Chemical implications for antitumor and antiviral prostaglandins: Reaction of Δ7-prostaglandin A1 and prostaglandin A1 methyl esters with thiols

Article abstract of DOI:10.1021/ja9628359

Prostaglandins (PGs) such as Δ¹²-PGJ₂ and Δ⁷-PGA₁ methyl ester that possess a cross-conjugated dienone unit exhibit unique antitumor and antiviral activities independent of intracellular cAMP levels. These compounds are transported reversibly into cultured cells and accumulate in nuclei via covalent interaction, eliciting growth inhibition. PGA₁ methyl ester, a simple cyclopentenone analog, is less potent. The unique cellular behavior of the dienone PGs correlates well with their chemical properties. The PGs react specifically with thiol nucleophiles such as glutathione. Michael addition of thiols to Δ⁷-PGA₁ methyl ester, an alkylidenecyclopentenone derivative, occurs facilely at the endocyclic C(11) position rather than at the C(7) position. This process is reversible, and in solution phase, the adducts are in equilibrium with considerable amounts of free PG and thiols. However, the reaction of this PG with Sepharose-bound thiols, regarded to be plausible mimics of protein thiols, is irreversible, and the resulting adducts are dissociated only by alkali treatment. On the other hand, PGA₁ methyl ester reacts with soluble or polymer-anchored thiols at lower rates than Δ⁷-PGA₁ methyl ester, but the resulting thiol adducts are substantially more stable than those of the dienone PGs. This tendency of the PGA₁ methyl ester causes its equilibrium to shift to the adduct formation. The reversibility of the Michael reaction of PGs with thiols is consistent with their intracellular behavior and biological activities. Since glutathione adducts of PGs have no antiproliferative activities for cancer cells, the intracellular free PGs are presumed to interact with target molecules to cause cell growth inhibition. The involvement of the ATP-dependent glutathione S-conjugate export pump (GS-X pump) in the efflux of PGs is discussed. Thus, the marked difference in potency of the dienone and enone PGs is explained by considering the combined kinetic and thermodynamic properties and the action of the GS-X pump.

Full text of DOI:10.1021/ja9628359

2376
J. Am. Chem. Soc. 1997, 119, 2376-2385
Chemical Implications for Antitumor and Antiviral
Prostaglandins: Reaction of ∆7-Prostaglandin A1 and
Prostaglandin A1 Methyl Esters with Thiols
Masaaki Suzuki,*,† Makoto Mori,‡ Terutake Niwa,† Ryu Hirata,‡ Kyoji Furuta,†
Toshihisa Ishikawa,§ and Ryoji Noyori*,‡
Contribution from the Department of Biomolecular Science, Faculty of Engineering,
Gifu UniVersity, Yanagido 1-1, Gifu 501-11, Japan, Department of Chemistry and Molecular
Chirality Research Unit, Nagoya UniVersity, Chikusa, Nagoya 464-01, Japan, and Section of
Molecular Therapeutics, Department of Experimental Pediatrics, The UniVersity of Texas
M. D. Anderson Cancer Center, Houston, Texas 77030
ReceiVed August 13, 1996X
Abstract: Prostaglandins (PGs) such as ∆12-PGJ2 and ∆7-PGA1 methyl ester that possess a cross-conjugated dienone
unit exhibit unique antitumor and antiviral activities independent of intracellular cAMP levels. These compounds
are transported reversibly into cultured cells and accumulate in nuclei via covalent interaction, eliciting growth
inhibition. PGA1 methyl ester, a simple cyclopentenone analog, is less potent. The unique cellular behavior of the
dienone PGs correlates well with their chemical properties. The PGs react specifically with thiol nucleophiles such
as glutathione. Michael addition of thiols to ∆7-PGA1 methyl ester, an alkylidenecyclopentenone derivative, occurs
facilely at the endocyclic C(11) position rather than at the C(7) position. This process is reversible, and in solution
phase, the adducts are in equilibrium with considerable amounts of free PG and thiols. However, the reaction of this
PG with Sepharose-bound thiols, regarded to be plausible mimics of protein thiols, is irreversible, and the resulting
adducts are dissociated only by alkali treatment. On the other hand, PGA1 methyl ester reacts with soluble or polymer-
anchored thiols at lower rates than ∆7-PGAl methyl ester, but the resulting thiol adducts are substantially more
stable than those of the dienone PGs. This tendency of the PGA1 methyl ester causes its equilibrium to shift to the
adduct formation. The reversibility of the Michael reaction of PGs with thiols is consistent with their intracellular
behavior and biological activities. Since glutathione adducts of PGs have no antiproliferative activities for cancer
cells, the intracellular free PGs are presumed to interact with target molecules to cause cell growth inhibition. The
involvement of the ATP-dependent glutathione S-conjugate export pump (GS-X pump) in the efflux of PGs is discussed.
Thus, the marked difference in potency of the dienone and enone PGs is explained by considering the combined
kinetic and thermodynamic properties and the action of the GS-X pump.
Recently, intense attention has been focused on the antine-
activities slowly after dosing,3 resulting in cell cycle arrest at
the G1 phase,3 and effect inhibition of viral replication at
noncytotoxic doses,4 providing possible new therapeutic strate-
gies.1,2 Some PGs of the A and J series indeed show potent
antitumor effects not only in vitro but also in vivo.5 Particularly,
∆7-PGA1 methyl ester (1), an unnatural dienone PG easily
prepared by the three-component synthesis,6 is now under
preclinical study for the treatment of chemotherapeutically
oplastic and antiviral properties of prostaglandins (PGs) and
related compounds.1,2 Certain cyclopentenone prostaglandins
are known to be involved in the regulation of the cell cycles3
and cellular defenses against viral infection.4 PGs express their
* Author to whom correspondence should be addressed at the follow-
ing: Professor Masaaki Suzuki, Department of Biomolecular Science,
Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-11, Japan,
phone/fax +81-58-293-2635, e-mail suzukims@apchem.gifu-u.ac.jp; Profes-
sor Ryoji Noyori, Department of Chemistry and Molecular Chirality
Research Unit, Nagoya University, Chikusa, Nagoya 464-01, Japan, phone
+81-52-789-2956, fax +81-52-783-4177, e-mail noyori@chem3.chem.nagoya-
u.ac.jp.
(3) (a) Bergman, M. D.; Sander, D.; Meyskens, F. L., Jr. Biochem.
Biophys. Res. Commun. 1982, 104, 1080. (b) Wiley, M. H.; Feingold, K.
R.; Grunfeld, C.; Quesney-Huneeus, V.; Wu, J. M. J. Biol. Chem. 1983,
258, 491. (c) Hughes-Fulford, M.; Wu, J.; Kato, T.; Fukushima, M. AdV.
Prostaglandin, Thromboxane Leukotriene Res. 1985, 15, 401. (d) Narumiya,
S.; Fukushima, M. Biochem. Biophys. Res. Commun. 1985, 127, 739. (e)
Bhuyan, B. K.; Adams, E. G.; Badiner, G. J.; Li, L. H.; Barden, K. Cancer
Res. 1986, 46, 1688. (f) Ishioka, C.; Kanamaru, R.; Sato, T.; Dei, T.;
Konishi, Y.; Asamura, M.; Wakui, A. Cancer Res. 1988, 48, 2813. (g)
Hughes-Fulford, M. J. Cell. Biochem. 1994, 54, 265. (h) Sasaguri, T.;
Masuda, J.; Shimokado, K.; Yokota, T.; Kosaka, C.; Fujishima, M.; Ogata,
J. Exp. Cell Res. 1992, 200, 351. (i) Kim, I.-K.; Lee, J.-H.; Sohn, H.-W.;
Kim, H.-S.; Kim, S.-H. FEBS Lett. 1993, 321, 209. For a review, see: (j)
Narumiya, S.; Fukushima, M. In Eicisanoids and Other BioactiVe Lipids
in Cancer and Radiation Injury; Honn, K. V., Marnett, L. J., Nigam, S.,
Walden, T., Jr., Eds.; Kluwer Academic Publishers: Boston, MA, 1989;
pp 439-448. For recent attention from cell differentiation and development
including osteogenesis and adipogenesis, see: (k) Ohta, T.; Azuma, Y.;
Kanatani, H.; Kiyoki, M.; Koshihara, Y. J. Pharmacol. Exp. Ther. 1995,
275, 450. (l) Odani, N.; Negishi, M.; Takahashi, S.; Ichikawa, A. Biochem.
Biophys. Res. Commun. 1996, 220, 264. (m) Forman, B. M.; Tontonoz, P.;
Chen, J.; Brun, R. P.; Spiegelman, B. M.; Evans, R. M. Cell 1995, 83, 803.
(n) Kliewer, S. A.; Lenhard, J. M.; Willson, T. M.; Patel, I.; Morris, D. C.;
Lehmann, J. M. Cell 1995, 83, 813.
† Gifu University.
‡ Nagoya University.
§ The University of Texas M. D. Anderson Cancer Center.
X Abstract published in AdVance ACS Abstracts, February 15, 1997.
(1) For reviews, see: (a) Fukushima, M. Eicosanoids 1990, 3, 189. (b)
Fukushima, M. Prostaglandins, Leukotrienes Essent. Fatty Acids 1992, 47,
1. (c) Sasaki, H.; Fukushima, M. Anti-Cancer Drugs 1994, 5, 131. For earlier
important papers which opened this fascinating scientific area, see: (d)
Prasad, K. N. Nature New Biol. 1972, 236, 49. (e) Santoro, M. G.; Philpott,
G. W.; Jaffe, B. Nature 1976, 263, 777. (f) Honn, K. V.; Cicone, B.; Skoff,
A. Science 1981, 212, 1270. (g) Fukushima, M.; Kato, T.; Ueda, R.; Ota,
K.; Narumiya, S.; Hayaishi, O. Biochem. Biophys. Res. Commun. 1982,
105, 956. (h) Fukushima, M.; Kato, T.; Ota, K.; Arai, Y.; Narumiya, S.;
Hayaishi, O. Biochem. Biophys. Res. Commun. 1982, 109, 626. (i)
Fukushima, M.; Kato, T. In AdVances in Prostaglandin, Thromboxane, and
Leukotriene Research; Hayaishi, O., Yamamoto, S., Eds.; Raven Press: New
York, 1985; pp 415-418.
(2) Prostaglandins in Cancer Research; Garaci, E., Paoletti, R., Santoro,
M. G., Eds.; Springer-Verlag: Berlin, 1987; monograph.
S0002-7863(96)02835-1 CCC: $14.00 © 1997 American Chemical Society

Antitumor and AntiViral Prostaglandins
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2377
resistant ovarian cancer by intraperitoneal administration.7 Since
the activities do not affect intracellular cAMP levels,3 these PGs
differ from the PGs known as local hormones.1,2 This paper
discloses the correlation of the fascinating cellular behavior and
chemical properties of antitumor PGs.8
Background
Certain cyclopentenone PGs act on intracellular targets to
inhibit the growth of cancer cells. An extensive investigation
of the structure-activity relationship5 indicated that (1) the
cross-conjugated dienone system is essential for potent antitumor
activity (simple enone PGs such as 2-cyclopentenone or
2-alkylidenecyclopentanone derivatives are less effective); (2)
the length of the ω-side chain is also important, the analogs
possessing short side chains are less effective; and (3) the
absolute configuration of C(12) and C(15) as well as the
presence or absence of the hydroxyl group at C(15) do not
influenece the activity. Among such unconventional PGs,9 1
and ∆12-PGJ2 (2),7c,10 the ultimate active metabolite of PGJ2,
are ideal examples. These two PGs display common biological
Figure 1. Cellular behavior of ∆12-PGJ2.
effects. IC50 values of 1 and 2 toward L1210 cells are 0.3 and
0.7 µg/mL,5a respectively, while ID50 in inhibiting primary
transcription of HSV-2 are 0.35 and 1.8 µg/mL, respectively.4c,e
These PGs cause inhibition of c-myc gene expression together
with the induction of heat-shock proteins and hemoxygenases11
prior to cell cycle arrest in the G1 phase.
