Synthesis and biological evaluation of 1-[2-cyano-3,12-dioxooleana-1,9(11)- dien-28-oyl]-4-ethynylimidazole. A novel and highly potent anti-inflammatory and cytoprotective agent

Article abstract of DOI:10.1016/j.bmcl.2011.03.018

To explore more potent N-acylimidazole analogues of CDDO than CDDO-Im, which is one of the most potent compounds in several widely used bioassays related to protection against inflammation and carcinogenesis; we have synthesized and evaluated five new N-a

Full text of DOI:10.1016/j.bmcl.2011.03.018

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2188–2191
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1-[2-cyano-3,12-dioxooleana-
1,9(11)-dien-28-oyl]-4-ethynylimidazole. A novel and highly
potent anti-inflammatory and cytoprotective agent
Tadashi Honda a,b, , Albena T. Dinkova-Kostova c,d, Emilie David b, Eric M. Padegimas b,
⇑
Chitra Sundararajan b, Melean Visnick e, Ron Bumeister e, W. Christian Wigley e
a Department of Chemistry, Institute of Chemical Biology and Drug Discovery, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
b Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA
c Biomedical Research Institute, University of Dundee, Dundee DD1 9SY, Scotland, UK
d Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
e Reata Pharmaceuticals Inc., Irving, TX 75063, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore more potent N-acylimidazole analogues of CDDO than CDDO-Im, which is one of the most
potent compounds in several widely used bioassays related to protection against inflammation and car-
cinogenesis; we have synthesized and evaluated five new N-acyl(acetylenic)imidazole analogues. Among
them, 4-ethynylimidazole 4 is nearly equivalent to CDDO-Im in potency in these bioassays. Remarkably,
the solid form of 4 is more stable than that of CDDO-Im. These findings suggest that 4 is a very promising
anti-inflammatory and cytoprotective agent and its further preclinical evaluation is warranted.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 January 2011
Revised 3 March 2011
Accepted 4 March 2011
Keywords:
Triterpene
Oleanolic acid
Acetylenicimidazole
Inhibitors of nitric oxide production
Inducers of heme oxygenase-1 and
NQO1
Over the past decade, we have been engaged in the improve-
ment of anti-inflammatory and cytoprotective activity of oleanolic
acid, a naturally occurring triterpenoid. This led to the discovery of
2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, bar-
doxolone).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 diabetes mel-
litus patients, for which there exists a significant unmet medical
need. Bardoxolone methyl significantly increases eGFR (estimated
glomerular filtration rate) in more than 90% of the patients.3
We have been exploring the mechanism of action of CDDO and
its derivatives. Towards that end, the identification of the protein
targets of CDDO is very important. The N-acylimidazole is known
to be very reactive towards various nucleophiles.
CDDO-Im was not observed, most likely because the N-acylimidaz-
ole group is located at C17, a much hindered position. However,
introduction of the N-acylimidazole group greatly enhanced po-
tency, and CDDO-Im is now one of the most potent compounds
in our pool of semisynthetic triterpenoids which we have previ-
ously evaluated in various bioassays in vitro and in vivo.4–6
CDDO-Im is more potent and less toxic in rodents than CDDO-
Me. However, during feasibility studies on CDDO-Im, it was found
that it is unstable upon incubation in human plasma7 and its solid
form undergoes decomposition within one year when stored at
4 °C.
To explore more potent and stable N-acylimidazole analogues
of CDDO, we have synthesized various N-acylimidazoles of CDDO
using commercially available imidazoles (e.g., 2-methyl, 4-methyl,
2,4-dimethyl, 2-ethyl, 4-bromo-, 2-phenyl, etc.) However, these
analogues did not show any satisfactory results when tested for
potency and stability. Recently, we found that TBE-31 (Fig. 1) is
the most potent compound amongst semisynthetic triterpenoids
and synthetic tricycles,7–10 and that the acetylene group is essen-
tial for strong potency. Therefore, we reasoned that N-acyl(acety-
lenic)imidazoles of CDDO, 1–5 might be interesting and
important (Fig. 2). Indeed, these compounds are more potent than
Therefore, we envisioned that the N-acylimidazole derivative of
CDDO (CDDO-Im) would be a useful tool for identification of the
protein targets. Unfortunately, formation of protein adducts with
⇑
Corresponding author at present address: Department of Chemistry, Institute of
Chemical Biology & Drug Discovery, State University of New York at Stony Brook,
Stony Brook, NY 11794 USA. Tel.: +1 631 632 7162; fax: +1 631 632 7942.
