Design and synthesis of new mitomycin dimers containing a seven-membered cyclic disulfide and a diol linkers

Article abstract of DOI:10.1007/s12272-012-0811-6

We report the design and synthesis of two new mitomycin dimers, 7-N,7′-N′-(1″,2″-dithiepanyl- 3″,7″- dimethylenyl)bismitomycin C (8) and 7-N,7′-N′-(2″,6″- dihydroxy-1″,7″-heptanediyl)bismitomycin C (9). Mitomycins 8 and 9 are dimers connected by a seven-membered cyclic disulfide (a 1,2-dithiepane) and a 2,6-dihydroxyheptane linkers, respectively. Mitomycin 8 was designed to undergo efficient nucleophilic activation and following alkylation to give DNA adducts such as DNA interstrand cross-link (DNA ISC) adducts. The key moiety in 8 is a seven-membered cyclic disulfide linker that can generate two thiol groups in a molecule through disulfide cleavage. The two thiols can serve as probes to activate two mitomycin rings by intramolecular cyclization to quinone rings. The mitomycin 8 was synthesized using mitomycin A (1) and the key intermediate, cyclic disulfide 11 that was prepared through a nine-step synthetic sequence from 1,6- heptadiene (12). The diol mitomycin 9 was also synthesized from 1 and diamine salt 15.

Full text of DOI:10.1007/s12272-012-0811-6

Arch Pharm Res Vol 35, No 8, 1413-1420, 2012
DOI 10.1007/s12272-012-0811-6
Design and Synthesis of New Mitomycin Dimers Containing a
Seven-Membered Cyclic Disulfide and a Diol Linkers
Jae Jin Kim1, Hyoung Rae Kim1, Hitoshi Arai2, and Sang Hyup Lee1
1College of Pharmacy, Duksung Women's University, Seoul 132-714, Korea and 2Chemical Process Research and
Development Laboratory, Kyowa Hakko Kirin Co., Osaka 590-8554, Japan
(Received March 3, 28, 2012/Revised April 21, 2012/Accepted April 23, 2012)
We report the design and synthesis of two new mitomycin dimers, 7-
3'',7''-dimethylenyl)bismitomycin C ( ) and 7- ,7'- '-(2'',6''-dihydroxy-1'',7''-heptanediyl)bismito-
mycin C ( ). Mitomycins and are dimers connected by a seven-membered cyclic disulfide (a
1,2-dithiepane) and a 2,6-dihydroxyheptane linkers, respectively. Mitomycin was designed to
undergo efficient nucleophilic activation and following alkylation to give DNA adducts such as
DNA interstrand cross-link (DNA ISC) adducts. The key moiety in is a seven-membered cyclic
N,7'-N'-(1'',2''-dithiepanyl-
8
N
N
9
8
9
8
8
disulfide linker that can generate two thiol groups in a molecule through disulfide cleavage. The
two thiols can serve as probes to activate two mitomycin rings by intramolecular cyclization to
quinone rings. The mitomycin
mediate, cyclic disulfide 11 that was prepared through a nine-step synthetic sequence from 1,6-
heptadiene (12). The diol mitomycin was also synthesized from and diamine salt 15
8 was synthesized using mitomycin A (1) and the key inter-
9
1
.
Key words: Disulfide mitomycin, Diol mitomycin, Mitomycin dimers, 1,2-Dithiepane, Disul-
fide formation, Antitumor agent
the reductive activation. Although
bifunctional alkylating agent due to the presence of
Mitomycins are known to be potent antitumor agents two reactive sites, the reactivity of the C(10) site has
as reductive alkylating agents. Mitomycin A (MMA, been expected to be 10-100 times lower than that of
(Hata et al., 1956) was isolated first in this series of the C(1) site (Hornemann et al., 1979). Nevertheless,
compounds, and mitomycin C (MMC, ), a representa- the reaction of and DNA could still provide both
2 is considered as a
INTRODUCTION
1)
2
2
tive example, was reported as a potent antitumor agent mono- and bis-alkylation adducts (Moore and Czerniak,
of major clinical significance (Carter and Crooke, 1979). 1981). Among them DNA interstrand cross-link (DNA
Mitomycins display chemical reactivity and correspond- ISC) products by bis-alkylation have been found to be
ing biological activity only after prior activation pro- the most lethal case (Keyes et al., 1991; Ramos et al.,
cess in which mitomycin is converted to a very good 1997; Tomasz and Palom, 1997).
electrophile. Generally, the activation of mitomycins
can be induced under reductive and acidic conditions.
