A reversible monoamine oxidase inhibitor, toloxatone: Structural and electronic properties

939

Eur J Med Chem (1992) 27,939-948 0 Elsevier,

Paris

A reversible monoamine oxidase inhibitor, toloxatone: structural and electronic properties* F Moureaul, J Woutersl, DP Vercauterenz, S Collinl, G Evrardl, F Durantl, F Ducrey3, JJ Koenig3**, FX Jarreau3 ‘Laboratoire de Chimie Moltkulaire Structurale; 2Laboratoire de Physico-Chimie Informatique, FacuMs Universitaires Notre-Dame-de-la-Pair, 61, rue de Bruxelles, B-5000 Namur, Belgium; 3Centre de Recherche Delalande, 10, rue des CarriPres, F-92500 Rueil-Malmaison, France (Received

24 April 1992; accepted 16 July 1992)

Summary - Toloxatone is a reversible MAO,-inhibitor, marketed as antidepressant (Humoryl@), with an original chemical structure. It differs from first generation irreversible MAOIs, known to induce covalent bonds with the enzyme active site. In order to understand the mechanism of the reversible inactivation of the MAO, as a first step, a detailed structural and electronic analysis was undertaken. An X-ray diffraction-crystallographic study showed that toloxatone is a planar molecule and brought to light hydrogen bonds and rt-rr interactions. MO calculations confirmed the planar structure as energetically favoured. Electronic analysis demonstrated a delocalization of both ring systems. The combined results give evidence for the potential of toloxatone to participate in reversible, long distance interactions with an appropriate partner. reversible

monoamine

oxidase inhibitors

/ toloxatone

/ SAR / stereo / electronic

Introduction Monoamine oxidase inhibitors (MAOIs) were introduced in psychiatry during the late 1950s and were the first of many antidepressants (ADS) to enter the clinical arena [ 11. However, their use became limited, to the profit of tricyclic-ADS, as they were found to induce severe food (tyramine rich/cheese effect) and drug interactions. The interest in MAOIs, however, remained because of the central role played by the MAO in the metabolism of monoamine neurotransmitters. As well as providing new drugs, pharmacological and neurochemical research has clarified how MAOIs work (for reviews see [2, 31). A second generation of MAOIs emerged with the appearance of selective inhibitors of the A (clorgyline) and B (deprenyl) forms. Since serotonin (5HT) and noradrenaline (NA) are preferentially deaminated by MAO, and since dopamine (DA)

*A preliminary report of this work was presented RICT Meeting in Caen, France, July 1991. **Correspondence and reprints

at the 27th

conformational

properties

/ STO-3G

/ PCILO

and tyramine are substrates of both forms, selective MAOIs appeared to open new vistas for the use of MAOIs as antidepressants: they should represent an improvement on the earlier drugs, as more selective in terms of both target symptoms and adverse side effects. However, in fact, clorgyline engenders covalent bonds with the active site of the enzyme and induces irreversible inhibition; it tends to lose its initial selectivity at high doses or with repeated administration. ln spite of these drawbacks, MAOIs, compared to tricyclic-ADS, appeared to have a wide spectrum of action that included the relief of anxiety disorders [4]. These results encouraged the pursuit of research on MAOIs. During the last decade a third generation of MAOIs appeared: the reversible and selective inhibitors, which from theoretical considerations were expected to have higher selectivity over a wide dose range and during chronic use, thereby inducing minimal adverse side effects. Toloxatone (fig 1) is the first drug that emerged from the reversible and selective MAOIs; it was marketed as an antidepressant (HumoryP) in France

940 H

.a+ ...’ @f.

Ccl3

--OH

r:--‘r/’

I R.Toloxarone

Ck,

II S-Toloxatonc

Fig 1. Structural formulae of R-toloxatone I and S-toloxatone II.

in 1985 [5]. Following the successful verification of HumoryP as a safe antidepressant, other MAOIs with this biochemical profile have been developed. At the most advanced stage are moclobemide (Roche) which entered its first market (Switzerland) in 1990 [6], and two drugs in clinical development, brofaromine (Ciba-Geigy) [7] and befloxatone (MD 370503, Delalande), a follow-up of toloxatone [8]. However, in biochemical studies with moclobemide and brofaromine discrepancies have been reported concerning their reversibility/irreversibility [9]. On the contrary, toloxatone and befloxatone are pure, reversible and competitive and selective inhibitors of MAO,. These distinct biochemical profiles can be related to a fundamental difference in chemical structure: moclobemide and brofaromine present an amino function which can behave as a substrate of the MAO, whereas toloxatone is an aryloxazolidinone devoid of any amino function. In order to rationalize these differences and to explain the reversibility of toloxatone’s MAO inhibition, a program was engaged to elucidate the mechanism of action at the molecular level. This paper describes the first part of this approach: a thorough analysis of the structural (X-ray diffraction of monocrystals), conformational, and electronic properties (ab initio and semi-empirical molecular orbital calculations) of toloxatone. First, the structural and electronic properties of toloxatone will be analyzed. Then the conformational space of toloxatone will be studied in comparison with some active or inactive rigid derivatives. Finally, a computerized graphic superimposition of these compounds will be performed in order to try to determine the active conformation of toloxatone.

