RENAL-SELECTIVE PRODRUGS FOR THE TREATMENT
OF HYPERTENSION
Related Application
This application is a continuation-in-part of U.S. Application Ser. No. 07/386,527 filed 27 July 1989. Field of the 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 in 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. Physiol., 245, 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. J. 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 intermediates 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., 20, 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 sulfamethoxazole 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 in vitro by γ- glutamyl transpeptidase [S.D.J. Magnan et al, J. Med.
Chem., 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. Sci. 69, 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. J. 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 R1 through R3 is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxy, aryloxy, aralkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino,
monoalkylamino, dialkylamino, carboxyl, carboxyalkyl, alkanoyl, alkenyl, cycloalkenyl and alkynyl; wherein R4 selected from hydrido, alkyl, cycloalkyl, hydroxyalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, aralkyl, aryl, alkanoyl, alkoxycarbonyl, carboxyl, amino, cyanoamino, monoalkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfinyl and arylsulfonyl; wherein R5 is selected from -CR6 and
wherein R
6 is selected from hydrido, alkyl.
cycloalkyl, cycloalkylalkyl, aralkyl and aryl, and wherein each of R7 and R8 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 R9 through R13 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 R13 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, 1H-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, 2,1,3-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-[1,5-a]pyrid-7-yl;
and wherein A may be selected from
wherein each of R14 through R20 is independently selected from hydrido, alkyl, hydroxy, hydroxyalkyl, alkoxy, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, aryloxy, alkoxycarboxyl, aryl, aralkyl, cyano, cyanoalkyl, amino, monoalkylamino and dialkylamino, wherein each of R21 and R22 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:
wherein each of R
1 and R
2 is hydrido; wherein m is one or two; wherein R
3 is selected from alkyl, alkenyl and
alkynyl; wherein R4 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 R5 is selected from -OR6 and
wherein R6 is selected from
hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenalkyl and phenyl, and wherein each of R7 and R8 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 R9 through R13 is independently selected from hydrido, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxycarbonyl, alkoxycarbonyl, alkoxy, arykoxy, aralkoxy, alkpxyalkyl, haloalkyl, hydroxyalkyl, halo, cyano, amino, monoalkylamino, 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 R9 through R13 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, 1H-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-6-yl, 2,1,3-benzothiadiazol-5-yl, 1,3- dihydro-2,2-dioxo-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-1,4- benzoxazin-7-yl; wherein R3 is -CH=CH2 or -C≡CH; wherein R5 is selected from -OR6 and
wherein R6 is selected from
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-1-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-1H-indol-4-yl)-α-methylalanine;
α-methyl-3-(1H-2-oxindol-5-yl) alanine;
3-[1-(N-benzoylcarbamimidoyl)-2,3-dihydro-1Hindol-5-yl)-α-methylalanine;
3-(1-carbamimidoyl-2,3-dihydro-1H-indol-5-yl-α-methylalanine;
3-(1H-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-6-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-1,3-dimethylbenzo-2,1,3-thiadiazol-5yl-)-2-methylalanine 2,2-dioxide;
α-methyl-3-[4-methyl-2(1H)-oxoquinolin-6-yl] alanine;
3-[4-methyl-2(1H)-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-(1,4-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)phenylalanine;
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-methyl-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-dihydroxyphenylalanine;
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-dihydroxyphenylalanine
L-α-ethyl-β-4-trifluoromethyl-2,3-dihydroxyphenylalanine
L-α-propyl-β-4-trifluoromethyl-2,3-dihydroxyphenylalanine 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-dihydroxyphenyl alanine;
L-α-ethyl-β-4-trifluoromethyl-3,5-dihydroxyphenylalanine; L-α-propyl-β-4-trifluoromethyl-3,5-dihydroxyphenylalanine;
L-α-butyl-α-4-trifluoromethyl-3,5-dihydroxyphenylalanine;
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-dihydroxyphenyl alanine;
L-α-ethyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalanine; L-α-propyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalanine;
L-α-butyl-β-4-trifluoromethyl-2,5-dihydroxyphenylalanine;
L-α-methyl-β-3,4,5-trihydroxyphenylalanine;
