J Neural Transm [GenSect] (1992) 88:177-185
_ Journal o f Neural Transmission 9 Springer-Verlag 1992 Printed in Austria
Effects of 3,N,N'-trimethyl-2-phenyl-l,4-piperazine diastereomers on monoamine uptake and monoamine oxidase in rat brain D. F. Smith1, P. N. Jensen1, M. Gelbcke2, and D. Tytgat2
1PsychopharmacologyResearch Unit, Psychiatric Hospital, Risskov, Denmark 2Institut de Pharmacie, Universit6 Libre de Bruxelles, Bruxelles, Belgium Accepted January 20, 1992
Summary. The diastereomers of 3,N,N'-trimethyl-2-phenyl-l,4-piperazine dihydrochloride (TPP) were tested for their effects on NA, DA and 5-HT uptake in synaptosomes prepared from hypothalamus, corpus striatum, and frontal cortex, respectively. The diastereomers differed with respect to their inhibitory properties. (2 R, 3 R)-TPP was more potent than the other diastereomers on NA and DA uptake, whereas (2 S, 3 S)-TPP was least potent. In contrast, the (2 S, 3 S)- and (2 S, 3 R)-diastereomers of TPP were more potent than (2 R, 3 R)and (2 R, 3 S)-TPP as inhibitors of 5-HT uptake. None of the diastereomers affected monoamine oxidase activity. The findings show that the diastereomers of TPP interact stereoselectively with neuronal mechanisms for monoamine uptake, and that the (S)-configuration at the 2 carbon is important for inhibitory actions of TPP on 5-HT uptake. Keywords: Phenylpiperazine, monoamine uptake, NA, DA, 5-HT, MAO, phenylethylamine, rat. Introduction
Spatial features often play an important role in effects of drugs (Smith, 1984, 1989a). Of particular interest for the present study is the stereoselectivity of neuronal uptake processes for monoamines. Since the removal of monoamines from the synaptic cleft by reuptake processes may be involved in the etiology of some psychic disorders and in therapeutic effects of some drugs (Koe, 1976; Richelson and Pfenning, 1984), it is of interest to study relations between spatial features of drugs and their effects on those processes. Previous studies have shown that spatial features in the phenylethylamine moiety of stereoisomeric compounds are involved in the inhibition of monoamine uptake (Maxwell et al., 1976; Tuomisto et al., 1976; Tuomisto and Smith, 1986). However, many previous studies have been carried out using only stereo-
178
D . F . Smith etal.
isomeric mixtures, so there are still uncertainties concerning effects of individual stereoisomers on m o n o a m i n e uptake processes (Smith, 1989 a). Recently, limited a m o u n t s of the individual diastereomers of 3,N,N'-trimethyl-2-phenyl-l,4-piperazine (TPP) became available (Tytgat et al., 1990). Since those c o m p o u n d s contain the phenylethylamine moiety in various spatial orientations (Fig. 1), it was of interest to determine whether those c o m p o u n d s have effects on the u p t a k e of m o n o a m i n e s into rat brain synaptosomes. In addition, the effects of the c o m p o u n d s on the activity of m o n o a m i n e oxidase in brain was measured in order to determine whether they influence that enzyme.
Materials and methods
Animals Male Wistar rats (250-310 g) were used. They were housed individually in clear plastic cages in a thermostatically controlled room (20 ~ on a 12 hour light-dark cycle (lights on 6 a.m.) for at least 2 weeks before tests. They had free access to food pellets and tap water prior to tests.
Compounds The cis- and trans-diastereomers of 1,3,4-trimethyl-2-phenyl-piperazines were obtained from optically pure erythro- and threo-N,N'-dimethyl-l-phenylpropane-l,2-diamines, respectiyely, as described in detail elsewhere (Tytgat etal., 1990; Tytgat, 1991). Briefly, dihydrochloride salt of erythro- or threo-N,N'-dimethyl-l-phenylpropane-l,2-diamine was dissolved in water, and the solution was raised to pH 11 with stirring by addition of 1 N NaOH. Chloroacetic anhydride was added, with the solution maintained at pH 11 by automatic addition of 1 N NaOH. After stirring for one hour, the solution was extracted with CHC13. This procedure gave a mixture of eis-l,3,4-trimethyl-2-phenylpiperazin-5- and
H
1-4
H
OH 3
(2S,3S)
{2_R,3S)
H
CH 3 (2S,3__R)
N
H (2R,3R)
Fig. 1. Structural drawing of the diastereomers of 3,N,N' -trimethyl-2-phenyl-l,4-piperazine
3,N,N'-trimethyl-2-phenyl-l,4-piperazine diastereomers
179
-6-one or trans-l,3,4-trimethyl-2-phenylpiperazin-5- and -6-one respectively, with a yield of about 90%. The compounds, dissolved in ether, were gradually added to a stirred, icecold suspension of LiA1H4 in ether, and the resulting mixture was stirred overnight at room temperature. Aqueous NaOH (30%) was added gradually and the ether layer was separated, was washed with water, and was extracted with aqueous H 2 SO4 (10%). The extraction solution was made alkaline, and the liberated base was extracted with ether, was dried with Na2 SO4, and was treated with HC1. The precipitated dihydrochloride eis-compound (2 R, 3 S or 2 S, 3 R) was recrystallized from ethanol, whereas the trans-compound (2 S, 3 S or 2 R, 3 R) was recrystallized from a mixture of ethanol/ether (80 : 20). The purity of the compounds was confirmed by N M R spectroscopy (Tytgat, 1991).
