Synthesis and biological evaluation of santacruzamate A and analogs as potential anticancer agents

Article abstract of DOI:10.1039/c4ra13889a

Santacruzamate A, a recently discovered natural product from a Panamanian marine cyanobacterium Symploca sp., features a similar structure to the clinically used histone deacetylase (HDAC) inhibitor vorinostat (SAHA). We have synthesized the natural product and a small set of analogues for SAR studies. To our surprise, the synthetic natural product santacruzamate A (1a) and the analogues did not show an obvious inhibition even at 2 μM in HDAC enzyme assays while the IC₅₀ value was 0.12 nM in the original report. However, a novel compound, 5, containing a terminal thiourea motif was found to inhibit the growth of malignant cells at submicromolar concentrations. Moreover, 5 was not cytotoxic to normal human colonic epithelial cells CCD841, suggesting that its cytotoxicity was specific to cancer cells. Further investigation indicated that the compound induced apoptosis, affected cell cycle progression and increased ROS production. We believe its mechanism of action is unrelated to HDAC inhibition and the original activity reported for santacruzamate needs to be reevaluated.

Full text of DOI:10.1039/c4ra13889a

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Synthesis and biological evaluation of
santacruzamate A and analogs as potential
Cite this: RSC Adv., 2015, 5, 1109
anticancer agents†
Qi Liu,‡ab Wenhua Lu,‡a Mingzhe Ma,a Jianwei Liao,a A. Ganesan,c Yumin Hu,a
Received 5th November 2014
Accepted 26th November 2014
ab
Shijun Wen
and Peng Huanga
*
DOI: 10.1039/c4ra13889a
www.rsc.org/advances
Santacruzamate A, a recently discovered natural product from a agents, focusing on the search of novel chemical entities for the
Panamanian marine cyanobacterium Symploca sp., features a similar successful treatment of cancer.8,9
structure to the clinically used histone deacetylase (HDAC) inhibitor
Nature has been continuously providing humans with
vorinostat (SAHA). We have synthesized the natural product and a important leads and natural product medicines for the treat-
small set of analogues for SAR studies. To our surprise, the synthetic ment of a wide spectrum of diseases.10–12 Natural products have
natural product santacruzamate A (1a) and the analogues did not show been a particularly important source of anticancer chemother-
an obvious inhibition even at 2 mM in HDAC enzyme assays while the apeutic medicines including taxanes, Vinca alkaloids and
IC50 value was 0.12 nM in the original report. However, a novel camptothecin that act upon the mitotic spindle.13 This trend is
compound, 5, containing a terminal thiourea motif was found to likely to continue as natural products are identied that
inhibit the growth of malignant cells at submicromolar concentrations. modulate specic signaling pathways in cells. One example is
Moreover, 5 was not cytotoxic to normal human colonic epithelial the relatively new eld of epigenetics relating to chromatin
cells CCD841, suggesting that its cytotoxicity was specific to cancer modelling via structural modications of the DNA and histone
cells. Further investigation indicated that the compound induced proteins. A variety of natural products have already been
apoptosis, affected cell cycle progression and increased ROS reported that inhibit the enzymes involved in epigenetics.14
production. We believe its mechanism of action is unrelated to HDAC Among these, the histone deacetylase (HDAC) family of
inhibition and the original activity reported for santacruzamate needs enzymes has received much attention. HDACs are the enzymes
to be reevaluated.
that hydrolyse acetyl-lysine amino acid residues in proteins
back to lysine and play crucial roles in diverse cellular func-
tions, while their overexpression or mutation is widely observed
in cancer cells.15–17 A number of potent natural product HDAC
inhibitors such as trichostatin A and apicidin are used as bio-
logical tools while the depsipeptide FK228 (romidepsin) has
received FDA approval for the treatment of cutaneous T-cell
lymphoma (CTCL).
