Macrolide analogues of the novel immunosuppressant sanglifehrin: New application of the ring-closing metathesis reaction

Article abstract of DOI:10.1002/(SICI)1521-3773(19990816)38:16<2443::AID-ANIE2443>3.0.CO;2-B

Macrocycles containing a conjugated 1,3-diene moiety have been synthesized for the first time in good yields by the ring-closing metathesis reaction [Eq. (1)]. The new compounds represent cyclophilin-binding, simplified analogues of the macrocyclic core of sanglifehrin A, an immunosuppressant which binds with high affinity to cyclophilin.

Full text of DOI:10.1002/(SICI)1521-3773(19990816)38:16<2443::AID-ANIE2443>3.0.CO;2-B

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Ziegler Catalysts (Eds.: G. Fink, R. Mülhaupt, H. H. Brintzinger),
Springer, Berlin, 1995, pp. 111 ± 147.
Macrolide Analogues of the Novel
Immunosuppressant Sanglifehrin: New
Application of the Ring-Closing Metathesis
Reaction**
[9] a) P. Jutzi, A. Seufert, J. Organometal. Chem. 1979, 169, 327; b) P. Jutzi,
A. Seufert, J. Organometal. Chem. 1979, 169, 373; c) M. Bernheim, G.
Boche, Angew. Chem. 1980, 92, 1043, Angew. Chem. Int. Ed. Engl.
1980, 19, 1010; d) C. M. L. Atkinson, R. Dietz, Macromol. Chem.
1976, 177, 213.
Luisa M. Martin Cabrejas, Stefan Rohrbach,
Dieter Wagner, Jörg Kallen, Gerhard Zenke, and
Jürgen Wagner*
[10] Crystal structure analysis of 1: Bruker-Smart-CCD area detector
system, MoKa irradiation, graphite monochromator, Rigaku rotating-
anode M18HF (operating at 50 kV, 100 mA). The reflections were
recorded at 153 K on a shock-cooled crystal with the dimensions 0.4 Â
0.3 Â 0.3 mm3 under an inert oil (RS3000; Riedel-deHaen) up to
2qmax ˆ 618. The data were processed with the programs SAINT (data
reduction, SAINT-NT V5.0) and SADABS (absorption correction).
The structure solution and refinement were carried out with
SHELXTL (NT-Version V5.1). C12H16B1Cl4P1Zr1, Mr ˆ 433.03, mono-
clinic, space group P21/c, a ˆ 14.6429(5), b ˆ 8.3296(3), c ˆ
More than two decades ago, the discovery of cyclosporin A
(CsA) 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 been intensified.[2] Besides its im-
portant therapeutic use, CsA has also proven to be a powerful
tool for dissecting signal transduction pathways at the
molecular level.[3] It has been shown that the biological
activity of CsA is mediated by an intracellular binding protein
called cyclophilin (CyP). However, although CyP binding is
required, it is not sufficient for the immunosuppressive
activity of this drug. Full biological activity is obtained only
once the CyP ± CsA complex binds to and inhibits the serine/
threonine phosphatase activity of calcineurin, thereby block-
ing the production of cytokines including interleukin-2.[4]
We wondered whether other ligands for cyclophilin might
exist which would interfere with signaling pathways not
involving calcineurin. The screening of microbial broth
extracts for CyP-binding substances led to the isolation from
Streptomyces flaveolus of a new class of compounds named
sanglifehrins.[5a] Among the 20 different sanglifehrins isolated
so far from this strain, sanglifehrin A (SFA, Figure 1) is the
most abundant component. The affinity of SFA for cyclophilin
is remarkably high (IC50 ˆ 2 ± 4nm),[6] approximately 20-fold
higher than that of CsA (Ki ˆ 82nm). Sanglifehrin A displays
potent immunosuppressive activity in the mixed lymphocyte
reaction (IC50 ˆ 170nm), an in vitro immune response assay.[5a]
However, SFA does not affect T-cell receptor-mediated
cytokine production, indicating a mode of action different
from that of CsA. Moreover, in contrast to the T-cell-selective
drug CsA, SFA inhibits mitogen-induced B-cell proliferation
(IC50 ˆ 90nm). These data clearly indicate that the immuno-
suppressant SFA acts by a new mode of action. However, the
details of the mechanism by which this compound exerts its
immunosuppressive activity at the molecular level are un-
known.