Early mechanistic studies have shown that the activa-
tion is initiated by reduction at the quinone ring and
subsequently, leads to the generation of electrophilic
sites at C(1) and C(10) that may react with nucleophiles
such as DNA (Iyer and Szybalski, 1964; Szybalski and
Iyer, 1967; Moore and Czerniak, 1981), which comprises
Continuing efforts to overcome the drug resistance
Correspondence to: Sang Hyup Lee, College of Pharmacy,
and side effects (Carter and Crooke, 1979) caused by
Duksung Women's University, Seoul 132-714, Korea
long-term administration of
ment of disulfide-containing mitomycins
2
have led to the develop-
(KW-2149)
Tel: 82-2-901-8393, Fax: 82-2-901-8386
E-mail: sanghyup@duksung.ac.kr
3
1413

1414
J. J. Kim et al.
(Kono et al., 1989) and
1985). These mitomycins
4
3
(BMS-181174) (Vyas et al.,
and feature the presence
of C(7) aminoethylene disulfide unit compared to the
C(7) amino unit in , and display improved pharma-
4
2
cological properties, which should be related to the ad-
ditional structural unit, a disulfide group (Vyas et al.,
1985; Kono et al., 1989; Kobayashi et al., 1993). Mech-
anistic studies implied that disulfide cleavage of
by thiol (e.g., glutathione (GSH) or L-dithiothreitol (L-
DTT)) generated a key intermediate, C(7)-aminoethylene
thiol that could trigger the activation of the mitomycin
3 and
4
5
ring (He et al., 1994; Kohn and Wang, 1996; Wang and
Kohn, 1999; Na et al., 2002).
These results prompted us to investigate the effect
of ring size of cyclic disulfide linker. So, we report herein
the design and synthesis of new mitomycin dimers
8
and , and expect that would be transformed to the
9
8
dithiol species 10 upon disulfide cleavage that could
trigger activation processes.
MATERIALS AND METHODS
General
Melting points were measured in open capillary
With the observed advantages of disulfide mitomycins tubes using a Buchi B-545 melting point apparatus
in mind we became interested in the dimerization of and are uncorrected. FT-IR spectra were obtained on
1
mitomycin units as a good strategy to enhance the a Perkin-Elmer Spectrum GX spectrometer. H- (300
ability of the compound to generate DNA ISC. As MHz) and 13C- (75 MHz) NMR spectra were recorded
mentioned above, although the mitomycin unit contains on a Bruker DRX 300 spectrometer. Mass spectra
two reactive sites (C(1) and C(10)), the reactivity of were obtained by CI, EI or FAB ionization method.
the C(10) site is very low. So, a mitomycin dimer would UV-vis spectra were obtained on a Shimatzu UV-1800
provide two C(1) sites of higher reactivity and, as a Spectrophotometer. pH Measurements of aqueous solu-
result, lead to higher probability for DNA ISC formation, tions were conducted using an IQ-240 pH meter. The
presumably by bis-alkylation at the two distal C(1) nonaqueous effective pH (‘pH’) of the buffered metha-
sites. Accordingly, we had previously started a program nolic solutions was similarly measured. Thin layer
to study disulfide mitomycin dimers, in which the dimers chromatography was run on silica gel plates (20
× 20
were connected by a cyclic disulfide linker. In this pro- cm; Aldrich No. Z12272-6). HPLC analyses were con-
gram, we had already reported the studies on two ex- ducted using the following Waters Associate Units:
amples,
et al., 2011). We observed that
faster activation and generated higher levels of DNA column (4.6
ISC adducts under nucleophilic conditions than did performed using linear gradient condition: 90% A
which might be the effects of both cyclic disulfide unit (aqueous 0.025 M triethylammonium acetate, pH 6.5),
and dimeric structure. However, when we compared 10% B (acetonitrile) isocratic for 5 min, then from 90%
and , we found that underwent ~ three times faster A, 10% B to 45% A, 55% B in 30 min. The flow rate was
activation than , but provided similar levels of DNA 1 mL/min, and the eluent was monitored from 200 to
ISC to (Lee and Kohn, 2005; Park et al., 2011). This 400 nm. The HPLC solvents were filtered (aqueous
6
(Lee, 2003; Lee and Kohn, 2005) and
and underwent detector, 717 plus autosampler, and Hypersil ODS
300 mm). The product analyses were
7 (Park 515 A pump, 515 B pump, dual λ absorbance 2487
6
7
×
2,
6
7
7
6
6
discrepancy between the activation rates and DNA solution with Millipore HVLP, 0.45 mm; acetonitrile
ISC formation was surprising. Considering that the with Millipore HV, 0.45 mm) and degassed before use.
only difference between
6
and
7
is the ring size of the
The key intermediate cyclic disulfide 11 including
cyclic disulfide linker in the dimers, we recognized the related intermediates were synthesized from diene
that the ring size of the linker may be a significant 12 as shown in Scheme 1. Compound 14 were prepared
factor in the activation and the corresponding forma- from 12 according to our previous procedures (Lee and
tion of DNA ISC.
Kohn, 2002). Mitomycins 8 and 9 were synthesized

Design and Synthesis of New Mitomycin Dimers
1415
EtOH (19 mL) was added NH2NH2·H2O (0.26 mL, 5.3
mmol), and the resulting mixture was heated under
reflux (3.5 h). After cooling to room temperature (1 h),
the solvent was removed in vacuo and the residue was
treated with dilute hydrochloric acid (10%, 30 mL).
