17S,20S-Methanofusidic acid, a new potent semi-synthetic fusidane antibiotic.

Article abstract of DOI:10.1016/S0960-894X(02)00797-7

A novel fusidic acid type antibiotic having the side chain linked to the tetracyclic ring system via a spiro-cyclopropane system is described. 17S,20S-Methanofusidic acid is obtained by an efficient synthetic route including cyclopropanation of the Delta17(20) bond with attack solely from the least hindered alpha-face. The spiro-cyclopropane system orients the side chain into a bioactive conformational space. The new 17S,20S-methanofusidic acid exerts antibacterial activity against several Gram-positive species with potency essentially equal to natural fusidic acid.

Full text of DOI:10.1016/S0960-894X(02)00797-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3569–3572
17S,20S-Methanofusidic Acid, a New Potent Semi-synthetic
Fusidane Antibiotic
Tore Duvold,* Anne Jørgensen, Niels R. Andersen, Anne S. Henriksen,
Morten Dahl Sørensen and Fredrik Bjorkling
LEO Pharma, Industriparken 55, DK-2750 Ballerup, Denmark
Received 10 June 2002; revised 23 August 2002; accepted 16 September 2002
Abstract—A novel fusidic acid type antibiotic having the side chain linked to the tetracyclic ring system via a spiro-cyclopropane
system is described. 17S,20S-Methanofusidic acid is obtained by an efficient synthetic route including cyclopropanation of the
Á17(20) bond with attack solely from the least hindered a-face. The spiro-cyclopropane system orients the side chain into a
bioactive conformational space. The new 17S,20S-methanofusidic acid exerts antibacterial activity against several Gram-positive
species with potency essentially equal to natural fusidic acid.
# 2002 Elsevier Science Ltd. All rights reserved.
Fusidic acid (Fucidin1) is a well-known antibiotic with
unique structural features including a tetracyclic ring
system with an unusual chair–boat–chair conformation
and a carboxylic acid bearing side chain attached by a
double bond. Fusidic acid is the most potent of a small
family of steroidal antibiotics, the fusidanes, and is iso-
lated from the fungus Fusidium coccineum.1,2 Although
fusidic acid is commonly used against staphylococci, it
is also efficient against several other Gram-positive spe-
cies.3 The excellent distribution in various tissues, low
degree of toxicity and allergic reactions and the absence
cross-resistance with other clinically used antibiotics has
made fusidic acid a highly valuable antibiotic, especially
for skin and eye infections.4 The structure–activity rela-
tionship (SAR) of fusidic acid has been extensively
studied, and although a large number of analogues have
been prepared, only a few have shown activities com-
parable with that of natural fusidic acid.5À8 In spite of
the extensive SAR studies, the potential of side chain
modifications has not been explored until very recently.9
been assumed to be essential for antibacterial activity.8
We recently found that a flexible side chain having a
single bond between C-17 and C-20 surprisingly led to a
derivative with the same potency as natural fusidic
acid.9 Only one of the four possible stereoisomers,
17S,20S-dihydrofusidic acid (2), showed potent activity
whereas the other three were virtually inactive.
The study demonstrated the crucial importance of the
orientation of the fusidic acid side chain in a limited
bioactive space above the ring plane. This prompted us
to explore alternative structural side chain modifications
resulting slightly altered orientations of the side chain
by means of different linkages to the ring system.
Replacement of the Á17(20) bond by a spiro-cyclopro-
pane ring seemed to fulfil the requirements of orienting
the side chain correctly without interfering sterically
with the carboxylic acid functionality and the C-16
acetoxy group. Furthermore, absence of the double
bond between C-17 and C-20 should also create a more
stable environment for the pharmacokineticaly labile
C-16 acetoxy group. In accordance with the SAR
requirement of orienting the essential carboxylic acid
group to the same side as the important C-16 acetoxy
group, only two alternative orientations of the cyclopro-
pane ring remained possible, 17S,20S-methanofusidic
acid 3 and 17R,20R-methanofusidic acid 4 (Scheme 1).
As part of our renewed interest in improving the anti-
bacterial and pharmacokinetic properties of fusidic acid
type antibiotics, we focused on this relatively unex-
plored side chain. In particular, we wanted to investi-
gate the role of the Á17(20) double bond which has
Conformational analysis of the side-chain derivatives 3
and 4 were carried out using molecular modelling and
*Corresponding author. Tel.: +45-72-262225; fax: +45-44-945510;
e-mail: tore.duvold@leo-pharma.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00797-7

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T. Duvold et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3569–3572
carboxyl group of 4 is in fact close to that of fusidic
acid, the COOH RMS value with respect to the crystal
structure being 1.5 A. These observations hold for all
conformations of 3 and 4 within 3 kcal/mol of the glo-
bal minimum (average COOH RMS values are 1.4Æ0.1
and 1.6Æ0.2 A, respectively, none above 2 A).
