in synthesis of the neurotransmitter norepinephrine. Stepwise, these catecholamines are synthesized in the following manner: (1) tyrosine is converted to dopa by the enzyme tyrosine hydroxylase; (2) dopa is converted to dopamine by the enzyme dopa decarboxylase; and (3) dopamine is converted to norepinephrine by the enzyme dopamine-β-hydroxylase. Inhibition of dopamine-β-hydroxylase activity, in particular, would increase the renal vasodilatory, diuretic and natriuretic effects due to dopamine. Inhibition of the action of any of these enzymes would decrease the renal vasoconstrictive, .antidiuretic and antinatriuretic effects of norepinephrine. Therapeutically, these effects oppose chronic sodium retention.

Many compounds are known to inhibit the action of the catecholamine-cascade-converting enzymes. For example, the compound a-methyltyrosine inhibits the action of the enzyme tyrosine hydroxylase. The compound a- methyldopa inhibits the action of the enzyme dopa- decarboxylase, and the compound fusaric acid inhibits the action of dopamine-β-hydroxylase. Such inhibitor compounds often cannot be administered systemically because of the adverse side effects induced by such compounds. For example, the desired therapeutic effects of dopamine-β- hydroxylase inhibitors, such as fusaric acid, may be offset by hypotension-induced compensatory stimulation of the

renin-angiotensin system and sympathetic nervous system, which promote sodium and water retention.

To avoid such systemic side effects, drugs may be targetted to the kidney by creating a conjugate compound that would be a renal-specific prodrug containing the targetted drug modified with a chemical carrier moiety. Cleavage of the drug from the carrier moiety by enzymes predominantly localized in the kidney releases the drug in the kidney. Gamma glutamyl transpeptidase and acylase are examples of such cleaving enzymes found in the kidney which have been used to cleave a targetted drug from its prodrug carrier within the kidney.

Renal targetted prodrugs are known for delivery of a drug selectively to the kidney. For example, the compound L-γ-glutamyl amide of dopamine when administered to dogs was reported to generate dopamine in vivo by specific enzymatic cleavage by γ-glutamyl transpeptidase [J. J. Kyncl et al. Adv. Biosc .. 2£, 369-380 (1979)]. In another study, γ-glutamyl and N-acyl-γ-glutamyl derivatives of the anti-bacterial compound sulfamethoxazole were shown to deliver relatively high concentrations of sulfame¬ thoxazole to the kidney which involved enzymatic cleavage of the prodrug by acylamino acid deacylase and γ-glutamyl transpeptidase [M. Orlowski et al, ____ Pharmacol. Exp. Ther.. 212., 167-172 (1980)]. The N-γ-glutamyl derivatives of 2-, 3-, or 4-aminophenol and p-fluoro-L-phenylalanine have been found to be readily solvolyzed ____ vitro by γ- glutamyl transpeptidase [S.D.J. Magnan et al, ____. Med. aai./ 25, 1018-1021 (1982)]. The hydralazine-like vasodilator 2-hydrazino-5-g-butylpyridine (which stimulates guanylate cyclase activity) when substituted with the N- acetyl-γ-glutamyl residue resulted in a prodrug which provided selective renal vasodilation [K. G. Hofbauer et

al, J. Pharmacol. Exp. Ther., 212, 838-844 (1985)] . The dopamine prodrug γ-L-glutamyl-L-dopa ("gludopa") has been shown to be relatively specific for the kidney and to increase renal blood flow, glomerular filtration and urinary sodium excretion in normal subjects [D. P. Worth et al, Clin. $____ . £&, 207-214 (1985)]. In another study, gludopa was reported to an effective renal dopamine prodrug whose activity can be blocked by the dopa-decarboxylase inhibitor carbidopa [R. F. Jeffrey et al, Br. __, Clin. Pharmac.. 25, 195-201 (1988)].

BRIEF DESCRIPTION OF THE DRAWING FIGURES

Figure 1 shows the acute effects of i.v. injection of vehicle and Example #3 conjugate on mean arterial pressure in rats.

Figure 2 shows the acute effects of i.v. injection of vehicle and Example #3 conjugate on renal blood flow in rats.

Figure 3 shows the chronic effects of i.v. infusion of vehicle and Example #464 conjugate on mean arterial pressure in spontaneously hypertensive rats.

Figure 4 shows time-dependent formation of the dopamine-β-hydroxylase inhibitor fusaric acid from the Example #859 conjugate incubated with rat kidney homogenate.

Figure 5 shows time-dependent formation of fusaric acid from the Example #859 conjugate incubated with a mixture of purified acylase I and gamma-glutamyl transpeptidase at pH 7.4 and 8.1.

Figure 6 shows the concentration-dependent effect of fusaric acid and the Example #859 conjugate on norepinephrine production by dopamine-β-hydroxylase in vitro.

Figure 7 shows dopamine-β-hydroxylase inhibition in vitro by fusaric acid, the Example #859 conjugate and possible metabolites at a concentration of 20 μM.

Figure 8 shows the acute effects of i.v. injection of fusaric acid and Example #859 conjugate on mean arterial pressure in spontaneously hypertensive rats.

Figure 9 shows the acute effects of i.v. injection of fusaric acid and Example #859 conjugate on renal blood flow in spontaneously hypertensive rats.

Figure 10 shows the effects of chronic i.v. infusion of vehicle, fusaric acid, and Example #859 conjugate for 5 days on mean arterial pressure in spontaneously hypertensive rats.

Figure 11 shows the effects of chronic i.v. infusion of vehicle and Example #863 conjugate for 4 days on mean arterial pressure in spontaneously hypertensive rats.

Figure 12 shows the heart tissue concentrations of norepinephrine following the 5 day infusion experiment described in Figure 10.

Figure 13 shows the kidney tissue concentrations of norepinephrine following the 5 day infusion experiment described in Figure 10.

Figure 14 shows the effects of Example #859 conjugate on mean arterial pressure in anesthetized dogs after i.v. injection at two doses.

Figure 15 shows the effects of Example #859 conjugate on renal blood flow in anesthetized dogs after i.v. injection at two doses.

DESCRIPTION OF THE INVENTION

Treatment of chronic hypertension or sodium- retaining disorders such as congestive heart failure, cirrhosis and nephrosis, may be accomplished by administering to a susceptible or afflicted subject a therapeutically-effective amount of a renal-selective prodrug capable of causing selective blockage of heightened sympathetic nervous system effects on the kidney. An advantage of such renalselective prodrug therapy resides in reduction or avoidance of adverse side effects associated with systemically-acting drugs.

A renal-selective prodrug capable of providing renal sympathetic nerve blocking action may be provided by a conjugate comprising a first residue and a second residue connected together by a cleavable bond. The first residue is derived from an inhibitor compound capable of inhibiting formation of a benzylhydroxyamine intermediate in the biosynthesis of an adrenergic neurotransmitter, and wherein said second residue is capable of being cleaved from the

first residue by an enzyme located predominantly in the kidney.

The first and second residues are provided by precursor compounds having suitable chemical moieties which react together to form a cleavable bond between the first and second residues. For example, the precursor compound of one of the residues will have a reactable carboxylic acid moiety and the precursor of the other residue will have a reactable amino moiety or a moiety convertible to a reactable amino moiety, so that a cleavable bond may be formed between the carboxylic acid moiety and the amino moiety. An* inhibitor compound which provides the first residue may be selected from tyrosine hydroxylase inhibitor compounds, dopa-decarboxylase inhibitor compounds, dopamine-β-hydroxylase inhibitor compounds, and mimics of any of these inhibitor compounds.

It is understood that the inhibitor compounds described herein have been classified as tyrosine hydroxylase inhibitors, or as dopa-decarboxylase inhibitors, or as dopamine-β-hydroxylase inhibitors, for convenience of description. Some of the inhibitor compounds may be classifiable in more than one of these classes. For example, 2-vinyl-3-phenyl-2-aminopropionic acid derivatives are classified herein as tyrosine hydroxylase inhibitors, but such derivatives may also act as dopa-decarboxylase inhibitors.

A class of compounds from which a suitable tyrosine hydroxylase inhibitor compound may be selected to provide the conjugate first residue is represented by Formula I:

wherein each of R ¹ through R ³ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aryloxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carbox alkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; wherein R - selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein R~ is selected from -CR ⁶ and

^' '

\ , wherein R ⁶ is selected from hydrido, alkyl.

R ^ε

cycloalkyl, cycloalkylalkyl, aralkyl and aryl, and wherein each of R^ and R~ is independently selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein m* is a number selected from zero through six;

wherein A is a phenyl ring of the formula

wherein each of R^ through R---' is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, carboxyl, cyano, thiocarbamoyl, aminomethyl, alkylsulfanamido, nitro, alkylsulfonyloxy, carboxyalkoxy, formyl and a substituted or unsubstituted 5- or 6-membered heterocyclic ring selected from the group consisting of pyrrol-1-yl, 2- carboxypyrrol-1-yl, imidazol-2-ylamino, indol-1-yl, carbozol9-yl, 4,5-dihydro-4-hydroxy-4- trifluoromethylthiazol3-yl, 4-trifluoromethylthiazol-2-yl, imidazol-2-yl and 4,5-dihydroimidazol-2-yl; wherein any two of the R9 through R---" groups may be taken together to form a benzoheterocylic ring selected from the group consisting of indolin-5-yl, 1-(N-benzoylcarbamimidoyl) indolin5-yl, 1- carbamimidoylindolin-5-yl, lH-2-oxindol-5-yl, insol-5-yl, 2-mercaptobenzimidazol-5(6)-yl, 2-aminobenzimidazol-5- (6) - yl, 2-methanesulfonamidobenzimidazol-5 (6)-yl, 1H- benzoxanol-2-on-6-yl, 2aminobenzothiazol-6-yl, 2-amino-4- mercaptobenzothiazol6-yl, 2,l,3-benzothiadiazol-5-yl, 1,3- dihydro-2,2-dioxo-2,1,3-benzothiadiazol-5-yl, 1,3-dihydro- 1,3-dimethyl2,2-dioxo-2,1,3-benzothiadiazol-5-yl, 4-methyl- 2(H) oxoquinolin-6-yl, quinoxalin-6-yl, 2-hydroxyqμinoxalin- 6-yl, 2-hydroxquinoxalin-7-yl, 2, 3-dihydroxyquinoxalin6-yl and 2,3-didydro-3 (4H)-oxo-1,4-benzoxazin-7-yl; 5-hydroxy-

4H-pyran-4-on-2-yl, 2-hydroxypyrid-4-yl, 2-aminopyrid-4-yl, 2-carboxypyrid-4-yl and tetrazolo- [l, 5-a]pyrid-7-yl; and wherein A may be selected from

R ²¹

/ and -N

\

R ²² wherein each of R-- - through R* ² ^ is independently selected from hydrido, alkyl, hydroxy, hydroxyalkyl, alkoxy, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, aryloxy, alkox carboxyl, aryl, aralkyl, cyano, cyanoalkyl, amino, monoalkylamino and dialkylamino, wherein each of R^-**- and R ² ^ is independently selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; or a pharmaceutically-acceptable salt thereof.

A preferred class of tyrosine hydroxylase inhibitor compounds within Formula I is provided by compounds of Formula II:

(H)

wherein each of R ¹ and R ² is hydrido; wherein m is one or two; wherein R3 is selected from alkyl, alkenyl and alk nyl; wherein R - is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein R-5 is selected from -OR6 and

^"N , wherein R ~ is selected from R ^ε hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenalkyl and phenyl, and wherein each of R ⁷ and R^ is independently selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein each of R^ through R1 i _s independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxycarbonyl, alkoxycarbonyl, alkoxy, arykoxy, aralkoxy, alkpxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkyl¬ amino, dialkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, pyrrol-1-yl 2- carboxypyrrol-1-yl, imidazol-2-ylamino, indol-1-yl, carbazol-9-yl, 4,5-dihydro-4-trifluoromethyithiazol-3-yl, 4-trifluoromethylthiazol-2-yl, imidazol-2-yl and 4,5- dihydroimidazol-2-yl, and wherein any two of the R^ through R ¹³ groups may be taken together to form a benzoheterocyclic ring selected from the group consisting of indolin-5-yl, 1-(N-benzoylcarbamimidoyl) indolin-5-yl, 1- carbamimidoylindolin-5-yl, lH-2-oxindol-5-yl, indol-5-yl, 2-mercaptobenzimidazol-5(6)-yl, 2-aminobenzimidazol5- (6) - yl, 2-methanesulfonamidobenzimidazol-5(6)-yl, 1H-

benzoxanol-2-on-6-yl, 2-amino-benzothiazol-6-yl, 2-amino-4- mercaptobenzothiazol-β-yl, 2, 1, 3-benzothiadiazol-5-yl, 1, 3- dihydro-2, 2 -di oxo-2, 1, 3-benzothiadiazol-5-yl, 1, 3-dihydro - 1, 3-dimethyl-2, 2-dioxo-2, 1, 3benzothiadiazol-.5-yl, 4-methyl - 2 (H) -oxoquinolin-β-yl, quinoxalin-6-yl, 2 - hydroxyquinoxalin-6-yl, 2-hydroxquinoxalin-7-yl, 2, 3- dihydroxyquinoxalin-6-yl and 2, 3-didydro-3 (4H) -oxo-l, 4- benzoxazin-7-yl; wherein R ³ is -CH=CH*2 or -C≡CH; wherein R~ is selected from -OR ⁶ and

R ⁷ / ^"N , wherein R ⁶ is selected from

R ^ε hydrido, alkyl, hydroxy, hydroxyalkyl, alkoxy, halo, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, amino, monoalkylamino, dialkylamino; and wherein each of R7 and R8 independently is selected from hydrido, alkyl, hydroxyalkyl, cycloalkyl, cycloalkylalkyl, aryl and aralkyl; or a pharmaceutically-acceptable salt thereof.

A first sub-class of preferred tyrosine hydroxylase inhibitor compounds consists of the following specific compounds within Formula II:

4-cyanoamino-α-methylphenyalanine;

3-carboxy-α-methylphenylalanine;

3-cyano-α-methylphenylalanine methyl ester; α-methyl-4-thiocarbamoylphenylalanine methyl ester; 4-(aminomethyl) -α-methylphenylalanine;

4-guanidino-α-methylphenylalanine;

3-hydroxy-4-methanesulfonamido-α-methylphenylalanine;

3-hydroxy-4-nitro-α-methyIphenylalanine;

4-amino-3-methanesulfonyloxy-α-methylphenylalanine; 3-carboxymethoxy-4-nitro-α-methyIphenylalanine; α-methyl-4-amino-3-nitrophenylalanine;

3,4-diamino-α-methylphenylalanine; α-methyl-4-(pyrrol-1-yl)phenylalanine;

^■ 4-(2-aminoimidazol-l-yl) -α-methyIphenylalanine;

4- (imidazol-2-ylamino) -α-methyIphenylalanine;

4-(4,5-dihydro-4-hydroxy-4-trifluoromethyl-thiazol-2yl)-a - methylphenylalanine methyl ester; α-methyl-4- (4-trifluoromethylthiazol-2-yl)phenylalanine; α-methyl-3- (4-trifluoromethylthiazol-2-yl)-phenylalanine;

4- (imidazol-2-yl)-α-methyIphenylalanine;

4-(4,5-dihydroimidazol-2-yl)-α-methylphenylalanine;

3-(imidazol-2-yl)-α-methyIphenylalanine; 3-(4,5-dihydroimidazol-2-yl)-a-methylphenylalanine;

4-(imidazol-2-yl)phenylalanine;

4,5-dihydroimidazol-2-yl)phenylalanine;

3-(imidazol-2-yl)phenylalanine;

3-(2,3-dihydro-lH-indol-4-yl)-α-methylalanine; α-methyl-3-(lH-2-oxindol-5-yl)alanine;

3-[1-(N-benzoylcarbamimidoyl)-2,3-dihydro-lHindol-5-yl)-� �- meth lalanine;

3-(l-carbamimidoyl-2,3-dihydro-lH-indol-5-yl-α- meth lalanine; 3-(lH-indol-5-yl-α-methylalanine;

3-(benzimidazol-2-thione-5-yl) -α-methylalanine;

3-(2-aminobenzimidazol-5-yl-2-methylalanine;

2-methyl-3-(benzoxazol-2-on-6-yl)alanine;

3-(2-aminobenzothiazol-6-yl) -2-methylalanine; 3-(2-amino-4-mercaptobenzothiazol-ό-yl) -2methylalanine;

3-(2-aminobenzothiazol-6-yl)alanine;

2-methyl-3- (2,1,3-benzothiadiazol-5-yl)alanine;

3-(1,3-dihydrobenzo-2,1,3-thiadiazol-5-yl)-2methylalanine -

2,2-dioxide; 3-(1,3-dihydrobenzo-2,1,3-thiadiazol-5-yl)-2-methylalanine-

2,2-dioxide methyl ester;

3-(1,3-dihydrobenzo-2, 1,3-thiadiaxol-5-yl) alanine 2,2- dioxide;

3-(1,3-dihydro-l,3-dimethylbenzo-2,1,3-thiadiazol-5yl-) -2- methylalanine 2,2-dioxide;

α-methyl-3- [ -methyl -2 (IH) -oxoquinolin-6-yl] alanine;

3- [4-methyl-2 (IH) -oxoquinolin- 6-yl] alanine;

2 -methyl -3- (quinoxalin-6-yl) alanine;

2-methyl-3- (2-hydroxyquinoxalin-6-yl) alanine; 2-methyl-3- (2-hydroxyquinoxalin-7-yl) alanine;

3- (2, 3-dihydroxyquinoxalin-6-yl) -2-methylalanine;

3- (quinoxalin-6-yl) alanine;

3-(2,3-dihydroxyquinoxalin-6-yl)alanine;

3-(1,4-benzoxazin-3-one-6-yl)-2-methylalanine; 3-(1, -benzoxazin-3-one-7-yl)alanine;

3-(5-hydroxy-4H-pyran-4-on-2-yl) -2-methylalanine;

3-(2-hydroxy-4-pyridyl) -2-methylalanine;

3- (2-carboxy-4-pyridyl)-2-methylamine; α-methyl-4- (pyrrol-1-yl)phen lalanine; α-ethyl-4- (pyrrol-1-yl)phenylalanine; α-propyl-4- (pyrrol-1-yl)phenylalanine;

4-[2-(carboxy)pyrrol-1-yl)phenylalanine; α-methyl-4-(pyrrol-1-yl)phenylalanine;

3-hydroxy-α-4-(pyrrol-1-yl)phenylalanine; 3-methoxy-α-4-(pyrrol-1-yl)phenylalanine;

4-methoxy-α-3-(pyrrol-1-yl)phenylalanine;

4- (indol-1-yl)-α-methyIphenylalanine;

4- (carbazol-9-yl)-α-methyIphenylalanine;

2-methyl-3-(2-methanesulfonylamidobenzimidazol-5- yl)alanine;

2-methyl-3-(2-amino-4-pyridyl)alanine;

2-methyl-3[tetrazolo-(1,5) -α-pyrid-7-yl]alanine;

D,L-α-β-(4-hydroxy-3-methyl)phenylalanine;

D,L-α-β-(4-hydroxy-3-phenyl)phenylalanine; D,L-α-β-(4-hydroxy-3-benzyl)phenylalanine;

D,L-α-β-(4-methoxy-3-cyclohexyl)phenylalanine; α, β, β trimethyl-β-(3,4-dihydroxyphenyl)alanine; α, β, β trimethyl-β-(4-hydroxyphenyl)alanine;

N-methyl α, β, β trimethyl-β-(3,4-dihydroxphenyl) alanine; D,L α, β, β trimethyl-β-(3,4-dihyroxyphenyl)alanine;

trimethyl-β- (3,4-dimethoxyphenyl)alanine;

L-α-methyl-β-3,4-dihydroxyphenylalanine;

L-α-ethyl-β-3,4-dihydroxyphenylalanine;

L-α-propyl-β-3,4-dihydroxyphenylalanine; L-α-butyl-β-3,4-dihydroxyphenylalanine;

L-α-methyl-β-2,3-dihydroxphenylalanine;

L-α-ethyl-β-2,3-dihydroxphenylalanine;

L-α-propyl-β-2,3-dihydroxphenylalanine;

L-α-butyl-β-2,3-dihydroxphenylalanine; L-α-methyl-4-chloro-2,3-dihydroxyphenylalanine;

L-α-ethyl-4-chloro-2,3-dihydroxyphenylalanine;

L-α-propyl-4-chloro-2,3-dihydroxyphenylalanine;

L-α-butyl-4-chloro-2,3-dihydroxyphenylalanine;

L-α-ethyl-β-4-methyl-2,3-dihydroxyphenylalanine; L-α-methyl-β-4-methy1-2,3-dihydroxyphenylalanine;

L-α-propyl-β-4-methyl-2,3-dihydroxyphenylalanine;

L-α-butyl-β-4-methyl-2,3-dihydroxyphenylalanine;

L-α-methyl-β-4-fluoro-2,3-dihydroxyphenylalanine;

L-α-ethyl-β-4-fluoro-2,3-dihydroxyphen lalanine; L-α-propyl-β-4-fluoro-2,3-dihydroxyphenylalanine;

L-α-butyl-β-4-fluoro-2,3-dihydroxyphenylalanine;

L-α-methyll-b-4-trifluoromethyl-2,3-dihydroxyphenylalani ne

L-α-ethyl-β-4-trifluoromethyl-2,3-dihydroxyphenylalanin e

L-α-propyl-β-4-trifluoromethyl-2,3-dihydroxyphenylalani ne L-α-butyl-β-4-trifluoromethyl-2,3-dihydroxyphenylalanine

L-α-methyl-β-3,5-dihydroxyphenylalanine;

L-α-ethyl-β-3,5-dihydroxyphenylalanine;

L-α-propyl-β-3,5-dihydroxyphenylalanine;

L-α-butyl-β-3,5-dihydroxyphenylalanine; L-α-methyl-β-4-chloro-3,5-dihydroxphenylalanine;

L-α-ethyl-β-4-chloro-3,5-dihydroxphenylalanine;

L-α-propyl-β-4-chloro-3,5-dihydroxphenylalanine;

L-α-butyl-β-4-chloro-3,5-dihydroxphenylalanine;

L-α-methyl-β-4-fluoro-3,5-dihydroxyphenylalanine; L-α-ethyl-β-4-fluoro-3,5-dihydroxyphenylalanine;

L-α-propyl-β-4-fluoro-3,5-dihydroxyphenylalanine;

L-α-butyl-β-4-fluoro-3,5-dihydroxyphenylalaninei

L-α-methyl-β-4-trifluoromethyl-3,5-dihydroxyphenylalani ne;

L-α-ethyl-β-4-trifluoromethyl-3,5-dihydroxyphenylalanin e; L-α-propyl-β-4-trifluoromethyl-3,5-dihydroxyphenylalanine;

L-α-butyl-α-4-trifluoromethyl-3,5-dihydroxyphenylalanin e;

L-α-methyl-2,5-dihydroxphenylalanine;

L-α-ethyl-2,5-dihydroxphenylalanine;

L-α-propyl-2,5-dihydroxphenylalanine; L-α-butyl-2,5-dihydroxphenylalanine;

L-α-methyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-ethyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-propyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-butyl-β-4-chloro-2,5-dihydroxyphenylalanine; L-α-methyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-ethyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-propyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-butyl-β-4-chloro-2,5-dihydroxyphenylalanine;

L-α-methyl-β-methyl-2,5-dihydroxyphenylalanine; L-α-ethyl-β-methyl-2,5-dihydroxyphenylalanine;

L-α-propyl-β-methyl-2,5-dihydroxyphenylalanine;

L-α-butyl-β-methyl-2,5-dihydroxyphenylalanine;

L-α-methyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalani ne;

L-α-ethyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalanin e; L-α-propyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalanine;

L-α-butyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalanin e;

L-α-methyl-β-3, ,5-trihydroxyphenylalanine;