The mixture was heated under reflux (1 h) and sub-
sequently cooled to 0oC, leading to the formation of
precipitates. The precipitates were filtered off and the
filtrate was concentrated in vacuo. The residue was
dissolved in H2O (25 mL) and insoluble matter was
removed by filtration. The clear filtrate was concen-
trated in vacuo to afford the title compound (460 mg,
93%, dr = 1.4:1, 13C-NMR analysis) as a white solid.
M.p. 225-226oC. Rf 0.52 (2:1 EtOAc-hexanes). IR (KBr)
3399, 2917, 1677, 1428, 1383, 1202, 1112, 559 cm−1.
1H-NMR (300 MHz; DMSO-d6):
H2 H2CH), 2.55-2.70 (m, 2 H, C
2 H, CH 'N), 3.67 (br s, 2 H, C
NH2·HCl), the signals for the CHO
detected and believed to overlap with the observed
peaks. 13C-NMR (75 MHz; CD3OD):
22.6 ( H2CH2CH),
36.0 (CH2CH2CH), 46.4 (CH2N), 68.9 (CHOH), for the
δ
1.20-1.58 (m, 6 H,
HH'N), 2.75-2.90 (m,
C
C
H
HOH), 8.10 (br s, 6 H,
H
protons were not
δ
C
Scheme 1. Synthesis of cyclic disulfide 11. Reagents and
conditions: (a)
m
-CPBA, CH2Cl2, 0oC
→
room temperature, 1
minor diastereomer: δ 22.5 (CH2CH2CH), 46.5 (CH2N).
d, 79%; (b) PhtNH, DMF, 100oC/1 h
→
115oC/1 h 135oC/1 h,
→
MS m/z 163 [M-2HCl+H]+. HRMS (+FAB) calcd for
C7H19N2O2 [M-2HCl+H]+: 163.1446; found 163.1447.
61%; (c) NH2NH2·H2O, EtOH, reflux, 3.5 h; then HCl, reflux, 1
h, 93%; (d) Boc2O, DMF-H2O (1:1), room temperature, 1 d,
76%; (e) MsCl, Et3N, CH2Cl2, 0oC, 1 h, 90%; (f) KSAc, DMF,
60oC, 8 h, 74%; (g) K2CO3, MeOH-H2O (5:1), room tempera-
ture, 1 h, 50%; (h) I2, Et3N, CHCl3, room temperature, 3 h,
79%; (i) K2CO3, MeOH-H2O (5:1), room temperature, 1 h;
then I2, Et3N, room temperature, 3 h, 71%; (j) TFA, room tem-
perature, 1 h, 100%.
1,7-Bis(tert-butyloxycarbonylamino)-2,6-hepta-
nediol (16)
To a stirred solution of 1,7-diamino-2,6-heptanediol
dihydrochloride (15, 250 mg, 1.1 mmol, dr = 1.4:1) and
Et3N (0.9 mL, 6.6 mmol) in H2O-DMF (1:1, 21 mL) was
added a solution of di-tert-butyl dicarbonate (Boc2O)
using mitomycin A (1, Kyowa Hakko Kirin Co.) and (589 mg, 2.7 mmol) in DMF (2 mL). After stirring at
the corresponding intermediates as shown in Scheme room temperature (1 d), the solvent was removed in
2.
vacuo. The remaining residue was treated with H2O
(80 mL) and then the mixture was extracted with
EtOAc (2
× 80 mL). The combined organic layers were
1,7-Diamino-2,6-heptanediol dihydrochloride (15)
To a stirred mixture of 1,7-bis(phthalimido)-2,6- successively washed with aqueous 0.1 N HCl (80 mL),
heptanediol (14, 900 mg, 2.1 mmol, dr = 1.3:1) and saturated aqueous NaHCO3 (80 mL) and H2O (80 mL).
Scheme 2. Synthesis of mitomycin dimers
8 and 9. Reagents and conditions: (a) Et3N, MeOH, room temperature, 3 d, 70%;
(b) Et3N, MeOH, room temperature, 2 d, 80%.