presented in figure 2. Bond lengths and valence angles are identical for both isomers (fig 3). Characteristic torsion angles of the (R) enantiomer (I, fig 1) are reported in table I; those of the (S) enantiomer (II, fig l), sign apart, are identical. Final atomic coordinates are given in table II. The oxazolidinone ring is practically planar. Only the C, and C., atoms are slightly out of the plane (table III). N, is trigonal as shown by the small deviation from the mean plane (C,-C&J and the sum of the valence angles around N, (table III). The C,-O, and C,-N, bond length values, 1.346(2) A and 1.354(2) A respectively (fig 3) fall between standard single and double bonds (C-O = 1.43 A; C = 0 = 1.23 A; C-N = 1.47 A; C=N = 1.27 A) and indicate an -I

Fig 2. Stereoview of the molecular conformation with vibrational ellipsoids (probability 50%) of a) R-toloxatone I and b) S-toloxatone II.

2*,. ,,8,6 ,,,, 5,2*,gf-Gs P120.6

Results and discussion Structural

and electronic study

The structural analysis by X-ray diffraction was carried out on toloxatone (fig l), the racemate. The molecular conformation for both enantiomers is

119.8

22.7*26.P"1~:,o.y

1182121.7

n1.7

120.1

O

129 3 Y

120.6

0

Fig 3. Atom numbering, bond lengths (A), and valence angles (“) for toloxatone; maximum estimated standard errors (esd’s) 0.005 A and 0.3”.

941 Table I. Main torsion

angles of R-toloxatone

c,-O&-C,

I (“).

this study, the internal co-ordinates of the heavy atoms of R-toloxatone I were taken from the crystallographic resolution except for the torsion angles. Coplanarity between the phenyl and oxazolidinone rings was imposed, as was a torsion angle 0,-C&-O, of 200” (see Structure-activity relationships). The atomic and interatomic populations calculated according to Mulliken (fig 4) confirm the delocalization within the oxazolidinone ring. The C,-0, and C-N, bonds are characterized by a 7~overlap population (contribution of interatomic rr overlap with respect to total overlap) of 13.3% and 11.2%, respectively. It should be remembered that the 7c contribution in benzene is 22%. The 7c type contribution for N,-C,, 7.2%, is much weaker. These observations confirm the hypothesis, deduced from the crystallographic analysis, as to the electronic delocalization within the oxazolidinone ring, but invalidate that of a conjugation between the phenyl and oxazolidinone rings. The diagram proposed in figure 5 takes these observations into account.

-118.4(2)

o,-C&-O,

67.3(2)

C,-N&,-C,,

-177.2(2)

C,-N,-C,-C,,

2.4(3)

electronic delocalization within the oxazolidinone ring. The N,-C, intercyclic bond length, 1.414(3) A, is also intermediate between a single and a double C-N standard bond. Such a value had also been observed in a series of previous crystallographic investigations of other 3-phenyl oxazolidinones [ 10-211 and considered to indicate some delocalization. In order to improve the description of the electronic structure of toloxatone, ab i&o STO-3G molecular orbital (MO) calculations were undertaken for the more active isomer I, R-toloxatone (table IVa). For

Table II. Final atomic co-ordinates

(X

104) and B,

values with esd’s in parentheses

I.

for R-toloxatone

(Be4 = 8 7~2U,, (A”) and Ueq = 1/3~i~ju,ai*aj*a,aj)

xla 0

(1)

c 0 c c

(2) (3) (4) (5)

0

(6)

c

(7)

N

(8)

c C C C c C C H H H H H H H H H H H H H

6399( 5878( 4891(l) 4401(l) 3022( 2892(

YJb 1) 1) 1) 1)

