Enantioselective Synthesis and Biological Evaluation of Sanglifehrin A and B and Analogs

Article abstract of DOI:10.1002/anie.202103022

Sanglifehrin A and B are immunosuppressive macrocyclic natural products endowed with and differentiated by a unique spirocyclic lactam. Herein, we report an enantioselective total synthesis and biological evaluation of sanglifehrin A and B and analogs. Access to the spirocyclic lactam was achieved through convergent assembly of a key pyranone intermediate followed by a stereo-controlled spirocyclization. The 22-membered macrocyclic core was synthesized by ring-closing metathesis in the presence of 2,6-bis(trifluoromethyl) benzeneboronic acid (BFBB). The spirocyclic lactam and macrocycle fragments were united by a Stille coupling to furnish sanglifehrin A and B. Additional sanglifehrin B analogs with variation at the C40 position were additionally prepared. Biological evaluation revealed that the 2-CF₃ analog of sanglifehrin B exhibited higher anti-proliferative activity than the natural products sanglifehrin A and B in Jurkat cells. Both natural products induced higher-order homodimerization of cyclophilin A (CypA), but only sanglifehrin A promoted CypA complexation with inosine-5′-monophosphate dehydrogenase 2 (IMPDH2). The synthesis reported herein will enable further evaluation of the spirolactam and its contribution to sanglifehrin-dependent immunosuppressive activity.

Full text of DOI:10.1002/anie.202103022

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Enantioselective Synthesis and Biological Evaluation of
Sanglifehrin A and B and Analogs
Authors: Chia-Fu Chang, Hope A Flaxman, and Christina Woo
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202103022
Link to VoR: https://doi.org/10.1002/anie.202103022

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
Enantioselective Synthesis and Biological Evaluation of
Sanglifehrin A and B and Analogs
Chia-Fu Chang[a], Hope A. Flaxman[a], Christina M. Woo*,[a]
[a]
Dr. C.F. Chang, H. A. Flaxman, Dr. C. M. Woo
Department of Chemistry and Chemical Biology
Harvard University
12 Oxford St
Cambridge, MA 02138
E-mail: cwoo@chemistry.harvard.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: Sanglifehrin A and B are immunosuppressive macrocyclic
natural products endowed with and differentiated by a unique
spirocyclic lactam. Here, we report an enantioselective total synthesis
and biological evaluation of sanglifehrin A and B and analogs. Access
to the spirocyclic lactam was achieved via convergent assembly of a
= 102 nM), indicating that these compounds may decrease the
toxic side effects of CsA (mixed lymphocyte IC50 = 10.6 nM).[6]
However, although sanglifehrin A (1) and B (2) are potent ligands
for several members of the cyclophilin family,[4a] further
mechanistic investigation revealed that sanglifehrin
A (1)
key pyranone intermediate followed by
a
stereo-controlled
possesses a mechanism of action that is distinct from CsA. In
contrast to CsA, sanglifehrin A (1) does not inhibit calcineurin
activity upstream of IL-2 expression and instead blocks late-stage
IL-2-dependent proliferation of T cells.[7] Mechanistic studies have
further shown that sanglifehrin A (1) induces NF-κB-mediated p53
activation, stalls cell proliferation at the G1–S phase transition,[8]
and induces mitochondrial dysfunction.[9] These effects may occur
through a united target or through several targets, including
binding interactions with CypA or cyclophilin D (CypD),[9a] which
may be accompanied by the formation of higher-order complexes
such as a sanglifehrin A (1)–CypA homodimer[5c] or a ternary
complex between CypA and IMPDH2 that is stabilized by
sanglifehrin A (1).[10] However, whether the immunosuppressive
spirocyclization. The 22-membered macrocyclic core was
synthesized by ring-closing metathesis in the presence of 2,6-
bis(trifluoromethyl) benzeneboronic acid (BFBB). The spirocyclic
lactam and macrocycle fragments were united by a Stille coupling to
furnish sanglifehrin A and B. Additional sanglifehrin B analogs with
variation at the C40 position were additionally prepared. Biological
evaluation revealed that the 2-CF3 analog of sanglifehrin B exhibited
higher anti-proliferative activity than the natural products sanglifehrin
A and B in Jurkat cells. Both natural products induced higher-order
homodimerization of cyclophilin A (CypA), but only sanglifehrin A
promoted CypA complexation with inosine-5'-monophosphate
dehydrogenase 2 (IMPDH2). The synthesis reported here will enable
further evaluation of the spirolactam and its contribution to
sanglifehrin-dependent immunosuppressive activity.
activity of sanglifehrin
A (1) requires these higher-order
complexes or cyclophilin binding itself is inconclusive.