13.3791(5) Š,
b ˆ 103.6560(10)8,
Vˆ 1585.71(10) Š3,
1calcd
ˆ
3
1
1.814 Mgm
,
m ˆ 1.449 mm
,
17987 measured reflections, 4834
independent (Rint ˆ 0.0264), and 4245 observed (Fo > 4s(Fo)). The
hydrogen atoms were refined with isotropic temperature factors, all
other atoms with anisotropic ones. R1 ˆ 0.0171, wR2 ˆ 0.0436 (all
data), GOF ˆ 1.020 for 228 parameters. Max./min. residual electron
density 0.436/ 0.305 eŠ3. Crystallographic data (excluding structure
factors) for the structures reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC- CCDC-118714. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union Road,
Cambridge CB21EZ, UK (fax: (‡44)1223-336-033; e-mail: deposit@
ccdc.cam.ac.uk).
[11] a) K. A. Ostoja Starzewski, H. tom Dieck, Phosphorus 1976, 6, 177;
b) K. A. Ostoja Starzewski, J. Witte, Angew. Chem. 1985, 97, 610;
Angew. Chem. Int. Ed. Engl. 1985, 24, 599; c) K. A. Ostoja Starzewski,
J. Witte, Angew. Chem. 1987, 99, 76; Angew. Chem. Int. Ed. Engl. 1987,
26, 63; d) K. A. Ostoja Starzewski, J. Witte in Transition Metal
Catalyzed PolymerizationsÐZiegler Natta and Metathesis Polymer-
ization (Ed.: R. P. Quirk), Cambridge University Press, Cambridge,
1988; e) K. A. Ostoja Starzewski, J. Witte, K. H. Reichert, G. Vasiliou
in Transition Metals and Organometallics as Catalysts for Olefin
Polymerization (Eds.: W. Kaminsky, H. Sinn), Springer, Berlin, 1988;
f) W. C. Kaska, K. A. Ostoja Starzewski in Ylides and Imines of
Phosphorus (Ed.: A. W. Johnson), Wiley, New York, 1993; g) K. A.
Ostoja Starzewski in Ziegler Catalysts (Eds.: G. Fink, R. Mülhaupt,
H. H. Brintzinger), Springer, Berlin, 1995, pp. 497 ± 505, and refer-
ences therein.
[12] a) K. A. Ostoja Starzewski, W. M. Kelly, A. Stumpf (Bayer AG),
WO-A 98/01455 (patent registered July 2, 1997) [Chem. Abstr. 1998,
128, 115367]; b) K. A. Ostoja Starzewski, W. M. Kelly, A. Stumpf
(Bayer AG), WO-A 98/01487 (patent registered July 2, 1997) [Chem.
Abstr. 1998, 128, 141901]; c) K. A. Ostoja Starzewski, W. M. Kelly, A.
Stumpf (Bayer AG), PCT WO 98/01484 (patent registered July 2,
1997) [Chem. Abstr. 1998, 128, 141177]; d) K. A. Ostoja Starzewski,
W. M. Kelly, A. Stumpf, unpublished results.
[*] Dr. J. Wagner, Dr. L. M. Martin Cabrejas, S. Rohrbach, D. Wagner,
Dr. J. Kallen, Dr. G. Zenke
Novartis Pharma AG
S-350.2.07
CH-4002 Basel (Switzerland)
Fax : (‡ 41)61-324-45-13
E-mail: juergen.wagner@pharma.novartis.com
[**] We thank Richard Sedrani for reading the manuscript, Julien France
for performing the NMR measurements, and Serge Fuchs for
performing the CyP-binding experiments. We are very grateful to
the Novartis Kilolab Facility for the supply of multigram quantities of
3.
