Efficient synthesis of [3H]-sanglifehrin A via selective oxidation/reduction of alcohols at C31 and C35

Article abstract of DOI:10.1021/jo051112h

Sanglifehrin A is a novel complex natural product showing strong immunosuppressive activity and remarkably high affinity for cyclophilin A. To assess its pharmacokinetic properties in vivo, an efficient synthetic route was developed to introduce a tritium

Full text of DOI:10.1021/jo051112h

Efficient Synthesis of [3H]-Sanglifehrin A
via Selective Oxidation/Reduction of
Alcohols at C31 and C35
structural attractiveness of sanglifehrin, combined with
its biological activity, generated broad interest in indus-
trial and academic laboratories. Their efforts resulted in
the preparation of several fragments3 and finally culmi-
nated in the total syntheses by Nicolaou et al.4 and
Paquette et al.5
Ju¨rgen Wagner,* Hendrik Andres,‡ Stefan Rohrbach,
Dieter Wagner, Lukas Oberer, and Julien France
The promising immunosuppressive properties of san-
glifehrin A prompted us to undertake a complete study
of its pharmacokinetic properties. Toward this purpose,
a tritium or carbon-14 radiolabeled isotopomer of san-
glifehrin A was needed. Typically, for such complex
natural products, the radiolabel could be introduced
either by a synthetic approach6 or via a fermentation
process.7 We decided to explore tritium labeling via a
synthetic route first. Indeed, we reasoned that a straight-
forward two-step oxidation/reduction strategy could be
applied to either position C31 or C35. The reduction of one
or both positions was expected to proceed stereoselec-
tively due to the steric effect of the adjacent methyl
groups.
Novartis Institutes for BioMedical Research Basel,
Transplantation Research, Novartis Pharma AG,
Basel, Switzerland
juergen.wagner@novartis.com
Received June 2, 2005
Herein, a detailed account of the regioselective oxida-
tion of the hydroxyl groups at either C31 or C35 as well as
the stereoselective outcome of the reduction of the cor-
responding ketones is described. This study led us to a
straightforward and high-yielding synthesis of sangli-
fehrin A tritium labeled in position C35 on large scale.
The overall synthetic route, which allows the pre-
paration of [3H]-labeled sanglifehrin A, is shown in
Scheme 1.
Intramolecular cyclization of the C53 ketone of san-
glifehrin A with both hydroxy groups at C15 and C17 under
mild acidic conditions provided in quantitative yield ketal
1. The stable ketal moiety was used as a protecting group
for this sensitive portion of the molecule. Attempts to
oxidize directly intermediate 1 bearing a free phenol
moiety were unsuccessful. Therefore, m-tyrosine was
selectively acetylated in the presence of acetic anhydride
and catalytic amounts of DMAP to afford 2 quantita-
tively. The CH2Cl2/pyridine ratio was critical for the
Sanglifehrin A is a novel complex natural product showing
strong immunosuppressive activity and remarkably high
affinity for cyclophilin A. To assess its pharmacokinetic
properties in vivo, an efficient synthetic route was developed
to introduce a tritium label in position C35 of sangliferin A
via an oxidation/reduction strategy. The synthetic approach
is particularly attractive, because the C35-oxo intermediate
7 is available in good yield on large scale and the reducing
agent, lithium tri-sec-butylborotritide, is readily available.
An attempt to apply a similar strategy to the alcohol in
position C31 led primarily to C31-epi-hydroxy sanglifehrin A
under a variety of conditions.
Sanglifehrin A (SFA) was isolated from Streptomyces
flaveolus in 1995.1 The substance is a potent immuno-
suppressant and has a remarkably high affinity for an
intracellular binding protein called cyclophilin A (IC50
)
2-4 nM).2 In addition to its interesting biological activity,
SFA has a unique and complex molecular structure,
which consists of a 22-membered macrocycle, bearing in
position 23 a nine-carbon tether terminated by a highly
substituted spirobicyclic moiety. The macrolide contains
an (E,E)-diene, a short polypropionate fragment, and a
tripeptide unit composed of valine and two unusual
amino acids, piperazic acid and m-tyrosine. One of the
most remarkable features of the molecule is certainly the
complex spirobicyclic oxaazaspiro[5.5]undecanone system
extending from C33 to N42. This substructure, which
exhibits seven stereocenters, six of which are contiguous,
is unique for the sanglifehrin class of compounds. The
(3) (a) Ba¨nteli, R.; Brun, I.; Hall, P.; Metternich, R. Tetrahedron Lett.
