7-N,7′,N′-(1″,2″-Dithianyl-3″, 6″-dimethylenyl)bismitomycin C: Synthesis and nucleophilic activation of a dimeric mitomycin

Article abstract of DOI:10.1039/b414806a

Dimeric alkylating agents that modify complementary DNA strands have engendered significant interest. We have prepared the novel dimeric mitomycin, 7-N,7′-N′-(1″,2″-dithianyl-3″,6″- dimethylenyI)bismitoinycin C (9), in which the mitomycins are bridged by a dithiane unit. Dimer 9, like the clinically tested acyclic disulfides KW-2149 (3) and BMS-181174 (4), was designed to activate under nucleophilic and reductive conditions. Successive nucleophile-mediated disulfide cleavage transformations of 9 are expected to generate thiol species ideally positioned to render the two mitomycin systems vulnerable to nucleophilic attack and permit DNA interstrand cross-link formation. The dithiane linker, strategically positioned between the two mitomycins, distinguished 9 from 3 and 4. Nucleophilic activation of this cyclic disulfide permitted both activated mitomycins to remain tethered to one another. We report the synthesis of 9, and show that the nucleophile Et,P markedly enhances the activation and consumption of 9, compared with the reference compound 7-N, 7′-N′-(cyclohexanyl- trans-1″,4″-duiiethylenyl)bismitomycin C (27). We further demonstrated that 9 provides higher levels of DNA interstrand cross-links than either the dimeric reference compounds, 27 and 7-N,7-N′-(2″, 5″-dihydroxy-1″,6″-hexanediyl)bismitomycin C (28), or the monomeric mitomycins, 1 and 3, when Et₃P is added to solutions containing EcoRI-linearized pBR322 DNA.

Full text of DOI:10.1039/b414806a

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A R T I C L E
7-N,7ꢀ-Nꢀ-(1ꢀꢀ,2ꢀꢀ-Dithianyl-3ꢀꢀ,6ꢀꢀ-dimethylenyl)bismitomycin C:
synthesis and nucleophilic activation of a dimeric mitomycin†
Sang Hyup Lee and Harold Kohn*
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of
North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA.
E-mail: harold_kohn@unc.edu; Fax: (919) 843-7835; Tel: (919) 966-2680
Received 23rd September 2004, Accepted 8th December 2004
First published as an Advance Article on the web 6th January 2005
Dimeric alkylating agents that modify complementary DNA strands have engendered significant interest. We have
prepared the novel dimeric mitomycin, 7-N,7ꢀ-Nꢀ-(1ꢀꢀ,2ꢀꢀ-dithianyl-3ꢀꢀ,6ꢀꢀ-dimethylenyl)bismitomycin C (9), in which
the mitomycins are bridged by a dithiane unit. Dimer 9, like the clinically tested acyclic disulfides KW-2149 (3) and
BMS-181174 (4), was designed to activate under nucleophilic and reductive conditions. Successive
nucleophile-mediated disulfide cleavage transformations of 9 are expected to generate thiol species ideally positioned
to render the two mitomycin systems vulnerable to nucleophilic attack and permit DNA interstrand cross-link
formation. The dithiane linker, strategically positioned between the two mitomycins, distinguished 9 from 3 and 4.
Nucleophilic activation of this cyclic disulfide permitted both activated mitomycins to remain tethered to one another.
We report the synthesis of 9, and show that the nucleophile Et3P markedly enhances the activation and consumption
of 9, compared with the reference compound 7-N, 7ꢀ-Nꢀ-(cyclohexanyl-trans-1ꢀꢀ,4ꢀꢀ-dimethylenyl)bismitomycin C (27).
We further demonstrated that 9 provides higher levels of DNA interstrand cross-links than either the dimeric
reference compounds, 27 and 7-N,7-Nꢀ-(2ꢀꢀ,5ꢀꢀ-dihydroxy-1ꢀꢀ,6ꢀꢀ-hexanediyl)bismitomycin C (28), or the monomeric
mitomycins, 1 and 3, when Et3P is added to solutions containing EcoRI-linearized pBR322 DNA.
mitomycin C(7) amino moieties with a hydrocarbon, alkyl ether,
or a dialkylamine unit linker. DNA cross-linking experiments
using a denaturing-gel electrophoresis-based assay showed that
the extent of DNA ISC for select dimeric mitomycins exceeded
those of 1 and that the reaction proceeded, in part, at the two
distal C(1) sites in the dimer. Recently, information was reported
Introduction
Mitomycin C (1) is a clinical anticancer agent of significant
importance.1 Bioreduction of 1 leads to the sequential activation
of the C(1) and C(10) sites necessary for the generation of
mono- and bis-(cross-linked) alkylated DNA adducts.2 While
both DNA lesions contribute to the antitumor activity of 1, it is
the DNA interstrand cross-link adducts (DNA ISC) that have
concerning the site(s) of DNA ISC bonding for this class of
dimeric mitomycins.7 Moreover, evidence was presented that
attracted the most interest. The cross-linking of complementary
these dimeric mitomycins were activated by the same reductases
DNA strands by 1 (1–DNA) is expected to inhibit DNA
that catalyze 1 utilisation and that DNA adduction proceeded
replication and subsequent cell proliferation.2 Consistent with
with the apparent 5ꢀ-CpG sequence specificity previously docu-
this notion 1–DNA ISC have been shown to be ca. 60 times
mented for 1.8,9
more lethal than the corresponding 1–DNA monoadducts.3
The clinical successes and limitations of 1 has led to the
syntheses and evaluation of more than 1000 analogues.10 In
the 1980s, two mitomycins were disclosed, 3 (KW-2149)11 and
4 (BMS-181174),12 where each contained a C(7) aminoethyl-
ene disulfide moiety in place of the C(7) amino unit in 1.
Significantly, 3 was active in 1-resistant P388 and non-hypoxic
There has been an increased interest in the design and
evaluation of novel dimeric agents that target DNA.4,5 Among
these compounds are intercalating and irreversible alkylating
agents that cross-link complementary strands of DNA. We have
prepared a series of dimeric mitomycins (2) designed to take
advantage of the enhanced reactivity of the C(1) site in 1,
compared with the C(10) position.6 Modification of DNA by
cells.13 Both 3 and 4 were entered into clinical trials, and 3
progressed to phase II testing. Mechanistic studies using 3, 4,
and structurally related compounds indicated that these agents
likely underwent nucleophile-assisted cleavage [e.g. glutathione
(GSH)] of the C(7) aminodisulfide unit to provide 5, and that 5
these dimeric mitomycins at the distal C(1) positions would lead
to DNA ISC. These dimeric mitomycins were tethered at the
initiated drug–DNA adduction.14–16
Compounds 3 and 4 are members of an emerging class of an-
ticancer agents that contain a multi-sulfide linkage. Cytotoxicity
† Dedicated to Laurence H. Hurley on the occasion of his 60th birthday.
in these agents is associated, in part, with a nucleophile-assisted,
T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2
4 7 1
©

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sulfur–sulfur cleavage transformation.11,12,17–22 These reports led
us to prepare C(8) iminoporfiromycin 6 that contained a cyclic
disulfide unit and its hydrocarbon counterpart 7.23 Porfiromycin
2a
(8) is the N(1a) methyl derivative of 1. We showed that
phosphine nucleophiles accelerated 6 activation and solvolysis,
compared with 7, and that it also provided increased levels of
DNA ISC, compared with 7.23
In this study, we report the synthesis of the dimeric mitomycin
9, which contains a cyclic disulfide unit. Dimer 9 was designed
to undergo facile DNA ISC by a nucleophile-assisted disulfide
cleavage process permitting successive modification of the two
mitomycin distal C(1) sites that remain tethered together.
We demonstrated that phosphines dramatically accelerate the
activation and utilisation of 9 compared with 1 and provide
enhanced levels of DNA ISC adducts.
Results and discussion
Selection of dimeric mitomycin 9
Compound 9 retained the key structural features found for 1
and for many semi-synthetic mitomycins. Thus, we expected
that this dimeric mitomycin would likely be activated by both
reductases and acids, pathways previously documented for 1.2
Four structural elements are, however, unique to 9 and were
incorporated into our design of this mitomycin to promote
nucleophile-assisted DNA ISC. First, 9 had two mitomycin units
to permit DNA adduction at the reactive C(1) site.6 Second, the
disulfide unit was strategically placed three atoms away from
the C(7) position in both mitomycins. This arrangement has
been important for intramolecular thiol-mediated mitomycin
activation processes.16 Third, the dithiane linker in 9 ensured that
the two mitomycin units would remain together after activation
and DNA adduction. This feature differentiated 9 from 3 and 4
where disulfide cleavage leads to a monomeric species. Fourth,
the dithiane linker is of sufficient size to permit modification of
the two distal mitomycin C(1) sites in 9 by guanine residues on
complementary DNA strands. We estimate with computational
programs (Sybyl 6.0, HyperChem 7.1) that the distance for 9
Scheme 1 Proposed nucleophile-mediated activation pathway for 9.
