Propargylic sulfones possessing a 2-nitroimidazole function: Novel hypoxic-cell radiosensitizers with intracellular non-protein thiol depletion ability

Article abstract of DOI:10.1016/j.bmcl.2004.02.060

Propargylic sulfones (1a-c) containing a 2-nitroimidazole structure were synthesized, and their non-protein thiol (NPSH) depletion abilities were investigated. Propargylic sulfones 1a,c containing an electron withdrawing p-nitrophenyl group showed high reactivity toward capturing glutathione (GSH), a typical intracellular NPSH, in phosphate buffer. Among the three propargylic sulfones 1a-c, carboxylic acid derivative 1c showed the most potent radiosensitizing activity toward hypoxic EMT6/KU tumor cells. In view of these results and the partition coefficients between 1-octanol and water, we concluded that appropriate NPSH-depletion ability and lipophilicity are both important in achieving potent hypoxic-cell radiosensitization by propargylic sulfones possessing a 2-nitroimidazole function in biological systems.

Full text of DOI:10.1016/j.bmcl.2004.02.060

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2633–2635
Propargylic sulfones possessing a 2-nitroimidazole function: novel
hypoxic-cell radiosensitizers with intracellular non-protein thiol
depletion ability
Kazuhito Tanabe,* Ryusuke Kojima, Hiroshi Hatta and Sei-ichi Nishimoto*
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus,
Kyoto 615-8510, Japan
Received 16 December 2003; revised 17 February 2004; accepted 17 February 2004
Abstract—Propargylic sulfones (1a–c) containing a 2-nitroimidazole structure were synthesized, and their non-protein thiol (NPSH)
depletion abilities were investigated. Propargylic sulfones 1a,c containing an electron withdrawing p-nitrophenyl group showed high
reactivity toward capturing glutathione (GSH), a typical intracellular NPSH, in phosphate buffer. Among the three propargylic
sulfones 1a–c, carboxylic acid derivative 1c showed the most potent radiosensitizing activity toward hypoxic EMT6/KU tumor cells.
In view of these results and the partition coefficients between 1-octanol and water, we concluded that appropriate NPSH-depletion
ability and lipophilicity are both important in achieving potent hypoxic-cell radiosensitization by propargylic sulfones possessing a
2-nitroimidazole function in biological systems.
Ó 2004 Elsevier Ltd. All rights reserved.
Endogenous non-protein thiols (NPSH) play a crucial
function in determining the response of biological cells
to several types of radiation.1 The reduced form of
glutathione (GSH), which is a typical endogenous
NPSH and exists abundantly in cells, is known to pro-
tect intracellular molecules from radiation.2–6 Therefore,
depletion of GSH in tumor cells is recognized as being
effective for radiation therapy.7–9 Several strategies have
been proposed to enhance the effect of radiation ther-
apy, including oxidation of GSH to the oxidized form
(GSSG),10 conjugation of functional compounds to
GSH through a covalent bond,11 and inhibition of
intracellular GSH synthesis.7 Previously, we reported a
series of nitroazole derivatives containing an a,b-
unsaturated carbonyl group in the side chains that are
able to deplete GSH in tumor cells, thereby resulting in
more enhanced hypoxic-cell radiosensitization in vitro
relative to a well-documented nitroimidazole radiosen-
sitizers, such as misonidazole.12–14
On the other hand, propargylic sulfones have been
identified as prodrugs that undergo isomerization to
allenic sulfones with alkylating reactivity for a variety of
nucleophiles including DNA bases and thiols.15–18 In
view of such an alkylating mechanism, propargylic
sulfones may be applicable to a family of NPSH-
depleting agents. To expand our field of developing
hypoxic-cell radiosensitizers with NPSH-depletion abil-
ity, we synthesized three propargylic sulfones (1a–c)
possessing a 2-nitroimidazole structure, which has a
well-identified radiosensitizing function (Fig. 1). Among
the propargylic sulfones synthesized, carboxylic acid
derivative 1c exhibited efficient intracellular GSH-
depletion ability and radiosensitizing activity toward
hypoxic EMT6/KU cells. The propargylic sulfone 1c
had a very low lipophilicity as measured by the partition
coefficient between 1-octanol and water, which should
be a key property for achieving efficient radiosensitizing
activity.
