Total Synthesis and Stereochemical Assignment of Sunshinamide and Its Anticancer Activity

Article abstract of DOI:10.1021/acs.orglett.0c00070

Total synthesis of cyclodepsipeptide sunshinamide has been achieved for the first time using a convergent approach. The key features of this synthesis comprise Crimmins acetate aldol, Shiina esterification, amide coupling, macrolactamization, and an I

Full text of DOI:10.1021/acs.orglett.0c00070

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Letter
Total Synthesis and Stereochemical Assignment of Sunshinamide
and Its Anticancer Activity
Joyanta Mondal, Ruma Sarkar, Prosenjit Sen,* and Rajib Kumar Goswami*
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ABSTRACT: Total synthesis of cyclodepsipeptide sunshinamide has been achieved
for the first time using a convergent approach. The key features of this synthesis
comprise Crimmins acetate aldol, Shiina esterification, amide coupling, macro-
lactamization, and an I2-mediated deprotection with concomitant disulfide-bridge
formation. This synthetic study enabled the unambiguous determination of the
stereochemistry of the unassigned stereocenter of the isolated sunshinamide. The
cytotoxicity of sunshinamide and one of its analogues was evaluated against different
cancerous and noncancerous human cell lines, which revealed their attractive and
selective activities toward cancer cells at very low concentrations.
cyclic scaffolds: one 15-membered and the other 8-membered.
icyclic natural products containing a disulfide linkage
Bmake up an important class of molecules that exhibit a D-Phenylalanine and two consecutive L-cysteines are embedded
in the peptide backbone, whereas the nonpeptidic part is 3-
hydroxydecanoic acid.4 The stereochemistry of the hydroxy
center remained unestablished. Sunshinamide is architecturally
novel; the consecutive L-cysteines are connected through a
disulfide bond. The unique architectural features and
promising bioactivity of sunshinamide together with our
interest5 in the chemical synthesis of bioactive natural products
prompted us to develop a total synthesis. In this work, we
report a convergent and flexible synthetic route for
sunshinamide and one of its analogues for the first time. We
also disclose herein their cytotoxicity against different human
cancerous and noncancerous cell lines with the aim of
determining their pharmaceutical relevance.
The retrosynthesis analysis of sunshinamide is shown in
Scheme 1. The stereochemistry of the hydroxy center in the
nonpeptidic segment of sunshinamide remained undisclosed
during the determination of the structure. Thus, we planned to
synthesize both possible stereoisomers 1a and 1b to compare
them with the reported data of the isolated natural product.
Compounds 1a and 1b could be constructed from compounds
2a and 2b, respectively, by S−S bond formation. There are
several possible sites in compounds 1a and 1b for macro-
cyclization. We relied on macrolactamization and planned to
disconnect compounds 2a and 2b between two cysteine
residues to realize compounds 3a and 3b, respectively, which
broad range of biological activities and pharmacological
properties. Many of these natural products showed striking
anticancer1 and immunosuppressant2 activities. Considerable
efforts have been made toward the synthesis of this class of
natural products and their analogues by the synthetic
chemistry community,3 which led some of them into a very
advanced stage of a drug discovery program.1g Thus, searching
for new members of this class of natural products and chemical
synthesis and evaluation of their biological efficacies is a
subject of great importance. Piel and co-workers in 2018
discovered first the disulfide-containing cyclodepsipeptide
sunshinamide (1) (Figure 1) from a plant-associated marine
bacterium Gynuella sunshinyii YC6258, using a genome-based
identification method.4 Sunshinamide showed potent cytotox-
icity against HeLa cells with an IC50 value of 0.59 μM. The
structure of the molecule was proposed on the basis of
spectroscopic investigations that revealed that it comprises two
Received: January 7, 2020
Figure 1. Proposed structure of sunshinamide (1).
https://dx.doi.org/10.1021/acs.orglett.0c00070
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Scheme 1. Retrosynthetic Analysis of Sunshinamide
Scheme 2. Synthesis of Compounds 7a and 7b
could further be made by esterification of compound 5 with
compounds 4a and 4b, respectively. Compounds 4a and 4b
would be constructed separately from compounds 7a and 7b,
respectively, by amide coupling with compound 6. Com-
pounds 7a and 7b could be synthesized from octanal using
Crimmins acetate aldol as one of the key steps.
