PAF-receptor. III. Conformational and electronic properties of PAF-like agonists and antagonists

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PAF-receptor *. III. Conformational and electronic properties of PAF-like agonists and antagonists J o s e t t e L a m o t t e - B r a s s e u r L2, G e o r g e s D i v e t,3 A a z d i n e L a m o u r i 4, Fran~oise Heymans 4 and Jean-Jacques Godfroid 4 I Laboratoire de Mk'robiologie, Institut de Chimie. l i~ge (Be(~ium). 2 Laboratoire tie Cristallographie, Institut de Physique Liege (Belgium), 3 Laboratoire de Chimie Pharmaceutique. Institut de Pharmacie. Li~'ge (Belgium) and 4 Laboratcire de Pharmacochimie Mol&'ulaire. Unirersitt; Pans, Paris (France)

tReceived 6 Februar-/ tqt)l)

Key words: PAF agonist:PAF antagonist:Confi)rmation:Electrostaticmap. three-dimensional:Synthesis in order to compare electronic and conformational properties of PAF-agonists and PAF-antagonists, 14 analogues structurally related to PAF were studied. A common conformation of the glycerol backbone was present in all agonists and all constrained or flexible antagonists. The distinction between agonists and antagonists appears to be casted on position-2 where the folded conformation of the substituent for agonists should be the most probable. In position-3 the gauche conformation can be adopted by all the analysed compounds. The electrostatic potential weU at --30 k c a l / m o l stretches to the carbonyl group in position-2 in the folded conformation of the agonists. On the contrary, in constrained antagonists, a second negative zone appears around the carbamate group. Given the modelling results, the triethylammonium PAl? analogue considere~l in literature as a weak agunist, was resynthesized and proved to be more potent than previously reported. These experimental results confirm our hypothesis in terms of a common conformation of agonist and antagonist PAF-iike molecules.

Introduction

Platelet activating factor (PAF) 1 is an autacoid phospholipidie mediator involved in a large range of pathological conditions including inflammation, vascular disorders and shock (for a review see Ref. 1,2). Its systemic effects by i.v. injection (rats, guinea-pigs, dogs, baboons) are hypotension, bronchoconstriction, pulmonary hypertension, increased vascular permeability, neutropenia, thrombocytopenia and death. After elucidation of its chemical structure [3-5] and its total synthesis [6-9] attention was first focused on

* PAF, platelet-activatingfactor. Part I, see Ref. 16; part 2. see ReL

20. Abbreviations: MNDO, modified neglect of differential overlap; STO-3G, Slater type orbital-3 Gaussians; CNDO, complete neglect of differential overlap; (Q)SAR, (Quantitative)structure-activityrelationships. Correspondence: J.-J. Godfroid, Laboratoire de Pharmacoch!mie Mol~culaire. Universit~Paris 7, 2 place Jussieu. 75251 Paris. Cedex 05, France.

structaral requirements inducing agonistic activities (platelet aggregation, hypotension, thrombocytopenia, bronchoconstrlction) [10-12]. In parallel, antagonistic activities based on functional modifications of glyc,:~rol skeleton were investigated (see Ref. 2, 11, 13, 14). Simultaneously, new antagonists, natural or synthetic, appeared in the literature; research into these compounds is witnessing rapid development in recent years [!,2,13]. Considerable attention has also been focused on the existence of specific binding sites which have been confirmed in rabbit, dog, guinea-pig and human platelets, leukocytes and in various tissues (lung, brain) [15]. The unusually low number of receptors per cell for this mediator explains the difficulties in attempting to isolate the maeromolecule(s) representing this site of high affinity, differences of functionality probably depending on the cellular origin. In initial papers [16,17] we compared the common electronic features generated by non-PAF-like molecules belonging to heterogeneous series, enabling us to propose a mirror image of the receptor. These compounds (gingkolides, kadsurenone, 2,5-diaryltetra-

hydrofurans, triazolothienobenzodiazepins) presented a major common electronic feature, the so called 'Cache-oreilles' ('ear-muffs') effect, i.e., two negative electrostatic potential clouds generated at 180 °C from each other. As a first approximation, these results suggested an interaction of the molecule with a multipolarized cylinder of 10-12 A, diameter in the platelet membrane. A moderate hydrophobic appendage could constitute a third point of anchorage which could better position the 'Cache-oreilles' system in the receptor. Dealing with rigid highly potent antagonists [18,19], this study only refers to negative electrostatic potential generating functions and does not include any zwitterionic molecules. Before further proposals can be made concerning the structure of the PAF receptor, the electronic properties and conformation of PAF itself and its structurally related agonists and antagonists need to be investigated. In this paper we attempt to define the common conformational and electronic features which could explain the agonistic or antagonistic activities of PAF analogous molecules. SAR and QSAR studies have previously been used to determine the following major structural and functional requirements for PAF receptor agonists, [10,12]: (i) the lipophilicity of the fatty chain in position-1 is the key factor explaining PAF interactions in a hydrophobic pocket of the receptor. This feature has been revealed by a parabolic curve between the chain's hydrophobic constant and the platelet stimulation [20]; (ii) at this position-l, an ether oxide function is an absolute requirement for a potent agonistic activity [21]; (iii) the steric effect in the glycerol skeleton dramatically decreases platelet stimulation [10-12]; (ix') at position-2, chain length and steric hindrance are strictly limited to small groups [10-12]; (v) at position-3, all functional modifications decrease agonistic activities, except the variation of the onium function including bulky alkyl or cycloalkyl groups [10-13]. Taking account of these qualitative results, the conformational and electronic distribution will be discussed in terms of the biological activity of some PAFagonists and antagonists. Moreover, well k:,own and new PAF analogues have been synthesized in order to verify our hypothesis. Materials and Methods Selected compounds

14 molecules with PAF agonist and antagonist activity have been chosen. Tables I - I V describe the four natural variations and differences in functionalit.j related to the three sites of substitution on glycerol and the type of ammonium head [1,2,10-14].

