CHIRALITY 2:90-95 (1990)

(R)- and (S)-5-Hydroxy-Z-( dipropy1amino)tetralin (5-OH DPAT):

Assessment of Optical Purities and Dopaminergic Activities

ANDERS KARLSSON, LENA WORK, CURT PEITERSSON, NILS-ERIK A N D ~ N , AND ULI HACKSELL

Departments of Analytical Pharmaceutical Chemistry (AX., C.P.) and Organic Pharmaceutical Chemistry (U.H.), Uppsala Biomedical Center, Box 574, University of Uppsala, 5-751 23 Uppsala, Sweden; Department of Medical Pharmacology, Uppsala Biomedical Center, University of Uppsala,

Box 593, S-75124 Uppsala, Sweden (LB.); and Department of Pharmology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden (N.-EA.)

ABSTRACT Racemic 5-hydroxy-2-(dipropylamino)tetralin (5-OH DPAT), a potent and selective dopamine (DA) D,-receptor agonist, was resolved into the enantiomers by a new method. The enantiomers of 5-OH DPAT were determined by chiral ion-pair chromatography using N-benzyloxycarbonylglycyl-L-proline as the counter ion. The enantiomeric purity of (R)-5-OH DPAT was found to be > 99.7%. The ability of the enantiomers to change the rat brain DOPA levels was evaluated in vivo. The results indicate that (RM-OH DPAT is a weakly potent DA D2-receptor antagonist.

KEY WORDS: stereoselectivity, 5-OH DPAT, dopamine D2-receptor antagonist, chiral ion pair chromatography, N-benzyloxycarbonylglycyl- L-proline (L-ZGP)

INTRODUCTION 5-Hydroxy-2-(dipropylamino)tetralin (5-OH DPAT)l

is a potent dopamine (DA) D2-receptor agonist.2 It has served as a lead compound in several structure- activity relationship studies (see, for example, refs. 2- 7) and certain analogues have been claimed to be of potential therapeutic use.' 5-OH DPAT is chiral, C2 being a center of asymmetry. The racemate has been resolved into the enantiorners' and the absolute con- figuration of the enantiomer which is dextrarotatory when dissolved in methanol has been assigned the R configuration on the basis of X-ray crystallography of the hydrochloride salt.' Pharmacological studies have demonstrated that (S)-5-OH DPAT is much more po- tent than the antipode; the eudismic ratio" of 5-OH DPAT varies from 10 to 176 in a series of pharmaco- logical assays (Table l).l,11-15 The activity of (R)- 5-OH-DPAT has, however, never been established since its optical purity has not been determined. In fact, it might be possible that the various activities of (R)-5-OH DPAT shown in Table 1 are due to the pres- ence of small amounts of the S-enantiomer; for exam- ple, Wikstrom et al.15 estimated the purity of (RM-OH DPAT to be 97.8% ee. Thus, (R)-5-OH DPAT might be inactive, a DA D2-receptor agonist or an antagonist.

During the preparation of this study, Gerding et a1.16 reported an indirect separation method for determina- tion of the enantiomeric purity of (R)- and (S)- 2-(N-propyl-N-2-thienylethylamino)-5-hydroxy-

0 1990 Wiley-Liss, Inc.

OM

(2R)b-OH DPAT

tetralin, which are structurally related to the enanti- omers of 5-OH DPAT. The determination was based on formation of diastereomeric glucuronides by incuba- tion with uridine 5'-diphosphoglucuronyl transferase (UDPGT) and the cosubstrate uridine 5'-diphospho- glucuronic acid for 2 h at 37°C.

In the present study, we have (1) prepared the enan- tiomers of 5-OH DPAT by use of a new resolution method, (2) determined the enantiomeric excess of (R)- 5-OH DPAT by a rapid and effective ion-pair chromato- graphic separation achieved by addition of an optically active counter ion" to the mobile phase, and (3) eval- uated the effect of (R)-5-OH DPAT in an assay which detects DA D2-receptor agonists as well as antagonists.

Received for publication November 27, 1989; accepted December 20, 1989. Address reprint requests to Uli Hacksell, at the address given above.

