Synthesis of a major mitomycin C DNA adduct via a triaminomitosene

Article abstract of DOI:10.1016/j.bmcl.2012.09.052

We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O⁶-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N² and O⁶ positions.

Full text of DOI:10.1016/j.bmcl.2012.09.052

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7198–7200
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a major mitomycin C DNA adduct via a triaminomitosene
Elise Champeil a, , Manuel M. Paz b, Elaan Lukasiewicz a, Wan S. Kong a, Stephanie Watson a,
⇑
Anne-Marie Sapse a
a John Jay College of Criminal Justice, 524 West 59 Street, New York, NY 10019, USA
b Departamento de Quimica Organica, Faculdade de Farmacia, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2012
Revised 12 September 2012
Accepted 17 September 2012
Available online 25 September 2012
We report here the synthesis of two amino precursors for the production of mitomycin C and 10-deca-
rbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precur-
sors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA
adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O6-(2-p-nitrophenyleth-
yl)deoxyinosine followed by deprotection at the N2 and O6 positions.
Published by Elsevier Ltd.
Keywords:
Nucleoside
Anticancer
Mitomycin C
Stereoisomer
Antibiotic
The mitomycins are a family of antitumor antibiotics made by
Streptomyces. One member of this family, mitomycin C (MC), is cur-
rently used to treat certain cancers.1 Its cytotoxic and antitumor
activity is attributed to its ability to alkylate DNA monofunctional-
ly and bifunctionally, resulting in DNA monoadducts as well as
interstrand and intrastrand cross-links (ICL).2 10-Decarbamoylmi-
tomycin C (DMC) is a derivative of MC lacking the carbamate
groups on C-10 (Fig. 1). As such, DMC was originally thought to
only alkylate DNA monofunctionally. However, when EMT6 mouse
mammary tumor cells were treated with DMC, both ICL and DNA
monoadducts were produced.3 The major adducts generated by
MC and DMC have different stereochemistry at carbon 1: trans
(or alpha, a) for MC and cis (or beta, b) for DMC. A rationale for this
stereochemical preference of alkylation has recently been pro-
posed.4 In addition, biochemical responses to the two drugs are
strikingly different.5 Individual structure–activity relationships of
multiple DNA adducts generated by a single agent have been inves-
tigated mostly in the case of organic mutagens and carcinogens.6 In
general, such studies utilize synthetic oligonucleotides bearing a
specific adduct at a unique position of their base sequences.7 The
postoligomerization method8 has been previously used to access
such adducts. The MC and DMC adducts present an opportunity
for similar studies, enabling in this case direct comparisons of
biological effects induced by a different stereochemistry at carbon
1 (Fig. 1).
Figure 1. Major Mitomycin C (MC, R = CONH2) adduct 1a = 1-alpha-Monoadduct
(trans) and major Decarbamoyl Mitomycin C (DMC, R = H) adduct 1b = 1-beta-
Decarbamoyl monoadduct (cis).
We have previously reported the synthesis of an MC metabolite
(2,7-diaminomitosene) adduct both on the nucleoside and oligonu-
cleotide levels by the postoligomerization method, the latter
resulting in the synthesis of a site-specifically modified oligode-
oxyribonucleotide.9 This was the first application of this method
to a DNA adduct of a complex natural product. We report here
the synthesis of two amino precursors for the production of
adducts 1a and 1b. In addition, the synthesis of monoadduct 1a
⇑
Corresponding author. Tel.: +1 646 557 4502; fax: +1 212 237 8318.
E-mail address: echampeil@jjay.cuny.edu (E. Champeil).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.09.052

