Synthesis of dammarane-type triterpenoids with anti-inflammatory activity in vivo

Article abstract of DOI:10.1016/j.bmcl.2004.02.104

The 17-α-substituted triterpene 1 [(17α)-23-(E)-dammara-20,23- diene-3β,25-diol] showed promising activity in animal models of immunosuppression and inflammation. Using a mouse model for inflammatory skin diseases (oxazolone-induced allergic contact derma

Full text of DOI:10.1016/j.bmcl.2004.02.104

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2983–2986
Synthesis of dammarane-type triterpenoids with
anti-inflammatory activity in vivo
Dieter Scholz,a,* Karl Baumann,a Max Grassberger,a Barbara Wolff-Winiski,a
Grety Rihs,b Hansrudolf Walterb and Josef G. Meingassnera
aNovartis Institute for Biomedical Research, A-1230 Vienna, Austria
bNovartis Pharma AG, CH-4002 Basle, Switzerland
Received 3 December 2003; revised 4 February 2004; accepted 23 February 2004
Abstract—The 17-a-substituted triterpene 1 [(17a)-23-(E)-dammara-20,23-diene-3b, 25-diol] showed promising activity in animal
models of immunosuppression and inflammation. Using a mouse model for inflammatory skin diseases (oxazolone-induced allergic
contact dermatitis, ACD) as the directing in vivo test system, Structure–activity-relationship studies with the aim to understand the
necessary structural requirements for the biological activity of 1 were conducted. Furthermore, we anticipated to identify biolog-
ically active compounds with the 17b configuration, which are thermodynamically more stable and much easier to synthesize. This
was achieved by identifying the 17-b substituted dammarane 5B and its analogues.
Ó 2004 Elsevier Ltd. All rights reserved.
The novel triterpene 1 ([(17a)-23-(E)-dammara-20,23-
diene-3b, 25-diol], Fig. 1) has been elucidated as the
active principle1 by tracking the observation that the
flour of the palmyrah palm (Borassus flabellifer) induces
immunosuppression.2
ical application (Table 1). Furthermore, efficacy in the
mouse model could also be demonstrated after oral
administration (data not shown).
As 1 is inactive in the bis-pyridyl-N-oxide-disulfide
(BPNOD)-induced edema model in the rat,1 its potent
anti-inflammatory effect in the ACD models occurs by a
mechanism distinct from that of the structurally related
glucocorticoids.2 Based on the promising biological
profile we initiated a medicinal chemistry program with
the aim to identify new highly active anti-inflammatory
triterpenoids starting with 1 as new lead. Compound 1
features the thermodynamically less stable a-configura-
tion at C-17, which is unique for triterpenoids of the
dammarane type Table 2.
Compound 1 shows a very promising immunosuppres-
sive profile in vitro and in vivo. It is active in assays for
mixed lymphocyte reaction and murine delayed type
hypersensitivity,1 and efficacious in models of acute skin
inflammation (oxazolone-induced allergic contact der-
matitis, ACD) in mouse3 and domestic pigs3 upon top-
18
H
17
Due to the very low concentration of 1 in its natural
source (only 0.5 mg of 1 had been isolated from 50 kg of
flour) an efficient synthetic access was mandatory.
Starting from dipterocarpol 2, the major constituent of
commercially available Dammar resin, an 11-step syn-
thesis via the 17b-substituted key intermediate 3 has
been developed (Scheme 1).1
OH
H
HO
3
H
1
Figure 1. Structure of the immunosuppressant triterpene.
Compound 3, which can be conveniently obtained from
2, served as the common starting material for our pro-
gram. Since all efforts to identify an in vitro marker
predictive for the anti-inflammatory in vivo activity of 1
were unsuccessful, we used the results of the well
Keywords: Anti-inflammatory; Immunosuppressive; Allergic contact
dermatitis; Triterpene; Triterpenoids; Dammar resin; Dammarane;
Wittig.
* Corresponding author. Tel.: +43-1-86634-584; fax: +43-1-86634-354;
e-mail: dieter.scholz@pharma.novartis.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.104