(4) (a) Santoro, M. G.; Banedetto, A.; Garruba, G.; Garaci, E. Science
1980, 209, 1039. (b) Santoro, M. G.; Fukushima, M.; Benedetto, A.; Amici,
C. J. Gen. Virol. 1987, 68, 1153. (c) Yamamoto, N.; Fukushima, M.;
Tsurumi, T.; Maeno, K.; Nishiyama, Y. Biochem. Biophys. Res. Commun.
1987, 146, 1425. (d) Yamamoto, N.; Rahman, M. M.; Fukushima, M.;
Maeno, K.; Nishiyama, Y. Biochem. Biophys. Res. Commun. 1989, 158,
189. (e) Fukushima, M.; Kato, T.; Narumiya, S.; Mizushima, Y.; Sasaki,
H.; Terashima, Y.; Nishiyama, Y.; Santro, M. G. In AdVances in Prostag-
landin, Thromboxane, and Leukotriene Research; Samuelsson, B., Wong,
P. Y. K., Sun, F. F., Eds.; Raven Press: New York, 1989; pp 415-418.
For a review, see: (f) Parker, J.; Ahrens, P. B.; Ankel, H. AntiViral Res.
1995, 26, 83.
(5) (a) Kato, T.; Fukushima, M.; Kurozumi, S.; Noyori, R. Cancer Res.
1986, 46, 3538. (b) Fukushima, M. In Role of Lipids in Cancer Research.
III. The Pharmacological Effect of Lipids; Kabera, J. J., Ed.; American Oil
Chemist Society: Chicago, IL, 1989; pp 215-221. (c) Sasaki, H.; Mah, J.;
Terashima, Y.; Fukushima, M. Proc. Am. Assoc. Cancer Res. 1989, 30,
582. (d) Sasaki, H.; Takada, K.; Terashima, Y.; Ekimoto, H.; Takahashi,
K.; Tsuruo, T.; Fukushima, M. Gynecol. Oncol. 1991, 41, 36. See also:
refs 4b, c, and e.
(6) (a) Suzuki, M.; Kawagishi, T.; Yanagisawa, A.; Suzuki, T.; Okamura,
N.; Noyori, R. Bull. Chem. Soc. Jpn. 1988, 61, 1299. (b) Suzuki, M.; Morita,
Y.; Koyono, H.; Noyori, R. Tetrahedron 1990, 46, 4809. For a review,
see: (c) Noyori, R.; Suzuki, M. Chemtracts: Org. Chem. 1990, 3, 173.
(7) (a) Fukushima, M. In Eicosanoids and Other BioactiVe Lipids in
Cancer, Inflammation and Radiation Injury; Nigam, S., Marnett, L. J., Honn,
K. V., Walden, T. L., Jr., Eds.; Kluwer Academic Publishers: Boston, MA,
1993; pp 737-739. (b) Kikuchi, Y.; Kita, T.; Hirata, J.; Fukushima, M.
Cancer Metastasis ReV. 1994, 13, 309. (c) Fukushima, M.; Sasaki, H.;
Fukushima, S. Ann. N.Y. Acad. Sci. 1994, 744, 161.
It is also known that these PGs are actively and selectively
transported into cultured cells and accumulate in their nuclei.12
Detailed pharmacokinetic studies on the behavior of the A or J
type PGs using L1210 murine leukemia cells revealed the
presence of a unique cellular traffic mechanism comprising
cellular uptake and nuclear accumulation.12 The feature is
schematically illustrated in Figure 1, where several character-
istics are seen: (1) Carrier-mediated incorporation of PG 2 into
the cells13 is very rapid but this process is reversible above 20
°C, establishing an influx-efflux system. (2) There is a
considerable medium/cell concentration gradient, where cellular
uptake is favored by a factor of 20. (3) The intracellular PG is
incorporated into nuclei at 37 °C without metabolism. (4) The
PG eventually binds to nuclear proteins. This binding is
covalent in nature and irreversible under physiological condi-
tions; the PG does not dissociate with hypotonic washing or by
treatment with Triton-X. However, treatment with alkali such
as a 0.05 M NaOH solution can break this interaction to liberate
the free PGs. The structurally related compounds 1 and 2 show
the same characteristics.
(8) For a preliminary report, see: (a) Noyori, R. Suzuki, M. Science
1993, 259, 44. (b) Noyori, R.; Koyano, H.; Mori, M.; Hirata, R.; Shiga, Y.;
Kokura, T.; Suzuki, M. Pure Appl. Chem. 1994, 66, 1999.
(9) For naturally-occurring clavulones (claviridenones) or punaglandins
as ∆7-PGA type compounds, see: (a) Kikuchi, H.; Tukitani, Y.; Iguchi,
K.; Yamada, Y. Tetrahedron Lett. 1982, 23, 517. (b) Kobayashi, H.;
Yasuzawa, T.; Yoshikawa, M.; Akatsu, H.; Kyogoku, Y.; Kitagawa, I.
Tetrahedron Lett. 1982, 23, 5331. (c) Baker, B. J.; Okuda, R. K.; Yu, P.
T.; Scheuer, P. J. J. Am. Chem. Soc. 1985, 107, 2976. See also: (d) Suzuki,
M.; Morita, Y.; Yanagisawa, A.; Baker, B. J.; Scheuer, P. J.; Noyori, R. J.
Org. Chem. 1988, 53, 286. For antiproliferative activity, see: ref 1i.
(10) (a) Fitzpatrick, F. A.; Wynalda, M. A. J. Biol. Chem. 1983, 258,
11713. (b) Kikawa, Y.; Narumiya, S.; Fukushima, M.; Wakatsuka, H.;
Hayaishi, O. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1317. (c) Hirata, Y.;
Hayashi, H.; Ito, S.; Kikawa, Y.; Ishibashi, M.; Suda, M.; Miyazaki, H.;
Fukushima, M.; Narumiya, S.; Hayaishi, O. J. Biol. Chem. 1988, 263, 16619.
See also: ref 3b.
No cellular uptake was seen with cyclopentanone PGs, such
as PGD2 or PGE2. PGA2 (3), a simple enone analog, is
transported into the cell and its nuclei in a similar fashion but
is more weakly bound to the nuclei by a factor of >3. Recently,
(11) (a) Ohno, K.; Fukushima, M.; Fujiwara, M.; Narumiya, S. J. Biol.
Chem. 1988, 263, 19764. (b) Santro, M. G.; Garaci, E.; Amici, C. Proc.
Natl. Acad. Sci. U.S.A. 1989, 86, 8407. (c) Koizumi, T.; Yamauchi, R.;
Irie, A.; Negishi, M.; Ichikawa, A. Biochem. Pharmacol. 1991, 42, 777.
(d) Koizumi, T.; Negishi, M.; Ichikawa, A. Prostaglandins 1992, 43, 121.
(e) Amici, C.; Sistonen, L.; Santro, M. G.; Morimoto, R. I. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 6227. (f) Amici, C.; Palamara, A. T.; Santro,
M. G. Exp. Cell Res. 1993, 207, 230. (g) Amici, C.; Giorgi, C.; Rossi, A.;
Santro, M. G. J. Virol. 1994, 68, 6890. (h) Santro, M. G. Experientia 1994,
50, 1039. (i) Rossi, A.; Santro, M. G. Biochem. J. 1995, 308, 455. (j)
Koisumi, T.; Odani, N.; Okuyama, T.; Ichikawa, A.; Negishi, M. J. Biol.
Chem. 1995, 270, 21779.
(12) (a) Narumiya, S.; Fukushima, M. J. Pharmacol. Exp. Ther. 1986,
239, 500. (b) Narumiya, S.; Ohno, K.; Fujiwara, M.; Fukushima, M. J.
Pharmacol. Exp. Ther. 1986, 239, 506. (c) Narumiya, S.; Ohno, K.;
Fukushima, M.; Fujiwara, M. J. Pharmacol. Exp. Ther. 1987, 242, 306.
(d) Narumiya, S.; Fukushima, M. AdV. Prostaglandin, Thromboxane,
Leukotriene Res. 1987, 17, 972. See also: (e) Narumiya, S.; Ohno, K.;
Fukushima, M.; Fujiwara, M. In Prostaglandins in Cancer Research; Garaci,
E., Paoletti, R., Santoro, M. G., Eds.; Springer-Verlag: Berlin, 1987; pp
86-96.
(13) In cultured cells, the possibility of fatty acid binding protein (FABP)
as an intracellular carrier of PGA2 and PGJ2 has been proposed: Khan, S.
H.; Sorof, S. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9401.

2378 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Suzuki et al.
it was claimed that the efflux phenomenon may be mediated
by the ATP-dependent glutathione S-conjugate export pump
(GS-X pump), probably associated with the multidrug resistance
of cancer cells.14 In any event, such cellular uptake and
extrusion mechanisms are closely correlated to growth inhibition
caused by the PGs.
Results
The dienone PGs 1 and 2 act directly in cell nuclei to express
their growth inhibitory effects. We have been interested in the
chemical implications of such biological properties. Our major
concerns were the cellular behavior, the nature of the target
molecules, and the origin of the difference in potency between
the dienone and enone compounds. Thus, ∆7-PGA1 methyl ester
(1) and PGA1 methyl ester (4), both readily accessible by the
three-component PG synthesis,6 were selected as representative
substrates, and their chemical reactions were studied.
1. Reaction with Thiols in Solution. The susceptibility of
R,â-unsaturated ketones15 to nucleophilic addition prompted us
to screen nucleophilic compounds. Thiols are particularly strong
nucleophiles and certain unsaturated A- and J-type PGs are,
indeed, known to react enzymatically or nonenzymatically with
thiols such as gluthathione and cysteine to give the thiol
conjugates.16,17 Therefore, we first examined chemical reactions
of the dienone 1 with various thiols under conditions that mimic
the physiological system. NMR monitoring revealed that 1
reacts readily with methanethiol, butanethiol, mercaptoethanol,
methyl mercaptoacetate, cysteine, and glutathione, giving the
conjugate addition products of type 5 (1/thiol ) 1:0.5-1.0, 2:1
CD3OD-deuterio phosphate buffer, pH 7.4, 20 °C, 5 h). 1H
NMR signals of the vinylic protons of C(10) and C(11) observed
at δ 6.34 and 7.36, respectively, diminished as the reaction
Figure 2. LUMO coefficients and the charge distribution of 4-methyl-
5-ethylidene-2-cyclopentenone.
proceeded. The signal intensity of the vinylic proton at C(7)
was also reduced, but the compensatory new vinylic signal
appeared adjacently at δ 6.75 which was assigned to the C(7)
proton of the adduct 5. These results confirmed that the addition
reaction occurs selectively at the C(11) position. NOE mea-
surements in the methanethiol adduct 5a, which gave a 5%
increase of the C(12) and C(13) proton signals (δ 3.53 and 5.68)
upon irradiation at SCH3 (δ 2.13) and the C(11) proton (δ 3.17),
respectively, indicates that the adduct has the 11,12-trans
stereochemistry. Other thiol adducts also gave a single set of
signals, suggesting stereochemical homogeneity, probably the
11,12-trans relationship. The reaction with glutathione, a
common endogenous thiol in cells, also occurred at C(11) and
afforded a single monoadduct under the given conditions. In
the presence of excess thiol, such as methanethiol or glutathione,
the monoadduct 5 reacted slowly with thiols at its C(7) position
to form the 7,11-bis-thiol adduct of type 6.16a
(14) Involvement of the GS-X pump has been suggested in the efflux of
alkylating agents: (a) Ishikawa, T. Trend Biochem. Sci. 1992, 17, 463. (b)
Ishikawa, T. In Structure and Function of Glutathione S-Transferases; Tew,
K. D., Pickett, C. B., Mantle, T. J., Mannervik, B., Hayes, J., Eds.; CRC
Press: Boca Raton, FL, 1993; pp 211-221. (c) Ishikawa, T.; Akimaru, K.