Eg:j 7102(l) (9) (10) 7163(l) (11) 8090(2) (12) 8932(2) (13) 8888(l) (14) 7968(l) (15) 9801(2) 4427(20) (4) (71) 5837(19) (72) 4779(19) (51) 2569(20) (52) 2648(20) 3090(20) (6) (10) 6539(27) (11) 8095(24) (12) 9521(27) (14) 7975(20) (151) 10614(31) (152) 9501(31) (153) 9861(28)

5030( 1) 4110(l) 4225( 1) 3007( 1) 2904( 1) 2875( 1) 2073( 1) 2879( 1) 2379( 1) 1071(l) 555(2) 1309(4) 2620( 2) 3129(l) 3471(5) 2879( 19) 1488(20) 1441(20) 3586(21) 2164(20) 3725(23) 474(28) A56(28) 1083(24) 3974(20) 3478(27) 4247(31) 3259(27)

Z/C

1060( 1) 1474( 1) 2267( 1) 2616(l) 1980( 1) 471(l) 2006( 1) 1270( 1) 504(l) 372(2) -403(2) -1028(2) -882( 1) -113(l) -1563(4) 3643(24) 2666(23) 1326(23) 2319(24) 2322(24) 117(26) 777(30) -383(30) -1608(31) 9(27) -1033(34) -1704(35) -2581(38)

B -7

UII

5.1 l(2) 3.86(2) 4.49(2) 4.35(3) 4.97(3) 5.14(2) 4.22(3) 3.88(2) 4.17(2) 5.47(3) 7.29(5) 7.37(6) 6.25(5) 5.02(3) 8.54(8) 4.66 4.58 4.58 5.21 5.21 5.29 6.71 7.11 7.19 5.21 7.90 7.90 7.90

756(9) 588(9) 692(8) 705( 10) 659( 10) 723(9) 634(9) 562(8) 546(9) 742( 12) 910(16) 758(15) 502(10) 557(9) 633( 13)

u22

397(5) 384(8) 441(5) 529(9) 662( 10) 662( 8) 408(8) 373(6) 544(9) 573( 10) 885( 16) 1315(24) 1281(20) 751(12) 1764(35)

u33

789(9) 485(8) 592(8) 430(9) 593( 10) 553(8) 549(9) 540( 8) 476(9) 723( 12) 929( 17) 716(13) 579(12) 590(10) 868( 19)

u23

UI3

lOO(6) ‘g; -76(5) 27(6)

128) W5) 87(6) 47(5) -9(6) -109(9) -311(13) -227(15) A2(12) 31(9) 109(21)

136)

155(5) 119(8) 188(9) -l(5)

WV 75(5) -16(6) -78(9) -86(13) 40(12) 12(9)

2W)

179( 12)

UI2

:;:g; -26(5) -86(8)

-42@)

-126(5) I::#

W6)

141(9) 344(13) 392(16) 142( 10)

W9)

24( 16)

942 Table III. Compared values of main structural features for R-toloxatone, I and previously published oxazolidinones [ 10-12, 14, 15, 17-211.

Deviations from mean plane [N&-0,-0,]

R-toloxatone I

Other oxazolidinones

-0.030 0.043

0.017-0.270 0.016-0.211

(A)

2 Sum of valence angles around N, (“)

359.9

Deviation of N, from mean plane [C&,-C,] q-0, (‘Q G-N, (A>

(A)

359.4-360.0

-0.014 1.346 1.354

0.0094.065 1.337-1.366 1.335-1.393

2.9

4.4-24.3

Dihedral angle between the mean plane [N&,-0,-0,] and the phenyl ring (“) Number of compounds having torsion angle O&&,-X, (with X = 0 or N) equal to: -% Cl 180”

1

Table IV. In vitro MAO1 activity, Experimental protocols for details).

a. Toloxatone

b.

:I III IV V

C.

GI VIII

IC,,

(M)

(see

MAO, (5-HT)

MAO, (PEA)

3.0 lo-6 1.5 lo-6 > l&5

6.0 1O-5 > l&5 > 10-s

1.8 10-s > 10-s >1&5

> > > 2.9 > 1.1

1.8 1O-s > lct5 1.2 l&s

72 1

10-5 lck5 10-S 1e7 1cks 10-7

Fig 4. Ab initio MO STO-3G Mulliken population analysis for toloxatone: a) atomic charges of heavy atoms (e) and b) tt overlap percentages (%).

