Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents

Article abstract of DOI:10.1021/jm101445p

Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the trea

Full text of DOI:10.1021/jm101445p

ARTICLE
pubs.acs.org/jmc
Tricyclic Compounds Containing Nonenolizable Cyano Enones.
A Novel Class of Highly Potent Anti-Inflammatory and
Cytoprotective Agents79
Tadashi Honda,*,† Hidenori Yoshizawa,† Chitra Sundararajan,† Emilie David,† Marc J. Lajoie,†
Frank G. Favaloro, Jr.,† Tomasz Janosik,† Xiaobo Su,† Yukiko Honda,† Bill D. Roebuck,‡ and
Gordon W. Gribble†
†Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States
‡Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, United States
S Supporting Information
b
ABSTRACT: Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed
and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is
currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in
diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A
and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with
interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and
NAD(P)H:quinone oxidoreductase-1. Among these compounds, (()-(4bS,8aR,10aS)-10a-ethynyl-
4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((()-31) is the most potent in these
bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest
that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic
tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.
oxidants such as heme, hydrogen peroxide, oxidized lipids, and
heavy metals.16 HO-1 and its breakdown products possess potent
anti-inflammatory and cytoprotective properties.17-19 Currently,
there is major interest in stimulating HO-1 as a protective enzyme
in many chronic disease conditions in which inflammation and
oxidative stress play an important role. Thus, we have evaluated
our compounds, whose potency in the iNOS assay is sufficient, for
induction of HO-1 in RAW cells (the HO-1 assay).
NAD(P)H:quinone oxidoreductase (NQO1) is also a repre-
sentative phase 2 enzyme. It is a widely distributed FAD-
dependent flavoprotein that catalyzes the obligatory two-elec-
tron reduction of a broad range of substrates, including quinones,
quinoneimines, and nitro compounds by using either NADPH or
NADH as the hydride donor.20 In addition and independent of
its catalytic mechanism, NQO1 also has a “gatekeeping” role in
regulating the proteasomal degradation of specific proteins, and
this function appears to be important in the stabilization of p53, a
broadly functioning tumor suppressor gene. NQO1 is transcriptionally
induced in response to various agents, including xenobiotics, oxidants,
antioxidants, as well as ultraviolet and ionizing radiation.21-24 NQO1,
therefore, is important as a cytoprotective enzyme. We have evaluated
the potency of our compounds for induction of NQO1 in Hepa1c1c7
murine hepatoma cells (the NQO1 assay).
1. INTRODUCTION
The concept that inflammation and carcinogenesis are related
phenomena has been the subject of many studies that have
attempted to link these two processes in a mechanistic fashion.1,2
The enzyme that mediates the constitutive synthesis of nitric
oxide (NO) from arginine has relatively little significance for
either inflammation or carcinogenesis. In contrast, inducible
nitric oxide synthase (iNOS) has critical roles in the response
of tissues to injury or infectious agents and is an essential
component of the inflammatory response, the ultimate repair
of injury, and carcinogenesis.3-5 Although the physiological
activity of iNOS may provide a definite benefit to the organism,
aberrant or excessive expression of iNOS has been implicated in
the pathogenesis of many disease processes, particularly in
Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, rheu-
matoid arthritis, inflammatory bowel disease, chronic kidney
disease, and carcinogenesis.6-10 Therefore, we adopted inhibi-
tion of NO production in primary mouse macrophages11 and/or
RAW 264.7 cells stimulated with interferon-γ (IFN-γ) (the
iNOS assay) as initial screening systems for the development
and evaluation of our potential anti-inflammatory and cytopro-
tective agents that we have designed and synthesized.
Heme oxygenase is the rate-limiting phase 2 enzyme in the
catabolism of heme, producing biliverdin, iron, and carbon mono-
xide. Three heme oxygenase isoforms have been described:12-14
HO-1, HO-2, and HO-3 [the last two recently described as a
pseudogene15 are constitutively expressed]. HO-1 is induced by a
variety of stimuli, including growth factors, cytokines, NO, and
Over the past decade, we have been engaged in the improve-
ment of the anti-inflammatory and cytoprotective activity of
Received: November 9, 2010
Published: March 01, 2011
r
2011 American Chemical Society
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Table 1. Inhibitory Activity of New TCEs 1-12 on NO
Production Induced by IFN-γ in Primary Mouse
Macrophagesa
Figure 1. Structures of CDDO, CDDO-Me, and CDDO-Im and
general structures of TCEs.
oleanolic acid, a ubiquitous naturally occurring triterpenoid. This
led to the discovery of CDDO (Figure 1).25-27 CDDO and its
related compounds are multifunctional agents. CDDO shows
high inhibitory activity in the iNOS assay25,26 and induces HO-1
in vitro28 and in vivo and NQO1 in vitro.29 CDDO induces
differentiation of human myeloid leukemia cells and mouse 3T3-
L1 fibroblasts and enhances nerve-growth-factor-induced neu-
ronal differentiation of rat PC12 cells,27,30 inhibits the prolifera-
tion of human myeloid leukemia and carcinoma cell lines,27
blocks de novo synthesis of iNOS and inducible cyclooxygenase
(COX-2) in mouse macrophages, microglia, and fibroblasts,27
and induces apoptosis of human myeloid leukemia,31-33
osteosarcoma,34 lung cancer,35 and CLL cells.36 The acylimida-
zole of CDDO (CDDO-Im, Figure 1) inhibits aflatoxin-induced
tumorigenesis in rats.37 The methyl ester of CDDO (CDDO-
Me, bardoxolone methyl, Figure 1) prevents lung cancer induced
by vinyl carbamate in A/J mice.38 Presently, bardoxolone methyl
is being developed in late phase II clinical trials for the treatment
of severe chronic kidney disease in type 2 diabetes mellitus
patients. It significantly increases the estimated glomerular
filtration rate in more than 90% of diabetic patients.39
compd
IC50 (nM)
compd
(()-9
IC50 (nM)
(()-1
(()-2
(()-3
(()-4
(()-5
(-)-5
(þ)-5
(()-6
(()-7
(()-8
310
480
53
2.1
14
(-)-9
(þ)-9
(()-10
1.3
>60
>600
19
75
61
(()-11
64
(()-12
58
(-)-12
26
91
(þ)-12
CDDO
19
1600
61
0.5
0.2
10
CDDO-Me
hydrocortisone
a IC50 values of TCEs, CDDO, CDDO-Me, and hydrocortisone were
determined in the range 0.1 pM to 10 μM (10-fold dilutions). Values are
an average of two separate experiments. None of the compounds were
toxic to primary mouse macrophages at 10 μM. The experimental
protocol is in the Supporting Information. These data have been
published and presented in refs 79a-79d.
We have chemically demonstrated that the nonenolizable
cyano enone in ring A of CDDO gives reversible Michael adducts
with the SH group of DTT by UV and NMR studies.40 We
speculate that this characteristic reactivity may imply a molecular
mechanism of action. Indeed, our mechanism studies suggest
that CDDO and its related compounds regulate proteins affect-
ing inflammation, oxidative stress, differentiation, apoptosis, and
proliferation, including Keap1,29 IKKβ, and JAK1, to name a few,
by reversible Michael addition between their nonenolizable
cyano enone functionality in ring A and the SH groups of
cysteine moieties on these proteins. Recently, Cys179 in the
kinase domain on IKKβ was identified as a target of CDDO-Me
and CDDO-Im.41,42 By binding to this site on IKKβ, CDDO-Me
inactivates the kinase and ultimately results in blocking of the
binding of NF-κB to DNA and thus inhibits transcriptional
activation of NF-κB-dependent target genes. It has also been
reported that CDDO-Me inhibits the JAK1 f STAT3 path-
way by directly binding to JAK1 at Cys1077 and STAT3 at
Cys259.43 Small molecule inhibitors of the STAT3 pathway are
known to be effective as anticancer agents in vitro and in animal
models.