1999, 40, 2109-2112. (b) Hall, P.; Brun, I.; Denni, D.; Metternich, R.
Synlett 2000, 3, 315-318. (c) Nicolaou, K. C.; Ohshima, T.; Murphy,
F.; Barluenga, S.; Xu, J.; Winssinger, N. Chem. Commun. 1999, 9, 809.
(d) Paquette, L. A.; Konetzki, I.; Duan, M. Tetrahedron Lett. 1999, 40,
7441-7444. (e) Duan, M.; Paquette, L. A. Tetrahedron Lett. 2000, 41,
3789-3792. (f) Gurjar, M. K.; Chaudhuri, S. R. Tetrahedron Lett. 2002,
43, 2435-2438. (g) Metternich, R.; Denni, D.; Thai, B.; Sedrani, R. J.
Org. Chem. 1999, 64, 9632-9639.
(4) (a) Nicolaou, K. C.; Xu, J.; Murphy, F.; Barluenga, S.; Baudoin,
O.; Wei, H.-X.; Gray, D. L. F.; Ohshima, T. Angew. Chem., Int. Ed.
1999, 38, 2447-2451. (b) Nicolaou, K. C.; Murphy, F.; Barluenga, S.;
Ohshima, T.; Wei, H.; Xu, J.; Gray, D. L. F.; Baudoin, O. J. Am. Chem.
Soc. 2000, 122, 3830-3838.
(5) (a) Duan, M.; Paquette, L. A. Angew. Chem., Int. Ed. 2001, 40,
3632-3636. (b) Paquette, L. A.; Duan, M.; Konetzki, I.; Kempmann,
C. J. Am. Chem. Soc. 2002, 124, 4257-4270.
(6) For rapamycin, see: Curran, D. P.; Somayajula, K. V.; Yu, H.
Tetrahedron Lett. 1992, 33, 2295-2298.
(7) (a) For rapamycin analogues, see: Mo¨nius, Th.; Voges, R.;
Mahnke, M.; Burtscher, P.; Metz, Y.; Guenat, Ch. J. Labelled Compd.
Radiopharm. 1999, 42, 29-41. (b) For cyclosporine analogues, see:
Mo¨nius, Th.; Voges, R.; Susan, A.; Jones, L.; Tarapata, R.; Shapiro,
M.; Jarema, M.A. C. Synth. Appl. Isot. Labeled Compd. 1991, 149-
154. (c) For a review, see: Mo¨nius, Th.; Baumann, K.; Bulusu, M.;
Schweitzer, A.; Voges, R. Synth. Appl. Isot. Labeled Compd. 2001, 424-
429.
* To whom correspondence should be addressed. Phone: +41-61-
324-26-83. Fax: +41-61-324-89-18
‡ Present address: Novartis Pharma AG, Exploratory Development,
DMPK, Isotope Laboratories, Basel, Switzerland.
(1) (a) Sanglier, J.-J.; Quesniaux, V.; Fehr, T.; Hofmann, H.;
Mahnke, M.; Memmert, K.; Schuler, W.; Zenke, G.; Gschwind, L.;
Maurer, C.; Schilling, W. J. Antibiot. 1999, 52, 466. (b) Fehr, T.; Kallen,
J.; Oberer, L.; Sanglier, J.-J.; Schilling, W. J. Antibiot. 1999, 52, 474.
(2) Zenke, G.; Strittmatter, U.; Fuchs, S.; Quesniaux, V. F. J.;
Brinkmann, V.; Schuler, W.; Zurini, M.; Enz, A.; Billich, A.; Sanglier,
J.-J.; Fehr, T. J. Immunol. 2001, 166, 7165-7171.
10.1021/jo051112h CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/14/2005
9588
J. Org. Chem. 2005, 70, 9588-9590

a
SCHEME 1. Synthetic Approach toward Sanglifehrin A Tritium Labeled in Position C35
a Reagents and conditions: (a) TFA (catalytic), CH3CN, quantitative; (b) for (2): Ac2O, DMAP (catalytic), CH2Cl2/pyridine 27:1,
quantitative; for (3): Ac2O, DMAP (catalytic), CH2Cl2/pyridine 9:1, 65%; for (4): (i) same as for (2), (ii) (Cl3CO)2O, DMAP (catalytic),
CH2Cl2/pyridine 31:1, 67% (two steps); (c) Dess-Martin’s periodinane, CH2Cl2, (5, 91% and 6, quantitative); (d) 1.0 M aqueous NaHCO3/
MeOH 10:1, (7, 55% and 8, 30%); (e) L-Selectride, THF, -20 °C, 50%; (f) see ref 3g.
