The first total synthesis of iPF4α-VI and its deuterated analog

Article abstract of DOI:10.1016/S0040-4039(02)00398-2

The total and stereospecific syntheses of iPF_4α-VI and d₄-iPF_4α-VI have been accomplished. iPF_4α-VI is a docosahexanenoic acid (DHA)-derived isoprostane. DHA is the most abundant polyunsaturated fatty acid in the brain. Different synthetic designs have been used for the two syntheses. In the d₄-iPF_4α-VI design, the deuterated part of the molecule was introduced last in the synthesis.

Full text of DOI:10.1016/S0040-4039(02)00398-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2801–2805
The first total synthesis of iPF4a-VI and its deuterated analog
Seongjin Kim,a John A. Lawson,b Domenico Pratico`,b Garret A. FitzGeraldb and Joshua Rokacha,*
aClaude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 W. University Blvd., Melbourne,
FL 32901, USA
bThe Center for Experimental Therapeutics, The University of Pennsylvania, Philadelphia, PA 19104, USA
Received 14 January 2002; accepted 19 February 2002
Abstract—The total and stereospecific syntheses of iPF4a-VI and d4-iPF4a-VI have been accomplished. iPF4a-VI is a docosahex-
anenoic acid (DHA)-derived isoprostane. DHA is the most abundant polyunsaturated fatty acid in the brain. Different synthetic
designs have been used for the two syntheses. In the d4-iPF4a-VI design, the deuterated part of the molecule was introduced last
in the synthesis. © 2002 Elsevier Science Ltd. All rights reserved.
Isoprostanes (iPs) are a class of natural products gener-
ated by the action of free-radicals on polyunsaturated
fatty acids (PUFA).1,2 We have previously reported on
the total synthesis of isoprostanes and metabolites
derived from free-radical peroxidation of arachidonic
acid (AA) 5.3–7 We have used these synthetic standards
to discover and characterize four classes of iPs in
biological fluids2,8,9 and designed a GC and LC/MS/MS
methodology to analyze for the distribution of iPs
within each class.10,11
3 using GC/MS in AD and control brains.13 The levels
of both iPs were markedly elevated in both frontal and
temporal poles of AD brains compared to the corre-
sponding cerebella and controls. The next phase was to
attempt to show this correlation in a non-invasive
fashion by measuring iPs in urine. Our group has made
some preliminary measurements of iPs level in urine
of AD patients and was able to show a substantial
increase of 8,12-iso-iPF2a-VI 4 (Scheme 1).14
Human phospholipids normally contain large amounts
of arachidonic acid with only low levels of v−3 polyun-
saturated fatty acids. The brain, however, is extremely
rich in DHA, which makes up to 37% of its total fatty
acids. Because of the preponderance of DHA in brain
tissue, we decided to take a closer look at iPs derived
from DHA, with the ultimate goal of providing a
selective index of the severity of AD. Some GC/MS
studies have in fact provided evidence for the formation
of DHA-derived isoprostanes.15,16
We wish to report here on the first synthesis of an iP
derived from docosahexaenoic acid (DHA) 18, namely
iPF4a-VI. DHA is a 22:6v3 fatty acid (v3=the first
double bond from the v end of the molecule, 22=the
number of carbons, 6=the number of double bonds).12
Alzheimer’s disease (AD) includes a group of dement-
ing neurodegenerative disorders that have diverse eti-
ologies but the same hallmark brain lesions. Since
oxidative stress may play a role in the pathogenesis of
AD and iPs are chemically stable peroxidation products
of AA, our lab measured both iPF2a-III 2 and iPF2a-VI
Because of the preponderance of DHA in the brain, the
iPs derived from this v-3 n−6 PUFA are expected to be
OH
OH
OH
OH
OH
OH
CO2H
CO2H
CO2H
CO2H
OH
OH
OH
iPF2α−III
OH
PGF2α
OH
OH
4
8,12-iso-iPF2α−VI
1
iPF2α−VI
3
2
Scheme 1.
Keywords: isoprostanes, iP; arachidonic acid, AA; docosahexaenoic acid, DHA; iPF4a-VI; d4-iPF4a-VI.
* Corresponding author.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00398-2