During the development of CDDO, we found the very
important structure-activity relationships (SARs) that the
nonenolizable cyano enone in ring A and the enone in ring C
are essential for the extremely high potency of CDDO [see
Figure S1 in the Supporting Information].26 Therefore, we
reasoned that the entire oleanane skeleton might not be neces-
sary for potency. Consequently, we have designed tricycles
containing nonenolizable cyano enones (TCEs, i.e., compounds
1-44, Figure 1). A literature survey revealed that these com-
pounds were previously unknown.
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Scheme 1. Synthesis of TCEs 10 and 11a
Figure 2. Structures of 45-47 and 55.
Our rationale for pursuing the synthesis of these compounds is
as follows. Since they could be synthesized from commercially
available small molecules, these compounds with various func-
tionalities at different positions would be easily obtained. Such
diversity-oriented synthesis could lead to new potential anti-
inflammatory and cytoprotective agents that have high oral
potency and high water solubility for ease of administration
and formulation, as well as high biological selectivity for avoiding
possible side effects. Accordingly, we have synthesized various
new TCEs and evaluated them in the iNOS, HO-1, and NQO1
assays. As a result, we have found that TCE 31 (code number in
house: TBE-31, (()-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-
3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbo-
nitrile) is the most potent in our collection of our semisynthetic
triterpenoids and synthetic tricycles. We herein describe the full
account of our synthetic work with these tricycles and their
interesting biological results.
a Reagents and yields: (a) ethylene glycol, PPTS, PhH, 94%; (b) CrO3,
t-BuOOH, CH2Cl2, 55%; (c) p-TsCN, LDA, THF; (d) DDQ, PhH,
71% from 49; (e) aqueous HCl, MeOH, 65%; (f) MMC, DMF, 100%;
(g) CH2N2, Et2O, 71%; (h) PhSeCl, pyridine, CH2Cl2, 30% H2O2, 28%;
(i) aqueous KOH, MeOH, 72%.
We efficiently synthesized (-)-9, with the same configuration
as the naturally occurring oleanolic acid, and its antipode (þ)-9
in seven steps from known bicyclic enones (-)-47 and (þ)-47,47
respectively (structure of (-)-47, see Figure 2; syntheses, see
Scheme S4 in the Supporting Information).79c
2. CHEMISTRY
2.1. Initial Set of Tricycles. Our initial targets 1-5
(structures, see Table 1) were synthesized in racemic form from
known compound 4544 (structure, see Figure 2) by synthetic
sequence that has been published (syntheses, see Scheme S1 in
the Supporting Information).79a
Because 5 in racemic form shows good potency in the iNOS
assay among the initial set of the tricycles (Table 1), we have
synthesized both (-)-5, with the same configuration as CDDO,
and its antipode (þ)-5 from the known bicyclic enones (-)-46
and (þ)-4645 (structure of (-)-46, see Figure 2), respectively,
according to the improved synthetic route that is specifically
directed toward (-)-5 and (þ)-5 (syntheses, see Scheme S2 in
the Supporting Information).79c
2.2. Insertion of Electron-Withdrawing Groups at C2
Position of Tricycles. Since 5 shows good potency in the iNOS
assay, this compound was thought to be a good scaffold from
which to discover new, more potent tricycles. Thus, we targeted
6-9 (structures, see Table 1), analogues of 5 with electron-
withdrawing groups at the C2 position, to discern the influence of
substituents at the C2 position on biological activity, because we
previously found that substitution at the R position of an R,β-
unsaturated ketone strongly affects the potency of semisynthetic
triterpenoids.46
Racemic 6-9 were synthesized from 45 according to the
synthetic route that has been published (syntheses, see Scheme
S3 in the Supporting Information).79a Of these TCEs, 9 showed
high potency (IC50 = 1 nM level) and it is approaching the
potency of CDDO in the iNOS assay (see Table 1).
2.3. Synthesis of TCEs 10 and 11. We designed 10 and 11 in
racemic form as analogues of 9 because 11 and its salts would be
soluble in water. TCEs 10 and 11 were synthesized from 4879a,c
by the sequence shown in Scheme 1 (also see Scheme S5 in the
Supporting Information). Tricycle 49 was prepared by ketaliza-
tion of 48, followed by a chromium-mediated allylic oxidation48
with CrO3 and tert-butyl hydroperoxide (t-BuOOH) in CH2Cl2
(52% yield). Cyanation of the enolate of 49, generated using
LDA in THF, with p-toluenesulfonyl cyanide (p-TsCN),49 fol-
lowed by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxi-
dation, gave 50 in 71% yield. Methyl ester 51 was obtained by
removal of the ketal of 50, followed by carboxylation at the C6
position with Stiles’ reagent50 and subsequent methylation with
diazomethane (46% yield). TCE 10 was prepared in 28% yield
from 51 by the addition of PhSeCl in the presence of pyridine
and subsequent oxidation/elimination with H2O2.51 Cleavage of
the methyl ester under basic conditions gave 11 in 72% yield.
2.4. Synthesis of TCE 12. We have designed and synthesized
12, which has the same A, B, and C ring system as that of CDDO
(Schemes 2 and S6 in the Supporting Information). Imidazole
1-thiocarboxylate 52 was prepared in 77% yield from 45 with
1,10-thiocarbonyldiimidazole in THF. Reduction of 52 with tri(n-
butyl)tin hydride in toluene gave 48 in 91% yield.52 This tricycle
48, which is derived from 45 (whose structure has already been
confirmed),44 is identical with that obtained by reductive methyl-
ation of 5553 (structure, see Figure 2; preparation, see Scheme S4
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Scheme 2. Synthesis of Racemic and Optically Active TCE
12a
Scheme 3. Synthesis of TCE 13a
a Reagents: (a) HCO2Et, NaOMe, PhH; (b) NH2OH HCl, aqueous
3
EtOH; (c) CrO3, t-BuOOH, CH2Cl2; (d) NaOMe, MeOH, Et2O; (e)
DDQ, PhH.
Scheme 4. Synthesis of TCE 15a
a Reagents: (a) 1,10-thiocarbonyldiimidazole, THF; (b) (n-Bu)3SnH,
toluene; (c) HCO2Et, NaOMe, PhH; (d) NH2OH HCl, aqueous
EtOH; (e) CrO3, t-BuOOH, CH2Cl2; (f) NaOMe, MeOH, Et2O; (g)
DDQ, PhH.
3
a Reagents and yields: (a) HCO2Et, NaOMe, PhH, 45%; (b) NH2OH
HCl, aqueous EtOH, 100%; (c) SEMCl, EDIA, CH2Cl2, 80%; (d) CrO3,
t-BuOOH, CH2Cl2, 67%; (e) NaOMe, MeOH, Et2O, 93%; (f) DDQ,
PhH, 64%; (g) 48% aqueous HF-CH3CN (1:9), 87%.
3
in the Supporting Information). Consequently, it was proven
that the reductive methylation product 48 has the thermodyna-
mically stable trans-A/B ring juncture. Isoxazole 53 was obtained
in 70% yield by formylation of 48 with ethyl formate in the
presence of NaOMe in benzene,54 followed by treatment with
hydroxylamine hydrochloride in aqueous EtOH.55 A chromium-
mediated allylic oxidation of 53 with t-BuOOH produced 54 in
64% yield. The desired TCE 12 was prepared by cleavage of the
isoxazole moiety of 54 with NaOMe,55 followed by DDQ
oxidation (79% yield). We have also synthesized optically active
(þ)-12 ([R]25D þ58° (c 1.3, CHCl3), 18% yield in five steps)
Substitution of functionalities at the C8a position of 12 would be
expected to improve the potency and pharmacokinetics because
the balance between hydrophilicity and hydrophobicity is shifted.
2.5. Functionality Substitutions at the C8a Postion of TCE
12. We synthesized various C8a functionalized TCE analogues
using tricycles 56a-c (structures, see Schemes 3, 4, and 6) as
starting materials, whose efficient synthesis we have already
established for our projected synthesis.56 These analogues in-
clude typical electron-withdrawing, electron-releasing, hydrophi-
lic, hydrophobic, and bulky groups.
