Chemistry and Physics o f Lipids, 55 (1990) 155--161 Elsevier Scientific Publishers Ireland Ltd.
155
Synthesis of azathia analogues of platelet activating factor with polyheterosidechains* J.M.
Zeidler**, W. Zimmermann
and H.J.
Roth
Pharmazeutisches lnstitut der Universit~t T~bingen, A u f der Morgenstelle 8, 7400 T~bingen (F.R.G.) (Received January 29th, 1990; revision received and accepted March 23rd, 1990)
The synthesis of azathia analogues of the platelet activating factor with oxygen and sulphur-containing sidechains is reported. The starting point is 1-acetylthio-3-hydroxy-2-propaneamine-HCl, which permits the formation of the thioether and the acetamido linkage in one step. The phosphocholine part is introduced via 2-chloro-2-oxo-l,3,2-dioxaphospholane and subsequent ring opening with trimethylamine under pressure.
Keywords: phospholipid synthesis; platelet activating factor analogues; azathia analogues; ether lipids.
Introduction
Platelet activating factor (PAF) is an alkyl ether phospholipid (structure see Fig. l a) composed primarily of the C~6 and Cls homologues. It is a potent activator of platelet aggregation and one of the important mediators of anaphylaxis and inflammation [1]. The ~taturally occurring lipid also exhibits a strong hypotensive quality. Over the last few years a number of structure analoga have been prepared and tested [2--7]. Wissner et al. [8] synthesized some analogues with multiple oxygen substitution of the alkoxy chain (Fig. lb). Incorporation of ether linkages results in a significant reduction of biological activity of these compounds. In 1982, a new analogous structure with an acetamide function at the chiral C-atom was prepared by Chandrakumar and Hajdu [9]. This modification ieads to a lipid which is stable against enzymatic degradation by acetylhydrolase and exhibits potent platelet activation [9]. The next step was Correspondence to: W. Zimmermann. *Dedicated to H. Mthrle on the occasion of his 60th birthday. **Part of dissertation, Ref. H.J. Roth.
the exchange of the ether linkage by a thioether group (Fig. lc) [10]. The sulfur substitution at the sn-1 position seems to lower the plateletaggregating potency and to enhance the tumorcytotoxicity [ll,12]. The thioether phosphocholine analogous BM 41.440 (Ilmofosine) [13] has been tested in clinical phase I/II trials. It showed antitumor potency in a variety of tumor models in vitro and in vivo. For that reason, it is of interest to synthesize new potent analogues which in addition help to understand the structure-activity-relationship of cytotoxicity and tumor selectivity. As described earlier [14], it is possible to synthesize azathia analogues containing two oxygens in the alkylchain at sn-1 (Fig. l d). The homologues line and one thiaanalogue were now prepared. The oxygen-atoms are posed near the backbone so that the sidechain has a hydrophobic end, which seems to be necessary for the affinity to the cell membrane. Materials and Methods
2-Chloro-2-oxo- 1,3,2-dioxaphospholane, 97% pure; trimethylamine anhydrous, 98°/o pure,
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
156
0/C16H33
II
0
/P~n~N~
Io
I_OIe
0~O~0(CH2)9
O
CH3
k~O/P~o~N~ h
I
I
iol e
s/ClaH37 H
H3C~]~N~ j ~ I. O k~o/P~n~N~ c. I I IO_I e s~O~/~0/C12 H2s H3C~]~N
°
II
0 d, IOI e
Fig. 1. PAF (a) and synthetic analogues (b--d).
ethyleneglycol, >99070 pure; triethyleneglycol, > 99°7o pure; tetraethyleglycol, > 99070 pure; pentaethyleneglycol > 9707o pure; 1-bromo-hexadecane, >9807o pure; 1-bromo-decane, >97070 pure and 1-bromo-octane, >98o70 pure; were from Fluka AG (Neu-Ulm, F.R.G.). 4-Nitrobenzenesulfonylchloride, >9807o pure; sodium amide, > 9707o pure and 1,8-dihydroxy-3,6dithiaoctane were purchased from Merck-Schuchardt (Hohenbrunn, F.R.G.). LiBr, anhydrous, > 9907o pure was purchased from Merck (Darmstadt, F.R.G.) 1-Acetylthio-3hydroxy-2-propaneamine-HCl was prepared as described elsewhere [14].
Analytical and preparative methods 'H- and '3C-NMR-spectra: Brucker AC-80spectrometer (80 MHZ), internal standard: TMS; IR-spectra: Perkin-Elmer FT-IR spectrometer 1750 (KBr); melting points: Gallenkamp, melting point apparatus, uncorrected; column chromatography: silica gel 60 (7734) from Merck (Darmstadt, F.R.G.) with a particle size of 0.063 --0.2 mm; phospholipids were purified by MPLC (Labomat MD 80/100, Labomatic) on silica gel (Labogel NP 30--60, 30/~m/60A from Labomatic); column: vol. 410 ml, diameter 37 mm; polyalcohols and the related bromides were distilled in a kugelrohr apparatus (Biichi) under reduced pressure; solvents were removed under vacuum at 40°C. The purity of the lipids was checked by thin-layer chromatography (Polygram Sil G/UV 254, Macherey-Nagel, layer: 0.25 mm silica gel, 40 × 80 mm). Lipids were detected by spraying with Molybdenum Blue (Sigma)/0.1 mol H2SO4 (3 : 1). Solvent, chloroform/methanol/NH 3 25°70 (65 : 35 : 6, by vol.), without saturation. Benzene, acetonitrile and tetrahydrofuran were distilled from calciumhydride directly into the reaction vessels. Triethylamine was dried over potassiumhydroxide and then distilled. Acetone was distilled from P205. General procedure for the polyoxabromide synthesis 1-bromo-3-oxanonadecane (A). Sodium amide (25 mmol) was suspended in 150 ml dry tetrahydrofuran. Ethyleneglycol (50 retool) was added drop by drop for 30 min and the reaction mixture was heated to reflux for 30 min. 1-Bromohexadecane (25 mmol) was then added and heating was continued for further 5 h. Thereafter, the solvent was removed and the residue was poured into 100 ml water and extracted with n-hexane. After evaporation the crude product was distilled under reduced pressure. Yield: 38.6 g (54070); mp: 37°--39°C; C18H380 2 (286.5). 3-Oxanonadecane-l-ol (25 mmol), obtained as described above, was dissolved together with triethylamine (26 retool) in 100 ml waterfree tetrahydrofuran. At 0 °C p-nitrobenzene-
157 TABLE I Homologeous polyoxabromides. Yields are calculated over all steps. Bromide (B) 1-bromo-3,6,9-trioxanonadecane CI6H3303Br (353.3) (C) l-bromo-3,6,9,12-tetraoxaeicosane CI6H330,Br (369.3) (D) l-bromo-3,6,9,12,15-pentaoxapentaeicosane C20H,iOsBr (441.5) (E) 1-bromo-3,6-dithio-9-oxanonadecane Ct6H33OS~Br (385.5)
~
bp.