Monoamine uptake Monoamine uptake was measured by methods modified from Magnussen etal. (1982). Rats were decapitated and brain was removed and placed on ice. Samples of hypothalamus (55-100 rag), corpus striatum (80-170 mg) and fr6ntal cortex (80-160mg) were obtained for measurement of NA, DA, and 5-HT uptake, respectively. The brain regions were homogenized immediately in 2ml of ice cold 0.32M sucrose in a Potter-Elvehjelm homogenizer with a teflon pestle, and centrifuged at 1000 g, 4 ~ for 10 min. The supernatant consisting of the crude mitochondrial-synaptosomal fraction was used. Uptake was measured in a modified Krebs-Ringer bicarbonate buffer of the following composition (in mM): Glucose, 5.6; NaC1, 118.5; KCL, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 24.9; ascorbic acid, 1.7; and pargyline, 0.08. Buffer was prepared fresh daily. It was gassed immediately before use with a mixture of 95% O2 and 5% CO2 and adjusted to pH between 7.3 and 7.4. The diastereomers of TPP salts (2 HC1) were dissolved in water and added to buffer at final concentrations between 0.005 and 1.0mM. Crude mitochondrial-synaptosomal fraction was added and the samples were placed in waterbaths at either 0 ~ or 37 ~ for 12 min (preincubation period). Then, either 3H-NA (final concentration 2 • 10-9 M), 14CDA (final concentration 2 • 10-8 M) or 14C-5-HT (final concentration 2 • 10-8 M) was added to the synaptosomal suspensions from hypothalamus, corpus striatum, and frontal cortex, respectively. (-)-7,8-3H-NA (12.0 Ci/mmol) and [7-14C]-DA (57mCi/mmol) were obtained from Amersham, whereas 5-[2-14C]-5-HT (54.6 mCi/mmol) was from New England Nuclear. Mixing was performed by gently bubbling with a gas mixture of 95% 02 and 5% CO2 in order to maintain pH, and vials were capped during incubation. The incubation was carried out for 15rain at either 0"C or 37~ Uptake was stopped by filtration under vacuum onto Millipore filter (1.2 ~m), and the vials were rinsed twice with 1.5 ml buffer. After addition of scintillation fluid, radioactivity was measured. Active uptake was calculated by subtracting the uptake at 0 ~ from that at 37 ~ (Tuomisto and Smith, 1986). Uptake in the presence of an inhibitor was compared with that o f control, and percent inhibition was calculated. The concentration of each diastereomer that inhibited uptake by 50% (ICs0) was calculated by linear regression. Computerized analysis of variance and Student-Newman-Keuls tests (Norusis, 1988) were used to determine whether the diastereomers influenced monoamine uptake in a dosedependent fashion and whether there were significant differences between the effects of the diastereomers on the uptake of NA, DA and 5-HT.
Monoamine oxidase The activity ofmonoamine oxidase in rat brain (minus brainstem) was measured as described previously (Smith, 1989 b). Briefly, the brain was homogenized in 100 mM sodium phosphate buffer (pH 7.4). The homogenate was centrifuged at 1,000 g for 10 min at 4 "C. The pellet
180
D.F. Smith etal.
was discarded and the supernatant was centrifugedat 33,500g for 20 min at 4 ~ The pellet was suspended in buffer and MAO activity was measured using either 2-phenyl[1-14C]ethylamineHC1 (PEA, specific activity 54.6mCi/mmol, Amersham) (33 gM final concentration) or 5-[2-14C]-hydroxytryptamine binoxalate (5-HT, specific activity 54.5mCi/mmol, NEN) as substrate. Racemic tranylcypromine (r-TCP, 1mM), which is known to inhibit PEA metabolism by MAO (Smith, 1989b), was used for comparison to test compounds. Compounds were incubated with the enzyme source for 2 min at 37 ~ substrate was added, and the incubation was continued for 20 min. The reaction was stopped with 2 N HCI. Deaminated products were extracted in ethyl acetate and were counted in Opti-Fluor according to conventional scintillation techniques. Paired t-tests were used for determining whether the compounds influenced MAO activity. Results
Monoamine uptake Figure 2 shows the effects of the diastereomers of TPP on the uptake of NA, DA and 5-HT in corpus striatum, hypothalamus and frontal cortex, respectively. Each of the diastereomers inhibited the uptake of NA, DA and 5-HT in a dosedependent fashion (p's < 0.02). Furthermore, there were reliable differences between the inhibitory effects of the diastereomers on the uptake of NA ((2 R, 3 R) > (2 S, 3 S)) (p < 0.05), of DA ((2 R, 3 R), (2 S, 3 R) > (2 S, 3 S)) (p's < 0.05) and of 5-HT ((2 S, 3 R), (2 S, 3 S), (2 R, 3 S) > (2 R, 3 R)) (p's < 0.05) (Table 1). The diastereomers of TPP were more potent inhibitors of NA uptake than of either DA or 5-HT uptake (p's < 0.05). Furthermore. (2 S, 3 S)- and (2 S, 3 R)TPP were more potent inhibitors of 5-HT uptake than of DA uptake (p's < 0.05), whereas (2 R, 3 R)-TPP was a more potent inhibitor of DA uptake than of 5-HT uptake (p < 0.05).
Monoamine oxidase Table 2 shows the results of experiments carried out to determine whether the diastereomers of TPP influence the activity of monoamine oxidase in rat brain. None of the diastereomers of TPP affected the metabolism of either PEA or 5-HT by MAO, whereas racemic tranylcypromine exerted an inhibitory effect on rat brain MAO, as expected (Smith, 1989 b). Discussion
Previous studies have shown both neuronal monoamine uptake and metabolism of monoamines by MAO to be stereoselective processes (Patil etal., 1975), in that they depend in part on spatial features in the molecules. Stereoselective effects of drugs on monoamine uptake and monoamine oxidase are of interest in light of possible relations between such effects and antidepressant actions of drugs (Smith, 1989 a). The aim of the present study was to determine whether the individual diastereomers of TPP, which are novel compounds containing the phenylethylamine moiety in various conformations, influenced monoaminergic processes in a stereoselective manner.