In Glutathione S-Transferases: Structure, Function and Clinical Implica-
tions; Vermeulen, N. P. E., Mulder, G. J., Nieuwenhuyse, H., Peters, W.
H. M., van Bladeren, P. J., Eds.; Tayler and Francis: London, 1996; pp
199-211.
(15) Warning, A. J. ComprehensiVe Organic Chemistry; Stoddart, A. J.,
Ed.; Pergamon: Oxford, 1979; Vol. 1, Part 5.2, pp 1017-1104.
(16) For ∆12-PGJ2 (2), see: (a) Atsmon, J.; Sweetman, B. J.; Baertschi,
S. W.; Harris, T. M.; Roberts, L. J., II Biochemistry 1990, 29, 3760. (b)
Atsmon, J.; Freeman, M. L.; Meredith, M. J.; Sweetman, B. J.; Roberts, L.
J., II Cancer Res. 1990, 50, 1879. For PGA1, see: (c) Cagen, L. M.; Pisano,
J. J.; Ketley, J. N.; Habig, W. H.; Jakoby, W. B. Biochim. Biophys. Acta
1975, 398, 205. (d) Smith, J. B.; Silver, M. J.; Ingerman, C. M.; Kocsis, J.
J. Prostaglandins 1975, 9, 135. (e) Ham, E. A.; Oien, H. G.; Ulm, E. H.;
Kuehl, F. A., Jr. Prostaglandins 1975, 10, 217. (f) Cagen, L. M.; Fales, H.
M.; Pisano, J. J. J. Biol. Chem. 1976, 251, 6550. (g) Cagen, L. M.; Pisano,
J. J. Biochim. Biophys. Acta 1979, 573, 547. (h) Heasley, L. E.; Watson,
M. J.; Brunton, L. L. J. Biol. Chem. 1985, 260, 11520. (i) Couin, B.;
Normand, B. V.-L.; Couyette, A.; Heidet, V.; Nagel, N.; Dray, F. Biochem.
Biophys. Res. Commun. 1986, 141, 1254. (j) Normand, B. V.-L.; Couyette,
A.; Heidet, V.; Nagel, N.; Dray, F. Prostaglandins 1988, 35, 515. See
also: (k) Grudzinkas, C. V.; Weiss, M. J. Tetrahedron Lett. 1973, 141. For
PGA2 (3), see: (l) Parker, J.; Ankel, H. Biochem. Pharmacol. 1992, 43,
1053.
(17) For the reaction of thiols such as alkanethiols, glutathione, or cysteine
with R,â-unsaturated ketones other than PGs, see: (chemical reactions under
physiological conditions) (a) Esterbauer, H.; Zollner, H.; Scholz, N. Z.
Naturforsch. 1975, 30C, 466. (b) Dimmock, J. R.; Smith, L. M.; Smith, P.
J. Can. J. Chem. 1980, 58, 984. (enzymatic or biomimetic reactions). (c)
Boyland, E.; Chasseaud, L. F. Biochem. J. 1968, 109, 651. (d) Boyland,
E.; Chasseaud, L. F. In AdVances in Enzymology; Nord, F. F., Ed.;
Interscience Publishers: New York, 1969; Vol. 32, p 173. (e) Lillehang, J.
R.; Kleppe, K.; Siegel, C. W.; Kapchan, S. M. Biochem. Biophys. Acta
1973, 327, 92. (f) Nicholas, A. W.; Rosazza, J. P. Bioorg. Chem. 1976, 5,
367. (g) Fujita, E.; Nagao, Y.; Kaneko, K.; Nakazawa, S.; Kuroda, H. Chem.
Pharm. Bull. (Tokyo) 1976, 24, 2118. (h) Kapchan, S. M.; Siegel, C. W.;
Matz, M. J.; Gilmore, C. J.; Bryan, R. F. J. Am. Chem. Soc. 1976, 98,
2295. (i) Hall, I. H.; Lee, K.-H.; Mar, E. C.; Starnes, C. O. J. Med. Chem.
1977, 20, 333. For the reaction of oridomin, enmein, and tenulin, see: (j)
Fujita, E.; Nagao, Y. Bioorg. Chem. 1977, 6, 287.
The MO characteristics of (E)-4-methyl-5-ethylidene-2-cy-
clopentenone, a model for dienone PG 1, are consistent with
the regioselectivity of the thiol addition (Figure 2). The LUMO
has a higher coefficient at the 3-position compared to the
exocyclic â-carbon, while a positive charge develops more at
the endocyclic â-position. The difference in the 13C NMR
chemical shift of C(11) and C(7), δ 160.4 vs 137.0, may reflect
such electronic characteristics.18 This argument is also ap-
plicable to the reaction of 2 with thiols, which has the same
dienone component as 1 in its upside-down structure.16a
The dienone 1, however, is totally inert to other biomolecules
having a phosphate, hydroxyl, carboxylate, or amino function.
Inactive compounds examined include nucleotides (CMP, AMP,
GMP, UMP, dGMP, and dAMP), carbohydrates (glucose and
2′-deoxyglucose), and amino acids (lysine, histidine, and
glutamine).17g,i
(18) For addition to the exocyclic double bond in a cross-conjugated
dienone system, see: Siwapinyoyes, T.; Thebaranoth, Y. Tetrahedron Lett.
1984, 25, 353.

Antitumor and AntiViral Prostaglandins
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2379
Table 1. Equilibrium Constants for the Reaction of PGs with
The simple cyclopentenone 4 also reacts with thiols to afford
the adducts of type 7 as a mixture of epimers at C(11).16c,f For
example, 4 and methanethiol formed a 9:1 mixture of the trans
and cis isomers. In the NMR spectrum of the trans isomer, a
+9% NOE effect was seen with the C(11) proton upon
irradiation at the C(13) proton. Reaction with mercaptoethanol
affords a 1:1 mixture of the trans and cis adducts. The decrease
or absence of stereoselectivity in the formation of 7 is presum-
ably due to the destabilization of the 11R-isomer caused by the
1,3-interaction between the C(8) and C(11) substituents.
The PG-thiol adducts were fully characterized by spectral
analyses (see Experimental Section). The authentic monothiol
adducts 5 and 7 were prepared by the reaction of equimolar
amounts of thiols with 1 or 4 in alkaline phosphate buffer (pH
7.4).
Thiolsa
equilibrium constant (M-1
)
PG
RSH
CH3SH
n-C4H9SH
HOCH2CH2SH
CH3OOCCH2SH
cysteine
glutathione
CH3SH
addudt
10-3K (Kd ) 1/K, mM)
1
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
1.6 (0.63)
1.4 (0.71)
0.3 (3.3)
0.5 (2.0)
0.24 (4.2)
0.35 (2.9)
10.2 (0.10)
10.8 (0.09)
9.3 (0.11)
10.0 (0.11)
9.3 (0.11)
6.1 (0.16)
4
n-C4H9SH
HOCH2CH2SH
CH3OOCCH2SH
cysteine
glutathione
a The reactions were performed in a 2:1 mixture of CD3OD and
deuterio phosphate buffer (0.1 M, pH 7.4) at 25 °C. The equilibrium
constants are the mean values of 3-5 experiments conducted with
different initial concentrations of the substrates.
2. Kinetics and Thermodynamics. Significantly, the
Michael additions depicted in eqs 1 and 2 are reversible under
homogeneous conditions such as these, establishing an equi-
librium with the free PG and thiol.17a
Figure 3. Competitive reaction of 1 and 4 with mercaptoethanol. An
equimolar mixture of PGs and mercaptoethanol was stirred at 25 °C in
a 2:1 mixture of CD3OD and deuterio phosphate buffer (0.1 M, pH
7.4). The change of the product distribution (2, 1; b, 4; 4, 5c; O, 7c)
with the passage of time was monitored by NMR.
(a) Rates. The reaction was conducted at 25 °C in a 2:1
CH3OH/0.1 M phosphate buffer (pH 7.4) under argon atmo-
sphere.19 The reaction rates of 1 and 4 were found by
monitoring the decrease of the UV absorptions at 270 and 220-
230 nm, respectively. The second-order rate constant, k1, could
be determined when the substrate consumption was as low as
10%.
Notably, addition of thiols to the dienone 1 takes place 6-8
times faster than to the simple enone 4. With mercaptoethanol,
for example, the k1 values for 1 and 4 were determined to be
5.3 and 0.92 M-1 s-1, respectively. Similarly, reaction with
methanethiol gave the rate constant of 4 vs 0.5 M-1 s-1 for 1
and 4, respectively. The higher reactivity of 1 correlates well
with its lower reduction potential of Ep ) -1.64 eV, vs Ep )
-1.96 eV for 4, obtained by cyclic voltammetry.
7 were determined by comparing signal intensities of the C(10)
or C(11) vinylic protons of 4 and total vinylic protons at C(13)
and C(14) of 4 and 7. The formation of the bis-thiol adduct
was negligible. The equilibrium constants, K ) [adduct]/
[PG][RSH], thus obtained are summarized in Table 1, with the
dissociation constants, Kd ) 1/K, given in parentheses.
Interestingly, the dienone-thiol adducts 5 are substantially
more labile than the enone-thiol adducts 7. The Kd values of
the mercaptoethanol adducts 5c and 7c, for example, are 3.3
and 0.11 mM, respectively. The same tendency was seen for
the glutathione-PG adducts 5f and 7f; Kd ) 2.9 vs 0.16 mM.
For a given thiol, the K value for the dienone 1 appears to always
be smaller than that for the enone 4. The formation of the thiol
adducts of 1 and 4 is 3.5-4.4 and 5.2-5.5 kcal mol-1
exothermic, respectively.
Figure 3 illustrates the profile of a competitive experiment.
When a 1:1:1 mixture of 1, 4, and mercaptoethanol in CD3OD
and 0.1 M deuterio phosphate buffer (pH 7.4) was allowed to
stand at 25 °C, the product distribution varied as the reaction
proceeded. At a very early stage of the reaction, the dienone-
thiol adduct 5c was prevalent in the mixture, while after a
prolonged reaction the enone adduct 7c became dominant. Thus,
the dienone 1 reacts with thiols faster than the enone 4, but
under thermodynamic conditions, the former exists in its free
state to a greater extent than does the latter.
The addition of mercaptoethanol to the dienone 1 in a
temperature range of 15-30 °C gave ∆Hq ) 0.13 kcal mol-1
and ∆Sq ) -24 eu. In a similar manner, the reaction of the
enone 4 proceeded with ∆Hq ) 0.09 kcal mol-1 and ∆Sq )
-35 eu. Thus, the activation energies ∆Gq at 25 °C for 1 and
4 were calculated to be 7.39 and 10.8 kcal mol-1, respectively.