Supporting information for this article is available on the WWW
under http://www.wiley-vch.de/home/angewandte/ or from the author.
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Figure 1. The structure of sanglifehrin A (SFA) and of the designed macrolides 1 and 2 (atoms forming hydrogen bonds to cyclophilin are shown in red). The
possible macrocyclization strategies are also included.
Herein we describe the design and the syntheses of the first
synthetic analogues of the SFA macrolide which bind to
cyclophilin. The simplified macrocycles 1 and 2 (Figure 1)
were designed in order to study the relevance of the CyP ±
SFA interaction, which is important for a better understand-
ing of the mechanism. We also disclose the first example of the
construction of macrocyclic conjugated 1,3-dienes by the ring-
closing metathesis (RCM) reaction.
The structure of SFA has been determined by spectroscopic
analysis, and its stereochemistry has been confirmed by X-ray
analysis of the Cyp ± SFA complex.[5b, c] The sanglifehrins have
a unique structure: A novel, highly substituted spirolactam
system is linked to a 22-membered macrolide through a rigid,
partially unsaturated alkyl chain. The macrolide contains a
tripeptide unit composed of piperazic acid (according to
IUPAC the acid should be called hexahydropyridazine-3-
carboxylic acid), meta-tyrosine, and valine. (For a synthesis of
the macrolide, see reference [18].) Interestingly, the peptidic
backbone extends through the b-nitrogen atom of piperazic
acid rather than its a-nitrogen atom. This feature, unique to
the sanglifehrins, is a key element for optimal binding to
cyclophilin.
role in locking the macrolide into the optimal conformation
for binding to cyclophilin.
Having established the part of the macrolide which is
important for binding, we chose, for our first-generation
macrolide analogues an intact recognition domain, except
that meta-tyrosine was replaced by phenylalanine in 1. The
difference in binding between 1 and 2 should reflect the
strength of the phenol-mediated hydrogen bond. Several
functionalities of the spacer region were removed, but the
conjugated (E,E)-diene was conserved in order to provide the
required conformational stability.
A retrosynthetic analysis showed that several routes can be
considered for the construction of the macrocycles: macro-
lactonization, macrolactamization, Stille coupling, and RCM
(Figure 1). The development of a new methodology based on
the RCM reaction promised to be particularly attractive,
because 1) the macrolides can be rapidly accessed in a
convergent manner, 2) the strategy has potential for the
creation of analogue libraries, and 3) the formation of simple
macrocyclic olefins by RCM has already proven to be one of
the most powerful techniques in organic synthesis.[8]
The syntheses of the macrocycles 1 and 2 are shown in
Scheme 1. At first, two different tripeptide fragments had to
be assembled. The synthesis of tripeptide fragment 5 began
with the optically pure piperazic acid derivative 3, which was
obtained according to Hale et al. in multigram quantities.[9]
On the other hand, the first step in the construction of
tripeptide 11 involved the synthesis of benzyl-protected meta-
tyrosine in a chiral form. The alkylation of the bis-lactim ether
(Schöllkopf auxiliary) 8 with 3-benzyloxybenzyl bromide
provided intermediate 9 with 89% diastereoselectivity.[10]
The undesired isomer was readily separated by chromatog-
raphy. With the two unnatural amino acids 3 and 9 in hand, the
tripeptides 5 and 11 were assembled by using carbodiimide-
based coupling procedures or activation of the acids by the
mixed anhydride methodology. It is noteworthy that the
coupling of 3 occurred selectively at the less hindered b-
nitrogen atom.[11] At that stage, the correct choice of
orthogonal protecting groups was critical for the further
transformation of 11. Therefore, the benzyl group was
removed by hydrogenolysis in isopropyl alcohol and replaced
by the TBDMS group to afford 12. Use of a less hindered
Degradation studies on SFA and the X-ray crystallographic
structure of SFA bound to cyclophilin provided the basis for
the design of compounds 1 and 2. Cleavage of SFA at the
C26 C27 double bond by a two-step procedure (Sharpless
dihydroxylation, periodate oxidation) allowed selective re-
moval of the complex spirolactam.[7] The resulting macrolide
and SFA have comparable affinities for cyclophilin. There-
fore, the whole side chain attached to the macrolide at C23 was
not incorporated into the target compounds. As observed
from the degradation studies, the X-ray crystal structure
showed that the macrocycle alone is responsible for the
binding to cyclophilin.