TABLE l PAF analogues: variation of tire oninm group in position 3

Compound Ia 2b 3 h.c

Z N(CHs)3 NEt2(CH3) NEt3

PAF N-diethyl PAF N-triethyl PAF

4 h'c

N~

N-pyrroPAF

CH3 " Platelet Activating Factor (PAF). b These compounds must be considered as new PAF agonists,c These derivatives were described by Ohno et al. [32], but with an l-O-hexadecyl (instead of l-O-octadecyl) ether oxide function in position 1 (cf. the text for discussions). The conformational space and electronic properties of molecules 1-4, which possess very high agonist activity (cf. Biological Results), is influenced by the bulkiness of the onium group (Table I). At position-1 of the glycerol backbone, the ether linkage is essential [12,20]. 1-phenoxy analogues with a meta and ortho side chain (1-m-phenoxy PAF 5a, 1-ophenoxy PAF 5b) as well as the 1,l-dimethyl PAF analogue (6) have thus been selected (Table II) in order to investigate the sterie hindrance at this position. Other compounds resulting from a unique modification of the reference molecule, i.e., 2.1yso PAF (7), 2-methoxy PAF (8) and 2-butanoyl PAF (9), were selected to assess the role of the substitution at position-2 (Table I11). TABLE I1 PAF analogues." variation in position I

R

R

~-O-R1

I.

CHs'CO'OT ~ N (CHs)3 e.,-W Compound R H Sa " 5b a 6b

RI O ~

CnH2n+l

H n ffi 13 meta H n = 15 ortho CH3 -O-CIBH37

a Ref. 34; b 35; for a reviewsee Refs. 12, 21.

1- m-phenoxyPAF I - o-phenoxyPAF l,l-dimethyl PAF

TABLE Ill

constraint ~itl'~ a five m e m b e r e d ring at position-2. O t h e r cyclisation of the modified glycerol backbone of P A F also leads to a tetrahydropyran (13) analogue (Table IV). Finally, two non-zwitterionic derivatives with an ether function (10) and a carbamoyl group in CV 6209 (14)_ instead of the phosphate have been investigated. The biological activities of the selected molecules are presented in Table V and examined in the Results Section.

PAF analogues: cariation in position 2

CHz~)-C18 n3.;, +

CH2.O.P.OJ~I N ICH3)3

#'o Compour~d

Y

1 a

CH 3-CO-O-OH CH 3-OCH 3(CH2)2"CO'O"

7~ 8 9

PAF 2-1ysoPAF 2-methox'y PAF 2-butanoyl PAF

Platelet aggregation

The re-examination of platelet aggregation (agonist and antagonist effect) in rabbit platelet ~ieh plasma (PRP) has been performed by the methods described in previous publications [18,21], as well as the calculation of the relative platelet stimulation (RPS) [!0,21]RPS of an analogue is defined as the ratio of EDso using Ct~ P A F (R or R , S ) v e r s u s E D s . of this analogue (R or R,S).

a Platelet Activating Factor (PAF), b ~LysoPAF'. For instance see Ref. 31; for a review see Refs. IO-12.

t~anong antagonists, SRI 63072 (11) and SR! 63441 (12,) introduce two important variations, with a carbamoyi side chain at position-1 and a conformational

TABLE IV Antagonists structurally related to platelet acticating ]'actor

o

t

CH2-O-C-NH-CI$ H37

Ha-OCleH~r

CH20~,: N

11"

CH:rO-iCI-NH-Cls H:7 I o

CH:O T H

~"

O

&~o

a 3-Ether PAl= deletion of phosphate group see Ref. 37. b SR! 63072, Refs. 42, 43. c SRi 63441, Ref. 44. d PYRAN see Ref. 43. c CV 6209, Ref. 40.

Xa"

N~,~,~'S

Conformational analysis In order to explore the energetically accessible conformational space of the flexible molecules, nine torsional angles of their common backbone (Fig. 1) were considered. Initially, all the degrees of freedom of the molecules were fully optimized within the quantum chemistry MNDO framework [22]. Next, using the optimized geometry as starting point, conformational analysis was performed in the molecular mechanics MM2 framework [24], by steps of 10 degrees around pairs of consecutive dihedral angles. The conformers corresponding to each minimum of relative energy less than 3 kcal/mol were selected from the two-dimensional energy maps. The energy of each conformer thus obtained has also been calculated at the ab initio STO-3G level [23]. All the relative energies given in this paper refer to the ab initio results.

CH~O-~-O(CH~)~Br

HN(RI)2 CH20CI8H37

c+.sc,2o+.