L-α-ethyl-β-3,4,5-trihydroxyphenylalanine;
L-α-propyl-β-3,4,5-trihydroxyphenylalanine;
L-α-butyl-β-3,4,5-trihydroxyphenylalanine;
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-2,4,5-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-methylphenylalanine;
3-iodotyrosine;
3, 5-diiodotyrosine;
L-α-methylphenylalanine;
D,L-α-β-(4-hydroxy-3-methylphenyl) 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-cyclohexylphenyl) alanine;
D,L-α-β-(4-methoxy-3-methylphenyl) alanine;
D,L-α-β-(4-hydroxy-3-methylphenyl) alanine;
N,O-dibenzyloxycarbonyl-D,L-α-β-(4-hydroxy-3-methylphenyl) alanine;
N, O-dibenzyloxycarbonyl-D,L-α-β-(4-hydroxy-3-methylphenyl) alanine amide;
D,L-α-β-(4-hydroxy-3-methylphenyl) 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-methylphenylalanine;
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-methyl-β-(2,5-dihydroxyphenyl) alanine;
α-ethyl-β-(2,5-dimethoxyphenyl) alanine;
α-ethyl-β-(2,5-dihydroxyphenyl) alanine;
α-methyl-β-(2,4-dimethoxyphenyl) alanine;
α-methyl-β-(2,4-dihydroxyphenyl) alanine;
α-ethyl-β-(2,4-dimethoxyphenyl) alanine;
α-ethyl-β-(2,4-dihydroxyphenyl) 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-1-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 R10, R11 and R12 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-butyl-m-tyrosine;
Lα-p-trifluoromethyl-m-tyrosine;
L-3-iodotyrosine;
L-3-chlorotyrosine;
L-3,5-diiodotyrosinei
L-α-methyltyrosine;
D,L-α-methyltyrosine;
D,L-3-iodo-α-methyltyrosine;
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-hydroxyphenyl)propionic acid.
Another preferred class of tyrosine hydroxylase inhibitor compounds within Formula I consists of compounds
wherein R
3 is selected from alkyl, alkenyl and alkynyl;
wherein R4 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 R5 is selected from OR6 and
wherein R6 is selected from
hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenalkyl and phenyl, and wherein each of R7 and R8 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 R9 through R13 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 R10, R11 and R12 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
1 and R
2 is hydrido; wherein m is a number selected from zero through five, inclusive; 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 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, alkylsulfonyloxy, 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
wherein R6 is selected from
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 R23 and R24 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 R25 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 R26 through R35 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 R43 and R44 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 R43 and R44 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 R43 and R44 cannot both be carboxyl at the same time, and with the further proviso that at least one of R43 through R44 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 R42 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 R43 and R44 is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxyalkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl and alkanoyl; and wherein any R43 and R44 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 R36 through R42 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 R43 and R44 is independently selected from hydrido, alkyl, benzyl, phenyl, alkoxyalkyl, haloalkyl,
hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyl, carboxyalkyl and alkanoyl; and wherein any R43 and R44 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 R36 through R42 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 R43 and R44 is independently selected from hydrido, alkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl and carboxyalkyl; and wherein any R43 and R44 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 R36 and R37 is hydrido and n is one; wherein each of R38 through R42 is independently selected from hydroxy, alkyl, alkoxy, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl, carboxyalkyl, aminomethyl, carboxyalkoxy and formyl; wherein each of R43 and R44 is independently selected from hydrido, alkyl, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl and carboxyalkyl; and wherein any R43 and R44 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 R36 and R37 is independently selected from hydrido, alkyl and amino and n is two; wherein each of R38 through R42 is independently selected from hydroxy, alkyl, alkoxy, haloalkyl,
hydroxyalkyl, amino, monoalkylamino, carboxyl,
carboxyalkyl, aminomethyl, carboxyalkoxy and formyl;
wherein each of R43 and R44 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-dihydroxyphenyl) propionic acid (Carbidopa), α-(monofluoromethyl) 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
45 through R
48 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
