Stereocontrolled synthesis of the PPAR-γ agonist 10-nitrolinoleic acid

Article abstract of DOI:10.1021/jo1007493

(Figure presented) The naturally occurring PPAR-γ ligand 10-nitrooctadeca-9(E),12(Z)-dienoic acid (10-nitrolinoleic acid) (2a) was prepared as a single regio- and geometrical isomer in a practical eight-step, convergent sequence. The synthetic route featured a nitro aldol reaction between 9-oxononanoic acid methyl ester (3) and 1-nitronon-3(Z)-ene (4) in the key carbon-carbon bond forming step. The ability of 2a (and its methyl ester 9) to bind to PPAR-γ in a ligand-binding assay is reported.

Full text of DOI:10.1021/jo1007493

pubs.acs.org/joc
Stereocontrolled Synthesis of the PPAR-γ Agonist
10-Nitrolinoleic Acid
Elizabeth Dunny and Paul Evans*
Centre for Synthesis and Chemical Biology, School of
Chemistry and Chemical Biology, University College Dublin,
Dublin 4, Ireland
paul.evans@ucd.ie
Received April 18, 2010
FIGURE 1. Structures of linoleic acid (1) and 10-nitrolinoleic acid
(2a) and its retrosynthesis based on the nitro aldol reaction.
appreciable activation.4 More recently, it has been shown
that unsaturated nitrated lipids (for example, 2a and 2b,
Figure 1), which have been postulated to form from nitric
oxide by an unknown biochemical pathway, are potent
PPAR-γ activators at concentrations below those detected
within cells.5 In 2008, an X-ray crystallographic study de-
monstrated that a mixture of 10-nitrolinoleic acid (2a) and
12-nitrolinoleic acid (2b) bind within the same groove in
PPAR-γ as the TZD rosiglitazone (for further discussion,
see below).6 Syntheses of the regioisomers and geometrical
isomers of the monounsaturated nitro olefins stemming from
oleic acid have been reported.7 However, to date, a regio-
defined and stereoselective synthesis of the linoleic acid
derived congener has not. The reported synthesis, relying
on the mercury(II)- and selenium-based nitration of 1, is
cumbersome and requires preparative HPLC separation of
the regioisomeric 9-, 10-, 12-, and 13-nitro olefinic products.5b,8
As a consequence of the potent reported biological activ-
ities of 2, our interest9 in the activation of PPAR-γ, and the
unmet need for chemically pure samples of 2a, we designed
a synthesis based on the classic nitro aldol (Henry) reaction.
Thus, it was envisaged that 2 might be assembled in a
straightforward manner from aldehyde 3 and (Z)-olefin 4.
The naturally occurring PPAR-γ ligand 10-nitroocta-
deca-9(E),12(Z)-dienoic acid (10-nitrolinoleic acid) (2a)
was prepared as a single regio- and geometrical isomer in
a practical eight-step, convergent sequence. The synthetic
route featured a nitro aldol reaction between 9-oxonona-
noic acid methyl ester (3) and 1-nitronon-3(Z)-ene (4)
in the key carbon-carbon bond forming step. The ability
of 2a (and its methyl ester 9) to bind to PPAR-γ in a
ligand-binding assay is reported.
