Probing cytochrome P450-mediated activation with a truncated azinomycin analogue

Article abstract of DOI:10.1039/c4md00411f

A deactivated alkene precursor (IC₅₀ = 81 μM) to the azinomycin epoxide natural product can be bioactivated by several cytochromes P450 (CYP) to generate antiproliferative metabolites with increased potency (IC₅₀ = 1-30 μM) in CHOwt cells. CYP1A1 and 3A4 were shown to generate exclusively the unnatural and the natural-configured azinomycin epoxide diastereoisomer respectively, while CYP1B1 produced both epoxides in a 3:1 mixture. The antiproliferative activity is linked to DNA damage as demonstrated using the comet assay. This journal is

Full text of DOI:10.1039/c4md00411f

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Probing cytochrome P450-mediated activation
with a truncated azinomycin analogue†
Cite this: DOI: 10.1039/c4md00411f
Victoria Vinader,a Maria Sadiq,a Mark Sutherland,a Mengying Huang,b
Paul M. Loadman,a Lina Elsalem,a Steven D. Shnyder,a Hongjuan Cui,b
a
c
a
a
*
*
Kamyar Afarinkia, Mark Searcey, Laurence H. Patterson and Klaus Pors
A deactivated alkene precursor (IC50 ¼ 81 mM) to the azinomycin epoxide natural product can be
bioactivated by several cytochromes P450 (CYP) to generate antiproliferative metabolites with increased
potency (IC50 ¼ 1–30 mM) in CHOwt cells. CYP1A1 and 3A4 were shown to generate exclusively the
unnatural and the natural-configured azinomycin epoxide diastereoisomer respectively, while CYP1B1
produced both epoxides in a 3 : 1 mixture. The antiproliferative activity is linked to DNA damage as
demonstrated using the comet assay.
Received 15th September 2014
Accepted 14th October 2014
DOI: 10.1039/c4md00411f
www.rsc.org/medchemcomm
The azinomycins A (1), B (2) and the azinomycin epoxide (3) are that the alkene derivative has utility as a bioprecursor that could
natural products isolated from Streptomyces griseofuscus (strain increase the therapeutic utility of the azinomycins in malignant
S42227) that have been extensively studied due to their potent tissues that (over) express enzymes capable of epoxidising
antitumour activity, their novel structures and their mode of xenobiotics notably the cytochrome P450 (CYP) mono-
action.1–4 The complete azinomycin A and B structures 1 and 2 oxygenases. Several drug metabolising CYPs have been shown
(Fig. 1) bind to the major groove of DNA and form interstrand to be expressed at high frequency in cancer tissues10 and we
crosslinks through alkylation by the epoxide and aziridine have focused our attention on several CYP1 and 2 family
moieties.5,6 The truncated epoxide structure 3 has been shown members as tumour selective prodrug activating enzymes. We
to alkylate at the N7 of guanine and promote a depurination developed AQ4N, a Phase II clinical trial candidate, to undergo
reaction that leads to single strand breaks.7 The naphthalene bioreductive activation by CYP1A1 and 3A4 selectively in
moiety is important in 3 due to an ability to intercalate into the hypoxic tumours.11,12 More recently we demonstrated that bio-
DNA duplex following alkylation.8
precursors of the duocarmycins can be activated selectively to
Although the azinomycins possess potent cytotoxicity in ultrapotent anti-proliferative metabolites by tumours express-
vitro, they have variable in vivo activity, most likely as a conse- ing CYP1A1 (ref. 13) and 2W1 (ref. 14) and we are proposing this
quence of the metabolic instability of the epoxide and aziridine approach to address the failure of the duocarmycins in the
moieties. In studies to dene the biosynthetic pathways to the clinic due to unacceptable normal tissue toxicity. Similarly,
azinomycins, Watanabe and co-workers have demonstrated that despite the promise of the azinomycins as cancer chemother-
valine is the precursor to the epoxide.9 This amino acid apeutics, they have thus far failed to progress clinically.
undergoes a series of transformations to generate 3-methyl-2-
Towards the aim of developing a class of chemotherapeutics
oxobutenoic acid, which is predicted to be reduced to the based on the azinomycins we describe the synthesis and bio-
alcohol and incorporated into the azinomycin structure. Enzy- logical evaluation of a library of azinomycin epoxide bio-
matic epoxidation of the alkene amide also occurs in this precursors with potential for CYP bioactivation.
sequence of events. This incorporation of an alkene is similar to
the synthetic sequence described previously and adapted by us
to generate all four diastereoisomers of 3.7 We hypothesised
aInstitute of Cancer Therapeutics, University of Bradford, West Yorkshire BD7 1DP, UK.