1416
J. J. Kim et al.
The organic layer was dried (MgSO4) and concentrated ring was continued (8 h) and then the solvent was re-
in vacuo. Purification by column chromatography (1:1 moved in vacuo. H2O (80 mL) was added to the residue
→
2:1 EtOAc-hexanes) afforded the title compound and the resulting mixture was extracted with EtOAc
(300 mg, 76%, dr = 1.7:1, 13C-NMR analysis) as a white (2
80 mL). The combined organic layers were dried
solid. M.p. 82-84oC. Rf 0.23 (2:1 EtOAc-hexanes). IR (MgSO4) and concentrated in vacuo. Purification by
(KBr) 3368, 3977, 2929, 1687, 1519, 1366, 1251, 1170, column chromatography (1:4 1:3 EtOAc-hexanes)
876, 781 cm−1. 1H-NMR (300 MHz; CDCl3):
1.40-1.75 afforded the title compound (142 mg, 74%, dr = 1.7:1,
(m, 24 H, CH2
H2CH, OC(CH3)3), 2.88 (br s, 2 H, 13C-NMR analysis) as a white solid. M.p. 88-90oC. Rf
CHO ), 2.97-3.11 (m, 2 H, C H'N), 3.20-3.35 (m, 2 H, 0.33 (1:2 EtOAc-hexanes). IR (KBr) 3366, 2977, 2927,
CH 'N), 3.69 (br s, 2 H, C
OH), 5.02 (br s, 2 H, NHCO). 1694, 1519, 1366, 1250, 1170 cm−1. 1H-NMR (300 MHz;
13C-NMR (75 MHz; CDCl3):
21.2 ( H2CH2CH), 28.6 CDCl3): 1.35-1.75 (m, 24 H, CH2 H2CH, OC(CH3)3),
(OC( H3)3), 34.3 (CH2CH2CH), 46.8 (CH2N), 71.5 (CHOH), 2.34 (s, 6 H, SAc), 3.18-3.43 (m, 4 H, CH2N), 3.50-3.61
79.9 (O (CH3)3), 157.1 (NHCO), for the minor dias- (m, 2 H, C
SAc), 4.78 (br s, 2 H, NHCO). 13C-NMR (75
tereomer: 21.4 ( H2CH2CH), 34.2 (CH2CH2CH), 46.3 MHz; CDCl3): 24.2 ( H2CH2CH), 28.6 (OC( H3)3),
H3), 31.7 (CH2CH2CH), 44.4 (CH2N), 45.1
HSAc), 79.6 (O (CH3)3), 156.1 (NHCO), 195.8 ( OCH3),
×
→
δ
C
H
H
H
H
δ
C
δ
C
C
C
H
δ
C
δ
C
C
(CH2N), 71.2 (CHOH). MS m/z 363 [M+H]+. HRMS 31.0 (CO
C
(+FAB) calcd for C17H35N2O6 [M+H]+: 363.2495; found
363.2488.
(C
C
C
for the minor diastereomer:
δ 24.3 (CH2CH2CH), 31.6
(CH2CH2CH). MS m/z 479 [M+H]+. HRMS (+FAB) calcd
for C21H39N2O6S2 [M+H]+: 479.2249; found 479.2249.
1,7-Bis(tert-butyloxycarbonylamino)-2,6-hepta-
nediol dimethanesulfonate (17)
To a cooled (0oC) solution of 1,7-bis(tert-butyloxycar-
bonyl)amino-2,6-heptanediol (16, 290 mg, 0.80 mmol,
dr = 1.7:1) in CH2Cl2 (3.9 mL) was added Et3N (0.33
1,7-Bis(tert-butyloxycarbonylamino)-2,6-dimer-
captoheptane (19)
To a stirred solution of 1,7-bis(tert-butyloxycarbonyl-
mL, 2.4 mmol) and methanesulfonylchloride (0.15 mL, amino)-2,6-bis(acetylthio)heptane (18, 30 mg, 0.062
1.9 mmol). After stirring at the same temperature (1 mmol) in MeOH-H2O (5:1, 4.1 mL) was added K2CO3
h) the solvent was removed in vacuo. H2O (70 mL) (52 mg, 0.37 mmol). After stirring at room tempera-
was added to the residue and then the mixture was ture (1 h), H2O (50 mL) was added to the residue and
extracted with EtOAc (2
× 70 mL). The combined the mixture was extracted with EtOAc (2 × 50 mL). The
organic layers were washed with saturated aqueous combined organic layers were washed with H2O (50
NaHCO3 (70 mL) and H2O (70 mL). The organic layer mL), dried (MgSO4) and concentrated in vacuo. Purifi-
was dried (MgSO4) and concentrated in vacuo. Crystal- cation by column chromatography (1:4
→ 1:3 EtOAc-
lization of crude product in EtOAc-hexanes mixture hexanes) gave the title compound (12 mg, 50%, dr =
afforded the title compound (370 mg, 90%, dr = 1.8:1, 1.2:1, 13C-NMR analysis) as a yellow solid. M.p. 97-
13C-NMR analysis) as a white solid. M.p. 132-133oC. 104oC. Rf 0.54 (1:2 EtOAc-hexanes). IR (KBr) 3358,
Rf 0.55 (2:1 EtOAc-hexanes). IR (KBr) 3399, 1693, 2977, 2929, 1694, 1514, 1366, 1251, 1169, 861, 732
1518, 1366, 1171, 909 cm−1. 1H-NMR (300 MHz; CDCl3):
1.44 (s, 18 H, OC(CH3)3), 1.60-1.85 (m, 6 H, CH2 H2CH),
3.06 (s, 6 H, OMs), 3.22-3.38 (m, 2 H, C H'N), 3.40- 3.01-3.14 (m, 2 H, C
3.57 (m, 2 H, CH 'N), 4.65-4.80 (m, 2 H, C
OMs), 4.92- 5.03 (br s, 2 H, NHCO). 13C-NMR (75 MHz; CDCl3):
5.07 (m, 2 H, NHCO). 13C-NMR (75 MHz; CDCl3):
H2CH2CH), 28.5 (OC( H3)3), 31.9 (CH2CH2CH), 38.7 41.5 (CH2N), 48.0 (
(OMs), 44.1 (CH2N), 80.1 (O (CH3)3), 81.6 (
156.1 (NHCO), for the minor diastereomer:
δ
cm−1. 1H-NMR (300 MHz; CDCl3):
H2 H2CH, OC(CH3)3, SH), 2.80-2.97 (m, 2 H, C
H'N), 3.30-3.50 (m, 2 H, CH
δ
1.35-1.73 (m, 26 H,
SH),
'N),
C
C
C
H
H
H
H
H
H
δ
δ
20.7 21.2 (
CH2CH2CH), 28.5 (OC(CH3)3), 35.6 (CH2CH2CH),
(C
C
CHSH), 79.7 (OC(CH3)3), 156.0
C
C
HOMs), (NHCO), for the minor diastereomer:
δ 24.4 (CH2CH2CH),
δ
20.4 35.5(CH2CH2CH). MS m/z 417 [M+Na]+.
