5-F2t-isoprostane, a human hormone?

Article abstract of DOI:10.1021/ja990859k

Syntheses of the four enantiomerically pure diastereomers of 5-F_2t-isoprostane (5-8) are described. The key step is the lipase-catalyzed chemo-enzymatic resolution of the racemic diol 40 to give the mono-acetates 41 and 42. The enantiomerically

Full text of DOI:10.1021/ja990859k

J. Am. Chem. Soc. 1999, 121, 7773-7777
7773
5-F2t-Isoprostane, A Human Hormone?
Douglass F. Taber,* Kazuo Kanai, and Richard Pina
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Delaware,
Newark, Delaware 19716
ReceiVed March 17, 1999
Abstract: Syntheses of the four enantiomerically pure diastereomers of 5-F2t-isoprostane (5-8) are described.
The key step is the lipase-catalyzed chemo-enzymatic resolution of the racemic diol 40 to give the mono-
acetates 41 and 42. The enantiomerically pure diastereomers of 5-F2t-isoprostane (5) may be human hormones.
Introduction
In 1990, prostaglandin (PG) F2-like compounds were dis-
covered to be produced in abundance in vivo by free radical-
induced peroxidation of arachidonic acid (1), independent of
the cyclooxygenase enzymes.1 Because these compounds are
isomeric to the PGF2R derived by the action of cyclooxygenase,
they were named F2-isoprostanes.2 Subsequently, it was dem-
onstrated that D2-isoprostanes and E2-isoprostanes are also
produced in vivo as products of this pathway.3 Four different
regioisomers of each of these classes of isoprostanes are formed
(e.g., 8-F2t-isoprostane 2, 12-F2t-isoprostane 3, 15-F2t-isoprostane
4, 5-F2t-isoprostane 5). 15-F2t-Isoprostane (4), prepared by total
synthesis,4-11 has been shown to have hormonal activity, with
a receptor in the kidney vasculature.12 To investigate the
biological activity4 of the other isoprostanes, it will be necessary
to devise synthetic routes to them. We report herein the first
preparation of each of the four enantiomerically pure isomers
of 5-F2t-isoprostane (5-8).
(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) (a) For a summary of isoprostane nomenclature, see Taber, D. F.;
Morrow, J. D.; Roberts, L. J., II. Prostaglandins 1997, 53, 63. (b) For an
alternative nomenclature system for the isoprostanes, see Rokach, J.;
Khanapure, S. P.; Hwang, S. W.; Adiyaman, M.; Lawson, J. A.; FitzGerald,
G. A. Prostaglandins 1997, 54, 853.
(3) Morrow, J. D.; Minton, T. A.; Mukundan, C. R.; Campbell, M. D.;
Zackert, W. E.; Daniel, V. C.; Badr, K. F.; Blair, I. A.; Roberts, L. J., II.
J. Biol. Chem. 1994, 269, 4317.
(4) Morrow, J. D.; Roberts, L. J., II. Biochem. Pharmacol. 1996, 51, 1.
(5) For synthetic routes to 15-F2t-isoprostane: (a) Corey, E. J.; Shih,
C.; Shih. N.-Y.; Shimoji, K. Tetrahedron Lett. 1984, 25, 5013. (b) Hwang,
S. W.; Adiyaman, M.; Khanapure, S.; Schio, L.; Rokach, J. J. Am. Chem.
Soc. 1994, 116, 10829. (c) Taber, D. F.; Herr, R. J.; Gleave, D. M. J. Org.
Chem. 1997, 62, 194. (d) Taber, D. F.; Kanai, K. Tetrahedron 1998, 54,
11767.
(6) For synthetic routes to 15-F2c-isoprostane: (a) Larock, R. C.; Lee,
N. H. J. Am. Chem. Soc. 1991, 113, 7815. (b) Vionnet, J.-P.; Renaud, P.
HelV. Chim. Acta 1994, 77, 1781. (c) Hwang, S. W.; Adiyaman, M.;
Khanapure, S. P.; Rokach, J. Tetrahedron Lett. 1996, 37, 779.
(7) (a) For a synthetic route to 15-E2t-isoprostane: Taber, D. F.; Hoerrner,
R. S. J. Org. Chem. 1992, 57, 441. (b) See also ref 5d.
Results and Discussion
The 5-isoprostanes are produced in vivo as racemic mixtures
of C-5 diastereomers. Rather than design an independent
synthesis of each of the four enantiomerically pure diastereomers
of a particular isoprostane, it seemed more sensible to develop
a stereodivergent synthesis that would lead to each of the four
from a common intermediate.
