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

Article abstract of DOI:10.1021/jm025565f

Novel tricyclic compounds with enone functionalities in rings A and C, which were designed on the basis of the structure of a synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid, have been synthesized. Among them, 10 shows high inhib

Full text of DOI:10.1021/jm025565f

©
Copyright 2002 by the American Chemical Society
Volume 45, Number 22
October 24, 2002
Letters
Design a n d Syn th esis of Tr icyclic
Com p ou n d s w ith En on e F u n ction a lities
in Rin gs A a n d C: A Novel Cla ss of
High ly Active In h ibitor s of Nitr ic Oxid e
P r od u ction in Mou se Ma cr op h a ges
synthase (iNOS) and inducible cyclooxygenase (COX-
2
) in mouse macrophages. These potencies are found at
-
6
-9
concentrations ranging from 10
to 10
M in cell
culture. Furthermore, CDDO shows inhibitory activity
against mouse peritoneal inflammation induced by
thioglycollate and IFN-γ (ip administration).6
However, our recent studies of CDDO show that the
in vivo potency (po administration) of CDDO is not
optimal because of its low bioavailability. Therefore, we
have modified CDDO to improve this problem and
†
,†
Frank G. Favaloro, J r., Tadashi Honda,*
†
,†
‡
Yukiko Honda, Gordon W. Gribble,* Nanjoo Suh,
Renee Risingsong, and Michael B. Sporn
‡
‡
7
obtained some interesting analogues. However, struc-
Department of Chemistry, 6128 Burke Laboratory,
Dartmouth College, Hanover, New Hampshire, and
Department of Pharmacology and Toxicology, Dartmouth
Medical School, Hanover, New Hampshire 03755
tural modifications of CDDO are severely limited be-
cause CDDO has only one functionality at C-17 that can
be modified. In addition, CDDO and its analogues would
have a serious cost problem for large-scale synthesis
because the synthesis of CDDO requires 11 steps from
oleanolic acid, an expensive triterpenoid isolated from
natural sources.
Because our earlier structure-activity relationships
(SARs) show that a 2-cyano-1-en-3-one functionality in
ring A and a 9(11)-en-12-one functionality in ring C are
essential for the extremely high potency of CDDO, we
reasoned that the entire oleanane skeleton might not
be necessary for potency. Therefore, we have focused
our attention on tricyclic compounds having general
formula I [tricyclic-bis-enone derivatives (TBEs)], which
have the same A, B, and C rings as CDDO. A literature
survey revealed that they are previously unknown
compounds.
Received J uly 24, 2002
Abstr a ct: Novel tricyclic compounds with enone functional-
ities in rings A and C, which were designed on the basis of
the structure of a synthetic triterpenoid, 2-cyano-3,12-dioxo-
oleana-1,9(11)-dien-28-oic acid, have been synthesized. Among
them, 10 shows high inhibitory activity (IC50 ) 1 nM level)
against production of nitric oxide induced by interferon-γ in
mouse macrophages and is orally active at 15 mg/kg (once) in
a preliminary in vivo study using mouse peritoneal inflam-
mation induced by thioglycollate and interferon-γ.
1
In tr od u ction . In a series of previous papers, we
reported that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-
oic acid (CDDO) (1) shows high inhibitory activity (IC50
)
0.1 nM level) against production of nitric oxide (NO)
2
induced by interferon-γ (IFN-γ) in mouse macrophages.
We have also reported that CDDO is a potent, multi-
functional agent in various in vitro assays.3 For ex-
ample, CDDO induces monocytic differentiation and
apoptosis of human myeloid leukemia cells and adipo-
genic differentiation of mouse 3T3-L1 fibroblasts. CDDO
4
5
inhibits proliferation of many human tumor cell lines.
CDDO blocks de novo synthesis of inducible nitric oxide
*
To whom correspondence should be addressed. For T. H.: (phone)
6
03-646-1591; (fax) 603-646-3946; (e-mail) th9@dartmouth.edu. For
Our rationale for exploring these TBEs is as follows.
1) Because TBEs could be synthesized from com-
mercially available small molecules, TBEs with various
functionalities at different positions can be obtained.
G.W.G: (phone) 603-646-3118; (fax) 603-646-3946; (e-mail) Grib@
dartmouth.edu.