E-mail: k.pors1@bradford.ac.uk
bState Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing,
400715, China
cSchool of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4
7TJ, UK. E-mail: M.Searcey@uea.ac.uk
† Electronic supplementary information (ESI) available: Experimental details for
representative compounds synthesised and description of biological assays. See
DOI: 10.1039/c4md00411f
Fig. 1 Structure of azinomycins A (1) and B (2) and the azinomycin
epoxide (3).
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We previously7 adopted the stereoselective synthesis of
the epoxide 4 using Shipman's methodology (i.e. a Sharpless
dihydroxylation) in order to set the stereochemistry of the
subsequent alkene (in this case, compound 6). In this study, we
reasoned that as a proof-of-concept, synthesis of the racemic
alkene is appropriate, as it is not predictable as to whether the
stereochemistry of the naphthoate ester amide fragment has an
inuence on enzymic oxidation of the alkene by selected CYPs.
The benzyl ester of commercially available 3,3-dimethylacrylic
acid was epoxidised directly using m-CPBA to give an excellent
yield (93%) of the racemic compound, a reaction which can be
carried out on a multigram scale. Acid catalysed ring opening to
give the alkene was followed by direct treatment with ammonia
to give the amide 6.
Table 1 Antiproliferative activity of azinomycin analogues in wild-type
and CYP1A1-transfected CHO cell lines
Structure of
R group
IC50 CHO
(mM)
IC50 CHO(1A1)
(mM)
Compound ID
3a
4a
5a
2.8
5.7
85
3.2
6.0
6.3
It has previously been shown that changes in substitution on
the naphthalene ring maintain antitumour activity.15 In this
study we wanted to establish whether the substitution pattern
inuenced CYP mediated epoxidation of the alkene. To facili-
tate this structure activity relationship (SAR) for CYP-catalysed
epoxidation, we introduced a number of different analogues of
the naphthalene moiety alongside the natural product struc-
ture. The aromatic substituents were introduced via DCC
mediated coupling with fragment 6 (Scheme 1). Subsequent
chemical oxidation of the alkenes gave a mixture of diastereo-
isomers that were separable into the racemic mixtures. The
relative stereochemistry was assigned through reference to the
H2 proton (-proton) in the 1H NMR which was shied upeld
(5.32 from 5.21 ppm) in the conguration opposite to that
found in the natural azinomycin analogue.
3b
4b
5b
10
10
>100
5
9
>100
3c
4c
5c
7
7
>100
3
2
>100
3d
4d
5d
6
3
>100
3
9
>100
To determine the cytotoxicity of the truncated azinomycin
precursors, chemosensitivity studies were carried out using a
Chinese Hamster Ovary (CHO) cell line +/ꢀ transfected with
CYP1A1. The anti-proliferative activity of the various naphtha-
lene derivatives against this isogenic pair of CHO cell lines was
assessed using the MTT cell viability assay (Table 1). The
substitution pattern of the naphthalene moiety in the epox-
yamide compounds 3 and 4 has little effect on the biological
activity, with IC50 values between 2 and 10 mM for all
compounds tested. However, incubation of the alkenes 5a–e
with the cell lines demonstrated that only the natural product
naphthalene structure (5a) was bioactivated in the CYP1A1
expressing cell line, indicating that the substitution pattern on
the naphthalene moiety affects the binding mode with CYP1A1.
Since the naphthoate substitution pattern of compound 5a is
that of the natural azinomycin we explored whether other CYPs
important in drug metabolism could inuence its cytotoxicity.