3,N,N' -trimethyl-2-phenyl-1,4-piperazine diastereomers
g
100
100
80
80
60 /"
,'
60
T/
[./
.-s
4-0
40
rl/ I
'F, "-
{
I
[
t
10-2
10 - t
100
Log ( 2 S , 3 S ) - T P P
g
I
ill
20
10-3
conc.
20
10-3
I
I
I
10-2
10 -1
10 0
Log ( 2 R , 3 S ) - T P P
(mM)
100
100
80
80
60
g
?-2 ..Q "7" .E
181
-/
/
60
.a
-~
40
4o
/
o D
(mM)
conc.
20
10 - 3
20
[
I
I
10-2
10-1
10 o
Log ( 2 S , 3 R ) - T P P
conc.
(mM)
1 -5
I
/l
i
I
r
I
10-2
10-t
10 o
Log ( 2 R , 3 R ) - T P P
conc.
(raM)
Fig. 2. Dose-response curves for effects of TPP diastereomers on the synaptosomal uptake of NA in hypothalamus (circles), DA in corpus striatum (squares) and 5-HT in frontal cortex (triangles) from rat brain
The spatial orientation of the aromatic ring with respect to the nitrogen atom in the phenylethylamine moiety of conformationally-restricted molecules has been found to play an important role in effects of such compounds on uptake processes (for review, see Rutledge etal., 1984). Previous studies on structural features in compounds containing a phenylalkylamine moiety have left questions unanswered concerning the role of spatial features in monoamine uptake and monoamine oxidase, due in part to the use of compounds with flexible side-chains which can assume an infinite number of conformations (Horn and Snyder, 1972; Tuomisto, 1978; Tenne and Youdim, 1984). Horn and Snyder (1972), for example, used conformationally-restricted 2-phenylcy-
182
D.F. Smith et al. Table 1. Estimated ICs0 values for the effects of TPP diastereomers (mM) on inhibition of synaptosomal uptake of NA, DA and 5-HT in rat brain regions (hypothalamus, corpus striatum and frontal cortex respectively) Treatment
Substrate
(2 S, 3 S)-TPP (2 R, 3 S)-TPP (2 S, 3 R)-TPP (2R, 3 R)-TPP
NA
DA
5-HT
0.06 0.02 0.03 0.01
0.43 0.27 0.26 0.18
0.19 0.25 0.08 0.68
Table2. Effects of the diastereomers of 3,N,N'-trimethyl-2phenyl-1,4-piperazine (TPP) (0.17 mM) on monoamine oxidase activity (nmol/min/mg protein) in rat brain. Racemic tranylcypromine (r-TCP) (0.17raM) was used for assuring that the enzyme was responsive to drug inhibition. Values shown are means • S.D. for 5-6 assays Treatment
Control (2 S, 3 S)-TPP (2 R, 3 S)-TPP (2 S, 3 R)-TPP (2 R, 3 R)-TPP r-TCP
Substrate a PEA
5-HT
0.57 4- 0.03 0.57 • 0.02 0.57 • 0.03 0.59 4- 0.03 0.57 • 0.02 0.002 • 0.001"
1.71 + 0.22 1.79 4- 0.23 1.77 • 0.26 1.77 • 0.23 1.77 • 0.24 0.04 • 0.01"
* indicates significant difference from buffer control values (p < 0.001) 1 PEA beta-Phenylethylamine; 5-HT 5-Hydroxytryptamine
clopropylamines and a m i n o i n d a n e s in studies on m o n o a m i n e uptake. They f o u n d that the ant•177 c o n f o r m a t i o n of a m p h e t a m i n e analogs was favorable for interactions with the N A uptake site in h y p o t h a l a m i c tissue. T u o m isto et al. (1974) used conformationally-restricted decalin derivatives for investigating d o p a m i n e uptake and f o u n d that a synelinal c o n f o r m a t i o n was favorable for a t t a c h m e n t to the D A carrier in the corpus striatum. Later, T u o m i s t o and Smith (1986) f o u n d that the + anticlinal c o n f o r m a t i o n of (+)-tranylcypromine, a conformationally-restricted c o m p o u n d containing a phenylethylamine moiety, favored actions on 5-HT uptake, whereas the - a n t i c l i n a l conform a t i o n of ( - ) - t r a n y l c y p r o m i n e favored inhibitory actions on D A and N A
3,N,N' -trimethyl-2-phenyl-1,4-piperazine diastereomers
183
uptake. In the present piperazine series, steric factors prevent the phenyl group from occupying an axial position, so the antiperiplanar conformation is favored by all four diastereomers of TPP. Thus, the diastereomers of TPP are suitable for studying the role of other spatial features in monoaminergic processes. The present findings support and extend previous ones in showing that synaptosomal uptake of monoamines is influenced by spatial features in the phenylethylaminemoiety of conformationally-restrictedcompounds. We found, for example, that the diastereomers of TPP were more potent inhibitors of NA uptake than of either DA or 5-HT uptake. This finding is of interest in light of previous reports which show selective blockade of synaptosomal uptake of norepinephrine to be a property shared by most antidepressant drugs (Richelson and Pfenning, 1984). It is also of interest to note that phenylpiperazine derivatives similar to TPP diastereomers have been found to have antidepressant properties in some neuropharmacological tests (French Patent 2585702). It is noteworthy, however, that the diastereomers of TPP are relatively weak inhibitors compared to the enantiomers of tranylcypromine, which are structurally related to TPP diastereomers, and which were 10-100 times more potent under comparable test conditions (Tuomisto and Smith, 1986). It is, therefore, unlikely that the diastereomers of TPP will prove to be effective antidepressant agents, although they may be valuable lead compounds. The present findings show that there were marked differences between effects of the diastereomers of TPP on monoaminergic uptake into rat brain synaptosomes. With regard to 5-HT uptake, the greatest inhibitory effect was produced by the (2 S, 3 R)-and (2 S, 3 S)- diastereomers of TPP, while the (2 R, 3 R)-form had only a weak inhibitory action on 5-HT uptake. These findings indicate that the (S)-configuration at the 2 carbon atom in TPP played an important role for inhibition of 5-HT uptake. The present findings agree with previous reports on the stereoselectivity of 5-HT uptake (Smith, 1986), and may provide further clues concerning topographical features in the 5-HT transport sites (Smith etal., 1991). The diastereomers of TPP exerted inhibitory effects on the synaptosomal uptake of DA and NA; the (2 R, 3 R)-diastereomer of TPP tended to be more potent than the other diastereomers as inhibitor of both NA and DA uptake, whereas (2 S, 3 S)-TPP was the least potent inhibitor of both NA and DA uptake. Although the compounds inhibited the uptake of NA and DA, no marked differences were noted between the stereoselectivity of NA and DA uptake processes towards the diastereomers of TPP. Thus, spatial features of the individual diastereomers of TPP fail to provide new insight into specific requirements for stereoselective interactions with catecholaminergic uptake processes. Finally, none of the diastereomers of TPP had effects on the activity of monoamine oxidase. Evidently, the molecular structure of TPP is inappropriate for effective interactions with stereoselective sites of MAO (Tenne and Youdim, 1984). In conclusion, the diastereomers of TPP and related compounds appear
184
D . F . Smith et al.
