Sanglifehrin-cyclophilin interaction: Degradation work, synthetic macrocyclic analogues, x-ray crystal structure, and binding data

Article abstract of DOI:10.1021/ja021327y

Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC₅₀ = 6.9 ± 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C₂₆ = C₂₇ exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC₅₀ = 29 ± 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the x-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The x-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.

Full text of DOI:10.1021/ja021327y

Published on Web 03/06/2003
Sanglifehrin-Cyclophilin Interaction: Degradation Work,
Synthetic Macrocyclic Analogues, X-ray Crystal Structure, and
Binding Data
Richard Sedrani,† Jo¨rg Kallen,† Luisa M. Martin Cabrejas,‡
Charles D. Papageorgiou,§ Francesco Senia,| Stefan Rohrbach,† Dieter Wagner,†
Binh Thai,† Anne-Marie Jutzi Eme,† Julien France,† Lukas Oberer,† Grety Rihs,†
Gerhard Zenke,† and Ju¨rgen Wagner*,†
Contribution from the Transplantation Research, NoVartis Pharma AG, S-507.312,
CH-4002 Basel, Switzerland, Centro de InVestigacio´n Lilly S. A., AVda. de la Industria 30,
28108 Alcobendas, Madrid, Spain, UniVersity of Cambridge, Lensfield Road,
Cambridge CB2 1EW, U.K., and Glaciology Center, UniVersity of Bristol, Bristol BS8 1SS, U.K.
Received November 4, 2002; E-mail: Juergen.wagner@pharma.novartis.com
Abstract: Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces
sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC50 ) 6.9 ( 0.9 nM) but is structurally
different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA
has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-
carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the
C26dC27 exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity
of 2 for cyclophilin (IC50 ) 29 ( 2.1 nM) is essentially identical to SFA, which indicates that the interaction
between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This
observation was confirmed by the X-ray crystal structure resolved at 2.1 Å of cyclophilin A complexed to
macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the
same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity
relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or
(2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key
step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination
of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide
unit, which together provide an extremely strong affinity for cyclophilin A.
pathways at the molecular level.3 It has been shown that the
Introduction
biological activity of CsA, FK506, and rapamycin is mediated
by intracellular binding proteins called immunophilins. CsA
binds to cyclophilins (CyP), whereas FK506 and rapamycin bind
to FK506-binding proteins (FKBP) (Figure 2). However,
although immunophilin binding is required, it is not sufficient
for the immunosuppressive activity of these drugs. Biological
effects are observed only upon interaction of the drug/immu-
nophilin complexes with a third partner, the effector protein.
Thus, the CyP-CsA and FKBP-FK506 complexes inhibit the
serine/threonine phosphatase activity of calcineurin, thereby
blocking the production of cytokines including interleukin-2.4
On the other hand, the FKBP-rapamycin complex inhibits a
kinase called FRAP (also known as RAFT or mTOR)5 that is
involved in interleukin-2 receptor-mediated T cell proliferation.6
More than two decades ago, the discovery of cyclosporin A
(CsA, Figure 1) allowed a spectacular progress in the field of
organ transplantation.1 Since then, the number of transplanted
organs has grown continuously and the search for novel
immunosuppressants has intensified.2 These efforts resulted in
the isolation of several immunosuppressants from natural sources
which interfere with T cell activation or proliferation. The most
prominent examples are FK506 and rapamycin (Figure 1).
Besides their important therapeutic use, these drugs have also
proven to be powerful tools for dissecting signal transduction
† Novartis Pharma AG.
‡ Centro de Investigacio´n Lilly S. A.
§ University of Cambridge.
| University of Bristol.
(1) For a review on cyclosporin, see: Kallen, J.; Mikol, V.; Quesniaux, V. F.
J.; Walkinshaw, M. D.; Schneider-Scherzer, E.; Scho¨rgendorfer, K.; Weber,
G.; Fliri, H. G. In Biotechnology, Vol. 7; Kleinkauf, H., von Do¨hren, H.,
Eds.; VCH: Weinheim, Germany, 1997; Chapter 12, pp 535-591.
(2) (a) Dumont, F. J. Exp. Opin. Ther. Patents 2001, 11, 377-404. (b)
PerspectiVes in Drug DiscoVery and Design, Vol. 2; Anderson, P. S.,
Kenyon, G. L., Marshall, G. R., Eds.; ESCOM Science Publishers: Leiden,
The Netherlands, 1994; pp 3-248.
(3) (a) Schreiber, S. L.; Albers, M. W.; Brown, E. J. Acc. Chem. Res. 1993,
26, 412-420. (b) Schreiber, S. L.; Crabtree, G. R. Immunol. Today 1992,
13, 136-142.
(4) (a) Liu, J.; Farmer, J. D., Jr.; Lane, W. S.; Friedman, J.; Weissman, I.;
Schreiber, S. L. Cell 1991, 66, 807-815. (b) Friedman, J.; Weissman, I.
Cell 1991, 66, 799-806.
9
10.1021/ja021327y CCC: $25.00 © 2003 American Chemical Society
J. AM. CHEM. SOC. 2003, 125, 3849-3859
3849

A R T I C L E S
Sedrani et al.
Figure 1. Molecular structures of the immunosuppressive natural products interfering with T cell signaling pathways: cyclosporin A, FK506, and rapamycin.
Figure 2. Mode of action of the immunosuppressants and the discovery of the sanglifehrins.
