Synthesis, chemical reactivity as michael acceptors, and biological potency of monocyclic cyanoenones, novel and highly potent anti-inflammatory and cytoprotective agents(1)

Article abstract of DOI:10.1021/jm3003922

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g

Full text of DOI:10.1021/jm3003922

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Article
Synthesis, Chemical Reactivity as Michael Acceptors, and
Biological Potency of Monocyclic Cyanoenones, Novel and
Highly Potent Anti-inflammatory and Cytoprotective Agents
Suqing Zheng, Santosh Laxmi Y. R., Emilie David, Albena T. Dinkova-Kostova,
Katherine H. Shiavoni, Yanqing Ren, Ying Zheng, Isaac Trevino, Ronald Bumeister,
Iwao Ojima, W. Christian Wigley, James Bliska, Dale F. Mierke, and Tadashi Honda
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3003922 • Publication Date (Web): 25 Apr 2012
Downloaded from http://pubs.acs.org on April 30, 2012
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Synthesis, Chemical Reactivity as Michael Acceptors, and Biological Potency of
Monocyclic Cyanoenones, Novel and Highly Potent Anti-inflammatory and
Cytoprotective Agents1
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Suqing Zheng,✝ Santosh Laxmi, Y. R.,✝ Emilie David,‡ Albena T. Dinkova-Kostova,§,

‡
Katherine H. Shiavoni, Yanqing Ren, Ying Zheng, Isaac Trevino, Ronald Bumeister,
‡
⊥
#
#
✝,⊥
‡
Iwao Ojima,✝, ¶ W. Christian Wigley, James B. Bliska, Dale F. Mierke, and Tadashi
#
Honda*,✝,¶
✝Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook,
New York 11794, United States
‡Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United
States
§Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee
DD1 9SY, Scotland, United Kingdom
Department of Pharmacology and Molecular Sciences, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205, United States
⊥
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony
Brook, New York 11794, United States
#
Reata Pharmaceuticals Inc., Irving, Texas 75063, United States
¶Department of Chemistry, Stony Brook University, Stony Brook, New York 11794,
United States
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ABSTRACT
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Novel monocyclic cyanoenones examined to date display unique features regarding
chemical reactivity as Michael acceptors and biological potency. Remarkably, in some
biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g.,
CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Amongst monocyclic
cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles.
Furthermore an important feature of 1 is that its Michael addition is reversible. For the
inhibition of NO production, 1 shows the highest potency. Notably, its potency is about
three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in
phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest
potency. These results suggest that the reactivity of these Michael acceptors is closely
related to their biological potency. Interestingly, in LPS–stimulated macrophages, 1
causes apoptosis and inhibits secretion of TNF-α and IL-1β with potencies that are higher
than those of bardoxolone methyl and TBE-31.
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1. INTRODUCTION
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Over the past decade, we have been engaged in the modifications of oleanolic
acid, a ubiquitous naturally occurring triterpenoid, which is almost inactive in our
bioassays related to inhibition of inflammation and carcinogenesis.2–9 Our intensive
efforts led to the discovery of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
(CDDO, Figure 1), which has high anti-inflammatory and cytoprotective potency.3,4,6,10
CDDO and its related compounds are multi–functional agents. CDDO blocks the de
novo synthesis of inducible nitric oxide synthase,3,4,6 and inducible cyclooxygenase
(COX-2) in mouse macrophages, microglia, and fibroblasts.10 CDDO induces the
production of the cytoprotective enzymes such as HO-1 in U937 cells11 and NQO1 in
Hepa1c1c7 murine hepatoma cells.12 Presently, the methyl ester of CDDO (bardoxolone
methyl, BARD, Figure 1) is in Phase III clinical trials for the treatment of severe chronic
kidney disease in type 2 diabetes mellitus patients. In a multi–center, double–blind,
placebo–controlled Phase IIb clinical trial, patients treated with bardoxolone methyl
experienced a significant increase in estimated glomerular filtration rate (eGFR),
compared with no change in the placebo group.13
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During the development of CDDO, we found that the tricyclic compound, (±)-
(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-
octahydrophenanthrene-2,6-dicarbonitrile (TBE-31, Figure 1) having the nonenolizable
cyanoenones in rings A and C is also a novel class of potential anti-inflammatory, growth
suppressive, and propapoptotic compound. TBE-31 inhibits nitric oxide (NO) production
at low nanomolar concentrations in RAW cells and mouse primary macrophages
stimulated with IFN-γ.14,15 TBE-31 induces cytoprotective enzymes HO-1in RAW
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cells15 and in mice14 and NQO1 in Hepa1c1c7 murine hepatoma cells.1b,15 These
potencies are much higher than those of CDDO and bardoxolone methyl. Incorporation
of small quantities of TBE-31 in the diet robustly induces NQO1 and GST in the liver,
skin, and stomach in mice.1b TBE-31 is orally highly active against aflatoxin–induced
liver cancer in rats.15,16
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We have chemically demonstrated that the nonenolizable cyanoenones in ring A
of CDDO and in rings A and C of TBE-31 give reversible Michael adducts with the SH
group of DTT by UV and NMR studies.1b,16,17 We speculate that this characteristic
reactivity may imply a molecular mechanism of action. Indeed, our mechanism studies
suggest that bardoxolone methyl and TBE-31 regulate proteins affecting inflammation,
oxidative stress, differentiation, apoptosis, and proliferation, including Keap1, IKKβ, and
JAK1, to name a few, by reversible Michael addition between the nonenolizable
cyanoenone functionalities and the SH groups of cysteine moieties on these proteins.
