Design, synthesis, and mode of action studies of a mitomycin tetramer inducing double activations with a single probe

Article abstract of DOI:10.1016/j.bmc.2016.06.041

We report design, synthesis, and mechanistic studies of a new mitomycin tetramer 9 along with a new mitomycin dimer 10. Mitomycin 9 is a tetramer connected by the disulfide linker 11, and easily undergoes disulfide cleavage to provide two dimeric structures 9r that each contains a single thiol probe for activations. So, tetramer 9 as a precursor of 9r was specifically targeted to undergo double activations with a single probe. A tetramer 9 was synthesized using 1 and key intermediate 11, and a dimer 10 was synthesized from 1 and diamine 12. Activation studies revealed that 9 underwent effective double activations with a single probe by nucleophiles while the reference 10 did not. Evaluations of DNA ISC formations showed that 9 generated substantial levels of DNA ISC by nucleophilic activation while the references 10 and 2 did not. The effectiveness of 9 in activation and formation of DNA ISC per probe was verified by comparing with dimers 5–8 of double activations with two probes. These findings highlighted the role of a single thiol in 9r and demonstrated the intended double activations with a single probe, which marks the first case in mitomycin studies.

Full text of DOI:10.1016/j.bmc.2016.06.041

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and mode of action studies of a mitomycin
tetramer inducing double activations with a single probe
⇑
Hyoung Rae Kim, Yeon Kyeong Park, Sang Hyup Lee
College of Pharmacy and Innovative Drug Center, Duksung Women’s University, 419 Ssangmun-dong, Dobong-gu, Seoul 132-714, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We report design, synthesis, and mechanistic studies of a new mitomycin tetramer 9 along with a new
mitomycin dimer 10. Mitomycin 9 is a tetramer connected by the disulfide linker 11, and easily
undergoes disulfide cleavage to provide two dimeric structures 9r that each contains a single thiol probe
for activations. So, tetramer 9 as a precursor of 9r was specifically targeted to undergo double activations
with a single probe. A tetramer 9 was synthesized using 1 and key intermediate 11, and a dimer 10 was
synthesized from 1 and diamine 12. Activation studies revealed that 9 underwent effective double
activations with a single probe by nucleophiles while the reference 10 did not. Evaluations of DNA ISC
formations showed that 9 generated substantial levels of DNA ISC by nucleophilic activation while the
references 10 and 2 did not. The effectiveness of 9 in activation and formation of DNA ISC per probe
was verified by comparing with dimers 5–8 of double activations with two probes. These findings
highlighted the role of a single thiol in 9r and demonstrated the intended double activations with a single
probe, which marks the first case in mitomycin studies.
Received 14 May 2016
Revised 21 June 2016
Accepted 21 June 2016
Available online xxxx
Keywords:
Mitomycin tetramer
Double activations
Disulfide cleavage
DNA alkylation
DNA interstrand cross-link
Ó 2016 Elsevier Ltd. All rights reserved.
1
. Introduction
nucleophilic activation mechanisms that differ from traditional
and/or reductive activation mechanisms.
2
a,d
Mitomycins constitute a class of antitumor agents that display
Among several types of DNA adducts of mitomycin, the DNA
interstrand cross-link (DNA ISC) adducts by bis-alkylation of DNA
by mitomycin have drawn special interest because they are ꢀ60
1,2
antiproliferative activity by DNA alkylation, and recently, studies
on synthesis and mode of actions of mitomycinoid alkaloids were
2
b,c
9
broadly reviewed.
Despite the reports of mitomycin A (MMA,
times more lethal than the corresponding monoadducts of 2. Con-
1
) and its numerous derivatives, only mitomycin C (MMC, 2) has
sidering that the reactivity of C(1) site toward DNA was 10–100
times higher than that of C(10) site, we focused on the dimeriza-
1,2a
10
been a useful agent of clinical importance
efforts to overcome the drug resistance and side effects of 2 led
to the findings of disulfide mitomycins 3 (KW-2149) and 4
(Fig. 1). Continuing
2
a
tion of mitomycin units that would provide two C(1) sites of higher
reactivity, which could eventually provide higher levels of DNA ISC
by bis-alkylation of DNA by both C(1) sites. So, aiming to enhance
the formation of DNA ISC, we previously reported the design, syn-
thesis, and activation studies of strategically-designed disulfide
3
4
(
BMS-181174). In general, mitomycins have to undergo proper
activation processes by which they are converted to good elec-
trophiles for chemical reactivity and biological activities. For 3
and 4, it was believed that a different activation mechanism
through the key function of the disulfide group5 in 3 and 4 led
to their improved pharmacological properties and DNA adduc-
1
1
12
13,14
15
mitomycin dimers, 5, 6, 7,
and 8 (Scheme 1). Interest-
–7
ingly, dimers 5–8 all contained a medium-sized cyclic disulfide
group as a linker to connect two mitomycin units. Cleavage of
disulfide group in dimers 5–8 would provide 5r–8r (reduced form)
containing two thiols that could serve as good probes to initiate the
mitomycin activation, leading to double activations with two thiol
probes. As a result, dimers 5–8 underwent fast activations (kobs
6
–8
tion
compared to 2. The thiol generated by disulfide cleavage
of 3 and 4 would trigger the activation of the mitomycin ring by
intramolecular cyclization to a quinone ring, which leads to the
generation of highly electrophilic C(1) site (activation) that pro-
ceeds to react with DNA (DNA adduction). Hence, the thiol group
could serve as a good probe to activate the mitomycin ring by
ꢁ1
(d ): 2.7–16) and produced high levels of DNA ISC (83–98%)
adducts, which represented the important function of thiol probes.
Based on the results of double activations with two thiol probes,
we became interested in the strategy of double activations with a
single thiol probe.
⇑
Corresponding author. Fax: +82 2 901 8386.
E-mail address: sanghyup@duksung.ac.kr (S.H. Lee).
http://dx.doi.org/10.1016/j.bmc.2016.06.041
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
0
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2
H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
10
9
OC(O)NH2
O
O
best ways to create the specifically-designed dimer (e.g., 9r) for
our purpose. As a result, we envisioned dimerization of the same
dimeric unit 9r to afford dimeric dimer 9. Second, compound 9
OC(O)NH2
RS
H
N
R
7
6
9
OMe
8
OMe
a
1
N
NH
N
O
NH
(
and 9r) contained a 2-mercaptoethylamino unit at C(7), which is
O
an important structural requirement for mitomycin activation.
Only in this structural condition could the thiol easily attack the
quinone ring by forming a six-membered cyclic structure. Taken
together, compound 9 was aimed to undergo efficient nucleophilic
double activations with a single probe and corresponding facile
DNA adduction. On the other hand, hydroxy mitomycin 10 con-
tains hydroxy linker instead of disulfide linker with the same car-
bon skeleton as in 9r and could therefore serve as a good reference
to identify the net effect of thiol probe (or disulfide) in 9r (or 9).
This is the first case in mitomycin studies where double activations
with a single thiol probe were tested.
1
R = OCH3
3 R =
4 R =
S
S
CO H
2
N
H
NH2
2
R = NH2
NO2
Figure 1. Structures of mitomycins 1–4.
2
2
. Results and discussion
.1. Design of 9 and study objectives
We also attempted to compare target mitomycin 9 with 5–8 in
terms of the function of thiol probes and the distances and flexibil-
ities of the linker. Although compounds 5r–8r with two thiol
probes seemed to be more advantageous for double activations
of mitomycin rings, the two thiols in 5r–8r could easily revert back
to disulfide to form 5–8, which might retard the activations
because activations could be induced by thiol itself. In this regard,
it was simply presumed that compound 9r with a single probe
could be sufficient for double activations as there was no possibil-
ity of intramolecular disulfide formation. In addition, the flexibility
and distance between the two C(1) sites in 9r would be lower and
shorter, respectively, than those in 5r–8r, which might affect the
mitomycin activations and subsequent DNA adductions.
