iPF2α-III metabolism. First total synthesis of 2,3-dinor iPF2α-III, a primary β-oxidation metabolite

Article abstract of DOI:10.1016/S0040-4039(01)01794-4

The total synthesis of a primary iPF_2α-III metabolite, namely dinor iPF_2α-III 8, is described. The successful synthesis was finally accomplished by generating the carboxylic acid group at the end of the synthesis so as to avoid formation of an α,β-unsaturated carboxylic acid, which is prone to undergo side reactions.

Full text of DOI:10.1016/S0040-4039(01)01794-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8277–8280
iPF2a-III metabolism. First total synthesis of 2,3-dinor iPF2a-III,
a primary b-oxidation metabolite
Seongjin Kim,a Young-Ju Jung,a John A. Lawson,b Garret A. FitzGeraldb and Joshua Rokacha,*
aClaude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 W. University Boulevard,
Melbourne, FL 32901, USA
bThe Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA
Received 25 July 2001; accepted 17 September 2001
Abstract—The total synthesis of a primary iPF2a-III metabolite, namely dinor iPF2a-III 8, is described. The successful synthesis
was finally accomplished by generating the carboxylic acid group at the end of the synthesis so as to avoid formation of an
a,b-unsaturated carboxylic acid, which is prone to undergo side reactions © 2001 Elsevier Science Ltd. All rights reserved.
Isoprostanes (iPs), a new class of natural products are
produced in vivo as a result of free-radical initiated
oxygenation of polyunsaturated fatty acids.1,2 Over the
past few years we have performed the first total synthe-
ses of iPF2a-III,3 iPF2a-IV,4 iPF2a-V,5 and iPF2a-VI2
(Scheme 1). Using these synthetic standards, we have
discovered the existence of these isoprostanes in biolog-
ical fluids.6,7,5,8 We have developed GC–MS6,7,9 and
LC–MS10,11 methodologies to measure isoprostanes in
biological fluids. We have used these sensitive methods
to measure increased urinary levels of isoprostane for-
mation and, in particular, iPF2a-VI, 8,12-iso-iPF2a-VI
and iPF2a-III in disease states such as atherosclerosis12,2
and Alzheimer’s disease13 and shown that the severity
of the diseases correlates with the increase in urinary
isoprostane levels.
provide a better index of the isoprostane formation
than the original parent.
The metabolism of iPs has not been examined, except
for a few published studies with iPF2a-III. GC–MS
studies have provided evidence for the formation of
metabolites such as 6,14 7,15 8,16 and 916 (Scheme 2). 6
14,17
has attracted the most attention,
and a synthesis
has also been reported.18 We wish to report here on the
first total and stereospecific synthesis of 2,3-dinor
iPF2a-III metabolite 8.
We focused on this metabolite because there is a good
chance that this is the initial b-oxidation product
formed. If this is the case, it will allow us to study its
conversion in vivo to 10, 6 and 9 (Scheme 3). Scheme 3
shows a proposed metabolic sequence to interpret the
formation of these products. We propose the intermedi-
acy of the a,b-unsaturated derivative 10 in order to
explain the reduction step of 8 to 6. The transformation
sequence shown in the brackets is not unlike earlier
work on the metabolism of polyunsaturated fatty
Before iPs are excreted in urine they undergo a
metabolic process. The amount of intact isoprostanes
measured in urine represent only a part of the total
isoprostanes produced by cells. It is of interest to study
the metabolism of these iPs since a metabolite can
OH
OH
HO
HO
HO
HO
HO
CO2H
CO2H
CO2H
CO2H
CO2H
HO
HO
HO
HO
HO
OH
OH
OH
iPF2α-IV
3
iPF2α-VI
PGF2α
1
5
iPF2α-III
2
iPF2α-V
4
Scheme 1.
Keywords: isoprostanes; iPF2a-III; dinor iPF2a-III; dinor dihydro-iPF2a-III; arachidonic acid.
* Corresponding author. E-mail: jrokach@fit.edu
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01794-4

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S. Kim et al. / Tetrahedron Letters 42 (2001) 8277–8280
HO
HO
HO
HO
HO
HO
CO2H
CO2H
OH
CO2H
CO2H
HO
HO
OH
O
OH
2,3,4,5-tetranor-15-keto
-5,6.13,14-tetrahydro-iPF2α-III
2,3-dinor-iPF2α-III
8
2,3-dinor-5,6-dihydro-iPF2α-III
6
2,3,4,5-tetranor-5,6-dihydro-iPF2α-III
9
7
Scheme 2.
