BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 180, No. 3, 1991
Pages 1416-1421
November 14, 1991
INHIBITION
OF L E U K O T R I E N E
LIPOXIN T a k H.
Lee,
NEUTROPHIL
MIGRATION
A~! S T R U C T U R E - F U N C T I O N
RELATIONSHIPS
Lympany,
Crea,
Penny
Department
B471NDUCED
Attilio
E.G.
and
Bernd
BY
W.
of A l l e r g y and A l l i e d R e s p i r a t o r y D i s o r d e r s , H o s p i t a l , L o n d o n SEI 9RT, UK
Spur
Guy's
Received September 24, 1991
L i p o x i n s are t r i h y d r o x y k e t r a e n e m e t a b o l i t e s w h i c h are d e r i v e d from a r a c h i d o n i c acid t h r o u g h an i n t e r a c t i o n b e t w e e n d i f f e r e n t l i p o x y g e n a s e pathways. P r e v i o u s w o r k has shown that lipoxin A t (LXA~) i n h i b i t s the chemotactic responsiveness of n e u t r o p h i l s (PMN) to l e u k o t r i e n e B~. We have now a s s e s s e d the s t r u c t u r a l d e t e r m i n a n t s of the lipoxin A 4 m o l e c u l e w h i c h are n e c e s s a r y for its i n h i b i t o r y activity, using structural analogs of LXA~ prepared by chemical synthesis. Our r e s u l t s indicate the i m p o r t a n c e of two a d j a c e n t free h y d r o x y l groups in e i t h e r the R or the S c o n f i g u r a t i o n ; one h y d r o x y l group has to be in the C - 6 p o s i t i o n , but the other h y d r o x y l group can be in e i t h e r the C-5 or the C-7 p o s i t i o n for the c o n f e r m e n t of i n h i b i t o r y activity. © 1991 A c a d e m i c
Press,
Lipoxins
are
arachidonic
of
lipoxin
trihydroxy acid
lipoxygenases structure
Inc.
B~
tetraene
through
with the
C5
and
two
(LXB4)
an C15
major
are
ll-cis-eicosatetraenoic
recently
isomers,
5S,
6R,
acid
and
inhibits
demonstrated
the c h e m o t a c t i c
(LTB4) and to the p e p t i d e (FMLP)
lipoxin
5S,
14R,
The
At
and
(LXA4)
5S-trihydroxy-
respectively
prior
exposure
responsiveness formyl-
(1,2,3).
15S-trihydroxy-7,9,13-trans-
acid,
that
from
different
of PMN
of
6,10,
(1,2,3). PMN
to
We LXA 4
£o l e u k o t r i e n e
B4
methionyl-leucyl-phenylalanine
(4).
We n o w report
on the s t r u c t u r a l
w h i c h are i m p o r t a n t towards
derived
between
specificities
12-trans-8-cis-eicosatetraenoic have
metabolites
interaction
cellular
determinants
in the e x p r e s s i o n
migration
in r e s p o n s e
of its
of the LXA 4 m o l e c u l e inhibitory
activity
to LTB 4.
MATERIALS AND METHODS Lipoxin A t and lipoxin a convergent approach
B 4 were starting
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
synthesized with the
1416
chemically acetonide
of
by way of 2-deoxy-
Vol. 180, No. 3, 1991
CHs
CHs
O
O
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
- -
'
~A~ 3
RO OR I
U
IE
OH
OH "OH
OH __J_~.,j,,,,,,v~ CH3
OH
CH3 / ~
CH3
X
o OH~
1
~
~
~
"
COOCH3 OH
OH
V
IV
1
COOCH.
~
Vl
CH3 OR
o
COOCH3
, ~ C H 3
~
CH
,
RO OR
IX
Vlll
VII
OH O
H
~
~
:
COOCH:I
COOCHm
OH 3
OH / V
V
~ CH3
CH3 ~
COOCH3
OH OH
XI
X
OH
Xll
OH
OH
OH '
OH
COOCH~,
~
COOCH3 3
OH OH
~
COOCH3 OH
XlV
XIll
XV
OH OH ~
CH3 OH
XVl
Figure 1. "a" denotes methylester
D-ribose LXA4 a n d
Structure of each of the molecules noted in the text. the free acid of the compound instead of the as shown in the figure.
since it t h e 14R,
already possesses 15S configuration
t h e 5 S , 6R o f LXB 4.