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D. Scholz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2983–2986
Table 1. In vivo activity of 1 in comparison to dexamethasone in the murine and porcine allergic contact dermatitis (ACD) models after topical
application
% Inhibition versus controlsa
ACD mouse
ACD pig
Concn (lM)
1
Dexamethasone
Concn (lM)
1
Dexamethasone
40
4
35 ꢀ 4.5ꢁꢁ
44 ꢀ 3.5ꢁꢁ
41 ꢀ 3.3ꢁꢁ
16 ꢀ 3.1 ns
65 ꢀ 4.1ꢁꢁ
37 ꢀ 3.7ꢁꢁꢁ
6 ꢀ 2.9 ns
NT
400
130
40
32 ꢀ 13.6ꢁꢁ
35 ꢀ 6.7ꢁꢁꢁ
20 ꢀ 9.6ꢁꢁꢁ
NT
43 ꢀ 5.3ꢁꢁ
0.4
0.04
28 ꢀ 7.4ꢁꢁ
ꢁꢁꢁ: p < 0:001, ꢁꢁ: p < 0:01, ns: not statistical significant (p > 0:05), ANOVA followed by Tukey post hoc test used; NT: not tested.
a Means ꢀ SEM of P2 experiments with eight animals per group.
Table 2. Yields for the preparation of 4a–f from 3
Anti-inflammatory active derivatives with the 17b con-
Yielda of 4 (%)
figuration are synthetically attractive, because this con-
figuration is thermodynamically more stable and
therefore much easier accessible. Therefore, we investi-
gated the influence of the configuration of the C-17
substituent on the biological activity together with side
chain variations. Reductive amination reactions of 3
with a series of primary amines (a–f) in presence of
NaCNBH3 produced an array of different amines (4a–f,
Scheme 2) and after acylation of amides (6a–c, Scheme
3) at C-18 (all with the 17b configuration), as their
mixtures of diastereoisomers, which could be separated
by conventional chromatography on silica gel. The
absolute configurations of the newly created chiral
center at C-18 could not be determined via NMR
methods due to the unhindered rotation at the C-17 C-
18 bond. We designated therefore the isomer with the
higher Rf value with the letter A and the one with the
lower Rf value with B (solvent systems: 4a, 4f, 5, 13, 15:
CH2Cl2/methanol: 9/1; 4a: toluene/ethyl acetate: 1/2; 4d:
toluene/ethyl acetate: 2/1; 11, 12: toluene/ethyl acetate:
9/1).
Entry
R1
a
b
c
d
e
f
(CH2)3CH3
CH2C6H5
93
83
92
89
31
79
2-Benzimidazolylmethyl
CH2CO2C(CH3)3
(4-Aminosulfonylphenyl)ethyl
2-(1-Imidazolyl)-2-phenylethyl
a Sum of both isomers [all isomers were obtained as 1:1 mixture with
the exception of 4d (4dA:4dB ¼ 2:1)].
OH
O
H
H
7 steps
4 steps
H
H
1
HO
O
H
H
2
3
Scheme 1. Synthesis of 1.
Catalytic hydrogenation of 4b gave 5AB in high yield,
which were again separated via conventional chroma-
tography. Crystals of the hydrochloride salt of 5A
established ACD/mouse model3 (topical application),
for guidance in the design of synthetic analogues.
NH2
H
NHR1
H
R1NH2
H2/Pd-C
H
H
3
4b
NaCNBH3
EtOH, 20°C;
80%
H
EtOH/AcOH
H
pH: 6.5; 20°C
HO
HO
H
H
4a - f
5
Scheme 2. Reductive aminations of 3 furnishing 4a–f.
NH2
H
NHCOR2
H
R2CO2H; EDC.HCl
H
Hydroxybenztriazol
H
DMF; 20°C
H
H
HO
HO
H
H
6aA-cA
5A
a: R2: (CH3)2CH2CH2: 82%
b: R2:N-BOC-3-(4-pyridyl)alanyl: 89%
c: R2:2-(E)-phenylethenyl:79%
Scheme 3. Preparation of 6aA–cA.