(C
H2CH2CH), 31.6 (CH2CH2CH). MS m/z 519 [M+H]+.
HRMS (+FAB) calcd for C19H39N2O10S2 [M+H]+: 519.2046;
found 519.2042.
3,7-Bis(tert-butyloxycarbonylaminomethyl)-1,2-
dithiepane (20).
Method A: To a stirred solution of 1,7-bis(tert
butyloxycarbonylamino)-2,6-bis(acetylthio)heptane
18, 34 mg, 0.071 mmol, dr = 1.7:1) in MeOH-H2O (5:1,
-
1,7-Bis(tert-butyloxycarbonylamino)-2,6-bis(ace-
tylthio)heptane (18)
(
To a stirred solution of 1,7-bis(tert-butyloxycarbonyl- 4.8 mL) was added K2CO3 (59 mg, 0.43 mmol). After
amino)-2,6-heptanediol dimethanesulfonate (17, 210 stirring at room temperature (1 h) Et3N (21 L, 0.15
µ
mg, 0.40 mmol, dr = 1.8:1) in DMF (5.5 mL) was added mmol) was added. A saturated CHCl3 solution of
KSAc (114 mg, 1.0 mmol). After warming to 60oC, stir- iodine was then added dropwise at room temperature

Design and Synthesis of New Mitomycin Dimers
1417
until a slight excess of iodine was evidenced by its C7H17N2S2 [M-2TFA+H]+: 193.0833; found 193.0833.
color. After stirring at room temperature (3 h) the
solution was treated with saturated aqueous Na2S2O3
(50 mL) and the mixture was extracted with EtOAc (2
7-N,7'-N'-(1'',2''-Dithiepanyl-3'',7''-dimethylenyl)
bismitomycin C (8)
×
50 mL). The combined organic layers were washed
with saturated aqueous NaHCO3 (50 mL) and H2O (50
mL). The organic layer was dried (MgSO4) and concen- in MeOH (0.5 mL) was added mitomycin A (
trated in vacuo. Purification by column chromatography 14 mol). The reaction solution was stirred at room
To a solution of 3,7-bis(aminomethyl)-1,2-dithiepane
·
2TFA (11, 2.7 mg, 7
µmol) and Et3N (5.8
µ
L, 42
µmol)
1, 5 mg,
µ
(1.5
→ 1:4 EtOAc-hexanes) provided the title compound temperature (3 d) and then the solvent was removed
(20 mg, 71%, dr = 2.1:1, 13C-NMR analysis) as a white in vacuo. Purification of the reaction mixture by PTLC
solid. M.p. 79-81oC. Rf 0.56 (1:2 EtOAc-hexanes). IR (2:8 MeOH-CHCl3) gave the title compound (4.1 mg,
(KBr) 3357, 2977, 2926, 1693, 1514, 1250, 1170, 911, 70%, dr = 1.4:1, 13C-NMR analysis) as a dark blue solid.
732 cm−1. 1H-NMR (300 MHz; CDCl3):
OC(CH3)3), 1.53-2.05 (m, 6 H, CH2
(m, 2 H, CHS), 3.17-3.27 (m, 2 H, C
(m, 2 H, CH
'N), 4.92 (br s, 2 H, NHCO). 13C-NMR (75 C(6)CH3), 2.75 (d,
MHz; CDCl3): 23.7 ( H2CH2CH), 28.5 (OC( H3)3), 4.3 Hz, 2 H, C(1)H), 3.22 (s, 6 H, C(9a)OCH3), 3.61 (d,
29.8 (CH2CH2CH), 45.1 (CH2N), 54.8 (CHS), 79.7 = 12.6 Hz, 2 H, C(3)
(O (CH3)3), 155.9 (NHCO), for the minor diastereomer: 4.02 (dd, = 10.9, 3.8 Hz, 2 H, C(9)H), 4.54 (d,
21.7 ( H2CH2CH), 33.6 (CH2CH2CH), 43.8 (CH2N), 12.6 Hz, 2 H, C(3)H '), 5.08 (t, = 10.9 Hz, 2 H,
156.0 (NHCO) H'), 5.41 (dd, = 10.3, 3.8 Hz, 2 H, C(10)H '),
MS m/z 393 [M+H]+. HRMS (+FAB) calcd C(10)
for C17H33N2O4S2 [M+H]+: 393.1881; found 393.1872.