E-mail address: tadashi.honda@stonybrook.edu (T. Honda).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.018

T. Honda et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2188–2191
2189
Table 1
Inhibitory activity of new compounds 1–5 on NO production induced by IFN-
RAW cells and their NQO1–inducing potency in Hepa1c1c7 cells
c
in
21
19
13
O
E
17
12
C
22
11
9
R
D
1
CO2H
NC
O
2
16
10
4
H
A
15
B
6
7
HO
3
H
H
Oleanolic acid
O
CDDO (bardoxolone): R = CO2H
CDDO-Me (bardoxolonemethyl):
CN
Compound
R1
R2
IC50 (nM)
CDb (nM)
a
R = CO2Me
NC
O
N
1
2
3
4
–C„CH
–C„C–Ph
–C„C–Me
H
H
H
H
H
Me
–C„CH
–C„CPh
H
2.3 0.7
4.0 1.0
2.0 0.1
1.4 0.3
7.8 1.0
0.6 0.2
11.0 1.0
0.9
1.2
0.4
0.4
CDDO-Im: R =
N
H
O
TBE-31
5
CDDO-Imc
CDDO
0.2
3.0
Figure 1. Structures of oleanolic acid, CDDO, CDDO-Me, CDDO-Im, and TBE-31.
a
RAW 264.7 mouse macrophages were plated in 96-well plates at 30,000 cells/
well in triplicate. On the next day cells were pre-treated with DMSO or test com-
pounds (0–200 nM dose range) for 2 h, followed by IFN- treatment for an addi-
c
tional 24 h. NO concentration in media was determined using the Griess reagent
system (Promega). Cell viability was assessed using WST-1 reagent (Roche). IC50
values were determined based on suppression of IFN-c-induced NO production
normalized to cell viability.
b
Hepa1c1c7 cells (10,000 cells/well) were grown in 96-well plates for 24 h and
then treated with serial dilutions of compounds for 48 h. The concentration
required to double (CD) the specific enzyme activity of NQO1 was used to quantity
inducer potency. The value is based on the activity from eight replicate wells at
each concentration. The standard deviation in each case was between 5 and 10%.
c
Freshly prepared CDDO-Im was used for this evaluation. Its CD value (0.2 nM) is
16 times lower than the one that was previously reported (3.3 nM).7,17 The latter
assay was done with a DMSO solution of CDDO-Im that had been stored, and the
discrepancy in inducer potency can be explained by the now known instability of
CDDO-Im. The new CD value is in good agreement with the potency in the iNOS
assay.
Figure 3. Correlation of potencies of new compounds 1–4, CDDO and CDDO-Im as
inducers of NQO1 in Hepa1c1c7 murine hepatoma cells, expressed as CD values,
and for suppression of iNOS induction by IFN-
c in RAW cells, expressed as IC50
Figure 2. Structures of 1–5 and chemical shifts (d) of their imidazole protons. 1H
NMR spectra of 1–4 were measured in DMSO-d6 and 5 in CD3OD.
values. The linear correlation coefficient is r2 = 0.98.
The acetylenic-1H-imidazoles 6–10 are not commercially avail-
able, and were previously unknown except for 1013 (structure, see
Scheme 2). These acetylenic-1H-imidazoles were synthesized by
the sequence shown in Scheme 1. 2-Ethynyl-1H-imidazole (6)14
could not be obtained from the known imidazole 1113 under two
classic conditions, tetra(n-butyl)ammonium fluoride (TBAF) in
THF and 49% aqueous hydrofluoric acid in acetonitrile. After survey
of various conditions, we have found that trifluoroacetic acid (TFA)
in THF (at room temperature, overnight) is very efficient for the
CDDO for inhibition of nitric oxide (NO) production in RAW 264.7
cells and induction of the cytoprotective enzymes, NAD(P)H: qui-
none oxidoreductase 1(NQO1)11 and heme oxygenase-1 (HO-1)12
(Table 1 and Figs. 3 and 4). Particularly, 4-ethynylimidazole 4 is
nearly equivalent to CDDO-Im in potency in these bioassays. More-
over, the solid state of 4 is more stable than that of CDDO-Im. Here-
in we report the synthesis, anti-inflammatory and cytoprotective
potency of these new derivatives.

2190
T. Honda et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2188–2191
Figure 4. Compounds 1–4 induce HO-1. RAW 264.7 cells were plated in a 24-well
dish at 100,000 cells/well. On the next day cells were treated with DMSO or test
compounds overnight for 16 h and harvested in tricine sample buffer with b-ME.
Lysates were separated by SDS–PAGE and probed with the antibodies against HO-1
(Santa Cruz) or GAPDH (Sigma).
