Leukotriene B4 photoaffinity probes: design, synthesis and evaluation of new arylazide-1,3-disubstituted cyclohexanes.

Article abstract of DOI:10.1016/S0960-894X(00)00103-7

The synthesis and the binding affinities of new leukotriene B4 receptor photoaffinity probes, where a 1,3-disubstituted cyclohexane ring replaces the conjugated delta6,7 and delta8,9 double bonds of the natural eicosanoid, are described. One enantiomeric compound, 4b alpha, is specifically cross-linked upon photolysis to the recombinant leukotriene B4 receptor from human origin (h-BLTR) solubilized in a micellar medium. This probe appears as a good candidate for identifying the ligand binding site of this receptor.

Full text of DOI:10.1016/S0960-894X(00)00103-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 811±814
Leukotriene B4 Photoanity Probes: Design, Synthesis and
Evaluation of New Arylazide-1,3-Disubstituted Cyclohexanes
Denis Durand, Pierre Hullot, Jean-Pierre Vidal, Jean-Pierre Girard,* Jean Louis Baneres,
Joseph Parello, Agnes Muller, Claude Bonne and Jean-Claude Rossi
 Â
Laboratoire de Chimie Biomoleculaire et des Interactions Biologiques, UPRESA-CNRS 5074, Faculte de Pharmacie,
15 Avenue Flahault, 34060 Montpellier Cedex 2, France
Received 6 December 1999; accepted 10 February 2000
AbstractÐThe synthesis and the binding anities of new leukotriene B4 receptor photoanity probes, where a 1,3-disubstitued
cyclohexane ring replaces the conjugated Á6,7 and Á8,9 double bonds of the natural eicosanoid, are described. One enantiomeric
compound, 4bꢀ, is speci®cally cross-linked upon photolysis to the recombinant leukotriene B4 receptor from human origin (h-BLTR)
solubilized in a micellar medium. This probe appears as a good candidate for identifying the ligand binding site of this receptor.
# 2000 Elsevier Science Ltd. All rights reserved.
Leukotriene B4 (LTB4), a product of 5-lipoxygenase
induced arachidonic acid metabolism, has been shown
to be a potent mediator of the in¯ammatory process
and to be involved in the progression of a variety of
in¯ammatory diseases.1 12 At the cellular level, LTB4
exerts its e€ect through recruitment and activation of
neutrophils and eosinophils, through binding to a
cell-surface receptor. Recently, the cloning of the
complementary DNA (cDNA) encoding a cell-surface
receptor of LTB4 that is highly expressed in human
leukocytes has been reported.13 The amino acid
sequence of this protein h-BLTR14 is suggestive of a
G-protein coupled receptor with seven transmembrane
helices. We were particularly interested in mapping the
BLTR ligand binding site through photoanity label-
ling. We have recently reported15 the synthesis and
structure±activity relationship of a novel series of com-
pounds where a 1,3-disubstituted cyclohexane ring
replaces two of the conjugated double bonds of the
natural eicosanoid. The synthesized enantiomeric com-
pounds, 1bꢀ and 2bꢀ, are more stable and more rigid
mimics of the natural ligand and display good anities
(IC50=0.3 and 0.04 mM, respectively) for the LTB4
receptor in human neutrophils (see Table 1).
We have now focused on the use of aryl azides16 as
photoreactive nitrene precursors which are structurally
related to the LTB4 antagonists 1bꢀ and 2bꢀ with the
terminus of their lower chains replaced by a benzyl
azide group. It was anticiped that these probes would
bind to h-BLTR and establish a covalent crosslink with
the receptor upon photolysis of the aryl azide, thus
allowing us to map the h-BLTR ligand binding site. We
describe here our initial e€orts in this respect with the
synthesis of the racemic diastereoisomers 3, 4, 5 and 6,
and the isolation of the enantiomers 4bꢀ, 4bꢁ and 6bꢀ,
6bꢁ.