D. Scholz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2983–2986
2985
showing appropriate diffracting properties allowed us to
determine its 3D structure by X-ray crystallography
(Fig. 2).
Bromination of 3-O-tert-butyl diphenylsilyl (TBDPS)
protected 3 with Br2 in CCl4 gave a mixture of starting
material together with mono-, di- and tribrominated
products. N-Bromosuccinimide did not lead to the
desired 7 either. Finally we succeeded by applying
dioxane dibromide5 as the halogenating agent affording
7 in excellent yield. The yield of the side products
(dibromide and tribromide) amounted to less than 3%.
Straightforward treatment of 7 with triphenylphosphine
gave the expected phosphonium salt, which was trans-
formed into the stabilized Wittig ylide under alkaline
conditions. Reaction with benzaldehyde produced
smoothly the olefin 8. Hydrogenation of 8 gave 9 to-
gether with a diastereoisomeric mixture of 10. Removal
of the TBDPS group from the unsaturated and the
saturated ketones, 8 and 9, with TBAF failed. However,
deprotection of the alcohol 10 could be achieved leading
to 11. The two diastereomers again could be separated
via chromatography (toluene/ethylacetate: 9/1). Reduc-
tive amination of 9 and removal of the TBDPS group
furnished after separation the desired benzylamine
derivatives 12A and 12B. Hydrogenolytic cleavage of the
N-benzyl residue resulted in 13A and 13B. Compound 9
served also as suitable starting material for the intro-
duction of a methyl group instead of an amino group.
The complete set of crystallographic data has been
deposited in the Cambridge Crystallographic Data
Base.4 It is reasonable to assume, that compounds 6aA–
cA, which were obtained from 5A have the same absolute
stereochemistry.
Next we aimed at compounds more closely related to 1.
Compound 1 carries a methylene substituent and an
aliphatic side chain at C-18. Scheme 4 shows our suc-
cessful synthetic strategy using a Wittig approach to
obtain analogues of 1 with –NH2, –OH, –CH3 and a
methylene group at C-18 together with a phenethenyl
side chain. This Wittig strategy, which will allow pre-
paring an array of different side chains, made it neces-
sary to monobrominate protected 3, a reaction that
proceeded in high yield.
NH2
H
H
H
Thus, as outlined in Scheme 5, Petasis olefination6 of 9
followed by cleaving off the TBDPS group gave the exo-
methylene compound 14 in 81% yield. Catalytic hydro-
genation of 14 with H2/Pd–C resulted in 15A and 15B as
diastereomeric mixture. Chromatographic separation
gave 15A and 15B.
HO
H
5A
Figure 2. Structure and ORTEP plot of 5A.
1. tBuPh2SiCl
O
Br
imidazole
O
DMF; 20°C
1. Ph3P/toluene; reflux
H
H
3
2. Na2CO3
3. C6H5CHO
CH2Cl2; 20°C
2. dioxane/Br2
H
H
H
H
tBuPh2SiO
tBuPh2SiO
7 (80%)
8 (85%)
H
H
O
H
HO
H
H2/Pd-C
H
EtOH; 20°C
H
+
H
H
tBuPh2SiO
H
tBuPh2SiO
H
9(75%)
10AB (10%)
1. PhCH2NH2/ NaCNBH3
Bu4NF, THF; reflux
EtOH/AcOH, pH:6.5; 20°C
2. Bu4NF, THF; reflux
H
N
HO
H
H2N
H
H
H
H
H
H
H
H2/Pd-C
EtOH; 20°C
H
H
HO
HO
HO
H
H
11A(23%);11B (55%)
13A (85%); 13B (80%)
12A (39%);12B(21%)
Scheme 4. Wittig strategy for the preparation of 10–13.