wherein R
51 is selected from hydroxy, alkoxy,
aryloxy, aralkoxy, amino, monoalkylamino and dialkylamino with the proviso that R
49 and R
50 cannot both be carboxyl at the same time, and with the further proviso that at least one of R
45 through R
48 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
45 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
49 and R
50 is independently selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyalkyl and alkanoyl and
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 R45 through R48 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
50 s independently selected from hydrido, alkyl, benzyl, phenyl, alkoxyalkyl, haloalkyl, hydroxyalkyl, cyano, amino, monoalkylamino, dialkylamino, carboxyalkyl and alkanoyl and
wherein R
51 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 R45
through R48 is independently selected from hydrido, hydroxy, alkyl, alkoxy, haloalkyl, hydroxyalkyl, amino, monoalkylamino, carboxyl, carboxyalkyl aminomethyl, carboxyalkoxy and formyl; wherein each of R49 and R50 is independently selected from hydrido, alkyl, amino,
monoalkylamino, carboxyalkyl and
wherein R
51 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
45 through R
48 is independently selected from hydrido, hydroxy, alkyl, alkoxy and hydroxyalkyl; wherein each of R
49 and R
50 is independently selected from alkyl, amino, monoalkylamino, and
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; ethylendo-2-amino-1,2,3,4-tetra-hydro-1,4-ethano-naphthalene-2- carboxylate hydrochloride; exo-2-amino 1,2,3,4-tetrahydro- 1,4-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(4-ethylphenyl)-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-dihydroxyphenyl) lactate;
D,L-β-(5-hydroxyindolyl-3) lactate;
2,4-dihydroxy-5-(1-oxo-2-propenyl) benzoic acid;
2,4-dimethoxy-5-[1-oxo-3-(2,3,4-trimethoxyphenyl-2-propenyl]benzoic acid;
2,4-dihydroxy-5-[1-oxo-3-(2-thienyl)-2-propenyl] benzoic acid;
2,4-dihydroxy-5-[3-(4-hydroxyphenyl)-1-oxo-2-propenyl] benzoic acid;
5-[3-(4-chlorophenyl)-1-oxo-2-propenyl]-2,4-dihydroxy benzoic acid;
2,4-dihydroxy-5-(1-oxo-3-phenyl-2-propenyl) benzoic acid;
2,4-dimethoxy-5-[1-oxo-3-(4-pyridinyl)-2-propenyl] benzoic acid;
5-[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]-2,4 dimethoxy benzoic acid;
2,4-dimethoxy-5-(1-oxo-3-phenyl-2-propenyl) benzoic acid;
5-[3-(2-furanyl)-1-oxo-2-propenyl]-2,4-dimethoxy benzoic acid;
2,4-dimethoxy-5-[1-oxo-3-(2-thienyl)-2-propenyl] benzoic acid;
2,4-dimethoxy-5-[3-(4-methoxyphenyl)-1-oxo-2-propenyl] benzoic acid;
5-[3-(4-chlorophenyl)-1-oxo-2-propenyl]-2,4-dimethoxy benzoic acid; and
5-[3-[4-(dimethylamino)phenyl]-1-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
wherein R64is selected from
hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenalkyl and phenyl, and wherein each of R65 and R66 is independently selected from hydrido, alkyl, alkanoyl, amino,
monoalkylamino, dialkylamino, phenyl and phenalkyl; wherein each of R53, R54 and R57 through R63 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 R55 and R56 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 R52 is OR64 wherein R64
is selected from hydrido, alkyl, cycloalkyl, cycloalkylalkyl, benzyl and phenyl; wherein each of R53, R54 and R57 through R63 is independently selected from hydrido, alkyl, cycloalkyl, hydroxy, alkoxy, benzyl and phenyl; wherein each of R55 and R56 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 R52 is OR64 wherein R64 is selected from hydrido and lower alkyl;
wherein each of R53 through R58 is hydrido; wherein each of R59 through R63 is independently selected from hydrido, alkyl, hydroxy and alkoxy, with the proviso that two of the R59 through R63 substituents are hydroxy; wherein each of m 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-3hydroxyphenylpropionic 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-4',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-8-1H-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
68 is independently selected from hydrido, alkyl, alkenyl and alkynyl; wherein R
69 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 R67is 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 R67, R68 and R69 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'-thienyl)propene; 3-amino-2-(2'furanyl) propene; 3-amino-2- (3'-furanyl)propene; 1-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-(4'-hydroxyphenyl)-1-butyne; (±)4-amino3-phenyl-1-butene; (+) 4-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:
wherein W is selected from alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl and heteroaryl; wherein Y is selected from
wherein R
70 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 R70 is selected from hydrido, alkyl, amino, monoalkylamino, dialkylamino, phenyl and phenalkyl; wherein each of R71 and R72 is independently selected from hydrido, hydroxy, alkyl, phenalkyl, phenyl, alkoxy, benzyloxy, phenoxy, alkoxyalkyl, hydroxyalkyl, halo, amino,