DIPEA11 coupling conditions were screened. HATU/HOAt/
DIPEA was found to function efficiently to produce the
corresponding coupling product that was subsequently treated
with TBAF to obtain compound 4a in 67% yield over three
steps. Compound 4b was also synthesized from compound 6
using acid 7b in very good overall yield following exactly the
same chemistry. Next, both compounds 4a and 4b were
esterified separately with the known acid 512 in the presence of
MNBA (2-methyl-6-nitrobenzoic anhydride)/DMAP/Et3N
following the Shiina protocol13,5b to obtain compounds 3a
and 3b, respectively
The completion of the total synthesis of compounds 1a and
1b is depicted in Scheme 4. Compound 3a was treated
separately with TFA (40% in CH2Cl2) to obtain the
corresponding Boc- and tert-butyl-deprotected product. The
stage was now set to perform the crucial macrolactamization.
Different conditions have been screened at a concentration of
∼10−3 M at this stage (Table 1). HATU/HOAt/DIPEA was
found to be the best condition in our case to obtain compound
2a in 72% yield. No epimerization or cyclodimerization was
observed during this reaction. Compound 2b was also
prepared with a similar yield from compound 3a following
The synthesis of compounds 7a and 7b is depicted in
Scheme 2. Octanal was subjected to Crimmins acetate
aldol,5a−c,6 with the known thiazolidinethione 96b,c in the
presence of TiCl4/DIPEA to obtain aldol adducts 10a and
10b, respectively, in 70% yield (dr = 7:3), which were
separated by silica gel column chromatography. The absolute
stereochemistry of the newly generated hydroxy center of
compound 10a was determined by converting it to (S)- and
(R)-MTPA esters 11a and 11b, respectively.5c,7 All of the
1
protons of the pairs of Mosher’s esters were assigned by H
NMR. The negative Δδ values (Δδ = δS − δR) (Scheme 2)
found for H-2 protons from esters 11a and 11b clearly
confirmed the desired (R)-configuration of the originated
hydroxy center. Compound 10a was then reacted with
TBSOTf/2,6-lutidine followed by LiOH/H2O2 to obtain the
known acid 7a.8 Similarly, the absolute stereochemistry of the
hydroxy center of compound 10b was established as the (S)-
configuration from (S)- and (R)-MTPA esters 12a and 12b,
respectively [Δδ = (δS − δR) = +ve], which was transformed
finally to the known acid 7b.8
the chemistry of compound 2a. Both compounds 2a and 2b
3a,b
were then reacted with I2/MeOH-CH2Cl2
to access the
The synthesis of compounds 3a and 3b is described in
Scheme 3. Known amine 139 and acid 1410 prepared from L-
cysteine and D-phenylalanine, respectively, following literature
procedures, were coupled together using EDCI/HOBT/
DIPEA11 to realize compound 6 in 86% yield.
disulfide bridge-containing targeted compounds 1a and 1b,
respectively, in excellent yield (90%). The spectroscopic data
1
of both compounds 1a and 1b were recorded. H and 13C
NMR data of compound 1a were in very good agreement with
the data of isolated natural products, whereas discrepancies in
chemical shifts between the synthesized compound 1b and
isolated sunshinamide were observed (see the NMR
comparisons in Tables S1 and S2). The major anomalies
Compound 6 was then treated with Et2NH5d to obtain the
corresponding Fmoc-deprotected amine that was then coupled
with acid 7a. EDCI/HOBt/DIPEA and HATU/HOAt/
B
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Scheme 3. Synthesis of Compounds 3a and 3b
the splitting patterns of H2-2 and H2-4 were also quite different
from the reported spectra. The 13C signals of C-1, C-3, C-1′,
C-1′″, and C-2′″ of compound 1b also differ from the isolated
values. The 2D NMR correlations of compound 1a were also
in accordance with the reported data. These observations
clearly confirmed that the structures of isolated sunshinamide
and synthesized compound 1a were identical. However, the
Scheme 4. Completion of the Synthesis of Compounds 1a
and 1b
observed difference in the specific rotation [observed [α]26
D
−37.1 (c 0.015, MeOH); reported [α]26 −1.5 (c 0.015,
D
MeOH)] of synthesized sunshinamide could not be reconciled
at this point.