Scheme 2. Synthesis of new imidazole analogues 1–5.
New imidazole analogues 1–516 were synthesized by condensa-
tion of known acyl chloride of CDDO 17 (1 equiv) and the corre-
sponding imidazoles 6–10 (2 equiv) in benzene (at room
temperature, overnight) (Scheme 2, yields are shown in the
scheme).4 Regiochemistry of the ethynyl group at imidazole moi-
ety of 4 was determined by comparison of chemical shifts of imid-
azole protons of 4 with those of 1 (Fig. 2). The C4 imidazole proton
of 1 appears at d 7.12 ppm (d, J = 1.5 Hz) and the C5 proton appears
at d 8.10 ppm (d, J = 1.5 Hz) due to deshielding effect of carbonyl
group. On the other hand, two imidazole protons of 4 appear at d
8.26 and 8.59 ppm (broad singlet each) due to deshielding effect
of carbonyl group. Therefore, the ethynyl group is placed at C4 of
imidazole moiety of 4. Because two imidazole protons of 5 show
similar chemical shift (d 8.17 and 8.43 ppm) to those of 4, we have
assigned the structure of 5 as shown in Figure 2. This regiochemist-
ry is kinetically supported because the acyl chloride moiety of 17
would preferentially attack the less hindered nitrogen of imida-
zoles. An imidazole proton of 3 is observed at d 7.74 ppm. We have
assigned the structure of 3 as shown in Figure 2 for the following
reasons: (1) The chemical shift value is close to C5 protons on
the imidazoles of 1 and 2 (d8.10 and 8.16 ppm) rather than the
C4 protons (d7.12 and 7.20 ppm). (2) The regiochemistry is kineti-
cally supported as described above.
We evaluated the potency of the new compounds 1–5 for inhi-
bition of NO production in RAW 264.7 cells stimulated with inter-
feron-c. The IC50 values are shown in Table 1. All compounds 1–5
Scheme 1. Synthesis of new imidazoles 6–9. Reagents and conditions: (a) TFA, THF
(rt, overnight); (b) MeI, MeLi, THF; (c) HC„C-TMS, Pd(PPh3)2Cl2, CuI, Et3N (60 °C,
overnight); (d) aqueous NaOH, MeOH (rt); (e) PhC„CH, Pd(PPh3)2Cl2, CuI, Et3N
(60 °C, overnight).
are more potent than CDDO. Notably, 4 is nearly equivalent to
CDDO-Im in potency. Compounds 1 and 3 are slightly less potent
than 4. Two (phenylethynyl)imidazoles 2 and 5 are much less po-
tent than 4. These results indicate that a bulky phenyl group de-
creases potency.
We evaluated compounds 1–4 for induction of the phase 2 cyto-
protective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells. All
compounds 1–4 are more potent than CDDO. Notably, compounds
3 and 4 are nearly as potent as CDDO-Im. We previously demon-
strated a linear correlation between inhibitory activity on NO pro-
duction (IC50) and NQO1 inducer potency (CD) of oleanolic acid
derivatives.17 In this series of N-acylimidazole derivatives of CDDO,
we observed an even more striking correlation (r2 = 0.98, Fig. 3)
than the previously reported one (r2 = 0.91).17
cleavage of trimethylsilylethoxymethyl (SEM) group from N-SEM-
imidazoles. Imidazole 6 was obtained in 90% yield from 11 by this
method. 4-Methyl-2-(1-propynyl)-1H-imidazole (7) was synthe-
sized as follows. Imidazole 12 was prepared in 38% yield by the
treatment of 11 with MeI and MeLi. The removal of SEM group
by TFA in THF gave 7 in 99% yield. 4-Acetylenic-1H-imidazoles 8
and 9 were synthesized as follows. Sonogashira coupling between
known 1315 and trimethylsilylacetylene (TMS-acetylene) in the
presence of Pd(PPh3)2Cl2 and CuI in triethylamine afforded 14 in
51% yield. The TMS group of 14 was removed in a mixture of aque-
ous NaOH solution and methanol to give 15 in 88% yield. The re-
moval of SEM group yielded 4-ethynyl-1H-imidazole (8) in 99%
yield. Imidazole 16 was prepared in 38% yield by Sonogashira cou-
pling between 13 and phenylacetylene under the same conditions
as for 14. Removal of the SEM group gave 4-(phenylethynyl)-1H-
imidazole (9) in 78% yield.