L-α-ethyl-β-3,4,5-trihydroxyphenylalanine;

L-α-propyl-β-3, ,5-trihydroxyphenylalanine; L-α-butyl-β-3,4,5-trihydroxyphen lalanine;

L-α-methyl-β-2,3,4-trihydroxyphenylalanine;

L-α-ethyl-β-2,3,4-trihydroxyphenylalanine;

L-α-propyl-β-2,3,4-trihydroxyphenylalanine;

L-α-butyl-β-2,3,4-trihydroxyphenylalanine; L-α-methyl-β-2, ,5-trihydroxyphenylalanine;

L-α-ethyl-β-2,4,5-trihydroxyphenylalanine;

L-α-propyl-β-2,4,5-trihydroxyphenylalanine;

L-α-butyl-β-2,4,5-trihydroxyphenylalanine;

L-phenylalanine; D,L-α-methyIphenylalanine;

D,L-3-iodophenylalanine;

D,L-3-iodo-α-methylphen lalanine;

3-iodotyrosine;

3,5-diiodotyrosine; L-α-methylphenylalanine;

D,L-α-β-(4-hydroxy-3-meth lpheny1)alanine;

D,L-α-β-(4-methoxy-3-benzylphenyl)alanine;

D,L-α-β-(4-hydroxy-3-benzylphenyl)alanine;

D,L-α-β-(4-methoxy-3-cyclohexylphenyl)alanine; D,L-α-β-(4-hydroxy-3-cyclohexylpheny1)alanine;

D,L-α-β-( -methoxy-3-methylpheny1)alanine;

D,L-α-β-(4-hydroxy-3-methylphen 1)alanine;

N,O-dibenzyloxycarbonyl-D,L-α-β-(4-hydroxy-3- meth lpheny1)alanine; N,O-dibenzyloxycarbonyl-D,L-α-β- (4-hydroxy-3- methylpheny1)alanine amide;

D,L-α-β-(4-hydroxy-3-meth lpheny1) alanine amide;

N,O-diacetyl-D,L-α-β-(4-hydroxy-3-methylphenyl)alanine;

D,L-N-acetyl-α-β-(4-hydroxy-3-methylphenyl)alanine; L-3,4-dihydroxy-α-meth lphenylalanine;

L-4-hydroxy-3-methoxy-α-methyIphenylalanine;

L-3 ,4-methylene-dioxy-α-methyIphenylalanine;

2-vinyl-2-amino-3-(2-methoxyphenyl)propionic acid;

2-vinyl-2-amino-3-(2,5-dimethoxyphenyl)propionic acid; 2-vinyl-2-amino-3-(2-imidazolyl)propionic acid;

2-vinyl-2-amino-3-(2-methoxyphenyl)propionic acid ethyl ester; α-methyl-β-(2,5-dimethoxyphenyl)alanine; α-methy1-β-(2,5-dihydroxyphenyl) alanine; α-ethyl-β-(2,5-dimethoxyphenyl)alanine;

α-ethyl-β-(2,5-dihydroxyphenyl)alanine; α-methyl-β-(2, -dimethoxyphenyl)alanine; α-methy1-β-(2,4-dihydroxypheny1)alanine; α-ethyl-β- (2,4-dimethoxyphenyl)alanine; α-ethyl-β- (2, -dihydroxypheny1)alanine; α-methyl-β-(2,5-dimethoxyphenyl)alanine ethyl ester;

2-ethynyl-2-amino-3-(3-indolyl)propionic acid;

2-ethynyl-2,3- (2-methoxyphenyl)propionic acid;

2-ethynyl-2,3- (5-hydroxyindol-3-yl)propionic acid; 2-ethynyl-2-amino-3- (2,5-dimethoxyphenyl)propionic acid;

2-ethynyl-2-amino-3- (2-imidazolyl)propionic acid;

2-ethynyl-2-amino-3-(2-methoxyρhenyl)propionic acid ethyl ester;

3-carbomethoxy-3- (4-benzyloxybenzyl)-3-aminoprop-l-yne; α-ethynyltyrosine hydrochloride; α-ethynyltyrosine; α-ethynyl-m-tyrosine; α-ethynyl-β-(2-methoxyphenyl)alanine; α-ethynyl-β-(2,5-dimethoxyphenyl)alanine; and α-ethynylhistidine.

A second sub-class of preferred tyrosine hydroxylase inhibitor compounds consists of compounds wherein at least one of R ¹ ^, R-*-- ¹ and R ¹² is selected from hydroxy, alkoxy, aryloxy, aralkoxy and alkoxycarbonyl. More preferred compounds of this second sub-class are α-methyl-3- (pyrrol-1-yl)tyrosine; α-methyl-3-(4-trifluoromethylthiazol-2-yl)tyrosine;

3- (imidazol-2-yl)-α-methyItyrosine; Lα-m-tyrosine;

L-α-ethyl-m-tyrosine;

L-α-propyl-m-tyrosine;

L-α-butyl-m-tyrosine;

Lα-p-chloro-m-tyrosine; L-α-ethyl-p-chloro-m-tyrosine;

L-α-butyl -p-chloro-m-tyrosine;

Lα-p-bromo-m-tyrosine;

L-α-ethyl-p-bromo-m-tyrosine;

L-α-butyl-p-bromo-m-tyrosine; Lα-p-fluoro-m-tyrosine;

Lα-p-iodo-m-tyrosine;

L-α-ethyl-p-iodo-m-tyrosine;

Lα-p-methyl-m-tyrosine;

Lα-p-ethyl-m-tyrosine; L-α-ethyl-p-ethyl-m-tyrosine;

L-α-ethyl-p-methyl-m-tyrosine;

Lα-p-buty1-m-tyrosine;

Lα-p-trifluoromethyl-m-tyrosine;

L-3-iodotyrosine; L-3-chlorotyrosine;

L-3,5-diiodotyrosine;

L-α-methyltyrosine;

D,L-α-meth ltyrosine;

D,L-3-iodo-α-meth ltyrosine; L-3-bromo-α-methyltyrosine;

D,L-3-bromo-α-methyltyrosine;

L-3-chloro-α-methyltyrosine;

D,L-3-chloro-α-methyltyrosine; and

2-vinyl-2-amino-3-(4-hydroxypheny1)propionic acid.

Another preferred class of tyrosine hydroxylase inhibitor compounds within Formula I consists of compounds

R ³ (in)

wherein R ³ is selected from alkyl, alkenyl and alkynyl; wherein R ⁴ is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein m is a number selected from zero through five, inclusive; wherein R ⁵ is selected from OR ⁶ and

R ⁷ / , wherein R ~ is selected from R ^ε hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenalkyl and phenyl, and wherein each of R ⁷ and R-^ is independently selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein each of R^ through R1 is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxycarbonyl, alkoxy, aryloxy, aralkoxy, alkoxyalkyl, haloalkyl, alkoxycarbonyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl.

A preferred sub-class of compounds within Formula III consists of compounds wherein at least one of R---, RH and R***- ² is selected from hydroxy, alkoxy, aryloxy, aralkoxy and alkoxycarbonyl. More preferred compounds of this sub-class are methyl(+)-2-(4-hydroxyphenyl)glycinate; isopropyl and 3-methyl butyl esters of (+)-2-(4- hydroxyphenyl)glycine; (+)-2-(4-hydroxyphenyl)glycine; (-)- 2-(4-hydroxyphenyl)glycine; (+)-2- (4-methoxyphenyl-glycine; and (+)-2-(4-hydroxyphenyl)glycinamide.

Still another preferred class of tyrosine hydroxylase inhibitor compounds within Formula I is provided by compounds of Formula IV:

wherein each of R- and R ² is hydrido; wherein m is a number selected from zero through five, inclusive; wherein R~ is selected from alkyl, alkenyl and alkynyl; wherein R ⁴ is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein each of R ¹⁴ through R ¹⁷ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, carboxyl, cyano, thiocarbamoyl, aminomethyl, alkylsulfanamido, nitro, alk lsulfonyloxy, carboxyalkoxy and formyl.

A preferred sub-class of compounds within Formula IV consists of L-α-methyltryptophan; D,L-5- methyltryptophan; D,L-5-chlorotryptophan; D,L-5- bromotryptophan; D,L-5-iodotryptophan; L-5-- hydroxytryptophan; D,L-5-hydroxy-α-methyltryptophan; α- ethynyltryptophan; 5-methoxymethoxy-α-ethynyltryptophan; and 5-hydroxy-α-ethynyltryptophan.

Still another preferred class of tyrosine hydroxylase inhibitor compounds within Formula I is provided by compounds wherein A is

^■ N . wherein R - is selected from

R ²² three, inclusive. More preferred compounds in this class are 2-vinyl-2-amino-5-aminopentanoic acid and 2-ethynyl-2- amino-5-aminopentanoic acid.

Still another preferred class of tyrosine hydroxylase inhibitor compounds within Formula I is provided by compounds of Formula V:

wherein each of R ²³ and R ²⁴ is independently selected from hydrido, hydroxy, alkyl, cycloakyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, aryloxy, alkoxyalkyl.

haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxy, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; wherein R ²⁵ is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein each of R ²⁶ through R ³ -5 is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, carboxyl, cyano, thiocarbamoyl, aminomethyl, alkylsulfanamido, nitro, alkylsulfonyloxy, alkoxy and formyl; wherein n is a number selected from zero through five, inclusive; or a pharmaceutically-acceptable salt thereof. A more preferred compound of this class is benzoctamine.

A class of compounds from which a suitable dopa- decarboxylase inhibitor compound may be selected to provide the conjugate first residue is represented by Formula VI:

wherein each of R ³⁶ through R ⁴² is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl.

hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, cyano, thiocarbamoyl, aminomethyl, alkylsulfanamido, nitro, alkylsulfonyloxy, carboxyalkoxy and formyl; wherein n is a number from zero through four; wherein each of R ⁴³ and R ⁴⁴ is independently selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, monoalkylcarbonylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, alkenyl, cycloalkenyl and alkynyl, wherein any R ⁴³ and R - - substituent having a substitutable position may be further substituted with one or more groups selected from hydroxyalkyl, halo, haloalkyl, carboxyl, alkoxyalkyl, alkoxycarbonyl; with the proviso that R ⁴³ and R ⁴⁴ cannot both be carboxyl at the same time, and with the further proviso that at least one of R ⁴³ through R ⁴⁴ is a primary or secondary amino group; or a pharmaceutically-acceptable salt thereof.

A preferred class of compounds within Formula VI consists of compounds wherein each of R36 through R ⁴² is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, cyano, aminomethyl, carboxyalkoxy and formyl; wherein n is a number from one through three; wherein each of R ⁴³ and R ⁴⁴ is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxyalkyl, haloalkyl, hydroxyalkyl, amino, mono¬ alkylamino, dialkylamino, carboxyl, carboxyalkyl and alkanoyl; and wherein any R ⁴³ and R ⁴⁴ substituent having a

substitutable position may be further substituted with one or more groups selected from hydroxyalkyl, halo, haloalkyl, carboxyl, alkoxyalkyl, alkoxycarbonyl.

A more preferred class of compounds within

Formula VI consists of those compounds wherein each of R~~ through R ⁴² is independently selected from hydrido, hydroxy, alkyl, benzyl, phenyl, alkoxy, benzyloxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, cyanoamino, cyano, aminomethyl, carboxyl, carboxyalkoxy and formyl; wherein n is one or two; wherein each of R ⁴³ and R ⁴⁴ is independently selected from hydrido, alkyl, benzyl, phenyl, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl and alkanoyl; and wherein any R ⁴³ and R ⁴⁴ substituent having a substitutable position may be further substituted with one or more groups selected from hydroxyalkyl, halo, haloalkyl, carboxyl, alkoxyalkyl, alkoxycarbonyl.

An even more preferred class of compounds within Formula VI consists of those compounds wherein each of R ³⁶ through R ⁴² is independently selected from hydrido, hydroxy, alkyl, alkoxy, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl, carboxyalkyl, aminomethyl, carboxyalkoxy and formyl; wherein n is one or two; wherein each of R ⁴³ and R ⁴⁴ is independently selected from hydrido, alkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl and carboxyalkyl; and wherein any R ⁴³ and R ⁴⁴ substituent having a substitutable position may be further substituted with one or more groups selected from hydroxyalkyl, halo, haloalkyl, carboxyl, alkoxyalkyl, alkoxycarbonyl.

A more highly preferred class of compounds within Formula VI consists of those compounds wherein each of R~ - and R ³⁷ is hydrido and n is one; wherein each of R-~ through R ⁴² is independently selected from hydroxy, alkyl, alkoxy, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl, carboxyalkyl, aminomethyl, carboxyalkoxy and formyl; wherein each of R ⁴³ and R ⁴⁴ is independently selected from hydrido, alkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl and carboxyalkyl; and wherein any R ⁴³ and R ⁴⁴ substituent having a substitutable position may be further substituted with one or more groups selected from hydroxyalkyl, halo, haloalkyl, carboxyl, alkoxyalkyl, alkoxycarbonyl. Compounds of specific interest are (2,3,4-trihydroxy)-benzylhydrazine, 1-(D,L- seryl-2 (2,3,4-trihydroxybenzyl)hydrazine (Benserazide) and 1-(3-hydroxylbenzyl)-1-methylhydrazine.

Another more highly preferred class of compounds consists of those compounds wherein each of R ³⁶ and R ³⁷ is independently selected from hydrido, alkyl and amino and n is two; wherein each of R~"~ through R ⁴² is independently selected from hydroxy, alkyl, alkoxy, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl, ^' carboxyalkyl, aminomethyl, carboxyalkoxy and formyl; wherein each of R ⁴³ and R ⁴⁴ is independently selected from hydrido, alkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl and carboxyalkyl. Compounds of specific interest are 2-hydrazino-2-methyl-3-(3,4- dihydrox phenyl)propionic acid (Carbidopa) , α-(monofluoro- methyl)dopa and α- (difluoromethyl)dopa.

Another class of compounds from which a suitable dopa-decarboxylase inhibitor compound may be selected to provide the conjugate first residue is represented by Formula VII

wherein each of R ⁴ *-* ⁵ through R -~ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, cyano, thiocarbamoyl, aminomethyl, alkylsulfanamido, nitro, alkylsulfonyloxy, carboxyalkoxy and formyl; wherein each of R ⁴⁹ and R ⁵⁰ is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl and

-CR ⁵¹ wherein R ^* ** ⁵ ***- is selected from hydroxy, alkoxy, aryloxy, aralkoxy, amino, monoalkylamino and dialkylamino with the proviso that R - and R^O cannot both be carboxyl at the same time, and with the further proviso that at least one of R ⁴⁵ through R ⁴ ^ is a primary or secondary amino group or a carboxyl group; or a pharmaceutically- acceptable salt thereof.

A preferred class of compounds within Formula VII consists of those compounds wherein each of R ^4* -*-" through R ⁴⁸ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl,- aryl, alkoxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, cyano, aminomethyl, carboxyalkoxy and formyl; wherein each of R --- and R-50 is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyalkyl and alkanoyl and

O

-CR ⁵¹ wherein R-51 is selected from hydroxy, alkoxy, phenoxy, benzyloxy, amino, monoalkylamino and dialkylamino.

A more preferred class of compounds within Formula VII consists of those compounds wherein each of R ⁴ *5 through R -~ is independently selected from hydrido, hydroxy, alkyl, benzyl, phenyl, alkoxy, benzyloxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, cyanoamino, cyano, aminomethyl, carboxyalkoxy and formyl; wherein each of R ⁴⁹ and R ⁵ s independently selected from hydrido, alkyl, benzyl, phenyl, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyalkyl and alkanoyl and

0

-C HR51 wherein R ^OJ - is selected from hydroxy, alkoxy, amino and monoalkylamino.

An even more preferred class of compounds of Formula VII consists of those compounds wherein each of R ⁴ ^

through R -~ is independently selected from hydrido, hydroxy, alkyl, alkoxy, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl, carboxyalkyl aminomethyl, carboxyalkoxy and formyl; wherein each of R--- and R^O is independently selected from hydrido, alkyl, amino, monoalkylamino, carboxyalkyl and

O

-CR ⁵¹ wherein R^l is selected from hydroxy, alkoxy, amino and monoalkylamino.

A highly preferred class of compounds within Formula VII consists of those compounds wherein each of R-- ¹ through R -- is independently selected from hydrido, hydroxy, alkyl, alkoxy and hydroxyalkyl; wherein each of R ⁴⁹ and R^O is independently selected from alkyl, amino, monoalkylamino, and

O

-C IIR ⁵¹ wherein R-51 is selected from hydroxy, methoxy. ethoxy, propoxy, butoxy, amino, methylamino and ethylamino.

A more highly preferred class of compounds within Formula VII consists of those compounds wherein said inhibitor compound is selected from endo-2-aminol,2,3,4- tetrahydro-1,2-ethanonaphthalene-2-carboxylic acid; ethyl- endo-2-amino-l,2,3,4-tetra-hydro-l,4-ethano-naphthalene-2- carboxylate hydrochloride; exo-2-amino1,2,3,4-tetrahydro- l,4-ethanonaphthalene-2-carboxylic acid; and ethyl-exo-2- amino-1,2,3,4-tetrahydro-1,4-ethano-naphthalene-2- carboxylate hydrochloride.

Another family of specific dopa-decarboxylase inhibitor compounds consists of 2,3-dibromo-4,4-bis( -ethylpheny1)-2-butenoic acid;

3-bromo-4-(4-methoxyphenyl) -4-oxo-2-butenoic acid;

N-(5'-phosphopyridoxyl) -L-3,4-dihydroxyphenylalanine;

N-(5'-phosphopyridoxyl) -L-m-aminotyrosine;

D,L-β-(3,4-dihydroxypheny1) lactate; D,L-β-(5-hydroxyindolyl-3)lactate;

2,4-dihydroxy-5- (l-oxo-2-propenyl)benzoic acid;

2,4-dimethoxy-5-[l-oxo-3- (2,3,4-trimethoxyphenyl-2- propenyl]benzoic acid;

2,4-dihydroxy-5-[l-oxo-3- (2-thienyl)-2-propenyl] benzoic acid;

2,4-dihydroxy-5-[3- (4-hydroxyphenyl)-l-oxo-2-propenyl] benzoic acid;

5-[3-(4-chlorophenyl) -l-oxo-2-propenyl]-2,4-dihydroxy benzoic acid; 2,4-dihydroxy-5-(l-oxo-3-phenyl-2-propenyl)benzoic acid;

2,4-dimethoxy-5-[l-oxo-3-(4-pyridinyl)-2-propenyl] benzoic acid;

5-[3-(3,4-dimethoxyphenyl)-l-oxo-2-propenyl]-2,4 dimethoxy benzoic acid; 2,4-dimethoxy-5-(l-oxo-3-phenyl-2-propenyl)benzoic acid;

5-[3-(2-furanyl)-l-oxo-2-propenyl]-2,4-dimethoxy benzoic acid;

2,4-dimethoxy-5-[l-oxo-3-(2-thienyl)-2-propenyl] benzoic acid; 2,4-dimethoxy-5-[3-(4-methoxyphenyl) -l-oxo-2-propenyl] benzoic acid;

5-[3-(4-chlorophenyl)-l-oxo-2-propenyl]-2,4-dimethoxy benzoic acid; and

5-[3-[4-(dimethylamino)phenyl]-l-oxo-2-propenyl]-2,4 dimethoxy benzoic acid.

Another class of compounds from which a suitable dopa-decarboxylase inhibitor may be selected to provide the conjugate first residue is represented by Formula VIII:

wherein R ⁵² is selected from hydrido, OR ⁶⁴ and

_R 65

/

^" ^ , wherein R~ - is selected from R* hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenalkyl and phenyl, and wherein each of R ⁶⁵ and R ⁶⁶ is independently selected from hydrido, alkyl, alkanoyl, amino, monoalkylamino, dialkylamino, phenyl and phenalkyl; wherein each of R ⁵³ , R ⁵⁴ and R ⁵⁷ through R ⁶³ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxycarbonyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; wherein each of R-'-' and R^δ is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxyalkyl, halo, haloalkyl, hydroxyalkyl and carboxyalkyl; wherein each of m and n is a number independently selected from zero through six, inclusive; or a pharmaceutically-acceptable salt thereof.

A preferred class of compounds of Formula VIII consists of those compounds wherein R^ is OR ⁶⁴ wherein R ~ -

is selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, benzyl and phenyl; wherein each of R-^ , R ⁵⁴ and R ^*57 through R ⁶³ is independently selected from hydrido, alkyl, cycloalkyl, hydroxy, alkoxy, benzyl and phenyl; wherein each of R ⁵ ^ and R-56 is independently selected from hydrido, alkyl, cycloalkyl, benzyl and phenyl; wherein each of m and n is a number independently selected from zero through three, inclusive.

A more preferred class of compounds of Formula

VIII consists of those compounds wherein R^ ² is OR ⁶⁴ wherein R ⁶⁴ is selected from hydrido and lower alkyl; wherein each of R ⁵³ through R~"~ is hydrido; wherein each of R59 through R -~ is independently selected from hydrido, alkyl, hydroxy and alkoxy, with the proviso that two of the R- 9 through R ⁶³ substituents are hydroxy; wherein each of and n is a number independently selected from zero through . two, inclusive.

A preferred compound within Formula IX is 3-

(3,4-dihydroxyphenyl)-2-propenoic acid, also known as caffeic acid.

Another class of compounds from which a suitable dopa-decarboxylase inhibitor compound may be selected to provide the conjugate first residue is a class of aromatic amino acid compounds comprising the following subclasses of compounds:

- amino-haloalkyl-hydroxyphenyl propionic acids, such as 2-amino-2-fluoromethyl-3hydroxy- phenylpropionic acid;

- alpha-halomethyl-phenylalanine derivatives such as alpha-fluoroethylphenethylamine; and

- indole-substituted halomethylamino acids.

Still other classes of compounds from which a suitable dopa-decarboxylase inhibitor compound may be selected to provide the conjugate first residue are as follows:

- isoflavone extracts from fungi and streptomyces, such as 3' ,5,7-trihydroxy-4* , 6- dimethoxyisoflavone, 3*,5,7-trihydroxy-4' , 8- dimethoxyisoflavone and 3', 8-dihydroxy- ',6,7- trimethoxyisoflavone;

- sulfinyl substituted dopa and tyrosine derivatives such as shown in U.S. Patent No. 4,400,395 the content of which is incorporated herein by reference;

- hydroxycoumarin derivatives such as shown in

U.S. Patent No. 3,567,832, the content of which is incorporated herein by reference;

- 1-benzylcyclobutenyl alkyl carbamate derivatives such as shown in U.S. Patent No.

3,359,300, the content of which is incorporated herein by reference;

- arylthienyl-hydroxylamine derivatives such as shown in U.S. Patent No. 3,192,110, the content of which is incorporated herein by reference; and

- β-2-substituted-cyclohepta-pyrrol-8-lH-on-7-yl alanine derivatives.

Suitable dopamine-β-hydroxylase inhibitors may be generally classified mechanistically as chelating-type inhibitors, time-dependent inhibitors and competitive inhibitors.

A class of compounds from which a suitable dopamine-β-hydroxylase inhibitor may be selected to provide the conjugate first residue consists of time-dependent inhibitors represented by Formula IX:

wherein B is selected from aryl, an ethylenic moiety, an acetylenic moiety and an ethylenic or acetylenic moiety substituted with one or more radicals selected from substituted or unsubstituted alkyl, aryl and heteroaryl; wherein each of R ⁶⁷ and R ~~ is independently selected from hydrido, alkyl, alkenyl and alkynyl; wherein R-9 is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; and wherein n is a number selected from zero through five.

A preferred class of compounds of Formula IX consists of those compounds wherein B is phenyl or hydroxyphenyl; wherein R ⁶⁷ is ethenyl or ethynyl; or an

acetylenic moiety substituted with an aryl or heteroaryl radical; and wherein n is a number from zero through three.