(R)- AND (S)-5-HYDROXY-2-(DIPROPYLAMINO)TETFtALIN 91

TABLE 1. Previously reported dopaminergic activities of the enantiomers of 5-OH DPAT

Assay Ref. (2S)-5-OH DPAT (2R1-5-OH DPAT Eudismic ratioa

[3HlDopamine displacement IC,,, nM 11 19 1.200 63.2 12 4 200 50.0

13 80 2.200 27.5 [3H]Apomorphine displacement IC,,, nM 12 18 360 20.0

displacement IC,,, nM 14 5.8 200 34.5 [3H]-N-Propylnorapomorphine

[3HlSpiroperidol displacement IC,,, nM 11 340 3.600 10.6 12 190 2.050 10.8 14 600 70.800 118

Minimum emetic dose (pgkg) 1 2 >80 >40.0 Minimum dose producing stereotyped

behaviour (mgkg) 1 0.005 >1.8 >36.0 Reduction of DOPA accumulation in the

Reduction of DOPA accumulation in the limbic system (ED,,, nmolkg) 15 3.7 410 110

striatal system (ED,,, nmolkg) 15 3.7 650 176

T h e ratio of the potencies of the two enantiomers.

The results indicate that (R)-5-OH DPAT is a weakly potent DA D2-receptor antagonist.

MATERIALS AND METHODS General

Melting points (uncorrected) in open glass capillaries were determined on a Thomas-Hoover apparatus. Op- tical rotations were measured on a Perkin-Elmer 241 polarimeter. 'H and 13C NMR spectra (recorded on a JEOL FX 90 Q spectrometer) were in accordance with the assigned structures.

Resolution of (~)-2-BenzylaminoS-methoxytetralin [(+)-11

Racemic 2-benzylamino-5-methoxytetralin [(+)-111 (10.29 g, 38.5 mmol) and (+ 1-di-p-toluoyl-L-tartaric acid (15.56 g, 38.5 mmol) were added to 900 ml of hot 95% ethanol. The solution was left to reach room tem- perature and crystals started to form. After 4 days, the precipitate (11.48 g) was removed by filtration and re- crystallized from 95% ethanol (600 ml). An additional recrystallization gave 8.20 g of tartrate, which was con- verted into hydrochloride. The hydrochloride was re- crystallized once from methanol/ether to give 3.31 g (57%) of (S)-1 . HC1, which had mp 245°C dec, and -63.8" (c 2.0, MeOH). Lit.' mp 246-247°C dec, -61" (c 2.0, MeOH).

The combined mother liquors from the above recrys- tallizations were concentrated and the residue was par- titioned between aqueous sodium hydroxide and ether to afford the partially resolved base (R)-1 (7.01 g). The base was dissolved in hot 95% ethanol and the solution was treated with an equimolar amount of (-1-di-p- toluoyl-D-tartaric acid. Two recrystallizations from 96% ethanol gave 8.50 g of tartrate, which was con- verted into the hydrochloride. One recrystallization from methanol/ether afforded 3.41 g (58%) of

(R)-1 * HCl; mp 245°C dec, Lit.' mp 246-247°C dec, [a];' +61" (c 2.0, MeOH).

+ 64.0" (c 2.0, MeOH).

(S)-2-AminoS-methoxytetralin Hydrochloride [(S)-2 * HCl]

Hydrogenolysis [methanol, H2 (1 atm.), Pd/C (lo%)] of (S)-1 * HC1 produced a crude product which was re- crystallized from methanol/ether to give 69% of (S)-1 * HC1, mp 270°C dec, [a];' - 71.7" (c 2.0, MeOH). Lit. mp 260°C decl and 278-28O0C,l5 [albO - 61" (c 2.0, MeOH)l and [a];' -63.0" (c 1.0, MeOH).15

(R)-Z-AminoS-methoxytetmlin Hydrochloride [(R)-Z * HCl]

Hydrogenolysis of (R)-1 - HCl by the above procedure gave a crude product which was recrystallized from methanol/ether to give 69% of (R)-2 - HC1, mp 270°C dec, + 71.6" (c 2.0, MeOH). Lit. mp 260°C decl and 278-280"C,15 [a];' +61" (c 2.0, MeOH)l and +69.7" (c 1.0, MeOH).15