(b) Equilibria. The equilibrium constants of the reactions
1
of eqs 1 and 2 were determined by H NMR measurements of
a mixture of a free PG, thiol, and a PG-thiol adduct in a
methanol phosphate buffer solution (pH 7.4) in a sealed tube
under argon atmosphere. Equilibration was determined by the
absence of change of the signal intensities of the PG and its
thiol adduct caused by saturation. The concentrations of 1 and
the adduct 5 were determined by the comparison of the
intensities of their C(7) vinylic protons. The amounts of 4 and
The PG-thiol adducts could generally be isolated by flash
chromatography or preparative HPLC on a silica gel column.
The PG-glutathione adduct 5f was isolated by combination of
the Sep-Pak and reversed-phase TLC under carefully controlled
conditions.16 The stability of the PG-glutathione conjugate was
(19) Reactions were performed under pseudo-second-order conditions
with a large excess of phosphate buffer.

2380 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Suzuki et al.
Figure 4. Reaction of PGs with polymer-supported thiols in phosphate
buffer (pH 7.4) at 25 °C. (A) Reaction with the reduced form of
Thiopropyl-Sepharose 6B (8) (initial concentrations of the PG and thiol
function are 0.89 and 1.78 mM, respectively): O, ∆7-PGA1 methyl
ester (1); 0, PGA1 methyl ester (4). (B) Reaction with the reduced
form of activated Thiol-Sepharose 4B (9) (initial concentration of the
PG and thiol function are 0.26 and 0.33 mM, respectively): O, ∆7-
PGA1 methyl ester (1); 0, PGA1 methyl ester (4).
studied from pH 6.0 to 7.4 by dissolving the conjugate (3-6
mM) into a 1:1 mixture of 0.1 M deuterio phosphate buffer/
CD3OD. Consequently, the glutathione adduct 5f readily
liberated a free PG and glutathione at pH 7.4 as described above,
and equilibrium was established after approximately 15 min.
Although the dissociation rate at pH 7.0 decreased slightly
compared to the behavior at pH 7.4, the equilibrium was
established after 60 min, giving a 59:41 mixture of the adduct
adduct. The presence of amide moieties in 9 disturbs structural
elucidation of 10b by the FT-IR technique, but it can be
presumed that the latter could also have a similar structure.
The rate of the 1,4-addition was examined by measuring the
concentration of unreacted PG that remained in aqueous media.
The results, given in Figure 4, indicate that, as seen in solution
phase reactions, the dienone 1 reacted with the solid thiols
approximately 3 times faster than the enone 4. The rate
constants, k, of the reaction between 8 and 1 or 4 are 7.7 and
2.7 M-1 s-1, respectively. In a similar manner, the reaction
with 9 gave the k value of 3.7 and 1.2 M-1 s-1 for 1 and 4,
respectively.
The stability of the resin-anchored products is shown in Figure
5. According to the K values in Table 1, it is expected that
free 1 would exist in 92 and 97% in the reactions with
mercaptoethanol and glutathione, respectively, under the given
concentrations. The same calculation for 4 resulted in 35 and
68%. However, both the dienone and enone adducts 10 and
11 are stable at pH 7.4 (0.1 M phosphate buffer, 20 °C) and do
not liberate free PGs, as judged by UV analysis. They dissociate
into free thiols and PGs only under basic conditions at pH >
ca. 9.5.12 Such behavior is in sharp contrast to the above
described properties of the small thiol adducts 5 and 7, and this
is probably due to the decreased molecular motion in the solid
state.
1
and free PG as judged by H NMR measurement. At pH 6.0,
the adduct 5f was fairly stable and only 7% of the PG was
dissociated after 20 h.
3. Reaction with Solid Thiols. Polymer-anchored thiols
may mimic biological thiols (proteins) in nuclei.20 Reduced
forms of thiopropyl-Sepharose 6B (8) and activated thiol-
Sepharose 4B (9) were selected as representative models for
macromolecular thiols. The gels 8 and 9 contain 20 and 1 µmol
of SH group/mL, respectively.21 When a mixture of the
cyclopentenone PGs and 8 or 9 was stirred in a methanol
phosphate buffer mixture at 25 °C, the PGs were smoothly
incorporated into the gel. Thus, the reaction of 1 with the solid
thiols gave the corresponding adducts of type 10 and PG 4 gave
11.
The alkylidenecyclopentanone structures of the adducts 10
were verified by FT-IR spectroscopy using a diffuse reflectance
measurement technique.22 Thus, 10a displayed the carbonyl
stretching frequency due to the 9-keto function at 1730 cm-1
,
which is close to the value of 5a (1725 cm-1). In comparison,
the starting dienone 1 and its bis-thiol adducts 6a exhibited
ketonic carbonyl absorption at 1702 and 1740 cm-1, respec-
tively. This analysis led us to conclude that the dienone binds
covalently to the solid thiol as a form of the mono-Michael
(20) For involvement of sulfhydryl components in the binding with the
target protein in the nuclei, see: Ohno, K.; Hirata, M. Biochem. Pharmacol.
1993, 46, 661.
Discussion
The cellular behavior displayed by anticancer PGs can be
projected from the above chemical phenomena. Table 2 shows
the correlation of the chemical reactions of the PGs 1 and 4 in
(21) Affinity Chromatography-Principles and Methods; Pharmacia Bio-
tech.
(22) Spragg, R. A. Appl. Spectrosc. 1984, 38, 604.

Antitumor and AntiViral Prostaglandins
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2381
reaction is barely reversible compared with the solution phase,27
due to the decrease in the molecular motion leading to the
accumulation of PG into the nuclei. The accumulated PG can
be dissociated only by alkali treatment. This situation was
mimicked by the chemical models forming 10. The PG is
obviously not metabolized during these processes.
The PG-glutathione adducts have no antiproliferative activi-
ties for cancer cells and cannot be transported into nuclei. Our
recent study has suggested that the glutathione adducts of 1
might be effluxed from the cells through the membrane protein
MRP/GS-X pump.28 This view is consistent with the fact that
5f and 6f strongly inhibit the ATP-dependent leukotriene C4
(LTC4) transport through this pump in a competitive manner
with IC50 values of 0.70 and 0.56 µM, respectively; the
experiments were conducted by incubating plasma membrane
vesicles with the glutathione adducts and 10 nM LTC4 in the
presence of 1 mM ATP at 37 °C.28 Thus, the PG-glutathione
conjugate is considered to be a good substrate for the MRP/
GS-X pump and may be eliminated from the cell,29 resulting in
the reduction of intracellular accumulation of the anticancer PG.
Actually, the action of the MRP/GS-X pump was clearly
demonstrated as a drug resistance phenomenon of human
leukemia-60/R-CP (cisplatin-resistant) cells30 after being incu-
bated with 1.28 The decreasing antiproliferative activity of PGs
with increasing cellular glutathione concentration is also
consistent with this molecular mechanism.16b,16l,25a,31
On the other hand, the simple enone PGs such as 3 or 4
incubated to the cells also react with thiols, but the equilibrium
favors the adduct formation. Thus, in cytoplasm, the glutathione
adducts of such PGs are expected to be dominant and only 6%
of 4 could be free, for example, in L1210 cells. The glutathione
conjugate of PGs could be effluxed by the MRP/GS-X pump,28
and the minor unbound PG could react irreversibly with thiols
in nuclei but at a lower rate than 1. This interpretation is
consistent with the cellular behavior of PGA2 using L1210
murine leukemia cells:20 (1) PGA2 was incorporated into cells
over 20 °C and existed as a mixture of the glutathione conjugate
form (predominant), the protein-bound form32 (second major
component), and the free form (only small portions) in cytosol;
(2) PGA2 was transported into nuclei at 37 °C, but the PG-
glutathione conjugate and the free PG were also detected in
the medium during such incubation and the PG-glutathione
conjugate increased time-dependently in the medium; (3) when
the glutathione level was lowered by treating cells with
Figure 5. Dissociation of PGs from polymer-supported thiol-PG
adducts, 10a (spotted bar), 10b (white bar), 11a (solid bar), and 11b
(gray bar), under alkaline conditions. The results of the reaction of
10a at pH 8.4 were drawn in the column of pH 8.5 for convenience.
a mixture of methanol phosphate buffer at pH 7.4 and the
cellular behavior of these and related PGs.
Chemical investigation suggests that the cross-conjugated
dienone PGs ∆7-PGA1 (1) and ∆12-PGJ2 (2) in cells easily
undergo Michael reaction with small, soluble thiols such as
glutathione or cysteine in a reversible manner to give an
equilibrium mixture.17a,23,24 Glutathione is the major nonprotein
cellular thiol with concentrations of 1-10 mM14b and plays
important roles in a variety of physiological functions such as
oxidative stress, detoxification, inflammation, and drug resis-
tance.14 Glutathione reacts with a wide range of electrophiles
including alkylating anticancer drugs to form glutathione
conjugates.14 In relation to this, there is accumulating evidence
that the active export of anticancer drugs as glutathione
conjugates from cells via an ATP-dependent export pump (MRP/
GS-X pump) is one of the major mechanisms of drug resis-
tance.14 The fact that the enone PGs can react with glutathione
to form PG-glutathione conjugates is important for probing
the actions of anticancer PGs in cells. The intracellular state
of PGs in L1210 cells were estimated using the equilibrium
constants determined by the chemical reactions. When 0.01
mM of dienone PG 1 is incubated at 37 °C to L1210 cells, its
intracellular concentration may approach a maximum of 0.2
mM.12a Since the cytoplasm of L1210 cells contains about 2.8
mM of glutathione,12a,25 it is calculated that 52% of 1 exists in
a free state and the remaining 48% as the Michael adduct 5f.26
The intracellular free PG is thought to bind to target proteins
in nuclei (or cytosol) to reveal biological actions leading to the
growth inhibition of cancer cells accompanying the suppression
of c-myc gene expression, the induction of heat-shock proteins
and hemoxygenases,11 and the cell cycle arrest in the G1 phase.
The free PGs shift to nuclei directly or by the aid of transport
proteins in the cytosol20 and interact with specific nuclear
proteins there. The newly formed high molecular weight thiol
adduct of the PG 1, in which the 11-position is bound covalently
to the biomatrix, in turn remains stable. This solid state Michael
(27) This type of stabilization is reminiscent of the “message-address”
concept which has been used for designing ligand molecules: (a) Schwyzer,
R. Ann. N.Y. Acad. Sci. 1977, 297, 3. (b) Chavkin, C.; Goldstein, A. Proc.
Natl. Acad. Sci. U.S.A. 1981, 78, 6543. (c) Sayre, L. M.; Larson, D. L.;
Takemori, A. E.; Portoghese, P. S. J. Med Chem. 1984, 27, 1325. (d)
Takayanagi, I.; Konno, F.; Goromaru, N.; Koike, K.; Kanematsu, K.; Fujii,
I.; Togame, H. Arch. Int. Pharmacodyn. 1988, 294, 71. For a review, see:
(e) Kanematsu, K. Kagaku 1990, 45, 385, (in Japanese, Kagakudojin,
Kyoto).
(28) (a) Akimaru, K.; Kuo, M. T.; Furuta, K.; Suzuki, M.; Noyori, R.;
Ishikawa, T. Cytotechnology 1996, 19, 221. (b) Akimaru, K.; Nakanishi,
M.; Suzuki, M.; Furuta, K.; Noyori, R.; Ishikawa, T. In Eicosanoids and
Other BioactiVe Lipids in Cancer, Inflammation, and Radiation Injury;
Honn, K. V., Nigam, S., Jones, R., Marnett, L. J., Wong, P. Y-K., Eds.;
Plenum Press: New York, 1996.