Recently, Balunas and co-workers reported the isolation of
santacruzamate A (1a) from a marine Panamanian cyanobac-
terium resembling the genus Symploca.18 Santacruzamate A
shares some structural similarity with the synthetic HDAC
inhibitor vorinostat (SAHA), the rst clinically approved drug in
this class (Fig. 1). It was reported that santacruzamate A
specically inhibited the isoform HDAC2 with an IC50 of 0.12
nM. This was a surprising result given the established SAR of
HDAC inhibitors in which a zinc-binding group, such as the
hydroxamic acid in vorinostat, is important for reversible
binding to the enzyme active site.19 Instead, santacruzamate A
contains a carbamate and amide-functional groups that have
not been previously associated with potent HDAC inhibition.
The natural product azumamide A, for example, contains a
Tumors, the result of abnormal cells with uncontrolled, rapid
and pathological proliferation, cause one of the most formi-
dable afflictions globally.1,2 Apart from the use of surgical
treatment and irradiation, chemotherapy still remains the main
therapeutic strategy to treat cancer.3,4 However, one of the major
hurdles in cancer chemotherapy is attributed to the prevalence
of drug and multidrug resistance and the need for selectivity
against normal cells.5–7 Therefore, considerable efforts have
been made on the design and discovery of new anticancer
aSun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South
China, Collaborative Innovation Center for Cancer Medicine, 651 Dongfeng East
Road, Guangzhou 510060, China. E-mail: wenshj@sysucc.org.cn
bSchool of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road,
Guangzhou 510006, China
cSchool of Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
† Electronic supplementary information (ESI) available: Synthesis and
characterisation of the compounds. See DOI: 10.1039/c4ra13889a
‡ These authors contributed equally to this work.
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Fig. 1 The structures of santacruzamate A and SAHA.
carboxamide zinc-binding group and is only micromolar in
HDAC inhibition.20,21 Furthermore, the high selectivity of san-
tacruzamate A for HDAC2 was intriguing as SAHA itself is a non-
selective inhibitor active against both Class I and Class II HDAC
isoforms. With this background, it was of interest to synthesize
santacruzamate A as well as a series of analogues to investigate
the structure–activity relationships (SAR) of this new lead or the
development of anticancer agents. This has led to the identi-
cation of a potent cytotoxic compound 5 that we nevertheless
believe acts by a HDAC-independent mechanism of action.
To investigate the importance of the linker in santacruza-
mate A between the carbamate and amide structural features,
our SAR strategy was to move around the position of the amide
and also to replace the terminal ethoxycarbonyl group with
Fig. 2 The strategy to design analogues of santacruzamate A.
showed signicant HDAC2 inhibition at the concentration of 2
mM while the IC50 value of santacruzamate A was reported to be
0.12 nM.18 The assay was repeated three times on different
dates, and the results were consistent. We have also double
1
checked the spectrum of H NMR of our synthetic santacruza-
mate A (1a), and veried that our synthetic sample matches the
reported data for the natural product (Fig. 3). To exclude a
possibility that 1a might be decomposed under the enzymatic
assay conditions, a solution of 1a in DMSO was diluted in the
buffer employed for the enzymatic assay and incubated for
several hours. The sample was nally taken for mass spectros-
copy detection, and the clean major peak (m/z 301.0)
other bioisosteric functional groups. Thus,
a series of
compounds were designed and prepared alongside with the
natural product santacruzamate A itself (1a) (Fig. 2).
The synthetic route to obtain these compounds is shown in
Scheme 1. Conventional acylation of the terminal amino groups
of commercial available amino acids 6 with ethyl chloroformate
gave compounds 7a–d, while protection of 6c with di(tert-butyl)
carbonate (Boc2O) afforded 8. Amidation of the carboxylic acid
groups of 7a–d and 8 with amines or aniline afforded the
desired target products 1a–d, 2 and 3. Aer removal of the Boc
group from 3 under acidic conditions, the obtained free amine 9
underwent subsequent treatment with either isocyanatoethane
or ethyl isothiocyanate to give two additional analogues 4 and 5.