‰onoalkylamino, 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 R70 is selected from hydrido, alkyl, amino and monoalkylamino; wherein each of R71 and R72 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 R70 is selected from hydrido, alkyl and amino; wherein each of R71 and R72 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 R70 is hydrido; wherein each of R71 and R72 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:
wherein E is selected from alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl and heteroaryl; wherein F is selected from
wherein Z is selected from O, S and N-R
78; 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 R75 and R76 may form oxo or thio; wherein r is a number selected from zero through six, inclusive; wherein each of R77 and R78 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 R82 through R85 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 R79 and R80 is independently selected from hydrido and alkyl;
wherein R81 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 R82 through R85 is independently selected from hydrido, alkyl and
haloalkyl; wherein Y is selected from oxygen atom or sulfur atom; wherein each of R79, R80 and R81 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;
aminomethyl-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)propyl-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) propyl-5-n-butylpicolinate;
2'-aminopropyl-5-n-butylthiopicolinate;
2'-aminopropyl-5-n-butylpicolinate;
4'-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
86, R
87 and R
90 through R
93 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 R86 and R87 together may form oxo or thio;
wherein r is a number selected from zero through six, inclusive; wherein each of R88 and R89 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 R90 through R93 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 R88 and R89 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 R86, R87 and R90 through R93 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 R88 and R89 is selected from hydrido, alkyl, amino and monoalkylamino. Most preferred are compounds wherein each of R90 through R93 is independently selected from hydrido and alkyl; wherein each of R86 and R87 is hydrido; wherein r is selected from zero, one and two; wherein R88 is selected from hydrido, alkyl and amino; and wherein R89 is selected from hydrido and alkyl.
Especially preferred within this class is the compound 5-n- 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
98 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
98 is
wherein A' is or wherein R
99 is selected
from hydrido, alkyl, hydroxy, alkoxy, alkylthio, phenyl, phenoxy, benzyl, benzyloxy,
-OR100 and wherein R100 is selected from
hydrido, alkyl, cycloalkyl, cycloalkylalkyl, phenyl and benzyl; each of R101' R102,R103 and R104 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 R98 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 R100 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
106 is 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
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 R105 is selected from hydroxy and lower alkoxy; wherein R106 is hydrido; wherein R107 is selected from hydrido and lower alkyl;
wherein R
108 is hydrido; wherein R
109 is selected from hydrido and
with R
110 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:
wherein R
111 is selected from hydroxy and lower alkyl;
wherein R107 is selected from hydrido and lower alkyl;
wherein R
109 is selected from hydrido and
with R
110 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 R111 is hydroxy; wherein R107 is hydrido or methyl; wherein R109 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-1- 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 R112 through R119 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 XVIII consists of those compounds wherein R112 is selected from mercapto and alkylthio; wherein each of R113 and R114 is independently selected from hydrido, amino, aminoalkyl, monoalkylamino, monoalkylaminoalkyl, carboxyl and carboxyalkyl; wherein each of R115 and R119 is hydrido; and wherein each of R116, R117 and R118 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 R112 is selected from amino, aminoalkyl, monoalkylamino,
monoalkylaminoalkyl, carboxy and carboxyalkyl; wherein each of R113, R114, R115 and R119 is hydrido; and wherein each of R116, R117 and R118 is independently selected from hydrido, hydroxy, alkyl, halo and haloalkyl; or a pharmaceutically-acceptable salt thereof. Compounds which fall within any of the aforementioned 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: 1-(3,5-dihaloaryl) imidazol-2-thione derivatives such as 1-(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 (designatedas "T").