Keywords: Propargylic sulfone; NPSH depletion; Radiosensitizer;
Hypoxic-cell.
* Corresponding authors. Tel.: +81-75-383-2500; fax: +81-75-383-25-
01; e-mail addresses: tanabeka@scl.kyoto-u.ac.jp; nishimot@scl.
kyoto-u.ac.jp
Figure 1. Structure of propargylic sulfones possessing a 2-nitroimid-
azole function.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.060

2634
K. Tanabe et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2633–2635
corresponding rates were lower than those at pH 8.5. It
has been demonstrated that both the electron-with-
drawing sulfonyl and 2-nitrophenyl groups increase the
acidity of the propargylic proton, thereby resulting
in efficient conversion to the corresponding allenic
isomer.23 Thus, introduction of electron-withdrawing
groups to the propargylic sulfones is indispensable for
obtaining efficient GSH-depletion ability.
Upon 60 h incubation of 1a and GSH in phosphate
buffer (pH 8.5) at 37 °C, the reaction mixture was ana-
lyzed by mass spectroscopy. The ESI/MS indicated the
formation of 1a-GSH adduct ([M–H]ꢀ, 742).
We next examined the effect of propargylic sulfones 1a
and 1c on intracellular GSH-depletion and radiosensi-
tization of hypoxic EMT6/KU cells.24 The cells were
exposed to varying X-ray doses under hypoxic condi-
tions and survival was determined by colony assay. The
Scheme 1. Reagents and conditions: (a) n-BuLi, boron trifluoride
diethyl etherate, THF, )78 °C, and then epichlorohydrin, )78 °C, 58%;
(b) NaOH, H2O–EtOH, rt, 67%; (c) 2-nitroimidazole, K2CO3, EtOH,
70 °C, 72%; (d) Ac2O, DMAP, CH2Cl2, rt, 88%; (e) PPTS, MeOH, rt,
94%; (f) (i) MsCl, Et3N, CH2Cl2, rt; (ii) 4-nitrothiophenol (for 7a),
thiophenol (for 7b), NaH, rt, 80% (for 7a), 26% (for 7b); (g) mCPBA,
CH2Cl2, 0 °C, 80% (for 1a), 73% (for 1b), 30% (for 1c); (h) NaOH,
CH2Cl2–MeOH–H2O, rt, 88%; (i) succinic anhydride, DMAP,
CH2Cl2, rt, 94%.
survival data were fitted to a single-hit multi-target
n
model: S ¼ 1 ꢀ ð1 ꢀ eꢀD=D Þ , where D is the radiation
0
dose, n is the extrapolation number, and D0 is the mean
lethal dose. Figure 2 shows the profile of the dose–sur-
vival curve for EMT6/KU cells incubated with 1a or 1c
for 4 h followed by X-irradiation. The mean lethal dose
was decreased by the treatment of hypoxic EMT6/KU
cells with 1a or 1c from 3.6 Gy for the control to 1.6 and
1.5 Gy, respectively. A shoulder observed at lower dose
as a nonlinear portion of the survival curve almost dis-
appeared, indicating that 1a and 1c acted as efficient
sensitizers of hypoxic cells even at a low radiation dose.
The value of n for 1a and 1c decreased from 5.6 for the
control to 3.4 and 2.6, respectively. To get molecular
structural insights into intracellular GSH-depletion
reactivity, we characterized radiosensitization of hyp-
oxic cells in the presence of a reference compound 7a.
While 7a could sensitize hypoxic cells, alteration of n
value was not achieved in the dose–survival curve (data
not shown). As reported previously, depletion of the
intracellular NPSH level upon treatment with NPSH
reactive species and irradiation leads to a decrease in the
n value.14 In view of this, it is reasonable to conclude
that propargylic sulfones 1a and 1c can decrease the
GSH level in the cell, as in phosphate buffer, thereby
resulting in efficient radiosensitization. The sensitizer
The synthetic route to propargylic sulfones possessing a
nitroimidazole function is shown in Scheme 1. The
propargylic sulfides 7a,b were readily obtained from
acetylene 2. Oxidation of 7a,b by mCPBA produced the
propargylic sulfones 1a,b.19;20 Carboxylic acid derivative
1c21 was obtained by the coupling of succinic anhydride
with alcohol 8, which was prepared by hydrolysis of 7a
followed by oxidation.