(29) ATP-dependent transport of the PG-glutathione conjugate by the
MRP/GS-X pump across the plasma membrane was verified by using the
inside-out vesicles prepared from cisplatin-resistant HL-60 cells: Ishikawa,
T.; Suzuki, M.; Furuta, K.; Noyori, R. Manuscript in preparation
(30) The MRP/GS-X pump is overexpressed in this cell 4-5 times more
than in usual HL-60 cells: (a) Ishikawa, T.; Wright, C. D.; Ishizuka, H. J.
Biol. Chem. 1994, 269, 29085. (b) Ishikawa, T.; Bao, J.-J.; Yamane, Y.;
Akimaru, K.; Frindrich, K.; Wright, C. D.; Kuo, M. T. J. Biol. Chem. 1996,
271, 14981.
(23) For the reversible reaction of Navelbine, an antitumor vinblastine
analog, with the nucleophilic entity, see: (a) Potier, P. Pure Appl. Chem.
1986, 58, 737. (b) Potier, P. Semin. Oncol. 1989, 16, 2. For the reaction of
leucodaunomycins, the intermediary compounds in the redox chemistry of
an antitumor daunomycin, with cysteine derivatives, see: (c) Bird, D. M.;
Gaudiano, G.; Koch, T. H. J. Am. Chem. Soc. 1991, 113, 308. See also:
(d) Ishikawa, T.; Akimaru, K.; Kuo, M. T.; Priebe, W.; Suzuki, M. J. Natl.
Cancer Inst. 1995, 87, 1639.
(24) The dienone PGs should be substantially different from the common
alkylating anticancer agents, which react with cellular nucleophiles in an
irreversible manner.
(25) (a) Ohno, K.; Hirata, M.; Narumiya, S.; Fukushima, M. Eicosanoids
1992, 5, 81. See also: (b) Kosower, N. S.; Kosower, E. M. Int. ReV. Cytol.
1978, 54, 109. (c) Meister, A.; Anderson, M. E. Annu. ReV. Biochem. 1983,
52, 711.
(31) Koizumi, T.; Negishi, M.; Ichikawa, A. Biochem. Pharmacol. 1992,
44, 1597.
(26) At this concentration of glutathione, the formation of the bis-
glutathione adduct 6f can be excluded on the basis of the kinetic and
thermodynamic data.
(32) The transport of PGA2 into nuclei is supposed to be mediated by
the PG-binding proteins in cytosol. Two proteins of 100-150 and 25-35
kDa were identified so far.20

2382 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Suzuki et al.
Table 2. Correlation of Chemical Reactions and Cellular Behavior of PGs
chemical reactiona
behavior of PG in cells
reversible reaction with thiols including glutathione
Kd(1)/Kd(4) ≈ 10:1
transport into nuclei in the presence of excess glutathione and/or other soluble thiols
effective transport of free ∆12-PGJ2 into nuclei
higher reactivity of 1 to thiols than that of 4
small thiols: k(1)/k(4) ≈ 8:1
polymer thiols: k(1)/k(4) ≈ 3:1
formation of stable adducts with polymer
supported thiols
faster accumulation of ∆12-PGJ2 into nuclei
irreversible accumulation in nuclei through covalent binding with nuclear proteins
a Kd(1), dissociation constant of the type 5 compounds; Kd(4), dissociation constant of the type 7 compound; k(1), rate constant for the reaction
of 1 with thiols; k(4), rate constant for the reaction of 4 with thiols.
Figure 6. Molecular mechanism for cellular behavior of anticancer PGs.
buthionine sulfoximine, the PG-glutathione conjugate in cytosol
markedly decreased, while the free PG increased, and the PG-
glutathione conjugate in medium decreased. These phenomena
can be explained by combining the chemical reaction of 4 with
glutathione and the operation of the function of the GS-X pump
to extrude the PG-glutathione conjugate.
Thus, the combined thermodynamic/kinetic properties derived
from pure chemical reactions and the GS-X pump clearly
illustrate the circumstances under which the simple enone PGs
demonstrate insufficient potency for tumor cell growth inhibition
compared with the cross-conjugated dienone analogs. The mode
of action of PGs for viruses may be interpreted in a similar
manner.4 Overall, a tentative molecular mechanism of anti-
cancer PGs influx-efflux phenomena and subsequent transport
into nuclei are illustrated in Figure 6. Next, the target proteins
of anticancer PGs in the nuclei must be identified, which would
reveal the molecular mechanisms involved in the regulation of
c-myc expression33 and the induction of specific proteins such
as heat-shock proteins and hemoxygenases.11
for the treatment of chemotherapeutically resistant ovarian
cancer as a promising anticancer drug at noncytotoxic doses.7,34
Experimental Section
Chemicals. Unless otherwise noted, the reagents were commercial
grade. Mercaptoethanol, n-butanethiol, and methyl thioglycolate were
freshly distilled under argon atmosphere prior to use. Tetrahydrofuran
(THF) was freshly distilled from sodium-benzophenone ketyl. Thio-
propyl-Sepharose 6B and activated thiol-Sepharose 4B were purchased
from Pharmacia Fine Chemicals and were used after the reduction to
thiol forms according to the reported procedure.21 ∆7-PGA1 methyl
ester (1) and PGA1 methyl ester (4) were synthesized by the three-
component coupling process described previously.6
General. Analytical and preparative thin-layer chromatography
(TLC) were performed on precoated silica gel plates (silica gel 60 F254
,
0.25 mm, Merck 5715) or reversed-phase silica gel plates (RP-18 F254S
,
0.25 mm, Merck 15685). Visualization of the products, developed as
the chromatogram, was performed by UV light, an ethanolic phospho-
molybdic acid solution, or an ethanolic solution of p-anisaldehyde
containing sulfuric acid. Column chromatography was performed on
Merck silica gel 60 (No. 9385, 230-400 mesh), Fuji Devison BW-
820-MH (70-200 mesh), or Fuji Devison BW-300 (200-400 mesh).
Recycle preparative high-performance liquid chromatography (HPLC)
was conducted on a GPC column (JAIGEL-2H, 20 mm × 60 cm) or
a GS column (JAIGEL-GS310, 20 mm × 50 cm) with a JAI LC-908
instrument. Nuclear magnetic resonance (1H and 13C) spectra were
recorded on a JEOL JNM-A400, FX-90Q, GX-270, or GSX-270
spectrometer. Chemical shifts are reported in parts per million (δ) with
tetramethylsilane (in CDCl3) or the deuterium lock signal of the solvent
(CDCl3, CD3OD, D2O) as an internal standard. The signal assignment
of a glutathione adduct was done on the basis of the H,H-COSY
measurement. Mass spectra (MS and HRMS) were measured on a
The cellular behaviors of anticancer PGs can be explored
using the above chemical implications, which will enable the
rational design of effective anticancer drugs. The synthetic
anticancer PG, ∆7-PGA1 methyl ester (1), described here has a
structure that differs considerably from natural PGs; therefore,
the expression of local hormone activity can be avoided which
arises when it binds to cell membrane receptors.33 ∆7-PGA1
methyl ester (1) and its analogs are now under preclinical study
(33) Elucidation of the molecular mechanism of the cancer cell growth
inhibition effect by anticancer PGs, based on the use of 1 as an efficient
biochemical probe for HL-60 human leukemia cells and cisplatin resistant
HL-60/R-CP cells, has recently been reported. See: Reference 28b.
Ishikawa, T.; Akimaru, K.; Nakanishi, M.; Suzuki, M.; Furuta, K.; Noyori,
R. Submitted.
(34) The combined use of cisplatin and lipo-∆7-PGA1 methyl ester reveals
additive antitumor effects on ovarian cancer cells resistant to cisplatin in
vivo.1c

Antitumor and AntiViral Prostaglandins
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2383
JEOL TMS-DX 300 (EI) and a SHIMADZU GCMS-QP1000A (EI)
instruments. Fast atom bombardment mass spectra (FAB MS) were
recorded on a JEOL JMS-DX705L instrument (Xe, 5 keV). Infrared
spectra (IR) were taken on a JASCO IRA-1, IR-810, or DR-81 (FT-
IR) spectrometer. Ultraviolet (UV) spectra were obtained on a Hitachi
228 model spectrometer. Cyclic voltammetry was performed with a
Yanako Polarographic Analyzer P-1100 equipped with the platinum
wire counter electrodes, the C-glassy working electrode, and the SCE
reference electrode in DMF (0.08 M tetrabutylammonium perchlorate
as a supporting electrolyte).
All reactions were performed under a positive pressure of argon with
deoxygenated solvents in the dark. Deoxygenation of organic solvents
and phosphate buffer was conducted by freeze-pump-thaw cycles
using argon as an inert gas.
The MO calculations were performed at Nagoya University Com-
putation Centre with FACOM VP-200 with Monstergauss 8135 (STO-
3G36). Geometry of model compounds was decided on the basis of
the X-ray date of the compound Shikoccin.37
Screening of Biomolecules Reactive to PGs. Typical procedure.
A mixture of ∆7-PGA1 methyl ester (1) (2.0 mg, 5.74 µmol) and
glutathione (1.76 mg, 5.73 µmol) placed in an NMR tube was dissolved
with CD3OD (0.4 mL) and deuterio phosphate buffer (0.1 M, pH 7.4,
0.2 mL) under argon atmosphere. The solution was allowed to stand
at 20 °C for 5 h and then subjected to the NMR measurement. The
formation of adducts was judged by analysis of the NMR spectra (see
text). Screening of other biomolecules was conducted by a similar
procedure using 0.5-1.0 equiv of a biomolecule for 1 or PGA1 methyl
ester (4).
Kinetics and Thermodynamics. The initial rate of the reactions
was measured by monitoring the changes of UV absorbance at 220-
230 nm for PGA1 methyl ester and at 270 nm for ∆7-PGA1 methyl
ester, respectively. The reaction of a PG with a thiol was performed
in a 2:1 mixture of methanol and phosphate buffer (0.1 M, pH 7.4)
under the pseudo-second-order condition ([phosphate buffer] . [PG],
[RSH]) over a period of 10-15 min at 25 °C with various initial
concentrations of 0.1-0.15 mM (PG) and 0.01-0.24 mM (thiol). The
rate constant was determined from the slope of the plot of UV
absorbance vs time.
The enthalpy and entropy of activation, ∆Hq and ∆Sq, respectively,
were calculated by the Eyring equation, ln k1/T ) -∆Hq/R(1/T) +
∆Sq/R + ln kB/h, where R, kB, and h are the gas constant, the Boltzmann
constant, and the Planck constant, respectively.
Decision of the establishment of equilibrium was conducted as
follows. In an NMR tube were placed a mixture of PG (3-20 µmol)
and thiol (1-20 µmol) dissolved in CD3OD (0.4 mL) and deuterio
phosphate buffer (0.1 M, pH 7.4, 0.2 mL) under argon. The course of
the reaction was monitored by the NMR measurement of the solution
at 25 °C (see text).
with diluted hydrochloric acid (0.1 M, 1 mL). The product was
extracted with ether, and the organic layer was dried over magnesium
sulfate. The solvent was removed under reduced pressure, and the
residue was subjected to recycle preparative HPLC using chloroform
as eluant to give the adduct 5b (17.9 mg, 73%): TLC Rf ) 0.35 (2:1
1
hexane/ethyl acetate); H NMR (CDCl3) δ 0.85-0.94 (m, 6H, CH3),
1.2-1.7 (m, 19H, CH2 and OH), 2.14 (q, J ) 7.4 Hz, 2H, C(6)H2),
2.30 (t, J ) 7.4 Hz, 2H, C(2)H2), 2.35 (dd, J ) 2.5, 18.3 Hz, 1H,
C(10)HaHb), 2.54 (t, J ) 7.4 Hz, 2H, SCH2), 2.75 (dd, J ) 6.9, 18.3
Hz, 1H, C(10)HaHb), 3.25 (dt, J ) 6.9, 2.5 Hz, 1H, C(11)H), 3.51 (br
d, J ) 6.4 Hz, 1H, C(12)H), 3.66 (s, 3H, OCH3), 4.09 (m, 1H, C(15)H),
5.51 (dd, J ) 5.9, 15.3 Hz, 1H, vinyl), 5.66 (dd, J ) 6.4, 15.3 Hz, 1H,
vinyl), 6.75 (dt, J ) 2.0, 7.4 Hz, 1H, C(7)H); 13C NMR (CD3OD) δ
14.0, 14.4, 23.0, 23.7, 25.8, 26.3, 29.1, 29.9, 30.0, 31.6, 32.7, 33.0,
34.6, 38.4, 44.2, 44.6, 49.8, 52.0, 73.0, 130.6, 136.1, 139.1, 141.2, 175.8,
205.9; HRMS m/z calcd for C25H42O4S 438.2804, found 438.2794 (M+).