Another preferred class of compounds of Formula IX consists of those compounds wherein B is an ethylenic or acetylenic moiety incorporating carbon atoms in the beta- and gamma-positions relative to the nitrogen atom; and wherein n is zero or one. More preferred are compounds wherein the ethylenic or acetylenic moiety is substituted at the gamma carbon with an aryl or heteroaryl radical.

Even more preferred are compounds wherein said aryl radical is selected from phenyl, 2-thiophene, 3-thiophene, 2- furanyl, 3-furanyl, oxazolyl, thiazolyl and isoxazolyl, any one of which radicals may be substituted with one or more groups selected from halo, hydroxyl, alkyl, haloalkyl, cyano, alkoxy, alkoxyalkyl and cycloalkyl. More highly preferred are compounds wherein said aryl radical is selected from phenyl, hydroxyphenyl, 2-thiophene and 2- furanyl; and wherein each of R ⁶⁷ , R -~ and ~~ is hydrido.

A family of specifically-preferred compounds within Formula IX consists of the compounds 3-amino-2-(2'- thienyl)propene; 3-amino-2- (2'-thienyl) butene; 3-(N- methylamino)-2- (2'-thienyl)propene; 3-amino-2- (3'- thienyDpropene; 3-amino-2- (2'furanyl) propene; 3-amino-2- (3'-furanyl)propene; l-phenyl-3aminopropyne; and 3-amino-2- phenylpropene. Another family of specifically-preferred compounds of Formula VIII consists of the compounds (±)4- amino-3-phenyl-lbutyne; (±)4-amino-3- (3'-hydroxyphenyl)-1- butyne; (±)4-amino-3- ( '-hydroxyphenyl)-1-butyne; (±)4- amino3-phenyl-l-butene; (+) -amino-3- (3'-hydroxyphenyl)-1- butene; and (±) 4-amino-3- (4'-hydroxyphenyl) -1-butene.

Another class of compounds from which a suitable dopamine-β-hydroxylase inhibitor may be selected to provide the conjugate first residue is represented by Formula X:

O

II

W-C-Y (X)

wherein W is selected from alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl and heteroaryl; wherein Y is selected from

wherein R ⁷ ^ is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein each of Q and T is one or more groups independently selected from

wherein each of R ⁷¹ through R ⁷⁴ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, aryloxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino.

monoalkylamino, dialkylamino, carboxy, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; or a pharmaceutically-acceptable salt thereof.

A preferred class of compounds within Formula X consists of compounds wherein W is heteroaryl and Y is

wherein R ⁷ 0 is selected from hydrido, alkyl, amino, monoalkylamino, dialkylamino, phenyl and phenalkyl; wherein each of R ⁷¹ and R ⁷² is independently selected from hydrido, hydroxy, alkyl, phenalkyl, phenyl, alkoxy, benzyloxy, phenoxy, alkoxyalkyl, hydroxyalkyl, halo, amino, monoalkylamino, dialkylamino, carboxy, carboxyalkyl and alkanoyl; and wherein each of p and q is a number independently selected from one through six, inclusive.

A more preferred class of compounds of Formula X consists of wherein R ⁷ ^ is selected from hydrido, alkyl, amino and monoalkylamino; wherein each of R ⁷¹ and R ⁷² is independently selected from hydrido, hydroxy, alkyl, alkoxy, amino, monoalkylamino, carboxy, carboxyalkyl and alkanoyl; and wherein each of p and q is a number indpendently selected from two through four, inclusive. Even more preferred are compounds wherein R ⁷ ^ is selected from hydrido, alkyl and amino; wherein each of R ⁷¹ and R ⁷² is independently selected from hydrido, amino.

monoalkylamino and carboxyl; and wherein each of p and q is independently selected from the numbers two and three. Most preferred are compounds wherein R ⁷ ^ is hydrido; wherein each of R ⁷¹ and R ⁷² is hydrido; and wherein each of p and q is two.

Another class of compounds from which a suitable dopamine-β-hydroxylase inhibitor may be selected to provide the conjugate first residue is represented by Formula XI:

O

II

E-C-F (XI)

wherein E is selected from alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl and heteroaryl; wherein F is selected from

⁷

wherein Z is selected from 0, S and N-R ⁷ -**; wherein each of R ⁷⁵ and R ⁷⁶ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, aryloxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, minoalkylamino, dialkylamino, carboxy, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; wherein R ⁷ 5 and R ⁷⁶ may form oxo or thio; wherein r is a number selected from zero through six, inclusive; wherein each of R ⁷⁷ and R ⁷⁸ is independently selected from hydrido, alkyl, cycloalkyl.

hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; or a pharmaceuticallyacceptable salt thereof.

Another class of compounds from which a suitable dopamine-β-hydroxylase inhibitor may be selected to provide the conjugate first residue is represented by Formula XII:

wherein each of R^ through R-- ¹ is independently selected from hydrido, alkyl, haloalkyl, mercapto, alkylthio, cyano, alkoxy, alkoxyalkyl and cycloalkyl; wherein Y is selected from oxygen atom and sulfur atom; wherein each of R ⁷⁹ and R80 is independently selected from hydrido and alkyl; wherein R-~- is selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; and wherein m is a number from one through six; or a pharmaceutically- acceptable salt thereof.

A preferred family of compounds of Formula XII consists of those compounds wherein each of R^ through R-~ is independently selected from hydrido, alkyl and haloalkyl; wherein Y is selected from oxygen atom or sulfur atom; wherein each of R ⁷ 9, R^0 and R~^- is independently

hydrido and alkyl; and wherein m is a number selected from one through four, inclusive.

A family of preferred specific compounds within Formula XII consists of the following compounds: aminomethyl-5-n-butylthiopicolinate; aminomethy1-5-n-butylpicolinate;

2'-aminoethyl-5-n-butylthiopicolinate;

2'-aminoethyl-5-n-butylpicolinate; (2'-amino-1',1'-dimethyl)ethyl-5-n-butylthiopicolinate;

(2'-amino-1' ,1'-dimethyl)ethyl-5-n-butylpicolinate;

(2'-amino-1'-methyl)ethyl-5-n-butylthiopicolinate;

(2'-amino-1'-methyl)ethyl-5-n-butylpicolinate;

3'-aminopropyl-5-n-butylthiopicolinate; 3'-aminopropyl-5-n-butylpicolinate;

(2'-amino-2'-methyl)propyl-5-n-butylthiopicolinate;

(2'-amino-2'-methyl)propyl-5-n-butylpicolinate;

(3'-amino-1',1'-dimethyl) ropyl-5-n-butylthiopicolinate;

(3'-amino-1',1'-dimethyl)propyl-5-n-butylpicolinate; (3*-amino-2',2'-dimethyl)propyl-5-n-butylthiopicolinate;

(3'-amino-2*,2'-dimethyl)propy1-5-n-butylpicolinate;

2'-aminopropyl-5-n-butylthiopicolinate;

2'-aminopropyl-5-n-butylpicolinate; '-aminobutyl-5-n-butylthiopicolinate; 4 ^• -amino-3'-methyl)butyl-5-n-butylthiopicolinate;

(3'-amino-3'-methyl)butyl-5-n-butylthiopicolinate; and (3'-amino-3'-methyl)butyl-5-n-butylpicolinate.

Another preferred class of compounds within Formula XII consists of those compounds of Formula XIII:

wherein each of R- ~, R^ ⁷ and R^O through R-^ ³ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, aryloxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxy, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; wherein R^δ and R-***- ⁷ together may form oxo or thio; wherein r is a number selected from zero through six, inclusive; wherein each of R^8 and R--* is independently selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl.

A more preferred class of compounds within Formula XIII consists of those compounds wherein each of R86, R87 and ^90 through R ⁹ is independently selected from hydrido, hydroxy, alkyl, phenalkyl, phenyl, alkoxy, benzyloxy, phenoxy, alkoxyalkyl, hydroxyalkyl, halo, amino, monoalkylamino, dialkylamino, carboxy, carboxyalkyl and alkanoyl; wherein r is a number selected from zero through four, inclusive; wherein each of R-~ and R^9 is

independently selected from hydrido, alkyl, amino, monoalkylamino, dialkylamino, phenyl and phenalkyl.

An even more preferred class of compounds within Formula XIII consists of those compounds wherein each of _R 86 _{/ R} 87 and R**-"*-- ¹ through R^ is independently selected from hydrido, hydroxy, alkyl, alkoxy, amino, monoalkylamino, carboxy, carboxyalkyl and alkanoyl; and wherein r is a number selected from zero through three, inclusive; and wherein each of R-~ and R-^ is selected from hydrido, alkyl, amino and monoalkylamino. Most preferred are compounds wherein each of R-® through R ⁹ is independently selected from hydrido and alkyl; wherein each of R^ and R ⁸⁷ is hydrido; wherein r is selected from zero, one and two; wherein R&~ is selected from hydrido, alkyl and amino; and wherein R ~ is selected from hydrido and alkyl. Especially preferred within this class is the compound 5-π- butylpicolinic acid hydrazide (fusaric acid hydrazide) shown below:

Another class of compounds from which a suitable dopamine-β-hydroxylase inhibitor compound may be selected to provide the conjugate first residue is represented by Formula XIV:

wherein each of R ⁹⁴ through R ~ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, aryloxy, alkoxy, alkylthio, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, amido, alkylamido, hydroxyamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, cyanoamino, carboxyl, tetrazolyl, thiocarbamoyl, aminomethyl, alkylsulfanamido, nitro, alkylsulfonyloxy, formoyl and alkoxycarbonyl; with the proviso that at least one of R ⁹⁴ through R ~ is

_R 101 wherein A' is -CR or -N wherein R ⁹⁹ is selected

^N _R 102 from hydrido, alkyl, hydroxy, alkoxy, alkylthio, phenyl, phenoxy, benzyl, benzyloxy,

_R 103

-O lOO and -N , wherein R ¹⁰⁰ is selected from

\ R ¹⁰⁴ hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenyl and benzyl; each of R ¹⁰¹ ' R ¹⁰² , R ¹⁰³ and R ¹⁰⁴ is independently

selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein t is a number selected from zero through four, inclusive; or a pharmaceutically-acceptable salt thereof.

A preferred family of compounds within Formula XIV consists of those compounds characterized as chelating- type inhibitors of Formula XV:

wherein each of R95 through R ⁹⁸ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, phenyl, benzyl, alkoxy, phenoxy, benzyloxy, alkoxyalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, amido, alkylamido, hydroxyamino, carboxyl, carboxyalkyl, alkanoyl, cyanoamino, carboxyl, thiocarbamoyl, aminomethyl, nitro, formoyl, formyl and alkoxycarbonyl; and wherein R-W is selected from hydrido, alkyl, phenyl and benzyl.

A class of specifically-preferred compounds of Formula XV consists of

5-n-butylpicolinic acid (fusaric acid) ;

5-ethylpicolinic acid; picolinic acid;

5-nitropicolinic acid; 5-aminopicolinic acid;

5-N-acetylaminopicolinic acid;

5-N-propionylaminopicolinic acid;

5-N-hydroxyaminopicolinic acid;

5-iodopicolinic acid; 5-bromopicolinic acid;

5-chloropicolinic acid;

5-hydroxypicolinic acid

5-methoxypicolinic acid;

5-N-propoxypicolinic acid; 5-N-butoxypicolinic acid;

5-cyanopicolinic acid;

5-carboxylpicolinic acid;

5-n-butyl-4-nitropicolinic acid;

5-n-butyl-4-methoxypicolinic acid; 5-n-butyl-4-ethoxypicolinic acid;

5-n-butyl-4-aminopicolinic acid;

5-n-butyl-4-hydroxyaminopicolinic acid; and

5-n-butyl-4-methylpicolinic acid.

Especially preferred of the foregoing class of compounds of Formula XV is the compound 5-n-butylpicolinic acid (fusaric acid) shown below:

Another class of compounds from which a suitable dopamine-β-hydroxylase inhibitor may be selected to provide the conjugate first residue consists of azetidine-2- carboxylic acid derivatives represented by Formula.XVI:

wherein R ¹⁰⁵ is hydrido, hydroxy, alkyl, amino and alkoxy; wherein R ¹ -^ i _s selected from hydrido, hydroxy and alkyl; wherein each of R ¹⁰⁷ and R ¹⁰⁸ is independently selected from hydrido, alkyl and phenalkyl; wherein R ¹⁰⁹ is selected from hydrido and O

II

RIIOC- with R ¹¹⁰ selected from alkyl, phenyl and phenalkyl; wherein u is a number from one to three, inclusive; and wherein v is a number from zero to two, inclusive; or a pharmaceutically-acceptable salt thereof.

A preferred class of compounds within Formula

XVT consists of those compounds wherein R^ ^* -* ⁵ is selected from hydroxy and lower alkoxy; wherein R ¹⁰⁶ is hydrido; wherein R^ ⁷ is selected from hydrido and lower alkyl; wherein R ¹⁰⁸ is hydrido; wherein R ¹⁰⁹ is selected from hydrido and O

II

RIIOC- with R ¹¹ -**- ¹ selected from lower alkyl and phenyl; wherein u is two; and wherein v is a number from zero to two, inclusive.

A more preferred class of compounds within Formula XVT consists of those compounds of Formula XVII:

107

0

R ¹⁰⁹ S—-CH, :H- :-N- CR ¹¹¹ (XVII)

wherein R*-*-*!**** is selected from hydroxy and lower alkyl; wherein RIO ⁷ is selected from hydrido and lower alkyl; wherein Rl^ ⁹ is selected from hydrido and 0

II

RIIOC- with RHO selected from lower alkyl and phenyl and v is a number from zero to two, inclusive.

A more preferred class of compounds within Formula XVII consists of those compounds wherein R****-***-***- is hydroxy; wherein R107 is hydrido or methyl; wherein R^ ⁹ is hydrido or acetyl; and wherein n is a number from zero to two, inclusive.

Most preferred within the class of compounds of Formula XVII are the compounds 1-(3-mercapto-2-methyl-l- oxopropyl)-L-proline and 1- (2-mercaptoacetyl)-L-proline (also known as captopril) .

Another class of compounds from which a suitable dopamine-β-hydroxylase inhibitor compound may be selected to provide the conjugate first residue is represented by Formula XVIII:

wherein each of R ¹¹² through R ¹¹⁹ is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, alkoxy, alkoxyalkyl, aralkyl, aryl, alkoxycarbonyl, hydroxyalkyl, halo, haloalkyl, cyano, amino, aminoalkyl, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl, alkynyl, mercapto and alkylthio; or a pharmaceutically-acceptable salt thereof.

A first preferred class of compounds within Formula XVTII consists of those compounds wherein R ¹¹² is selected from mercapto and alkylthio; wherein each of R ¹¹³ and R ¹¹⁴ is independently selected from hydrido, amino, aminoalkyl, monoalkylamino, monoalkylaminoalkyl, carboxyl and carboxyalkyl; wherein each of R ¹ *-*- ⁵ and R ¹¹⁹ is hydrido; and wherein each of R ¹¹⁶ , R ¹¹⁷ and R ¹¹⁸ is independently selected from hydrido, hydroxy, alkyl, halo and haloalkyl; or a pharmaceutically-acceptable salt thereof.

A second preferred class of compounds within Formula XVIII consists of those compounds wherein R ¹¹² is selected from amino, aminoalkyl, monoalkylamino, monoalkylaminoalkyl, carboxy and carboxyalkyl; wherein each of R ¹¹³ , R ¹¹⁴ , R ¹¹⁵ and R ¹¹⁹ is hydrido; and wherein each of R***- ¹⁶ , R***-***- ⁷ and R ¹¹⁸ is independently selected from hydrido, hydroxy, alkyl, halo and haloalkyl; or a pharmaceutically- acceptable salt thereof.

Compounds which fall within any of the afore¬ mentioned inhibitor compounds, but which lack a reactive acid or amino moiety to form a cleavable bond, may be modified or derivatized to contain such acid of amino moiety. Examples of classes of such compounds lacking an amino on acidic moiety are the following: l-(3,5- dihaloaryl) imidazol-2-thione derivatives such as l-(3,5- difluorobenzyl) imidazol-2thione; and hydroxyphenolic derivatives such as resorcinol.

The first component used to form the conjugate of the invention provides a first residue derived from an inhibitor compound capable of inhibiting formation of a benzylhydroxylamine intermediate involved in the biosynthesis of an adrenergic neurotransmitter. This inhibitor compound must contain a moiety convertible to a primary or secondary amino terminal moiety. An example of a moiety convertible to an amino terminal moiety is a carboxylic acid group reacted with hydrazine so as to convert the acid moiety to carboxylic acid hydrazide. The hydrazide moiety thus contains the terminal amino moiety which may then be further reacted with

the carboxylic acid containing residue of the second component to form a hydrolyzable amide bond. Such hydrazide moiety thus constitutes a "linker" group between the first and second components of a conjugate of the invention.

Suitable linker groups may be provided by a class of diamino-terminated linker groups based on hydrazine as defined by Formula XIX:

wherein each of R ²⁰⁰ and R ²⁰¹ may be independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, hydroxyalkyl, aralkyl, aryl, haloalkyl, amino, monoalkylamino, dialkylamino, cyanoamino, carboxyalkyl, alkylsulfino, alkylsulfonyl, arylsulfinyl and arylsulfonyl; and wherein n is zero or a number selected from three through seven, inclusive. In Table I there is shown a class of specific examples of diamino-terminated linker groups within Formula XIX,

identified as Linker Nos. 1-73. These linker groups would be suitable to form a conjugate between a carbonyl moiety of an All antagonist (designated as "I") and a carbonyl moiety of a carbonyl terminated second residue such as the carbonyl moiety attached to the gamma carbon of a glutamyl residue (designated *T") .

TABLE I

I = inhibitor

T = acθtyl-γ-glutamyl

|| LINKER _R 200 R201 NO.

1 0 H H

2 0 CH3 H

0 C2H5 ^' H

0 C3H7 H

5 CH(CH3)2 H

6 C4H9 H

CH(CH3)CH2CH3 H

8 0 C(CH3)3 H

0 C5H9 H

10 C6Hn(cyclo) H

11 0 C6H5 H

12 0 CH2C6H5 H

13 H CH3

LINKER R200 R201 NO.

14 H C2H ₅

H C3H7

H CH(CH3)2

H C4H9

18 H CH(CH3)CH2CH3

19 H C(CH3)3

20 H C5-H9

21 H C6H13

H C6H5

H CH2C6H5

H ^• C6H11 (cyclo)

C6-H13 H

CH3 CH3

C2-H5 C2H5

C3H7 C3H7

CH(CH3)2 CH ⁽ CH3)2

LINKER _R 200 R201 NO.

30

31

32

33

34

35

CH3 H

H CH3

C6-H5 H

H C6H5

CH3 C6H5

C6H5 CH3

LINKER R200 R201 NO .

CH2C6H5 H

H CH2C6H5

H H

CH3 H

49 H CH3

50 C6H5 H

51

52

53

54

55 H CH2C6H5

H H

CH3 H

H CH3

C6H5 H

H C6H5

LINKER 200 R201 NO.

61 CH3 C6-H5

62 C6-H5 CH3

63 CH2C6H5 H

64 H CH2C6H5

65 H H

66 CH3 H

67 H CH3

68 6 C6-H5 H

69 6

70 6

71

72

73 H CH2C6H5

Another class of suitable diamino terminal linker groups is defined by Formula XX:

wherein each of Q and T is one or more groups independently selected from

wherein each of R202 through R ² 05 _s independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aralkoxy, aryloxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, dialkylamino, carboxy, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl.

A preferred class of linker groups within Formula IV is defined by Formula XXI:

(XXI)

wherein each of R202 and R203 is independently selected from hydrido, hydroxy, alkyl, phenalkyl, phenyl, alkoxy, benzyloxy, phenoxy, alkoxyalkyl, hydroxyalkyl, halo, amino, monoalkylamino, dialkylamino, carboxy, carboxyalkyl and alkanoyl; and wherein each of p and q is a number independently selected from one through six, inclusive; with the proviso that when each of R ²⁰² and R ²⁰³ is selected from halo, hydroxy, amino, monoalkylamino and dialkylamino, then the carbon to which R ²⁰² or R ²⁰³ is attached in Formula XXI is not adjacent to a nitrogen atom of Formula XXI.

A more preferred class of linker groups of Formula V consists of divalent radicals wherein each of R ² ^ ² and R ^ is independently selected from hydrido, hydroxy, alkyl, alkoxy, amino, monoalkylamino, carboxy, carboxyalkyl and alkanoyl; and wherein each of p and q is a number independently selected from two through four, inclusive. Even more preferred are linker groups wherein each of R ² 02 _-_ R203 is independently selected from hydrido, amino, monoalkylamino and carboxyl; and wherein each of p and q is independently selected from the numbers two and three. Most preferred is a linker group wherein each of R202 a^ R203 i _S hydrido; and wherein each of p and q is two; such most preferred linker group is derived from a piperazinyl group and has the structure r—

-N N- ^•

In Table II there is shown a class of specific examples of cyclized, diamino-terminated linker groups within Formula XXI. These linker groups, identified as Linker Nos. 74-95, would be suitable to form a conjugate between a carbonyl moiety of an All antagonist (designated as "I") and a carbonyl moiety of carbonyl terminated second residue such as the carbonyl moiety attached to the gamma carbon of a glutamyl residue (designated as "T") .

H H H H H H H

3 H H H H H H H

H H H CH3 H H H

77 CH3 H H H CH3 H H H

78 ' CH3 H CH3 H H H H H

H H H CH3 H

H H H H H

H CH3 CH3 H H

82 CH3 CH3 H H CH3 CH3 H H

83 CH3 CH3 CH3 CH3 H H H H

84 CH3 CH3 H H H H CH3 CH3

85 H H H H CH3 CH3 CH3 CH3

86 6H5 H H H H H H H

87 H H H H C6H5 H H H

88 C6H5 H H H C6H5 H H H

89 C6H5 H H H H H C6H5 H

90 C6H5 H C6H5 H H H H H

91 CH2C6H5 H H H H H H H

92 H H H H CH2C6H5 H H H

93 CH2C6H5 H H H CH2C6H5 H H H

94 CH C6H5 H H H H H CH2C6H5 H

95 CH2C6H5 H CH2C6H5 H H H H H

Another class of suitable diamino terminal linker groups is defined by Formula XXII:

wherein each of R ²¹⁴ through R ²¹⁷ is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, alkoxyalkyl, aralkyl, aryl, haloalkyl, amino, monoalkylamino, dialkylamino, cyanoamino, carboxyalkyl, alkylsulfino, alkylsulfonyl, arylsulfinyl and arylsulfonyl; and wherein p is a number selected from one through six inclusive.

A preferred class of linker groups within Formula VI consists of divalent radicals wherein each of R ²¹⁴ and R ²¹⁵ is hydrido; wherein each of R ⁶² and R ⁶³ is independently selected from hydrido, alkyl, phenalkyl, phenyl, alkoxyalkyl, hydroxyalkyl, haloalkyl and carboxyalkyl; and wherein p is two or three. A more preferred class of linker groups within Formula XXII consists of divalent radicals wherein each of R214 and R215 s hydrido; wherein each of R ²¹⁶ and R ²¹⁷ is independently selected from hydrido and alkyl; and wherein p is two. A specific example of a more preferred linker within Formula XXII is the divalent radical ethylenediamino. In Table III there is shown a class of specific examples of diamino-terminated linker gorups within Formula XXII. These linker groups, identified as Linker Nos. 96-134, would be suitable to form a conjugate between a carbonyl moiety of an All antagonist (designated as "I") and a carbonyl moiety of carbonyl terminated second residue such as the carbonyl moiety attached to the gamma carbon of a glutamyl residue (designated as "T") .

TABLE III

_R 220 _R 222

I I

N - C - C - N - 7

_R 218 ^221 _R 223 _R 219

I = inhibitor

G = acetyl-γ-glutamyl

LINKER R218 R219 R220 _R 221 _R 222 R223 NO .

