2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride. A novel and highly potent anti-inflammatory and cytoprotective agent

Article abstract of DOI:10.1016/j.bmcl.2010.02.007

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized, which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflammation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride is in progress.

Full text of DOI:10.1016/j.bmcl.2010.02.007

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2275–2278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride. A novel
and highly potent anti-inflammatory and cytoprotective agent
a
a
a
b
Tadashi Honda a, , Eric M. Padegimas , Emilie David , Chitra Sundararajan , Karen T. Liby ,
*
Charlotte Williams b, Michael B. Sporn b, Melean Visnick c
a Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA
b Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA
c Reata Pharmaceutical Inc., Dallas, TX 75063, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized,
which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar
to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflam-
mation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride
levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride
is in progress.
Received 18 December 2009
Revised 1 February 2010
Accepted 2 February 2010
Available online 6 February 2010
Keywords:
Triterpene
Ó 2010 Elsevier Ltd. All rights reserved.
Oleanolic acid
Acid anhydride
Inhibitors of nitric oxide production
Inducers of heme oxygenase-1
Over the past decade, we have been engaged in the improve-
ment of anti-inflammatory and antiproliferative activity of olean-
olic acid, a naturally occurring triterpenoid. This led to the
discovery of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
(CDDO, bardoxolone).1,2 Its methyl ester (CDDO-Me, bardoxolone
methyl) is presently being developed in late Phase II clinical trials
for the treatment of severe chronic kidney disease in type 2 diabe-
tes mellitus patients. Although CDDO was also evaluated in Phase I
clinical trials for the treatment of leukemia and solid cancer,3 due
to its low bioavailability, the evaluation has been suspended. To in-
crease the bioavailability, in particular through oral administration
(po), we designed some possible pro-drugs, but unfortunately they
were not successful as pro-drugs for the following reasons: (1)
Some analogues did not generate CDDO because the linkage be-
tween CDDO and the leaving groups was too robust. (2) Some ana-
logues did not increase the bioavailability through po because of
their high polarity.
that oleanane and ursane triterpenoid anhydrides have not been
reported. Although only acetoxybetulinic acid anhydride with a lu-
pane triterpenoid skeleton was reported,5 it was produced as a by-
product (2% yield) during acetylation of betulinic acid (Fig. 1).
Herein we report the first synthesis of oleanane triterpenoid anhy-
drides and the promising biological results for CDDO anhydride 1.
Since a carboxyl group at C17 of oleanolic acid is very hindered,
we anticipated that the anhydride synthesis would be difficult.
However, classical conditions (Et3N, THF, rt) gave the desired anhy-
dride6 from CDDO and CDDO-Cl7 in 96% yield (Scheme 1). Because
a naturally occurring oleanane triterpenoid anhydride has not been
reported as we described above, we have applied the same condi-
tions for the synthesis of oleanonic acid anhydride6,8 (Fig. 2). The
conditions gave the anhydride in 85% yield from oleanonic acid9
and oleanonyl chloride.9 We have also tried to synthesize a new lu-
pane triterpenoid anhydride, 2-cyano-3-oxolupa-1,20(29)-dien-
28-oic acid anhydride (lupane anhydride 2, Fig. 2)6 to compare bio-
logical properties of 1 with those of 2. However, these reaction
conditions gave 2 in low yield (14%) from 2-cyano-3-oxolupa-
1,20(29)-dien-28-oic acid (3)10 and its acyl chloride.10 These re-
sults suggest that a carboxyl group at C17 of lupane skeleton is
more hindered than that of oleanane skeleton.
After several attempts, we have envisioned that CDDO anhy-
dride 1 would have better bioavailability through po4 than CDDO
because an anhydride is generally less polar than the correspond-
ing acid. Thus, we have decided to investigate properties of CDDO
anhydride including the possibility that CDDO anhydride would
work as a pro-drug of CDDO. Our literature survey has disclosed
We have evaluated the potency of new anhydrides 1 and 2 in
comparison with their corresponding acids, for inhibition of NO
production in RAW 264.7 cells stimulated with interferon-
assay, Table 1). The IC50 values are shown in Table 1. Notably,
c (iNOS
* Corresponding author. Tel.: +1 603 646 8104; fax: +1 603 646 3946.
E-mail address: th9@dartmouth.edu (T. Honda).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.007

