Total Synthesis of Gombamide A

Article abstract of DOI:10.1021/acs.orglett.6b01825

The first total synthesis of Gombamide A (1), a cytotoxic cyclic thiopeptide from the sponge Clathria gombawuiensis, has been achieved. Highlights of the convergent synthesis feature a disulfide bond forming cascade to close the 17-membered macrocycle and a selenoazidylation procedure to access the unusual para-hydroxystyrlyamide (pHSA) moiety. The synthesis required 18 steps, 11 steps in its longest linear sequence, and proceeded in 9.1% overall yield. This work will facilitate the study of the biological effects of Gombamide A and provide groundwork to explore the structure-activity relationship around this rare natural product.

Full text of DOI:10.1021/acs.orglett.6b01825

Letter
pubs.acs.org/OrgLett
Total Synthesis of Gombamide A
Pedro M. Garcia-Barrantes and Craig W. Lindsley*
Departments of Chemistry and Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: The first total synthesis of Gombamide A (1), a
cytotoxic cyclic thiopeptide from the sponge Clathria
gombawuiensis, has been achieved. Highlights of the convergent
synthesis feature a disulfide bond forming cascade to close the
17-membered macrocycle and a selenoazidylation procedure to
access the unusual para-hydroxystyrlyamide (pHSA) moiety.
The synthesis required 18 steps, 11 steps in its longest linear
sequence, and proceeded in 9.1% overall yield. This work will
facilitate the study of the biological effects of Gombamide A
and provide groundwork to explore the structure−activity
relationship around this rare natural product.
ombamide A (1, Figure 1) was recently isolated from the
red encrusting marine sponge Clathria gombawuiensis. The
lipopeptides6 and in the malformins,7 metabolites from
Aspergillus niger with antibiotic and cytotoxic activity. An
homologue 18-membered macrocycle is present in antitumor
antibiotics related to quinomycin, DNA polymerase, and
topoisomerase inhibitors.8 Quinomycin and malformins are
bicyclic structures, and one could argue that the monocyclic
Gombamide A would be a simplified pharmacophore for
cytotoxic activity. In order to perform experiments to explore
the biological effects and elucidate the cytotoxic mechanism of
action of 1, enough material must be available. An efficient
chemical access of 1 is likely the best option, as 1 is produced in
scarce amounts in C. gombawuiensis, making its isolation
prohibitive. Also, we believed that a natural product guided
synthesis would pave the way for structure−activity relationship
(SAR) studies around 1 that can lead to the optimization of its
biological activity. Therefore, synthetic intrigue and interest to
further explore the biology of this unique thiopeptide motivated
us to embark on the total synthesis of Gombamide A.
G
Figure 1. Gombamide A, 1, a thiohexapeptide with unusual amino acids
and a rare disulfide-containing, 17-membered macrocycle.
cyclic hexapeptide 1 displayed modest cytotoxic activity against
K562 chronic myelogenous leukemia and A549 epithelial lung
carcinoma cell lines (LC50s of 6.9 and 7.1 μM, respectively),
along with weak inhibitory activity for Na+/K+-ATPase (IC50 =
17.8 μM).1
In our retrosynthetic analysis (Scheme 1), we proposed to
install the disulfide bond from two protected cysteine residues
within an acyclic peptide; enabling a deprotection/disulfide bond
forming cascade to close the 17-membered macrocycle. Further
amide cleavage at the L-Cys 1 and L-Phe bond liberates Fragment
A (2) and Fragment B (3), enabling a convergent synthetic
approach. Fragment A (2) would be readily constructed from a
linear series of amide couplings employing protected variants of
L-pyroglutamic acid, L-phenylalanine, L-cysteine, and L-proline.