96 H H H H H H

97 H H H H H CH3

98 H H H CH3 H H

99 H H H CH3 H CH3

100 CH3 H H H H H

101 H CH3 H H H H

102 H H H CH3

H CH3 H

CH3 H H

H H H H C6H5

H H C6H5 H H

107 H H H C6H5 H C6H5

108 C6H5 H H H H H

LINKER R218 R219 R220 R221 > 222 R223 NO .

109 H C6H5 H H H H

110 H H H H C6H5 C6H5

111 H H C6-H5 C6-H5 H H

112 C6H5 C6H5 H H H H

113 H H H H H C2H5

114 H H H C2H5 H H

115 H H H C2H5 H C2-H5

116 C2H5 H H H H H

117 H C2H5 H H H H

118 H H H H C2H5 C2H5

H C2H5 C2H5 H H

C2H5 H H H H

H C6-H5 H H H

H H H C6H5 H

CH3 C6-H5 H H H

LL]INKER R218 R219 R220 R221 ■ 222 R223 NO .

124 H CH3 H H C6H5 H

125 CH3 CH3 H C6H5 H H

126 CH ₃ CH3 H H H C6H5

127 H H H H H CH2C6H5

128 H H H CH2C6H5 H H

H H

H H

H H

CH2C6H5 H

H H

CH2C6H5 H

The term "hydrido" denotes a single hydrogen atom (H) which may be attached, for example, to an oxygen atom to form a hydroxyl group. Where the term "alkyl" is used, either alone or within other terms such as "haloalkyl", "aralkyl" and "hydroxyalkyl", the term "alkyl" embraces linear or branched radicals having one to about ten carbon atoms unless otherwise specifically described. Preferred alkyl radicals are "lower alkyl" radicals having one to about five carbon atoms. The term "cycloalkyl" embraces radicals having three to ten carbon atoms, such as cyclopropyl, cyclobutyl, cyclohexyl and cycloheptyl. The term "haloalkyl" embraces radicals wherein any one or more of the carbon atoms is substituted with one or more halo groups, preferably selected from bromo, chloro and fluoro. Specifically embraced by the term "haloalkyl" are monohaloalkyl, dihaloalkyl and polyhaloalkyl groups. A monohaloalkyl group, for example, may have either a bromo, a chloro, or a fluoro atom within the group. Dihaloalkyl and polyhaloalkyl groups may be substituted with two or more of the same halo groups, or may have a combination of different halo groups. Examples of a dihaloalkyl group are dibromomethyl, dichloromethyl and bromochloromethyl. Examples of a polyhaloalkyl are trifluoromethyl, 2,2,2- trifluoroethyl, perfluoroethyl and 2,2,3,3tetrafluoroρropyl groups. The term "alkoxy", embraces linear or branched oxy- containing radicals having an alkyl portion of one to about ten carbon atoms, such as methoxy, ethoxy, isopropoxy and butoxy. The term "alkylthio" embraces radicals containing a linear or branched alkyl group, of one to about ten carbon atoms attached to a divalent sulfur atom, such as a ethythio group. The term "aryl" embraces aromatic radicals such as phenyl, naphthyl and biphenyl. The term "aralkyl" embraces aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, phenylbutyl and diphenylethyl. The terms "benzyl" and "phenylmethyl" are interchangeable. The terms "aryloxy" and "arylthio"

denote radical respectively, aryl groups having an oxygen or sulfur atom through which the radical is attached to a nucleus, examples of which are phenoxy and phenylthio. The terms "sulfinyl" and "sulfonyl", whether used alone or linked to other terms, denotes respectively divalent radicals ^SO and ^S0 ₂ The term "acyl" whether used alone, or within a term such as acyloxy, denotes a radical provided by the residue after removal of hydroxyl from an organic acid, examples of such radical being acetyl and benzoyl. "Lower alkanoyl" is an exairple of a more preferred sub-class of acyl.

Within the classes of conjugates of the invention described herein are the pharmaceutically- acceptable salts of such conjugates including acid-addition salts and base addition salts. The term "pharmaceutically- acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts of conjugates of the invention may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, p-hydroxybenzoic, salicyclic, phenylacetic, mandelic, embonic (pamoic) , methansulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxy-

butyric, malonic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts of the conjugates include metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'dibenzylethylenediamine, chloroprocaine, choline, diethanola ine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding conjugates described herein by reacting, for example, the appropriate acid or base with the conjugate.

Conjugates of the invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non-racemic mixtures thereof. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid and then separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts. A .different process for separation of optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by reacting conjugates with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure compound. The optically active conjugates can likewise be obtained by utilizing optically active starting materials. These isomers may be

in the form of a free acid, a free base, an ester or a salt.

Synthetic Procedures -

Conjugates of the invention are synthesized by reaction between precursors of the first and second residues. One of such precursors must contain a reactive acid moiety, and the other precursor must contain a reactive amino moiety, so that a conjugate is formed having a cleavable bond. Either precursor of the first and second residues may contain such reactive acid or amino moieties. Preferably, the precursors of the first residue are inhibitors of benzylhydroxyamine biosynthesis and will contain a reactive amino moiety or a moiety convertible to a reactive amino moiety. Many of the tyrosine hydroxylase inhibitors and dopa-decarboxylase inhibitors are characterized in having a reactive amino moiety. Inhibitor compounds lacking a reactive amino moiety, such as the dopamine-β-hydroxylase inhibitor fusaric acid, may be chemically modified to provide such reactive amino moiety. Chemical modification of these inhibitor compounds lacking a reactive amino group may be accomplished by reacting an acid or an ester group on the inhibitor compound with an amino compound, that is, a compound having at least one reactive amino moiety and another reactive hetero atom selected from 0, S and N. A suitable amino compound would be a diamino compound such as hydrazine or urea. Hydrazine, for example, may be reacted with the acid or ester moiety of the inhibitor compound to form a hydrazide derivative of such inhibitor compound.

The dopamine-β-hydroxylase inhibitor compound 5- butyl-n-butylpicolinic acid (fusaric acid) may be used as a model compound to illustrate the chemical modification of an acid-containing inhibitor compound to make a reactive

amino-containing precursor for synthesizing a conjugate of the invention. In the following General Synthetic Procedures, the substituents and reagents are defined as follows: each of R ⁷⁹ , R ⁸ 0, R ⁸¹ , R ⁸⁶ , R ⁸⁷ , R ⁸⁸ , R ⁸⁹ and R115 is as defined above; W is selected from alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl and heteroaryl; and Z is selected from oxygen and sulfur. DCC is an abbreviation for dicyclohexylcarbodiimide.

General Synthetic Procedures

Procedure 1;

Procedure 2:

Procedure 3:

Ac^O.base

Procedure 4:

O

HO-C-CH,CH ₂ -

Procedure 5:

O C0 ₂ H

I I

WC-N" -C-CH,— CH,— CH

.-- N-H

I c=o

,115

ProeeriiiTo ft-

HO-C— CH ₂ — CH ₂ —

O _R i< _R u ₀ C0 ₂ H

II I I I I

W-C — N — (C)-N— C-CH ₂ — CH ₂ — CH

I c=o

_R ιιs

Procedure 7 :

O

8

HO-C— CH ₂ — CH ₂ — 115

The following Examples 1-1857 shown in Tables IV- XVII are highly preferred conjugates of the invention. These conjugates fall within three classes, namely, conjugates of tyrosine hydroxylase inhibitors of Tables IV-VI, conjugates of dopa-decarboxylase inhibitors of Tables VII-XI, and conjugates of dopamine-β-hydroxylase inhibitors of Tables XII-XVII. These conjugates may be prepared generally by the procedures outlined above in Schemes 1-7. Also, specific procedures for preparation of Examples 1-1857 are found in the conjugate preparations described in the examples appearing with the tables of conjugates.

The following Examples #1-#461 comprise three classes of highly preferred conjugates formed from tyrosine hydroxylase inhibitor compounds and glutamic acid derivatives. Examples #l-#3 are descriptions of specific preparations of such conjugates. Examples #4-#461, as shown in Tables IV-VI, may be prepared by procedures shown in these specific examples and in the foregoing general synthetic procedures of Schemes 1-7.

Example 1

4-ami no-4-carboxy-4-oxobntγl-«-methvl-L-tvrosine. me yl ester.

Step . 1. Preparation of methvl ra-methvl-L-tvrosinate, hydroπhloridfi.

A solution of 11.0 g (56.4 mmol) of α-methy1-L- tyrosine in 100 mL of absolute methanol was cooled to 0°C and treated with 20.1 g (169 mmol) of thionyl chloride under a nitrogen atmosphere. The reaction was allowed to warm to ambient temperature and stir at reflux for 2 days. Concentration followed by trituration with 150 mL of ether gave 13.3 g (96%) of colorless product: NMR (DMSO-dg) δ 1.49 (s, 3H), 3.02 (s, 2H), 3.73 (s, 3H) , 6.73 (d, J = 11 Hz, 2H) , 6.97 (d, J = 11 Hz, 2H), 8.50-8.70 (br s, 3H) , 9.50 (s, IH) .

Step. 2. Preparation of 4-aτnino-4-carhoxγ-4-oxobnty1-n_- methvl-L-r.yrnsine. methyl ester.

Under nitrogen, a solution of 35.1 g (116 mmol) of N-Boc-L-γ-glutanic acid-α-£-butyl ester (BACHEM) in 200 mL of methylene chloride was treated with 11.95 g (58 mmol) of solid dicyclohexylcarbodiimide (DCC) . The reaction was allowed to stir for 2 hr prior to filtration under a nitrogen atmosphere. The methylene chloride was removed n vacuo and the residue

dissolved in 100 mL of anhydrous dimethylformamide (DMF) . The anhydride solution was slowly added to a solution of 7.0 g (29 mmol) of the α-methyl tyrosine ester from step 1 and 18.73 g (145 mmol) of diisopropylethylamine (DIEA) in 100 mL of anhydrous DMF. The reaction was allowed to stir overnight and was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with cold 1M K2CO3 followed by water, dried (MgSU4) , and concentrated in vaςuo to give the protected coupled product; a solution of this material in 150 mL of methylene chloride was cooled to 0°C and treated with 150 mL of trifluoracetic acid (TEA) under nitrogen. The reaction was allowed to warm to ambient temperatures and stir overnight. Concentration in vacuo gave 4-amino-4-carboxy-4-oxobutyl-α- methyl-L-tyrosine, methyl ester: NMR (DMSO-dg) δ 1.20 (s, 3H), 1.90-2.20 (m, 2H) , 2.23-2.38 (m, 2H) , 2.95 (d,* J = 13 Hz, IH), 3.26 (d, J = 13 Hz), 3.57 (s, 3H) , 3.92-4.06 (m, IH) , 7.06 (d, J = 9 Hz, 2H), 7.12 (d, J = 9 Hz, 2H) .

Example 2

N- T4- (ar.er.y1 amino . -4-carboxy-4-oxohυt_γ] 1 -n_-met_hγl-L-t,vrosine. methyl ester.

The compound of Example 1 was dissolved in 100 mL of water and the pH adjusted to 9 wit 1 M K2CO3. The solution was cooled to 0°C and 3.30 mL (35 mmol) of acetic anhydride and 35 mL (35 mmol) of 1 M K2&D3 was added every 30 min. for 5 h; the pH was maintained at 9 and the reaction temperature kept below 5°C. After the last addition, the reaction was allowed to warm to ambient temperature overnight. The pH was adjusted to 4 with 6 M HC1 and concentrated to 100 mL. Purification by reverse phase chromatography (Waters Deltaprep-3000) using isocratic 25% acetonitrile/water (0.05% TFA) gave 9.0 g (82%) of colorless product: NMR (DMSO-dg) δ 1.18 (s, 3H), 1.72-2.03 (m, 2H) , 1.85 (s, 3H) , 2.15 (t, J = 8 Hz, 2H), 2.93 (d, J = 13 Hz, IH) , 3.38 (d, J = 13 Hz, IH) , 3.57 (s, 3H), 4.12-4.23 (m, IH) , 7.02 (d, J. = 9 Hz, 2H) , 7.09 (d, J = 9 Hz, 2H), 8.06 (s, IH) , 8.12 (d, J = 8 Hz, IH) .

Example 3

N- r4- far:e ylamino -4-carboxy-4-oxobut.yn -rt-methyl-L-tyrosine .

A solution of 9.0 g (23.7 mmol) of the compound of Example 2 in 225 mL of water was cooled to 0°C and treated with 3.3 g (82.5 mmol) of solid NaOH in portions over 15 min. The reaction was stirred at 0-5°C overnight, the pH adjusted to pH 5 with 6N HCl, and concentrated to 100 mL. Purification by reverse phase chromatography (Waters Deltaprep-3000) using isocratic 15% acetonitrite/water (0.05% TFA) gave 5.50 g (63%) of colorless product: NMR (DMSO-dβ) δ 1.17 (s, 3H) , 1.70-2.00

(m, 2H), 1.85 (s, 3H) , 2.14 (t, J = 8 Hz, 2H) ,' 2.83 (d, J = 13 Hz, IH), 3.14 (d, J = 13 Hz, IH) , 4.12-4.23 (m, IH) , 6.56 (d, J = 9 Hz, 2H), 6.85 (d, J = 9 Hz, 2H) , 7.69 (s, IH) , 8.12 (d, J = 8 Hz, IH); MS (FAB) m/e (rel intensity) 367 (70), 196 (52), 179 (58) 150 (100), 130 (80); HRMS. Calcd for M + H: 367.1505. Found: 367.1547. Anal. Calcd for C ₁₇ H22N2θ7-H ₂ 0- ^» 0.125 TFA: C, 52.00; H, 6.03; N, 7.03; F,

1.60. Found: C, 51.96; H, 6.25; N, 7.12; F, 1.60.

The following Examples #4-#109 of Table IV are highly preferred conjugates formed from tyrosine hydroxylase inhibitor compounds and glutamic acid derivatives. These tyrosine hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula I and II, above.

TfiFT.F. TV

_R 10 Rll Rl2 R5

H OH H OCH3 CH3 COCH3

H OH H OH H H

H OH H OCH3 CH3 H

H CH H OH CH3 H

H OH H OH CH3 COCH3

H OH H OCH3 H H

H OH H OCH3 H COCH 3

H OH H OCH3 CH3 H

H OH H OCH3 CH3 COCH3

H OH H OH H H

H OH H OH H COCH3

EXAMPLE Rl R9 _R 10 Rll Rl2 R5

NO .

15 CH2F H H OH H OH CH3 H

16 CH2F H H OH H OH CH3 COCH3

17 CHF2 H H OH H OCH3 H H

18 CHF2 H H OH H OCH3 H COCH3

19 CHF2 H H OH H OCH3 CH3 H

20 CHF2 H H OH H OCH3 CH3 COCH3

21 CHF2 H H OH H OH H H

22 CHF2 H H OH H OH H COCH 3

23 CHF2 H H OH H OH CH3 H

24 CHF2 H H OH H OH CH3 COCH3

25 CF3 H H OH H OCH3 H H

26 CF3 H H OH H OCH3 H COCH3

27 CF3 H H OH H OCH3 CH3 H

28 CF3 H H OH H OCH 3 CH3 COCH 3

29 CF3 H H OH H OH H H

30 CF3 H H OH ^H . CH H COCH 3

EXAMPLE R ¹ R ⁹ Rl Rll Rl2 _R 5 E

NO .

31 CF3 H H OH H OH CH3 H

32 CF3 H H OH H OH CH3 COCH 3

33 C2H5 H H OH H OCH3 H H

34 C2H5 H H OH H OCH3 H C CH3

35 C2H5 H H OH H OCH3 CH3 H

36 C2H5 H H OH H OCH3 CH3 COCH3

37 C2H5 H H OH H OH H H

38 C2H5 H H OH H OH H COCH3

39 C2H5 H H OH H CH CH3 H

40 C2H5 H H OH H OH CH3 COCH3

41 C3H7 H H OH H OCH3 H H

42 C3H7 H H OH H OCH3 H COCH 3

43 C3-H7 H H OH H OCH3 CH3 H

44 C3H7 H H OH H OCH3 CH3 COCH3

45 C3H7 H H OH H OH H H

EXAMPLE R ¹ R ⁹ _R 10 Rll Rl2 _R 5 E

NO .

46 C3H7 H H OH H OH H COCH 3

47 C3H7 H H OH H OH CH3 H

48 C3H7 H H OH H OH CH3 COCH3

49 CH3 H H NHCN H OH H COCH3

50 CH3 H C02H H H H OH COCH3

51 CH3 H CN H H* OH H COCH3

52 CH3 H H CH2NH2 H OH H COCH3

53 CH3 H H CH2CH2C H OH H COCH3

54 CH3 H OH CH3SO2NH H OH • H COCH3

55 CH3 H OH N02 H OH H COCH3

56 CH3 H CH3SO3 NH2 H OH H COCH3

57 CH3 H CO2CH3 N02 H OH H COCH3

58 CH3 H N02 NH2 H OH H COCH 3

59 CH3 H NH2 NH2 H OH H COCH3

60 CH3 H CH3 OH H OH H COCH3

61 CH3 H C6H5 OH H OH ^' H COCH 3

62 CH3 H CH2C6H5 OH H OH H COCH 3

63 CH3 H C6H11 (cyclo) CH3O H OH H COCH3

64 CH3 OH OH H H OH H COCH3

65 CH3 CH OH Cl H CH H COCH 3

66 CH3 OH OH CH3 H OH H COCH 3

67 CH3 CH OH H OH H COCH3

68 CH3 CH OH CF3 H OH H COCH3

69 CH3 H OH H CH OH H COCH3

70 CH3 H OH Cl OH CH H COCH3

71 CH3 H OH OH CH H COCH3

72 CH3 H OH CF3 CH OH H COCH3

73 CH3 OH H H OH OH H COCH3

74 CH3 OH H Cl OH OH H COCH3

75 CH3 OH H CH3 CH OH H COCH3

76 CH3 OH H CF3 CH* OH H COCH3

89

EXAMPLE R ¹ R$ RlO R" Rl2 R5 E

NO.

77 CH3 H OH OH OH OH H COCH3

78 CH3 OH OH OH H OH H COCH3

79 CH3 OH H OH OH OH H COCH3

80 CH3 H H H H OH H COCH3

81 H H H H H CH H COCH3

82 H H H H H H COCH3

83 CH3 H H H H H COCH3

84 H H OH H H H COCH3

85 H H H H H COCH3

86 CH3 H CH3 OH H H H COCH3

87 CH3 H C6H5CH2 CH3O H H H COCH3

88 CH3 H C6H5CH2 OH H H H COCH3

89 CH3 H C6H11 (cyclo) CH3O H H H COCH3

90 CH3 H C6H11 (cyclo) OH H H H COCH3

91 CH3 H CH3 CH3O H H H COCH3

EXAMPLE R ¹ R ⁹ RlO Rll Rl2 R5

NO .

92 CH3 H CH3 CH H H H COCH3

93 CH3 H CH3 C6H5CH2CO2 H H H COCH3

94 CH3 H CH3 OH H H H COCH3

95 CH3 H CH3 C6H5CH2CO2 H H H COCH3

96 CH3 H CH3 CH3CO2 H H H COCH3

97 CH3 H CH3O OH H H H COCH3

98 CH3 H -OCH2O- H H H COCH3

99 CH3 CH3O H H CH3O H H COCH3

100 CH3 CH H H OH H H COCH3

101 CH3 CH3O H CH3O H H H COCH3

102 CH3 OH H OH H H H COCH3

103 CH3 CH3O H H CH3O OC2H5 H COCH3

104 C ^β CH CH3O H H H H H COCH3

105 C ^β CH CH3O H H CH3O H H COCH3

106 C ^β CH H H CH H H H COCH 3

EXAMPLE R ¹ R^ R ¹⁰ R ¹¹ R ¹² 5 E

NO .

107 C ^β ECH H CH H H H Η COCH3

108 CH =CH ₂ CH3O H H H H H COCH3

₁₀ CH =CH ₂ CH3O H H CH3O H H COCH3

The following Examples #110-#413 of Table V are highly preferred conjugates formed from tyrosine hydroxylase inhibitor compounds and glutamic acid derivatives. These tyrosine hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula I, above.

EXAMPLE R3 R5 E NO.

118 CH3 0CH3 H H

EXAMPLE A R ³ R ⁵ E

NO.

124 CH3 OH CH3 H

EXAMPLE A R ⁵ R5 E

NO.

135 OCH3 H COCH3

¹⁴¹ II J_H. -™s CH3 CH CH ₃ COCH3

^{1 3} CH3 OCH3 H COCH3

150 CH3 OCH3 H H

157 CH3 OH CH3 COCH3

163 CH3 OH H COCH3

EXAMPLE R3 R5 E NO.

^Y

168 Jl NH ₂ CH ₃ OCH3 CH3 H

169 CH3 OCH3 CH3 COCH3

X ^N-H

174 OCH3 H H

175 . , ^s CH3 OCH3 H COCH3

177 OCH3 CH3 COCH3

EXAMPLE A R ³ R5

NO.

181 OH CH3 COCH3

183 OCH3 H COCH3

EXAMPLE R3 5 NO.

EEXX*AMPLE Rϊ R5 E NO.

192 H OCH3 CH3 H

EXAMPLE R5 E NO.

205 XX ⁾ CH3 OH CH3 COCH3

CH3 CH H H

*• _*

221 CH3 OH CH3 COCH3

228 CH CH3 H

EXAMPLE R5 E NO.

EXAMPLE A R ³ R ⁵ E

NO.

EXAMPLE R3 R5 E NO.

EXAMPLE E NO.

91/01724

122

EXAMPLE A R ³ R5 E

NO.

i^

290 _& JL \ / CH3 OH H H

I

H

EXAMPLE R5 E NO.

302 < & C ^β CH OCH3 H H

EXAMPLE ^j R5 E NO.

313 O C ^β CH 0CH3 CH3 COCH3

I

H

317 C ^β CH CH CH3 COCH3

319 -O CβCH

N > , OCH3 H COCH3

I

H

321 C ^β CH ₂ 0QJ3 _CH3 COCH3

327 ^"° xχ C ^β CH 0CH3 H COCH3

I

H

EXAMPLE A R ³ 5 E

NO.

337 CsCH OCH3 CH3 COCH3

EXAMPLE A R ³ R5 E

NO.

EXAMPLE R ^3" R-5 E NO.

351 H OCH3 H COCH3

356 H OH CH3 H

EXAMPLE A R R5 E

NO.

358 H OCH3 H H

H

EXAMPLE A R ³ R ⁵ E

NO.

EXAMPLE A R ³ R5 E

NO.

369 ^,s 0 H OCH3 CH3 COCH3

I

H

371 H H3

H^ OH H COC

H OCH3 H H

EXAMPLE R5 E NO.

EXAMPLE R5 E NO.

393 0 1 7 -2-3 00*3 ^CH 3 COCH3

I H

398 C2H CH=CH2 CH3 H H

399 C2H5 CH=CH2 OCH3 H COCH3

EXAMPLE R3 R5 E NO.

400 C2H5 CH=CH2 OCH3 CH3 H

401 C2H5 CH=CH2 OCH3 CH3 COCH3

402 C2H5 CH=CH2 OH H H

403 C2H5 CH=CH ₂ OH H COCH3

404 C2H5 CH=CH2 CH H COCH3

405 C2H5 CH=CH2 CH CH3 COCH3

407 C2H5 C ^β CH 0CH3 H COCH3

408 C2H5 CsCH 0CH3 CH3 H

409 C2H5 CsCH 0CH3 CH3 COCH3

EXAMPLE R5 E NO.

413 C ₂ H ₅ CsCH OH CH3 COCH3

The following Examples #414-# 61 of Table VI are highly preferred conjugates formed from tyrosine hydroxylase inhibitor compounds and glutamic acid derivatives. These tyrosine hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula III, above.

TOMB VT

EXAMPLE Rll R3 R5 E NO.