In continuing efforts to identify the function of a thiol probe in
mitomycin activations, we wished to see if the intended double
activations with a single probe indeed occurred, and if so, to assess
the effectiveness of the activations and DNA adductions. In addi-
tion, we were interested in whether the distance and flexibility
of the linker would have any effect on those activations. As part
of our studies for this purpose, we report herein new mitomycins
9
and 10 in the context of the strategy of double activations with
a single probe (Scheme 2), and their comparisons with the prece-
dent examples (e.g., 5–8). Compound 9 contains several structural
features associated with mechanistic aspects. First, although com-
pound 9 is a tetramer, it could be seen as a ‘dimeric dimer’ since it
can easily undergo disulfide cleavage (or reduction) to provide two
dimeric species 9r with a single thiol. The thiol–disulfide exchange
So, the objectives of this work were studies on design, synthesis,
activation mechanism, and DNA ISC formations for 9 compared to
10, and elucidation of the strategy of double activations with a sin-
gle probe in comparison with 5–8. Notably, considering 9 and 10
still contained units of mitomycin C (2), it was believed that these
and, in particular, the disulfide cleavage by a proper nucleophile
were observed in our previous studies1
1–15
and thus, expected to
16
occur in compound 9. As mentioned above, our primary interest
lay in double activations with a single probe and we therefore
aimed to create an appropriate mitomycin dimer with a single thiol
probe (e.g., 9r). However, mitomycin 9r was believed to be very
unstable due to the facile and reversible attack of the thiol to qui-
none ring and it was very difficult to isolate, which was consistent
with the previous observation that the thiol species generated by
disulfide cleavage of 3 or 4 was also very unstable.7 Accordingly,
instant generation from a disulfide precursor could be one of the
mitomycins would undergo activations under reductive and
1
7
acidic conditions, and thus exhibit basic levels of cytotoxicity.
Furthermore, since our main interest lay in the activation mecha-
nism of these peculiar mitomycins, the cytotoxicity data of 9 and
10 were not included in the scope of our present studies. Despite
comprehensive investigations of mitomycin activations, much still
remains unknown about the precise activation processes and the
structures of intermediates and activated products.
2
2
.2. Chemistry
.2.1. Synthesis of disulfide intermediate 11
Prior to synthesis of the target mitomycins 9 and 10, we estab-
H
2
N(O)CO
MeO
O
8'
O
8
OC(O)NH
2
S
S
H
N
H
N
OMe
1
(
)
n
lished the synthesis of the required key intermediate, disulfide
11 as shown in Scheme 3.
1
'
18
HN
N
N
NH
5
6
7
8
n= 1
n= 2
n= 3
n= 4
2
At first, we treated amine 12 with di-t-butyl dicarbonate (Boc -
O
O
19
O) to afford Boc-protected derivative 13 in 99% yield. Next, we
reacted 13 with methanesulfonyl chloride (MsCl) to obtain mesyl
1
9
derivative 14 in 92% yield. Subsequent replacement of the mesyl
group with acetylthio group using potassium thioacetate (KSAc) in
Disulfide cleavage
2
0
DMF gave acetylthio derivative 15 along with substantial forma-
tion of a side product with higher R value (0.80, 1:2 EtOAc/hex-
f
SH SH
anes) than that (0.70) of product 15. The optimized yield of this
step was 61% and further efforts to improve the yield and identify
the side product were not successful. It was believed that the adja-
cent Boc-amino group might interfere with the replacement pro-
cess, which was also observed in the previous example of 1,5-bis
H
2
N(O)CO
O
O
8
OC(O)NH
2
H
H
N
N
MeO
8'
( )
n
OMe
1
1'
HN
N
N
NH
5
r n= 1
O
6r n= 2
O
7
8
r n= 3
r n= 4
(tert-butyloxycarbonylamino)-2,4-pentanediol
dimethanesul-
1
1
fonate. Hydrolysis of 15 by potassium carbonate (K CO ) was
2
3
Double activations
with
20
then conducted to afford thiol derivative 16. Although the reac-
tion seemed to proceed smoothly, the yield was generally low (less
than 50%) probably due to the instability of thiol species, which is
consistent with our previous results that thiol could undergo easy
two probes
Scheme 1. Disulfide cleavage of mitomycin dimers 5–8 to give 5r–8r.
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H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
O
HN
MeO
N(O)CO
N
N
NH
OMe
OC(O)NH2
N
H
N
H
H N(O)CO
OH
O
O
OC(O)NH
OMe
2
O
O
2
H
2
O
O
O
O
H
N
H
N
S
S
MeO
HN
H N(O)CO
OC(O)NH
OMe
1
2
2
N
N
NH
H
N
H
N
MeO
HN
10
1
N
N
NH
O
9
O
disulfide cleavage
H N(O)CO
SH
OC(O)NH
OMe
2
O
O
O
2
H
N
H
N
MeO
HN
2
DNA cross-links
1
N
N
NH
O
9r
Double activations
with
a single probe
Scheme 2. Disulfide cleavage of mitomycin tetramer 9 to give 9r.
OH
OR
of the key intermediate, disulfide 11, was achieved through a six-
step synthetic sequence from 12 in a good overall yield (43%).
a
H N
2
NH
2
BocHN
b
NHBoc
12
13 R = H
4 R = OMs
2.2.2. Synthesis of mitomycins 9 and 10
1
New target mitomycins 9 and 10 were synthesized using mito-
mycin A (MMA, 1, Kyowa Hakko Kirin Co.) and the corresponding
intermediates 11 and 12, respectively, as shown in Scheme 4.
Treatment of 1 with intermediate 11 in MeOH provided the target
mitomycin 9 in 32% yield. Further efforts to improve the results of
this reaction were inconclusive, probably due to the complexity of
the reaction. In reality, this step is composed of four consecutive
reactions of a tetraamine 11 with four molecules of 1, affording tet-
ramer 9 that is very polar and long-tailing on the TLC. During the
reaction, the reaction media could turn into a kind of buffer solu-
tion, due to the presence of excess amount of amine and TFA,
and therefore, the reaction rate seemed to gradually decrease. In
addition, disulfide could undergo further reactions including disul-
fide cleavage and/or polymerization. These phenomena hampered
us to improve the results. The synthesis of tetramer 9 seemed to be
more difficult than those of dimers 5–8. In addition, treatment of 1
with amine 12 in MeOH afforded hydroxy mitomycin 10 in 86%
yield, which was employed as a reference for the studies of 9.
c
SH
SAc
d
BocHN
NHBoc
BocHN
NHBoc
1
6
15
f
e
TFA .H
N
NH2 .TFA
2
BocHN
BocHN
NHBoc
NHBoc
S
S
S
g
S
TFA .H N
NH
.TFA
2
2
1
1
1
7
Scheme 3. Synthesis of key intermediate, disulfide 11. Reagents and conditions: (a)
Boc O, Et N, DMF–H O (1:1), room temperature, 3 h, 99%; (b) MsCl, Et N, CH Cl
°C, 5 h, 92%; (c) KSAc, Et N, DMF, 60 °C, 1 d, 61%; (d) K CO , MeOH–H O (5:1),
room temperature, 1 h, 57%; (e) I , Et N, CHCl , room temperature, 3 h, 67%; (f)
CO , MeOH–H O (5:1), room temperature, 1 h, then I , Et N, MeOH–H O–CHCl
5:1:2), room temperature, 6 h, 77% (for two steps); (g) TFA, room temperature, 2 h,
100%.
2
3
2
3
2
2
,
0
3
2
3
2
2
3
3
K
(
ꢀ
2
3
2
2
3
2
3
TFA. H N
.
.
2
NH
NH
2
2
TFA
TFA
a
S
S
9
.
2
TFA H N
O
O
OC(O)NH2
OMe
oxidation and polymerization.11,13 Hence, we conducted careful
hydrolysis and work-up procedure under a purge of nitrogen gas
to afford the thiol 16 in 57% yield. We then performed the disulfide
MeO
11
N
NH
2 3
formation (oxidation) using I /Et N condition, leading to the gener-
ation of disulfide 17 in 67% yield. In order to improve these two
reactions by avoiding the complexity related to the thiol species,
we performed the two reactions, hydrolysis and disulfide forma-
tion, in situ without isolation of thiol 16, finally affording disulfide
1 (MMA)
OH
H
2
N
NH
2
10
b
12
1
7 in good yield (77%). Deprotection of Boc group in 17 was
achieved by employing trifluoroacetic acid (TFA) to afford ami-
Scheme 4. Synthesis of mitomycins 9 and 10. Reagents and conditions: (a) Et
3
N,
neꢂTFA salt 11 in near quantitative yield. Consequently, synthesis
MeOH, room temperature, 4 d, 32%; (b) Et N, MeOH, room temperature, 2 d, 86%.