Competitive displacement of sanglifehrin A (1) from CypA has no
effect on immunosuppressive activity[7] and oxidative cleavage of
the C26–C27 olefin affords a macrocycle that maintains CypA
binding, but is non-immunosuppressive.[8a] These sanglifehrin-
Introduction
derived CypA macrocyclic ligands are under evaluation as non-
By contrast, beyond the
initial reports,[6] additional mechanistic studies with sanglifehrin B
The natural products cyclosporin A (CsA), FK506, and rapamycin
heralded transformative improvements in organ transplant
success rates and remediation of autoimmune diseases in the
clinic[1] via unique mechanisms of action involving formation of
higher-order protein complexes.[2] For example, CsA mediates a
ternary complex between cyclophilin A (CypA) and calcineurin,
which inhibits signal transduction during T cell activation.[1c, 3] In
screening for novel immunosuppressants that target CypA, the
sanglifehrin class of macrolides were discovered from isolates of
Streptomyces sp. A92-308110 in 1999.[4] Sanglifehrin A (1) and B
(2) are composed of a 22-membered macrocycle linked to a
structurally unique and highly substituted [5,5] spirolactam
(Figure 1A). The macrocycle mediates the binding interaction
with CypA,[5] and consists of an exocyclic (E,E)-1,3-diene
polyketide backbone fused to a tripeptide, which is composed of
valine, m-tyrosine, and an unusual β-substituted piperazic acid
residue.
11]
immunosuppressive anti-virals.[8c,
(2) have not been reported to date.
Sanglifehrin A (1) and B (2) possess differential cyclophilin
binding profiles and anti-proliferative effects,[4a] indicating that the
structural differences in their respective spirolactams play a
crucial role in tuning the activity of the sanglifehrins. While the
structure-activity relationship (SAR) of the sanglifehrin
macrocycle has been explored,[5a, 5b] SAR of the spirolactam has
not been evaluated. Since the discovery of the sanglifehrins, total
synthesis of sanglifehrin A (1) has been reported by the
Nicolaou[12] and Paquette[13] groups, along with many other
efforts.[14] Here, we report the development of a versatile
approach to the spirolactam, which culminated in the total
synthesis of sanglifehrin A (1) and B (2) and biological evaluation
of the protein–protein interactions that are differentially mediated
by these compounds. Furthermore, we utilized this synthetic route
to develop analogs at the C40 position and report a more potent
analog of sanglifehrin B, 2-CF3. This versatile route will enable
Sanglifehrin A (1) and B (2) possess mild anti-proliferative effects
against T cells and B cells (mixed lymphocyte IC50, 1 = 170 nM; 2
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
further investigation into the mechanistic contribution of the
by prior art[12-13] to afford two key fragments, the spirolactam 3 and
the iodide 4 (Figure 1A). Prior approaches to access the
spirolactam 3a employed a linear route[12-13] that may limit access
to spirolactam analogs like 3b or those with alternative
substitution patterns about positions C33–C40. We therefore
envisioned that the spirolactam structures 3a and 3b could be
elaborated from the common pyranone intermediate 5 through a
6-exo-trig Michael cyclization followed by stereo-controlled
reduction at C35 (Figure 1B).
spirolactam
and
drive
the
development
of
novel
immunosuppressants based on the sanglifehrin family.
Results and Discussion
In designing a synthetic approach to sanglifehrin A (1) and B (2),
we adopted a retrosynthetic disconnection of C25–C26 inspired
Figure 1. A. Structures and retrosynthetic analysis of sanglifehrin A (1) and B (2). B. Retrosynthetic approach to the spirolactam 3a and 3b.
Scheme 1. A. Synthesis of the ketone 6. B. Synthesis of the aldehyde 7a.
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
The pyranone
5
is easily accessed by assembly of the
yield with diastereoselectivity (74%, dr 10:1). After silyl protection
of the resulting alcohol with triethylsilane triflate (TESOTf), an
optimized NaIO4-mediated oxidative cleavage[18] of the bulky
alkene was performed to provide the aldehyde 12 in good yield
(74%, two steps). Grignard addition of an ethyl group to the
aldehyde 12 followed by oxidation using Dess-Martin periodinane
provided the ketone 6 in a good yield (75%) over two steps.
functionalized ketone 6 and the aldehyde 7a. A convergent
strategy that unites 6 and 7a at a later stage decouples installation
of each stereocenter, such that each stereocenter may be
strategically elaborated independently, to maximize overall
synthetic flexibility for derivatization of the substitution pattern
about the spirolactam 3 in the future.