I

Chemistry Cm-PAF 1 and common PAF analogues 8 (2methoxy PAF) and 9 (2-butanoyl PAF) were prepared following methods described previously [8,29]. Compounds 2 (N-diethyl PAF), 3 (N-triethyl PAF) and 4 (N-pyrro PAF) were synthesized from 1-O-octadecyl 2-O-benzylglycero-3-phosphoryl-2'-bromoethanol 15 intermediate in the synthesis of PAF 1 [8] following Scheme 1. Substitution of the bromo atom of 15 with pyrrolidine gave the 1-O-octadecyl 2-O-benzyl glycero-3-phosphoryl-2'-N-pyrrolidino ethanol 16a as a bromhydrate which gave the l-O-octadecyl 2-O-benzyl glycero-3phosphoryl-2'-N-(N-methyl pyrrolidinio)-ethanol 17a when quaternized with methyl iodide. Hydrogenolysis of the benzyl group led to the corresponding lyso compound which was acetylated as usual into the l-Ooctadecyl 2-O-acetyl glycero-3-phosphoryl-2'-N(Nmethyl pyrrolidinio)-ethanol 4 (N-pyrro PAF). The same treatment of 15, with diethylamine followed by quaternization with methyl or ethyl iodides, hydrogenolysis and acetylation, led to the corresponding 1-O-octadecyl 2-O-acetyl glyeero-3-phosphoryl-2'(N,N-diethyl, N-methylammonio)-ethanol (N-tdethyl

~ "(R,)2: " ~

1) Na2CO 3 2) R2!

CH20CI8H37 17b RI = CH2CH ~- , R2 = CH3

o o-

R2

17c RI : RZ : CHzCH3

11 H2/PO 2) (CH3CO)20/NFt3

c+.o.i;'°c'+H~'

Electrostatic potential calculation [23-27] The electrostatic potential was calculated within a complete CNDO scheme [25], after deorthogonalisation of the zigenvectors [26]. The nuclear attraction integrals were approximated by minus the repulsion integrals between S orbitals [27]. A regular 3-D grid was calculated around each motecule. The dimensions of the box were defined as the dimensions of the molecules increased by 5 A in each direction. From these data sets, a contouring algorithm [28] was applied joining the points at the same energy level.

+

+

°

R2

~

g ~.2oi~-m~"2)2~c",)2 [ 0 o-

.~.,,.=0..,+~.~ " , : ~"2~"i . "z o ~"3 RI : R2 : CH2CH3

Scheme I

PAF) 2 and 1-O-octadecyl 2-O-acetyl glycero-3-phosphoryl-2'-N-triethylammonio ethanol 3 (N-triethyl PAl=). Solvents were all reagent grade and were used as received. Column chromatography was performed with silica gel 60 from Merck without special treatment. The purity of each compound was checked by thin-layer chromatography performed on TLC-ready plastic sheets (silicage160 F 254 precoated 20 x 20 cm, layer thickness 0.2 mm) from Merck using CHCI3/MeOH/NH40H (80:20:2 or 70:35:7, v/v) as eluent. Spots were revealed with iodine or molybdenum spray according to the method described in Rcf. 30. HPLC was conducted on a Beckman high-pressure chromatograph Model 110 A equipped with a differential refractometer. Microporasil columns were used and elution was performed with CH 2CI2/MeOH/H 20 (60:50:5, v/v). Infrared spectra were recorded on a Pye-Unicam SP3-200 apparatus and tH-NMR spectra were obtained using a 250 MHz Brtiker spectrometer in CD3OD with tetramethyl silane as an internal standard. Chemical ionization (CI) mass spectra were measured at 200-260 ° C and at a gas (isobutane) pressure of 0.5-0.6 torr on a modified AEI M S9 spectrometer

t3t]. l-O-octadecyl 2-O-benzyl glycero-3-phosphoryl-2'-N#vdiethylamino ethanol 16b A mixture of 2 . 5 T ( 4 mmol) of 15 and 5 ml of diethylamine in 50 ml of CHCI3/isopropanol/dimethyl-formamide (3:5:5, v/v) was stirred overnight

at room temperhture. After evaporation of solvents and excess of amine, the crude residue was purified on a silica gel column using 50% MeOH in CHCI:, as eluent to recover 0.85 g (85%) of the title compound as a hygroscopic hromhydrate. IR (film): 3450 (O-H,N-H), 3090, 3070, 3040 (aromatic C-H), 2950, 2870 (C-H), 1250 ( P = O ) , 1125, 1050, 1030 (C-O-C, P-O) cm -I. IH-NMR (250 MHz) #:7,23 (m, 5H, C6Hs), 4.60 (s, 2H, CH2~), 4.01 (m, 2H, alp spin coupling, POCH2), 3.90 (m, 2H, alp spin coupling, CH:OP), 3.71 (t, 1H, CHOBz), 3.56 (t, 2H, CH2OR), 3.16 (t, 2H, OCH2CH2N+), 3.08 (4H, N+CH2), 1.47 (m, 2H, OCHECH2), 1.28 (m, 36H, (CH2)Ls +CH3CH2N+), 0.81 (t, 3H, CH3). Exchangeable protons were not detectable in this solvent.