3
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H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
2
2
.3. Mode of action studies of mitomycins 9 and 10
nucleophiles to induce disulfide cleavage and sequential activa-
tions. The activation reactions were conducted in buffered
methanolic solutions (0.1 M TrisꢂHCl, ‘pH’ 7.4) and monitored by
UV–vis spectroscopy (200–500 nm) for more than two half-lives.
The absorption was periodically measured at ꢀ370 nm for starting
mitomycins and at ꢀ313 nm for activated mitosene products. Gen-
erally, the reactions followed pseudo first-order kinetics, and the
reaction constant (kobs) and half-lives (t1/2) were calculated using
the absorption values at 370 nm. The reactions were run in dupli-
cate, and the results averaged, which were summarized in Table 1.
First, we measured the activation rates of 9 and 10 without
nucleophile and observed no distinct decrease (less than 10% of
original amount) of starting mitomycins in 5 d, which means no
noticeable activation without nucleophile. Next, we checked the
.3.1. Activation of mitomycin 9 using acidic methanolysis
Prior to the nucleophilic activation of 9, the activation of 9
under acidic conditions was evaluated (Scheme 5). In order to imi-
tate the activation and subsequent DNA adduction for 9 in real bio-
logical system, we applied methanolysis reaction as a chemical
model system where 9 was induced to undergo activation and
react with methanol. So, we conducted the methanolysis of 9 in
3
acidic media (1:1 MeOH–CHCl solution at nonaqueous effective
pH (‘pH’) 2.5–3.0) for ꢀ3 d, expecting the formation of tetra-acti-
vated mitosene product, C(1)-methoxymitosene 18. We monitored
the reaction using TLC and UV–vis spectroscopy, and in particular,
the UV–vis spectra were diagnostic in identifying the activation
process. The established UV–vis absorption pattern of mitomycin
effect of thiol nucleophiles,
L-DTT and GSH, on the activation of 9
1
7,21
activations
implied that activated mitosene products such as
and 10. We observed modest activations of mitomycin 9 with L-
ꢁ
1
ꢁ1
1
8 display absorption maximum at ꢀ313 nm and the starting mit-
DTT (kobs: 0.69 d with 10 equiv; 1.3 d with 20 equiv). Similarly,
ꢁ1
omycin such as 9 at ꢀ370 nm. We observed the gradual disappear-
ance of starting mitomycin 9 along with gradual and stepwise
generation of multi-activated products on TLC and UV–vis spec-
troscopy, which enabled us to confirm the progress of the activa-
tion of 9. However, the activated products were highly polar and
we found comparable activation of 9 with GSH (kobs: 1.0 d with
20 equiv). However, we observed no appreciable activation of ref-
erence mitomycin dimer 10 with these thiol nucleophiles. It was
surprising to see the activation of 9 with thiol nucleophiles since
we did not observe noticeable activation of disulfide mitomycin
dimers 5–8 with these thiols in our previous studies.1
1,12
Mito-
displayed long tailing with low R
f
value on TLC, which made them
difficult to purify. HPLC chromatograms also showed multiple
mycin 9 (and its reduced form 9r) differs from mitomycin dimers
5–8 (and their reduced forms 5r–8r), and in particular, the longer
lifetime of thiol in 9r compared to 5r–8r was believed to induce
weak activations of 9. Taken together, the target mitomycin 9
underwent modest activation by thiol nucleophiles, which was
not previously demonstrated. Thus, we concluded that the weak
double activations with a single probe for 9 were induced by thiol
nucleophiles.
peaks between ꢀ32 and ꢀ35 min with absorption maxima at
3
13 nm. We believe that the multi-activated products including
1
8 are highly polar due to the presence of multiple primary amines
generated at each C(2) site and also exist as a mixture of many
diastereomers due to the uncontrolled stereochemistry at each C
(
1) site, which could all cause the complexity of the reaction. Nev-
ertheless, we attempted to isolate the products by PTLC with par-
tial success of low yields (ꢀ20%).
3
We then checked the effects of Et P as a phosphine nucleophile
on activations of tetramer 9 compared to reference 10. We
observed a significant decrease of 9 in the range of 5–50 equiv of
2
.3.2. Kinetic studies of nucleophilic activations of 9 and 10
Using the methanolysis reactions, we performed kinetic studies
Et
Et
3
P, which represented the rapid nucleophilic activation of 9 by
ꢁ
1
of nucleophilic activations induced by appropriate nucleophiles to
see if the target mitomycin 9 efficiently undergoes nucleophilic
activation compared to reference hydroxy mitomycin 10, and more
significantly, if the intended double activations with a single probe
for 9 (or 9r) indeed occurred by the thiol group in 9r. In general, the
rates of activation of mitomycins under the methanolysis reaction
as a chemical model system were believed to directly reflect the
3
P. The kobs values of activation reactions ranged from 0.72 d
ꢁ1
(5 equiv) to 6.9 d (50 equiv) and, in general, the activation rates
were proportional to the amount of Et P employed. However, the
reference hydroxy mitomycin 10 showed no appreciable decrease
in 5 d even with 50 equiv of Et P, which indicated that 10 did
not undergo appreciable activation by Et P. Considering that the
3
3
3
only structural difference of 9 (or 9r) compared to 10 is the pres-
ence of a disulfide (or thiol) group, the nucleophilic activation by
1
1
real efficiency of activation and adduction of the mitomycins.
So, we conducted kinetic studies of nucleophilic activations by
measuring the rate of methanolysis of mitomycins, employing
3
Et P for only 9 but not 10 must be directly attributed to the
function of the disulfide (thiol) unit. Consequently, we confirmed
3
Et P,
L
-dithiothreitol (
L-DTT), and glutathione (GSH) as proper
the intended double activations with a single probe for 9 in the
Table 1
9
a
Activation rates for 9 and 10 at ‘pH’ 7.4
Reagents
equiv
9
10
activation
MeOH / H+
ꢁ1
ꢁ1
Nu
kobs (d
)
t
1/2 (d)
kobs (d
)
t1/2 (d)
b
b
b
b
b
b
b
b
b
b
H N
O
O
No Nu
2
NH2
N
L
-DTT
10
0.69
1.3
1.0
0.53
N
20
MeO
OMe
GSH
Et
20
2
5
1.0
0.69
N
H
N
H
b
b
3
P
—
—
H N(O)CO
O
O
O
O
2
S
S
OC(O)NH
2
0.72
1.3
2.9
0.96
0.53
0.24
0.10
—
—
b
b
10
H
2
N(O)CO
OC(O)NH2
b
b
b
b
H
N
H
N
20
50
6.9
MeO
OMe
1
'
1
a
Reactions were run in buffered methanolic solution (0.1 M TrisꢂHCl, ‘pH’ 7.4) at
N
N
NH
2
25 °C. The reactions were run in duplicate and the values averaged. The results were
obtained using a Shimadzu UV-1800 spectrophotometer and the reactions moni-
tored at ꢀ370 nm unless otherwise indicated. The concentrations of the mito-
mycins were 0.015 mM for tetramer 9 and 0.030 mM for dimer 10.
H N
O
O
2
18
b
Scheme 5. Activation of 9 to give mitosene product 18.
No appreciable change in 5 d (less than 10% of the original amount).
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H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
presence of Et
3
P. When the activation reactions by Et
3
P were mon-
could serve as an activation probe. We believed that the involve-
ment of a thiolate instead of a thiol in the activation process is also
itored, we observed similar phenomena as in acidic activations: a
gradual disappearance of starting mitomycin 9 at 370 nm and a
continual appearance of products at 313 nm, and the formation
2
3
a possibility. The thiol is expected to attack C(8) carbon of qui-
none ring by intramolecular cyclization to provide hemi-thioketal
19, thereby destabilizing the nonbonding electron pair at N(4).
Here we showed that intramolecular cyclization occurs at the C
(8); however, we recognized that the C(6) or C(7) may be another
of multiple peaks (t
R
32–35 min, kmax ꢀ313 nm) on HPLC, which
again confirmed the formation of activated mitosene products.
However, intensive efforts to identify these multiple peaks were
inconclusive, probably due to the formation of many polar
diastereomers even at low concentrations (ꢀ0.015 mM).