The peroxisome proliferator activated receptor-γ (PPAR-γ),
first identified in the late 1980s, is a transcription factor
belonging to the steroid, thyroid, and retinoid receptor
super family.1 Members of this class control lipid and glu-
cose cellular homeostasis, and as such, PPAR-γ has clinical
relevance since it is the biological target for the thiazolidin-
dione (TZD) class of drugs, which are widely prescribed
to combat the symptoms of type 2 diabetes.2 In 1995,
two groups independently suggested that its natural ligand
was the cyclopentenone prostanoid Δ12,14-15-deoxy-PGJ2.3
However, this proposal has not been universally accepted
since the in vivo detectable levels of this prostanoid
are significantly lower than the dose required to affect its
(5) (a) Lim, D. G.; Sweeney, S.; Bloodsworth, A.; White, C. R.; Chumley,
P. H.; Krishna, N. R.; Schopfer, F.; O’Donnell, V. B.; Eiserich, J. P.;
Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15941. (b) Baker,
P. R. S.; Schopfer, F. J.; Sweeney, S.; Freeman, B. A. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 11577. (c) Schopfer, F. J.; Lin, Y.; Baker, P. R. S.; Cui, T.;
Garcia-Barrio, M.; Zhang, J.; Chen, K.; Chen, Y. E.; Freeman, B. A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 2340. (d) Baker, P. R. S.; Lin, Y.; Schopfer,
F. J.; Woodcock, S. R.; Groeger, A. L.; Batthyany, C.; Sweeney, S.; Long,
M. H.; Iles, K. E.; Baker, L. M. S.; Branchaud, B. P.; Chen, Y. E.; Freeman,
B. A. J. Biol. Chem. 2005, 280, 42464. (e) Gorczynski, M. J.; Huang, J.; Lee,
H.; King, S. B. Bioorg. Med. Chem. Lett. 2007, 17, 2017. (f) For a review, see:
Freeman, B. A.; Baker, P. R. S.; Schopfer, F. J.; Woodcock, S. R.;
Napolitano, A.; d’Ischia, M. J. Biol. Chem. 2008, 283, 15515.
(6) Li, Y.; Zhang, J.; Schopfer, F. J.; Martynowski, D.; Garia-Barrio,
M. T.; Kovach, A.; Suino-Powell, K.; Baker, P. R. S.; Freeman, B. A.; Chen,
Y. E.; Xu, H. E. Nat. Struct. Mol. Biol. 2008, 15, 865.
(7) (a) Gorczynski, M. J.; Huang, J.; King, S. B. Org. Lett. 2006, 8, 2305.
(b) Woodcock, S. R.; Marwitz, A. J. V.; Bruno, P.; Branchaud, B. P. Org.
Lett. 2006, 8, 3931. (c) Gorczynski, M. J.; Smitherman, P. K.; Akiyama,
T. E.; Wood, H. B.; Berger, J. P.; King, S. B.; Morrow, C. S. J. Med. Chem.
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ꢀ
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5334 J. Org. Chem. 2010, 75, 5334–5336
Published on Web 07/01/2010
DOI: 10.1021/jo1007493
r
2010 American Chemical Society

Dunny and Evans
JOCNote
SCHEME 1. Nitration of Alcohol 5 and Preparation of
Aldehyde 3
the characteristic vinylic chemical shift for the nitro olefin
proton.7b Following chromatographic purification, the in-
termediate acetoxy derivative was also isolated (26%) and
could be resubmitted to the elimination process. In relation
to this process, the use of 3 equiv of DMAP in the second
step, acetoxy elimination, gave 9 in diminished yield (30%).
The sensitive methyl ester 9 was then converted into 10-
nitrolinoleic acid (2a) following an enzyme-mediated ester
hydrolysis protocol in 73% yield.9
Apart from the issues surrounding the previously de-
scribed methods for the synthesis of nitro olefins derived
from linoleic acid, inspection of the X-ray crystal structure
of PPAR-γ bound to a mixture of 2a and 2b6 indicated
that 2a was oriented in an arrangement in which the nitro
group interacted electrostatically with an arginine residue
(Arg288). This in turn served to place the electrophilic
β-carbon of 2a in close proximity to a cysteine residue
(Cys285) within the binding cleft. Since it is widely appre-
ciated that nitro olefins undergo rapid conjugate addition
with nucleophilic reagents such as thiol-containing species,
SCHEME 2. Formation of 10-Nitrolinoleic Acid (2a)
etc.,11,12 the fact that the crystal structure, solved at 2.4 A
˚
resolution, does not detect a covalent linkage between
PPAR-γ and 2a seems surprising (Scheme 3). In relation to
this point, it is argued that the formation and reservoir of
such nitrated lipids reside in the cell membrane, which
protects them from reaction with adventitious nucleophilic
species such as water and glutathione.5,8 The nature of this
type of ligand-PPAR-γ binding mode is extremely relevant
since it has been suggested that the conformation adopted by
the ligand and the way it interacts with PPAR-γ, on a
molecular scale, may determine the precise nature and level
of the subsequent gene transcription.6,13
With samples of 9 and 2a in hand, the ability of these lipids
to bind to human PPAR-γ was investigated in a cell-free
PPAR-γ binding assay.14 On the basis of the displacement of
radiolabeled rosiglitazone, IC50 values of 0.22 and 0.11 μM
were calculated for 2a and 9, respectively.