˚
and its expected intermediates to be between 7–25 A depending
C(9a) and mitosene formation, permitting the successive DNA
adduction at the two distal C(1) sites in the dimer. If correct,
9 nucleophile-mediated activation in the presence of DNA will
lead to DNA ISC.
upon their conformation. This distance can cross-link guanines
on different DNA strands separated by as many as five base
pairs.
The envisaged nucleophile-mediated route unique to 9 is
shown in Scheme 1. Activation of 9 begins with intra-
cellular thiol- or serum albumin (CSH)-mediated disulfide
cleavage14–16,24–26 to give 10, which is then converted to 11
through intramolecular cyclization. In Scheme 1, we show
that intramolecular cyclization occurs at the C(8); however,
we recognize that alternative cyclization sites [C(7), C(6)]14,16,23
may initiate mitomycin activation. A second round of disulfide
cleavage beginning with 11 provides 12, which is then converted
to 13 by intramolecular cyclization. Formation of 11 and 13
disrupts the N(4)–C(4a)–C(8a)–C(8)–O conjugated system and
is expected to lead to the rapid loss of methanol from C(9) and
Thus, three objectives were established for this study: the
synthesis and characterization of 9, the documentation of the
nucleophile-mediated activation pathway for 9, and the assess-
ment of the 9 DNA ISC efficiencies under nucleophile-mediated
conditions using linearized pBR322 DNA. We also determined
the in vitro cellular cytotoxicities of 9 against human lung
adenocarcinoma cell line A549. While we found it interesting,
the cytotoxicity information for 9 was not used in our assessment
of the structural requirements for nucleophile activation of this
mitomycin since neither the effective concentration of 9 within
the A549 cells nor the factors that contributed to the mitomycin
inhibition of cell replication were determined.
4 7 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2

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Synthesis of the cyclic disulfide bridge: (3R,6S)-meso-
3,6-bis(aminomethyl)-1,2-dithiane di-trifluoroacetate salt (14)
acid produced no reaction (data not shown). Next, we treated
19 (meso : dl = 3.5 : 1) with MsCl in pyridine to give 20 in
a moderate yield (67%). 13C NMR analysis showed that 20
was isolated as a single diastereomer. Once again, the observed
yield for this transformation indicated that this compound was
the meso-isomer. We suspect that conditions employed in the
isolation work-up of this reaction (addition of H2O) led to the
selective precipitation of the meso-20. Displacing mesylate units
in 20 with KSAc in EtOH gave 21 in a 60% yield. 13C NMR
analysis showed only a single isomer, thus providing evidence
that substitution proceeded with stereochemical control by an
SN2 pathway. We have, therefore, assigned 21 as the meso-
stereoisomer. Hydrolysis of the thioacetate groups in 21 with
aqueous methanolic K2CO3 gave the dithio derivative 22, which
was directly oxidized in basic methanol with O2 to yield cyclic 23
(55% yield). Cyclic disulfide 23 was also obtained in 89% yield
from 21 by sequential hydrolysis and oxidation without isolation
of 22. The BOC groups in 23 were removed in near quantitative
yield by trifluoroacetic acid (TFA) to give cyclic disulfide 14. The
overall yield for 14 from 15 was 11% (9 steps) and our reaction
sequence provided 14 as a single diastereomer (meso).
Synthesis of 9 required the preparation of (3R,6S)-meso-
3,6-bis(aminomethyl)-1,2-dithiane di-trifluoroacetate salt (14)
(Scheme 2). We began by converting 1,5-hexadiene (15) to
diepoxide 16 using m-chloroperbenzoic acid.27,28 Epoxide cleav-
age with phthalimide in DMF gave 17.28 Three stereoisomers
of 17 exist: meso-(R,S) and the enantiomeric pair, threo-(R,R),
and threo-(S,S). We found that the isolated product existed
as a mixture of diastereomers (2.7 : 1, 13C NMR analysis).28
Recrystallization (twice) of 17 in DMF (80 ◦C) provided a
sample enriched in one isomer (9.4 : 1, 13C NMR analysis) in 64%
yield.28 X-Ray crystallographic analysis identified this adduct
as meso-17,28 indicating that the major product in the initially
isolated 17 mixture (2.7 : 1 diastereomeric mixture) was the
meso-adduct. Hydrazine deprotection of the phthalimide units
in 17 (meso : dl = 2.7 : 1) gave 1829 (meso : dl = 2.7 :
1, 13C NMR analysis) in near quantitative yields after acid
work-up. Subsequent protection of the amino groups in 18
with BOC2O and Et3N afforded 19 in moderate yield (66%)
as a mixture of diastereomers (meso : dl = 3.5 : 1, 13C NMR
analysis). Recrystallization of 19 from ethyl acetate provided
a single isomer (≥10 : 1, 13C NMR analysis) (75% yield).
The product yields and the ratio of diastereomers obtained
for the conversion of 17 to 19 indicated that the major 19
diastereomer in the 3.5 : 1 mixture was the meso-isomer. Several
methods were explored to replace the hydroxyl groups in 19
by thioacetate units. Mitsunobu reaction30 of 19 with thioacetic
Synthesis of mitomycin dimers 9, 27, and 28
Treatment of mitomycin A31 (MMA, 24) with 14 in the presence
of Et3N gave 9 (53% yield) along with the mono-substituted
products 25a and 25b (21% yield). Several reaction conditions
and solvents (i.e. dichloromethane, ethanol, methanol, chloro-
form) were examined for this transformation, and we found that
methanol (room temperature, 1 d) gave the best results. In the
synthesis of 9, the solution color changed from violet to dark
blue as the reaction proceeded (1 d), with monomers 25a and 25b
being formed initially and then converted to dimer 9 (TLC and
HPLC analyses). On TLC, 25a and 25b exhibited lower Rf values
(0.15) than dimer 9 (0.47). After 1 day the product ratio of dimer
9 to monomers 25 was ca. 2.5. Using HPLC, we observed two
peaks of equal intensity for the monomers (tR 21.8, 22.3 min) that
were consistent with the formation of two diastereomers (25a,
25b); only one HPLC peak (tR 29.8 min) was observed for 9.
Using similar reaction conditions, we prepared the two
dimeric mitomycins, 27 (70% yield) and 28 (59% yield), upon
treatment of 24 with 1,4-bis(aminomethyl)cyclohexane (26) and
18, respectively. Compound 27 was chosen as the reference
compound for the chemical studies. In 27, we replaced the
dithiane moiety in 9 with a cyclohexyl moiety to deter-
mine the importance of the disulfide cleavage processes for
Scheme 2 Synthesis of cyclic disulfide meso-14.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2
4 7 3

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nucleophile-mediated mitomycin activation. We recognized that
27 existed as the trans-isomer while 9 was the cis-isomer. Our
decision to prepare 27 was based on the commercial availability
of trans-26. While this structural difference should not affect
the compounds’ reactivities with nucleophiles, it can impact
their DNA adduction and cytotoxicity properties. Thus, we
used 28 along with 27 as reference compounds for 9 for the
biochemical studies. Significantly, the hydroxyl groups in diol
28 are spatially positioned at the same sites as the thiol units
in projected intermediates 10 and 12 (Scheme 1). We prepared
diastereomeric 28 (1.6 : 1 mixture) by treating 18 (meso : dl =
2.7 : 1) with 24. On the basis of the yield obtained for 28 (59%)
and the starting composition of 18 we have termed the major
isomer in the 1.6 : 1 diastereomeric mixture (13C NMR analysis)
as (2ꢀꢀR,5ꢀꢀS)-28.
Chemical reactivity of dimeric mitomycins
We determined the rate of methanolysis of 9 and 27 in the
absence of nucleophiles and in the presence of a phosphine and
thiols to learn if this mitomycin is activated by nucleophiles
(see later in Tables 1 and 2). The kinetic measurements were
monitored by UV-vis spectroscopy (200–600 nm) or HPLC using
UV-vis detection for greater than two half-lives, when possible,
and then the absorbance of the starting mitomycin (374 nm)
was plotted against time. We used non-linear regression analysis
to fit the observed exponential decay of 9 using the SigmaPlot
Program 2001 to provide pseudo-first-order rate constants. The
reactions were conducted in duplicate, and the results averaged.
The products were verified, when possible, by HPLC and TLC
analyses with authentic samples.
The methanolysis of 9 was first monitored by HPLC using a
MeOH–CHCl3 (1 : 1) solution (effective ‘pH’ 3, room tempera-
ture, 3 d). The HPLC chromatograms for 9, 29 and 30 are shown
in Fig. 1(a)–(c), respectively, and are distinguished by their
distinctive UV-vis profiles, which show either the mitomycin
(374 nm, e.g. 9) or mitosene (316 nm, e.g. 30) chromophore32
or both (e.g. 29). As the reaction proceeded, we observed that 9
disappeared (tR 29.8 min) and four peaks that corresponded to
the four diastereomers for 29 [tR 29.6, 30.0, 30.3 and 30.8 min (1 :
1.1 : 0.9 : 1)] appeared with the subsequent emergence of three
peaks for 30 (tR 30.6, 31.3 and 32.1 min) in an approximate 1 : 2 :
1 ratio. We have assumed that two of the four 30 diastereomeric
adducts overlapped in the HPLC.