2
3b
alternative site.
This destabilization would induce the
elimination of the adjacent methoxide at C(9a) to give an
iminium ion at N(4)–C(9a) and then mitosene structure 20. In
structure 20, formation of a highly conjugate allylic-type carbon
at C(1) along with electron push from N(4) would facilitate
opening of the aziridine ring of high ring-strain. Accordingly, the
Based on the information obtained by methanolysis using
nucleophiles, we propose a mechanism for nucleophilic activation
of tetramer 9 by Et
goes disulfide cleavage by Et
thiophosphonium ion by H
3
P as shown in Scheme 6. Mitomycin 9 under-
P and subsequent decomposition of
3
22
2
O (or MeOH) to give a thiol in 9r that
C(1) site becomes electrophilic enough to react with a
O
O
HN
MeO
N
N
NH
OMe
disulfide
cleavage
N
H
N
H
H2N( O)CO
MeO
O
8'
SH
O
8
OC(O)NH2
OMe
H2N(O)CO
O
O
OC(O)NH2 Et P / H+
H
N
H
N
S
S
3
Et3P
H2N(O)CO
MeO
O
8'
O
8
OC(O)NH2
OMe
H O
1'
N
1
NH
H
N
H
N
2
HN
N
-Et3P=O
+
O
O
-
H
9
r
HN
1
'
N
N
1
NH
O
O
9
H2N( O)CO
MeO
O
H2N(O)CO
MeO
O
8'
NH
S
OH
OC(O)NH2
-MeOH
NH
S
OH
OC(O)NH2
OMe
8
'
HN
HN
1
'
N
8
HN
1'
N
8
HN
1
N
1
NH
N
NH
O
O
2
0
O
1
9
O
NuH
H2N( O)CO
O
H2N(O)CO
O
SH
O
O
OC(O)NH2
NH
S
OH
OC(O)NH2
H
MeO
H
8
'
N
N
HN
MeO
8'
HN 1'
N
8
Nu
Nu
1
HN 1'
N
N
1
N
O
O
NH2
O
NH2
2
2
2
1
C(1) Activation
O
O
OC(O)NH2
O
OC(O)NH2
H2N(O)CO
HO
S
HN
H2N(O)CO
HN
-
MeOH
HO S
Nu
Nu
NH
NH
8
'
N
MeO
HN 1'
8'
1
Nu
N
NuH
1
'
NH2
N
N
NH2
O
H2N
O
O
2
4
23
C(1') Activation
H2N
O
O
NH2
Nu
1
'
N
1
N
N
Nu
H2N(O)CO
O
SH
O
O
OC(O)NH2
8'
N
H
N
H
N
disulfide
N
H
H
formation
O
O
O
OC(O)NH2
OC(O)NH2
H2N(O)CO
H2N(O)CO
S
S
8
'
Nu
Nu
1
'
N
1
N
O
8'
O2/-H2
H
N
H
N
NH2
H2N
O
25
Nu
Nu
1
'
N
1
O
NH2
H2N
O
2
6
where NuH: MeOH, DNA
3
Scheme 6. Proposed mechanism of nucleophilic activation of 9 by Et P.
Please cite this article in press as: Kim, H. R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.041

6
H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
nucleophile (e.g., MeOH or DNA) to afford 21, which constitutes C
1) activation and adduction. Then, reverting the hemi-thioketal in
1 back to quinone would regenerate the thiol in 22 by which a
2
10
9
6
(
2
2
3kb -
second round of activation would be initiated. Similar sequential
transformations (22 ? 23 ? 24) would afford 24, which
constitutes another C(1 ) activation and adduction. Again,
9
6
.4kb -
.6kb -
0
reverting the hemi-thioketal back to quinone would give thiol
species 25 that could undergo proper oxidation or disulfide
formation reaction, finally affording 26. Taken together,
mitomycin 9 successfully induced double activations at C(1) and
4.4kb -
0
C(1 ) sites with a single thiol probe. This proposed pathway
seems to be consistent with our previous studies on the
activations of 5–8.1
1–15
To the best of our knowledge, this marks
3
Figure 2. Denaturing 1.2% alkaline agarose gel for 2, 10, 9 and 6 using Et P. DNA
cross-linking experiments using 0.2 mM (monomer 2), 0.1 mM (dimers 10 and 6),
and 0.05 mM concentrations of mitomycins (tetramer 9), and EcoRI-linearized
the first study of double activations with a single probe in a series
of mitomycin compounds.
3
pBR322 plasmid DNA with 5 equiv of Et P. All reactions were incubated at room
temperature (2 h). Lane 1: k Hind III DNA molecular weight marker. Lane 2: 2 + Et
3
P.
2
.4. Studies on the formation of DNA ISC for 9 and 10
Lane 3: 10 + Et P. Lane 4: 9 + Et P. Lane 5: 6 + Et P.
3
3
3
We wished to evaluate the ability of the target mitomycin 9 to
by the proposed process of double activations with a single probe.
Furthermore, in this experiment we also used dimer 6 of double
activations with two probes for comparison with 9. The levels of
DNA ISC for dimer 6 were 81%, which was similar to the values
produce DNA ISC compared to reference 10 highlighting the func-
tion of disulfide in 9, and significantly, to demonstrate the strategy
of double activations with a single probe. So, we applied the meth-
2
4
25
ods by Cech, and Tepe and Williams with minor modifications
for our studies. Linearized pBR322 DNA obtained by linearization
of pBR322 plasmid DNA with EcoRI enzyme was treated with mit-
omycins in nucleophilic activation conditions. Then, the purified
DNA mixture was applied to denaturing alkaline agarose gel elec-
trophoresis to identify the generated DNA ISC along with k DNA
digested with HindIII as a molecular weight marker.
1
2
(
83%) reported in our previous studies. Although the levels of
DNA ISC for tetramer 9 were nearly half of those for 6, the levels
of DNA ISC per probe were nearly the same (ꢀ41%), which supported
the strategy of double activations with a single probe.
2
.5. Reflections on the strategy of double activations with a
single probe
We first needed to determine the proper range of concentra-
tions of mitomycin at which the ability of target compound to gen-
erate DNA ISC would be noticeably differentiated. Our previous
As discussed above, one of our main interests in this study lay in
the strategy of double activations with a single probe. We wished
to see if the intended double activations with a single probe indeed
occurred and, if so, to assess the effectiveness of the activations
and DNA adductions. To assess this strategy, we compared 9 (or
1
1
studies indicated that the proper concentrations for dimeric
compounds 5–8 were 0.1 mM whereas that for monomeric com-
pound 2 was 0.2 mM, which keeps the concentrations of mito-
mycin unit the same, regardless of the number of units in a
molecule. Considering this, we applied a range of concentrations
of 0.025–0.20 mM at room temperature for 2 h in the presence of
9
r) with an appropriate reference. Among mitomycins 5–8 that
represent double activations with two probes, we chose 6 (or 6r)
as a reference considering the ring size of the cyclic disulfide linker
and the distances between the two mitomycin rings. Thus, we
compared 9 with 6 in terms of activation rates and the levels of
DNA ISC as shown in Table 3. In the activation rate studies, the tar-
get mitomycin 9 definitely underwent activations through double
activations with a single probe, and moreover, the activation rate
Et
1
3
P (5 equiv), and found that the levels of DNA ISC ranged from
7% to 68%, as shown in Table 2. Thus, we chose 0.05 mM (41%
DNA ISC) for the target mitomycin 9, in order to sufficiently differ-
entiate the levels of DNA ISC and to compare the results in parallel
with those of compounds 5–8 while keeping the concentration of
mitomycin units the same.
First, we measured the formation of DNA ISC in the presence of
3
Et P (5 equiv), and the results were shown in Figure 2. The target
mitomycin 9 produced modest levels of DNA ISC (ꢀ41%) under
these conditions compared to references 10 (5%) and 2 (2%).
Accordingly, we concluded that mitomycin 9 underwent more effi-
ꢁ
1
(
e.g., kobs = 1.3 d ) per probe of 9 (or 9r) was nearly the same as
ꢁ1
that (e.g., kobs = 1.35 d ) per probe of mitomycin 6 (or 6r). As dis-
cussed above, the levels of DNA ISC per probe for both 9 and 6 were
also nearly identical (41% for 9; 40.5% for 6), which seemed reason-
able and consistent based on the function of each thiol.