Considering that different ligands for FKBP exert their
biological effects by different mechanisms, we wondered
whether besides CsA other ligands of cyclophilin might exist
and whether these ligands would exhibit different modes of
action than CsA. Screening of microbial broth extracts for
cyclophilin-binding substances led to the isolation from Strep-
tomyces sp. A92-308110 of a new class of compounds named
sanglifehrins.7 Among the 20 different sanglifehrins isolated so
far, sanglifehrin A (SFA) is the most abundant component
(Scheme 1). The affinity of SFA for cyclophilin in a cell-free
competitive binding assay is remarkably high (IC50 ) 6.9 (
0.9 nM),8 approximately 60-fold higher than that of CsA (IC50
) 420 ( 56 nM). SFA displays potent immunosuppressive
activity in the murine mixed lymphocyte reaction (IC50 ) 170
( 30 nM), an in vitro immune response assay. The details of
the mechanism by which this compound exerts its immunosup-
(5) (a) Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith, C. T.;
Lane, W. S.; Schreiber, S. L. Nature 1994, 369, 756-758. (b) Sabers, C.
J.; Martin, M. M.; Brunn, G. J.; Williams, J. M.; Dumont, F. J.; Wiederrecht,
G.; Abraham, R. T. J. Biol. Chem. 1995, 270, 815-822. (c) Sabatini, D.
M.; Erdjument Bromage, H.; Lui, M.; Tempst, P.; Snyder, S. H. Cell 1994,
78, 35-43.
(6) (a) Bierer, B. E.; Mattila, P. S.; Standaert, R. F.; Herzenberg, L. A.;
Burakoff, S. J.; Crabtree, G.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 9231-9235. (b) Dumont, F. J.; Staruch, M. J.; Koprak, S. L.;
Melino, M. R.; Sigal, N. H. J. Immunol. 1990, 144, 251-258.
(7) (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, 466-473. (b) Fehr, T.; Kallen, J.; Oberer, L.;
Sanglier, J.-J.; Schilling, W. J. Antibiot. 1999, 52, 474-479.
(8) 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.
9
3850 J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003

Sanglifehrin−Cyclophilin Interaction
A R T I C L E S
Scheme 1. Structure of Sanglifehrin A and Oxidative Cleavage of
the C26dC27 Bond13
two-step procedure (Sharpless asymmetric dihydroxylation,
periodate oxidation) (Scheme 1).13 This approach allowed an
efficient preparation of the spirolactam containing fragment 1
and macrolide 2. Fragment 1 by itself does not bind to
cyclophilin (IC50 g 5000 nM). On the other hand, the removal
of the C27-N42 subunit has only a minor effect on the affinity
of macrolide 2 for cyclophilin (IC50 ) 29 ( 2.1 nM). But
surprisingly, neither of the two fragments exhibits immunosup-
pressive activity. Based on these initial observations, we initiated
a two-phase synthetic effort. First, we aimed at further reducing
the complexity of the macrocycle while maintaining its affinity
for cyclophilin. During this phase, particular emphasis was put
on reducing the overall polarity of the molecule. Indeed, it is
believed that the high number of polar functional groups of the
sanglifehrin class of substances is responsible for their poor cell
permeation.14 In the second phase, we planned to recombine
suitable macrolides with various side chains in order to restore
the immunosuppressive activity.15
Herein, we will discuss in detail our findings related to the
interaction between the macrolide fragment of sanglifehrin A
and cyclophilin. An overview of the structure-activity relation-
ship (SAR) was obtained either by degradation work on 2 or
by synthesis of a library of macrolide analogues based on the
ring-closing metathesis reaction. In addition, we disclose the
first X-ray crystal structure of a sanglifehrin derivative, mac-
rolide 16, bound to cyclophilin A.
pressive activity at the molecular level is currently under
investigation, and the first results were published recently.9
These data clearly indicate that SFA represents a novel type of
immunosuppressant whose mode of action is different from that
of all other known immunophilin-binding compounds, namely
CsA, FK506, and rapamycin.
Chemistry
Degradation Work on Macrolide 2. The syntheses of the
new derivatives are summarized in Schemes 2 and 3. To assess
the role of the ketone in the side chain attached at C14,
sanglifehrin A was reduced with NaBH4. The resulting mixture
of epimeric alcohols could be selectively converted into the
epimeric iodo derivatives 3, which were not separated and were
obtained in 48% yield over two steps. The other hydroxy groups
in the molecule did not react under these conditions, most
probably due to steric hindrance. Compound 3 could be
selectively dihydroxylated at the C26dC27 double bond, and the
resulting diol could be oxidatively cleaved under the same
conditions used for sanglifehrin A to afford macrolide 4.
We then concentrated our efforts on modifying the conjugated
C18-C21 (E,E)-diene system of the macrocyclic fragment.
Hydrogenation of 2 in the presence of Pd/C resulted in reduction
of both the C18-C21 (E,E)-diene system and the C24dC25
exocyclic double bond, affording the fully saturated macrocycle
5 as an epimeric mixture at C24. Selective hydrogenation of the
(E,E)-diene system only could not be achieved under a variety
of conditions. The lack of selectivity during the hydrogenation
of macrolide 2 might be associated with the electron-withdraw-
ing effect of the aldehyde at C26. In the case of sanglifehrin A,
it is indeed possible to obtain selectively the C18-C21 tetrahydro
derivative using Wilkinson’s rhodium catalyst.
In addition to its fascinating biological activity, sanglifehrin
A has a complex and unique molecular structure. The compound
consists of a 22-membered macrocycle, bearing in position 23
a nine-carbon tether terminated by a highly substituted spiro-
bicyclic moiety. The macrolide contains an (E,E)-diene, a short
polypropionate fragment, and a tripeptide unit composed of
valine and two rather unusual amino acids, piperazic acid and
meta-tyrosine. Interestingly, it is the â-nitrogen of piperazic acid
that is involved in the amide bond formation. This stands in
contrast to all other piperazic acid containing natural products
isolated so far. The structural attractiveness of sanglifehrin,
combined with its immunosuppressive activity, generated broad
interest in industrial and academic laboratories. Their efforts
resulted in the preparation of several fragments10 and finally
culminated in the total syntheses by Nicolaou et al.11 and
Paquette et al.12
Despite the structural complexity of SFA, selective cleavage
of the C26dC27 exocyclic double bond could be achieved by a
(9) (a) Zhang, L.-H.; Liu, J. O. J. Immunol. 2001, 166, 5611-5618. (b) Zhang,
L.-H.; Youn, H.-D.; Liu, J. O. J. Biol. Chem. 2001, 276, 43534-43540.
