Total Synthesis and Biological Evaluation of Apratoxin e and Its C30 Epimer: Configurational Reassignment of the Natural Product

Article abstract of DOI:10.1021/acs.orglett.6b02780

Apratoxin E provided the inspiration for the design of apratoxin A/E hybrids under preclinical development. Through total synthesis using two different strategies, it was determined that the originally proposed configuration of the thiazoline at C30 is op

Full text of DOI:10.1021/acs.orglett.6b02780

Letter
pubs.acs.org/OrgLett
Total Synthesis and Biological Evaluation of Apratoxin E and Its C30
Epimer: Configurational Reassignment of the Natural Product
Ping Wu,§,† Weijing Cai,§,‡,∥ Qi-Yin Chen,‡,∥ Senhan Xu,† Ruwen Yin,† Yingxia Li,† Wei Zhang,*,†
and Hendrik Luesch*,‡,∥
†School of Pharmacy, Fudan University, Shanghai, China
‡Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610, United States
∥Center for Natural Products, Drug Discovery and Development (CNPD3), University of Florida, Gainesville, Florida 32610, United
States
S
* Supporting Information
ABSTRACT: Apratoxin E provided the inspiration for the
design of apratoxin A/E hybrids under preclinical develop-
ment. Through total synthesis using two different strategies, it
was determined that the originally proposed configuration of
the thiazoline at C30 is opposite from that in apratoxin A, in
contrast to previous assumptions on biosynthetic grounds. The
epimer and true natural apratoxin E were synthesized, and the
biological activities were evaluated.
pratoxins are potent cytotoxins derived from marine
1
Acyanobacteria.
Their cytotoxicity is due to potent
inhibition of cotranslational translocation, leading to down-
regulation of various receptor tyrosine kinases and reduced
growth factor secretion.1g Apratoxin A (1, Figure 1) possesses
broad-spectrum differential in vitro activity;2 however, it also
showed irreversible toxicity in vivo and was not well tolerated.3a
We suspected earlier that the Michael acceptor in the modified
cysteine (moCys) unit could be a liability in vivo. Apratoxin E
(2, 6; Figure 1) has been the only natural apratoxin to date
without the Michael acceptor in this position; however, the
C34−C35 double bond in this compound reduced the
antiproliferative activity against different colon cancer cells by
15- to 31-fold.1c,3a In order to overcome the drawbacks of
apratoxin A and develop lead compounds with better activity,
we previously designed and synthesized apratoxin S4, a hybrid
of apratoxins A and E, containing the hydroxylated polyketide
unit (from A) and saturated moCys unit (from E), as well as a
series of analogues, including published apratoxins S5−S9.3
Apratoxin S4 (3, Figure 1) showed greater potency and efficacy
in a colorectal tumor xenograft model than the parent molecule
(apratoxin A) and was much better tolerated in vivo.3a
Apratoxin S9 (5, Figure 1), the C30 epimer of apratoxin S4,
exhibited even greater in vitro potency with subnanomolar IC50
Figure 1. Structures of apratoxins A and E and related synthetic hybrid
values. The C30 configuration is central to the present report.
In the synthesis of apratoxins S4−S7 and S9, we noticed that
the hydroxy group at C35 tended to dehydrate to form a
conjugated system with the thiazoline.3 Specifically, this
tendency is higher for apratoxin S7 (4, Figure 1) than other
analogues. Initially, we expected that the dehydration product
in the synthesis of apratoxin S7 should be apratoxin E
(Supporting Information, Figure S1).1c Contrary to our
compounds.
expectation, the NMR spectra of the dehydration product did
not match with those of the natural product apratoxin E (SI,
Received: September 14, 2016
© XXXX American Chemical Society
A
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Figure S2). In order to resolve the discrepancy, we aimed to
synthesize apratoxin E using a novel strategy (Scheme 1). We
apratoxin E in which a new synthetic strategy was utilized,
which could be used for general route for the synthesis of other
apratoxins.