We have also evaluated compounds 1–4 for induction of the
anti-inflammatory and cytoprotective enzyme, heme oxygenase-
1 (HO-1) in RAW 264.7 cells (Fig. 4). 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

T. Honda et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2188–2191
2191
role.8 Even in this assay, compounds 1–4 are much more potent
than CDDO. Particularly, compound 4 is as potent as or slightly
more potent than CDDO-Im.
Notably, the solid forms of compounds 1–4 are more stable than
that of CDDO-Im. Whilst the solids of 1–4 were stable for at least
three years at 4 °C, the solid of CDDO-Im was decomposed one year
after storage under the same conditions. We speculate that the
acetylenicimidazoles of these compounds can prevent nucleophilic
attack to a greater extent than the imidazole of CDDO-Im, which
has no substituents.
In summary, we have found a new promising compound, 1-[2-
cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4-ethynylimidazole
(4), which is nearly as potent as CDDO-Im in three bioassays and
whose solid is more stable than that of CDDO-Im. Presently further
preclinical studies of this compound are in progress.
8. Honda, T.; Sundararajan, C.; Yoshizawa, H.; Su, X.; Honda, Y.; Liby, K. T.; Sporn,
M. B.; Gribble, G. W. J. Med. Chem. 2007, 50, 1731.
9. Dinkova-Kostova, A. T.; Talalay, P.; Sharkey, J.; Zhang, Y.; Holtzclaw, W. D.; Xiu
Jun Wang, X. J.; David, E.; Schiavoni, K. H.; Finlayson, S.; Dale, F.; Mierke, D. F.;
Honda, T. J. Biol. Chem. 2010, 285, 33747.
10. Honda, T.; Yoshizawa, H.; Sundararajan, C.; David, E.; Lajoie, M. J.; Favaloro, F.
G. Jr.; Janosik, T.; Su, X.; Honda, Y.; Roebuck, B. D.; Gribble, G. W. J. Med. Chem.,
in press. doi:10.1021/jm101445p.
11. Ross, D.; Kepa, J. K.; Winski, S. L.; Beall, H. D.; Anwar, A.; Siegel, D. Chem. Biol.
Interact. 2000, 129, 77.
12. Ryter, S. W.; Alam, J.; Choi, A. M. Physiol. Rev. 2006, 86, 583.
13. Paul, D.; Wytko, J. A.; Koepf, M.; Weiss, J. Inorg. Chem. 2002, 41, 3699.
14. Cosford, N. D.; Kamenecka, T.; Roppe, J. R. U.S. Patent 0085523 A1, 2005. The
inventorsprepared an inseparable mixture of 6 and 2-formyl-1H-imidazole from
2-formyl-1H-imidazole with dimethyl (1-diazo-2-oxopropyl)phosphonate.
15. He, Y.; Chen, Y.; Du, H.; Schmid, L. A.; Lovely, C. J. Tetrahedron Lett. 2004, 45,
5529.
16. All new compounds 1–5 provided acceptable HRMS data ( 5 ppm) and 1H NMR
spectra that exhibit no discernible impurities. 13C NMR was not measured
because of the low solubility in various solvents (e.g., CDCl3, DMSO-d6 etc.).
Instead of the optical rotations ([
were measured. 1-(2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-2-
ethynylimidazole (1): CD: e228 = +1.39, e247 = +1.32
e237 = À0.58, and
(c = 0.0027, CH2Cl2); 1H NMR (DMSO-d6)
8.65 (1H, s), 8.10 (1H, d,
J = 1.5 Hz), 7.12 (1H, d, J = 1.5 Hz), 6.26 (1H, s), 4.50 (1H, s), 3.09 (1H, m),
2.82 (1H, d, J = 4.8 Hz), 1.43, 1.26, 1.16, 1.05, 0.96, 0.95, 0.91 (each 3H, s); MS
(ESI+) m/z 566 [M+H]+; HRMS: (ESI+) calcd for C36H43N3O3 + H 566.3383,
found 566.3372. 1-(2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-2-
a]D), circular dichroism (CD) values
Acknowledgments
D
D
D
d
We thank Isaac Trevino (Reata Pharmaceuticals) for expert
technical assistance and Jeff Cui (Dartmouth College) for his helpful
assistance. This investigation was supported by funds from John
Zabriskie ’61 Undergraduate Research Fellowship, Reata Pharma-
ceuticals, and Cancer Research UK (C20953/A10270). E.M.P. was a
James O. Freedman Presidential Scholar at Dartmouth College.