Keap1 is the electrophile sensor protein in the cellular Keap1/Nrf2/ARE pathway that
controls a network of cytoprotective genes which defend against the damaging effects of
oxidative and electrophilic stress and inflammation.1b,12,18 Murine Keap1 is a thiol–rich
protein possessing 25 cysteine residues, some of which are highly reactive.18 Briefly,
bardoxolone methyl induces Nrf2–dependent transcription by binding to the SH groups
of Keap1 in the cytoplasm. Consequently, Keap1 loses its ability to target Nrf2 for
ubiquitination and proteosomal degradation, leading to Nrf2 nuclear accumulation and
transcriptional activation of ARE–dependent genes, including HO-1, NQO1, and GST.
The Keap1/Nrf2/ARE pathway is considered to be of major importance for suppressing
kidney disease progression.13 Recently, Cys179 in the activation domain of the kinase
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IKKβ was identified as a target of bardoxolone methyl.19,20 By binding to the site of
IKKβ, bardoxolone methyl inactivates the kinase and ultimately results in blocking of the
binding of NF-κB transcription factors to DNA and thus inhibits transcriptional
activation of NF-κB–dependent target genes. It has also been reported that bardoxolone
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methyl inhibits the JAK1 à STAT3 pathway by directly binding to JAK1 at Cys1077
and STAT3 at Cys259.21 Small molecule inhibitors of the STAT3 pathway are known to
be effective as anticancer agents in vitro and in animal models.
O
O
1
CN
E
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3
C
2
R1
D
NC
O
R2
A
B
1: R2 =
2: R2 =
C CH
H
C C-Me
3: R2 = C=CH2
4: R2 = Me
5: R2 = CN
6: R2 = CO2Me
CDDO: R1 = CO2H
BARD: R1 = CO2Me
O
CN
O
O
C
NC
O
CN
CN
A
B
H
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8
TBE-31
Figure 1. Structures of CDDO, bardoxolone methyl (BARD), TBE-31, and
monocyclic cyanoenones 1–8.
Therefore, on the basis of the mechanism studies, we concluded that the
pharmacophores in semisynthetic pentacyclic triterpenoids and synthetic tricycles are
nonenolizable cyanoenones which work as Michael acceptors. Thus, we were interested
in the chemical reactivity as Michael acceptors and biological potency of monocyclic
cyanoenones, which represent the reactive moiety in more complex tri- and penta-cycles.
We have synthesized a series of monocyclic cyanoenones 1–8 (Figure 1) to delineate the
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structural features important for the unique reactive properties. For example, 1 has the
same structure as that of ring C in TBE-31, 8 has the same structure as that of ring A in
bardoxolone methyl and TBE-31, and 5 and 6 have typical electron–withdrawing groups
at quaternary carbon.
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We have evaluated the chemical reactivity of monocyclic cyanoenones using UV
and 1H NMR spectroscopy and their biological potency in cell–based assays related to
inhibition of inflammation and carcinogenesis. Monocyclic cyanoenones 1 and 5 are
very reactive Michael acceptors, but more importantly, the addition is reversible. For the
inhibition of NO production induced by IFN-γ in RAW cells, 1 shows the highest potency,
most notably, 1 is three times more potent than CDDO. For the induction of the
cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells, 1 also demonstrates
the highest potency. These results suggest that the reactivity of these Michael acceptors
is closely related to the biological potency. Monocyclic cyanoenone 1 causes apoptosis
and inhibits secretion of IL-1β and TNF-α in LPS–stimulated macrophages and these
potencies are higher than those of bardoxolone methyl and TBE-31. Remarkably, in this
assay, the simple monocyclic structure is more potent than penta- and tricycles. We
herein describe the full account of our synthetic work with monocyclic cyanoenones and
their unique and interesting features with respect to the chemical reactivity as Michael
acceptors and biological potency.
2. CHEMISTRY
Monocyclic cyanoenone 1 was synthesized from commercially available ethyl 4-
oxocyclohexanone-carboxylate (9) by the sequence shown in Scheme 1. Alcohol 1022
was prepared in 83% yield from 9 by ketalization with ethylene glycol in the presence of
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(+)-CSA in benzene and subsequent methylation, followed by reduction with LiAlH4.
Ratcliffe oxidation of 10 with CrO323 gave aldehyde 11 in 95% yield. Acethylene 12 was
obtained in 68% yield from 11 by Wittig reaction with
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(chloromethyl)triphenylphosphonium chloride,24 followed by dehydrochlorination with
MeLi and subsequent treatment with TMSCl.25 The ketal of 12 was removed under
acidic conditions to afford 13 in 98% yield. Conversion of 13 into enone 14 was
achieved by addition of phenylselenyl group to lithium enolate of 13 and subsequent
oxidation/elimination with 30% aqueous H2O2 solution (36% yield) after several
oxidation methods failed (for example, Br2–HBr in acetic acid, PTAB and DBU,26
PhSeCl in ethyl acetate and subsequent oxidation/elimination with 30% H2O2,27 PhSeCl–
pyridine in CH2Cl2 and subsequent oxidation/elimination with 30% H2O228). Formylation
of 14 with ethyl formate in the presence of NaOMe in benzene,29 followed by the
condensation with hydroxylamine hydrochloride in aqueous EtOH,30 gave isoxazole 15 in
36% yield. The cleavage of the isoxazole ring of 15 with NaOMe in MeOH and Et2O30
produced 16 in quantitative yield. Monocyclic cyanoenone 1 was obtained in 47% yield
by oxidation of 16 with DDQ in benzene under reflux (3.2% overall yield from 9).
Although we could synthesize 10 mg of 1 in a single batch by this sequence, it was quite
difficult to provide more than 100 mg of 1 in a single batch (the amount necessary for
further biological evaluation) because this synthesis requires 12 steps and the yield of
some steps is pretty low. In particular, conversion of 13 into 14 (insertion of a double
bond into a cyclohexanone ring) was problematic. The yield decreased according to the
increasing amount of 13.