2802
S. Kim et al. / Tetrahedron Letters 43 (2002) 2801–2805
The new synthon 29 was selected and prepared as
shown in Scheme 3. 1,2,4-Butanetriol 19 was converted
to the aldehyde 21 in two steps. Variable amounts of
the six-membered ring acetonide were formed. These
can be purified by flash chromatography. Alternatively,
the oxidation of the six-membered isopropylidene alco-
hol is much slower than the required 20 and can be
purified at that stage. Then the Wittig reaction with
commercial (3-carboxylpropyl)triphenylphosphonium
bromide in THF gave the cis olefin 23 in 78% yield.
After removal of the 7,8-isopropylidene group in 23
with 4% aqueous H2SO4, the 7,8-dihydroxy compound
major iPs. In addition, the types and distribution of iPs
derived from this PUFA will be different. It is antici-
pated that eight groups of iPs can be derived from
DHA (Scheme 2).17 We have devised a formula to
predict the number of groups of F2 iPs as a function of
PUFA double bonds (n−2)×2=number of groups, n
being the number of skipped double bonds. For exam-
ple, DHA has six skipped double bonds. This translates
to eight groups of iPs (Scheme 2). As a first step in this
area we performed the total and stereospecific synthesis
of iPF4a-VI (Scheme 4) and its tetradeutero derivative
(Scheme 7).
Scheme 2. Expected iPs from AA and DHA. (ROS: reactive oxygen species).
BrPh3P
CO2H
O
HO
a
O
O
b
22
a)
O
CHO
HO
CO2H
O
O
OH
c
OH
23
19
21
20
O
S
g
f
HO
HO
d
SMe
O
e
O
I
O
CO2H
CO2H
24
25
CO2H
26
O
HO
O
O
i
h
MeO P
OMe
I
I
CO2Me
CO2Me
CO2Me
29
27
28
b)
j
l
I
IPh3P
k
HO
TsO
33
32
30
31
Scheme 3. Reagents and conditions: (a) 2,2-dimethoxy propane, TsOH, CH3CN, 1 h 65%; (b) PCC, camphorsulfonic acid, CH2Cl2,
3 h, 74%; (c) LiHMDS, HMPA, −78°C, 3 h, 78%; (d) 4% H2SO4, THF, rt, 8 h; (e) SꢀC(Im)2, CH3CN, rt, 8 h, 51%; (f) MeI,
ClCH2CH2Cl, 70°C, 6 h, 87%; (g) DIBAL-H, CH2Cl2, −78°C, 30 min, CH2N2, ether, 97% (two steps); (h) periodinane, CH2Cl2,
rt, 6 h; (i) P(OMe)3, THF, rt, 6 h, 62% (two steps); (j) TsCl, pyridine, rt, 8 h, 60%; (k) NaI, CH3CN, 70°C, 3 h; (l) Ph3P, CH3CN,
80°C, 8 h, 87%.

S. Kim et al. / Tetrahedron Letters 43 (2002) 2801–2805
2803
24 was treated with thiocarbonylbis(imidazole) in ace-
tonitrile to afford the thionocarbonate 25. The conver-
sion of thionocarbonate 25 to the iodohydrin 26 was
performed by a method described by us recently.18 The
reduction of the methylthiocarbonyl group in 26 with
DIBAL-H, followed by an acidic work-up and dia-
zomethane treatment furnished the iodohydrin 27 in
97% yield. After oxidation with Dess–Martin periodi-
nane, the treatment of the b-keto iodide 28 with
trimethylphosphite in THF at room temperature
afforded the b-keto phosphonate 29 in 62% yield for
the two steps.
We also needed to synthesize the tetradeutero deriva-
tive of iPF4a-VI for its eventual quantitation by GC
and LC/MS in biological fluids. With the extra double
bonds in the molecule, as compared with AA-derived iP
(Scheme 2), our choices for the position of four deu-
terium atoms in the molecule were limited. Scheme 5
shows some choices of deuterated derivatives we have
considered. The tetradeutero compound 43 is not
appropriate because the deuterium would be positioned
a to a carboxylate group. This could result in a loss of
deuterium during the synthesis. Analog 44, while practi-
cally feasible, appeared more challenging. We elected to
proceed with the synthesis of the tetradeutero analog 45
(Scheme 5). The synthesis of the deuterated synthon 56
(Scheme 6) was our initial target. We planned to use 56
in a synthesis similar to that shown in Scheme 4.
The precursor 35 for the cyclization was prepared in six
steps from diacetone
D
-glucose 34.3 The lactol 37 was
reacted with the phosphonium derivative 33 to afford
the alcohol 38 in 78% yield. Dess–Martin oxidation
gave aldehyde intermediate 39. The b-keto phosphonate
29 was reacted in a Horner–Emmons reaction with
aldehyde 39 to introduce the upper side chain, afford-
ing derivative 40. As shown in Scheme 4, the (S)-
BINAL-H reduction was carried out at −100°C to
afford 41 in 87% yield. We have been unable to detect
any of the R-isomer, indicating that the e.e. is >95%.
iPF4a-VI 42 was obtained after the deprotection of silyl
and hydrolysis.19 We can perform steps g and h as a
one-pot reaction. We found the use of formic acid in
the deprotection step convenient20 and, in some cases,
competitive with TBAF or HF methods.
The synthesis of 56 as it turned out is not without
problems. The main problem is the small size of alcohol
49 which is very volatile. In addition, we could not find
any precedent for the deuteration of a primary acetyl-
ene. We were worried that the acetylenic proton, being
acidic, would cause partial hydrogen–deuterium
exchange.21 The synthon 51 we needed for the synthesis
was prepared from the O-THP derivative of propagyl
alcohol 46. The transformation of 47 to 48 proceeded
smoothly and a high incorporation of D2 (>99.7%) has
been achieved, no scrambling was observed. The depro-
tection of the O-THP, however, was difficult (low yield)
O
O
S
TBDMSO
TBDMSO
O
O
OH
CO2Me
b
a
O
O
O
O
6steps
OTBDMS
TBDMSO
O
OH
O
TBDMSO
CHO
TBDMSO
37
36
D-glucose
35
34
O
O
-IPh3+P
TBDMSO
MeO P
CO2Me
TBDMSO
TBDMSO
d
OMe
29
e
33
OH
c
TBDMSO
OH
39
38
OH
O
HO
HO
TBDMSO
TBDMSO
TBDMSO
g, h
f
CO2H
CO2Me
CO2Me
TBDMSO
42
iPF4α-VI
40
41
Scheme 4. Reagents and conditions: (a) n-Bu3SnH, AIBN, benzene, reflux, 5 h, 52%; (b) DIBAL-H, CH2Cl2, −78°C, 97%; (c)
t-BuOK, HMPA, THF, −78°C to rt, 3 h, 78%; (d) periodinane, CH2Cl2, rt, 4 h, 97%; (e) LiHMDS, THF, −78°C, 69%; (f)
S-BINAL-H, THF, −100°C, 4 h, 87%; (g) 6:3:1=THF:formic acid:H2O, rt, 3 h, 72%; (h) 5% KOH, THF, rt, 2 h, 97%.
OH
OH
OH
D
D
D
HO
HO
HO
HO
HO
HO
CO2H
CO2H
D D
CO2H
D
D
D
D
D
D
D
16,17,19,20-d4-iPF4α-VI 44
45
2,2,3,3-d4-iPF4α-VI
21,21,22,22-d4-iPF4α-VI
43
Scheme 5.