I-O-octadecyl 2-O-benzyl glycero-3-phosphoryl-2'-(N. pyrrolidino)-ethanol 16a The title compound was prepared from 15 following the same process as for 16__bbabove but using pyrrolidine instead of diethylamine. 16a presented the same IR spectrum as 16b but the'r-H'H-NMR spectrum differed for the signals of pyrrolidinium 8 1.90 (m, 4H, N+CH2CH2 ).

l-O-octadecyl 2-O-benzyl glycero-3-phosphoryl-2'-(N,Ndiethyl, N-methyl ammonio)-ethanol 17b A solution of 0.89 g (1.3 mmol) of 16b in 20 ml dry (CH3)2CO was added with 0.8 g (7.5 mmol) of Na2CO 3 and 1.8 g (13 mmol) of methyl iodide. The mixture was stirred until disappearance of starting material on TLC ( C H C I a / M e O H / N H 4 O H , 80: 20: 2, v/v). After filtration and evaporation, the crude residue was purified on a silica gel column using 70% MeOH in CHCI 3 as eluent to give 0.75 g (yield 92%) of the title compound as a wax. 1R: the same as 16b. IH-NMR (250 MHz): 8 7.28 (m, 5H, C6Hs), 4.64 (s, 2H, CH2d~), 4.11 (m, 2H, 3tp spin coupling, POCH2), 3.91 (m, 2H, 3~p spin coupling, CH2OP) , 3.73 (quintet, 1H, CHOBz), 3.53 (t, 2H, CH2OR), 3.40 (t, 2H, OCH2), 3.31 (m, 6H, CH2N+), 2.91 (s, 3H, CH3N+), 1.50 (quintet, 2H, OCHzCH2), 1.23 (m, 36H, (CH2)15 + CH3CH2N+), 0.84 (t, 3H, CH3).

l-O-octadecyl 2-O-benzyl glycero-3-phosphoryl-2'-N-(Nmethyl pyrrolidinio )-ethanol 17a Quaternization of 16a with methyl iodide was conducted as described for 17b above yielding 17a as a wax. IH-NMR:8 1.93 (m, 4H, CHzCH2NZ--oof the pyrrolidinium cycle), 1.23 (large s, 30H, (CH2)~s).

l-O-octadecyl 2-O-benzyl glycero-3-phosphoryl-2'-N-triethylammonio ethanol 17c Quaternization of 16b with ethyl iodide was condueted as described above for 17b yielding 17c as a wax. tH-NMR : 8 3.31 (m, 8H, CH-~2N+), 1.23 ~ 39H, (CH2)~s + CH3CH2N+).

Hydrogenolysis and acetylation. Compounds 17a, 17b, 17c were debenzylated with H2/Pd and acetylated with A ~ 2 0 / N E t 3 as described previously [8]. Purification on HPLC permitted elimination of traces of the benzol and lyso derivatives to give the 2, 3 and 4 PAF analogues as waxy compounds. IR: 1735 (C = 0), 1240 (C-O-C ester, P = 0). 1090 (C-O-C ether. P-O) cm- I. ~H-NMR ( 4 0 MHz):common signals for ttle three compounds: 8 5.t3 (quintet, IH, CHOCO), 4.27 (m, 2H, alp spin coupling, POCH2), 4.01 (m, 2H, 31p spin coupling, CH2OP), 3.59 (d, 2H, CH2OR), 3.46 (t, 2H, OCH 2), 2.06
Biological actil'ilies (i) It is well known that several agonists containing a cyclic ammonium moiety are more potent than PAF TABLE V

Biologicaldata of selectedPAFanalogues No. Cpd

Configuration

Ib 2 3 4 5a Sb 6 7c 8 9 tO II a 12 c 13 14 f

R or rac rac rac rac rac rac rac R rac rac rac rac rac rac rac

Agonist RPS (%) ~ t00 200 s 200 40(}0 g 36 0.02 0.5 0.01 0.1 0.5 0.1 -

Antagonist IC5o,~M a 47% atO.l/zM s 30 4.7 0.20 (K)) 8.4 0.02

For definitions and measurememssee Material and Methods, paragraph 'platelet aggregation'. b C~a PAF (R) as reference for an (R) analogue and Cas PAF (R,S) for a racemic mixtureof analogue. ¢ LysoPAF. d SRI 63072. ¢ SRI 63441. f CV 6209. New data; for referencesand reexaminationof biologicaldata, see the text and Tables I to IV.

itself [32,33], (see Table V). The N-methyl pyrrolidinium 4 (N-pyrro PAF) and the N-triethylammonium 3 (N-triethyl PAF) analogues were resynthesized (cf. paragraph Materials and Methods: chert istry) in order to investigate their platelet stimulating effects in our biological tests. The N-triethyl PAF ar, alogue 3 is a highly potent agonist, in accordance wi h the conformatiznal and electronic results (cf. rcsuh s). These findings contrast with the biological activit) previously reported [33]. Although the PAF analogues synthesized and used in the present study are l-O-octadecyl compounds and those synthesized by Ohno et al. are 1-Ohexadecyl derivatives, it is well known that agonistic activities of Cj~ and Ct8 compounds are similar [12,21]. With the new N-diethylmethylammonium PAF agonist 2 (N-diethyl PAF), the data in Table V confirm that all compounds bearing a bulky onium group are more potent than PAF, up to 40-times so in the case of the N-pyrro PAF 4 analogue. (ii) At position-1 a phenyl group bearing a meta branched fatty chain does not significantly modify the platelet stimulation (m-phenoxy PAF, compound 5a), however an ortho substitution (o-phenoxy PAF, compound 5b) completely abolishes [21,35] the agonistic activity (Table V). The platelet aggregating activity was also abolished with methyl substitution on the glycerol backbone (compound 6) [36] as well as in the 2-1yso PAF (compound 7) [37]. The 2-methoxy PAF analogue 8 is notably less active [37] (cf. Table V). The 2-butanoyl derivative _9 shows a weak agonistic activity [37] and an antagonistic activity at l0 "5-10-6 M (Table V). It should be noted that the antagonistic effect is also apparent: the balance between those two activities depending on the batch of platclets used. (iii) Antagonists. As reviewed in Refs. 10, 12, 18, the deletion of the phosphate group nullifies the agonistic activity but induces an antagonistic activity observed with compounds 10 and 14 [37,38,39]. All the constrained derivatives SRI 63072 (11) [41,42], 63441 (12) [43] reviewed in Ref. 2, tetrahydropyran analogue 13 [43] exhibit a potent antagonistic effect.