H OH H H

H OH H COCH3

H H

417 OH H COCH3

418 OH H H

419 OH H COCH3

420 OH H H

421 OH H COCH3

422 OH CH3 H

423 OH CH3 CH H COCH3

424 OH CH ₃ OH CH3 H

The following Examples # 62-#857 comprise five classes of highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives _. - Examples #462-# 64 are descriptions of specific preparations of such conjugates. Examples #465-#857, as shown in Tables VII-XI, may be prepared by procedures shown in these specific examples and in the foregoing general synthetic procedures of Schemes 1-7.

Example 462

-amino-4-carboxy-4-oxobutyl-3-hydroxy-α-methy1-L-tyrosin e, methyl ester.

Step. 1: Preparation of α-methvl-L-DOPA. methvl ester. hydrochloride .

A suspension of 29.7 g (141 mmol) of α-methy1-L-DOPA in 300 mL of absolute methanol was cooled to -15°C and treated with 125.8 g (1.06 mol) thionyl chloride under a nitrogen atmosphere. The reaction was allowed to warm to ambient temperature and stir at reflux for 3 days. Concentration followed by trituration with ether gave 31.7g (97%) as an off- white solid: NMR (DMSO-dβ) δ 1.47 (s, 3H) , 2.92 (d, J = 12 Hz,

IH), 2.98 (d, J = 12 Hz, IH) , 3.74 (s, 3H) , 6.41 (d of d, J = 9 Hz AND 2 Hz, IH) , 6.54 (d, _J = 2 Hz, IH) , 6.68 (d, J = 9 Hz, IH) , 8.46-8.90 (br s, 3H) , 8.93 (s, IH) , 8.96 (s, IH) .

Step 2 : Preparation of 4-amino-4- arboxy-4-oxobutyl-3-hvdroxy- α-methyl-L-tyrosine. methyl ester.

Under nitrogen, a solution of 32.7 g (108 mmol) of N- Boc-L-γ-glutamic acid-α-£-butyl ester (BACHEM) in 150 L of methylene chloride was treated with 11.14 g (54 mmol) of solid dicyclohexylcarbodiimide (DCC) . The reaction was allowed to stir for 2 hr prior to filtration under a nitrogen atmosphere. The methylene chloride was removed in vacuo and the residue dissolved in 110 mL of dimethylformamide (DMF) . The anhydride solution was slowly added to a solution of 12.9 g (49 mmol) of the α-methyl- DOPA ester from step 1 and 12.6 g (98 mmol) of diisopropylethylamine (DIEA) in 50 mL of anhydrous DMF. The reaction was allowed to stir overnight and was concentrated in. vacuo. The residue was dissolved in ethyl acetate, washed with 1U citric acid, IE NaHCC*3, water, and brine, dried (Na2S0 ), and concentrated in vacuo to give the protected coupled product; a solution of this material in 100 mL of methylene chloride was cooled to 0°C and treated with 400 mL of trifluoroacetic acid (TFA) under nitrogen. The reaction was allowed to warm to ambient temperature and stir for 72 hr. Concentration in vacuo gave 4-amino-4-carboxy-4-oxobutyl-3-hydroxy-α-methyl-L-tyrosine, methyl ester: NMR (DMSO-dg) δ 1.40 (s, 3H) , 1.85-2.30 (m, 2H) ,

2.30-2.50 (m, 2H) , 2.77 (d, J = 12 Hz, IH) , 3.00 (d, J = 12 Hz, IH), 3.58 (s, 3H), 3.85-4.10 (m, IH) , 6.29 (d of d, J. = 9 Hz and 2 Hz, IH), 6.45 (d, jl = 2 Hz, IH) , 6.62 (d, J = 9 Hz, IH) ; MS (FAB) m/e (rel intensity) 355 (92), 225 (51), 148 (35).

Example 463

N-[4-(acetylamino)-4-carboxy-4-oxobutyl]-3-hydroxy-α-met hyl-L- tyrosine, methyl ester.

The compound of Example 462 was dissolved in 100 mL of degassed water and under nitrogen the pH adjusted to 9 with 1 M K2CO3. The solution was cooled to 0°C and 12 mL (127 mmol) of acetic anhydride and 180 mL (180 mmol) of 1 M K2CO3 was added every 30 min. for 5h; the pH was maintained at 9 and the reaction temperature kept ^" below 5°C. After the last addition, the reaction was allowed to warm to ambient temperature overnight. The pH was adjusted to 3 with 3M HCl and concentrated to 100 mL. Purification by reverse phase chromatography (Waters Deltaprep- 3000) using a 5-15% gradient of acetonitrile/water (0.05% TFA) gave 14.Og (49%) of colorless product: NMR (DMSO-dg) δ 1.15 (s,

3H), 1.70-1.83 (m, 2H) , 1.85 (s, 3H) , 1.87-2.00 (m, 2H) , 2.15 (t, J = 7 Hz, 2H), 2.75 (d, _J = 12 Hz, IH) , 3.00 (d, J = 12 Hz, IH) , 3.55 (s, 3H), 4.10-4.22 (m, IH) , 6.29 (d of d, J = 9 Hz and 2Hz, IH), 6.43 (d, J * 2Hz, IH) , 6.60 (d, _J = 9 Hz, IH) , 7.96 (s, IH) , 8.12 (d, J = 8 Hz, IH); MS (FAB) m/e (rel intensity) 397 (100), 365 (10), 226 (70), 166 (90), 153 (22), 130 (72), 102 (28).

Example 464

N-T4-(acetvlamino)-4-carboxv-4-oxobutvπ-3-hvdroxv-α-met hvl-L- tyrosin .

A solution of 13.5 g (102 mmol) of the compound of Example 463 in 34 mL of water was cooled to 0°C and treated with 102 mL (102 mmol) of lϋ NaOH (all solutions were degassed n vacuo and flushed with nitrogen prior to use) . The reaction was stirred at ambient temperature for 5 hr and the pH adjusted to pH 1 with 6M HCl. Purification by reverse phase chromatography (Waters Deltaprep-3000) using a 2-10% gradient of acetonitrile/water (0.05% TFA) gave 8.9 g (68%) of colorless product: NMR (DMSO-dg) δ l.18 (s, 3H), 1.70-1.83 (m, 2H), 1.85

(s, 3H), 1.87-2.00 (m, 2H), 2.15 (t, _J = 7 Hz, 2H), 2.75 (d, J = 12 Hz, IH), 3.05 (d, J = 12 Hz, IH), 4.10-4.23 (m, IH) , 6.31 (d of d, J = 9 Hz and 2 Hz, IH), 6.47 (d, J = 2 Hz, IH) , 6.60 (d, _J = 9 Hz, IH), 7.71 (s, IH) , 8.15 (d, J = 8 Hz, IH); MS (FAB) m/e

(rel intensity) 383 (23), 212 (10), 166 (18), 130 (21), 115 (23); HRMS. Calcd for M + H: 383.1454. Found: 383.1450. ______]_ :

Calcd for Ci7^2^03*1.06 H2O-0.85 TFA: C, 48.67; H, 5.59; N,

6.46; F, 3.73. Found: C, 49.02; H, 5.73; N, 6.40; F, 3.70.

The following Examples #465-#541 of Table VII are highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. These dopa- decarboxylase inhibitors utilized to make these conjugates are embraced by generic Formula IV, above.

TftfiT.ff VTT

I

P

I EXAMPLE A R ¹ E NO.

465 H CH3 COCH3

EXAMPLE E NO.

H H COCH

EXAMPLE Rl E NO.

EXAMPLE Rl NO.

NH2 CH3 COCH3

H CH3 COCH3

C0 ₂ H

495 H H C0CH3

NH2 CH3 COCH3

CH ₂ F , OH

I

505 -C-CH ₂ 3 COCH3

C0 ₂ CH ₃ ^OH H CH

EXAMPLE Rl E NO.

EXAMPLE Rl E NO.

EXAMPLE Rl E NO.

CF, OH

521 -C-CH. :* -OH H CH3 COCH3 C0 ₂ CH ₃

EXAMPLE Rl E NO.

ΓF ^OH

525 1 ^* _ OH H ^■ Cf CH3 COCH3 O°gHH __/ ^~

EXAMPLE Rl E NO.

EXAMPLE Rl NO.

537 H CH3 C0CH3

(EXAMPLE A R ¹ E

NO.

The following Examples #5 _. 42-#577 of Table VIII are highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. These dopa- decarboxylase inhibitors utilized to make these conjugates are embraced by generic Formula VIII, above.

TABLE VΪU

OH

543 NHNH / ⁼ \ ^H H ^" H COCH3

- ^" ° ^H

EXAMPLE M R56 ^~ R55 NO.

PCH,

547 NHNH ' r__- Br H H COCH3

^■ -

r H CH3 COCH3

lEEXXAAMMPPLE M R 6 R55 NO.

EXAMPLE M ;56 R p ^' 5O5D NO.

557 NHCH ₂ CH ₂ NH H H CH3 COCH3

Br H CH3 H

561 HCH2CH2NH Br H CH3 COCH3

563 NHCH2CH2NH Br Br H COCH3

564 NHCH2CH2NH Br CH3 H

565 NHCH2CH2NH Br CH3 COCH3

EXAMPLE M R56 R55 NO.

566 piperazinyl H H H H

567 piperazinyl H H H COCH3

568 piperazinyl H H CH3 H

569 piperazinyl H H CH3 COCH3

OCH,

O /

570 piperazinyl || / — \ Br H H H

^■ -AJ

OCH,

571 piperazinyl || / ^s=s \ Br H H COCH3

- J

572 piperazinyl Br H CH3 H

573 piperazinyl Br H CH3 COCH3

574 piperazinyl Br H H

575 piperazinyl Br H COCH3

EXAMPLE M R5 R55 E NO.

576 piperazinyl "OH- Λ -CH. Br Br CH3 H

577 piperazinyl Br Br CH3 COCH3

The following Examples #578-#757 of Table IX are highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. , These dopa- decarboxylase inhibitors utilized to make these conjugates are benzoic acid type derivatives based on the list of similar compounds described earlier.

EXAMPLE L R ¹³⁰ R ¹³¹ R ¹³²

NO .

578 NHNH H H OH H H

579 NHNH H OH OH H COCH3

580 NHNH H OH OH CH3 H

581 NHNH H OH OH CH3 COCH3

583 NHNH - —O λ OH OH H COCH3

584 NHNH -O λ CH OH CH3 H

585 NHNH -O OH OH CH3 COCH3

591 NHNH ) — OCH3 OCH3 H COCH3

OCH/ OCH ₃

593 NHNH OCH3 CH3 COCH3

594 NHNH -O OCH3 OCH3 H H

595 NHNH " OCH3 OCH3 H COCH3

596 NHNH - OCH3 OCH3 CH3 H

597 NHNH -O 0CH3 0CH3 CH3 C0CH3

[EXAMPLE L R ¹³⁰ R ¹³¹ R1 ³²

NO .

603 NHNH OCH3 OCH3 H COCH3

605 NHNH ^~ 0 // OCH3 OCH3 CH3 COCH3

606 NHNH ∞ OH H H

EXAMPLE (1.30 _R 131 _R 132 _E NO .

611 NHNH OCH3 OCH3 H COCH3

613 NHNH OCH3 OCH3 CH3 COCH3

^0CH 3 OCH3 H H

615 NHNH 0CH3 OCH3 H COCH3

619 NHNH ^0CΑ ~ ^0CΑ 3 ^H COCH3

621 NHNH OCH3 OCH3 CH3 COCH3

622 NHNH OH OH H H

626 NHNH ~ J 0GH ₃ OCH3 H H

627 _N HN _H — ₀ 0,3 ^ _{H CQa} _ ₃

_{628 NHNH} ~ J ^ ^ ₃ ^' ^ _R

629 _N H _N H —ζj ^ ^3 ^ _coαj3

630 NHNH -^ OCH3 OCH3 H H

63 _{1 N} H _N H — ₀ ^3 ^3 _{R C0CH3}

EXAMPLE _R 130 _R 131 _R Ϊ32 E NO.

633 NHNH -O OCH3 OCH3 CH3 COCH3

634 NHNH -O OH OH H H

635 NHNH -ό OH OH H COCH3

636 NHNH -ό OH OH CH3 H

637 NHNH - CH OH CH3 COCH3

638 NHCH2CH2NH H OH OH H H

639 NHCH2CH2NH H OH OH H COCH3

640 NHCH2CH2NH H OH OH CH3 H

641 NHCH2CH2NH H OH OH CH3 COCH3

EXAMPLE L R ¹³⁰ R131 _R 132

NO .

643 NHCH2CH2NH -O OH OH H COCH ₃

644 NHCH ₂ CH ₂ NH -o ( QH OH CH3 H

645 NHCH2CH2NH OH OH CH ₃ COCH3

647 NHCH2CH2NH CH OH H COCH3

648 NHCH2CH2NH OH OH CH3 H

649 NHCH ₂ CH ₂ NH CH OH CH ₃ COCH3

651 NHCH2CH2NH OCH3 H COCH3

652 NHCH2CH2NH OCH3 CH3 H

653 NHCH2CH2NH OCH3 CH3 COCH3

654 NHCH2CH2NH " OCH3 OCH3 H H

655 NHCH2CH2NH -G ^N OCH3 OCH3 H COCH3

656 NHCH2CH2NH - OCH3 OCH3 CH3 H

658 NHCH2CH2NH OCH3 H H

660 NHCH2CH2NH OCH3 CH3 H

661 NHCH2CH2NH OCH3 CH3 COCH3

662 NHCH2CH2NH -O OCH3 OCH3 H H

663 NHCH2CH2NH -O / OCH3 OCH3 H COCH3

EXAMPLE L R ¹³ R131 _R 132 _E

NO.

664 NHCH2CH2NH -O // OCH3 OCH 3 CH3 H

665 NHCH2CH2NH OCH3 OCH3 CH3 COCH3

667 NHCH2CH2NH OH OH H COCH3

668 NHCH2CH2NH OH OH CH3 H

669 NHCH2CH2NH ~-~- ^{0H CH} 3 COCH3

670 NHCH2CH2NH OCH3 OCH3 H H

671 NHCH2CH2NH OCH 3 OCH 3 H COCH3

672 NHCH2CH2NH OCH3 OCH3 CH3 *H

673 NHCH ₂ CH ₂ NH 0CH3 OCH3 CH3 COCH3

674 NHCH2CH2NH OCH3 OCH3 H H

675 NHCH2CH2NH _{H C0CR 3}

0CH ₃ OCH3 CH ₃ H

677 NHCH2CH2NH OCH 3 OCH 3 CH3 COCH3

678 NHCH2CH2NH OCH3 OCH3 H H

679 NHCH2CH2NH OCH3 OCH3 H COCH3

680 NHCH2CH2NH OCH3 OCH3 CH3 H

EXAMPLE L R ¹³ 0 _R 131 _R 132

NO.

681 NHCH2CH2NH OCH3 OCH3 CH3 COCH3

683 NHCH2CH2NH OH H COCH3

684 NHCH2CH2NH ff OH OH CH3 H

685 NHCH2CH2NH OH OH CH3 COCH3

686 NHCH2CH2NH ff OCH3 OCH3 H H

687 NHCH2CH2NH OCH3 CCH3 H COCH3

688 NHCH2CH2NH OCH 3 OCH 3 CH3 H

689 NHCH2CH2NH ff OCH 3 OCH 3 CH3 COCH3

690 NHCH2CH2NH OCH3 OCH3 H H

691 NHCH2CH2NH fl OCH3 OCH3 H COCH3

692 NHCH2CH2NH fl OCH3 OCH3 CH3 H

693 NHCH2CH2NH fl OCH3 OCH3 CH3 COCH3

694 NHCH2CH2NH fl OH OH H H

695 NHCH2CH2NH OH OH H COCH3

696 NHCH2CH2NH fl CH OH CH3 H

697 NHCH2CH2NH fl Orl OH CH3 COCH3

698 piperazinyl H OH OH H H

IEXAMPLE Rl30 _R 131 _R 132 E NO.

699 piperazinyl H OH OH H COCH3

700 piperazinyl H CH OH CH3 H

701 piperazinyl H OH OH CH3 COCH3

702 piperazinyl -O CH OH H H

703 piperazinyl -O OH OH H COCH3

704 piperazinyl OH OH CH3 H

705 piperazinyl CH OH CH3 COCH3

706 piperazinyl CH OH H H

707 piperazinyl OH OH H COCH3

I E EXXAAMMPPLE Rl30 RΪ31 _R 132 _E N NOC .

708 piperazinyl OH OH CH3 H

709 piperazinyl OH OH CH3 COCH3

710 piperazinyl OCH ₃ H H

711 piperazinyl OCH 3 H COCH3

712 piperazinyl OCH3 CH ₃ H

713 piperazinyl OCH3 CH3 COCH3

714 piperazinyl " OCH3 OCH3 H H

(EXAMPLE L R ¹³ 0 131 _R 132 _E

NO .

715 piperazinyl OCH3 OCH3 H COCH3

716 piperazinyl - ^Q /} OCH 3 OCH 3 CH3 H

717 piperazinyl OCH3 OCH3 CH3 COCH3

719 piperazinyl OCH 3 H COCH3

720 piperazinyl OCH 3 CH3 H

721 piperazinyl OCH3 CH3 COCH3

EXAMPLE R130 131 _R 132 E NO .

722 piperazinyl OCH 3 OCH 3 H H

723 piperazinyl OCH3 OCH3 H COCH3

724 piperazinyl -O OCH 3 OCH 3 CH3 H

725 piperazinyl OCH3 OCH3 CH3 COCH3

726 piperazinyl OH OH H H

727 piperazinyl °H OH H COCH3

728 piperazinyl OH OH CH3 H

729 piperazinyl CH OH CH3 COCH3

730 piperazinyl OCH 3 OCH 3 H H

EXAMPLE 130 _R 131 _R 132 E NO .

731 piperazinyl OCH3 0CH3 H COCH3

732 piperazinyl OCH3 OCH3 CH3 H

733 piperazinyl OCH3 OCH3 CH3 COCH3

734 piperazinyl OCH 3 OCH 3 H H

735 piperazinyl 0CH3 OCH 3 H COCH3

736 piperazinyl 0CH3 OCH 3 CH3 H

737 piperazinyl 0CH3 OCH 3 CH3 COCH3

738 piperazinyl 0CH3 OCH3 H H

EXAMPLE L R-^O _R 131 _R 132

NO.

739 piperazinyl OOCCHH 33 OOCCHH 33 HH COCH3

740 piperazinyl OCH 3 OCH 3 CH3 H

741 piperazinyl OCH 3 OCH 3 CH3 COCH3

742 piperazinyl fl OH OH H H

743 piperazinyl OH OH H COCH3

744 piperazinyl OH OH CH3 H

745 piperazinyl fl OH OH CH3 COCH3

746 piperazinyl fl OCH 3 OCH 3 H H

747 piperazinyl fl OCH 3 OCH 3 H COCH3

748 piperazinyl fl OCH 3 OCH 3 CH3 H

749 piperazinyl OCH3 OCH 3 CH3 COCH3

750 piperazinyl fl OCH 3 OCH 3 H H

751 piperazinyl fl OCH 3 OCH 3 H COCH3

752 piperazinyl OCH 3 OCH 3 CH3 H

753 piperazinyl OCH 3 OCH 3 CH3 COCH3

754 piperazinyl fl OH OH H H

755 piperazinyl fl OH OH H COCH3

EXAMPLE L R ¹³ 0 131 _R 132

NO .

756 piperazinyl OH OH CH3 H

757 piperazinyl OH OH CH3 COCH3

The following Examples #758-#809 of Table X are highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. These dopa- decarboxylase inhibitors utilized to make these conjugates are propenoic acid derivatives based on the list of similar compounds described earlier.

758 H -O H H H

759 H ^~ ύ H H COCH3

760 H -O H CH3 H

762 CH3 -ό H H H

EXAMPLE Rl33 _R 134 _R 135

NO.

764 CH3 ~ H CH3 H

770 H H H H

I EXAMPLE R 133 _R 134 _R 135 NO.

772 H H CH3 H

^

774 CH3 -ύ H H H

775 CH3 ^~ ύ H H COCH3

777 CH3 -ύ CH3 COCH3

779 H _m \\ // H H COCH3

780 H _m \\ II H CH3 H

783 CH3 _m \\ // H H COCH3

784 CH3 H CH3 H

791 CH3 H H COCH3

794 H -ύ CH3 H H

795 H -ύ CH3 H COCH3

796 H -Ό CH3 CH3 H

798 H -O H H H

EXAMPLE R ¹³³ R ¹³⁴ R ¹³⁵

NO. ^■

805 CH3 H CH3 COCH3

EXAMPLE R ¹³³ R ¹³⁴ R ¹³⁵

NO.

The following Examples #810-#833 of Table XI are highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. These dopa- decarboxylase inhibitors utilized to make these conjugates are embraced by generic Formula IX, above.

TRFTiTi XT

823 CH3 H H COCH3

824 CH3 H CH3 H

825 CH3 H CH3 COCH3

826 CH3 CH H H

827 CH3 OH H COCH3

828 CH3 OH CH3 H

829 CH3 OH CH3 COCH3

830 CH3 OCH3 H H

831 CH3 OCH3 H COCH3

832 CH3 OCH3 CH3 H

833 CH3 OCH3 CH3 COCH3

The following Examples #834-#857 of Table XII are highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. These dopa- decarboxylase inhibitors utilized to make these "conjugates are embraced by generic Formula IX, above.

TABLE XII

EXAMPLE R138 _R 139 ■67 E NO.

H H

H COCH3

H COCH3

H H

844 H OH C CH CH3 H

EXAMPLE _R 138 _R 139 _R 67 NO .

846 H H CH=CH H H

847 H H CH=CH2 H COCH 3

848 H H CH=CH2 CH3 H

849 H H CH=CH ₂ CH3 COCH3

850 OH H CH=CH ₂ H H

851 OH H CH=CH2 H COCH 3

852 OH H CH=CH ₂ CH3 H

853 CH H CH=CH ₂ CH3 COCH 3

854 H OH CH=CH2 H H

855 H OH CH=CH2 H COCH3

856 H OH CH=CH ₂ CH3 H

857 H OH CH=CH2 CH3 COCH 3

The following Examples #858-#1857 comprise five classes of highly preferred conjugates composed of dopamine-β- hydroxylase inhibitor compounds and glutamic acid derivatives. Examples #858-#863 are descriptions of specific preparations of such conjugates. Examples #864-#1857, as shown in Tables XIII- XVTI, may be prepared by procedures shown in these specific examples and in the foregoing general synthetic procedures of Schemes 1-7.

Example 858

L-glutamic acid, 5- [ (5-butyl-2-pyridinyl) carbonyl] -hydrazide

Step. 1 : Preparation of 5-n-Butvlpicolinic (Fusaric Acid

Hydrazide.

A solution of 36.0 g (0.20 mol) of fusaric acid (Sigma) in 800 ml of absolute methanol was cooled to -10°C by means of an ice/methanol bath and 120 ml (199 g, 1.67 mol) of SCO2 was added dropwise over a 1 hr period. The reaction was allowed to slowly warm to ambient temperature and then stirred at reflux for 72 hr. The reaction was concentrated; the addition of 100 ml of toluene (twice) followed by reconcentration insured the complete removal of any unreacted SOC12- The viscous syrup thus formed was dried in vacuo (0.01mm) overnight prior to treatment with cold NaHCθ3(sat) . The ester was extracted with ether and dried (MgSθ4) . Concentration gave 32.3 g (83%) of crude methyl fusarate which was redissolved in 100 ml of absolute methanol and cooled to 0°C. Under a nitrogen atmosphere, 5.5 ml (0.174 mol) of anhydrous hydrazine was slowly added by syringe. The reaction was allowed to slowly warm to ambient temperature and stir

overnight. The methanol was removed and the yellow-brown residue was dried in vacuo (0.01 mm) overnight where it solidified producing 31.7g (98%) based on ester) of crude hydrazide. Recrystallization from ether/hexane gave colorless needles: mp 51-53°C NMR (CDCI3) δ 0.95 (t, J = 7 Hz, 3H, CH CH ₃ ); 1.30-1.45 (m, 2H, CH2CH3); 1.55-1.70 (m, 2H, CH ₂ CH ₂ CH2); 2.67 (t, J = 7 Hz, 2H, ArCH2); 7.65 (d of d, ^3,4 = 7 Hz and J _4/ g = 2 Hz, IH, ArH); 8.05 (d, J3 ₄ = 7 Hz, IH, ArH); 8.37 (d, IH, ArH, _J _4f ζ = 2 Hz); HRMS. Calcd for M + H: 194.1270. Found: 194.1293.