The synthesized sunshinamide (1a) and its configurational
isomer (1b) were evaluated for their in vitro cytotoxic effects
against MDA-MB-231 (human metastatic breast adenocarci-
noma), MCF7 (human breast adenocarcinoma), HeLa
(human cervical cancer), and HepG2 (human liver cancer)
cells using the MTT reduction assay. The effects of both of the
compounds were also evaluated on noncancerous cell lines,
specifically CHOK1 (Chinese hamster ovary), WI38 (human
lung fibroblast) cells, to check whether they have differential
cytotoxic effects on cancer and noncancer cells. The results are
listed in Table 2, which revealed that the synthesized
compounds are selective toward cancer cell lines and possessed
attractive cytotoxic activity. Next, to study the mechanism
behind the cytotoxic effects, we have treated the cancer cells
with synthesized compounds (1a and 1b) and systematically
analyzed the mode of killing by measuring several cellular
assays. Confocal microscopic examination of the cancer cells
(MDA-MB-231) treated with the compounds and nuclear
DNA stained with propidium iodide showed typical apoptotic
features, like fragmented nuclei, chromatin condensation, and
formation of apoptotic bodies (Figure 2A). This study also
showed that the exposure of the synthesized compounds (1a
and 1b) to the cancer cells increased the activity of the caspase
3 (Figure 2D), which is known as the key signature regulator of
the apoptotic process. To further confirm apoptosis, treated
MDA-MB-231 cells were subjected to flow cytometric analysis
to evaluate the fragmented apoptotic DNA. Exposure of MDA-
MB-231 cancer cells to sunshinamide (1a) and its analogue
(1b) caused significant accumulation of fragmented DNA at
the sub G0/G1 phase of the cell cycle (Figure 2B,C), which is
also a characteristic feature of apoptosis. Overall, these results
Table 1. Optimization of Macrolactamization for
Compound 2a
entry
reagents
conditions
time (h) yield
1
2
3
4
EDCI/HOBt, CH2Cl2
0 °C to rt
0 °C to rt
0 °C to rt
0 °C to rt
14
16
14
3
14
72
9
HATU, HOAt/DIPEA, CH2Cl2
PyBOP/DIPEA, CH2Cl2
COMU/DIPEA, DMF
15
1
were observed in the H NMR signals of H2-2, H2-4, 2′-NH,
H-2″, 2″-NH, 2′″-NH, and H2-3″ of the synthesized
compound 1b with respect to the reported values. Moreover,
C
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Table 2. Evaluation of the Cytotoxic Activities of Sunshinamide (1a) and Its Congener (1b) with Respect to Cancer and
Noncancerous Human Cell Lines
IC50 value (μM)
MDA-MB-231
MCF-7
HeLa
HepG2
CHOK1
WI38
1a
1b
0.11 0.041
0.10 0.087
0.08 0.019
0.09 0.013
0.11 0.005
0.13 0.017
0.10 0.018
0.13 0.20
0.72 0.099
0.47 0.008
0.36 0.031
1.05 0.645
Figure 2. Induction of apoptosis by the synthesized compounds 1a and 1b. (A) MDA-MB-231 cells were incubated with IC50 doses of compounds
1a and 1b for 6 h and then stained with PI. Images were captured by confocal microscopy. Images are representative of three independent
experiments. (B and C) MDA-MB-231 cells were treated with IC50 doses of the compounds for 6 and 12 h and subjected to cell cycle analysis by
flow cytometry, following staining with PI. The percentage of the sub G1 phase is graphically represented. Values are expressed as the means the
standard deviation (SD) of three independent experiments. (D) MDA-MB-231 cells were exposed to IC50 doses of the compounds, and caspase 3
activities were assessed. Values are expressed as the means the SD of triplicate samples. The scale bar is 10 μM.
suggest that sunshinamide showed cytotoxicity by inducing
apoptosis in the cancer cells.
convergent synthetic route we have developed allows easy
access to a large array of analogues of sunshinamide.
Evaluation of the detailed signaling cascades involved in
inducing apoptosis by sunshinamide and its structure−activity
relationship studies are in progress and will be disclosed in due
course.
In summary, we have achieved the first total synthesis of
sunshinamide in nine linear steps from octanal with an overall
yield of 13.7%. The previously unassigned C-3 stereocenter of
sunshinamide has been established unambiguously, and the
absolute stereochemistry was determined as R. The cytotox-
icity of synthesized sunshinamide and that of its C-3 epimer
were evaluated against a number of human cancerous and
noncancerous cell lines, which revealed their promising and
selective activities with respect to cancer cells with very
encouraging IC50 values. Notably, the stereochemistry of the
C-3 center of sunshinamide has no such differential cytotoxic
effect against human cancerous (MDA-MB-231, MCF-7,
HeLa, and HepG2) cell lines, but such differences were
observed in the case of human noncancerous (CHOK1 and
WI38) cell lines. Further study revealed that sunshinamide
(1a) and its analogue (1b) have cytotoxic effects on human
cancer cells through the induction of apoptosis. The
ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c00070.
