[54] Synthesis of cyclopentanoid analogs of diacylglycerophosphate

640

SUBSTRATES, ANALOGS, AND INHIB[TORS

[54]

[54] S y n t h e s i s o f C y c l o p e n t a n o i d A n a l o g s o f Diacylglycerophosphate

By ANTHONY J. HANCOCK This chapter describes methods for the preparation of a series of glycerophospholipid analogs whose conformationally restricted nature allows study of the dependency of their biochemical and biophysical properties on their molecular conformation. The analogs are derived from the diastereoisomeric cyclopentane-l,2,3-triols (1-3), which are formal analogs of glycerol. Therefore, comparative studies of the conformationally restricted cyclopentano-lipid analogs and their natural glycerol counterparts should allow assessment of the conformational state (rotameric state) of the glycerol backbone during physiological involvement. The rationale has been presented in some detail in a communication describing the synthesis and properties of the cyclopentanoid analogs of homologous triacylglycerols (C8-Cla). 1 The synthetic protocols to be described allow the preparation of gram quantities of six stable cyclopentano analogs of diacylglycerophosphate (phosphatidic acid) from cyclopentadiene. Routes to these compounds generally involve procedures well established in the field of lipid synthesis, but thus far have been limited to the preparation of derivatives possessing only one type of fatty acid (bis-homo-acyl derivatives). Accounts of this work have been published. 1-3 NOTE: Cyclic compounds described in this paper are named according to the Tentative Rules for Nomenclature of Cyclitols. 4 The names are derived from those of the parent cyclanes of which they are formal derivatives; the location and disposition of the hydroxyl groups are indicated by a configurational fraction in which all the substituent on one side of the plane of the cyclane ring are assembled in the denominator. Thus the configurations depicted in Fig. 1 are denoted by 1,2,3/0, 1,2/3, and 1,3/2 for structures 1, 2, and 3, respectively. The lowest possible numbers are used in each case, and no absolute configuration is implied by this fraction. When absolute configuration must be specified, a separate convention relates the lowest-numbered asymmetric center to D- or L-glyceraldehyde. For example, the enantiomer of compound 2 depicted A. J. Hancock, S. M. Grecnwald, and H. Z. Sable, J. LipidRes. 16, 300 (1975). 2 A. J. Hancock, M. H. Stokes, and H. Z. Sable, J. LipidRes. 18, 81 (1977). 3 A. J. Hancock and M. D. Lister, J. LipidRes. 20, 271 (1979). 4 I U P A C - I u a Commission on Biochemical Nomenclature. Tentative Rules for Nomenclature of Cyclitols. Arch. Biochem. Biophys. 128, 269 (1968).

METHODS IN ENZYMOLOGY,VOL. 72

Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

OH OH (1)

OH

641

OH (3)

(2)

FIG. 1. Diastereoisomeric cyclopentane-l,2,3-triols: (1) 1,2,3/0; (2) 1,2/3; (3) 1,3/2.

'c°

'

b@

(4)

o®

(5)

RCOO~

.coo

o®

.coo

(6)

OCOR

(8) R= C15H31

o®

(7)

RCOO~ O(,~)OCOR (9) (~= -

P(O)(OH)2

FIG. 2. Cyclopentanoid analogs of diacylglycerophosphate: (4) 1,3/2-1P: ( ~ 1,2/3-3P; (6) 1,2,3/0-1P; (7) 1,2/3-1P; (8) 1,3/2-2P; (9) 1,2,3/0-2P.

ttol

HO

'"~22~H

119)

O1'O 1161

CH3~ ,. IP:CH3,,C~ FIG. 3. Synthesis of syn- and anti-anhydrocyclopentanetriols; syn- and anti-triol acetonides

642

SUBSTRATES, ANALOGS, AND 1NHIBll-ORS

HO~

°eF,,°

[54]

BzCI OH 0

~.lpS0

(23) ~H+

O6)

HO~

~OH BxCI Bz O ~ .

(12)

(27)

KMnO4~oBHz O ~ OH OH (24)

~ RCOCI pyridine (~)O ~

(C6HsO)2POCI~H O ~ pyridine

OCOROCOR (5) 0 ®---~-~oc..,~, BzmC6H$CH 2R:C15H31-

Pd-C B z O ~ H2 OCOROCOR OCOROCOR (26)

1251

Fro. 4. Synthesisof (l,2/3-3P)-cyclopentanophosphatidicacid. in Fig. 1 would be designated as 1-D-(1,2/3)-cyclopentane-l,2,3-triol. It is to be emphasized that in this work only m e s o - or racemic substances are involved. For the phosphorylated compounds, the 1-phosphate and 2-phosphate positional isomers are designated by -1P or -2P, respectively, immediately following the cyclane nomenclature (see Fig. 2). Cyclopentane- 1,2,3-triols (1,2,3) •These compounds are not available commercially, but excellent routes for their synthesis have been established by the elegant pioneering efforts of Sable and co-workers ~ during a systematic study of cyclitols and their derivatives during the last two decades. Much of our understanding of the conformational analysis of these systems derives from these studies. In some cases in the present work, advantage has been taken of these routes directly to synthesize suitably protected intermediates from the triols themselves (1, 2, 3). In other cases, however, new routes have been established that allow the generation of protected intermediates without recourse to the expensive cyclopentanetriol itself. In all procedures described in this chapter, the intermediates employed were ultimately derived from cyclopentadiene; the structures in 5 H. Z. Sable, T. A d a m s o n , B. Tolbert, and T. Posternak, Helv. Chirn. Acta 46, 1157 (1963).

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

643

~r m- Chloroperbenzoic acid 1

j',

HO OH

..

P 2.

OBz

(30)

l.coo

.coo

pyridine

frsct, cryst.

..~

H

3...pon,,,(O.-~

OBz

(33)

l,coo

O

pyrldine

(~): - ; = (OC6H5) 2 R: -C1s H31 RC Bz = -CH2C6H5

'c~3~°'"

KO~

RC~

"c°%2)°"

I

(3,f'"

(a~l °"

.c..,o,,,oc.

.coo(7)o®

I

~c°°(4~)o~

FIG. 5. Synthesis o f ( 1,3/2-1P)- and ( 1,2/3- I P)-cyclopentanophosphatidic acids.

the reaction schemes are numbered (Figs. 3-8) in such a way that intermediates common to more than one synthetic route can be identified. Section I deals with the synthetic procedures leading to 3-chlorocyclopentene and the corresponding allylic alcohol 1-cyclopentenol. Subsequent sections include the procedures by which the diastereoisomeric dipalmitoylcyclopentanetriol phosphates [referred to in this chapter as cyclopentanophosphatidic acids or cyclo-PA(s)] are prepared. Analytical thin-layer chromatography was performed using silica gel G-coated glass plates, adsorbent thickness 250 /zm (Brinkmann, Inc., 5763). Preparative thin-layer chromatography was performed on silica gel G-coated glass plates, adsorbent thickness 1500/am (Analtech, Inc. Newark, Delaware).

644

SUBSTRATES, ANALOGS, AND INHIBITORS HO

pyriclinium

[54]

P//H2

chJorochromafe

Bz

O~lp.O

O-ipsO

(le)

(ae)

"91---

OCOROCOR pyridlne

IIz

O-ip'O

12~)

"1""

OH OH

(3e)

gz

BzCI oH

O~lp, 0

(aa)

(zT)

PclC ~ H2

'~*H'-~L'°c' ~

~

OH OCOR

,¢..,o,,,~,

OCOROCOR (40)

ff .

(41)

(9)

®O~--'--f

Bz- C6H, CH2-

ocomocoR

® = -p= (OC~Hs)2

~e)

o RB C15H31-

Ft~. 6. Synthesis of ( 1,2,3/0-1 P); and ( 1,2,3/0-2P)-cyclopentanophosphatidic acids.

HO~7#

- NO 2 C6 H4CHO

OH

(3)

~

02NC'H4 ~

0

~

OH

(44)

o® (45)

R=ClsH31-

® : -, :,oc°.,), o

H O ~

o® (46)

o® (8) FIG. 7. Synthesis of (1,3/2-2P)-cyclopentanophosphatidic acid.

[54]

C Y C L O P E N T A N O I D A N A L O G S OF D 1 A C Y L G L Y C E R O P H O S P H A T E

~l~ 0

=

OH

-DPPC ~

(19)

0

645

0@ H÷

(42)

oII -P=(OC6H5)2

R=-C15H31

I 0H(43) I RCOCI RCO0~ @ OCOR (4)

FIG. 8. Synthesis of (l,3/2-1P)-cyclopentanophosphatidicacid. I. Synthesis of Chlorocyclopentene and Cyclopentenol A. 3-Chlorocyclopentene (! 1) The hydrochlorination of cyclopentadiene (10) at low temperatures to give good yields of 11 has been described elsewhere. 6 The authors observe that 3-chlorocyclopentene is unstable and should not be stored for prolonged periods even at low temperatures. Our experience is that it is prudent to perform the hydrochlorination and either to epoxidize or to hydrolyze the product during the same day. B. DL-2-Cyclopenten- 1-ol (12) A suspension of sodium bicarbonate (450 g) in 2 liters of water was mechanically stirred and cooled to 0 °. A dry ice/acetone bath at a temperature of approximately - 1 5 ° was more effective than an ice-salt mixture. Freshly prepared 3-chloro-l-cyclopentene ( l l ; 140 g) was added o v e r a period o f 1 hr, and the hydrolysis mixture was stirred at 0 ° for an additional 2 hr. Cyclopentenol 12 was salted out by addition of sodium chloride (500 g) and obtained by thorough extraction with diethyl ether. The extract was dried (anhydrous sodium sulfate), the ether was removed by evaporation, and the remaining oil was purified by distillation (bp 62-65°/36 mm); n D z8 = 1.4692). Thin-layer chromatographic (TLC) analysis ( C H C I ~ E t 2 0 , 20 : 1, v/v) of the distillate showed a major component (R e 0.30) and traces of dicyclopentenyl ether (Re 0.75). R. B. Moffett, Org. Synth., Collect. Vol. 4, 779 (1963).

646

SUBSTRATES, ANALOGS, AND INHIBITORS

[54]

NOTES: l. Dicyclopentenyl ether invariably codistilled with cyclopentenol and formed up to 5% of all fractions. However, the contaminant did not interfere with subsequent reaction steps. The presence of dicyclopentenyl ether may also be detected by characteristic absorption bands in the infrared spectrum (CS2) at 1160 cm -~, 1315 cm -~, and 1360 cm -1, none of which were present in the spectrum of cyclopentenol. The alcohol gave prominent bands at 965 cm -1 and 775 cm -~ that were weak or absent in the spectrum of the ether. 2. The proportion of ether side product in the reaction products was minimized both by maintaining reaction temperature below 2° (freezing of the mixture begins at about -2°), and by adding the 3-chlorocyclopentene at a rate not exceeding 2.5 ml/min. 3. Dr. Henry C. Stevens is thanked for details of this procedure.