As shown in Scheme 1, treatment of the commercially
available and inexpensive (−)-citronellal with tert-butyl lithium
gave the alcohol 7 in 95% yield. Oxidation of the alcohol and
simultaneous bromination of the olefin with 1,3-dibromo-5,5-
dimethylhydantoin (DDH)4 afforded ketone 8 in good yield.
Reduction of this ketone by using (R)-CBS5 in the presence of
BH3-Me2S followed by treatment with KOH gave alcohol 10 in
high yield. The configuration of the newly generated stereo-
center and the diastereoselectivity (dr = 11:1) in the reduction
step were determined by conversion of 10 to the corresponding
(R)- and (S)-Mosher’s esters6 (see SI). Installation of proline
residue, ozonolysis, and then exposure of the resulting aldehyde
to allyl 2-(diethoxyphosphoryl)acetate under basic conditions
furnished ester 12. Cleavage of the allyl ester, followed by
coupling with the free amine of modified cysteine 133 gave 14
in a yield of 56% over three steps. Next, the thiazoline unit was
assembled using a tandem deprotection/cyclodehydration
strategy developed by Kelly.7 Removal of the allyl ester
protection of 15 and coupling with the free amine from 16 gave
the linear peptide 17 in 73% yield. Cleavage of allyl ester by
Pd(PPh3)4/N-methylaniline (NMA) and the removal of Fmoc
by Et2NH/CH3CN from 17 smoothly gave unmasked
cyclization precursor. However, only trace amount of the
expected product 2, (30S)-apratoxin E, was obtained under
various coupling conditions. It is possible that the preformed
double bond prevented the formation of macrocyclization.
Similarly, in our previous work with the dehydrated
intermediate 15, only 5% cyclized final product was obtained
(SI, Figure S1, Route A).
Scheme 1. Attempt To Synthesize Apratoxin E with Pre-
Assembled Carbon−Carbon Double Bond
The polyketide domain of 2 was simpler than that of 1 (and
many of its analogues), having a trans carbon−carbon double
bond instead of a flexible saturated chain. We aimed to
introduce a hydroxy as a latent group for the olefin at this site.
As we disclosed previously, the hydroxy at C35 of apratoxin A
was prone to elimination to E-dehydroapratoxin A even when
exposed to weak acidic CDCl3.1b Additionally, we also observed
similar dehydration in the synthesis of apratoxin A/E hybrids.3
However, when the hydroxy group was masked with an
electron-withdrawing group (2,2,2-trichloroethoxycarbonyl,
Troc), the elimination was accelerated and occurred as soon
as the thiazoline ring formed, according to Doi’s research.8
Taking inspiration from these known side-reactions, we
speculated that masking this free hydroxy with an electron-
donating group might avoid the dehydration in the preparation
of the intermediates. However, the protecting group should
also be easily removed to release the free hydroxy, which could
be converted to the desired trans carbon−carbon double bond
after the macrocyclization. Forsyth and Chen9 converted the
linear peptide bearing the triethylsilyl (TES) ether at the C35
position to the apratoxin A scaffold and then safely removed the
TES protecting group in the last step.
found that the product obtained via the new total synthesis was
the same as the dehydration product from the initial synthesis
of apratoxin S7 and also did not match the published apratoxin
E (SI, Figures S12−S14).
With these ideas in mind, we then advanced intermediate 11
to alcohol 18 via Reformatsky reaction10 (Scheme 2).