configuration
of
Condensation of the acetonide of 2-deoxy-D-ribose with methyl[triphenylphosphoranylidene] acetate (1.2 eq.) in tetrahydrofuran (60oC, 3h) followed by catalytical hydrogenation of the double bond (10% Pd/C methylacetate) afforded alcohol (I) (87%) (Fig. 1) ( 5 ) . S w e r no x i d a t i o n (1.5 eq. COCI], 2.0 eq. DMSO, 5 eq. Et3N, CH2CI2 -75 ° -25°C, 15 min) gave i n almost q u a n t i t a t i v e y i e l d the aldehyde which was c o n v e r t e d t o t h e key i n t e r m e d i a t e ( I I ) i n >90% y i e l d by the f o l l o w i n g sequence: W i t t i g r e a c t i o n ([C6HsJ 3 P=CH- CH=CH-Cm C-Si[CH3]3, 1.1 e q . , -78 ° -0°C, THF, l h ) . I o d i n e i s o m e r i s a l i o n t o the t r a n s , t r a n s enyne (I 2, CH2CI2 6H) and removal of t h e t r i m e t h y l s i l y l group with f l u o r i d e (KF, 18 crown 6 DMF, l h , r.t.). The second fragment III was o b t a i n e d as described in the literature (6). The final Pd ( 0 ) , Cu(1) coupling was c a r r i e d out as described in the literature (6) to give the protected triple b o n d i s o m e r o f LXA4. Removal of the protective g r o u p s (1N HC1, THF, CH3OH, KF) y i e l d e d IV, and final hydrogenation of the triple bond (Pd- lindlar) ( 6 ) g a v e LXA4 methylester (V) a n d s m a l l a m o u n t s o f a l l trans LXA# m e t h y l e s t e r (VI), which were readily separated b y R P - HPLC ( 6 5 : 3 5 , CH30H:H20; Waters Delta-Pak Cls, 30 x 3 0 0 mm; 51 m l / m i n ) . 1417
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The s y n t h e s i s of LXB 4 was a c c o m p l i s h e d b y the c o n d e n s a t i o n of the a c e t o n i d e of 2 - d e o x y - d - r i b o s e w i t h n - p r o p y l i d e n e - t r i p h e n y l phosphorane (2 eq.) in THF (0oC, 2h). This was f o l l o w e d by catalytical hydrogenation (10% Pd / C, m e t h y l a c e t a t e 100%) to give VII. VII was o x i d i z e d (Cr03. 2 p y r i d i n e , CH2CI 2, r.t.) and c o n d e n s e d w i t h the p h o s p h o r a n e ([C6H5] 3 P = C H CH=CHCmCSi (CH3) 3, THF - 78 ° - 0°C) f o l l o w e d by iodine i s o m e r i s a t i o n and c l e a v a g e of the silyl g r o u p w i t h f l u o r i d e as d e s c r i b e d above for LXA# g i v i n g the i n t e r m e d i a t e (VIII) in 70% overall yield. The fragment (IX) was s y n t h e s i z e d as p r e v i o u s l y described (7). C o u p l i n g of the f r a g m e n t s VIII and IX f o l l o w e d by removal of the p r o t e c t i v e groups gave X. Semi-hydrogenation of X r e s u l t e d in LXB 4 methylester(XI) and small a m o u n t s of the a l l - t r a n s LXB 4 m e t h y l e s t e r (XII).