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 R205 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.
A preferred class of linker groups within Formula IV is defined by Formula XXI:
wherein each of R
202 and R
203 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 R202 and R203 is selected from halo, hydroxy, amino, monoalkylamino and dialkylamino, then the carbon to which R202 or R203 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 R202 and R203 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 R202 and 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 and R203 is 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
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 AII 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").
Another class of suitable diamino terminal linker groups is defined by Formula XXII:
wherein each of R214 through R217 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 R
214 and R
215 is 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").
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 methythio 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
and
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 pharmaceuticallyacceptable salts of such conjugates including acid- addition salts and base addition salts. The term "pharmaceuticallyacceptable 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, diethanolamine, 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 O, 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 R79, R80, R81, R86, R87, R88, R89 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.
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 #1-#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.
4-amino-4-carboxy-4-oxobntyl-α-methyl-L-tyrosine, methyl ester.
Step. 1. Preparation of methyl α-methyl-L-tyrosinate, hydrochloride .
A solution of 11.0 g (56.4 mmol) of α-methyl-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-d6) 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, 1H).
Step. 2. Preparation of 4-amino-4-carhoxy-4-oxobntyl-α-methyl-L-tyrosine, methyl ester.
Under nitrogen, a solution of 35.1 g (116 mmol) of N-Boc-L-γ-glutanic acid-α-t-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 in 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 (MgSO4), and concentrated 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 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-d6) δ 1.20 (s, 3H), 1.90-2.20 (m, 2H), 2.23-2.38 (m, 2H) , 2.95 (d, J = 13 Hz, 1H), 3.26 (d, J = 13 Hz), 3.57 (s, 3H) , 3.92-4.06 (m, 1H), 7.06 (d, J = 9 Hz, 2H), 7.12 (d, J = 9 Hz, 2H) .
N-[4-(acetylamino)-4-carboxy-4-oxobutyl]-α-methyl-L-tyrosine, methyl ester. The compound of Example 1 was dissolved in 100 mL of water and the pH adjusted to 9 with 1 M K
2CO
3. The solution was cooled to 0°C and 3.30 mL (35 mmol) of acetic anhydride and 35 mL (35 mmol) of 1 M K
2CO
3 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 HCl 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-d
6) 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, 1H), 3.38 (d, J = 13 Hz, 1H), 3.57 (s, 3H), 4.12-4.23 (m, 1H), 7.02 (d, J. = 9 Hz, 2H) , 7.09 (d, J = 9 Hz, 2H), 8.06 (s, 1H), 8.12 (d, J = 8 Hz, 1H).
N-[4-(acetylamino)-4-carboxy-4-oxobutyl]-α-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-d6) 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, 1H), 3.14 (d, J = 13 Hz, 1H) , 4.12-4.23 (m, 1H), 6.56 (d, J = 9 Hz, 2H), 6.85 (d, J = 9 Hz, 2H) , 7.69 (s, 1H), 8.12 (d, J = 8 Hz, 1H); 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
C17H22N2O7·H2O·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.
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.
The following Examples #414-#461 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.
The following Examples #462-#857 comprise five classes of highly preferred conjugates composed of dopa-decarboxylase inhibitor compounds and glutamic acid derivatives. Examples #462-#464 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.
4-amino-4-carboxy-4-oxobutyl-3-hydroxy-α-methyl-L-tyrosine, methyl ester.
Step. 1: Preparation of α-methyl-L-DOPA, methyl ester,
hydrochloride.
A suspension of 29.7 g (141 mmol) of α-methyl-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-d6) δ 1.47 (s, 3H), 2.92 (d, J = 12 Hz, 1H), 2.98 (d, J = 12 Hz, 1H), 3.74 (s, 3H), 6.41 (d of d, J = 9 Hz AND 2 Hz, 1H), 6.54 (d, J = 2 Hz, 1H), 6.68 (d, J = 9 Hz, 1H), 8.46-8.90 (br s, 3H), 8.93 (s, 1H) , 8.96 (s, 1H).