Scheme 1a
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O
O
O
O
O
a, b, c
e, f
d
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7
CH2OH
CHO
CO2Et
8
9
10 (83%)
11 (95%)
9
O
O
O
O
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g
h
TMS
TMS
CN
TMS
O
12 (68%)
13 (98%)
14 (36%)
N
O
O
CN
i, j
k
l
TMS
15 (36%)
16 (100%)
1 (47%)
aReagents: (a) ethylene glycol, (+)-CSA, PhH; (b) LDA, CH3I; (c) LiAlH4, Et2O; (d) CrO3, pyridine
CH2Cl2; (e) Ph3PCH2Cl(Cl), n-BuLi, THF; (f) MeLi, THF; TMSCl; (g) aqueous HCl, acetone; (h)
LDA, PhSeCl; 30% H2O2, CH2Cl2; (i) HCO2Et, NaOMe, PhH; (j) NH2OH·HCl, aqueous EtOH; (k)
NaOMe, MeOH, Et2O; (l) DDQ, PhH.
Scheme 2a
OTMS
O
OTMS
O
O
O
O
c
a
b
d
MeO
MeO
Cl
Cl
CHO
CHO
17 (100%)
18 (52%)
19 (75%)
20 (90%)
N
O
O
OH
O
O
O
CN
CN
e
f
g
h
i
16 (100%)
1 (71%)
21 (90%)
22 (100%)
23 (35%)
aReagents: (a) toluene; (b) (Ph)3PCH2Cl(Cl), n-BuLi, THF; (+)-CSA, Et2O; (c) ethylene glycol,
PPTS, PhH; (d) MeLi, THF; (e) aqueous HCl, acetone; (f) HCO2Et, NaOMe, PhH; (g) NH2OH·HCl,
aqueous EtOH; (h) NaOMe, MeOH, Et2O; (i) PhSeCl, pyridine, CH2Cl2; 30% H2O2, CH2Cl2.
Thus, we have explored an entirely new synthesis as shown in Scheme 2. To
avoid insertion of a double bond to a cyclohexanone ring, we envisioned that a
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cyclohexenone ring should be constructed by Diels-Alder reaction initially. Known
Diels–Alder adduct 1731 was obtained as a mixture of two diasteromers (6.5:1.0) in
quantitative yield from Danishefsky’s diene and methacrolein (2.1 eq.) in toluene in a
sealed tube at 110 °C. Wittig reaction on 17 with (chloromethyl)triphenylphosphonium
chloride, followed by deprotection with (+)-CSA in Et2O gave 18 as a mixture of E/Z
chlorovinyl isomers (E:Z = 2:1) in 52% yield. Ketalization of 18 with ethylene glycol in
the presence of PPTS in benzene afforded ketal 19 in 75% yield. Dehydrochlorination of
19 with MeLi in THF, followed by quenching of the acetylide with aqueous NH4Cl
solution provided 20 in 90% yield.24 The ketal of 20 was removed under acidic conditions
to produce 21 in 90% yield. Formylation of 21 with ethyl formate gave 22 in quantitative
yield. Isoxazole 23 was obtained in 35% yield by condensation of 22 with
hydroxylamine hydrochloride. The cleavage of the isoxazole ring of 23 with NaOMe
afforded nitrile 16 in quantitative yield. Monocyclic cyanoenone 1 was prepared in 71%
yield from 16 by addition of PhSeCl in the presence of pyridine and subsequent
oxidation/elimination of the selenated intermediate with 30% aqueous H2O2 solution.28
This oxidation method (71% yield) was better than the DDQ method (47% yield) for this
oxidation with an unprotected acetylene group. Overall, 1 was synthesized in 7.9% yield
in 9 steps from Danishefsky’s diene. The scheme results in three less steps and an overall
yield of two times more than the previous scheme. Accordingly, we were able to
synthesize 300 mg of 1 in a single batch (see the Supporting Information).
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Scheme 3a
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O
O
O
O
O
a
b
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Cl
19
24 (96%)
25 (63%)
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O
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CN
e, f
c, d
2 (68%)
26 (22%)
aReagents: (a) MeI, n-BuLi, THF; (b) aqueous HCl, acetone; (c) HCO2Et, NaOMe, PhH; (d)
NH2OH·HCl, aqueous EtOH; (e) NaOMe, MeOH, Et2O; (f) PhSeCl, pyridine, CH2Cl2; 30% H2O2,
CH2Cl2.
New monocyclic cyanoenone 2 was synthesized from 19 by the sequence shown
in Scheme 3. Ketal 24 was prepared in 96% yield by the treatment of 19 with MeI in the
presence of n-BuLi in THF. Removal of the ketal of 24 under acidic conditions gave
enone 25 in 63% yield. Monocyclic cyanoenone 2 was obtained in 15 % yield via
isoxazole 26 from 25 by the same sequence as for 1 from 21.
Scheme 4a
OTMS
O
O
OH
c
a, b
MeO
CHO
27 (53%)
17
28 (84%)
N
O
O
CN
d
e, f
3 (59%)
29 (91%)
aReagents: (a) Ph3PCH3I, n-BuLi, THF; (b) (+)-CSA, Et2O; (c) HCO2Et, NaOMe, PhH; (d)
NH2OH·HCl, aqueous EtOH; (e) NaOMe, MeOH, Et2O; (f) DDQ, PhH.