Geometry Geometries of PAF and analogues have been generated with the R configuration at position 2. In all the studied molecules, the fatty chain was restricted to an ethyl group. Two conformers of PAF (1), N-pyrro PAF (4_) and 2-butanoyl PAF (_9) were optimized with an extended and a folded conformation of the ester group at position-2. In each case, the extended form is the most stable one (1 to 4 kcal/mol). Consequently, the geometry of the other compounds was optimized with

o. !

.->f '°'~AS°'N 2~ '~-oA'°'~c ~o nj

\'o

%

Fig. 1. Numberingof atoms and definition of the dihedral angles involvedin the conformationalanalysis.

the extended conformation of the acetate at this position 2. The two enantiomers generated by spiro substitution of the tetrahydrofuran ring in SR1 63072 (11) were analysed. In pyran compound (1_3), the flexibility of the saturated cycle gives rise to a transoid conformation more stable than the cisoid one (4.5 kcal/mol).

Conformational analysis The results are presented in two sections, corresponding firstly to the conformation of the phosphate group substituent (q~ 1 to to 5), and secondly to that of the rest of the molecule (to 6 to ~p 9), hereafter referred to as the 'ammonium group' and 'position 1 and 2 group', respectively. (a) Ammonium group. The position of the ammonium head with regard to the phosphate is determined by five dihedral angles. The first two (to 1 and tO 2, noted 'OPO'), involve rotation around the P7-O1 and P7-O8 bonds. For each selected O P O conformer, with a relative MM2 energy less than 3 kcai/mol, an additional two-dimensional map was calculated by rotation around tO 3 and tO 4 (O8-C9 and C9-C10 bonds). The fifth dihedral angle, tO 5, only refers to the rotation around the C10-Nll bond. Table VI summarizes the O P O results. In most cases, the gauche conformation (~p 1 and ~p 2 equal to - 6 0 " , - 6 0 °), is the stable one. The position of a second minimum, depends strongly on the conformation of the ester group at position-2. This second minimum appears in the MM2 map at (60", 180 ° ) with the extended form of the acetate (Fig. 2a), but is not present in the folded one (Fig. 2b), giving rise to another minimum at (180 °, 180"). This result is confirmed by the MM2 conformational map of 2-1yso PAF (7) (Fig. 2c), in which both these secondary minima also exist. From the - 6 0 ° , - 6 0 ° conformer, the rotations around ~p 3, ~p 4 and tO 5 angles corroborate the relative stability of the gauche conformation in which the torsion angles values are - 60 °, - 60 ° and 180 °, respectively. This theoretical result can be correlated to the experimental N M R conformation [28] in agreement with a preferred gauche conformation of the phosphorylcholine fragment. It should also be stressed that the gauche conformation is also found in the

TABLE Vl STO-3G relatice energy (local~tool) of the stable confomwrs of compounds I to 12. dependi,,g on ~t and ~: cahws

The missing values refer to conformers which were not four.d as minima in the MM2 map. 04 ~P6

I

9,

...,..c:o.... y o , x." Zo,~...-,f~c.A,,o, /

\

o Compound

o ¢2

60 °

180 °

S°l

180 °

60 °

180 °

+ 3.0

+ 6. I + 7.5 + 14.5 + 5.6 + 6. l + 7.0 + 7.5 + 7.6 + 6.0 +0.0

PAF I a PAF I b N-pyrro PAF 4 a N-pyrro PAF 4 b 101-DimethylPAF 6 a I ,l-Dimethyl PAF 6 t, I-Lyso PAF 7 N-dimethyl PAF 2 t, N-tricthyl PAF 3 b SRI 63072 !1 SR! 63441 12

+ 5.5 + 9.5 0.0 + 10.4 + 7.0 + 7.2 +11.6

+ 6.4 + 2.0 +4.2 + 4.5 + 5.7 + 4.2 -

-60 ° -60 ° + 5.0

+ 4.4 + 3.6 + 5.8 + 3.9

180°

-60 °

+ 2.2 + 6.4 + 1.0 + 3.4

0.0 0.0 0.0 0.0 0.0 + 1.7 (|.0 0.0 0.0 0.0 + 1.q

+ 4.5 +,L6

a Folded conformation of acetate group. b Extended conformation of acetate group.