II. Synthesis of (1,3/2)-, (1,2/3)-, and (1,2,3/0)-Cyclopentane-l,2,3-triols A. 1,3/2-Cyclopentanetriol (3) The 1,3/2-triol (all-trans configuration) is generated by acid-catalyzed ring opening (Note 1 below) of both the trans-anhydrotriol 19 [major product 1,3/2-triol (3), minor product 1,2/3-triol (2)] and the cis-anhydrotriol 13 [major product 1,2/3-triol (2), minor product 1,3/2-triol (3)] ~'r (for the synthetic procedure leading to epoxides 13 and 19, see Notes 4 and 5). In each case, treatment of the triol mixture product with acetone, dimethoxypropane, and trifluoroacetic acid (TFA) (Note 2) allowed removal of the 1,2/3-triol as its acetonide (isopropylidene derivative 16). NOTES: 1. Ten to twenty grams of anhydrotriol were refluxed with 0.2 N sulfuric acid (200-400 ml) with vigorous stirring until the oil dissolved (usually 2-3 hr). Polymerization of the anhydrotriol was minimized by the use of an oil bath maintained between 105° and 110°. The cooled solution was neutralized with excess barium carbonate and filtered through Celite; the filtrate was concentrated to an oil. 2. The ratio of triols formed after hydrolysis is conveniently determined by gas-liquid chromatography of their trimethylsilyl derivatives (6% SE-30 on Chromosorb W). 3. Mixtures of triols 14 and 15, dried by repeated evaporation of benzene-ethanol and high vacuum treatment, were resolved by suspending the anhydrous mixture in acetone, diluting with 2,2-dimethoxypropane (molar ratio 2,2-DMP:diol = 4: 1, w/w), and stirring overnight with a r R. Steyn and H. Z. Sable, Tetrahedron 25, 3579 (1969).

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

647

catalytic amount of trifluoroacetic acid (large-scale preparations, containing 0.3-0.5 mol of triol, required up to 1 ml of TFA). The solution was neutralized with barium carbonate, filtered over Celite, and evaporated to dryness (the bath temperature was maintained below 40 ° to minimize loss o f volatile acetonide). The resultant oil was partitioned between hexane and aqueous methanol ( C H a O H - H 2 0 ; 9 • l, v/v); the free triol 14 remained in the methanol, and the 1,2/3-acetonide 16 was obtained from the upper hexane phase for subsequent recrystallization from acetone (mp 49--500). 7 4. trans- Anhydrotriol 19 ~ was obtained from 3-chlorocyclopentene II by epoxidation to give 3-chlorocyclopentene-l,2-oxide 17, hydrolysis to chlorocyclopentanediol 18 (not isolated), and base-catalyzed ring closure to 19 as follows: A solution of 22 g of 85-87% m-chloroperbenzoic acid (108-111 mmol) in 450 ml of chloroform was chilled and stirred at 3-5 ° in a 2-liter wide-mouthed brown bottle, and a solution of l0 g (98 mmol) of II in 20 ml of chloroform was added dropwise over 30 min, with magnetic stirring. Stirring was continued for l hr, and m-chlorobenzoic acid precipitated during this time. Usually, four such preparations were carried out simultaneously. The mixtures were stored overnight at 4 ° then filtered, and the filtrate was washed with 5% sodium bicarbonate (4 x 250 ml) and water (1 z 250 ml) and dried over sodium sulfate. Chloroform was removed in a rotary evaporator, and the chloroepoxide 17 was purified by vacuum distillation (bp 50-52°/17 torr); nD25 = 1.4775; yield 7.1-7.7 g (6065%). Ten grams of 3-chlorocyclopentene-l,2-oxide 17 (85 mmol) was hydrolyzed by heating to reflux in 500 ml of 0.2 N sulfuric acid for 2.5 hr. The cooled solution was neutralized with 5 N potassium hydroxide and cooled again to approximately 5°; 29 g of solid potassium hydroxide were added (to give a solution 1 N w i t h respect to potassium hydroxide), and the solution was stirred overnight at room temperature. The solution was cooled to about 5° and carefully neutralized with 6 N sulfuric acid (about 86 ml required). The neutral solution was continuously extracted with diethyl ether for 36 hr; the extract was dried with anhydrous sodium sulfate and evaporated to give 6.9 g of an oil (81% based on chlorocyclopentene oxide) with Rr 0.60 in c h l o r o f o r m - m e t h a n o l - w a t e r , 90: 10: 1, v/v/v. The oil was purified by vacuum distillation (bp 60-62°/0.5 torr). 5. cis-Anhydrotriol 137 was obtained by direct epoxidation of cyclopentenol 12. The procedure was analogous to that described (Note 4 above) for the peracid epoxidation of 3-chlorocyclopentene. B.

1,2/3-Cyclopentanetriol (2)

The 1,2/3-triol (cis-trans configuration) is obtained by hydrolysis of eithercis- ortrans-anhydrotriol (13 or 19) as described in Section II, A and

648

SUBSTRATES, ANALOGS, AND INHIBITORS

[54]

is isolated as its acetonide 16. The acetonide is used as starting material for the henzylation reaction, which serves in the synthesis of (1,2/3)cyclo-PA 5. C. 1,2,3/0-Cyclopentanetriol (1) The 1,2,3/0-triol (all-cis configuration) was formerly synthesized by hydride reduction of ( 1,2,3,4/0)- 1,2-anhydropentane- 1,2,3,4-tetrol, ~ but is now more readily available from the stereospecific reduction of ketoacetonide 36. The ketone 36 is synthesized by mild oxidation (pyridinium chlorochromate) of 1,2/3-triol acetonide 16 (see Note that follows). NOTE: 1,2/3-Triol acetonide 16 (10 g, 63.2 mmol) in methylene chloride (20 ml) was added over 1 hr at room temperature to a mechanically stirred suspension of sodium acetate (15.6 g, 190.1 mmol), and pyridinium chlorochromate (20 g, 92.8 mmol) in methylene chloride (100 ml). After 12 hr the dark suspension was filtered over Celite and concentrated to an oil. Dilution of the oil with diethyl ether (50 ml) gave a brown precipitate that was removed by filtration over Celite. The fltrate was evaporated to a purple oil, TLC analysis of which (CHCla-Et20, 20 : l, v/v) indicated the ketone product 36 (Rt 0.45) and approximately 20% unchanged acetonide (Rs 0.30). The percentage yield of ketone was raised by increasing the molar ratio of oxidant, but since traces of acetonide invariably persisted in the reaction mixture, regardless of the reaction conditions, the ketone was isolated by silicic acid column chromatography of the mixture obtained from the molar ratio specified above. Hexanediethyl ether (95:5, v/v) elution gave pure ketone (5.92 g, 37.9 mmol, 60%; rap, after crystallization from acetone, 37-38°). Further elution with hexane-diethyl ether (90 : 10, v/v) gave acetonide 16 with traces of ketone 36. These mixtures were subsequently treated again with pyridinium chlorochromate. Analysis of ketone 16: calculated for CsH12Oa (156.18): C, 61.52; H, 7.75; found: C, 61.69; H, 7.68. D. (1,2,3/0)-l,2,-Di-O-isopropylidenecyclopentane-l,2,3-triol (22) NOTE: Catalytic reduction of keto-diol acetonide 36 proceeded stereospecifically and quantitatively in ethyl acetate or ethanol to give all-cis-(1,2,3/O)-triol acetonide 22. Gas-liquid chromatographic analysis (6% SE-30 on Chromosorb W) of the trimethylsilyl (TMS) derivative showed that <0.4% of the unwanted 1,2/3-triol acetonide was formed [retention of TMS-1,2,3/0-acetonide (22) 0.85, relative to TMS-1,2/3acetonide (16)]

[54]

C Y C L O P E N T A N O 1 D A N A L O G S OF D I A C Y L G L Y C E R O P H O S P H A T E

649

A solution of keto-diol acetonide 36 (10.0 g) in ethyl acetate (100 ml) was agitated with platinum oxide (50 rag) in a Parr apparatus (60 psi hydrogen). Reduction to 22 was complete after 2 hr, as shown by TLC analysis (chloroform-diethyl ether, 3: 1, v/v; Rs 36 and 22, 0.80 and 0.30, respectively). After filtration through Celite, removal of solvent gave 22 as a colorless oil that could not be induced to crystallize.

III. Synthesis of Monobenzylcyclopentanetriols NOTES: 1. The benzyl protecting group was used extensively during subsequent acylation reactions, since it is not itself prone to migrate during its protective role, and subsequent debenzylation under netural hydrogenolytic conditions minimized concomitant acyl migration. 2. Good yields of benzylated triol acetonides were obtained by basecatalyzed alkylation in refluxing benzene using benzyl chloride as alkylating agent and finely powdered potassium hydroxide as catalyst. A domestic blender was useful for the rapid pulverization of potassium hydroxide. Monobenzyl triols in the 1,2/3 series (24), 1,2,3/0 series (38), and the benzyl ether of cyclopentenol (27) were obtained in this way. Typically, 10-20 g amounts of the triol acetonide (or cyclopentenol) were used in the reaction; larger amounts tended to char in the initial alkoxide-forming reaction between base and the triol derivative.

A. DL-(I,2/3)- 1,2-O-Isopropylidene-3-O-benzylcyclopentane- 1,2,3-triol (23) Isopropylidenetriol 16 (3.0 g, 19.0 mmol) and benzyl chloride (4.8 g, 38 mmol) were allowed to react in refluxing benzene (100 ml) in the presence of powdered potassium hydroxide (3.0 g) for 8 hr. A Dean-Stark phase separator was used to retain the water produced. During this time the mixture darkened and 0.35 ml of water collected (theory 0.34 ml). The cooled mixture was added to 5 volumes each of chloroform and icewater, shaken vigorously, and phase-separated. The lower chloroform phase was washed successively with water (2 × 1 volume), 2.0N sulfuric acid (2 × 1 volume), and saturated sodium bicarbonate (1 x 1 volume). Removal of solvents and excess benzyl chloride after drying over anhydrous sodium sulfate gave a yellow oil whose TLC (CHCIs-EhO, 20 : 1, v/v) showed one major spot (Rr 0.80) and two minor contaminants (Rs 0.20, 0.90). Distillation (bp 126--129°, 0.5 torr) gave 23 as a colorless oil (3.60 g, 14.5 mmol 77%), which crystallized on storage at 5°.