Fig. 1 HPLC profiles of 9 (a), 29 (b) and 30 (c). The reactions were
monitored at 374 nm for 9 (tR 29.8 min) and 316 nm for 29 (tR 29.6,
30.0, 30.3 and 30.8 min) and 30 (tR 30.6, 31.3 and 32.1 min).
Solvolytic products 30 were identified by their HPLC, UV-vis,
1
1H NMR and mass spectrometric properties. In the H NMR
Purification of the product mixture by preparative thin layer
chromatography (PTLC) afforded an authentic sample of the
C(1) methoxymitosenes 30 as a mixture of diastereomers.
spectra for 30 we observed the expected resonance (d 3.51) for
the C(1) methoxy units and the downfield resonances (d 5.73 and
5.78) for the C(10) methylene unit.33 The expected molecular ion
4 7 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2

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Table 1 Methanolysis rates for 9 at effective ‘pH’ 7.4, 5.5, and 4.0a
either 9 or 27 after 6–10 d, compared with solutions without
L-DTT. Likewise, when GSH was used as the nucleophile
(20 equiv.) the activation rates of 9 and 27 were not affected
(<5%) after 10 d. The similarity of the 9 and 27 rate data
and the lack of detectable rate enhancements as the thiol and
the thiol concentration were varied led us to conclude that
L-DTT and GSH did not contribute to the methanolysis of
mitomycins 9 and 27 at pH 7.4. This finding mirrored the low
activation level observed for C(8) iminoporfiromycin 6 when L-
DTT was added.23 For 6, we suggested that the thiol-cleaved
C(8) iminoporfiromycin product 31, if formed, can revert back
‘pH’
k
obs/d−1
t1/2/d
7.4
5.5
4.0
0.0347
0.0533
1.65
20b
13
0.42
a Reactions were run in buffered methanolic solution (0.1 M Tris·HCl,
pH 7.4; 0.1 M bis-Tris·HCl, pH 5.5; 0.1 M bis-Tris·HCl, pH 4.0) at
25 ◦C. All reactions were run in duplicate and the values averaged. The
concentration of 9 was 0.03 mM and the data were obtained using a Cary
3Bio Varian UV-visible spectrophotometer and the reactions monitored
at 374 2 nm unless otherwise indicated. b The data were obtained using
HPLC.
to 6 due to the ease of formation of the 6-membered ring.23
A
similar pathway may have occurred with the L-DTT–9 cleaved
product 32.
peak (m/z 813 [M + 1]+) for the C(1) methoxymitosenes 30 was
observed in the low-resolution mass spectrum.
We next determined the rate of methanolysis for mitomycin
dimer 9 in buffered methanolic solutions at effective ‘pH’ 7.4,
5.5 and 4.0 in the absence of nucleophiles at 25 ◦C (Table 1).
We observed that reduction of pH led to enhanced solvolysis
rates. The observed rate increases were not proportional to the
pH. We observed only a modest increase in the conversion of
9 to 29 and to 30 as we reduced the pH from 7.4 to 5.5. The
observed half-life (t1/2) for 9 at pH 5.5 was comparable with that
reported for 1 (t1/2 = 13.7 d).34 These results suggested that 9
underwent acid-catalyzed activation along a pathway similar to
that of 1.32,35 A more substantial rate increase was observed as the
pH was lowered from 5.5 to 4.0. We observed a clear isosbestic
point at 337 nm (data not shown). Analysis of the rate data was
consistent with our assumption that the methanolysis followed
pseudo-first-order kinetics and that the two mitomycin units
underwent change independent of one another. Using HPLC we
observed the steady depletion of 9, the appearance of 29 followed
by appearance of 30, and the concomitant disappearance of 29
over a 75 h period (data not shown). When we examined the
solvolysis of 27 at pH 7.4 by UV-vis spectroscopy we observed
little solvolysis after 10 d.
We next asked if phosphines affected the activation of 9 and
27 (Table 2). Previously, Gates and co-workers showed that
Ph3P triggered the activation of leinamycin.22 We subsequently
showed that Et3P increased the activation of C(8) iminopor-
firomycin 6.23 We found that Et3P (10–50 equiv.) markedly
enhanced 9 activation. Rate increases for 9 were only observed
upon addition of ≥10 equiv. of Et3P; a similar finding was
observed for 6.23 We suspect that this represented the threshold
level of Et3P needed for mitomycin nucleophilic activation
under our conditions, and that sub-threshold levels of Et3P are
consumed by the trace amounts of O2 not purged by Ar. For
10, 20 and 50 equiv. of Et3P we observed a 77-, 154- and 345-
fold increase, respectively, in the rate of mitomycin consumption
and the formation of mitosene products (Table 2), documenting
that the activation rate was linear with Et3P concentration. In
contrast to 9, no appreciable rate enhancements (<5%) were
observed for 27 upon addition of Et3P (10–50 equiv.) (Table 2).
Our findings that Et3P affected only the consumption of 9
and not of 27 led us to conclude that the disulfide group in 9
underwent cleavage to thiol 33 thereby activating the appended
mitomycin units.
Since 9 and 27 underwent little change at pH 7.4, we deter-
mined if the solvolysis rates of these dimers were increased in
the presence of thiols [L-dithiothreitol (L-DTT), and glutathione
(GSH)] at pH 7.4 (Table 2). Adding L-DTT (10, 20 equiv.)
gave no significant increases (≤5%) in the consumption of
Table 2 Methanolysis rates for 9 and 27 in the presence of nucleophiles
at effective ‘pH’ 7.4a
Reagents
Nu−
9
27
Equiv.
k
obs/d−1
t
1/2/d
k
obs/d−1
t
1/2/d
c
c
c
c
c
c
None
L-DTT
—
10
20
0.0347b
20
d
d
d
d
c
c
d
c
c
d
c
c
GSH
Et3P
20
2
5
—
—
—
—
cb
cb
cb
c
c
c
10
20
50
2.67
5.33e
12.0
0.26
0.13
0.058
a Reactions were run in buffered methanolic solution (0.1 M Tris·HCl,
pH 7.4) at 25 ◦C. The reactions were run in duplicate and the values
averaged. The data were obtained using a Cary 3Bio Varian UV-visible
spectrophotometer and the reactions monitored at 374 3 nm unless
otherwise indicated. The concentration of the mitomycin was 0.03 mM.
b The data were obtained using HPLC. c No appreciable change in
10 d (less than 5% of original amount). The unreacted mitomycin was
identified by HPLC and TLC. d No appreciable change in 6 d (less than
5% of original amount). The unreacted mitomycin was identified by
HPLC and TLC. e The data were obtained by UV spectroscopy and are
in agreement with the HPLC data (t1/2 = 0.13 d).
When the reactions of 9 with excess Et3P were monitored
by UV-vis spectroscopy we observed the progressive increase in
the 314 nm absorption with time, which was consistent with the
formation of mitosene products. Careful inspection of the HPLC
chromatograms for the 9 plus Et3P (10 equiv.) reaction revealed
minor amounts of the C(1) substituted products 29 and 30
during the initial stages (0–45 min) of the reaction. The products
disappeared with time with the concomitant appearance of
a new, unidentified peak (tR 24.0 min) that showed UV-vis
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2
4 7 5

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absorption maxima at 233 and 314 nm (data not shown). When
9 was treated with 20 equiv. of Et3P we observed only 29 in
the HPLC at the initial stages of the reaction followed by the
appearance of the unidentified 24 min peak. Several attempts to
identify this new peak by LC–MS and after chemical treatment
(e.g. hydrolysis, oxidation) gave inconclusive results (data not
shown).
Our finding that 9 consumption was nearly linear with Et3P
concentration and that Et3P mediated the conversion of 9 to
mitosenes 29 and then to 30 led us to conclude that phosphine
cleavage of the disulfide unit in 9 provided thiol(s) (i.e. 33) capa-
ble of activating the quinone ring in 33. The kinetic data further
suggest that the disulfide cleavage is the rate-limiting step. In
Scheme 3, we propose a phosphine-mediated activation pathway
for 9 that is consistent with these findings. This mechanism is
similar to that suggested for C(8) iminoporfiromycin 6.23 The
generation of thiol 33 is key to this pathway. We expect that
33 undergoes intramolecular cyclization to give hemi-thioketal
34 leading to the disruption of the N(4)–C(4a)–C(8a)–C(8)–
O conjugated system. Mitosene formation and aziridine ring-
opening then occur to give the C(1)-activated product 35. A
second round of activation is initiated after the decomposition
of the thiophosphonium species 35 by H2O (or MeOH)36,37
to provide thiol 36. Similar to the conversion of 33 to 35,
intramolecular cyclization of thiol 36 leads to 37 and then 38, in
which both C(1) sites in the two distal mitomycin units have been
modified. The reaction is completed when 38 converts to 40. Our
pathway derives support from recent studies showing that Et3P
rapidly promotes L-DTT disulfide cleavage,37 and comparable
activation results obtained for C(8) iminoporfiromycin 6 with
phosphines.23
DNA bonding profiles for mitomycin dimers 9, 27, and 28
The ability of dimeric mitomycins 9, 27 and 28 to cross-link com-
plementary EcoRI-linearized pBR322 DNA was determined
using denaturing alkaline agarose gel electrophoresis as reported
38a
38b
by Cech and Tepe and Williams. The size of the DNA
product(s) was estimated using k DNA digested with HindIII as
a molecular weight marker. The extent of DNA ISC formation
for 9, 27 and 28 was determined in the absence and the presence
of Et3P and L-DTT. We also included 1 and 3 in this study.