10 step 2 : Preparation of L-ςrlut.arπic acid, 5- r (5-butyl-2 - pvridinγl) carbonyl1hydrazide.

A solution of 7.27 g (24.0 mmol) of Boc-L-γglutamic

15 acid-α-i-butyl ester (BACHEM) in 150 ml of anhydrous THF was cooled to 0°C under static nitrogen and treated with 2.7 ml (2.46 g, 24.4 mmol) of anhydrous N-methyl morpholine. The mixture was then slowly treated with 3.1 ml (3.26 g, 23.9 mmol) of isobutyl chloroformate and allowed to stir for 1 hr prior to the dropwise

20 addition of a solution of 3.86 g (20.0 mmol) of fusaric acid hydrazide from step 1 in 30 ml of anhydrous THF. The reaction mixture was stirred at 0°C for 2 hr and then allowed _. to warm to ambient temperature and stir overnight. The N-methylmorpholine hydrochloride was removed by filtration and the filtrate

25 concentrated in vacuo to give 11.5 g of crude product which was a colorless glass. This material was dissolved in 50 ml of CH2CI2 and treated with 50 ml of CF3CO2H. After 4 hr at ambient temperataure, the volitiles were removed in vacuo. The addition of acetonitrile caused the product to precipitate producing 3.97

30 g (46%). of colorless material: mp 162-164°C (dec); NMR (DMSO- dg) δ 1.90 (t, J = 7 Hz, 3H, CH ₂ CH ₃ ); 1.30-1.45 (m, 2H, CH. ₂ CH ₃ );

<* 1.50-1.65 (m, 2H, CH ₂ CH ₂ CH ₂ ); 2.00-2.20 (m, IH, Cfl ₂ CH) ; 2.30-2.50 (m, IH, CH ₂ CH); 2.70 (t, J = 7 Hz, 2H, ArCH2> 3.60 (t, _J = 7 Hz, 2H, C0CH ₂ ); 3.95-4.05 (M, IH, CH ₂ CH.); 7.85 (d of d, ^4 = 7 Hz

and J _{4r 6} = 2 Hz, IH, ArH) ; 7.95 (d, 3, = 7 Hz, IH, ArH) ; 8 .55 (d, J _4f g = 2 Hz, IH, ArH) .

Example 859

N-acetyl-L-glutamic a_niύ__ 5- r (5-frutyl-2-pyridinyl) - arbonvl1hydrazide

A suspension of 2.85 g (6.54 mmol) of the compound of Exairple 858 in CH3CN/H2O (1:1) was treated with 2 equiv. of 1 M K2CO3 at 0°C. With efficient stirring, 1 ml (10.6 mmol) of acetic anhydride and 11 ml (11 mmol) of IM K2CO3 were added every 10 min for 1 hr; since the product is soluble, the mixture became homogenous as the reaction proceeded. The reaction mixture was stirred for 1 hr, filtered, and the filtrate cooled to 0°C. The pH was adjusted to pH 4 by the careful addition of cold dilute HCl. All volitile≤ were removed in vacuo and the product dissolved in ethanol. Recrystallization from ethanol/petroleum ether produced 2.16g (69%) of colorless material: mp 191.5- 192.0°C; NMR (D ₂ 0 and NaOD)δ 1.85 (t, _l = 7 Hz, 3H, CH ₂ CH ₃ ); 1.20-1.35 (m, 2H, CII2CH3); 1.55-1.70 (m, 2H, CH2CII2CH2); 1.95- 2.10 (m, IH, C2 ₂ CH); 2.05 (s, 3H, COCH3); 2.20-2.35 (m, IH, CH ₂ CH); 2.45 (t, J = 7 Hz, 2H, COC22- ¹ ; ² - ⁷⁵ _* -> 2H, rCU2); 3.45- 3.55 (m, IH, CH ₂ Cfi) ; 8.05 (s, 2H, ArH); 8.55 (s, IH, ArH); HRMS. Calcd for M + H: 365.1825. Found 365.1860. Anal .

Calcd. for C17H24N O5: C, 55.98; H, 6.58; N, 15.36. Found: C, 55.96; H, 6.64; N, 15.30.

Exam le 86Q

N-[2-[ f (5-butyl-2-pyridinyl)carbonyl]amino]ethyl]-L-glutamine.

step l: Preparation of the ethylene diamine amide of fusaric acid .

A solution of 7.8 g (130 mmol) of ethylene diamine in 400 mL of anhydrous THF under nitrogen was treated with 27 mmol of n-butyllithium at 0°C. The solution was allowed to stir for 30 min and was treated with 5.0 g (26 mmol) of neat methyl fusarate (from step 1 of Example 690) by syringe. The reaction was kept at 0°C for 2 hr and stirred at ambient temperature overnight. The reaction was quenched with water, filtered, and concentrated in vacuo. Purification by silica gel chromatography gave 3.8 g (66%) of pure amide: NMR (DMSO-d ₆ ) δ 0.90 (t, J = 8 Hz, 3H), 1.23-1.38 (m, 2H) , 1.52-1.64 (m, 2H) , 2.67 (t, J = 8 Hz, 2H), 2.74 (t, J = 8 Hz, 2H) , 3.18-3.30 (br s, 2H) , 3.34 (q, *J = 8 Hz, 2H), 7.82 d of d, J = 9 Hz and 2 Hz, IH) , 7.96 (d, J = 9 Hz, IH), 8.47 (d, J = 2 Hz, IH) , 8.75 (t, J = 8 Hz, IH) .

step 2: Preparation of N-T2-rr(5-fr tyl-2- pyridinyl)carbonyl1aminolethyl 1-L-ςrl-uat.mine.

Under nitrogen, a solution of 26.8 g (88.5 mmol) of N- Boc-L-γ-glutamic acid-α-£-butyl ester (BACHEM) in 125 mL of

methylene chloride was treated with 9.14 g (44.3 mmol) of solid dicyclohexylcarbodiimide (DCC) . The reaction was allowed to stir for 2 hr prior to filtration under a nitrogen atmosphere. The anhydride solution was slowly added to a solution of 8.5 g (38.5 mmol) of the ethylene diamine amide from step 1 in 100 mL of methylene chloride. The reaction was allowed to stir overnight and was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with IM K2CO3 followed by water, dried (MgSθ4) and reconcentrated in vacuo to give the protected coupled product; a solution of this material in 250 mL of methylene chloride was cooled to 0°C and treated with 250 mL of trifluoroacetic acid (TFA) . The reaction was allowed to warm to ambient temperature and stir overnight; the course of the reaction was monitored by analytical LC. Concentration in vacuo gave N-[2-[ [ (5-butyl-2-pyridinyl)carbonyl]amino]ethyl]-L- glutamine.

Example 861

N2-acetyl-N- \Z- . r(5-b.f.yJ-2-pyridinyl)carbonyl1-aminolethyll-I- glu amine .

The compound of Example 860 was dissolved in 150 mL of acetonitrile/water (1:1) and the pH adjusted to 9 with 2 M K2CO3.

The solution was cooled to 0°C and 2.27 mL (24 mmol) of acetic anhydride and 12 mL (24 mmol) of 2 M K2CO3 was added every 30

min. for 5 h; the pH was maintained at 9 and the reaction temperature kept below 5°C. After the last addition, the reaction was allowed to warm to ambient temperature overnight. The pH was adjusted to 3 with 3 M HCl and concentrated to 300 mL, Purification by reverse phase chromatography (Waters Deltaprep- 3000) using isocractic 30% acetonitrile/water (0.05% TFA) gave 7.8 g (52% overall yield from the amide of step 1) of colorless product; an analytical sample was recrystallized from acetonitrile and then water: mp 156-158°C; Anal . Calcd for C29H28 4O5-0.83 TFA: C, 57.32; H, 7.00; N, 13,96; F, 1.14%.

Found: C, 57.22; H, 7.07; N, 13.88; F, 1.07.

Example 862

2-amino-5-[4-[ (5-butyl-2-pyridinyl)carbonyl]-1-piperazinyl]-5- oxopentanoic acid.

Step 1: Preparation of the pipe izine amide of fusaric acid.

A solution of 11.20 g (130 mmol) of piperazine in 400 mL of anhydrous THF under nitrogen was treated with 27.3 mmol of n-buytyllithium at 0°C. The solution was allowed to stir for 30 min and was treated with 5.0 g (26 mmol) of neat methyl fusarate (from step 1 of Example 690) by syringe. The reaction was kept at 0°C for 2 hr and stirred at ambient temperature overnight. The reaction was quenched with water, filtered, and concentrated in vacuo. Purification by silica gel chromatography using chloroform/methanol (70:30) gave 5.82 g (90%) of pure amide: NMR (CDCl3)δ 0.94 (t, _2 = 8 Hz, 3H) , 1.28-1.45 (m, 2H) , 1.55-1.67 (m,

2H), 1.66-1.72 (br s, IH) , 2.64 (t, J = 8 Hz, 2H) , 2.86 (t, J = 6

Hz, 2H) , 2.97 (t _r. J = 6 Hz, 2H) , 3.58 (t, _J = 6 Hz, 2H) 3.77 (t, J = 6 Hz, 2H) , 7 .54-7. 63 (m, 2H) , 8.37-8.43 (br s, IH) .

Step 2: Preparation of 2-amino- -r4-r ( -hn yl-?- vridinyl)πarbonvll-1-pipera?invl1- -oxoper.t-_ano-i r. acid.

Under nitrogen, a solution of 17.4 g (57 mmol) of N- Boc-L-γ-glutamic acid-α-£-butyl ester (BACHEM) in 100 mL of anhydrous THF was treated with 5.57 g (27 mmol) of solid dicyclohexylcarbodiimide (DCC) . The reaction was allowed to stir for 2 hr prior to filtration under a nitrogen atmosphere. The anhydride solution was slowly added to a solution of 5.82 g (23.5 mmol) of the piperazine amide from step 1 in 50 mL of anhydrous THF. The reaction was allowed to stir overnight and was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with IM K2CO3 followed by water, dried (MgSθ ), and reconcentrated in. vacuo to give the protected coupled product; a solution of this material in 150 mL of methylene chloride was cooled to 0°C and treated with 150 mL of trifluoroacetic acid (TFA) under nitrogen. The reaction was allowed to warm to ambient temperature and stir overnight; the course of the reaction was monitored by analytical LC. Concentration in vacuo gave 2-amino-5-[4-[ (5-butyl-2- pyridinyl)carbonyl]-1-piperazinyl]-5-oxopentanoic acid.

Example 863

2-(acetylamino)-5- (4-[ (5-butyl-2-pyridinyl)carbonyl]-1- piperazinyl]-5-oxopentanoic acid.

The compound of Exairple 862 was dissolved in 150 mL of acetonitrile/water (1:1) and the pH adjusted to 9 with 1 M K2CO3. The solution was cooled to 0°C and 2.36 mL (25 mmol) of acetic anhydride and 25 mL (25 mmol) of 1 M K2CO3 was added every 30 min. for 5 h; the pH was maintained at 9 and the reaction temperature kept below 5°C. After the last addition, the reaction was allowed to warm to ambient temperature overnight. The pH was adjusted to 4 with 3 M HCl and concentrated to 300 mL. Purification by reverse phase chromatography (Waters Deltaprep- 3000) using isocratic 25% acetonitrile/water (0.05% TFA) gave 8.13 g (78%) of colorless product: MS (FAB) m/e (rel intensity) 419 (100), 258 (10), 248 (37), 205 (28); HRMS. Calcd for M+H: 419.2294. Found: 419.2250.

The following Examples #864-#1097 of Table XIII are highly preferred conjugates composed of dopamine-β-hydroxylase inhibitor compounds and glutamic acid derivatives. These dopamine-β-hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula XIV and XV, above.

TfiFT.F. XTTT

EXAMPLE R9 E NO.

864 NHNH H

865 NHNH COCH3

866 NHNH H

867 NHNH COCH3

868 NHNH H

869 NHNH COCH3

870 NHNH H

871 NHNH COCH3

872 NHNH H

873 NHNH COCH3

874 NHNH H

875 NHNH COCH3

EXAMPLE R97 E NO.

876 NHNH

877 NHNH

878 NHNH

879 NHNH

880 NHNH

881 NHNH

882 NHNH

883 NHNH

884 NHNH

885 NHNH

886 NHNH

887 NHNH

888 NHNH

889 NHNH

890 NHNH

891 NHNH

EXAMPLE R97 E NO .

892 NHNH

893 NHNH

894 NHNH

895 NHNH

896 NHNH

897 NHNH

898 NHNH

899 NHNH

900 NHNH

901 NHNH

902 NHNH

903 NHNH

904 NHNH

905 NHNH

906 NHNH

907 NHNH

EXAMPLE R97 NO.

908 NHNH SC3H7 CH3 H

909 NHNH SC3H7 CH3 COCH3

910 NHNH H H

911 NHNH H COCH3

912 NHNH CH3 H

913 NHNH CH3 COCH3

914 NHNH Cl H H

915 NHNH Cl H COCH3

916 NHNH Cl CH3 H

917 NHNH Cl CH3 COCH3

918 NHNH Br H H

919 NHNH Br H COCH3

920 NHNH Br CH3 H

921 NHNH Br CH3 COCH3

922 NHNH H H

923 NHNH H COCH3

EXAMPLE R97 E NO.

924 NHNH CH3 H

925 NHNH CH3 COCH 3

926 NHNH CN H H

927 NHNH CN ,H COCH3

EXAMPLE L R ⁹⁷

NO.

940 NHCH2CH2NH CH3 CH3 H

941 NHCH2CH2NH CH3 CH3 COCH3

942 NHCH2CH2NH C2H5 H H

943 NHCH2CH2NH C2H5 H COCH3

944 NHCH2CH2NH C2H5 CH3 H

945 NHCH2CH2NH C2H5 CH3 COCH3

946 NHCH2CH2NH C3H7 H H

947 NHCH2CH2NH C3H7 H COCH3

948 NHCH2CH2NH C3H7

949 NHCH2CH2NH C3H7

950 NHNH CH3

951 NHNH CH3

952 NHCH2CH2NH C4H9

953 NHCH2CH2NH C4H9

954 NHCH2CH2NH C5H11 H H

955 NHCH2CH2NH C5H11 H COCH 3

956 NHCH2CH2NH C5H11

957 NHCH2CH2NH C5H11

958 NHCH2CH2NH C6H13

959 NHCH2CH2NH C6H13

960 NHCH2CH2NH C6H13

961 -NHCH2CH2NH C6H13

962 NHCH2CH2NH OCH 3 H H

963 NHCH2CH2NH OCH 3

964 NHCH2CH2NH OCH3

965 NHCH2CH2NH OCH3

966 NHCH2CH2NH OC2H5 H H

967 NHCH2CH2NH OC2H5 H COCH3

968 NHCH2CH2NH OC2H5

969 NHCH2CH2NH OC2H5

970 NHCH2CH2NH OC3H7

971 NHCH2CH2NH OC3H7

EEXXAAMft PLE R97 E NNCO .

972 NHCH2CH2NH OC3H7 CH3 H

973 NHCH2CH2NH OC3H7 CH3 COCH3

974 NHCH2CH2NH OC4H9 H H

975 NHCH2CH2NH OC4H9 H COCH3

976 NHCH2CH2NH OC4H9 CH3 H

977 NHCH2CH2NH OC4H9 CH3 COCH 3

978 NHCH2CH2NH SCH3 H H

979 NHCH2CH2NH SCH3 H COCH3

980 NHCH2CH2NH SCH3 CH3 H

981 NHCH2CH2NH SCH3 CH3 COCH 3

982 NHCH2CH2NH SC2H5 H H

983 NHCH2CH2NH SC2H5 H COCH3

984 NHCH2CH2NH SC2H5 CH3 H

985 NHCH2CH2NH SC2H5 CH3 COCH3

986 NHCH2CH2NH SC3H7 H H

987 NHCH2CH2NH SC3H7 H COCH3

EXAMPLE R97 E NO .

988 HCH2CH2NH SC3H7 CH3 H

989 NHCH2CH2NH SC3H7 CH3 COCH3

H H

H COCH3

CH3 H

CH3 COCH3

Cl H H

H COCH3

Cl CH3 COCH3

Br H H

Br H COCH3

Br CH3 H

Br CH3 COCH3

H H

H COCH3

1004 NHCH2CH2NH I CH3 _. H

1005 NHCH2CH2NH. I CH3 COCH3

1006 NHCH2CH2NH CN H H

1007 NHCH2CH2NH CN H COCH3

1008 NHCH2CH2NH CN CH3 H

1009 NHCH2CH2NH CN CH3 COCH3

1010 NHCH2CH2NH N02 H H

1011 NHCH2CH2NH N02 H COCH3

1012 NHCH2CH2NH N02 CH3 H

1013 NHCH2CH2NH N02 CH3 COCH3

1014 NHCH2CH2NH OH H H

1015 NHCH2CH2NH OH H COCH3

1016 NHCH2CH2NH OH CH3 H

1017 NHCH2CH2NH CH CH3 COCH3

1018 piperzinyl CH3 H H

1019 piperzinyl CH3 H COCH3

EXAMPLE R97 E NO.

1020 piperzinyl CH3 CH3 H

1021 piperzinyl CH3 CH3 COCH3

1022 piperzinyl C2H5 H H

1023 piperzinyl C2H5 H COCH3

1024 piperzinyl C2H5 CH3 H

1025 piperzinyl C2H5 CH3 COCH3

1026 piperzinyl C3H7 H H

1027 piperzinyl C3H7 H COCH3

1028 piperzinyl C3H7 CH3 H

1029 piperzinyl C3H7 CH3 COCH3

1030 NHNH C2H5 H H

1031 NHNH C2H5 H COCH3

1032 piperzinyl C4H9 CH3 H

1033 piperzinyl C4H9 CH3 COCH3

1034 piperzinyl C5H11 H H

1035 piperzinyl C5H11 H CCCH3

EXAMPLE R9 E NO .

1036 piperzinyl C5H11 CH3 H

1037 piperzinyl C5H11 CH3 COCH3

1038 piperzinyl CβHi3 H H

1039 piperzinyl C6H13 H COCH3

1040 piperzinyl C6H13 CH3 H

1041 piperzinyl C6H13 CH3 COCH3

1042 piperzinyl OCH3 H H

1043 piperzinyl OCH3 H COCH3

1044 piperzinyl OCH3

1045 piperziny1 OCH3

1046 piperzinyl OC2H5

1047 piperzinyl OC2H5

1048 piperzinyl OC2H5

1049 piperzinyl OC2H5

1050 piperzinyl OC3H7 H H

1051 piperzinyl OC3H7 H COCH3

IEXAMPLE R9 E NO.

1052 piperzinyl OC3H7 CH3 H ^■

1053 piperzinyl OC3H7 CH3 COCH3

1054 piperzinyl OC4H9 H H

1055 piperzinyl OC4H9 H COCH3

1056 piperzinyl OC4H9 CH3 H

1057 piperzinyl OC4H9 CH3 COCH3

1058 piperzinyl SCH3 H H

1059 piperzinyl SCH3 H COCH3

1060 piperzinyl SCH3 CH3 H

1061 piperzinyl SCH3 CH3 COCH3

1062 piperzinyl SC2H5 H H

1063 piperzinyl SC2H5 H COCH3

1064 piperzinyl SC2H5 CH3 H

1065 piperzinyl SC2H5 CH3 COCH3

1066 piperzinyl SC3H7 H H

1067 piperzinyl SC3H7 H CCCH3

1084 piperzinyl CH3 H

1085 piperzinyl CH3 COCH3

1086 piperzinyl CN H H

1087 piperzinyl CN H COCH3

1088 piperzinyl CN CH3 H

1089 piperzinyl CN CH3 COCH3

1090 piperzinyl N02 H H

1091 piperzinyl N02 H COCH3

1092 piperzinyl Nθ2 CH3 H

1093 piperzinyl NO2 CH3 COCH3

1094 piperzinyl OH H H

1095 piperzinyl OH H COCH3

1096 piperzinyl OH CH3 H

1097 piperzinyl OH CH3 COCH3

EXAMPLE R9 E NO.

The following Examples #1098-#1137 of Table XIV are highly preferred conjugates composed of dopamine-β-hydroxylase inhibitor compounds and glutamic acid derivatives. These dopamine-β-hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula XIV, above.

The following Exairples #1138-#1377 of Table XV are highly preferred conjugates composed of dopamine-β-hydroxylase inhibitor compounds and glutamic acid derivatives. These dopamine-β-hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula XVIII, above.

I&B E X2

(EXAMPLE _R ll _R 114 _R 116 _R 117 _R 118 _E NO.

The following Examples #1378-#1497 of Table XVI are highly preferred conjugates composed of dopamine-β-hydroxylase inhibitor compounds and glutamic acid derivatives. These dopamine-β-hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula XVIII, above.

EXAMPLE R116 _R 117 Rll8 NO .

H CH H H H

H CH H H COCH3

H OH H CH3 H

H OH H CH3 COCH3

H H H H

H H H COCH3

H H CH3 H

H H CH3 COCH3

H CF3 H H H

1387 H CF3 H H COCH3

1388 H CF3 H CH3 H

EXAMPLE n _R 116 _R 117 Rll8 E NO.

H

H

H

1392 OH CH H

1393 OH OH H

H

H H COCH3

H H

H COCH3

H H

H COCH3

H H

H COCH3

1402 H CH H H H

1403 H CH H H COCH3

1404 H CH H CH3 H

j EXAMPLE R116 R117 Rll8 E NO .

1405 H OH H CH3 COCH3

1406 H H H H

1407 H H H COCH3

1408 H H CH3 H

1409 H H CH3 COCH3

1410 H CF3 H H H

1411 H CF3 H H COCH3

1412 H CF3 H CH3 H

1413 H CF3 H CH3 CCCH3

1414 OH OH H H H

1415 OH OH H H COCH3

1416 OH OH H CH3 H

1417 OH OH H CH3 COCH3

1418 H H H

1419 H H COCH3

1420 H CH3 H

1421 H CH3 COCH3

1422 CF3 H CF3 H H

1423 CF3 H CF3 H COCH3

1424 CF3 H CF3 CH3 H

CF3 H CF3 CH3 COCH3

H OH H H H

H OH H H COCH3

H OH H CH3 H

H OH H CH3 COCH3

H H H H

H H H COCH3

1432 H H CH3 H

1433 H H CH3 COCH3

1434 H CF3 H H H

1435 H CF3 H H COCH3

1436 H CF3 H CH3 H

1437 H CF3 H CH3 COCH3

1438 OH CH H H H

1439 OH CH H H CCCH3

1440 OH CH H CH3 H

1441 OH OH H CH3 COCH3

1442 2 H H H

1443 2 H H COCH3

1444 H CH3 H

CH3 COCH3

CF3 H H

CF3 H COCH3

CF3 CH3 H

CF3 CH3 COCH3

H H H

H H COCH3

H CH3 H

EXAMPLE n Rll ⁶ RU ⁷ RH 8 E

NO.

