Syntheses of the marine alkaloids 6-oxofascaplysin, fascaplysin and their derivatives

Article abstract of DOI:10.1016/j.tetlet.2018.01.023

A simple approach towards the pyrido[1,2-a:3,4-b′]diindole system via the reaction of indigo with methylene active compounds was used for the syntheses of the marine alkaloids 6-oxofascaplysin, fascaplysin, and their derivatives. It was also demonstrated that the reaction with ketones led to indigo decomposition and the formation of isatin derivatives. The derivative of fascaplysin with a phenyl substituent at C-7 demonstrated 2–3 times greater inhibitory activity against selected cancer cell lines than fascaplysin.

Full text of DOI:10.1016/j.tetlet.2018.01.023

Accepted Manuscript
Syntheses of the marine alkaloids 6-oxofascaplysin, fascaplysin and their de-
rivatives
Maxim E. Zhidkov, Alexey V. Kantemirov, Alexey V. Koisevnikov, Alexander
N. Andin, Alexandra S. Kuzmich
PII:
S0040-4039(18)30031-5
https://doi.org/10.1016/j.tetlet.2018.01.023
TETL 49610
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 October 2017
23 December 2017
5 January 2018
Please cite this article as: Zhidkov, M.E., Kantemirov, A.V., Koisevnikov, A.V., Andin, A.N., Kuzmich, A.S.,
Syntheses of the marine alkaloids 6-oxofascaplysin, fascaplysin and their derivatives, Tetrahedron Letters (2018),
doi: https://doi.org/10.1016/j.tetlet.2018.01.023
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Syntheses of the marine alkaloids 6-oxofascaplysin, fascaplysin and their
derivatives
Maxim E. Zhidkov,†,* Alexey V. Kantemirov,† Alexey V. Koisevnikov,† Alexander
N. Andin,† Alexandra S. Kuzmich‡
†
School of Natural Sciences, Far Eastern Federal University, 8 Sukhanov Str.,
Vladivostok 690950, Russian Federation
‡
Pacific Institute of Bioorganic Chemistry, 159 Prospect 100-Let Vladivostoku,
Vladivostok 690022, Russian Federation
*Corresponding author: e-mail: zhidkov.me@dvfu.ru; phone: +79247353506
Abstract: A simple approach towards the pyrido[1,2-a:3,4-b’]diindole system via
the reaction of indigo with methylene active compounds was used for the syntheses
of the marine alkaloids 6-oxofascaplysin, fascaplysin, and their derivatives. It was
also demonstrated that the reaction with ketones led to indigo decomposition and
the formation of isatin derivatives. The derivative of fascaplysin with a phenyl
substituent at C-7 demonstrated 2-3 times greater inhibitory activity against
selected cancer cell lines than fascaplysin.
Dedicated to the memory of Dmitriy A. Maiboroda.
Keywords: Fascaplysin; Indigo; 6-Oxofascaplysin, Total synthesis; Isatin
derivatives
Introduction
The 12H-pyrido[1,2-a:3,4-b’]diindole ring system (1, Fig. 1) forms the
framework of several marine alkaloids, such as fascaplysin, homofascaplysins A-
C, and their brominated analogues.1 The red pigment fascaplysin (2), isolated in
1988 from the sponge Fascaplysinopsis sp., is the most investigated representative
alkaloid.2 This compound exhibits a broad range of bioactivities including
antibacterial, antifungal, antiviral, HIV-1-RT, p56 tyrosine kinase, and antimalarial
properties, as well as suppressing the proliferation of numerous cancer cell lines.3
In addition, fascaplysin inhibits the growth of S180 cell-implanted tumors and