2804
S. Kim et al. / Tetrahedron Letters 43 (2002) 2801–2805
because of the volatility of tetradeutero propanol. We
have managed to obtain the phosphonium derivative 51
in 15–37% yield, as shown in Scheme 6.
The bicyclic lactol 37 was coupled with 3-(tetrahydro-
pyran-2-yloxy)-propyl-phosphonium bromide 57 by Wit-
tig reaction and 58 was obtained in 82% yield. The
synthesis of 59 was achieved by a Dess–Martin oxidation.
The aldehyde 59 was used in a Horner–Emmons reaction
at −78°C to introduce the upper side chain using the
anion of b-ketophosphonate 29 and afforded 60 in 76%
yield. The enantioselective reduction of the C7 keto
group 60 with chiral reducing agent (S)-BINAL-H pro-
ceeded well and afforded the desired pure 7(S) derivative
61 in 97% yield. Compound 61 was protected with the
TBDMS group, and then the tetrahydro pyranyl group
deprotection was carried out selectively using Me2AlCl
in 72% yield. Aldehyde 64 was reacted with the tetra-
deutero phosphonium derivative 51 as shown. The
deprotection of tris-silyl groups in 65 was carried out
using formic acid. Finally, hydrolysis with aqueous KOH
afforded the desired d4-iPF4a-VI 45.22
In addition, we have tried different protections on
propagyl alcohol such as silyl groups. The reduction with
Wilkinson’s catalyst was very sluggish and mostly start-
ing material was recovered. It is possible that the bulky
silyl group interferes with the reduction.
As mentioned above, the most difficult step was the
isolation of deuterated propanol 49. The continuation of
the synthesis to obtain the phosphonium derivative 56,
beyond alcohol 54, was impractical at that point because
of the dwindling yield due to the multi-step synthesis. We
elected instead to redesign the synthesis of 45, which is
shown in Scheme 7. The major advantage of the new
design is the fact that we performed the addition of the
three-carbon synthon 51 at the last step of the synthesis.
Wilkinson's
D
D
D
D
D
D
DHP, TsOH
catalyst
TPP
I2, Im,TPP
Me2AlCl
HO
THPO
THPO
I
HO
D
D
D
D
99.98% D2
CH2Cl, RT, 92%
D
D
46
47
99.7% pure
50
Benzene, 99%
49
48
D
D
D
D
D
D
D
D
CHO
D
D
TBAF
I2, Im,TPP
TPP
TBDMSO
TBDMSO
IPh3P
IPh3P
HO
I
D
D
D
D
D
D
D
D
D
D
52
53
56
54
55
37% yield
3 steps
51
Scheme 6.
TBDMSO
TBDMSO
TBDMSO
CHO
a
O
b
OH
-
+
OH
BrPh3P
OTHP
OTHP
OTHP
TBDMSO
TBDMSO
O
TBDMSO
OH
57
37
58
59
O
O
TBDMSO
TBDMSO
MeO P
OMe
TBDMSO
TBDMSO
CO2Me
e
29
CO2Me
d
CO2Me
c
OTHP
OTHP
60
61
OTBDMS
OTBDMS
OTBDMS
TBDMSO
TBDMSO
TBDMSO
TBDMSO
g
f
CO2Me
CO2Me
CO2Me
CHO
OTHP
OH
HO
HO
TBDMSO
TBDMSO
64
62
63
OH
D
D
OTBDMS
TBDMSO
TBDMSO
IPh3P
CO2H
D
51
CO2Me
D
i,j
D
D
D
D
h
45
D
D
D
D
65
21,21,22,22-d4-iPF4α-VI
Scheme 7. Reagents and conditions: (a) t-BuOK, THF, −20°C, 5 h, 82%; (b) periodinane, CH2Cl2, rt, 2 h, 97%; (c) LiHMDS,
THF, −78°C, 76%; (d) S-BINAL-H, THF, −100°C, 5 h, 97%; (e) TBDMSCl, Im, CH2Cl2, rt, 8 h, 75%; (f) Me2AlCl, CH2Cl2,
−20°C to rt, 3 h, 72%; (g) periodinane, t-BuOH, CH2Cl2, rt, 5 h; (h) LiHMDS, HMPA, −78°C, 3 h, 68%; (i) 6:3:1=THF:formic
acid:H2O, rt, 3 h; (j) KOH, THF, 1.5 h 85% (two steps).