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T. Honda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2275–2278
Figure 2. Structures of oleanonic acid anhydride and lupane anhydride 2.
Table 1
Inhibition of NO production in RAW cells stimulated with interferon-
anhydrides 1 and 2
c by new
Figure 1. Structures of oleanolic acid, CDDO, CDDO-Me, betulinic acid and
acetoxybetulinic acid anhydride.
Compd
IC50 (nM)
1
2
14.7 3.8a
>10,000a
28.3 11.6a
200b
CDDO
3
a
RAW 264.7 cells were treated with various concentrations of compounds and
interferon- (10 ng/mL) for 24 h. Supernatants were analyzed for NO by the Griess
c
reaction. IC50 values are an average of three experiments.
b
This data was previously reported in Ref. 10.
Figure 3. CDDO and CDDO anhydride 1 induce HO-1 in RAW 264.7 cells. Cells were
incubated with compounds (100 nM) for 6 h. Total cell lysates were analyzed by
SDS–PAGE, probed with an HO-1 antibody, and developed by ECL. The HO-1 in the
negative control lane is basal expression of HO-1.
is inactive while the corresponding acid
potency.
3 shows moderate
If CDDO anhydride 1 is immediately converted to CDDO in the
RAW cell growth medium (RMPI supplemented with 10% fetal bo-
vine serum) at 37 °C, because the cells take in CDDO, we have
merely evaluated the potency of CDDO instead of the potency of
1 in two assays above. Thus, we have evaluated the lifetime of
CDDO anhydride 1 in the cell growth medium. The results are
shown in Figure 4. Even after 24 h, the percentage of material
remaining of 1 is much higher than that of CDDO. Therefore we
have concluded that the cells take in 1 in the two assays above.
Subsequent to the in vitro assay, we have evaluated the potency
of CDDO anhydride 1 for induction of HO-1 in the liver (in vivo, ip
injection4). CDDO anhydride 1 is as potent as CDDO in the liver at
Scheme 1. Synthesis of CDDO anhydride.
CDDO anhydride 1 is about 2 times more potent than the corre-
sponding acid, CDDO, but lupane anhydride 2 is inactive whilst
the corresponding acid 3 shows moderate potency at 200 nM.
We have also evaluated 1 and 2 for induction of the anti-inflamma-
tory and cytoprotective enzyme, heme oxygenase-1 (HO-1) in RAW
cells (HO-1 assay, Fig. 3). There is major interest in stimulating HO-
1 as a protective enzyme in many chronic disease conditions in
which inflammation and oxidative stress play a key role.11 CDDO
anhydride 1 shows the same potency as CDDO. To the contrary, 2
2 lmol dosage (Fig. 5).

