M. N. Semenova et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3914–3918
3915
Figure 1. Structure of podophyllotoxin and its heterocyclic analogues.
in anticancer activity of PT (Fig. 1).8 The complex stereochemistry of
PT renders SAR studies of its derivatives to be challenging. Introduc-
tion of a heteroatom in ring C allowed several research groups to
address both epimerization and SAR issues associated with this
compound class. For instance, the ‘simplified’ aza- and oxa-PT ana-
logues (Fig. 1) are potent microtubule destabilizing agents exhibit-
ing significant cytotoxicity.13,14 It was shown that methylenedioxy
ring A could be successfully replaced by a 7-methoxy moiety. Such
modification together with the derivatization of the ring E with 3,5-
dimethoxy and 3,4,5-trialkoxy groups proved to be advantageous
for compound activity.13,14 Notably, oxa-PTs were reported to be
more stable towards oxidation as compared to the respective aza-
derivatives.14 However, the synthetic approach to 7-methoxy-oxa-
PTs (Fig. 1) resulted in low overall yields of the targeted molecules
(3–6%), presumably due to the ambiguous reactivity of tetronic acid
with 3-methoxyphenol.14,15
4-Aryl-4H-chromenes (Fig. 1) could be regarded as structural
analogues of oxa-PTs where the lactone ring D is replaced with
CN and NH2-groups (Fig 1). These compounds were reported to
be microtubule destabilizing agents interacting with or adjacent
to the colchicine binding site, and cytotoxic against a panel of
human cancer cells.16 Their effects on cancer cells, including mito-
tic arrest, caspase-dependent apoptotic cell death, and tumor vas-
culature disruption were described in detail.16–23 4-Aryl-chromene
EPC2407 (Fig. 1) is currently in the phase I/II clinical trials as a
vasculature targeting anticancer agent and apoptosis inducer for
the treatment of patients with advanced solid tumors.23–25
4-Aryl-4H-chromenes are easily accessed via the three-
component reaction of aldehydes, malononitrile, and phenols
(Scheme 1).26–29 Interestingly, this procedure involving 3-
methoxyphenol affords low yields of the targeted chromenes 2
(7–24%),30 whereas application of resorcinol followed by methyla-
tion of the 7-hydroxy group in 1 furnishes 2 in 70–90% yields.26–29
Diversity of starting materials, synthetic feasibility of the con-
densation protocol, and potential for antimitotic properties turned
our attention to 7-methoxy-4-aryl-4H-chromenes 2 as analogues
of highly active 7-methoxy-PT derivatives.14 It was speculated that
the optimized substitution pattern for ring E may further improve
antiproliferative activity of the targeted compounds.8,31,32 In addi-
tion, small hydrophobic moieties at C7 in ring B (methoxy, amino,
dimethylamino, and ethylamino groups) were found to be benefi-
cial for inhibition of cancer cell growth and caspase-dependent
apoptosis.19,30 Considering the above, our initial aim was the bio-
logical evaluation of 4-aryl-4H-chromenes 1 and 2 with different
alkoxy substituents in ring E33 as antimitotic microtubule destabi-
lizing agents in the phenotypic sea urchin embryo assay.6 The
results are presented in Table 1. Typical effects of 7-methoxy-4-
aryl-4H-chromenes on the sea urchin embryos, as exemplified by
compound 2a, are demonstrated in Figure 2.
It was found that 7-hydroxy-4H-chromenes (1a–t) displayed
weak to no activity. Only chromene 1g with a 3-methoxy-4,5-
methylenedioxy ring E caused embryo spinning, indicating antimi-
totic microtubule destabilizing activity. A similar compound with
an ethylenedioxy group in ring E (1h) as well as molecules contain-
ing an unsubstituted (1t) or a 3-methoxyphenyl ring E (1o) were
identified as weak antitubulin agents, as suggested by the forma-
tion of tuberculate eggs. In contrast, 7-methoxy-4H-chromenes
(2) markedly influenced sea urchin embryo development, produc-
ing cleavage alteration/arrest and embryo spinning. They exhibited
significant antimitotic microtubule destabilizing activity in the low
nanomolar concentration range, and were 100-fold or more potent
than the corresponding 7-hydroxychromenes (1). Methylenedioxy
ring A in 4H-chromenes, as well as in aza-PTs,13 was not essential
and could be substituted by a 7-methoxy group yielding active
molecules (compare I and 2g, II and 2o). Our tests further demon-
strated that the 3-methoxy substituent in ring E was critical to the
antitubulin effect of 7-methoxy-4H-chromenes. The replacement
of the 3-methoxy moiety with an –NO2 or especially an –NH2
group (2h vs 2i and 2ii) markedly reduced the activity. Compounds
2m and 2n featuring a 2-methoxyphenyl ring E showed moderate
activity. The increase of the number of methoxy substituents from
three to four (2g and 2h vs 2a) was unfavourable. However, it was
impossible to establish precisely the relationship between the
number of methoxy groups in ring E and antimitotic potency, since
chromenes with one (2o) or three (2g and 2h) substituents exhib-
ited similar effects. Instead, the position of the methoxy groups in
the ring E apparently played the more important role (compare 2j
and 2n, 2k and 2m, 2o and 2p). Chromene 2j with a 3,5-dime-
thoxyphenyl ring E was identified as the most active antitubulin
agent in the sea urchin embryo assay.
The stereochemistry of microtubule destabilizing agents based
on the PT core was reported to significantly affect their antiprolif-
erative activity.8,34 For example, the R(À)-isomer of 4-aryl-4H-
chromene EPC2407 (Fig. 1) was shown to activate caspases, induce
Scheme 1. Synthesis of 7-substituted 4-aryl-4H-chromenes (1 and 2). Reagents and conditions: (a) MW, DBU (5 mol %), EtOH, 50 °C, 2–4 min; (b) CH3CN, CH3I, K2CO3, 20 °C,
48 h. (3): Respective 7-oxa-podophyllotoxins.
Semenova, Marina N.