7.20-7.35 (m, 2 H, C(7)NH), the signals for the N(1a)H,
δ
1.45 (s, 18 H, HPLC tR 29.7 min. UV-vis (MeOH)
λ
max: 221, 376 nm. Rf
H2CH), 2.88-3.06 0.54 (2:8 MeOH-CHCl3). H-NMR (300 MHz; pyridine-
H'N), 3.34-3.46 d5): 1.48-1.98 (m, 6 H, C(4'')H2 C(5'')H2), 2.11 (s, 6 H,
1
C
H
δ
H
J
= 3.5 Hz, 2 H, C(2)H), 3.14 (d,
J
=
δ
C
C
J
H
H'), 3.65-3.90 (m, 4 H, C(7)NHCH2),
C
J
J =
δ
C
H
J
J
.
H
H
Method B: To a stirred solution of 1,7-bis(tert-butyl- C(10)OC(O)NH2, C(3'')H protons were not detected
oxycarbonylamino)-2,6-dimercaptoheptane (19, 20 mg, and are believed to overlap with the observed peaks.
0.051 mmol) in CHCl3 (3.3 mL) was added Et3N (15 13C-NMR (75 MHz; pyridine-d5):
δ
9.5 (C(6)
L, 0.11 mmol). A saturated CHCl3 solution of iodine (C(5'')), 29.4 (C(4'')), 32.1 (C(2)), 36.2 (C(1)), 43.8 (C(9)),
was added dropwise to the resulting solution at room 47.5 (C(3'')), 48.5 (C(7)NH H2), 49.1 (C(9a)O H3), 50.1
CH3), 22.4
µ
C
C
temperature until a slight excess of iodine was evi- (C(3)), 61.9 (C(10)), 104.0 (C(6)), 106.4 (C(9a)), 110.4
denced by its color. After stirring at room temperature (C(8a)), 146.7 (C(7)), 155.2 (C(5a)), 157.5 (C(10a)), 176.3
(3 h) the solution was treated with saturated aqueous (C(8)), 178.7 (C(5)), for the minor diastereomer:
Na2S2O3 (50 mL) and the mixture was extracted with (C(9)), 146.5 (C(7)). MS m/z 849 [M+Na]+. HRMS
EtOAc (2
50 mL). The combined organic layers were (+FAB) calcd for C37H46N8O10S2Na [M+Na]+: 849.2676;
δ 43.9
×
washed with saturated aqueous NaHCO3 (50 mL) and found 849.2674.
H2O (50 mL). The organic layer was dried (MgSO4) and
concentrated in vacuo. Purification by column chroma-
tography (1:4 EtOAc-hexanes) afforded the title com-
pound (16 mg, 79% from 19) as a white solid that was
7-N,7'-N'-(2'',6''-Dihydroxy-1'',7''-heptanediyl)bis-
mitomycin C (9)
To a solution of 1,7-diamino-2,6-heptanediol dihydro-
identified as the same compound obtained in method chloride (15, 1.0 mg, 4.3
A. mol) in MeOH (0.3 mL) was added mitomycin A (
mg, 8.6 mol). The reaction solution was stirred at
room temperature (2 d) and then the solvent was re-
µmol) and Et3N (3.6
µ
L, 26
µ
1, 3
µ
3,7-Bis(aminomethyl)-1,2-dithiepane·2TFA (11)
3,7-Bis(tert-butyloxycarbonylaminomethyl)-1,2-dithi- moved in vacuo. Purification of the reaction mixture
epane (20, 10 mg, 0.025 mmol) was dissolved in trifluo- by PTLC (3:7 MeOH-CHCl3) afforded the title compound
roacetic acid (1 mL) and stirring was continued at room (2.7 mg, 80%, dr = 1.1:1, 13C-NMR analysis) as a dark
temperature (1 h). The reaction was concentrated in blue solid. HPLC tR 22.0 min. UV-vis (MeOH) λmax
vacuo to afford the title compound (10 mg, 100%) as a 223, 367 nm. Rf 0.55 (3:7 MeOH-CHCl3). 1H-NMR (300
brown solid. M.p. 92-94oC. Rf 0.15 (4:6 MeOH-CHCl3). MHz; pyridine-d5):
1.45-1.90 (m, 6 H, C(3'')H2 C(4'')H2),
IR (KBr) 3433, 2924, 2853, 1637, 1462, 1204, 1139, 801, 2.19 (s, 6 H, C(6)CH3), 2.70-2.74 (m, 2 H, C(2)H), 3.14
723 cm−1. 1H-NMR (300 MHz; CD3OD):
1.60-2.30 (m, (br s, 2 H, C(1)H), 3.21 (s, 6 H, C(9a)OCH3), 3.49-3.54
6 H, CH2 H2CH), 2.87-2.96 (m, 2 H, CHS), 3.04-3.20 (m, 2 H, C(3) H'), 3.59-3.73 (m, 2 H, C(1'')H '), 3.81-
(m, 4 H, NCH2). 13C-NMR (75 MHz; CD3OD):
22.1 3.88 (m, 2 H, C(1'') H'), 3.96-4.09 (m, 4 H, C(2'')H,
H2CH2CH), 24.5 (CH2CH2CH), 35.2 (CH2N), 43.3 (CHS). C(9)H), 4.56 (d, = 13 Hz, 2 H, C(3)H '), 5.00 (2 H,
MS m/z 193 [M-2TFA+H]+. HRMS (+FAB) calcd for C(10)
H', overlapped with H2O peak), 5.39 (dd,
:
δ
δ
C
H
H
δ
H
(C
J
H
H
J =