HO
CO2H
HO
HO
HO
HO
HO
enoyl-CoA
isomerase
6
OH
CO2H
β-oxidation
CO2H
CO2H
HO
CO2H
HO
OH
HO
OH
OH
10
8
2
iPF2α-III
HO
9
OH
Scheme 3.
acids19 and our own work20 and others21 on the
metabolism of LTE4 (Scheme 4). Note that the trans-
formation of 16 to 18 and 19 parallels the transforma-
tion of 8 to 6 and 9, as shown in Scheme 3. The
formation of 9 can be the result of a b-oxidation cycle
from the C-16 derivative 6, or directly from the a,b-
unsaturated derivative 10. In other words, the a,b-
unsaturated derivative 10 is in equilibrium with the
reduced form 6. Hydration of 10, leading eventually to
9, will probably draw the equilibrium toward the tetra-
nordihydro derivative 9. Other metabolic transforma-
tions of many of these intermediates, for example
oxidation of the hydroxy group to a ketone, are likely
also to occur to an unknown degree.15
not successful. The use of Ph3P+X−CH2CH2CH2O−
affords a variable amount of trans double bond we
would rather avoid.24 The final design of the synthesis
relies on the oxidation of the C-1 alcohol late in the
synthesis. The alternative, which was to establish the
carboxylic acid and its protected ester earlier, causes
migration of the double bond into conjugation with the
carboxyl group during the subsequent steps of the
synthesis, resulting in mixtures which are difficult to
handle. The S-BINAL-H reduction of the carbonyl
derivative 27 to afford 28 worked in 96% e.e. and in
excellent yield. We found that the deprotection of the
three TBDMS groups in 3125 to yield the target
metabolite 826 was best achieved using formic acid.
The synthesis of metabolite 8 was performed in 18 steps
A word of caution on the metabolism of iPF2a-III: as
mentioned earlier, iPF2a-III produces a dinor dihydro
metabolite 6 which appears more abundant than the
parent iPF2a-III.14,16 As we have shown in a recent
report,17 g-linolenic acid 32, which is a C-18 PUFA
with three conjugated double bonds, on free radical
peroxidation in vitro gives rise to an isoprostane identi-
cal to 6 (Scheme 6).
and is described in Scheme 5. The synthesis of the
precyclization synthon 21 was prepared from L-glucose
as we reported earlier.3,22,23
The cyclization of 21 to 22 was performed at reflux in
benzene or reflux in toluene. The use of toluene mini-
mizes byproduct formation at the higher temperature
by providing a more efficient carbon oxygen homolysis
in the radical-generating step. The addition of a three-
carbon unit to the top side chain proved difficult. The
use of a three-carbon Wittig reagent such as Ph3P+X−
CH2CH2COOH and Ph3P+X−CH2CH2COOCH3 was
The experiment we did which precludes metabolism is
as follows. A blood sample was subjected to a freeze,
thawing process in order to allow a peroxidation pro-
cess. Under these conditions a substantial amount of 6
NH2
Cytochrome P-450
NADPH / O2
β-oxidation
S
CO2H
CO2H
Cytochrome P-450
NADPH / O2
CO2H
CO2H
ATP/CoASH
ADH / AldDH
NAD+
OH
OH
14
12
13
LTE4
11
acyl-CoA
dehydrogenase
enoyl-CoA
isomerase
2,4-dienoyl-CoA
reductase
CO2H
18
CO2H
CO2H
CO2H
17
16
CO2H
15
19
Scheme 4.