Chemistry
The general synthetic route to compounds 3±6 is illu-
strated in Schemes 1 and 2. The o chain of these com-
pounds was introduced in good yields on the cyclohexylic
*Corresponding author. Fax: +33-6754-8625; e-mail: girard@
pharma.univ-montpl.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00103-7

812
D. Durand et al. / Bioorg. Med. Chem. Lett. 10 (2000) 811±814
ring by the Wadsworth±Horner±Emmons reaction
between the crude unstable aldehyde15 12 and the ylide
of the suitable b-ketophosphonate 11 (Scheme 1). This
reaction gives 13 in 75% yield.
hydroxyl group at C1 on the proton chemical shifts15 of
the cyclohexane ring.
We also report the synthesis of two iodine substituted
compounds 5 and 6 from precursor 15 (Scheme 2). The
latter was treated with Chloramine T and sodium
iodide, in a mixture of acetic acid/water, to give 17 in
55% yield. The phenyl amine was oxidized with sodium
nitrite in aqueous HCl and methanol at 0 ꢀC and treated
with NaN3 to a€ord 18 in 95% yield. Using the same
strategy, we previously reported that the condensation
of the ethyl acetate carbanion (LDA at 78 ꢀC) to 16
a€orded four diastereoisomeric compounds, two trans
and two cis compounds (5a±d) as racemic mixtures, in
70% yield. Similarly, the condensation of the N,N-
dimethylacetamide carbanion (LDA at 60 ꢀC) gave
four diastereoisomers 6a±d in 89% yield. Each dia-
stereoisomer was isolated by preparative HPLC18a
chromatography.
The reduction of the known keto-ester15 to the keto-
aldehyde 12 with diisobutylaluminium hydride at
78 ꢀC was monitored by gas chromatography in order
to control the formation of alcohol (overreduction). As
soon as traces of alcohol appeared (after 30 min), the
resulting crude aldehyde 12 was added at 40 ꢀC on the
ylide of the b-ketophosphonate 11 (NaH, THF). Com-
pound 11 was prepared in ®ve steps (Scheme 1). The ®rst
step, a Wittig reaction between p-nitrobenzaldehyde and
the phosphonium salt 7, a€orded compound 8 in 62%
yield. After esteri®cation in acidic conditions, the pro-
tected amine 10 was obtained by successive hydrogena-
tion and protection with trityl chloride in 90% yield.
Addition of 10 to the ylide of methyldiethylphos-
phonate, generated in the presence of n-butyllithium, in
a mixture of toluene/THF, gave the diethyl 2-oxo-6-(4-
tritylamino)phenylhexylphosphonate 11 in 85% yield. The
enone 13 was then reduced to the enol 14 by treatment
with sodium borohydride in the presence of cerium
chloride,17 to avoid simultaneous hydroboration of the
double bond. The latter was used as a precursor for
synthesizing compounds 3 to 6.
At this stage, all the racemic compounds 3a±d and 5a±d
were converted into their sodium salts (NaOH, methanol)
for biochemical/pharmacological testing (see below).
The racemic compounds 4b and 6b were resolved by
chiral HPLC18b to a€ord by order of elution the enan-
tiomers 4bꢀ19 and 4bꢁ for the former, 6bꢀ and 6bꢁ for
the latter.
After deprotection of both carbonyl and amine func-
tions under acidic conditions (Scheme 2), 15 reacted
with NaNO2 and NaN3 to give, in a mixture of MeOH/
HCl at 0 ꢀC, the azido compound 16 in 95% yield.
Biological Results
The diastereoisomers 3a±d to 6a±d, used here as racemic
mixtures, were tested for their ability to inhibit the
binding of LTB4 to human neutrophil membranes.20±22
In each series (1 to 6), the HPLC more polar trans dia-
stereoisomers b exhibited the highest competitive e€ect
(Table 1) compared to compounds a, c and d, which
also competed for [3H]LTB4 but with lower anities
(IC50 values not given).
The condensation of the ethyl acetate carbanion (LDA
at 78 ꢀC) a€orded four diastereoisomers, as a mixture
in 62% yield of two trans con®guration (racemate 3a
and 3b) and two cis con®guration (racemate 3c and 3d).