o p t i m i z e d g e o m e t r y o f t h e S R I 63072 (11) a n d P Y R A N derivative (13). (b) Conformation at position 1 and 2. A t the previously selected gauche c o n f o r m a t i o n , torsion a n g l e s 6, tp 7 a n d ¢ 8 w e r e varied. T w o stable c o n f o r m e r s with ,p 9 e q u a l to 180 ° o r - 9 0 °, differing by 1 k c a i / m o l f r o m e a c h o t h e r a r e f o u n d in the N - p y r r o P A F derivative (4_). U s i n g this m o l e c u l e as r e f e r e n c e c o m p o u n d , c o n f o r m e r s with q~ 6 e q u a l to 180 ° a n d n i n e p a i r s o f (¢, 7, ,p 8) a n g l e s v a r y i n g by steps o f 120 ° c a n b e l o c a t e d in t h e M M 2 e n e r g y r a n g e o f 3 k c a l / w o l f o r the m o l e c u l e s w h e r e n o steric h i n d r a n c e o c c u r s at position 1 a n d 2. T h e relative e n e r g i e s o f the fully e x t e n d e d c o n f o r m a t i o n s a r o u n d C 2 - C 3 a n d C3-C5, (180 °, 180 ° ) a r e very close to t h o s e c a l c u l a t e d for the o p t i m i z e d g e o m e tries, Gp 7 = 180 ° , ,p 8 = - 6 0 ° ) as also a d o p t e d by the semi-rigid t e t r a h y d r o p y r a n c o m p o u n d ( 1 3 ) w h e r e ~p 8 is c o n s t r a i n e d to - 60 °. In the positively c h a r g e d m o l e c u l e s e t h e r - P A F (10) a n d C V 6209 (14), t h e r o t a t i o n a r o u n d ,p 6 is e n e r g e t i cally free, a n d v a l u e s o f d i h e d r a l a n g l e s ~p 7 a n d ~ 8 ( T a b l e VII) in the s t a b l e c o n f o r m e r s a r e similar to the c o r r e s p o n d i n g o n e s f o u n d for the p h o s p h a t e g r o u p containing compounds. In S R I 63441 (12), t h e connectivity o f a t o m s bet w e e n C3 a n d 0 6 is different. T h e c o n f o r m a t i o n a l analysis w a s t h u s c a r r i e d o u t a r o u n d t h e C - C b o n d s p r e c e d i n g a n d following t h e five m e m b e r e d ring. T h e

e n e r g e t i c a l l y accessible c o n f o r m a t i o n s are restricted to five roughly e q u i p r o b a b l e ones. C o m m o n conformation A m o n g the previous c o n f o r m a t i o n a l results, the choice o f a c o m m o n c o n f o r m a t i o n is g u i d e d by the e n e r g e t i c a l l y accessible c o n f o r m a t i o n involved in the semi-rigid c o m p o u n d s . R e m a r k a b l y , except in S R I 63441, the oxygen a t o m s 1, 4 a n d 6 (Fig. 1) a r e linked to e a c h o t h e r by a similar connectivity d e f i n e d by t h r e e c a r b o n atoms. It c a n thus be s u g g e s t e d t h a t a c o m m o n c o n f o r m a t i o n h a s to fit o n the p l a n e o f these t h r e e oxygen a t o m s as o r i e n t e d in the rigid a n d flexible c o m p o u n d s . This c o m m o n c o n f o r m a t i o n , labelled ( * ) in T a b l e VII, e.~tphasizes the similarity o f the position 1 a n d 2 e n v i r o n m e n t as illust r a t e d by the g e o m e t r i c d a t a in T a b l e VII. T h i s table c o n t a i n s the torsion a n g l e v a l u e s f o u n d in the selected c o n f o r m a t i o n , the pairwise d i s t a n c e s b e t w e e n the oxyg e n a t o m s a n d the deviations f r o m the plane. T h i s analysis allows the selection o f the only active S R I 63072 e p i m e r as d i s p l a y e d in Fig. 3. R e g a r d i n g the l - p h e n o x y P A F derivatives 5 a a n d 5b, the two e n e r g e t ically e q u i p r o b a h l e (0.22 kcal) mere c o n f o r m e r s explain t h e i r agonistic effect, w h e r e a s the ortho substitution c a u s e s a r o t a t i o n o f 90 ° o f the p h e n y l g r o u p thus i n d u c i n g the lack o f activity. T h e consistency o f the c o m m o n c o n f o r m a t i o n is s t r e n g t h e n e d by the results o b t a i n e d by 1,l-dimethyl

\.~

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,,

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x

,,

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~

x.

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-4 ' .y-- •

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/'

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90

o

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310

b

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,

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.

.

.

.

:u

a Fig. 2. MM2 energy maps as a function of ~1 (O8-P7-O1-C2) and ',0z (C9-O8-P7-O1) for P A F with an exte~,nded acetate chain (a); a folded one (b); as well as the corresponding conformalional map of lyso-PAF (c). Contour levels are: 0.5, - - - - - - 3 . 0 . . . . . . 5.0 and - 10,1) k c a l / m o l above the minimum.

Fig. 3. 3-D structure of the studied compounds in their common conformation. The point of view is perpendicular to the plane passing through oxygen atoms O1, 0 4 and 0 6 . P: P A F with the acetate extended conformation and 1~ with the folded acetate conformation; _5a: both conformations of the m-phenoxy PAF; _9~': 2-butanoyl PAF with the extended butanoyl conformation 9 h with the folded butanoyl conformation; L3a: pyran cisoid and 13b pyran transoid.

~16

NI

0%+

o+

MI c

%

04

c~

.i

olo

o+

c3

o: olo

o~o

•i

o*

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oe

04

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cl

ols

.l

i

;

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c

....

os

o+

a

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l" ++'+.++' ++~y_ 6

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7

8

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~ sl

.

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I

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olo

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100 T A B L E VII

STO-3G relatice energy (kCal/mol) of the stable conformers of PAF like antagonists 10 to 14, as a ~mction of ~o7 and qs(¢,:'), c¢~mpared to the corresponding ones of PAF N-pyrro (4) chosen as a model of agonisL ¢t and ~2 remain equal to - 60 o * Refers to the selected conformer.

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o

Compound

.