TCE 13 with a methoxycarbonyl group at C8a was synthesized
in 42% overall yield via 57, 58, and 59 from 56a by the same
sequence as for 12 from 48 (Scheme 3 and Scheme S7 in the
Supporting Information). We attempted several methods [KOH,
aqueous MeOH (reflux, overnight); LiI, DMF (reflux, 30 min);57
KOSiMe3, THF (room temp, overnight);58 AlBr3, Me2S (room
and (-)-12 ([R]25 -66° (c 2.0, CHCl3), 14% yield in five
D
steps), from (þ)-4879c and (-)-48,79c respectively, by the same
sequence as for (()-12.
Since 12 is about 3 times more potent than TCE 5 in the iNOS
assay (Table 1), we considered that the 7,8-double bond in ring C
is not always necessary for potency. Therefore, we designed
and synthesized various C8a functionalized analogues of 12.
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Scheme 5. Synthesis of TCE 16a
Scheme 6. Synthesis of TCEs 17-20a
a Reagents and yields: (a) (COCl)2, CH2Cl2, 90%; (b) NH3, PhH, 80%;
(c) HCO2Et, NaOMe, PhH; (d) NH2OH HCl, aqueous EtOH; (e)
CrO3, t-BuOOH, CH2Cl2, 50%; (f) NaOMe, MeOH, Et2O; (g) DDQ,
3
a Reagents and yields: (a) TBSCl, imidazole, DMF, 98%; (b) HCO2Et,
1,4-dioxane, 68% in two steps (f) and (g).
NaOMe, PhH, 98%; (c) NH2OH HCl, NaOAc, aqueous AcOH, 85%;
3
(d) CrO3, t-BuOOH, CH2Cl2, 69% (e) NaOMe, MeOH, Et2O; (f)
DDQ, 1,4-dioxane, 100% in two steps (e) and (f); (g) 48% aqueous HF,
CH3CN, 82%; (h) Ac2O, pyridine, 86%; (i) CrO3, pyridine, CH2Cl2, 93%.
temp, overnight);59 LiS(n-Pr), HMPA (room temp, 1 h)60] for
the cleavage of methyl ester 13 to the corresponding acid 15, but
these methods failed to give 15.
Therefore, we synthesized acid 15 according to the alternative
sequence shown in Scheme 4 (Scheme S8 in the Supporting
Information). Isoxazole 60 was prepared by formylation of 56b
and subsequent isoxazole ring construction, followed by protec-
tion of the hydroxyl group with 2-((chloromethoxy)ethyl)-
trimethylsilane (SEMCl) in the presence of EDIA in CH2Cl2
(36% yield). TCE 14 was obtained in 40% yield from 60 by the
same sequence as for 13 from 58. The (trimethylsilyl)-
ethoxymethyl (SEM) group was removed from 14 with 48%
aqueous HF and CH3CN (1:9) to give 15 (87% yield). Although
we were concerned that 15 might be unstable toward decarboxy-
lation in a medium used for biological testing, in fact, 15 is both
soluble and stable in Dulbecco’s modified Eagle’s medium (10
mM) at room temperature for 4 days at least.
Scheme 7. Synthesis of TCE 21a
a Reagents and yields: (a) HCO2Et, NaOMe, PhH, 100%; (b)
NH2OH HCl, aqueous EtOH, 100%; (c) CrO3, t-BuOOH, CH2Cl2,
3
68%; (d) NaOMe, MeOH, Et2O, 97%; (e) DDQ, 1,4-dioxane, 58%.
TCE 16 with an aminocarbonyl group was synthesized from
56b (Scheme 5 and Scheme S9 in the Supporting Information).
Amide 61 was prepared in 72% yield from 56b by chlorination
with oxalyl chloride, followed by the treatment with NH3 in
benzene. Unexpectedly, the formylation of 61 with ethyl formate
in the presence of NaOMe gave 62 (52% yield) as a major
product and the desired compound 63 (16% yield) as a minor
one. However, treatment of 62 with hydroxylamine hydrochlo-
ride cleaved the N-formyl group to give the desired isoxazole 64
in 75% yield. Under the same conditions, 63 gave 64 in 75% yield.
TCE 16 was obtained by allylic oxidation of 64, followed by the
cleavage of the isoxazole under basic conditions and subsequent
DDQ oxidation in 1,4-dioxane (34% yield). For the DDQ
oxidation step, because the precursor of 16 was not soluble in
benzene, 1,4-dioxane was used as the solvent.
Schemes 6 and 7 (Schemes S10 and S11 in the Supporting
Information). The starting material 56c was protected with tert-
butylchlorodimethylsilane (TBSCl) to give 65 in 98% yield. Iso-
xazole 66 was prepared by formylation of 65, followed by treatment
with hydroxylamine hydrochloride in the presence of NaOAc in
aqueous AcOH (83% yield). At the isoxazole construction step,
NaOAc and aqueous AcOH were used instead of aqueous EtOH
because the latter conditions gave 66 in low yield. We considered that
1 equiv of HCl, which is produced from the latter conditions, would
disrupt this conversion. TCE 17 was obtained in 69% yield from 66
by the same sequence as for 16 from 64. The TBS group of 17 was
removed by 48% aqueous HF and CH3CN (1:9) to give 18 in 82%
yield. Acetylation of 18 with acetic anhydride in pyridine afforded 19
in 86% yield. Aldehyde 20 was obtained in 93% yield by Ratcliffe
oxidation61 of 18 with CrO3 and pyridine in CH2Cl2. Methyl ether
21 was synthesized in five steps from a known compound 6762 by the
same sequence as for 16 from 61 (38% yield, Scheme 7).
TCE 18 with a hydroxymethyl group and its derivatives 17 and
19-21 were synthesized according to the sequence shown in
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We tried to synthesize fluoro derivative 71 from 66 (Scheme 8
and Scheme S12 in the Supporting Information). Tricycle 68 was
obtained in 68% yield by allylic oxidation of 66, followed by
removal of the TBS group with 48% aqueous HF and CH3CN
(1:9). However, fluorination of 68 with DAST did not give 70
but a ring expansion product 69 in 52% yield. TCE 22 was
prepared in 66% yield by the cleavage of the isoxazole of 69,
followed by DDQ oxidation. The structures of 69 and 22 were
fully characterized by 1H and 13C NMR, high and low MS, and
elemental analysis. We speculate that 69 is produced from 68
according to the mechanisms shown in the box in Scheme 8. The
configuration of the fluorine atom has not been determined.
Dicarbonitrile 23 was synthesized from 56b and 56c by the
different sequence shown in Scheme 9 (Scheme S13 in the
Supporting Information). Amide 72 was prepared by chlorina-
tion of 56b with oxalyl chloride, followed by the treatment with
tert-butylamine (37% yield). Carbonitrile 73 was obtained by the
treatment of 72 with POCl3 (100% yield, 37% overall yield from
56b).63 However, since this step requires vigorous conditions,
the yield varies. Thus, we explored an improved synthesis of 73
from alcohol 56c. Ketalization of 56c, followed by Swern
oxidation64 gave aldehyde 74 (100% yield). Oxime 75 was
obtained in 86% yield by the condensation between hydroxyl-
amine and 74 in CH2Cl2. Dehydration of 75 with 1,1-carbonyl-
diimidazole in CH2Cl2 provided carbonitrile 76 in 89% yield.65
The ketal of 76 was removed under acidic conditions to afford 73
in 100% yield (77% overall yield from 56c). The overall yield of
this sequence is about twice that of 73 from 56b. Moreover,
importantly, the yield of this sequence is reproducible. Dicarbo-
nitrile 23 was synthesized in five steps from 73 by the same
sequence as for 16 from 61 (42% yield).
Scheme 8. Synthesis of TCE 22a
Amine hydrochloride 25 was synthesized from 76 by the
sequence shown in Scheme 10 (Scheme S14 in the Supporting
Information). Reduction of the cyano group of 76 with LiAlH4
gave only a complex mixture. In contrast, reduction of 76 with a
mixture of NaBH4 and CoCl2 (5:1),66 followed by workup with
10% aqueous HCl solution and subsequent protection of the
amino group with Boc2O gave 77 in 54% yield. TCE 24 was
obtained in five steps from 77 by the same sequence as for 16
from 61 (41% yield). Removal of the Boc group of 24 with 4 M
HCl in 1,4-dioxane provided 25 in 95% yield.