Fig 5. Delocalization scheme proposed for toloxatone. Despite the poor conjugation between the two rings, toloxatone adopts a nearly planar conformation in the crystalline state. The angle between the phenyl ring and the (N&-0,-0,) plane is 2.9” (table III). This coplanarity allows for optimal crystal stacking. In fact, the mean distance between the phenyl ring of one molecule of toloxatone and the oxazolidinone ring of another molecule stacked parallel to the first is shorter than the sum of the van der Waals radii (3.3-3.5 8, with respect to 3.5-3.7 A). This is indicative of important ~-rr type forces between the phenyl ring of one molecule and the oxazolidinone ring of the other (fig 6). The conformation obtained by X-ray diffraction would seem to be influenced by the stacking forces observed in the crystalline state. Besides the van der Waals forces between the different rings of the molecules, the crystal packing is further assured by intermolecular hydrogen bonds (fig 7). The CH,OH chain is folded, 0,-C&-O, = 67.3(2)“, so as to enable the formation of a hydrogen bond between its OH group and the 0, oxygen of the oxazolidinone ring of another molecule and vice versa

943 Table V. Characterization of the intermolecular hydrogen bond between toloxatone molecules in the crystalline state. D-H...A

D...A (A)

H...A (A)

D-H . ..A (“)

0,-H,. . .O,*

2.797 (2)

1.842 (25)

160.2 (2.2)

*1-x, l-Y, -z.

Fig 6. Stereoview of the crystal stacking showing K-X interactions between toloxatone molecules.

-I Fig 8. Stereoview of the crystal packing showing intermolecular hydrogen bonds between toloxatone molecules. 10

a i

Fig 7. Stereoview of the crystal packing of toloxatone. Dotted lines indicate hydrogen bonds.

(table V). The presence of dimers in the crystal is due to the formation of two symmetrical hydrogen bonds per pair of molecules (fig 8). The conformational analysis of the R-toloxatone enantiomer in the isolated state, carried out in parallel by MO calculations (see Experimental protocols) showed that the stability of the planar conformation is not merely due to crystal stacking; there is also a clear stabilization for C,-N,-C,-C,, torsion angles close to 0” and 180” (fig 9). The relative stability of these geometries should be governed by the formation of an intramolecular pseudo-hydrogen bond between the hydrogen on C,, or C,, of the phenyl group and 0, of the carbonyl group as observed in the crystalline state. Indeed the 0,. . .C,,, 2.903(2) A, and 0,. . .H,,, 2.310(24) A, bond lengths are shorter than the corresponding van der Waals contact distances, 3.35 A and 3.15 A, respectively. The stability of the planar conformation is thus an intrinsic property of the molecule.

0

60

120 180 C(2)-N(a)-C(9)-C(lo)

240

300

360

Fig 9. Ab initio MO STO-3G conformational energy (kcal/mol) for toloxatone as a function of the torsion angle C,-N&,-C,,.

The structural and electronic properties of toloxatone brought to light in this work have already been observed in previous studies of analogous oxazolidinone derivatives [lO-211. To summarize, these derivatives present: 1) a more or less planar oxazolidinone ring; 2) an electronic delocalization within the oxazolidinone ring; 3) coplanarity between the phenyl and oxazolidinone rings; and 4) three privileged conformations of the CH,OR (R = H, CH,) chain corresponding to O&&,-O, torsion angles of + 60”, - 60”, and 180” (table III).

944

Hypothetical tive site

interaction

scheme with the enzyme ac-

The detailed analysis of the forces responsible for the crystal packing of toloxatone brings to light the functional groups of the molecule which could participate in the formation of weak and reversible bonds with the enzyme: 1) the phenyl and oxazolidinone rings could contribute to the stabilization of the complex between the MAO, active site and toloxatone by means of rc-rr type interactions; 2) the 0, oxygen could play the role of proton acceptor in an intermolecular hydrogen bond; and 3) the 0,H function could participate in a hydrogen bond either as proton donor as observed in the crystalline state or as proton acceptor. This latter possibility is confirmed since the O-methylated derivative of toloxatone retains the affinity towards the MAO, [22]. MAO,-1 structure-activity

relationships

In order to ascertain the relative orientation of the functional groups necessary for the inhibitory activity of toloxatone, three bridged oxazolidinones were also considered (fig 10). In two of the three cases, an important loss of the affinity for the MAO, is observed (table IVb). Only the oxazolidinone III bridged by two carbons shows a very high affinity. The crystal structures of these three oxazolidinones were resolved by X-ray diffraction (Moureau et al, manuscript in preparation). The two carbon bridge between the phenyl and oxazolidinone rings forces the molecule III to adopt a planar structure. Figure 11

. 8’

a

,__--:

i

‘. ‘.

#ii!?

