J . Med. Chem. 1995,38,,4087-4097
4087
In Vivo and in Vitro Studies on the Neurotoxic Potential of 6-Hydroxydopamine Analogs Su Ma, Lorrie Lin, R. Raghavan, Pat Cohenour, Peter Y. T. Lin, Jennifer Bennett, Russell J. Lewis, Eric L. Enwall, Richard Kostrzewa,t Roland E. Lehr, and C. LeRoy Blank* Department of Chemistry & Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, and Department of Pharmacology, East Tennessee State University, Johnson City, Tennessee 37614 Received September 7, 1994@
In an attempt to determine which physical and biological properties could best be correlated (11,better with neurotoxic potential, seven analogs of 1-(2,4,5-trihydroxypheny1)-2-aminoethane known as 6-hydroxydopamine, were synthesized and compared to 1 in a variety of ways both in vivo and in vitro. The analogs, in combination with the standard 1, include all eight of the 2,4,5-trisubstituted-phenylderivatives of phenethylamine and a-methylphenethylamine in which the substitution is of the trihydroxy or aminodihydroxy form. Low (60 nmol) and high (300nmol) intracerebroventricular doses of all analogs produced long-term (7 day) reduction of mouse whole brain norepinephrine (NE) and lesser depletions of dopamine (DA), and effects on serotonin were varied. The analog 1-(5-amino-2,4-dihydroxyphenyl)-2-aminopropane (8) was both more complete and more selective than the standard 1 in depleting NE. Using a histofluorometric glyoxylic acid method and Fink-Heimer silver degeneration stain, it was determined that overt neural degeneration was produced by 8. In vitro, the ease of oxidation of the eight analogs was found to be represented by a formal potential range of -130 to -212 mV vs SCE. However, there was no obvious relationship between ease of oxidation and the extent of monoamine depletion from mouse brain. Using kinetic analysis of synaptosomal accumulation of L3H1NE and [3HlDA, it was found that the standard 1 is more potent in its interaction with the DA uptake site (Ki= 12 f 0 pM)than the NE uptake site (Ki= 51 f 1 pM). A correlation analysis was used to determine that differences in NE and DA depletion by each analog could not be explained by differences in potency for in vitro uptake blockade. However, there was a correlation between the Ki for [3HlNE uptake blockad&and the EC50 for synaptosomal release of preloaded r3H1NE for the eight analogs (R2= 0.96; for 1og:log plot, R2 = 0.541,indicating that the results for these two in vitro tests both reflect interaction with the same NE neuronal membrane transport site. A similar correlation between Ki and EC50 was shown for all eight analogs using r3H]DA (R2 = 0.92;for 1og:log plot, R2 = 0.52),indicating interaction with the same DA neuronal membrane transport site. These findings demonstrate that there is no single property that can account for selectivity of action and/or potency of catecholamine neurotoxins related t o 6-hydroxydopamine.
Introduction 6-Hydroxydopamine ( l p 4originally reported as a product of dopamine found in urine,5 was shortly thereafter found to produce long-term depletion of catecholamines6and actual degeneration of catecholaminergic neuronal pathways.' There are a number of striking similarities between the actions of 1 and the pathology observed in Idiopathic Parkinson's Disease, as recently summarized by Zigmond et aL4 One would hope that a more complete understanding of the mode of action of 1would be capable of being employed in the alleviation and/or delay of onset of such neurodegenerative disorders; for example, knowledge of the mode of action of another dopaminergic neurotoxin, l-methyl4-phenyl-1,2,3,6-tetrahydr0pyridine,~ was partly responsible for the recent finding that L-deprenyl, a monoamine oxidase blocking agent, was effective in delaying the onset of Idiopathic Parkinson's D i ~ e a s e . ~ Not being able to pass the blood-brain barrier in adults, 1 is normally administered intraventricularly or into the parenchyma in a specific brain region when central nervous system (CNS) effects are desired.1° + @
East Tennessee State University. Abstract published in Advance ACS Abstracts, September 1,1995.
And, while being a quite selective agent for noradrenergic or dopaminergic pathways under appropriate conditions,l it is neither complete nor specific with regard to its neurodegenerative action on either of these systems in the CNS. The mode of action of 1in eliciting degeneration has been shown to clearly involve uptake of this agent into the targeted neurons via the transport system of the endogenous neurotransmitter located on the cell membrane, since prior administration of uptake inhibitors completely eliminates the subsequent degeneration observed.ll Presumably a minimal intraneuronal concentration of 1 must be achieved before it can elicit its degenerative effects; Jonsson and Sachs have estimated that concentrations as high as 50 mM are required.12 But, beyond the absolute requirement for transport, the subsequent means by which 1 leads to degeneration of neuronal processes is not well-kn0wn.l The ease of oxidation of 1 has frequently been invoked in the mode of action; indeed, 1is much easier to oxidize than the endogenous transmitters norepinephrine and dopamine.13J4 One of three major hypotheses concerning the mode of action of 1 implicates the toxic oxygen byproducts resulting from oxidation of 1 by molecular 0 2 (includingHzOz, O$-,and OH') as being the primary cytotoxins; this is supported by the ability of catalase
0022-2623/95/1838-4087$09.00/00 1995 American Chemical Society
4088 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 20
R R4 H " 5 2
Cmpd 1
2 3 4
R2
R4
R5
OH NH2 OH OH
OH OH NH2 OH
OH OH OH NHz
R H H H H
C W R 2 5 6 7 8
% & J R
OH OH OH NH, OH OH OH NH2 OH OH OH NHz
CH:, CH, CH3 CH3
Figure 1. The eight 6-hydroxydopaminerelated neurotoxins.