to be useful tools for d e t e r m i n i n g further spatial requirements for selective p h a r m a c o l o g i c a l actions on m o n o a m i n e r g i c process.
Acknowledgements We thank E. Kristensen, E. Poulsen and H. Smith for skillful technical assistance and H. Hildebrandt for help with typing the manuscript.
References Horn AS, Snyder SH (1972) Steric requirements for catecholamine uptake by rat brain synaptosomes: studies with rigid analogs of amphetamine. J Pharmacol Exp Ther 180: 523-530 Koe BK (1976) Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J Pharmacol Exp Ther 199: 649661 Magnussen I, Tonder K, Engbaek F (1982) Paroxetine, a potent selective long-acting inhibitor of synaptosomal 5-HT uptake in mice. J Neural Transm 55:217-226 Maxwell RA, Ferris RM, Burcsu JE (1976) Structural requirements for inhibition of noradrenaline uptake by phenethylamine derivatives, desipramine, cocaine and other compounds. In: Paton DM (ed) The mechanism of neuronal and extraneuronal transport of catecholamines. Raven Press, New York, pp95-153 Norusis MJ (ed) (1988) SPSS/PC+ TM V2.0 Base Manual for the IBM PC/XT-/AT and PS/2. SPSS Inc., Chicago, Ill Patil PN, Miller DD, Trendelenburg U (1975) Molecular geometry and adrenergic drug activity. Pharmacol Rev 26:323-392 Richelson E, Pfenning M (1984) Blockade by antidepressant and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake. Eur J Pharmacol 104:277-286 Rutledge CO, Walters DE, Grunewald GL (1984) Noradrenergic drugs: conformational preferences in uptake mechanisms. In: Smith DF (ed) CRC Handbook of stereoisomers. Drugs in psychopharmacology. CRC Press, Boca Raton, pp 79-92 Smith DF (1984) CRC Handbook of stereoisomers. Drugs in psychopharmacology. CRC Press, Boca Raton Smith DF (1986) The stereoselectivity of serotonin uptake in brain tissue and blood platelets: the topography of the serotonin uptake area. Neurosci Biobehav Rev 10:37-46 Smith DF (1989 a) Psychotropic drugs. In: Smith DF (ed) Handbook of stereoisomers. Therapeutic drugs. CRC Press, Boca Raton, pp 323-393 Smith DF (1989 b) Lithium alters the stereoselectivity of monoamine oxidase in rat brain. J Neural Transm 76:205-210 Smith DF, Jensen PN, Poulsen SH, Mikkelsen EO, Elbaz E, Glaser R (1991) Effects of pyrroloisoquinoline enantiomers (( + )- and ( - ) McN-5652-Z) on behavioral and pharmacological serotonergic mechanisms in rats. Eur J Pharmacol 196:85-92 Tenne M, Youdim MBH (1984) Mao-inhibiting drugs: stereochemical aspects. In: Smith DF (ed) Handbook of stereoisomers. Drugs inpsychopharmacology. CRC Press, Boca Raton, pp 285-296 Tuomisto J (1978) Some structural features of amine uptake mechanisms. In: Simon P (ed) Adv Pharmacol Therapeutics, vol 2. Neurotransmitters. Pergamon Press, Oxford, pp 231-248 Tuomisto J, Smith DF (1986) Effects of tranylcypromine enantiomers on monoamine uptake and release and imipramine binding. J Neural Transm 65:135-145 Tuomisto J, Walaszek EJ, Smissman EE, Pazdernik TL (1974) Decalin analogs of phen-
3,N,N' -trimethyl-2-phenyl-1,4-piperazine diastereomers
185
ethylamines as inhibitors of amine uptake by rabbit platelets. II. Uptake of 5-hydroxytryptamine. J Pharm Sci 63:1714-1718 Tuomisto J, Tuomisto L, Pazdernik TL (1976) Conformationallyrigid amphetamine analogs as inhibitors of monoamine uptake by brain synaptosomes. J Med Chem 19:725-727 Tytgat D (1991) Synth6ses de ph6nylpropanediamines isost6res de l'6ph6drine: 6tude configurationnelle et conformationnelle par R.M.N. et essais pharmacologiques preliminaires. Ph.D. thesis, Facult+ de M6dicine et de Pharmacie, Universit~ Libre de Bruxelles Tytgat D, Gelbcke M, Smith DR (1990) Convenient synthesis of 1-phenyl-l,2-propanediamines. Preliminary pharmacological results. Pharmazie 45:835-836 Authors' address: D. F. Smith, Ph.D., Psychopharmacology Research Unit, Psychiatric Hospital, Skovagervej 2, DK-8240 Risskov, Denmark. Received June 10, 1991
_ Journal o f Neural Transmission 9 Springer-Verlag 1992 Printed in Austria
Effects of 3,N,N'-trimethyl-2-phenyl-l,4-piperazine diastereomers on monoamine uptake and monoamine oxidase in rat brain D. F. Smith1, P. N. Jensen1, M. Gelbcke2, and D. Tytgat2
1PsychopharmacologyResearch Unit, Psychiatric Hospital, Risskov, Denmark 2Institut de Pharmacie, Universit6 Libre de Bruxelles, Bruxelles, Belgium Accepted January 20, 1992
