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A new approach to phospholipase A2 inhibition
wo-4039192 $3 00 + 00 Pergamon Press plc
TeoaJ~cdron Lerters Vol 33, No 4, pp 551.554,1992 !‘mtcd m Great Br~tam
A New Approach to Phospholipase Juhan M C Golee*, Charles J R. Hedgecock, and W_ Roger Tully Roussel Laboratories
Ltd, KingfIsher
Swindon, Wiltshire,
A2 Inhibition Robert Murdoch,
Drive, Covingham,
SN3 5B2, England
Several approaches have been adopted m the ratlonal design of phosphohpase A, mhlbltors The most extensively studied have been phosphohpld analogues where the enzyme susceptible ester linkage has been replaced by an amide’ or carbamate’, and the analogues of the transition state, mlmlcked by the hydrated or the phosphate group 4 As an alternatlve approach to phosphohpase mhlbltlon, we fluoroketones3, envisaged compounds which, upon hydrolysis by the enzyme would form a bldentate llgand towards the calcium ion at the enzymes active site, thus glvmg a tightly bmdmg product The synthesis of such compounds5 1 and 2 are described,, as well as the compounds of type 3, analogues of the de Haas amide’
0
0
“rx
oC16H33
O%H37
(2) (a) R = Me (b) R = Ph(CH-J3 (c) R =
C&17
(d) R = %3H37
(e) R = Bn (f)R=H
The phosphohpld 1 was prepared from ethyl a-bromomethactylate 46 accordmg to Scheme I Treatment of 4 with sodium octadecanoxlde and s&urn lodlde m THF at room temperature gave the ether to the 5 Reduction of 5 with DIBAL gave the allyhc alcohol 6 (86%), which was oxldlsed (mCPA/CH,Cl,) 551
552
epoxlde 7 (86%), and protected as the t-butyldlmethylsllyl ether 8 The lactone 9 was prepared by use of Danlshefskys procedure’ Thus, meatment of 8 with the llthlo-damon of acetic acid m DME at 60’ gave a mixture of lactone 9 and the correqpondmg r-hydroxycarboxyllc acid The mixture was heated with toslc acid m benzene effectmg nng closure to the lactone 9 (75% overall) Removal of the sllyl protection with 10% HCl/acetone gave the alcohol 10 which was treated with 2-chloro-2-oxo-1,3,4-ox+phospholane m benzene, followed by mmethylamme m acetomtrlle at 60’ m a pressure bottle for 17h8, giving the desired phosphohpld 1(44%)
SCHEME I
==s Br
0
a
0 Et
C1&37
o cl
b
OEt
0
sH37
O-H
0
(4)
(5)
(6) c,d
Cl . P” O 0’
I
‘0
i-J (1)
0
O
o
e,f,g
C18H37
4
hi
“c (9) R= TBDMS (10) R= H
a) NaH/ C18H370H/ NaI/ THF I b) DIBAL-Hj d) TBDMSCY lmldazole/ g) 10% HCl/ acetone,
OR
(7) R= H (8) R= TBDMS
toluene, c) mCPA/ CH,Cl,
DMF, e) AcOZI/ LDA/ DME, f) TosOW C,H,
h) 2-chloro-2-oxo-1,3,2-oxdphospholane/
C&d
TEA
1) NMe$ MeCN/ hOa/ 17 h
Phosphohplds of types 2 and 3 were prepared from the common mtermehate 13, accordmg to Scheme II The dlethyl acetal 11 was prepared from dlethyl malondte dnd 2-bromo-l,l-dlethoxyethane Treatment of the dlethoxyacetal 11 In acetone afforded the cychc acetal 12 (99%) Oxldatlon to the lactone 13 was accomplished (95%) with mCPA/ BF, Et,0 9V or m equally high yield by Jones oxldatlon The hydroxyamlde 15 was obtained (76%) from the lactone 13 by tTeatmg It with octadecylamme and sodium cyanide m refluxmg ethanol lo The methyl ester 2a was obtained (23%) by heatmg the lactone 13 with methanol and sulphunc acid -4 supenor method of r-hydroxyester synthesis was Illustrated by the preparation of 2b Treatment of 13 with one equivalent of sodturn hydroxide m ethanol gave the sodium salt, whzh was thoroughly dned then heated with Lbromo-3-phenylpropane m DMF affordmg the ester 2b