We also examined the position of the lipophilic moiety
of the side chain as illustrated in Figure 2. The position
of this moiety of the side chain is shown for all low
energy conformations of compounds 1–4 where the
RMS value of the carboxyl group with respect to the
corresponding group in the crystal structure of fusidic
acid methyl ester 3-p-bromobenzoate is less than 2 A
(see ref 9 for further details). The position of the lipo-
philic part of the side chain differs significantly for 3 and
4 as can clearly be seen in Figure 2. Thus, compound 3
occupies a conformational space above the ring plane
whereas the side chain of compound 4 is found below
the ring plane.
Scheme 1. Fusidic acid 1 and related derivatives 2–4.
compared with natural fusidic acid 1 and four stereo-
isomers with a single bond between C-17 and C-20 from
our previous study.9 The conformation of the tetracyclic
ring system was kept constant according to crystal
structures of fusidic acid derivatives10,11 in all calcula-
tions since it appears virtually unaffected by side chain
modifications. Therefore, only the side chain and the C-
16 acetoxy group were allowed to change.12 Super-
position of the lowest energy conformations (global
minimum) of fusidic acid, 17S,20S-dihydrofusidic acid
(2), 17S,20S-methanofusidic acid (3) and 17R,20R-
methanofusidic acid (4) on the crystal structure of fusi-
dic acid methyl ester 3-p-bromobenzoate is shown in
Figure 1. It is clear from this superposition that the
position of the essential carboxyl group in the global
minimum conformation of 3 nicely emulates the posi-
tion of the carboxyl group in 1 and 2. The RMS value
of the carboxyl group of the global minimum con-
formation of 3 with respect to the crystal structure of
fusidic acid methyl ester 3-p-bromobenzoate is 1.3 A.
Although less clear from Figure 1, the position of the
The stereoselective synthesis of the desired 17S,20S-
methanofusidic acid 3 is outlined in Scheme 2. Sulfur
ylides13,14 seemed to be the reagents of choice for the
cyclopropanation of the tetrasubstituted conjugated
Á17(20) bond. Direct cyclopropanation of fusidic acid 1
using excess dimethylsulfoxonium methylide proved
unsuccessful whereas cyclopropanations of fusidic acid
esters resulted in poor yields and lactonisation. On the
other hand, the reaction proceeded smoothly when
using fusidic acid lactone 5 as substrate with attack of
the double bond solely from the least hindered a-face
yielding lactone 6 with no additional cyclopropanation
of the Á24(25) bond.15 We were unable to open the very
stable lactone ring in 7 and therefore employed an
alternative stepwise strategy as previously reported9 in
order to restore the free C-21 carboxyl group and the
C-16 acetoxy group. Lactone 7 was reduced to the cor-
responding diol 8, and the C-21 primary hydroxy group
in 8 was selectively protected with DPMS followed by
acetylation of the C-16 hydroxy group. The DPMS in
Figure 1. Superposition of the global minimum conformations of
fusidic acid and compounds 2–4 on the crystal structure of fusidic acid
methyl ester 3-p-bromobenzoate. The carbon atoms of the tetracyclic
ring system were superimposed. The crystal structure is shown in atom
colours, 1: green, 2: magenta, 3: cyan, and 4: yellow. The lipophilic
part of the side chain and the 3-p-bromobenzoate moiety of the crystal
structure have been omitted for clarity.
Figure 2. Superposition of all conformations within 3 kcal/mol of the
global minimum where the RMS value of the carboxyl group is less
than 2 A. Only the lipophilic part of the side chain is shown. The
colour Scheme is the same as in Figure 1.

T. Duvold et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3569–3572
3571
Table 1. Antibacterial activity (MIC) of fusidic acid (1) and 17S,20S-
methanofusidic acid (3)20
Organism/strain
MIC (mg/mL)a
Fusidic acid 1 17S,20S-Methanofusidic
acid 3
Staphylococcus aureus
ATCC 2977
0.006
0.015
0.006
>125
0.001
0.02
0.04
4
0.003
0.014
0.003
>125
0.001
0.02
Staphylococcus aureus
CJ 232 (MRSA)
Staphylococcus aureus
CJ 234 (R) (MRSA)
Staphylococcus aureus
CJ 234 (F) (MRSA)
Staphylococcus epidermidis
ATCC 12228
Propionibacterium acnes
NCTC 737
Corynebacterium xerosis
NCTC 9755
0.04
Entrococcus faecalis
ATCC 10541
16
Entrococcus faecium
EI 119 (P)
0.02
0.02
Streptococcus sp. EF 6
Streptococcus EG 14
Streptococcus pyogenes
EC
Lactobacillus plantarum
ATCC 8014
Clostridium perfringens
KT 13
Micrococcus luteus
ATCC 9341
Bacillus cereus
ATCC 10876
Bacillus stearothermophilus
KG 5
Escherichia coli HA 44
Pseudomonas aeruginosa
ATCC 10145
4
16
4
16
63
16
15À17
Scheme 2.