650

SUBSTRArES, ANALOGS, AND INHIBITORS

[54]

Analysis: calculated for C15H2oOz (248.3): C, 72.55; H, 8.12; found: C, 72.46; H, 8.29. NMR: 6 1.28, 6 1.39 (6) doublet, endo- and exo-CHa; 6 1.85 (4) singlet, ring CH~; 8 4.50 (2) singlet, benzyl CH2; 6 7.30 (5) singlet, C6H5; 6 3.87 (1) broad singlet, CH--CH2C6Hs; 8 4.42-4.82 (2) multiplet, ring CH--O---Ip. IR (CS2): 3030, 3060, 3090, 735,695 cm -~ (aromatic); 1360, 1370, 1375 cm -~ (gem-dimethyl). Hydroxyl absorption was absent.

B. oL-(1,2,3/O)- l-O-Benzyl-2,3-O-isopropylidenecyclopentane- l,2,3-triol (37) Isopropylidenetriol 22 (12.0 g, 75.9 mmol) was benzylated in refluxing benzene (120 ml) with benzyl chloride (20 g; 158 mmol) and powdered potassium hydroxide (14 g) as described for the 1,2/3-isomer 23. The reaction mixture remained colorless during the 8-hr reflux; 1.5 ml of water were collected (theory, 1.4 ml). The crude product was isolated as described for 23; distillation (bp 122-125 °, 0.4 torr) gave 37 as a colorless oil (16.9 g, 68.1 mmol, 90%) which showed one spot on TLC (hexane-EhO, 60:40), Rs 0.65, and one peak on GLC, retention 1.I0, relative to the 1,2/3-isomer 23. For analytical purposes, a sample was redistilled (bp 126-129°/0.5 torr). Analysis: calculated for C1~H2003 (248.3): C, 72.55; H, 8.12; found: C, 72.82; H, 8.50. NMR; 6 1.32, 1.53 (6) doublet, endo- and exo-CH3; ~ 4.66 (2) singlet, benzyl CH~; 6 1.55-2.08 (4) multiplet, ring CH2; 8 7.33 (5) singlet, C6H5; 8 4.45-4.60 (2) multiplet, ring C H - - O - - I p ; ~ 3.36-3.75 (1), unresolved multiplet, ring ( C H ) - - O - - CH2C6Hs. IR (liquid film): 3030, 3060, 3090, 1495, 735,700 cm -1 (aromatic); 1365, 1377 (doublet, shoulder at 1360 cm-1), gem-dimethyl. Hydroxyl absorption was absent. C. 1-O-Benzyl-2-cyclopentenol (27) Powdered potassium hydroxide (18 g) was added to a solution of 2-cyclopenten-l-ol (12) (21 g, 25 mmol) and benzyl chloride (68.3 g, 50 mmol) in benzene (100 ml) and heated at reflux for 5 hr when 5.0 mi of water had collected (theory 4.5 ml). The cooled solution was worked up as described for 23. The resulting yellow oil was freed from excess benzyl chloride by evaporation at room temperature under reduced pressure (60 tort). Thin-layer chromatography showed that the residual oil (50 g) contained unchanged 12 and traces of dibenzyl ether. Distillation (bp 72-75°/ 0.6 torr) gave the desired product 27 (27.2 g, 15.6 mmol 62.5%). Spectro-

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHAI'E

651

scopic data were consistent with the proposed structure, but acceptable elemental analyses could not be obtained. Similar difficulties in the case of an O-isopropylidene cyclopentenediol were reported by Young et al. ~ NMR: 8 7.30 (5), aromatic; 8 5.95 (2) multiplet, olefinic; 6 4.68 (1) multiplet, ring O - - C - - H ; 8 4.54 (2) singlet, benzyl CH2; 6 1.65-2.60 (4) multiplet, ring CH2. D. (1,2/3)-3-O-Benzylcyclopentane-l,2,3-triol (24) NOTE: (1,2/3)-3-O-benzyltriol was synthesized by two independent routes. The first route involved acid-catalyzed de-acetonation of benzylacetonide (23) and gave isomerically pure (1,2/3)-3-O-benzyltriol (24) in good yield, as described in detail below. The second route involved direct cis-hydroxylation by alkaline potassium permanganate, a reaction that we have reported 2 to give approximately 1% of the unwanted 1,2,3/0-(all-cis)diol isomer (38). However, subsequent reexamination of the reaction (S. M. Greenwald, unpublished observations) showed that the reaction conditions may affect the stereoselectivity of the hydroxylation and that up to 6% of all-cis-(1,2,3/0)-diol (38) may be formed. The two benzylated triois can be separated by fractional crystallization of theirp-nitrobenzoate esters in a similar manner to that reported for the (1,3/2)-l-benzyl and (l,2/3)-l-benzyl isomers (33 and 30), but the procedure is unreasonably tedious, especially in view of the facile synthesis via benzyl acetonide 23. Method I (via Acetonide)

Isopropylidenebenzyltriol (23) (2.36 g, 9.50 mmol) was hydrolyzed by stirring for 2 hr in refluxing 0.1 N sulfuric acid (200 ml). The cooled mixture was stirred with excess barium carbonate (10 g) for 1 hr, filtered through Celite, and evaporated to dryness to give 24 as an oil (2.22 g, 8.93 mmol, 94%). TLC (CHC13-MeOH-H~O, 90: 10: 1, v/v/v) showed one spot, R s 0.50; GLC of the trimethylsilyl derivative showed that diol 24 was 98% pure, the impurities including 1% of the 1,2,3/0-isomer (38) and 1% unidentified impurities. An analytical sample was obtained by preparative TLC on silica with CHCI~-MeOH-H~O (90:10: 1, v/v/v) as developing solvent and C H C I ~ M e O H - E t 2 0 (1 : 1 : 1, v/v/v) as eluent. Analysis: calculated for C12H1603 (208.3): C, 69.21; H, 7.75; found: C, 68.98; H, 7.87. NMR: 8 7.30 (5) singlet, C6H~; 6 4.54 (2) singlet, benzyl CH2; 6 3.10 (2) broad singlet, disappearing on exchange with D20, OH; 8 1.30-2.40 (4) multiplet, ring CH2; 8 3.80-4.20 (3) multiplet, ring O---C--H. W. G. Young, H. K. Hall, Jr., and S. Winstein, J. A m . Chem. Soc. 78, 4338 (1959).

652

SUBSTRATES, ANALOGS, AND INHIBITORS

[54]

Method H (via Benzylcyclopentenol) 1-O-benzylcyclopentenol 27 (30 g, 0.17 tool) was dissolved in 900 ml of a mixture of ethanol-dioxane-water (1 : 1 : 1, V/v/v) containing sodium hydroxide (12.5 g, 0.31 mol), and the solution was maintained at - 2 0 ° while a cool solution of a potassium permanganate (19 g, 0.2 tool) in water (700 ml) was added during a 4-hr period. The solution rapidly became green; the color became deeper as addition of the oxidant proceeded. Stirring was continued for 1 hr after addition was complete, and the suspended manganese dioxide was then reduced by treatment with a stream of sulfur dioxide gas. After filtration the solution was concentrated in vacuo to approximately half volume and then extracted continuously with dichloromethane for 2 days. The extract was dried with anhydrous sodium sulfate and evaporated, leaving a yellow oil (21.0 g, 0.11 tool, 64%). TLC (CHClz-MeOH-HzO, 90: 10: 1, v/v/v) showed a major spot (Rr 0.50); GLC analysis of the trimethylsilyl derivative showed a major peak due to 24, about 1% of the 1,2,3/0-isomer 38 and unchanged 12. Purification was achieved via the isopropylidene derivative 23: the impure diol (21.0 g) was vigorously stirred with acetone (100 ml), dimethoxypropane (100 ml), and trifluoroacetic acid (0.75 ml) overnight. The solution was neutralized with barium carbonate (5 g), filtered through Celite, and evaporated. The oily product (25 g) was fractionally distilled; the fraction at bp 95-100°/0.5 torr (16.5 g) was shown by GLC to be > 96% pure and to be free from the 1,2,3/0-isomer. For analytical purposes, a portion was converted to its bis-(p-nitrobenzoate) derivative (mp 117-118°). Analysis: calculated for C26H22OgN2 (506.5) bis-(p-nitrobenzoate): C, 61.66; H, 4.38; N, 5.53; found: C, 61.70; H, 4.32; N, 5.31.

E. (1,2,3/O)- l-O-Benzylcyclopentane- l,2,3-triol (38) Isopropylidenebenzyltriol 37 (6.2 g, 25 mmol) was hydrolyzed during 3 hr in refluxing 0.1 N H2SO 4 (300 ml). Work-up as described for the 1,2/3isomer 23 gave the desired diol 38 in quantitative yield as a yellow oil. TLC (CHCI~MeOH-H~O, 90: 10: 1, v/v/v) showed one spot (Rf 0.60) and GLC of the trimethylsilyl derivative showed that the diol was 97% pure and free of isomeric diol impurities. For analytical purposes, the bis-(p-nitrobenzoate) derivative was prepared and recrystallized from ethyl acetate-methanol (1 : 1, v/v), mp 107-109 °. Analysis: calculated for C~sH22OgN2 (506.5) bis-(p-nitrobenzoate): C, 61.66; H, 4.38; N, 5.53; found: C, 61.73; H, 4.38; N, 5.61. NMR: 6 7.30 (5) singlet, C6H5; 6 4.52 (2) singlet, benzyl CH2; 8 1.80 (4) broad singlet, ring CH2; 6 3.17 (2) broad singlet disappearing on exchange with D20, OH; 8 3.85 (3), ring O---C--H.