TCE 26 with an ethyl group was synthesized from 74
(Scheme 11 and Scheme S15 in the Supporting Information).
A Grignard reaction of 74 with MeMgBr in THF gave 78 in 87%
yield as a mixture of diastereomers. Ratcliffe oxidation of 78
afforded 79 in 73% yield. A forced Wolff-Kishner reduction67 of
79 followed by deketalization under acidic conditions provided
a Reagents: (a) CrO3, t-BuOOH, CH2Cl2; (b) HF, CH3CN; (c) DAST,
CH2Cl2; (d) NaOMe, MeOH, Et2O; (e) DDQ, 1,4-dioxane.
Scheme 9. Synthesis of TCE 23a
a Reagents and yields: (a) (COCl)2, CH2Cl2; (b) t-BuNH2, Et3N, CH2Cl2, 37% from 56b; (c) POCl3, 100%; (d) ethylene glycol, PPTS, PhH, 100%; (e)
Swern oxidation, 100%; (f) NH2OH HCl, NaOAc, MeOH, CH2Cl2, 86%; (g) 1,1-carbonyldiimidazole, CH2Cl2, 89%; (h) aqueous HCl, MeOH, 100%;
3
(i) HCO2Et, NaOMe, PhH, 72%; (j) NH2OH HCl, aqueous EtOH, 100%; (k) CrO3, t-BuOOH, CH2Cl2, 83%; (l) NaOMe, MeOH, Et2O, 88%; (m)
DDQ, 1,4-dioxane, 79%.
3
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Scheme 10. Synthesis of TCEs 24 and 25a
Scheme 12. Synthesis of TCE 27a
a Reagents and yields: (a) NaBH4, CoCl2, MeOH, 71%; 10% aqueous
HCl, Boc2O, THF, 76%; (b) HCO2Et, NaOMe, PhH, 100%; (c)
NH2OH HCl, aqueous EtOH, 76%; (d) CrO3, t-BuOOH, CH2Cl2,
3
62%; (e) NaOMe, MeOH, Et2O, 100%; (f) DDQ, 1,4-dioxane, 86%; (g)
4 M HCl in 1,4-dioxane, 95%.
a Reagents and yields: (a) Ph3PCH2Cl2, n-BuLi, THF, HMPA, 80%; (b)
MeLi, THF; aq NH4Cl, 95%; (c) 10% aqueous HCl, MeOH, 84%; (d)
Scheme 11. Synthesis of TCE 26a
HCO2Et, NaOMe, PhH, 76%; (e) NH2OH HCl, aqueous EtOH, 70%;
3
(f) CrO3, t-BuOOH, CH2Cl2, 46%; (g) NaOMe, MeOH, Et2O, 93%;
(h) DDQ, 1,4-dioxane, 37%.
Scheme 13. Synthesis of TCE 28a
a Reagents and yields: (a) MeMgBr, THF, 87%; (b) CrO3, pyridine,
CH2Cl2, 73%; (c) NH2NH2, KOH, diethylene glycol, 51%; (d) aqueous
a Reagents: (a) CrO3, t-BuOOH, CH2Cl2; (b) p-TsCN, LDA, THF; (c)
DDQ, 1,4-dioxane.
HCl, MeOH, 100%; (e) HCO2Et, NaOMe, PhH, 75%; (f) NH2OH
3
HCl, aqueous EtOH, 95%; (g) CrO3,t-BuOOH, CH2Cl2,69%;(h) NaOMe,
functionalized derivative of TCE 9, was synthesized by the
sequence shown in Scheme 13 (Scheme S17 in the Supporting
Information). A chromium-mediated allylic oxidation of 73 with
CrO3 and t-BuOOH in CH2Cl2 gave 84 in 63% yield. TCE 28
was obtained by double cyanation of 84 with LDA and p-TsCN,
followed by DDQ oxidation in 1,4-dioxane (29% yield). TCE 29
with an ethyl group (structure, see Table 3) was synthesized in
three steps from 80 by the same sequence as for 28 from 73
(preparation, see Scheme S18 in the Supporting Information).79e
TCE 30 with a vinyl group (structure, see Table 3) was obtained
in five steps from 74 (preparation, see Scheme S19 in the
Supporting Information).79e
MeOH, Et2O, 100%; (i) DDQ, 1,4-dioxane, 39%.
80 in 51% yield. TCE 26 was obtained in five steps from 80 by the
same sequence as for 16 from 61 (19% yield).
TCE 27 with an ethynyl group was synthesized from 74
(Scheme 12 and Scheme S16 in the Supporting Information). A
Wittig reaction on 74 with (chloromethyl)triphenylphosphonium
chloride68 gave 81 as a mixture of E/Z chlorovinyl isomers (E/Z =
4:1) in 80% yield. Dehydrochlorination of 81 with MeLi followed
by quenching of the acetylide with aqueous NH4Cl solution
provided 82 in 95% yield.68 The ketal of 82 was removed under
acidic conditions to afford 83 in 84% yield. TCE 27 was obtained in
8% yield from 83 by the same sequence as for 16 from 61.
2.6. Functionality Substitutions at the C10a position of
TCE 9. TCE 28 with a cyano group at C10a, which is a C10a
TCE 31 with an ethynyl group was synthesized in six steps
from 81 (Scheme 14 and Scheme S20 in the Supporting
Information). Dehydrochlorination of 81 with MeLi, followed
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Scheme 14. Synthesis of TCEs 31 and 32a
Scheme 16. Synthesis of TCE 36a
a Reagents: (a) MeLi, THF; TMSCl; (b) aqueous HCl, MeOH; (c)
CrO3, t-BuOOH, CH2Cl2; (d) p-TsCN, LDA, THF; (e) DDQ, PhH; (f)
TBAF, THF.
a Reagents: (a) MeLi, THF, TBSCl; (b) aqueous HCl, MeOH; (c)
CrO3, t-BuOOH, CH2Cl2; (d) p-TsCN, LDA, THF; (e) DDQ, PhH.
Optically active (-)- and (þ)-31 and 32 (structures, see Table 3)
were synthesized by the sequence that has been published
(syntheses, see Scheme S21 in the Supporting Information).79e
2.7. Improved Synthesis of TCE 31. Currently, since TCE
31 is the most potent compound in our pool of semisynthetic
triterpenoids and synthetic tricycles that we have evaluated in our
bioassays (see Biological Results and Discussion), it is essential
for further evaluation to prepare at least 1 g of 31 in a single batch.
Although the sequence shown in Scheme 14 is acceptable for a
small-scale synthesis (10-200 mg), it is not adequate for a
medium-scale synthesis (500 mg to 2 g). Particularly, the double
cyanation step using p-TsCN is not feasible because p-TsCN is a
very expensive reagent and the yield of this reaction drastically
decreases on a large scale. Thus, we have developed an improved
synthesis of 31 (Scheme 15 and Scheme S22 in the Supporting
Information) by adopting Johnson’s isoxazole method55 for the
double cyanation, which we used for the synthesis of TCE 12
analogues. With this sequence, 31 is consistently obtained in 30%
yield in 10 steps from 56c.
Scheme 15. Improved synthesis of TCE 31a
Removal of the ketal of 82 under acidic conditions gave 83 in
99% yield. Enone 87 was prepared in 65% yield from 83 by allylic
oxidation. Formyl groups were successfully inserted into the C3
and C7 positions of 87 simultaneously to afford 88 (99% yield)
using twice the amount of ethyl formate and NaOMe (11 equiv
each) than was used for monoformylation (5.5 equiv each).