We considered several factors such as the number and lifetime
3
cient activations and DNA ISC formation by the action of Et P on the
0
of thiol, the distance between C(1) and C(1 ) sites, and the flexibil-
disulfide unit, compared to references 10 and 2 that do not contain
the disulfide unit. In addition, when comparing the DNA ISC data
with the rate data of kinetic studies (Table 1), we found reasonable
correlations based on the observation that only mitomycin 9 under-
1
1–15
ity of two mitomycin rings. Our previous studies
using mito-
mycins 6–8 indicated that as the ring size of the cyclic disulfide
linker increased from six-membered ring (6) to eight-membered
ring (8), the lifetime of thiol(s) increased due to slower cyclization
to cyclic disulfide and the activation rates increased. Thus, in the
ꢁ1
3
went facile activations by Et P (5 equiv: kobs = 0.72 d ) while refer-
ences 10 and 2 did not. Thus, 9 was confirmed to produce DNA ISC
Table 3
Table 2
a
3
Comparison of 9 with 6 in activation rates and DNA ISC formations using Et P
The levels of DNA ISC depending on concentrations of 9
Activation ratesa
kobs (d
ꢁ1
)
DNA ISCb (%)
Compounds
Drug concentration (mM)
DNA ISC (%)
0.025
17
0.050
41
0.10
51
0.20
68
9
6
1.3
2.7
41
81
c
a
DNA cross-linking experiments for 9 using EcoRI-linearized pBR322 plasmid
a
b
c
DNA with Et
3
P (5 equiv). All reactions were incubated at room temperature (2 h).
3
Activation rates with 10 equiv of Et P.
DNA ISC formations with 5 equiv of Et
Ref. 12.
TM
The percentage of DNA ISC was obtained by Bio-Rad Smartspec 3000 and/or UV
Trans Illuminator with a digital camera.
3
P.
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H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
case of 9, the lifetime of the generated thiol was believed to be
longer than those for 6–8 since there is no possibility for
intramolecular cyclic disulfide formation, which might facilitate
the activations. However, the number of thiols should be consid-
ered at the same time, and the single thiol in 9r has to activate
two mitomycin rings while each thiol in 6r has to activate one mit-
omycin ring, which might influence the whole activation rates.
Thus, it seems reasonable that the activation rates and DNA ISC
per probe of 9 would be similar to those of 6. Regarding the dis-
spectra were recorded on a Perkin–Elmer Spectrum GX spectrom-
1
13
eter. H (300 MHz) and C (75 MHz) NMR spectra were obtained
by a Bruker DRX 300 spectrometer. Mass spectra were obtained
by CI, EI or FAB ionization method. UV–vis spectra were recorded
on a Shimadzu UV-1800 Spectrophotometer. The measurements
of pH in aqueous solutions were conducted using a IQ Scientific
Instruments IQ-240 meter, and the measurements of nonaqueous
effective pH (‘pH’) in buffered methanolic solutions were similarly
conducted with long equilibration time. Thin layer chromatogra-
phy was conducted on silica gel plates (20 ꢃ 20 cm; Aldrich No.
Z12272-6). HPLC analyses were executed using the following
Waters Associate Units: 515 A pump, 515 B pump, dual k absor-
bance 2487 detector, 717 plus autosampler, and Hypersil ODS col-
umn (4.6 ꢃ 300 mm). The products were analyzed by linear
gradient condition: 90% A (aqueous 0.025 M triethylammonium
acetate, pH 6.5), 10% B (acetonitrile) isocratic for 5 min, then from
90% A, 10% B to 45% A, 55% B in 30 min. The flow rate was 1 mL/
min, and the eluent was monitored from 200 to 400 nm. The HPLC
solvents were filtered (aqueous solution with Millipore HVLP,
0.45 mm; acetonitrile with Millipore HV, 0.45 mm) and degassed
immediately before use.
0
tance and flexibility, the distances between C(1) and C(1 ) in 9 ran-
ged from 7 to 20 Å according to their conformations (Sybyl 6.0,
HyperChem 7.1) and were a little shorter than those (7–25 Å) in
1
2
dimer 6. Therefore, the distances between the two reaction sites,
in a limited range, did not significantly affect the formation of DNA
ISC. Flexibility did not significantly influence DNA ISC based on the
observations using 9 and 6. However, much still remains unknown
and many other factors must be associated with the activation and
DNA adduction, and thus, advanced elucidation of a working mech-
anism must be further pursued. Taken together, despite the com-
plexities discussed above, double activations with a single probe
for 9 and the effectiveness of this strategy were successfully
demonstrated, which constitutes a new approach in mitomycin
activation studies.
4.2. Procedures for synthesis of intermediates and mitomycins
4
.2.1. 1,3-Bis(t-butyloxycarbonylamino)-2-propanol (13)19
To a stirred solution of 1,3-diamino-2-propanol (12, 500 mg,
3
. Conclusion
5
.6 mmol) and Et
3
N (4.6 mL, 33 mmol) in DMF–H
2
O (1:1, 32 mL)
O) (3.0 g,
2
O (15 mL) was added
We present the design, synthesis, and mode of action of novel
was added a solution of di-tert-butyl dicarbonate (Boc
4 mmol) in DMF. After stirring at rt (3 h), H
2
mitomycins 9 and 10. Mitomycin 9 features a unique structural
motif of a tetramer tethered through the disulfide linker 11 and
could therefore be viewed as a dimeric dimer; this motif specifi-
cally induces double activations with a single probe. Compound 9
easily undergoes disulfide cleavage (or reduction) to provide dimer
r containing a single probe in a dimeric structure. So, tetramer 9
was aimed to undergo efficient nucleophilic activations and corre-
sponding facile DNA adduction.
Synthesis of the key intermediate, disulfide 11, was achieved
through an efficient six-step sequence from 12 in an excellent over-
all yield (43%). Treatment of the intermediate 11 with 1 in MeOH
provided new target mitomycin 9 in 32% yield. Similarly, use of
the amine 12 and 1 in MeOH afforded new reference mitomycin
0 in 86% yield. Then, through mechanistic studies of the nucle-
ophilic activation of 9 compared to reference 10, we found that mit-
omycin 9 underwent effective activation by nucleophiles (e.g.,
1
and the mixture was extracted with EtOAc (2 ꢃ 15 mL). The com-
bined organic layers were washed with aqueous 0.1 N HCl
(
25 mL), saturated aqueous NaHCO
and dried (MgSO ) and concentrated in vacuo. Purification by col-
umn chromatography (1:2 EtOAc/hexanes) afforded the product as
a white solid (1.6 g, 99%). Mp 88–91 °C; R 0.45 (1:2 EtOAc/hex-
anes); IR (KBr) 3358, 2977, 1545, 1515, 1366, 1251, 1170, 989,
3 2
(25 mL) and H O (25 mL),
4
9
f
ꢁ1 1
8
60, 782, 615 cm
CH ), 3.17 (1/2ABMXqt, J = 14.3, 5.7 Hz, 2H, CHHCH), 3.28
ABMXqt, J = 14.3, 5.7 Hz, 2H, CHHCH), 3.71 (br s, 2H, OH), 3.75
3
; H NMR (300 MHz, CDCl ) d 1.46 (s, 18H, OC
(
(
(
(
3 3
)
13
quin, J = 4.9 Hz, 1H, CH
75 MHz, CDCl ) d 28.6 (OC(CH
0.0 (OC(CH ), 157.4 (NHCO); MS m/z 291 [M+H] ; HRMS
+FAB) calcd for C13
2
CH), 5.16 (br s, 2H, NHCO); C NMR
3
3
)
3
), 43.7 (CH CH), 71.1 (CH CH),
2
2
1
+
8
(
3 3
)
+
27 2 5
H N O [M+H] : 291.1920, found: 291.1924.