S. Kim et al. / Tetrahedron Letters 43 (2002) 2801–2805
2805
Acknowledgements
11. Lawson, J. A.; Rokach, J.; FitzGerald, G. A. J. Biol.
Chem. 1999, 274, 24441–24444.
12. Durand, T.; Guy, A.; Vidal, J. P.; Viala, J.; Rossi, J. C.
Tetrahedron Lett. 2000, 41, 3859–3862. A methyl ester of
an iP from Group VIII has been reported.
13. Pratico, D.; Lee, V. M.-Y.; Tojanowski, J. Q.; Rokach,
J.; FitzGerald, G. A. FASEB J. 1998, 12, 1777–1783.
14. Pratico, D.; Clark, C. M.; Lee, V. M. Y.; Trojanowski, J.
Q.; Rokach, J.; FitzGerald, G. A. Ann. Neurol. 2000, 48,
809–812.
We wish to acknowledge the NIH for support under
Grants DK-44730 (J.R.), HL-54500, and HL-62250
(G.A.F.); the AHA for support under Grant 0030211
(D.P.); and the NSF for an AMX-360 NMR instru-
ment (Grant CHE-90-13145).
15. Nourooz-Zadeh, J.; Liu, E. H. C.; Anggard, E. E.; Halli-
well, B. Biochem. Biophys. Res. Commun. 1998, 242,
338–344.
16. Roberts, L., II; Montine, T. J.; Markesbery, W. R.;
Tapper, A. R.; Hardy, P.; Chemtob, S.; Dettbarn, W. D.;
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17. Rokach, J.; Khanapure, S. P.; Hwang, S. W.; Adiyaman,
M.; Lawson, J. A.; FitzGerald, G. A. Prostaglandins
1997, 54, 853–873.
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127) base peak, 391 (M−217), 365 (M−243), 301 (M−
307), 275 (M−333).
The NMR data of compound 42: 1H NMR (CDCl3) l
5.67–5.22 (m, 8H), 4.14 (q, 1H, J=5.4 and 12.2), 4.05–
3.93 (m, 2H), 2.76 (m, 4H), 2.46–2.28 (m, 7H), 2.16 (m,
1H), 2.03 (m, 4H), 0.96 (t, 3 H, J=7.5).
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22. Mass spec. analysis of compound 45: d4 content (LC/
MS): 99.85%.
1
The NMR data of compound 45: H NMR (CD3OD): l
5.55–5.23 (m, 8H), 4.04 (q, 1H, J=5.3 and 10.3), 3.93 (q,
1H, J=6.6 and 11.8), 3.82 (q, 1H, J=6.9 and 11.3), 2.76
(m, 2H), 2.65 (m, 2H), 2.44 (m, 1H), 2.30 (m, 4H), 2.03
(m, 2H), 1.58 (m, 3H), 1.21 (s, 1H).