Summary. The diastereomers of 3,N,N'-trimethyl-2-phenyl-l,4-piperazine dihydrochloride (TPP) were tested for their effects on NA, DA and 5-HT uptake in synaptosomes prepared from hypothalamus, corpus striatum, and frontal cortex, respectively. The diastereomers differed with respect to their inhibitory properties. (2 R, 3 R)-TPP was more potent than the other diastereomers on NA and DA uptake, whereas (2 S, 3 S)-TPP was least potent. In contrast, the (2 S, 3 S)- and (2 S, 3 R)-diastereomers of TPP were more potent than (2 R, 3 R)and (2 R, 3 S)-TPP as inhibitors of 5-HT uptake. None of the diastereomers affected monoamine oxidase activity. The findings show that the diastereomers of TPP interact stereoselectively with neuronal mechanisms for monoamine uptake, and that the (S)-configuration at the 2 carbon is important for inhibitory actions of TPP on 5-HT uptake. Keywords: Phenylpiperazine, monoamine uptake, NA, DA, 5-HT, MAO, phenylethylamine, rat. Introduction
Spatial features often play an important role in effects of drugs (Smith, 1984, 1989a). Of particular interest for the present study is the stereoselectivity of neuronal uptake processes for monoamines. Since the removal of monoamines from the synaptic cleft by reuptake processes may be involved in the etiology of some psychic disorders and in therapeutic effects of some drugs (Koe, 1976; Richelson and Pfenning, 1984), it is of interest to study relations between spatial features of drugs and their effects on those processes. Previous studies have shown that spatial features in the phenylethylamine moiety of stereoisomeric compounds are involved in the inhibition of monoamine uptake (Maxwell et al., 1976; Tuomisto et al., 1976; Tuomisto and Smith, 1986). However, many previous studies have been carried out using only stereo-
178
D . F . Smith etal.
isomeric mixtures, so there are still uncertainties concerning effects of individual stereoisomers on m o n o a m i n e uptake processes (Smith, 1989 a). Recently, limited a m o u n t s of the individual diastereomers of 3,N,N'-trimethyl-2-phenyl-l,4-piperazine (TPP) became available (Tytgat et al., 1990). Since those c o m p o u n d s contain the phenylethylamine moiety in various spatial orientations (Fig. 1), it was of interest to determine whether those c o m p o u n d s have effects on the u p t a k e of m o n o a m i n e s into rat brain synaptosomes. In addition, the effects of the c o m p o u n d s on the activity of m o n o a m i n e oxidase in brain was measured in order to determine whether they influence that enzyme.
Materials and methods
Animals Male Wistar rats (250-310 g) were used. They were housed individually in clear plastic cages in a thermostatically controlled room (20 ~ on a 12 hour light-dark cycle (lights on 6 a.m.) for at least 2 weeks before tests. They had free access to food pellets and tap water prior to tests.
Compounds The cis- and trans-diastereomers of 1,3,4-trimethyl-2-phenyl-piperazines were obtained from optically pure erythro- and threo-N,N'-dimethyl-l-phenylpropane-l,2-diamines, respectiyely, as described in detail elsewhere (Tytgat etal., 1990; Tytgat, 1991). Briefly, dihydrochloride salt of erythro- or threo-N,N'-dimethyl-l-phenylpropane-l,2-diamine was dissolved in water, and the solution was raised to pH 11 with stirring by addition of 1 N NaOH. Chloroacetic anhydride was added, with the solution maintained at pH 11 by automatic addition of 1 N NaOH. After stirring for one hour, the solution was extracted with CHC13. This procedure gave a mixture of eis-l,3,4-trimethyl-2-phenylpiperazin-5- and
H
1-4
H
OH 3
(2S,3S)
{2_R,3S)
H
CH 3 (2S,3__R)
N
H (2R,3R)
Fig. 1. Structural drawing of the diastereomers of 3,N,N' -trimethyl-2-phenyl-l,4-piperazine
3,N,N'-trimethyl-2-phenyl-l,4-piperazine diastereomers
179
-6-one or trans-l,3,4-trimethyl-2-phenylpiperazin-5- and -6-one respectively, with a yield of about 90%. The compounds, dissolved in ether, were gradually added to a stirred, icecold suspension of LiA1H4 in ether, and the resulting mixture was stirred overnight at room temperature. Aqueous NaOH (30%) was added gradually and the ether layer was separated, was washed with water, and was extracted with aqueous H 2 SO4 (10%). The extraction solution was made alkaline, and the liberated base was extracted with ether, was dried with Na2 SO4, and was treated with HC1. The precipitated dihydrochloride eis-compound (2 R, 3 S or 2 S, 3 R) was recrystallized from ethanol, whereas the trans-compound (2 S, 3 S or 2 R, 3 R) was recrystallized from a mixture of ethanol/ether (80 : 20). The purity of the compounds was confirmed by N M R spectroscopy (Tytgat, 1991).