(a) aq NaOH in EtOH, reflux, (96%); (b) TBDMSCl,
imidazole in DMF, rt, overnight, (93%); (c) trimethylsulfox-
oniumiodide, NaH, rt, 5 h, (92%); (d) LiAlH4, THF, reflux, (quant);
(e) DPMSCl, Et3N, CH2Cl2, À20 ꢀC, (98%); (f) Ac2O/pyridine, (90%);
(g) TBA+FÀ, AcOH, THF, (90%); (h) (i) Dess–Martin periodinane,
CH2Cl2, 0 ꢀC, 3 h, (88%); (ii) NaClO2, tert.BuOH, (81%); (i) aq HF in
THF, rt, 24 h (82%).
4
16
1
63
16
0.25
1
10 was then selectively cleaved, and the resulting pri-
mary hydroxy group in 11 was oxidised to the corre-
sponding carboxylic acid. In a final step TBDMS of 12
was cleaved yielding 17S,20S-methanofusidic acid 3.16
4
0.03
0.03
>125
1
>125
1
We also attempted to synthesise the opposite stereo-
isomer, 17R,20R-methanofusidic acid 4. However, all
attempts to synthesise this stereoisomer were unsuc-
cessful due to the high stereoselectivity of dimethylsul-
foxonium methylide, in our hands the only successful
reagent for the cyclopropanation of the Á17(20) bond.19
MRSA, methicillin resistant S. aureus; R, rifampicin resistant; P,
penicillin resistant; F, fusidic acid resistant.
aMIC values were determined by a single run of dilutions.
The linkage of the side chain to the tetracyclic rings system
is decisive for the orientation of both the lipophilic part
and the essential carboxylic acid group. The successful
structural modifications of the fusidic acid side chain
underline the importance of this moiety and suggest a new
approach in the ongoing effort to design derivatives with
improved antibacterial and pharmacokinetical properties.
Antimicrobial evaluation20 of 17S,20S-methanofusidic
acid 3 revealed potent activity against several Gram
positive bacteria (Table 1), and in particular against
staphylococcal species including methicillin and peni-
cillin resistant S. aureus. However, the spectrum of
activity of the new derivative was similar to that of the
natural compound with cross-resistance observed for
fusidic acid resistant S. aureus.
Acknowledgements
In this present study, we locked the side chain by means
a spiro-cyclopropane system reducing the conforma-
tional freedom compared with that of the saturated
derivatives. Based on conformational analysis, we pre-
dicted that such a linkage should direct the lipophilic
part of the side chain into different regions depending
on the insertion of the cyclopropane ring between C-17
and C-20 (Fig. 2). Analysis of low energy conformations
suggested that only 17S,20S-methanofusidic acid would
give rise to side chain conformations above the ring
plane, that is the expected bioactive space. In excellent
agreement with this hypothesis, the 17S,20S-methano-
fusidic acid (3) revealed potent antibacterial activity.
The authors wish to thank Ms. A. G. Møller and Ms.
K. Hvidtfeldt Hansen for excellent technical assistance.
References and Notes
1. Godtfredsen, W. O.; Jahnsen, S.; Lorck, H.; Roholt, K.;
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2. Godtfredsen, W. O.; Roholt, K.; Tybring, L. The Lancet
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3. Kuchers, A.; Crove, S.; Grayson, M. L., Hoy, J. In The Use

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of Antibiotics, 5th ed.; Butterworth Heinemann: Oxford, 1997,
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4. Collignon, P.; Turnidge, J. Int. J. Antimicrob. Agents 1999,
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6. Godtfredsen, W. O.; von Daehne, W.; Tybring, L.; Vange-
dal, S. J. Med. Chem. 1966, 9, 15.
7. Janssen, G.; Vanderhaeghe, H. J. Med. Chem. 1966, 10,
205.
