Full text of DOI:10.1039/d1md00020a

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RESEARCH ARTICLE
New prodrugs and analogs of the phenazine 5,10-
dioxide natural products iodinin and myxin
promote selective cytotoxicity towards human
acute myeloid leukemia cells†
Cite this: DOI: 10.1039/d1md00020a
ab
c
Elvar Örn Viktorsson,
Reidun Aesoy,c Sindre Støa,a Viola Lekve,
d
c
a
*
Stein Ove Døskeland, Lars Herfindal and Pål Rongved
Novel chemotherapeutic strategies for acute myeloid leukemia (AML) treatment are called for. We have
recently demonstrated that the phenazine 5,10-dioxide natural products iodinin (3) and myxin (4) exhibit
potent and hypoxia-selective cell death on MOLM-13 human AML cells, and that the N-oxide
functionalities are pivotal for the cytotoxic activity. Very few structure–activity relationship studies dedicated
to phenazine 5,10-dioxides exist on mammalian cell lines and the present work describes our efforts
regarding in vitro lead optimizations of the natural compounds iodinin (3) and myxin (4). Prodrug strategies
reveal carbamate side chains to be the optimal phenol-attached group. Derivatives with no oxygen-based
substituent (–OH or –OCH3) in the 6th position of the phenazine skeleton upheld potency if alkyl or
carbamate side chains were attached to the phenol in position 1. 7,8-Dihalogenated- and 7,8-dimethylated
analogs of 1-hydroxyphenazine 5,10-dioxide (21) displayed increased cytotoxic potency in MOLM-13 cells
compared to all the other compounds studied. On the other hand, dihalogenated compounds displayed
high toxicity towards the cardiomyoblast H9c2 cell line, while MOLM-13 selectivity of the 7,8-dimethylated
analogs were less affected. Further, a parallel artificial membrane permeability assay (PAMPA) demonstrated
the majority of the synthesized compounds to penetrate cell membranes efficiently, which corresponded
to their cytotoxic potency. This work enhances the understanding of the structural characteristics essential
for the activity of phenazine 5,10-dioxides, rendering them promising chemotherapeutic agents.
Received 21st January 2021,
Accepted 29th March 2021
DOI: 10.1039/d1md00020a
rsc.li/medchem
(3) possesses potent and selective cytotoxic properties towards
cell lines derived from patients with acute myeloid leukemia
Introduction
Phenazine 5,10-dioxides (1, Fig. 1) constitute an oxidized
subclass of phenazines (2, Fig. 1), often endowed with anti-
infective1–4 and tumor growth-inhibiting properties.5–9
Iodinin (1,6-dihydroxyphenazine 5,10-dioxide, 3) and myxin
(1-hydroxy-6-methoxyphenazine 5,10-dioxide, 4) are well-
known examples of natural origin, both containing N-oxide
functionalities rarely observed in natural products.10
Herfindal et al. demonstrated that the purple pigment iodinin
(AML).11 Moreover, iodinin (3) showed lower toxicity than the
standard anti-AML drug daunorubicin for non-cancerous cells
like hepatocytes, cardiomyoblasts (H9c2) and normal rat
kidney (NRK) epithelial cells.11
a School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo,
PO Box 1068 Blindern, N0316 Oslo, Norway. E-mail: pal.rongved@farmasi.uio.no
b School of Health Sciences, Faculty of Pharmaceutical Sciences, University of
Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland
c Centre for Pharmacy, Department of Clinical Science, University of Bergen, Jonas
Lies vei 87, N-5021 Bergen, Norway
d Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5021
Bergen, Norway
† Electronic supplementary information (ESI) available: Synthetic procedures,
characterization data, NMR spectra, detailed results from PAMPA assay, KPLS-
data. See DOI: 10.1039/d1md00020a
Fig. 1 The structure of phenazine 5,10-dioxide (1), phenazine (2) with
numbered positions and the natural products iodinin (3) and myxin (4).