1418
J. J. Kim et al.
10.4, 4.1 Hz, 2 H, C(10)HH'), 7.42-7.56 (m, 2 H, C(7)NH), (Wang and Kohn, 1999), we expected the thiol 10 would
the signals for the N(1a)H, C(10)OC(O)NH2 protons also be unstable. So the reasonable way to generate
were not detected and are believed to overlap with the thiol would be instant cleavage of the disulfide unit.
observed peaks. 13C-NMR (75 MHz; pyridine-d5):
δ
10.7 Instant generation of two thiols in a molecule could
H3), 27.8 (C(4'')), 30.1 (C(3'')), 32.5 (C(2)), 36.2 only be achieved from a cyclic disulfide, which is a key
(C(1)), 44.6 (C(9)), 50.0 (C(9a)O H3), 51.1 (C(3)), 51.7 factor in our design. In addition, the thiol probes were
(C(6)C
C
(C(1'')), 62.9 (C(10)), 70.6 (C(2'')), 103.8 (C(6)), 107.3 positioned three atoms away from the mitomycin ring,
(C(9a)), 110.9 (C(8a)), 148.2 (C(7)), 156.9 (C(5a)), 158.6 which easily induces the thiol to undergo intramole-
(C(10a)), 177.1 (C(8)), 179.3 (C(5)), for the minor dias- cular cyclization to the quinone ring and subsequent
tereomer:
δ 27.4 (C(4'')), 44.7 (C(9)), 70.7 (C(2'')), 179.4 activation (Na et al., 2002; Lee and Kohn, 2004, 2005).
(C(5)). MS m/z 797 [M+H]+. HRMS (+FAB) calcd for As a result, dimer
8 would provide double activations
C37H49N8O12 [M+H]+: 797.3469; found 797.3479.
RESULTS AND DISCUSSION
at the two C(1) sites of higher reactivity and be ex-
pected to increase the formation of DNA ISC. The two
mitomycin units are still connected by a carbon linkage
even after disulfide cleavage (e.g.
,
10) since the linker
(or 10
in is a cyclic disulfide. Furthermore, dimer
8
8
)
Design and structural features of 8 and 9
We designed a new mitomycin dimer
a seven-membered cyclic disulfide (a 1,2-dithiepane) the two C(1) sites. We measured the maximum distance
linker. We also designed another new mitomycin dimer between the two C(1) sites in (or 10) to be ~ 26 Å de-
that containes a diol linker instead of a cyclic disul- pending on their conformations (Sybyl 6.0, HyperChem
fide linker. Mitomycin keeps the same carbon skeleton 7.1), and therefore (or 10) could reach a range of nu-
as except that two hydroxy groups were introduced cleophilic sites in ds-DNA. This distance was found to
in place of the disulfide unit, and therefore, was be slightly longer than that predicted for dimer (or
adopted as a reference compound to identify the net dithiol structure generated by disulfide cleavage in
effect of the disulfide unit in (~ 25 Å) (Lee and Kohn, 2005), and slightly shorter
than that for (or dithiol structure generated by di-
sulfide cleavage in ) (~ 28 Å) (Park et al., 2011), which
differentiates (or 10) from and
8 that contains may provide enough flexibility and distance between
8
9
9
8
8
9
6
6)
8.
7
7
8
6
7.