(10) (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, 809-810.
(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.
(11) (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.
The reduction of both carbonyl groups of 2 could be achieved
in quantitative yield using NaBH4 to afford the tetraol 6. Under
acidic conditions (aqueous HCl, dioxane), 6 was converted into
(13) Metternich, R.; Denni, D.; Thai, B.; Sedrani, R. J. Org. Chem. 1999, 64,
9632-9639.
(14) Ba¨nteli, R.; Wagner, J.; Zenke, G. Bioorg. Med. Chem. Lett. 2001, 11,
(12) Duan, M.; Paquette, L. A. Angew. Chem., Int. Ed. 2001, 40, 3632-3636.
Paquette, L. A.; Duan, M.; Konetzki, I.; Kempmann, C. J. Am. Chem. Soc.
2002, 124, 4257-4270.
1609-1612.
(15) The results of the second part of the program will be disclosed in a separate
publication.
9
J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003 3851

A R T I C L E S
Sedrani et al.
Scheme 2. Degradation Studies on Sanglifehrin Aa
a Reagents and conditions: (a) (i) NaBH4, MeOH, 0 °C, (ii) PPh3, imidazole, I2, CH2Cl2, 0 °C to rt, 48% (two steps); (b) (i) (DHQ)2PHAL, OsO4,
CH3SO2NH2, K3Fe(CN)6, K2CO3, t-BuOH/H2O 1:1, (ii) NaIO4, THF/H2O 2:1, 32% for 4 (two steps), see ref 13 for the preparation of 2; (c) Pd/C 10%, H2,
EtOH, 59%; (d) NaBH4, MeOH, 0 °C, quantitative; (e) 2.0 M aqueous HCl, dioxane, 30%.
the conjugated (E,E,E)-triene 7 by elimination of the allylic
alcohol at C17. The fully conjugated tetraene, which could
potentially be generated by the additional elimination of the
alcohol at C15 of 7 was not detected even after prolonged acidic
treatment. Triene 7 proved to be unstable even when stored at
-4 °C.
We next focused our attention on modifying the potentially
reactive R,â-unsaturated aldehyde (Scheme 3). The reaction of
macrolide 2 with phosphonoacetates under modified Wittig-
Horner conditions provided the esters 8 and 9 in 50% and 34%
yield, respectively. In this case, no protection of the other
functionalities in the macrolide was required. But for further
derivation of the aldehyde under strongly basic conditions, the
keto-diol was transformed quantitatively into the ketal 10 under
mild acidic conditions. For the olefination of the aldehyde with
aliphatic groups, the procedure developed by Sylvestre Julia et
al.16 and later modified by Kocienski et al.17 was applied to 10
as described previously for the resynthesis of sanglifehrin A
from degradation products.13 The desired 5-(alkane-1-sulfonyl)-
1-phenyl-1H-tetrazoles were prepared from the commercially
available primary alcohols in two steps according to the
procedure outlined by Kocienski et al.18 In the presence of 2
equiv. of NaHMDS, the olefination procedure afforded the ketal-
protected forms of dienes 11 and 12 in 74% and 49% yield,
respectively. In both cases, the (E,E)-diene products were formed
with very high selectivity (g10:1). Acidic hydrolysis of the
intramolecular ketal afforded the desired macrolides 11 and 12.
Typically, the latter reaction reached an equilibrium at 50%
conversion.13 Finally, the reaction between the macrolide and
an unstabilized Wittig reagent required an additional protection
of the phenol with a tert-butyldimethylsilyl group. The fully
protected aldehyde 13 was conveniently obtained using N-(tert-
(18) The sulfones were prepared according to following scheme:
(16) (a) Baudin, J. B.; Hareau, G.; Julia, S. A.; Ruel, O. Tetrahedron Lett. 1991,
32, 1175-1178. (b) Baudin, J. B.; Hareau, G.; Julia, S. A.; Ruel, O. Bull.
Soc. Chim. Fr. 1993, 130, 336-357. (c) Baudin, J. B.; Hareau, G.; Julia,
S. A.; Lorne, R.; Ruel, O. Bull. Soc. Chim. Fr. 1993, 130, 856-878.
(17) (a) Blakemore, P. R.; Cole, W. J.; Kocienski, P. J.; Morley, A. Synlett
1998, 26-28. (b) Kocienski, P. J.; Bell, A.; Blakemore, P. R. Synlett 2000,
365-366.
9
3852 J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003

Sanglifehrin−Cyclophilin Interaction
A R T I C L E S
Scheme 3. Derivation of the Aldehyde in Macrolide 2a
a Reagents and conditions: (a) RO2CCH2PO(OCH3)2, K2CO3, CH3CN, 50% for 8, 34% for 9; (b) HF‚pyridine, CH3CN, 0 °C, 92%; (c) (i) 5-(butane-
1-sulfonyl)-1-phenyl-1H-tetrazole, NaHMDS, DME, -78 °C, (ii) TsOH, B(OH)3, THF/H2O 1:1, 15% for 11 (two steps); d) (i) 5-[3-(tert-butyldimethylsi-
lyloxy)propane-1-sulfonyl)]-1-phenyl-1H-tetrazole, NaHMDS, DME, -78 °C, (ii) TBAF, THF, (iii) TsOH, B(OH)3, THF/H2O 1:1, 7% for 12 (three steps);
(e) MTBSTFA, CH3CN, 50 °C, 88%; (f) (i) Methyltriphenylphosphonium bromide, LiHMDS, THF, -78 °C, (ii) TBAF, THF, (iii) TsOH, THF/H2O 1:1, 6%
(three steps); (g) NaBH4, MeOH, 0 °C, 90%; (h) (i) TBAF, THF, (ii) TsOH, THF/H2O 1:1, 15% for 16 (two steps); (i) (i) CF3SO3CH3, 2,6-di-tert-butylpyridine,
toluene, (ii) TBAF, THF, (iii) TsOH, THF/H2O 1:1, 8% for 17, 7.5% for 18 (three steps).
butyldimethylsilyl)-N-methyltrifluoroacetamide as the silylating
agent. Aldehyde 13 was then allowed to react with methyl-
triphenylphosphonium bromide in the presence of LiHMDS at
-78 °C to afford the fully protected form of 14 in 39% yield.