These propargylic sulfones possessing a 2-nitroimid-
azole function 1a–c and a control compound without a
sulfonyl group 7a were subjected to evaluation of their
reactivity with a typical NPSH, namely glutathione, in
phosphate buffer at pH 8.5. The second-order rate con-
stants (k2) for the nucleophilic addition of GSH to 1a–c
and 7a are listed in Table 1.22 As expected, propargylic
sulfones 1a and 1c containing an electron-withdrawing
p-nitrophenyl group reacted efficiently with GSH. In
contrast, propargylic sulfone without a nitro group 1b
and control compound 7a showed low GSH-depletion
reactivity. We also investigated the reactivity of 1a–c
toward GSH in phosphate buffer at pH 7.5 where the
effect of propargylic sulfones on intracellular GSH-
depletion was evaluated. Both 1a and 1c showed higher
GSH-depletion reactivity than 1b at pH 7.5, while the
Table 1. Second-order rate constants (k2) for the reaction of propar-
gylic sulfones with GSH in phosphate buffer and 1-octanol-to-water
partition coefficients (Po=w) for propargylic sulfones
Propargylic
sulfones
k2 (mMꢀ1 hꢀ1
)
Po=w
pH 8.5
pH 7.5
1a
1b
1c
7a
17.0
0.7
8.6
0.8
3.0
0.2
1.1
––a
15.0
22.2
2.2
Figure 2. Survival of EMT6/KU cells upon X-irradiation after 4 h
incubation with (j) 0.5 mM of 1c, (N) 0.5 mM of 1a, and ( ) no
––a
d
propargylic sulfones under hypoxic conditions (95% N2+5% CO2).
a Not determined.

K. Tanabe et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2633–2635
2635
14. Zhou, L.; Ino, A.; Dai, W.-M.; Nishimoto, S. Bioorg.
Med. Chem. 1999, 7, 2591.
enhancement ratios in vitro (SERin vitro) at 0.5 mM for 1a
and 1c were 2.4 and 2.6, respectively (see Fig. 2).
15. Nicolaou, K. C.; Skokotas, G.; Maligres, P.; Zuccarello,
G.; Schweiger, E. J.; Toshima, K.; Wendeborn, S. Angew.
Chem., Int. Ed. Engl. 1989, 28, 1272.
16. Haruna, K.; Tanabe, K.; Ishii, A.; Dai, W.-M.; Hatta, H.;
Nishimoto, S. Bioorg. Med. Chem. 2003, 11, 5311.
17. Dai, W.-M.; Lai, K. W.; Wu, A.; Hamaguchi, W.; Lee,
Y. H. M.; Zhou, L.; Ishii, A.; Nishimoto, S. J. Med. Chem.
2002, 45, 758.
It is also seen from Figure 2 that carboxylic acid
derivative 1c showed higher hypoxic-cell radiosensitiz-
ing activity than acetate 1a, although the GSH-depletion
reactivity of 1c as measured by the second-order rate
constant k2 for nucleophilic addition (Table 1) was
about half that of 1a. By reference to the partition
coefficient (Po=w) between 1-octanol and water listed in
Table 1, 1a is of much higher lipophilicity (Po=w ¼ 15:0)
than carboxylic acid derivative 1c (Po=w ¼ 2:2). It seems
that 1c may readily permeate the cell membrane because
of its appropriately low lipophilicity, while highly lipo-
philic 1a is subject to trapping by the cell membrane.
Thus, appropriate lipophilicity and GSH-depletion
reactivity are important factors in the molecular design
of a family of propargylic sulfones containing a 2-
nitroimidazole function for hypoxic-cell radiosensitizers
with cellular NPSH-depletion ability.
18. Dai, W.-M.; Li, Q.; Fong, K. C.; Chow, C. W.; Zhou, L.;
Hamaguchi, W.; Nishimoto, S. Bioorg. Med. Chem. Lett.
1999, 9, 2789.
