Total synthesis of cyclosporine: Access to N-methylated peptides via lsonitrile coupling reactions

Article abstract of DOI:10.1021/ja100517v

Recent developments in the use of isonitriles to furnish secondary and tertiary amide bond formations have been applied to a novel total synthesis of the important cyclic polypeptide cyclosporine A. Specifically, the disclosed synthetic route demonstrates the utility of microwave-mediated carboxylic acid isonitrile couplings, thioacid isonitrile couplings at ambient temperature, and isonitrile-mediated couplings of carboxylic acids and thioacids with amines to form challenging amide bonds. Copyright

Full text of DOI:10.1021/ja100517v

Published on Web 03/03/2010
Total Synthesis of Cyclosporine: Access to N-Methylated Peptides via
Isonitrile Coupling Reactions
Xiangyang Wu,† Jennifer L. Stockdill,† Ping Wang,† and Samuel J. Danishefsky*,†,‡
Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York AVenue,
New York, New York 10065, and Department of Chemistry, Columbia UniVersity, HaVemeyer Hall, 3000 Broadway,
New York, New York 10027
Received January 20, 2010; E-mail: s-danishefsky@ski.mskcc.org
Scheme 1
Recently, we described a series of findings using isonitriles in
the formation of amide bonds.1-4 These early works suggested that
isonitrile-mediated bond constructions might be applicable to the
synthesis of tertiary amides. There is currently a great deal of
interest in such systems, particularly with respect to improving the
pharmacoavailability of biologically active polypeptides.5
We thought that our findings in this area were sufficiently
promising that it would be appropriate to explore various ways
to generate tertiary amides in the context of a total synthesis of
a challenging target system. In view of these considerations, it
was only natural to turn to cyclosporine A (1) as a worthy goal.
Cyclosporine A is a reversible inhibitor of cytokines in T helper
cells6 that was isolated from the fungus Tolypocladium inflatum
gams.7 Its structure was confirmed by chemical degradation,
NMR, and X-ray crystallographic studies,8 and its total synthesis
was reported by Wenger in 1984.9 The immunosuppressive
properties of cyclosporine A, which enable otherwise nonsus-
tainable transplantations, are very well established.10 In addition,
cyclosporine A has been applied to the treatment of Bec¸het’s
syndrome, endogenous uveitis, psoriasis, atopic dermatitis,
rheumatoid arthritis, active Crohn’s disease, and nephrotic
syndrome.10 Indeed, its bioavailability with respect to proteolysis
is highly dependent on the pattern of N-methylation present in
7 of the 11 amino acid residues of the cyclic polypeptide. As
established by Wenger, Rich, and Galpin through analogue
studies,11 the unusual amino acid (2S,3R,4R,6E)-3-hydroxy-4-
methyl-2-methylamino-6-octenoic acid (MeBmt) is of particular
importance to the biological activity of 1 as well as the sequence
MeBmt, Abu, Sar, MeLeu. Herein, we demonstrate the emerging
versatility and power of isonitrile chemistry in the context of a
total synthesis of cyclosporine A.
sensitive nature of the thioformyl amide. Ultimately, it was found
that the reduction proceeded well under free radical conditions.
Thus, treatment of the crude N-thioformyl amide with Bu3SnH and
azobis(isobutyronitrile) (AIBN) at 100 °C in toluene afforded the
desired N-methyl dipeptide 7 in 62% yield over two steps.
Following cleavage of the carbamate, the elegant retro-aza-
Diels-Alder methylation approach described by Greico et al.
provided access to the corresponding N-methylated derivative 8,
without epimerization, in 52% yield over three steps.16
Retrosynthetic planning began by disconnecting the macrocycle
at the Ala-(D-Ala) junction and on either side of the MeBmt residue,
thus revealing MeBmt acetonide 2, tetrapeptide 3, and hexapeptide
4 (Scheme 1). The protected MeBmt residue 2 is a known
compound that is obtainable via the route charted by Evans and
co-workers.12 Tetrapeptide 3 was to be assembled from its
component dipeptides, each of which could be reached via isonitrile-
mediated coupling methods. Hexapeptide 4 was seen as arising from
dipeptide and tetrapeptide fragments.
Scheme 2
We began by targeting dipeptide 8 en route to tetrapeptide 3.
Leucine-derived thioacid 513 was smoothly coupled with valine
isonitrile 614 to afford the corresponding N-thioformyl amide
(Scheme 2). Subsequent conversion to the N-methyl amide was
challenging. Treatment with Raney Ni as reported by Chupp and
co-workers15 was unsuccessful, presumably because of the base-
A similar strategy was applied for the second key dipeptide
fragment. Thus, D-alanine thioacid 913 underwent coupling with
leucine isonitrile 1014 in CHCl3 at ambient temperature, followed
by radical reduction, to afford the desired N-methylated dipeptide
† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
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4098 J. AM. CHEM. SOC. 2010, 132, 4098–4100
10.1021/ja100517v  2010 American Chemical Society