chemoselectivity. By increasing the concentration of
pyridine, the C31,C61-diacetylated product 3 is formed in
65% yield. Oxidation of the alcohol at C35 with Dess-
Martin’s periodinane8 afforded ketone 5 in excellent yield
(91%). Deprotection of 5 under mildly basic conditions
led to rapid removal of the phenolic acetate. Unfortu-
nately, under prolonged alkaline treatment, amide 8 was
isolated in 30% yield instead of the desired fully deacety-
lated ketone 7. The ring-opening of the spirobicyclic
subunit through R,â-keto elimination appeared to be
faster than deprotection of the sterically hindered C31
acetate. Removal of this protecting group under reductive
conditions, e.g. NaBH4 in EtOH or L-Selectride in THF,
was not successful either, because the macrolide ester
was reduced more rapidly than the C31 acetate. Therefore,
we turned our attention to the more labile trichloroac-
etate protecting group. Starting from mono-acetate 2,
selective trichloroacetylation followed by oxidation af-
forded intermediate 6 in excellent yield. Deprotection of
6 under mildly alkaline conditions led to the desired
ketone 7, the precursor for the stereoselective reduction,
in 55% yield. As predicted by the model, all reducing
agents reacted exclusively from the less hindered face of
the molecule producing the desired equatorial alcohol.
The highest yield (51%) was obtained with L-Selectride.
The 1H NMR spectra of the compound obtained by
reduction of 7 were identical with the spectra of inter-
mediate 1 obtained previously by acidic treatment of
sanglifehrin A. Final deprotection of the intramolecular
ketal 1 was performed as reported previously.3g
The synthetic route described in Scheme 1 was used
to produce successfully sanglifehrin A labeled with tri-
tium in position C35. The approach is particularly attrac-
tive for two reasons. The precursor 7 is readily available
from sanglifehrin A in five steps and 37% overall yield
and the radiolabeled material needs to be carried through
two synthetic steps only. The reducing agent of choice
for tritium labeling was lithium tri-sec-butylborotritide,
the tritiated analogue of L-Selectride, because it is readily
available from lithium tritide and tri-sec-butylborane.9,10
In parallel, we tested a similar two-step strategy on
the alcohol in position C31. To this end, we submitted
mono-acetate 2 to a series of oxidative conditions
(Scheme 2).
Under Swern conditions or with pyridinium dichro-
mate as an oxidant, the reaction was sluggish and led
mostly to decomposition products. On the other hand, the
C31,C35-diketone 10 could be obtained by using either
tetrapropylammonium perruthenate11 (TPAP) in the
presence of 4-methylmorpholine N-oxide as co-oxidant
(42% yield) or Dess-Martin’s periodinane8 (72% yield).
Finally, selective oxidation of the C31 alcohol was ob-
(9) Andres, H.; Morimoto, H.; Williams, P. G. J. Chem. Soc., Chem.
Commun. 1990, 8, 627-628.
(10) Andres, H.; Morimoto, H.; Williams, P. G. Synthesis and
Applications of Isotopically Labelled Compounds; Buncel, E., Kabalka,
G. W., Eds.; Elsevier Science Publishers: New York, 1991.
(11) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P. Synthesis
1994, 639-666.
(8) (a) Dess, B. D.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-
4156. (b) Dess, B. D.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7287.
J. Org. Chem, Vol. 70, No. 23, 2005 9589

SCHEME 2. Selective Oxidation of Alcohols at C31
Herein, we report on an efficient two-step oxidation/
reduction strategy to introduce a tritium label in position
C35 of complex natural product sanglifehrin A. In this
case, the synthetic approach is particularly attractive
because (1) the precursor for the reduction step, inter-
mediate 7, is available in five steps and 37% overall yield
from sanglifehrin A on large scale, (2) the reduction is
fully stereoselective, (3) the reducing agent, lithium tri-
sec-butylborotritide, used to introduce the tritium is
cheap and readily available at almost theoretical specific
activity, and (4) the radioactive material is involved in
two synthetic steps only. In addition to this approach,
possibilities to apply a similar strategy to position C31 of
sanglifehrin A were explored. The alcohol at position C31
could be regioselectively oxidized with IBX in DMF
containing 5% DMSO. Unfortunalely, reduction of the
corresponding ketone led either to an epimeric mixture
or, in the case of L-Selectride, selectively to the C31-epi-
hydroxy sanglifehrin A.
a
and C35
a Reagents and Conditions: (a) DMF, DMSO (5%), IBX, (9,
60%), or Dess-Martin’s periodinane, CH2Cl2, (10, 72%).