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New monocyclic cyanoenone 3 was synthesized from Diels-Alder adduct 17 by
the sequence shown in Scheme 4. Wittig reaction on 17 with
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9
methyltriphenylphosphonium iodide in the presence of n-BuLi in THF, followed by
deprotection with (+)-CSA, gave 27 in 53% yield. Monocyclic cyanoenone 3 was
prepared in 45% yield via 28 and 29 from 27 by the same sequence as for 1 from 14 (see
Scheme 1).
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Known monocyclic cyanoenone 4 (crystalline solid, mp 105–106 °C) was
prepared according to the methods in the literature.32
Scheme 5a
OTMS
O
OTMS
CN
O
O
CN
a
d, e
b
+
MeO
MeO
MeO
CN
CN
CN
CN
30
31
32
5
c
aReagents and yields: (a) toluene, 93%; (b) (+)-CSA, Et2O, 35: 19%, 36: 46%; (c) (+)-CSA, THF,
86%; (d) p-TsCN, LDA, THF, 55%; (e) DDQ, PhH, 38%
New monocyclic cyanoenone 5 was synthesized from the known Diels–Alder
adduct 3033 by the sequence as shown in Scheme 5. Diels–Alder adduct 30 was obtained
as a mixture of two separable diastereomers in 93% yield from Danishefsky’s diene and
acrylonitrile (3 eq.) in toluene in a sealed tube at 120 °C. Deprotection of 30 with (+)-
CSA in Et2O at rt gave 31 (19% yield as a mixture of two separable diastereomers) and
32 (46% yield). The treatment of 31 with (+)-CSA in THF at 60 °C produced 32 in 86%
yield. Overall, 32 was obtained in 62% yield from 30. Since Johnson’s isoxazole
method30 which was used for the synthesis of 1, 2, and 3, followed by DDQ oxidation
afforded 5 from 32 in very low yield (< 5%), we attempted to introduce a cyano group to
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32 using p-TsCN.34 Cyanation of the enolate of 32 generated using LDA in THF, with p-
TsCN, followed by DDQ oxidation gave 5 in 21% yield.
7
8
9
Monocyclic cyanoenone 6 was previously reported35 but we have explored a new
synthesis of 6 as shown in Scheme 6. Known compound 3436 was prepared by Diels-
Alder reaction between Danishefsky’s diene and methyl acrylate, followed by
deprotection with (+)-CSA (86% yield). New compound 35 was obtained in quantitative
yield from 34 by cyanation of the enolate with p-TsCN. DDQ oxidation of 35 gave 6 in
56% yield.
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Scheme 6a
OTMS
OTMS
a
b
MeO
MeO
O
CO2Me
CO2Me
33 (100%)
O
O
CN
d
CN
c
CO2Me
34 (86%)
CO2Me
35 (100%)
CO2Me
6 (56%)
aReagents: (a) toluene, (b) (+)-CSA, Et2O, (c) p-TsCN, LDA, THF; (d) DDQ, PhH.
New monocyclic cyanoenone 7 was synthesized from 13 as shown in Scheme 7.
Cyanoenone 36 was prepared in 78% yield from 13 by cyanation with p-TsCN, followed
by DDQ oxidation. Removal of TMS group from 36 gave 7 in 50% yield.
Scheme 7a
O
O
O
CN
CN
c
a, b
TMS
TMS
13
7 (50%)
36 (78%)
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aReagents: (a) p-TsCN, LDA, THF; (b) DDQ, PhH; (c) TBAF, THF.
3
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5
6
Monocyclic cyanoenone 8 was synthesized in 5 steps from 37 (Scheme 8), which
7
8
9
was prepared by Robinson annulation with isobutyraldehyde and ethyl vinyl ketone.37
Known compound 38 was prepared in 50% yield by reductive methylation of 37.38
Formylation of 38, followed by the treatment with hydroxylamine, gave isoxazole 40 in
78% yield. The cleavage of the isoxazole ring of 40 with NaOMe afforded 41 in 90%
yield. Monocyclic cyanoenone 8 was obtained by DDQ oxidation of 41 in 31% yield.
Scheme 8a
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N
O
O
OH
O
O
O
O
NC
NC
a
b
c
d
e
37
38 (50%)
39 (88%)
40 (89%)
41 (90%)
8 (31%)
aReagents: (a) MeI, NH3, Li, t-BuOH; (b) HCO2Et, NaOMe, PhH; (c) NH2OH·HCl, aqueous EtOH;
(d) NaOMe, Et2O, MeOH; (e) DDQ, PhH.
3. RESULTS AND DISCUSSION
3-1. Chemical Reactivity of Monocyclic Cyanoenones as Michael Acceptors
UV Studies on Monocyclic Cyanoenones
We have evaluated the chemical reactivity of 1–8 as Michael acceptors using UV
spectroscopy. Monocyclic cyanoenones 1–6, 7, and 8 with DTT give Michael adducts I,
II, and III, respectively (Scheme 9). Since adducts I, II, and III have different UV
absorption spectra from those of the corresponding monocyclic cyanoenones, UV
spectroscopy can clarify whether or not the Michael adducts are produced.
Scheme 9
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O
OH
4
5
6
7
8
9
CN
HS
CN
HS
CN
OH
SH
OH
S
SH
R
R
OH
I
1–6
OH
O
OH
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CN
S
OH
SH
OH
SH
OH
OH
7
II
O
OH
CN
HS
CN
S
OH
SH
OH
SH
OH
8
III
OH
(1) UV studies on 1, 2, and 6 with DTT
Monocyclic cyanoenone 1 has a UV absorption at 334 nm (absorbance (A) =
0.227 with 1 eq. of DTT and 0.406 with 10 eq. of DTT) upon the addition of DTT under
dilute (0.1 mM of 1) and neutral aqueous conditions (pH 7.4 phosphate buffered saline–
1% ethanol), which are similar to physiological conditions (Figure 2 (A)). The
absorption at 334 nm, which increases with the addition of DTT, is identical to the
absorption of 16 at 330–332 nm (Figure 2 (B)), which increases with the addition of
NaOH (presumably due to enhanced H–bonding to the enol hydroxyl). This observation
strongly indicates that the absorption at 334 nm comes from the Michael adduct of DTT
with 1, because the chromophore of the Michael adduct is the same as the enol form of
16, which shows the absorption at higher wavelength than the keto form of 16.