C O M M U N I C A T I O N S
Scheme 3
methyl-leucine residue was attached via a HATU coupling.
Acidic removal of the Boc protecting group afforded trifluoro-
acetic acid (TFA) salt 18, which bears the secondary amide bond
at the valine residue, in 85% yield over two steps.
We were now well-positioned to test additional isonitrile-
mediated amide bond formations.4 Thus, thioacid 19 was treated
with a “sacrificial isonitrile”4 to provide a putative formimidate
(thio)carboxylate mixed anhydride (thio-FCMA) that was inter-
cepted by amine 20, thereby producing dipeptide 21 in 80% yield
(Scheme 5). Hydrogenolysis of the benzyl ester was achieved
quantitatively, providing known dipeptide 22. We recently disclosed
a method by which carboxylic acids could be treated with
Lawesson’s reagent under microwave conditions or at room
temperature to afford the corresponding thioacids.20 With this
approach, 22 was converted to thioacid 23 (65% yield), which was
then coupled with secondary amine 18. Following cleavage of the
Boc group, hexapeptide 24 was obtained in 63% yield.
11 in 59% yield over two steps (Scheme 3). At this point, we
were poised to join the two dipeptide fragments. Hydrogenolysis
of the benzyl ester afforded the corresponding acid, and
subsequent coupling with fragment 8 was accomplished upon
treatment with DEPBT17 and Hu¨nig’s base in THF. Using these
optimized conditions,18 we were able to synthesize the desired
tetrapeptide 3 in 90% yield over the two steps with no observable
epimerization.
Scheme 5
With tetrapeptide fragment 3 in hand, we turned our attention
to the synthesis of hexapeptide 4. Microwave irradiation of azido
acid 1219 and leucine isonitrile 1314 afforded N-formyl amide
14 in 85% yield (Scheme 4). We reasoned that the presence of
the N-formyl group could be exploited to decrease the extent of
oxazolone formation, which might otherwise lead to epimeriza-
tion during the C-terminal extension with a suitably protected
alanine.1c,d Maintenance of the N-formyl group during acidic
removal of the tert-butyl ester and during coupling with alanine
benzyl ester was possible, thereby providing tripeptide 15 in
81% yield and >20:1 dr. The reduction of the N-formyl amide
was very challenging because of its propensity to undergo either
nonselective reduction, resulting in destruction of the peptide,
or deformylation, resulting in formation of the native amide bond.
Substantial optimization was required to identify conditions
under which the required transformation could be performed
chemoselectively. Ultimately, a combination of lithium boro-
hydride and 0.5 equiv of acetic anhydride in a mixture of CH2Cl2
and n-propanol at -50 °C was used to provide the relatively
unstable N-hydroxymethyl intermediate. Immediate reduction of
the crude product with triflic anhydride and triethylsilane in
CH2Cl2 afforded the desired N-methylated tripeptide 16 in 62%
yield over two steps. Selective reduction of the azide functional-
ity was readily accomplished by treatment of 16 with triph-
enylphosphine in the presence of water, forming 17. The final
We next began the final series of fragment couplings of the seco
system. The protected MeBmt residue, 2, was coupled to hexapep-
tide 24 using N,N′-dicyclohexylcarbodiimide (DCC) and hydroxy-
benzotriazole (HOBt). Subsequent treatment with aqueous HCl
provided heptapeptide 25 in 75% yield over two steps. At this point,
we sought to couple the final two fragments: tetrapeptide 3 and
heptapeptide 25. Hydrogenolysis of the C-terminus of 3 followed
by smooth coupling with fragment 25 afforded undecapeptide 26
in 52% yield (Scheme 6). Basic hydrolysis of 26 exposed the
C-terminus, and subsequent acidic treatment cleaved the tert-butoxy
carbamate from the N-terminus.
Scheme 4
The stage was now set for applying the logic of our recently
developed4 coupling method to a macrolactamization. This was no