yield
1.4579
190°C (1.8 x 10-~ mbar)
13.8070
1.4588
150°C (1.3 × 10-~ mbar)
27.3070
1.4610
175°C (7.2 x 10-2 mbar)
10.5%
mp: 37--38°C
19.7%
sulfonylchloride (25 mmol) was added in three parts and the mixture was stirred for 6 h at room temperature. The precipitated salt was filtered, the solvent removed, and the residue was dissolved together with dry lithium bromide (30 retool) in water-free acetone. After 30 h, the acetone was distilled off under reduced pressure and the residue was stirred up in 100 ml n-hexane for 15 rain. After filtration, the solvent was dried over sodium sulfate, then evaporated and the crude 1-bromo-3-oxanonadecane (A) was purified by distillation. CIsH37OBr (349.4); n~0d: 1.4600, bp: 175°C (3.7 X 10-~ mbar); yield: 29°7o. ~H-NMR (chloroform-d~): 6 (ppm): 0.88 (t, J = 5 Hz, 3H, CH3); 1.22--1.60 (m, 28H, C H 2 ) ; 1.72--2.08 (m, 2H, CH2Br); 3.26--3.93 (m, 4H, CH20).
13C-NMR (chloroform-dr): tt (ppm): 14.14 (CH~); 22.84 (CH2--CH3); 26.29, 28.39, 28.69, 29,14, 29.37, 29.62, 29.90, 31.06 (13CH2); 32.13 (CH2Br); 70.66, 71.31 ( 2 C H 2 0 ) .
The bromides B--E were prepared in the same manner. The data are summarized in Tables I and II.
N-[1-(3-oxanonadecylthiomethyl)-2-hydroxyethyl]acetamide (1). 1-Acetylthio-3-hydroxy-2propanamine-HC1 (4.0 g, 0.0215 tool) was heated to reflux together with 5.9 g (0.0426 tool) potassium carbonate and 8.0 g (0.0228 tool) 1bromo-3-oxanonadecane, in a mixture of 80 ml methanol and 10 ml water, for 3 h. Then 100 ml cool water was added and the thioether was extracted with dichloromethane. The solvent was removed, the residue diluted in ethylacetate and frozen at - 1 8 °C. The crude product was purified by column chromatography using chloroform/methanol (9 : 1, by vol.) as a solvent. Yield: 6.6 g (73.5e70) white solid; rap: 68-69°C; C23H47NO3S (417.7); N: calculated: 3.35, found: 3.35. IH-NMR (chloroform-dl): d (ppm): 0.87 (t, J = 7.5 Hz, 3H, CH3); 1.25--1.58 (m, 28H, CH2); 2.00 (s, 3H, C O C H 3 ) ; 2.67--2.82 (m, 4H,
TABLE II ~3C-NMR-shifts of the polyoxabromides d (ppm). Solvents: B--D = DMSO-d6; E = CDCi r Bromide
CH2--Br
CH20
CH2S
CH 2
C__H2--CH3
CH 3
B C D E
32.01 31.40 31.57 43.05
70.44--65.52 71.54--68.11 70.41--69.50 71.31, 70.85
---34.38--31.67
31.32--25.66 30.19--25.59 31.30---25.63 29.71--26.13
22.09 22.50 22.06 22.67
13.84 14.04 13.65 14.08
158 TABLE III Experimental data. 4
6
mp. (°C)
7
9
184---185 1.4842 60.1 •,d 3.32 3.41
yield (%) N: calculated N: found IR (cm"l) NH, OH
50.8 b 4.77 4.82
3420--3280 2926 2856
CH 2
C=O
3758 2964 2932 2862 1698
1656
10
168--172 1.4836 80.6 =,d 3.20 3.29 3421-3258 2927 2859 1657
1.4856 41.4•.d 2.75 2.69
51.9 b 4.65 4.71 3428
12
13
15
219--221
69--70
161--163
40.4 b 4.15 4.25
44.9 c 3.09 3.15
32.6 b 4.53 4.42
3420
3400-3280 2964 2924 2860 1680
3402
2928 2858
3432-3281 2925 2857
2904 2852
1652
1665
1653
2954 2924 2854 1651
• Purified by column chromatography, solvent: CHCI/CH3OH (9 : 1, v/v). bpurified by MPLC, solvent: CHCI/CH3OH/NH 3 25% (65 : 35 : 6, v/v/v). ° Ice water was added to the reaction mixture, the formed precipitate was filtered and recrystallized from hot n-hexane. a After removing the solvent in a rotary evaporator the residue was dissolved in dichloromethane, filtered and then purified by column chromatography.