The latter was sequentially deprotected using tetrabutylammo-
nium fluoride followed by standard acidic conditions.
with cyclophilin A (see X-ray crystal structure section). Ma-
crolide 15 was also used to modify the free R-nitrogen of
piperazic acid and probe its role in mediating the binding to
cyclophilin. Methylation of macrolide 15 using trifluoromethane-
sulfonic acid methyl ester in the presence of a hindered base,
2,6-di-tert-butylpyridine, afforded a mixture of di- and mono-
methylated products, which, upon deprotection, gave 17 and
18, respectively. Interestingly, while all sanglifehrin derivatives
in DMSO solution contain approximatively 20% of the hemiket-
The primary alcohol 15 was obtained in excellent yield by
reduction of aldehyde 13 with NaBH4. Complete deprotection
of 15 led to alcohol 16, which was used for cocrystallization
9
J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003 3853

A R T I C L E S
Sedrani et al.
Figure 3. Building blocks used for the preparation of a library of analogues of the sanglifehrin macrolide.
al form resulting from addition of the C15 hydroxyl to the
carbonyl in the side chain,13 the content of the hemiketal form
of 17 and 18 was increased to ∼50%.
center. The comparison between the final products resulting from
alkene 22 and diene 23 would allow us to assess the relative
importance of the C18-C21 diene. The possible role of the side
chain attached at C23 in stabilizing the 3-dimensional conforma-
tion of the macrolide could be tested using fragment 24. Finally,
four fragments were prepared as analogues of the polypropionate
portion extending from C13 to C18. Based on the results obtained
by degradation of macrolide 2, the hydroxy group at C17 was
not considered during the planning phase. For similar reasons,
the side chain attached at C14 was simplified to a methyl group.
The stereochemistry of the methyl group at C16 was maintained
in fragments 25-27, because this substituent, directed toward
the internal face of the macrolide (see discussion of the X-ray
crystal structure), was thought to play an important role in
stabilizing the three-dimensional structure of the macrolide.
Fragments 25-27 were synthesized in order to analyze the
influence of the stereochemistry at C14 and C15 on the affinity
of the macrolides for cyclophilin. Fragment 27 corresponds to
the original stereochemistry found in these positions for SFA.
Commercially available 6-heptenoic acid 28 was added to the
list in order to study the overall impact of the substituents of
the polypropionate fragment on cyclophilin binding.
The details of the synthesis of the tripeptide unit 19 are
described in Scheme 4.19,20 The selection of the three orthogonal
protecting groups (Tce, TBDMS, and Boc) was crucial to allow
the desired synthetic flexibility during the final stage of
macrolide assemblage. The synthesis of the tripeptide fragment
19 began with the preparation of benzyl-protected meta-tyrosine
in a chiral form. The alkylation of the bis(lactim) ether
(Scho¨llkopf auxiliary) 29 with 3-benzyloxybenzyl bromide
provided intermediate 30 with 89% diastereoselectivity.21 The
undesired isomer was readily separated by flash chromatogra-
Design and Construction of a Library of Macrolide
Analogues. Further manipulation of the functional groups
present in macrolide 2 was difficult. Therefore, we switched to
a fully synthetic approach to prepare a library of simplified
macrolides.19 For our first generation of analogues, we planned
to keep the tripeptide fragment, which forms all the hydrogen
bonds with cyclophilin, mostly intact. In addition, at least one
double bond was maintained in the C13-C23 subunit to reduce
the conformational flexibility of the 22-membered macrocycle.
With this basic scaffold in mind, a retrosynthetic analysis
showed that several routes can be considered for the construction
of the macrocycles: macrolactonization, macrolactamization,
Stille coupling, and ring-closing metathesis (RCM). The latter
approach was particularly attractive because the macrolides can
be rapidly assembled from relatively simple building blocks in
a highly convergent manner.
For the selection of the building blocks, the macrolide was
divided into three fragments: the tripeptide, the polypropionate
fragment C13-C18, and the carbon skeleton extending from C19
to C26. An overview of the 10 selected building blocks is shown
in Figure 3.
Three different protected tripeptide fragments were prepared.
Tripeptide 19 corresponds to the original motive found in the
sanglifehrins. In the two other tripeptides, 20 and 21, the unusual
amino acids meta-tyrosine and piperazic acid were replaced by
phenylalanine and/or nipecotic acid, respectively. These modi-
fications were introduced to investigate the influence of the
phenolic hydroxyl and of the R-nitrogen of piperazic acid on
cyclophilin binding. Three building blocks were considered for
the C19-C26 fragment. In two of them, 22 and 23, the side chain
attached at C23 was neglected, thus eliminating one asymmetric
(20) The syntheses of the tripeptides 20 and 21 were described previously:
Wagner, J.; Martin Cabrejas, L. M.; Grossmith, C. E.; Papageorgiou, C.;
Senia, F.; Wagner, D.; France, J.; Nolan, S. P. J. Org. Chem. 2000, 65,
9255-9260.
(21) Scho¨llkopf, U.; Groth, U.; Deng, C. Angew. Chem., Int. Ed. Engl. 1981,
20, 798-799.
(19) For a preliminary communication on this appraoch, see: Martin Cabrejas,
L. M.; Rohrbach, S.; Wagner, D.; Kallen, J.; Zenke, G.; Wagner, J. Angew.
Chem., Int. Ed. 1999, 38, 2443-2446.