The s y n t h e s e s of 5S, 6S and 5S, 6R di-HETE ( X I I I and XIV, r e s p e c t i v e l y ) were c a r r i e d out s t a r t i n g from LTA~ and 6 epi-LTA4 as a l r e a d y d e s c r i b e d ( 8 ) . Compound XV was o b t a i n e d from I v i a deprotecting with IN HCI. VII was converted to XVI via deprotecting w i t h iN HCI. 5S, 6S L X A ~ (XVII) was o b t a i n e d a c c o r d i n g to the p r e v i o u s l y d e s K r i b ~ d m e t h o d from LTA~ (8). The free acids (Va, Via, XIa) were o b t a i n e d from the r e s p e c t i v e m e t h y l e s t e r s by m i l d a l k a l i n e h y d r o l y s i s , u s i n g IM L i O H in THF at 0oC~in q u a n t i t a t i v e y i e l d (7). LXA~, LXB4 a n d a l l i n t e r m e d i a t e s were fully characterized by 2-DNMR s p e c t r o s c o p y , ultraviolet spectroscopy, mass spectrometry and infrared spectroscopy. LXA4 a n d LXB 4 p r e p a r e d as free acids and methyl esters were identical in all respects with material synthesized by published routes (8-11). FMN were isolated as described (4) and resuspended at a concentration of 2 x 101/ml in H B S S - O A (Hank's B a l a n c e d Salt S o l u t i o n s u p p l e m e n t e d w i t h 0 . 4 % o v a l b u m i n pH 7.4). 1 x i0 ? cells in 500 pl H B S S OA were w a r m e d at 37°C ior 5 m i n u t e s and then m i x e d w i t h 500 pl of p r e - w a r m e d solutions of the r e s p e c t i v e lipoxins or structural a n a l o g s of lipoxins in H B S S - O A or b u f f e r alone; final concentrations were l0 -IU mol/l £o i0 -6 mol/l for e a c h compound. The m i x t u r e was i n c u b a t e d at 37°C from 15 m i n u t e s before cooling to 4°C. 50 ~I of e a c h of £he cell s u s p e n s i o n s was then u s e d for a s s e s s m e n t of c e l l u l a r m i g r a £ i o n in r ~ s p o n s e to an o p t i m a l l y effective chemotactic concentration (10-'mol/l) of LTB~ as described (4) The percentage inhibition of c h e m o t a x i s was c a l c u l a t e d is: l - ( c h e m o t a c t i c r e s p o n s e of cells pretreated with lipoxin + c h e m o t a c t i c r e s p o n s e of control cells) x I00. The r e s u l t s were e x p r e s s e d as the c o n c e n t r a t i o n p r o d u c i n g 50% i n h i b i t i o n (ICb0) and w e r e o b t a i n e d by linear i n t e r p o l a t i o n of the dose r e s p o n s e curve.
Statistical
analysis
This data was a n a l y s e d u s i n g are expressed as m e a n + SEM.
analysis
of variance.
The
results
RESULTS AND DISCUSSION Preincubation
of PMN w i t h
resulted
in
a
migration
induced
increasing
concentrations
concentration-dependent by 10-?M LTB~,
(p
Vol. 180, No. 3, 1991
Pages 1416-1421
November 14, 1991
INHIBITION
OF L E U K O T R I E N E
LIPOXIN T a k H.
Lee,
NEUTROPHIL
MIGRATION
A~! S T R U C T U R E - F U N C T I O N
RELATIONSHIPS
Lympany,
Crea,
Penny
Department
B471NDUCED
Attilio
E.G.
and
Bernd
BY
W.
of A l l e r g y and A l l i e d R e s p i r a t o r y D i s o r d e r s , H o s p i t a l , L o n d o n SEI 9RT, UK
Spur
Guy's
Received September 24, 1991
L i p o x i n s are t r i h y d r o x y k e t r a e n e m e t a b o l i t e s w h i c h are d e r i v e d from a r a c h i d o n i c acid t h r o u g h an i n t e r a c t i o n b e t w e e n d i f f e r e n t l i p o x y g e n a s e pathways. P r e v i o u s w o r k has shown that lipoxin A t (LXA~) i n h i b i t s the chemotactic responsiveness of n e u t r o p h i l s (PMN) to l e u k o t r i e n e B~. We have now a s s e s s e d the s t r u c t u r a l d e t e r m i n a n t s of the lipoxin A 4 m o l e c u l e w h i c h are n e c e s s a r y for its i n h i b i t o r y activity, using structural analogs of LXA~ prepared by chemical synthesis. Our r e s u l t s indicate the i m p o r t a n c e of two a d j a c e n t free h y d r o x y l groups in e i t h e r the R or the S c o n f i g u r a t i o n ; one h y d r o x y l group has to be in the C - 6 p o s i t i o n , but the other h y d r o x y l group can be in e i t h e r the C-5 or the C-7 p o s i t i o n for the c o n f e r m e n t of i n h i b i t o r y activity. © 1991 A c a d e m i c
Press,
Lipoxins
are
arachidonic
of
lipoxin
trihydroxy acid
lipoxygenases structure
Inc.