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

653

F. (1,3/2)-l-O-Benzylcyclopentane-l,2,3-triol (33) and (1,2/3)- 1- O-Benzylcyclopentane-1,2,3-triol (30) NOTES: 1. The (1,3/2)-l-benzyl and (l,2/3)-l-benzyltriols were prepared by acid-catalyzed ring opening of the syn- and anti-epoxides 28 and 29. These epoxides were generated as an isomeric mixture (syn/anti ratio, 1 : 3) by epoxidation of 1-O-benzylcyclopenten-l-ol (27). 2. For analytical NMR and IR study, the syn- and anti-epoxides were separated by preparative TLC on silica (CHC13-Et20, 20: 1, v/v) as developing solvent and (CHCla-MeOH-Et20, 1 : 1 : 1, v/v/v) as eluent; R~ syn-epoxide and anti-epoxide 0.65 and 0.75, respectively. 3. Spectroscopic data on syn-epoxide (28) and anti-epoxide (29). a. IR data: Structural and conformational assignments of the two epoxides were based on earlier studies of Steyn and Sable. 9 It was established that, in pairs ofsyn or anti substituted cyclopentane epoxides, the frequency of a strong band in the 830-855 cm -I region of the infrared spectrum is structurally diagnostic. The bands were found at 854 cm -1 and 843 cm 1, which established the configurations indicated. b. N M R data: Another diagnostic test proposed involved the coupling of the cyclopentanoid ring proton on C-I with the vicinal oxirane proton on C-2 as measured in the NMR spectrum, t° This coupling was 1.2 and 0.6 Hz, respectively, for 28 and 29, in agreement with the proposed stereochemical assignment. In each of these compounds, the conformation may be determined from the coupling of the cyclopentanoid ring proton on C-I with the vicinal methylenic protons on C-5; in both cases the endo-, or boat, conformation was found, in agreement with previous findings for a series of related compounds. These assignments were possible only because of selective changes in chemical shift caused by the solvent hexadeuterobenzene. 4. The chemical shifts of the benzylic methylene proton resonances in the syn- and anti-benzyl epoxides (28, 29) differed, respectively, by 0.19 ppm (C6D6) and 0.08 ppm (CDCla). A similar difference in chemical shift was observed for the benzylic methylene resonances in the bis-pnitrobenzoates (0.14 ppm in CDC13). These differences permitted the determination of the isomeric composition of mixtures and the isomeric purity of each of the four compounds after separation. G. DL-(1,2,3/0)-l-O-Benzyl-2,3-epoxy-l-cyclopentanol(28) and DL-(1/2,3)- 1- O-Benzyl-2,3-epoxy-1-cyclopentanol (29) m-Chloroperoxybenzoic acid (19 g, 110 mmol) was dissolved in chloroform (300 ml) in a 2-liter wide-mouth brown bottle; the solution was 9 R. Steyn and H. Z. Sable, Tetrahedron 27, 4429 (1971).

~0H. Z. Sable, K. A. Powell,H. Katchian, C. B. Niewoehner,S. B. Kadlec,Tetrahedron 26, 1509 (1970).

654

SUBSTRATES, ANALOGS, AND INHIBITORS

[54]

stirred magnetically and cooled to 3-5 ° while a solution of DL-1-Obenzyl-2-cyclopenten-1-ol (27) (15 g, 86 mmol) in 15 ml of chloroform was added in 2-3 ml portions during 30 min. It was expedient to perform four such epoxidations simultaneously. The mixtures were stored in the dark at 5° overnight and filtered to remove m-chlorobenzoic acid; the solutions were combined and concentrated to small volume. The oily solid was extracted with cool benzene (100 ml); the extract was filtered to remove m-chlorobenzoic acid and washed with saturated sodium bicarbonate solution (6 × I00 ml) and water (l × 100 ml), dried over anhydrous sodium sulfate, and then evaporated to an oil. TLC (CHCIa-Et20, 20: 1, v/v) showed two epoxide-positive spots (R¢ 0.70 and 0.80) and traces of fastmoving material. Distillation (117-120°/0.5 torr) gave a mixture of 28 and 29 as a colorless oil (45 g, 24 mmol; 69%), GLC and NMR analysis of which showed that the anti- and syn-epoxides 29 and 28 were present in a ratio of 3 : 1. (See Note 4.) Analysis: calculated for C12H1402 (190.2): C, 75.76; H, 7.42, found: C, 75.84; H, 7.58.

H. (1,3/2)-l-O-Benzylcyclopentane-l,2,3-triol (33) and (1,2 /3 )- l-O-Benzylcyclopentane-1,2,3-triol (30) A mixture of the epoxy benzyl ethers 28 and 29 (25 g, 0.13 retool) was suspended by stirring in 500 ml of 0. I N sulfuric acid and heating at reflux temperature for 3 hr. The clear solution was neutralized with excess barium carbonate (I 5 g), filtered through Celite, and continuously extracted with dichloromethane for 24 hr. (See Note that follows). The extract was dried with anhydrous sodium sulfate and concentrated under reduced pressure to give a yellow oil (19.8 g; 73%). TLC (CHCIa-MeOH-HsO, 90: I0: I, v/v/v) showed two major components (Rf 0.40 and 0.55) and minor fast-moving components. GLC analysis of the trimethylsilyl derivatives showed that hydrolysis of the mixture of epoxides gave the diols 33 and 30 in a ratio of 65 : 35, respectively. The mixture of diols was separated by fractional crystallization of the bis-p-nitrobenzoates as described in what follows. NOTE: The monobenzyltriol products 30 and 33 are appreciably soluble in water. Simple manual extraction with methylene chloride or chloroform is sufficient to isolate the isomeric products in reasonable yield, although additional extraction losses of up to 5-6% were observed with either solvent if continuous extraction was not employed.

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

655

I. (1,2/3)- l-O-Benzyl-2,3-bis-O-(p-nitrobenzoyl)cyclopentane- l,2,3-triol and (1,3 /2 )- l-O-Benzyl-2,3-bis-O-(p-nitrobenzoyl)cyclopentane1,2,3-triol (p-Nitrobenzoates of 30 and 33) The mixed benzyl diols 30 and 33 (20 g, 0.10 mol) were dissolved in anhydrous pyridine (50 ml) and chilled to 0°;p-nitrobenzoyi chloride (55 g, 0.30 mol) was added with vigorous stirring. The mixture was left overnight. Ice (100 g) was added, and after 1 hr the slurry was partitioned between chloroform (200 ml) and water (200 ml). The phases were separated, the aqueous phase was further extracted with chloroform (200 ml), and the combined extracts were washed with 2 N sulfuric acid (3 × 100 ml), 5% sodium bicarbonate (200 ml), dried (anhydrous sodium sulfate), and evaporated to give a brown solid product (55 g). TLC (hexane-ether; 60 : 40, v/v) showed a single spot (Rs0.40) corresponding to the unresolved derivatives of 30 and 33, as well as a minor spot (R~0.35) corresponding to p-nitrobenzoic anhydride. The isomers were separated by fractional recrystallization from ethyl acetate; the rate of crystallization was adjusted for optimum separation (several crops of approximately 2 g each were collected over a period of a week). Isomeric distribution in each crop was assayed by NMR analysis of signal of the benzyl CH2 protons. The derivative of 30 (1,2/3) is less soluble in ethyl acetate than that of 33 (1,3/2); the early crops were almost pure 1,2/3-isomer (mp 159-162 °) whereas later crops were enriched in 1,3/2-isomer. Subsequent recrystallization of the later crops from acetonitrile gave pure 1,3/2-isomer (mp 122-123°). Analysis: calculated for C~H22OgN (506.5): C, 61.66; H, 4.38; N, 5.53; found (1,3/2): C, 61.75; H, 4.15; N, 5.50; found (1,2/3): C, 61.76; H, 4.48; N, 5.48. NMR: (1,3/2)-isomer: benzyl CH2, 8 4.6, phenyl ring H, 8 7.3; (1,2/3)isomer: benzyl CH2, 8 4.5, phenyl ring H, 8 7.2 ppm.

J. (1,3/2)- 1- O-Benzylcyclopentane- 1,2,3-triol (33) (1,3/2)-Bis-(p-nitrobenzoate) ester (10 g, 19.8 mmol) was refluxed for 2 hr in 250 ml of 10% potassium hydroxide in methanol-water (40 : 60, v/v). After cooling, the pH of the solution was adjusted to 7 with 6 N sulfuric acid; potassium sulfate was removed by filtration, and the filtrate was concentrated to remove methanol. The aqueous solution was extracted continuously with dichloromethane for 24 hr; the extract was dried with anhydrous sodium sulfate, filtered, and concentrated to give a yellow oil (4.1 g, 19.7 mmol, 99%). The diol was 95% pure and free from i,2/3isomer (GLC analysis of the trimethylsilyl derivative).

656

SUBSTRATES, ANALOGS, AND INHIBITORS

[54]

K. (1,2/3)-l-O-Benzylcyclopentane-l,2,3-triol (30) (1,2/3)-Bis-(p-nitrobenzoate) ester (3.55 g; 7.0 mmol) was saponified as described for the 1,3/2-isomer. The light yellow oil obtained (1.38 g, 6.63 mmol; 95%) was 98% pure and was free from 1,3/2-isomer. IV. Acylation of Protected Triols The general methodology is summarized below in Notes 1-6, and details peculiar to specific compounds are described under the compound heading. NOTES: 1. The efficiency of the acylation is markedly dependent on the anhydricity of the reaction. Since the triols (1-3) and their monosubstituted (benzyl and diphenylphosphoryl) derivatives are oils and difficult to dry exhaustively, the vacuum-dried diols (56°) were subjected to several evaporations of anhydrous pyridine immediately prior to acylation. 2. Monobenzyltriols were acylated in anhydrous pyridine by stirring under anhydrous conditions with palmitoyl chloride in anhydrous diethyl ether (molar ratio acyl chloride : diol, 1.0: 2.1) for 1 hr at 0° and then for a further 16 hr at room temperature. Pyridine-acyl chloride complex precipitated during the addition of the acyl chloride; after approximately 30 rain it dispersed and was replaced by a crystalline precipitate of pyridinium chloride as the solution underwent a series of color changes. 3. At the end of the reaction, ice was stirred with the suspension (30 rain) and then chloroform (10 volumes) and water (10 volumes) were added. The phases were separated and the aqueous phase was extracted three times with chloroform. The combined chloroform solutions were washed with water (2 × 5 volumes), dilute sulfuric acid (2.0 N, 2 x 5 volumes), and saturated sodium bicarbonate solution (1 x 1 volume) and were dried over anhydrous sodium sulfate. 4. The dipalmitoylmonobenzyl ethers were purified by a combination of recrystallization and column chromatography. The 1,3/2-, 1,2/3-, and 1,2,3/0-isomers (34, 31, 39) required purification by silicic acid chromatography prior to recrystallization from methanol. However, the 1,2/3isomer 25 was usually obtained pure after two recrystallizations from methanol without recourse to chromatography. 5. Acylations of the above protected triols using commercial palmitoyl chloride gave yields of product in the range 70-85%. The anhydride method of Van DenBerg 11 was also satisfactory, although no improvement in yield was achieved for the isomers studied (34 and 39). n E. Cubero Robles and D. Van DenBerg, Biochim. Biophys. Acta 187, 520 (1969).