Experimental procedure, spectroscopic data, Tables S1
and S2, copies of NMR (1H and 13C) and HRMS
spectra of representative compounds, and 2D NMR data
(COSY, HSQC, HMBC, NOESY, and ROESY) of
compounds 1a and 1b (PDF)
D
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K.; Ganesan, A.; Takahashi, T. Tetrahedron Lett. 2006, 47, 1177−
1180. (l) Narita, K.; Fukui, Y.; Sano, Y.; Yamori, T.; Ito, A.; Yoshida,
M.; Katoh, T. Eur. J. Med. Chem. 2013, 60, 295−304. (m) Di Maro,
S.; Pong, R. C.; Hsieh, J. T.; Ahn, J. M. J. Med. Chem. 2008, 51, 6639−
6641. (n) Yurek-George, A.; Cecil, A.; Mo, A. H. K.; Wen, S.; Rogers,
H.; Habens, F.; Maeda, S.; Yoshida, M.; Packham, G.; Ganesan, A. J.
Med. Chem. 2007, 50, 5720−5726. (o) Benelkebir, H.; Marie, S.;
Hayden, A. L.; Lyle, J.; Loadman, P. M.; Crabb, S. J.; Packham, G.;
Ganesan, A. Bioorg. Med. Chem. 2011, 19, 3650−3658.
AUTHOR INFORMATION
Corresponding Authors
■
Prosenjit Sen − School of Biological Sciences, Indian Association
for the Cultivation of Science, Kolkata 700032, India;
orcid.org/0000-0002-1233-1822; Email: bcps@iacs.res.in
Rajib Kumar Goswami − School of Chemical Sciences, Indian
Association for the Cultivation of Science, Kolkata 700032,
India; orcid.org/0000-0001-7486-0618; Email: ocrkg@
iacs.res.in
(4) Ueoka, R.; Bhushan, A.; Probst, S. I.; Bray, W. M.; Lokey, R. S.;
Linington, R. G.; Piel, J. Angew. Chem. 2018, 130, 14490.
(5) (a) Guchhait, S.; Chatterjee, S.; Ampapathi, R. S.; Goswami, R.
K. J. Org. Chem. 2017, 82, 2414−2435. (b) Das, S.; Paul, D.;
Goswami, R. K. Org. Lett. 2016, 18, 1908−1911. (c) Das, S.;
Goswami, R. K. J. Org. Chem. 2013, 78, 7274−7280. (d) Das, S.;
Goswami, R. K. J. Org. Chem. 2014, 79, 9778−9791. (e) Kuilya, T. K.;
Goswami, R. K. Org. Lett. 2017, 19, 2366−2369. (f) Paul, D.; Das, S.;
Goswami, R. K. J. Org. Chem. 2017, 82, 7437−7445. (g) Paul, D.;
Saha, S.; Goswami, R. K. Org. Lett. 2018, 20, 4606−4609.
Other Authors
Joyanta Mondal − School of Chemical Sciences, Indian
Association for the Cultivation of Science, Kolkata 700032,
India
Ruma Sarkar − School of Biological Sciences, Indian Association
for the Cultivation of Science, Kolkata 700032, India
Complete contact information is available at:
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(6) (a) Crimmins, M. T.; King, B. W.; Tabet, E. A.; Chaudhary, K. J.
Org. Chem. 2001, 66, 894−902. (b) Crimmins, M. T.; Shamszad, M.
Org. Lett. 2007, 9, 149−152. (c) Cochrane, S. A.; Surgenor, R. R.;
Khey, K. M. W.; Vederas, J. C. Org. Lett. 2015, 17, 5428−5431.
(7) (a) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092−4096. (b) Dale, J. A.; Mosher, H. S. J.
Am. Chem. Soc. 1973, 95, 512−519. (c) Mosher, H. S.; Dull, D. L.;
Dale, J. A. J. Org. Chem. 1969, 34, 2543−2549. (d) Seco, J. M.;
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
J.M. and R.S. thank the Council of Scientific and Industrial
Research, New Delhi, and DST for the research fellowship.
Financial support from the Science and Engineering Research
Board (Project EMR/2016/000988) and the Council of
Scientific & Industrial Research [Project 02(0294)/17/EMR-
II], India, to carry out this work is gratefully acknowledged.
Quinoa, E.; Riguera, R. Chem. Rev. 2004, 104, 17−117.