As mentioned above, our key interest lies in both non-
traditional nucleophilic activation induced by disulfide
unit and the double C(1) activation provided by the
dimeric structure, which could ensure the enhanced
formation of DNA ISC. Compound
8 was designed to
sufficiently meet these requirements and expected to
undergo nucleophilic activation at two C(1) sites. Com-
pound
8 that differs from 6 and 7 in the ring size of
Synthesis of cyclic disulfide 11
For the synthesis of designed mitomycins
cyclic disulfide linker also features several structural
8
and 9, the
characteristics. First, the linker contains a seven- cyclic disulfide 11 and amine salt 15 were synthesized
membered cyclic disulfide (a 1,2-dithiepane), and so, as shown in Scheme 1. We analyzed the diastereomeric
disulfide cleavage in
e.g., 10) that has two thiols in the linker moiety. Both to our previous procedures (Lee and Kohn, 2002), we
of the thiols in 10 would serve as good probes to acti- prepared diphthalimide derivative 14 from diene 12
vate the two mitomycin rings in a dimer, respectively. in which we modified the procedures to improve the
However, based on the instability of the thiol species results. Treatment of 1,6-heptadiene (12) with -chlo-
8
may provide an acyclic dimer ratio (dr) of compounds by 13C-NMR. First, according
(
,
5
m

Design and Synthesis of New Mitomycin Dimers
1419
roperbenzoic acid (m-CPBA) gave diepoxide 13 (dr = Consequently, the key intermediate, cyclic disulfide
1.2:1) (Lee and Kohn, 2002) in 79% yield. Next, reac- 11 was synthesized from 12 through a nine-step syn-
tion of 13 with phthalimide (PhtNH) gave diphthali- thetic sequence in 16% overall yield. Amine salt 15 was
mide derivative 14 (Lee and Kohn, 2002) in very low also obtained as an intermediate in this sequence.
yield (27%). We therefore applied stepwise temperature
control and modified the work-up procedures to im-
Synthesis of mitomycin dimers 8 and 9
prove the yield of 14 (61%, dr = 1.3:1). Deprotection of
Then, we have synthesized the mitomycin dimers 8
phthalimide groups in 14 was conducted by employing and
hydrazine hydrate to afford amine salt 15 (dr = 1.4:1) ate 11 obtained above and mitomycin A (
in 93% yield, and subsequent protection of amines provided the target mitomycin dimer
with di-
-butyl dicarbonate (Boc2O) gave 16 (dr = 1.7:1) and 13C-NMR analysis indicated that
9
as shown in Scheme 2. Treatment of intermedi-
1) in MeOH
8
in 70% yield,
t
8 existed as a
in 76% yield. We then treated 16 with MsCl to obtain 1.4:1 mixture of diastereomers. The diol mitomycin
9
dimesyl derivative 17 (dr = 1.8:1) in 90% yield. Fol- was also synthesized using amine salt 15 and
1
in
lowing substitution of the mesylate units with acetylthio 80% yield, and 13C-NMR analysis showed that
groups using potassium thioacetate (KSAc) in DMF 1.1:1 mixture of diastereomers.
9 was a
afforded diacetylthio derivative 18 (dr = 1.7:1) in 74%
yield. Then, hydrolysis of 18 using potassium carbonate mitomycin dimers
In conclusion, we report the design and synthesis of
and . Compound is composed
8
9
8
(K2CO3) led to the generation of dithiol derivative 19 of a dimeric structure connected through linker of a
in relatively low yield (50%) probably due to the for- seven-membered cyclic disulfide (a 1,2-dithiepane),
mation of disulfide or polysulfide. In fact, several polar which may play a key role in drug functions. Mitomycin
compounds with lower Rf value than 19 on TLC were
8 was designed for the purpose of combination of
gradually generated as the reaction proceeded. We nucleophilic activation and C(1) and C(1') double acti-
presume that the species with lower Rf value are the vations aiming to improve the formation of DNA ISC.
disulfide or polysulfide compounds, which were also Miotmycin
observed in our previous studies using dithiol com- identify the net effect of disulfide unit in
9
was also designed as a reference one to
. We developed
8
pounds (Lee et al., 2005; Park et al., 2011). For intra- a synthetic procedure for the key intermediate, cyclic
molecular cyclization of dithiol units in 19 through disulfide (11) through a nine-step synthetic sequence,
oxidation process, we applied both O2-KOH and I2- and then synthesized the target mitomycin dimer
Et3N in varying concentrations. While the desired using mitomycin A ( ) and 11. We also synthesized
cyclic disulfide 20 was obtained in low yield (~20%) diamine salt 15 and then, mitomycin dimer using
using O2-KOH condition along with the generation of and 15
8
1
9
1
.
substantial amount of side products that were believed
to be polysulfide compounds, the desired product 20
was obtained in good yield (79%) employing I2-Et3N
condition at around 15 mM concentrations without
ACKNOWLEDGEMENTS
This work was supported by National Research
major side products. Furthermore, when the two reac- Foundation of Korea Grant funded by the Korean
tions, hydrolysis and oxidation, were performed in situ Government (2009-0066477).
without isolation of dithiol compound 19 we obtained
the desired cyclic disulfide 20 (dr = 2.1:1) in good yield
(71%, for two steps). Compound 20 was identified by
NMR (1H and 13C), Mass, and IR spectroscopy, and
more importantly, 20 was found to display an approp-
riate Rf value (0.56, EtOAc-hexanes = 1:2) that is very
similar to the values of 3,6-bis(tert-butyloxycarbonyl-
aminomethyl)-1,2-dithiane (0.55) (Lee and Kohn, 2005)
and 3,8-bis(tert-butyloxycarbonylaminomethyl)-1,2-di-
thiocane (0.56) (Park et al., 2011). Then the Boc groups
in 20 were deprotected by trifluoroacetic acid (TFA)
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