T. Honda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2275–2278
2277
yield of 2 is much lower than that of 1 from each corresponding
acid, a carboxyl group at C17 of lupane skeleton is considered to
be more hindered than that of oleanane skeleton. This speculation
would lead us to consider that the conversion of 2 to 3 is much
slower than that of 1 to CDDO in the cells. Lupane anhydride 2 is
deemed to be too large to affect protein targets. If this is the case,
CDDO anhydride 1 is also too large to affect protein targets. How-
ever, since 1 is converted to CDDO in the cells and affects protein
targets, 1 is seemingly potent. To the contrary, 2 is not converted
to 3 in the cells, 2 is inactive. Further pharmacokinetics studies
on CDDO anhydride are in progress. Through the studies, it would
be clarified which scenario is correct.
In summary, we have found the following interesting features
about CDDO anhydride. These features demonstrate that CDDO
anhydride is more promising than CDDO.
Figure 4. Lifetime of CDDO anhydride in the cell culture medium.
(1) CDDO anhydride is the first example of oleanane triterpe-
noid anhydride, which is easily synthesized in good yield
from the corresponding acid and acyl chloride.
(2) CDDO anhydride is more potent than CDDO in the iNOS
assay and is similar in potency to CDDO in the HO-1 assay
in vitro. CDDO anhydride is stable in the RAW cell growth
medium.
(3) CDDO anhydride is as potent as CDDO for induction of HO-1
in the liver (in vivo).
(4) Notably, CDDO anhydride levels in mouse tissues and blood
are higher than CDDO levels. As we expected, the bioavail-
ability of CDDO anhydride is improved in comparison with
CDDO.
(5) We have not clarified whether CDDO anhydride works as a
pro-drug of CDDO or whether CDDO anhydride itself is
potent in the iNOS assay and the HO-1 assay (in vitro and
in vivo).
Figure 5. CDDO anhydride 1 and CDDO induce HO-1 in the liver. CD-1 mice (4 per
group) were injected ip with 2 lmol of CDDO anhydride or CDDO. Six hours later,
livers were harvested and homogenized. Lysates were separated by SDS–PAGE,
probed with HO-1 antibodies, and developed by ECL. The tubulin blot is a loading
control.
To compare the bioavailability of CDDO anhydride and CDDO,
we have performed pharmacokinetics studies using mice (ip injec-
tion,4 Table 2). Notably, CDDO anhydride levels in mouse tissues
and blood are higher than CDDO levels. Particularly, CDDO anhy-
dride level in plasma is much higher than CDDO level. These data
demonstrate that bioavailability of CDDO anhydride is definitely
better than CDDO. CDDO was not detected in the tissues and blood
of mice that were injected with CDDO anhydride.
We have two possible scenarios that would account for the high
potency of CDDO anhydride in the three assays (iNOS assay and
HO-1 assay in vitro and in vivo). The first one is that CDDO anhy-
dride itself is active. The other possibility is that CDDO anhydride
works as a pro-drug of CDDO. The preliminary phamacokinetics
studies did not give any evidence that CDDO anhydride is con-
verted to CDDO in tissues and blood. This result would support
the former scenario. Lupane anhydride 2 is inactive in two bioas-
says (iNOS and HO-1 in vitro) while its corresponding acid 3 is ac-
tive in both. To the contrary, CDDO anhydride is more potent than
CDDO in the iNOS assay and is similar to CDDO in the HO-1 assay.
The difference between 1 and 2 in potency would support the lat-
ter scenario. As we described in the chemistry section, because the
Further preclinical evaluation of CDDO anhydride is in progress.
Acknowledgments
We thank Renee Risingsong and Darlene Royce (Dartmouth
Medical School) for expert technical assistance. This investigation
was supported by funds from NIH Grant R01-CA78814, from John
Zabriskie ’61 Undergraduate Research Fellowship, and from Reata
Pharmaceuticals. E.M.P. is a James O. Freedman Presidential Scho-
lar at Dartmouth College. M.B.S. is Oscar M. Cohn Professor.
References and notes
1. Honda, T.; Gribble, G. W.; Suh, N.; Finlay, H. J.; Rounds, B. V.; Bore, L.; Favaloro,
F. G., Jr.; Wang, Y.; Sporn, M. B. J. Med. Chem. 2000, 43, 1866.
2. Honda, T.; Rounds, B. V.; Bore, L.; Finlay, H. J.; Favaloro, F. G., Jr.; Suh, N.; Wang,
Y.; Sporn, M. B.; Gribble, G. W. J. Med. Chem. 2000, 43, 4233.
3. CDDO was evaluated as an injection despite its low water solubility, because
the bioavailability through oral administration is quite poor.
4. In preliminary stages, we used ip injection instead of po administration
because CDDO anhydride 1 is deemed to be readily converted to CDDO in the
stomach. In advanced stages, we will use duodenum injection and/or we will
administer 1 as an entric coated pill.
Table 2
5. Urban, M.; Sarek, J.; Klinot, J.; Korinkova, G.; Hajduch, M. J. Nat. Prod. 2004, 67,
1100.
Preliminary pharmacokinetics of CDDO anhydride and CDDO in mouse tissues and
blood
6. All new anhydrides provided acceptable HRMS data ( 5 ppm) and 1H NMR
spectra that exhibit no discernible impurities. CDDO anhydride 1: 1H NMR
(CDCl3) d 8.04 (1H, s), 5.95 (1H, s), 2.88 (1H, s), 1.49 (3H, s), 1.37 (3H, s), 1.25
(3H, s), 1.17 (3H, s), 1.01 (3H, s), 0.99 (3H, s), 0.91 (3H, s); 13C NMR d 198.3,
196.7, 174.5, 169.1, 165.9, 124.0, 114.8, 114.6, 49.9, 48.8, 47.9, 46.1, 45.2, 42.7,
42.3, 35.8, 34.3, 33.3, 32.0, 31.9, 31.8, 30.8, 27.9, 27.1, 26.8, 25.0, 23.03, 22.96,
21.8, 21.7, 18.4; MS (ESI+) m/z 966 [M+H]+; HRMS (ESI+) calcd for
C62H80N2O7+H: 965.6044, found: 965.6033. Oleanonic acid anhydride: 1H
NMR (CDCl3) d 5.34 (1H, s), 3.11 (1H, m), 2.84 (1H, m), 2.56 (1H, m), 2.36 (1H,
m), 1.49 (3H, s), 1.42 (2H, s), 1.16 (3H, s), 1.09 (3H, s), 1.05 (3H, s), 1.04 (3H, s),
0.94 (3H, s), 0.92 (5H, s), 0.85 (2H, s); 13C NMR (CDCl3) d 217.9, 173.1, 143.5,
123.0, 55.5, 48.6, 47.7, 47.1, 46.0, 45.9, 42.1, 41.5, 39.6, 39.4, 36.9, 34.4, 33.8,
CDDO anhydride
CDDO
Liver (
l
mol/kg)
mol/kg)
0.045 0.013
0.031 0.009
52 45
0.041 0.035
0.018 0.022
19 12
Lung (
l
Whole blood (
Plasma ( M)
lM)
l
43 29
2
1
Female CD-1 mice were injected ip with 2 lmol of compounds in DMSO–cremo-
phor–PBS (1:1:8). Six hours later, the mice were sacrificed and blood and tissues
were collected. Levels were quantified by HPLC/MS using compound added to
control blood and tissues for standard.