Interestingly, while the UV spectrum of 1 (c = 0.1 mM) in the phosphate buffer
shows two absorption bands at 230 nm (A = 0.915) and 322 nm (A = 0.083), the one at
the higher wavelength (322 nm) is not observed in ethanol and deionized water (Figure 2
(C)). The phosphate buffer saline–1% ethanol (pH 7.4) solution includes 1 mM KH2PO4,
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5.6 mM Na2HPO4, and 154 mM NaCl. Although the phosphate and chloride anions are
much weaker nucleophiles than the SH group, because the concentration of NaCl is 1,540
times more than the concentration of DTT (0.1 mM), we expect that 1 reacts with
chloride anion to give a Michael adduct.39
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We have calculated an approximate equilibrium constant (K) of this Michael
reaction in the solution of 1 and DTT (each initial concentration, 0.1 mM). The value of
the constant is approximately 2.0 × 103 (Liters/mol, see the calculation in the Supporting
Information), implying that this addition is very strongly favored.
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Figure 2. UV spectra of monocyclic cyanoenones with DTT at rt. (A), (D), (E), (G)
and (H): UV spectra of 0.1 mM monocyclic cyanoenones (red lines) in phosphate buffer
saline–1% ethanol (pH 7.4) and their corresponding reaction mixtures with 0.1 mM DTT
(blue lines) and 1 mM DTT (black lines). (B) and (I): UV spectra of 16 (0.1 mM) and 41
(0.16 mM) (orange lines) in phosphate buffer saline–1% ethanol (pH 7.4) and their
corresponding reaction mixtures with 10 mM NaOH (green lines). (C) and (F): UV
spectra of 0.1 mM monocyclic cyanoenones (red lines) in phosphate buffer saline–1%
ethanol (pH 7.4), in water (light blue lines), and in ethanol (gray lines).
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While 2 with DTT in the same phosphate buffer gives the similar spectra at 333
nm (A = 0.237 with 1 eq. of DTT and 0.395 with 10 eq. of DTT) to those of 1 with DTT,
the spectrum of 2 without DTT gives a much smaller absorption at 319 nm (A = 0.042)
than that of 1 (see Figure S1 (A) in the Supporting Information). These results indicate
that 2 has the similar reactivity with DTT to that of 1 but is less reactive with the chloride
anion than 1.
Monocyclic cyanoenone 6 with DTT in the phosphate buffer gives absorptions at
328 nm (A = 0.171 with 1 eq. of DTT) and 330 nm (A = 0.360 with 10 eq. of DTT),
respectively. Monocyclic cyanoenone 6 without DTT in the same buffer does not show
clear absorption at the higher wavelength. These spectra are almost identical to those of
2 (Figure S1 (B) in the Supporting Information).
(2) UV studies on 3 and 4 with DTT
Neither 3 nor 4 gives the absorption at around 333 nm upon the addition of DTT
in the phosphate buffer (Figures 2 (D) and S1 (C) in the Supporting Information). These
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spectra indicate that both monocyclic cyanoenones do not react with DTT under these
conditions.
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(3) UV studies on 5 with DTT
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Monocyclic cyanoenone 5 without DTT in the phosphate buffer gives the strong
absorption at 323 nm (A = 0.561), which is considered to come from an adduct of 5 with
chloride anion analogous to that observed for 1.39 Upon the addition of DTT, the UV
spectra are similar [325 nm (A = 0.571 with 1 eq. of DTT) and 327 nm (A = 0.569 with
10 eq. of DTT)] to that of 5 without DTT. These observations indicate that 5 is highly
reactive with both chloride anion and DTT (Figure 2 (E)). Monocyclic cyanoenone 5 in
ethanol also shows the absorption at 302 nm (A = 0.171), which comes from a Michael
adduct of 5 with ethanol. The molecular ion peak [m/z 203 (M–H)–] of the adduct is
observed in the ethanolic solution of 5 by negative MS (ESI–). Surprisingly, 5 can react
with ethanol although ethanol is a weaker nucleophile than chloride anion. In deionized
water, an absorption band at the higher wavelength is not observed (Figure 2 (F)).
(6) UV studies on 7 with DTT
Monocyclic cyanoenone 7 (an acetylenic enolizable cyanoenone) with 1 eq. of
DTT in the phosphate buffer gives the same spectrum as that of 7 without DTT. The
small red shift, which is considered to come from a small amount of the adduct, is
observed when 10 eq. of DTT is added (Figure 2 (G)).
(7) UV studies on 8 with DTT
The UV spectrum of 8 shows the weak absorption at 275 nm upon the addition of
10 eq. of DTT while any changes are not observed upon the addition of 1 eq. of DTT
(Figure 2 (H)). The absorption at 275 nm is identical with the absorption of 42 at 260–
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265 nm (Figure 2 (I)), which increases with the addition of NaOH. This observation
strongly indicates that the absorption at 275 nm comes from the Michael adduct of 8 with
DTT, because the chromophore of the Michael adduct is the same as the enol form of 42,
which shows the absorption at the higher wavelength than the keto form of 42.