Scheme 3 Proposed phosphine-mediated activation pathway for 9.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2
4 7 6

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We first examined the extent of 9 DNA ISC induced by
Et3P for varying concentrations of 9 (0.1–0.01 mM) and Et3P
(5, 10 equiv.). The experiments were run at room temperature
(2 h) in aqueous buffered solutions (pH 7.4). We found that
treatment of 0.1 mM concentrations of 9 with Et3P provided
appreciable levels of DNA ISC adducts, and we used this
mitomycin concentration in our subsequent studies. Next, we
added Et3P (5 equiv.) and compared the extent of DNA ISC for
9 with those obtained for 1, 27 and 28 (Fig. 2). We observed that 9
efficiently generated DNA ISC (83%) while 27 and 28 generated
14 and 21%, respectively. Under these conditions, 1 gave 5% of
DNA ISC. These findings differentiate dimeric mitomycins 9,
27, and 28 from their monomeric counterpart 1, and distinguish
dimeric mitomycins that contain a disulfide-containing linker (9)
from those that do not (27, 28). Dimers 9, 27, and 28 all showed
higher levels (14–83%) of DNA ISC than 1 (5%). Since 1 can only
generate DNA ISC by sequential adduction at C(1) and C(10)
but 9, 27, and 28 can cross-link complementary strands by DNA
adduction processes that occur at the two mitomycins, we have
tentatively attributed the increased levels of DNA ISC for the
dimers, compared with 1, in part, to C(1) modification processes.
This finding is in agreement with our earlier studies that 2 gave
enhanced levels of DNA ISC compared with 1 under reductive
(Na2S2O4) conditions6 and was supported by the recent study of
Tomasz and co-workers.7 Significantly, when we compared the
extent of DNA ISC for 9, 27, and 28 we observed a 4.0–5.9-fold
increase in DNA ISC upon inclusion of a disulfide unit in the
linker. We have attributed the enhanced level of DNA ISC for
9, in part, to the functional role provided by the disulfide unit
in the Et3P-mediated mitomycin activation process (Scheme 3).
Significantly, the extent of DNA ISC for 9 (0.1 mM) under
Et3P-mediated conditions exceeded those reported for dimeric
mitomycins 2 (0.2 mM) under reductive conditions (Na2S2O4).6
to substantial levels of DNA ISC (Fig. 3) yet provided only trace
levels of mitosene products (Table 2) documented the sensitivity
of the denaturing agarose gel electrophoresis assay. Generation
of only a single DNA ISC within the 4361 bp EcoRI pBR322
DNA leads to a marked change in the electrophoretic mobility
of the DNA adduct.
Fig. 3 Denaturing 1.2% alkaline agarose gel for 1, 9, 27 and 28
(0.1 mM) using L-DTT (5 equiv.). DNA cross-linking experiments using
0.1 mM mitomycins and EcoRI-linearized pBR322 plasmid DNA with
L-DTT (5 equiv.). All reactions were incubated at room temperature
(2 h). Lane 1: k HindIII DNA molecular weight marker. Lane 2:
control (only linearized pBR322). Lane 3: 1 + L-DTT (5 equiv.). Lane 4:
28 + L-DTT (5 equiv.). Lane 5: 27 + L-DTT (5 equiv.). Lane 6: 9 +
L-DTT (5 equiv.). Lane 7: only L-DTT (5 equiv.).
Similar DNA-bonding profiles were obtained when the re-
action time was reduced from 2 h to 6 min and the amount
of nucleophile was increased from 5 to 10 equiv. For Et3P, we
observed that 9 gave moderate levels of DNA ISC (58%), and
the extent of DNA ISC for 27, 28, and 1 was 8, 12%, and 3%,
respectively (data not shown). For L-DTT (10 equiv.), we found
that 9 gave moderate levels of DNA ISC (30%), and 27, 28,
and 1 gave reduced levels, 13, 16, and 3%, respectively (data not
shown).
Mitomycin 9 was more efficient than 1 in generating DNA ISC
upon treatment with Et3P (Fig. 2). But 9 is a dimeric mitomycin
with an appended disulfide unit and 1 is a monomeric mitomycin
without an attached disulfide moiety. Thus, we compared 1
with 3 to determine the importance of the C(7) aminoethylene
disulfide unit for DNA ISC. Significantly, McAdam et al.
25b
showed that 3 upon treatment with thiols provided DNA ISC.
Treatment (6 min) of 0.1 mM solutions of 1 and 3 with Et3P
(10 equiv.) gave levels of DNA ISC corresponding to 5 and 14%,
respectively (data not shown). Since the effective monomeric
mitomycin unit concentration of a 0.1 mM solution of dimeric 9
is twice that of a 0.1 mM solution of 3, the observed 14% DNA
ISC for 3 will likely increase with increasing 3 concentration.
Nonetheless, the 58% DNA ISC found for 9 indicates that the
two mitomycin units and the cyclic disulfide bridge contributed
to the DNA ISC efficiency of this agent.
The effect of Et3P on 9 and 27 DNA ISC transformations
paralleled the observed consumption rates for these mitomycins
(Table 2). However, the effect of Et3P on the kinetic data
appeared greater than its effect on the extent of DNA ISC
formation. The DNA ISC experiments did not permit us
to measure the number of ISC adducts within EcoRI DNA
(4631 bp) for 9, 27 or 28. Thus, the true effect of Et3P in
these experiments remains unknown. Moreover, we have not
determined the site(s) of mitomycin covalent adduction [i.e.
C(1), C(10)]. Dimeric mitomycin activation may have given tri-
and tetra-functionalized adducts,7 and DNA products where
linkages exist between duplexes. These questions await further
study. Nonetheless, we concluded that the enhanced levels of
DNA ISC for 9 compared with 27, 28, and 3 stemmed, in
part, from nucleophile-mediated mitomycin activation processes
leading to DNA adduction (Scheme 3).
Fig. 2 Denaturing 1.2% alkaline agarose gel for 1, 9, 27 and 28
(0.1 mM) using Et3P (5 equiv.). DNA cross-linking experiments using
0.1 mM concentrations and EcoRI-linearized pBR322 plasmid DNA
with Et3P (5 equiv.). All reactions were incubated at room temperature
(2 h). Lane 1: k HindIII DNA molecular weight marker. Lane 2: control
(only linearized pBR322). Lane 3: 1 + Et3P (5 equiv.). Lane 4: 28 + Et3P
(5 equiv.). Lane 5: 27 + Et3P (5 equiv.). Lane 6: 9 + Et3P (5 equiv.). Lane
7: only Et3P (5 equiv.).
We also examined the effect of L-DTT on mitomycin 9–DNA
ISC and compared these findings with 1, 27 and 28. Using the
conditions established for Et3P, we treated aqueous buffered
solutions (pH 7.4) containing 1, 9, 27 and 28 (0.1 mM) and
DNA with L-DTT (5 equiv.) at room temperature (2 h) (Fig. 3).
We found that L-DTT (5 equiv.) led to detectably higher levels of
DNA ISC for 9 (77%) than 27 (24%) or 28 (31%). Significantly,
we observed noticeable amounts of DNA ISC for dimers 27
(24%) and 28 (31%) using L-DTT, and these values exceeded
those seen with Et3P (Fig. 2). Dimers 27 and 28 cannot undergo
disulfide activation yet DNA ISC adducts were observed. We
are uncertain if the observed differences in the DNA ISC levels
for 9 and 27 (28) are due to L-DTT-mediated cleavage of the
disulfide unit in 9. The kinetic experiments showed that the
consumption of 9 and 27 were not affected by L-DTT (10 equiv.)
(Table 2). Thus, we have attributed the 27 and 1 DNA ISC
products, in part, to trace levels of L-DTT activation pathways
other than the one outlined for 9, which proceeded through
disulfide cleavage (Scheme 3). Among these, the most likely
routes are quinone reductive and base-mediated C(9) proton
abstraction pathways. Our finding that L-DTT treatment of 9 led
Cytotoxicities
Using an antiproliferative activity assay we tested whether
mitomycin dimer 9 inhibited tumor cell line replication.39 As
discussed earlier, we did not use the IC50 values as a measure
of the extent and efficiency of DNA ISC for the mitomycins
in this test group since we did not determine the relative
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2
4 7 7

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Table 3 Antiproliferative activities for mitomycin dimers 9, 27, and 28 a
IC50/lmol L−1
9 consumption and mitosene product formation but not for 27.