Treatment of 88 with hydroxylamine hydrochloride provided
diisoxazole 89 in quantitative yield. Each isoxazole ring of 89 was
converted to a cyano group under basic conditions, which gave
31 upon the addition of PhSeCl and subsequent oxidation/
elimination with H2O2 (62% yield). This oxidation method was
better than the DDQ method for this oxidation with an un-
protected acetylene group.
a Reagents: (a) 10% aqueous HCl, MeOH; (b) CrO3, t-BuOOH,
CH2Cl2; (c) HCO2Et, NaOMe, PhH; (d) NH2OH HCl, aqueous
3
EtOH; (e) NaOMe, MeOH, Et2O; (f) PhSeCl, pyridine, CH2Cl2,
30% H2O2, CH2Cl2.
by trapping of the acetylide with chlorotrimethylsilane (TMSCl)
gave 85 in 93% yield.69 Deketalization of 85 under acidic
conditions followed by a chromium-mediated allylic oxidation
provided 86 in 63% yield. TCE 32 was obtained by double
cyanation of 86 with LDA and p-TsCN, followed by DDQ
oxidation in benzene (61% yield). The trimethylsilyl (TMS)
group was removed by tetra(n-butyl)ammonium fluoride (TBAF)70
to afford 31 in 71% yield (nine steps from 56c, 21% overall yield).
2.8. Synthesis of TCE 31 Analogues. The high potency of 31
and 32 in the iNOS assay (see Table 3) encouraged us to explore the
modifications of the ethyne group of TCE 31. TCEs 33-35 (struc-
tures, see Table 3) were synthesized in five steps from 82 (syntheses,
see Schemes S23-S25 in the Supporting Information).79e
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Scheme 17. Synthesis of TCE 37a
Scheme 18. Synthesis of TCEs 38 and 39a
a Reagents and yields: (a) ClCO2Me, MeLi, THF, 64%; (b) 10%
aqueous HCl, MeOH, 93%; (c) CrO3, t-BuOOH, CH2Cl2, 60%; (d)
p-TsCN, LDA, THF; (e) DDQ, PhH, 26% in two steps (d) and (e); (f)
aqueous KOH, MeOH, 95%.
Scheme 19. Synthesis of TCEs 40 and 41a
a Reagents and yields: (a) n-BuLi, BF3 Et2O, DMF, THF, 91%; (b)
3
Ph3PCH2Cl2, n-BuLi, HMPA, THF, 76%; (c) MeLi, THF, TMSCl,
97%; (d) 10% aqueous HCl, MeOH, 74%; (e) CrO3, t-BuOOH,
CH2Cl2, 50%; (f) p-TsCN, LDA, THF; (g) DDQ, PhH, 22% in two
steps (f) and (g); (h) TBAF, THF, 28%.
TCE 36 was synthesized in five steps from 82 (Scheme 16 and
Scheme S26 in the Supporting Information). Insertion of the
TBS group into the acetylene moiety was achieved by treating 82
with MeLi and trapping the resulting anion with TBSCl, to give
90 in 91% yield. The ketal 90 was subjected to acidic conditions
to give 91 in 94% yield. Allylic oxidation of 91 afforded 92 in 66%
yield. Double cyanation of 92, followed by DDQ oxidation in
benzene, gave 36 in 27% yield.
TCE 37 with a buta-1,3-diynyl group was synthesized in eight
steps from 82 (Scheme 17 and Scheme S27 in the Supporting
Information). The treatment of acetylide 82 with DMF in the
presence of BF3 Et2O in THF provided aldehyde 93 in 91%
3
yield. Tricycle 94 was prepared by a Wittig reaction of 93 with
(chloromethyl)triphenylphosphonium chloride and subsequent
treatment with MeLi and TMSCl, followed by deketalization
(55% yield). Tricycle 95 was obtained in three steps from 94 by
the same sequence as for 36 from 91 (11% yield). The TMS
group of 95 was removed with TBAF to give 37 in 28% yield.
TCE 38 with a methoxycarbonyl group and the corresponding
acid 39 were synthesized from 82 (Scheme 18 and Scheme S28 in
the Supporting Information). Methyl ester 96 was obtained by
the treatment of acetylide 82 with methyl chloroformate, fol-
lowed by deketalization (60% yield). TCE 38 was prepared in
three steps from 96 by the same sequence as for 36 from 91 (20%
yield). Alkaline hydrolysis of 38 gave 39 in 95% yield.
Acetylenic imidazoles 40 and 41 were synthesized in six steps
from 82 (Scheme 19 and Scheme S29 in the Supporting
Information). A Sonogashira coupling71 between 2-iodo-1-
((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole72 and 82 in
the presence of Pd(PPh3)2Cl2 and CuI in Et3N gave 97 in
73% yield. Deketalization of 97 provided 98 in 90% yield. TCE
40 was obtained by allylic oxidation of 98 and subsequent
isoxazole opening under basic conditions, followed by addition
of PhSeCl in the presence of pyridine, and subsequent oxidation/
a Reagents and yields: (a) 2-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-imidazole, Pd(PPh3)2Cl2, CuI, Et3N, 73%; (b) 10% aqueous HCl,
MeOH, 90%; (c) CrO3, t-BuOOH, CH2Cl2, 43% (d) p-TsCN, LDA,
THF, 88%; (e) PhSeCl, pyridine, CH2Cl2; 30% H2O2, CH2Cl2, 75%; (f)
CF3CO2H-THF (10:1).
elimination with H2O2 (28% yield). In this conversion, the
PhSeCl and H2O2 were used instead of DDQ because the
DDQ oxidation gave 40 in a very low yield. Removal of the
SEM group of 40 was successfully achieved by CF3CO2H-THF
(10:1) to afford 41 in 50% yield, after the deprotection using
several well-known methods (TBAF, HF-CH3CN, HF-pyr-
idine, BF3 Et2O etc.) was unsuccessful.
3
Acetylenic thiazole 42 was synthesized in seven steps from 82
(Scheme 20 and Scheme S30 in the Supporting Information).
Tricycle 99 was obtained by a Sonogashira coupling between
2-bromothiazole and 82, followed by removal of the ketal and
subsequent allylic oxidation (13% yield). Formylation of 99 gave
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Scheme 20. Synthesis of TCE 42a
Scheme 21. Synthesis of TCE 43a
a Reagents and yields: (a) 2-iodo-4-methylthiazole, Pd(PPh3)2Cl2, CuI,
Et3N, PhH, 48%; (b) 10% aqueous HCl, MeOH, 80%; (c) CrO3, t-BuOOH,
CH2Cl2, 49%; (d) p-TsCN, LDA, THF; (e) PhSeCl, pyridine, CH2Cl2,
30% H2O2, CH2Cl2, 13% in two steps (d) and (e).
Scheme 22. Synthesis of TCE 44a
a Reagents and yields: (a) 2-bromothiazole, Pd(PPh3)2Cl2, PPh3, CuI,
Et3N, PhH, 32%; (b) 10% aqueous HCl, MeOH, 100%; (c) CrO3,
t-BuOOH, CH2Cl2, 41%; (d) HCO2Et, NaOMe, PhH, 88%; (e) NH2-
OH HCl, aqueous EtOH, 94%; (f) NaOMe, MeOH, Et2O; (g) PhSeCl,
3
pyridine, CH2Cl2, 30% H2O2, CH2Cl2, 17% in two steps (f) and (g).
bis(hydroxymethylene) 100 in 88% yield. Treatment of 100 with
hydroxylamine provided diisoxazole 101 in 94% yield. TCE 42
was prepared in 17% yield from 101 by the same methods as for
31 from 89 (see Scheme 15). Because double cyanation of 99
with p-TsCN followed by DDQ oxidation gave impure 42, which
we could not sufficiently purify, we adopted the Johnson’s
isoxazole method for this conversion.
Acetylenic methylthiazole 43 was synthesized by a Sonoga-
shira coupling between 2-iodo-4-methylthiazole and 82 and
subsequent deketalization, followed by the same sequence from
102 as for 40 from 98 (2% overall yield, Scheme 21 and Scheme
S31 in the Supporting Information).
a Reagents and yields: (a) iodobenzene, Pd(PPh3)2Cl2, CuI, Et3N, THF,
64%; (b) 10% aqueous HCl, MeOH, 77%; (c) CrO3, t-BuOOH,
CH2Cl2, 81%; (d) HCO2Et, NaOMe, PhH, 92%; (e) NH2OH HCl,
3
aqueous EtOH, 97%; (f) NaOMe, MeOH, Et2O, 32%; (g) PhSeCl,
pyridine, CH2Cl2, 30% H2O2, CH2Cl2, 50%.