As shown in Scheme 1, the required (Z)-nitro olefin 4 was
prepared in two steps from the commercially available
alcohol 5. Thus, bromination of 5 followed by treatment
with NaNO2 in DMF (often termed the Kornblum reaction)
gave 4. In relation to this sequence, negligible amounts of the
nitrite ester 6 were detected under these reaction conditions,
and only minor amounts of alkene isomerization were
detected. However, this observation was in contrast with
the corresponding AgNO2-mediated process (Victor Meyer
reaction) which gave both 4 and 6, which proved chromato-
graphically separable, and more significantly double-bond
isomerization ((Z)-4:(E)-4; 3:1).
Resubmission of 6 to the NaNO2-DMF conditions did
not result in formation of 4, suggesting a lack of reversibility
of this process under the reaction conditions (Scheme 1).
The aldehyde aldol reaction partner 3 was prepared from
mono methyl azelate 7by either a borane reduction-oxidation
sequence, or following a reduction-esterification-oxidation
sequence (Scheme 1).
The pivotal nitro aldol reaction was performed according
to Denmark’s protocol using substoichiometric amounts
of KO-t-Bu.10 This gave 8 in reasonable yield as a mixture
of diastereoisomers (Scheme 2). Optimal conditions for the
formation of 9 (R = Me) from 8 were found to be acetylation
followed by solvent exchange and addition of 1.2 equiv of
DMAP to facilitate acetoxy elimination.7 Thus, 9 could be
isolated in 45% yield as a single geometrical isomer based on
Experimental Section
(Z)-Methyl 9-hydroxy-10-nitrooctadec-12-enoate (8). Under
nitrogen, potassium tert-butoxide (0.02 g, 0.18 mmol) was
added to a solution of methyl 9-oxononanoate (3) (0.38 g,
2.04 mmol), (Z)-1-nitronon-3-ene (4) (0.34 g, 1.99 mmol) in
THF (1 mL), and 2-methyl-2-propanol (1 mL) at 0 °C. The
mixture was stirred for 15 min at 0 °C and then allowed to stir for
(11) (a) Baker, L. M. S.; Baker, P. R. S.; Golin-Bisello, F.; Schopfer, F. J.;
Fink, M.; Woodcock, S. R.; Branchaud, B. P.; Radi, R.; Freeman, B. A. J.
Biol. Chem. 2007, 282, 31085. (b) Alexander, R. L.; Bates, D. J. P.; Wright,
M. W.; King, S. B.; Morrow, C. S. Biochemistry 2006, 45, 7889.
(12) See, for example: Spanton, S. G.; Prestwich, G. D. Tetrahedron 1982,
38, 1921.