Monoamine uptake Monoamine uptake was measured by methods modified from Magnussen etal. (1982). Rats were decapitated and brain was removed and placed on ice. Samples of hypothalamus (55-100 rag), corpus striatum (80-170 mg) and fr6ntal cortex (80-160mg) were obtained for measurement of NA, DA, and 5-HT uptake, respectively. The brain regions were homogenized immediately in 2ml of ice cold 0.32M sucrose in a Potter-Elvehjelm homogenizer with a teflon pestle, and centrifuged at 1000 g, 4 ~ for 10 min. The supernatant consisting of the crude mitochondrial-synaptosomal fraction was used. Uptake was measured in a modified Krebs-Ringer bicarbonate buffer of the following composition (in mM): Glucose, 5.6; NaC1, 118.5; KCL, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 24.9; ascorbic acid, 1.7; and pargyline, 0.08. Buffer was prepared fresh daily. It was gassed immediately before use with a mixture of 95% O2 and 5% CO2 and adjusted to pH between 7.3 and 7.4. The diastereomers of TPP salts (2 HC1) were dissolved in water and added to buffer at final concentrations between 0.005 and 1.0mM. Crude mitochondrial-synaptosomal fraction was added and the samples were placed in waterbaths at either 0 ~ or 37 ~ for 12 min (preincubation period). Then, either 3H-NA (final concentration 2 • 10-9 M), 14CDA (final concentration 2 • 10-8 M) or 14C-5-HT (final concentration 2 • 10-8 M) was added to the synaptosomal suspensions from hypothalamus, corpus striatum, and frontal cortex, respectively. (-)-7,8-3H-NA (12.0 Ci/mmol) and [7-14C]-DA (57mCi/mmol) were obtained from Amersham, whereas 5-[2-14C]-5-HT (54.6 mCi/mmol) was from New England Nuclear. Mixing was performed by gently bubbling with a gas mixture of 95% 02 and 5% CO2 in order to maintain pH, and vials were capped during incubation. The incubation was carried out for 15rain at either 0"C or 37~ Uptake was stopped by filtration under vacuum onto Millipore filter (1.2 ~m), and the vials were rinsed twice with 1.5 ml buffer. After addition of scintillation fluid, radioactivity was measured. Active uptake was calculated by subtracting the uptake at 0 ~ from that at 37 ~ (Tuomisto and Smith, 1986). Uptake in the presence of an inhibitor was compared with that o f control, and percent inhibition was calculated. The concentration of each diastereomer that inhibited uptake by 50% (ICs0) was calculated by linear regression. Computerized analysis of variance and Student-Newman-Keuls tests (Norusis, 1988) were used to determine whether the diastereomers influenced monoamine uptake in a dosedependent fashion and whether there were significant differences between the effects of the diastereomers on the uptake of NA, DA and 5-HT.
Monoamine oxidase The activity ofmonoamine oxidase in rat brain (minus brainstem) was measured as described previously (Smith, 1989 b). Briefly, the brain was homogenized in 100 mM sodium phosphate buffer (pH 7.4). The homogenate was centrifuged at 1,000 g for 10 min at 4 "C. The pellet
180
D.F. Smith etal.
was discarded and the supernatant was centrifugedat 33,500g for 20 min at 4 ~ The pellet was suspended in buffer and MAO activity was measured using either 2-phenyl[1-14C]ethylamineHC1 (PEA, specific activity 54.6mCi/mmol, Amersham) (33 gM final concentration) or 5-[2-14C]-hydroxytryptamine binoxalate (5-HT, specific activity 54.5mCi/mmol, NEN) as substrate. Racemic tranylcypromine (r-TCP, 1mM), which is known to inhibit PEA metabolism by MAO (Smith, 1989b), was used for comparison to test compounds. Compounds were incubated with the enzyme source for 2 min at 37 ~ substrate was added, and the incubation was continued for 20 min. The reaction was stopped with 2 N HCI. Deaminated products were extracted in ethyl acetate and were counted in Opti-Fluor according to conventional scintillation techniques. Paired t-tests were used for determining whether the compounds influenced MAO activity. Results
Monoamine uptake Figure 2 shows the effects of the diastereomers of TPP on the uptake of NA, DA and 5-HT in corpus striatum, hypothalamus and frontal cortex, respectively. Each of the diastereomers inhibited the uptake of NA, DA and 5-HT in a dosedependent fashion (p's < 0.02). Furthermore, there were reliable differences between the inhibitory effects of the diastereomers on the uptake of NA ((2 R, 3 R) > (2 S, 3 S)) (p < 0.05), of DA ((2 R, 3 R), (2 S, 3 R) > (2 S, 3 S)) (p's < 0.05) and of 5-HT ((2 S, 3 R), (2 S, 3 S), (2 R, 3 S) > (2 R, 3 R)) (p's < 0.05) (Table 1). The diastereomers of TPP were more potent inhibitors of NA uptake than of either DA or 5-HT uptake (p's < 0.05). Furthermore. (2 S, 3 S)- and (2 S, 3 R)TPP were more potent inhibitors of 5-HT uptake than of DA uptake (p's < 0.05), whereas (2 R, 3 R)-TPP was a more potent inhibitor of DA uptake than of 5-HT uptake (p < 0.05).
Monoamine oxidase Table 2 shows the results of experiments carried out to determine whether the diastereomers of TPP influence the activity of monoamine oxidase in rat brain. None of the diastereomers of TPP affected the metabolism of either PEA or 5-HT by MAO, whereas racemic tranylcypromine exerted an inhibitory effect on rat brain MAO, as expected (Smith, 1989 b). Discussion
Previous studies have shown both neuronal monoamine uptake and metabolism of monoamines by MAO to be stereoselective processes (Patil etal., 1975), in that they depend in part on spatial features in the molecules. Stereoselective effects of drugs on monoamine uptake and monoamine oxidase are of interest in light of possible relations between such effects and antidepressant actions of drugs (Smith, 1989 a). The aim of the present study was to determine whether the individual diastereomers of TPP, which are novel compounds containing the phenylethylamine moiety in various conformations, influenced monoaminergic processes in a stereoselective manner.