3
Et P), while reference 10 did not, implying that the generated thiol
in 9 activated both mitomycin rings. Thus, we demonstrated the
intended double activations with a single probe for 9 in the presence
of Et P. Based on these observations, we also proposed a nucle-
3
ophilic activation pathway for 9. Through the evaluation of the abil-
ity of mitomycin 9 to produce DNA ISC, we found that 9 produced
4
.2.2. 1,3-Bis(t-butyloxycarbonylamino)-2-propanol
19
methanesulfonate (14)
To a cooled (0 °C) solution of 1,3-bis(t-butyloxycarbonylamino)-
-propanol (13, 500 mg, 1.7 mmol) in CH Cl (5 mL) was added
Et N (0.50 mL, 3.4 mmol) and methanesulfonylchloride (0.2 mL,
.1 mmol). After stirring at the same temperature (5 h) the solvent
was removed in vacuo. H O (80 mL) was added to the residue and
then the mixture was extracted with EtOAc (2 ꢃ 80 mL). The com-
bined organic layers were washed with saturated aqueous NaHCO
80 mL) and H O (80 mL). The organic layer was dried (MgSO ) and
2
2
2
3
2
substantial levels of DNA ISC (ꢀ41%) in the presence of Et
3
P while
2
the references 10 and 2 yielded trace levels (5% and 2%, respec-
tively), which again confirmed the double activations with a single
probe and highlighted the key role of the thiol group. The effective-
ness in activations and formation of DNA ISC per probe for 9 were
verified by comparing with dimers 5–8. This strategy using specif-
ically-designed 9 marks the first case among the mitomycin activa-
tions and constitutes a new approach for the activations of
mitomycins and other compounds that require prior activations.
3
(
2
4
concentrated in vacuo. Crystallization of crude product in EtOAc/
hexanes mixture afforded the product as a white solid (580 mg,
9
2%). Mp 138–141 °C; R
976, 1704, 1511, 1366, 1265, 1175, 916, 738, 528 cm
) d 1.44 (s, 18H, OC(CH ), 3.09 (s, 3H, OS
), 3.31 (1/2ABMXqt, J = 14.7, 5.8 Hz, 2H, CHHCH), 3.50
(1/2ABMXqdd, J = 14.7, 7.2, 4.5 Hz, 2H, CHHCH), 4.66 (quin,
f
0.60 (1:2 EtOAc/hexanes); IR (KBr) 3446,
ꢁ1
1
2
;
H
NMR (300 MHz, CDCl
O) CH
3
3 3
)
(
2
3
4
4
. Experimental
1
3
2
J = 5.0 Hz, 1H, CH CH), 5.17 (br s, 2H, NHCO); C NMR (75 MHz,
DMSO-d ) d 28.5 (OC(CH ) ), 38.1 (OS(O) CH ), 41.7 (CH CH),
.1. General
6
3 3
2
3
2
7
8.5 (OC(CH
3
)
3
), 79.9 (CH
2
CH), 156.1 (NHCO); MS m/z 369 [M
+
+
Melting points were measured by Buchi B-545 melting point
+H] ; HRMS (+FAB) calcd for C14H29N O S [M+H] : 369.1695,
2
7
apparatus using open capillary tubes and are uncorrected. FT-IR
found: 369.1704.
Please cite this article in press as: Kim, H. R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.041

8
H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4
.2.3. 1,3-Bis(t-butyloxycarbonylamino)-2-acetylthiopropane
stirring at room temperature (1 h), Et
saturated CHCl
wise at room temperature until a slight excess of iodine was evi-
denced by its color. After stirring at room temperature (6 h) the
3
N (59 lL, 0.43 mmol) and a
solution of iodine (ꢀ2.0 mL) was then added drop
2
0
(
15)
To a stirred solution of 1,3-bis(t-butyloxycarbonylamino)-2-
propanol methanesulfonate (14, 300 mg, 0.81 mmol) in DMF
5 mL) was added KSAc (112 mg, 0.97 mmol) and Et N (82 mg,
.81 mmol). After warming to 60 °C, stirring was continued (1 d)
3
(
0
3
2 2 3
solution was treated with saturated aqueous Na S O (100 mL)
and the mixture was extracted with EtOAc (2 ꢃ 70 mL). The com-
and then the solvent was removed in vacuo. H
was added to the residue and the resulting mixture was extracted
2
O (2 ꢃ 80 mL)
bined organic layers were washed with aqueous 0.1 N HCl
(70 mL), saturated aqueous NaHCO
3
2
(70 mL) and H O (70 mL).
with EtOAc (2 ꢃ 80 mL). The combined organic layers were dried
The organic layer was dried (MgSO
4
) and concentrated in vacuo.
(
4
MgSO ) and concentrated in vacuo. Purification by column chro-
Purification by column chromatography (1:5 EtOAc/hexanes)
afforded the product as a white solid (40 mg, 77%), which was
identified as the same compound obtained in method A.
matography (1:2 ? 1:1 EtOAc/hexanes) afforded the product as a
white solid (175 mg, 61%). Mp 92–93 °C; R 0.70 (1:2 EtOAc/hex-
anes); IR (KBr) 3366, 2977, 1720, 1511, 1366, 1251, 1167, 956,
f
ꢁ
1
1
18
8
(
3
64, 737, 632 cm
CH ), 2.34 (s, 3H, SC(O)CH
.45 (m, 2H, CHHCH), 3.62 (quin, J = 5.7 Hz, 1H, CH
;
H NMR (300 MHz, CDCl
), 3.21–3.34 (m, 2H, CHHCH), 3.35–
CH), 5.18 (br
) d 28.4 (OC(CH ), 30.9
CH), 79.7 (OC(CH ), 156.3
); MS m/z 349 [M+H] ; HRMS (+FAB) calcd
3
) d 1.44 (s, 18H, OC
4.2.6. Bis(1,3-diamino-2-propyl)disulfideꢂ4TFA (11)
3
)
3
3
Bis(1,3-bis(t-butyloxycarbonylamino)-2-propyl)disulfide (17,
13 mg, 0.021 mmol) was dissolved in trifluoroacetic acid (0.5 mL)
and stirring was continued at room temperature (2 h). The reaction
mixture was concentrated in vacuo to give the product as a brown
2
1
3
s, 2H, NHCO); C NMR (75 MHz, CDCl
), 40.8 (CH
NHCO), 194.8 (SC(O)CH
3
3 3
)
)
3 3
(
SC(O)CH
3
2
CH), 45.4 (CH
2
+
(
3
+
solid (13 mg, ꢀ100%). Mp 185–186 °C; R
f 2 2
0.10 (1:2 MeOH/CH Cl );
ꢁ1
for C15
H
29
N
2
O
5
S [M+H] : 349.1797, found: 349.1794.
IR (KBr) 3433, 1673, 1522, 1431, 1392, 1206, 837, 721, 598 cm ;
1
H NMR (300 MHz, MeOD-d
overlapped with H O peak), 3.31 (dd, J = 13.9, 5.4 Hz, 4H, C(1)
6
HH), 3.50–3.62 (m, 2H, C(2)H); C NMR (75 MHz, DMSO-d ) d
4
) d 3.11–3.23 (m, 4H, C(1)HH, partly
4
.2.4. 1,3-Bis(t-butyloxycarbonylamino)-2-mercaptopropane
2
2
0
13
(
16)
To a stirred solution of 1,3-bis(t-butyloxycarbonylamino)-2-
acetylthiopropane (15, 60 mg, 0.17 mmol) in MeOH–H O (5:1,
mL) was added K CO (140 mg, 1.0 mmol). After stirring at room
temperature (1 h), H O (50 mL) was added to the residue. The mix-
ture was extracted with EtOAc (2 ꢃ 50 mL). The combined organic
layers were washed with H O (50 mL). The organic layer was dried
MgSO ) and concentrated in vacuo. Purification by column chro-
matography (1:4 ? 1:3 EtOAc/hexanes) afforded the product as a
white solid (30 mg, 57%). Mp 91–93 °C; R 0.72 (1:2 EtOAc/hex-
anes); IR (KBr) 2925, 1692, 1505, 1390, 1252, 1168, 780 cm ; H
NMR (300 MHz, CDCl ) d 1.22–1.30 (m, 1H, SH), 1.45 (s, 18H, OC
CH ), 2.92–3.20 (m, 3H, CHHCH), 3.40–3.62 (m, 2H, CHHCH),
.27 (br s, 2H, NHCO); C NMR (75 MHz, CDCl
CH ), 40.6 (CH CH), 44.2 (CH CH), 79.9 (OC(CH
+
46.9 (CH
(+FAB) calcd for
211.1055.