(13) (a) Itoh, T.; Fairall, L.; Amin, K.; Inaba, Y.; Szanto, A.; Balint, B. L.;
Nagy, L.; Yamamoto, K.; Schwabe, J. W. R. Nat. Struct. Mol. Biol. 2008, 15,
924. For a commentary, see: Nettles, K. W. Nat. Struct. Mol. Biol. 2008, 15,
893. (b) Waku, T.; Shiraki, T.; Oyama, T.; Fujimoto, Y.; Maebara, K.;
Kamiya, N.; Jingami, H.; Morikawa, K. J. Mol. Biol. 2009, 385, 188. This
nitro lipid-PPAR-γ activation concept has recently been investigated:
Schopfer, F. J.; Cole, M. P.; Groeger, A. L.; Chen, C.-S.; Khoo, N. K. H.;
Woodcock, S. R.; Golin-Bisello, F.; Motanya, U. N.; Li, Y.; Zhang, J.;
Garcia-Barrio, M. T.; Rudolf, T. K.; Rudolf, V.; Bonacci, G.; Baker, P. R. S.;
Xu, H. E.; Batthyany, C. I.; Chen, Y. E.; Hallis, T. M.; Freeman, B. A. J. Biol.
Chem. 2010, 285, 12321.
(14) Performed by Cerep, France (www.cerep.fr) according to: Ferry, G.;
Bruneau, V.; Beauverger, P.; Goussard, M.; Rodriguez, M.; Lamamy, V.;
Dromaint, S.; Canet, E.; Galizzi, J. -P.; Boutin, J. A. Eur. J. Pharmacol.
2001, 417, 77.
(10) Denmark, S. E.; Marcin, L. R. J. Org. Chem. 1993, 58, 3850.
J. Org. Chem. Vol. 75, No. 15, 2010 5335

Dunny and Evans
JOCNote
SCHEME 3. Plausible Mechanism for the Activation of PPAR-γ by the Electrophilic Nitro Lipid 2a
24 h at room temperature. Water (10 mL) and diethyl ether
(10 mL) were added, and the solution was transferred to a
separating funnel where it was washed with a saturated aqueous
solution of NaHCO3 (10 mL), brine (10 mL), and diethyl ether
(2 Â 10 mL). The combined organic layers were dried with
MgSO4 and filtered, and the solvent was removed in vacuo.
Purification by silica gel column chromatography (pentane/
diethyl ether 19:1 to 9:1) yielded the product 8 (0.45 g, 62%)
as a mixture of diastereoisomers. Nitro compound 4 (0.11 g,
30%) was also recovered: Rf = 0.03 (pentane/diethyl ether 9:1);
IR (film) 3450, 3100, 2929, 2857, 1739, 1551, 1438, 1377, 1201,
1169, 1107, 1030, 877 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.89
(t, J = 8 Hz, 3H), 1.29-1.63 (m, 18H), 2.03 (broad q, J = 8 Hz,
2H), 2.28 (t, J = 8 Hz, 2H), 2.54-2.61 (m, 1H), 2.78-2.92 (m,
1H), 3.67 (s, 3H), 3.85-3.94 (m, 0.6H), 4.03-4.09 (m, 0.4H),
(q, J = 8 Hz, 2H), 2.31 (t, J = 8 Hz, 2H), 3.34 (d, J = 8 Hz, 2H),
3.67 (s, 3H), 5.21-5.29 (m, 1H), 5.46-5.54 (m, 1H), 7.08 (t, J =
8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.0, 22.5, 24.82,
24.83, 27.4, 27.9, 28.4, 28.93, 28.95, 29.0, 29.1, 31.5, 34.0, 51.4,
123.2, 133.0, 136.4, 150.6, 174.1; HRMS C19H34NO4 calcd
340.2488, found 340.2471.
(9E,12Z)-10-Nitrooctadeca-9,12-dienoic Acid (2a). Lipozyme
TL 100 L (400 mg) was added to a solution of nitrated linoleic
acid methyl ester 9 (16 mg, 0.049 mmol) in acetone (0.6 mL) and
aqueous phosphate buffer (6 mL, pH 7.4). The solution was
vigorously stirred at room temperature for 24 h before acidifica-
tion with 1 M HCl solution (pH 3) and extraction with diethyl
ether (5 Â 10 mL). The combined organic layers were dried with
MgSO4 and filtered, and the solvent was removed in vacuo.