Synthesis of the ketone 6 was initiated by a one-pot Leighton
allylation[15] of the readily accessible aldehyde 8[16] with the (R,R)-
Leighton ligand 9 to provide the alcohol 10 in 91% yield with
excellent diastereoselectivity (dr > 20:1, Scheme 1A). Silylation
of the alcohol 10 with t-butyldimethylsilane triflate (TBSOTf) and
an oxidative cleavage sequence afforded the desired aldehyde 11
in high yield over three steps (74%). Subsequent Brown
crotylation[17] established the requisite chiral centers in 12 in good
An efficient sequence for synthesis of the corresponding aldehyde
7a was developed using Myers asymmetric alkylation[19] (Scheme
1B). Accordingly, the readily accessible iodide 13[20] was reacted
with the lithiated pseudoephedrine propionate 14, followed by
removal of the chiral auxiliary by reaction with lithium
amidohydroborate generated in situ to afford the primary alcohol
15 in good yield and excellent diastereoselectivity (80%, dr >
20:1). The alcohol 15 was oxidized to give the aldehyde 7a.
Scheme 2. Synthesis of the spirolactam 3a and 3b of sanglifehrin A (1) and B (2), respectively.
With the two precursors in hand, we next turned to constructing
the spirolactam moiety. Aldol cross-coupling of the ketone 6 with
the aldehyde 7a followed by oxidation afforded a 1,3-diketone
intermediate, which was immediately subjected to acid-catalyzed
cyclization[21] at 50 ˚C to furnish the 4-pyranone 16 in good yield
(37%, Scheme 2). A regioselective debenzylation of 16 was
performed under one atm pressure of hydrogen gas in the
presence of Raney nickel to unmask the primary alcohol, which
was then converted to the corresponding primary amide 5 through
2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO) mediated
oxidation,[22] and an one-pot amide transformation promoted by
N,N'-disuccinimidyl carbonate (DSC). Attempts at direct
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
spirocyclization via Michael addition from the amide 5 proved
unproductive. Therefore, the enone in 5 was subjected to 1,2-
reduction with NaBH4 in the presence of CeCl3•7H2O to afford the
vinyl alcohol 17, which we observed spontaneously cyclize to the
With the spirolactam core synthesized, our focus moved to
functionalization of the spirolactam 18 with a stannane for Stille
cross coupling via regioselective hydrostannation of a terminal
alkyne. Therefore, oxidative removal of p-methoxybenzyl (PMB)
was carried out using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) to afford the primary alcohol 19 in good yield (83%). After
oxidation, the resulting aldehyde was subjected to Seyferth-
Gilbert homologation[25] using the Bestmann-Ohira reagent 20 to
afford the terminal alkyne 21 (66%, two steps). Following
deprotection of the TBS group, palladium-catalyzed
hydrostannation was performed using tricyclohexylphosphine
(Cy3P)[26] as a ligand to provide the stannane 3b towards
sanglifehrin B (2) (55% yield, two steps).
spirolactam 18 as
a
single diastereomer via Ferrier
rearrangement[23] in low yield (35%), along with recovery of the
alcohol 17. The 2,6-cis stereochemistry was comfired by a
Nuclear
Overhauser
Effect
(NOE)
experiment.
The
spirocyclization was subsequently optimized by subjection of the
crude 17 to catalytic BF3•OEt2 at –78 ˚C, which greatly improved
the yield without compromising stereocontrol (77%). The
excellent diastereoselectivity may be as a result of both kinetic
and thermodynamic control. The facial selectivity of
oxocarbenium in the transition states is favored for the desired
diastereomer.[24] In the transition state T1, the steric clash of the
side chain and A 1,3-strain interactions between the two methyl
groups are minimized on electrophilic trapping with the primary
amide when approaching from the Si face, in contrast to T2, where
steric interactions arise during approach of the primary amide
from the Re face. The less stable twist boat carbocation as
alternative intermediate in transition state also leads to the
desired stereochemical outcome (Figure S1). In addition, the
Access to the corresponding sanglifehrin A spirolactam 3a was
achieved by functionalization of the olefin across positions C35–
C36 of 19 to install the secondary alcohol in 22 (Scheme 2).
Accordingly, a stereo-controlled hydroboration-oxidation[27] tactic
installed the desired stereochemistry on C36 exclusively, albeit
with inverted stereochemistry on the hydroxyl group on C35.
Subsequently, a two-step sequence of oxidation and reduction
using L-selectride resulted in the desired stereochemistry on C35
exclusively (60%, two steps).[14i] After TEMPO-mediated oxidation
using (diacetoxyiodo)benzene (BAIB),[28] the aldehyde 22 was
converted to the stannane 3a in a similar manner to that used to
access the stannane 3b.
desired
diastereomer
as
2,6-cis-dihydropyran
is
thermodynamically favored (Figure S2, Scheme S1).