^{1453 3 H} OH H CH ₃ COCH3

1454 3 H F ■ H _H H

¹⁴⁵⁵ 3 H F H H COCH3

¹⁴⁵⁶ 3 H F H CH3 H

^{1 57 3 H} F H CH3 C0CH3

1458 3 H CF ₃ H H H

^{1 59 3} H CF ₃ H H C0CH3

^{1460 3} H CF3 H CH3 H

^{1461 3} H CF3 H CH3 C0CH ₃

1462 3 QH CH H H H

^{1 63 3} OH OH H H COCH3

¹⁴⁶ 3 OH ^Q H H CH ₃ H

^{1 65} 3 CH CH H CH ₃ COCH3

1466 3 F H F H H

¹⁴⁶⁷ 3 F H F H COCH3

¹⁴⁶⁸ 3 F H F CH3 H

j EXAMPLE Rll6 Rl Rllβ E NO.

1469 H CH3 COCH3

1470 CF3 H CF3 H H

1471 CF3 H CF3 H COCH3

CF3 H CF3 CH3 H

CF3 H CF3 CH3 COCH3

H OH H H H

H OH H H COCH3

1476 H OH H CH3 H

1477 H OH H CH3 COCH3

H H H H

H H H COCH3

H H CH3 H

H H CH3 COCH3

H CF3 H H H

H CF3 H H COCH3

H CF3 H CH3 H

EXAMPLE Rll6 _R 117 118 E NO.

1485 H CF3 H CH3 COCH3

1486 OH OH H H H

1487 QH OH H H COCH3

1488 OH OH H CH3 H

1489 OH OH H CH3 COCH3

1490 H H ^' H

1491 H H CCCH3

1492 H

1493 H

1494 CF3 H

1495 CF3 H

1496 CF3 H

1497 C 3 H

The following Exanples #1498-#1857 of Table XVII are highly preferred conjugates composed of dopamine-β-hydroxylase inhibitor compounds and glutamic acid derivatives. These dopamine-β-hydroxylase inhibitors utilized to make these conjugates are embraced by generic Formula XVIII, above.

TAB E XVII

I EXAMPLE _R 116 _R 117 .118 E NO .

NHNH H OH H H H

NHNH H OH H H COCH3

NHNH H OH H CH3 H

NHNH H OH H CH3 COCH3

NHNH H H H H

NHNH H H H COCH3

NHNH H H CH3 H

NHNH H H CH3 COCH3

NHNH H CF3 H H H

NHNH H CF ₃ H H COCH3

NHNH H CF3 H CH3 H

EXAMPLE n _R 116 _R 117 _R 118 E NO.

1509 NHNH H CF ₃ H* CH3 COCH3

1510 NHNH ^' CH QH H H H

1511 NHNH QH QH H H COCH3

1512 NHNH OH QH H CH3 H

1513 NHNH OH OH H CH3 COCH3

1514 NHNH H H H

1515 NHNH H H COCH3

1516 NHNH H CH3 H

1517 NHNH H CH3 COCH3

1518 NHNH CF3 H CF3 H H

1519 NHNH CF3 H CF3 H COCH3

1520 NHNH CF3 H CF3 CH3 H

1521 NHNH CF3 H CF3 CH3 COCH3

1522 0 NHCH2CH2NH H CH H H H

1523 0 NHCH ₂ CH ₂ NH _H OH H H COCH3

1524 0 NHCH2CH2NH H OH H CH3 H

1525 0 NHCH2CH2NH H CH H CH3 COCH3

1526 0 NHCH2CH2NH H H H H

1527 0 NHCH2CH2NH H H H COCH3

1528 0 NHCH2CH2NH H H CH3 H

1529 0 NHCH2CH2NH H H CH ₃ COCH3

1530 0 NHCH2CH2NH- H CF3 H H H

1531 0 NHCH2CH2NH H CF3 H H COCH3

1532 0 NHCH2CH2NH H CF3 H CH3 H

1533 0 NHCH2CH2NH H CF3 H CH3 COCH3

1534 0 NHCH2CH2NH OH OH H H H

1535 0 NHCH2CH2NH OH OH H H COCH3

1536 0 NHCH2CH2NH OH OH H CH3 H

1537 0 NHCH2CH2NH OH OH H CH ₃ COCH3

1538 0 NHCH2CH2NH F H H H

1539 0 NHCH2CH2NH F H H COCH3

1540 0 NHCH2CH2NH F H CH3 H

IEXAMPLE n ,116 •117 Rll ^β E NO.

1541 0 NHCH2CH2NH H CH3 COCH3

1542 0 NHCH2CH2NH CF3 H CF3 H H

1543 0 NHCH2CH2NH CF3 H CF3 H COCH3

1544 0 NHCH2CH2NH CF3 H CF3 CH3 H

1545 0 NHCH2CH2NH CF3 H CF3 CH3 COCH3

1546 0 piperazinyl H CH H H H

1547 0 piperazinyl H OH H H . COCH3

1548 0 piperazinyl H OH H CH3 H

1549 0 piperazinyl H OH H CH3 COCH3

1550 0 piperazinyl H H H H

1551 0 piperazinyl H H H COCH3

1552 0 piperazinyl H H CH3 H

1553 0 piperazinyl H H CH3 COCH3

1554 0 piperazinyl H CF3 H H H

1555 0 piperazinyl H CF3 H H COCH3

1556 0 piperazinyl H CF3 H CH3 H

1557 0 piperazinyl H CF ₃ H CH3 COCH3

1558 0 piperazinyl OH QH H H H

1559 0 piperazinyl CH QH H H COCH3

1560 0 piperazinyl CH QH H CH3 H

1561 0 piperazinyl CH OH H CH3 COCH3

1562 0 piperazinyl F H H H

1563 0 piperazinyl F H H COCH3

1564 0 piperazinyl F H CH3 H

1565 0 piperazinyl F H CH3 COCH3

1566 0 piperazinyl CF3 H CF3 H H

1567 0 piperazinyl CF3 H CF3 H COCH3

1568 0 piperazinyl CF3 H CF3 CH3 H

1569 0 piperazinyl CF3 H CF3 CH3 COCH3

1570 NHNH H CH H H H

1571 1 NHNH H OH H H COCH3

1572 NHNH H OH H CH3 H

1573 1 NHNH H QH H ^{• "'} CH3 COCH3

1574 1 NHNH H H H H

1575 1 NHNH H H H COCH3

1576 1 NHNH H H CH3 H

1577 1 NHNH H H CH3 COCH3

1578 NHNH H CF ₃ H H H

NHNH H CF3 H H COCH3

NHNH H CF3 H CH3 H

NHNH H CF ₃ H CH3 COCH3

NHNH CH CH H ^" H H

NHNH CH OH H H COCH3

NHNH CH OH H CH3 H

NHNH CH OH H CH3 COCH3

NHNH H F H H

NHNH H F H COCH3

NHNH H F CH3 H

1589 NHNH H CH3 COCH3

1590 1 NHNH CF3 H CF3 H H

1591 NHNH CF3 H CF3 H COCH3

1592 NHNH CF3 H CF3 CH3 H

1593 NHNH CF3 H CF3 CH3 COCH3

1594 1 NHCH2CH2NH H OH H H H

1595 1 NHCH2CH2NH H OH H H COCH3

1596 1 NHCH2CH2NH H OH H CH3 H

1597 1 NHCH2CH2NH H OH H CH3 COCH3

1598 1 NHCH2CH2NH H H- H H

1599 1 NHCH2CH2NH H H H COCH3

1600 1 NHCH2CH2NH H H CH3 H

1601 1 NHCH2CH2NH H H CH3 COCH3

1602 1 NHCH2CH2NH H CF3 H H H

1603 1 NHCH2CH2NH H CF3 H H COCH3

1604 1 NHCH2CH2NH H CF3 H CH3 H

EXAMPLE Rll ^β (117 ,118 E NO.

1605 1 NHCH2CH2NH H CF3 ^' H CH3 COCH3

1606 1 NHCH2CH2NH QH CH H H H

1607 1 NHCH2CH2NH CH QH H H COCH3

1608 1 NHCH2CH2NH CH QH H CH3 H

1609 1 NHCH2CH2NH CH QH H CH3 COCH3

1610 1 NHCH2CH2NH H H H

1611 1 NHCH2CH2NH H H COCH3

1612 1 NHCH2CH2NH H CH3 H

1613 1 NHCH2CH2NH H CH3 COCH3

1614 1 NHCH2CH2NH CF3 H CF3 H H

1615 1 NHCH2CH2NH CF3 H CF3 H COCH3

1616 1 NHCH2CH2NH CF3 H CF3 CH3 H

1617 1 NHCH2CH2NH CF3 H CF3 CH3 COCH3

1618 1 piperazinyl H QH H H H

1619 1 piperazinyl H QH H H COCH3

1620 1 piperazinyl H OH H CH3 H

1621 1 piperazinyl H CH H CH3 COCH3

1622 1 piperazinyl H H H H

1623 1 piperazinyl H H H COCH3

1624 1 piperazinyl H H CH3 H

1625 1 piperazinyl H H CH3 COCH3

1626 1 piperazinyl H CF3 H H H

1627 1 piperazinyl H CF3 H H COCH3

1628 1 piperazinyl H CF3 H CH3 H

1629 1 piperazinyl H CF3 H CH ₃ COCH3

1630 1 piperazinyl CH CH H H H

1631 1 piperazinyl CH . CH H H COCH3

1632 1 piperazinyl CH CH H CH3 H

1633 1 piperazinyl OH OH H CH ₃ COCH3

1634 1 piperazinyl F H H H

1635 1 piperazinyl F H H COCH3

1636 1 piperazinyl F H CH3 H

1637 1 piperazinyl F H CH3 COCH3

1638 1 piperazinyl CF3 H CF3 H H

1639 1 piperazinyl CF3 H CF3 H COCH3

1640 1 piperazinyl CF3 H CF3 CH3 H

1641 1 piperazinyl CF3 H CF3 CH3 COCH3

1642 NHNH H QH H H H

1643 2 NHNH H QH H H COCH3

NHNH H QH H CH3 H

NHNH H QH H CH3 COCH3

NHNH H H H H

NHNH H H H COCH3

NHNH H H CH3 H

1649 NHNH H H CH3 COCH3

1650 NHNH H CF3 H H H

1651 NHNH H CF3 H H COCH3

1652 NHNH H CF3 H CH3 H

1653 NHNH H CF3 H CH3 CCCH3

R116 R117 R118

NHNH CH OH H H H

NHNH CH OH H H COCH3

NHNH OH OH H CH3 H

NHNH OH OH H CH3 COCH3

NHNH H H H

NHNH H H CCCH3

NHNH H CH3 H

NHNH H CH3 COCH3

NHNH CF3 H CF3 H H

NHNH CF3 H CF3 H COCH3

NHNH CF3 H CF3 CH3 H

NHNH CF3 H CF3 CH3 COCH3

1666 2 NHCH2CH2NH H OH H H H

1667 2 NHCH2CH2NH H CH H H COCH3

1668 2 NHCH2CH2NH H CH H CH3 H

1669 2 NHCH2CH2NH H CH H CH3 COCH3

(IEEXXAAMftPLE n L Rllβ _R 117 R118 _E NO.

1670 2 NHCH2CH2NH H H H H

1671 2 NHCH2CH2NH H H H COCH3

1672 2 NHCH2CH2NH H H CH3 H

1673 2 NHCH2CH2NH H H CH3 COCH3

1674 2 NHCH2CH2NH H CF3 H H H

1675 2 NHCH2CH2NH H CF3 H H COCH3

1676 2 NHCH2CH2NH H CF3 H CH3 H

1677 2 NHCH2CH2NH H CF3 H CH3 COCH3

1678 2 NHCH2CH2NH CH OH H H H

1679 2 NHCH2CH2NH OH OH H H COCH3

1680 2 NHCH2CH2NH OH QH H CH3 H

1681 2 NHCH2CH2NH QH QH H CH3 COCH3

1682 2 NHCH2CH2NH F H H H

1683 2 NHCH2CH2NH F H H COCH3

1684 2 NHCH2CH2NH F H CH3 H

1685 2 NHCH ₂ CH ₂ NH F H CH3 COCH3

1686 2 NHCH2CH2NH CF3 H CF3 H H

1687 2 NHCH2CH2NH CF3 , H CF3 H COCH3

1688 2 NHCH2CH2NH CF3 H CF3 CH3 H

1689 2 NHCH2CH2NH CF3 H CF3 CH3 COCH3

1690 2 piperazinyl H OH H H H

1691 2 piperazinyl H OH H H COCH3

1692 2 piperazinyl H CH H CH ₃ H

1693 2 piperazinyl H OH H CH ₃ COCH3

1694 2 piperazinyl H F H H H

1695 2 piperazinyl H F H H COCH3

1696 2 piperazinyl H F H CH3 H

1697 2 piperazinyl H F H CH3 COCH3

1698 2 piperazinyl H CF3 H H H

1699 2 piperazinyl H CF3 H H COCH3

1700 2 piperazinyl H CF3 H CH3 H

1701 2 piperazinyl H CF3 H CH3 COCH3

1702 2 piperazinyl CH CH H H H

1703 2 piperazinyl CH CH H H COCH3

1704 2 piperazinyl QH CH H CH3 H

1705 2 piperazinyl CH CH H CH3 COCH3

1706 2 piperazinyl F H F H H

1707 2 piperazinyl F H F H COCH3

1708 2 piperazinyl F H F CH3 H

1709 2 piperazinyl F H F CH3 COCH3

1710 2 piperazinyl CF3 H CF3 H H

1711 2 piperazinyl CF3 H CF3 H COCH3

1712 2 piperazinyl CF3 H CF3 CH3 H

1713 2 piperazinyl CF3 H CF3 CH3 COCH3

1714 3 NHNH H OH H H H

1715 3 NHNH H OH H H COCH3

1716 3 NHNH H QH H CH3 H

1717 3 NHNH H OH H CH3 COCH3

I EXAMPLE Rllβ .117 Rll ^β E NO.

NHNH H H H H

NHNH H H H COCH3

NHNH H H CH3 H

1721 NHNH H H CH3 COCH3

1722 NHNH H CF3 H H H

1723 NHNH H CF3 H H COCH3

NHNH H CF3 H CH3 H

NHNH H CF3 H CH3 COCH3

NHNH OH CH H H H

NHNH OH OH H H COCH3

NHNH OH OH H CH3 H

NHNH OH OH H CH3 COCH3

NHNH H H H

NHNH H H COCH3

NHNH H CH3 H

NHNH H CH3 COCH3

1734 NHNH CF3 H CF3 H H

1735 NHNH CF ₃ H CF3 H COCH3

1736 NHNH CF ₃ H CF3 CH3 H

1737 NHNH CF3 H CF3 CH3 CCCH3

1738 3 NHCH2CH2NH H OH H H H

1739 3 NHCH2CH2NH H OH H H COCH3

1740 3 NHCH2CH2NH H OH H CH3 H

1741 3 NHCH2CH2NH H OH H CH3 COCH3

1742 3 NHCH2CH2NH H H H H

1743 3 NHCH2CH2NH H H H COCH3

1744 3 NHCH2CH2NH H H CH3 H

1745 3 NHCH2CH2NH H H CH3 COCH3

1746 3 NHCH2CH2NH H CF3 H H H

1747 3 NHCH2CH2NH H CF3 H H COCH3

1748 3 NHCH2CH2NH H CF3 H CH3 H

1749 3 NHCH2CH2NH H CF3 H CH3 COCH3

EXAMPLE _R llβ _R 117 _R 118 E NO.

1750 3 NHCH2CH2NH CH OH H H H

1751 3 NHCH2CH2NH ^' OH CH H H COCH3

1752 3 NHCH2CH2NH CH OH H CH3 H

1753 3 NHCH2CH2NH CH OH H CH3 COCH3

1754 3 NHCH2CH2NH F H H H

1755 3 NHCH2CH2NH F H H COCH3

1756 3 NHCH2CH2NH F H CH3 H

1757 3 NHCH2CH2NH F H CH3 COCH3

1758 3 NHCH2CH2 H CF3 H CF3 H H

1759 3 NHCH2CH2NH CF3 H CF3 H COCH3

1760 3 NHCH2CH2NH CF3 H CF3 CH3 H

1761 3 NHCH2CH2NH CF3 H CF3 CH3 COCH3

1762 3 piperazinyl H OH H H H

1763 3 piperazinyl H OH H H COCH3

1764 3 piperazinyl H QH H CH3 H

1765 3 piperazinyl H QH H CH3 COCH3

1766 3 piperazinyl H F ^" H- ' H H

1767 3 piperazinyl H F H H COCH3

1768 3 piperazinyl H F H CH3 H

1769 3 piperazinyl H F H CH3 COCH3

1770 3 piperazinyl H CF3 H H H

1771 3 piperazinyl H CF3 H H COCH3

1772 3 piperazinyl H CF3 H CH3 H

1773 3 piperazinyl H CF3 H CH3 COCH3

1774 3 piperazinyl QH QH H H H

1775 3 piperazinyl OH OH H ^" H COCH3

1776 3 piperazinyl OH OH H CH3 H

1777 3 piperazinyl OH OH H CH3 COCH3

1778 3 piperazinyl F H F H H

1779 3 piperazinyl F H F H COCH3

1780 3 piperazinyl F H F CH3 H

1781 3 piperazinyl F H F CH3 COCH3

H 1EEXXAAKMPLE Rllβ R117 R118 E NO.

1782 3 piperazinyl CF3 H CF3 H H

1783 3 piperazinyl CF3 H CF3 H COCH3

1784 3 piperazinyl CF3 H CF3 CH3 H

1785 3 piperazinyl CF3 H CF3 CH3 COCH3

1786 NHNH H OH H H H

NHNH H OH H H COCH3

NHNH H OH H CH3 H

NHNH H QH H CH3 COCH3

NHNH H H H H

NHNH H H H COCH3

NHNH H H CH3 H

NHNH H H CH3 COCH3

NHNH H CF3 H H H

NHNH H CF3 H H COCH3

NHNH H CF3 H CH3 H

NHNH H CF3 H CH3 COCH3

1798 NHNH CH QH - H H H

1799 NHNH CH QH H H COCH3

1800 NHNH CH OH H CH ₃ H

1801 NHNH CH OH H CH3 COCH3

1802 NHNH H H H

1803 NHNH H H COCH3

1804 NHNH H CH3 H

1805 NHNH H CH3 COCH3

1806 4 NHNH CF3 H CF3 H H

1807 NHNH CF ₃ H CF3 H COCH3

1808 NHNH CF ₃ H CF3 CH3 H

1809 NHNH CF3 H CF3 CH3 COCH3

1810 4 NHCH2CH2NH H QH H H H

1811 4 NHCH2CH2NH _H QH H H COCH3

1812 4 NHCH2CH2NH H OH H CH3 H

1813 4 NHCH2CH2NH H CH H CH3 COCH3

EXAMPLE Rllβ » 117 Rll ^β E NO .

1814 4 NHCH2CH2NH H H H H

1815 4 NHCH2CH2NH H H H COCH3

1816 4 NHCH2CH2NH H H CH ₃ H

1817 4 NHCH2CH2NH H H CH ₃ COCH3

1818 4 NHCH2CH2NH H CF3 H H H

1819 4 NHCH2CH2NH H CF3 H H COCH3

1820 4 NHCH2CH2NH H CF3 H CH3 H

1821 4 NHCH2CH2NH H CF3 H CH3 COCH 3

1822 4 NHCH2CH2NH OH OH H H H

1823 4 NHCH2CH2NH CH OH H H COCH 3

1824 4 NHCH2CH2NH OH OH H CH3 H

1825 4 NHCH2CH2NH OH OH H CH3 COCH 3

1826 4 NHCH2CH2NH F H H H

1827 4 NHCH2CH2NH F H H COCH3

1828 4 NHCH2CH2NH F H CH3 H

1829 4 NHCH2CH2NH F H CH3 COCH3

1830 4 NHCH2CH2NH CF3 H - CF3 H H

1831 4 NHCH2CH2NH CF3 H CF3 H COCH3

1832 4 NHCH2CH2NH CF3 H CF3 CH3 H

1833 4 NHCH2CH2NH CF3 H CF3 CH3 COCH3

1834 4 piperazinyl H QH H H H

1835 4 piperazinyl H OH H H COCH3

1836 4 piperazinyl H QH H CH3 H

1837 4 piperazinyl H OH H CH3 COCH3

1838 4 piperazinyl H F H H H

1839 4 piperazinyl H F H ^• H COCH3

1840 4 piperazinyl H F H CH3 H

1841 4 piperazinyl . H F H CH3 COCH3

1842 4 piperazinyl H CF3 H H H

1843 4 piperazinyl H CF3 H H COCH3

1844 4 piperazinyl H CF3 H CH3 H

1845 4 piperazinyl H CF3 H CH3 COCH3

EXAMPLE _R llβ Rll7 ,118 NO .

1846 4 piperazinyl QH OH H H H

1847 4 piperazinyl OH OH H H CCCH3

1848 4 piperazinyl QH OH H CH ₃ H

1849 4 piperazinyl OH OH H CH3 COCH3

1850 4 piperazinyl F H H H

1851 4 piperazinyl H H COCH3

1852 4 piperazinyl H CH3 H

1853 4 piperazinyl H CH3 COCH3

1854 4 piperazinyl CF3 H CF3 H H

1855 4 piperazinyl CF3 H CF3 H COCH3

1856 4 piperazinyl CF3 H CF3 CH3 H

1857 4 piperazinyl CF3 H CF3 CH3 COCH3

Rτnτ,nπτr.ΑT, KVΆLTTATION

Conjugates of the invention were evaluated biologically by in vitro and in vivo assays to determine the ability of the conjugates to selectively inhibit renal sympathetic nerve activity and lower blood pressure. Three classes of conjugates of the invention were evaluated for their ability to inhibit the enzymes of the catecholamine cascade selectively within the kidney. These inhibitor conjugates variously inhibit tyrosine hydroxylase, dopa- decarboxylase and dopamine-β-hydroxylase in order to interfere ultimately with the synthesis of norepinephrine in the kidney.

Assays I and II evaluate in vivo the acute and chronic effects of Ex. #3 conjugate (a tyrosine hydroxylase inhibitor conjugated with N-acetyl-γ-glutamyl) in rats. Assay III evaluates the chronic effects of Ex. #464 conjugate (a dopa-decarboxylase inhibitor conjugated with N-acetyl-γ-glutamyl) in rats.

Assay IV and V describes in vitro experiments performed to determine if the Ex. #859 conjugate was capable of being specifically metabolized by enzymes known to be abundant in the kidney. In Assay IV, the Ex. #859 conjugate was incubated with either rat kidney homogenate or a solution containing purified kidney enzymes to characterize resulting metabolites. In Assay V, experiments were performed to determine the potency of the Ex. #858 and Ex. #859 conjugates and potential metabolites as inhibitors of purified dopamine-β-hydroxylase.

Assays VI through IX describe in vivo experiments performed to characterize and compare the effects of fusaric acid and various conjugates of fusaric acid (Ex. #859, Ex.

#861 and Ex. #863) on spontaneously hypertensive rats (SHR) by

acute administration i.v. and i.d. and by chronic administration i.v. Assay X describes analysis of catecholamine levels in tissue from rats used in the chronic administration experiment of Assay VIII. Assays XI and XII describe in vivo experiments in dogs to determine the renal and mean arterial pressure effects of fusaric acid and Ex. #859 conjugate.

Assay I; Acute In Vivo Effaπ s of Ex. #3 nn-hiςrat

Sprague-Dawley rats were anesthetized with inactin (100 mg/kg, i.p.) and catheters were implanted into a carotid artery for measurement of mean arterial pressure (Gould model 3800 chart recorder; Statham pressure transducer model no. P23DB) and into a jugular vein for compound administrations (i.v.) . In addition, a flow probe was implanted around the left renal artery for measurement of renal blood flow using Carolina Medical Electronics flow probes. Rats were allowed 60 min to stabilize before 10 minutes of control recordings of mean arterial pressure and renal blood flow were obtained. Control measurements were followed by intravenous injection of Ex. #3 conjugate and saline vehicle. As shown in Table XVIII and in Figs. 1 and 2, the Ex. #3 conjugate had no acute effects on mean arterial pressure (MAP) , but increased renal blood flow (RBF) .