Protecting group manipulations followed by installation of
the modified cysteine residue via HATU-mediated condensa-
tion gave 19 in 72% yield over three steps. The thiazoline unit
was assembled, and the TES protecting group was exchanged
for Troc immediately to avoid undesired elimination at this
stage. Removal of the allyl group of 22 followed by coupled
with the free amine from 16 with the assistance of 3-
We suspected that apratoxin E must have 30R configuration
for the thiazoline because C30 was the only stereocenter that
was not experimentally proven but only assumed to be 30S, as
the same with other natural apratoxins from the same
location.1c We embarked on the synthesis of naturally occurring
apratoxin E. Since apratoxin A/E hybrids are now in preclinical
development, it was imperative to unambiguously determine
the absolute configuration. The final results confirmed our
speculation. In this letter, we report the corrected structure of
B
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trans configuration. However, the NMR data of 2 did not match
that of the natural product, but were identical to the data of
dehydrated product of apratoxin S7, which we previously
prepared (SI, Figures S2 and S12).
Scheme 2. Successful Synthesis of Apratoxin E and Its
Epimer with Late-Stage Formation of the Carbon−Carbon
Double Bond
After the successful synthesis of (30S)-apratoxin E (2), we
then turned to the preparation of its C30 epimer (6) by
following the same procedure but using ent-13 instead of 13
(Scheme 2).
With these two isomers in hand, we were able to compare
their spectroscopic data with those of natural apratoxin E and
unambiguously confirmed that (30R)-apratoxin E (6) is the
natural product (and thus has the same configuration as
apratoxin S9) and that (30S)-apratoxin E is actually 30-epi-
apratoxin E (SI, Figures S3−S14). NMR data was also obtained
in benzene-d6 in order to confirm (E)-configuration of the
double bond (SI, Figures S8 and S14).1c
Both isomers were tested for their antiproliferative activities
in HCT116 cells. Consistent with the reduced potency of
natural apratoxin E compared with apratoxin A in colon cancer
cells,1c,3a (30R)-apratoxin E (6) exhibits about 9-fold less
activity (IC50 52.1 nM, SI, Figure S15) than apratoxin A (IC50
5.97 nM)3a in HCT116 cells. While (30S)-apratoxin E (2)
showed 17-fold less activity with IC50 value of 100 nM (SI,
Figure S15), indicating the positive impact of 30R on activity.
Importantly, this trend is in concert with the SAR study of
apratoxin S4 (3) and S9 (5).3b
In summary, we accomplished the first total synthesis of
apratoxin E and its C30 epimer, unambiguously assigning and
correcting the originally proposed absolute configuration. This
result indicates that apratoxin S9 is the truest apratoxin A/E
hybrid with identical configurations of the stereocenters “taken”
from the respective portions of the natural products.3b The
formal epimerization of C30 for apratoxin A versus apratoxin E
also fuels the interest in the biosynthesis of these compounds.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b02780.
Experimental procedures, analytical data, and 1H and 13
NMR spectra for all compounds (PDF)
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: zhangw416@fudan.edu.cn.
*E-mail: luesch@cop.ufl.edu.
Author Contributions
§P.W. and W.C. contributed equally to this work.
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
(DEPBT)11 furnished 23 in 66% yield.
With the advanced intermediate 23 in hand, we embarked on
the final steps of the total synthesis of apratoxin E. Releasing of
the carboxylic acid from allyl ester and removal of the Fmoc
moiety on N-terminus followed by DEPBT-mediated macro-
cyclization furnished the apratoxin scaffold. Notably, part of the
TES was destroyed during the coupling steps. Treatment of the
mixture of 24/25 with HF-pyridine removed the TES group
thoroughly and generated a small amount of dehydrated
product (2) (monitored by LC−MS). Finally, exposure of the
mixture of 25 and 2 to Martin’s reagent12 afforded the
originally proposed apratoxin E (30S), with the carbon−carbon
double bond generated at the expected position and desired
Notes
The authors declare the following competing financial
interest(s): H.L. is co-founder of Oceanyx Pharmaceuticals,
Inc., which has licensed patents and patent applications related
to the subject matter.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (81573340), the “Zhuo
Xue” Talent Plan of Fudan University, and National Institutes
of Health grant R01CA172310.