Unless otherwise stated, the following monothiol adducts of ∆7-
PGA1 methyl ester were synthesized by a similar procedure to the
synthesis of 5b.
5a. Reaction of sodium thiomethoxide and 1 gave 5a in 33%
yield: TLC Rf ) 0.44 (1:1 hexane/ethyl acetate); 1H NMR (CDCl3) δ
0.88 (t, J ) 6.4 Hz, 3H, CH3), 1.2-1.7 (m, 15H, CH2 and OH), 2.13
(s, 3H, SCH3), 2.15 (q, J ) 7.6 Hz, 2H, C(6)H2), 2.30 (t, J ) 7.4 Hz,
2H, C(2)H2), 2.38 (dd, J ) 3.0, 18.5 Hz, 1H, C(10)HaHb), 2.75 (dd, J
) 7.3, 18.3 Hz, 1H, C(10)HaHb), 3.17 (dt, J ) 7.3, 3.0 Hz, 1H, C(11)H),
3.53 (br d, J ) 6.3 Hz, 1H, C(12)H), 3.67 (s, 3H, OCH3), 4.15 (m, 1H,
C(15)H), 5.52 (ddd, J ) 1.0, 6.6, 15.5 Hz, 1H, vinyl), 5.68 (ddd, J )
0.8, 6.3, 15.3 Hz, 1H, vinyl), 6.75 (dt, J ) 2.0, 7.6 Hz, 1H, C(7)H);
MS m/z 396 (M+).
5c. Reaction of mercaptoethanol and 1 gave 5c in 56% yield: TLC
Rf ) 0.22 (1:2 hexane/ethyl acetate); IR (CHCl3) 3300-3700, 3000,
1
2930, 2850, 1720, 1645 cm-1; H NMR (CDCl3) δ 0.88 (t, J ) 6.4
Hz, 3H, CH3), 1.2-1.7 (m, 14H, CH2), 2.0-2.3 (m, 2H, OH), 2.17 (q,
J ) 7.4 Hz, 2H, C(6)H2), 2.31 (t, J ) 7.4 Hz, 2H, C(2)H2), 2.36 (dd,
J ) 3.5, 18.3 Hz, 1H, C(10)HaHb), 2.74 (dt, J ) 13.9, 5.9 Hz, 1H,
SCHaHb), 2.78 (dd, J ) 6.9, 18.3 Hz, 1H, C(10)HaHb), 2.83 (dt, J )
13.9, 5.9 Hz, 1H, SCHaHb), 3.27 (dt, J ) 3.5, 6.9 Hz, 1H, C(11)H),
3.52 (m, 1H, C(12)H), 3.67 (s, 3H, OCH3), 3.76 (t, J ) 5.9 Hz, 2H,
OCH2), 4.10 (q, J ) 6.4 Hz, 1H, C(15)H), 5.56 (dd, J ) 5.9, 15.3 Hz,
1H, vinyl), 5.66 (dd, J ) 6.4, 15.3 Hz, 1H, vinyl), 6.75 (dt, J ) 2.0,
7.4 Hz, 1H, C(7)H); 13C NMR (CD3OD) δ 14.4, 23.8, 25.8, 26.3, 19.1,
30.0, 30.1, 33.0, 33.3, 34.6, 38.4, 44.3, 44.7, 49.9, 52.0, 62.5, 73.0,
130.6, 136.1, 139.0, 141.3, 175.8, 205.8; HRMS m/z calcd for
C23H36O4S (M - H2O) 408.2334, found 408.2327 (M+ - H2O).
5d. Reaction of methyl thioglycolate and 1 gave 5d in 47% yield:
1
TLC Rf ) 0.63 (1:1 chloroform/ethyl acetate); H NMR (CDCl3) δ
0.88 (t, J ) 6.6 Hz, 3H, CH3), 1.2-1.7 (m, 14H, CH2), 1.83 (br, 1H,
OH), 2.15 (q, J ) 7.1 Hz, 2H, C(6)H2), 2.30 (t, J ) 7.4 Hz, 2H, C(2)H2),
2.36 (dd, J ) 3.2, 18.5 Hz, 1H, C(10)HaHb), 2.78 (dd, J ) 7.1, 18.5
Hz, 1H, C(10)HaHb), 3.25 (d, J ) 14.7 Hz, 1H, SCHaHb), 3.33 (d, J )
14.7 Hz, 1H, SCHaHb), 3.41 (dt, J ) 3.2, 7.1 Hz, 1H, C(11)H), 3.54
(br, 1H, C(12)H), 3.67 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 4.10 (q, J
) 6.4 Hz, 1H, C(15)H), 5.56 (dd, J ) 6.3, 15.6 Hz, 1H, vinyl), 5.66
(dd, J ) 6.4, 15.6 Hz, 1H, vinyl), 6.76 (dt, J ) 1.7, 7.1 Hz, 1H, C(7)H);
13C NMR (CD3OD) δ 14.0, 22.6, 24.6, 25.1, 28.0, 28.8, 28.9, 31.7,
32.8, 33.8, 37.2, 42.7, 44.0, 48.1, 51.6, 52.6, 72.2, 129.2, 135.3, 136.7,
140.7, 170.4, 174.1, 202.8; MS m/z 454 (M+).
The competitive reaction of mercaptoethanol with 1 and 4 was
performed using equimolar amounts (2.9 µmol) of 1, 4, and mercap-
toethanol dissolved into a mixture of CD3OD (0.5 mL) and deuterio
phosphate buffer (0.1 M, pH 7.4, 0.25 mL) in an NMR tube under
argon. The product distribution with the passage of the time was
1
monitored by the H NMR measurement at 25 °C.
Synthesis of PG-Thiol Adducts. The PG-thiol adducts were
synthesized and fully characterized by spectral analyses. The authentic
thiol adducts 5 and 7 were prepared by the reaction of the corresponding
PGs and thiols in THF or methanol in the presence of phosphate buffer
(pH 7.4). The glutathione adducts 5f and 6f were isolated and purified
by combination of the Sep-Pak and reversed-phase TLC technique under
carefully controlled conditions.16a,b
Preparation of 10,11-Dihydro-11r-butylthio-∆7-prostaglandin A1
Methyl Ester (5b). To a solution of 1 (19.5 mg, 56.0 µmol) in THF
(0.5 mL) and phosphate buffer (0.1 M, pH 7.4, 0.1 mL) was added
butanethiol (11.2 µL, 0.105 mmol) at 15 °C. The solution was stirred
for 30 min at the same temperature, and then the reaction was quenched
Preparation of 10,11-Dihydro-11-(2-hydroxyethylthio)prosta-
glandin A1 Methyl Ester (7c). Mercaptoethanol (24.2 mg, 0.31 mmol)
was added to a solution of 4 (54.2 mg, 0.155 mmol) in THF (1.5 mL)
and phosphate buffer (0.1 M, pH 7.4, 0.1 mL) at 15 °C. The mixture
was stirred for 1 h and then diluted hydrochloric acid (0.1 M, 4 mL)
was introduced to the solution. The product was extracted with ether,
and the organic layer was dried over magnesium sulfate. After removal
of the solvent under reduced pressure, the residue was subjected to
column chromatography on silica gel (hexane/ethyl acetate, 1:1) to
afford two stereoisomers of adduct 7c (1:1, 57.7 mg, 87%). For 7c
(35) Peterson, M. R.; Poirier, R. A. University of Toronto. This program
is based on parts of Pople’s GAUSSIAN 80, QCPE 406, Indiana University,
Bloomington, Ind.
(36) (a) Hehre, W. J.; Stewart, R. F.; Pople, A. J. J. Chem. Phys. 1969,
51, 2657. (b) Hehre, W. J.; Ditchfield, R.; Stewart, R. F.; Pople, J. A. J.
Chem. Phys. 1970, 52, 2769.
1
(11,12-trans): TLC Rf ) 0.21 (1:2 hexane/ethyl acetate); H NMR
(CDCl3) δ 0.88 (t, J ) 6.4 Hz, 3H, CH3), 1.1-1.7 (m, 18H, CH2),
2.0-2.5 (m, 2H, OH), 2.00 (dt, J ) 11.1, 5.6 Hz, 1H, C(8)H), 2.18
(dd, J ) 11.4, 18.3 Hz, 1H, C(10)HaHb), 2.29 (t, J ) 7.4 Hz, 2H,
C(2)H2), 2.39 (ddd, J ) 8.4, 11.1, 11.4 Hz, 1H, C(12)H), 2.79 (dd, J
) 7.9, 18.3 Hz, 1H, C(10)HaHb), 2.89 (t, J ) 7.4 Hz, 2H, SCH2), 2.99
(37) Taga, T.; Osaki, K.; Ito, N.; Fujita, E. Acta Crystallogr. 1982, B38,
2941.

2384 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Suzuki et al.
(dt, J ) 7.9, 11.4 Hz, 1H, C(11)H), 3.66 (s, 3H, OCH3), 3.6-3.8 (m,
2H, OCH2), 4.15 (q, J ) 6.9 Hz, 1H, C(15)H), 5.54 (dd, J ) 8.4, 15.3
Hz, 1H, vinyl), 5.72 (dd, J ) 6.9, 15.3 Hz, 1H, vinyl); 13C NMR
(CD3OD) δ 14.5, 23.8, 26.0, 26.3, 27.7, 28.5, 30.0, 30.6, 33.0, 34.7,
34.8, 38.4, 45.2, 47.4, 52.0, 54.4, 56.4, 62.7, 72.4, 132.3, 137.9, 175.9,
218.2; MS m/z 428 (M+). For 7c (11,12-cis): TLC Rf ) 0.14 (1:2
and the desired compound was eluted with methanol (3 mL). Evapora-
tion of the solvent in vacuo afforded the PG-glutathione adduct as a
colorless oil (10.1 mg, 33%). Analysis of the compound by FAB MS
and NMR confirmed the structure of the monoglutathione adduct of
∆7-PGA1 methyl ester at the C(11) position. For 5f: TLC (reversed-
phase) Rf ) 0.49 (3:1 methanol/phosphate buffer (0.1 M, pH 7.4)); 1H
NMR (CD3OD) δ 0.89 (t, J ) 6.8 Hz, 3H, CH3), 1.23-1.42 (m, 6H,
CH2), 1.4-1.55 (m, 6H, CH2), 1.61 (tt, J ) 7.4, 7.7 Hz, 2H, C(3)CH2),
2.02-2.2 (m, 2H, Glu(CâH2)), 2.19 (dt, J ) 7.6, 7.3 Hz, 2H,
CdCHCH2), 2.31 (t, J ) 7.4 Hz, 2H, CH2COOCH3), 2.34 (m, 1H,
COCHaHb), 2.53 (m, 2H, Glu(CγH2)), 2.82 (dd, J ) 6.5, 19 Hz, 1H,
COCHaHb), 2.86 (dd, J ) 8.9, 14.0 Hz, 1H, Cys(SCHaHb)), 3.07 (dd,
J ) 5.0, 14.0 Hz, 1H, Cys(SCHaHb)), 3.41 (br m, 1H, SCH), 3.55 (br
m, 1H, C(12)H), 3.64 (s, 3H, COOCH3), 3.65 (m, 1H, Glu(CRH)), 3.90
(br s, 2H, Gly(CH2COO)), 4.02 (br dt, J ) 6.2, 6.8 Hz, 1H, CHOH),
4.58 (br dd, J ) 5.0, 8.9 Hz, 1H, Cys(CRH)), 5.51 (dd, J ) 6.8, 15.4
Hz, 1H, CHdCHCHOH), 5.70 (dd, J ) 6.9, 15.4 Hz, 1H,
CHdCHCHOH), 6.69 (dt, J ) 1.6, 7.6 Hz, 1H, CdCH); 13C NMR
(CD3OD) δ 14.4, 23.7, 25.8, 26.3, 27.7, 29.1, 30.0, 30.0, 32.9, 33.0,
33.7, 34.7, 38.4, 42.2, 44.2, 44.9, 49.8, 52.0, 54.4, 55.3, 73.1, 130.6,
136.3, 138.9, 141.4, 173.0, 173.1, 173.7, 175.1, 175.8, 205.6; MS (FAB,
glycerol matrix) m/z 656.3 (MH+).