phenylethynylimidazole
(2):
CD:
D
e224 = –3.40
and
De247 = +3.69
(c = 0.00078, CH2Cl2); 1H NMR (DMSO-d6)
d
8.62 (1H, s), 8.16 (1H, d,
J = 1.5 Hz), 7.48 (5H, m), 7.20 (1H, d, J = 1.5 Hz), 6.23 (1H, s), 3.11 (1H, d,
J = 6.0 Hz), 2.88 (1H, d, J = 3.0 Hz), 1.31, 1.18, 1.13, 1.01 (each 3H, s), 0.96 (6H,
s), 0.92 (3H, s); MS (ESI+) m/z 642 [M+H]+; HRMS (ESI+) calcd for
References and notes
C
42H48N3O3 + H 642.3696, found 642.3711. 1-(2-Cyano-3,12-dioxooleana-
1,9(11)-dien-28-oyl)-4-methyl-2-(1-propynyl)imidazole (3): CD: e217 = –
2.34 and
e235 = +1.75 (c = 0.0013, CH2Cl2); 1H NMR (DMSO-d6) d 8.66 (1H,
D
D
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. J. Med. Chem. 2000, 43, 4233.
3. Schwartz, S. L.; Denham, D. S.; Hurwitz, C. A.; Meyer, C. J.; Pergola, P.E.
American Diabetes Association Meeting, New Orleans, LA, Abstract No. 112-OR
(2009).
4. 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.
5. Place, A. E.; Suh, N.; Williams, C. R.; Risingsong, R.; Honda, T.; Honda, Y.;
Gribble, G. W.; Leesnitzer, L. M.; Stimmel, J. B.; Wilson, T. M.; Rosen, E.; Sporn,
M. B. Clin. Cancer Res. 2003, 9, 2798.
6. Yates, M. S.; Kwak, M.-K.; Egner, P. A.; Groopman, J. D.; Bodreddigari, S.; Sutter,
T. R.; Baumgartner, K. J.; Roebuck, B. D.; Liby, K. T.; Yore, M. M.; Honda, T.;
Gribble, G. W.; Sporn, M. B.; Kensler, T. W. Cancer Res. 2006, 66, 2488.
7. Liby, K.; Yore, M. M.; Roebuck, B. D.; Baumgartner, K. J.; Honda, T.;
Sundararajan, C.; Yoshizawa, H.; Gribble, G. W.; Williams, C. R.; Risingsong,
R.; Royce, D. B.; Dinkova-Kostova, A. T.; Stephenson, K. K.; Egner, P. A.; Yates, M.
S.; Groopman, J. D.; Kensler, T. W.; Sporn, M. B. Cancer Res. 2008, 68, 6727.
s), 7.74 (1H, s), 6.25 (1H, s), 3.05 (1H, d, J = 13 Hz), 2.87 (1H, d, J = 5.0 Hz), 2.10,
2.00, 1.43, 1.28, 1.16, 1.05, 0.95, 0.94, 0.91 (each 3H, s); MS (ESI+) m/z 594
[M+H]+; HRMS: (ESI+) calcd for C38H47N3O3 + H 594.3696, found 594.3680. 1-
(2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-4-ethynylimidazole (4): CD:
D
e248 = +1.08 (c = 0.0030, CH2Cl2); 1H NMR (DMSO-d6) d 8.66 (1H, s), 8.59 (1H,
s), 8.26 (1H, s), 6.24 (1H, s), 4.25 (1H, s), 3.09 (1H, d, J = 4.5 Hz), 2.96 (1H, d,
J = 13 Hz), 1.42, 1.19, 1.16, 1.05, 0.97 (each 3H, s), 0.91 (6H, s); MS (ESI+) m/z
566 [M+H]+; HRMS: (ESI+) calcd for C36H43N3O3 + H 566.3383, found 566.3391.
1-(2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-4-phenylethynylimidazole
(5): CD:
D
e274 = –0.76 (c = 0.0034, CH2Cl2); 1H NMR (CD3OD) d 8.61 (1H, s), 8.43
(1H, s), 8.17 (1H, s), 7.53 (2H, m), 7.42 (3H, m), 6.18 (1H, s), 3.17 (1H, m), 1.54,
1.34, 1.24 1.17, 1.11, 1.03, 0.97 (each 3H, s); MS (ESI+) m/z 642 [M+H]+; HRMS:
(ESI+) calcd for C42H47N3O3 + H 642.3696, found 642.3684.
17. Dinkova-Kostova, A. T.; Liby, K. T.; Stephenson, K. K.; Holtzclaw, W. D.; Gao, X.;
Suh, N.; Williams, C.; Risingsong, R.; Honda, T.; Gribble, G. W.; Sporn, M. B.;
Talalay, P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4584.