Synthesis and comparative evaluation of 4-oxa- and 4-aza-podophyllotoxins as antiproliferative microtubule destabilizing agents

Article abstract of DOI:10.1016/j.bmcl.2012.01.128

A series of novel 4-oxa-podophyllotoxin derivatives 7 featuring the intact lactone ring D and various substituents in rings B and E has been synthesized and evaluated in a phenotypic sea urchin embryo assay along with the representative 4-aza-analogs 5 for their antimitotic and microtubule destabilizing activity. The most active compounds exhibited myristicin-derived or a 3′,5′-dimethoxy substitution pattern in the ring E and a 6-methoxy moiety replacing the methylenedioxy ring A. Compounds 5xb, 5xe, 5yb, 7xa, 7xb, and 7xc showed potent antiproliferative effects in the NCI60 cytotoxicity screen. Notably, growth of the multi-drug resistant NCI/ADR-RES cells was more affected by these agents than the parent OVCAR-8 cell line. Although generally 4-oxa-podophyllotoxins were less potent than the respective 4-aza-derivatives in these assays, stability of the former series towards oxidation may prove to be of interest for the development of anticancer agents with in vivo activity.

Full text of DOI:10.1016/j.bmcl.2012.01.128

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2590–2593
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and comparative evaluation of 4-oxa- and 4-aza-podophyllotoxins
as antiproliferative microtubule destabilizing agents
Natalia B. Chernysheva a, Dmitry V. Tsyganov a, Alex A. Philchenkov b, Michael P. Zavelevich b,
Alex S. Kiselyov c, Roman V. Semenov a, Marina N. Semenova d,e, Victor V. Semenov a,
⇑
a N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991 Moscow, Russia
b R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU, Vasyl’kivska Street, 45, 03022 Kyiv, Ukraine
c CHDI Foundation, 6080 Center Drive, Suite 100, Los Angeles, CA 90045, USA
d Institute of Developmental Biology, RAS, Vavilov Street, 26, 119334 Moscow, Russia
e Chemical Block Ltd, 3 Kyriacou Matsi, 3723 Limassol, Cyprus
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 4-oxa-podophyllotoxin derivatives 7 featuring the intact lactone ring D and various sub-
stituents in rings B and E has been synthesized and evaluated in a phenotypic sea urchin embryo assay
along with the representative 4-aza-analogs 5 for their antimitotic and microtubule destabilizing activity.
The most active compounds exhibited myristicin-derived or a 30,50-dimethoxy substitution pattern in the
ring E and a 6-methoxy moiety replacing the methylenedioxy ring A. Compounds 5xb, 5xe, 5yb, 7xa, 7xb,
and 7xc showed potent antiproliferative effects in the NCI60 cytotoxicity screen. Notably, growth of the
multi-drug resistant NCI/ADR-RES cells was more affected by these agents than the parent OVCAR-8 cell
line. Although generally 4-oxa-podophyllotoxins were less potent than the respective 4-aza-derivatives
in these assays, stability of the former series towards oxidation may prove to be of interest for the devel-
opment of anticancer agents with in vivo activity.
Received 9 December 2011
Revised 26 January 2012
Accepted 30 January 2012
Available online 9 February 2012
Keywords:
Oxa-podophyllotoxins
Aza-podophyllotoxins
Microtubule destabilizing agents
Sea urchin embryo
Cytotoxicity
Ó 2012 Elsevier Ltd. All rights reserved.
Podophyllotoxin (PT) (Fig. 1) is a natural aryltetralin lignan
found in plants of genus Podophyllum as well as in other species.