Step 2: Preparation of 4-amino-4-carboxy-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-t-butyl ester (BACHEM) in 150 mL 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 1N citric acid, 1N NaHCC3, water, and brine, dried (Na2SO4), 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-d6) δ 1.40 (s, 3H), 1.85-2.30 (m, 2H),
2.30-2.50 (m, 2H), 2.77 (d, J = 12 Hz, 1H), 3.00 (d, J = 12 Hz, 1H), 3.58 (s, 3H), 3.85-4.10 (m, 1H) , 6.29 (d of d, J = 9 Hz and 2 Hz, 1H), 6.45 (d, J = 2 Hz, 1H) , 6.62 (d, J = 9 Hz, 1H) ; MS (FAB) m/e (rel intensity) 355 (92), 225 (51), 148 (35).
N- [4-(acetylamino)-4-carboxy-4-oxobutyl]-3-hydroxy-α-methyl-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 14.0g (49%) of colorless product: NMR (DMSO-d6) 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, 1H) , 3.00 (d, J = 12 Hz, 1H) , 3.55 (s, 3H), 4.10-4.22 (m, 1H) , 6.29 (d of d, J = 9 Hz and 2Hz, 1H), 6.43 (d, J = 2Hz, 1H) , 6.60 (d, J = 9 Hz, 1H) , 7.96 (s, 1H), 8.12 (d, J = 8 Hz, 1H); MS (FAB) m/e (rel intensity) 397 (100), 365 (10), 226 (70), 166 (90), 153 (22), 130 (72), 102 (28).
N-[4-(acetylamino)-4-carboxy-4-oxobutyl]-3-hydroxy-α-methyl-L-tyrosine.
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 1N NaOH (all solutions were degassed in 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-d6) 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, 1H), 3.05 (d, J = 12 Hz, 1H) , 4.10-4.23 (m, 1H), 6.31 (d of d, J = 9 Hz and 2 Hz, 1H) , 6.47 (d, J = 2 Hz, 1H), 6.60 (d, J = 9 Hz, 1H), 7.71 (s, 1H), 8.15 (d, J = 8 Hz, 1H); 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. Anal:
Calcd for C17H22N2O3·1.06 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.
The following Examples #542-#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.
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.
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.
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.
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.
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-XVII, may be prepared by procedures shown in these specific examples and in the foregoing general synthetic procedures of Schemes 1-7.
L-glutamic acid, 5-[(5-butyl-2-pyridinyl)carbonyl]-hydrazide Step. 1: Preparation of 5-n-Butylpicolinic (Fusaricl 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 SOCl2 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 SOCl2. The viscous syrup thus formed was dried in vacuo (0.01mm) overnight prior to treatment with cold NaHCO3(sat). The ester was extracted with ether and dried (MgSO4). 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, CH2CH3); 1.30-1.45 (m, 2H, CH2CH3); 1.55-1.70 (m, 2H, CH2CH2CH2); 2.67 (t, J = 7 Hz, 2H, ArCH2); 7.65 (d of d, J3,4 = 7 Hz and J4,6 = 2 Hz, 1H, ArH); 8.05 (d, J3,4 = 7 Hz, 1H, ArH); 8.37 (d, 1H, ArH, J4,6 = 2 Hz); HRMS. Calcd for M + H: 194.1270. Found: 194.1293. step 2: Preparation of L-glutamic acid, 5-[(5-butyl-2-pyridinyl) carbonyl]hydrazide.