2CH2S); 3.36--3.69 (m, 6H, 2OCH2, CH 2 OH), 4.02 (m, 2H, CH, OH) 6.66 (s, IH, NH). 13C=NMR (chloroform-dl): 6 (ppm): 14.09 (CH3); 22.66 (CH2--CH3); 23.25 (COCH3); 26.13, 29.36, 29.69, 31.94 (13CH2); 32.26, 33.05 (2CH2S); 51.30 (CH); 62.98 (CH2OH); 70.49, 71.31 (2CH20); 170.90 (C=O). IR: 3400--3295 (NH, OH); 2978, 2920, 2850 (CH2); 1670
(C = O) cm -t. The homologues compounds were prepared in a similar manner: n-[1-(3,6,9-trioxanonadecylthiomethyl)-2-hydroxyethyl]acetamide (4) C2tH43NOsS (421.6); n-[1-(3,6,9,12-tetraoxaeicosylthiomethyl)-2-hydroxyethyl]acetamide (7) C2~H43NO6S (437.6); n-[1-(3,6,9,12,15-pentaoxapentaeicosylthiomethyl)-2-hydroxyethyl]acetamide (10) C25HslNOTS (509.7); n-[1-(3,6-dithio-
TABLE IV IH-NMR-shifts 8 (ppm). Solvents: 4,7,10,13: chloroform-d1; 6,9,12,15: methanol-d4.
CH~ CH 2 COCH 3 CH2S CH20 CHzOH CH)OH NH ÷NCH~ CH CH2N
CH20
4
6
7
9
10
12
13
15
0.88 1.27--1.57 2.00 2.67--2.83 3.37--3.77 3.95---4.12
0.89 1.28--1.63 1.96 2.66---2.82
0.87 1.27--1.64 1.98 2.65--2.80 3.28--4.21
0.89 1.30---1.55 1.96 2.66--2.82
0.87 1.27--1.66 1.98 2.65--2.82 3.33--3.82 3.87--4.07
0.90 1.30--1.67 1.98 2.76--2.96
0.87 1.26--1.69 2.01 2.64--2.70 3.35--3.78 3.93--4.08
0.89 1.29--1.66 1.96 2.64---2.78
6.91
7.03
6.68
6.34
3.24
3.24
3.27
3.23
3.30--4.24
3.46---4.29
3.41--4.49
3.34---4.37
159 T~LEV s3C-NMR-shifisd~pm).Solvents:4,7,10,13:chlorofo~:6,9,12,1$:m~hanol~4. 4
6
7
9
10
12
CH 3 CH2--CH 3 COCH 3 CH 2
14.02 22.61 23.14 26.13 29.26 29.57 29.71 31.90
14.42 22.73 23.60 27.11 30.34 30.46 30.62 32.67
13.41 21.93 22.42 25.40 28.53 28.71 28.93 31.13
14.42 22.73 23.63 27.12 30.34 30.46 30.64 32.69
14.07 22.61 23.14 26.02 29.25 29.53
14.43 22.80 23.66 27.13 30.36 30.59 32.62
CH2S
32.26 33.56
32.94 33.92
31.22 32.54
32.91 33.93
31.89 33.21
32.95 33.89
CH *NCH 3 CHzN CH2OH CH20
51.63
51.05 54.69 60.41
50.73
51.07 54.73 60.46
51.35
51.06 54.77 60.53
CH=O
63.12 70.15 70.39 70.67 71.37 71.54
170.72
66.83 67.17 71.03 71.14 71.42 71.83 72.23 172.60
61.94 69.33 69.55 69.85 70.50 70.72
170.11
66.77 67.41 71.03 71.i6 71.42 71.63 72.29 172.92
9-oxanonadecylthiomethyl)-2-hydroxyethyl]acetamide (13) C21H43NO3S3 (453.8). The data are summarized in Tables III, IV and V.
N-[1-(3-oxanonadecylthiomethyl)-2-(2-oxa1,3,2-dioxaphospholane-2-yloxy)ethyl] acetamide (2). Thioether (1) (2.4 g, 0.0057 tool) was dissolved together with 0.92 g (0.0064 mol) 2chloro-2-oxo-l,3,2-dioxaphospholane in 25 ml dry benzene. At 0oC, 0.67 g (0.0066 mol) of dry triethylamine was added drop by drop and the mixture was stirred at room temperature for 5 h. After filtration of the precipitated salt, the solvent was removed by reduced pressure. The obtained residue was used without further purification. C25Hs0NO~PS (523.7); the following homologues were prepared: n-[1-(3,6,9-trioxanonadecylthiomethyl)-2-(2-oxo- 1,3,2-dioxaphospholane-2-yloxy)ethyl]acetamide (5) C23H4~NOsPS
62.77 69.98 70.21 70.51 71.27 71.43
170.66
66.86 70.30 70.96 71.10 71.27 71.71 72.30 172.99
13
14.07 22.64 23.31 26.08 29.29 29.43 29.54 29.64 31.79 32.21 32.36 32.67 32.94 50.95
62.91 70.57 71.31
170.67
15
14.47 22.74 23.66 27.22 30.40 30.52 30.72 32.54 33.02 33.10 33.27 33.53 33.69 51.11 54.74 60.45
66.68 67.27 72.01
172.92
(527.7); n-[1-(3,6,9,12-tetraoxaeicosylthiomethyl)-2-(2-oxo-l,3,2-dioxaphospholane-2-yloxy)ethyllacetamide (8) C23H,sNO9PS (543.7); n-[1(3,6,9,12,15-pentaoxapentaeicosylthiomethyl)-2(2-oxo- 1,3,2-dioxaphospholane-2-yloxy)ethyl]acetamide (11) C2~Hs4NO~0PS (615.8); n-[l(3,6,dithio-9-oxanonadecylthiomethyl)-2-(2-oxo1,3,2-dioxaphospholane-2-yloxy)ethyl]acetamide (14) C23H46NO6PS 3 (559.8).