The kinetic studies indicated that Et3P-mediated 9 activation
proceeded by phosphine attack at the disulfide unit to give 33
(Scheme 3) and was supported by a previous investigation using
DTT and Et3P. 37
Compound
Aerobic
Hypoxic
IC50 (hypoxic)/IC50 (aerobic)
The efficiency of mitomycin nucleophile-mediated activation
processes for DNA ISC was determined using pBR322 DNA
and a denaturing alkaline agarose gel electrophoresis assay.38
We found that 9 generated higher levels of DNA ISC adducts
upon Et3P addition than did the reference compounds 1, 3,
27, and 28. The extent of DNA ISC for 9 with Et3P exceeded
that of dimeric mitomycins 2 under reductive conditions.6 The
higher levels of DNA ISC adducts for 9 compared with dimeric
mitomycin 27 paralleled the rates of mitomycin consumption
observed in the kinetic studies. Taken together, these findings
indicated that the proposed nucleophile-mediated mechanism
for disulfide cleavage (Scheme 3) contributed to the increased
levels of DNA ISC. This pathway complements the reductive and
acid-catalyzed DNA ISC pathways previously established for the
mitomycins.2,40 The DNA ISC efficiency of this non-reductive
activation route for 9 warrants the further investigation of this
transformation where the sequence specificity, site of mitomycin
adduction [i.e. C(1), C(10)], and the extent of inter-duplex cross-
linking products are determined.
9
41
51
63
2.4
0.12
>100
>100
>100
20
>2.4
>2.0
>1.6
8.2
27
28
1
3
0.41
3.4
a A549 cell lines were used. The IC50 (lmol/L) value is the concentration
that inhibits cell replication by 50% under the assay condition.
levels of the mitomycins within the cells and the factors that
contributed to the inhibition of cell replication. The in vitro
antiproliferative activity tests were conducted at the Kyowa
Hakko Pharmaceutical Company (Shizuoka, Japan) using the
human tumor cell line A549 (lung adenocarcinoma). This cell
line was chosen because of its sensitivity to KW-2149 (3). Since
compound 9 was designed to undergo non-reductive, acidic,
and nucleophile-mediated activation, we were also interested
in evaluating the antiproliferative activities of this compound
under both aerobic and hypoxic conditions and determining the
activity ratio [IC50 (hypoxic)/IC50 (aerobic)] of 9 compared with
1, 3, 27 and 28.
Experimental
The results for in vitro antiproliferative activity tests for
mitomycin dimer 9 and reference compounds 1, 3, 27 and
28 are listed in Table 3. We found that under aerobic and
hypoxic conditions, 9 was 17-fold and >5-fold, respectively, less
potent than its monomer prototype, mitomycin C (1). Similar
differences were previously observed for dimeric mitomycins
2 tethered by alkyl linkers with a central heteroatom [N(H),
O] against the A549 tumor cell line.6 The activity ratio (hy-
poxic/aerobic) for 9 was >2.4 while for 1 and 3 it was 8.2 and
3.4, respectively. When 9 was compared with 27 and 28, we
found small differences under aerobic and hypoxic conditions.
The activity ratios (hypoxic/aerobic) for 9, 27 and 28 appeared
to be similar (>1.6 to >2.4) and these values did not permit us to
differentiate the compounds. Consequently, we have concluded
that for the A549 cell line incorporating the disulfide unit within
the dimeric assembly did not measurably contribute to cell death
and that the advantages of this unit observed for nucleophile-
mediated mitomycin activation and DNA adduction processes
did not extend to cell cytotoxicity.
General
Mitomycins A (24) and C (1) used in this study were generously
provided by Kyowa Hakko Co., Ltd. (Shizuoka, Japan). 1H and
13C NMR spectra were recorded on a General Electric QE-
300 spectrometer. Mass spectral (MS) data were obtained by
Dr Mehdi Moini at the University of Texas at Austin. The
low-resolution MS studies were run on a Finnegan TSQ-70
triple quadrupole mass spectrometer, and the high-resolution
MS studies were conducted on a Micromass ZAB-E mass spec-
trometer. FT-IR spectra were run on a Mattson Galaxy Series
FT-IR 5000 spectrometer. Melting points were determined in
open capillary tubes using a Thomas-Hoover melting point
apparatus and are uncorrected. pH Measurements of aqueous
solutions were determined on a Radiometer pHM26 meter using
a Radiometer pHC4000 glass electrode. The effective ‘pH’ of the
buffered methanolic solutions was similarly determined and the
meter and electrode was standardized against aqueous buffer
solutions.
LC–MS analyses were conducted with Agilent 1100 LC/MSD
by Dr Voyksner (LCMS Limited, Raleigh, NC). The products
were analyzed with a Zorbak C18 SB column (2.1 × 50 mm,
3.5 lm particles) using the following linear gradient condition:
80% A [0.025 M ammonium acetate in H2O–CH3CN (95 : 5),
pH 6.5], 20% B [0.025 M ammonium acetate in H2O–CH3CN
(5 : 95), pH 6.5] isocratic for 1 min, and then from 80% A, 20%
B to 20% A, 80% B for 30 min. The flow rate was 0.3 mL min−1,
and the eluent was monitored at 365 and 313 nm. The mass
spectral mode of operation was positive ion electrospray (+ESI)
and scan range was 300–1900 Da with 45 psi of nebulization
pressure.
Conclusions
In this study, the synthesis and evaluation of the novel mit-
omycin dimer 9 is reported. This mitomycin contained four
key structural elements that were expected to improve drug
activation and DNA adduction upon nucleophilic activation.
First, there was the inclusion of two mitomycin units in 9.
This feature permits DNA adduction processes to occur at
the two reactive mitomycin C(1) sites. Second, there was the
disulfide moiety. Cleavage of this unit in 3 and 4 is likely to be
responsible for the activities of these mitomycins in 1-resistant
and non-hypoxic cells.14–16 Similarly, nucleophilic cleavage of the
disulfide unit in 9 generates a thiol species ideally positioned
to render the ring system vulnerable to nucleophilic attack
(Scheme 1, 10→11, 12→13). Third, the disulfide unit was
incorporated within a dithiane permitting the two mitomcyin
moieties to remain tethered upon disulfide cleavage. This feature
differentiated 9 from 3 and 4, in which disulfide cleavage leads
to monomeric mitomycins. Fourth, the dithiane linker allowed
for guanines on complementary DNA strands to modify the two
distal mitomycin C(1) sites to give DNA ISC adducts.
1,6-Diamino-2,5-hexanediol dihydrochloride (18) (ref. 29).
A
mixture of 1,6-bis(phthalimido)-2,5-hexanediol (17, meso : dl =
2.7 : 1, 867 mg, 2.1 mmol), EtOH (19 mL) and NH2NH2·H2O
(0.26 mL, 5.3 mmol) was heated under reflux (3 h). After cooling
at room temperature, the solvent was removed in vacuo. H2O
(22 mL) and concentrated HCl (11 mL) was added to the
residue and the mixture was heated under reflux (1 h). After
◦
cooling to 0 C, the precipitate was removed by filtration. The
filtrate was then concentrated under reduced pressure and the
wet residue was dissolved in H2O (30 mL). A small amount
of insoluble matter was removed by filtration and the clear
filtrate was concentrated under reduced pressure to afford 18
We compared the rate of methanolysis of 9 with the
cyclohexyl-linked dimeric mitomycin 27 in the presence and
absence of nucleophiles. Et3P markedly increased the rate of
4 7 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2

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(yellow solid) as a mixture of diastereomers (meso : dl = 2.7 : 1,
successively washed with H2O (10 mL) and EtOH (2 mL) to
afford 20 (white solid) as an apparent single diastereomer (meso,
13C NMR analysis): yield, 135 mg (67%); mp 235–243 ◦C; Rf
0.51 (2 : 1 EtOAc/hexanes); mmax(KBr)/cm−1 3345, 2983, 2937,
1685, 1533, 1347, 1173, and 911; dH (300 MHz; CDCl3) 1.42 [s,
18 H, OC(CH3)3], 1.82 (br s, 4 H, CH2CH2), 3.06 (s, 6 H, OMs),
3.25–3.48 (m, 4 H, CH2N), 4.72 (br s, 2 H, CHOMs), and
5.04 (br s, 2 H, NHCO); dC (75 MHz; CDCl3) 27.3 (CH2CH),
28.3 [OC(CH3)3], 38.4 (OMs), 43.5 (CH2N), 80.0 [OC(CH3)3],
80.9 (CHOMs), and 156.0 (NHCO); m/z (+CI) 505 ([M +
1]+, 100%); HRMS (+CI) C18H37N2O10S2 [M + 1]+ requires
505.18896; found 505.18777.
◦
13C NMR analysis): yield, 469 mg (ca. 100%); mp 227–240 C
[lit.,29 bp (free amine) 130 ◦C (2 mm Hg)]; mmax (KBr)/cm−1
3381, 3069, 2952, 1560, 1260, 1042 and 977; dH (300 MHz;
CD3OD–D2O) 1.43–1.74 (m, 4 H, CH2CH2), 2.77–2.88 (m,
2 H, CHHꢀN), 3.07 (app d, J = 12.6 Hz, 2 H, CHHꢀN), and 3.81
(br s, 2 H, CHOH), the 1H NMR data were in agreement with
the COSY spectrum; dC (75 MHz; CD3OD–D2O) for the major
diastereomer, 31.4 (CH2CH), 45.7 (CH2N), and 68.7 (CHOH);
dC (75 MHz; CD3OD–D2O) for the minor diastereomer, 31.1
(CH2CH), and 68.3 (CHOH), one signal was not detected and
is believed to overlap with one of the observed peaks; m/z (+CI)
149 ([M + 1]+, 100%).