9
3854 J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003

Sanglifehrin−Cyclophilin Interaction
A R T I C L E S
Scheme 4. Synthesis of Building Block 19a
Scheme 5. Synthesis of Building Block 24a
a Reagents and conditions: (a) TMSI, HMDS, pentane, -20 °C to rt,
48%; (b) AD-mix-â, t-BuOH/H2O 1:1, 0 °C to rt, 89% ee, 56%; (c) (i)
TBDMSCl, imidazole, DMF, 0 °C to rt, (ii) NaH, triethylphosphonoacetate,
toluene, rt to 60 °C, 59% (two steps); (d) (i) DIBAL-H, CH2Cl2, 0 °C to rt,
(ii) NaH, MeI, THF, 0 °C to rt, (iii) TBAF, THF, 0 °C to rt, 69% (three
steps).
a Reagents and conditions: (a) n-BuLi, 3-benzyloxybenzylbromide, THF,
-78 °C, 62%; (b) (i) 0.5 M HCl, CH3CN, (ii) L-Boc-Val-OH, NMM,
isobutylchloroformate, THF, -15 °C to rt, 50% (two steps); (c) (i) LiOH,
THF/H2O 3:1, (ii) H2N-Pip-OCH2CCl3, NMM, isobutylchloroformate,
THF, -15 °C to rt, 62% (two steps); (d) (i) Pd/C 10%, H2, i-PrOH, (ii)
TBDMSCl, imidazole, DMF, 40 °C, 95% (two steps).
building block 24 in 69% yield over three steps. This synthetic
route allowed straightforward access to 24 on a multigram scale.
Finally, we focused our attention on the synthesis of building
blocks 25-27 for the polypropionate fragment C13-C18. The
preparation of all three fragments was based on the same chiral
aldehyde, (S,E)-2-methyl-hex-4-enal 37, which was available
in multigram quantities.26 The corresponding (R)-enantiomer of
37 is a crucial intermediate for the synthesis of a unique amino
acid, MeBmt, which is part of the structure of cyclosporin A.27
The additional methyl group attached to the double bond would
be lost during the final ring-closing metathesis step. Building
blocks 25 and 26 with the syn stereochemistry between C15 and
C16 were prepared using Evans’ aldol methodology (Scheme
6).28 The reaction between the (R)-imide 38 and aldehyde 37
in the presence of freshly prepared dibutylboron triflate29 at -78
°C gave the desired aldol adduct 39 as a single diastereoisomer
in excellent yield (92%). On the other hand, the reaction of the
(S)-imide 40 with 37 resulted in a sluggish reaction, which
afforded 41 in 43% yield. HPLC analysis showed that 41
contained approximately 10-20% of the C3 epimer.30 The slow
reaction is probably caused by the mismatch situation between
the R-methyl group of the aldehyde and the benzyl of the imide.
The epimers could be separated by reverse phase HPLC, but
the bulk material was carried through and separated only at the
macrolide stage. The chiral auxiliary was removed under
standard conditions to afford the acids 25 and 26 in 76% and
64% yield, respectively. While the formation of syn aldol
adducts is well documented, only few efficient methodologies
allowing the formation of anti aldol products in high diastereo-
selectivity and yield have been published. In the case of
aldehydes possessing a chiral center in the R position, examples
are very scarce. The synthesis of fragment 27, possessing the
phy. The bis(lactim) ether 30 was cleaved under strongly acidic
conditions, and the resulting amine was coupled to Boc-protected
valine to afford the dipeptide 31 in 50% yield over two steps.
The latter was saponified and coupled to 2,2,2-trichloroethyl-
protected piperazic acid using the mixed anhydride method to
provide tripeptide 32. Optically pure piperazic acid was obtained
in multigram quantities by the procedure described by Hale et
al.22 The benzyl group of 32 was removed by hydrogenolysis
in 2-propanol and replaced by the TBDMS group to afford the
desired fragment 19. Use of a less hindered alcohol during the
debenzylation resulted in partial transesterification of the
trichloroethyl ester. Tripeptide 19 was thus prepared in seven
steps and 18% overall yield.
5-Hexen-1-ol 22 is commercially available, and 3,5-hexadien-
1-ol 23 was prepared according to published procedures.23 The
synthesis of optically pure 24 is described in Scheme 5.24
7-Octen-2-one 33 was prepared according to known procedures
on a multigram scale.25 Sharpless dihydroxylation of the
intermediate silylenolether 34 afforded the highly volatile
R-hydroxy ketone 35 in 27% yield from 33. The reaction
proceeded in 89% ee as determined by 19F NMR of the Mosher
ester derivative of 35. Protection of 35 with a tert-butyldi-
methylsilyl group followed by a Wittig-Horner reaction with
triethyl phosphonoacetate in the presence of sodium hydride
afforded the ester 36 in good yield. The ester was reduced with
DIBAL-H to the primary alcohol, which was methylated. Final
deprotection of the secondary alcohol afforded the desired
(22) Hale, K. J.; Cai, J.; Delisser, V.; Manaviazar, S.; Peak, S. A.; Bhatia, G.
S.; Collins, T. C.; Jogiya, N. Tetrahedron 1996, 52, 1047-1068.
(23) (a) Stevens, R. V.; Cherpeck, R. E.; Harrison, B. L.; Lai, J.; Lapalme, R.
J. Am. Chem. Soc. 1976, 98, 6317-6321. (b) Hoye, T. R.; Magee, A. S.;
Trumper, W. S. Synth. Commun. 1982, 12, 183-187.
(24) A very short racemic route based on the alkylation of commercially available
(E)-3-methyl-4-oxo-but-2-enoic acid ethyl ester with the required Grignard
reagent was evaluated first. But attempts to resolve the secondary alcohol
with chiral auxiliaries, for example, camphanic acid, or separation of the
diastereoisomers at the macrolide stage were unsuccessful.