(S)S-Hydroxy-Z-(dipropyopylamino)tetralin Hydrochloride [(S)-ti-OH DPAT * HCl]

Dipropylation of (S)-2 - HC1 (iodopropane, potassium carbonate, acetonitrile) followed by demethylation (aqueous hydrobromide, 120°C) according to literature procedures'*2 produced (S)-2 - HCl: mp 231-233"C, [a1F2 -67.2' (c 2.0, MeOH). Lit. mp 228-229°C dec' and 229-23loC, [alFO -71" (c 2.0, MeOHY and - 63.7" (c 1.0, MeOH).15

(R)s-Hyd~y-2-(dipropylamino)tetralin Hydrochloride

(R)-2 * HC1 was converted into (R)-5-OH DPAT - HC1 by the above procedure: mp 231-233"C, [a1F2 - 66.9" (c 1.0, MeOH). Lit . mp 228-229.5"C dec' and 226-229"C,15 [a]?0 +70" (c 2.0, MeOH)l and + 67.2" (c 1.0, MeOH).15

[(R)S-OH DPAT - HCl]

92 KARLSSON ET AL.

Chromatogmph y

The HPLC system used was a ConstaMetric I11 pump (LDC, Riviera Beach, FL) and a Rheodyne Model 7125 injector (Cotati, CA) equipped with a 20 ~1 loop. A Hi- tachi 655 A-22 (Merck, Darmstadt, F.R.G.) was used to monitor the absorbance (278 nm) of the eluent.

The column (100 x 4.7 mm i.d.1 was of stainless steel and packed with porous graphitic carbon (7 Fm, PGC Batch 93). The column was kindly supplied by Prof. J.H. Knox, Department of Chemistry, Edinburgh, U.K. N-Benzyloxycarbonylglycyl-L-proline (L-ZGP) (1.58 g) (Fluka, Buchs, Switzerland) was dissolved in 500 ml of dichloromethane (LiChrosolv, Merck). The water con- tent of the mobile phase was adjusted to about 80 ppm by mixing water saturated dichloromethane (2,100 ppm H20) and dry dichloromethane (<30 ppm H20).18

The column and the solvent reservoir were thermo- statted at 25°C & 0.1"C with a HETO type 02 pt 923 C water-bath (Birkergd, Denmark). The flow rate was 0.50 ml min-'. All samples of 5-OH DPAT were dis- solved in the mobile phase.

Pharmacology

Male Sprague-Dawley rats (Alab, Stockholm) weighing 165-220 g were used. The test substances were dissolved in 0.9% NaCl. Throughout, injection volumes were 5 mlkg. Substances to be tested were given to the rats as the hydrochlorides, subcutaneously in the neck region.

The tyrosine hydroxylase activities were determined from the accumulation of 3,4-dihydroxyphenylalanine (DOPA) following inhibition of the aromatic L-amino acid decarboxylase by 3-hydroxybenzylhydraine (NSD 1015; 60 mgkg, i.e., 287 Fmolkg sc, 30 min before killing).lg The experimental drugs were given sc 30 min prior to NSD 1015.

The rats were killed by decapitation. The brain was taken out quickly and placed on an ice-cooled Petri dish. The corpus striatum and the limbic system were separated. The rhinal fissures were used as a landmark in the dissection of the limbic system. The concentra- tions of DOPA were determined electrochemically fol- lowing ion-pair, reversed-phase HPLC (for additional details, see ref. 20).

RESULTS AND DISCUSSION Resolution

The enantiomers of 5-OH DPAT have been prepared previously: Wikstrom et al.15 resolved a derivatized precursor, the diastereomeric mixture of (R)-O-methyl- mandeloylamides of (?)-2-amino-5-rnethoxytetralin [( *)-21, by chromatographic separation. This procedure gave, after amide hydrolysis, (R)- and (S)-2 with 97.8% ee and 98.6% ee, respectively. The enantiomers of 2 were then converted into those of 5-OH DPAT.

McDermed et al.' resolved racemic 2-benzyla- mino-5-methoxytetralin [(k )-13 by fractional crystalli-

The enantiomers of 1 were then converted to the target compounds.