A solution of 7.27 g (24.0 mmol) of Boc-L-γglutamic acid-α-t-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 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
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 g (46%). of colorless material: mp 162-164°C (dec); NMR (DMSO-d6) δ 1.90 (t, J = 7 Hz, 3H, CH2CH3); 1.30-1.45 (m, 2H, CH2CH3); 1.50-1.65 (m, 2H, CH2CH2CH2); 2.00-2.20 (m, 1H, CH2CH) ; 2.30-2.50 (m, 1H, CH2CH); 2.70 (t, J = 7 Hz, 2H, ArCH2); 3.60 (t, J = 7 Hz, 2H, COCH2); 3.95-4.05 (M, 1H, CH2CH); 7.85 (d of d, J3 ,4 = 7 Hz
and J4,6 = 2 Hz, 1H, ArH); 7.95 (d, J3,4 = 7 Hz, 1H, ArH); 8.55 (d, J4,6 = 2 Hz, 1H, ArH) .
N-acetyl-L-glutamic acid, 5-[(5-brutyl-2-pyridinyl)-carbonyl]hydrazide
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 1M 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 volitiles 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 (D2O and NaOD)δ 1.85 (t, J = 7 Hz, 3H, CH2CH3);
1.20-1.35 (m, 2H, CH2CH3); 1.55-1.70 (m, 2H, CH2CH2CH2); 1.95-2.10 (m, 1H, CH2CH); 2.05 (s, 3H, COCH3); 2.20-2.35 (m, 1H, CH2CH); 2.45 (t, J = 7 Hz, 2H, COCH2); 2.75 (t. 2H, ArCH2); 3.45-3.55 (m, 1H, CH2CH) ; 8.05 (s, 2H, ArH); 8.55 (s, 1H, ArH); HRMS. Calcd for M + H: 365.1825. Found 365.1860. Anal.
Calcd. for C17H24N4O5: C, 55.98; H, 6.58; N, 15.36. Found: C, 55.96; H, 6.64; N, 15.30.
N-[2-[[(5-butyl-2-pyridinyl) carbonyl] amino]ethyl]-L-glutamine. step 1: 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-d6) 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, 1H), 7.96 (d, J = 9 Hz, 1H), 8.47 (d, J = 2 Hz, 1H), 8.75 (t, J = 8 Hz, 1H). step 2: Preparation of N-[2-[[(5-frutyl-2-pyridinyl) carbonyl] aminolethyl]-L-gluatmine.
Under nitrogen, a solution of 26.8 g (88.5 mmol) of N-Boc-L-γ-glutamic acid-α-t-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 1M K2CO3 followed by water, dried (MgSO4) 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.
N2-acetyl-N-[2-[[(5-butyl -2-pyridinyl) carbonyl] -aminol ethyl] -1-glutamine.
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 C19H28N4O5·0.83 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.
2-amino-5-[4-[(5-butyl-2-pyridinyl) carbonyl]-1-piperazinyl]-5-oxopentanoic acid.
Step 1: Preparation of the piperizine 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, J = 8 Hz, 3H), 1.28-1.45 (m, 2H), 1.55-1.67 (m,
2H), 1.66-1.72 (br s, 1H) , 2.64 (t, J = 8 Hz, 2H), 2.86 (t, J = 6
Hz, 2H), 2.97 (t, 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, 1H).
Step 2: Preparation of 2-amino-5-[4-[(5-butyl-2-pyridinyl) carbonyl]-1-piperazinyl]-5-oxopentanoic acid.
Under nitrogen, a solution of 17.4 g (57 mmol) of N-Boc-L-γ-glutamic acid-α-t-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 1M K2CO3 followed by water, dried (MgSO4), 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.
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.
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.
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.
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.
BIOLOGICAL EVALUATION
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 Effacts of Ex. #3 Conjugate
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 30 45 60 Vehicle (0.5 ml 0.9% NaCl i.v.)
MAP (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 Chronic Tn Vivo Effects of Ex. #3 Conjugate Time After Injection (days)
Zero 1 2 3 4
Vehicle (300 μL/hr)
MAP(mm Hg) 181±8 172±6 170±7 174±6 182±3
Ex. #3 Conjugate (5 mg/hr) MAP (mm Hg) 164±3 175±5 174±5 172±2 N.A.