TCE 44 with a phenylethynyl group was synthesized in seven
steps from 82 (Scheme 22 and Scheme S32 in the Supporting
Information). Enone 103 was obtained by a Sonogashira cou-
pling between iodobenzene and 82 and subsequent deketaliza-
tion, followed by allylic oxidation (40% yield). TCE 44 was
prepared in four steps from 103 by the same sequence as for 42
from 99 (14% yield).
about 4 times less potent than CDDO. A nitrile group at C2
enhances potency among the typical electron-withdrawing
groups surveyed at C2. (2) Since 3 and 4 are more potent than
2 and 1, respectively, the importance of the bis(enone) structure
for high potency, even in tricycles, is confirmed. (3) A nitrile
group at C6 is essential for high potency (9 vs 10 and 11). (4)
Both enantiomers of 5 and 12 show similar potency, while (þ)-9
having the same configuration as the CDDO antipode is about 10
times more potent than (-)-9 having the same configuration
as CDDO.
The inhibitory potency of 9 in this assay was not blocked by
the glucocorticoid antagonist mifepristone (RU486, at 1 μM),
which reverses the action of hydrocortisone (see Figure S2 in the
Supporting Information). This strongly implies that the actions
of TCEs in this assay are not mediated by their interaction with
the glucocorticoid receptor.
3. BIOLOGICAL RESULTS AND DISCUSSION
3.1. TCEs 1-12 Inhibit NO Production Induced by IFN-γ in
Primary Mouse Macrophages. We have evaluated inhibitory
activities of the initial set of TCEs on NO production induced by
IFN-γ in mouse macrophages. The inhibitory activities [IC50
(nM)] of racemic 1-12, optically active 5, 9, and 12, triterpe-
noids [CDDO and CDDO-Me], and hydrocortisone (a positive
control) are shown in Table 1. Important results, which were
obtained from these compounds, are as follows: (1) Among all of
the synthetic TCEs, 9 has the highest potency, and next is 12.
TCE 9 approaches the potency of CDDO in this assay; it is only
3.2. TCEs 13-44 Inhibit NO Production Induced by IFN-γ
in RAW 264.7 Cells. Our results from the initial set of TCEs
encouraged us to explore additional TCEs. Thus, we first designed
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Table 2. Inhibitory Activity of New TCEs 13-27 on NO Production Induced by IFN-γ in RAW Cellsa
compd (racemic)
R
IC50 (nM)
compd (racemic)
R
IC50 (nM)
13
14
15
16
17
18
19
20
21
22
CO2Me
CO2SEM
CO2H
290
150
83
23
CN
64
64
24
CH2NHBoc
25
CH2NH2 HCl
240
80
3
CONH2
CH2OTBS
CH2OH
CH2OAc
CHO
>10000
480
350
85
26
Et
27
CtCH
Me
83
12
57
5
Table 1
Table 1
Table 1
87
83
9
23
CH2OMe
40
CDDO
17
360
hydrocortisone
61
a RAW 264.7 cells were treated with various concentrations of compounds and IFN-γ (10 ng/mL) for 24 h. Supernatants were analyzed for NO by the
Griess reaction.11 IC50 values are an average of two separate experiments. These data have been published and presented in refs 79f and 79h.
and synthesized the new TCEs 13-27, which have various
functionalities at C8a on the basis of 12. These analogues include
typical electron-withdrawing, electron-releasing, hydrophilic,
hydrophobic, and bulky groups (Table 2). Among them, the
lead compound 12 having a methyl group at C8a was still the
best. However, we found that hydrocarbon groups are slightly
better than functional groups containing heteroatoms.
against inhibitors than RAW cells. The IC50 values of 31 and
CDDO are 0.056 and 4.3 nM, respectively. Thus, TCE 31 is
about 80 times more potent than CDDO in primary mouse
macrophages, whereas 31 is about 20 times more potent than
CDDO in RAW cells.
Next, we designed and synthesized acetylenic TCEs 33-44
and observed that 31 is still the most potent compound in this
series (Table 3). However, TCEs 33, 35, and 37 are nearly
equivalent to 31 in potency. Moreover, TCEs 34, 38, 42, and 43
are still more potent than or at least as potent as 9, CDDO, and
dexamethasone. TCEs containing a hydrophilic group are much
less potent than those with a hydrophobic group (39 vs 38 and
41 vs 40). A TCE with a bulky TBS group is much less potent
than with a TMS group (36 vs 32).
3.3. TCEs Induce NQO1 in Hepa1c1c7 Murine Hepatoma
Cells. We evaluated some TCEs for induction of the phase 2
cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma
cells. TCEs induce NQO1 at 1-100 nM (Table 3). Notably, (þ)-
and (()-31 double the specific enzyme activity of NQO1 at 0.9
nM. The potency is higher than the potencies of CDDO and
CDDO-Im. Remarkably, even in this assay, (þ)-enantiomers of 9,
31, and 32, having the same configuration as the CDDO antipode
are more potent than (-)-enantiomers of 9, 31, and 32, having the
same configuration as CDDO, respectively. We previously demon-
strated a linear correlation between NQO1 inducer potency (CD)
and inhibitory activity on NO production (IC50) of oleanolic acid
derivatives.29 In this series of TCEs, we also observed a similar
correlation (Figure 3), and 31 is the most potent compound in
both assays. Most recently, we have demonstrated that in-
corporation of 31 into the diet of SKH-1 hairless mice dose-
dependently induces NQO1 enzyme activity in liver, skin, and
stomach.74
Second, we designed new TCEs 28-32 on the basis of TCE 9.
From the SARs above, we mainly synthesized compounds with
hydrocarbon groups at C10a. Also, we prepared optically active 31
and 32 to compare the differences in potency between the enantio-
mers. Notably and importantly, these compounds have two different
nonenolizable cyano enones in rings A and C, which represent two
theoretically possible monocyclic nonenolizable cyano enones.
The IC50 (nM) values of these TCEs are shown in Table 3.
Remarkably, TCEs 29-32 having hydrocarbon groups are more
potent than the lead compound 9, as well as the positive controls
CDDO and dexamethasone. In particular, acetylene groups
dramatically enhance potency. TCE 31 is much more potent
than 9, CDDO, and dexamethasone and is as potent as CDDO-
Im, which is the most potent CDDO analogue. Importantly,
TCEs having two cyano enones are much more potent than
TCEs having only one cyano enone (9 vs 12, 29 vs 26, 31 vs 27).
These results suggest that the cyano enone is a very important
factor for potency. TCE 28 having a nitrile group at C10a is much
less potent than others. The fact that an electron-withdrawing
group at C10a decreases potency is identical with the previous
results shown in Table 2.
Interestingly, the racemic form and the (þ)- and (-)-en-
antiomers of 32 are slightly less potent than those of 31, although
32 has a much more bulky group than 31.73 Notably, (þ)-
enantiomers of 31 and 32, having the same configuration as the
CDDO antipode, are more potent than (-)-enantiomers of 31
and 32, having the same configuration as CDDO, respectively.
These results are identical with those of optically active 9 (Table 1).
We have evaluated 31 and CDDO in the iNOS assay using
primary mouse macrophages which are much more sensitive
3.4. TCEs Inhibit the Induction of iNOS in RAW Cells
Stimulated with IFN-γ. CDDO blocks de novo synthesis of
iNOS protein. We have confirmed that TCEs also inhibit de novo
synthesis of iNOS protein (Figure 4). TCEs 31 and 32 signifi-
cantly inhibit the induction of iNOS at 30 nM. These abilities are
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Table 3. Inhibitory Activity of New TCEs 28-44 on NO Production Induced by IFN-γ in RAW Cells and NQO1-Inducing
Potency of Some TCEs in Hepa1c1c7 Cells
a RAW 264.7 cells were treated with various concentrations of compounds and IFN-γ (10 ng/mL) for 24 h. Supernatants were analyzed for NO by the
Griess reaction.11 IC50 values are an average of two separate experiments. b These data have been published and presented in refs 79e-79h. c Hepa1c1c7
cells were grown 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.