We proposed (Scheme 1) to prepare 5-F2t-isoprostane (5),
5-epi-5-F2t-isoprostane (6), ent-5-F2t-isoprostane (7), and 5-epi-
ent-5-F2t-isoprostane (8) by aldol condensation of the diazo-
ketone 9 with the aldehyde 10, followed by cyclization of
the silyloxy ketone 11. The relative configuration of the alkyl
side chains on the ring would then be established by kinetic
opening of the activated cyclopropane of the bicyclic ketone
12 with thiophenol and BF3‚OEt2. A key question was whether
the cyclization of the diazoketone 11, having a trans,cis
conjugated side chain, would give the desired bicyclic ketone
12 efficiently.
(8) For a synthetic route to 5-F2t-isoprostane: Adiyaman, M.; Lawson,
J. A.; Hwang, S.-W.; Khanapure, S. P.; FitzGerald, G. A.; Rokach, J.
Tetrahedron Lett. 1996, 37, 4849.
(9) For a synthetic route to 5-F2c-isoprostane: Adiyaman, M.; Lawson,
J. A.; FitzGerald, G. A.; Rokach, J. Tetrahedron Lett. 1998, 39, 7039.
(10) For a synthetic route to 8-F2t-isoprostane: Adiyaman, M.; Li, H.;
Lawason, J. A.; Hwang, S.-W.; Khanapure, S. P.; FitzGerald, G. A.; Rokach,
J. Tetrahedron Lett. 1997, 38, 3339.
(11) For a synthetic route to 12-F2t-isoprostane: Pudukulathan, Z.;
Manna, S.; Hwang, S.-W.; Khanapure, S. P.; Lawson, J. A.; FitzGerald, G.
A.; Rokach, J. J. Am. Chem. Soc. 1998, 120, 11953.
(12) (a) Morrow, J. D.; Minton, T. A.; Roberts, L. J., II. Prostaglandins
1992, 44, 155. (b) Fukunaga, M.; Makita, N.; Roberts, L. J., II.; Morrow,
J. D.; Takahashi, K.; Badr, K. F. Am. J. Physiol (Cell Physiol. 33) 1993,
264, C1619. (c) Takahashi, K.; Nammour, T. M.; Fukunaga, M.; Ebert, J.;
Morrow, J. D.; Roberts, L. J., II; Hoover, R. L.; Badr, F. K. J. Clin. InVest.
1992, 990, 136. (d) Longmire, A. W.; Roberts, L. J., II.; Morrow, J. D.
Prostaglandins 1994, 48, 247. (e) Fukunaga, M.; Takahashi, K.; Badr, K.
F. Biochem. Biophys. Res. Commun. 1993, 195, 507.
10.1021/ja990859k CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/13/1999

7774 J. Am. Chem. Soc., Vol. 121, No. 34, 1999
Taber et al.
Scheme 2a
Scheme 1
a Reagents and conditions: (a) C4H9MgBr, Et2O, 0 °C ∼ rt; (b) CBr4,
Ph3P, CH2Cl2, 0 °C ∼rt; (c) benzoylacetone, K2CO3, nBu4NBr, toluene,
90 f 40 °C; (d) p-NBSA, DBU, CH2Cl2, 0 °C.
Scheme 3a
a Reagents and conditions: (a) KHMDS; ethyl 5-oxopentanoate,
THF, -78 ∼0 °C; (b) MnO2, CH2Cl2, rt.
Table 1. 13C Chemical Shifts of C-4 and C-9 Methylenes
The diazoketone 9 was prepared (Scheme 2) by homologa-
tion13 of the chloro alcohol 14 with butylmagnesium bromide,
to give the alcohol 15. Alkylation of benzoylacetone with the
derived bromide 16 gave the diketone 17. Diketone 17 was
smoothly converted to the diazoketone 9 on exposure to
p-nitrobenzenesulfonyl azide (p-NBSA) and DBU.14
The requisite (Z,E)-conjugated dienal 10 (Scheme 3) was
prepared by a modification of the method of Kobayashi.15 Wittig
reaction of the phosphonium salt 18 and ethyl 5-oxopentanoate16
with one molar equiValent of KHMDS proceeded smoothly to
give dienol 19 (Z,E/E,E ) >95:<5). Oxidation of 19 with MnO2
furnished the dienal 10.17
To be sure of the geometry of 19, we also prepared the
isomeric dienols 20-22 (Table 1). Dienol 20 was prepared
by oxidation of 19 with PCC followed by reduction with
NaBH4. A inseparable mixture of 21 and 22 was prepared
by condensation of the phosphonium salt derived from 14
with ethyl 4-oxopentanoate. The geometry of the conjugated
dienes was easily assigned by comparison of the 13C NMR
chemical shifts of the C-4 and C-9 methylenes.18 The 13C
chemical shift of the C-4 methylenes of compounds 20 and
22, having an (E)-double bond next to the C-4 methylene,
are downfield compared to the (Z)-isomers 19 and 21. The
13C chemical shifts for the C-9 methylenes of compounds
a In CDCl3.