Using the same strategy, the condensation of the N,N-
dimethylacetamide carbanion (LDA at 60 ꢀC) gave
four diastereoisomers 4a±d in 90% yield. These com-
pounds were isolated by column chromatography and
their relative trans and cis con®gurations were readily
established by 1H NMR, using the in¯uence of the
We note that incorporation of a p-azido group is sys-
tematically associated with a reduced anity. Further-
more, the iodination in the meta-position results in a
Scheme 1. Reagents and conditions (isolated yield): (i) p-nitrobenzaldehyde, t-BuOK, THF (62%); (ii) MeOH, H2SO4 (98%); (iii) H2, Pd/C 5%,
55 psi (100%); (iv) TrCl, Pyridine (90%); (v) (C2H5O)2P(O)±CH3, n-BuLi, Toluene/THF (85%); (vi) NaH, 40 ꢀC (75%); (vii) NaBH4, CeCl3,
Methanol (99%).

D. Durand et al. / Bioorg. Med. Chem. Lett. 10 (2000) 811±814
813
Scheme 2. Reagents and conditions (isolated yield): (viii) HCl 1N, THF (98%); (ix) NaNO2, NaN3, MeOH, 0 ꢀC (95%); (x) NaI, Chloramine T,
AcOH:H2O 9:1 (55%); (xi) Ethyl acetate, LDA, THF, 78 ꢀC (62 to 70%); (xii) N,N-dimethylacetamide, LDA, THF, 60 ꢀC (90%).
further decrease of the anity. This decrease is sig-
ni®cantly marked for the amide series 6a±d. The ®rst
eluted enantiomers from racemates 4b and 6b by chiral
HPLC (4bꢀ and 6bꢀ) resulted, as expected, in higher
anities than the racemic 4b and 6b. The other enan-
tiomers (4bꢁ and 6bꢁ) only demonstrated a very weak
anity for the receptor.
The enantiomer 4bꢀ was then used to de®ne the ligand
binding site of h-BLTR. This receptor has been recently
produced in our laboratory as a recombinant protein in
Escherichia coli and isolated as a micelle-solubilized
protein. The latter was characterized by circular
dichroism thus a€ording 45±50% of helical residues in
agreement with a protein including seven transmem-
brane helices (labelled TM-I to TM-VII). Photolysis23
of 4bꢀ in the presence of h-BLTR at 1:1 molar ratio
resulted in covalent cross-linking of about 40% of the
photoactivable antagonist to the receptor, as established
by electrospray mass spectrometry based on measure-
ments with the reaction mixture of the photoadduct and
the unreacted protein. Analysis combining amino acid
sequencing and mass spectrometry of the generated
h-BLTR/4bꢀ photolabelled peptides, after trypsin pro-
teolysis, establishes that cross-linking occurs at the level
of speci®c residues in two of the seven putative trans-
membrane helices, namely Cys 97, Ser 100, Met 101 and
Ser 104 in TM-III, as well as Trp 234 and Tyr 237 in
TM-VI. Our preliminary results with the isolated recep-
tor thus establish that compound 4bꢀ is a good candi-
date for mapping the ligand binding site of the LTB4
membrane receptor (full details to be published else-
where). The iodinated compound 6bꢀ, although dis-
playing a reduced anity (Table 1), is intended as a
radiolabelled probe to investigate the potential hetero-
geneity of the LTB4 receptor.
Table 1. Binding of the synthetic competitors to the LTB4 receptor in
human neutrophils
Compound
R
R2
IC50 (mM)a
1bꢀ
2bꢀ
3b
4b
4bꢀ
5b
OHb
N(CH3)2
OHb
N(CH3)2
N(CH3)2
OHb
N(CH3)2
N(CH3)2
(CH2)4Ph
(CH2)4Ph
0.315
0.0415
2
2
0.7
(CH2)4PhN3
(CH2)4PhN3
(CH2)4PhN3
(CH2)4PhN3I
(CH2)4PhN3I
(CH2)4PhN3I
>10
>10
6
6b
6bꢀ
aIC50 are extrapolated from mean competition curves obtained from at
least three di€erent experiments.
bTested as sodium salt.