Our preparation of the enantiomers of 5-OH DPAT is outlined in Scheme 1. Racemic 1 was resolved by frac- tional crystallization of the diastereomeric di- p-toluoyltartrates from ethanol. The N-benzyl group was removed by hydrogenolysis. (S)- and (R)-2 were converted into (S) - and (R1-5-OH DPAT, respectively, by NJV-dipropylation followed by O-demethylation.

Zon-Pair Chromatographic Determination of Enantiomeric Purity

Indirect separation of enantiomers by formation and separation of diastereomeric derivatives in a nonchiral chromatographic system has some serious limita- tions.'8*21 Racemization or presence of an enantiomeric impurity in the reagent will affect the accuracy when determining the enantiomeric excess (ee). Determina- tion of enantiomers by a direct separation method, that is, using a chiral stationary phase or a chiral mobile phase additive, has several advantages; for example, the accuracy of the ee is not affected by the enantio- meric purity of the selector.18*21

The use of L-ZGP as a chiral additive (counterion) in organic solvents of low polarity has allowed separa- tions of various enantiomeric amino alcohols" and amines (A. Karlsson and C. Pettersson, unpublished results). The technique was applied to determine the enantiomeric purity of (R)-5-OH DPAT. The chiral sep- aration (Fig. 1) was performed by using 10 mM L-ZGP in dichloromethane as the eluent and porous graphitic carbon as the absorbing phase. This gave high enanti- oselectivity (asnt = 1.30) which enabled a complete res- olution (Rs = 1.7) of (k)-5-OH DPAT within 8 min, thus facilitating the determinations of high optical pu- rities. Figure 2 gives the chromatograms of the authen- tic (R)-5-OH DPAT and mixtures of 0.12, 0.25, and 1.0% of (S)-5-OH DPAT in (R)-5-OH DPAT, respec- tively. In spite of the fact that the enantiomer of inter- est eluted on the tail of the main component, it was detectable in the lowest concentration investigated. Determinations of smaller impurities are preferably made by using D-ZGP in the mobile phase as this en- antiomer will give the reversed retention order of (S)- and (R)-5-OH DPAT (compare ref. 18). The optical pu- rity of (RWOH DPAT was estimated from chromato- graphic data (Fig. 2) to be larger than 99.7% ee.

Pharmacology

It is well known that agonists at dopamine (DA), 5-hydroxytryptamine, and norepinephrine (NE) recep- tors inhibit the biosynthesis of the corresponding monoamine.22 Thus, the monoamine synthesis can be used as an indicator of pre- and postsynaptic receptor activation. In the present study, the bioeynthesis was measured indirectly by determining the accumulation of (DOPA) in the DA-predominant (corpus striatum, limbic system) rat brain regions following aromatic L-

zation of the diastereomeric mandelates from- ether. amino -acid decarboxylase i n h i b i t i k with 3-

(R)- AND (S)-5-HYDROXY-2-(DIPROPYLAMINO)TETRALIN 93

Ib

( 5 1 - 5 - O H D P A l

OCI-I, I

OCH, I

(!)-5-OH D P A T

Reagents: (a) fractional crystallization of diastereomeric

di-p-toluoyltartrates; ( b ) H2, Pd/C; ( c ) C3H71,

K2CO3; (d) HBr ( 4 8

hydroxybenzylhydrazine (NSD 1015).19 Both DA D, re- ceptor agonists and antagonists can be detected in this biochemical model; the former induce a decrease of the DOPA levels via negative feedback systems, whereas the latter increase the DOPA levels. It may be noted that it is impossible to detect DA-receptor antagonists in biochemical models in which the monoamine stores have been depleted by reserpine pretreatment.

At a dose of 0.05 pmolkg sc, the potent D2-receptor agonist (S)-5-OH DPAT produced a significant de- crease (as compared to controls) of the DOPA levels in the striatal and limbic rat brain areas (Table 2). The effects of (R)-5-OH DPAT were investigated at doses of

% ) .

100- and 1,000-fold larger. At both doses tested, (R)- 5-OH DPAT induced a large increase in striatal DOPA levels. However, the limbic DOPA levels were not sig- nificantly affected by (R)-5-OH DPAT.