(8) UV studies on monocyclic cyanoenones with Keap1
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Murine Keap1 is a thiol–rich protein possessing 25 cysteine residues, some of
which are highly reactive.18 UV studies on 1, 4, 7, and 8 with Keap1 are completely
consistent with those with DTT (Figure S2 in the Supporting Information). Importantly,
1 is very reactive with both cysteine moieties on Keap1 as well as DTT, a small thiol
nucleophile. To the contrary, 4, 7, and 8 have greatly reduced reactivity towards Keap1.
Overall, our UV studies demonstrate that the order of the reactivity as Michael
acceptors with nucleophiles is 5 >1 > 2 = 6 > 7 > 8 > 3 = 4. We have observed a
tendency that electron–withdrawing groups at C3 position increase the reactivity while
electron–donating groups do not. Monocyclic cyanoenone 5 with a nitrile group, a strong
electron–withdrawing group, is the most reactive Michael acceptor among monocyclic
cyanoenones evaluated. The reactivity of 5 with chloride anion is almost the same as that
with DTT. Only 5 can react with ethanol, a weaker nucleophile, to produce an adduct.
Notably and interestingly, moderate electron–withdrawing groups, alkyne groups
enhance the reactivity equal to or slightly more than a methoxycarbonyl group.
Importantly, the enolizable cyanoenone 7 is less reactive than the corresponding
nonenolizable cyanoenone 1.
NMR studies on 1 and 5
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1H NMR (500 MHz) studies confirmed that 1 reacts in a dose–dependent fashion
with DTT to give the Michael adduct (Figure 3 (A)). In a 30 mM DMSO-d6 solution of
1, three olefinic protons HA, HB, and HC are observed at δ 8.17 ppm (d, J = 3 Hz), 7.21
ppm (dd, J = 3 and 10 Hz), and 6.35 ppm (d, J = 10 Hz), respectively. Upon increasing
amounts of DTT (0.25 eq. à 1.0 eq.), HA, HB, and HC decrease and an enol proton HD
and the new olefinic protons HE and HF appear and increase, which are derived from the
Michael adduct of 1 with DTT. The adduct formation was also confirmed by the
detection of molecular ion peaks at m/z 310 (ESI–) and 312 (ESI+ with formic acid) upon
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1
treating 1 with 1 eq. of DTT. H NMR (300 MHz) studies confirmed that 5 also reacts in
a dose–dependent fashion with DTT to give the Michael adduct (data not shown).
In a 30 mM DMSO-d6 solution of 1 with 1.0 eq. of DTT at 21 °C, three olefinic
protons HA, HB, and HC, which are derived from 1, and the enol proton HD and two
olefinic protons HE and HF, which are derived from the adduct, are observed. At elevated
temperatures (21 à 37 à 50 °C), the enol proton HD and olefinic protons HE and HF of
the adduct decrease and the olefinic protons HA, HB, and HC of 1 increase. At 37° C, the
ratio of the adduct and 1 is about 1:1. Upon decreasing the temperature (50 à 37 à
21 °C), the reverse reaction occurs and the protons of the adduct increase while the
protons of 1 decrease. This observation shows that the Michael addition is reversible
(Figure 3 (B)). Although the Michael adduct of 1 with DTT is observed by UV and 1H
NMR, the adduct cannot be isolated. These results indicate that the conversion of the
Michael adduct to 1 is very fast.
Such chemical reversibility has very significant biological implications: 1) it
enhances the bioavailability of 1; 2) it allows reversible cysteine modifications of the
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protein targets, which do not need to be permanently inactivated (and possibly
subsequently destroyed), but could be easily regenerated without requiring de novo
protein synthesis, and 3) it leads to a pulse of inhibitory action, rather than constitutive
downregulation of the target signaling pathways. Taken together these properties may
explain, at least in part, why TBE-31 containing 1 structure in ring C has extraordinary
high biological potency in vivo,1b,14–16 given their ability to react with protein targets at
nanomolar concentrations despite the presence of millimolar concentrations of
glutathione.
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A
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1
Figure 3. H NMR studies on 1 with DTT (A) 1H NMR changes upon increasing
amounts of DTT in a 30 mM DMSO-d6 solution of 1. (B) 1H NMR changes of 30 mM
DMSO-d6 solution of 1 with 1 equivalent of DTT at elevated and decreased temperatures.
The δ 2.50 signal of CD3SOCD2H was used as an internal standard.
3-2. BIOLOGICAL RESULTS
Monocyclic cyanoenones 1–8 inhibit NO production induced by IFN-γ in RAW
264.7 cells.
We evaluated the inhibitory activities of 1–8 on NO production induced by IFN-γ
in RAW 264.7 cells. The inhibitory activities [IC50 (nM) values] of 1–8, TBE-31, and
CDDO are shown in Table 1. Among monocyclic cyanoenones, 1 has the highest
potency, followed by 2. Notably, both 1 and 2, which are acetylenic monocyclic
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nonenolizable cyanoenones, are 2–3 times more potent than CDDO which is a
pentacyclic tritepenenoid. Remarkably, 1 approaches the potency of TBE-31 in this
assay, which is currently the most potent among semisynthetic pentacyclic triterpenoids
and synthetic tricycles; 1 is only about six times less potent than TBE-31.
Monocyclic cyanoenones 1–8 induce NQO1 in Hepa1c1c7 murine hepatoma cells.
Monocyclic cyanoenones 1–8 were next evaluated for the induction of the phase 2
cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells. Monocyclic
cyanoenones double the specific enzyme activity of NQO1 in the 20–2000 nM
concentration range (Table 1). Notably, even in this assay, 1 is the most potent and 2 is
the next.