1
hexane/ethyl acetate); H NMR (CDCl3) δ 0.88 (t, J ) 6.4 Hz, 3H,
CH3), 1.1-1.7 (m, 20H, CH2 and OH), 2.25 (dt, J ) 6.9, 10.4 Hz, 1H,
C(8)H), 2.29 (t, J ) 7.9 Hz, 2H, C(2)H2), 2.52 (ddd, J ) 1.0, 3.0, 18.8
Hz, 1H, C(10)HaHb), 2.64 (dd, J ) 6.9, 18.8 Hz, 1H, C(10)HaHb), 2.7-
2.9 (m, 3H, SCH2 and C(12)H), 3.55 (dt, J ) 3.0, 6.9 Hz, 1H, C(11)H),
3.66 (s, 3H, OCH3), 3.75 (t, J ) 6.4 Hz, 2H, OCH2), 4.15 (q, J ) 6.4
Hz, 1H, C(15)H), 5.64 (dd, J ) 6.4, 15.3 Hz, 1H, vinyl), 5.81 (dd, J
) 8.9, 15.3 Hz, 1H, vinyl); 13C NMR (CD3OD) δ 14.4, 23.8, 25.9,
26.3, 27.6, 29.1, 29.9, 30.5, 33.0, 34.8, 35.0, 38.4, 46.7, 47.4, 50.8,
52.0, 52.1, 62.4, 73.5, 131.5, 137.3, 175.9, 219.9; MS m/z 428 (M+).
Unless otherwise stated, the following monothiol adducts of PGA1
methyl ester were synthesized by a similar procedure to the synthesis
of 7c.
7a. Reaction of sodium thiomethoxide and 4 gave 7a in 85% yield
as a mixture of stereoisomers (9:1, 11,12-trans form predominant): TLC
Rf ) 0.46 (1:1 hexane/ethyl acetate); 1H NMR (CDCl3) δ 0.88 (t, J )
6.4 Hz, 3H, CH3), 1.1-1.7 (m, 19H, CH2 and OH), 2.01 (m, 1H,
C(8)H), 2.13 (s, 3H, SCH3), 2.18 (dd, J ) 10.5, 18.3 Hz, 1H,
C(10)HaHb), 2.29 (t, J ) 7.9 Hz, 2H, C(2)H2), 2.39 (ddd, J ) 8.0,
10.5, 11.4 Hz, 1H, C(12)H), 2.79 (ddd, J ) 1.0, 8.0, 18.3 Hz, 1H,
C(10)HaHb), 2.94 (dt, J ) 8.0, 10.5 Hz, 1H, C(11)H), 3.66 (s, 3H,
OCH3), 4.14 (m, 1H, C(15)H), 5.56 (dd, J ) 8.0, 15.3 Hz, 1H, vinyl),
5.69 (dd, J ) 6.2, 15.3 Hz, vinyl); 13C NMR (CD3OD) δ 14.1, 14.4,
23.8, 26.0, 26.3, 27.7, 28.6, 29.9, 30.5, 33.0, 34.8, 38.5, 46.3, 46.5,
52.0, 53.5, 56.5, 73.3, 132.2, 137.8, 175.9, 218.3; MS m/z 398 (M+).
Preparation of 10,11-Dihydro-7,11-bis(glutathion-S-yl)prosta-
glandin A1 Methyl Ester (6f). The bis-glutathione adduct was
synthesized in a similar manner to the synthesis of 5f except that an
excess amount of glutathione to ∆7-PGA1 methyl ester was used. A
solution of glutathione (39.8 mg, 130 µmol) in phosphate buffer (0.1
M, pH 7.4, 0.5 mL) was added to a solution of ∆7-PGA1 methyl ester
(15.0 mg, 43.0 µmol) in methanol (0.5 mL) at room temperature. After
the mixture was stirred for 6 h in the dark, a solution of acetic acid in
water (1.75 M, 0.8 mL) and then water (4 mL) was added. The mixture
was loaded on a C-18 Sep-Pak column washed in advance with
methanol (2 mL) and water (10 mL). The column was rinsed with
water (10 mL) and then treated with methanol (3 mL). The methanol
eluate was concentrated under reduced pressure, and the semisolid
residue was triturated in the presence of methanol (2 mL). The resulting
solid material was filtered off, washed with methanol (2 mL), and dried
in vacuo to give the desired product as a colorless solid (16.7 mg, 40%).
Analysis of the compound by FAB MS and NMR confirmed the
structure of the bis-glutathione adduct of ∆7-PGA1 methyl ester at C(7)
and C(11) positions. For 6f: TLC (reversed-phase) Rf ) 0.59 (2:1
7b. Reaction of butanethiol and 4 gave two stereoisomers of adduct
7b in 90% yield (1:1). For 7b (11,12-trans): TLC Rf ) 0.52 (2:1
hexane/ethyl acetate); IR (CHCl3) 3300-3600, 3015, 2950, 2925, 2850,
1
1735 cm-1; H NMR (CDCl3) δ 0.88 (t, J ) 6.4 Hz, 3H, CH3), 1.2-
1.7 (m, 21H, CH2 and OH), 2.00 (br dt, J ) 5.4, 10.9 Hz, 1H, C(8)H),
2.18 (dd, J ) 11.4, 18.3 Hz, 1H, C(10)HaHb), 2.29 (t, J ) 7.4 Hz, 2H,
C(2)H2), 2.37 (dt, J ) 8.4, 10.9 Hz, 1H, C(12)H), 2.57 (t, J ) 5.4 Hz,
2H, SCH2), 2.81 (br dd, J ) 7.9, 18.3 Hz, 1H, C(10)HaHb), 2.99 (dt,
J ) 7.9, 10.6 Hz, C(11)H), 3.68 (s, 3H, OCH3), 4.14 (q, J ) 6.4 Hz,
1H, C(15)H), 5.54 (dd, J ) 7.9, 15.3 Hz, 1H, vinyl), 5.68 (dd, J )
5.9, 15.3 Hz, 1H, vinyl); 13C NMR (CD3OD) δ 14.1, 14.5, 23.0, 23.8,
26.0, 26.3, 27.7, 28.5, 30.0, 30.6, 32.1, 33.1, 33.1, 34.8, 38.5, 45.2,
47.4, 52.0, 54.0, 56.4, 73.2, 132.0, 137.7, 175.9, 218.3; HRMS m/z
calcd for C25H44O4S 440.2960, found 440.2982 (M+). For 7b (11,12-
cis): TLC Rf ) 0.44 (2:1 hexane/ethyl acetate); IR (CHCl3) 3300-
3600, 3010, 3000, 2950, 2925, 2850, 1740 cm-1; 1H NMR (CDCl3) δ
0.8-1.0 (m, 6H, CH3), 1.2-1.7 (m, 21H, CH2 and OH), 2.2-2.4 (m,
3H, C(8)H and C(2)H2), 2.5-2.7 (m, 4H, C(10)H2 and SCH2), 2.79
(ddd, J ) 5.4, 8.4, 10.4 Hz, 1H, C(12)H), 3.51 (dt, J ) 3.5, 5.4 Hz,
1H, C(11)H), 3.66 (s, 3H, OCH3), 4.14 (q, J ) 6.9 Hz, 1H, C(15)H),
5.60 (dd, J ) 6.9, 15.3 Hz, 1H, vinyl), 5.81 (dd, J ) 8.4, 15.3 Hz, 1H,
vinyl); 13C NMR (CD3OD) δ 14.0, 14.4, 23.0, 23.8, 25.9, 26.3, 27.6,
29.1, 29.9, 30.5, 32.6, 32.8, 33.0, 34.8, 38.5, 46.5, 47.3, 50.8, 52.0,
52.0, 73.5, 131.5, 137.1, 175.9, 220.1; MS m/z 440 (M+).
Preparation of 10,11-Dihydro-11r-glutathion-S-yl-∆7-prosta-
glandin A1 Methyl Ester (5f). To a solution of ∆7-PGA1 methyl ester
(18.1 mg, 51.9 µmol) in methanol (0.5 mL) was added glutathione (14.4
mg, 46.8 µmol) dissolved in phosphate buffer (0.1 M, pH 7.4, 0.5 mL),
and the mixture was stirred at room temperature for 3.5 h in the dark.
Then, aqueous acetic acid (1.75 M, 0.8 mL) was added to the solution
followed by water (4 mL). The resulting mixture was applied to a
C-18 Sep-Pak (Waters Associates, WAT020515), which was precon-
ditioned by rinsing with methanol (2 mL) and water (10 mL). After
loading the sample, the Sep-Pak was rinsed with water (10 mL) and
then with methanol (3 mL). The methanol eluate was concentrated
under reduced pressure and subjected to reversed-phase preparative thin-
layer chromatography with methanol/phosphate buffer (0.1 M, pH 7.4)
(3:1, v/v) as eluent. The band at a Rf value of 0.49 was scraped, and
the adsorbate was washed out with methanol. After the solvent was
removed under reduced pressure, the residue was dissolved in methanol
(0.5 mL), diluted with water (5 mL), and then applied to a C-18 Sep-
Pak cartridge again. The cartridge was washed with water (10 mL),
1
methanol/phosphate buffer (0.1 M, pH 7.4)); H NMR (1:1 CD3OD/
D2O) δ 0.87 (t, J ) 6.8 Hz, 3H, CH3), 1.23-1.63 (br m, 16H, CH2),
2.15 (m, 4H, double Glu(CâH2)), 2.25 (dd, J ) 11.6, 18.6 Hz, 1H,
COCHaHb), 2.33 (t, J ) 7.3 Hz, 2H, CH2COOCH3), 2.53 (m, 4H, double
Glu(CγH2)), 2.61-2.67 (br, 1H, COCH), 2.68-2.77 (m, 1H, C(12)H),
2.82-3.0 (m, 4H, Cys(SCHaHb), Cys′(SCH2), COHaHb), 3.07-3.15 (m,
2H, Cys(SCHaHb), C(7)H), 3.19 (m, 1H, C(11)H), 3.66 (s, 3H,
COOCH3), 3.74 (m, 2H, double Glu(CRH)), 3.92 (br, 4H, double
Gly(CH2COO)), 4.06 (dt, J ) 6.2, 6.9 Hz, 1H, CHOH), 4.45 (dd, J )
4.7, 8.7 Hz, Cys′(CRH)), 4.56 (dd, J ) 5.4, 8.5 Hz, Cys(CRH)), 5.59
(dd, J ) 8.2, 15.2 Hz, C(13)HdCH), 5.66 (dd, J ) 6.9, 15.2 Hz,
CHdC(14)H); MS (FAB, glycerol matrix) m/z 963.3 (MH+).