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Iodinin (3) was one of the first phenazines to be
discovered, first isolated in 1939 by Davis et al., from
cultures of Brevibacterium iodinum.12,13 Its weak to moderate
antimicrobial activities have been known for decades,3,14,15
and early studies by Hano et al. also showed iodinin (3) to
expand the lifetime of mice with Ehrlich sarcoma relative to
placebo control.16 Although evidence from in vitro
experiments strongly suggest iodinin (3) to be a promising
candidate to pursue in new options for AML treatment,11,17
a major drawback is the very poor solubility in aqueous
solvents. This issue needed to be resolved in order to obtain
Fig. 2 The structure of compound 7, a highly cytotoxic analog of
iodinin (3) and myxin (4).
˙OH radicals such as tirapazamine (10, Fig. 3), as earlier
demonstrated by Gates and co-workers.25
a
preclinical evaluation of iodinin's potential as an
We have recently published an efficient synthesis of
iodinin (3), myxin (4) and several alkylated analogs, and
studied their cytotoxic effects on human AML-cells (MOLM-
13).26 The 1-hydroxy-6-(2-ethoxy-2-oxoethoxy) phenazine 5,10-
dioxide (7) was found to be the most potent analog with
ability to induce cell death, showing EC50 at sub-micromolar
concentrations (see Fig. 2).
anticancer drug. The poor aqueous solubility stems from the
two intramolecular ion–dipole bonds resulting in low
solute–solvent interactions (see 3, Fig. 1). Myxin (4) was
isolated from Sorangium sp. in 1966 (ref. 18) and has
received attention from the medicinal chemistry community
mainly because of its antimicrobial profile.3,4,19 In fact,
myxin (4) was earlier marketed as
(Unitrop®) in veterinary medicine for treatment of topical
infections.20
a
cupric complex
Several N-oxidized heterocyclic aromatic scaffolds have
been reported to act more potently in hypoxic than normoxic
environment. Examples are phenazine 5,10-dioxides (1),8,27
quinoxaline 1,4-dioxides28 (8) and 1,2,4-benzotriazine
1,4-dioxides (9).29 The latter class includes tirapazamine
(3-amino-1,2,4-benzotriazine 1,4-dioxide, 10), a lead prototype
of hypoxia selective prodrugs and a subject of several phase I,
II and III clinical trials for treatment of various cancers.30–32
The rapid cell growth and incomplete vasculature in tumors
generate a hypoxic environment33 where hypoxia selective
Two modes of action have been proposed for the
biological activity of myxin (4) and iodinin (3). Hollstein et al.
demonstrated that both of them are capable of DNA
intercalation, especially in guanine–cytosine rich regions of
the DNA-double strand at concentrations similar to those
required for other known intercalators like ethidium bromide
and actinomycin.21,22 They also showed myxin (4) to affect
the incorporation rate of CTP and GTP into RNA, but not of
ATP and UTP.21 The other proposed mechanism is by
generation of reactive oxygen species (ROS). Gates et al.23
showed phenazine 5,10-dioxides like myxin (4) being able to
undertake a bio reductive activation to form a radical
intermediate of the mother compound (Scheme 1), which
further collapses to unleash the cytotoxic hydroxyl radical
(˙OH).23 They found also that myxin (4) can induce cell death
of human colorectal HCT-116 cells under both aerobic and
anaerobic conditions.23 It is widely accepted that hydroxyl
radicals cause DNA damage and are able to abstract
hydrogens from the deoxyribose sugars in the DNA-double
strand helix.24 This is a typical result for drugs that unleash
drugs could play
a
role in antineoplastic treatment.34
Cimmino et al. reviewed phenazines and cancer, concluding
that phenazine 5,10-dioxides have the highest potential
among phenazines to be exploited for cancer therapy.5
We wanted to further exploit the anti-cancer potential of
the phenazines by (i) synthesis of bio-activatable prodrugs of
O-functionalized phenazine 5,10-dioxides with increased
aqueous solubility, (ii) increase their potency against cancer
cells by rationally optimize the ring substitution patterns, or
by utilizing other related scaffolds. Further we wanted to (iii)
assess cytotoxicity of the analogs on cancer cells and non-
Scheme 1 Discharge of the hydroxyl radical (˙OH) upon bio-reductive
activation of myxin (4).23
Fig. 3 The core structures of known hypoxia selective scaffolds and
tirapazamine.