2
CH), 54.8 (CH
2
CH); MS m/z 211 [Mꢁ4TFA+H] ; HRMS
+
2
C
H
6 19
N
S
4 2
[Mꢁ4TFA+H] : 211.1051, found:
6
2
3
2
0
0
00 00
00
4.2.7. (7-N,7 -N -Bis(1 ,3 -propanediyl)-2 -disulfide)
tetrakismitomycin C (9)
2
(
4
To a stirred solution of bis(1,3-diamino-2-propyl)disulfideꢂ4TFA
3
(11, 1.7 mg, 2.8 lmol) and Et N (4.0 lL, 29.0 lmol) in MeOH
f
(0.4 mL) was added MMA (1, 5.0 mg, 14 lmol). The reaction solu-
tion was stirred at room temperature (4 d) and then the solvent
was removed in vacuo. Purification of the reaction mixture by PTLC
ꢁ1 1
3
(
5
3
)
3
(1:3 MeOH/CHCl
(1.3 mg, 32%). R 0.68 (3:7 MeOH/CHCl
UV–vis (MeOH) kmax: 370 nm; H NMR (300 MHz, pyridine-d
2.19 (s, 12H, C(6)CH ), 2.72 (br s, 4H, C(2)H), 3.13 (br s, 4H, C(1)
H), 3.25 and 3.26 (2s, 12H, C(9a)OCH
J = 11.8 Hz, 4H, C(3)HH), 3.73 (app t, J = 6.4 Hz, 2H, C(2 )H)
3
) afforded the product as a dark blue solid
); HPLC t 38.6 min (96%);
) d
13
3
) d 28.6 (OC
), 156.0
f
3
R
1
(
(
[
)
3 3
2
2
3
)
3
5
+
NHCO); MS m/z 307 [M+H] ; HRMS (+FAB) calcd for C13
M+H] : 307.1692, found: 307.1686.
27
H N
2
O
4
S
3
+
0
3 3
and C(9a )OCH ), 3.56 (d,
00
,
3.90–
), 4.50 (app d,
J = 12.6 Hz, 4H, C(3)HH), 5.08–5.20 (m, 4H, C(10)HH, partly over-
lapped with H O peak), 5.32–5.45 (m, 4H, C(10)HH), 7.25–7.43
(m, 4H, C(7)NH), the signals for the N(1a)H and C(10)OC(O)NH
0
0
4
(
4
.2.5. Bis(1,3-bis(t-butyloxycarbonylamino)-2-propyl)disulfide
17)
.2.5.1. Method A.
4.09 (m, 4H, C(9)H), 4.11–4.38 (m, 8H, C(1 )H
2
To a stirred solution of 1,3-bis(t-butyloxy-
2
carbonylamino)-2-mercaptopropane (16, 30 mg, 0.098 mmol) and
Et N (34 L, 0.25 mmol) in CHCl (1.5 mL) was dropwise added a
saturated CHCl
2
)
3
l
3
protons were not detected and are believed to overlap with the
+
3
solution of iodine (ꢀ1.0 mL) at room temperature
observed peaks; MS m/z 1501 [M+Na] ; HRMS (+ESI) calcd for
+
until a slight excess of iodine was evidenced by its color. After stir-
ring at room temperature (3 h) the solution was treated with sat-
C H
66 78
N
16NaO20S
2
[M+Na] : 1501.4917, found: 1501.4913.
0
0
00
00 00
urated aqueous Na
with EtOAc (2 ꢃ 50 mL). The combined organic layers were washed
with aqueous 0.1 N HCl (50 mL), saturated aqueous NaHCO
50 mL) and H O (50 mL). The organic layer was dried (MgSO
S
2 2
O
3
(50 mL) and the mixture was extracted
4.2.8. 7-N,7 -N -(2 -Hydroxy-1 ,3 -propanediyl)bismitomycin C
(10)
3
To a stirred solution of 1,3-diamino-2-propanol (12, 0.50 mg,
(
2
4
)
5.5
l
3
mol) and Et N (4.8
lL, 35 lmol) in MeOH (0.2 mL) was added
and concentrated in vacuo. Purification by column chromatogra-
phy (1:5 EtOAc/hexanes) afforded the product as a white solid
MMA (1, 4.0 mg, 11
room temperature (2 d) and then the solvent was removed in
l
mol). The reaction solution was stirred at
(
f
20 mg, 67%). Mp 125–127 °C; R 0.55 (1:2 EtOAc/hexanes); IR
vacuo. Purification of the reaction mixture by PTLC (1:3 MeOH/
(
KBr) 3053, 2984, 2305, 1705, 1505, 1422, 1265, 1167, 895,
CHCl
3
) afforded the product as a dark blue solid (3.4 mg, 86%). R
); HPLC t 23.3 min (97%); UV–vis (MeOH)
max: 370 nm; H NMR (300 MHz, pyridine-d ) d 2.18 and 2.19
), 2.74 (d, J = 3.6 Hz, 2H, C(2)H),
f
ꢁ1
1
7
3
3
39 cm
.01 (quin, J = 5.5 Hz, 2H, CH
.48–3.65 (m, 4H, CHHCH), 5.52 (br s, 4H, NHCO); C NMR
;
H NMR (300 MHz, CDCl
3
) d 1.44 (s, 36H, OC(CH
3
)
3
),
0.44 (3:7 MeOH/CHCl
k
(2s, 6H, C(6)CH and C(6 )CH
3 3
3
R
1
2
CH), 3.07–3.20 (m, 4H, CHHCH),
5
13
0
(
7
(
75 MHz, CDCl
3
) d 28.4 (OC(CH
), 156.5 (NHCO); MS m/z 611 [M+H] ; HRMS
3
)
3
), 40.6 (CH
2
CH), 52.2 (CH
2
CH),
3.13 (app d, J = 3.6 Hz, 2H, C(1)H), 3.23 and 3.24 (2s, 6H, C(9a)
+
0
9.7 (OC(CH
3
)
3
OCH
4H, C(1 )H
3
and C(9a )OCH
3
), 3.54–3.65 (m, 2H, C(3)HH), 3.76–3.98 (m,
+
00
+FAB) calcd for C26
H
51
N
4
O
S
8 2
[M+H] : 611.3148, found: 611.3143.
2
), 3.99 (dd, J = 11.2, 3.9 Hz, 2H, (C(9)H), 4.27–4.37 (m,
0
0
1
H, C(2 )H), 4.53 (d, J = 12.6 Hz, 2H, C(3)HH), 4.96–5.12 (m, 2H, C
(10)HH, partly overlapped with H O peak), 5.37 (dd, J = 10.5,
3.9 Hz, 2H, C(10)HH), 7.36–7.71 (m, 6H, C(7)NH, C(10)OC(O)NH ),
the signal for the N(1a)H proton was not detected and is believed
4
.2.5.2. Method B.
carbonylamino)-2-acetylthiopropane (15, 60 mg, 0.17 mmol) in
MeOH–H O (5:1, 6 mL) was added K CO (140 mg, 1.0). After
To a stirred solution of 1,3-bis(t-butyloxy-
2
2
2
2
3
Please cite this article in press as: Kim, H. R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.041

H. R. Kim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
+
to overlap with the observed peaks; MS m/z 725 [M+H] ; HRMS
of EtOH, ꢁ70 °C (10 min)]. The mixture was centrifuged at 0 °C
+
(
+FAB) calcd for C33
H
41
N
8
O
11 [M+H] : 725.2895, found: 725.2902.
(15 min), and the EtOH was decanted off and evaporated in vacuo.
The remaining DNA was dissolved in 25
solution containing 1 mM EDTA (pH 8.0).