and various radical trapping agents to block the action of l.15 On the other hand, the originally proposed mode of action,' involving interaction of the oxidized, quinone form of 1 with nucleophilic components of functionally important cellular proteins or other nucleophiles, is equally viable and strongly supported by the correspondence between irreversible binding of 3H-labeled 1 t o endogenous proteins and the observed degeneration.16 Studies by Liang et al.17Jshave clearly demonstrated the nucleophilic attack of glutathione at the unsubstituted ring carbon atom ortho t o the side chain in 1; the isolation of the glutathionyl adduct of 1 following its injection into hypothalamus established that such reactions are possible in vivo. A third hypothesis concerning the mode of action of 1 focuses on the ability of this compound to interfere with the oxidative phosphorylation process; in fact, 1 has been claimed t o be equipotent to 2,4-dinitrophenol, a classic uncoupler of this process.lg Additionally, it is possible that the mode of action of this neurotoxin is different in noradrenergic neurons than it is in dopaminergic neurons and, perhaps, even different in subpopulations of individual neuronal types.' Studies by Borchardt et a1.20aimed primarily a t measuring the relative import of the first two hypotheses in the mode of action of 1 and reported on the properties of the two ring monomethylated and the one ring dimethylated derivatives of this agent. However, both of the monomethylated derivatives appeared to be capable of eliciting destructive actions; thus, one might reasonably conclude that both of the first two hypothesized actions and/or the third are involved, although to unknown degrees for each, in the ultimate degeneration process. In an attempt t o assess the relative importance of these different factors in the mode of action of 1, we have synthesized various congeners of this toxin and examined some of their properties. Additionally, we thought it possible that such agents might provide more complete and/or more selective degeneration for noradrenergic and/or dopaminergic pathways than the original 1; such agents could advantageously be employed in subsequent studies in place of 1. In fact, many of the analogs of 1 that we have selected have already been shown to be neurotoxic by Tranzer and Thoenen in peripheral tissues using electron microscopy.21 From this previous report, neurotoxic activity toward catecholaminergic neuronal systems is easily projected for or -a-meall the 2,4,5-trisubstituted-phenethylamines thylphenethylamines in which the ring substitution is of the trihydroxy or aminodihydroxy variety. As seen in Figure 1,this group of compounds encompasses eight possibilities, including 1. Synthesis. Synthesis of analogs 2,5, and 6 followed previously reported procedure^.^^-^^ The syntheses of analogs 3 and 4 are outlined in Schemes 1 and 2,
Ma et al.
respectively, while the syntheses of both 7 and 8 are sufficiently similar to allow incorporation into the single Scheme 3. Compounds 3,4, and 8 have previously been reported. However, in the two previous reports for 3, one21 provided no synthetic information, no physical/ spectral data, and no statement as to the salt form obtained, while the second25reported a different form of the final product (3.HC1 instead of 32HBr) and employed a different pathway. In the only previous report of 4,21 no synthetic information, no physic& spectral data, and no statement as to the salt form obtained were provided. In the only previous report of 8,26no synthetic information and no physicdspectral data were provided. The standard 1 is commercially available. Preparation of 3, as shown in Scheme 1,began with reduction of the nitro group of the previously reported2' 2,5-dimethoxy-4-nitrobenzylcyanide, yielding the desired 3a. Subsequent conversion of the acetonitrile side chain to the ethylamine was achieved by dissolution in an EtOWconcentrated HC1 solvent and hydrogenation under more forcing conditions, yielding 3b. Demethylation of the ring methoxy groups employed concentrated HBr, providing the desired 302HBr. The overall yield for the reaction sequence shown was 35%, with the lowest yield, 52%, occurring in the formation of 3b. In the synthesis of 4, as shown in Scheme 2, the 2,4dimethoxy-5-nitrobenzylcyanide starting material was prepared from 2,4-dichloronitrobenzene as described to the amino derivative 4a p r e v i o u ~ l y . ~Conversion ~,~~ was accomplished with hydrogenation under moderate conditions using Pd/C catalyst. The cyanide group of 4a was converted to the acetamide with concentrated HC1, and the resulting acetamide of 4b was reduced to the saturated ethylamine with BH3. The two ring methoxy groups