A detailed analysis of this structure suggested that the
macrolide can be viewed as the combination of a recognition
domain and a spacer domain (Figure 1). The recognition
domain corresponds to the tripeptide, where all the functional
groups forming hydrogen bonds with cyclophilin are located
(shown in red). The spacer domain, which extends from C14 to
C23, combines a short polypropionate chain (C14 ± C17) and a
conjugated (E,E)-diene. These two features might play a key
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Scheme 1. a) Trifluoroacetic acid, CH2Cl2 (100%); b) l-Boc-Phe-OH, EDC, HOBT, NMM, CH2Cl2 (66%); c) l-Boc-Val-OH, EDC, HOBT, NMM, CH2Cl2
(71%); d) BuLi, THF, 788C, 3-benzyloxybenzyl bromide (69%); e) HCl 0.5n, CH3CN (70%); f) l-Boc-Val-OH, NMM, isobutyl chloroformate
(ClC(O)OiBu), THF (70%); g) LiOH, H2O/THF 1:4 (100%); h) trichloroethyl piperazate, NMM, isobutyl chloroformate, THF (62%); i) H2, Pd/C, iPrOH
(96%); j) TBDMSCl, imidazole, DMF (99%); k) TMSOTf, CH2Cl2 (97%); l) 6-heptenoic acid, EDC, HOBT, NMM, CH2Cl2 (95%); m) 6-heptenoic acid,
NMM, isobutyl chloroformate, THF (70%); n) Zn, NH4OAc 1.0n, THF (90%); o) DEAD, PPh3, 3,5-hexadiene-1-ol, THF (64% (7); 82% (14)); p) [(PCy3)2
ˆ
-Cl2-Ru CHPh], CH2Cl2, 408C (57% (1); 47% (15)); q) Bu4NF, THF (75%). Bn ˆ benzyl, Boc ˆ tert-butoxycarbonyl, DEAD ˆ diethylazodicarboxylate,
EDC ˆ N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide, HOBt ˆ 1-hydroxy-1H-benzotriazole, NMM ˆ N-methylmorpholine, OTf ˆ trifluoromethane-
sulfonate, TBDMS ˆ tert-butyldimethylsilyl, Tce ˆ trichloroethyl, TMS ˆ trimethylsilyl.
alcohol during cleavage of the benzylic bond resulted in
partial transesterification of the trichloroethyl ester. Never-
theless, the Tce group was selected, because it could be
removed under very mild conditions even in the presence of
the TBDMS protecting group.[12]
Deprotection of the Boc group with TMSOTf in CH2Cl2
followed by coupling of the resulting amines to 6-heptenoic
acid afforded 6 and 13. Methyl ester hydrolysis of 6 or
selective deprotection of the trichloroethyl ester group of 13
with zinc provided the corresponding acids, which were
esterified with 3,5-hexadiene-1-ol under Mitsunobu condi-
tions.[13] The precursors 7 and 14 for the RCM reaction were
thus obtained in good overall yield (28% from 3 and 11%
from 8). The diene moiety had to be introduced at this late
stage of the synthesis, because partial isomerization of the
double bonds occurred under the acidic Boc-cleavage con-
ditions.