It has been described to be a strong microtubule destabilizing
agent that binds to the colchicine site of tubulin.1–4 Furthermore,
PT has been shown to be a bi-functional ligand interacting with
multiple tubulin sites similar to colchicine.5,6 Several semisyn-
thetic PT derivatives, including etoposide, teniposide, and etopo-
phos (Fig. 1), are antitumor agents currently in clinical use for
the treatment of various malignancies.3,7,8
There are four contiguous stereocenters in PT (Fig. 1). Epimeriza-
tion of the C1 center substituted with a thrimethoxyphenyl group in
the parent molecule proved to be an issue in the structure–activity
studies of PT derivatives.2 Several teams described heterocyclic
analogs of PT that do not undergo epimerization and modified in
the ring C. For example, 4-aza-PT9–19 and 4-oxa-PT13,20,21 deriva-
tives feature a single chiral center at C1 (Fig. 1) and are easily acces-
sible. They exhibited a broad spectrum of biological activities,
including insecticidal potential,13 cancer cell growth inhibi-
tion,9,11,12,14–18,20,21 cell cycle arrest in the G2/M phase,16,18,20 inhi-
bition of tubulin polymerization,14,16,17,20 cellular microtubule
disassembly,17,18 caspase-3 dependent apoptosis,11,16–18 and vascu-
lar disruption effect.14 Among 4-aza-PTs made from the easily
available plant polyalkoxybenzenes myristicin (1b) and apiol (1d)
(Scheme 1),18,22 the most potent analogs contained the myristi-
cin-derived ring E (II; Fig. 1).18 Notably, the methylenedioxy ring
A was not a prerequisite for their activity, since 6-methoxy deriva-
tives 5xb and 5xe displayed strong antiproliferative effect. These
molecules were more potent in the sea urchin embryo assay than
the parent PT.18 Compounds 5xa and 5xe also displayed pro-
nounced larvicidal effects.13 Notably, our previous studies showed
that while 4-aza-PTs I, II, and 5 were stable as solids during storage
at room temperature, they underwent rapid oxidation to furnish
inactive quinolines 4 in DMSO or ethanol.18 Therefore in this paper
we focused our attention on respective 4-oxa-analogs 7 that are less
likely to be unstable under these conditions.
Abbreviations: PT, podophyllotoxin.
Corresponding author. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
⇑
It was reported previously that 4-oxa-PT inhibited cancer cell
growth at concentrations of 0.1–1
l
M,21 whereas 4-oxa-2-aza-PT
E-mail addresses: info@chemblock.com (N.B. Chernysheva), tsyg@ioc.ac.ru (D.V.
Tsyganov), a_philch@onconet.kiev.ua (A.A. Philchenkov), butenko@onconet.kiev.ua
(M.P. Zavelevich), Alex.Kiselyov@chdifoundation.org (A.S. Kiselyov), rs@chemical-
block.com (R.V. Semenov), ms@chemical-block.com (M.N. Semenova), vs@zelins-
ky.ru (V.V. Semenov).
was cytotoxic with IC50 = 0.077
l
M.21,23 These compounds were
less active than the parent PT (IC50 = 10–40 nM (Table S1, Supple-
mentary data)). It is worth emphasizing that a replacement of
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.128

N. B. Chernysheva et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2590–2593
2591
Figure 1. Structures of podophyllotoxin and its analogs.
the dihydropyridine ring C in aza-PTs with pyran significantly de-
creased larvicidal activity.13 On the other hand, methylenedioxy-
benzopyran analogs of PT substituted at positions C2 and C3 versus
the lactone ring D inhibited tubulin polymerization, competitively
blocked colchicine binding to tubulin, and displayed cytotoxicity
against murine leukemia cells in a submicromolar concentration
range.20 In this respect, it should be emphasized that the lactone
ring D is essential for binding to tubulin,6 and its modifications
led to decreased cytotoxicity of the resulting derivatives.3,24,25
Therefore, the aim of this study was to prepare mainly 4-oxa-
and several 4-aza-PT derivatives with the intact lactone ring D,
while varying substituents in the rings B and E, and to compare
the resultant series in a panel of biological assays.