Bioactivation by CYP1A1 was compared to CYP1B1, known to be
overexpressed in many tumour types16 and three hepatic CYPs
(1A2, 2D6 and 3A4). NADPH dependent CYP/CYP reductase
expressing bactosomes were incubated for 1 h with 5a. Subse-
quently the incubation extract was added to CHO wild type cells
which were subjected to the MTT antiproliferative assay. This
revealed that all CYPs could signicantly (3–24 fold) potentiate
5a activity. Potentiation by CYP3A4 (24.4-fold) was much greater
than CYP1B1 (7-fold) or CYP1A1 (5.8-fold), Table 2. The bio-
activation of the CYP bactosomes brings the potency of the
inactive azinomycin precursor to the low micromolar region,
3e
4e
5e
6
5
>100
3
9
>100
Scheme 1 Synthesis of compounds 3–6.
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Table 2 Growth inhibition of bioprecursor 5a and CYP bactosomes generated metabolite fractions of 5a against CHO cell line
5a
5a + CYP1A1
IC50
5a + CYP1A2
IC50
5a + CYP1B1
IC50
5a + CYP2D6
IC50
5a + CYP3A4
IC50
PFb
5.8
PFb
4.0
PFb
7.0
PFb
2.9
PFb
a
IC50
81.61 ꢁ 4.23
14.15 ꢁ 3.07
20.30 ꢁ 1.46
11.75 ꢁ 2.36
28.31 ꢁ 2.06
3.34 ꢁ 0.92
24.4
a
IC50 ¼ mM. b Potentiation factor (IC50 of 5a in CHO cells/IC50 of 5a + CYP isoform metabolites).
which is similar to what we have observed for the four diaste- of metabolic activation to generate the requisite proximate
reoisomers of compound 3 in the NCI 60 cell line panel.7 The crosslinking alkylation sites. Furthermore, 5a alone produced
CYP-mediated potentiation observed here suggests the possible no DNA damage conrming that CYP-activation is required for
formation of an azinomycin epoxide metabolite capable of the generation of DNA damage.
causing DNA damage. We therefore explored the nature of this
activation further.
CYP2W1 has been reported to be expressed in both primary17
and metastatic18 colon cancer tissue but not healthy tissues and
To investigate if the antiproliferative activity could be we are engaged with discovering new pharmacophores that are
correlated with DNA damage, CHO cells were treated with bio- bioactivated by CYP2W1. In this context, we explored the
precursor 5a or the metabolite fraction generated from 5a potential for 5a to be bioactivated by CYP2W1 using our
incubation with the CYP3A4 bactosomes. Aer 1 h exposure to isogenic SW480 +/ꢀ CYP2W1 cell line pair, but we did not
cells, DNA damage was assessed using the comet assay. EO9, observe any signicant antiproliferative activity in the CYP2W1-
camptothecin and melphalan were used as positive controls for transfected cells (Fig. S4†). However, 5a displayed a dose-
DNA single strand breaks (SSBs), DNA double strand breaks dependent growth inhibition against two human gastric cancer
(DSBs) and DNA interstrand cross-links (ICLs), respectively. The cell lines, SGC-7901 and MKN-45, which expressed CYP1A1 and
results showed that CYP3A4-generated metabolites were 3A4 at both gene and protein level (Fig. 3).