Figure 4. TCEs 9, 31, and 34 inhibit the induction of iNOS in RAW
cells stimulated with IFN-γ. Cells were incubated with compounds
(30-300 nM) and IFN-γ (10 ng/mL) for 24 h. Total cell lysates were
analyzed by Western blot for iNOS. These data have been published in
ref 79h.
equivalent to that of CDDO-Im. TCE 9 also inhibits the
induction of iNOS at 300 nM.
3.5. TCE 31 Directly and Reversibly Reacts with Cysteine
Residues of Keap1. The sensor for inducers of NQO1 and of a
Figure 3. Correlation of potencies of TCEs as inducers of NQO1 in
network of more than 100 other cytoprotective genes is Keap1, a
protein endowed with highly reactive cysteine residues and an
essential component of the Keap1/Nrf2/ARE signaling pathway.75
Hepa1c1c7 murine hepatoma cells, expressed as CD values, and for
suppression of iNOS induction by IFN-γ in RAW cells, expressed as
IC50 values. The linear correlation coefficient is r2 = 0.91.
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Figure 7. SARs of TCEs 29, 30, acetylenic TCEs 36 and 38-44 in the
HO-1 assay. Cells were incubated with compounds (30 nM) for 6 h.
Total cell lysates were analyzed by Western blot for HO-1. These data
have been presented in ref 79g.
Figure 5. TCE 31 reacts with cysteine residues of Keap1. Absorption
spectra of 50 μM 31 (31, black line) and the reaction mixture of 50 μM
31 and 10 μM Keap1 (31 þ Keap1, gray line) in 20 mM Tris-HCl/
0.005% Tween 20 (pH 8.0) at 25 °C against Keap1 blank.
Figure 8. Racemic and optically active 31 and 32 induce HO-1 in RAW
cells. Cells were incubated with compounds (10-100 nM) for 6 h. Total
cell lysates were analyzed by Western blot for HO-1. These data have
been presented in ref 79f.
Figure 6. SARs of acetylenic TCEs in the HO-1 assay. Cells were
incubated with compounds (30-300 nM) for 6 h. Total cell lysates were
analyzed by Western blot for HO-1. These data have been presented and
published in refs 79f and 79h.
Recently, we have found that both cyano enones in rings A and C
of 31 react with cysteine residues of Keap1 to give Michael
adducts.74 The absorptions at 265 and 345 nm in the UV
spectrum of 31 with Keap1 correspond to a Michael adduct of
ring A and ring C with Keap1, respectively (Figure 5).74 Also, we
have demonstrated by NMR variable temperature studies40,76
that the Michael additions of ring A and ring C with DTT are
both reversible. The chemical reversibility of these reactions has
very significant biological implications: (1) it enhances inducer
bioavailability; (2) it allows reversible cysteine modifications of
the protein sensor Keap1, which does not need to be perma-
nently inactivated (and possibly subsequently destroyed) but
could be easily regenerated without requiring de novo protein
synthesis; (3) it leads to a pulse of activation rather than
constitutive up-regulation of the pathway; (4) it may explain,
at least in part, some of the reasons why TCE 31 is such a potent
inducer in vivo, given its ability to react with Keap1 at nanomolar
concentrations despite of the presence of millimolar concentra-
tions of glutathione.
3.6. TCEs Induce HO-1 in RAW Cells. We have evaluated
tricycles with two cyano enones in rings A and C (structures, see
Table 3) for induction of the anti-inflammatory and cytoprotec-
tive enzyme, HO-1 in RAW cells. These results are shown in
Figures 6 and 7. All compounds induce HO-1 at 30 nM. TCEs
31, 32, 37, 38, 42, 43, and 44 are higher inducers at 30 nM. They
are superior to CDDO-Im in potency at 30 nM. Although 38, 42,
43, and 44 are about 10-50 times less potent than 31 in the
iNOS assay, they are similar to 31 in potency in this HO-1 assay.
TCEs 29, 30, 35, and 40 are moderate inducers, and TCEs 36,
39, and 41 are weak inducers. A hydrophilic group and a bulky
silyl group decrease the potency. The SARs seen here correlate
nicely with the SARs obtained in the iNOS assay.
Figure 9. TCE 31 induces HO-1 in liver when given by gavage. Male
CD-1 mice (three mice per group) were gavaged with 1 μmol of the
following: TCE 9 or 31, CDDO or CDDO-Im in DMSO. After 6 h, the
livers were collected and analyzed by Western blot for HO-1. The
tubulin blot is a loading control. These data have been published and
presented in refs 79e, 79f, and 79h.
Figure 10. TCE 31 induces HO-1 in stomach when given by gavage.
Male CD-1 mice (three mice per group) were gavaged with 1 μmol of
the following: TCE 9 or 31, CDDO or CDDO-Im in DMSO. After 6 h,
the stomachs were collected and analyzed by Western blot for HO-1.
The tubulin blot is a loading control. These data have been published
and presented in refs 79e, 79f, and 79h.
between the two enantiomers. Even in this experiment, racemic
and optically active 31 are more potent inducers than CDDO-Im
at 10 nM.
3.7. TCE 31 Induces HO-1 in the Liver and Stomach of CD-1
Mouse When Given by Gavage. Subsequent to the HO-1
assay in vitro, we evaluated the potency of 9 and 31 for induction
of HO-1 in the liver and stomach using male CD-1 mice by
gavage. As shown in Figures 9 and 10, oral dosing of 1 μmol of 9
and 31 causes significant induction of HO-1 in the liver and
stomach while CDDO is markedly less potent at this low dose.
We have also compared optically active 31 and 32 in this assay
(Figure 8), but we do not observe any significant difference
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Notably, TCE 31 is as potent as CDDO-Im in the liver but clearly
more potent than CDDO-Im in the stomach, which is the most
potent semisynthetic triterpenoid we have developed for induc-
tion of HO-1.28 We have also examined optically active 31 in the
same in vivo assay, but we did not find a significant difference
between both enantiomers in the liver and stomach (see Figures
S3 and S4 in the Supporting Information).
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures and char-
b
acterization data for new compounds 1-103, experimental
procedures for biological evaluation, and Figures S1-S6. This
material is available free of charge via the Internet at http://pubs.
acs.org.
3.8. TCE 31 Reduces the Formation of Preneoplastic Foci
in the Livers of Rats Challenged with Aflatoxin B1
(AFB1). We have evaluated 31 in the short-term model of
hepatic tumorigenesis induced with AFB1 in rats.37 Prior to this
experiment, we confirmed that 31 inhibits the formation of
hepatic AFB1-DNA adducts in a dose-dependent manner.77
In the model of hepatic tumorigenesis, both 31 and CDDO-Im
were well-tolerated, as indicated by the growth rate and final
body weight being the same for dosed rats as the controls which
were not treated with AFB1. Both the number (p = 0.001) and
diameter (p = 0.024) of the glutathione S-transferase P (GST-P)
positive lesions in the liver were significantly decreased with
increasing doses of 31. The focal volume percent of foci
(analogous to tumor burden) was also significantly (p < 0.005)
reduced in a dose-dependent manner. There were no foci
detected in the AFB1-treated rats at the highest doses of 31
(i.e., 30 and 60 μmol/kg). Even the lowest dose of 31 (0.3 μmol/
kg) reduced the tumor burden by more than 90% compared to
the AFB1-treated positive control group. TCE 31 and CDDO-Im
showed the same potency (p = 0.6) at 10 μmol/kg. At these doses
the focal burden was reduced by greater than 99% (see Figure S5
and S6 in the Supporting Information).