Scheme 3. Alternative synthetic route to the spirolactam 3a and 3b of sanglifehrin A (1) and B (2), respectively.
Alternatively, we envisioned that elaborating the C26–C31 spacer
after construction of the spirolactam would allow access to
sanglifehrin analogs with different connectivity (Figure 1A). Thus,
the ketone 24 was elaborated via a similar sequence as described
above to afford the spirolactam 27 (Scheme 3). The spirolactam
27 was subjected to radical-mediated debenzylation using lithium
4,4’-di-tert-butylbiphenylide (LiDBB, Freeman’s reagent),[29]
followed by Dess-Martin oxidation to smoothly deliver the
aldehyde 28 in excellent yields (99%, 81%, respectively). The
aldehyde 28 was elaborated to install the stereocenters on C30–
C31 using Brown crotylation (65%, dr 3:1), and the resulting
terminal alkene was subjected to cross metathesis with
crotonaldehyde using Hoveyda-Grubbs Second Generation (H-G
II) catalyst to afford the α,β-unsaturated aldehyde 29 in a good
yield (78%). A chemoselective hydrogenation using Lindlar
catalyst afforded the hemiketal,[30] which was readily converted to
the stannane 3b. The sanglifehrin A stannane 3a was prepared in
a similar manner from 27 after manipulation of the oxidation state
of the olefin.
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
We next focused on synthesis of the macrocycle 32. Despite
efforts to access 32 via an intramolecular Stille coupling[12] or
macrolactonization,[13] we found that material throughput was
challenging. Therefore, we were attracted to the recent reports of
a ring-closing metathesis (RCM) strategy to provide sufficient
access to the macrocyclic core[11, 14a, 14f] for the preparation of
sanglifehrin A (1) and B (2). We further elected to use silyl
protection for the diol and a ketal to mask the ketone in 32, which
would allow for stepwise deprotection and avoid elimination of the
C17-hydroxyl group, which we observed in our early studies.
Retrosynthetic disconnection across C20–C21 yields the tetraene
33, which could be prepared by amide coupling between the
tripeptide 34 and the diene 35 (Figure 2).
The sequence leading to the polyketide-derived fragment 35
commenced from the reported allyl alcohol 36,[31] which was
subjected to Sharpless epoxidation[32] to yield the epoxide 37
(86%, dr = 8:1, Scheme 4). Ring opening of the epoxide[33] with
the bromide 38[34] in the presence of anhydrous copper iodide
successfully delivered the diol 39 in good yield (73%).[35]
Figure 2. Retrosynthetic analysis of the macrocycle 32.
Scheme 4. Synthesis of the macrocycle 32 and total synthesis of sanglifehrin A (1) and B (2).
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
After sequential protection of the diol 39, the intermediate 40 was
debenzylated and oxidized to afford the aldehyde 41. Installation
of the requisite diene on the aldehyde 41 was carried out through
B (2), which yielded a more potent analog 2-CF3 (IC50 = 0.24 µM),
in which the ethyl substituent was replaced by a trifluoroethyl
group (Figure 3A, 3B). These data indicate that structural
modification about the spirolactam, and specifically at the C40
position, may further enhance the activity of sanglifehrin analogs
against Jurkat cells.
a
Horner-Wadsworth-Emmons reaction (HWE) with diethyl
allylphosphonate in the presence of N,N
′
-
dimethylpropyleneurea (DMPU).[36] The resulting diene 42 was
functionalized to afford the acid 35, by a sequence of pivalate
deprotection and oxidation. The coupling partner tripeptide
34•TFA was prepared through
a sequence reported by
Nicolaou[12] from the corresponding allyl alcohol. Amide coupling
between the acid 35 and the tripeptide 34•TFA using
hexafluorophosphate azabenzotriazole tetramethyl uronium
(HATU) yielded the tetraene 33 (62%).
The tetraene 33 was converted to the macrocycle 32 via a RCM
reaction. In initial conditions, 33 was treated with the H-G II
catalyst in the presence of benzoquinone (BQ) at 80 ˚C to achieve
the macrocycle 32 in 12% yield (Scheme 4). Use of the modified
H-G II catalyst improved the yield of 32 by two-fold (24%).[37]
Notably, we observed oxidation of 33 or 32 across position C2–
N6' to the imine, which may be due to an intramolecular reaction
between C2–N6' moiety with the ruthenium carbene formed in situ.