possesses anti-angiogenesis properties.4, 5 The mechanisms of action include the
selective inhibition of cyclin-dependent kinase 4, which regulates the G0–G1/S
checkpoint of the cell cycle, the intercalation of DNA, and the induction of
apoptosis, in part, as a result of the activation of the TRAIL signaling pathway by
upregulating DR5 expression.6–8 Recent research has shown that selective CDK4/6
inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour
immunity.9 Besides its antitumor properties, fascaplysin triggers cell shrinkage and
phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part
due to Ca2+ entry, oxidative stress and ceramide abundance.10 It also could be used
as a P-gp inhibitor for the development of anti-Alzheimer agents and as a
“balanced agonist” of the opioid receptor with a signaling profile that resembles
endorphins.11, 12
These facts demonstrate the significant potential of fascaplysin and related
compounds for therapeutic assays. Recently, a novel representative of these
alkaloids named 6-oxofascaplysin (3) was isolated from the Australian marine
sponge Hyrtios sp.13 The absence of some correlations in the HMBC spectra led the
authors to utilise DFT NMR calculations to support their structural assignment.
Herein, we report the development of a simple approach for the first synthesis of 6-
oxofascaplysin and selected derivatives. A one-step transformation of 6-
oxofascaplysin and its derivatives into fascaplysin and its derivatives which are
inaccessible by other methods was also demonstrated.
O
R
Cl-
6
7
4
N+
5
3
N
4a
8
7a
N
7b
C
9
E
2
D
B
A
12b
12a
N
H
13a
N
H
10
1
11a
N
H
12
13
O
O
11
3: R = H
2
1
4a
: R = COOEt
4b: R = Ph
Figure 1. Structures of 12H-pyrido[1,2-a:3,4-b']diindole (1), fascaplysin (2), 6-oxofascaplysin
(3), and its known derivatives.
Fascaplysin and its naturally occurring analogues have been synthesized by
several groups and more than ten syntheses have been reported to date.14 However,
none can be considered effective for the synthesis of 6-oxofascaplysin. At the same
time, condensation of the well-known dye indigo with a malonic ester in the

presence of base followed by subsequent hydrolysis and decarboxylation of the
obtained product 4a would represent a simple approach towards 6-oxofascaplysin
3. The obtainment of mixtures of highly functionalized pyrazino[1,2-a:4,3-
a`]diindole, pyrido[1,2-a:3,4-b`]diindole and benzo[b]indolo[1,2-h]naphthyridine
heterocyclic systems via the base-induced propargylation of indigo was previously
reported.15 Compound 4a was previously obtained in 1923-1924 along with the
product of condensation with ethyl phenylacetate in order to confirm the structure
of indigo (Fig. 1).16, 17 Recently, compound 4b and series of related compounds
were obtained by the condensation of selected arylacetic chlorides with N,N-
diacetylindigo, a more soluble derivative of indigo.18
Results and Discussion
Our work began with the optimization of the preparation of compound 4a
using unsubstituted indigo as a starting material (Scheme 1). After preliminary
experiments, sodium hydroxide was replaced by sodium hydride and DMF was
used instead of nitrobenzene. For subsequent hydrolysis and decarboxylation
reactions, compound 4a was heated at reflux in 40% hydrobromic acid for two
hours. A single compound was isolated with spectral characteristics which were
identical to those of natural 6-oxofascaplysin. The overall yield of the target
compound was 70%.
O
R1
O
O
H
N
N
N
b
a
4a
for
95%
N
H
N
H
N
H
O
3
4a-d
O
O
a: R1 = COOEt 75%
: R1 = Ph
55%
c: R1 = CO-CH3 88%
d: R1 = CN
50%
a
Scheme 1. Syntheses of 6-oxofascaplysin (3) and its derivatives (4a-d) Reagents and conditions:
(a) R-CH2-COOEt (8 equiv.), NaH (4 equiv.), DMF, ∆, 0.5 h; (b) 40% HBr (excess), ∆, 2 h
After the successful synthesis of 6-oxofascaplysin 3, the condensation
reaction was applied to acetoacetic, cyanoacetic, phenylacetic and nitroacetic
esters. In most cases, the condensation was successful with yields ranging from 50-
88% (4b-d).19 However, when nitroacetic ester was used the reaction did not occur.