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

657

6. Since the reaction schemes employed to date for the cyclopentano analogs involve benzyl and phenyl protecting groups whose removal requires hydrogenolytic cleavage reactions and therefore preclude unsaturated fatty acyl groups, recourse to mild acylation conditions such as that recently reported by Patel et al. 12 seems not yet necessary in this work. A. DL-(1,2/3)-3-O-Benzyl- l,2-O-dipalmitoylcyclopentane- l,2,3-triol (25)

Triol monobenzyl ether 24 (3.33 g, 16 retool) in 20 ml of anhydrous pyridine at 0° was acylated with palmitoyl chloride (10 g, 36 retool) in 15 ml of anhydrous diethyl ether. After reaction and work-up (see Notes 2 and 3), removal of solvent under reduced pressure gave a yellow oil that crystallized on treatment with ethanol at 5° (I0.9 g, 15.9 retool, 99%). TLC (hexane-diethyl ether-acetic acid, 90 : I0 : l, v/v/v) showed a major component, R r 0.23. Trituration with cold acetone removed the color and gave 10 g of product 25, mp 50-53°; traces of fatty acid were removed by recrystallization at 4° (I g in 30 ml of methanol); mp 52-53 °. Analysis: calculated for C44Hv605 (685.1): C, 77.14; H, l I. 18; found: C, 77.27; H, II.26. NMR: 8 1.28 singlet, CH2 (chain); 8 0.89 singlet, CH3; 8 4.56 (2) sharp singlet, benzylic CH2; 8 5.22 (I), ring O - - C - - H ; 8 7.32 (5) singlet, aromatic; 8 1.86-2.47 multiplet (8), CH2 ring and CH2--COO---. B. DL-(l,3/2)-l-O-Benzyl-2,3-O-dipalmitoylcyclopentane-l,2,3-triol (34) Triol monobenzyl ether 33 (2.38 g, I 1.9 mmol) was acylated in 25 ml of anhydrous pyridine with palmitoyl chloride (5 g, 18 mmol) in diethyl ether (I0 ml), to give 6.30 g of an oil that could not be induced to crystallize. Analysis by TLC showed a relatively polar impurity, as well as traces of fatty acid. Purification was achieved by chromatography: 2.0 g of the crude product in 10 ml of hexane-benzene (l : I, v/v) were applied to 75 g of silica (50 cm × 2 cm column); elution with the same solvent (75-mi fractions) gave the desired product in fractions 2-6. Evaporation gave 34 as an oil (1.25 g) that crystallized in vacuo (mp 39-43 °) and was recrystallized from methanol (rap 42-43 °) (final yield of pure product was 3.54 g, 5.17 retool, 44%). Analysis: calculated for C44Hv~O5 (685.1): C, 77.14; H, I 1.18; found: C, 77.10; H, II.09. NMR: 8 1.28, CH2 chain; 8 0.85, CHz; 8 4.58, 4.60 (2) "doublet," benzylic CH2; 8 7.31 (5) singlet, aromatic; 8 1.73-2.38 (8), CHz ring and --CH2--COO---. iz K. M. Patel, J. D. Morrisett, and J. T. Sparrow, l . LipidRes. 20, 674 (1979).

658

SUBSrRATES, ANALOGS, AND |NHIBITORS

[54]

C. DL-(1,2/3)-l-O-Benzyl-2,3-di-O-palmitoylcyclopentane-l,2,3-triol (31) Triol monobenzyl ether 30 (1.38 g, 6.63 mmol) in 10 m! of anhydrous pyridine was acylated with palmitoyl chloride (5.0 g, 18 mmol) in diethyl ether (10 ml) to give 4.0 g of an oil, part of which crystallized on standing. Purification by column chromatography in hexane-benzene (1 : 1, v/v) as described for 34 gave 31 (3.67 g, 5.36 mmol; 81%) mp 44-45 ° (recrystallization from methanol). Analysis: calculated for C44H7605 (685.1): C, 77.14; H, 11.18; found: C, 77.22; H, 10.94. NMR: 8 1.28, CHz (chain); 6 0.85, CHz; 6 4.50 (2) sharp singlet, benzylic CH2; 8 7.30 (5) singlet, CsH~; ~ 1.55-2.40 (8), CH2 ring and - - C H 2 C O O - - ; 8 4.0-4.2 and 8 5.05-5.30 (3), ring O - - C - - H . D. DL-(1,2,3/0)- 1- O-Benzyl-2,3-di-O-palmitoylcyclopentane- 1,2,3-triol (39) Triol monobenzyl ether 38 (3.33 g, 16 mmol) in anhydrous pyridine (20 ml) at 0° was acylated with palmitoyl chloride (10 g, 36 mmol) in diethyl ether (15 ml). Work-up gave a brown oil (13 g), which was purified by column chromatography (6.0 g of oil in hexane-benzene 1 : 1, v/v; 195 g of silica acid). Elution with hexane-benzene (1 : 1, v/v), collecting 75-ml fractions, gave 39 in fractions 12-18. The total yield was 7.18 g (10.5 mmol, 66%). The pure product crystallized slowly in v a c t t o ; it was recrystallized from methanol at 4° (mp 38-39°). Analysis: calculated for C~H7605 (685.1); C, 77.14; H, 11.18; found: C, 77.16; H, 11.32. NMR: 8 1.28, singlet CH~ chain; 8 0.89 singlet, CHz; 6 1.86-2.47 (8) multiplet, CH2 ring and - - C H z - - C O O ; 8 4.50, 4.55 (2) "doublet," benzylic CH2; 8 5.38 (1), 8 7.30 (5) singlet, CrHs. E. (1,3/2)- 1- O-(Diphenylphosphoryl)-2,3-di- O-palmitoylcyclopentane1,2,3-triol (4) The phosphorylated triol 43 (2.63 g, 7.50 mmol) in 20 ml of anhydrous pyridine was acylated with palmitoyl chloride (5.0 g, 18.2 mmol) in anhydrous diethyl ether (10 ml) at 0°. Ice was added (10 g), and after 1 hr the product was isolated as described for the benzyl diacyl derivatives (e.g., 25) to give a brown oil. The oil crystallized on storage in v a c u o giving 5.71 g (6.90 mmol, 92%) of crude product. TLC (CC14-EtOAc, 80: 20, v/v) showed desired product (Rs 0.54), traces of diol starting material 43, and free palmitic acid. An analytical sample was obtained by preparative TLC of the crude product on silica gel using hexane-diethyl ether (60: 40, v/v) as developing solvent, and chloroform-methanol-

[54]

CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

659

diethyl ether (1 : 1 : 1, v/v/v) as eluent. The eluted product 4 was recrystallized from methanol (mp 69-70°). Analysis: calculated for C49H79OsP (827.1): C, 71.15; H, 9.63; P, 3.75; found: C, 71.25; H, 9.37; P, 3.86. Repeated crystallizations from methanol were subsequently found to give a chromatographically pure product of melting range 68-70 ° without prior column chromatography. F. (1,3/2)-2-O-(Diphenylphosphoryl)- 1,3-di-O-palmitoylcyclopentane1,2,3-triol (8) Phosphorylated triol 46 (2.63 g, 7.50 mmol) in 20 ml anhydrous pyridine was acylated with palmitoyl chloride (5.0 g, 18.2 mmol) at 0°. After work-up as described in Note 3, cyclo-PA 8 was isolated as described for benzyl diacyl derivative 25. The oil crystallized on standing (5.71 g, 6.90 mmol, 92%). TLC (CC14-EtOAc, 80:20, v/v) showed 8 (R~ 0.54), traces of 46, and traces of palmitic acid. Recrystallization from methanol gave chromatographically pure 8 (mp 69-70°). Analysis: calculated for C49H79OsP (827.1): C, 71.15; H, 9.63; P, 3.75; found: C, 71.39; H, 9.56; P, 3.61.

V. Debenzylation of Monobenzyldiacyltriols NOTES: 1. Debenzylation was achieved by hydrogenolysis over palladium-charcoal catalyst (10%) at room temperature and pressure, or at 2 arm in a Parr apparatus; 0.8-6.0 g of benzyl derivative were routinely processed. Ethyl acetate was generally used as solvent, but faster reactions were obtained with a solvent mixture of chloroform and ethanol (3 : 2, v/v). The weight of catalyst used was approximately one-quarter of the weight of the benzyl ether to be cleaved. The initial rate of uptake of hydrogen was typically 3-4 ml/min (at room pressure); the overall consumption was generally considerably more than theoretical, but TLC confirmed that the reaction was complete when the uptake diminished to less than about 0.5 ml/min. The catalyst was removed by filtration over Celite, and the solvent was removed under reduced pressure to give the product in quantitative yield. The residue was thoroughly washed with chloroform to remove combustible solvents and thus the risk of catalytic oxidation. 2. Isomers in which the newly generated hydroxyl group is cis- vicinal to an O-acyl group, e.g., 40, rarely showed acyl migration except when they were chromatographed on unbuffered silica. However, the facile acyl migration observed for the 1,2,3/0-isomer (40) can be used to generate the

660

SUBSTRATES, ANALOGS, AND INHIBITORS

[S &]

symmetric all-cis-1,3-diacylcyclopentane-1,2,3-triol (41) for conversion to the 1,3-diacyl-2-phosphate (9). 3

A. DL-(1,2 /3 )- l ,2-Di-O-palmitoylcyclopentane- l ,2,3-triol (26) A solution of diacylmonobenzyltriol 25 (8.0 g, 11.7 mmol) in 90 ml of chloroform-ethanol (75: 15, v/v) was vigorously stirred with 2 g of 10% palladium-charcoal in an atmosphere of hydrogen. Hydrogenolysis was complete after 2 hr; the uptake of hydrogen was 310 ml (theory 286 ml at 25°). After filtration and evaporation (Note 1 above) of solvent, the product 26 (6.87 g, l 1.6 mmol, 99%) readily crystallized. TLC in chloroformether (20: l, v/v) showed a single spot, R f 0.22. Recrystallization from hexane gave a white solid (mp 63-64°). Analysis: calculated for C37H7005 (594.9): C, 74.69; H, 11.86; found: C, 74.51; H, 11.89. B. DL-(1,2,3 /O)- l,2-Di-O-palmitoylcyclopentane- l ,2,3-triol (40)

Diacylmonobenzyltriol 39 (4.62 g, 6.75 mmol) in 20 ml of ethyl acetate was hydrogenolyzed as described for the 1,2/3-isomer (25) in the presence of 1.5 g of palladium-charcoal (10%). Absorption of H2:226 ml in 90 min (theory at 25°, 166 ml). Work-up (Note 1) gave a solid mp 60-63 ° (3.98 g; 99%), and after recrystallization from hexane almost quantitative recovery of product was obtained (mp 64-65°). Analysis: calculated for C37H7005 (594.9): C, 74.69; H, 11.86; found: C, 74.61; H, 11.74. C. DL-(1,2/3)-2,3-Di-O-palmitoylcyclopentane-l,2,3-triol (32) Diacylmonobenzyltriol 31 (3.67 g, 5.36 mmol) in 15 ml of ethyl acetate was hydrogenolyzed in the presence of 1 g of palladium-charcoal (10%) as described for 1,2/3-isomer 25. Absorption of hydrogen was 162 ml in 140 rain (theory 132 ml at 25°). Work-up (Note 1 above) gave a solid product whose TLC (CHC13-Et20, 20 : I, v/v) showed the absence of 31, but the major product 32, R I 0.55, was contaminated with a minor product, R I 0.65, probably the 1,3-diacyltriol resulting from acyl migration (see Note 4 below). The product 32 (3.11 g, 5.23 mmol; 98%) was recrystallized from hexane, mp 52-53 °. Analysis: calculated for C3rH7005 (594.9): C, 74.69; H, 11.86; found: C, 74.59; H, 11.66. NOTES: 3. Subsequent preparations of diacyltriol (32) showed that the 1,3-isomer was always formed (Rf 0.65 in chloroform-ether, 20: 1,