This position was previously reported to be oxidized on standing
in air.[14f] To circumvent the undesired imine formation, we
attempted to mask N6' using Lewis acids [e.g., chloride
dicyclohexylborane,[38] or titanium (IV) isopropoxide],[39] but these
additives led to decomposition. Serendipitously, we found that the
yield of the RCM reaction to afford the macrocycle 32 was
significantly improved in the presence of a catalytic amount of 2,6-
bis(trifluoromethyl)benzeneboronic acid (BFBB, 48%) on 260 mg
scales. Interestingly, addition of stoichiometric amounts of BFBB
to the reaction did not further improve the yield,[40] possibly due to
accelerating decomposition of the catalyst.[41]
Finally, the Stille-Migita cross coupling of the vinyl iodide 32 with
the stannane 3a under conditions reported by Fürster et al.[42] was
performed to successfully give the coupling product in 50% yield.
For the final deprotection of the ketal and silyl groups, the TBS
groups were first removed by tris(dimethylamino)sulfonium
difluorotrimethylsilicate (TASF)[43] in a good yield and the final
deprotection of the ketal was conducted using catalytic p-
toluenesulfonic acid (TsOH) in the presence of boric acid to afford
sanglifehrin A (1) along with the corresponding interchangable
hemiketal isomers in a 2:1 ratio (8.8 mg, 50% yield, Scheme 4,
Figure S3). The ratio of the ketal to hemiketal was in agreement
with previous reports.[30] In a similar manner, sanglifehrin B (2)
was obtained in 23% yield over three steps (3.0 mg). The
stepwise deprotection improved the overall yield as compared to
use of sulfuric acid[12] or stoichiometric TsOH[13, 30] for deprotection
of the intramolecular ketal. Synthetic sanglifehrin A (1) and
sanglifehrin B (2) are in agreement with the NMR data in the
literature.[4b, 30][44]
Figure 3: Biological evaluation of sanglifehrin A (1), B (2), and analogs. A.
Structures of the sanglifehrin B C40 analogs 2-H and 2-CF3. B. Antiproliferative
effects in Jurkat cells as measured in a 72 h MTT assay. C. Co-enrichment of
IMPDH2 with recombinant GST-CypA from a Jurkat lysate treated with the
indicated compounds. D. Characterization of CypA dimerization state in the
presence of the indicated compounds as determined by size-exclusion
chromatography.
We next investigated whether the higher-order protein complexes
that are mediated by sanglifehrin A (1) are recapitulated by
sanglifehrin B (2) and therefore potential targets for further
mechanistic investigation.[5c, 10] We first evaluated of the higher-
order protein complexe between Cyp A and IMPDH2, which have
previously been reported to form a ternary complex in the
presence of sanglifehrin A. The data revealed that while
sanglifehrin A (1) promotes an interaction between CypA and
IMPDH2 in vitro, sanglifehrin B (2) induces a minimal CypA–
IMPDH2 interaction (Figure 3C). By contrast, both sanglifehrin A
(1) and sanglifehrin B (2) drive nearly complete homodimerization
on interaction with CypA in vitro (Figure 3D). These data indicate
that the spirolactam plays a key role in mediating the higher-order
protein complexes formed by sanglifehrin A (1) and B (2), and
suggests additional targets may be involved in the anti-
We proceeded to evaluate the biological activity of sanglifehrin A
(1) and B (2) to probe whether the spirolactam promoted
differential activity. Sanglifehrin A (1) and sanglifehrin B (2) both
displayed moderate anti-proliferative effects in Jurkat cells, with
sanglifehrin B (2) being more potent (1: IC50 = 4.2 µM; 2: IC50
=
0.8 µM, Figure 3B). Enthused by this finding, we further
investigated additional analogs based on sanglifehrin B using our
synthetic route. We synthesized two C40 analogs of sanglifehrin
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
[6]
(a) M. Naesens, D. R. Kuypers, M. Sarwal, Clin J Am Soc Nephrol 2009,
4, 481-508; (b) H. Y. Lin, L. L. Rocher, M. A. McQuillan, S. Schmaltz, T.
D. Palella, I. H. Fox, N Engl J Med 1989, 321, 287-292; (c) B. Ryffel,
Toxicology 1992, 73, 1-22.
G. Zenke, U. Strittmatter, S. Fuchs, V. F. Quesniaux, V. Brinkmann, W.
Schuler, M. Zurini, A. Enz, A. Billich, J. J. Sanglier, T. Fehr, J Immunol
2001, 166, 7165-7171.
(a) M. A. Gregory, M. Bobardt, S. Obeid, U. Chatterji, N. J. Coates, T.
Foster, P. Gallay, P. Leyssen, S. J. Moss, J. Neyts, M. Nur-e-Alam, J.
Paeshuyse, M. Piraee, D. Suthar, T. Warneck, M. Q. Zhang, B.
Wilkinson, Antimicrob Agents Chemother 2011, 55, 1975-1981; (b) S.