Aer the synthesis of the designed compounds 1a–d, and 3–
5, they were subsequently screened for their cellular biological
activity. To our surprise, synthetic santacruzamate A, i.e. 1a did
not show cytotoxicity against human colon cancer cells HCT-116
even at 100 mM while most of the other synthetic analogues were
inactive (Table 1). However, compound 5 inhibited the growth
of HCT-116 cells and human myeloblastic leukemia cells ML-1
with IC50 values of 6.0 and 9.4 mM. To our delight, 5 was not
cytotoxic to normal cells (CCD841) even at 100 mM. Indeed, it is
high desirable to obtain a compound with a high selectivity to
kill malignant cancer cells because most of clinical anticancer
drug also kill normal cells during therapeutical treatment. For
example, SAHA was very cytotoxic to CCD841 cells at 20 mM
although with its lower IC50 value to cancer cells.
Scheme 1 Synthesis of santacruzamate A (1a) and the analogues.
Reagents and conditions: (i) ethyl chloroformate, K2CO3, THF/H2O,
Next, we performed mechanistic enzyme assays against both
total HDACs isolated from cell lysates and the individual
recombinant isoform HDAC2, using SAHA as a positive control. cat. DMAP, CH2Cl2, 0 ꢀC to rt., (v) TFA, CH2Cl2, rt., (vi) isocyanato-
0
ꢀC to rt., (ii) Boc2O, NaOH, THF/H2O, 0 ꢀC to rt., (iii) amine, Et3N,
EDCI, cat. DMAP, CH2Cl2, 0 ꢀC to rt., (iv) phenethylamine, Et3N, EDCI,
ethane, THF, rt., (vii) isothiocyanate, THF, rt. Note: EDCI, 1-ethyl-3-(3-
When SAHA showed the expected activity with IC50 79.7 nM
against HDAC2 (Fig. S1†), none of our synthesized compounds
dimethyllaminopropyl)carbodiimide
dimethylaminopyridine.
hydrochloride;
DMAP,
4-
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Table 1 MTS assay to evaluate the effect of compounds on the
proliferation of two cancer cell lines: HCT-116 and ML-1 and normal
colonic epithelial cell line CCD841
IC50a (mM) ꢁ SEM
Compound
HCT-116
ML-1
CCD841
1a
1b
1c
1d
2
>100
>100
>100
>100
>100
NT
NT
NT
NT
90.8 ꢁ 6.9
>100
94.3 ꢁ 13.8
>100
>100
NT
3
4
5
86.0 ꢁ 9.0
>100
NT
NT
>100
20.8 ꢁ 0.17
>100
79.5 ꢁ 3.8
9.4 ꢁ 3.8
2.9 ꢁ 1.1
6.0 ꢁ 1.2
1.4 ꢁ 0.0
SAHA
a
IC50 is the drug concentration effective in inhibiting 50% of the cell
growth measured by the MTS assay. NT, not tested.
Fig. 5 The effect of 5 on cell cycle progression of (A) HCT-116 cells
and (B) ML-1 cells; (C) and (D) the normalization of the effects above.
We then further explored whether this inhibition of cell
growth and cell cycle arrest by 5 was attributed to the induction
of apoptosis. Annexin V/PI double-staining assay was used to
study whether 5 could directly induce apoptotic cell death in
HCT-116 and ML-1 cells (Fig. 6). The results indicated that 5
signicantly increased the percentage of apoptotic cells
(Annexin-V-positive) in a dose-dependent manner. No obvious
change was observed in necrotic cells (only PI stained) as
compared to control at 48 h (data not shown).
It has been widely recognized that increased endogenous
reactive oxygen species (ROS) generation can selectively eliminate
cancer cells, mainly by raising oxidative stress over the threshold
of toxicity to abnormal cancer cells.22 Recently, Schreiber and co-
1
Fig. 3 The HNMR spectra of (a) the reported santacruzamate A and
(b) our synthetic 1a.
corresponded to 1a, indicating that 1a tolerated the enzymatic
assay conditions (Fig. S2†). These solid results lead us to believe
that the original report need to be further reexamined.