S. Kim et al. / Tetrahedron Letters 42 (2001) 8277–8280
8279
S
TBDMSO
TBDMSO
TBDMSO
TBDMSO
CH2OH
HO
O
O
OH
b
c
a
MeO2C
TBDMSO
O
-
+
O
O
HO(H)
OH
OH
BrPh3P
OTHP
8 steps
OTBDMS
24
22
23
L-glucose
20
21
TBDMSO
TBDMSO
TBDMSO
d
TBDMSO
TBDMSO
OTHP
OTHP
OTHP
OTHP
f
e
OH
CHO
TBDMSO
TBDMSO
TBDMSO
O
OH
25
26
27
28
HO
TBDMSO
TBDMSO
TBDMSO
OTHP
OH
CO2H
g
h
i, j
CO2H
k
HO
OH
8
TBDMSO
TBDMSO
TBDMSO
OTBDMS
OTBDMS
OTBDMS
29
31
2,3-dinor-iPF2α-III
30
Scheme 5. Reagents and conditions: (a) n-Bu3SnH, AIBN, benzene, reflux, 5 h, 52%; (b) DIBAL-H, CH2Cl2, −78°C, 97%; (c)
t-BuOK, THF, −20°C, 5 h, 85%; (d) periodinane, CH2Cl2, rt, 2 h, 97%; (e) (Ome)2POCH2CO(CH2)4CH3, LiHMDS, THF, −78°C,
91%; (f) S-BINAL-H, THF, −100°C, 4 h, 96%; (g) TBDMSCl, IM, CH2Cl2, rt, 8 h, 99%; (h) Me2AlCl, CH2Cl2, −20°C to rt, 3
h, 96%; (i) periodinane, t-BuOH, CH2Cl2, rt, 3 h, 97%; (j) NaClO2, KH2PO4, t-BuOH, 2-methyl-2-butene, H2O, rt, 96%; (k)
6:3:1=THF:formic acid:H2O, rt, 3 h, 78%.
CO2H
O
O
CO2H
CO2H
CO2H
HO
O
O2
reactive
oxygen
species
O
CO2H
O O
35
O2
34
33
γ-linolenic acid
32
HO
OH
HO
HO
2,3-dinor-5,6-dihydro-iPF2α-III
6
CO2H
OH
iPF2α-III
2
Scheme 6.
was formed. This indicates to us that the measurement
of metabolite 6, as an indicator of iPF2a-III production,
may be augmented with the peroxidation product of
g-linolenic acid 32. The measurement of metabolite 8,
the synthesis of which is described in this paper, may be
a better indicator of iPF2a-III metabolism. Clearly more
work is necessary in order to substantiate this idea.
3. Hwang, S. W.; Adiyaman, M.; Khanapure, S.; Schio, L.;
Rokach, J. J. Am. Chem. Soc. 1994, 116, 10829–10830.
4. Adiyaman, M.; Li, H.; Lawson, J. A.; Hwang, S. W.;
Khanapure, S. P.; FitzGerald, G. A.; Rokach, J. Tetra-
hedron Lett. 1997, 38, 3339–3342.
5. Pudukulathan, Z.; Manna, S.; Hwang, S. W.; Khana-
pure, S. P.; Lawson, J. A.; FitzGerald, G. A.; Rokach, J.
J. Am. Chem. Soc. 1998, 120, 11953–11961.
6. Lawson, J. A.; Li, H.; Rokach, J.; Adiyaman, M.;
Hwang, S. W.; Khanapure, S. P.; FitzGerald, G. A. J.
Biol. Chem. 1998, 273, 29295–29301.
Acknowledgements
7. Pratico, D.; Barry, O. P.; Lawson, J. A.; Adiyaman, M.;
Hwang, S. W.; Khanapure, S. P.; Iuliano, L.; Rokach, J.;
FitzGerald, G. A. Proc. Natl. Acad. Sci. USA 1998, 95,
3449–3454.
8. Adiyaman, M.; Lawson, J. A.; FitzGerald, G. A.;
Rokach, J. Tetrahedron Lett. 1998, 39, 7039–7042.
9. Adiyaman, M.; Lawson, J. A.; Khanapure, S. P.;
FitzGerald, G. A.; Rokach, J. Anal. Biochem. 1998, 262,
45–56.
10. Li, H.; Lawson, J. A.; Reilly, M.; Adiyaman, M.; Hwang,
S. W.; Rokach, J.; FitzGerald, G. A. Proc. Natl. Acad.
Sci. USA 1999, 96, 13381–13386.
11. Lawson, J. A.; Rokach, J.; FitzGerald, G. A. J. Biol.
Chem. 1999, 274, 24441–24444.
We wish to acknowledge the NIH for support under
Grants DK44730 (J.R.), HL54500 and HL62250
(G.A.F.), and the NSF for an AMX-360 NMR instru-
ment (Grant CHE-90-13145).