We envisioned multiple strategies to access Fragment B (3) and
form the enamide in pHSA, including dehydration of octopamine
derived amide 4, copper-catalyzed amidation of 7 with 8 and
Grieco elimination on substrate 9. In the latter, 9 will be derived
from the amide coupling of a protected L-cysteine 6 and the α-
Notably, 1 presents a number of unique chemical features in its
structure. Gombamide A is a rare, 17-membered macrocyclic
thiohexapeptide with two unusual exocyclic substitutions: an
(E)-para-hydroxystyrylamide (pHSA) and an L-pyroglutamic
amide (pyroGlu). The O-substituted pHSA feature has been
observed forming part of macrocyclic peptides, primarily in 14-
membered cyclopeptides in the Z configuration,2 while the only
E example has been found in the bastadins.3 The uncapped pHSA
has only been reported in small, linear natural products,4 and 1 is
the sole reported molecule where this moiety is present in a cyclic
peptide. There are few examples of cyclic peptides with disulfide
linkages originating from sponges.5 Moreover, the 17-membered
ring thiopeptide core has only been found in the eudistomide
Received: June 22, 2016
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.6b01825
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Scheme 1. Retrosynthetic Analysis of Gombamide A, 1
seleno amine produced from 10, an intermediate generated from
a selenoazidylation of a protected p-hydroxystyrene.
Scheme 3. Synthesis of Acyclic Intermediate 20
Initially, we pursued the dehydration approach for the
synthesis of 3 (Scheme 2). Here, HATU-coupling of ( )-octop-
Scheme 2. Synthesis of 13
amine 5 with 11, led to the isolation of 12 in 86% yield, without
the need for protecting group manipulation of the alcohols. The
stability of the benzylthioether protecting group allowed us to
explore a wide range of dehydration conditions. Burgess reagent
and Martin sulfurane did not provide desired dehydration
product 13, using different equivalents and temperatures.
Numerous conditions such as trifluoroacetic acid (TFA),
trifluoroacetic anhydride, p-toluensulfonic acid, p-toluensulfonic
anhydride, and p-toluensulfonyl chloride in pyridine or thionyl
chloride in pyridine led to either no conversion or decomposition
of the starting material. Finally, we were able to effectively
achieve this transformation by the use of BF3·OEt2 in N,N-
dimethylformamide (DMF), a procedure previously used in the
synthesis of mekaluvamine E.9 In our case, the reported
conditions (entry 1, Table S1) affected the desired dehydration
with concomitant Boc-deprotection of 12, but only in trace
amounts. In an effort to optimize this conversion, we sought
more mild conditions varying temperature and Lewis acid
equivalents. The use of 5 equiv of BF3·OEt2 allowed the isolation
of 13 in 27% yield (entry 2, Table S1); while lowering the
temperature led to trace amounts of the product. A great
improvement was observed when heating the reaction in the
microwave (entry 6, Table S1), as the yield increased almost 2-
fold to 52% after only 30 min, allowing an efficient way to access
13.
the Boc group and an EDCI/HOBt-mediated coupling with Boc-
L-Cys-SBn 11 gave tetrapeptide 17 in 63% over two steps.
Repetition of the same two-step sequence with 17 but employing
L-pyroglutamic acid afforded the key pentapeptide 18 in
quantitative yield for the two-step deprotection/coupling
sequence. Benzyl deprotection to give the desired fragment 19
proved challenging, as the majority of standard conditions were
not compatible with the sulfur containing starting material, due
to its ability to poison palladium-based deprotection conditions
and loss of optical purity during basic hydrolysis. Ultimately, we
found that Lewis acid conditions (AlCl3, anisole, CH3NO2/
DCM) were effective, generating key fragment 19 in 99%
isolated yield.
With amide 13 in hand, attention now focused on assembling
fragment A. The route to compound 19 began with an EDCI/
HOBt-mediated coupling between L-Phe-OBn 14 and Boc-L-Pro
to provide 15 in quantitative yield (Scheme 3). TFA
deprotection of the Boc group and a second EDCI/HOBt-
mediated coupling with another Boc-L-Pro delivered tripeptide
16 in 89% yield over two steps. Subsequent TFA deprotection of
The amide coupling between fragments 13 and 19 proceeded
smoothly to yield intermediate 20 in 82%, which, once
deprotected, should cyclize to give 1. Unfortunately, attempts
for deprotection/cyclization using iodine,10 CH3SiCl3/Ph2SO in
B
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TFA,11 or sodium in liquid ammonia12 failed to provide the
natural product. Due to the difficulties found in the deprotection
of the benzylthioether, we tried different more labile protecting
groups, including acetamidomethyl, p-methylbenzyl, p-methox-
ybenzyl, and trityl groups; however, none of these were
compatible with the dehydration conditions previously em-
ployed to obtain 13, thus impeding the evaluation of the new
protecting groups in the cyclization reaction.
