Design, synthesis, and anti-inflammatory activity both in vitro and in vivo of new betulinic acid analogues having an enone functionality in ring A

Article abstract of DOI:10.1016/j.bmcl.2006.09.012

Fifteen new betulinic acid analogues were designed, synthesized, and tested for anti-inflammatory activity. Many of these analogues effectively suppress nitric oxide (NO) production in RAW cells stimulated with interferon-γ. Analogue 10 is highly and orally active in vivo for induction of the anti-inflammatory and cytoprotective enzyme, heme oxygenase-1.

Full text of DOI:10.1016/j.bmcl.2006.09.012

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6306–6309
Design, synthesis, and anti-inflammatory activity both
in vitro and in vivo of new betulinic acid analogues having
an enone functionality in ring A
Tadashi Honda,a,*, Karen T. Liby,b, Xiaobo Su,a Chitra Sundararajan,a Yukiko Honda,a
Nanjoo Suh,b Renee Risingsong,b Charlotte R. Williams,b Darlene B. Royce,b
Michael B. Spornb and Gordon W. Gribblea,*
aDepartment of Chemistry, Dartmouth College, Hanover, NH 03755, USA
bDepartment of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA
Received 9 August 2006; revised 31 August 2006; accepted 6 September 2006
Available online 25 September 2006
Abstract—Fifteen new betulinic acid analogues were designed, synthesized, and tested for anti-inflammatory activity. Many of these
analogues effectively suppress nitric oxide (NO) production in RAW cells stimulated with interferon-c. Analogue 10 is highly and
orally active in vivo for induction of the anti-inflammatory and cytoprotective enzyme, heme oxygenase-1.
Ó 2006 Elsevier Ltd. All rights reserved.
Betulinic acid (BA, 1) is a pentacyclic lupane-type triter-
pene isolated from various plants.1,2 Betulinic acid selec-
tively inhibits the growth of human melanoma cells
in vitro and in vivo,3 suppresses proliferation of neuro-
blastoma4 and ovarian carcinoma cells,5 and enhances
differentiation and apoptosis of primary leukemia cells.6
Despite a lack of toxicity in animal studies even at high
concentrations,3 the relatively low potency of betulinic
acid itself lessens its clinical utility as an anti-cancer
drug.
Our ongoing efforts to improve the anti-inflammatory
and anti-proliferative activity of oleanolic acid (2), a
naturally occurring oleanane-type triterpene, led us to
discover 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic
acid (CDDO) and related compounds (e.g., CDDO–
Me, CDDO–Im, and TP-222, see Fig. 1).7–9 CDDO
and CDDO–Me are currently being evaluated in phase
I clinical trials for the treatment of leukemia and solid
tumors. In these investigations, we discovered two
Keywords: Triterpene; Betulinic acid; Inhibitors of nitric oxide pro-
duction; RAW 264.7 cells; Inducer of heme oxygenase-1.
*
Corresponding authors. Tel.: +1 603 646 1591; fax: +1 603 646 3946
(T.H.); tel.: +1 603 646 3118; fax: +1 603 646 3946 (G.W.G.); e-mail
addresses: th9@dartmouth.edu; ggribble@dartmouth.edu
Co-first authors, contributed equally to this work.
Figure 1. Structures of betulinic acid, oleanolic acid, CDDO, and
CDDO analogues.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.012