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cancerous cell lines to develop a SAR study and to be able to
understand why some analogs are superior leads compared
to others. Taken together, in the present study we show that
improvement of bioactivity as well as physico-chemical
properties of simple molecules like the phenazines gives new
insight in how this scaffold can be optimized for future drug
radical before or after hydrolysis of the side chain. We also
aim to investigate if other non-cleavable substituents than
those published earlier26 can alter the anti-AML activity of
the phenazine 5,10-dioxide scaffold.
Results and discussion
Synthesis of compounds
development. The phenazine 5,10-dioxides require
a
bioreductive process to discharge an ˙OH radical and can
therefore be viewed as prodrugs (Scheme 1). In this paper
however, the term prodrug is specifically used for analogs
with side chain functionalities attached to their phenols and
are generally regarded as labile towards aqueous or
enzymatic hydrolysis (esters, carbonates, carbamates). In
theory, such compounds could have better physicochemical
properties than the lead iodinin (3), improving factors such
as solubility and membrane crossover. Once entered the cell,
such prodrugs could either release their cytotoxic hydroxyl
Iodinin (3) and myxin (4), synthesized according to our
previously described procedure,26 served as scaffold for the
synthesis of a small catalogue of iodinin and myxin prodrugs.
The ester analogs 11 and 12 were prepared by reacting
iodinin (3) with pivaloyl chloride at −40 °C and valeric
anhydride at
0 °C respectively, under basic conditions
catalyzed by DMAP (Scheme 2). By similar means, mono- and
bis-substituted carbonates (13 and 14) were produced by
reacting iodinin (3) with ethyl chloroformate. The reactions
Scheme 2 Synthesis of iodinin (3) and myxin (4) prodrugs. Reagents and conditions: a) pivaloyl chloride, Et3N, DMAP, PhMe −40 °C, 30 min, 39%.
b) Valeric anhydride, Et3N, DMAP, PhMe, 0 °C, 16 h, 39%. c) Ethyl chloroformate, Et3N, DMAP, PhMe −40 °C, 30 min, 25%. d) Ethyl chloroformate,
Et3N, DMAP, PhMe, 0 °C, 90 min, 67%. e) A corresponding carbamoyl chloride (or HCl salt), DABCO, THF, rt.
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forming 11–14 occurred rapidly since the reaction mixture
quickly shifted color from deep purple towards brown yellow.
Next in line were carbamate functionalities. Attempts to
react iodinin (3) with a carbamoyl chloride in PhMe with
DMAP and Et3N only resulted in trace conversions (TLC
analysis). Iodinin (3) showed no nucleophilicity towards any
isocyanates in attempts to form primary carbamates.
However, when DABCO was exposed to iodinin (3) and a neat
carbamoyl chloride, the reaction mixture started to produce
fume. The cherry red carbamate analogs 15–17 were thus
synthesized as depicted in Scheme 2. The yields from these
reactions aimed to mono-functionalize iodinin (3) were
modest (9–33%). Myxin (4) was rapidly transformed into the
orange-colored carbamate-analogs 18–20 using the same
method in good yields (see Scheme 2). The higher yields for
myxin (4) can be explained by its far superior solubility in
organic media compared to iodinin (3) and the
functionalization of only a single phenol group instead of
two. Interestingly DABCO does not deprotonate phenols,
because if such a deprotonation did occur, the reaction
mixture would instantly switch color from red to dark green,
like for the K2CO3/18-crown-6 system used for alkylations of
iodinin (3).26 This suggests that DABCO activates the
carbamoyl chloride allowing the neutral phenol moiety of the
phenazine to attack the DABCO–carbamoyl chloride complex
as depicted in Scheme 3.