The ruthenium-based catalyst first described by Grubbs
et al.[8a] was used for the critical macrocyclization step. The
best yields of the macrocycles were obtained in CH2Cl2 under
reflux and at dilute concentrations (<5mm). Under these
conditions, the desired products 1 and 15 were obtained
selectively in 57% and 47% yield, respectively, after purifi-
cation by HPLC. At higher concentrations, dimerization
became an important side reaction, and in other solvents
(benzene, toluene) the yields were lower. The configuration of
the newly formed conjugated diene was assessed by 1H NMR
spectroscopy to be E,E in both cases.[14] The (E,Z)-diene was
present as a minor component (<5%), but we could not
detect any product resulting from metathesis of the internal
diene double bond. The macrocyclization also proceeded in
the presence of the free phenol, but at a slower rate. Removal
of the silyl protecting group from 15 provided the final
product 2. The affinity of the macrolides 1 and 2 for
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cyclophilin was assessed in a competitive ELISA format.[6]
They were found to bind with IC50 values of 7.3 and 5.7mm,
respectively. Several uncyclized tripeptide intermediates did
not show any activity when tested in the same assay.
H. Schneider, N. Charara, R. Schmitz, S. Wehrli, V. Mikol, M. G. M.
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Sedrani (Sandoz Ltd), WO-A 9702285 A1, 1997.
The synthesis of 1 and 2 is based on the RCM reaction,
which has already proven to be one of the most powerful
techniques for the construction of macrocyclic natural prod-
ucts in solution[15] or on solid support,[16] but whose use so far
has been limited to the formation of simple olefins. However,
various macrocyclic, biologically relevant, natural products
embody conjugated 1,3-dienes.[17] For the first time, macro-
cycles containing a cyclic 1,3-diene moiety have been synthe-
sized in good yields by a metathesis reaction, as exemplified
by 1 and 2. As this methodology is extendable to the creation
of analogue libraries, it should be of interest for organic
synthesis in general and natural product synthesis and
medicinal chemistry in particular.
Compounds 1 and 2 are the first synthetic analogues of the
macrocyclic core of sanglifehrin A which bind to cyclophilin.
Indeed, all the open-chain tripeptides tested so far are
inactive in the CyP-binding assay. The reduced affinity of 1
and 2 for cyclophilin compared to sanglifehrin A is a clear
indication that the conformation of these first-generation
macrolides is not yet optimal. In other words, the results
suggest that the conjugated diene is not sufficient to lock the
macrocycle into the ideal three-dimensional conformation.
Therefore, future work will include the preparation of a
library of macrolides based on the chemistry developed herein
and taking into account other functional features of the
sanglifehrins.
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1H NMR spectroscopy (DMSO, 400 MHz) on the basis of the coupling
constants: JH18,H19 ˆ 15.2 and 14.6 Hz, JH20,H21 ˆ 15.0 and 14.4 Hz for 1
and 2, respectively.
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[17] Some representative examples of macrocyclic 1,3-dienes are the
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3935 ± 3948; milbemycins-avermectins: M. T. Crimmins, R. S. Al-
Awar, I. M. Vallin, W. G. Hollis, Jr., R. OꢁMahony, J. G. Lever,
D. M. Bankaitis-Davis, J. Am. Chem. Soc. 1996, 118, 7513 ± 7528;
aplyronines: H. Kigoshi, K. Suenaga, T. Mutou, T. Ishigaki, T. Atsumi,
H. Ishiwata, A. Sakakura, T. Ogawa, M. Ojika, K. Yamada, J. Org.
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Hoffmann, Tetrahedron Lett. 1997, 38, 1001 ± 1004.
Received: March 15, 1999 [Z13162IE]
German version: Angew. Chem. 1999, 111, 2595 ± 2599
Keywords: immunosuppressants
´
metathesis
´
natural
products
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[6] The affinity of the substrates for cyclophilin A was assessed in a
competitive binding assay. The procedure was identical to that
described in the following reference, except that CsA coupled to
bovine serum albumin (BSA) was replaced by SFA coupled to BSA:
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Angew. Chem. Int. Ed. 1999, 38, No. 16