O-[2-acetylamino-3-(3-oxanonadecylthio)propyl]cholinephosphate (3). The obtained amount of 2 was dissolved in 80 ml of dry acetonitrile and transferred into a pressure bottle. A large excess of dry trimethylamine (10 ml) was added and the solution was stirred at 65°C for 36 h. After cooling, the solvent was removed and the remaining solid was purified by column chromatography on silica gel ( M P L C ) using
16o
0 S"~CH3
SAR
H, .yI Ele
I,,,~OH
0 "~OH 0 [1 II .cj/p~,?y H /
R = ~0--C16H33 R = -..-(-0-,,,,.~0~[10H21 R = -+-O-,v~O__EsH17
s~R
2. N(CH3~
[3.6.,~.1z'15.]
o
R = .-+-O-,v~O~C10H21
0e
R = .-~-S-,,,~0~£10H21 Fig. 2. Reaction scheme.
chloroform/methanol/NH3 25070 (65 : 35 : 6, by vol.) as a solvent. Yield: 2.1 g (63.2~70) white solid; mp: 187--189°C. C2sH59N206PS (582.8); N: calculated: 4.81, found: 4.75. ~H-NMR (methanol-d4): 6 (ppm): 0.89 (t, J = 7.5 3H, CH3); 1.28--1.55 (m, 28H, CH2); 1.96 (s, 3H, COCH3); 2.65--2.81 (m, 4H, 2CH2S), 3.23 (s, 9H'N(CH3)3); 3.34--4.32 (m, llH, CH, 4CH20, CH2N). 13C-NMR (methanol-d4): d (ppm): 14.42 (CH3); 22.71 (CH2--CH3); 23.65 (COCH3); 27.17, 30.40, 30.52, 30.70, 32.82 (13CH2); 33.00, 33.97 (2CH2S); 51.06 (CH); 54.70 ('N(CH3)3); 60.45 (CH,N); 67.20, 67.43 (2CH20); 71.43, 72.00 (2CH20); 172.92 (C=O). IR: 3440 (NH); 2964, 2921, 2850 (CH2); 1653 (C=O) cm -~. The same procedure was used for the preparation of the homologues lipids: (see also Fig. 2). o-[2Acetylamino-3-(3,6,9-trioxanonadecylthio)propylcholine phosphate (6) C2~HssN2OsPS (586.8); o-[2-acetylamino-3-(3,6,9,12-tetraoxae i c o s y l t h i o ) p r o p y l ] c h o l i n e p h o s p h a t e (9) C2~H55N2OsPS (602.8); o-[2-acetylamino-33,9,12,15-pentaoxapentaeicosylthio]propyl] choline phosphate (12) C30H63NzOsPS (674.9); o[2-acetylamino-3-(2,6-dithio-9-oxanonadecylthio)propyl]choline phosphate (15) (618.9). The
data are summarized in Tables III, IV and V. Results and Discussion
The results show that the use of l-acetylthio3-hydroxy-2-propaneamine-HCl (s-acetyl-cysteinol) as the starting point of the preparation permits the simultaneous formation of the acetamido and thioether linkages in the basic medium (Fig. 2). The fact that no disulfide formation was observed, although no inert atmosphere was used, leads to the hypothesis that the thioether is formed via a thiazolidine intermediate. It is known that the observed S ~ N acyl migration in cysteine derivatives [15] and in sacyl-amino-mercaptanes [161 leads intermediately to a thiazolidine compound, which forms the nacetyl derivative by ring opening. It seems permissable to assume the same behaviour for the sacetyl-cysteinol (Fig. 3a) under these conditions (Fig. 3b). In basis medium, it seems possible that the attack of the e-C-atoms of the polyoxabromides on the sulphur occurs simultaneously with the acetamide formation (Fig. 3c). This mechanism explains the absence of disul-
161
R
[ H2TBr /S',.,v/CH 3
/svR
~HO~NH 0..
c.
Fig. 3. Mechanism of thioether formation.
H3C~
fide, because there is consistently no thiolate present. Small amounts of s-acetyl-cysteinol perhaps show a thioether formation with a simultaneous S ~ O acetyl migration. This product suffers an ester cleavage in the basic aqueous solvent. The finished product is an amino-derivative, which is also obtained by acid hydrolysis of the acetamide compound. Indeed we detected this by-product by thiniayer chromatography (CHC1/CH3OH; 9 : 1, v/v) in the reaction mixture. Therefore, it is necessary to purify the polyethers by column chromatography, because this by-product reacts in the next step with 2chloro-2-oxo-l,3,2-dioxaphospholane to different compounds, which are difficult to separate from the finished phespholipids. The purification also allows recovery of the rest of the remaining commercially unavailable bromides. The described method is of general interest for the synthesis of thioacetamide analogs of the platelet activating factor without any disulfide formation. The acetamide linkage also presents an easily cleavable protecting group (methanol/ HC1 conc.; 3 : 1 ; v/v, reflux) and is therefore useful for the synthesis of other azathia-analogs. The phosphocholine part is introduced in a similar way to that described by Bhatia and Hajdu [17] via 2-chloro-2-oxo-l,3,2-dioxaphospholane and following ring opening with trimethylamine. This method leads directly to the inner salt and only small amounts of phosphorcontaining by-products.
edged. Furthermore we thank Mr. Helms for recording the NMR-spectra and Mr. Kahnt for accomplishing the IR-measuring.
Acknowledgements
15
Material support by the Fonds der chemischen Industrie, Frankfurt a. M. is gratefully acknowl-
References 1
2 3 4 5
6 7 8 9 10 11
12
13 14
16 17
J. Benveniste and B.B. Vargaftig (1983) in: H.K. Mangold and F. Paltauf (Eds.), Ether Lipids: Biochemical and Biomedical Aspects, Academic Press, New York, pp. 355--376. G. Ostermann, H-P. Kertscher, A. Lang and U. Till (1987) Chem. Phys. Lipids 43, 247--255. P. Hadv~ry and T. Weller (1986) Holy. Chim. Acta 69, 1862--1871. K.L. Meyer, S.W. Schwendner and R.E. Counsell (1989) J. Med. Chem. 32, 2142--2147. H. Miyazaki, N. Nakamura, T. lto, T. Sada, T. Oshima and H. Koike (1987) Sankyo Kenkynsho Nempo 39, 55--65. S.K. Bhatia and J. Hajdu (1987) Tetrahedron Lett. 28, 271--274. G. Grue-Sorensen, I.M. Nielsen and C.K. Nielsen (1988) J. Med. Chem. 31, 1174--1178. A. Wissner, C.A. Kohler and B.M. Goldstein (1986) J. Med. Chem. 29, 1315--1319. N.S. Chandrakumar nad J. Hajdu (1982) Tetrahedron Lett. 23, 1043--1046. B. Garrigues (1984) Synthesis, 870--872. S. Morris-Natschke, J.R. Surles, L.W. Daniel, M.E. Berens, E.J. Modest and C. Piantadosi (1986) J. Med. Chem. 29, 2114--2117. W.E. Berdel, R. Andreesen and P.G. Munder (1985) in: J.F. Kuo (Ed.), Phospholipids and Cellular Regulation, CRC Press, Boca Raton, FL, Vol. 2, pp. 42--72. E. Bosis, D.B.J. Herrmann, U. Bicker, R. Gall and W. Pahlke (1987) Lipids 22, 947--951. J.M. Zeidler, W. Zimmermann and H.J. Roth (1989) Sci. Pharm. 57, 417--422. R.G. Hiskey, T. Mizoguchi and T. Inui (1966) J. Org. Chem. 31, 1192--1341. T. Wieland and E. Bokelmann (1952) Ann. Chem. 576, 20--34. S.K. Bhatia and J. Hajdu (1989) Synthesis, 16--20.