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CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

661

v/v), but that glassware rinsed in 0.1 N sodium hydroxide after normal washing procedures appeared to reduce the proportion of isomerized product observed. Ethyl acetate was the solvent of choice for the hydrogenolysis in spite of the longer reaction times required; the presence of chloroform in the hydrogenolysis vessel appeared to favor acyl group migration. We speculate that the chloroform contained phosgene, which led to the formation of hydrogen chloride, which in turn induced acyl migration. 4. Attempts have been made to isolate the isomerized product (1,3diacyltriol) as its diphenyl phosphate ester, a procedure successfully adopted for the isomerization product of the 1,2,3/0-diacyltriol (i.e., sequence 40 ~ 41 ~ 9). 3 However, we were unsuccessful in separating the mixture of 1- and 2-diphenyl phosphates in the 1,2/3 series, and thus the (1,2/3)-2-phosphate analog remains to be isolated in an isomerically pure state. D. (1,3/2)-l,2-Di-O-palmitoylcyclopentane-l,2,3-triol (35) Diacylmonobenzyltriol 34 (3.12 g, 4.55 mmol) in 20 ml of ethyl acetate was hydrogenolyzed in the presence of 1 g of palladium-charcoal (10%) (absorption of H2 was 159 ml in 40 min; theory 112 ml at 25°) and worked up as described in Note 1. TLC analysis in CHCIa-EtzO (20:1, v/v) showed a single spot R e 0.40; the product crystallized as the solvent was removed (2.70 g, 4.56 mmol, 100%). Recrystallization from hexane gave an analytical sample, mp 57-59 °. Analysis: calculated for CarH7005 (594.9): C, 74.69; H, 11.86; found: C, 74.65; H, 11.72. VI. Phosphorylation of Diacylcyclopentanetriols NOTES: 1. Since the present work is confined to the preparation of saturated fatty acid derivatives, the most convenient phosphorylating agent was diphenylphosphoryl chloride (DPPC), a relatively cheap, stable, and yet reactive compound. Subsequent removal of the phenyl groups from the phosphate ester is quantitative under catalytic hydrogenation in glacial acetic acid, a practice, however, that is precluded when unsatu~ rated fatty acyl chains are present. 2. Essentially quantitative conversion of diacyltriol to the corresponding diphenylphosphoryl ester (diphenyl ester of cyclophosphatidic acid) was easily obtained by allowing anhydrous diacyltriol (dried by repeated evaporation of anhydrous pyridine) to react in anhydrous pyridine-diethyl either (1 : l, v/v) with 1-2 mol-equiv, of DPPC. In the most expedient and effective procedure, the stoppered reaction flask was cooled to 0-2 ° with

662

SUBSTRATES, ANALOGS, AND INHIBITORS

[54]

vigorous magnetic stirring and a calculated excess of DPPC was directly and swiftly added by means of a Pasteur pipette. The reaction mixture usually darkened a little, and on stirring for 6-8 hr a crystalline precipitate of pyridinium chloride generally formed. In humid laboratory conditions, a two-neck flask fitted with a calcium chloride drying tube and a rubber septum was beneficial. The DPPC was then injected by means of a hypodermic syringe. 3. The reaction work-up simply involved decomposing the excess acid chloride with ice-water (30 min stirring with a fivefold excess of water was sufficient), and a partitioning between chloroform and water. (Since a considerable excess of pyridine was used, some washing time can be saved by reducing the volume of the mixture before partition, but the pyridine-water-diethyl ether mixture is prone to froth and the evaporation is itself time-consuming and probably not advantageous.) The lower chloroform phase was washed with water (2 × 10 volumes), 2.0N sulfuric acid (3 × 10 volumes), and saturated sodium bicarbonate (1 × l0 volumes) and dried with anhydrous sodium sulfate. The phosphorylated product could usually be induced to crystallize during solvent removal when 1-2 volumes of methanol were added prior to evaporation under reduced pressure. A. DL-(1,2/3)-3- O-(Diphenylphosphoryl)- 1,2-di- O-palmitoyleyelopentane1,2,3-triol (5) Diacyltriol 26 (6.0 g, 11 mmol) in anhydrous pyridine-ethyl ether (50 ml, 1 : 1, v/v) was stirred at 0° while diphenylphosphoryl chloride (5.37 g, 20 mmol) was added directly. After reaction and work-up as described in Note 3, the solvent was removed under reduced pressure, leaving a light-brown oil. Addition of ethanol and reevaporation gave 7.64 g of solid 5 (9.24 mmol, 84%), whose TLC showed one spot (R~ 0.44 in CC14-EtOAc, 80:20, v/v). The crude solid melted at 53-57°; recrystallization (7.5 g dissolved in 90 ml of warm methanol) raised the mp to 56-57 °. Analysis: calculated for C4aH~9OsP (827.1): C, 71.15; H, 9.63; P, 3.75; found: C, 71.09; H, 9.50; P, 3.76. B. DL-(1,3/2)-l-O-(Diphenylphosphoryl)-2,3-di-O-palmitoylcyclopentane1,2,3-triol (4) Method 1 Diacyltriol 35 (2.60 g, 4.37 mmol) in anhydrous pyridine-diethyl ether 25 ml ( l : l , v/v) was phosphorylated with diphenylphosphoryl chloride (2.30 g, 8.56 mmol). The resulting product (3.39 g, 4.10 mmol; 94%),

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CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

663

worked up as described in Note ~ crystallized as the solvent was removed and was then chromatographically pure (Rs 0.46 in CCI4-EtO~;c, 80 : 20, v/v). Recrystallization from methanol gave crystals (mp 57.5-58°). Analysis: calculated for C49H79OaP (827.1): C, 71.15; H, 9.63; P, 3.75; found: (Method 1) C, 71.25, H, 9.37; P, 3.86; (Method 2) C, 70.98; H, 9.63; P, 4.00. Method 2

See under acylation of protected triols (Section IV, E). C. DL-(1,2/3)- 1- O-(Diphenylphosphoryl)-2,3-di- O-palmitoylcyclopentane1,2,3-triol (7) Diacyltriol 32 (2.77 g, 4.66 mmol) in anhydrous pyridine-diethyl ether 20 ml (1: 1, v/v) was phosphorylated with diphenylphosphoryl chloride (2.70 g, 10 mmol) and worked up as described in Note 3 above. The resulting colorless oil crystallized on storage in vacuo giving 3.62 g (6.09 mmol, 94%) of chromatographically pure product 7 (R r 0.40 in CC14EtOAc, 80:20, v/v). Recrystallization from methanol gave crystals (rap 43-44°). Analysis: calculated for C49H79OsP (827.1): C, 71.15; H, 9.63; P, 3.75; found: C, 71.33; H, 9.86; P, 3.72. D. DL-(1,2,3/0)- 1-O-(Diphenylphosphoryl)-2,3-di-O-palmitoylcyclopentane- 1,2,3-triol (6) Diacyltriol 40 (3.60 g, 6.05 mmol) in 50 ml of anhydrous pyridinediethyl ether ( l : l , v/v) was phosphorylated with diphenylphosphoryl chloride (3.80 g, 14.1 retool). After work-up as described in Note 3, the product crystallized as the solvents were removed, giving 4.80 g (5.81 mmol; 96%) of chromatographically pure 6 (Rr 0.36 in CC14-EtOAc, 80 : 20, v/v). An analytical sample was obtained by recrystallization from methanol (1 g in 25 ml at 60°; mp 52-53°). Analysis: calculated for C49H79OsP (827. l): C, 71.15; H, 9.53; P, 3.75; found: C, 71.43; H, 9.68; P, 3.73. E. DL-(1/2,3)- 1- O-(Diphenylphosphoryl)-2,3-anhydrocyclopentanetriol (42) Diphenylphosphoryl chloride (10.8 g, 40 mmol) in anhydrous diethyl ether (15 ml) was added slowly to a stirred ice-cold solution of DE-(I/2,3)2,3-anhydro-l-cyclopentanetriol 19 (3.0 g, 30 mmol) in 50 ml of anhydrous

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SUBSTRATES, ANALOGS, AND INHIBITORS

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pyridine. The solution was stirred for 1 hr and then diluted with 200 ml (I volume) each of chloroform and water. The aqueous phase was extracted with chloroform (4 x l volume), and the CHCIa solution was washed with water (2 × l volume), 1.0 N sulfuric acid (3 x l volume), and saturated sodium bicarbonate (2 × 1 volume) and dried over anhydrous sodium sulfate. Evaporation gave a colorless oil (9.30 g, 28 mmol, 93%); TLC (CHCla-Et20, 3 : 1, v/v) showed a major phosphate-positive spot (Re 0.80) and a trace of phosphate-positive material at the origin. An analytical sample was obtained by preparative TLC on silica with CHCla-Et20 (3 : l, v/v) as developing solvent and CHCla-MeOH-Et20 ( l : l : l , v/v/v) as eluent. Analysis: calculated for C17HlrOsP (332.3): C, 61.45; H, 5.16; P, 9.32; found: C, 61.63; H, 5.23; P, 9.58. The slightly impure product can be used without further purification for hydrolysis to diol 43. F. DL-(1,3/2)-l-O-(Diphenylphosphoryl)cyclopentane-l,2,3-triol (43) DL-(I/2,3)- 1- O-(Diphenylphosphoryl)-2,3-anhydrocyclopentanetriol 42 (5.55 g, 16.7 mmol), dissolved in a mixture of 0.4 N sulfuric acid (40 ml) arid dioxane (50 ml), was refluxed for 90 min, cooled, and stirred with sodium bicarbonate (1.40 g, 19.5 mmol) for 1 hr. The mixture was evaporated, the oily solid was triturated with CHCla, and inorganic salts were removed by filtration. The filtrate was dried over anhydrous sodium sulfate and evaporated to give an oil (43) (5.71 g, 16.3 mmol, 98%), which showed one spot on TLC (Ry 0.35 in CHCla-MeOH-H~O; 90: 10: 1, v/v/v). The product was used directly for the acylation to 4. For analytical purposes, a portion of 43 was benzoylated. The dibenzoate was recrystallized from aqueous 90% ethanol to give white needles (rap 132-133°). Analysis: calculated (dibenzoate) CalH27OaP (558.5): C, 66.66; H, 4.87; P, 5.55; found: C, 66.57; H, 4.73; P, 5.33. VII. Synthesis of Symmetrical Cyclophosphatidic Acid (1,3/2-2P) (8) NOTES: 1. The introduction of the 2-phosphate moiety required prior synthesis of a 1,3-protected all-trans-triol. The p-nitrobenzylidene acetal 44 offered the advantages of crystallinity and susceptibility to neutral deblocking by catalytic hydrogenolysis. 2. Intramolecular migration during hydrogenolysis is precluded since the diphenylphosphoryl group of 46 is anti to the regenerated hydroxyl groups.