Exploring other routes outlined in our retrosynthesis (Scheme
1), we tried the copper-catalyzed amidation to obtain Fragment
B in similar fashion to Porco’s synthesis of antitumor macrolide
lobatamide C.13 To assess this alternative, a S-Acm protected
carboxamide 8 was prepared and the amidation of E-4-(2-
halidevynyl)phenol 7 attempted. The reaction was performed
with the bromide and iodide vinyl halides and also their TBS
phenoxysilane protected versions; however, none of the
substrates yielded the desired product after using different
copper sources and ethylendiamine ligands.
in 80% yield, without observable production of the Z-enamide.
The selection of the trityl group was essential for this reaction as
it prevented the overoxidation of the cysteine, a feature that was
observed with its Acm protected congener; also, the steric bulk of
this protecting group probably contributes to the selective
formation of the E enamide. Removal of the Fmoc group of 27 to
afford 28 proved difficult as classical conditions (20% piperidine
in DMF) led to complex mixtures. We found that 28 was base
sensitive, and that ultimately morpholine, a weaker base (pKa ≈
8), was effective in removing the Fmoc protecting group cleanly
and in good isolated yield (72%).
With 28 in hand, attention now focused on assembling
fragment A and producing the acyclic precursor of 1 (Scheme 5).
Scheme 5. Synthesis of 1
Efforts then shifted toward the preparation of the required
intermediates to pursue the Grieco elimination procedure to
access the unusual pHSA moiety of 1 (Scheme 4). First, using the
Scheme 4. Synthesis of Enamide 28
Starting with compound 16, Boc deprotection followed by amide
coupling with Boc-L-Cys-S-Acm afforded 29 in 83% yield in two
steps. Subsequent TFA deprotection and HATU-mediated
coupling with L-pyroglutamic acid yielded 30 (87%). AlCl3-
facilitated benzyl deprotection produced 31 in 94% yield. Finally,
28 and 31 were coupled using HATU conditions providing
acyclic, bis-cysteine protected, OTBS-hexapeptide 32 in 51%
yield. This set the stage for the disulfide bond forming cascade to
close the 17-membered macrocycle and to deliver 1 for the first
time.
We envisioned an oxidative cyclization protocol employing
molecular iodine9 wherein the S-Trityl and S-Acm protecting
groups would be removed, followed by disulfide formation; as
their removal conditions should be milder with respect to the
benzyl thioether. The conversion of 32 to the TBS-protected
congener of 1 required optimization, as both the equivalents of I2
and concentration were key reaction parameters (Table S3).
Under optimized conditions, 5.0 equiv of I2 in DCM/MeOH
(12:1) (0.0008 M) provided 33 in 51% yield (entry 2, Table S3).
Deprotection of the phenolic TBS moiety also proved nontrivial,
as standard conditions caused product degradation (TBAF or
HCl/MeOH). Finally, HF-pyridine or KF in MeOH proved
effective, the latter being faster (6 h to completion, compared to
24 h), thus providing, for the first time, Gombamide A in 58%
yield.
amide derived from 5 and Boc-L-Cys-S-Acm, we tried to
substitute the alcohol with the selenide, which has been typically
used to generate endocyclic Z olefins in cyclic peptides,14 but this
approach led to no satisfactory result. Therefore, we envisioned
introducing the selenide prior the amide coupling. Here, a Wittig
reaction using p-hydroxybenzaldehyde 21 provides styrene 22 in
95% yield, and a subsequent TBS protection of the phenol
proceeded in quantitative yield to deliver 23. An anti-
Markovnikov regiospecific selenoazidylation, using phenyliodo-
(III) diacetate (PIDA), NaN3, and (PhSe)2,15 afforded the key α-
seleno azide 24, in low yields (30−40%) and with poor
scalability. The replacement of NaN3 with azidotrimethylsi-
lane,16 rendered the reaction homogeneous, more robust to scale
up, and improved the yield to 74%. LAH reduction of 24
produced the corresponding α-seleno amine 25 in 42% yield,
similar results were obtained with Staudinger reaction con-
ditions. A HATU-mediated amide coupling reaction between 25
and Fmoc-L-Cys-STr generated the elaborated scaffold 26,
poised for the key Grieco elimination. Treatment of 26 with
hydrogen peroxide in THF smoothly delivered the protected 27
In summary, we have completed the first total synthesis of
gombamide A (1), employing a convergent synthetic route that
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(9) Izawa, T.; Nishiyama, S.; Yamamura, S. Tetrahedron 1994, 48,
13593−13600.