Fig 11. Stereoview of the molecular superimposition of R-toloxatone I (solid lines) and compound III (dotted lines).

shows the close structural similarity between this compound and R-toloxatone (compound I) in their crystalline conformation. On the other hand, the three carbon bridge (compound IV) induces a 58.3” angle between the phenyl and oxazolidinone rings. A priori the activity of compound III and the inactivity of compound IV can be interpreted in terms of respective coplanarity and non-coplanarity between the phenyl and oxazolidinone ring (fig 12). However, derivative V bridged above the oxazolidinone ring is inactive despite an inter-ring angle close to 0”. The inactivity of this compound must thus be attributed to the orientation of the CH,OH chain (fig 13). The O,-C&-O, torsion angle fixed at 86.1” by the bridging should not correspond to the active conformation. A theoretical study was undertaken to analyse the conformational freedom of the CH,OH chain. Two

W OH

Fig 10. Planar structure formulae analogues III, IV, and V.

of bridged

oxazolidinone

Fig 12. Stereoview compound III (solid

of the molecular superimposition of lines) and compound IV (dotted lines).

945 W

\ C,-OCH,

/ G--c4 I

\

Fig 13. Stereoview of the molecular superimposition of R-toloxatone I (solid lines) compound V (dotted lines).

oxazolidinones substituted on C, by a l-methyl methoxymethyl group (fig 14) which present particularly high stereoselectivity were considered. The erythro pair VI (4R, 5S and 4S, 5R) shows good MAO,--1 activity whereas the threo pair VII (4R, 5R and 4S, 5s) is devoid of activity (table IVc). The structure of these compounds had previously been determined by X-ray diffraction [ 17, 201. As the (4R) enantiomer always shows a greater affinity for the MAO, active site than the (4s) enantiomer, the conformational study was carried out on the (4R, 5s) and the (4R, 5R) stereoisomers VIII and IX. For this analysis, the semi-empirical molecular orbital PCILO method, widely used as well adapted for conformational studies involving no particular electronic effects such as intramolecular hydrogen bonds and delocalization, was chosen. Figure 15 clearly shows critical values for the 0,-C&-O, torsion angle between 190” and 280”. The conformations corresponding to these angles are stable in the case of the active (4R, 5s) isomer VIII but forbidden in the case of the inactive (4R, 5R) isomer IX. Taken together, the stereoselectivity and the conformational properties of these molecules seem to indicate that the active conformation of the CH,OH chain requires a 0,-C&-O, torsion angle of 200°, which corresponds to the minimum energy conformer within the critical interval. This particular active conformation can easily be adopted by R-toloxatone (fig 16) and the energy of this conformation for R-toloxatone I is only 1 kcal mol-1 higher than the global minimum and moreover situated in an energy well. Conclusions The determination of the three-dimensional (3D) structure (X-ray diffraction) coupled with conforma-

Cti

Fig 14. Structural formulae of compounds VI, VII, VIII, and IX.

280

(4K3

(4R 5R) Ix

Fig 15. Values of the allowed (E I 5 kcal/mol, white zones) and forbidden (E 2 5 kcal/mol, dotted zone) 0,-C&-O, torsion angle (“) for VIII and IX calculated by the PCILO method. tional and electronic characterization (by the ab initio molecular orbital method) of toloxatone demonstrated the existence of an electron delocalization within the oxazolidinone ring and the stability of the planar conformation. The detailed analysis of the forces responsible for the crystal packing brought to light which functional

946 10

was used for all X-ray measurements using an Enraf-Nonius CAD-4 diffractometer (Cu K,, h = 1.54178 A). The lattice parameters were obtained from least-squares refinement of 25 medium angle reflections. Further data collection details are provided in table VI. The intensities collected were corrected for Lorentz and polarization effects. The structure was solved by the application of direct methods using SHELX86 [26] and refined by the method of full-matrix least-squares using SHEL X76 [27]. Hydrogen atoms of the methyl group were calculated, all other hydrogen atoms appeared in a difference Fourier map. Anisotropic temperature factors were used for all non-hydrogen atoms and isotropic ones were used for all hydrogen atoms (Ue4 of the carrier atom incremented by 0.01). The final weighted least-squares cycle gave R = 0.06 with W = 1.0/(02(F) + 0.002 F). The geometric analysis was performed with XRAY76 [28].