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63441

~o7

60 °

~Ps

60 °

180 °

_60 °

600

180 °

_60 °

80 °

180 °

_600

0.0 +1.55

+0.2 +0.5

+3.0 +1.1

+0.3 +1.1

+1.3 +0.4

+0.85* 0.0"

+4.5 +4.4

+1.8 +1.35

+1.3 +1.5 0.0

N-pyrro P A F 4 SRI 63072 11 Pyran 13 transom P/ran 13 cisoid 3-Ether P A F 10 C V 6209 14

180 °

o--

c3 cs.. "x_/-~<.c/

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-

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-

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-

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-

-

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-

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-

41.2 +0.8

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180 °

SRI 63441 12

0.0

+0.5 -~°

600

1~o

_600

+ 1.2

+0.6

+ I.I

80 ° 0.0

1~o +0.2 *

T A B L E VIII

Dihedral angles 'PI, ~z, 'P~, ~PT,~P~ pairwise distances between the oxygens 0 t, 04, Oo deviation .from 08 to the plane passing through atoms Oi, 04 and Oo 04

Cio

o I Cp~

OI

o Compound

N-pyrro PAF 4 1-m-phenoxy P A F 5 ~ 1-o-phenoxy P A F 5 b l-Dimethyl PAF 6 2-Lyso P A F 7 2-Methoxy P A F 8 2-Butanoyl P A F 2 a 2-Butanoyl PAF 2 b 3-Ether P A F 10 SRI 63072 11 SRI 63441 12 P / r a n 13 transoid P / r a n 13 cisoid C V 6209 14

I (p,

o Dihedral angles

Distances (A)

tPl

~2

~6

~P7

~'s

D

D

D

D8/

(o)

(o)

(o)

(o)

(o)

01-04

Ol-O 6

04-06

P146

176.4 - 176.2 - 174.5 - 174 177.7 - 177.7 178.7 176,1 177.~ 132 176,3 176.9 171.1 163.1 120

168.7 168.7 - 178.9 179.6 178.7 - 175 178.1 178.9 177.3 180 - 169.6 169,4 169.5 179.1

- 60 - 60 -71.5 -73.3 -61.3 - 70.5 - 72.7 -72.1 --73 -60 - 62.8 -89.6 -53 - 60

2.86 2.86 2.81 2.82 2.8 2.83 2.89 2.82 2.84 2.81 2.81 2.89 2.87 2.86 2.8

4.2 4.2 4.32 4.33 4.19 4.34 4.31 4.32 4.34 4.3 4,3 5.23 4.52 4.36 4.2

3.68 3.68 3.62 3,62 3.6 3.59 3.62 3.62 359 3.66 3.63 3.63 3.88 3.99 3.68

- 93 - 93.5 - 95.7 - 100.5 177.7 - 91.3 - 123.3 - 102.1 --88.1 -- 86.1 - 86.2 -89.2 -98

-

87.6 79.2 83.6 82 70.1 81.7 - 73.6 -81.4 --78.1 -- 82.9 - 105.2 -76.3 -69.5 -

" Folded conformation and b Extended conformation of the 2-botanoyl group.

0.58 0.59 0.51 0.67 2.16 0.35 1.48 0.81 0.5 0.4 - 2.26 0.9 1.38 -

101

a

b

Fig. 4. 3-D electrostatic potential negative cloudsof PAF, at the contouringlevels:(a) - 10 kcal/mol; (b) -20 kcal/mol; (c) -30 kcal/mol and (d) - 40 kcal/mol.

PAF (6). In this molecule, the ~ 6 to g, 9 values are in the same range as those round in the other compounds (Table VIII), but the methyl bulkiness gives rise to a rotation of the acetate around C3-C4 bond in such a way the conformation of the phosphate is significantly modified (~p 1 = 177.7 ° ).

Electrostatic potential map The 3-D electrostatic potential can be regarded as the best 'finger-print' of a molecule because it takes into account the volume, conformation and electronic distribution. Electrostatic potential calculation was car-

ried out for the above PAF-like molecules in their selected common conformation. Except for the 3-ether PAF (_8)and CV 6209 (12), all the molecules exhibit a zwitterionic form due ~ the ammonium and phosphate groups. Such a separation in the electronic distribution is directly cxpressed by a very high value of the dipole moment ( + 18 Debyes) as calculated by the CNDO method. As this electronic property is the first term of the serial development of the electrostatic potential itself, it is not surprising that the positive and negative volumes defined as a given contour level are very impressive.

102

a

d

e

[I Fig. 5. 3-D electrostatic negative clouds of PAF-like agonistsand antagonists(contouring level -30 kcal/mol): (a) PAF Ia with the extended acetate conformation;(b) PAF Ib with the folded acetate conformation;(c) N-pyrroPAF 4; (d) l,l-dimethyl PAF 6; (e) 2-1ysoPAF .7; (O SRI 63072 11; (g) SR163441 12; (h) pyran 13 transoM.