T. Honda et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6306–6309
6307
important structure–activity relationships (SARs). The
combination of enone functionalities with cyano and
carboxyl groups in ring A and an enone functionality
in ring C is an essential structural feature for high poten-
cy in various bioassays related to inflammation and can-
cer,8 and modifications of the C-17 carboxyl group affect
both potency and pharmacokinetics.9 Thus, we envi-
sioned incorporating the same structures into betulinic
acid for increasing its anti-inflammatory activity.
Compounds 4–12 with a cyano enone functionality in
ring A were synthesized by the sequence shown in
Scheme 1. Acid 4 was obtained in 61% yield by cleavage
of methoxycarbonyl group of 3 with AlBr3 in Me2S.13
Ethyl ester 5 was prepared in 78% yield from 4 using
EtI and DBU in toluene.14 Reaction of 4 with oxalyl
chloride gave acyl chloride 19 in quantitative yield.
Amides 6–9 and imidazolides 10–12 were synthesized
in moderate yields by condensation reactions between
19 and the corresponding amines and imidazoles
(Scheme 1 and Table 1).
We initially designed and synthesized methyl ester 310
and 15 new betulinic acid analogues 4–1811 without
the enone functionality in ring C (Table 1) because bet-
ulinic acid differs from oleanolic acid and is devoid of
functionality in ring C. We evaluated the potency of
the new analogues for inhibition against nitric oxide
(NO) production in RAW 264.7 cells (in vitro) and
induction of the anti-inflammatory, cytoprotective en-
zyme, heme oxygenase-1 (in vivo).12 We report here that
several new semi-synthetic betulinic acid analogues dis-
play highly potent anti-inflammatory activity in vitro,
and we show that 10 is also highly and orally active
in vivo.
Compounds 13–18 with a carboxyl or methoxycarbonyl
enone functionality in ring A were synthesized by the se-
quence shown in Scheme 2. Methyl ester 21 was pre-
pared in 85% yield from methyl betulonate (20), which
was easily synthesized in 95% yield in 2 steps (methyla-
tion with CH2N2 and Jones oxidation) from 1, by Stiles’
reagent (methoxymagnesium methyl carbonate) in
1
DMF,15 followed by methylation with CH2N2. The H
NMR spectrum showed that 21 is the single tautomer
in CDCl3 as depicted in Scheme 2. Initially, we expected
that addition of phenylselenyl chloride (PhSeCl, 2 equiv-
Table 1. Inhibition of NO production in RAW cells stimulated with interferon-c by methyl ester 3 and new betulinic acid analogues 4–18
R3
R2
H
R1
H
O
H
R1
R2
R3
Yield (%)
Inhibition of NO IC50 (lM)
c
Compound
3
4
5
6
7
8
9
CN
CN
CN
CN
CN
CN
CN
CO2Me
CO2H
CO2Et
H
H
H
H
H
H
H
Ref. 10
a
0.03
0.2
a
0.02
0.03
0.05
0.07
0.03
CONH2
CONHMe
CONHEt
CONMe2
74b
70b
79b
77b
O
10
CN
H
91b
0.03
N
N
O
O
11
12
CN
CN
H
H
62b
60b
0.05
0.03
N
N
N
N
Et
a
a
a
a
a
a
13
14
CO2Me
CO2Me
CO2Me
CO2H
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
H
0.6
OH
F
1
0.3
15
16
H
0.3
0.9
17
18
CO2H
CO2H
OH
F
0.3
Inactive
0.02
BA (1)
CDDO
a Yield is shown in the text.
b Yield from 19.
c RAW 264.7 cells were treated with various concentrations of compounds and interferon-c (10 ng/mL) for 24 h. Supernatants were analyzed for NO
by the Griess reaction.

6308
T. Honda et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6306–6309
Scheme 1. Reagents and conditions: (a) AlBr3, Me2S (rt); (b) (COCl)2,
CH2Cl2 (rt); (c) EtI, DBU, toluene (reflux); (d) NH3, PhH (rt); (e)
amine hydrochloride, NaHCO3, PhH, water (reflux); (f) Me2NH, PhH
(reflux); (g) imidazole, PhH (rt).
Scheme 3. Conversion of 13/21 to 14/22 using PhSeCl–H2O2.
followed by hydrolysis of 25 to give allylic alcohol 14
and/or 22.18 Allylic fluoride 15 was obtained in 65%
yield from 14 with DAST in CH2Cl2.19 Acids 16–18 were
prepared from the corresponding methyl esters 13–15 by
alkaline hydrolysis (86%, 100%, and 86% yield,
respectively).
alents) to 21 and subsequent oxidation/elimination of
the selenated intermediate with H2O2 would give only
13. However, these conditions16 afforded 14 (28% yield)
and 22 (14%) in addition to 13 (22%). We believe that
PhSeCl produces the rearranged allylic selenide 23 from
13 and/or 21 (Scheme 3).17 Oxidation of 23 with H2O2
forms allylic selenoxide 24, which is converted to allylic
selenenic ester 25 by a [2,3]sigmatropic rearrangement,
We have evaluated the potency of methyl ester 3 and
new analogues 4–18 for inhibition of NO production
in RAW 264.7 cells stimulated with interferon-c, and
induction of the anti-inflammatory, cytoprotective
Scheme 2. Reagents and conditions: (a) Stiles’ reagent, DMF (at 110 °C); (b) CH2N2, Et2O, THF (rt); (c) PhSeCl, pyr., CH2Cl2 (at 4 °C); 30% H2O2,
CH2Cl2 (at 4 °C); (d) aq KOH, MeOH (reflux); (e) DAST, CH2Cl2 (ꢀ78 °C).