Unfortunately, the poor solubility of 6-oxofascaplysin and its derivatives 4a-
d in both water and organic solvents such as DMSO and DMF prevented an
investigation of their bioactivities. At the same time, it can be expected that the
reaction of indigo and a wide range of ketones should allow the preparation of
fascaplysin derivatives with substitution at the C-6 and C-7 positions (Fig. 1). Due
to that fact, the reaction of indigo with acetophenone as a model compound was
investigated. According to the spectral data, this resulted in a mixture of Z-, and E-
isomers of compound 5a (Scheme 2). The E-isomer was unstable and
spontaneously converted into the Z-isomer. The reaction of more reactive
dimedone resulted in the formation of a mixture of indigo decomposition product
5b and its tautomers with 85% total yield (Scheme 2, see also the ESI).
O
N
H
Ph
O
O
O
O
13%
O
H
N
b
a
CH3
CH3
5a
+
N
H
N
H
O
O
O
Ph
5b + tautomers, 85%
N
H
7%
Scheme 2. Reactions of indigo with acetophenone (a) and dimedone (b). Reagents and conditions:
(a) acetophenone (1.5 equiv.), NaH (5 equiv.), DMF, ∆, 0.5 h; (b) dimedone (5 equiv.), NaH (7
equiv.), DMF, ∆, 0.5 h.
The obtained data can be easily explained within the framework of the
HSAB principle. However, when indigo reacts with ethyl phenylacetate both
variants of this reaction can be realized depending on the conditions utilized
(Scheme 3). Thus, upon the slow addition of ethyl phenylacetate to indigo and
excess sodium hydride, the product of indigo decomposition 5c was obtained. As
indicated above for compound 5a, a mixture of geometric isomers was obtained,
consisting of an unstable E-isomer that spontaneously transformed into the Z-
isomer. On the other hand when indigo and ethyl phenylacetate were mixed in
advance then heated in the presence of NaH, the main reaction product was
compound 4b. Thereby it can be assumed that the reaction of indigo with active
methylene compounds is determined by their methylene activities that result in the
prevalence of one of two different processes: i) Michael reaction between the

deprotonated methylene active compound and the activated double bond of indigo
followed by the decomposition of indigo and formation of products 5; ii) acylation
of the nitrogen atom of indigo followed by intramolecular nucleophilic attack on
the "opposite" carbonyl group with formation of the heterocyclic 12H-pyrido[1,2-
a:3,4-b’]diindole system.
O
Ph
Ph
O
O
O
N
H
COOEt
H
N
18%
N
a
b
5c
+
O
-EtOH
N
H
N
H
O
COOEt
Ph
O
N
H
O
Ph
NaH
-H2O
N
N
H
4b
O
55%
Scheme 3. Reactions of indigo with ethyl phenylacetate under different conditions. Reagents and
conditions: (a) NaH (4 equiv.), then slowly add PhCH2COOEt (10 equiv.), DMF, r.t., 30 min,
then ∆, 30 min; (b) Ph-CH2-COOEt (8 equiv.), then NaH (4 equiv.), ∆, 30 min.
Since the anticipated fascaplysin derivatives with substitution at the C-6 and
C-7 positions were not obtained, it was decided to investigate the potential
application of compounds 4 as starting materials for the preparation of fascaplysin
derivatives. For the development of this conversion, 6-oxofascaplysin was chosen
as a model compound and the formation of fascaplysin was explored (Scheme 4).
Initially lithium aluminum hydride was examined for the reduction of the 2-
pyridone fragment. The reaction of 6-oxofascaplysin with LiAlH4 was carried out
in THF for 24 hours at room temperature and followed by air oxidation, resulting
in a complex mixture of compounds containing trace amounts of fascaplysin.
Numerous attempts to optimize the reaction conditions were unsuccessful.
Replacement with the more selective BH3·THF complex in THF at reflux for two
hours followed by hydrolysis and air oxidation gave fascaplysin in 43% isolated
1
yield (see the ESI for a comparison of the H/13C NMR data from natural and
synthetic fascaplysin). The product of the condensation of indigo and ethyl

phenylacetate (4b) under the developed conditions afforded compound 6 in 44%
yield.
O
R
R
Cl-
N+
O
N
N
H
N
H
O
3: R = H
4b: R = Ph
2: R = H, 43%
6: R = Ph, 44%
Scheme 4. Syntheses of fascaplysin and its derivative 6. Reagents and conditions: BH3·THF (24
equiv.), THF, Ar, ∆, 2 h, then H2O/H+, air, ∆, 2 h.
Preliminary in vitro bioassays for compound 6 showed its activity against
HeLa (cervix cancer) and THP-1 (acute monocytic leukemia) cell lines with IC50
values of 290 and 320 nM, respectively, compared to 550 and 890 nM,
respectively, for fascaplysin. To explain the obtained data, the molecular docking
of fascaplysin and its derivative 6 with a model of cyclin-dependent kinase 4 was
carried out. The results of this experiment in the program Autodock 4 indicated the
larger binding energy for compound 6 in comparison with fascaplysin (-9.55 and -
8.67 kcal/mol, respectively). Based on the analysis of the theoretical complex (see
ESI), the large substituent does not violate the structure of intermolecular contacts
specific for fascaplysin, but allows the formation of additional interactions that
increases the affinity of this compound to the therapeutic target.20
Conclusion
In conclusion, a simple one-step approach was developed for the synthesis
of the heterocyclic 12H-pyrido[1,2-a:3,4-b’]diindole system from indigo. This
method was used for the first synthesis of the marine alkaloid 6-oxofascaplysin and
the confirmation of its proposed structure. Preliminary in vitro tests of the
fascaplysin C-7 derivative demonstrated the potential of related compounds for
therapeutic assays and will necessitate further investigations of their syntheses and
detailed studies of the structure activity relationships among these potentially
bioactive substances.