3,N,N' -trimethyl-2-phenyl-1,4-piperazine diastereomers
g
100
100
80
80
60 /"
,'
60
T/
[./
.-s
4-0
40
rl/ I
'F, "-
{
I
[
t
10-2
10 - t
100
Log ( 2 S , 3 S ) - T P P
g
I
ill
20
10-3
conc.
20
10-3
I
I
I
10-2
10 -1
10 0
Log ( 2 R , 3 S ) - T P P
(mM)
100
100
80
80
60
g
?-2 ..Q "7" .E
181
-/
/
60
.a
-~
40
4o
/
o D
(mM)
conc.
20
10 - 3
20
[
I
I
10-2
10-1
10 o
Log ( 2 S , 3 R ) - T P P
conc.
(mM)
1 -5
I
/l
i
I
r
I
10-2
10-t
10 o
Log ( 2 R , 3 R ) - T P P
conc.
(raM)
Fig. 2. Dose-response curves for effects of TPP diastereomers on the synaptosomal uptake of NA in hypothalamus (circles), DA in corpus striatum (squares) and 5-HT in frontal cortex (triangles) from rat brain
The spatial orientation of the aromatic ring with respect to the nitrogen atom in the phenylethylamine moiety of conformationally-restricted molecules has been found to play an important role in effects of such compounds on uptake processes (for review, see Rutledge etal., 1984). Previous studies on structural features in compounds containing a phenylalkylamine moiety have left questions unanswered concerning the role of spatial features in monoamine uptake and monoamine oxidase, due in part to the use of compounds with flexible side-chains which can assume an infinite number of conformations (Horn and Snyder, 1972; Tuomisto, 1978; Tenne and Youdim, 1984). Horn and Snyder (1972), for example, used conformationally-restricted 2-phenylcy-
182
D.F. Smith et al. Table 1. Estimated ICs0 values for the effects of TPP diastereomers (mM) on inhibition of synaptosomal uptake of NA, DA and 5-HT in rat brain regions (hypothalamus, corpus striatum and frontal cortex respectively) Treatment
Substrate
(2 S, 3 S)-TPP (2 R, 3 S)-TPP (2 S, 3 R)-TPP (2R, 3 R)-TPP
NA
DA
5-HT
0.06 0.02 0.03 0.01
0.43 0.27 0.26 0.18
0.19 0.25 0.08 0.68
Table2. Effects of the diastereomers of 3,N,N'-trimethyl-2phenyl-1,4-piperazine (TPP) (0.17 mM) on monoamine oxidase activity (nmol/min/mg protein) in rat brain. Racemic tranylcypromine (r-TCP) (0.17raM) was used for assuring that the enzyme was responsive to drug inhibition. Values shown are means • S.D. for 5-6 assays Treatment
Control (2 S, 3 S)-TPP (2 R, 3 S)-TPP (2 S, 3 R)-TPP (2 R, 3 R)-TPP r-TCP
Substrate a PEA
5-HT
0.57 4- 0.03 0.57 • 0.02 0.57 • 0.03 0.59 4- 0.03 0.57 • 0.02 0.002 • 0.001"
1.71 + 0.22 1.79 4- 0.23 1.77 • 0.26 1.77 • 0.23 1.77 • 0.24 0.04 • 0.01"
* indicates significant difference from buffer control values (p < 0.001) 1 PEA beta-Phenylethylamine; 5-HT 5-Hydroxytryptamine
clopropylamines and a m i n o i n d a n e s in studies on m o n o a m i n e uptake. They f o u n d that the ant•177 c o n f o r m a t i o n of a m p h e t a m i n e analogs was favorable for interactions with the N A uptake site in h y p o t h a l a m i c tissue. T u o m isto et al. (1974) used conformationally-restricted decalin derivatives for investigating d o p a m i n e uptake and f o u n d that a synelinal c o n f o r m a t i o n was favorable for a t t a c h m e n t to the D A carrier in the corpus striatum. Later, T u o m i s t o and Smith (1986) f o u n d that the + anticlinal c o n f o r m a t i o n of (+)-tranylcypromine, a conformationally-restricted c o m p o u n d containing a phenylethylamine moiety, favored actions on 5-HT uptake, whereas the - a n t i c l i n a l conform a t i o n of ( - ) - t r a n y l c y p r o m i n e favored inhibitory actions on D A and N A
3,N,N' -trimethyl-2-phenyl-1,4-piperazine diastereomers
183
uptake. In the present piperazine series, steric factors prevent the phenyl group from occupying an axial position, so the antiperiplanar conformation is favored by all four diastereomers of TPP. Thus, the diastereomers of TPP are suitable for studying the role of other spatial features in monoaminergic processes. The present findings support and extend previous ones in showing that synaptosomal uptake of monoamines is influenced by spatial features in the phenylethylaminemoiety of conformationally-restrictedcompounds. We found, for example, that the diastereomers of TPP were more potent inhibitors of NA uptake than of either DA or 5-HT uptake. This finding is of interest in light of previous reports which show selective blockade of synaptosomal uptake of norepinephrine to be a property shared by most antidepressant drugs (Richelson and Pfenning, 1984). It is also of interest to note that phenylpiperazine derivatives similar to TPP diastereomers have been found to have antidepressant properties in some neuropharmacological tests (French Patent 2585702). It is noteworthy, however, that the diastereomers of TPP are relatively weak inhibitors compared to the enantiomers of tranylcypromine, which are structurally related to TPP diastereomers, and which were 10-100 times more potent under comparable test conditions (Tuomisto and Smith, 1986). It is, therefore, unlikely that the diastereomers of TPP will prove to be effective antidepressant agents, although they may be valuable lead compounds. The present findings show that there were marked differences between effects of the diastereomers of TPP on monoaminergic uptake into rat brain synaptosomes. With regard to 5-HT uptake, the greatest inhibitory effect was produced by the (2 S, 3 R)-and (2 S, 3 S)- diastereomers of TPP, while the (2 R, 3 R)-form had only a weak inhibitory action on 5-HT uptake. These findings indicate that the (S)-configuration at the 2 carbon atom in TPP played an important role for inhibition of 5-HT uptake. The present findings agree with previous reports on the stereoselectivity of 5-HT uptake (Smith, 1986), and may provide further clues concerning topographical features in the 5-HT transport sites (Smith etal., 1991). The diastereomers of TPP exerted inhibitory effects on the synaptosomal uptake of DA and NA; the (2 R, 3 R)-diastereomer of TPP tended to be more potent than the other diastereomers as inhibitor of both NA and DA uptake, whereas (2 S, 3 S)-TPP was the least potent inhibitor of both NA and DA uptake. Although the compounds inhibited the uptake of NA and DA, no marked differences were noted between the stereoselectivity of NA and DA uptake processes towards the diastereomers of TPP. Thus, spatial features of the individual diastereomers of TPP fail to provide new insight into specific requirements for stereoselective interactions with catecholaminergic uptake processes. Finally, none of the diastereomers of TPP had effects on the activity of monoamine oxidase. Evidently, the molecular structure of TPP is inappropriate for effective interactions with stereoselective sites of MAO (Tenne and Youdim, 1984). In conclusion, the diastereomers of TPP and related compounds appear
184
D . F . Smith et al.