required 18 synthetic steps, with an overall yield of 2.5% from p-
hydroxybenzldehyde 21 (10 linear step sequence), and 9.1%
from benzyl L-phenylalaninate 14 (11 linear step sequence). This
synthetic route gives access to Gombamide A, allowing further
characterization of its biological properties.
(10) (a) Boger, D. L.; Ichikawa, S. J. Am. Chem. Soc. 2000, 122, 2956−
2957. (b) Boger, D. L.; Ichikawa, S.; Tse, W. C.; Hedrick, M. P.; Jin, Q. J.
Am. Chem. Soc. 2001, 123, 561−568.
(11) (a) Akaji, K.; Tatsumi, T.; Yoshida, M.; Kimura, T.; Fujiwara, Y.;
Kiso, Y. J. Chem. Soc., Chem. Commun. 1991, 3, 167−168. (b) Koide, T.;
Otaka, A.; Suzuki, H.; Fujii, N. Synlett 1991, 5, 345−346. (c) Akaji, K.;
Tatsumi, T.; Yoshida, M.; Kimura, T.; Fujiwara, Y.; Kiso, Y. J. Am. Chem.
Soc. 1992, 114, 4137−4143.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b01825.
(12) (a) Manning, M.; Lammek, B.; Bankowski, K.; Seto, J.; Sawyer, W.
H. J. Med. Chem. 1985, 28, 1485−1491. (b) Theodoropoulos, D.;
Poulos, C.; Gatos, D.; Cordopatis, P.; Escher, E.; Mizrahi, J.; Regoli, D.;
Dalietos, D.; Furst, A.; Lee, T. D. J. Med. Chem. 1985, 28, 1536−1539.
(13) (a) Shen, R.; Porco, J. A. Org. Lett. 2000, 2, 1333−1336. (b) Shen,
R.; Lin, C. T.; Porco, J. A. J. Am. Chem. Soc. 2002, 124, 5650−5651.
(c) Jiang, L.; Job, G. E.; Klapars, A.; Buchwald, S. L. Org. Lett. 2003, 5,
3667−3669.
1
Experimental procedures, characterization data, and H
and 13C NMR spectra for new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
(14) Xiao, D.; East, S. P.; Joullie,
9631−9632.
́
M. M. Tetrahedron Lett. 1998, 39,
*E-mail: craig.lindsley@vanderbilt.edu
Notes
(15) (a) Tingoli, M.; Tiecco, M.; Chianelli, D.; Balducci, R.;
Temperini, A. J. Org. Chem. 1991, 56, 6809−6813. (b) Tingoli, M.;
Tiecco, M.; Testaferri, L.; Andrenacci, R.; Balducci, R. J. Org. Chem.
1993, 58, 6097−6102. (c) Czernecki, S.; Randriamandimby, D.
Tetrahedron Lett. 1993, 34, 7915−7916. (d) Gupta, V.; Besev, M.;
Engman, L. Tetrahedron Lett. 1998, 39, 2429−2432.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.W.L. thanks the William K. Warren Family and Foundation for
funding the William K. Warren, Jr. Chair in Medicine and
support of our programs. P.M.G. thanks the VISP program for
their support.
(16) (a) Mironov, Y. V.; Sherman, A. A.; Nifantiev, N. E. Tetrahedron
Lett. 2004, 45, 9107−9110. (b) Mironov, Y. V.; Grachev, A. A.; Lalov, A.
V.; Sherman, A. A.; Egorov, M. P.; Nifantiev, N. E. Russ. Chem. Bull.
2009, 58, 284−290.
REFERENCES
■
(1) Woo, J.-K.; Jeon, J.; Kim, C.-K.; Sim, C. J.; Oh, D.-C.; Oh, K.-B.;
Shin, J. J. Nat. Prod. 2013, 76, 1380−1383.