’ AUTHOR INFORMATION
Corresponding Author
*Present address: Department of Chemistry and Institute of
Chemical Biology and Drug Discovery, State University of New
York at Stony Brook, Stony Brook, NY 11794. Phone: 631-632-
7162. Fax: 631-632-7942. E-mail: tadashi.honda@stonybrook.edu.
’ ACKNOWLEDGMENT
We especially thank Dr. Albena T. Dinkova-Kostova (The
University of Dundee, U.K.) for testing TCEs in the NQO1 assay
and her very helpful suggestions. We also thank Karen T. Liby,
Mark M. Yore, Charlotte Williams, Renee Risingsong, Darlene
Royce, Nanjoo Suh, Karen Baumgartner, and Michael B. Sporn
(Dartmouth Medical School) and Katherine K. Stephenson
(The Johns Hopkins University, MD) for expert technical
assistance with bioassays at the initiation of this project. This
investigation was supported by funds from NIH Grants R03-
CA105294 and R01-CA39416, from Reata Pharmaceuticals,
from the American Cancer Society (Grant RSG-07-157-01-
CNE), and from Cancer Research UK (Grant C20953/
A10270). H.Y. was a Visiting Scholar from Shionogi & Co.
Ltd. (Japan). M.J.L. was supported by the Arnold and Mabel
Beckman Foundation and John Zabriskie’ 61 Undergraduate
Research Fellowship.
TCE 31 significantly reduced formation of both AFB1-DNA
adducts and preneoplastic foci in the livers of rats challenged with
AFB1. The potency of 31 is similar to or better than that of
CDDO-Im for inhibiting hepatic tumorigenesis induced by
AFB1, and this chemoprevention is dependent on activation of
Nrf2.37 Notably, both CDDO-Im and 31 are 100 times more
potent than what has been reported for oltipraz, which inhibits
activation of aflatoxin in humans.78
’ ABBREVIATIONS USED
CDDO, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid; CDDO-
Me, methyl 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate;
CDDO-Im, 1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-
imidazole; DAST, N,N-diethylaminosulfur trifluoride; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DTT, 1,4-dithiothreitol; DXM,
dexamethasone; EDIA, ethyldiisopropylamine; HMPA, hexam-
ethylphosphoric triamide; IKKβ, inhibitor of nuclear factor κB
kinase β; JAK1, Janus kinase 1; Keap1, kelchlike ECH-associated
protein 1; MTPA, R-methoxy-R-(trifluoromethyl)phenylacetyl;
Nf-κB, nuclear factor κB; Nrf2, nuclear factor (erythroid-derived
2) related factor 2; STAT3, signal transducer and activator of
transcription 3
In conclusion, although a series of TCEs was designed based
on the ring A and C structures of CDDO initially, the ring C
structures of analogues of 9 are clearly different from that of
CDDO. It is noteworthy that the analogues of 9 have two
different kinds of nonenolizable cyano enones in rings A and
C, which work as strong Michael acceptors, while CDDO and its
analogues have one nonenolizable cyano enone in ring A. Indeed,
most of analogues of 9 are more potent than CDDO in the iNOS
assay. Conversely, the triterpenoid oleanolic acid, lacking any
cyano enone functional groups, is inactive. These facts strongly
suggest that the essential factor for potency is not the triterpe-
noid or the tricycle skeleton but the functional monocyclic cyano
enones that are positioned at a specific orientation relative to
each other. TCE 31, an analogue of 9 having an acetylene group at
C10a, is the most bioactive compound in both in vitro and in vivo
bioassays in our pool of drug candidates, including semisynthetic
triterpenoids and synthetic tricycles. Tricyclic compounds, diter-
penoids, and triterpenoids with an acetylene group at C10a (C8 in
terpenoid nomenclature) have not been reported prior to our
synthesis of 31. Therefore, 31 may represent a new class of
potential drug candidates having an entirely new structure for
prevention and/or treatment of inflammatorydiseases andcancers.
Further preclinical studies and detailed mechanism studies
including identification of the protein targets of 31 are in
progress.
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(77) Liby, K.; Yore, M. M.; Roebuck, B. D.; Baumgartner, K. J.;
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Journal of Medicinal Chemistry
ARTICLE
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. A novel acetylenic tricyclic bis-(cyano enone) potently induces
phase 2 cytoprotective pathways and blocks liver carcinogenesis induced
by aflatoxin. Cancer Res. 2008, 68, 6727–6732.
(78) Wang, J. S.; Shen, X.; He, X.; Zhu, Y.-R.; Zhang, B.-C.; Wang,
J.-B.; Qian, G.-S.; Kuang, S.-Y.; Zarba, A.; Egner, P. A.; Jacobson, L. P.;
Munoz, A.; Helzlsouer, K. J.; Groopman, J. D.; Kensler, T. W. Protective
alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in
residents of Qidong, People’s Republic of China. J. Natl. Cancer Inst.
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(79) Part of this work has been reported in preliminary form: (a)
Favaloro, F. G., Jr.; Honda, T.; Honda, Y.; Gribble, G. W.; Suh, N.;
Risingsong, R.; Sporn, M. B. Design and synthesis of tricyclic com-
pounds with enone functionalities in rings A and C: a novel class of
highly active inhibitors of nitric oxide production in mouse macro-
phages. J. Med. Chem. 2002, 45, 4801–4805.(b) Honda, T.; Favaloro,
F. G., Jr.; Honda, Y.; Gribble, G. W.; Suh, N.; Risingsong, R.; Sporn,
M. B. Design and Synthesis of Tricyclic Compounds with Enone
Functionalities in Rings A and C: A Novel Class of Highly Active
Inhibitors of Nitric Oxide Production in Mouse Macrophages. Pre-
sented at the 225th National Meeting of the American Chemical Society,
New Orleans, LA, March 23-27, 2003. (c) Honda, T.; Favaloro, F. G.,
Jr.; Janosik, T.; Honda, Y.; Suh, N.; Sporn, M. B.; Gribble, G. W. Efficient
synthesis of (-)- and (þ)-tricyclic compounds with enone functional-
ities in rings A and C. A novel class of orally active anti-inflammatory and
cancer chemopreventive agents. Org. Biomol. Chem. 2003, 1, 4384–4391.
(d) Honda, T.; Favaloro, F. G., Jr.; Gribble, G. W.; Sporn, M. B.; Suh, N.
Tricyclic-Bis-enone Derivatives and Methods of Use Thereof. U.S.
Patent 7,176,237 B2, February 13, 2007. (e) Honda, T.; Sundararajan,
C.; Yoshizawa, H.; Su, X.; Honda, Y.; Liby, K. T.; Sporn, M. B.; Gribble,
G. W. Novel tricyclic compounds having acetylene groups at C8a and
cyano enones in rings A and C: highly potent anti-inflammatory and
cytoprotective agents. J. Med. Chem. 2007, 50, 1731–1734.(f) Honda, T.;
Sundararajan, C.; Yoshizawa, H.; Su, X.; Honda, Y.; Liby, K. T.; Sporn,
M. B.; Gribble, G. W. Novel Tricyclic Compounds Having Acetylene
Groups at C-8a and Cyano Enones in Rings A and C: Highly Potent
Anti-Inflammatory and Cytoprotective Agents. Presented at the 234th
National Meeting of the American Chemical Society, Boston, MA,
August 19-23, 2007. (g) Lajoie, M. J.; Honda, T.; David, E.; Sundar-
arajan, C.; Liby, K. T.; Sporn, M. B.; Gribble, G. W. Design and Synthesis
of Novel Tricyclic Compounds Having Acetylene Groups at C-8a and
Cyano Enones in Rings A and C: Highly Potent Anti-Inflammatory and
Cytoprotective Agents. Presented at the 235th National Meeting of the
American Chemical Society, New Orleans, LA, April 6-10, 2008.
(h) Honda, T.; Sundararajan, C.; Gribble, G. W.; Sporn, M. B.; Liby,
K. T. Synthesis and Biological Activities of New Tricyclic-Bis-enones
(TBEs). U.S. Patent 7,714,012 B2, May 11, 2010.
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dx.doi.org/10.1021/jm101445p |J. Med. Chem. 2011, 54, 1762–1778