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T. Honda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2275–2278
33.2, 32.4, 31.6, 30.9, 27.7, 26.6, 26.0, 23.8, 23.7, 23.2, 21.7, 19.8, 17.3, 15.2, 8.8;
8. There are many publications, in which oleanonic acid was isolated from natural
sources. For example: Ikuta, A.; Itokawa, H. J. Nat. Prod. 1989, 52, 623.
9. Oleanonic acid was prepared in 95% yield from oleanolic acid by Jones
oxidation. Oleanonyl chloride was obtained in 87% yield by chlorination of
oleanonic acid with oxalyl chloride in CH2Cl2.
10. Honda, T.; Liby, K. T.; Su, X.; Sundararajan, C.; Honda, Y.; Suh, N.; Risingsong, R.;
Williams, C. R.; Royce, D. B.; Sporn, M. B.; Gribble, G. W. Bioorg. Med. Chem. Lett.
2006, 16, 6306.
MS (ESI+) m/z 892 [M+H]+; HRMS (ESI+) calcd for C60H90O5+H: 891.6867,
found: 891.6888. Lupane anhydride 2: 1H NMR (CDCl3) d 7.82 (1H, s), 4.76 (1H,
s), 4.66 (1H, s), 3.02 (1H, m), 2.37 (1H, m), 2.23 (1H, m), 2.00 (2H, m), 1.85 (1H,
m), 1.71 (3H, s), 1.27 (3H, s), 1.19 (3H, s), 1.13 (3H, s), 1.12 (3H, s), 1.08 (3H, s),
1.00 (3H, s). MS (ESI+) m/z 938 [M+H]+; HRMS (ESI+) calcd for C62H84N2O5+H:
937.6458, found: 937.6471.
7. Honda, T.; Honda, Y.; Favaloro, F. G., Jr.; Gribble, G. W.; Suh, N.; Place, A. E.;
Rendi, M. H.; Sporn, M. B. Bioorg. Med. Chem. Lett. 2002, 12, 1027.
11. Ryter, S. W.; Alam, J.; Choi, A. M. Physiol. Rev. 2006, 86, 583.