Syntheses and preliminary pharmacological evaluation of the two epimers of the 5-F2t-isoprostane

Article abstract of DOI:10.1016/S0960-894X(01)00473-5

The total synthesis of the 5-F_2t-isoprostane 1 and its 5-epimer 2 from diacetone-D-glucose is described. We report preliminary data on the vascular properties of these compounds.

Full text of DOI:10.1016/S0960-894X(01)00473-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2495–2498
Syntheses and Preliminary Pharmacological Evaluation of the
Two Epimers of the 5-F2t-isoprostane
Thierry Durand,a,* Jean-Luc Cracowski,b Alexandre Guya and Jean-Claude Rossia
a
´
´
UMR C.N.R.S. 5074, Universite Montpellier 1, Faculte de Pharmacie, 15 Av. C. Flahault, F-34060 Montpellier Cedex 2, France
b
´
Laboratoire de Pharmacologie, Faculte de Medecine de Grenoble, 38706 La Tronche Cedex, France
´
Received 13 April 2001; revised 15 June 2001; accepted 4 July 2001
Abstract—The total synthesis of the 5-F2t-isoprostane 1 and its 5-epimer 2 from diacetone-d-glucose is described. We report pre-
liminary data on the vascular properties of these compounds. # 2001 Elsevier Science Ltd. All rights reserved.
The isoprostanes are a family of compounds produced
in vivo by a free radical peroxidation of arachidonic
acid. Depending on which of the labile hydrogen atoms
is first abstracted by free radicals, three initial arachi-
donyl radicals can be formed (Fig. 1), leading to the
four prostaglandin H2-regioisomers. These four com-
pounds can then be fully reduced to form four
prostaglandin F2a regioisomers (5-, 8-, 12-, 15-series).
We report preliminary data on the vascular properties
of these compounds.
Chemistry
The synthesis of 5-F2t-isoprostane 1 and its 5(R)-epimer
2, from the commercially available diacetone-d-glucose
as starting material, is shown in Schemes 2 and 3. The
first nine steps leading to cyclopentane alkoxyester 3
were achieved in 27% overall yield by using iodo path-
way, according to our procedure.7
The first demonstration that 15 F2t-isoprostanes were
produced in vivo was made by Morrow in 1990.1 Since
that time, 15-F2t-isoprostanes have been extensively
quantified as markers of lipid peroxidation in vivo.2 In
addition, 15-F2t-isoprostanes have been shown to pos-
sess potent biological activity including vasoconstriction
and platelet aggregation,3 which may be implicated in
the pathophysiology of cardiovascular diseases. Aside
from 15-F2t-isoprostanes, 5-F2t-isoprostanes have
recently been synthesised.4 Indeed, 5-F2t-isoprostanes
appear to be the most abundant identifiable iso-
prostanes in vivo.5 However, since these regioisomers
are not widely available, their quantification remains
limited to a few research groups. Furthermore, these
compounds are not available to study their potential
biological effects. As a consequence, and in connection
with our program directed towards the synthesis of iso-
prostanes,6 we synthesised the 5-F2t-isoprostane 1 and
its 5(R)-epimer 2 from the alkoxyester 37 (Scheme 1) in
order to enable the study of their biological properties.
The alkoxyester 3 was converted into the aldehyde 4 by
treatment with DIBAL-H in anhydrous toluene with
93% yield (Scheme 2). The introduction of the o chain
of the isoprostane was achieved by using the commer-
cial hexyltriphenyl phosphonium bromide. The alde-
hyde 4 reacted with the ylide derived from this
phosphonium salt and sodium hexamethyldisilyl amide
as a base, in anhydrous THF at ꢀ80 ꢁC, to afford the
pure (Z) enic ether 5 in 89% yield.8 No trace of trans
1
compound could be detected by 13C and H NMR ana-
lysis. All the relative configurations were checked by
1
homonuclear H NOE experiments.
Deprotection of the triethylsilyl groups with ammonium
fluoride in a mixture of MeOH/THF (2:1) at 60 ꢁC gave
the diol 6 in 92% yield. The protection of the hydroxy
functions of 6 with benzoyl chloride in dry pyridine
gave the colourless diester 7 in 97% yield.
The tert-butyldiphenylsilyl ether 7 was converted into
the alcohol 8 with a solution of 3% hydrogen chloride9
*Corresponding author. Fax: +33-4-67-54-86-25; e-mail: froxy@
pharma.univ-montp1.fr
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00473-5

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T. Durand et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2495–2498
Figure 1.