(
†
Dartmouth College.
Dartmouth Medical School.
‡
1
0.1021/jm025565f CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/24/2002

4
802 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Letters
Sch em e 1. Synthesis of (()-2-6a
Ta ble 1. Inhibitory Activity of New Compounds 2-10
activitya
IC50 (nM)
activitya
IC50 (nM)
compd
compd
(()-2
(()-3
(()-4
(()-5
(()-6
(-)-6
(+)-6
310
480
53
75
61
(()-7
91
1600
61
2.1
0.5
(()-8
(()-9
(()-10
1 (CDDO)
oleanolic acid
hydrocortisone
64
58
>40000
10
a
IC50 values of compounds 1-10 and hydrocortisone were
determined in the range of 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.
Sch em e 2. Synthesis of TBEs (-)- and (+)-6a
a
Reagents: (a) HCO2Et, NaOMe, PhH; (b) NH2OH‚HCl, aque-
ous EtOH; (c) NaOMe, Et2O, MeOH; (d) DDQ, PhH; (e) Ac2O, pyr;
(f) CrO3 (cat.), t-BuOOH, CH2Cl2; (g) KOH, aqueous MeOH.
Such “diversity-oriented synthesis” of TBEs could lead
to novel drugs for the prevention and/or treatment of
cancer, Alzheimer’s disease, Parkinson’s disease, mul-
tiple sclerosis, rheumatoid arthritis, inflammatory bowel
disease, and other diseases whose pathogenesis may
involve excessive production of NO and/or prostaglan-
dins. (2) This structural flexibility might provide us with
new SARs and different compounds that are appropriate
and specialized for each therapeutic area. (3) Some of
the activities of CDDO might be absent because of the
delineation of the pharmacophore of CDDO. (4) The cost
of large-scale synthesis would be overcome because
TBEs could be synthesized more easily than CDDO from
cheap commercially available compounds.
a
Reagents: (a) Et3N, THF; (b) (R)-(+)-phenylalanine, d-CSA,
DMF; (b′) (S)-(-)-phenylalanine, d-CSA, DMF; (c) NaBH4, EtOH;
d) ethyl vinyl ketone, Na, MeOH; (e) Li, liquid NH3, THF, CH3I;
(f) Ac O, pyr; (g) CrO (cat.), t-BuOOH, CH Cl ; (h) DBU, CH Cl ;
(i) LDA, p-TsCN, THF; (j) DDQ, PhH.
(
2
3
2
2
2
2
yields, respectively. Alkaline hydrolysis of 4 gave 5 and
6 in 50% and 46% yields, respectively. TBEs 4-6 show
significant inhibitory activity (IC50 ) 0.01 µM level,
similar in potency to that of hydrocortisone) on NO
production induced by IFN-γ in mouse macrophages (see
Table 1).
It is often the case that one enantiomer of a drug has
greater potency and/or less toxicity than its antipode.
Therefore, we synthesized both optically pure (-)-6,
with the same configuration as naturally occurring
triterpenoids, and its antipode, (+)-6 (Scheme 2).13
Optically pure (+)- and (-)-17 were prepared via achiral
intermediate 16 from ethyl vinyl ketone and 2-methyl-
In this communication, we report our initial results
on the synthesis and biological activity of some new
8
TBEs.
Ch em istr y. Our initial targets 2-6 were synthesized
9
in racemic form from known compound 11 (Scheme 1).
Hydroxymethylene 12 was prepared in 92% yield from
1
1 with ethyl formate in the presence of sodium meth-
1
0
oxide in benzene. Isoxazole 13 was prepared in 73%
yield by condensation of 12 and hydroxylamine hydro-
chloride in aqueous ethanol.11 Cleavage of the isoxazole
moiety of 13 with sodium methoxide gave nitrile 14
quantitatively.11 Enone 2 was prepared quantitatively
by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxi-
dation of 14. Acetylation of 2 gave 3 in 97% yield. Allylic
oxidation of 3 with a catalytic amount of chromium
trioxide and tert-butyl hydroperoxide in methylene
chloride12 afforded 4 and 1,2-epoxide 15 in 47% and 29%
14
1,3-cyclohexanedione by a known method. Enantiomer
(+)-11 and its antipode (-)-11 were synthesized from
(-)- and (+)-17, respectively, according to the same
sequence as for racemic 11. The enantiomeric excess
of each enantiomer (+)- or (-)-11 was determined to be
90% by H and F NMR of the (-)-R-MTPA ester
derived from each enantiomer. TBE (-)-6 was synthe-
sized from (+)-11 according to the alternative route.