T. Honda et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6306–6309
6309
References and notes
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HO-1
Figure 2. Analogue 10 induces heme oxygenase-1 (HO-1) in vivo in
liver. Male CD-1 mice (three mice per group) were gavaged with
triterpenoids (2 lM) in DMSO. After 6 h, livers were collected and
analyzed by Western blot for heme oxygenase-1.20
3. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W.; Fong,
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reported methyl ester 3; You, Y.-J.; Kim, Y.; Nam, N.-H.;
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enzyme, heme oxygenase-1 in the liver (in vivo). In the
RAW cell assay (Table 1), compounds 3–12 with a cya-
no enone functionality in ring A are highly active, with a
potency which is similar to that of CDDO, whereas bet-
ulinic acid is inactive. The series of compounds 13–18
with a carboxyl or methoxycarbonyl enone functionality
in ring A is less active, with a potency more than 10-fold
less than that of CDDO. Most importantly, we have
found that one of the new analogues, 10, is significantly
more potent in vivo than both betulinic acid and the
oleanolic acid analogue, CDDO. Thus, as shown in
Figure 2, oral dosing of 2 lM of compound 10 caused
significant induction of the anti-inflammatory, cytopro-
tective enzyme, heme oxygenase-1, in the liver, while
betulinic acid and CDDO were both markedly less
potent at this low dose. There is major interest in
stimulating heme oxygenase-1 as a protective enzyme
in many chronic disease conditions in which inflamma-
tion and oxidative stress play a key role.12
11. All new compounds 4–18 provided acceptable HRMS
data ( 5 ppm) and 1H NMR spectra that exhibit no
discernible impurities. Compound 10: amorphous solid.
In summary, in a series of new betulinic acid ana-
logues, we have found the following interesting SARs:
(1) A cyano enone functionality in ring A is necessary
for exhibiting potent inhibitory activity against NO
production in RAW cells. (2) Noteworthy is that an
enone functionality in ring C is not necessary for
the potency. The SARs are entirely different from
those of oleanolic acid analogues.7,8 (3) The methoxy-
carbonyl and carboxyl enone functionalities in ring A
are not important for the potency. (4) C-17 modifica-
tions do not affect the potency in the RAW cell assay.
These results are also different from those of CDDO
analogues.9 However, interestingly, only the acyl imid-
azole increases the potency in vivo. (5) Isopropenyl
side chains do not affect the potency. Overall, it is
important to note that our present results are different
from those of the oleanolic acid analogues that we
have studied previously. Further syntheses and biolog-
ical evaluation of new betulinic acid analogues are in
progress.
25
½aꢁD +0.7° (c 0.57, CHCl3). CD (c 0.0016, EtOH) De265
1
+1.71 and De346 ꢀ2.32. H NMR (CDCl3) d 8.30 (1H, br
s), 7.82 (1H, s), 7.55 (1H, br s), 7.07 (1H, br s), 4.79 (1H, d,
J = 1.46 Hz), 4.68 (1H, t, J = 1.46 Hz), 2.98 (1H, ddd,
J = 4.76, 10.98, 10.98 Hz), 2.77 (1H, ddd, J = 3.66, 12.27,
12.27 Hz), 2.48 (1H, ddd, J = 3.29, 3.29, 13.91 Hz), 2.34
(1H, q, J = 6.59 Hz), 1.72, 1.18, 1.13, 1.12, 1.021, 1.016
(each 3H, s); 13C NMR (CDCl3) d 198.4, 173.1, 170.8,
149.5, 137.5, 130.0, 117.6, 115.2, 114.2, 110.7, 57.7, 52.8,
51.4, 45.3, 45.2, 44.2, 42.7, 42.1, 41.0, 37.2, 34.2, 33.6, 33.2,
30.7, 29.8, 27.9, 25.4, 21.6, 19.6, 19.0, 18.6, 16.7, 14.7. MS
(ESI+) m/z 528 [M+H]+; HRMS (ESI+) calcd for
C35H45N3O2 + H 528.3590, found 528.3580.
12. Ryter, S. W.; Alam, J.; Choi, A. M. Physiol. Rev. 2006, 86,
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Acknowledgments
This investigation was supported by funds from NIH
Grant 1 R01 CA78814, the Norris Cotton Cancer Cen-
ter, the Dartmouth College Class of 1934, and the
National Foundation for Cancer Research. M.B.S. is
Oscar M. Cohn Professor.