Scheme 1.
in methanol/diethyl ether (1:1, v/v), prepared freshly
from acetyl chloride and methanol, a method which
proved to be much milder and to give a higher yield
(83%) than TBAF in THF. Dess–Martin oxidation10 of
8 in CH2Cl2 gave the unstable aldehyde 9 which was
immediately used in the next stepwithout purification.
It is important to note that this Dess–Martin oxidation
gave higher yield and avoided any epimerization than
our first attempts using Swern conditions. The con-
densation of 9 with diethyl [5-(methoxycarbonyl)-2-
oxopentyl]phosphonate,11 in the presence of sodium
hexamethyldisilyl amide, in anhydrous THF at room
temperature, afforded the trans-a,b-enone ester 10 in
75% overall yield from the alcohol 7 (Scheme 2).
Scheme 2. (a) 1.1 equiv DIBAL-H (1 M in toluene), toluene, ꢀ80 ꢁC,
30 min, 93%; (b) 2.25 equiv hexyltriphenylphosphonium bromide, 2.2
equiv NaN(SiMe3)2, THF, ꢀ80 ꢁC to 20 ꢁC, 2 h, 89%; (c) 4 equiv
NH4F, THF–MeOH, 4 h, 92%; (d) 3 eq. BzCl, pyridine, 20 ꢁC, 1 h,
97%; (e) HCl 3% in MeOH, 20 ꢁC, overnight, 83%; (f) periodinane,
CH2Cl2, rt, 2 h; (g) 3.3 equiv diethyl[5-(methoxycarbonyl)-oxopentyl]-
phosphonate, 3 equiv NaN(SiMe3)2, THF, 20 ꢁC, 30 min, 75%.
The diastereoselective reduction of the C5 keto groupin
10 with the chiral reducing agent12 (S)-BINAL-H pro-
ceeded smoothly and gave the desired pure 5(S) deriva-
tive 11 in 85% yield. Similarly, reduction of 10 using the
(R)-BINAL-H gave the 5(R)-epimer 12 in 87% yield
(Scheme 3).
Rat Aorta Preparation
In accordance with the local ethical committee guide-
lines for animal research, male Wistar rats (270–410 g,
IFFA CREDO, Lyon, France) were housed in climate
controlled conditions and provided standard rat chow.
Animals were anaesthetised with sodium pentobarbital
Finally, deprotection of the benzoyl groups in 11 and
12, with 1 N NaOH at room temperature, followed by
excess of CH2N2, afforded the desired 5-F2t-isoprostane
1
13 and its 5-epimer 214 in 93 and 90% yields, respectively.

T. Durand et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2495–2498
2497
maximal contraction (EC50) was determined from each
curve by a logistic, curve-fitting equation. EC50 values
were expressed as pD2 (ꢀlog EC50). Results are expres-
sed as meanꢂSEM.
Results and Discussion
U46619 induced a vasoconstriction in a concentration-
dependent manner (pD2: 7.8ꢂ0.1, Emax: 189ꢂ31%). In
contrast, 5-F2t-IsoP and 5-epi-5-F2t-IsoP had no effect
upto 10 ꢀ5 M (n=4).
5-F2t-IsoP is more abundant than 15-F2t-IsoP in biolo-
gical fluids in humans.5 Both compounds are quantifi-
able in vivo, and currently used as clinical markers of
lipid peroxidation in cardiovascular diseases.3 However,
it remains undetermined whether the generation of spe-
cific F2-isoprostane regioisomers differs in clinical
situations of increased oxidative stress. Our preliminary
observations contrast with data of the 15-series. 15-F2t-
IsoP has been shown to be a vasoconstrictor with a
potency equivalent to prostaglandin F2a, but lower than
U46619.3 Our data suggest that, unlike the 15-series, the
5-series does not possess vascular activity. The 5-series
is therefore unlikely to contribute to the pathophysiol-
ogy of cardiovascular diseases, which could be the
major difference between both regioisomers. Such data
need to be implemented. The chemical syntheses we
described will enable a full pharmacological character-
isation of the effects (or the lack of effect) of the 5-F2t-
isoprostanes in comparison with 15-F2t-isoprostanes
and helpto better understand the hparmacological
differences between F2-isoprostane regioisomers.