Kroller-Schon, S. Steven, S. Kossmann, A. Scholz, S. Daub, M. Oelze,
N. Xia, M. Hausding, Y. Mikhed, E. Zinssius, M. Mader, P. Stamm, N.
Treiber, K. Scharffetter-Kochanek, H. Li, E. Schulz, P. Wenzel, T.
Munzel, A. Daiber, Antioxid Redox Signal 2014, 20, 247-266; (c) Z. K.
Sweeney, J. Fu, B. Wiedmann, J Med Chem 2014, 57, 7145-7159.
(a) S. J. Clarke, G. P. McStay, A. P. Halestrap, J Biol Chem 2002, 277,
34793-34799; (b) A. H. Giang, T. Raymond, P. Brookes, K. de Mesy
Bentley, E. Schwarz, R. O'Keefe, R. Eliseev, J Biol Chem 2013, 288,
33303-33311.
proliferative activity of sanglifehrin B (2). These data are the first
mechanistic efforts comparing sanglifehrin A (1) to sanglifehrin B
(2) and additional spirolactam analogs, revealing biologic effects
mediated by the structural differences about the spirolactam.
[7]
[8]
Conclusion
In conclusion, we developed a convergent and enantioselective
synthetic route toward sanglifehrin A (1) and the first total
synthesis of sanglifehrin B (2), which enabled preparation of
additional analogs and characterization of the effects of the
spirolactam on the resulting protein complexes underlying their
biological activity. Strategic assembly of the spirolactam via the
common pyranone 5 enables derivatization of novel spirolactam
analogs, such as those at position C40, to illuminate the
mechanism of action of the sanglifehrin class of natural products.
In addition, synthesis of the macrocycle 32 through RCM with the
addition of BFBB to suppress the formation of the imine byproduct
improved access to the natural product. Biological evaluation
reveals that although sanglifehrin B (2) is more potent than
sanglifehrin A (1) in Jurkat cells, sanglifehrin A (1) preferentially
forms higher-order protein complexes between CypA and
IMPDH2, while both natural products promote homodimerization
with CypA. In addition, a more potent analog was discoveried by
synthesizing two additional analogs of sanglifehrin B on C40,
suggesting the dervatization on C40 would serve as starting point
for further SAR investigation. Further insights into the mechanism
of action of the sanglifehrin family will be enabled by evaluation of
novel sanglifehrin analogs derivatized at other positions about the
spirolactam (C33–C40), which are now accessible via the
reported synthetic pathway.
[9]
[10] K. H. Pua, D. T. Stiles, M. E. Sowa, G. L. Verdine, Cell Rep 2017, 18,
432-442.
[11] V. A. Steadman, S. B. Pettit, K. G. Poullennec, L. Lazarides, A. J. Keats,
D. K. Dean, S. J. Stanway, C. A. Austin, J. A. Sanvoisin, G. M. Watt, H.
G. Fliri, A. C. Liclican, D. Jin, M. H. Wong, S. A. Leavitt, Y.-J. Lee, Y.
Tian, C. R. Frey, T. C. Appleby, U. Schmitz, P. Jansa, R. L. Mackman,
B. E. Schultz, J Med Chem 2017, 60, 1000-1017.
[12] (a) K. C. Nicolaou, J. Xu, F. Murphy, S. Barluenga, O. Baudoin, H. Wei,
D. L. Gray, T. Ohshima, Angew Chem Int Ed Engl 1999, 38, 2447-2451;
(b) K. C. Nicolaou, F. Murphy, S. Barluenga, T. Ohshima, H. Wei, J. Xu,
D. L. F. Gray, O. Baudoin, J Am Chem Soc 2000, 122, 3830-3838.
[13] (a) M. Duan, L. A. Paquette, Angew Chem Int Ed Engl 2001, 40, 3632-
3636; (b) L. A. Paquette, M. Duan, I. Konetzki, C. Kempmann, J Am
Chem Soc 2002, 124, 4257-4270.
[14] (a) L. M. Martin Cabrejas, S. Rohrbach, D. Wagner, J. Kallen, G. Zenke,
J. Wagner, Angew Chem Int Ed Engl 1999, 38, 2443-2446; (b) R. Banteli,
I. Brun, P. Hall, R. Metternich, Tetrahedron Lett 1999, 40, 2109-2112; (c)
L. A. Paquette, I. Konetzki, M. S. Duan, Tetrahedron Lett 1999, 40, 7441-
7444; (d) K. C. Nicolaou, T. Ohshima, F. Murphy, S. Barluenga, J. Y. Xu,
N. Winssinger, Chem Commun 1999, 809-810; (e) P. Hall, J. Brun, D.
Denni, R. Metternich, Synlett 2000, 315-318; (f) J. Wagner, L. M. Martin
Cabrejas, C. E. Grossmith, C. Papageorgiou, F. Senia, D. Wagner, J.