While the mechanism of action of our compound 5 does not
involve HDAC inhibition, further studies were carried out to
prole its biological activity. At a concentration of 5 mM, 5 was
able to effectively suppress colony formation of HCT-116 cells in
a concentration dependent manner (Fig. 4) and to induce cell
cycle arrest at the G2/M phase of HCT-116 cells but not ML-1
cells (Fig. 5).
Fig. 6 Appotosis of (A) HCT-116 and (B) ML-1 cells induced by 5; (C)
Fig. 4 Inhibition of colony formation of HCT-116 cells by 5.
and (D) the normalization of the induced appoptosis above.
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workers have discovered that a natural product piperlongumine
selectively killed cancer cells by targeting the stress response to
ROS.23 Thus, it was of our interest to examine ROS level in HCT-
116 and ML-1 cells treated with 5 (Fig. S3†). The data indicated
that a treatment with 5 at 10 mM induced a signicant increase in
ROS levels both in HCT-116 and ML-1 cells (P < 0.05). Our results
implied that selective killing of cancer cells but not normal cells
by 5 might result from the ROS generation. Further study to
investigate its exact mechanism is under way.
4 K. Juvale, J. Gallus and M. Wiese, Bioorg. Med. Chem., 2013,
21, 7858–7873.
˜
´
5 C. P. Reyes, F. Munoz-Martınez, I. R. Torrecillas,
˜
´
C. R. Mendoza, F. Gamarro, I. L. Bazzocchi, M. J. Nunez,
´
L. Pardo, S. Castanys, M. Campillo and I. A. Jimenez, J.
Med. Chem., 2007, 50, 4808–4817.
6 H. Y. Hung, E. Ohkoshi, M. Goto, K. F. Bastow, K. Nakagawa-
Goto and K. H. Lee, J. Med. Chem., 2012, 55, 5413–5424.
7 X. Tang, X. Gu, Z. Ren, Y. Ma, Y. Lai, H. Peng, S. Peng and
Y. Zhang, Bioorg. Med. Chem. Lett., 2012, 22, 2675–2680.
8 M. Nagaraju, E. Gnana Deepthi, C. Ashwini,
M. V. P. S. Vishnuvardhan, V. Lakshma Nayak, R. Chandra,
S. Ramakrishna and B. B. Gawali, Bioorg. Med. Chem. Lett.,
2012, 22, 4314–4317.
Conclusions
In summary, a novel series of compounds were designed and
synthesized based on the natural product santacruzamate A.
The SAR study demonstrated that most of these analogues as
well as synthetic santacruzamate A showed weak cytotoxicity
against the two tested cancer cell lines, HCT-116 and ML-1. It is
noteworthy that synthetic santacruzamate A did not inhibit
either total HDACs or HDAC2 in enzyme assays. While this is in
stark contrast to the original publication, it is consistent with
the known SAR of HDAC inhibitors and it is likely that the
earlier report was in error.18 However, one analogue, 5 was
found to exhibit anti-proliferative activity against HCT-116 (IC50
¼ 6.0 mM) and ML-1 (IC50 ¼ 9.4 mM) cell lines. In addition, 5 did
not cause damage to normal human colorectal cells, suggesting
that 5 selectively killed the abnormal cancer cells. It is
phenomenal that such a simple compound with a terminal
thiourea has gained submicromolar anticancer activity with low
toxicity to normal cells although the thiourea motif are reported
in some biologically active compounds.24 Further studies
showed that 5 inhibited colony formation of HCT-116, induced
apoptosis of both cancer cells HCT-116 and ML-1, and arrested
cell cycle of HCT-116 at G2/M phase. Finally, ROS generation
was observed in both cancer cell lines HCT-116 and ML-1,
implying that this might be the reason why 5 selectively elimi-
nated cancer cells. Further study to investigate its exact mech-
anism of action is underway. Due to its simple structure and
selective killing of cancer cells, 5 might provide a useful scaffold
for anticancer drug development.
¨
9 D. Roell, T. W. Rosler, S. Degen, R. Matusch and
A. Baniahmad, Chem. Biol. Drug Des., 2011, 77, 450–459.
10 D. J. Newman and G. M. Cragg, J. Nat. Prod., 2012, 75, 311–
335.