155
Synthesis of azathia analogues of platelet activating factor with polyheterosidechains* J.M.
Zeidler**, W. Zimmermann
and H.J.
Roth
Pharmazeutisches lnstitut der Universit~t T~bingen, A u f der Morgenstelle 8, 7400 T~bingen (F.R.G.) (Received January 29th, 1990; revision received and accepted March 23rd, 1990)
The synthesis of azathia analogues of the platelet activating factor with oxygen and sulphur-containing sidechains is reported. The starting point is 1-acetylthio-3-hydroxy-2-propaneamine-HCl, which permits the formation of the thioether and the acetamido linkage in one step. The phosphocholine part is introduced via 2-chloro-2-oxo-l,3,2-dioxaphospholane and subsequent ring opening with trimethylamine under pressure.
Keywords: phospholipid synthesis; platelet activating factor analogues; azathia analogues; ether lipids.
Introduction
Platelet activating factor (PAF) is an alkyl ether phospholipid (structure see Fig. l a) composed primarily of the C~6 and Cls homologues. It is a potent activator of platelet aggregation and one of the important mediators of anaphylaxis and inflammation [1]. The ~taturally occurring lipid also exhibits a strong hypotensive quality. Over the last few years a number of structure analoga have been prepared and tested [2--7]. Wissner et al. [8] synthesized some analogues with multiple oxygen substitution of the alkoxy chain (Fig. lb). Incorporation of ether linkages results in a significant reduction of biological activity of these compounds. In 1982, a new analogous structure with an acetamide function at the chiral C-atom was prepared by Chandrakumar and Hajdu [9]. This modification ieads to a lipid which is stable against enzymatic degradation by acetylhydrolase and exhibits potent platelet activation [9]. The next step was Correspondence to: W. Zimmermann. *Dedicated to H. Mthrle on the occasion of his 60th birthday. **Part of dissertation, Ref. H.J. Roth.
the exchange of the ether linkage by a thioether group (Fig. lc) [10]. The sulfur substitution at the sn-1 position seems to lower the plateletaggregating potency and to enhance the tumorcytotoxicity [ll,12]. The thioether phosphocholine analogous BM 41.440 (Ilmofosine) [13] has been tested in clinical phase I/II trials. It showed antitumor potency in a variety of tumor models in vitro and in vivo. For that reason, it is of interest to synthesize new potent analogues which in addition help to understand the structure-activity-relationship of cytotoxicity and tumor selectivity. As described earlier [14], it is possible to synthesize azathia analogues containing two oxygens in the alkylchain at sn-1 (Fig. l d). The homologues line and one thiaanalogue were now prepared. The oxygen-atoms are posed near the backbone so that the sidechain has a hydrophobic end, which seems to be necessary for the affinity to the cell membrane. Materials and Methods
2-Chloro-2-oxo- 1,3,2-dioxaphospholane, 97% pure; trimethylamine anhydrous, 98°/o pure,
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
156
0/C16H33
II
0
/P~n~N~
Io
I_OIe
0~O~0(CH2)9
O
CH3
k~O/P~o~N~ h
I
I
iol e
s/ClaH37 H
H3C~]~N~ j ~ I. O k~o/P~n~N~ c. I I IO_I e s~O~/~0/C12 H2s H3C~]~N
°
II
0 d, IOI e
Fig. 1. PAF (a) and synthetic analogues (b--d).
ethyleneglycol, >99070 pure; triethyleneglycol, > 99°7o pure; tetraethyleglycol, > 99070 pure; pentaethyleneglycol > 9707o pure; 1-bromo-hexadecane, >9807o pure; 1-bromo-decane, >97070 pure and 1-bromo-octane, >98o70 pure; were from Fluka AG (Neu-Ulm, F.R.G.). 4-Nitrobenzenesulfonylchloride, >9807o pure; sodium amide, > 9707o pure and 1,8-dihydroxy-3,6dithiaoctane were purchased from Merck-Schuchardt (Hohenbrunn, F.R.G.). LiBr, anhydrous, > 9907o pure was purchased from Merck (Darmstadt, F.R.G.) 1-Acetylthio-3hydroxy-2-propaneamine-HCl was prepared as described elsewhere [14].