1

-1. 06 P

Ab initio molecular orbital quantum calculations 0

Fig

60

120 180 O(3)-C(4)-C(5)-O(6)

240

300

360

16. PCILO conformational energy (kcal/mol) of I as a function of the 0,-C&,-O, torsion

R-toloxatone angle (“).

groups of toloxatone are likely to interact with the MAO active site: 1) the phenyl and oxazolidinone rings, characterized by their electron delocalization are able to interact by means of 7c--7~type van der Waals forces; 2) the carbonyl oxygen can play the role of proton acceptor in a hydrogen bond, as can 3) the hydroxyl oxygen. The importance of the coplanarity between the oxazolidinone and phenyl rings was confirmed by the comparative study of toloxatone and some rigid derivatives. The preference of this coplanarity for MAO inhibition and the potential of toloxatone to interact by means of 7c--7~type bonds leads to the hypothesis of a privileged fixation of toloxatone with the flavin cofactor of the MAO; this particular entity being characterized by a known electron acceptor planar structure [23]. This hypothesis is supported by spectrophotometric measurements. The results of this study will be further discussed in another paper [24]. We thus propose that toloxatone, like other inhibitors of MAO [25], should exert its inhibitory activity by binding to the cofactor of MAO but in the specific case of toloxatone this binding is of a completely different nature as resulting from non-covalent, ie long distance forces. This should be the mechanism of the reversible inhibition by this compound at the molecular level. Experimental

protocols

Crystallography Toloxatone crystallized from a toluene solution at room temperature. A colourless prismatic crystal (0.30 x 0.20 x 0.08 mm)

The RHF (restricted Hartree Fock) LCAO-MO-SCF (Linear Combination of Atomic Orbitals-Molecular Grbitals-Self Consistent Field) method was used to scan the conformational space and to characterize the electronic properties. Calculations were nerformed at the STO-3G degree of sonhistication of the LCAG expansion of the molecul& orbitals’as introduced by Pople [29, 301. Atomic charges and interatomic x-overlap percentages were calculated by-me Mulliken charge population analvsis 1311. The bi-electronic integral cut-off and convergence on-the density matrix threshol& were fixed at 1O-8 au (atomic units) and 1t7, respectively. Our experience has shown that within this basic set, the computed total energy has

Table VI. X-ray diffraction Molecular formula Molecular mass Crystal system Space group a (8) b (A) c @I R (9

207.2 Monoclinic

%c

(mm)

Diffractometer Absorption coefficient (cm-l) WJW 28 range (“) Unique data Unique data with I 2 2.0 d (I) R final wR final Max and min heights in final A-Fourier (e-.A3)

(4@,,

of toloxatone.

W-WO,

i’(A3) z 6, (g cm-3) Temperature (K) Crystal dimensions Radiation

data parameters

10.491(l) 10.649( 1) 9.403( 1) 96.452(3) 1042.9 4 1.32 293 0.30 x 0.20 x 0.08 Graphite monochromated cu k, (h = 1.54178 A) Enraf-Nonnius CAD-4 7.09 440.0 O-144 2047 1661 0.05 0.06 With w = l.O/ [oz (F) + 0.002 p) -0.22 and 0.14 0.30 for Z/C of C,,

947 at least seven significant digits; it corresponds in our calculations to a numerical error of about 0.1 kcal mol-1. Four digits were taken into account for the atomic charges and the overlap populations (in electrons). The internal co-ordinates of the heavy atoms of R-toloxatone I were taken from the crystallographic resolution except for the torsion angles. For the evaluation of the electronic properties, coplanarity between the phenyl and oxazolidinone rings was imposed, as was a torsion angle O&&-O, of 200°C (see Structure-activity relationships). The interatomic distances and valence angles of hydrogen atoms were fixed at 1.084 or 1.090 A and 120.0 or 109.471”, depending on the hybridization of the carrier atom. The phenyl and oxazolidinone rings were placed in the xz plane in order to differentiate the n-electron contribution (ie the 2P,, component) from the total contribution. For the conformational analysis, a systematic variation (step: ISo) of the C,-N&-C,, torsion angle was considered. All calculations were performed with the GAUSSIAN 86 program [32]. Semi-empirical

quantum conformational

calculations

The conformational analysis of the CH(CH,)OCH, chain of VIII and IX and of the CH,OH chain of R-toloxatone I was performed with PCILO (Perturbative Configuration Interaction using Localized Grbitals), a semi-empirical quantum method 133. 341. The use of this method in conformational analvsis has . already been widely described [35]. Crystal structure data were used as input data. The hydrogen positions were fixed as for the ab initio calculations. The potential energy curve was built by systematic variation (increment between successive step 10’) of the torsion angle using an enhanced DPCILO (Differential PCILO) version [36]. L