The effects of (S)-5-OH DPAT in the biochemical as- say are consistent with a powerful activation of DA D2 receptors. Thus, the present results support the classi- fication of (S)-5-OH DPAT as a potent DA D2-receptor agonist. The observation that (RM-OH DPAT dose- dependently increases the striatal. DOPA levels indi- cates that this enantiomer acts as a D,-receptor antag- onist in this brain area. However, none of the doses of the R-enantiomer was able to affect the limbic DOPA

94

SI

KARLSSON ET AL.

R

0.002A I

rnin

J

10 5

Fig. 1. Resolution of (*)&OH DPAT. Solid phase: Porous graphitic carbon. Mobile phase: 10 nMN-benzyloxycarbonyl-L-glycylproline (L- ZGP) in dichloromethane (80 ppm H,O).

0

-

rnin 10 5 0

.I I

min 5 0 5 0 5 0 Fig. 2. Estimation of enantiomeric purity in (R)-5-OH DPAT. Con-

ditions as in Figure 1. Authentic (R1-5-OH DPAT (top) and spiked samples (bottom) containing 0.12 (left), 0.25 (middle), and 1.00% (right) of the antipode. The Senantiomer appeared as a small peak after the major enantiomer in chromatograms of the spiked samples.

TABLE 2. Effects of (R)- and (S)&OH DPAT on the accumulation of DOPA induced by NSD 1015 in two parts

of the rat brain

Treat men t a DOPA (ng/gIb

Dose corpus Limbic Compound (pmoVkg, sc) striatum system

(R)-5-OH DPAT 5.0 1,558 f 72** 445 * 20 50 1,723 f 139** 498 f 32

(S)-5-OH DPAT 0.05 614 f 13** 242 ? 4.9** Control 1,162 f 32 450 f 18

"Compounds to be tested were given sc, 60 min before killing. NSD 1015 (287 pmol/kg, sc) was given 30 min before killing. bThe values are means * SEM (n = 7 and 6-7 in the control and experimental groups, respectively). Statistics: one-way analysis of variance followed by Dunnet's t test, **p < 0.01 vs controls.

levels. This indicates that (R)-5-OH DPAT is a less potent antagonist on limbic as compared to striatal D2 receptors.

CONCLUSIONS

In previous investigations of the pharmacology of the enantiomers of 5-OH DPAT, the stereochemical purity of the enantiomers has not been established. Therefore, it has not been possible to establish the profile of the distomer." In the present investigation we have estab- lished that the purity of (RM-OH DPAT is larger than 99.7% ee. Thus, the effect due to contaminating S- enantiomer should be minimal in a large dose range of (R)-5-OH DPAT. In fact, the observation that (R)-5-OH DPAT acts as an antagonist at a dose 1,000-fold larger than a dose of (S)-5-OH DPAT, which produces almost 50% reduction of the DOPA levels, indicates that the purity of the R-enantiomer is larger than 99.9% ee. It also indicates that (R)-5-OH DPAT is a weak antago- nist of DA D2-receptors in the striatum. Previously, it has been believed t h a t most dopaminergic 2- aminotetralins consist of one active and one inactive enantiomer. The present study indicates that dis- tomers which are structurally related to 5-OH DPAT may possess DA-receptor antagonistic properties. This conclusion is corroborated by recent studies on two related derivates: (lS,2R)-5-hydroxy-l-methyl-2-(di- pr~pylamino)tetralin,~~ a C1-methyl substituted deriv- ative of (RM-OH DPAT, is a moderately potent DA D2-receptor antagonist, whereas the antipode is a weakly potent DA D2-receptor a g ~ n i s t . ~ ~ Similarly, (S)- 2-~N-propyl-N-2-thienylethylamino)-5-hydrox~etralin stimulates DA D2 receptors, whereas the antipode be- haves as an a n t a g o n i ~ t . ~ ~

ACKNOWLEDGMENTS

Financial support obtained from the Swedish Natu- ral Science Research Council, the Swedish Medical Re- search Council, and the Swedish National Board for Technical Development is gratefully acknowledged.

(R)- AND (S)-5-HYDROXY-2-(DIPROPYLAMINO)TETRALIN 95

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