A two-steps synthesis of aromatic aldehydes (2b, 2d) from nat-
ural allylbenzenes (1b, 1d) and involving alkaline double bond
migration followed by ozonolysis was reported previously.26
Plant-derived (2b, 2d) and commercial (2a, 2c, 2e, 2f) aldehydes
served as key intermediates in our synthetic approach as described
in Scheme 1.22 In an effort to synthesize 4-oxa-PT derivatives 7 we
were unable to reproduce published synthetic strategy towards
key intermediates 6 using MeOH as a solvent.13 Gratifyingly, these
targeted Mannich bases were successfully accessed via a condensa-
tion reaction of aromatic aldehydes (2a–f), phenols, and morpho-
line in absolute toluene under N2 in a reactor equipped with a
moisture trap filled with an anhydrous Na2SO4 and a condenser.
In the next step, the molecules 7 (3.2–35% yield) were subse-
quently obtained by reacting 6 and tetronic acid.13,21,27 An alterna-
tive synthetic route to 7xe was reported earlier.13 4-Aza-PTs (5)
were synthesized as reported previously18 by cyclizing 4-Cl-aceto-
acetic acid with amines to afford anilinolactones (3).28 These inter-
mediates were further reacted with aldehydes (2a–e) in the
presence of p-chloranil as an oxidizing agent to furnish quinoline
derivatives 4. Quinolines 4 were subsequently hydrogenated with
NaBH3CN in a glacial acetic acid to afford the targeted 1,4-dihydro-
pyridines 5 (43–73% yield). Further experimental details describing
synthetic protocols and analytical data are summarized in the Sup-
plementary data.
All molecules were evaluated in a phenotypic sea urchin em-
bryo assay29 in order to assess their antimitotic and microtubule
destabilizing activities using PT as a benchmark reference com-
pound (Table 1). The assay includes (i) fertilized egg test for anti-
mitotic activity displayed by cleavage alteration/arrest, and (ii)
behavioral monitoring of a free-swimming blastulae treated imme-
diately after hatching. We have shown earlier that lack of forward
movement, settlement to the bottom of the culture vessel, and ra-
pid spinning of embryos around the animal-vegetal axis suggests a
microtubule destabilizing activity caused by a test article.29,30 In
our hands, data generated from the sea urchin embryo assay, puri-
fied tubulin polymerization screen, and microtubule disruption in
human cancer cells correlated well.18,31,32 The activity was quanti-
fied as an effective threshold concentration (EC) resulting in cleav-
age alteration, cleavage arrest, or embryo spinning. At these
concentrations, all tested molecules caused 100% effect, whereas
at twofold lower concentrations, the compounds failed to produce
any developmental changes. Further details on the sea urchin em-
bryo assay are summarized in the Supplementary data. The cyto-
toxicity of compounds 5xb and 7xb was further assessed in
Jurkat leukemia T-cells and the NCI60 human tumor cells.33
As shown in Table 1, all tested molecules except for 7zf caused a
noticeable cleavage alteration, arrest, and embryo spinning, sug-
gesting their direct microtubule destabilizing activity. 4-Aza-PTs
(II, 5xb, 5xc, 5xe, 5yb) and 4-oxa-PTs (7xa, 7xb, 7xc, 7xe) displayed
higher antimitotic activity than the parent PT. 4-Oxa-derivatives
were somewhat less potent than the respective aza-analogs. Myris-
ticin derivatives of both 4-aza and 4-oxa PTs were generally more
active than the corresponding trimethoxyphenyl analogs (com-
pare: I and II, 5xa and 5xb, 7xa and 7xb, 7ya and 7yb). Similarly,
4-aza-PT derivative endowed with the myristicin moiety afforded
higher antimitotic activity than the corresponding 3,5-dimethoxy-
benzene analog (compare 5xb and 5xe). Interestingly, the trend
was opposite for the respective 4-oxa-PTs 7xb and 7xe. The api-
ol-derived substituent at C1 afforded less active compound 7xd.