capable of producing DNA damage primarily through SSBs
(Fig. 2) with no evidence of DSBs as measured by increase in the DNA damage and CYP-associated antiproliferative activity,
mean tail moment (MTM) (Fig. S3†). metabolite fractions from CYP1A1, 1B1 and 3A4 were analysed
To identify the active metabolites likely to be responsible for
The results also indicated that a small amount of ICLs were by LC/MS/MS. All three CYPs oxidised the alkene to the epoxide,
generated. This is intriguing because it would require two sites the anticipated alkylating species of these truncated azinomy-
cins. However the epoxide stereochemistry generated by
different CYP isoforms was not the same. CYP1A1 and 3A4 were
shown to generate exclusively the ‘unnatural’ diastereoisomer
epoxide 3a and the naturally-congured epoxide 4a respectively,
while CYP1B1 produced both epoxides in a 3 : 1 mixture as
identied by co-elution with the respective authentic truncated
Fig. 2 DNA damage caused by 5a metabolite generated from incu-
bation with CYP3A4. DSBs and SSBs were measured by increase in
MTM (A) and ICLs by a decrease in MTM (B). Typical comet images
show SSBs with no drug (C), 20 mM E09 (D), 50 mM 5a (E) and 50 mM Fig. 3 CYP1A1 and 3A4 expression and antiproliferative activity in
CYP3A4-metabolised 5a (F). DNA ICLs with no drug (G), 20 mM gastric cell lines SGC-7901 and MKN-45. CYP1A1 and 3A4 expression
melphalan (H), 50 mM 5a (I) and 50 mM CYP3A4-metabolised 5a (J). The was measured by qRT-PCR (A) and Western blot (B). The growth
results are the mean ꢁ SD of at least three independent assays.
inhibition was measured using the MTT assay (C).
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Further development of such a bioprecursor approach could
provide a strategy to exploit the azinomycins for therapeutic
use. While CYP1A1 and 1B1 provide selective cancer drug
targets, the hepatic CYP3A4 enzyme could be exploited for liver
specic treatment of hepatitis B, hepatitis C and hepatocellular
carcinoma21 or with the reemergence of the oncolytic virus, a
‘suicide’ GDEPT approach also exists as
strategy.22,23
a therapeutic
Acknowledgements
The authors wish to gratefully acknowledge nancial support
from Yorkshire Cancer Research for Medicines Discovery Pro-
gramme grant number B209 and EPSRC Science Bridges China
for grant number EP/G042365/1. We thank Miss Gemma Mar-
ston and Miss Komal Nadeem for their contribution to gener-
ation of experimental data and the EPSRC NMSSC for HRMS
data.
Fig. 4 Metabolism profile of 5a incubated with CYP1A1, 1B1 or 3A4.
References
1 G. T. Kelly, C. Liu, R. Smith 3rd, R. S. Coleman and
C. M. Watanabe, Chem. Biol., 2006, 13, 485–492.
2 S. Ishizeki, M. Ohtsuka, K. Irinoda, K. Kukita, K. Nagaoka
and T. Nakashima, J. Antibiot., 1987, 40, 60–65.
3 K. Nagaoka, M. Matsumoto, J. Oono, K. Yokoi, S. Ishizeki and
T. Nakashima, J. Antibiot., 1986, 39, 1527–1532.
4 K. Yokoi, K. Nagaoka and T. Nakashima, Chem. Pharm. Bull.,
1986, 34, 4554–4561.
5 R. S. Coleman, R. J. Perez, C. H. Burk and A. Navarro, J. Am.
Chem. Soc., 2002, 124, 13008–13017.
6 R. S. Coleman, R. L. Woodward, A. M. Hayes, E. A. Crane,
A. Artese, F. Ortuso and S. Alcaro, Org. Lett., 2007, 9, 1891–
1894.
7 M. H. David-Cordonnier, M. Casely-Hayford, M. Kouach,
G. Briand, L. H. Patterson, C. Bailly and M. Searcey,
ChemBioChem, 2006, 7, 1658–1661.
8 M. A. Casely-Hayford, K. Pors, L. H. Patterson, C. Gerner,
S. Neidle and M. Searcey, Bioorg. Med. Chem. Lett., 2005,
15, 653–656.
Scheme 2 Generation of metabolites by CYP1A1, 1B1 and 3A4.
azinomycins (Fig. 4). The IC50 data and our CYP-metabolism
data from this study are consistent with the high potency of the
naturally-congured diastereoisomer epoxides previously
observed.7
The identication of a m/z 330 metabolite, consistent with
epoxide formation (Scheme 2), and DNA SSBs following CYP-
oxidation indicates the potential for bioactivation of the
precursor azinomycins prepared in this study.
9 V. Sharma, G. T. Kelly and C. M. Watanabe, Org. Lett., 2008,
10, 4815–4818.
10 C. Rodriguez-Antona and M. Ingelman-Sundberg, Oncogene,
2006, 25, 1679–1691.