814
D. Durand et al. / Bioorg. Med. Chem. Lett. 10 (2000) 811±814
isopropanol (70:30) a€orded successively the enantiomers 4bꢀ
References and Notes
(23 min) and 4bꢁ (33 min).
1. Ford-Hutchinson, A. W.; Bray, M.; Doig, M.; Schipley, M.;
Smith, M. J. Nature 1980, 286, 264.
2. Goldman, D. W.; Gi€ord, L. A.; Marotti, T.; Koo, C. H.;
Goetzl, E. J. Fed. Proc. 1987, 46, 200.
19. Compound 4bꢀ: (1RÃ,3RÃ)-1-Hydroxy-3-((3RÃSÃ, E)-3-
hydroxy-7-(4-azidophenyl)hept-1-en-1-yl)cyclohexane-1-N,N-
dimethyl acetamide: 1H NMR (CDCl3, 360 MHz): 0.90 (m,
0
0
0
0
0
0
1H, H4 a); 0.94 (t, 1H, H2 a J2 ,3 =12.6 Hz); 1.11 (m, 1H, H6 a);
00 00 0
1.33 (m, 2H, H5 ); 1.39 (m, 2H, H4 ); 1.54 (m, 1H, H5 e); 1.57
3. Palmblad, J.; Malmster, C.; Uden, A.; Radmark, O.;
Engstedt, L.; Samuelson, B. Blood 1981, 58, 658.
4. Showell, H. J.; Naccache, P. H.; Borgeat, P.; Picard, S.;
Vallerand, P.; Becher, E. L.; Sha'a®, R. I. J. Immunol. 1982,
128, 811.
00
0
0
0
0
0
0
0
(m, 2H, H6 ); 1.64 a 1.87 (m, 4H, H4 e H5 a H6 e H2 e); 2.35 (s,
0
2H, H2); 2.46 (m, 1H, H3 ); 2.56 (t, 2H, H7 , J7 ,6 =10.2 Hz);
00
00 00
00
2.94 (s, 3H, Me); 2.98 (s, 3H, Me); 3.97 (q, 1H, H3 , J3 ,2
00 00
=
00 00
J3 ,4 =6.5 Hz); 5.14 (s, 1H, OH); 5.40 (q, 1H, H2 , J2 ,1 =15.7
00
00 00
00 00
00
00 00
00
Hz, J2 ,3 =6.5 Hz); 5.52 (q, 1H, H1 , J1 ,2 =15.6 Hz, J1 ,3 =
0
5. Kragballe, K.; Voorhees, J. Acta Derm. Venereol. 1985,
(Suppl. 120), 12.
6. Stenson, D. W. J. Gastroenterol. 1990, 25 (Suppl. 172), 13.
7. Davidson, E. M.; Rae, S. A.; Smith, M. J. H. Annu. Rheum.
Dis. 1983, 43, 677.
00
6.5 Hz); 6.92 (d, 2H, H9 , J9 ,10 =8.6 Hz); 7.13 (d, 2H, H10
00 00 0
J10 ,9 =8.6 Hz). C NMR (CDCl3, 90 MHz): 20.8 (C5 ), 24.9
00
(C5 ), 31.3 (C6 ), 32.2 (C4 ), 35.0 (C3 ), 35.0 (N-CH3), 35.2
0
(C7 ), 37.1 (C4 ), 37.2 (C6 ), 37.2 (N-CH3), 43.5 (C2), 43.5 (C2 ),
00
00
00
,
13
00
0
0
00
00
0
0 00 00 00 00
69.9 (C1 ), 73.0 (C3 ), 118.8 (2C10 ), 129.7 (2C9 ), 130.1 (C2 ),
8. Rae, S. A.; Davidson, E. M.; Smith, M. J. H. Lancet 1982,
2, 1122.