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As described earlier, a strong correlation between reactivity of monocyclic
cyanoenones towards DTT and interaction with Keap1 has been established. Monocyclic
cyanoenone 1 is very reactive towards DTT, much higher than that observed for 8 and
CDDO,17 which has the same structure in ring A as that of 8. Monocyclic cyanoenone 1
is also very reactive towards cysteine moieties of Keap1 (Figure S2 in the Supporting
Information). This reactivity with Keap1 as monitored by UV absorption is a unique
property of 1, as the related compound 8 has greatly reduced reactivity towards Keap1.
Nevertheless, 1 is about 10 times less potent than CDDO in this assay although 1 is much
more potent than 8. It is possible that 1 would be as potent as CDDO or more potent with
respect to NQO1 inducing potency in the other cell lines, for example, macrophages,
because there may be differences between macrophages and hepatoma cells with respect
to their response to monocyclic cyanoenones.
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Table 1. Inhibitory activity of monocyclic cyanoenones on IFN-γ induced NO production
5
6
7
in RAW 264.7 cells and NQO1–inducing potency in Hepa1c1c7 cells
8
9
O
O
O
O
O
CN
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16
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CN
CN
CN
CO2H
NC
O
NC
O
R
H
H
TBE-31
1–6
7
8
CDDO
Compd
R
IC50
CD
Compd
IC50
CD
(nM)a
(nM)b
(nM)a
(nM)b
1
5.8±0.9
7.3±1.1
22
7
8
151.9±17.6
100
C≡CH
2
3
4
5
6
33
1901.4±309.8 1900
C≡C-Me
C=CH2
Me
169.9±26.7 150
304.8±21.4 150
219.4±63.4 58
TBE-31 1.0c
CDDO 16.5±0.5
0.9c
2.3c
CN
CO2Me
58.0±7.6
320
aRAW 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 compounds, using a
2 fold serial dilution (0–200 nM or 0–10 µM, as appropriate) for 2 h, followed by IFN-γ
(20 ng/mL) treatment for an additional 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-γ–induced
NO production normalized to cell viability. Results shown are the average of three
independent experiments. Error represents standard deviation.
bHepa1c1c7 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
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value is based on the activity from eight replicate wells at each concentration. The
standard deviation in each case was between 5 and 10%.
cThese data have been published in ref 15.
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We previously demonstrated a linear correlation between NQO1 inducer potency
(CD) and inhibitory activity on NO production (IC50) of semisynthestic triterpenoids9,12
and synthetic tricycles.15 In this series of monocyclic cyanoenones, although we
observed a similar general trend that the lower IC50 values (nM), the lower CD values
(nM), the correlation was not linear.
We have found that the reactivity of these Michael acceptors is closely related to
the biological potency except for 5 and that 1 has the highest biological potency amongst
all of the monocyclic cyanoenones so far. It is very interesting that an ethynyl group at
C3 position plays an important role for enhancing the reactivity as a Michael acceptor
and the biological potency in both bioassays. Curiously, although 5 has the highest
reactivity as a Michael acceptor, the biological potency is not so high. The reasons for
this can include: (i) due to its exceedingly high chemical reactivity, a large portion of 5
could be “quenched” by abundant cellular thiols, such as the cysteine residue of
glutathione, which is present at millimolar concentrations; (ii) monocyclic cyanoenone 5
could be inactivated by chloride anion in the cell culture medium used for biological
testing and/or (iii) in addition to the chemical reactivity, other factors such as cellular
uptake and export mechanisms may play a role in biological potency.
Monocyclic cyanoenone 1 strongly causes apoptosis and inhibits secretion of TNF-α
and IL-1β in LPS–stimulated macrophages.
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Figure 4. Monocyclic
5
6
7
8
cyanoenone 1 causes apoptosis
and inhibits secretion of TNFα
and IL-1β in LPS-induced
macrophages. Macrophages
were left untreated or treated
with TPCA-1 (0.5 µM),
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bardoxolone methyl (BARD, 0.5
µM), TBE-31 (0.1 µM), or 1
(0.5 µM). Some of the
macrophages were not exposed
to LPS (Compound 24 h) while
other macrophages were
exposed to LPS (100 ng/mL, 1 h
after the start of compound
treatment) for 8 h (+LPS 8 h) or
24 h (+LPS 24 h). Culture
supernatants collected at 8 h or
24 h were assayed for (A)
lactate dehydrogenase (LDH, %
LDH released) to measure cell death or (B) TNF-α (pg/mL) or (C) IL-1β (pg/mL) to
measure levels of secreted cytokines. Results shown are averages from 2 independent
experiments. Error bars represent standard deviation.
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Previous results show that inhibition of IKKβ with the ATP–competitive inhibitor
ML120B in LPS–stimulated macrophages results in apoptosis, caspase-1 activation, and
IL-1β secretion.40 We evaluated 1 as a representative of monocyclic cyanoenones, TBE-
31, bardoxolone methyl, and TPCA-141 which is an ATP-competitive IKKβ inhibitor in
LPS–stimulated macrophages.42 In order to measure LPS–stimulated production of
cytokines in macrophages after pretreatment with these compounds, it is important to use
maximal concentrations of the compounds that are not directly toxic to the cells
(maximum non-toxic dose; MNTD), otherwise lack of cytokine production could be an
artifact from rapid cell death. We determined the MNTDs for 1 (0.5 µM), TBE-31 (0.1
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µM), bardoxolone methyl (0.5 µM) and TPCA-1 (0.5 µM). In preliminary experiments,
1 and TPCA-1 strongly caused LPS-stimulated cell death but bardoxolone methyl and
TBE-31 did not (Figure 4A). Monocyclic cyanoenone 1 was more potent than TPCA-1
after macrophages were exposed to LPS for 8 h. Even 24 h after LPS–exposure, 1 was as
potent as TPCA-1. Monocyclic cyanoenone 1 was also more effective than TPCA-1,
bardoxolone methyl, and TBE-31 at inhibiting secretion of TNF-α from LPS stimulated
macrophages (Figure 4B). Importantly, unlike TPCA-1, 1 did not induce high levels of
secretion of IL-1β (Figure 4C) despite the fact that both inhibitors caused LPS-stimulated
cell death. Remarkably, in this assay, the simple monocyclic structure is more potent
than pentacycles and tricycles. These results suggest that 1 may be a new class of IKKβ
inhibitor for the treatment of inflammation and cancer.