553
(76%) The octadecyl-tc, and benzylL2d esters were prepared m high yield m the same way The hydroxyamlde and esters were converted to the correspondmg phosphatadylcholmes by the method described above, m yields of 25% to 56% after chromatography, followed by recryqtalhsatlon from acetone Hydrogenanon of the benzyl ester 2d (Pd/C /EtOH) afforded the acid 2e SCHEME II
f-c
oc16H33
(EO,
OH
o C16H33
a
b
-
Et0
L--f-
o
(11)
oC16H33
RQ
OH 0 (14)
a) TosOH/ acetone, b) BF3 Et@/ mCPA/ CH,Cl, or Jonec oxldatlon c) NaOH/ EtOH,
d) RBr/ DMF, e) RNH2/ NaCN/ EtOH
The compounds were evaluated as phosphohpase mhlbltors uqmg the naJa naja tqa venom enzyme in the muted mlcelle system desclbed by Gelb3 None of the compounds inhibited the mmal rate of the hydrolysis However, mhlbltlon was seen after a time delay, a consequence of the product of hydrolysis bemg the mhlbltor The lactone 1 and the ester 2d were the best compounds, having an actlvlty similar to the best of Gelb’s fluoro ketone transmon state analogues 3 It should be noted that the acid Zf, the proposed mhlbltor, was machve m this expenment Owmg to its dlsslmllanty to substrate, this result was not unexpected
0
*O- Nat
554
De Haas13 has recently described the crystal structure of a complex between an extracellular phosphohpase A, and the competmvely mhlbltmg substrdte analngue 16 The amide 16 binds the phosphohpase three orders of magnitude more strongly than the natural substrate De Haas proposed rhat a strong hydrogen bond exists between the NH of the mhlbltor’s amide bond and the NSl of the catalytlcally important as-48 m the active site This extra hydrogen bond 1s seen to conmbute to the higher affinity of the inhibitor compared to the natural substrate Molecular graphic studies mdcate that the amide analogue 3d should also form this hydrogen bond To our surprise, this compound 3d was found to be mactlve Acknowledgments:
We thank Mrs Kathy Woodward REFERENCES
1
2 3 4 5
6 7 8 9 10 11 12 13
for the enzyme kmetlc studies
AND NOTES
Bansen, P P M , deHaas, G H , Pleterson, W A, VanDeenen, L L M Btochrm Brophys Acta 1972, 270, 364, Chandrakumar, N S , HaJdu, J Tetrahedron Lett 1981,22, 2949, and J Org Chem 1982, 47,2144 Pluckthun, A , Rohlfs, R , Davidson, F F , Denms, E A Bzochemtstty 1985,24,4201 Gelb, M H .I Am Chem Sot 1986, IOh’, 3146, Yuan, W, Berman, R J, Gelb, M H I Am Chem Sot 1987,109,8071 Yuan, W , Gelb, M H J Am Chem Sac , 1988,110,2665 For another example of a lactone phosphohpld phosphohpase A, mhlbltor see Campbell, MM , Long-Fox, J , Osguthorpe, D J , Samsbury, M , Sessions, R B J Chem Sot , Chem Commun 1988, 1560 , Campbell, M M , Long-Fox, J , Sunsbury, M Tetrahedron 1982,924 Damshefsky, , Schuda, T , Etheredge, S J J Am Chem Sot 1977,99,6066 Chandrakumar, N S ,HaJdu, J J Org Chem 1983,48,1197 Hoppe, 0, Branneke, A Tetrahedron Lett 1983,24, 1687 Hogberg, T , Strom, P , Ebner, M , Ramsby, S J Org Chem 1987,52,2033 All phophohplds were first punfied by flash chromatography (H,O/CHCId MeOH, 4 60 25) Compounds were characten$ed by 200 MHz ‘H and 13C n m r , I R , and C,H,N, anyhsls Owmg to the character of phosphohplds, their analyses were calculated for hydrates Thunmssen, M M G M , Ab, E , Kalk, K H , Drenth, J , DiJkstra, B W , Kulpers, 0 P , Dqkman, R , de Haas, G H 1VerheiJ, H M Nature 1990,347,689