(2S,5R)-meso-1,6-Bis(tert-butyloxycarbonylamino)-2,5-bis(ac-
etylthio)hexane (meso-21). To a stirred solution of (2R,5S)-
meso-1,6-bis(tert-butyloxycarbonylamino)-2,5-hexanediol di-
methanesulfonate (meso-20, 107 mg, 0.21 mmol) in DMF
(2.5 mL) was added KSAc (54 mg, 0.48 mmol). After warming
to 60 ◦C, stirring was continued (3 h) and then the solvent
was removed in vacuo. H2O (40 mL) was added to the residue
and the mixture was extracted with EtOAc (2 × 40 mL). The
combined organic layers were dried (MgSO4) and concentrated
in vacuo. Purification by PTLC (1 : 2 EtOAc/hexanes) afforded
21 (white solid) as an apparent single diastereomer (meso, 13C
NMR analysis): yield, 59 mg (60%); mp 122–125 ◦C; Rf 0.50
(1 : 2 EtOAc/hexanes); mmax (KBr)/cm−1 3378, 2979, 2928, 1693,
1518, 1260, and 1169; dH (300 MHz; CDCl3) 1.41 [s, 18 H,
OC(CH3)3], 1.50–1.62 (m, 2 H, CHHꢀCH), 1.70–1.87 (m,
2 H, CHHꢀCH), 2.30 (s, 6 H, SAc), 3.15–3.39 (m, 4 H, CH2N),
3.43–3.58 (m, 2 H, CHSAc), and 4.89 (br s, 2 H, NHCO), the
1H NMR data were in agreement with the COSY spectrum;
dC (75 MHz; CDCl3) 28.3 [OC(CH3)3], 28.8 (CH2CH), 30.7
(COCH3), 43.8 (CH2N), 44.9 (CHSAc), 79.4 [OC(CH3)3], 155.9
(NHCO), and 195.2 (COCH3); m/z (+CI) 465 ([M + 1]+, 100%);
HRMS (+CI) C20H37N2O6S2 [M + 1]+ requires 465.20931, found
465.20927.
1,6-Bis(tert-butyloxycarbonylamino)-2,5-hexanediol (19). To
a stirred solution of 1,6-diamino-2,5-hexanediol dihydrochlo-
ride (18, 222 mg, 1.0 mmol, meso : dl = 2.7 : 1) and Et3N
(0.84 mL, 6.0 mmol) in H2O–DMF (1 : 1, 6 mL) was added
a solution of di-tert-butyl dicarbonate (BOC2O) (448 mg,
2.2 mmol) in DMF (2 mL). After warming to 50 ◦C, stirring
was continued (5 h) and then the solvent was removed in vacuo.
H2O (40 mL) was added to the residue and the mixture was
extracted with EtOAc (2 × 40 mL). The combined organic
layers were successively washed with aqueous 0.1 N HCl
(40 mL), saturated aqueous NaHCO3 (40 mL) and H2O (40 mL).
The organic layer was dried (MgSO4) and concentrated in vacuo.
Purification by PTLC (2 : 1 EtOAc/hexanes) afforded 19 (white
solid) as a mixture of diastereomers (meso : dl = 3.5 : 1, 13C NMR
analysis): yield, 230 mg (66%); mp 115–120 ◦C; Rf 0.15 (2 : 1
EtOAc/hexanes); mmax (KBr)/cm−1 3373, 2964, 2927, 1694, 1525,
1276, 1173, 1003, and 642; dH (300 MHz; CDCl3) 1.34–1.69 [m,
22 H, CH2CH2, OC(CH3)3], 2.95–3.16 (m, 2 H, CHHꢀN), 3.24
(app d, J = 13.5 Hz, 2 H, CHHꢀN), 3.55 (br s, 2 H, CHOH), 3.71
(br s, 2 H, CHOH), and 5.10 (br s, 2 H, NHCO), the 1H NMR
data were in agreement with the COSY spectrum; dC (75 MHz;
CDCl3–CD3OD) for the major diastereomer, 28.4 [OC(CH3)3],
30.4 (CH2CH), 46.5 (CH2N), 71.2 (CHOH), 79.7 [OC(CH3)3],
and 157.0 (NHCO); dC (75 MHz; CDCl3–CD3OD) for the minor
diastereomer, 31.3 (CH2CH), 46.7 (CH2N), and 71.5 (CHOH),
the other signals were not detected and are believed to overlap
with the observed peaks; m/z (+CI) m/z 349 ([M + 1]+, 100%);
HRMS (+CI) C16H32N2O6 [M + 1]+ requires 349.23386; found
349.23347.
(3R,6S)-meso-3,6-Bis(tert-butyloxycarbonylaminomethyl)-1,2-
dithiane (meso-23). To a stirred solution of (2S,5R)-meso-
1,6-bis(tert-butyloxycarbonylamino)-2,5-bis(acetylthio)hexane
(meso-21, 40 mg, 0.086 mmol) in MeOH–H2O (5 : 1, 4.4 mL)
was added K2CO3 (70 mg, 0.51 mmol). After stirring at room
temperature (30 min), KOH (10 mg, 0.18 mmol) was added and
O2 was bubbled through the solution (5 h). The solvent was
removed in vacuo and H2O (15 mL) was added to the residue.
The mixture was extracted with EtOAc (2 × 15 mL) and the
combined organic layers were dried (MgSO4) and concentrated
in vacuo. Purification by PTLC (1 : 2 EtOAc/hexanes) afforded
23 (white solid) as an apparent single diastereomer (cis, 13C
NMR analysis): yield, 29 mg (89%); mp 118–120 ◦C; Rf 0.55
(1 : 2 EtOAc/hexanes); mmax (KBr)/cm−1 3376, 2976, 2926, 1691,
1524, 1252, and 1169; dH (300 MHz; CDCl3) 1.45 [s, 18 H,
OC(CH3)3], 1.73–2.11 (m, 4 H, CH2CH2), 2.87–3.02 (m, 2 H,
CHS), 3.31–3.50 (m, 4 H, CH2N), and 4.88 (br s, 2 H, NHCO),
the 1H NMR data were in agreement with the COSY spectrum;
dC (75 MHz; CDCl3) 28.3 [OC(CH3)3], 28.5 (CH2CH), 42.5
(CH2N), 44.2 (CHS), 79.6 [OC(CH3)3], and 155.8 (NHCO);
m/z (+CI) 379 ([M + 1]+, 100%) HRMS (+CI) C16H31N2O4S2
[M + 1]+ requires 379.17253, found 379.17198.
Resolution of 1,6-bis(tert-butyloxycarbonylamino)-2,5-hex-
anediol (19) by fractional recrystallization. A mixture of the
purified product [19, 220 mg, a mixture of diastereomers (meso :
◦
dl = 3.5 : 1, 13C NMR analysis), mp 115–120 C] and EtOAc
(15 mL) was heated to 45 ◦C and then filtered to remove
the insoluble impurities. The solution was allowed to stand
overnight (room temperature) leading to the precipitation of a
white solid. The precipitate was filtered and washed with hexanes
to give resolved 19 as an apparent single diastereomer (meso,
13C NMR analysis): yield, 141 mg (64%); mp 124–126 ◦C; dH
(300 MHz; CDCl3) 1.32–1.68 [m, 22 H, CH2CH2, OC(CH3)3],
2.94–3.14 (m, 2 H, CHHꢀN), 3.24 (app d, J = 13.5 Hz, 2 H,
CHHꢀN), 3.54 (br s, 2 H, CHOH), 3.70 (br s, 2 H, CHOH),
and 5.12 (br s, 2 H, NHCO); dC (75 MHz; CDCl3–CD3OD) 28.1
[OC(CH3)3], 30.0 (CH2CH), 45.8 (CH2N), 70.5 (CHOH), 79.4
[OC(CH3)3], and 156.9 (NHCO).
(2R,5S)-meso-1,6-Bis(tert-butyloxycarbonylamino)-2,5-hex-
anediol dimethanesulfonate (meso-20). To a cooled (0 ◦C)
solution of 1,6-bis(tert-butyloxycarbonyl)amino-2,5-hexanediol
[19, 139 mg, 0.4 mmol, a mixture of diastereomers (meso : dl =
3.5 : 1)] in pyridine (1.5 mL) was slowly added methanesulfonyl
chloride (105 lL, 1.4 mmol) for 20 min. After warming to
room temperature, stirring was continued (3 h) and then the
mixture was poured into a cooled aqueous 2 M HCl solution
(15 mL) leading to the precipitation of a white solid. After
stirring at 0 ◦C (1 h), the reaction mixture was filtered and
(3R,6S)-meso-3,6-Bis(aminomethyl)-1,2-dithiane di-trifluoro-
acetate salt (meso-14). (3R,6S)-meso-3,6-Bis(tert-butyloxycar-
bonylamino)-1,2-dithiane (meso-23, 14 mg, 0.04 mmol) was
dissolved in trifluoroacetic acid (1.5 mL) and stirring was
continued at room temperature (30 min). The reaction was con-
centrated in vacuo to afford 14 as a viscous oil: yield, 15 mg (ca.