minor extension of our earlier work because, in the case at hand,
we would be attempting to apply the logic to a C-terminal acid
rather than to a thioacid.4 In the event, exposure of the unprotected
undecapeptide 27 to 10 equiv of cyclohexyl isonitrile at 70 °C under
microwave radiation resulted in isolation of cyclosporine A (1) in
30% yield over three steps. We note that ordinarily with carboxylic
acids (as opposed to thioacids), amide bond formation by 1,3-OfN
acyl transfer does not occur below ∼130 °C under microwave
mediation. We had conjectured that the FCMA actually forms at
lower temperature but that the usual 1,3-OfN acyl transfer requires
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C O M M U N I C A T I O N S
Scheme 6
Dr. Cindy Kan, and Dr. Karthik Iyer for helpful discussions. We
thank Professor Gregory Verdine for suggesting cyclosporine A as
a methodology-challenging molecule.
Supporting Information Available: Experimental procedures,
copies of spectral data, and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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higher temperature for efficient rearrangement. In the case at hand,
the substrate was likely preorganized as a result of intrastrand
hydrogen bonding.21 Thus, the FCMA of the seco acid, once
formed, had an increased proclivity for cyclization, and macrolac-
tamization was achieved at 70 °C, with only a trace amount of
1,3-OfN acyl transfer visible by LC-MS of the crude reaction
mixture.
While this finding was encouraging, the yield was nonetheless
disappointing. Fortunately, the yield could be significantly increased
through the addition of HOBt (1.5 equiv) to the cyclization medium.
Actually, the reaction seemed very clean and appeared to proceed
with high conversion. However, the isolated yield of cyclosporine
A is at this time 54%. It is tempting to suppose, but is certainly
not proVen, that an initial FCMA formed from 27 and cyclohexy-
lisonitrile is intercepted with HOBt, thereby generating an active
HOBt ester. The latter could well be the actual intermediate for
macrolactamization, thereby attenuating the tendency for either 1,3-
OfN acyl transfer or FCMA hydrolysis. Importantly, no dimer-
ization was observed in either the HOBt or HOBt-free cyclization
reactions.
In summary, we have shown how the chemistry of isonitriles
can be applied to the construction of a variety of tertiary amides.
These findings made possible a total synthesis of cyclosporine
A in a fashion that allows for a more detailed mapping of its
SAR.1a Further applications of isonitrile logic in peptide, cyclic
peptide, and glycopeptide settings will be disclosed in due course.
(18) Standard coupling conditions, including HATU/DIPEA/DMF, PyBOP/
NMM/CH2Cl2, and DCC/HOBt/NMM/THF, led to low yields (40-45%)
and high levels of epimerization (1:1 to 2:1 ratios of diastereomers).
(19) Prepared according to the following procedure: Goddard-Borger, E. D.;
Stick, R. V. Org. Lett. 2007, 9, 3797–3800.
(20) Rao, Y.; Li, X.; Nagorny, P.; Hayashida, J.; Danishefsky, S. J. Tetrahedron
Lett. 2009, 50, 6684–6686.
(21) This preorganization was originally proposed by Wenger in ref 9a. Our 1H
NMR data support this hypothesis. Resolution of the seven N-Me groups
demonstrated that 26 exists largely as one rotamer. The acetonide-protected
precursor to heptapeptide 25, which would possess only two potential
intrastrand hydrogen bonds, exists as a mixture of rotamers. See the
Supporting Information for images of these spectra.
Acknowledgment. Support was provided by the NIH (CA28824
to S.J.D.). The authors thank Dr. George Sukenick, Hui Fang, and
Sylvi Rusli of SKI’s NMR core facility for mass spectral and NMR
assistance and Rebecca Wilson and Dana Ryan for assistance with
the preparation of the manuscript. We also thank Dr. Pavel Nagorny,
JA100517V
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