were demethylated by treatment with concentrated HBr to yield 402HBrH20. The overall yield for the reaction sequence shown was 29%. Compounds 7 and 8 were synthesized as their dihydrobromide salts according to Scheme 3. The starting materials, 2,5-dimethoxyphenyl- and 2,4-dimethoxyphenyl-2-nitropropene for 7 and 8, respectively, were obtained through condensation of the corresponding benzaldehydes with nitroethane, as described previously.30>31 Ring nitration was position specific in both cases, giving an 85% yield for 8a and a 72% yield for 7a. In both cases, simultaneous reduction of the ring nitro group to an amine and the side chain t o an a-methylethylamine employed Raney nickel, while demethylation of the methoxy groups utilized concentrated HBr. The overall yield for the sequence shown was 44% for 72HBr and 50% for 8.2HBr. Long-Term Depletion of Endogenous Neurotransmitters. Absolute proof of degeneration by a neurotoxin like 1 requires verification of biochemical measurements by electron microscopy7and/or histochemical fluorescence technique^.^^ But, the use of either of these approaches to routine quantitative measures of degeneration is impractical. Thus, once degeneration has been demonstrated by these techniques, more rapid and readily available biochemical measurements are generally accepted to provide the desired q ~ a n t i t a t i o n . ~ ~ In the case of the compounds investigated, six (1-6) have been previously shown to produce degeneration of sympathetic innervation to the heart and/or vas defer-
Journal of Medicinal Chemistry, 1995, Vol. 38, No. 20 4089
Neurotoxic Potential of 6-Hydroxydopamine Analogs
Scheme 1
Scheme 2
Scheme 3
for 7, R,rOMe, RpH; for 8 . R i d , R2=0Me
Reagents: (a) Hfld-C, (b) HC1, Hfld-C, Raney Ni.
7 Cqa,R 2 4 4 2 ) or8 (Rt-NH2, R 2 - W )
78 ( R i a , Rp=NQ) or 8 8 (Rl=NOa RpOMe)
(c) concentrated HBr, (d) concentrated HC1, (e) BHflHF, and (0 HNOaOAc, (g) Hz/
ens 7 days following iv administration21 or in other preparation~,",~~-3~ and it would appear reasonable to expect a priori that the same catecholaminergic destructive capability exists for the remaining two compounds (7-8). The three most commonly employed biochemical measures for catecholaminergic degeneration following treatment with 1 and related compounds include (1) remaining levels of endogenous transmitter, (2) remaining activity levels of tyrosine hydroxylase, and (3) remaining transporter activity levels. Each of these measures has both advantages and disadvantages. For example, levels of neurotransmitters may be very rapidly depleted shortly &r treatment due to releasing effects of the agent. Enzyme activities may exhibit short-term and/or long-term enhancements in response t o neurotransmitter depletion and/or degeneration. Transporter density responses, if any, are unknown. A further complication arises in that neuronal pathways subjected to neurotoxic degeneration generally exhibit regeneration and/or sprouting shortly after the degenerative event. Degeneration following administration of 1 tends to predominate during the first 24-48 h following treatment, while regeneration slowly begins to predominate thereafter.32 Endogenous transmitter levels tend to decline more rapidly and recover more slowly than transporter activity levels, but both seem t o offer comparable results a t ca. 7 days following treatment.32 Given these factors, we decided to employ the simple measurement of remaining levels of pertinent neurotransmitters at 7 days following treatment as our quantitative determination of degeneration. The choice of 7 days provides some assurance, particularly for agents 7 and 8 , that the measurements were of degeneration rather than simple depletion. This approach also allows simultaneous assessment of the effects of the agents on three different transmitter systems, i.e., the noradrenergic, dopaminergic, and serotonergic systems; thus, an assessment of selectivity is additionally provided by the approach. Results and Discussion Table 1 shows the long-term depletion of norepinephrine (NE), dopamine (DA),and serotonin (5-HT)achieved in whole mouse brains following a low dose of the eight agents, while Table 2 shows the same for a higher dose.
Table 1. Depletion of Whole Mouse Brain Neurotransmitters 7 Days after a Low Dose (60 nmol) of Various 6-Hydroxydopamine Related Neurotoxinsa levels, % control x b l SEM compd n NE DA 5-HT 100 i 2 100 f 4 5 100f8 control 5 40f8*** 8 6 % 1*** 97f2 1 5 40f1*** 90f3* 78 f 1*** 2 6 37f2*** 100f5 73 f 9** 3 4 5 6 7 8
6
5 6
6 5
40f2*** 25f2*** 32f4*** 33 f 1*** 26f3***
96f 2 87fO*** 96f5 100f 7 100f4
91f4 93f8 72 f 4*** 79 i 6** 103 i 5
Significant differences compared to control values ( t test): *P 0.05; **P < 0.01; ***P< 0.001. a