Acknowledgments
The research described in this publication was carried out with the financial
support of the Far Eastern Federal University (FEFU), the FEFU Endowment
Foundation and the Genzo Shimadzu scholarship. The presented bioassays were
made possible by a grant from the Russian Scientific Fund (Project No. 14-50-
00034).
References
1. Bharate, S. B.; Manda, S.; Mupparapu, N.; Battini, N.; Vishwakarma, R. A.
MiniRev. Med. Chem. 2012, 12, 650–664.
2. Roll, D. M.; Ireland, C. M.; Lu, H. S.; Clardy, J. J. Org. Chem.1988, 53,
3276–3278.
3. For example, see: (a) Jimenez, C.; Quinoa, E.; Adamczeski, M.; Hunter, L.
M.; Crews, P. J. Org. Chem. 1991, 56, 3403–3410; (b) Schmidt, E. W.;
Faulkner, D. J. Tetrahedron Lett. 1996, 37, 3951–3954; (c) Kirsch, G.;
Konig, G. M.; Wright, A.D.; Kaminsky, R. J. Nat. Prod. 2000, 63, 825–829;
(d) Charan, R. D.; McKee, T. C.; Gustafson, K. R.; Pannell, L. K.; Boyd, M.
R. Tetrahedron Lett. 2002, 43, 5201–5204; (e) Popov, A. M.; Stonik, V. A.
Antibiot. Khimioter. 1991, 36, 12–14; (f) Hamilton, G. Mar. Drugs. 2014,
12, 1377-1389.
4. Yan, X.; Chen, H.; Lu, X.; Wang, F.; Xu, W.; Jin, H.; Zhu, P. Eur. J. Pharm.
Sci. 2011, 43, 251–259.
5. Zheng, Y.; Lu, X.; Lin, J.; Chen, H.; Yan, X.; Wang, F.; Xu, W. Biomed.
Pharmacother. 2010, 64, 527–533.
6. Soni, R.; Muller, L.; Furet, P.; Schoepfer, J.; Stephan, C.; Zunstein-Mecker,
S.; Fretz, H.; Chaudhuri, B. Biochem. Biophys. Res. Commun. 2000, 275,
877–884.
7. Hormann, A.; Chaudhuri, B.; Fretz, H. Bioorg. Med. Chem. 2001, 9, 917–
921.
8. (a) Lu, X.; Zheng, Y.; Chen, H.; Yan, X.; Wang, F.; Xu, W. Yao XueXueBao
2009, 44, 980–986; (b) Wang, F.; Chen, H.; Yan X.; Zheng Y. Chin. J.
Oceanol. Limnol. 2013, 31, 560-569.
9. Goel, S.; DeCristo M. J.; Watt A. C.; Jones H. B.; Sceneay J.; Li B. B.;
Khan N.; Ubellacker J. M.; Xie S.; Metzger-Filho O.; Hoog J.; Ellis M. J.;
Ma C. X.; Ramm S.; Krop I. E.; Winer E. P.; Roberts T. M.; Kim H.-J.;
McAllister S. S.; Zhao J. J. Nature 2017, 548, 471–475.