19 and 20, having an (E)-double bond next to the C-9 meth-
ylene, are also downfield compared to the (Z)-isomers 21 and
22.
With the requisite components 9 and 10 in hand, we embarked
on the preparation of the four enantiomerically pure diastere-
omers of 5-F2t-isoprostane (Scheme 4). Aldol condensation5d
of the potassium enolate of the diazoketone 9 with the dienal
10 in the presence of triethylchlorosilane (TESCl) in toluene
gave the TES-protected aldol 23 together with a small amount
of the free aldol 24 (hydrolysis on work up) in good yield. The
TES group of 23 does not survive under the conditions for
cyclopropane ring opening with thiophenol and BF3‚OEt2, so it
was necessary to change the protecting group from TES to tert-
butyldiphenylsilyl (TBDPS). The diazoketone 11 was then
(13) Taber, D. F.; Louey, J. P. Tetrahedron 1995, 51, 4495.
(14) Taber, D. F.; Gleave, D. M.; Herr, R. J.; Moody, K.; Hennessy, M.
J. J. Org. Chem. 1995, 60, 2283.
(15) Hosoda, A.; Taguchi, T.; Kobayashi, Y. Tetrahedron Lett. 1987,
28, 65.
(16) Ethyl 5-oxopentanoate was prepared by ethanolysis of δ-valerolac-
tone followed by PCC oxidation. For full characterization, see Penn, J. H.;
Liu, F. J. Org. Chem. 1994, 59, 2608.
(17) For a complementary approach to the methyl esters of 19 and 10,
see Pohnert, G.; Boland, W. Tetrahedron 1996, 52, 10073.
(18) For a detailed discussion of the 13C chemical shifts of allylic
methylenes, see Taber, D. F.; You, K. J. Org. Chem. 1995, 60, 139 and
references therein.

5-F2t-Isoprostane
J. Am. Chem. Soc., Vol. 121, No. 34, 1999 7775
Table 2. 13C Chemical Shifts of C-8 and C-12 Methines
Scheme 4a
a Reagents and conditions: (a) KHMDS, toluene, -78 °C; 10,
TESCl, toluene, -78 °C; (b) TBAF, NH4Cl (solid), THF, 0 °C; (c)
TBDPSCl, imidazole, 4-DMAP, CH2Cl2, rt; (d) Cu-Salen cat., toluene,
100 °C; (e) PhSH, BF3‚OEt2, CH2Cl2, -30 °C; (f) mCPBA, CH2Cl2,
-78 °C; (MeO)3P, EtOH, -78 °C ∼rt.
a References. b In CDCl3.
converting both 27 and 28 to the allylic alcohols 31 and 32. On
the basis of the data in Table 2,21 both 31 and 32 have side
chains cis to each other on the ring. These observations support
the structures of 27, 28, 29, and 30 depicted in Scheme 4. In
each case, we have assumed that the opening with thiophenol
proceeded with inversion at the reacting center, as we have
previously observed.7
Reduction of the ketone 27 produced the epimeric alcohols
37 and 38 (Scheme 5). Again, the relative configurations of 37
(1H NMR δ 4.74, dt, J ) 2.6, 6.2 Hz, 1H; 4.29, m, 1H) and 38
(1H NMR δ 4.44, dt, J ) 3.6, 6.6 Hz, 1H; 4.08, m, 1H) were
assigned by analogy to the chemical shifts of the H’s at C-9
and C-11 in the corresponding diastereomers of the 15-
isoprostane precursors (1H NMR δ 4.64, m, 1H; 4.24, m, 1H)
and (1H NMR δ 4.38, m, 1H; 3.99, m, 1H).5c,d The undesired
alcohol 37 was recycled by oxidation with Dess-Martin
periodinane,22 followed by reduction with NaBH4. Desilyla-
tion of the â-alcohol 38 with TBAF in THF afforded the diol
13.
cyclized19 with the Cu-Salen catalyst 2520 in toluene to provide
the bicyclic ketones 12 and 26.