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D. Scholz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2983–2986
1. Petasis olefination
2. Bu4F
H
H
H2/Pd-C
9
H
H
H
H
HO
HO
H
14 (81%)
H
15A(43%);15B (45%)
Scheme 5. Petasis olefination and hydrogenation of 9.
Table 3. Activity of analogues 3–15 of 1 in the inflammatory ACD/
mouse model after topical application
amino (12, 13) or hydroxyl (11) analogues are inactive.
The most active compound is 5B. For details see Table 3.
Compd R1 (for 4 and 5) or R2 (6)
ACD mouse; % inhib.a
In conclusion, starting from the 17-a-substituted triter-
pene lead 1 we have developed an efficient derivatization
platform based on dammarane 3 as the common starting
material. By this way, we identified compounds with the
thermodynamically more stable and synthetically better
accessible C-17b configuration, particularly 5B, which
exhibit promising in vivo activity in a well established
animal model of skin inflammation.
1
––
––
42 ꢀ 3.7ꢁꢁ
12 ꢀ 7.5 ns
30 ꢀ 4.9ꢁꢁ
28 ꢀ 4.3ꢁꢁ
28 ꢀ 8.1ꢁ
3
4aA
4aB
4bA
4bB
4cAB
4dA
4dB
4eA
4fAB
5A
(CH2)3CH3
(CH2)3CH3
CH2C6H5
CH2C6H5
15 ꢀ 6.5 ns
27 ꢀ 8.9ꢁ
2-Benzimidazolylmethyl
CH2CO2C(CH3)3
CH2CO2C(CH3)3
(4-Aminosulfonylphenyl)ethyl
31 ꢀ 6.3ꢁ
21 ꢀ 3.8ꢁ
16 ꢀ 8.1 ns
2-(1-Imidazolyl)-2-phenylethyl 13 ꢀ 9.1 ns
Acknowledgements
H38
H41
ꢀ 4.3ꢁꢁ
ꢀ 4.6ꢁꢁ
5B
6aA
6bA
6cA
11A
11B
12A
12B
13A
13B
14
(CH3)2CH2CH2
N-Boc-3-(4-pyridyl)alanyl
2-(E)-Phenylethenyl
33 ꢀ 6.6ꢁ
We would like to thank Sabine Weber-Roth, Hannelore
Schmidt for technical assistance, Gerhard Schulz and
Ewald Haidl for Spectra recording and assistance in their
interpretation and Trixi Wagner for depositing the X-ray
data in the CCDC and Erwin Schreiner for fruitful dis-
cussions and critically reading the manuscript.
28 ꢀ 7.7 ns
11 ꢀ 12.4 ns
12 ꢀ 15.7 ns
14 ꢀ 13.6 ns
22 ꢀ 6.9ꢁ
––
––
––
––
––
––
––
––
––
15 ꢀ 8.2 ns
30 ꢀ 9.1 ns
6 ꢀ 11.9 ns
36 ꢀ 6.0ꢁꢁ
30 ꢀ 7.4ꢁꢁ
29 ꢀ 6.9ꢁꢁ
References and notes
15A
15B
1. Revesz, L.; Hiestand, P.; La Vecchia, L.; Naef, R.; Naegeli,
H.-U.; Oberer, L.; Roth, H.-J. Bioorg. Med. Lett. 1999, 9,
1521.
2. Citations 1–3 in Ref. 1.
3. Meingassner, J. G.; Grassberger, M.; Farngruber, H.;
ꢁꢁ: p < 0:01, ꢁ: p < 0:05, ns: not statistical significant (p > 0:05), AN-
OVA followed by Tukey post hoc test used; for details see Ref. 2.
a One experiment; mean ꢀ SEM values of eight mice per group; applied
concn (0.1 M).
€
Moore, H. D.; Schuurman, H.; Stutz, A. Br. J. Dermatol.
1997, 137, 568.
The biological activities of the compounds 3–15 are
summarized in Table 3. Whereas the starting ketone 3 is
inactive, we were able to obtain, as desired, anti-
inflammatory active compounds with the 17b confor-
mation. Interestingly, the stereochemistry at C-18 has
nearly no influence on the biological activity. Alkyl-
amino analogues (4a–f) are in general less active then the
primary amine (5) and acylation of 5 abolishes the
activity (6a–c). More bulky side chains are allowed, but
in this case only methylene (14) and methyl (15) sub-
stituents at C-18 proved to be biologically active. The
4. Crystallographic data (excluding structure factors) for this
structure in the paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication number CCDC212085. Copies of the data can
be obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge, CB2 1EZ, UK [fax: +44(0)1223
336033 or e-mail: deposit@ccdc.cam.ac.uk].
5. Pettit, G. R.; Green, B.; Dasgupta, A. K.; Whitehouse,
P. A.; Yardley, J. P. J. Org. Chem. 1970, 35, 1381.
6. For a review concerning methenylation of C@O bonds with
titanium reagents see: Hartley, R. C.; McKiernam, G. J. J.
Chem. Soc., Perkin Trans. 2002, 2763.