19. Compound 1a: Mp 148–150 °C. 1H NMR (CDCl3,
300 MHz) d 8.43 (d, 2H, J ¼ 9:0 Hz), 8.17 (d, 2H,
J ¼ 9:0 Hz), 7.13 (s, 1H), 7.13 (s, 1H), 5.22–5.19 (1H),
4.88 (dd, 1H, J ¼ 14:8, 3.0 Hz), 4.49 (dd, 1H, J ¼ 14:8,
7.5 Hz), 4.02 (s, 2H), 2.63–2.45 (2H), 1.96 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 169.5, 151.2, 143.3, 130.1,
128.4, 126.6, 124.5, 82.8, 76.6, 70.7, 69.2, 51.1, 48.7, 22.0,
20.6, 14.2; FABMS (NBA) m=z 437 [(M+H)þ]; HRMS
calcd for C17H17O8N4S [(M+H)þ] 437.0767, found
437.0773.
1
20. Compound 1b: H NMR (DMSO-d6 400 MHz) d 7.93 (d,
In summary, we synthesized propargylic sulfones con-
taining a 2-nitroimidazole function to investigate their
GSH-depletion reactivity and hypoxic-cell radiosensi-
tizing activity. These compounds were confirmed to
reduce the intracellular NPSH level and exhibited a very
high value of SERin vitro, thus, they are promising can-
didates as novel radiosensitizers for the treatment of
hypoxic tumor cells.
2H, J ¼ 7:2 Hz), 7.75 (t, 1H, J ¼ 7:2 Hz), 7.65 (t, 2H,
J ¼ 7:2 Hz), 7.49 (s, 1H), 7.15 (s, 1H), 5.19 (m, 1H), 4.67
(dd, 1H, J ¼ 14:0, 3.2 Hz), 4.49 (s, 2H), 4.37 (dd, 1H,
J ¼ 14:0, 8.8 Hz), 2.66–2.55 (2H), 1.86 (s, 3H); 13C NMR
(DMSO-d6 100 MHz) d 169.1, 144.6, 138.0, 134.1, 129.1,
128.2, 128.1, 127.7, 82.0, 71.5, 68.9, 51.1, 47.3, 21.4, 20.3;
FABMS (NBA) m=z 392 [(M+H)þ]; HRMS calcd for
C17H18O6N3S [(M+H)þ] 392.0916, found 392.0923.
21. Compound 1c: Mp 64–66 °C. 1H NMR (DMSO-d6
300 MHz) d 8.45 (d, 2H, J ¼ 9:0 Hz), 8.20 (d, 2H,
J ¼ 9:0 Hz), 7.51 (s, 1H), 7.11 (s, 1H), 5.17 (m, 1H), 4.54
(dd, 1H, J ¼ 12:8, 3.0 Hz), 4.37 (dd, 1H, J ¼ 12:8, 7.5 Hz),
3.42 (s, 2H), 2.61 (m, 6H), 2.40–2.31 (4H); 13C NMR
(DMSO-d6 75 MHz) d 173.0, 171.2, 150.7, 143.2, 130.0,
128.3, 127.8, 124.4, 82.5, 71.0, 69.1, 56.0, 51.0, 47.1, 28.4,
21.4, 18.5; FABMS (NBA) m=z 495 [(M+H)þ]; HRMS
calcd for C19H19O10N4S [(M+H)þ] 495.0822, found
495.0802.
Acknowledgements
We are grateful to Dr. Wei-Min Dai (Department of
Chemistry, The Hong Kong University of Science and
Technology) for his valuable discussions.
22. The reactivity of GSH toward propargylic sulfones were
measured by the Ellmanꢀs method. See: Tietze, F. Anal.
Biochem. 1969, 27, 502. To an aqueous solution of 10 mM
GSH and 20 mM aqueous EDTAÆ2Na was added a
solution of propargylic sulfones in 1:9 v/v mixture of
acetonitrile and phosphate buffer. The resulting mixture
was incubated at 37 °C for the given periods of reaction.
Aliquot (0.1 mL) was withdrawn from the mixture and
was poured immediately into phosphate buffer (0.9 mL)
containing 0.2 mM of the Ellmanꢀs reagent (5,50-dithio-
bis(2-nitrobenzoic acid), DTNB). The concentration of
unreacted GSH was determined by UV absorption spec-
troscopy at the maximum absorption wavelength at
412 nm.
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tion. All propargylic sulfones for evaluation were dis-
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