(a) NegatiL'e clouds. A progressive decrease in the contours from - 1 0 to - 4 0 kcal/mol was apparent for PAF (1) (Fig. 4). At the - 10 kcal/mol, level used in Ref. 16, the 3-D surface totally surrounds the molecule (Fig. 4a). The bulky cloud due to the phosphate group decreases in volume at - 2 0 kcal/mol (Fig. 4b), and becomes more localized at - 3 0 kcal/mol (Fig. 4c). It is extended to the oxygens at positions 1 and 2, through two narrow funnels. At - 4 0 kcal/mol, the negative

volume is only located around the phosphate group (Fig. 4d). For comparison with the other molecules, the same isocontour at - 3 0 kcal/mol was chosen. Fig. 5 displays the molecules perpendicularly to the preceding plane. In this representation, oxygen atoms O1, 0 4 and 0 6 lie roughly along the same horizontal axis, while the fatty side chain is at right angles to this axis. The wells generated by the phosphate group remain im-

103

b

a Fig. 6. 30 keal/mol positive contours and their correspondingvolume:(a) PAF 1 3,2 ,~3;(b) N-diethyl PAF 2 402 ,~3;(c) N-triethyl PAF 3 417 ,~3;(d) N-pyrroPAF 4 396,4:~.

pressive for all compounds. Molecules (2) to (4) generate the same negative potential features (not shown) four~d in PAF (1) (Fig. 5a). If the acetate conformation is either folded or extended, an additional cloud appears around the carbonyl fragment which is fused to the phosphate cioud in the extended conformation. Consistently, this feature is not present in the inactive lyso PAF derivative (7) (Fig. 5). The modification of the phosphate conformation induced by the acetate rotation in l,l-dimethyl PAF (6)

is expressed by its electrostatic potential cloud which is not superimposable with that found with PAF. Regarding the antagonist derivatives, SRI 63072(11), SRi 63441(12), PYRAN(13), all are substituted by a (thio)carbamoyl moiety which generates its own negative capsule. (b) Posb e clouds. T h e positive contour completely surrounds the molecules. It is shown at 30 kcal/mol (Fig. 6) for agonists (1) to (4), and clearly indicates the bulkiness of the ammonium group. The numerical integration calculation of the positive volume enables a

104 quantification of these differences. Visual estimation confirms that the volume of the positive cloud increases in N-triethyl P A F (3) relative to P A F (1). Conclusion A common conformation of 14 agonist and antagonist P A F like molecules is assumed to be accessible to all flexible and semi-rigid studied comFounds. From the conformational results, s e v ~ d t biological data can be related to the geometric features of SRI 63072, (11) o-phenoxy P A F (5b) and 1,1-dimethyl P A F (6) analogue. At this stage, the distinction between agonistie and antagonistic activity is not unequivocally explained by the nature of the substituents at position 1 and 2 due to the occurence of carbamoyl in 1, e t h e r oxide in 2 or ether oxide in 1 and carbamoyl in 2. Although the extended conformation of the acetate group is slightly more stable than the folded one in the isolated molecule, it can be postulated that, at the receptor site, the folded conformation of agonists is the most probable in accordance with the cyclic fragment in the antagonistic molecules 11, 12, 13 a lower activity being a p p a r e n t for compounds bearing a long side chain at position 2. Concerning the electrostatic potential, information is now available on zwitterionic compounds (this study) and on antagonists generating only negative potentials ('cache oreilles' molecules described in Ref. 16). If both types of compounds fit into the same receptor, the adjustment of the negative clouds of the 'cacheoreilles' molecules to the negative one of the PAF-iike analogues is required, assigning the position of the positive onium environment. Further investigation is required to propose a multipolarized receptor model comprising an alternate sequence of positive and negative c o u n t e r p a r t s and to elucidate the uniqueness of the antagonistic and agonistic structural features at position 1 and 2. Finally, in a g r e e m e n t with modelling results, we have synthesized and found N-triethyl P A F (3) as a potent agonist, whereas it was previously considered as a weak platelet stimulant in the literature [33]. Acknowledgements We sincerely t h a n k Professor J.M. Ghuysen for his interest in this work and constructive c o m m e n t s and Joelle Gavard for typing the manuscript, Dr. O. Convert (Laboratoire de Chimie O r g a n i q u e Structurale, Universit6 Paris 6) for ~H-NMR 250 M H z spectra and Dr. P. V a r e n n e (CNRS, Institut des Substances Naturelles) for mass spectra.

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36 TenetS, M., Co, frier, E., Heymans, F., Polonsky, J., Godfroid. J.-J. and Benveniste, J. (1981) Biochimie 63, 723-727. 37 Heymans, F.. Borrel, M.-C., Broquet, C., Lefort, J. and Godf;'oid, J.,J. (1985) J. Med. Chem. 28. 1094-1096.' 38 Wissner, A., Schaub, R.E.. S7um, P.E., Kohler, C.A,. and Golstein, B.M. (1985)J. Med. Chem. 29, 328-333. 39 Tokumara A., Homma, M. and Hanahan. D.J. (1985) J. Biol. Chem. 260, 12711)-12714. 4(I Terashita. Z . Imura, Y.. Takatani, M.. Tsushima, S. and Nishikawa, F. (1987) J. Pharm. Exp. Ther. 242, 263-268. ~.i |iandley, D.A., Anderson, R.C. and Saunders, ~..N. (1987) Eur. J. Pharmacol. 141,409-416, 42 Handley. D.A., Van Valen, R.G. and Saunders, R,N. (1986) lmmunt, pharmacology I 1, 175-182, 43 Handley, D.A.. Van Valen, R.G., Tomeseh, J.C., Melden, M.K., Jaffe. J.M., Ballard, F.H. and Saunders, R.N. (1987) lmmunopharmacology 13, 125-132. ,ta. Nakamura, N., Ookawa, N., Koike, H., Sada, T.. Oshima, T. Izuka, Y. (1987) Eur. Pat. Appl. EP 210, 804.