E. Champeil et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7198–7200
7199
is described. The present alternative approach to adduct 1a opens
doors to the synthesis of MC and DMC-DNA adducts with beta ste-
reochemistry. This is all the more essential as the biomimetic
method developed for the production of these adducts is inefficient
for the production of adducts with beta stereochemistry.10
methanol/ammonium hydroxide mixture. The reaction worked
well for the trans isomer 6a, and afforded the trans amine 7a. How-
ever, attempts to convert the cis azide 6b to the cis amine 7b
yielded a mixture of unidentified products. Coupling of precursor
7a (trans) with 2-fluoro-O6-(2-p-nitrophenylethyl)deoxyinosine13
afforded intermediate 8a and subsequent deprotection at the O6
position using DBU yielded 9a (Scheme 3). However, final depro-
tection at the 2 position using penicillin amidase failed repeatedly
despite varied reaction conditions. Our focus shifted to another
protecting group, the trimethylsilylethoxycarbonyl (teoc) group
which can be removed under mild conditions.14 The cis and trans
teoc-protected 2,7-diamino-1-hydroxymitosene 10a (trans) and
10b (cis) (Scheme 1) were converted to the azido derivatives (using
similar conditions as for the synthesis of 6a and 6b from 3b) to give
11a and 11b. Reduction to the amines groups progressed efficiently
in the case of the alpha amino precursor 11a. However, the reac-
tion was challenging in the case of the beta amino mitosene 11b.
(as previously observed for the conversion of 6b to 7b). In this case,
the cis amine could be isolated but the yield and outcome of the
reaction were inconsistent. Evidently, the cis azides 6b and 11b
have a very different reactivity than the trans azides toward the
Staudinger reduction.
For the synthesis of suitable stereoisomeric amino precursors
6a (trans) and 6b (cis), we needed to first protect the 2-amino
position of the 2,7-diamino-1-hydroxymitosenes 2a and 2b. The
amino hydrin precursors 2a and 2b (Scheme 1) are single enantio-
mers and were synthesized from Mitomycin C.11 We initially used
the phenylacetyl group12 as a protective group (compounds 3a
(trans) and 3b (cis)). Direct conversion of the 1-hydroxyl group
to an azido group was first tested on compound 3b. The reaction
proved to be challenging. Use of the correct solvent was crucial.
In the presence of pyridine, the elimination product 4 was
obtained, as seen in Scheme 1. Use of DMF yielded the addition
product 5. However conversion to the azido group proceeded
smoothly in DMSO to give a mixture of stereoisomers 6a and 6b.
Reduction of the azido group to the amine was tried using different
reaction conditions (H2/Pd; Dithiothreitol/FeSO4; Zn/NH4Cl). The
conversion was finally achieved via a Staudinger reaction in a
We investigated the reason for the substrates’ different reactiv-
ity through DFT calculations. The syntheses of compounds 12a and
12b (Scheme 3) were studied with quantum chemical calculations,
using the Density Functional Theory method (DFT) as implemented
by the Spartan program with the B3LYP method using the 6–31G**
basis set.15 The energies of the azides 11a and 11b, of the interme-
diate iminophosphoranes 13a, 13b and of the amine products 12a
and 12b were calculated as well as the energies of each step. The
structures investigated are depicted in Figures S9–S18 (Supple-
mentary data). Table S1 (Supplementary data) shows the energies
of all the structures optimized. Table 1 shows the energies of each
step of the Staudinger reaction (Scheme 2). The cis compounds
(azide 11 b, amine 12b) are somewhat more stable than the trans
compounds (azide 11a, amine 12a), by 3.6 kcal/mol for the azides
and 2.2 kcal/mol for the amines. This stability is due to some
hydrogen bonds which are only present in the cis azides and
amines. However, as shown experimentally, the cis azide is less
reactive toward the Staudinger reduction. The thermodynamics
of the reaction, with an energy slightly more favorable to the trans
reaction is not sufficient to explain the experimental results. On
the other hand, the attack of a water molecule on the iminophos-
phoranes 13a and 13b would encounter steric hindrance in the
cis molecule. The resulting pseudo-transition states during the
final hydrolysis (Figs. S12 and S17), show the trans structure to
be more stable than the cis structure by approximately 5 kcal/
mol. This difference and the steric hindrance can justify the trans
reaction being more favorable than the cis reaction.
Next, we synthesized adduct 1a through coupling of 12a with 2-
fluoro-O6-(2-p-nitrophenylethyl)deoxyinosine. The nucleoside was
deprotected at the O6 position using DBU to afford 15a. We previ-
ously reported that ZnBr2 was a good reagent leading to complete
and rapid deprotection of the teoc group at the nucleoside level for
the synthesis of an MC metabolite (2,7-diaminomitosene).9 None-
theless, in the case of adduct 15a, these conditions also cleaved the
10-carbamate group resulting in a mixture of products. Several
other deprotection conditions were tried until finally, the use of
Table 1
Energies for the Staudinger reaction (kcal/mol)
Step 1
Step 2
Step 3
Step 4
Total
12 b
12 a
3.3
6
À52.2
À60.2
15.6
14.3
À52.2
À45.7
À83.8
À86.5
Scheme 1. Solvent effects for the synthesis of 6a and 6b. All trans compounds are
labeled a and all cis compounds are labeled b.

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E. Champeil et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7198–7200
Scheme 2. Staudinger reaction.
mycin C DNA adducts with opposite stereochemistry at C-1. We
also have synthesized the major DNA-mitomycin C adduct at the
nucleoside level. Adduct 1a had previously been isolated using bio-
mimetic methods but this is the first synthesis of this adduct.
Acknowledgments
This work was supported by the Program for Research Initia-
tives for Science Majors (PRISM) at John Jay College as well as a
PSC-CUNY research award 63117-00-41. We also thank Dr. Cliff
Soll for performing the mass spectroscopy measurements.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
052.
References and notes
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18
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The synthesis of the cis DNA
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nylethyl)deoxyinosine; however, it could not be successfully
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In summary, we have developed new routes toward two precur-
sors for the production of mitomycin C and 10-decarbamoylmito-
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