(Received m UK 19 November
1991)
TeoaJ~cdron Lerters Vol 33, No 4, pp 551.554,1992 !‘mtcd m Great Br~tam
A New Approach to Phospholipase Juhan M C Golee*, Charles J R. Hedgecock, and W_ Roger Tully Roussel Laboratories
Ltd, KingfIsher
Swindon, Wiltshire,
A2 Inhibition Robert Murdoch,
Drive, Covingham,
SN3 5B2, England
Several approaches have been adopted m the ratlonal design of phosphohpase A, mhlbltors The most extensively studied have been phosphohpld analogues where the enzyme susceptible ester linkage has been replaced by an amide’ or carbamate’, and the analogues of the transition state, mlmlcked by the hydrated or the phosphate group 4 As an alternatlve approach to phosphohpase mhlbltlon, we fluoroketones3, envisaged compounds which, upon hydrolysis by the enzyme would form a bldentate llgand towards the calcium ion at the enzymes active site, thus glvmg a tightly bmdmg product The synthesis of such compounds5 1 and 2 are described,, as well as the compounds of type 3, analogues of the de Haas amide’
0
0
“rx
oC16H33
O%H37
(2) (a) R = Me (b) R = Ph(CH-J3 (c) R =
C&17
(d) R = %3H37
(e) R = Bn (f)R=H
The phosphohpld 1 was prepared from ethyl a-bromomethactylate 46 accordmg to Scheme I Treatment of 4 with sodium octadecanoxlde and s&urn lodlde m THF at room temperature gave the ether to the 5 Reduction of 5 with DIBAL gave the allyhc alcohol 6 (86%), which was oxldlsed (mCPA/CH,Cl,) 551
552
epoxlde 7 (86%), and protected as the t-butyldlmethylsllyl ether 8 The lactone 9 was prepared by use of Danlshefskys procedure’ Thus, meatment of 8 with the llthlo-damon of acetic acid m DME at 60’ gave a mixture of lactone 9 and the correqpondmg r-hydroxycarboxyllc acid The mixture was heated with toslc acid m benzene effectmg nng closure to the lactone 9 (75% overall) Removal of the sllyl protection with 10% HCl/acetone gave the alcohol 10 which was treated with 2-chloro-2-oxo-1,3,4-ox+phospholane m benzene, followed by mmethylamme m acetomtrlle at 60’ m a pressure bottle for 17h8, giving the desired phosphohpld 1(44%)
SCHEME I
==s Br
0
a
0 Et
C1&37
o cl
b
OEt
0
sH37
O-H
0
(4)
(5)
(6) c,d
Cl . P” O 0’
I
‘0
i-J (1)
0
O
o
e,f,g
C18H37
4
hi
“c (9) R= TBDMS (10) R= H
a) NaH/ C18H370H/ NaI/ THF I b) DIBAL-Hj d) TBDMSCY lmldazole/ g) 10% HCl/ acetone,
OR
(7) R= H (8) R= TBDMS
toluene, c) mCPA/ CH,Cl,
DMF, e) AcOZI/ LDA/ DME, f) TosOW C,H,
h) 2-chloro-2-oxo-1,3,2-oxdphospholane/
C&d
TEA
1) NMe$ MeCN/ hOa/ 17 h
Phosphohplds of types 2 and 3 were prepared from the common mtermehate 13, accordmg to Scheme II The dlethyl acetal 11 was prepared from dlethyl malondte dnd 2-bromo-l,l-dlethoxyethane Treatment of the dlethoxyacetal 11 In acetone afforded the cychc acetal 12 (99%) Oxldatlon to the lactone 13 was accomplished (95%) with mCPA/ BF, Et,0 9V or m equally high yield by Jones oxldatlon The hydroxyamlde 15 was obtained (76%) from the lactone 13 by tTeatmg It with octadecylamme and sodium cyanide m refluxmg ethanol lo The methyl ester 2a was obtained (23%) by heatmg the lactone 13 with methanol and sulphunc acid -4 supenor method of r-hydroxyester synthesis was Illustrated by the preparation of 2b Treatment of 13 with one equivalent of sodturn hydroxide m ethanol gave the sodium salt, whzh was thoroughly dned then heated with Lbromo-3-phenylpropane m DMF affordmg the ester 2b