to be useful tools for d e t e r m i n i n g further spatial requirements for selective p h a r m a c o l o g i c a l actions on m o n o a m i n e r g i c process.
Acknowledgements We thank E. Kristensen, E. Poulsen and H. Smith for skillful technical assistance and H. Hildebrandt for help with typing the manuscript.
References Horn AS, Snyder SH (1972) Steric requirements for catecholamine uptake by rat brain synaptosomes: studies with rigid analogs of amphetamine. J Pharmacol Exp Ther 180: 523-530 Koe BK (1976) Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J Pharmacol Exp Ther 199: 649661 Magnussen I, Tonder K, Engbaek F (1982) Paroxetine, a potent selective long-acting inhibitor of synaptosomal 5-HT uptake in mice. J Neural Transm 55:217-226 Maxwell RA, Ferris RM, Burcsu JE (1976) Structural requirements for inhibition of noradrenaline uptake by phenethylamine derivatives, desipramine, cocaine and other compounds. In: Paton DM (ed) The mechanism of neuronal and extraneuronal transport of catecholamines. Raven Press, New York, pp95-153 Norusis MJ (ed) (1988) SPSS/PC+ TM V2.0 Base Manual for the IBM PC/XT-/AT and PS/2. SPSS Inc., Chicago, Ill Patil PN, Miller DD, Trendelenburg U (1975) Molecular geometry and adrenergic drug activity. Pharmacol Rev 26:323-392 Richelson E, Pfenning M (1984) Blockade by antidepressant and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake. Eur J Pharmacol 104:277-286 Rutledge CO, Walters DE, Grunewald GL (1984) Noradrenergic drugs: conformational preferences in uptake mechanisms. In: Smith DF (ed) CRC Handbook of stereoisomers. Drugs in psychopharmacology. CRC Press, Boca Raton, pp 79-92 Smith DF (1984) CRC Handbook of stereoisomers. Drugs in psychopharmacology. CRC Press, Boca Raton Smith DF (1986) The stereoselectivity of serotonin uptake in brain tissue and blood platelets: the topography of the serotonin uptake area. Neurosci Biobehav Rev 10:37-46 Smith DF (1989 a) Psychotropic drugs. In: Smith DF (ed) Handbook of stereoisomers. Therapeutic drugs. CRC Press, Boca Raton, pp 323-393 Smith DF (1989 b) Lithium alters the stereoselectivity of monoamine oxidase in rat brain. J Neural Transm 76:205-210 Smith DF, Jensen PN, Poulsen SH, Mikkelsen EO, Elbaz E, Glaser R (1991) Effects of pyrroloisoquinoline enantiomers (( + )- and ( - ) McN-5652-Z) on behavioral and pharmacological serotonergic mechanisms in rats. Eur J Pharmacol 196:85-92 Tenne M, Youdim MBH (1984) Mao-inhibiting drugs: stereochemical aspects. In: Smith DF (ed) Handbook of stereoisomers. Drugs inpsychopharmacology. CRC Press, Boca Raton, pp 285-296 Tuomisto J (1978) Some structural features of amine uptake mechanisms. In: Simon P (ed) Adv Pharmacol Therapeutics, vol 2. Neurotransmitters. Pergamon Press, Oxford, pp 231-248 Tuomisto J, Smith DF (1986) Effects of tranylcypromine enantiomers on monoamine uptake and release and imipramine binding. J Neural Transm 65:135-145 Tuomisto J, Walaszek EJ, Smissman EE, Pazdernik TL (1974) Decalin analogs of phen-
3,N,N' -trimethyl-2-phenyl-1,4-piperazine diastereomers
185
ethylamines as inhibitors of amine uptake by rabbit platelets. II. Uptake of 5-hydroxytryptamine. J Pharm Sci 63:1714-1718 Tuomisto J, Tuomisto L, Pazdernik TL (1976) Conformationallyrigid amphetamine analogs as inhibitors of monoamine uptake by brain synaptosomes. J Med Chem 19:725-727 Tytgat D (1991) Synth6ses de ph6nylpropanediamines isost6res de l'6ph6drine: 6tude configurationnelle et conformationnelle par R.M.N. et essais pharmacologiques preliminaires. Ph.D. thesis, Facult+ de M6dicine et de Pharmacie, Universit~ Libre de Bruxelles Tytgat D, Gelbcke M, Smith DR (1990) Convenient synthesis of 1-phenyl-l,2-propanediamines. Preliminary pharmacological results. Pharmazie 45:835-836 Authors' address: D. F. Smith, Ph.D., Psychopharmacology Research Unit, Psychiatric Hospital, Skovagervej 2, DK-8240 Risskov, Denmark. Received June 10, 1991