Scheme 3. (a) (S)-BINAL-H, ꢀ100 ꢁC, 2 h, 85%; (b) (R)-BINAL-H,
ꢀ100 ꢁC, 2 h, 87%; (c) 1 N NaOH, THF–MeOH, rt, 1 h, then CH2N2,
93%.
(Sanofi, Libourne, France, 60 mg kgꢀ1 intraperitoneal).
Heparin (150 IU, Sanofi Winthrop, Gentilly, France)
was injected intravenously. Then, the thoracic aorta was
quickly excised, cleaned of connective tissue and cut
into 4-mm lengths. Four rings were taken from each
thoracic aorta.
Measurement of isometric tension in rings of rat thoracic
aorta
The methods used for the measurement of isometric
tension in rings of rat thoracic aorta were as previously
reported.15 All the dose–response curves were made by
adding increasing concentrations of the agonist to the
organ bath in 0.5-log unit steps. The contractile effects
of 5-F2t-IsoP and 5-epi-5-F2t-IsoP were studied in com-
parison with U46619, a potent thromboxane A2/pros-
taglandin H2 receptor agonist. Concentration–
contraction curves were expressed as percentages of KCl
(90 mM)-induced contraction.
References and Notes
1. Morrow, J. D.; Hill, K. E.; Burk, R. F.; Nammour, T. M.;
Badr, K. F.; Roberts, L. J., II. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 9383.
2. Roberts, L. J., II; Morrow, J. D. Free Radic. Biol. Med.
2000, 28, 505.
3. Cracowski, J. L.; Devillier, P.; Durand, T.; Stanke-Labesque,
F.; Bessard, G. J. Vasc. Res. 2001, 38, 93.
4. Adiyaman, M.; Lawson, J. A.; Hwang, S. W.; Khanapure,
S. P.; FitzGerald, G. A.; Rokach, J. Tetrahedron Lett. 1996,
37, 4849.
5. Li, H.; Lawson, J. A.; Reilly, M.; Adiyaman, M.; Hwang,
S. W.; Rokach, J.; FitzGerald, G. A. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 13381.
6. Durand, T.; Guy, A.; Henry, O.; Vidal, J. P.; Rossi, J. C.
Eur. J. Org. Chem. 2001, 4, 809 and references therein.
7. Roland, A.; Durand, T.; Egron, D.; Vidal, J. P.; Rossi, J. C.
J. Chem. Soc., Perkin Trans. 2 2000, 245 and references therein.
8. Compound 5: 1H NMR (360 MHz, CDCl3) d ppm 7.64–
7.61 (m,4H, HAr); 7.43–7.24 (m, 6H, HAr); 5.33–5.29 (m, 2H,
H8 and H9); 4.09–4.05 (m, 1H, H4); 3.95–3.89 (m, 1H, H1);
3.59 (d, J=5.1 Hz, 2H, H6); 2.33 (dt, J=6.3 and 13.6 Hz, 1H,
H5); 2.20–1.87 (m, 6H, H2, H3, H7 and H10); 1.55–1.43 (m,
1H, H50); 1.31–1.18 (m, 6H, H11, H12 and H13); 1.05 (s, 9H,
HtBu); 0.93 (t, J=7.9 Hz, 9H, SiCH2CH3); 0.87–0.83 (m, 12H,
H14 and SiCH2CH3); 0.56 (q, J=7.9 Hz, 6H, SiCH2CH3);
0.46 (q, J=7.9 Hz, 6H, SiCH2CH3). 13C NMR (90 MHz,
CDCl3) d ppm 135.69 (CAr); 133.54 (CAr); 130.38 (C9); 129.59
Drugs
U46619
(9,11-dideoxy-9a,11a-methanoepoxy-prosta-
glandin F2a), l-phenylephrine, and acetylcholine were
purchased from Sigma (Saint Quentin Fallavier,
France).
Hydrolysis of methyl ester
The basic hydrolysis of methyl esters of 1 and 2 with
aqueous lithium hydroxide in THF at room temperature
yielded the desired free acids in 92% yields.
Data analysis
Maximal contraction (Emax) and potency (pD2) were
calculated to determine the arterial segment reactivity.