The structures of the bicyclic ketones 12 and 26 were assigned
1
by comparing the H and 13C NMR spectra to those for the
analogous bicyclic ketones that are intermediates in the synthesis
of the 15-F2t-isoprostanes.7 In particular, the oxygenated methine
1
of 12 (13C δ 69.3, H δ 4.46, d, J ) 4.9 Hz) is almost exactly
congruent with the analogous 15-F2t-isoprostane precursor 12
(13C δ 69.6, 1H δ 4.41, d), while the oxygenated methine of 26
1
(13C δ 68.0, H δ 4.59, dt, J ) 5.1, 7.8 Hz) is quite dif-
ferent.
Difficulties were initially encountered in the kinetic cyclo-
propane ring opening of 12. We eventually found that treatment
of 12 with an excess of thiophenol and BF3‚OEt2 in CH2Cl2
(0.4 M concentration) at -30 °C for 4 h gave the desired
thioether 27 in 79% yield, accompanied by three other isomers
(28, 29, and 30). 13C NMR has proven to be an effective tool
for the assignment of the relative configuration of the isopros-
tanes (Table 2).21 The ring methines (C-8 and C-12) are
particularly distinctive. Ketones 28 and 30 could easily be
assigned on the basis of these correlations. We confirmed that
the side chains of ketone 27 were cis one to another by
At first, we attempted the chemo-enzymatic resolution23 at
this stage. Unfortunately, under the conditions we screened, the
diol 13 was not resolved efficiently. We reasoned that the Z-side
chain might be presenting too much steric bulk to be accom-
modated easily in the binding site of the lipase. We therefore
(19) With Rh2(OAc)4, Rh2(oct)4, and Rh2(piv)4, the unstable tetraene
resulting from â-hydride elimination was the major product.
(20) (a) Charles, R. G. J. Org. Chem. 1957, 22, 677. (b) Sacconi, L.;
Ciampolini, M. J. Chem. Soc. 1964, 276. (c) Corey, E. J.; Myers, A. G.
Tetrahedron Lett. 1984, 25, 3559.
(22) (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155. (b)
Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899.
(23) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic
Chemistry; Pergamon Press: Oxford, U.K., 1994; and references therein.
(21) Taber, D. F.; Kanai, K. J. Org. Chem. 1998, 63, 6607.

7776 J. Am. Chem. Soc., Vol. 121, No. 34, 1999
Taber et al.
Scheme 5a
Scheme 6a
a Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) Dess-Martin
periodinane, CH2Cl2, rt; (c) TBAF, THF, rt; (d) mCPBA, CH2Cl2, -78
°C; (MeO)3P, EtOH, -78 °C ∼rt; (e) DDQ, 1,4-dioxane - CH2Cl2,
40 °C.
decided to change the substrate of the enzyme reaction. To this
end, we effected oxidation and Mislow rearrangement24 of the
thioether 13 to give the allylic alcohol 39, which on further
treatment with DDQ5a,5d,25 in 1,4-dioxane-CH2Cl2 (1:1) afforded
the enone 40.
a Reagents and conditions: (a) Amano lipase AK, vinyl acetate, rt;
(b) NaBH4, MeOH, 0 °C, (43, 37%, 44, 44%; 46, 42%, 47, 51%); (c)
BzCl, Et3N, 4-DMAP, CH2Cl2 (45, 89%, >98% ee; 48, 99%, 91% ee).
Scheme 7a
After some exploration, we found (Scheme 6) that the pseudo-
meso enone 40 was effectively resolved by Amano lipase AK
in neat vinyl acetate, to furnish the monoacetates 41 and 42.
Reduction of the enantiomerically enriched enone 41 with
NaBH4 gave the alcohols 43 and 44, which were separable by
TLC. Racemic 44 and 47 were prepared from 13 (Scheme 5)
by acetylation followed by oxidation and Mislow rearrangement.
After separation of 43 and 44 by silica gel chromatography,
alcohol 44 was converted to the dibenzoate 45. The dibenzoate
48 was also prepared in the same way. The ee’s of the
dibenzoates 45 and 48 were determined to be >98% and 91%,
respectively, by chiral HPLC analysis.26
To investigate the biological activity of the isoprostanes, it
is necessary to prepare each of these in high enantiomeric excess.