In summary, through these investigations, we have demonstrated that the essential
factor for potency is not the pentacyclic triterpenoid or the tricyclic skeleton, but the
functional monocyclic cyanoenones that are positioned at a specific orientation relative to
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each other. We have clarified that the reactivity as Michael acceptors of monocyclic
cyanoenones is closely related to the biological potency. Amongst monocyclic
cyanoenones evaluated, 1 is a very reactive Michael acceptor with thiol nucleophiles and
more importantly, the addition is reversible. Monocyclic cyanoenone 1 has the highest
potency for the inhibition of NO production induced by IFN-γ in RAW cells and the
induction of the cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells. In
LPS–stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and
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IL-1β. These potencies are higher than those of bardoxolone methyl, TBE-31, and
TPCA-1, which is an ATP-competitive IKKβ inhibitor. Importantly and interestingly, a
very small molecule such as 1 has high biological potency against inflammation and
carcinogenesis. Not only further biological in vivo studies on 1 but also drug design of
new compounds using monocyclic cyanoenones as fragments are very interesting and
promising. These investigations are in progress.
ASSOCIATED CONTENT
Supporting Information. Synthetic procedures and characterization data for new
compounds 1–41, materials and methods for LPS-induced macrophage assay, UV spectra
of monocyclic cyanoenones with DTT and Keap1 (Figures S1 and S2), and list of
elemental analyses for specifying the purity of 1–8. This material is available free charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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Department of Chemistry and Institute of Chemical Biology and Drug Discovery, Stony
Brook University, Stony Brook, NY 11794-3400. Phone: 631-632-7162. Fax: 631-632-
7942. E-mail: tadashi.honda@stonybrook.edu.
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ACKNOWLEDGEMENT
We especially thank Dr. David M. Lemal (Dartmouth College) for his helpful suggestions
and discussions. We thank Mr. Wayne Casey (Dartmouth College) for expert technical
assistance with 1H NMR variable temperature studies. We also thank Dr. Bela Ruzsicska
(Stony Brook University) for expert technical assistance with LC-MS. This investigation
was supported by funds from Reata Pharmaceuticals, Research Councils UK, and Cancer
Research UK (C20953/A10270). K.H.S. was a James O. Freedman Presidential Scholar
at Dartmouth College.
ABBREVIATIONS USED
ARE, antioxidant response element; (+)-CSA, (+)-10-camphersulfonic acid; TMSCl,
chlorotrimethylsilane; DDQ, 2,6-dichloro-3,5-dicyano-1,4-benzoquinone; DTT,
dithiothreitol; GST, glutathione S-transferases; HO-1, heme oxygenase 1; IKKβ, inhibitor
of nuclear factor κB kinase β; IFN-γ, interferon-γ; IL-1β, interleukin-1β; JAK1, Janus
kinase 1; Keap1, kelch-like ECH–associated protein 1; LPS, lipopolysaccharide; NQO1,
NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor (erythroid–derived 2) related
factor 2; NF-κB, nuclear factor κB; PhSeCl, phenylselenyl chloride; PTAB,
phenyltrimethylammonium tribromide; PPTS, pyridinium p-toluenesulfonate; p-TsCN, p-
toluenesulfonyl cyanide; TNF-α, tumor necrosis factor-α.
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REFFERENCES
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(1) Part of this work has been reported in preliminary form: (a) Honda, T.; David, E.;
Mierke, E. D. Monocyclic cyanoenones and methods of use thereof. Patent
Application, PCT/WO 2010/011782 A1 (Jan. 28, 2010), US 2011/0196007 A1 (Aug.
11, 2011). (b) 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. An exceptionally potent inducer of cytoprotective enzymes:
elucidation of the structural features that determine inducer potency and reactivity
with Keap1. J. Biol. Chem. 2010, 285, 33747–33755.
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(2) Honda, T.; Finlay, H. J.; Gribble, G. W.; Suh, N.; Sporn, M. B. New enone
derivatives of oleanolic acid and ursolic acid as inhibitors of nitric oxide production
in mouse macrophages. Bioorg. Med. Chem. Lett. 1997, 7, 1623–1628.
(3) Honda, T.; Rounds, B. V.; Gribble, G. W.; Suh, N.; Wang, Y.; Sporn, M. B. Design
and synthesis of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, a novel and highly
active inhibitor of nitric oxide production in mouse macrophages. Bioorg. Med.
Chem. Lett. 1998, 8, 2711–2714.
(4) Honda, T.; Rounds, B. V.; Bore, L.; Favaloro, Jr., F. G.; Gribble, G. W.; Suh, N.;
Wang, Y.; Sporn, M. B. Novel synthetic oleanane triterpenoids: A series of highly
active inhibitors of nitric oxide production in mouse macrophages. Bioorg. Med.
Chem. Lett. 1999, 9, 3429–3434.
(5) Honda, T.; Gribble, G. W.; Suh, N.; Finlay, H. J.; Rounds, B. V.; Bore, L.; Favaloro,
Jr., F. G.; Wang, Y.; Sporn, M. B. Novel synthetic oleanane and ursane triterpenoids
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