B~
tetraene
through
with the
C5
and
two
(LXB4)
an C15
major
are
ll-cis-eicosatetraenoic
recently
isomers,
5S,
6R,
acid
and
inhibits
demonstrated
the c h e m o t a c t i c
(LTB4) and to the p e p t i d e (FMLP)
lipoxin
5S,
14R,
The
At
and
(LXA4)
5S-trihydroxy-
respectively
prior
exposure
responsiveness formyl-
(1,2,3).
15S-trihydroxy-7,9,13-trans-
acid,
that
from
different
of PMN
of
6,10,
(1,2,3). PMN
to
We LXA 4
£o l e u k o t r i e n e
B4
methionyl-leucyl-phenylalanine
(4).
We n o w report
on the s t r u c t u r a l
w h i c h are i m p o r t a n t towards
derived
between
specificities
12-trans-8-cis-eicosatetraenoic have
metabolites
interaction
cellular
determinants
in the e x p r e s s i o n
migration
in r e s p o n s e
of its
of the LXA 4 m o l e c u l e inhibitory
activity
to LTB 4.
MATERIALS AND METHODS Lipoxin A t and lipoxin a convergent approach
B 4 were starting
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
synthesized with the
1416
chemically acetonide
of
by way of 2-deoxy-
Vol. 180, No. 3, 1991
CHs
CHs
O
O
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
- -
'
~A~ 3
RO OR I
U
IE
OH
OH "OH
OH __J_~.,j,,,,,,v~ CH3
OH
CH3 / ~
CH3
X
o OH~
1
~
~
~
"
COOCH3 OH
OH
V
IV
1
COOCH.
~
Vl
CH3 OR
o
COOCH3
, ~ C H 3
~
CH
,
RO OR
IX
Vlll
VII
OH O
H
~
~
:
COOCH:I
COOCHm
OH 3
OH / V
V
~ CH3
CH3 ~
COOCH3
OH OH
XI
X
OH
Xll
OH
OH
OH '
OH
COOCH~,
~
COOCH3 3
OH OH
~
COOCH3 OH
XlV
XIll
XV
OH OH ~
CH3 OH
XVl
Figure 1. "a" denotes methylester
D-ribose LXA4 a n d
Structure of each of the molecules noted in the text. the free acid of the compound instead of the as shown in the figure.
since it t h e 14R,
already possesses 15S configuration
t h e 5 S , 6R o f LXB 4.
configuration
of
Condensation of the acetonide of 2-deoxy-D-ribose with methyl[triphenylphosphoranylidene] acetate (1.2 eq.) in tetrahydrofuran (60oC, 3h) followed by catalytical hydrogenation of the double bond (10% Pd/C methylacetate) afforded alcohol (I) (87%) (Fig. 1) ( 5 ) . S w e r no x i d a t i o n (1.5 eq. COCI], 2.0 eq. DMSO, 5 eq. Et3N, CH2CI2 -75 ° -25°C, 15 min) gave i n almost q u a n t i t a t i v e y i e l d the aldehyde which was c o n v e r t e d t o t h e key i n t e r m e d i a t e ( I I ) i n >90% y i e l d by the f o l l o w i n g sequence: W i t t i g r e a c t i o n ([C6HsJ 3 P=CH- CH=CH-Cm C-Si[CH3]3, 1.1 e q . , -78 ° -0°C, THF, l h ) . I o d i n e i s o m e r i s a l i o n t o the t r a n s , t r a n s enyne (I 2, CH2CI2 6H) and removal of t h e t r i m e t h y l s i l y l group with f l u o r i d e (KF, 18 crown 6 DMF, l h , r.t.). The second fragment III was o b t a i n e d as described in the literature (6). The final Pd ( 0 ) , Cu(1) coupling was c a r r i e d out as described in the literature (6) to give the protected triple b o n d i s o m e r o f LXA4. Removal of the protective g r o u p s (1N HC1, THF, CH3OH, KF) y i e l d e d IV, and final hydrogenation of the triple bond (Pd- lindlar) ( 6 ) g a v e LXA4 methylester (V) a n d s m a l l a m o u n t s o f a l l trans LXA# m e t h y l e s t e r (VI), which were readily separated b y R P - HPLC ( 6 5 : 3 5 , CH30H:H20; Waters Delta-Pak Cls, 30 x 3 0 0 mm; 51 m l / m i n ) . 1417
Vol. 180, No. 3, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The s y n t h e s i s of LXB 4 was a c c o m p l i s h e d b y the c o n d e n s a t i o n of the a c e t o n i d e of 2 - d e o x y - d - r i b o s e w i t h n - p r o p y l i d e n e - t r i p h e n y l phosphorane (2 eq.) in THF (0oC, 2h). This was f o l l o w e d by catalytical hydrogenation (10% Pd / C, m e t h y l a c e t a t e 100%) to give VII. VII was o x i d i z e d (Cr03. 2 p y r i d i n e , CH2CI 2, r.t.) and c o n d e n s e d w i t h the p h o s p h o r a n e ([C6H5] 3 P = C H CH=CHCmCSi (CH3) 3, THF - 78 ° - 0°C) f o l l o w e d by iodine i s o m e r i s a t i o n and c l e a v a g e of the silyl g r o u p w i t h f l u o r i d e as d e s c r i b e d above for LXA# g i v i n g the i n t e r m e d i a t e (VIII) in 70% overall yield. The fragment (IX) was s y n t h e s i z e d as p r e v i o u s l y described (7). C o u p l i n g of the f r a g m e n t s VIII and IX f o l l o w e d by removal of the p r o t e c t i v e groups gave X. Semi-hydrogenation of X r e s u l t e d in LXB 4 methylester(XI) and small a m o u n t s of the a l l - t r a n s LXB 4 m e t h y l e s t e r (XII).
