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its related analog, CDDO-methyl ester (“CDDO-Me”, bardoxolone
methyl), which is currently in clinical trial for treatment of chronic
kidney disease and pulmonary arterial hypertension, CDDO-Im has
not been developed for clinical use, in spite of its potency and safety
in many animal experiments. Its poor stability in human plasma
and concerns about its pharmacokinetics have been the principal
impediments to the clinical development of CDDO-Im as a practi-
cal drug for use in patients. With this in mind, we have therefore
undertaken the synthesis and biological evaluation of more than
30 new substituted imidazole analogues of CDDO-Im, in order to
improve its stability in plasma and pharmacokinetics.
for 15 min with the reagents and then resuspended in 100 l bind-
ing buffer. Data were analyzed with FlowJo 9.6.2 software.
2.4. In vitro iNOS suppression
RAW264.7 cells were plated in 96-well plates on day one
(2 × 104 cells per well). The next day, cells were treated with various
concentrations of triterpenoids and then stimulated with 10 ng/ml
IFN-␥ (R&D Systems, Minneapolis, MN) for 24 h. The Griess reac-
tion was used on the third day to measure NO accumulation in the
medium as nitrite. Plates were read at 595 nm [15].
We report here the structures of all these new com-
pounds, and then focus in detail on two of the most
useful,
namely
1-[2-Cyano-3,12-dioxooleana-1,9(11)-dien-
(“CDDO-2P-Im”) and
2.5. Stability assays
28-oyl]-4(-pyridin-2-yl)-1H-imidazole
1-[2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-
3-yl)-1H-imidazole (“CDDO-3P-Im”), to describe their overall
biological activities, which closely resemble those of CDDO-Im
itself, with the additional new benefit of improved stability in
plasma and better pharmacokinetics. These benefits, as well as
their impressive ability to suppress the development of lung cancer
in a mouse model that is highly relevant to human adenocarci-
noma of the lung, now make these new triterpenoids potential
candidates for clinical development.
Pooled human plasma was acquired from Innovative Research
(Novi, MI). Stock solutions of triterpenoids were prepared at
10 mM in HPLC-grade acetonitrile (Honeywell Burdick & Jackson,
Muskegon, MI) and diluted in plasma to a final concentration of
10 M. The samples were incubated at 37 ◦C for 0, 0.5, 1, 2, 4 or 6 h.
They were then extracted with ACN, vortexed and centrifuged at
14,000 rpm for 5 min and analyzed by LC–MS (Waters 2695HPLC
coupled to Waters ZQ mass spectrometer; Milford, MA). The start-
ing materials remaining in the plasma were determined by HPLC
measurement (AUC) using Waters MassLynx 4.1 software.
2. Materials and methods
2.6. Tissue distribution study
2.1. Reagents
All animal studies were done in accordance with the Guide for
the Care and Use of Laboratory Animals under protocols approved
by the Institutional Animal Care and Use Committee at Dartmouth.
Female C57BL/6 mice (6 per group) were gavaged with DMSO vehi-
cle or 1 mole of triterpenoid in DMSO, and 6 or 24 h later, blood,
liver, kidney, pancreas and lungs were harvested. Tissues were
homogenized in PBS, and all samples were extracted with ACN, son-
icated and centrifuged at 14,000 rpm for 15 min. The supernatants
were diluted with 20 mM ammonium acetate (1:1) and centrifuged
at 14,000 rpm for 5 min at 4 ◦C. Samples (100 l) were analyzed
by reverse phase HPLC on a Waters XTerra MS C18 5 m particle
column (2.1 × 150 mm). Samples were separated over an 8 min gra-
dient starting with 40%ACN/60% 10 mM ammonium acetate pH 7.4
and ending at 90% ACN:10% 10 mM ammonium acetate with a flow
rate of 0.5 ml/min. Fractions containing the sample of interest were
eluted off the column, introduced into the mass spectrometer and
ionized. Triterpenoids and their metabolites were detected using a
single quadrupole mass spectrometer (Waters 2695HPLC coupled
to Waters ZQ mass spectrometer) under electrospray positive con-
ditions and a cone voltage of 60. Standard curves were generated by
serial dilutions of six known concentrations of drug added to blood
or to homogenized control tissue. Waters MassLynx 4.1 software
was used to calculate drug levels. All calculated values were within
the limits of the standards.
The synthesis of CDDO-Im (PubChem CID 9958995) has
(1H-imidazol-4-yl) pyridine were obtained from Acros Organic
(Bridgewater, NJ) and Aldrich (Milwaukee, WI) respectively, while
4-(1H-imidazol-4-yl) pyridine and 2-(1H-imidazol-4-yl) pyridine
were synthesized by known procedures [12,13]. All new CDDO
imidazolide derivatives were prepared by condensation of CDDO
acid chloride and the corresponding imidazoles, with a purity >98%
(assessed by NMR). All drugs were dissolved in DMSO so the final
concentration of solvent was ≤0.1%, and controls containing equiv-
alent concentrations of DMSO were included in all assays.
2.2. Cell culture
U937 leukemia cells were grown at 37 ◦C and 5% CO2 in RPMI
1640 supplemented with 5% FBS and 1% Pen/Strep. RAW264.7
macrophage-like cells were cultured in DMEM containing 10% FBS
and 1% Pen/Strep. Both cell lines were purchased from the American
Type Culture Collection (Manassas, VA), and all media and supple-
ments were acquired from Corning Cellgro (Mediatech, Manassas,
VA) and HyClone Laboratories (Logan, UT). VC1 mouse lung can-
cer cells, generated in our laboratory as described previously [14],
were cultured in DMEM with 10% FBS and 1% Pen/Strep.
2.7. Rna extraction and real-time reverse-transcription
polymerase chain reaction analysis
2.3. Flow cytometry
Monocytic differentiation and apoptosis were measured by
FACS using a MACSQuant® VYB analyzer (Miltenyi Biotec, San
Diego, CA). After 4 days of treatment with the triterpenoids, U937
cells were washed with PBS and then stained for 30 min with a
CD11b monoclonal antibody (DAKO, Carpinteria, CA). Cells were
resuspended in 100 l PBS/BSA/sodium azide prior to FACS analy-
sis. Apoptosis was assessed in U937 cells and VC1 cells 48 h after
treatment with drugs using the TACSTM Annexin V-FITC Apoptosis
Detection Kit (Trevigen, Gaithersburg, MD). Cells were incubated
Total RNA was isolated from mouse liver, kidney and lung
samples with TRIzol (Invitrogen, Carlsbad, CA). cDNA was synthe-
sized from 2 g of RNA using SuperScript III Reverse Transcriptase
(Invitrogen, Carlsbad, CA). Gene expression for each sample was
performed using iQ SYBR Green Supermix and a BioRad CFX96
Touch Real Time PCR Detection system (Bio-Rad Laboratories,
Hercules, CA). Validated NQO1, HO-1 and actin primers were
purchased from Qiagen (Valencia, CA). Relative expression was
determined using the ꢀꢀCt method [16]. After normalization to
Cao, Martine