TABLE XVIII

Acute In Vivo Effects of Ex. #3 Conjugate

Time After Injection (min) Zero 15 20 45. _s

Vehicle (0.5 ml 0.9% NaCl i.v.)

MA (mm Hg) 78 76 75 80 82 RBF (ml/min) 4.9 4.5 4.2 4.6 4.7

Ex . #3 Conjugate (100 mg/kg i . v . ) „

MAP (mm Hg) 76+5 77 +5 73+4 70±2 71±6

RBF (ml/min) 4.8±0.8 7.1 +0.1 6.2 +0.3 5.9 +0.1 5.9+0.1

Assay II; Chronic In Vivo Effects of Ex. #3 Conjugate

The Ex. #3 conjugate and saline vehicle were infused continuously for four days in spontaneously hypertensive rats. Mean arterial pressure was measured (Gould Chart Recorder, model 3800; Statham P23Db pressure transducer) via an indwelling femoral artery catheter between 10:00 a.m. and 2:00 p.m. each day. The Ex. #3 conjugate was infused at 5 mg/hr and the saline vehicle was infused at 300 μL/hr. via a jugular vein catheter with a Harvard infusion pump. Results are shown in Table XIX.

TABLE XIX

Chron c Tn Vi vo ec s of Ex . #3 Conjugate

Time After Injec i on (days)

Zero 1 2 3 4

Vehicle (300 μf./hri

MAP (mm Hg) 181 +8 172±6 170 +7 174 + 6 182 +3

Ex. #3 Conjugate (F> ma/hr)

MAP (mm Hg) 164+3 175+5 174+5 172+2 N.A.

ASSay ITT; Chronic Tn Vivo Effects of Ex. -#464 on u a e

The Ex. #464 conjugate and saline vehicle were infused continuously for four days in spontaneously hypertensive rats. Mean arterial pressure was measured (Gould Chart Recorder, model 3800; Statham P23Db pressure transducer) via an indwelling femoral artery catheter between 10:00 a.m. and 2:00 p.m. each day. The Ex. #464 conjugate was infused at 10 mg/hr and the saline vehicle was infused at 300 μL/hr. As shown in Table XX and in Fig. 3, mean arterial pressure was lowered significantly over the four-day period.

TABLE XX

Chronic Tn Vivo Effects of Ex. #464 Conjugate

Time After Injection (days)

Zero 1 2 3 4

Vehicle (3QQ UL/hr)

MAP (mm Hg) 181 +8 172 + 6 170±7 174 +6 182±3

Ex . #464 Conjugate (1Q mg/hr)

MAP (mm Hg) 179±6 169+5 161 +4 163 +5 159 +8

Assay IV; In Vitro Evaluation of Enzyme Metabolism Effects of Ex. #85Q Conjugate

A freshly excised rat kidney was homogenized in 10 ml cold buffer (100 mM Tris, 15mM glycylglycine, pH 7.4) with a Polytron Tissue Homogenizer (Brinkmann) . The resulting suspension, diluted with buffer, was incubated in the presence of the Ex. #859 conjugate at 37°C. At various times aliquots were removed, deproteinized with an equal volume of cold trichloroacetic acid (25%) and centrifuged. The supernatant was injected onto a C-18 reverse-phase HPLC column and eluted isocratically with a mixture of acetonitrile and water (20:80 v/v) containing trifluoroacetic acid (0.05%). Eluted compounds were monitored by absorbance at 254 nm and compared to standards run under identical conditions. In the assay using pure kidney enzyme homogenate,, the Ex. #859 conjugate was also

incubated under the same conditions as described except that 5 mg of gamma-glutamyl transpeptidase (Sigma, 23 units/mg) and 10 mg.of acylase I (Sigma, 4800 units/mg) were added in place of the homogenate. Analysis by HPLC was performed in a manner identical to that used for the kidney homogenate experiment. Following incubation of the

Ex. #859 conjugate with kidney homogenate, there was a linear increase in the amount of fusaric acid liberated, as shown in Figure 4. No fusaric acid hydrazide or gamma- glutamyl fusaric acid hydrazide was observed; nor was any metabolism observed in the buffer control incubations. These data (Table XXI, Figure 4) show that renal tissue is able to metabolize the Ex. #859 conjugate to fusaric acid, which then remains stable under these conditions. Data from experiments using the purified enzymes show results similar to those seen for the kidney homogenate experiment, with only fusaric acid and the unreacted compound being present (see Table XXII, Figure 5) .

TABLE XXT

Formation of Fusaric Acid From the Ex. #859

Conjugate Incubated with Kidney Homogenate

Time (hrs.) : 0.00 0.17 1.25 17.00 41.00

Fusaric

Acid (μg/ml): 0.00 0.27 0.57 2.37 5.94

TABLE XXII

Formation of Fusaric Acid From Ex. #859 Conjugate Incubated with Puri ied Transpeptidase and Acvlase

Time (hrs.) : 3 24 72 96 .120

Fusaric

Acid (μg/ml): 0.00 2.56 12.15 15.44 18.75 @ pH 7.4

Fusaric Acid (μg/ml): 0.00 1.12 4.46 5.22 6.55 6 pH 8.1

Assay V; Tn Vitro Evaluation of DBH Inhibition bv Ex. #859 Conjugate

In order to characterize the relative patency of the Ex. #859 conjugate and its various potential metabolites as inhibitors of dopamine beta-hydroxylase (DBH; EC 1.14.17.1), the enzyme activity was determined in vitro in the presence of these compounds. DBH, purified from bovine adrenals (Sigma) was incubated at 37°C in buffer containing 20 mM dopamine as substrate. The reaction was stopped by addition of 0.5 M perchloric acid. The precipitate was removed and the product of the enzyme activity (norepinephrine) , contained in the clear supernatant, was analyzed by HPLC. The chromatographic separation used a reversed phase C-18 column run isocratically with 0.2 M ammonium acetate (pH 5.2) as the mobile phase. The amount of norepinephrine produced by the ^" enzyme-substrate mixture was analyzed by measuring the peak intensity (absorbance) at 280 nm for norepinephrine as it was eluted at 4.5 minutes, using a photo-diode array detector. The result of adding either fusaric acid or the Ex. #859 conjugate to the incubate at various concentrations is shown in Table XXIII and Figure 6. Above concentrations of 1 uM, fusaric acid inhibits the enzyme, while at concentrations up to 100 uM the Ex. #859 conjugate has no appreciable activity (Table XXIII and Figure 6) . Fusaric acid and Ex. #859 and two more possible metabolites (Ex #858 and fusaric acid hydrazide) were tested at 20 uM. Only fusaric acid had significant inhibitory effects on dopamine-β-hydroxylase activity (Table XXIV and Figure 7) .

TABLE XXIII DBH Inhibition by Fusaric Acid and the Ex. #859 Conjugate

Concentration ( μM) 0.01 0.10 0.50 1.00 5.00 10.00 50.00 100.00

Norepinephrine Peak Intensity (Abs 280) in the presence of Fusaric Acid: 0.59 0.59 0.60 0.53 0.25 0.14 0.00 0.00

Norepinephrine Peak Intensity (Abs 280) in the presence of Ex. #859 Conjugate 0.51 0.52 0.61 0.53

WO 91/01724 _ _QD PCT/US90/04168

298

compound administrations (i.v. or i.d.). In addition, a flow

25 probe was implanted around the left renal artery for measurement of renal blood flow using pulsed Doppler

renal blood flow to a greater degree than fusaric acid (Table XXV and Figures 8 and 9) . Similar results were found when these compounds were administered through a catheter implanted into the duodenum (i.d.) . The Ex. #859 conjugate had no effect on mean arterial pressure at a dose of 100 mg/kg (n=4) during a 60 minute observation period. Renal blood flow (n=4) was unchanged 15 minutes after injection of the Ex. #859 conjugate but increased from 1.1 KHz (control period) to 3.5 KHz at 30 minutes postinjection. Renal blood flow remained at this level for the following 30 minute observation period.

These data indicate that the Ex. #859 conjugate is active and displays renal selectivity whether administered i.d. or i.v. Results for Ex. #863 conjugate were similar to Ex. #859 and are shown in Table XXV : Ex. #863 had no effect on mean arterial pressure, but increased renal blood flow, indicating renal selectivity.

TABUS XXV

Acute Effects of Fusaric Acid and Ex. #859 conjugate on Blood

Pressure and Renal Blood Flow

Time (min) Zero 15 30 ^" 45 60

Fusaric Acid (5Qmg/kg i.v.)

MAP (mm Hg) 155 111 106 103 99 RBF (KHz) 2.5 3.1 3.2 3.4 3.9

Ex. #859 Conjugate (50 mg/kg i.v.)

MAP (mm Hg) 156 163 164 157 159 RBF (KHz) 2.4 3.8 4.0 4.6 4.8

Table YXVT

Acute Effects of Ex. #863 Conjugate

Time (min)

Zero 15 ^' 30 45 60

Ex. #863 nOO mg/kg i.v.)

N.A. - Not Available

Assay VTT: Comparison of Fusaric Acid. Fusaric Acid Hydrazide and Ex. #859 Conjugate on Arterial Pressure in Spontaneously Hypertensive Rats fSHRI

Mean arterial pressure effects of fusaric acid hydrazide (100 mg/kg, i.v.), fusaric acid (100 mg/kg, i.v.) and Ex. #859 conjugate (250 mg/kg, i.v.) are shown in Table XXVTI during a vehicle control period and 60 min post- injection of compound in anesthetized SHR. Rats were prepared as described above, minus the renal artery flow probe.

Tabl e YXVTT

coMPOTTNn SEEΩ 6Q MIN

Fusaric Acid (n=4) 164 + 10 m Hg 110 ± 21 mmHg

Fusaric Acid 159 + 8 mmHg 104 + 13 mmHg Hydrazide (n=4)

Ex. #859 Conjugate 151 + 9 mmHg 146 ± 15 mmHg (n=4)

The data show that the hypotensive effects of the fusaric acid hydrazide is similar to fusaric acid. The Ex. #859 conjugate had no effect on mean arterial pressure (Table XXV and Figure 8) .

Assay VIII; Chronic Tn Vivo Effects of Ex. #859 Conjugate

The Ex. #859 conjugate and saline vehicle were infused continuously for 5 days in SHR. Mean arterial pressure was measured (Gould Chart Recorder, model 3800; Statham P23Db pressure transducer) via an indwelling femoral artery catheter between 10:00 a.m. and 2:00 p.m. each day. The Ex. #859 conjugate (5 mg/hr), fusaric acid (2.5 mg/hr), and saline (100 μl/hr) were infused via a jugular vein catheter with a Harvard infusion pump. Compared to the control vehicle fusaric acid and the Ex. #859 conjugate lowered mean arterial pressure similarly. Mean arterial pressure did not change in the saline vehicle group. Results are shown in Table XXVIII.and Figure 10.

TABLE XXVTTT

Chronic Effects of Fusaric Acid and Ex. #859 Conjugate on Blood Pressure

Time (days) Zero 1 2 3 4 5

Vehicle (25 μT,/hr)

MAP (mm Hg) 139±2 139 +4 143±4 146+4 145±7 146±4

(SE)

Fusaric Acid (2.5 mg/hr)

MAP (mm Hg) 148±6 118±5 114±7 122+5 114±6 114+3

(SE)

Ex . #859 Conjugate (5 mg/hr)

MAP (mm Hg) 146±5 122 +9 115±9 119 +11 121±7 115±8 (SE)

Assay IX; Chronic Tn Vivo Effects of Ex. #861 and Ex. #863

Conjugates

The conjugates of Ex. #861 and #863 and saline vehicle were infused continuously for 4 days in spontaneously hypertensive rats. Mean arterial pressure was measured (Gould Chart Recorder, model 3800; Statham P23Db pressure transducer) via an indwelling femoral artery catheter between 10:00 a.m. and 2:00 p.m. each day. The Ex. #861 and Ex. #863 conjugates were infused at 5 mg/hr and the saline vehicle was infused at 100 μl/hr via a jugular vein catheter with a Harvard infusion pump. Results are shown in Table XXIX. The Ex. #863 conjugate lowered mean arterial pressure as shown in Fig. 11. Mean arterial pressure did not change for the Ex. #861 conjugate and the saline vehicle group (Table XXIX) . It is believed that at a higher dose of the Ex. #861 conjugate, blood pressure lowering effects would be observed.

TABLE XTX

Chronic Effects of Ex. #861 and Ex. #863 Conjugates on Blood Pressure

Time (days) Zero 1 2

Αssay X: Catechol mine Analysis of Tissue from Rats Treated with Ttx..#859 Conjugate

In order to evaluate the renal selectivity of DBH inhibition by the Ex. #859 conjugate, the catecholamine levels of heart and kidneys, both of which have been shown to be highly sensitive to DBH inhibition [Racz, K. et al., Euro ■ A . Pharmacol., Qi, 1 (1985)], were measured following chronic infusion of the Ex. #859 conjugate, fusaric acid and saline vehicle in rats. Following 5 days of infusion, the kidney was exposed through a small flank incision, made in the anesthetized rat, and the renal artery and vein were ligated. Following this the kidney was rapidly excised distal to the ligation and frozen in liquid nitrogen. Similarly, the heart was excised and frozen subsequent to the removal of both kidneys. The frozen tissues were stored in closed containers at -80°C. Tissue samples were thawed on ice and their weight recorded prior to being placed in a flat bottom tube. The cold extraction solvent (2 ml/g tissue) was then added and the sample was homogenized with a Polytron. Extraction Solvent: 0.1 M perchloric acid (3 ml of 70% PCA to 500 ml); 0.4 mM Na metabisulphite (38 mg/500 ml) . The volume was then measured and 0.05 ml of a 1-uM/L solution of dihydroxybenzylamine (DHBA) in extraction solvent was added for every 0.95 ml of homogenate to yield a 50 nM/L internal standard concentration. The homogenate was then mixed and centrifuged at °C, 3000 rpm for 35 minutes. A 2 ml aliquot of the supernatant was then neutralized by adding 0.5 ml of 2 M Tris, pH 8.8 and mixing. The sample was then placed on an alumina column (40 mg, Spe-ed CAT cartridge; Applied Separations; Bethlehem, PA) and the catecholamines were bound, washed and eluted using a vacuum manifold system (Adsorbex SPU, EM Science, Cherry Hill, NJ) operating at ca. 4 ml/min. until the column was dry. Washes of 1 ml H2O - 0.5 ml MeOH - 1 ml H2O were followed by elution with 1 ml

of extraction solvent. A 200 μl sample of the eluant was injected onto a C-18 reversed phase analytical HPLC column, 5 urn, 4.6 mm x 250 mm (e.g., Beckman #235335, LKB 2134-630 Spherisorb ODS-2) and eluted with a recycled mobile phase run at ambient temperature and a flow rate of 0.5 ml/min (ca. 75 bar) .

Mobile Phase: 0.02 M Na2HP0 ₄ in 75/25(v/v) H 0/MeOH 0.007% SDS pH 3.5 (cone. H3PO4) . The separated catecholamines were detected with a LKB 2143 electrochemical detector at a potential setting of 500 mV using a teflon flow cell spacer of 2.2 μl and a time constant of 2 sec. Peak heights were measured and recorded along with the chromatogram tracing using a Spectra-Physics 4270 integrator. Sample runs were preceded by injection of a mixture of calibration standards (200 ul) containing 50 nM/L of epinephrine (Epi) , norepinephrine (NE) , dopamine (DA) , and DHBA in extraction solvent. The peak heights for each sample run were corrected by dividing the peak height of the DHBA in the standard by the peak height of the DHBA in each saπple. The resulting factor (calculated for each sample) was used to correct for losses due to dilution, non-specific binding to the tissue precipitate, incomplete elution, etc. Concentrations were calculated by multiplying the peak heights for Epi, NE and DA by that samples correction factor and then dividing this value by the peak height of the respective standard. When this number is multiplied by the concentration of the standard (in this case 50 nM/L) the concentration of the catecholamine in the homogenate is obtained. This value is multiplied by the volume of the homogenate (determined previously) to get the total catecholamine content of the tissue expressed in moles/g tissue. The resolution and retention times for a mixture of standards run under the conditions described in the previous section are shown in Table XXX.

TABLE XXX

Retention Time (min.) Compound

12.10 3,4-dihydroxylphenylacetic acid (DOPAC)

18.24 norepinephrine (NE)

21.82 epinephrine (Epi)

23.19 homovanillic acid (HVA)

30.56 dihydroxybenzylamine (DHBA)

42.58 dopamine (DA)

The linear response to various standards run over a 100 fold concentration range was excellent with values for both the correlation coefficient (r) and the coefficient of determination (r-squared) being >.9999 for all standards, while the rank correlation (Spearman's rho) was 1.0. To confirm the precision and accuracy of the values, tissue analysis was performed on a control group of Sprague-Dawley rats. The cumulative results are within the range of values reported in the literature [(e.g. Racz, K. et al, J. Cardiovasc. Pharmacol.. £, 676 (1986) ] . The precision in the efficiency of extraction measured by the addition of an internal standard (DHBA) was also excellent with a fractional efficiency of 0.779(SE=.066) for the kidney extraction and 0.771 (SE=.083) for the heart extracts. Relative to vehicle administration, both the Ex. #859 conjugate and fusaric acid decreased kidney norepinephrine concentration; however, only fusaric acid decreased heart norepinephrine concentration (see Table XXXI and Figures 12 and 13) . These data indicate that the Ex. #859 conjugate is renal selective with chronic infusion.

TABLE XXVT

Effect of Fusaric Acid and Ex. #859 conjugate on Tissue Norepinephrine Concentrat on Following 5 Days of Infusion

Tissue : Kidney Heart

Vehicle (25 μL/hr)

Norepinephrine: 889(72) 2,248(164) (pMol/g) (SD)

Fusaric Acid (2.5 mg/hr)

Norepinephrine: 519(42) 862(147) (pMol/g) (SD)

Ex. #859 Conjugate (5 mg/hr)

Norepinephrine: 589(54) 2,444(534) (pMol/g) (SD)

ASSa XI: Intrarenal Admin tra ion of Fusaric Acid in

Anesthet zed Dogs

In one anesthetized dog, bolus doses of fusaric acid (0.1-5.0 mg/kg) were administered into the renal artery. Mean arterial pressure (MAP), renal blood flow (RBF) and urinary sodium excretion (UNa ^v ) were measured.

Bolus intrarenal injection of isotonic saline or 0.1 mg/kg of fusaric acid had no effect on any measure; however, 0.5, 1.0, and 5.0 mg/kg fusaric acid caused dose-related increases in renal blood flow, but had no significant effect on mean arterial pressure or urinary sodium excretion (see Table XXXII) .

TABLE XXXII

Effec of Intrarenal Injection of Fusaric Acid on Blood Pressure. Sodium Excretion and Renal Blood Flow in the Dog

Dose (mg/kg): Saline 0.1 0.5 1.0 5.0

Δ RBF(ml/min) : 0 0 +46 +58 +132

U _Na V(μEq/min) : 42.8 21.2 23.8 21.1 34.8

MAP(mm Hg) 136 136 136 138 140

Similar results were also found in a second experiment where non-depressor doses of fusaric acid were infused into the renal arteries of two dogs (see Table XXXIII) .

TABLE XXXITT

Effec of Intrarenal Tnfusion of Fusaric Acid on Blood Pressure. Sodium Excretion and Renal

Blood Flow in the Dog

Dog #1 Do #

Infusion: Fusaric Acid Fusaric Acid Saline (1.25 mg/kg/min) Saline (0.75mg/kg/min)

Δ RBF(ml/min) : 140 240 236 315

U _Na V(μEqlmin) : 95 82 44 13

MAP(mm Hg) 136 136 140 148

These data indicate that intrarenal administration of fusaric acid increases renal blood flow in anesthetized dogs without altering systemic mean arterial pressure.

A s y xTi: Acute In Vivo Effects of Ex. #859 Conjugate

This experiment was run to determine the renal selectivity of conjugate of the invention in dogs. Male mongrel dogs (15-20 kg/ n=8; Antech, Inc., Barnhard, MO) were anesthetized with sodium pentobarbital (30 mg/kg as i.v. bolus, and 4-6 mg/kg/hr infusion) and catheters were placed in the femoral veins for compound injection or pentobarbital infusion, and the femoral artery for arterial pressure recording. An electromagnetic flow probe (Carolina Medical Electronics, Inc., King, NC) was placed around the left renal artery for measurement of renal blood flow. Renal blood flow and arterial pressure were recorded on a Gould chart recorder. After surgery, 20-30 minutes were allowed for variables to stabilize. Then a 20 minute control measurement was followed by injection of Ex. #859 conjugate at doses of 20 and 60 mg/kg, i.v., to two different groups of dogs. Variables were monitored for the next three hours. Results are shown in Table XXXIV and Figures 14 and 15.

TABLE XXXIV

Renal Selectivity of Ex. #859 Conjugate in Dogs

Time After TnJection of Ex. #859 Conjugate

'Zero 1 Hour 2 Hour 3 Hour

Compositions __£_ the Invention

Also embraced within this invention is a class of pharmaceutical compositions comprising one or more conjugates described above in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants (collectively referred to herein as "carrier" materials) and, if desired, other active ingredients. The conjugates of the present invention may be _ administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. Thera¬ peutically effective doses of the conjugates of the present invention required to prevent or arrest the progress of the medical condition are readily ascertained by one of ordinary skill in the art. The conjugates and composition may, for example, be administered intravascularly, intraperitoneally, subcutaneously, intramuscularly or topically.

For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are tablets or capsules. These may with advantage contain an amount of active ingredient from about 1 to 250 mg, preferably from about 25 to 150 mg. A suitable daily dose for a human may vary widely depending on the condition of the patient and other factors. However, a dose of from about 0.1 to 3000 mg/kg body weight, particularly from about 1 to 100 mg/kg body weight, may be appropriate.

The active ingredient may also be administered by injection as a composition wherein, for example, saline.

dextrose solutions or water may be used as a suitable carrier. A suitable daily dose is from about 0.1 to 100 mg/kg body weight injected per day in multiple doses depending on the disease being treated.

A preferred daily dose would be from about 1 to 30 mg/kg body weight. Conjugates indicated for prophylactic therapy will preferably be administered in a daily dose generally in a range from about 0.1 mg to about 100 mg per kilogram of body weight per day. A more preferred dosage will be a range from about 1 mg to about 100 mg per kilogram of body weight. Most preferred is a dosage in a range from about 1 to about 50 mg per kilogram of body weight per day. A suitable dose can be administered, in multiple sub-doses per day. These sub-doses may be administered in unit dosage forms. Typically, a dose or sub-dose may contain from about 1 mg to about 100 mg of conjugate per unit dosage form. A more preferred dosage will contain from about 2 mg to about 50 mg of conjugate per unit dosage form. Most preferred is a dosage form containing from about 3 mg to about 25 mg of active compound per unit 'dose.

The dosage regimen for treating a disease condition with the conjugates and/or compositions of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex and medical condition of the patient, the severity of the disease, the route of administration, and the particular compound employed, and thus may vary widely.

For therapeutic purposes, the conjugates of this invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the conjugates may be admixed with lactose, sucrose, starch powder, cellulose

esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of conjugate in hydroxypropylmethyl cellulose. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration. The conjugates may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride solutions, and/or various buffer solutions. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. Appropriate dosages, in any given instance, of course depend upon the nature and severity of the condition treated, the route of administration, including the weight of the patient.

Representative carriers, diluents and adjuvants include for example, water, lactose, gelatin, starches, magnesium stearate, talc, vegetable oils, gums, polyalkylene glycols, petroleum jelly, etc. The pharmaceutical compositions may be made up in a solid form such as granules, powders or suppositories or in a liquid form such as solutions, suspensions or emulsions. The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

Jib

Although this invention has been described with respect to specific embodiments, the details of these embodiments are not to be construed as limitations. Various equivalents, changes and modifications may be made without departing from the spirit and scope of this invention, and it is understood that such equivalent embodiments are part of this invention.