̃
(8) De Vleeschouwer, M.; Sinnaeve, D.; Van den Begin, J.; Coenye,
T.; Martins, J. C.; Madder, A. Chem. - Eur. J. 2014, 20, 7766−7775.
(9) (a) Jin, H.-J.; Lu, J.; Wu, X. Bioorg. Med. Chem. 2012, 20, 3465−
3469. (b) Masiukiewicz, E.; Rzeszotarska, B. Org. Prep. Proced. Int.
1999, 31, 571−572.
(10) Ishiyama, H.; Yoshizawa, K.; Kobayashi, J. Tetrahedron 2012,
68, 6186−6192.
(11) Liu, J.; Chen, W.; Xu, Y.; Ren, S.; Zhang, W.; Li, Y. Bioorg. Med.
Chem. 2015, 23, 1963−1974.
(12) Silvia, V.; Baldisserotto, A.; Scalambra, E.; Malisardi, G.; Durini,
E.; Manfredini, S. Eur. J. Med. Chem. 2012, 50, 383−392.
(13) Shiina, I.; Ibuka, R.; Kubota, M. Chem. Lett. 2002, 31, 286−
287.
REFERENCES
■
(1) (a) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.;
Goto, T.; Okuhara, M. J. Antibiot. 1994, 47, 301−310. (b) Nakajima,
H.; Kim, Y. B.; Terano, H.; Yoshida, M.; Horinouchi, S. Exp. Cell Res.
1998, 241, 126−133. (c) Shigematsu, N.; Ueda, H.; Takase, S.;
Tanaka, H.; Yamamoto, K.; Tada, T. J. Antibiot. 1994, 47, 311−314.
(d) Crabb, S. J.; Howell, M.; Rogers, H.; Ishfaq, M.; Yurek-George,
A.; Carey, K.; Pickering, B. M.; East, P.; Mitter, R.; Maeda, S.;
Johnson, P. W. W.; Townsend, P.; Shin-ya, K.; Yoshida, M.; Ganesan,
A.; Packham, G. Biochem. Pharmacol. 2008, 76, 463−475.
(e) Davidson, S. M.; Townsend, P. A.; Carroll, C.; Yurek-George,
A.; Balasubramanyam, K.; Kundu, T. K.; Stephanou, A.; Packham, G.;
Ganesan, A.; Latchman, D. S. ChemBioChem 2005, 6, 162−170.
(f) Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K.-H.; Nishiyama,
M.; Nakajima, H.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.;
Horinouchi, S. Cancer Res. 2002, 62, 4916−4921. (g) Vigushi, D. M.
Curr. Opin. Invest. Drugs 2002, 1396−1402.
(2) Skov, S. PCT Int. Appl. WO 0142282 A1 20010614, 2001.
(3) (a) Li, K. W.; Wu, J.; Xing, W.; Simon, J. A. J. Am. Chem. Soc.
1996, 118, 7237−7238. (b) Yurek-George, A.; Habens, F.; Brimmell,
M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030−
1031. (c) Greshock, T. J.; Johns, D. M.; Noguchi, Y.; Williams, R. M.
Org. Lett. 2008, 10, 613−616. (d) Chen, Y.; Gambs, C.; Abe, Y.;
Wentworth, P., Jr.; Janda, K. D. J. Org. Chem. 2003, 68, 8902−8905.
(e) Wen, S.; Packham, G.; Ganesan, A. J. Org. Chem. 2008, 73, 9353−
9361. (f) Benelkebir, H.; Donlevy, A. M.; Packham, G.; Ganesan, A.
Org. Lett. 2011, 13, 6334−6337. (g) Takizawa, T.; Watanabe, K.;
Narita, K.; Oguchi, T.; Abe, H.; Katoh, T. Chem. Commun. 2008,
1677−1679. (h) Calandra, N. A.; Cheng, Y. L.; Kocak, K. A.; Miller, J.
S. Org. Lett. 2009, 11, 1971−1974. (i) Narita, K.; Kikuchi, T.;
Watanabe, K.; Takizawa, T.; Oguchi, T.; Kudo, K.; Matsuhara, K.;
Abe, H.; Yamori, T.; Yoshida, M.; Katoh, T. Chem. - Eur. J. 2009, 15,
11174−11186. (j) Fuse, S.; Okada, K.; Iijima, Y.; Munakata, A.;
Machida, K.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Org.
Biomol. Chem. 2011, 9, 3825−3833. (k) Doi, T.; Iijima, Y.; Shin-ya,
E
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