(25) 7-Octen-2-one 33 was prepared by saponification and acidic decarboxylation
of the corresponding alkenylacetoacetate. See: (a) Huckin, S. N.; Weller,
L. J. Am. Chem. Soc. 1974, 96, 1082-1087. (b) Crombie, L.; Harper, S.
H. J. Chem. Soc. 1952, 869-875.
(26) Deyo, D. T.; Aebi, J. D.; Rich, D. H. Synthesis 1988, 8, 608-610.
(27) (a) Blaser, D.; Ko, S. Y.; Seebach, D. J. Org. Chem. 1991, 56, 6230-
6233. (b) Togni, A.; Pastor, S. D.; Rihs, G. HelV. Chim. Acta 1989, 72,
1471-1478. (c) Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1986, 108,
6757-6761.
(28) (a) Evans, D. A.; Dart, M. J.; Duffy, J. L.; Rieger, D. L. J. Am. Chem. Soc.
1995, 117, 9073-9074 and references therein. (b) For a review on the use
of oxazolidinones as chiral auxiliaries, see: Ager, D. J.; Prakash, I.; Schaad,
D. R. Aldrichimica Acta 1997, 30, 3-11.
(29) For the preparation of dibutylboron triflate, see: Evans, D. A.; Nelson, J.
V.; Vogel, E.; Taber, T. R. J. Am. Chem. Soc. 1981, 103, 3099-3111.
(30) Similar fragments in a deuterated form were prepared previously. See:
Hailes, H. C.; Handa, S.; Leadlay, P. F.; Lennon, I. C.; Ley, S. V.; Staunton,
J. Tetrahedron Lett. 1994, 35, 315-318.
9
J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003 3855

A R T I C L E S
Sedrani et al.
Scheme 6. Synthesis of Building Blocks 25 and 26a
Figure 4. X-ray crystal structure of the aldol product 44 possessing the
C14-C15 anti stereochemistry.
Scheme 8. Synthesis of a Library of Sanglifehrin Macrolide
Analogues via the Ring-Closing Metathesis Reactiona
a Reagents and conditions: (a) Et3N, Bu2BOTf, (S,E)-2-methyl-hex-4-
enal 37, CH2Cl2, -78 °C, 92% for 39, 43% for 41; (b) LiOH, H2O2, THF/
H2O 3:1, 0 °C to rt, 76% for 25, 64% for 26.
Scheme 7. Synthesis of Building Block 27a
a Reagents and conditions: (a) (i) TMSOTf, CH2Cl2, 0 °C or TFA,
CH2Cl2, 0 °C, (ii) NMM, isobutylchloroformate, building blocks 22-24,
THF, -10 °C to rt or NMM, EDC, HOBT; (b) (i) Zn, 1.0 M aqueous
NH4OAc, THF or LiOH, THF/H2O 5:1, (ii) PPh3, DEAD, building blocks
25-28, THF, (iii) TBDMSOTf, pyridine, CH2Cl2, 0 °C; (c) (i) (PCy3)2Cl2Rud
CHPh, CH2Cl2, (ii) TBAF, THF. Symbols: X ) C or N; P ) -CH3 or
-CH2CCl3; For R1 to R5, see Table 1.
a Reagents and conditions: (a) (i) Et3N, TBDMSOTf, CH2Cl2, (ii) (S,E)-
2-methyl-hex-4-enal 37, TiCl4, CH2Cl2, -78 °C, 91%; (b) LiOH, THF/
H2O 3:1, 60 °C, 53%.
lated chiral aldehyde. Cleavage of the chiral auxiliary under
standard basic conditions afforded the acid 27 in 53% yield.
With all the building blocks in our hands, we started to
assemble the macrolide library according to Scheme 8. Removal
of the Boc group from tripeptides 19-21 in the presence of
TFA or TMSOTf in CH2Cl2, followed by coupling of the
resulting amines to building blocks 25-28, afforded compounds
45a-j. Methyl ester hydrolysis or selective deprotection of the
trichloroethyl ester group with zinc provided the corresponding
acids, which were esterified with the alcohols 22-24 under
Mitsunobu conditions. The precursors 46a-j for the ring-closing
metathesis reaction were thus obtained in good overall yield.
At this stage, the precursors with a free hydroxy group at C15,
46e-i, were protected with a tert-butyldimethylsilyl group. In
the presence of this protecting group, the subsequent metathesis
reaction was faster and proceeded in higher yields. The
ruthenium-based catalyst first described by Grubbs et al. was
used for the critical macrocyclization step.33 The best yields
were obtained in CH2Cl2 under reflux and high dilution (<5
mM). Under these conditions, the alkene or diene macrolides
47a-j were obtained in good to excellent yields (50-80%) after
same stereochemistry as sanglifehrin, required an anti aldol
reaction. First, we tested the Heathcock modification of Evans’
aldol reaction.31 Unfortunately, complexation of the additional
Lewis acid, Et2AlCl or TiCl4, to aldehyde 37 caused racem-
ization. We then turned our attention to Oppolzer’s sultam as
the chiral auxiliary.32 The condensation of aldehyde 37 with
the intermediate silyl enolate 43 obtained from sultam 42 formed
the desired anti aldol product with excellent diastereoselective
control (Scheme 7). Indeed, the anti aldol adduct 44 was isolated
as a single diastereoisomer and in excellent yield (91%) after
flash chromatography. The relative stereochemistry was con-
firmed by X-ray crystallography (Figure 4). To the best of our
knowledge, this is the first example of the formation an anti
aldol adduct obtained by reacting sultam 42 with an R-methy-
(31) Walker, M. A.; Heathcock, C. H. J. Org. Chem. 1991, 56, 5747-5750.
The Heathcock procedure was used recently to synthesize a very similar
fragment contained in callipeltin A. The anti aldol product was obtained
in 27% yield. See: Guerlavais, V.; Carroll, P. J.; Joullie´, M. M.