France, S. P. Nolan, J Org Chem 2000, 65, 9255-9260; (g) R. Banteli, J.
Wagner, G. Zenke, Bioorg Med Chem Lett 2001, 11, 1609-1612; (h) M.
K. Gurjar, A. R. Chaudhuri, Tetrahedron Lett 2002, 43, 2435-2438; (i) J.
Wagner, H. Andres, S. Rohrbach, D. Wagner, L. Oberer, J. France, J Org
Chem 2005, 70, 9588-9590; (j) L. C. Dias, A. G. Salles, Tetrahedron
Letters 2006, 47, 2213-2216; (k) J. S. Yadav, K. V. R. Rao, A. Kavita, D.
K. Mohapatra, Eur J Org Chem 2013, 2013, 2849-2858; (l) L. Radhika,
S. Chandrasekhar, Synthetic Commun 2014, 44, 3602-3609; (m) K.
Suttisintong, J. D. White, J Org Chem 2015, 80, 2249-2262; (n) M. J.
Hansson, S. J. Moss, M. Bobardt, U. Chatterji, N. Coates, J. A. Garcia-
Rivera, E. Elmer, S. Kendrew, P. Leyssen, J. Neyts, M. Nur-E-Alam, T.
Warneck, B. Wilkinson, P. Gallay, M. A. Gregory, Chem Biol 2015, 22,
285-292.
Acknowledgements
Financial support from the Burroughs Wellcome Fund (C.M.W.),
NSF-GRFP (H.A.F.), and Harvard University is gratefully
acknowledged.
[15] L. M. Suen, M. L. Steigerwald, J. L. Leighton, Chem Sci 2013, 4, 2413-
2417.
Conflict of Interest
The authors declare no conflict of interest.
[16] A. Tanaka-Yanuma, S. Watanabe, K. Ogawa, S. Watanabe, N. Aoki, T.
Ogura, T. Usuki, Tetrahedron Lett 2015, 56, 6777-6781.
[17] H. C. Brown, K. S. Bhat, J Am Chem Soc 1986, 108, 5919-5923.
[18] W. Yu, Y. Mei, Y. Kang, Z. Hua, Z. Jin, Org Lett 2004, 6, 3217-3219.
[19] A. G. Myers, B. H. Yang, H. Chen, L. McKinstry, D. J. Kopecky, J. L.
Gleason, J Am Chem Soc 1997, 119, 6496-6511.
[20] A. N. Lowell, M. D. DeMars, 2nd, S. T. Slocum, F. Yu, K. Anand, J. A.
Chemler, N. Korakavi, J. K. Priessnitz, S. R. Park, A. A. Koch, P. J.
Schultz, D. H. Sherman, J Am Chem Soc 2017, 139, 7913-7920.
[21] M. Jin, R. E. Taylor, Org Lett 2005, 7, 1303-1305.
[22] M. Zhao, J. Li, E. Mano, Z. Song, D. M. Tschaen, E. J. J. Grabowski, P.
J. Reider, J Org Chem 1999, 64, 2564-2566.
Keywords: Sanglifehrin A • Sanglifehrin B • Cyclophilin• Total
Synthesis• Natural product
[1]
(a) J. Clardy, Proc Natl Acad Sci U S A 1995, 92, 56-61; (b) S. L.
Schreiber, Science 1991, 251, 283-287; (c) J. Liu, J. D. Farmer, Jr., W.
S. Lane, J. Friedman, I. Weissman, S. L. Schreiber, Cell 1991, 66, 807-
815.
[23] (a) R. J. Ferrier, in Glycoscience: Epimerisation, Isomerisation and
Rearrangement Reactions of Carbohydrates (Ed.: A. E. Stütz), Springer
Berlin Heidelberg, Berlin, Heidelberg, 2001, pp. 153-175; (b) R. J. Ferrier,
O. A. Zubkov, in Organic Reactions, pp. 569-736.
[24] R. Kartika, J. D. Frein, R. E. Taylor, J Org Chem 2008, 73, 5592-5594.
[25] G. J. Roth, B. Liepold, S. G. Muller, H. J. Bestmann, Synthesis-Stuttgart
2004, 59-62.
[26] A. Darwish, A. Lang, T. Kim, J. M. Chong, Org Lett 2008, 10, 861-864.
[27] S. J. Danishefsky, D. C. Myles, D. F. Harvey, J Am Chem Soc 1987, 109,
862-867.
[28] A. DeMico, R. Margarita, L. Parlanti, A. Vescovi, G. Piancatelli, J Org
Chem 1997, 62, 6974-6977.