9
1
19
15

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4803
Sch em e 4. Efficient Synthesis of TBE (()-10a
Sch em e 3. Synthesis of TBEs (()-7-10a
a
Reagents: (a) Li (4.5 equiv), H2O (1 equiv), liquid NH3, THF,
CH3I; (b) CrO3 (cat.), t-BuOOH, CH2Cl2; (c) p-TsCN (4 equiv), LDA
(2.4 equiv), THF; (d) DDQ (2.5 equiv), PhH.
hydroxylamine. Allylic oxidation of 25 gave dienone 26
in 58% yield. TBE 7 was obtained in 81% yield by
cleavage of the isoxazole moiety of 26 with base, followed
by DDQ oxidation. Base hydrolysis of 7 gave 8 in 77%
yield. Amidation of 7 with saturated ammonia in
methanol gave 9 in 64% yield. Dehydration of 9 with
thionyl chloride in toluene gave 10 in 40% yield. Of
these TBEs, 10 shows high potency (IC50 ) 1 nM level)
and is approaching the potency of CDDO in our bioassay
(see Table 1).
For some in vivo assays, we needed to synthesize more
than 1 g of 10. Since we synthesized 10 in 19 steps via
1 from ethyl vinyl ketone and 2-methyl-1,3-cyclo-
a
Reagents: (a) HOCH2CH2OH, PPTS, PhH; (b) CrO3, pyr,
CH2Cl2; (c) MMC, DMF; (d) CH2N2, Et2O, THF; (e) NaBH4, EtOH,
CH2Cl2; (f) PPTS, aqueous acetone; (g) MsCl, pyr, CH2Cl2; (h) DBU,
THF; (i) HCO2Me, NaOMe, PhH; (j) NH2OH‚HCl, aqueous EtOH;
k) CrO3 (21 equiv), pyr, CH2Cl2; (l) NaOMe, Et2O, MeOH; (m)
DDQ, PhH; (n) KOH, aqueous MeOH; (o) NH3, MeOH; (p) SOCl2,
toluene.
1
hexanedione (Scheme 3), this route is too long for a
practical synthesis of 10. Therefore, we have succeeded
in preparing 10 in six steps from commercially available
simple compounds (Scheme 4). This much shorter route
indicates that we could supply a large number of useful
TBE compounds at low cost. Noteworthy in this new
route is the reductive methylation step using 1 equiv of
(
Acetylation of (+)-11 gave (+)-18 in quantitative yield.
Allylic oxidation of (+)-18 gave (+)-19 in 79% yield.
Deacetylation of (+)-19 with DBU gave (-)-20 in 97%
yield.16 Cyanation of the enolate of (-)-20 with p-TsCN
2
0
water to give the new compound 28 in 63% yield from
21
27, which is easily synthesized in two steps, and in
1
7
in THF gave (-)-21 in 93% yield. DDQ oxidation of
good yield from 2-methylcyclohexanone and 1-chloro-3-
pentanone. Interestingly, the reductive methylation of
27 without a proton donor or with proton donors other
than water gave no 28. Allylic oxidation of 28 gave 29
in 64% yield. Double cyanation of the enolate of 29 with
p-TsCN in THF successfully gave dinitrile 30 (isomeric
mixture). Finally, 10 was prepared in 67% yield (from
29) by DDQ oxidation of 30.
(
-)-21 gave (-)-6 ([R]D -115° (CHCl3)) in 73% yield. The
overall yield of (-)-6 from (+)-11 was 52%. This latter
route is much improved over our original one (Scheme
1
) in which the overall yield of 6 from 11 was only 14%.
Similarly, (+)-6 ([R]D +115° (CHCl3)) was synthesized
from (-)-11 by this sequence.