553
(76%) The octadecyl-tc, and benzylL2d esters were prepared m high yield m the same way The hydroxyamlde and esters were converted to the correspondmg phosphatadylcholmes by the method described above, m yields of 25% to 56% after chromatography, followed by recryqtalhsatlon from acetone Hydrogenanon of the benzyl ester 2d (Pd/C /EtOH) afforded the acid 2e SCHEME II
f-c
oc16H33
(EO,
OH
o C16H33
a
b
-
Et0
L--f-
o
(11)
oC16H33
RQ
OH 0 (14)
a) TosOH/ acetone, b) BF3 Et@/ mCPA/ CH,Cl, or Jonec oxldatlon c) NaOH/ EtOH,
d) RBr/ DMF, e) RNH2/ NaCN/ EtOH
The compounds were evaluated as phosphohpase mhlbltors uqmg the naJa naja tqa venom enzyme in the muted mlcelle system desclbed by Gelb3 None of the compounds inhibited the mmal rate of the hydrolysis However, mhlbltlon was seen after a time delay, a consequence of the product of hydrolysis bemg the mhlbltor The lactone 1 and the ester 2d were the best compounds, having an actlvlty similar to the best of Gelb’s fluoro ketone transmon state analogues 3 It should be noted that the acid Zf, the proposed mhlbltor, was machve m this expenment Owmg to its dlsslmllanty to substrate, this result was not unexpected
0
*O- Nat
554
De Haas13 has recently described the crystal structure of a complex between an extracellular phosphohpase A, and the competmvely mhlbltmg substrdte analngue 16 The amide 16 binds the phosphohpase three orders of magnitude more strongly than the natural substrate De Haas proposed rhat a strong hydrogen bond exists between the NH of the mhlbltor’s amide bond and the NSl of the catalytlcally important as-48 m the active site This extra hydrogen bond 1s seen to conmbute to the higher affinity of the inhibitor compared to the natural substrate Molecular graphic studies mdcate that the amide analogue 3d should also form this hydrogen bond To our surprise, this compound 3d was found to be mactlve Acknowledgments:
We thank Mrs Kathy Woodward REFERENCES
1
2 3 4 5
6 7 8 9 10 11 12 13
for the enzyme kmetlc studies
AND NOTES
Bansen, P P M , deHaas, G H , Pleterson, W A, VanDeenen, L L M Btochrm Brophys Acta 1972, 270, 364, Chandrakumar, N S , HaJdu, J Tetrahedron Lett 1981,22, 2949, and J Org Chem 1982, 47,2144 Pluckthun, A , Rohlfs, R , Davidson, F F , Denms, E A Bzochemtstty 1985,24,4201 Gelb, M H .I Am Chem Sot 1986, IOh’, 3146, Yuan, W, Berman, R J, Gelb, M H I Am Chem Sot 1987,109,8071 Yuan, W , Gelb, M H J Am Chem Sac , 1988,110,2665 For another example of a lactone phosphohpld phosphohpase A, mhlbltor see Campbell, MM , Long-Fox, J , Osguthorpe, D J , Samsbury, M , Sessions, R B J Chem Sot , Chem Commun 1988, 1560 , Campbell, M M , Long-Fox, J , Sunsbury, M Tetrahedron 1982,924 Damshefsky, , Schuda, T , Etheredge, S J J Am Chem Sot 1977,99,6066 Chandrakumar, N S ,HaJdu, J J Org Chem 1983,48,1197 Hoppe, 0, Branneke, A Tetrahedron Lett 1983,24, 1687 Hogberg, T , Strom, P , Ebner, M , Ramsby, S J Org Chem 1987,52,2033 All phophohplds were first punfied by flash chromatography (H,O/CHCId MeOH, 4 60 25) Compounds were characten$ed by 200 MHz ‘H and 13C n m r , I R , and C,H,N, anyhsls Owmg to the character of phosphohplds, their analyses were calculated for hydrates Thunmssen, M M G M , Ab, E , Kalk, K H , Drenth, J , DiJkstra, B W , Kulpers, 0 P , Dqkman, R , de Haas, G H 1VerheiJ, H M Nature 1990,347,689
(Received m UK 19 November
1991)