Assay III: Chronic In Vivo Effects of Ex. #464 Conjugate
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 (300 μL/hr) MAP(mmHg) 181±8 172±6 170±7 174±6 182±3
Ex. #464 Conjugate (10 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. #859 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 XXI 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 Purified 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 @ pH 8.1
Assay V; In Vitro Evaluation of DBH Inhibition by 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
TABLE XXIV
DBH Inhibition by Fusaric Acid, Ex. #859 Conjugate
and Various Potential Metabolites
Test Ex. Ex. Fusaric Acid Fusaric
Compound (20μM) : #859 #858 Hydrazide Acid
% Inhibition : 1.5 0.0 13.8 75.4
Assay VI; Acute In Vivo Effects of Ex. #859 and Ex. #863 Conjugates
Spontaneously hypertensive 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. or i.d.). In addition, a flow probe was implanted around the left renal artery for
measurement of renal blood flow using pulsed Doppler
flowmetry. 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 50 mg/kg of fusaric acid or the Ex. #859 conjugate. As shown in Figures 8 and 9 and Table XXV, fusaric acid (a systemic dopamine-β-hydroxylase inhibitor) decreased mean arterial pressure and increased renal blood flow throughout the 60 minute post-injection observation period. In sharp contrast, the Ex. #859 conjugate had no acute effects on mean arterial pressure, but increased
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 XXVI: Ex. #863 had no effect on mean arterial pressure, but increased renal blood flow, indicating renal selectivity.
TABLS 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 (50mg/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 XXVI Acute Effects of Ex. #863 Conjugate
Time (min)
Zero 15 30 45 60
Ex. #863 (100 mg/kg i.v.)
MAP (mm Hg) 149±14 N.A. N.A. N.A. 147±14
RBF (KHz) 1.6±0.2 N.A. N.A. N.A. 4.3±0.3
N.A. = Not Available
Assay VTT: Comparison of Fusaric Acid. Fusaric Acid Hydrazide and Ex. #859 Conjugate on Arterial Pressure in Spontaneously Hypertensive Rats (SHR)
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 XXVII 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.
Table XXVII
COMPOUND ZERO 60 MIN
Fusaric Acid (n=4) 164 ± 10 mmHg 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 XXVIII Chronic Effects of Fusaric Acid and Ex. #859 Conjugate on Blood Pressure
Time (days)
Zero 1 2 3 4 5
Vehicle (25 μL/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 (mmHg) 146±5 122±9 115±9 119±11 121±7 115±8 (SE)
Assay IX; Chronic In 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 XXIX
Chronic Effects of Ex. #861 and Ex. #863 Conjugates
on Blood Pressure
Time (days)
Zero 1 2 3 4
Vehicle 171±6 172±6 164±6 169±4 162±4
Ex. #861 177±3 173±3 172+4 172±3 163±9
Ex. #863 177±5 152±6 146±7 142±7 154±7
Αssay X: Catecholamine Analysis of Tissue from Rats
Treated with Ex. #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., Europ. J. Pharmacol., 109. 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 4°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 × 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 Na2HPO4 in 75/25 (v/v) H2O/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., 8, 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 XXXI
Effect of Fusaric Acid and Ex. #859 conjugate on Tissue Norepinephrine Concentration 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)
Assay XI: Intrarenal Administration of Fusaric Acid in
Anesthetized 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 (UNaV) 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
Effect 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
UNa 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 XXXIII
Effect of Intrarenal Tnfusion of Fusaric Acid on Blood Pressure. Sodium Excretion and Renal
Blood Flow in the Dog
Dog #1 Dog #2
Infusion: Fusaric Acid Fusaric Acid
Saline (1.25 mg/kg/min) Saline (0.75mg/kg/min)
Δ RBF (ml/min): 140 240 236 315 UNa 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.
Assay XII: 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 Injection of Ex. #859 Conjugate Zero 1 Hour 2 Hour 3 Hour
Mean Arterial
Pressure (mmHg)
20 mg/kg 138±6 139±7 139±8 140±8
60 mg/kg 123±3 124±1 126±3 127±10
Renal Blood
Flow (ml/min)
20 mg/kg 88±19 107±23 123±29 125±29
60 mg/kg 131±21 145±21 168±28 176±32
Compositions of 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. Therapeutically 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.
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.