Because 6 shows good potency in our assay, this
compound may be a good scaffold from which to discover
new, more potent TBEs. Thus, we targeted 7-10,
analogues of 6 with electron-withdrawing groups at
C-13, to discern the influence of substituents at C-13
on biological activity because we found previously that
substitution at the R-position of an R,â-unsaturated
ketone strongly affects the potency of synthetic triter-
penoids.1 Racemic 7-10 were synthesized according to
the synthetic route shown in Scheme 3. Ketal 22 was
synthesized in 93% yield by ketalization of 11, followed
by oxidation. Ester 23 (epimeric mixture) was prepared
in 71% yield from 22 by carboxylation with Stiles’
reagent, methylation with diazomethane, and reduc-
tion with NaBH4. Deketalization of 23, and subsequent
mesylation of the hydroxyl group at C-14, followed by
dehydration with DBU gave 24 in 74% yield.19 Isoxazole
5 was obtained in 97% yield by formylation at C-2 of
4 with methyl formate, followed by condensation with
Biologica l Resu lts a n d Discu ssion . The inhibitory
activities [IC50 (nM)] of racemic 2-10, (-)- and (+)-6,
triterpenoids [1 (CDDO) and oleanolic acid], and hydro-
cortisone (a positive control) on NO production induced
by IFN-γ in mouse macrophages are shown in Table 1.
Important results are the following. (1) TBE 10 ap-
proaches the potency of CDDO in this assay; it is only
about 4 times less than CDDO and about 5 and 30 times
greater than hydrocortisone and 6, respectively. A
nitrile group enhances potency among the typical elec-
tron-withdrawing groups surveyed at C-13. (2) Since 4
and 5 are more potent than 2 and 3, the importance of
the bis-enone structure for high potency even in rela-
tively small molecules is confirmed. (3) Both enanti-
omers (-)-6 and (+)-6 show the same potency. For
example, it is known that for CC-1065 and some related
compounds both natural (+) and unnatural (-) enan-
b
1
8
2
2
2
2
tiomers are active.

4
804 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Letters
inhibitors of nitric oxide production in mouse macrophages. J .
Med. Chem. 2000, 43, 1866-1877. (c) Honda, T.; Rounds, B. V.;
Bore, L.; Finlay, H. J .; Favaloro, F. G., J r.; Suh, N.; Wang, Y.;
Sporn, M. B.; Gribble, G. W. Synthetic oleanane and ursane
triterpenoids with modified rings A and C: A series of highly
active inhibitors of nitric oxide production in mouse macro-
phages. J . Med. Chem. 2000, 43, 4233-4246.
(
2) (a) Ding, A.; Nathan, C. F.; Graycar, J .; Derynck, R.; Stuehr, D.
J .; Srimal, S. Macrophage deactivating factor and transforming
growth factors-â1, -â2, and -â3 inhibit induction of macrophage
nitrogen oxide synthesis by IFN-γ. J . Immunol. 1990, 145, 940-
9
44. (b) Bogdan, C.; Paik, J .; Vodovotz, Y.; Nathan, C. Contrast-
ing mechanisms for suppression of macrophage cytokine release
by transforming growth factor-â and interleukin-10. J . Biol.
Chem. 1992, 267, 23301-23308.
(
3) Suh, N.; Wang, Y.; Honda, T.; Gribble, G. W.; Dmitrovsky, E.;
Hickey, W. F.; Maue, R. A.; Place, A. E.; Porter, D. M.; Spinella,
M. J .; Williams, C. R.; Wu, G.; Dannenberg, A. J .; Flanders, K.
C.; Letterio, J . J .; Mangelsdorf, D. J .; Nathan, C. F.; Nguyen,
L.; Porter, W. W.; Ren, R. F.; Roberts, A. B.; Roche, N. S.;
Subbaramaiah, K.; Sporn, M. B. A novel synthetic oleanane
triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid,
with potent differentiating, anti-proliferative, and anti-inflam-
matory activity. Cancer Res. 1999, 59, 336-341.
4) Ito, Y.; Pandey, P.; Place, A.; Sporn, M. B.; Gribble, G. W.; Honda,
T.; Kharbanda, S.; Kufe, D. The novel triterpenoid 2-cyano-3,12-
dioxooleana-1,9(11)-dien-28-oic acid induces apoptosis of human
myeloid leukemia cells by a caspase-8-dependent mechanism.
Cell Growth Differ. 2000, 11, 261-267.