I

Reaction mixture MAO activity was measured in test-tubes containing 500 pl of a reaction mixture of the following composition: 100 pl homogenate, 100 pl substrate, 50 pl test compound (or 50 pl H,O for controls and blanks), 250 pl buffer (sufficient quantity for 500 ~1). For the results given in table IVa and IVb, the homogenate was preincubated 20 min at 37°C with the buffer, with or without (controls and blanks) the test compound and the reaction started by adding the substrate and incubating with stirring at 37°C for 5 min ([14C]-5-HT, 65 pM) or 1 min ([14C]-PEA, 40 pM). For the results given in table IVc, the substrate was preincubated with the test compound for 5 min at 37°C and the reaction started by adding the homogenate and incubating with stirring for 10 min (substrate [14C]-5-HT, 480 pM) or 2 min (substrate [t4C]-PEA, 12 pM). Acidification and extraction of metabolites The -reaction was stopped by precipitating the proteins with 200 ul 4 N HCI and bv olacine the test-tubes in ice. Blanks were’ obtained by precipit’ating The proteins before adding the substrate (table IVa and IVb) or homogenate (table IVc). The metabolites formed were extracted with 7 ml of a (v/v) toluene/ethyl acetate mixture. The test-tubes were shaken 10-15 min. frozen, and 7 ml of the unoer (oreanic) ohase removed. This sample was shaken 5 min.&id the;adioa&ivity counted by liquid scintillation in 8-10 ml of a toluene/2.5 diphenyloxazole mixture (4 g 1-l).

~

Molecular

graphics

Real-time interactive superimpositions of molecular models were done using IFMFIT (Imoroved or Interactive Flexible Molecular Fittigg) [37, 38] implemented into KEMIT [39]. KEMIT is an in-house device-independent molecular graphics system developed in Fortran with the IBM graPHICS software [40]. All calculations, ab initio and semi-empirical, were done using the IBM 9377/90 - FPS M64 computer system of the Scientific Computing Centre of the University of Namur.

Results The mean values for controls and assays with test compound were calculated after substraction of the mean values of the corresponding blanks. The percentages inhibition was calculated by comparing the mean values of assays with test compound with those of controls and the graphic representation of percentage inhibition versus concentration of test compound gave the I&, (concentration of test compound that reduces MAO activity by 50%).

Acknowledgments We thank the National Belgian Foundation for Scientific Research (FNRS), IBM-Belgium, and the Facultes Universitaires Notre-Dame de la Paix for the use of the Namur Scientific Computing Facility. SC thanks the ENRS for her Research Associate position and JW for his Research Assistant position.

Biochemistry

References The in vitro MAO activity was measured in rat whole brain homogenates using [i4C]-5-HT (serotonin) as substrate for MAO, or [14C]-PEA (phenylethylamine) as substrate for MAO,. Preparation

of homogenates

Male Sprague-Dawley rats weighing 125-300 g were used. After killing by decapitation, the brain was rapidly removed, weighed and homogenized in a buffer solution (Na,HPOd NaH,PO,, 0.1 M, pH 7.4) at 4°C using an Ultra Turrax (or Polytron) homogenizer at maximum speed for 10 s. The composition of the homogenates was 1 g tissue/20 ml buffer for compounds III-V (table IVa and IVb) or 1 g tissue/l6 ml buffer for compounds VI-VIII (table IVc).

Rudorfer MV, Potter WZ (1989) Drugs 37,713-738 Youdim MBH, Findberg JPM, Tipton KF (1988) Handb Exp Pharmacol90,119-192 Dostert PL, Strolin Benedetti M, Tipton KF (1989) Med Res Rev 9,45-89 Tyrer P, Harrison-Read P (1990) Znt Rev Psychiatry 2, 33 I-340 Ferrey G, Rovei V, Strolin Benedetti M, Gomeni C, Languillat JM (1985) Clinical and Pharmacological Studies in Psychiatric Disorders (Burrows GD, Norman TR, Dennerstein L, eds) J Libbey, Paris, 83-86 Stab1 M, Biziere K, S&mid-Burgk W, Amrein R (1989) J Neural Transm S28,77-89 Schiwy W, Heath WR, Delini-Stula A (1989) J Neural Transm S28,33-44