11 G. M. Cragg, P. G. Grothaus and D. J. Newman, J. Nat. Prod.,
2014, 77, 703–723.
12 A. Ganesan, Curr. Opin. Chem. Biol., 2008, 12, 306–317.
13 A. Ganesan, The Impact of Natural Products Upon Cancer
Chemotherapy, in Natural Products and Cancer Drug Discovery,
ed. F. E. Koehn, Springer, Heidelberg, 2012, pp. 3–15.
14 F. L. Cherblanc, R. W. M. Davidson, P. D. Fruscia,
N. Srimongkolpithak and M. J. Fuchter, Nat. Prod. Rep.,
2013, 30, 605–624.
15 M. Haberland, R. L. Montgomery and E. N. Olson, Nat. Rev.
Genet., 2009, 10, 32–42.
16 P. Zhu, E. Martin, J. Mengwasser, P. Schlag, K. P. Janssen and
¨
M. Gottlicher, Cancer Cell, 2004, 5, 455–463.
17 A. Vaquero, R. Sternglanz and D. Reinberg, Oncogene, 2007,
26, 5505–5520.
18 C. M. Pavlik, C. Y. B. Wong, S. Ononye, D. D. Lopez,
N. Engene, K. L. McPhail, W. H. Gerwick and
M. J. Balunas, J. Nat. Prod., 2013, 76, 2026–2033.
19 M. Paris, M. Porcelloni, M. Binaschi and D. Fattori, J. Med.
Chem., 2008, 51, 1505.
20 Y. Nakao, S. Yoshida, S. Matsunaga, N. Shindoh, Y. Terada,
K. Nagai, J. K. Yamashita, A. Ganesan, R. W. M. van Soest
and N. Fusetani, Angew. Chem., Int. Ed., 2006, 45, 7553–7557.
21 S. Wen, K. L. Carey, Y. Nakao, N. Fusetani, G. Packham and
A. Ganesan, Org. Lett., 2007, 9, 1105–1108.
Acknowledgements
The work was supported by Doctoral Program of Higher
Education of China (grant 20110171120098), National Basic
Research Program of China (973 Program grant 2012CB967004),
and Guangdong Provincial International Project of Science and
Technology (2013B051000034).
22 D. Trachootham, J. Alexandre and P. Huang, Nat. Rev. Drug
Discovery, 2009, 8, 579–591.
23 L. Raj, T. Ide, A. U. Gurkar, M. Foley, M. Schenone, X. Li,
N. J. Tolliday, T. R. Golub, S. A. Carr, A. F. Shamji,
A. M. Stern, A. Mandinova, S. L. Schreiber and S. W. Lee,
Nature, 2011, 475, 231–234.
24 (a) A. Solinas, H. Faure, H. Roudaut, E. Traiffort,
A. Schoenfelder, A. Mann, F. Manetti, M. Taddei and
M. Ruat, J. Med. Chem., 2012, 55, 1559–1571; (b) A. Mishra
and S. Batra, Curr. Top. Med. Chem., 2013, 13, 2011–2025;
(c) H. Nishiyama, M. Ono, T. Sugimoto, T. Sasai,
N. Asakawa, S. Ueno, Y. Tominaga, T. Yaegashi,
M. Nagaoka, T. Matsuzaki, N. Kogure, M. Kitajima and
H. Takayama, Med. Chem. Commun., 2014, 5, 452–458.
Notes and references
1 O. O. Fadeyi, S. T. Adamson, E. L. Myles and C. O. Okoro,
Bioorg. Med. Chem. Lett., 2008, 18, 4172–4176.
2 S. A. F. Rostom, Bioorg. Med. Chem., 2006, 14, 6475–6485.
3 W. Liu, J. Zhou, T. Zhang, H. Zhu, H. Qian, H. Zhang,
W. Huang and R. Gust, Bioorg. Med. Chem. Lett., 2012, 22,
2701–2704.
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