5) Adipogenic differentiation of mouse 3T3-L1 fibroblasts is due
to CDDO being a ligand for the peroxisome proliferator-activated
receptor γ: Wang, Y.; Porter, W. W.; Suh, N.; Honda, T.; Gribble,
G. W.; Leesnitzer, L. A.; Plunket, K. D.; Mangelsdorf, D. J .;
Blanchard, S. G.; Willson, T. M.; Sporn, M. B. A synthetic
triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
F igu r e 1. TBE 10 (30 and 15 mg/kg) and hydrocortisone (HC)
30 mg/kg) suppress both NO production and iNOS protein
(
synthesis in vivo. Female CD-1 mice were injected ip with
thioglycollate 3 days before IFN-γ stimulation. On day 3, mice
(
1
6 per group) were injected ip with IFN-γ (0.5 µg/mouse). TBE
(
0 and hydrocortisone were gavaged once, 1 h before IFN-γ
injection. Nitric oxide was measured by the Griess reaction.
Cell lysates were obtained for Western blot analysis of iNOS
protein.
(
The inhibitory activity of 10 on NO production
induced by IFN-γ in mouse macrophages was not
blocked by the glucocorticoid antagonist mifepristone
2
3
(
RU486) (at 1 µM), which reverses the action of
(
CDDO), is a ligand for the peroxisome proliferator-activated
hydrocortisone. This strongly implies that the actions
of TBEs on the iNOS system are not mediated by their
interaction with the glucocorticoid receptor.
In a preliminary in vivo study using mouse peritoneal
inflammation induced by thioglycollate and IFN-γ, 10
is orally active at 30 and 15 mg/kg. Toxicity was not
observed at either dosage. The potency at 15 mg/kg is
still much better than hydrocortisone (30 mg/kg), which
is clinically used as an orally active antiinflammatory
drug (see Figure 1).
In conclusion, these biological results suggest that
TBEs may be a new class of potential drug candidates
for inflammatory diseases. Further syntheses and bio-
logical evaluation (in vitro and in vivo) of new TBEs,
including water-soluble derivatives, are in progress.
receptor γ. Mol. Endocrinol. 2000, 14, 1550-1556.
(6) Suh, N.; Wang, Y.; Williams, C.; Risingsong, R.; Honda, T.;
Rounds, B. V.; Bore, L.; Gribble, G. W.; Sporn, M. B.CDDO, a
novel synthetic oleanane triterpenoid, suppresses nitric oxide
production and synthesis of inducible nitric oxide synthase
(iNOS) in female CD-1 mice. Proceedings of the 91st Annual
Meeting of the American Association for Cancer Research, San
Francisco, CA, April 1-5, 2000; American Association of Cancer
Research: Philadelphia, PA; p 663.
(7) Honda, T.; Honda, Y.; Favaloro, F. G., J r.; Gribble, G. W.; Suh,
N.; Place, A. E.; Rendi, M. H.; Sporn, M. B. A novel dicyano-
triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile,
active at picomolar concentrations for inhibition of nitric oxide
production. Bioorg. Med. Chem. Lett. 2002, 12, 1027-1030.
8) Throughout this article, for the ease of comparison, the same
atom numbering as used with the oleanane skeleton is used for
TBEs. Racemic TBEs and their intermediates are shown by the
enantiomers with the same configuration as naturally occurring
triterpenoids.
9) Hirota, H.; Nakamura, T.; Tsuyuki, T.; Takahashi, T. Stereo-
selective total synthesis of (()-labdane-8R,15-diol and (()-
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(
(
Ack n ow led gm en t. We thank Dr. Carl Nathan for
expert advice on the preparation of macrophages and
the nitric oxide assay. We also thank Dr. Steven Mullen
(
(
University of Illinois) for the mass spectra. This
investigation was supported by funds from NIH Grant
R01-CA78814, the Norris Cotton Cancer Center, and
1
(
(
(
the National Foundation for Cancer Research. M.B.S.
is Oscar M. Cohn Professor. F.G.F., J r. is an Oscar M.
Cohn Scholar.
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chromium-mediated allylic oxidation by 70% tert-butylhydro-
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expected to be unstable because of their â-hydroxyketone
structures.
Su p p or tin g In for m a tion Ava ila ble: Spectral data and
elemental analyses of new compounds and experimental
details of biological evaluation, including data with RU486.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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