9. Wardlaw, J. J.; Hay, H.; Cromwell, O.; Collins, J. V.; Kay,
A. B. J. Allergy Clin. Immunol. 1989, 84, 12.
10. Cromwell, O.; Walport, M. J.; Morris, H. R.; Taylor,
G. W.; Hodson, M. E.; Batten, J.; Kay, A. B. Lancet 1981, 2,
164.
11. Antonelli, M.; Bu, M.; De Blasi, R. A.; Crimi, G.; Conti,
G.; Mattia, C.; Vivino, G.; Lenti, L.; Lombardi, D.; Dotta, A.
Intensive Care Med. 1989, 15, 296.
12. Katsura, K.; Minamisawa, H.; Katayama, Y.; Shimizu, J.;
Goto, T.; Urushiyama, K.; Terashi, A.; Kanda, Y.; Yoshino,
Y. Prostaglandins 1988, 36, 655.
13. Yokomizo, T.; Izumi, I.; Chang, K.; Takuwa, Y.; Shimizu,
T. Nature 1997, 387, 620.
14. Alexander, S. P. H.; Peters, J. A. Trends Pharmacol. Sci.
(Ion Channel Nomenclature Supplement) 1999, 53.
15. Poudrel, J. M.; Hullot, P.; Vidal, J. P.; Girard, J. P.; Rossi,
J. C.; Muller, A.; Bonne, C. J. Med. Chem. 1999, 42, 5289.
16. For review: Bayley, H. Photogenerated Reagents in Bio-
chemistry and Molecular Biology; Elsevier: New York, 1983.
17. Gemal, A. L.; Luche, J. L. J. Am. Chem. Soc. 1981, 103,
5454.
18. (a) Column used: Licrospher1 5 mm, 250Â10 mm. UV
detection at 250 nm. Elution (6 mL/min) with cyclohexane:
ethyl acetate (25:75) a€orded successively the diastereoisomers
4a (22 min), 4b (30 min), 4c (36 min) and 4d (45 min); (b)
Column used: Chiralcel1 OD (Daicel), 5 mm, 250Â10 mm. UV
detection at 250 nm. Elution (1.35 mL/min) with heptane:
00
00
00
137.3 (C1 ), 138.3 (C11 ), 139.4 (C8 ), 182.1 (C1). Mass spec-
trometry (FAB+; GT; PM=414): m/z=437 (M+Na+; 10);
415 (M+H+; 30); 397 (M-H2O+H+; 20); 307 (M-2H2O-
CH2CONMe; 15); 91 (PhCH+2 ; 35); 72 (CONMe2+, 55). UV
lmax (EtOH)nm (E): 250 (13600).
20. Neutrophil isolation: Polymorphonuclear leukocytes
(PMN) were puri®ed from freshly drawn human blood by
standard techniques using Dextran T500 sedimentation and
centrifugation on Ficoll/Paque (Pharmacia) followed by
hypotonic lysis of erythrocytes. The puri®ed PMN were
washed in HBSS (Hank's balanced salt solution, Sigma) without
Ca++ and Mg++ and cell viability was assessed by trypan
blue exclusion. [3H] LTB4 binding assays: Binding to PMN
was performed in HBSS without Ca++ and Mg++ containing
5 mmol/lꢀHEPES bu€er. PMN (106 cells) were incubated 20
min at 4 C with 1 nmol/L [3H] LTB4 (7.4 TBq/mmol from
Amersham) in the presence or absence of competitors at vari-
ous concentrations. Binding was determined by the ®ltration
technique previously described.22
21. Lin, A. H.; Ruppel, P. L.; Gorman, R. R. Prostaglandins
1984, 28, 837±849.
22. Muller, A.; Ghiglieri-Bertez, C.; Modat, G.; Bonne, C.
Prostaglandin Leukotriene Med. 1987, 26, 233.
23. Photolysis experiment: A mixture of h-BLTR and 4bꢀ at
1 mM each was incubated at 4 ꢀC for 30 min and then irra-
diated for 20 min at 4 ꢀC using a 150-watt ultraviolet lamp
(Heraeus TQ 150 Z3) with a 3 mm thick glass plate as a ®lter
(active emission spectrum: l>315 nm).