SCHEME 3. Stereoselectivity of the Reduction of
a
the Ketone at C31
Experimental Section
Tritiated Sanglifehrin A. Lithium tritide was obtained from
252 µL of n-butyllithium (1.6 M in hexane, 0.4 mmol) and 90 µL
of TMEDA (0.6 mmol) at an initial pressure of 812 hPa of tritium
gas. After stirring at 23 °C for 1 h the LiT had formed and the
pressure dropped to 650 hPa indicating the uptake of 0.4 mmol
of tritium. The mixture was frozen to -196 °C and the excess
tritium was taken back. Hydrogen was admitted to a pressure
of 400 hPa and the mixture was warmed. Then 400 µL of 1.0 M
tri-sec-butylborane in hexane (0.4 mmol) was added, whereupon
the LiT went into solution. After 30 min, 170 mg of 35-Oxo
Sanglifehrin A Ketal 7 in 1.5 mL of dry THF was added dropwise
at -15 °C. After stirring for 1 h the reaction was quenched with
3 mL of acetonitrile/water 1:1. After 5 min the mixture was
frozen to -196 °C and the tritium-hydrogen mixture formed
by hydrolysis of the reagent was taken back into a uranium trap.
After evaporation of the solvents the residue was lyophilized
with methanol to remove volatile and exchangeable tritium.
The total activity of the crude reaction mixture was 82.58 GBq
(19.4% based on butyllithium, 47.9% based on 7). Preparative
HPLC separation on RP18 column with use of a mixture of
acetonitrile and water (37 to 90% of acetonitrile within 10 min)
gave 43.29 GBq of radiochemically pure tritiated Sanglifehrin
A Ketal 2.
a Reagents and Conditions: (a) 1.0 M aqueous NaHCO3/MeOH
10:1, quantitative; (b) NaBH4, EtOH, 0 °C, (1, 16% and 11, 39%).
served predominantly when 2-iodoxybenzoic acid (IBX),12
the precursor of Dess-Martin’s periodinane, was used
as an oxidant in DMF containing 5% DMSO. Under these
mild conditions, ketone 9 was isolated in 60% yield. After
alkaline deprotection of the phenol, several reductive
conditions were tested on the C31 ketone 9 (Scheme 3).
Reduction with NaBH4 in EtOH afforded a 1:2 mixture
in favor of C31-epi-hydroxy sanglifehrin A 11 as assessed
by comparing the 1H NMR spectra with the spectra of 1.
Running the NaBH4 reduction in the presence of cerium
trichloride (Luche’s conditions) did not modify the iso-
meric ratio, even though an effect of organolanthanide
reagents on the stereochemical outcome of ketone reduc-
tion has been reported.13 The more sterically hindered
reducing agent, L-Selectride, afforded exclusively 11
based on HPLC analysis. No reaction was observed by
using DIBAL-H at low temperature. As no conditions
were found to reduce the C31 ketone to the alcohol with
the desired stereoselectivity, sanglifehrin A tritium
labeled in position C31 or doubly labeled in positions C31
and C35 is not accessible via the oxidation/reduction
strategy.
Ketal (3.7-GBq) was then deprotected with a mixture of 100
µL of THF, 100 µL of water, and 20 µL of 2 N HCl. After 30%
conversion the reaction mixture was submitted to preparative
HPLC and the unchanged starting material was resubmitted
to the deprotection. After 5 cycles a total yield of 1.095 GBq of
[3H]-Sanglifehrin A (29.6%) at a specific activity of 0.943 TBq/
mmol and a radiochemical purity of 97.34% was obtained.
Acknowledgment. We thank U. Zandona for her
assistance in typing the manuscript.
In conclusion, the availability of radiolabeled material
is essential to do a full study of the pharmacokinetic
properties of promising new development compounds.
Supporting Information Available: Full experimental
details and spectral characterization of all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(12) Frigerio, M.; Santagostino, M.; Sputore, S. J. Org. Chem. 1999,
64, 4537-4538.
(13) (a) Leclaire, M.; Jean, P. Bull. Soc. Chim. Fr. 1996, 133, 801-
803. (b) Krief, A.; Surleraux, D. Synlett 1991, 273-275.
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