Agarose loading dye (5 L) 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 aqueous 100 mM Tris pH 7.0
buffer containing 150 mM NaCl, which was refreshed every
15 min. The gel was stained with aqueous 100 mM Tris pH 7.5 buf-
lL of aqueous 10 mM Tris
4
.2.9. C(1) methoxymitosenes (18)
Mitomycin 9 (2.0 mg, 1.4 mol) was dissolved in a solution of
MeOH–CHCl (1:1, 2 mL) and then the ‘pH’ was carefully adjusted
l
l
3
to 2.5–3.0 with a dilute methanolic HCl solution (0.02 M). The reac-
tion solution was stirred at room temperature while the pH of the
solution was maintained at near 3.0 by periodically adding the
methanolic HCl solution. After stirring for 3 d, the reaction solution
was neutralized (pH ꢀ7) with a dilute methanolic solution of Et
3
N
fer (100 mL) containing ethidium bromide [20 lL of an aqueous
ethidium bromide stock solution (10 mg/10 mL)] and 150 mM
(
0.02 M). Purification of the reaction mixture by PTLC (1:1 MeOH/
3
CHCl ) afforded the product 18 as a red solid (0.40 mg, 20%). HPLC
NaCl for 20 min. The background staining was then removed by
t
R
32.0–35.0 min (multiple peaks); UV–vis (MeOH) kmax: 313 nm;
4
soaking the gel in aqueous 50 mM NH OAc and 10 mM b-mercap-
+
MS m/z 1479 [M+H] .
toethanol solution (3 h). The gel was analyzed with a Bio-Rad
TM
Smartspec 3000 and/or UV Trans Illuminator with a digital cam-
4
.3. General procedure for the mitomycin activation studies
era, and quantitative analyses of each band were also performed.
To a buffered methanolic solution (0.1 M TrisꢂHCl ‘pH’ 7.4) (final
Acknowledgement
volume 1.5 mL) containing the mitomycins (for tetramer 9: 23 lL
of 1.0 mM methanolic solution, final concentration 0.015 mM; for
dimer 10: 45 L of 1.0 mM methanolic solution, final concentration
.03 mM) was added a methanolic solution (18–113 L) of the
This work was supported by the Duksung Women’s University
Research Grant 2014.
l
0
l
nucleophile of choice (stock solution: 5–20 mM, final nucleophile
concentration 0.06–1.5 mM). The reaction was monitored by UV–
vis spectroscopy (200–500 nm), and generally followed for greater
than two half-lives. The ‘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 solutions were analyzed by HPLC
and unreacted staring materials and products (e.g., 9, 10, and 18)
were determined by coinjection of authentic samples in the HPLC
and cospotting of authentic samples in the TLC. The kmax of mito-
mycin (ꢀ370 nm) was plotted versus time and found to decrease
in a first-order decay (exponential decay) process. The nonlinear
regression analysis to fit the observed exponential decay by Sigma-
Plot 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.
References and notes
1
.
.
Hata, T.; Hoshi, T.; Kanamori, K.; Matsumae, A.; Sano, Y.; Shima, T.; Sugawara,
R. J. Antibiot. 1956, 9, 141.
2
(a) Carter, S. K.; Crooke, S. T. Mitomycin C: Current Status and New Developments;
Academic Press: New York, 1979; (b) Bass, P. D.; Gubler, D. A.; Judd, T. C.;
Williams, R. M. Chem. Rev. 2013, 113, 6816; (c) Paz, M. M.; Pritsos, C. A. Adv.
Mol. Toxicol. 2012, 6, 243; (d) Paz, M. M. Bioorg. Chem. 2013, 48, 1.
Kono, M.; Saitoh, Y.; Kasai, M.; Sato, A.; Shirahata, K.; Morimoto, M.; Ashizawa,
T. Chem. Pharm. Bull. 1989, 37, 1128.
Vyas, D. M.; Chiang, Y.; Benigni, D.; Rose, W. C.; Brander, W. T. In Recent
Advances in Chemotherapy. Anticancer Section; Tshigami, J., Ed.; University of
Tokyo Press: Tokyo, 1985; p 485.
3
.
.
4
5. Kobayashi, E.; Okabe, M.; Kono, M.; Arai, H.; Kasai, M.; Gomi, K.; Lee, J.-H.;
Inaba, M.; Tsuruo, T. Cancer Chemother. Pharmacol. 1993, 32, 20.
6
7
8
.
.
.
He, Q.-Y.; Maruenda, H.; Tomasz, M. J. Am. Chem. Soc. 1994, 116, 9349.
Wang, S.; Kohn, H. J. Med. Chem. 1999, 42, 788.
(a) Masters, J. R. W.; Know, R. J.; Hartley, J. A.; Kelland, L. R.; Hendricks, H. R.;
Conners, T. Biochem. Pharmacol. 1997, 53, 279; (b) McAdam, S. R.; Knox, R. J.;
Hartley, J. A.; Masters, J. R. W. Biochem. Pharmacol. 1998, 55, 1777.
(a) Keyes, S. R.; Loomis, R.; DiGiovanna, M. P.; Pritsos, C. A.; Rockwee, S.;
Sartorelli, A. C. Cancer Commun. 1991, 3, 351; (b) Ramos, L. A.; Lipman, R.;
Tomasz, M.; Basu, A. K. Proc. Am. Assoc. Cancer Res. 1997, 38, 182; (c) Tomasz,
M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73.
9
.
4
.4.
General
procedure
for
alkaline
agarose
gel
2
4,25
electrophoresis
1
1
0. Hornemann, U.; Keller, P. J.; Kozlowski, J. F. J. Am. Chem. Soc. 1979, 101, 7121.
1. Kim, H. R.; Kim, J. J.; Park, J. J.; Lee, S. H. Bioorg. Med. Chem. 2012, 20, 5720.
Agarose gels were prepared by adding 1.20 g of agarose to
00 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 hot solution was
poured and then 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.
1
(
12. Lee, S. H.; Kohn, H. Org. Biomol. Chem. 2005, 3, 471.
13. Kim, J. J.; Kim, H. R.; Arai, H.; Lee, S. H. Arch. Pharm. Res. 2012, 35, 1413.
14. Kim, J. J.; Kim, H. R.; Lee, S. H. Arch. Pharm. Res. 2012, 35, 1629.
15. Park, H. J.; Kim, J. J.; Kim, H. R.; Lee, E. K.; Kim, E. S.; Jeong, C. S.; Moon, A.; Lee, S.
H. Bioorg. Med. Chem. 2011, 19, 4004.
16. (a) Iyer, V. N.; Szybalski, W. Science 1964, 145, 45; (b) Moore, H. W.; Czerniak, R.
Med. Res. Rev. 1981, 1, 249.
(
1
7. (a) Stevens, C. L.; Taylor, K. G.; Munk, M. E.; Marshall, W. S.; Noll, K.; Shah, G. D.;
Shah, L. G.; Uzu, K. J. Med. Chem. 1965, 8, 1; (b) Tomasz, M.; Lipman, R. J. Am.
Chem. Soc. 1979, 101, 6063; (c) McClelland, R. A.; Lam, K. J. Am. Chem. Soc. 1985,
1
07, 5182.
To an aqueous solution of ꢀ80
M TrisꢂHCl (pH 7.4) was added a solution of linearized pBR322
L, 5 g) in 10 mM Tris solution containing 1 mM EDTA (pH
.0). After deaeration with N gas (15 min), the mitomycin (2–
L of 5 mM DMSO solution, final concentration 0.1–0.2 mM)
L of 5–25 mM DMSO solution, final
concentration 0.5 mM) were added and the resulting solution (final
volume 100 L) was incubated at room temperature (2 h). The
solution was washed with 1:1 PhOH/CHCl
2 ꢃ 100 L), and precipitated [12.1 L of 3 M NaOAc and 250
2
lL of H O (sterile) and 2.5 lL of
18. For HCl salt of 11: see, Pickering, D. S.; Krishna, M. V.; Miller, D. C.; Chan, W.
1
W.-C. Arch. Biochem. Biophys. 1985, 239, 368.
(
8
4
5
l
l
19. Ramalingam, K.; Raju, N.; Nanjappan, P.; Nowotnik, D. P. Tetrahedron 1995, 51,
875.
2
2
2
2
0. Jamila, N. (Genfit Co.) Fr. Pat. 2,850,869 (A1), 2004.
1. Tomasz, M.; Lipman, R. Biochemistry 1981, 20, 5056.
l
and the nucleophile (2–10
l
22. Lee, S. H.; Kohn, H. Heterocycles 2003, 60, 47.
23. (a) Paz, M. M. Chem. Res. Toxicol. 2009, 22, 1663; (b) Lee, S. H.; Kohn, H. J. Am.
Chem. Soc. 2004, 126, 4281.
l
24. Cech, T. R. Biochemistry 1981, 20, 1431.
3
(100
l
L) and CHCl
3
25. Tepe, J. J.; Williams, R. M. J. Am. Chem. Soc. 1999, 121, 2951.
(
l
l
lL
Please cite this article in press as: Kim, H. R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.041