Therefore, the enantiomerically enriched acetate 42 (only 91%
ee), after conversion (Scheme 7) to the diol 40a by reduction
of the enone, hydrolysis of the acetyl group, and oxidation of
the allylic alcohol with DDQ,5a,5d,25 was again subjected to the
enzymatic resolution23 to give the monoacetate 42a. Reduction
of the enone 42a with NaBH4 gave the epimeric alcohols 46a
and 47a. Diol 47a was converted to the dibenzoate 48a, the ee
of which was determined to be >99% by chiral HPLC
analysis.26
a Reagents and conditions: (a) K2CO3, EtOAc, MS 4A, EtOH, 50
°C; (b) DDQ, 1,4-dioxane - CH2Cl2, 40 °C; (c) amano lipase AK,
vinyl acetate, rt; (d) NaBH4, MeOH, 0 °C; (e) BzCl, Et3N, 4-DMAP,
CH2Cl2, rt.
defined dibenzoate 45b (Scheme 8). This dibenzoate 45b was
prepared by acetylation of the racemic alcohol 38 to give the
acetate 49, which was then subjected to oxidation and Mislow
rearrangement24 to give the alcohol 50. After oxidation of the
alcohol with Dess-Martin periodinane,22 the enone 51 was
reduced enantioselectively with (S)-BINAL-H under conditions
already reported,5d,6a,6c,8,9,10,11,27 to give the alcohols 52 and 53
as an inseparable mixture. Desilylation followed by acid
treatment gave the diols 44b and 46b, which were separated.
Diol 44b was converted to the dibenzoate 45b, the ee of which
The absolute configuration of the enzymatically resolved
compounds was determined by comparison of the chiral HPLC
retention time of the dibenzoate 45 with that of the structually
(24) (a) Bickart, P.; Carson, F. W.; Jacobus, J.; Miller, E. J.; Mislow, K.
J. Am. Chem. Soc. 1968, 90, 4869. (b) Tang, R.; Mislow, K. J. Am. Chem.
Soc. 1970, 92, 2100.
(25) (a) Becker, H.-D.; Bjo¨rk, A.; Alder, E. J. Org. Chem. 1980, 45,
1596. (b) Tsubuki, M.; Kanai, K.; Keino, K.; Kakinuma, N.; Honda, T. J.
Org. Chem. 1992, 57, 2930.
(26) The ee’s were determined by HPLC analyses with a CHIRALCEL
OD column (Daicel Chemical Industries Ltd.): detector, UV (254 nm);
flow rate, 1 mL/min; mobile phase, hexane/2-PrOH ) 95/5 for 45 and 45b,
hexane/2-PrOH ) 99/1 for 48 and 48a.
(27) Noyori, R.; Tomino, I.; Tanimoto, Y.; Nishizawa, M. J. Am. Chem.
Soc. 1984, 106, 6709.

5-F2t-Isoprostane
J. Am. Chem. Soc., Vol. 121, No. 34, 1999 7777
Scheme 8a
Scheme 9a
a Reagents and conditions: (a) LiOH‚H2O, THF - H2O (1:1), rt.
F2t-isoprostane (6), 5-F2t-isoprostane (5), ent-5-epi-F2t-isopros-
tane (8), and ent-5-F2t-isoprostane (7).
Conclusion
We have developed a practical synthesis of the potential
human hormones 5-8 using the chemo-enzymatic resolution
of the pseudo-meso diol 40 as a key step. We have also
established what should be a general strategy for the assignment
of relative configurations in this series. This synthesis will make
5-8 available in sufficient quantity to allow the detailed
assessment of their physiological activity.
a Reagents and conditions: (a) Ac2O, Py, 4-DMAP, CH2Cl2, rt,
(99%); (b) mCPBA, CH2Cl2, -78 °C; (MeO)3P, EtOH, -78 °C ∼rt;
(c) Dess-Martin periodinane, rt, (84%); (d) (S)-BINAL-H, THF, -78
°C; (e) TBAF, THF, rt; cat. H2SO4, EtOH, rt; (f) BzCl, Et3N, 4-DMAP,
CH2Cl2, rt.
was determined to be 43% by chiral HPLC.26 The major
enantiomer of the dibenzoate 45b had a retention time of
10.8 min, and its minor enantiomer had a retention time of
8.0 min. The dibenzoate 45, derived by the enzymatic resolu-
tion, had a retention time of 10.8 min. Thus, we established
the absolute configurations of the enzymatically resolved
acetates 41 and 42 to be as shown in Scheme 6. This result is
consistent both with our previous observations5d and with those
of others.23
Acknowledgment. We thank the National Institute of Health
(GM42056) for support of this work. We also thank Dr. L.
Jackson Roberts II and co-workers at Vanderbilt University for
collaborative efforts and Amano Pharmaceutical Co., Ltd. for
a gift of the lipases.
Supporting Information Available: Detailed experimental
procedures and spectroscopic characterization for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
The acetates 43, 44, 46, and 47 were separately hydrolyzed
(Scheme 9) with LiOH in THF-H2O (1:1) to furnish 5-epi-
JA990859K