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CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

665

A. (1,3 /2 )- l,3-O-(p-Nitrobenzylidene)cyclopentane- l,2,3-triol (44) (1,3/2)-Cyclopentane-1,2,3-triol 3 (15 g, 127 mmol) in toluene (250 ml) was heated to reflux temperature with p-nitrobenzaldehyde (22.6 g, 150 mmol) and p-toluenesulfonic acid (50 mg) in an apparatus fitted with a phase-separating head. The solution discolored considerably during the heating, the triol slowly dissolved, and after 2.5 hr, 1.8 ml of water was collected (theory 2.3 ml). The hot solution was decanted from the tarry residue; the solution crystallized (25 g) on cooling. A second crop (1.5 g) ofp-nitrobenzylidene acetal 44 was obtained by concentrating the mother liquor to 100 ml. The crystals were dissolved in 120 ml of toluene at 80-85°; the clear solution was decanted from a small quantity of dark oil and allowed to crystallize. The p-nitrobenzylidene acetal 44 formed lightbrown rosettes (14.6 g); TLC showed a major spot (Rf 0.35 in CHC13EhO; 3 : 1, v/v) and traces of impurity at the origin. Second and third crops of crystals of equivalent purity were obtained by concentrating the mother liquors and seeding (total yield 20.4 g, 68%). Recrystallization from toluene failed to remove the contaminant seen at the origin in TLC analysis, but the product was sufficiently pure at this stage to proceed. For elemental analysis, the 2,2,2-trichloroethoxycarbonyl ester was prepared as follows: 1,3-p-Nitrobenzylidene acetal 44 (200 mg, 0.80 retool) in anhydrous diethyl ether-pyridine (5: 1, v/v) was stirred at 0° with 2,2,2trichloroethoxychloroformate (200 rag, 0.94 mmol) for 16 hr. Ice was added (2 g); the reaction mixture was stirred for 30 min and was then diluted with 50 ml each of water and diethyl ether. The phases were separated, and the ethereal solution was washed with ice-cold 2 N sulfuric acid, water, saturated sodium bicarbonate solution, and water and dried with anhydrous sodium sulfate. Evaporation gave a brown solid (340 mg, 0.80 mmol, 100%); TLC in CHCI3-Et20 (20 : 1, v/v) showed a single spot, R s 0.70. Recrystallization from ether gave yellow crystals (mp 143-144°). Analysis: calculated for C~sH14OrNCla (426.6): C, 42.23; H, 3.31; N, 3.28; C1, 24.99; found: C, 42.31; H, 3.44; N, 3.18; C1, 24.90. NMR: acetal 4 4 : 8 2.15 (4) singlet, ring CH2; 6 1.90 (1), broad, exchangeable with D20, OH; 8 4.20 (1) singlet (broad), ring HOCH; 8 4.52 (2) singlet (broad), ring ethereal-O--CH; 6 5.82 (1) singlet, benzylidene CH; 8 7.55-8.26 (4) quartet, aromatic, 2,2,2-trichloroethoxycarbonyl ester: 8 2.23 (4) broad singlet, ring CH2; 8 5.88 (1) singlet, benzylidene CH; 8 5.05 (1), ester-O-CH; 8 4.78 (4), COOCH~CCIa and ring ethereal O--C--H. IR: acetal 44: (CC10, 3640 cm -1, OH (KBr) 3350 cm -1 (broad), OH; 1350, 1525 cm -t, NO2; 3060, 1610, 750, 700 cm -I.

666

SUBSTRATES, ANALOGS, AND |NH|BITORS

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B. (1,3 /2)- l,3-O-(p-Nitrobenzylidene)-2-O-(diphen ylphosphoryl)cyclopentane- 1,2,3-triol (45)

A solution ofp-nitrobenzylidene acetal 44 (4.0 g, 16.2 mmol) in anhydrous pyridine-diethyl ether (30 ml; !: 1, v/v) was stirred at 0° for 1 hr with diphenylphosphoryl chloride (6.46 g, 24 mmol); the solution darkened, and stirring was continued at room temperature for 16 hr. Ice was added (5 g), and after stirring for 1 hr the product was isolated as described earlier for the diphenylphosphatidates. The brown oil (7.73 g, 16 retool; 99%) was decolorized by treatment with charcoal in hot benzene; evaporation gave an oil that crystallized in vacuo (mp 83-88°). TLC (CHC13-Et20, 20:1, v/v) showed a major spot (R e 0.60). Recrystallization from methanol removed traces ofp-nitrobenzaldehyde (1.0 g in 10 ml at 50°), and gave 550 mg of yellow plates (mp 88-89°). Analysis: calculated for C24H2zOsPN (483.4): C, 59.63; H, 4.59; P, 6.41; N, 2.90; found: C, 59.96; H, 4.57; P, 6.27; N, 2.80. NMR: 45:8 7.55-8.26 (4) quartet, C6H4NOz;/5 7.30 (10) singlet, C6H5; 8 5.82 (2) singlet, benzyl CH2; 8 4.70 (2) singlet, CH--O--CHC6H4NO2; /5 4.86, 4.98 (1) doublet of triplets, CHOP; 8 2.10 (4) singlet, ring CH2.

C. (1,3/2)-2-O-(Diphenylphosphoryl)cyclopentane-l,2,3-triol (46) The phosphorylated acetal 45 (2.75 g, 5.69 mmol) in 50 ml of ethanolchloroform (3:2, v/v) was hydrogenolyzed with 4.0 g of palladiumcharcoal (10%). After 90 min (uptake of hydrogen, 810 ml) TLC analysis showed the reaction to be complete. The suspension was filtered through Celite and concentrated to give a yellow solid, which was then partitioned between chloroform and ice-cold 1 N sulfuric acid. The chloroform phase was washed with saturated sodium bicarbonate, dried with anhydrous sodium sulfate, and evaporated to give an oily solid (1.75 g, 88%). TLC (CHCla-MeOH-H20; 90 : 10 : 1, v/v/v) showed a major spot (R f 0.50) and a trace of impurity at the solvent front. For analytical purposes, a sample was converted to the bis-(p-nitrobenzoate) derivative. Recrystallization from ethyl acetate gave yellow needles (mp 195-196°; partial sublimation at I70°). Analysis: calculated for CalH2~OI2PN2 (648.5): C, 57.41; H, 3.89; N, 4.32; P, 4.78; found: C, 57.64; H, 3.80; N, 4.31; P, 4.79. The diol discolored on standing and was therefore used at once for the acylation to 8. NOTE: The acylation of 46 to cyclo-PA 8 is described in detail in Section IV, F.

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CYCLOPENTANOID ANALOGS OF DIACYLGLYCEROPHOSPHATE

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VIII. Isomerization of all-cis-Diacylcyclopentanetriol (40) NOTES: I. In this procedure, advantage is taken of the facile acidcatalyzed isomerization of ( 1,2,3/0)- 1,2-diacylcyclopentanetriol (40).3 Debenzylation of (1,2,3/0)-monobenzyldiacyltriol 39 proceeds readily in a hydrogen atmosphere using palladium as catalyst, and if acid is rigorously excluded nonisomerized product (40) is almost exclusively obtained. However, incubation of the 1-hydroxy product (40) in aqueousmethanolic HC1 promotes an equilibration of the l-hydroxy isomer with its 2-hydroxy isomerization product (41). 2. The two positional isomers 40 and 41 are difficult to separate by chromatographic means owing to their readiness to isomerize, but diphenylphosphorylation of the isomeric mixture gives a stable pair of cyclophosphatidic acid isomers, which can easily be separated by column chromatography or preparative thin-layer chromatography. A. Isomerization of DL-(1,2,3/0)- 1,2 -Dipalmitoylcyclopentane-1,2,3-triol (41)) Diacyltriol 40 (500 mg) was in part isomerized to 41 when incubated with 10 ml of 0.1 N methanolic-HC1 containing 5% H20 (15 min, 45°). Treatment of 40 with anhydrous 2.5% methanolic-HC1, however, rapidly transesterified the lipid, TLC and GLC analysis revealing the presence of methyl palmitate and almost no desired product 41. The reaction mixture was cooled, treated with excess sodium bicarbonate and dried over anhydrous sodium sulfate. Evaporation of solvent from the filtrate gave an approximately equivalently proportioned mixture of isomers 40 and 41 (rap range 49-58 °) as estimated by TLC. B. Phosphorylation of Isomeric Mixture of Diacyltriols 40 and 4 I The mixture of isomeric diacyltriols 40 and 41 was phosphorylated with DPPC under anhydrous conditions as described previously. TLC analysis showed two phosphate positive spots, R s 0.45 and 0.55 (CHCI~ EtOAc, 10 : 1, v/v). The faster moving isomer (R r 0.55) was shown to be the new compound 9 by TLC comparison of the mixture with the authentic 1-phosphate (6). The diphenyl esters were isolated by preparative TLC (developing solvent: hexane-diethyl ether, 60:40, v/v; eluting solvent: CHCI3-MeOH-EhO, 1 : 1 : 1, v/v/v), and each isomer was recrystallized from MeOH (mp 6, 50-51 °, lit. 2 52-53°; mp 9, 61-62°). Analysis: calculated for 9: C49Hr9OsP (827.1), C, 71.15; H, 9.63; P, 3.75; found: C, 71.09; H, 9.72; P, 3.70.