Analytical and preparative methods 'H- and '3C-NMR-spectra: Brucker AC-80spectrometer (80 MHZ), internal standard: TMS; IR-spectra: Perkin-Elmer FT-IR spectrometer 1750 (KBr); melting points: Gallenkamp, melting point apparatus, uncorrected; column chromatography: silica gel 60 (7734) from Merck (Darmstadt, F.R.G.) with a particle size of 0.063 --0.2 mm; phospholipids were purified by MPLC (Labomat MD 80/100, Labomatic) on silica gel (Labogel NP 30--60, 30/~m/60A from Labomatic); column: vol. 410 ml, diameter 37 mm; polyalcohols and the related bromides were distilled in a kugelrohr apparatus (Biichi) under reduced pressure; solvents were removed under vacuum at 40°C. The purity of the lipids was checked by thin-layer chromatography (Polygram Sil G/UV 254, Macherey-Nagel, layer: 0.25 mm silica gel, 40 × 80 mm). Lipids were detected by spraying with Molybdenum Blue (Sigma)/0.1 mol H2SO4 (3 : 1). Solvent, chloroform/methanol/NH 3 25°70 (65 : 35 : 6, by vol.), without saturation. Benzene, acetonitrile and tetrahydrofuran were distilled from calciumhydride directly into the reaction vessels. Triethylamine was dried over potassiumhydroxide and then distilled. Acetone was distilled from P205. General procedure for the polyoxabromide synthesis 1-bromo-3-oxanonadecane (A). Sodium amide (25 mmol) was suspended in 150 ml dry tetrahydrofuran. Ethyleneglycol (50 retool) was added drop by drop for 30 min and the reaction mixture was heated to reflux for 30 min. 1-Bromohexadecane (25 mmol) was then added and heating was continued for further 5 h. Thereafter, the solvent was removed and the residue was poured into 100 ml water and extracted with n-hexane. After evaporation the crude product was distilled under reduced pressure. Yield: 38.6 g (54070); mp: 37°--39°C; C18H380 2 (286.5). 3-Oxanonadecane-l-ol (25 mmol), obtained as described above, was dissolved together with triethylamine (26 retool) in 100 ml waterfree tetrahydrofuran. At 0 °C p-nitrobenzene-
157 TABLE I Homologeous polyoxabromides. Yields are calculated over all steps. Bromide (B) 1-bromo-3,6,9-trioxanonadecane CI6H3303Br (353.3) (C) l-bromo-3,6,9,12-tetraoxaeicosane CI6H330,Br (369.3) (D) l-bromo-3,6,9,12,15-pentaoxapentaeicosane C20H,iOsBr (441.5) (E) 1-bromo-3,6-dithio-9-oxanonadecane Ct6H33OS~Br (385.5)
~
bp.
yield
1.4579
190°C (1.8 x 10-~ mbar)
13.8070
1.4588
150°C (1.3 × 10-~ mbar)
27.3070
1.4610
175°C (7.2 x 10-2 mbar)
10.5%
mp: 37--38°C
19.7%
sulfonylchloride (25 mmol) was added in three parts and the mixture was stirred for 6 h at room temperature. The precipitated salt was filtered, the solvent removed, and the residue was dissolved together with dry lithium bromide (30 retool) in water-free acetone. After 30 h, the acetone was distilled off under reduced pressure and the residue was stirred up in 100 ml n-hexane for 15 rain. After filtration, the solvent was dried over sodium sulfate, then evaporated and the crude 1-bromo-3-oxanonadecane (A) was purified by distillation. CIsH37OBr (349.4); n~0d: 1.4600, bp: 175°C (3.7 X 10-~ mbar); yield: 29°7o. ~H-NMR (chloroform-d~): 6 (ppm): 0.88 (t, J = 5 Hz, 3H, CH3); 1.22--1.60 (m, 28H, C H 2 ) ; 1.72--2.08 (m, 2H, CH2Br); 3.26--3.93 (m, 4H, CH20).
13C-NMR (chloroform-dr): tt (ppm): 14.14 (CH~); 22.84 (CH2--CH3); 26.29, 28.39, 28.69, 29,14, 29.37, 29.62, 29.90, 31.06 (13CH2); 32.13 (CH2Br); 70.66, 71.31 ( 2 C H 2 0 ) .
The bromides B--E were prepared in the same manner. The data are summarized in Tables I and II.
N-[1-(3-oxanonadecylthiomethyl)-2-hydroxyethyl]acetamide (1). 1-Acetylthio-3-hydroxy-2propanamine-HC1 (4.0 g, 0.0215 tool) was heated to reflux together with 5.9 g (0.0426 tool) potassium carbonate and 8.0 g (0.0228 tool) 1bromo-3-oxanonadecane, in a mixture of 80 ml methanol and 10 ml water, for 3 h. Then 100 ml cool water was added and the thioether was extracted with dichloromethane. The solvent was removed, the residue diluted in ethylacetate and frozen at - 1 8 °C. The crude product was purified by column chromatography using chloroform/methanol (9 : 1, by vol.) as a solvent. Yield: 6.6 g (73.5e70) white solid; rap: 68-69°C; C23H47NO3S (417.7); N: calculated: 3.35, found: 3.35. IH-NMR (chloroform-dl): d (ppm): 0.87 (t, J = 7.5 Hz, 3H, CH3); 1.25--1.58 (m, 28H, CH2); 2.00 (s, 3H, C O C H 3 ) ; 2.67--2.82 (m, 4H,
TABLE II ~3C-NMR-shifts of the polyoxabromides d (ppm). Solvents: B--D = DMSO-d6; E = CDCi r Bromide
CH2--Br
CH20
CH2S
CH 2
C__H2--CH3
CH 3
B C D E
32.01 31.40 31.57 43.05
70.44--65.52 71.54--68.11 70.41--69.50 71.31, 70.85
---34.38--31.67
31.32--25.66 30.19--25.59 31.30---25.63 29.71--26.13
22.09 22.50 22.06 22.67
13.84 14.04 13.65 14.08
158 TABLE III Experimental data. 4
6
mp. (°C)
7
9
184---185 1.4842 60.1 •,d 3.32 3.41
yield (%) N: calculated N: found IR (cm"l) NH, OH
50.8 b 4.77 4.82
3420--3280 2926 2856
CH 2
C=O
3758 2964 2932 2862 1698
1656
10
168--172 1.4836 80.6 =,d 3.20 3.29 3421-3258 2927 2859 1657
1.4856 41.4•.d 2.75 2.69
51.9 b 4.65 4.71 3428
12
13
15
219--221
69--70
161--163
40.4 b 4.15 4.25
44.9 c 3.09 3.15
32.6 b 4.53 4.42
3420
3400-3280 2964 2924 2860 1680
3402
2928 2858
3432-3281 2925 2857
2904 2852
1652
1665
1653
2954 2924 2854 1651
• Purified by column chromatography, solvent: CHCI/CH3OH (9 : 1, v/v). bpurified by MPLC, solvent: CHCI/CH3OH/NH 3 25% (65 : 35 : 6, v/v/v). ° Ice water was added to the reaction mixture, the formed precipitate was filtered and recrystallized from hot n-hexane. a After removing the solvent in a rotary evaporator the residue was dissolved in dichloromethane, filtered and then purified by column chromatography.