The s y n t h e s e s of 5S, 6S and 5S, 6R di-HETE ( X I I I and XIV, r e s p e c t i v e l y ) were c a r r i e d out s t a r t i n g from LTA~ and 6 epi-LTA4 as a l r e a d y d e s c r i b e d ( 8 ) . Compound XV was o b t a i n e d from I v i a deprotecting with IN HCI. VII was converted to XVI via deprotecting w i t h iN HCI. 5S, 6S L X A ~ (XVII) was o b t a i n e d a c c o r d i n g to the p r e v i o u s l y d e s K r i b ~ d m e t h o d from LTA~ (8). The free acids (Va, Via, XIa) were o b t a i n e d from the r e s p e c t i v e m e t h y l e s t e r s by m i l d a l k a l i n e h y d r o l y s i s , u s i n g IM L i O H in THF at 0oC~in q u a n t i t a t i v e y i e l d (7). LXA~, LXB4 a n d a l l i n t e r m e d i a t e s were fully characterized by 2-DNMR s p e c t r o s c o p y , ultraviolet spectroscopy, mass spectrometry and infrared spectroscopy. LXA4 a n d LXB 4 p r e p a r e d as free acids and methyl esters were identical in all respects with material synthesized by published routes (8-11). FMN were isolated as described (4) and resuspended at a concentration of 2 x 101/ml in H B S S - O A (Hank's B a l a n c e d Salt S o l u t i o n s u p p l e m e n t e d w i t h 0 . 4 % o v a l b u m i n pH 7.4). 1 x i0 ? cells in 500 pl H B S S OA were w a r m e d at 37°C ior 5 m i n u t e s and then m i x e d w i t h 500 pl of p r e - w a r m e d solutions of the r e s p e c t i v e lipoxins or structural a n a l o g s of lipoxins in H B S S - O A or b u f f e r alone; final concentrations were l0 -IU mol/l £o i0 -6 mol/l for e a c h compound. The m i x t u r e was i n c u b a t e d at 37°C from 15 m i n u t e s before cooling to 4°C. 50 ~I of e a c h of £he cell s u s p e n s i o n s was then u s e d for a s s e s s m e n t of c e l l u l a r m i g r a £ i o n in r ~ s p o n s e to an o p t i m a l l y effective chemotactic concentration (10-'mol/l) of LTB~ as described (4) The percentage inhibition of c h e m o t a x i s was c a l c u l a t e d is: l - ( c h e m o t a c t i c r e s p o n s e of cells pretreated with lipoxin + c h e m o t a c t i c r e s p o n s e of control cells) x I00. The r e s u l t s were e x p r e s s e d as the c o n c e n t r a t i o n p r o d u c i n g 50% i n h i b i t i o n (ICb0) and w e r e o b t a i n e d by linear i n t e r p o l a t i o n of the dose r e s p o n s e curve.
Statistical
analysis
This data was a n a l y s e d u s i n g are expressed as m e a n + SEM.
analysis
of variance.
The
results
RESULTS AND DISCUSSION Preincubation
of PMN w i t h
resulted
in
a
migration
induced
increasing
concentrations
concentration-dependent by 10-?M LTB~,
(p