(m, 1H), 3.00 (m, 1H), 2.70 (m, 1H), 1.67 (bs, 3H), 1.63 (bs,
3H), 1.38 (s, 3H), 1.22 (d, J=4.5 Hz, 1H), 1.14 (d, J=4.5 Hz,
1H), 0.96 (bs, 3H), 0.92 (bs, 3H), 0.89 (s, 9H), 0.80 (d, J=6.8
Hz, 3H), 0.02 (s, 3H), 0.01 (s, 3H), 13C NMR (d/CDCl3),
178.2, 132.1, 123.7, 84.5, 71.6, 68.0, 50.4, 50.0, 44.1, 40.3, 38.3,
37.1, 36.6, 36.4, 36.0, 33.1, 32.8,30.7, 30.5, 30.3, 30.2, 25.9,
25.9, 25.9, 25.6, 25.5, 23.6, 22.7, 20.4, 18.1, 17.9, 17.6, 17.5,
16.5, À4.6, À5.1
16. 17S,20S-Methanofusidic acid 3: Mp 243–245 ꢀC. 1H
NMR (d/CDCl3), 5.11 (t, J=6.0 Hz, 1H), 4.98 (d, J=7.6 Hz,
1H), 4.26 (m, 1H), 3.65 (m, 1H), 2.78 (m, 1H), 1.92 (s, 3H),
1.66 (bs, 3H), 1.61 (d, J=4.6 Hz, 1H), 1.60 (bs, 3H), 1.38 (s,
3H), 1.12 (s, 3H), 0.99 (s, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.69 (d,
J=4.6 Hz, 1H). 13C NMR (d/CDCl3), 177.03, 172.33, 132.67,
125.22, 81.09, 72.50, 68.64, 50.86, 49.93, 45.22, 41.85, 41.05,
39.80, 38.20, 38.13, 37.93, 36.99, 36.57, 33.17, 32.65, 31.09,
31.06, 27.37, 25.89, 24.22, 23.67, 22.40, 21.48, 20.51, 18.00,
17.71, 16.52.
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Commun. 1972, 46, 871.
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A. S.; Rastrup-Andersen, N. J. Med. Chem. 2001, 28, 3125.
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11. Søtofte, I.; Duvold, T. Acta. Cryst. 2001, E57, 829.
12. All calculations were performed on a Silicon Graphics O2
R10000 workstation. The conformational analyses were car-
ried out using the Monte Carlo (Mcrlo) routine of Macro-
Model 7.0 (Schrodinger Inc.). Structures were visualized with
Sybyl 6.8 (Tripos Inc.)
The configuration of C-17 and C-20 in lactone (7) and
17S,20S-methanofusidic acid (3) was determined by means of
NOESY experiments. In both compounds H-31a showed
strong NOE to H-13 and medium NOE to H-16, whereas H-
31b showed strong NOE to H-13 and no NOE to H-16.
17. Conventional 1H, 13C and DEPT135 spectra were
obtained on all compounds. HMQC, HMBC, COSY,
HHTOCSY and CHTOCSY experiments were performed on
compounds 3 and 7 in order to make total assignments.18
18. Rastrup-Andersen, N.; Duvold, T. Magn. Reson. Chem.
2002, 40, 471.
19. Various cyclopropanation methods including Simmons-
Smith type reactions and dichlorocarbene proved unsuccess-
ful. For a recent review see Donaldson, W. A. Tetrahedron
2001, 57, 8589, and references cited therein.
20. MIC values were determined according to Hewitt, W.;
Vincent, S. The Agar Diffusion Assay. In Theory and Appli-
cation of Microbiological Assay; Academic: San Diego, 1988;
p 38.
13. Corey, E. J.; Chaykovski, M. J. Am. Chem. Soc. 1962, 84,
3782.
14. Landor, S. R.; Punja, N. J. Chem. Soc. 1967, 2495.
15. Cyclopropanation of lactone 613 NaH (60% in mineral
oil, 20 mg, ca. 0.5 mmol) was washed with pentane and dis-
solved in DMF (0.5 mL) followed by addition of neat tri-
methylsulfoxoniumiodide (108 mg, 0.48 mmol) in one portion.
The suspension was stirred for 30 min and lactone 6 (190 mg,
0.34 mmol) in DMF (1 mL) was then added dropwise to the
ylide. The reaction mixture was stirred for 5 h at rt and then
poured into ice-cold aq HCl (10%, 10 mL). The aq suspension
was extracted with EtOAc, dried (Na2SO4) and evaporated
under reduced pressure yielding a white powder. Pure lactone
7 (183 mg, 92%) was obtained after crystallisation from
MeOH–water. Mp 186–187 ꢀC. H NMR (d/CDCl3), 5.10 (t,
1
J=7.2 Hz, 1H), 4.39 (d, J=9.3 Hz, 1H), 4.29 (m, 1H), 3.68