Introduction of ethylenedioxy fragment to the ring E decreased
the antimitotic activity (compare: 5xb and 5xc, 7xb and 7xc).

2592
N. B. Chernysheva et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2590–2593
Scheme 1. Reagents and conditions: (a) (i) step 1: powdered KOH, (n-Bu)4N+Brꢀ, heat, 100 °C, 40 min; (ii) step 2: O3, CHCl3–MeOH–pyridine (80:20:3 v/v), ꢀ15 °C, 1–2 h;26
(b) C6H6–AcOH, reflux, 8 h; (c) CF3COOH, rt, 24 h; (d) AcOH–NaBH3CN, rt, 15 h; (e) CH2Cl2–AcOH–AcOK, 80–85 °C, 5 h;35 (f) toluene–N2, Na2SO4, reflux, 12–15 h; (g) (i) AcOH–
water (1:1 v/v), reflux, 30–40 min; (ii) AcOH–H2SO4, 110 °C, 0.5 h. Reactions (a)–(d) were performed as reported previously.18
Importantly, it was found that the methylenedioxy ring A could
be replaced by methoxy groups to result in retention of activity in
both 4-aza- and 4-oxa-series. The position of a methoxy substitu-
ent in the ring B played an important role: 6-methoxy derivatives
5xb, 7xa, and 7xb were consistently more potent antimitotic
agents than the corresponding 7-methoxy analogs 5yb, 7ya, and
7yb. Three methoxy groups in the B ring (7zf) resulted in a dra-
matic decrease of antiproliferative activity.
Data obtained from the sea urchin embryo assay have been fur-
ther supported by the Jurkat leukemia cells cytotoxicity test (for
experimental details see Supplementary data). Compounds 5xb
and 7xb were found to be cytotoxic exhibiting IC50 values of
18 nM and 55 nM, respectively, versus 25 nM for the parent PT.
These molecules, as well as II, 5xe, 5yb, 7xa, and 7xc, were further
selected for the NCI60 anticancer drug screen (Tables S1 and S2,
Supplementary data). Compounds 7xb and 7xc containing the
methylenedioxy and ethylenedioxy ring E, respectively, showed
similar cytotoxicity. Noticeably, 5xe was the most potent cell
growth inhibitor with GI50 <10 nM against 43 cancer cell lines.
When comparing the respective 4-oxa- and 4-aza-PTs 7xb and
5xb, it was found that both molecules displayed strong cytotoxicity
against a diverse set of cancer cell lines with GI50 values in the
nanomolar concentration range (Table S1, Supplementary data;
PT (NSC 24818) used as a standard). Dose–response profiling fur-
ther revealed that the aza-derivative 5xb was more potent than
the corresponding oxa-derivative 7xb. Moreover, 5xb was more ac-
tive than the parent PT against a panel of 25 cancer cell lines. Non-
small cell lung carcinoma (NCI-H522) and melanoma (MDA-MB-
435) were the most sensitive cell lines towards both 5xb and
7xb (GI50 <10 nM). In addition, 5xb caused total cell growth inhibi-
tion (TGI) of colon (COLO 205) and ovarian cancer (OVCAR-3) cells
at concentrations of 26.8 nM and 12.2 nM, respectively. TGI values
for 7xb were 69.2 nM (COLO 205) and 44.6 nM (OVCAR-3). Impor-
tantly, both 4-oxa- and 4-aza-derivatives demonstrated high activ-

N. B. Chernysheva et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2590–2593
2593
Table 1
7xc by the Developmental Therapeutics Program at NCI (Anti-can-
cer Screening Program; http://dtp.cancer.gov).
Effects of 4-oxa- and 4-aza-podophyllotoxins on the sea urchin embryos
Compound
ECa (nM)
Supplementary data
Cleavage alteration
Cleavage arrest
Embryo spinning
PT
20
50
5
50
500
50
200
2000
50
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.128.