948

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

23 24 25 26

Jarreau FX, Rovei V, Koenig JJ, Schoofs AR (1989) French Pat 2653017 Waldmeier PC, Stiicklin K (1990) Eur J Pharmacol 180, 297-304 Durant F, Lefevre F, Evrard G, Michel A (1982) Cryst Struct Commun 11,975-98 1 Durant F, LeRvre F, Norberg B, Evrard G (1982) Cryst Struct Commun 11,983-990 Durant F, Lefevre F, Evrard G, Michel A (1982) Bull Sot Chin-t Belg 91,83 l-832 Durant F. Butkens F. Evrard G. Lamotte C (1982) ~ , Bull Sot dhim Belg 9 1,825-830 ’ Durant F, Lef&vre F, Evrard G, Lamotte C (1982) Bull Sot Chim Belg 9 1,949-950 Durant F, LeRvre F, B&ens F, Evrard G, Michel A (1982) Crvst Struct Commun 11.1825-1831 Dun&t F: Bufkens F, Lef&vre’ F, Evrard G, Michel A (1985) Acta Ctystallogr C41,243-246 Durant F, Bufkens F, Lefevre F, Lamotte C, Evrard G (1982) Cryst Struct Commun 11, 1833-1839 Durant F, Lefevre F, Bufkens F, Evrard G, Michel A (1982) Bull Sot Chim Belg 91,925-929 Durant F. Lefevre F. Bufkens F. Norberu B. Evrard G (1982) Cryst Struct Cammun 11, 1925-1932 ’ Durant F, Norberg B, Bufkens F, Evrard G (1984) Bull Sot Chim Fr II, 183-186 Durant F, Norberg B, B&kens F, Evrard G (1985) Acta Crystallogr C41,247-249 Dostert P, Strolin Benedetti M, Boucher T, Langlois M (1984) Monoamine Oxidase and Disease (Tipton KF, Dostert P, Strolin Benedetti M, eds) Academic Press, New York, 582-584 Slifken MA (1971) Charge Transfer Interactions of Biomalecules (Slifken MA, ed), Academic Press, London, 132-172 Moureau F, Wouters J, Vercauteren DP, Evrard G, Durant F, Ducrey F, Koenig JJ, Jarreau FX (1991) Poster N-17,27th RICT Meeting, Caen, France, July 1991 Silverman RB. Hoffman SJ (1984) Med Res Rev 4. 415-447 ’ Sheldrick GM (1986) In: SHELX86. A Program for Crystal Structure Determination. Institut fur Anorganische Chemie der Universitlt, Gijttingen ~

I

27 28

29 30

33 34 35 36

37 38 39 40

Sheldrick GM (1976) SHELX76. A Program for Crystal Structure Determination. Univ Cambridge, Cambridge Stewart JM, Machin PA, Dickinson CW, Ammon HL, Heck M, Flack H (1976) XRAY76, Tech Rep TR-445. Computer Science Center, University of Maryland, MD Hehre WJ, Stewart RF, Pople JA (1969) J Chem Phys 51, 2657-2664 Pople JA (1977) Applications of Electronic Structure Theorv. Modern Theoretical Chemistrv Series. Vol 4 (Schaefer ‘III HF, ed) Plenum Press, New York; l-27 Mulliken RS (1955) J Chem Phys 23, 1833-1846 Frish MJ, Binkley JS, Schlegel HB, Raghavachari K, Melius CF. Martin RL. Stewart JJP. Bobrowicz FW. Rohlfing CM, Kahn ‘LR, Defrees ’ DJ, Seeger R; Whiteside RA, Fox DJ, Fleuder EM, Pople JA (1988) GAUSSZAN86. Carnegie-Mellon Quantum Chemistry Publishing Unit, Pittsburgh, PA Diner S, Mahieu JP, Jordan F, Gilbert M (1969) Theor Chim Acta 15, 100-l 10 Jordan F. Gilbert M. Mathieu JP. Pincelli U (1969) \ I Theor Chim Acta 15,21 l-224 ’ Tollenaere JP, Moreels H, Raymaeckers LA (1980) Drug Design Vol 10 (Ariens EJ, ed) Academic Press, NY, 72-l 18 Daudey JP, Malrieu JP, Rojas 0 (1975) Localization and Delocalization in Quantum Chemistry Vol 1 (Chavet 0, Daudel R, Diner S, Mahieu JP, eds) Reidel, Dordrecht, 155-205 Lejeune J, Michel AG, Vercauteren DP (1986) J Mol Graphics 4, 194-199 Lejeune J, Michel AG, Vercauteren DP (1986) J Comp Chem 7,739-744 Vanderveken DJ, Vercauteren DP (1989) KEMIT, a Molecular Graphics System, Rel 1.2. Facultes Universitaires Notre-Dame de la Paix, Namur Chin S. Vercauteren DP. Luken WL. Re M. Scateni R. Tagliavmi R, Vanderveken DJ, Baudoux’ G (1989) Modern Techniques in Computational Chemistry: MO TECC 89 (Clementi E, ed) ESCOM Publ, Leiden, 499-546