(2) Reviews on cyclopeptide alkaloids: (a) Gournelis, D. C.; Laskaris,
́
G. G.; Verpoorte, R. Nat. Prod. Rep. 1997, 14, 75−82. (b) Joullie, M. M.;
Richard, D. J. Chem. Commun. 2004, 2004, 2011−2015. (c) Tan, N.-H.;
Zhou, J. Chem. Rev. 2006, 106, 840−895.
(3) Kazlauskas, R.; Lidgard, R. O.; Murphy, P. T.; Wells, R. J.; Blount, J.
F. Aust. J. Chem. 1981, 34, 765−786.
(4) (a) Andersen, J. A.; Stonard, R. J. Can. J. Chem. 1979, 57, 2325−
2328. (b) Stonard, R. J.; Andersen, J. A. J. Org. Chem. 1980, 45, 3687−
3691. (c) Oltz, E. M.; Bruening, R. C.; Smith, M. J.; Kustin, K.;
Nakanishi, K. J. Am. Chem. Soc. 1988, 110, 6162−6172. (d) Casapullo,
A.; Finamore, E.; Minale, L.; Zollo, F. Tetrahedron Lett. 1993, 34, 6297−
6300. (e) Palermo, J. A.; Rodriguez Brasco, M. F.; Seldes, A. M.
Tetrahedron 1996, 52, 2727−2734. (f) Tincu, J. A.; Taylor, S. W. J. Nat.
Prod. 2002, 65, 377−378. (g) Rao, M. R.; Falukner, D. J. J. Nat. Prod.
2004, 67, 1064−1066. (h) McKay, M. J.; Carroll, A. R.; Quinn, R. J. J.
Nat. Prod. 2005, 68, 1776−1778. (i) Guella, G.; Frassanito, R.; Mancini,
I.; Sandron, T.; Modeo, L.; Verni, F.; Dini, F.; Petroni, G. Eur. J. Org.
Chem. 2010, 2010, 427−434. (j) Yin, S.; Cullinane, C.; Carroll, A. R.;
Quinn, R. J.; Davis, R. A. Tetrahedron Lett. 2010, 51, 3403−3405.
(5) (a) Davis, R. A.; Mangalindan, G. C.; Bojo, Z. P.; Antemano, R. R.;
Rodriguez, N. O.; Concepcion, G. P.; Samson, S. C.; De Guzman, D.;
Cruz, L. J.; Tasdemir, D.; Harper, M. K.; Feng, X.; Carter, G. T.; Ireland,
C. M. J. Org. Chem. 2004, 69, 4170−4176. (b) Williams, D. E.; Austin, P.;
Diaz-Marrero, A. R.; Van Soest, R.; Matainaho, T.; Roskelley, C. D.;
Roberge, M.; Andersen, R. J. Org. Lett. 2005, 7, 4173−4176. (c) Li, H.;
Bowling, J. J.; Fronczek, F. R.; Hong, J.; Jabba, S. V.; Murray, T. F.; Ha,
N.-C.; Hamman, M. T.; Jung, J. H. Biochim. Biophys. Acta, Gen. Subj.
2013, 1830, 2591−2599.
(6) Whitson, E. L.; Ratnayake, A. S.; Bugni, T. S.; Harper, M. K.;
Ireland, C. M. J. Org. Chem. 2009, 74, 1156−1162.
(7) (a) Sugawara, F.; Kim, K.-W.; Uzawa, J.; Yoshida, S.; Takahashi, N.;
Curtis, R. W. Tetrahedron Lett. 1990, 31, 4337−4340. (b) Kim, K.-W.;
Sugawara, F.; Uzawa, J.; Yoshida, S.; Murofoshi, N.; Takahashi, N.;
Curtis, R. W.; Kanai, M. Tetrahedron Lett. 1990, 32, 6715−6718.
(8) Nakaya, M.; Oguri, H.; Takahashi, K.; Fukushi, E.; Watanabe, K.;
Oikawa, H. Biosci., Biotechnol., Biochem. 2007, 71, 2969−2976.
D
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Org. Lett. XXXX, XXX, XXX−XXX