The effective concentration of agent that caused 50% of

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T. Durand et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2495–2498
(CAr); 128.59 (C8); 127.59 (CAr); 76.41 (C1); 73.26 (C4); 62.64
(C6); 50.68(C3); 48.29 (C2); 45.14 (C5); 31.50 (C12); 29.30
(C11); 27.31 (C10); 26.92 (CtBu); 25.59 (C7); 22.56 (C13); 19.15
(C(CH3)3); 14.04 (C14); 6.83 (SiCH2CH3); 6.76 (SiCH2CH3);
4.93 (SiCH2CH3); 4.46 (SiCH2CH3). FAB-MS m/z 709 (M+.
+1). Anal. calcd for C42H72O3Si3: C, 71.12; H, 10.23. Found:
C, 71.07; H, 10.53.
(C7); 126.40 (C15); 124.00 (C6); 122.55 (C14); 70.61 (C1 and
C4); 67.01 (C8); 48.36 (C3); 46.32 (OCH3); 45.61 (C2); 37.15
(C5); 31.32 (C9); 28.43 (C11); 26.28 (C18); 24.04 (C17); 22.18
(C13); 21.83 (C16); 17.30 (C19); 15.53 (C10); 8.78 (C20). FAB-
MS m/z 369 (M+ꢃ +1). Anal. calcd for C21H36O5: C, 68.44; H,
9.85. Found: C, 68.38; H, 9.73. IR (NaCl) n: 3520, 1720.
14. Compound 2: UV (ethanol) lmax: 203 nm. 1H NMR
(360 MHz, CDCl3) d ppm 5.61–5.33 (m, 4H, H6, H7, H14 and
H15); 4.09–3.95 (m, 3H, H1, H4 and H8); 3.66 (s, 3H, OCH3);
2.78–2.74 (m, 1H, H3); 2.41 (dt, J=6.1, 14.1 Hz, 1H, H5); 2.33
(t, J=7.2 Hz, 2H, H11); 2.18–2.05 (m, 1H, H2); 2.01–1.93 (m,
4H, H13 and H16); 1.71–1.51 (m, 5H, H50, H9 and H10);
1.43–1.25 (m, 6H, H17, H18 and H19); 0.87 (t, J=6.5 Hz, 3H,
H20). 13C NMR (90 MHz, CDCl3) d ppm 174.06 (C12); 135.64
(C7); 131.60 (C15); 129.60 (C6); 127.75 (C14); 76.36 (C1 and
C4); 72.32 (C8); 53.57 (C3); 51.55 (OCH3); 50.94 (C2); 42.28
(C5); 36.56 (C9); 33.71 (C11); 31.51 (C18); 29.27 (C17); 27.39
(C13); 27.00 (C16); 22.53 (C19); 20.72 (C10); 14.02 (C20). FAB-
MS m/z 369 (M+. +1). Anal. calcd for C21H36O5: C, 68.44; H,
9.85. Found: C, 68.54; H, 9.87. IR (NaCl) n: 3520, 1720.
9. Nashed, E. M.; Glaudemans, C. P. J. J. Org. Chem. 1987,
52, 5255.
10. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
Ireland, R. E.; Liu, L. J. Org. Chem. 1983, 58, 2899.
11. Delamarche, I.; Mosset, P. J. Org. Chem. 1994, 59, 5453.
12. Noyori, R.; Tomino, I.; Tanimoto, Y.; Nishizawa, M. J.
Am. Chem. Soc. 1984, 106, 6709.
13. Compound 1: UV (ethanol) lmax: 203 nm. 1H NMR
(360 MHz, CDCl3) d ppm 5.62–5.34 (m, 4H, H6, H7, H14 and
H15); 4.11–3.95 (m, 3H, H1, H4 and H8); 3.65 (s, 3H, OCH3);
2.80–2.75 (m, 1H, H3); 2.41 (dt, J=6.2, 14.4 Hz, 1H, H5); 2.33
(t, J=7.1 Hz, 2H, H11); 2.19–2.08 (m, 1H, H2); 2.02–1.96 (m,
4H, H13 and H16); 1.69–1.51 (m, 5H, H50, H9 and H10);
1.41–1.24 (m, 6H, H17, H18 and H19); 0.87 (t, J=6.7 Hz, 3H,
H20). 13C NMR (90 MHz, CDCl3) d ppm 170.06 (C12); 130.39
15. Stanke-Labesque, F.; Devillier, P.; Veitl, S.; Caron, F.;
Cracowski, J. L.; Bessard, G. Cardiovasc. Res. 2001, 49, 152.