11 A. Yakkundi, V. McErlane, M. Murray, H. O. McCarthy,
C. Ward, C. M. Hughes, L. H. Patterson, D. G. Hirst,
S. R. McKeown and T. Robson, Cancer Gene Ther., 2006, 13,
598–605.
Conclusions
We have demonstrated that the putative bioprecursor 5a to the
natural azinomycin product 3 can be activated in cells
expressing a number of CYPs known to be responsible for drug
metabolism. Certain isoforms, notably CYP1A1, 1B1 and 3A4 12 L. H. Patterson, S. R. McKeown, T. Robson, R. Gallagher,
are identied as highly expressed in certain tumours10 indi-
cating the value of truncated azinomycin analogues as potential
S. M. Raleigh and S. Orr, Anti-Cancer Drug Des., 1999, 14,
473–486.
bioprecursors with chemotherapeutic efficacy. We have 13 M. Sutherland, J. H. Gill, P. M. Loadman, J. P. Laye,
demonstrated proof of principle with bioprecursors of the
duocarmycin natural products targeting CYP1A1 (ref.13 and 19)
and 2W1 (ref. 14 and 20) enzymes. Uniquely, we have demon-
H. M. Sheldrake, N. A. Illingworth, M. N. Alandas,
P. A. Cooper, M. Searcey, K. Pors, S. D. Shnyder and
L. H. Patterson, Mol. Cancer Ther., 2013, 12, 27–37.
strated that CYP selective bioactivation in vivo can be obtained 14 S. Travica, K. Pors, P. M. Loadman, S. D. Shnyder,
with no overt toxicity to normal tissues including the liver.13,14
I. Johansson, M. N. Alandas, H. M. Sheldrake,
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2014

View Article Online
Concise Article
MedChemComm
S. Mkrtchian, L. H. Patterson and M. Ingelman-Sundberg, 20 H. M. Sheldrake, S. Travica, I. Johansson, P. M. Loadman,
Clin. Cancer Res., 2013, 19, 2952–2961.
15 T. J. Hodgkinson, L. R. Kelland, M. Shipman and F. Suzenet,
Bioorg. Med. Chem. Lett., 2000, 10, 239–241.
16 M. C. McFadyen and G. I. Murray, Future Oncol., 2005, 1,
259–263.
17 K. Stenstedt, M. Hallstrom, I. Johansson, M. Ingelman-
Sundberg, P. Ragnhammar and D. Edler, Anticancer Res.,
2012, 32, 3869–3874.
M. Sutherland, L. Elsalem, N. Illingworth, A. J. Cresswell,
T. Reuillon, S. D. Shnyder, S. Mkrtchian, M. Searcey,
M. Ingelman-Sundberg, L. H. Patterson and K. Pors, J.
Med. Chem., 2013, 56, 6273–6277.
21 M. D. Erion, K. R. Reddy, S. H. Boyer, M. C. Matelich,
J. Gomez-Galeno, R. H. Lemus, B. G. Ugarkar, T. J. Colby,
J. Schanzer and P. D. Van Poelje, J. Am. Chem. Soc., 2004,
126, 5154–5163.
18 K. Stenstedt, M. Hallstrom, F. Ledel, P. Ragnhammar, 22 H. Lu, C. S. Chen and D. J. Waxman, Cancer Gene Ther., 2009,
M. Ingelman-Sundberg, I. Johansson and D. Edler, Acta
Oncol., 2014, 53, 885–891.
19 K. Pors, P. M. Loadman, S. D. Shnyder, M. Sutherland,
H. M. Sheldrake, M. Guino, K. Kiakos, J. A. Hartley,
M. Searcey and L. H. Patterson, Chem. Commun., 2011, 47,
12062–12064.
16, 393–404.
23 H. O. McCarthy, A. Yakkundi, V. McErlane, C. M. Hughes,
G. Keilty, M. Murray, L. H. Patterson, D. G. Hirst,
S. R. McKeown and T. Robson, Cancer Gene Ther., 2003, 10,
40–48.
This journal is © The Royal Society of Chemistry 2014
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