2CH2S); 3.36--3.69 (m, 6H, 2OCH2, CH 2 OH), 4.02 (m, 2H, CH, OH) 6.66 (s, IH, NH). 13C=NMR (chloroform-dl): 6 (ppm): 14.09 (CH3); 22.66 (CH2--CH3); 23.25 (COCH3); 26.13, 29.36, 29.69, 31.94 (13CH2); 32.26, 33.05 (2CH2S); 51.30 (CH); 62.98 (CH2OH); 70.49, 71.31 (2CH20); 170.90 (C=O). IR: 3400--3295 (NH, OH); 2978, 2920, 2850 (CH2); 1670
(C = O) cm -t. The homologues compounds were prepared in a similar manner: n-[1-(3,6,9-trioxanonadecylthiomethyl)-2-hydroxyethyl]acetamide (4) C2tH43NOsS (421.6); n-[1-(3,6,9,12-tetraoxaeicosylthiomethyl)-2-hydroxyethyl]acetamide (7) C2~H43NO6S (437.6); n-[1-(3,6,9,12,15-pentaoxapentaeicosylthiomethyl)-2-hydroxyethyl]acetamide (10) C25HslNOTS (509.7); n-[1-(3,6-dithio-
TABLE IV IH-NMR-shifts 8 (ppm). Solvents: 4,7,10,13: chloroform-d1; 6,9,12,15: methanol-d4.
CH~ CH 2 COCH 3 CH2S CH20 CHzOH CH)OH NH ÷NCH~ CH CH2N
CH20
4
6
7
9
10
12
13
15
0.88 1.27--1.57 2.00 2.67--2.83 3.37--3.77 3.95---4.12
0.89 1.28--1.63 1.96 2.66---2.82
0.87 1.27--1.64 1.98 2.65--2.80 3.28--4.21
0.89 1.30---1.55 1.96 2.66--2.82
0.87 1.27--1.66 1.98 2.65--2.82 3.33--3.82 3.87--4.07
0.90 1.30--1.67 1.98 2.76--2.96
0.87 1.26--1.69 2.01 2.64--2.70 3.35--3.78 3.93--4.08
0.89 1.29--1.66 1.96 2.64---2.78
6.91
7.03
6.68
6.34
3.24
3.24
3.27
3.23
3.30--4.24
3.46---4.29
3.41--4.49
3.34---4.37
159 T~LEV s3C-NMR-shifisd~pm).Solvents:4,7,10,13:chlorofo~:6,9,12,1$:m~hanol~4. 4
6
7
9
10
12
CH 3 CH2--CH 3 COCH 3 CH 2
14.02 22.61 23.14 26.13 29.26 29.57 29.71 31.90
14.42 22.73 23.60 27.11 30.34 30.46 30.62 32.67
13.41 21.93 22.42 25.40 28.53 28.71 28.93 31.13
14.42 22.73 23.63 27.12 30.34 30.46 30.64 32.69
14.07 22.61 23.14 26.02 29.25 29.53
14.43 22.80 23.66 27.13 30.36 30.59 32.62
CH2S
32.26 33.56
32.94 33.92
31.22 32.54
32.91 33.93
31.89 33.21
32.95 33.89
CH *NCH 3 CHzN CH2OH CH20
51.63
51.05 54.69 60.41
50.73
51.07 54.73 60.46
51.35
51.06 54.77 60.53
CH=O
63.12 70.15 70.39 70.67 71.37 71.54
170.72
66.83 67.17 71.03 71.14 71.42 71.83 72.23 172.60
61.94 69.33 69.55 69.85 70.50 70.72
170.11
66.77 67.41 71.03 71.i6 71.42 71.63 72.29 172.92
9-oxanonadecylthiomethyl)-2-hydroxyethyl]acetamide (13) C21H43NO3S3 (453.8). The data are summarized in Tables III, IV and V.
N-[1-(3-oxanonadecylthiomethyl)-2-(2-oxa1,3,2-dioxaphospholane-2-yloxy)ethyl] acetamide (2). Thioether (1) (2.4 g, 0.0057 tool) was dissolved together with 0.92 g (0.0064 mol) 2chloro-2-oxo-l,3,2-dioxaphospholane in 25 ml dry benzene. At 0oC, 0.67 g (0.0066 mol) of dry triethylamine was added drop by drop and the mixture was stirred at room temperature for 5 h. After filtration of the precipitated salt, the solvent was removed by reduced pressure. The obtained residue was used without further purification. C25Hs0NO~PS (523.7); the following homologues were prepared: n-[1-(3,6,9-trioxanonadecylthiomethyl)-2-(2-oxo- 1,3,2-dioxaphospholane-2-yloxy)ethyl]acetamide (5) C23H4~NOsPS
62.77 69.98 70.21 70.51 71.27 71.43
170.66
66.86 70.30 70.96 71.10 71.27 71.71 72.30 172.99
13
14.07 22.64 23.31 26.08 29.29 29.43 29.54 29.64 31.79 32.21 32.36 32.67 32.94 50.95
62.91 70.57 71.31
170.67
15
14.47 22.74 23.66 27.22 30.40 30.52 30.72 32.54 33.02 33.10 33.27 33.53 33.69 51.11 54.74 60.45
66.68 67.27 72.01
172.92
(527.7); n-[1-(3,6,9,12-tetraoxaeicosylthiomethyl)-2-(2-oxo-l,3,2-dioxaphospholane-2-yloxy)ethyllacetamide (8) C23H,sNO9PS (543.7); n-[1(3,6,9,12,15-pentaoxapentaeicosylthiomethyl)-2(2-oxo- 1,3,2-dioxaphospholane-2-yloxy)ethyl]acetamide (11) C2~Hs4NO~0PS (615.8); n-[l(3,6,dithio-9-oxanonadecylthiomethyl)-2-(2-oxo1,3,2-dioxaphospholane-2-yloxy)ethyl]acetamide (14) C23H46NO6PS 3 (559.8).