Purification by silica gel column chromatography (pentane/
diethyl ether 5:1) yielded the title compound 2a (11 mg, 73%)
as a yellow oil.8 Methyl ester 9 (4 mg, 25%) was also recovered:
Rf = 0.07 (pentane/diethyl ether 5:1); IR (film) 3278, 3017, 2918,
2850, 1710, 1522, 1336, 911 cm-1; 1H NMR (400 MHz, CDCl3) δ
0.83-0.95 (m, 3H), 1.18-1.67 (m, 16H), 2.13 (q, J = 8 Hz, 2H),
2.24 (q, J = 8 Hz, 2H), 2.36 (t, J = 8 Hz, 2H), 3.34 (d, J = 8 Hz,
2H), 5.21-5.30 (m, 1H), 5.46-5.54 (m, 1H), 7.08 (t, J = 8 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.6, 24.6, 24.9, 27.4,
27.9, 28.4, 28.9, 28.95, 29.0, 29.1, 31.6, 33.4, 123.2, 133.0, 136.4,
150.7, 176.9; HRMS C18H32NO4 calcd326.2331, found326.2318.
4.41-4.47 (m, 1H), 5.25-5.33 (m, 1H), 5.54-5.60 (m, 1H); 13
C
NMR (100 MHz, CDCl3) δ 14.0, 22.5, 24.8, 25.3, 26.3, 27.2,
27.25, 28.6, 28.95, 29.0, 29.05, 29.10, 29.15, 31.45, 31.50, 33.2,
33.6, 34.0, 51.5, 71.6, 72.1, 91.9, 92.2, 121.5, 122.1, 135.0, 135.3,
174.3; HRMS C19H36NO5 calcd 358.2593, found 358.2579.
(9E,12Z)-Methyl 10-Nitrooctadeca-9,12-dienoate (9). Under
nitrogen, a solution of (Z)-methyl 9-hydroxy-10-nitrooctadec-
12-enoate (8) (169 mg, 0.49 mmol), DMAP (6 mg, 0.05 mmol),
and acetic anhydride (0.06 mL, 0.63 mmol) was stirred in diethyl
ether (1 mL) for 6 h and then concentrated. DMAP (73 mg,
0.60 mmol) was added to a solution of the crude diastereoiso-
meric nitroacetates in CH2Cl2 (2 mL), and the solution was
stirred at room temperature for 24 h. The solution was diluted
with CH2Cl2 (10 mL) followed by washing with water (10 mL),
0.1 M HCl (10 mL), brine (10 mL), and CH2Cl2 (2 Â 10 mL). The
combined organic layers were dried with MgSO4 and filtered,
and the solvent was removed in vacuo. Purification by silica gel
column chromatography (pentane/diethyl ether 15:1) yielded
the product 9 (72 mg, 45%) as a clear oil. Further elution yielded
the nitroacetates (42 mg, 26%) (HRMS C21H38NO6 calcd
400.2699, found 400.2679): Rf = 0.5 (pentane/diethyl ether;
9:1); IR (film) 3050, 2928, 2857, 1742, 1643, 1556, 1436, 1371,
1227, 1162, 1026 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.90 (t,
J = 8 Hz, 3H), 1.26-1.66 (m, 16H), 2.12 (q, J = 8 Hz, 2H), 2.24
Acknowledgment. We thank University College Dublin
for financial support and the Irish Research Council for
Science Engineering and Technology (IRCSET) for a post-
graduate scholarship (E.D.). Dr. Jimmy Muldoon is thanked
for assistance with NMR spectroscopy, and Dr. Cornelius
O’Connor is acknowledged for helpful advice concerning the
nitro aldol reaction reported.
Supporting Information Available: Experimental details and
copies of proton, carbon NMR spectra and biological data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
5336 J. Org. Chem. Vol. 75, No. 15, 2010