The next step was to construct phenazine 5,10-dioxides
lacking an oxygen substituent in position 6. 1-Hydroxyphenazine
5,10-dioxide (21) was synthesized by two separate strategies (see
Scheme 4). First, commercially obtained 1-hydroxyphenazine
(22) was oxidized to 21 using pulse-wise addition of mCPBA in
toluene over a period of 5–6 hours, as reported previously for
the synthesis of iodinin (3).26 This method yielded the dioxide
(21) in 49% yield at a single gram scale. In an alternative
approach, a Beirut reaction35 was undertaken as cyclohexane-
1,2-dione and benzofuroxan (23) were condensed in neat
diethylamine. This gave a filtered bright red crude that was
oxidized using mCPBA. This 2-step procedure gave 2 g of
isolated 1-hydroxyphenazine 5,10-dioxide (21) in 40% yield.
The reaction of 21 with ethyl bromoacetate in the presence
of K2CO3 and 18-crown-6 gave compound 24 in 84% yield.
This molecule was synthesized to serve as an analog to 7 (see
Fig. 2), a highly cytotoxic compound in MOLM-13 cells.26 The
same step, when repeated with only tert-butyl bromoacetate,
produced 25, and again with 2-chloro-N,N-diethylacetamide
(in addition to KI) gave 26 in 25% yield after recrystallization
from hot EtOH. The main building block 21 was also exposed
to carbamoyl chlorides and DABCO forming carbamate
analogs 27–29 in 73–87% yields (see Scheme 4).
7,8-Disubstituted 1-methoxyphenazines 36–39 were
synthesized via the classic condensation procedure using
1,2-benzoquinones and O-phenylenediamines as starting
materials. Oxidation of 3-methoxycatechol (30) using
ortho-chloranil in cold ether afforded the corresponding
1,2-benzoquinone 31 after filtration of the reaction mixture
which was immediately transferred to an acidic toluene
solution containing a corresponding ortho-phenylenediamine
(32–35) (see Scheme 5). These efforts afforded
7,8-disubstituted-1-methoxyphenazines 36–39 isolated in
moderate to good yields.
Similar work had been performed earlier by Cushman
et al.36 and Huigens III and co-workers.1 Next, each
1-methoxyphenazine (36–39) was individually reacted in BBr3
neat
under
reflux
to
afford
the corresponding
1-hydroxyphenazines 40–43 in yields 87% and higher.
The 1-hydroxyphenazines 40–43 were then exposed to a
pulse-wise addition of mCPBA in toluene at 80 °C to give
7,8-disubstituted-1-hydroxyphenazine 5,10-dioxides 44–47 in
non-optimized, yet acceptable yields for further use (see
Scheme 5). Especially, 7,8-dibromo and 7,8-dichloro
substituted analogs 45 and 46 were difficult to purify due to
poor solubility and high lipophilicity resulting in tailing on
the silica gel. They were thus not subjected to biological
testing but the crudes after flash column chromatography
could be used for further synthesis.
7,8-Disubstituted-1-hydroxyphenazine 5,10-dioxides 44–47
were subsequently functionalized with ethyl-bromoacetate
(Scheme 6). These compounds were also synthesized to serve
as mimics for the cytotoxic compound 7 (see Fig. 2). In
addition, compounds 44–47 were also reacted with the
hydrochloride salt of 4-methyl-1-piperazinecarbonyl chloride
in order to give piperazine analogs 52–55 in good yields
Scheme 3 A proposed reaction mechanism for DABCO enforced carbamoylation of myxin (4).
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