Tetrahedron: Asymmetry 2002, 13, 675-680.
(32) (a) Oppolzer, W.; Starkemann, Ch.; Rodriguez, I.; Bernardinelli, G.
Tetrahedron Lett. 1991, 32, 61-64. (b) Oppolzer, W.; Rodriguez, I. HelV.
Chim. Acta 1993, 76, 1275-1281.
(33) Schwab, P.; France, M. B.; Ziller, J. W.; Grubbs, R. H. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 2039-2041.
9
3856 J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003

Sanglifehrin−Cyclophilin Interaction
A R T I C L E S
Table 1. Macrocyclic Analogues and Yields of the Ruthenium-Catalyzed Ring-Closing Metathesis Reaction
HPLC purification (Table 1). The additional methyl group
attached to the alkene (entries 5-9) did not affect the yield of
the RCM reaction. Macrolide 47g, the C15 epimer of 47f, could
be separated as a side product (<5%) by reverse-phase HPLC
from 47f after macrocyclization and desilylation. This product
arises from the presence of a minor isomer present in the
macrocyclization substrate 46f. This isomer has its origin in
the Evans aldol reaction leading to acid 26, as indicated
previously.
His126) make intermolecular hydrogen bonds through their side
chains, while one amino acid (Asn102) forms an antiparallel
â-sheet interaction with the ligand. In detail, these hydrogen
bonds are located between NE2-His126 and the meta-tyrosine
hydroxy group (2.69 Å/2.65 Å), between the main chain nitrogen
of Asn102 and the main chain carbonyl oxygen of meta-tyrosine
(2.97 Å/2.92 Å), between the main chain carbonyl oxygen of
Asn102 and the main chain nitrogen of meta-tyrosine (2.75
Å/2.83 Å), between NE2-Gln63 and the main chain carbonyl
oxygen of valine (3.04 Å/3.01 Å), between OE1-Gln63 and the
R-nitrogen of the piperazic acid (3.03 Å/3.00 Å), and between
NH2-Arg55 and the carbonyl oxygen of the ester moiety (3.31
Å/3.20 Å). In the latter list, distances are given between
heteroatoms in the two independently refined complexes of the
asymmetric unit (the averaged values are displayed in Figure
5b). The piperazic acid moiety of macrolide 16 occupies the
same deep hydrophobic groove of cyclophilin A that is used
by the side chain of Me-Val11 of cyclosporin A1 or by the proline
ring of the model substrate Ala-Ala-Pro-Ala.34 The hydrophobic
pocket is formed by Phe60, Met61, Phe113, and Leu122. The 3-oxo-
butyl side chain attached at C14 extends on the surface of the
complex and does not form any obvious interaction with the
protein. The C15 hydroxy group points toward a pocket in the
cyclophilin structure occupied by water molecules. As men-
tioned earlier, the methyl group at C16 is directed toward the
interior surface of the macrolide and the C17 hydroxyl is exposed
to the solvent. Altogether, it appears that the spacer domain is
essential to induce the optimal 3-dimensional conformation of
the macrolide, which would allow a perfect fit of the recognition
domain in the catalytic site of cyclophilin A.
X-ray Crystal Structure of Macrolide 16 Bound to
Cyclophilin. The X-ray crystal structure of macrolide 16 bound
to cyclophilin was determined and allowed further insight into
the ligand-protein interactions. The resolution of the complex
was 2.1 Å, and the final R-factor, 16.4%. The crystal structure
revealed the details of the interaction between the sanglifehrin
macrolide and cyclophilin (Figure 5a and b). Similarly to
cyclosporin A, the macrocyclic subunit of sanglifehrin A binds
to the active site of cyclophilin A, which is an enzyme that
catalyzes the cis-trans isomerization of prolyl peptide bonds.1
Accordingly, both CsA and SFA inhibit the catalytic activity
of the enzyme.8 The tripeptide Pip-meta-Tyr-Val is located deep
in the binding pocket and forms all hydrogen bonds with the
protein. This portion of the ligand will be referred to as the
recognition domain. The C13-C26 fragment, that we would call
the spacer domain, extends on the surface of the complex and
does not undergo any direct interactions with cyclophilin. The
spacer domain might play a critical role in maintaining the
molecule in the optimal three-dimensional conformation.
Six direct hydrogen bonds (cutoff 3.4 Å) could be identified
between the recognition domain and four amino acids of the
protein. Three amino acids of the protein (Arg55, Gln63, and
(34) Kallen, J.; Walkinshaw, M. D. FEBS Lett. 1992, 300, 286-290.
9
J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003 3857

A R T I C L E S
Sedrani et al.
Figure 5. (a) Final 2Fobs - Fcalc electron density map covering macrolide 16 and the refined model. Direct hydrogen bonds (cutoff 3.4 Å) between 16 and
cyclophilin A are indicated as dashed lines. (b) Schematic view of the direct intermolecular hydrogen bonds between macrolide 16 and cyclophilin A in the
binding site. The distances are for the separation between heteroatoms, averaged over the two independently refined complexes in the asymmetric unit. The
representation of 16 was rotated in order to match the view in part a.