O-[2-acetylamino-3-(3-oxanonadecylthio)propyl]cholinephosphate (3). The obtained amount of 2 was dissolved in 80 ml of dry acetonitrile and transferred into a pressure bottle. A large excess of dry trimethylamine (10 ml) was added and the solution was stirred at 65°C for 36 h. After cooling, the solvent was removed and the remaining solid was purified by column chromatography on silica gel ( M P L C ) using
16o
0 S"~CH3
SAR
H, .yI Ele
I,,,~OH
0 "~OH 0 [1 II .cj/p~,?y H /
R = ~0--C16H33 R = -..-(-0-,,,,.~0~[10H21 R = -+-O-,v~O__EsH17
s~R
2. N(CH3~
[3.6.,~.1z'15.]
o
R = .-+-O-,v~O~C10H21
0e
R = .-~-S-,,,~0~£10H21 Fig. 2. Reaction scheme.
chloroform/methanol/NH3 25070 (65 : 35 : 6, by vol.) as a solvent. Yield: 2.1 g (63.2~70) white solid; mp: 187--189°C. C2sH59N206PS (582.8); N: calculated: 4.81, found: 4.75. ~H-NMR (methanol-d4): 6 (ppm): 0.89 (t, J = 7.5 3H, CH3); 1.28--1.55 (m, 28H, CH2); 1.96 (s, 3H, COCH3); 2.65--2.81 (m, 4H, 2CH2S), 3.23 (s, 9H'N(CH3)3); 3.34--4.32 (m, llH, CH, 4CH20, CH2N). 13C-NMR (methanol-d4): d (ppm): 14.42 (CH3); 22.71 (CH2--CH3); 23.65 (COCH3); 27.17, 30.40, 30.52, 30.70, 32.82 (13CH2); 33.00, 33.97 (2CH2S); 51.06 (CH); 54.70 ('N(CH3)3); 60.45 (CH,N); 67.20, 67.43 (2CH20); 71.43, 72.00 (2CH20); 172.92 (C=O). IR: 3440 (NH); 2964, 2921, 2850 (CH2); 1653 (C=O) cm -~. The same procedure was used for the preparation of the homologues lipids: (see also Fig. 2). o-[2Acetylamino-3-(3,6,9-trioxanonadecylthio)propylcholine phosphate (6) C2~HssN2OsPS (586.8); o-[2-acetylamino-3-(3,6,9,12-tetraoxae i c o s y l t h i o ) p r o p y l ] c h o l i n e p h o s p h a t e (9) C2~H55N2OsPS (602.8); o-[2-acetylamino-33,9,12,15-pentaoxapentaeicosylthio]propyl] choline phosphate (12) C30H63NzOsPS (674.9); o[2-acetylamino-3-(2,6-dithio-9-oxanonadecylthio)propyl]choline phosphate (15) (618.9). The
data are summarized in Tables III, IV and V. Results and Discussion
The results show that the use of l-acetylthio3-hydroxy-2-propaneamine-HCl (s-acetyl-cysteinol) as the starting point of the preparation permits the simultaneous formation of the acetamido and thioether linkages in the basic medium (Fig. 2). The fact that no disulfide formation was observed, although no inert atmosphere was used, leads to the hypothesis that the thioether is formed via a thiazolidine intermediate. It is known that the observed S ~ N acyl migration in cysteine derivatives [15] and in sacyl-amino-mercaptanes [161 leads intermediately to a thiazolidine compound, which forms the nacetyl derivative by ring opening. It seems permissable to assume the same behaviour for the sacetyl-cysteinol (Fig. 3a) under these conditions (Fig. 3b). In basis medium, it seems possible that the attack of the e-C-atoms of the polyoxabromides on the sulphur occurs simultaneously with the acetamide formation (Fig. 3c). This mechanism explains the absence of disul-
161
R
[ H2TBr /S',.,v/CH 3
/svR
~HO~NH 0..
c.
Fig. 3. Mechanism of thioether formation.
H3C~
fide, because there is consistently no thiolate present. Small amounts of s-acetyl-cysteinol perhaps show a thioether formation with a simultaneous S ~ O acetyl migration. This product suffers an ester cleavage in the basic aqueous solvent. The finished product is an amino-derivative, which is also obtained by acid hydrolysis of the acetamide compound. Indeed we detected this by-product by thiniayer chromatography (CHC1/CH3OH; 9 : 1, v/v) in the reaction mixture. Therefore, it is necessary to purify the polyethers by column chromatography, because this by-product reacts in the next step with 2chloro-2-oxo-l,3,2-dioxaphospholane to different compounds, which are difficult to separate from the finished phespholipids. The purification also allows recovery of the rest of the remaining commercially unavailable bromides. The described method is of general interest for the synthesis of thioacetamide analogs of the platelet activating factor without any disulfide formation. The acetamide linkage also presents an easily cleavable protecting group (methanol/ HC1 conc.; 3 : 1 ; v/v, reflux) and is therefore useful for the synthesis of other azathia-analogs. The phosphocholine part is introduced in a similar way to that described by Bhatia and Hajdu [17] via 2-chloro-2-oxo-l,3,2-dioxaphospholane and following ring opening with trimethylamine. This method leads directly to the inner salt and only small amounts of phosphorcontaining by-products.
edged. Furthermore we thank Mr. Helms for recording the NMR-spectra and Mr. Kahnt for accomplishing the IR-measuring.
Acknowledgements
15
Material support by the Fonds der chemischen Industrie, Frankfurt a. M. is gratefully acknowl-
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2 3 4 5
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13 14
16 17
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