C
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REFERENCES
■
(1) (a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett,
T. H. J. Am. Chem. Soc. 2001, 123, 5418−5423. (b) Luesch, H.;
Yoshida, W. Y.; Moore, R. E.; Paul, V. J. Bioorg. Med. Chem. 2002, 10,
1973−1978. (c) Matthew, S.; Schupp, P. J.; Luesch, H. J. Nat. Prod.
́
2008, 71, 1113−1116. (d) Gutierrez, M.; Suyama, T. L.; Engene, N.;
Wingerd, J. S.; Matainaho, T.; Gerwick, W. H. J. Nat. Prod. 2008, 71,
1099−1103. (e) Tidgewell, K.; Engene, N.; Byrum, T.; Media, J.; Doi,
T.; Valeriote, F. A.; Gerwick, W. H. ChemBioChem 2010, 11, 1458−
1466. (f) Thornburg, C. C.; Cowley, E. S.; Sikorska, J.; Shaala, L. A.;
Ishmael, J. E.; Youssef, D. T. A.; McPhail, K. L. J. Nat. Prod. 2013, 76,
1781−1788. (g) Liu, Y.; Law, B. K.; Luesch, H. Mol. Pharmacol. 2009,
76, 91−104.
(2) Luesch, H.; Chanda, S. K.; Raya, R. M.; De Jesus, P. D.; Orth, A.
P.; Walker, J. R.; Belmonte, J. C. I.; Schultz, P. G. Nat. Chem. Biol.
2006, 2, 158−167.
(3) (a) Chen, Q.; Liu, Y.; Luesch, H. ACS Med. Chem. Lett. 2011, 2,
861−865. (b) Chen, Q.; Liu, Y.; Cai, W.; Luesch, H. J. Med. Chem.
2014, 57, 3011−3029.
(4) (a) Taneja, S. C.; Sethi, V. K.; Andotra, S. S.; Koul, S.; Qazi, G. N.
Synth. Commun. 2005, 35, 2297−2303. (b) Khazaei, A.; Abbasi, F.;
Kianiborazjani, M.; Saednia, S. J. Braz. Chem. Soc. 2014, 25, 361−364.
(5) (a) Corey, E. J.; Bakshi, R. K.; Shibata, S. J. Am. Chem. Soc. 1987,
109, 5551−5553. (b) Corey, E. J.; Bakshi, R. K. Tetrahedron Lett. 1990,
31, 611−614. (c) Corey, E. J.; Helal, C. J. Angew. Chem., Int. Ed. 1998,
37, 1986−2012.
(6) (a) Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Org. Chem. 1973,
38, 2143−2147. (b) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H.
J. Am. Chem. Soc. 1991, 113, 4092−4096.
(7) You, S.; Razavi, H.; Kelly, J. W. Angew. Chem., Int. Ed. 2003, 42,
83−85.
(8) Doi, T. Chem. Pharm. Bull. 2014, 62, 735−743.
(9) (a) Chen, J.; Forsyth, C. J. J. Am. Chem. Soc. 2003, 125, 8734−
8735. (b) Chen, J.; Forsyth, C. J. Proc. Natl. Acad. Sci. U. S. A. 2004,
101, 12067−12072.
(10) (a) Reformatsky, S. Ber. Dtsch. Chem. Ges. 1887, 20, 1210−1211.
(b) Fuerstner, A. Synthesis 1989, 1989, 571−590. (c) Ocampo, R.;
Dolbier, W. R., Jr. Tetrahedron 2004, 60, 9325−9374.
(11) (a) Li, H.; Jiang, H.; Ye, Y.-H.; Fan, C.; Romoff, T.; Goodman,
M. Org. Lett. 1999, 1, 91−93. (b) Ye, Y.-H.; Li, H.; Jiang, X.
Biopolymers 2005, 80, 172−178.
(12) Martin, J. C.; Arhart, R. J. J. Am. Chem. Soc. 1971, 93, 4327−
4329.
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