[2]
[3]
C. J. Gerry, S. L. Schreiber, Nat Chem Biol 2020, 16, 369-378.
J. Liu, M. W. Albers, T. J. Wandless, S. Luan, D. G. Alberg, P. J. Belshaw,
P. Cohen, C. MacKintosh, C. B. Klee, S. L. Schreiber, Biochemistry 1992,
31, 3896-3901.
[4]
[5]
(a) J. J. Sanglier, V. Quesniaux, T. Fehr, H. Hofmann, M. Mahnke, K.
Memmert, W. Schuler, G. Zenke, L. Gschwind, C. Maurer, W. Schilling,
J Antibiot (Tokyo) 1999, 52, 466-473; (b) T. Fehr, J. Kallen, L. Oberer, J.
J. Sanglier, W. Schilling, J Antibiot (Tokyo) 1999, 52, 474-479.
(a) M. J. Hansson, S. J. Moss, M. Bobardt, U. Chatterji, N. Coates, J. A.
Garcia-Rivera, E. Elmer, S. Kendrew, P. Leyssen, J. Neyts, E. A. M. Nur,
T. Warneck, B. Wilkinson, P. Gallay, M. A. Gregory, Chem Biol 2015, 22,
285-292; (b) R. Sedrani, J. Kallen, L. M. Martin Cabrejas, C. D.
Papageorgiou, F. Senia, S. Rohrbach, D. Wagner, B. Thai, A. M. Jutzi
Eme, J. France, L. Oberer, G. Rihs, G. Zenke, J. Wagner, J Am Chem
Soc 2003, 125, 3849-3859; (c) J. Kallen, R. Sedrani, G. Zenke, J.
Wagner, J Biol Chem 2005, 280, 21965-21971.
[29] R. R. Hill, S. D. Rychnovsky, J Org Chem 2016, 81, 10707-10714.
[30] R. Metternich, D. Denni, B. Thai, R. Sedrani, J Org Chem 1999, 64, 9632-
9639.
[31] M. Miyashita, M. Hoshino, A. Yoshikoshi, J Org Chem 1991, 56, 6483-
6485.
7
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
[32] J. G. Hill, B. E. Rossiter, K. B. Sharpless, J Org Chem 1983, 48, 3607-
3608.
[33] M. Alam, C. Wise, C. A. Baxter, E. Cleator, A. Walkinshaw, Org Process
Res Dev 2012, 16, 435-441.
[34] R. P. Hsung, Synthetic Commun 1990, 20, 1175-1179.
[35] C. F. Chang, E. Stefan, R. E. Taylor, Chem–Eur J 2015, 21, 10681-
10686.
[36] Y. Wang, F. G. West, Synthesis-Stuttgart 2002, 99-103.
[37] I. C. Stewart, T. Ung, A. A. Pletnev, J. M. Berlin, R. H. Grubbs, Y. Schrodi,
Org Lett 2007, 9, 1589-1592.
[38] A. D. Abell, N. A. Alexander, S. G. Aitken, H. Y. Chen, J. M. Coxon, M.
A. Jones, S. B. McNabb, A. Muscroft-Taylor, J Org Chem 2009, 74, 4354-
4356.
[39] A. K. Ghosh, J. Cappiello, D. Shin, Tetrahedron Letters 1998, 39, 4651-
4654.
[40] The RCM reaction performed in the presence of one equivalent of BFBB
resulted in a lower yield of 29%.
[41] C. Simocko, K. B. Wagener, Organometallics 2013, 32, 2513-2516.
[42] A. Furstner, J. A. Funel, M. Tremblay, L. C. Bouchez, C. Nevado, M.
Waser, J. Ackerstaff, C. C. Stimson, Chem Commun 2008, 2873-2875.
[43] K. A. Scheidt, H. Chen, B. C. Follows, S. R. Chemler, D. S. Coffey, W.
R. Roush, J Org Chem 1998, 63, 6436-6437.
[44] T. Fehr, L. Oberer, V. Q. Reffel, J-J. Sanglier; W. Schuler, Macrolides.
U. S. Patent, US-6124453-A, December 24, 1996.
8
This article is protected by copyright. All rights reserved.

10.1002/anie.202103022
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
The immunosuppressive natural products, sanglifehrin A and B, bearing a 22-membered macrocycle appended with a unique highly-
substituted spirolactam, pose formidable challenges to synthetic accessibility and derivatization. A versatile strategy toward the
spirolactam via convergent assembly of fully-functionalized fragments culminated in the total synthesis of sanglifehrin A and sanglifehrin
B and preparation of additional analogs at position C40 for evaluation of biological activity in Jurkat cells and stabilization of protein–
protein interactions.
9
This article is protected by copyright. All rights reserved.