100%); mmax (neat)/cm−1 2924, 1677, 1194, 1136, 838, and 722; dH
(300 MHz; D2O) 1.80–2.42 (m, 4 H, CH2CH2), and 3.15–3.70
(m, 6 H, NCH2CH); dC (75 MHz; D2O) 29.5 (CH2CH), 42.1
(CH2N), and 42.6 (CHS); m/z (+CI) 179 ([M − 2TFA + 1]+,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2
4 7 9

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100%); HRMS (+CI) C6H15N2S2 [M − 2TFA + 1]+ requires
7-N,7ꢀ-Nꢀ-(2ꢀꢀ,5ꢀꢀ-Dihydroxy-1ꢀꢀ,6ꢀꢀ-hexanediyl)bismitomycin C
(28). Using the procedure for 9 and MeOH (1.5 mL), 18 (a
mixture of diastereomers: 2.7 : 1, 13C NMR analysis, 4.8 mg,
0.022 mmol), triethylamine (18 lL, 0.13 mmol), and 24 (15 mg,
179.06767, found 179.06685.
Procedure for mitomycins 9 and 25. To an anhydrous
methanolic solution (1 mL) of meso-14 (2.0 mg, 0.005 mmol)
and triethylamine (4 lL, 0.03 mmol) was added 24 (3.4 mg,
0.01 mmol). The reaction solution was stirred at room tempera-
ture (1 d) and then the solvent was removed in vacuo. Purification
of the reaction mixture by PTLC (20% MeOH–CHCl3) afforded
the desired products.
0.043 mmol) gave 28 as a mixture of diastereomers (1.6 : 1, 13
C
NMR analysis) in 59% yield (10 mg) after PTLC purification
(30% MeOH–CHCl3); HPLC tR 22.8 min; Rf 0.40 (30% MeOH–
CHCl3); kmax (CH3CN–H2O)/nm 222 and 371; dH (300 MHz;
pyridine-d5) 1.83–2.03 [m, 4 H, C(3ꢀ)H2], 2.17 [s, 6 H, C(6)CH3],
2.73 [d, J = 3.9 Hz, 2 H, C(2)H], 3.12 [d, J = 3.9 Hz, 2 H,
C(1)H], 3.20 [s, 6 H, C(9a)OCH3], 3.60 [br d, J = 12.7 Hz,
2 H, C(3)HHꢀ], 3.67–3.78 [m, 2 H, C(1ꢀ)HHꢀ], 3.79–3.93
[m, 2 H, C(1ꢀ)HHꢀ], 3.97 [dd, J = 10.5, 3.8 Hz, 2 H,
C(9)H], 4.03–4.15 [m, 2 H, C(2ꢀ)H], 4.55 [d, J = 12.7 Hz, 2 H,
C(3)HHꢀ], 5.03 [dd, J = 10.5, 10.2 Hz, 2 H, C(10)HHꢀ], 5.37 [dd,
J = 10.2, 3.8 Hz, 2 H, C(10)HHꢀ], and 7.48 [br s, 2 H, C(7)NH],
the signals for the C(10)OC(O)NH2 and N(1a)H protons were
not detected and are believed to overlap with the observed
7-N,7ꢀ-Nꢀ-(1ꢀꢀ,2ꢀꢀ-Dithianyl-3ꢀꢀ,6ꢀꢀ-dimethylenyl)bismitomycin C
(9). Yield, 2.2 mg (53%); HPLC tR 29.8 min; Rf 0.47 (20%
MeOH–CHCl3); kmax (CHCN–H2O)/nm 222, and 374; dH
(300 MHz; pyridine-d5) 1.83–2.04 [m, 4 H, C(3ꢀ)H2], 2.14 [s, 6 H,
C(6)CH3], 2.75 [d, J = 3.9 Hz, 2 H, C(2)H], 3.15 [d, J = 3.9 Hz,
2 H, C(1)H], 3.22 [s, 6 H, C(9a)OCH3], 3.62 [br d, J = 12.6 Hz,
2 H, C(3)HHꢀ], 3.83–3.95 [m, 4 H, C(1ꢀ)H2], 4.02 [dd, J = 10.8,
3.9 Hz, 2 H, C(9)H], 4.56 [d, J = 12.6 Hz, 2 H, C(3)HHꢀ], 5.06
[dd, J = 10.8, 10.2 Hz, 2 H, C(10)HHꢀ], and 5.41 [dd, J = 10.2,
3.9 Hz, 2 H, C(10)HHꢀ], the signals for the C(2ꢀ)H, C(7)NH,
C(10)OC(O)NH2 and N(1a)H protons were not detected and
1
peaks, the H NMR data were in agreement with the COSY
spectrum; dC (75 MHz; pyridine-d5) for the major diastereomer,
10.2 [C(6)CH3], 32.3 [C(3ꢀ)], 32.8 [C(2)], 36.7 [C(1)], 44.4 [C(9)],
49.6 [C(9a)OCH3], 50.6 [C(3)], 51.3 [C(1ꢀ)], 62.5 [C(10)], 70.6
[C(2ꢀ)], 103.6 [C(6)], 107.0 [C(9a)], 110.6 [C(8a)], 147.8 [C(7)],
156.3 [C(4a)], 158.1 [C(10a)], 176.8 [C(8)], and 179.0 [C(5)]; dC
(75 MHz; pyridine-d5) for the minor diastereomer, 32.0 [C(3ꢀ)],
32.7 [C(2)], 44.5 [C(9)], 70.3 [C(2ꢀ)], 147.9 [C(7)], and 156.2
[C(5a)], the other signals were not detected and are believed
to overlap with the observed peaks; m/z (+FAB) 783 ([M +
1]+, 100%); HRMS (+FAB) C36H47N8O12 [M + 1]+ requires
783.33135, found 783.33250.
1
are believed to overlap with the observed peaks, the H NMR
data were in agreement with the COSY spectrum; dC (75 MHz;
pyridine-d5) 11.7 [C(6)CH3], 30.2 [C(3ꢀ)], 34.4 [C(2)], 38.4 [C(1)],
46.0 [C(9)], 46.7 [C(2ꢀ)], 48.1 [C(1ꢀ)], 51.3 [C(9a)OCH3], 52.3
[C(3)], 64.1 [C(10)], 106.3 [C(6)], 108.6 [C(9a)], 112.6 [C(8a)],
148.7 [C(7)], 157.4 [C(4a)], 159.8 [C(10a)], 178.5 [C(8)], and
180.9 [C(5)]; m/z (+FAB) 813 ([M + 1]+, 100%); HRMS (+FAB)
C36H45N8O10S2 [M + 1]+ requires 813.27001, found 813.26906.
7-N-(6ꢀ-Aminomethyl-1ꢀ,2ꢀ-dithianyl-3ꢀ-methylenyl)mitomycin
C (25a and 25b). Yield, 0.5 mg (21%); HPLC tR 21.8, 22.3 min
(two peaks, 1 : 1); Rf 0.15 (20% MeOH–CHCl3); kmax (CH3CN–
H2O)/nm 222 and 374; dH (300 MHz; pyridine-d5) 1.75–2.06
[m, 4 H, C(3ꢀ)H2, C(4ꢀ)H2], 2.13 [s, 3 H, C(6)CH3], 3.14 [d, J =
4.8 Hz, 1 H, C(1)H], 3.21 [s, 3 H, C(9a)OCH3], 3.22–3.38 [m, 3 H,
C(2ꢀ)H, C(6ꢀ)H2], 3.62 [br d, J = 12.9 Hz, 2 H, C(3)HHꢀ],
3.78–3.95 [m, 2 H, C(1ꢀ)H2], 3.99 [dd, J = 10.8, 3.9 Hz, 1 H,
C(9)H], 4.56 [d, J = 12.9 Hz, 1 H, C(3)HHꢀ], 5.06 [dd,
J = 10.8, 10.2 Hz, 1 H, C(10)HHꢀ], and 5.41 [dd, J = 10.2,
3.9 Hz, 1 H, C(10)HHꢀ], the signals for the C(5ꢀ)H, C(7)NH,
C(10)OC(O)NH2 and N(1a)H protons were not detected and
are believed to overlap with the observed peaks, the signal for
the C(2)H proton overlapped with the (CH3CH2)3N peak; m/z
(+FAB) 496 ([M + 1]+, 100%); HRMS (+FAB) C21H30N5O5S2
[M + 1]+ requires 496.16884, found 496.17002.