Ib
IIb
5xa
7xa
5xbb
7xb
5xc
7xc
7xd
5xeb
7xe
7ya
5ybb
7yb
7zf
50
5
0.5
2
10
10
100
2
500
50
5
10
50
50
500
10
5
2000
50
500
200
50
100
1000
500
4000
200
50
5000
500
2000
>4000
References and notes
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a
The sea urchin embryo assay was conducted as described in Ref. 29. Fertilized eggs
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pounds. Duplicate measurements showed no differences in effective threshold con-
centration (EC) values. Experimental details are presented in Supplementary data.
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Table 2
Growth inhibition of OVCAR-8 and NCI/ADR-RES cell lines
Compound
Cell growth inhibitiona (GI50, nM)
OVCAR-8b NCI/ADR-RESc
II
232.0
788.0
<10
103.0
334.0
<10
7xa
5xb
7xb
7xc
5xe
5yb
55.6
49.5
20.4
18.6
23.5
<10
629.0
595.0
a
b
c
GI50: Concentration required for 50% cell growth inhibition.
OVCAR-8: Ovarian cancer cell line 8.
NCI/ADR-RES: P-glycoprotein-overexpressing multi-drug resistant cell line
17. Magedov, I. V. F. L.; Manpadi, M.; Bhoga, U. D.; Tang, H.; Evdokimov, N. M.;
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derived from OVCAR-8.34
18. Semenova, M. N.; Kiselyov, A. S.; Tsyganov, D. V.; Konyushkin, L. D.; Firgang, S.
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ity against colon cancer cells (Figure S1, Supplementary data). As
shown in Table 2, all tested 4-oxa- and 4-aza-PTs were more cyto-
toxic against NCI/ADR-RES cells overexpresing P-glycoprotein than
against the parent OVCAR-8 cell line suggesting a potential for
these agents to overcome multi-drug resistance.
In summary, a series of novel 4-oxa-PT derivatives displayed sig-
nificant microtubule destabilizing activity in the sea urchin embryo
assay with EC values in the 10–100 nM range (vs 20 nM for PT).
Notable structural features of these active compounds included
myristicin-derived or 30,50-dimethoxy substitution pattern in the
ring E and a 6-methoxy moiety in the ring B, replacing the methyl-
enedioxy ring A. Several 4-oxa-PT derivatives and their aza-analogs
(7xb, 7xe, 7yb, 5xa, 5xb, 5xc) displayed pronounced inhibition of
human cancer cell growth in the NCI60 cytotoxicity screen. More-
over, these agents were determined to be active against multi-drug
resistant NCI/ADR-RES cells. Although generally 4-aza-PTs were
more potent than the respective 4-oxa- derivatives, stability of
the latter series towards oxidation may prove to be of importance
for the development of anticancer agents with in vivo activity.
28. Hitotsuyanagi, Y.; Kobayashi, M.; Fukuyo, M.; Takeya, K.; Itokawa, H.
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31. Semenova, M. N.; Kiselyov, A. S.; Titov, I. Y.; Raihstat, M. M.; Molodtsov, M.;
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33. NCI60 Anti-cancer Screening Program is available at http://dtp.cancer.gov.
Both sea urchin embryo assay and DTP NCI60 cell line activity data are
available free of charge via the Internet at http://www.zelinsky.ru.
34. For the description of NCI/ADR-RES cancer cell line, see: http://dtp.nci.nih.gov/
docs/misc/common_files/NCI-ADRres.html.
Acknowledgments
This work was supported by a Grant from Chemical Block Ltd.
We thank the National Cancer Institute (NCI) (Bethesda, MD,
USA) for screening compounds II, 5xb, 5xe, 5yb, 7xa, 7xb, and
35. Momose, T.; Toyooka, N.; Takeuchi, Y. Heterocycles 1986, 24, 1429.