Biological Evaluation of Macrolides and Discussion. The
macrolides obtained by derivatization of 2 as well as the
synthetic analogues were screened for their affinity for cyclo-
philin A in a competitive binding assay. In this assay, the
interaction between solid-phase coated SFA-BSA conjugates
and biotinylated cyclophilin is inhibited by free SFA in
solution.35 The IC50 value for sanglifehrin A in this assay is
6.9 ( 0.9 nM.8 The results obtained with the new derivatives,
summarized in Table 2, reveal a number of important structure-
activity relationships. Macrolide 16 exhibits the same affinity
to cyclophilin as the parent compound, clearly indicating that
the binding of sanglifehrin A is fully mediated by the macro-
cyclic portion. Various modifications of the side chain attached
at C23 (derivatives 8, 9, 11, 12, and 14) have little effect on the
binding affinity. Sufficient space is available to accommodate
side chains of different lengths and containing ester or hydroxy
functionalities. This is in agreement with the fact that removal
of the spirolactam moiety of SFA results only in the loss of
immunosuppressive activity but not in affinity for cyclophilin
A. In contrast, full hydrogenation of all double bonds to form
the hexahydro derivative 5 results in a 7- or 35-fold loss in
affinity in comparison to 2 and 16, respectively. Thus, the diene
system is important for fine-tuning the three-dimensional
conformation of the macrocycle and confirms our initial working
hypothesis. The C54 carbonyl group, which is in equilibrium
with its intramolecular hemiketal form in solution, is not
important for the binding activity. Indeed, reduction to the
alcohol, as in macrolide 6, does not affect the affinity.
Nevertheless, the reduced affinity of 4 over 2 suggests that
(35) For details of the ELISA binding assay, see ref 8.
9
3858 J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003

Sanglifehrin−Cyclophilin Interaction
A R T I C L E S
Table 2. Cyclophilin Binding Data for Selected Analogues of the
so far. Finally, the addition of a C23 side chain and the meta-
tyrosine hydroxyl to 47b, leading to 47j, did not significantly
improve the affinity.
Sanglifehrin Macrocyclea
compd
cyclophilic bindinga
compd
cyclophilin bindinga
2
4
5
6
7
8
9
11
12
14
16
29 ( 2.1
86 ( 28
200 ( 40
12 ( 0.8
49 ( 15
16 ( 1.8
11 ( 2.0
21 ( 1.7
8.9 ( 2.9
6.5 ( 1.0
5.7 ( 1.0
17
18
>5000
Conclusions
>5000
47a
47b
47c
47d
47e
47f
47h
47i
47j
>5000
The interaction between the macrocyclic moiety of sanglifeh-
rin A and cyclophilin was studied in detail. The X-ray crystal
structure of the cyclophilin A-macrolide 16 complex was
resolved at 2.1 Å and provided detailed insight into the ligand-
protein interactions. In addition, a series of macrolide analogues
was prepared either by derivatization of macrolide 2, resulting
from selective degradation of sanglifehrin A, or via a synthetic
approach using the ring-closing metathesis reaction as the key
step. The latter work confirmed the potential of the RCM
reaction for rapidly assembling a library of macrolides of
variable complexity. Altogether, the X-ray crystal structure and
the synthetic work proved that the high affinity of sanglifehrin
A for cyclophilin A is mediated exclusively by the macrocyclic
portion of the molecule. More surprising was the finding that
the complex macrocyclic structure is a highly optimized
combination of multiple functionalities including a rather
unusual tripeptide, an (E,E)-diene unit, and a polypropionate
portion, which together provide an extremely strong affinity for
cyclophilin. The “original” sanglifehrin A macrocycle 2 allows
for some, mostly single or double, modifications which do not
greatly affect binding. On the other hand, simultaneous modi-
fications of several positions, including removal of substituents,
configurational changes, and functional group modifications, as
in the synthetic macrolides 47a-j, result in a significant loss
of affinity for cyclophilin.
10 270 ( 405
7130 ( 640
5870 ( 320
9370 ( 950
12 930 ( 970
6430 ( 710
3830 ( 740
5100 ( 530
a The cyclophilin binding data were measured as described elsewhere.8
IC50 values [nM] represent mean ( SD of 3-4 independent experiments.
The IC50 value for SFA is 6.9 ( 0.9 nM.
oxygen is preferred over iodine in this position. Finally, the
good affinity retained by triene 7 shows that the C17 hydroxyl
is not critical for binding. This conclusion is supported by
analysis of the X-ray crystal structure, which shows that this
hydroxyl is exposed to the solvent. Furthermore, it appears that
the overall conformation of the macrocycle is not greatly
affected by the additional double bond. Derivatives 17 and 18,
in which the free R-nitrogen of piperazic acid is methylated,
are inactive. This observation confirms that the hydrogen bond
formed between the free R-nitrogen of piperazic acid and OE1-
Gln63 is crucial for binding to cyclophilin. It is interesting to
note that this interaction is mediated by a structural feature,
which is unique to the sanglifehrins. Therefore, it is not
surprising that the replacement of piperazic acid by nipecotic
acid in compound 47a also leads to a substance that does not
bind to cyclophilin.
All the synthetic macrolide analogues, 47b-47j, retain only
modest affinity in the micromolar range. Among those, 47d
combines the complete recognition domain with the (E,E)-diene
unit. The binding data of 47d prove that these two fragments
of the macrolide are certainly necessary but not sufficient to
achieve high affinity for cyclophilin as hypothesized initially.
The role of the three stereocenters at C14, C15, and C16 of the
polypropionate fragment was assessed with compounds 47e-
47i. To our surprise, the stereochemistry at C14 and C15 had
only a minor impact on the binding to cyclophilin. Compound
47i, which combines the absolute configurations of the C14-
C16 stereocenters found in sanglifehrin A with the (E,E)-diene
moiety, remained the most potent synthetic derivative prepared
Acknowledgment. We thank Beatrix Wagner for her help
with the X-ray crystal structure, Serge Fuchs of his technical
assistance, Francis Roll for the HRMS measurements, and
Fre´de´ric Zecri for his contribution to the paper.
Supporting Information Available: Details of crystallization
conditions and structure resolution. Complete experimental
procedures and spectral data for all compounds generated in
the course of the investigation reported here, including copies
1
of the H NMR spectra of 3-9, 11-12, 14-19, 24-27, 30-
32, 34-36, 39, 41, 44, 45d-f, h, 46d-f, h-j, and 47d-j. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA021327Y
9
J. AM. CHEM. SOC. VOL. 125, NO. 13, 2003 3859