Methanolysis of 9 to give cis- and trans-C(1) methoxymitosenes
30. Mitomycin 9 (3.5 mg, 0.0043 mmol) was dissolved in
MeOH–CHCl3 (1 : 1, 3 mL) and then the effective pH was
adjusted to ca. 3.0 with a methanolic 2 M HCl solution. The
reaction solution was stirred at room temperature (3 d) and then
the solvent was removed under reduced pressure. Purification of
the reaction mixture by PTLC (20% MeOH–CHCl3) afforded
30 as a red solid: yield, 1.5 mg (40%); HPLC tR 30.6, 31.3,
32.1 min (three peaks, 1 : 2 : 1);Rf 0.33 (20% MeOH–CHCl3); kmax
(CH3CN–H2O)/nm 214, 255, and 316; dH (300 MHz; pyridine-
d5) 1.84–2.09 [m, 4 H, C(3ꢀ)H2], 2.24 [br s, 6 H, C(6)CH3], 3.24
[br s, 2 H, C(2ꢀ)H], 3.51 [br s, 6 H, C(1)OCH3], 3.84–4.05 [m, 6 H,
C(2)H, C(1ꢀ)H2], 4.14–4.32 [m, 2 H, C(3)HHꢀ], 4.41–4.80 [m, 4 H,
C(3)HHꢀ, C(1)H], 5.73 [1/2ABq, J = 13.0 Hz, 2 H, C(10)HHꢀ],
5.78 [1/2ABq, J = 13.0 Hz, 2 H, C(10)HHꢀ], and 6.77–6.89 [m,
2 H, C(7)NH], the signals for the C(10)OC(O)NH2 and C(2)NH2
protons were not detected and are believed to overlap with the
observed peaks, the 1H NMR data were in agreement with the
COSY spectrum; m/z (+ESI) 813 ([M + 1]+, 100%); (LC–MS)
835 [M + Na]+ (tR 15.4–15.6 min), 835 [M + Na]+ (tR 15.6–16.2
min), 835 [M + Na]+ (tR 16.2–16.3 min).
7-N,7ꢀ-Nꢀ-(Cyclohexanyl-trans-1ꢀꢀ,4ꢀꢀ-dimethylenyl)bismitomy-
cin C (27). Using the procedure employed for 9 and MeOH
(2 mL), 26 (a mixture of diastereomers: cis : trans = 1 : 10, 13
C
NMR analysis, 3.1 mg, 0.02 mmol), and 24 (15 mg, 0.043 mmol)
gave 27 (12 mg, 70%) after PTLC purification (10% MeOH–
CHCl3); HPLC tR 30.1 min; Rf 0.14 (10% MeOH–CHCl3);
kmax (CH3CN–H2O)/nm 223 and 374; dH (300 MHz; CDCl3)
0.98–1.09 [m, 4 H, C(3ꢀ)HHꢀ], 1.58–1.78 [m, 2 H, C(2ꢀ)H], 1.86
[d, J = 7.5 Hz, 4 H, C(3ꢀ)HHꢀ], 2.00 [s, 6 H, C(6)CH3], 2.82 [d,
J = 3.9 Hz, 2 H, C(2)H], 2.90 [d, J = 3.9 Hz, 2 H, C(1)H], 3.21
[s, 6 H, C(9a)OCH3], 3.38 [t, J = 5.8 Hz, 4 H, C(1ꢀ)H2], 3.52
[br d, J = 13.0 Hz, 2 H, C(3)HHꢀ], 3.60 [dd, J = 10.8, 4.3 Hz,
2 H, C(9)H], 4.30 [d, J = 13.0 Hz, 2 H, C(3)HHꢀ], 4.49 [dd, J =
10.8, 10.2 Hz, 2 H, C(10)HHꢀ], 4.70 [dd, J = 10.2, 4.3 Hz,
2 H, C(10)HHꢀ], and 6.44 [t, J = 5.8 Hz, 2 H, C(7)NH], the
signals for the C(10)OC(O)NH2 and N(1a)H protons were not
detected and are believed to overlap with the observed peaks; dC
(75 MHz; CDCl3) 9.9 [C(6)CH3], 30.0 [C(3ꢀ)], 32.6 [C(2)], 36.5
[C(1)], 39.2 [C(2ꢀ)], 42.8 [C(9)], 49.8 [C(1ꢀ), C(9a)OCH3], 51.1
[C(3)], 62.6 [C(10)], 103.6 [C(6)], 106.2 [C(9a)], 109.7 [C(8a)],
147.2 [C(7)], 155.9 [C(4a)], 156.4 [C(10a)], 176.0 [C(8)], and
178.7 [C(5)]; m/z (+CI) 777 ([M + 1]+, 100%); HRMS (+CI)
C38H49N8O10 [M + 1]+ requires 777.35717, found 777.35877.
General procedure for the solvolysis of mitomycins
(kinetic studies)
To a buffered methanolic solution (0.1 M Tris·HCl, pH 7.4;
0.1 M bis-Tris·HCl, pH 5.5 and 4.0) (final volume 1.5 mL)
maintained at 25 ◦C containing the mitomycins (10–60 lL of
4 mM methanolic solution, final concentration 0.015–0.03 mM)
was added a methanolic solution (5–50 lL) of the nucleophile
of choice (stock solution: 4–20 mM, final nucleophile concen-
tration 0.03–0.6 mM). The reaction was monitored by UV-
visible spectroscopy (200–600 nm), and typically followed for
greater than two half-lives. The effective pH of the solution was
determined at the conclusion of the reaction and found to be
within
0.1 pH units of the original solution. The reaction
products were identified by coinjection of authentic samples
where possible in the HPLC and cospot of authentic samples
where possible in the TLC. The absorbance for the kmax of
mitomycin (ca. 374 nm) was plotted versus time and found to
4 8 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 7 1 – 4 8 2

View Article Online
decrease in a first-order decay (exponential decay) process. The
non-linear regression analysis to fit the observed exponential
decay by SigmaPlot Program (SigmaPlot, 2001) yielded pseudo-
first-order rate constants (kobs) and half-lives (t1/2). The reactions
were done in duplicate and the results averaged.
Yoshino Yamada (Kyowa Hakko Kogyo Co., Shizuoka, Japan)
for conducting the in vitro antiproliferative test and Dr Masaji
Kasai and Dr Hitoshi Arai (Kyowa Hakko Co., Ltd., Shizuoka,
Japan) for generously supplying mitomycins A (24) and C (1),
and KW-2149 (3).
General procedure for alkaline agarose gel electrophoresis23,38
References
The agarose gels were prepared by adding 1.2 g of agarose to
100 mL of an aqueous 100 mM NaCl and 2 mM EDTA solution
(pH 8.0). The suspension was heated in a microwave oven until
all of the agarose was dissolved (1 min). The gel was poured and
was allowed to cool and solidify at room temperature (1 h). The
gel was soaked in an aqueous alkaline running buffer solution
(50 mL) containing 40 mM NaOH and 1 mM EDTA (1 h) and
then the comb was removed. The buffer solution was refreshed
prior to electrophoresis. To an aqueous solution of ca. 85 lL of
H2O (sterile) and 2.5 lL of 1 M Tris·HCl (pH 7.4) was added
a solution of EcoRI-linearized pBR322 (5 lL, 5 lg) in 10 mM
Tris solution containing 1 mM EDTA (pH 8.0). After deaeration
with Ar (15 min), the mitomycin (1–5 lL of 1–2 mM DMSO
solution, final concentration 0.01–0.1 mM) and a nucleophile of
choice (1–5 lL of 1–20 mM DMSO solution, final concentration
0.05–1.0 mM) were added and the resulting solution (final vol-
ume 100 lL) was incubated at room temperature (2 h). The so-
lution was washed with 1 : 1 PhOH/CHCl3 (100 lL) and CHCl3
(2 × 100 lL), and precipitated [12.1 lL of 3 M NaOAc and
250 lL of EtOH, −70 ◦C (10 min)]. The mixture was centrifuged
at 0 ◦C (15 min), and the EtOH was decanted off and evaporated
in vacuo. The remaining DNA was dissolved in 25 lL of 10 mM
Tris solution containing 1 mM EDTA (pH 8.0). Agarose loading
dye (5 lL) was added to the sample (5 lL) and the samples were
loaded onto the wells. The gel was run at 75 mA/25 V (30 min)
and then at 145 mA/38 V (3–4 h). The gel was then neutralized
for 45 min in an aqueous 100 mM Tris pH 7.0 buffer solution
containing 150 mM NaCl, which was refreshed every 15 min.
The gel was stained with an aqueous 100 mM Tris pH 7.5 buffer
solution (100 mL) containing ethidium bromide [20 lL of an
aqueous ethidium bromide stock solution (10 mg in 10 mL)]
and 150 mM NaCl for 20 min. The background staining was
then removed by soaking the gel in an aqueous 50 mM NH4OAc
and 10 mM b-mercaptoethanol solution (3 h). The gel was then
analyzed by two methods. In one method, the gel was visualized
by UV and photographed using Polaroid film 667. In the second
method, the gel was analyzed with a StormTM 860 phosphorim-
ager operating in the blue fluorescence mode and ImageQuant
5.0 software (Molecular Dynamics, Sunnyvale, CA).
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In vitro antiproliferative tests were conducted using human
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Acknowledgements
The authors gratefully acknowledge the NIH (CA29756) for
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