References
1. Morrow, J. D.; Hill, K. E.; Burk, R. F.; Nammour, T.
M.; Badr, K. F.; Roberts, II, L. J. Proc. Natl. Acad. Sci.
USA 1990, 87, 9383–9387.
2. Adiyaman, M.; Lawson, J. A.; Hwang, S. W.; Khana-
pure, S. P.; FitzGerald, G. A.; Rokach, J. Tetrahedron
Lett. 1996, 37, 4849–4852.

8280
S. Kim et al. / Tetrahedron Letters 42 (2001) 8277–8280
12. Pratico, D.; Tangirala, R. K.; Rader, D. J.; Rokach, J.;
FitzGerald, G. A. Nature Med. 1998, 4, 1189–1192.
13. Pratico, D.; Clark, C. M.; Lee, V. M. Y.; Trojanowski, J.
Q.; Rokach, J.; FitzGerald, G. A. Ann. Neurol. 2000, 48,
809–812.
14. Roberts, II, L. J.; Moore, K. P.; Zackert, W. E.; Oates, J.
A. J. Biol. Chem. 1996, 271, 20617–20620.
15. Basu, S. FEBS Lett. 1998, 428, 32–36.
16. Chiabrando, C.; Valagussa, A.; Rivalta, C.; Durand, T.;
Guy, A.; Zuccato, E.; Villa, P.; Rossi, J.-C.; Fanelli, R. J.
Biol. Chem. 1999, 274, 1313–1319.
Issue: Nat. Prod. Synth. 1998, 569–580.
23. Hwang, S. W.; Adiyaman, M.; Lawson, J. A.; FitzGer-
ald, G. A.; Rokach, J. Tetrahedron Lett. 1999, 40, 6167–
6170.
24. Kim, S.; Adiyaman, Y.; Saha, G.; Powell, W. S.; Rokach,
J. Tetrahedron Lett. 2001, 42, 4445–4448.
25. NMR data for compound 31: 1H NMR (360 MHz,
CDCl3) l 5.59 (m, 2H), 5.46 (dd, J=5.4 and 15.4 Hz,
1H), 5.29 (dd, J=6.1 and 15.5 Hz, 1H), 4.07 (q, J=5.1
and 5.8 Hz, 1H), 3.92 (m, 1H), 3.79 (q, J=7.4, 1H), 3.12
(d, J=6.3 Hz, 2H), 2.54 (t, J=6.8 Hz, 1H), 2.37 (m,
J=6.5, 6.9 and 7.9 Hz, 1H), 2.15 (m, 2H), 2.09-1.81 (m,
3H), 1.73-1.08 (m, 6H), 0.89 (m, 30H), 0.05 (m, J=6.5
and 8.4 Hz, 18H).
17. Burke, A.; Lawson, J. A.; Meagher, E. A.; Rokach, J.;
FitzGerald, G. A. J. Biol. Chem. 2000, 275, 2499–2504.
18. Taber, D. F.; Kanai, K. J. Org. Chem. 1999, 64, 7983–
7987.
1
26. The NMR and mass spec data of compound 8: H NMR
19. Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of
Biochemistry, 2nd ed.; Worth Publishers: New York,
1993.
20. Delorme, D.; Girard, Y.; Rokach, J. J. Org. Chem. 1989,
54, 3635–3640.
21. Sala, A.; Voelkel, N.; Maclouf, J. J. Biol. Chem. 1990,
265, 21771–21778.
22. Rokach, J.; Khanapure, S. P.; Hwang, S. W.; Adiyaman,
M.; Schio, L.; FitzGerald, G. A. Synthesis, First Special
(360 MHz, d6-acetone) l 5.57 (m, 2H), 5.50 (m, 2H), 4.01
(q, J=5.0 Hz, 1H), 3.94 (m, J=2.9 and 3.6 Hz, 1H), 3.88
(q, J=6.3, 1H), 3.06 (br, 2H), 2.65 (m, 1H), 2.41(m,
J=7.1, 7.2, and 14.3 Hz, 1H), 2.16 (m, 2H), 1.59–1.22
(m, 6H), 0.87 (t, J=6.3, 3H). MS(NCI), PFB, TMS deriv.
m/z 541. MS(EI) methyl ester, TMS deriv. m/z 556 (M+),
541 (M−15), 485 (M−71), 466 (M−90), 443 (M−113), 395
(M−90−71) base peak, 353 (M−113−90), 305 (M−90−90−
71).