10.Mischitelli, M.; Jemaà, M.; Almasry, M.; Faggio, C.; Lang, F. Cell Physiol
Biochem. 2016, 39, 1638-1647.
11.Manda, S.; Sharma, S.; Wani, A.; Joshi, P.; Kumar, V.; Guru, S. K.; Bharate,
S. S.; Bhushan, S.; Vishwakarma, R. A.; Kumar, A.; Bharate, S. B. Eur J
Med Chem. 2016, 107, 1-11.
12.Johnson, T. A.; Milan-Lobo, L.; Che, T.; Ferwerda, M.; Lambo, E.;
McIntosh, N. L.; Li, F.; He, L.; Lorig-Roach, N.; Crews, P.; Whistler, J. L.
ACS Chem Neurosci. 2017, 8, 473-485.
13.Khokhar, S.; Feng, Y.; Campitelli, M. R.; Ekins, M. G.; Hooper, J. N. A.;
Beattie, K. D.; Sadowski, M. C.; Nelson, C. C.; Davis, R. A. Bioorg. Med.
Chem. Lett. 2014, 24, 3329–3332.
14. (a) Gribble, G. W.; Pelcman, B. J. Org. Chem. 1992, 57, 3636–3642; (b)
Rocca, P.; Marsais, F.; Godart, A.; Quéguiner, G. Tetrahedron Lett. 1993,
34, 7917–7918; (c) Molina, P.; Fresneda, M.; Garciazafra, S.; Almendros, P.
Tetrahedron Lett. 1994, 35, 8851–8854; (d) Radchenko, O. S.; Novikov, V.
L.; Elyakov, G. B. Tetrahedron Lett. 1997, 38, 5339–5342; (e) Dubovitskii,
S. V. Tetrahedron Lett. 1997, 37, 5207–5208; (f) Zhidkov, M. E.; Baranova,
O. V.; Balaneva, N. N.; Fedorov, S. N.; Radchenko, O. S.; Dubovitskii, S.
V. Tetrahedron Lett. 2007, 48, 7998–8000; (g) Waldmann, H.; Eberhardt,
L.; Wittstein, K.; Kumar, K. Chem. Commun. 2010, 46, 4622–4624; (h)
Zhidkov, M. E.; Baranova, O. V.; Kravchenko, N. S.; Dubovitskii, S. V.
Tetrahedron Lett. 2010, 51, 6498–6499; (i) Bharate, S. B.; Manda, S.; Joshi,
P.; Singh, B.; Vishwakarma, R. A. Med. Chem. Commun. 2012, 3, 1098-
1103; (j) Zhidkov, M. E.; Kaminskii, V. A. Tetrahedron Lett. 2013, 54,
3530-3532; (k) Zhu, Y. P.; Liu, M.; Cai, Q.; Jia, F. C., Wu, A. X. Chem.
Eur. J. 2013, 19, 10132 – 10137.
15.Shakoori, A.; Bremner, J. B.; Willis, A. C.; Haritakun, R.; Keller, P. A. J.
−7647.
Org. Chem. 2013, 78, 7639
16.Posner, T.; Pyl, G. Ber. 1923, 56, 31-44.
17.Posner, T.; Kemper, W. Ber. 1924, 57, 1311-1315.
18.Kolaczkowski, M. A.; He, B.; Liu, Y. Org Lett. 2016, 18, 5224-5227.
19.General procedure for the condensation of indigo with methylene active
compounds. Sodium hydride (75 mg, 60% dispersion in mineral oil) was
added to a suspension of powdered indigo (131 mg, 0.5 mmol) in dry DMF
(10 mL). When the evolution of hydrogen was finished, the methylene
active compound (4 mmol) was added gradually. The reaction mixture was
heated at reflux and stirred for 30 min, then cooled, diluted with H2O (50
mL), acidified with hydrochloric acid and filtered. The solid residue was
washed with Et2O (2 x 3 mL) and dried. The crude product was purified by

MPLC using chloroform as eluent. Compound samples for recording NMR
and mass spectra were repurified by a Shimadzu HPLC system.
20.Morris, G.; Huey, R.; Lindstrom, W.; Sanner, M.; Belew, R.; Goodsell, D.;
Olson, A. J. Comput. Chem 2009, 30, 2785-2791.

The alkaloid 6-oxofascaplysin and its derivatives were synthesized from indigo.
6-Oxofascaplysin was converted into the marine alkaloid fascaplysin in one step.
Reaction of indigo with ketones led to the formation of some of isatin derivatives.
Introduction of phenyl at C-7 of fascaplysin increase its activity up to 2-3 times.

Syntheses of the marine alkaloids 6-oxofascaplysin, fascaplysin and their
derivatives
O
R
N
R
O
H
N
N+
O
N
H
O
6-oxofascaplysin (R = H),
four examples
N
H
N
H
O
O
+
fascaplysin (R = H),
two examples
methylene active
compound
N
H
O
three examples