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SUBSTRATES, ANALOGS, AND INHIBITORS

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IX. Dephenylation of Diphenylphosphoryldiacylcyclopentane-1,2,3-triols (Cyclo-PA Diphenyl Esters) NOTE: Each of the six cyclo-PA diphenyl esters was treated in the manner described in what follows, to give a quantitative yield of the corresponding free acid form of the "cyclophosphatidic acid." Freshly recrystallized diphenyl esters were converted into the free cyclophosphatidates by hydrogenolysis over platinum in glacial acetic acid either at atmospheric pressure or at 2 atm in a Parr apparatus. Platinum was generated in situ by reduction of platinum dioxide. Approximately 50 mg of platinum dioxide were used to dephenylate 830 mg (I mmol) of phospholipid at room pressure, or 25 mg of platinum dioxide at 2 atm; the rate of absorption of hydrogen was usually about 2.5 ml/min at room pressure, and at least 25% more hydrogen than theoretical was absorbed. Some free cyclo-PA usually separated as a solid toward the end of the reaction, but this did not interfere with hydrogenolysis, and the yield of product was quantitative. No impurities were detected by TLC. Each PA isomer was freed of ions by partitioning the lipid according to a modification13 of the method of Bligh and Dyer14: The lipid was dissolved in 5 ml of chloroform, the solution was diluted with 5 ml of CH~OH, and then 4.5 ml of 0.2 N aqueous hydrochloric acid were added. The mixture was vortexed and briefly centrifuged. The chloroform phase was removed by Pasteur pipette, washed with an approximately equal volume of methanol-water (10: 9, v/v), and evaporated to dryness under a stream of nitrogen. The free cyclophosphatidic acid isomer was obtained thus as a sharply melting white solid (Table I).

X. Preparation and Interconversion of Salt Forms of Diacylcyclopentanetriol Phosphate Isomers (Cyclo-PA Isomers) Free cyclophosphatidic acid (isomers 4--9) (about 50 rag) was dissolved in chloroform, 1 drop of 1% ethanolic phenolphthalein solution was added, and the mixture was titrated with potassium hydroxide solution (or sodium hydroxide) (about 0.2 N, 2% water in methanol) to a permanent faint, pink color. The solution was concentrated under a stream of nitrogen to about 0.5 ml, cleared by the addition of 1 or 2 drops of chloroform, and centrifuged strongly. The clear supernatant was diluted with l0 ml of acetone with vigorous vortex treatment and cooled to 5°; the precipitated potassium salt (or sodium salt) was centrifuged down, and the acetone ~3 M. K a t e s , C. E. Park, B. Palameta, and C. N. Joo, Can. J. Biochem. 49, 275 (1971). ~4 E. G. Bligh and W. J. Dyer, Can. J. Biochern. Physiol. 37, 911 (1959).

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TABLE I MELTINGPOINTSOF ISOMERICCYCLOPENTANOIDANALOGSOF PHOSPHAT1DICACID(FREE ACIDSAND ESTERS) Melting point (°C) Compound~ 4 5 6 7 8 9

Configuration 1,3/2-(IP) 1,2/3-(3P) 1,2,3/0-(1P) 1,2/3-( 1P) 1,3/2-(2P) 1,2,3/0-(2P)

Free acid

Diphenyl ester

Dimethyl ester

82-83.5 77.5-78.5 76-78 78-80 73-74.5 80-81

57-58 56-57 52-53 43-44 69-70 61-62

59-60 53-54 34-36 68-71 61-63 --

" Identification numbers refer to Fig. 2. supernatant was r e m o v e d by Pasteur pipette. The salt was redissolved in 0.5 ml of C H C l a - M e O H (1 : l, v/v) and reprecipitated by the addition of 10 ml of acetone at 5°. The precipitate was again centrifuged, r e m o v e d , and dried in vacuo. After three such precipitations, the yield o f salt was 9096%. NOTE: The potassium and sodium salts of the cyclophosphatidic acid isomers were indefinitely stable in the dry state at 5° . Prolonged storage at r o o m t e m p e r a t u r e , however, led to slight decomposition, as evidenced by the a p p e a r a n c e o f a slower-moving spot on T L C (Rs, relative to the cyclo-PA isomer, 0.50 in C H C l z - M e O H - H 2 0 , 65 : 25 : 4, v/v/v). Storage in chloroform is not r e c o m m e n d e d ; even at - 1 5 °, considerable decomposition is evident in some stored samples after approximately one month.

XI. Physical Constants of Cyclo-PA Analogs A. Analytical Data Free A c i d Forms

Elemental analyses for the free acid forms of each of the cyclo-PA analogs were consistent with those calculated for anhydrous c o m p o u n d s . The equivalent weight of each isomer, determined by titration of the free acid with 0.05 N aqueous-methanolic potassium hydroxide (90% methanol) to the phenolphthalein end point, also agreed with the theoretical values; 45-50 mg samples of the free acid (0.067-0.074 mmol) con-

670

SUBSTRATES, ANALOGS, AND INHIBITORS

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sumed 0.64--0.75 ml (0.128-0.150 meq) of potassium hydroxide, giving equivalent weight values of 346 _ 11.5 (theory 337.4). P o t a s s i u m Salt F o r m s

The potassium salts obtained by neutralization of the free acids were purified by at least three acetone precipitations. After drying in v a c u o at room temperature, their elemental analyses correspond to trihydrated dipotassium salts. The water of hydration was particularly strongly bound: overnight drying at 100°/0.5 torr removed only 1 mol of water, the residual solid giving analytical figures corresponding to a dihydrate. In a separate analysis for water content, the hydrated dipotassium salt of the all-cisisomer 6 was dried overnight at 65°/0.5 torr; the data then corresponded to a dihydrate in both elemental analysis and in Karl Fischer analysis for water content. B. Melting Point Range The PA analogs are stable solids whose melting points lie in the range 73-84 °. In comparison, the melting points of dipalmitoylglycerophosphoric acid and its distearyl ether analog are 70-71 °1~ and 69°, respectively.13 Configurational differences among the analogs have a greater effect on the melting points of the dimethyl and diphenyl esters than on the melting points of the free acids themselves. Similarly, the chromatographic behavior of the esters is more affected than is that of the free acids. The 1,2,3/0-isomers have the lowest melting points in these series, and in this respect follow the pattern seen in the diacylcyclopentanetriols (benzyl ethers) and in the group of t r i s h o m o a c y l c y c l o p e n t a n e t r i o l s 1 (Table I). XII.

Spectral Characteristics of the Cyclo-PA Analogs

A. Free Acid Forms The NMR spectra of PA analogs showed the high field resonance signals expected for the aliphatic chains: 8 0.89, CHa; 6 1.26, CH2. Signals for ring methylene and ring O - - C - - H protons were not resolved, the spectra showing only broad lines in the appropriate regions. We believe that much of the line broadening is due to phosphate-chelated paramagnetic ions derived from the platinum catalyst used for cleavage of the phenyl groups. 1~ E. Baer and J. Maurukas, J. Biol. Chem. 212, 39 (1955).

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CYCLOPENYANO|D ANALOGS OF DIACYLGLYCEROPHOSPHATE

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B. Methyl and Phenyl Esters The spectra of the corresponding methyl and phenyl diesters of PA are significantly sharper, and the signals of some of the ring O---C--H protons are resolved. For example, in the case of the a l l - t r a n s - i s o m e r 4 and the corresponding dimethyl phosphoric ester, a signal representing the ring proton (C-2)--H appears as a well-resolved triplet, "]average = 4.0 Hz. From this coupling value one readily calculates that the conformational equilibrium of the ring backbone of this lipid analog is identical with that deduced earlier 9 for the tribenzoate of 3. The implication of this finding is that conformational preferences deduced for the simpler compounds may be used as a reasonable basis for predicting the conformation of ring skeletons of the various phospholipid analogs whose syntheses are the current object of our efforts. Another diagnostic and empirical feature in the NMR spectra of the cyclo-PA analogs was the appearance of fine structure in the signals of the P--O---CH3 protons centered at 8 3.80 ppm. In addition to the splitting of the methyl signal by 31p coupling (J= 11.3 Hz), some isomers (e.g., the dimethyl esters of 5, 6, and 7) clearly showed further splitting. The spectra of the dimethyl esters of 4 and 8 obtained at the same resolution do not exhibit this fine structure. An analogous phenomenon has been observed in the spectrum of a synthetic phosphatidylglycerosulfate.TM We believe that this phenomenon reflects the chemical nonequivalence of the diastereotopic methyl groups 17 in the case of the cyclopentanoid analogs.

C. Thin-Layer Chromatography The chromatographic mobility of the cyclo-PA analogs was, in general, slightly greater in acid or neutral solvent systems than that of dipalmitoylglycerophosphoric acid; in an alkaline system the analogs, in common with glycero-PA, did not migrate (Table II). The dependence of mobility on the ring configuration of the isomers was marginally evident in the chromatography of the free acids, but more marked for the dimethyl and diphenyl esters, where the (1,2,3/0)-isomers, e.g., 6, showed greater apparent polarity (Table II). The PA analogs gave well defined spots on chromatography in acid or neutral solvent systems, unlike other PA analogs, such as the diether glycero analogs reported by Kates e t al. 1~ for which these systems were less suitable.

~6 A. J. H a n c o c k and M. Kates, J. Lipid Res. 14, 430 (1973). ~7 A. A. Gallo, A. J. H a n c o c k , and H. Z. Sable, J. Lipid Res. 18, 77 (1977).

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SVBSTRATES, ANALOGS, AND INHIBITORS

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TABLE II CHROMATOGRAPHIC MOBILITY OF ISOMERIC CYCLOPENTANOID ANALOGS OF PHOSPHATIDIC ACID (FREE ACIDS AND ESTERS) a Rf Free acid Solvent

Diphenyl ester

Dimethyl ester

Compound ~

Configuration

A

B

C

Solvent D

Solvent E

4 5 6 7 8 9 --

1,3/2-(1P) 1,2/3-(3P) 1,2,3/0-(1P) 1,2/3-(1P) 1,3/2-(2P) 1,2,3/0-(2P) Dipalmitoylglycerophosphoric acid

0.44 0.39 0.36 0.44 0.46 0.45 0.36

0.70 0.67 0.62 0.70 0.71 0.70 0.62

0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.46 0.44 0.40 0.40 0.54 0.55 --

0.41 0.41 0.29 0.39 0.48 0.35 --

On silica gel G-coated plates, adsorbent thickness is 250 ~zm. b Identification numbers refer to Fig. 2. c Solvent systems: (A) C H C I 3 - M e O H - H ~ O , 6 5 : 3 5 : 5 ; (B) CHC13-(CH3)2CO-MeOHH O A c - H 2 0 , 6 : 8 : 2 : 2 : 1 ; (C) CHC13-MeOH-30% NH4OH, 65 : 35 : 5; (D) CCI4EtOAc, 8 : 2; (E) CHCI3-Et20, 3 : 1.

Acknowledgments This work was supported by grants AM-07719 and 5TO-GM-35 (from the National Institutes of Health and from the American Heart Association) to Dr. Henry Z. Sable and by a Grant-in-aid (Missouri Heart Association) and Faculty Research grants (University o f Missouri, Kansas City) to Dr. Anthony J. Hancock. The collaboration with Dr. Sable, with whom the work originated, is warmly acknowledged. The author also acknowledges the skilled assistance o f Dr. Steven M. Greenwald and of Mark D. Lister in preparing synthetic intermediates, o f Maureen B. O'Connell, who typed the manuscript, and of Thomas O. Mueller in preparing the figures.