Preparation of 10,11-Dihydro-7,11-bis(methylthio)prostaglandin
A1 Methyl Ester (6a). To a solution of 1 (12.0 mg, 34.4 µmol) in
THF (0.5 mL) was added an aqueous solution of sodium thiomethoxide
(1.55 M, 0.2 mL, 0.31 mmol) at 0 °C, and the mixture was stirred for
30 min at the same temperature. Saturated brine (1 mL) was added to
the solution, and the product was extracted with benzene. The organic
layer was dried over sodium sulfate and evaporated under reduced
pressure. The residue was subjected to column chromatography on
silica gel using a 4:1 mixture of hexane and ethyl acetate as eluent,
giving the bis-thiol adduct 6a (10.1 mg, 66%) as a mixture of
stereoisomers. For 6a: TLC Rf ) 0.47 (1:1 hexane/ethyl acetate); IR
1
(KBr) 3500, 1740, 1650 cm-1; H NMR (CDCl3) δ 0.89 (t, J ) 6.6
Hz, 3H, CH3), 1.2-1.7 (m, 16H, CH2), 2.07 and 2.075 (each s, 3H,
CH3SC(7) of two isomers), 2.13 and 2.14 (each s, 3H, CH3SC(11) of
two isomers), 2.30 and 2.33 (each t, J ) 7.4 Hz, 2H, C(2)H2 of two
isomers), 2.2-3.2 (m, 6H, C(7)H, C(8)H, C(11)H, C(12)H, and
C(10)H2), 3.67 (s, 3H, OCH3), 4.15 (m, 1H, C(15)H), 5.53-5.63 (m,
1H, C(13)H), 5.66-5.80 (m, 1H, C(14)H).
Synthesis of 7-(Methylthio)prostaglandin A1 Methyl Ester (the
monothiol adduct of 1 at the C(7) position). In order to confirm the

Antitumor and AntiViral Prostaglandins
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2385
first addition of a thiol to the C(11) position of ∆7-PGA1 methyl ester
(1), 7(-methylthio)PGA1 methyl ester was prepared independently.
Synthesis of the adduct was accomplished in two steps as follows: To
a solution of (7E)-7,8-didehydro-11,15-O-bis(tert-butyldimethylsilyl-
)prostaglandin E1 methyl ester6 (38.5 mg, 64.7 µmol) in THF (0.5 mL)
was added aqueous sodium thiomethoxide (1.55 M, 0.23 mL, 0.356
mmol) at 0 °C, and the mixture was stirred for 1.5 h. Then, saturated
brine (4 mL) was added, and the product was extracted with ether.
The combined ethereal extracts were dried over anhydrous magnesium
sulfate, and the solvent was removed under reduced pressure. The
residue was subjected to column chromatography on silica gel using
hexane/ethyl acetate (gradient from 30:1 to 20:1) as eluent, giving
7-(methylthio)-11,15-O-bis(tert-butyldimethylsilyl)prostaglandin E1 meth-
yl ester (13.9 mg, 33%) as a mixture of stereoisomers: TLC Rf ) 0.35
(10:1 hexane/ethyl acetate); 1H NMR (CDCl3) δ 0.02, 0.03, 0.04, and
0.05 (each s, 12H, CH3Si of 4 isomers), 0.81-0.89 (m, 21H, CH3 and
C(CH3)3), 1.2-1.7 (m, 16H, CH2), 2.04, 2.05, 2.06, and 2.07 (each s,
3H, CH3S of 4 isomers), 2.2-2.42 (m, 2H, C(10)H2), 2.29 (t, J ) 7.6
Hz, 2H, C(2)H2), 2.5-3.1 (m, 3H, C(7)H), C(8)H, and C(12)H), 3.66
(s, 3H, OCH3), 4.0 (m, 1H, C(11)H), 4.1 (m, 1H, C(15)H), 5.50 (m,
1H, C(13)H), 5.62 (m, 1H, C(14)H). The resulting 7-(methylthio)PGE1
derivative (9.0 mg, 14 µmol) was dissolved in a mixture of CH3COOH/
H2O/THF (6:3:1, 1 mL) and then heated at 65 °C for 21 h. Then, the
mixture was concentrated under vacuum, and the residue was subjected
to column chromatography on silica gel using hexane/ethyl acetate (4:
1) as eluent, affording 7-(methylthio)PGA1 methyl ester (1.5 mg, 27%)
as a mixture of four stereoisomers at the C(7) and C(8) positions: TLC
Reaction with 9. A methanol solution of PG (57 mM, 70 µL, 4
µmol) was added to a mixture of the reduced form of activated thiol-
Sepharose 4B (5 mL, 5 µmol as SH) and phosphate buffer (0.1 M, pH
7.4, 10 mL) under argon at 25 °C. Samples (1 mL) were withdrawn
at certain time intervals (indicated in Figure 4) and filtered through a
glass filter (3G). The filtrate was diluted with phosphate buffer (0.1
M, pH 7.4, 2 mL) and then subjected to UV measurement.
FT-IR Spectral Measurement. After the reaction of 8 (20 µmol)
with ∆7-PGA1 methyl ester (1) (4.5 mg, 12.9 µmol) as above, the
mixture was centrifuged at 3000 rpm for 10 min. The precipitate was
separated, washed with phosphate buffer (0.1 M, pH 7.4), and then
dried in Vacuo (10-2 mmHg) at room temperature for 18 h, giving 10a.
The resulting resin 10a was subjected to FT-IR measurement using
the diffuse reflectance method.22 For 10a: IR (KBr) 1730 and 1650
cm-1; the starting polymer thiol 8 indicated 1650 cm-1 as a broad small
band and the difference spectrum between 10a and 8 indicated 1730
and 1650 cm-1. In a similar manner, the FT-IR spectra of 1 and its
derivatives, 5a and 6a, were measured for the spectral comparison.
For 1: 1740 (ester), 1702 (ketone), 1655 (double bond) cm-1. For
5a: 1735 (ester), 1725 (ketone), 1650 (double bond) cm-1. For 6a:
1740 (ester and ketone), 1650 (small, double bond) cm-1
.
Dissociation of PGs from the Polymer-Supported Thiol-PG
Adducts. The resin 8 (1 mL, 20 µmol as SH) was mixed with PG 1
(10 µmol) in phosphate buffer (0.1 M, pH 7.4, 2.0 mL) at 20 °C. After
24 h, the mixture was filtered through a glass filter (3G) to afford the
PG-polymer adduct 10a. The reacton of 4 with 8 gave 11a. The
PG-9 adducts 10b and 11b were also prepared in a similar manner by
mixing PGs (0.75 µmol) with 9 (1.5 mL, 1.5 µmol as SH) in phosphate
buffer (0.1 M, pH 7.4, 2.0 mL).
The effect of pH for the dissociation of PGs from the PG-polymer
adducts was investigated as follows: A mixture of the PG-polymer
adduct (0.03 mL for 10a and 11a and 0.2 mL for 10b and 11b) obtained
above and phosphate buffer (0.1 M, pH 7.4, 2 mL) was stirred at 25
°C for 5 min and then centrifuged at 3000 rpm for 10 min. The
supernatant was removed pipette and subjected to UV analysis to
determine the concentration of the dissociated PG from the PG-
polymer thiol adduct. Studies on other pH conditions (pH 7.4-11.5)
were conducted in a similar manner.
1
Rf ) 0.35, 0.29, 0.23, and 0.17 (1:2 hexane/ethyl acetate); H NMR
(CDCl3) δ 0.88 (m, 3, CH3), 1.2-1.8 (m, 17, 8 CH2 and OH), 2.045,
2.068, 2.080, and 2.083 (each s, 3, CH3S), 2.3-2.4 (m, 3, C(8)H, and
C(2)H2), 3.13 (m, 1, C(7)H)), 3.58 (m, 1, C(12)H), 3.67 (s, 3, OCH3),
4.0-4.16 (m, 1, C(15)H), 5.64 (m, 2, C(13)H and C(14)H), 6.22 (m,
1, C(10)H), 7.53 (m, 1, C(11)H); HPLC tR ) 14.1, 17.07, 17.93, and
18.85 min for four stereoisomers, respectively (YMC Pack A002 +
A003, 1:3 hexane/ether, detection 254 nm, flow rate ) 1 mL/min).
Influence of pH on the Stability of the PG-Glutathione Con-
jugate 5f. The monoglutathione conjugate 5f (2.0 to 2.5 mg, 3.0 to
3.8 µmol) was dissolved independently into a 1:1 mixture of CD3OD
(0.3 mL) and pH 6.0, 7.0, 7.4 deuterio phosphate buffers (0.1 M, 0.3
mL each), respectively, in an NMR tube at 0 °C under argon
atmosphere. The sample tube was placed in an NMR probe im-
Acknowledgment. We thank Dr. M. Fukushima, Aichi
Cancer Center, and Professor S. Narumiya, Kyoto University,
for their helpful discussion on correlation of the chemical
reactions of PGs with biological behaviors. We are indebted
to Professor Y. Nakanishi of Osaka University for his kind
suggestion on the preparation of polymer-supported thiols. We
are also grateful to Dr. T. Kondo and Mr. S. Kitamura of Nagoya
University and Dr. F. Kaneuchi, Japan Spectroscopic Co., for
measurement of NOE, FAB MS, and FT-IR spectra, respec-
tively. Special thanks go to Professors K. Hirao and S.
Funahashi of Nagoya University for their helpful guidance in
conducting MO calculations and kinetic studies. We thank
Messrs. M. Kokura and Y. Shiga for preliminary experiments.
This work was partly supported by Grant-in-Aid for International
Scientific Research Program No. 06044095 and Cancer Research
No. 03152057 from Ministry of Education, Science, and Culture
of Japan. Financial support from Teijin Co. and Ono Pharma-
ceutical Co. is also acknowledged.
1
mediately, and H NMR spectra were measured at 18.7 °C at certain
intervals. The dissociation percentage of glutathione was determined
by comparison of the signal intensity of the the vinylic protons at C(7)
for 5f and 1.
Preparation of Polymer-Supported Thiols. Sepharose-anchored
thiols 8 and 9 were prepared according to the reported methods.21
Commercially available thiopropyl-Sepharose 6B (200 mg, 16 µmol)
was placed on a glass filter (G-3 type) and washed with phosphate
buffer (0.1 M, pH 7.4, 20 mL) with stirring. The resulting resin was
treated with carbonate buffer (0.3 M, pH 8.4, 2 mL) containing
mercaptoethanol (0.5 M) and EDTA disodium salt (0.1 mM), and the
mixture was stirred at 20 °C for 40 min. The resin was filtered and
washed with aqueous acetic acid (0.1 M, 80 mL) containing NaCl (0.5
M) and EDTA disodium salt (0.1 mM) and subsequently with phosphate
buffer (0.1 M, 60 mL), giving the swelling gel 8 (0.8 mL, 16 µmol of
SH group). The gel 9 was prepared similarly from the commercially
available activated thiol-Sepharose 4B.
Reaction of Polymer-Supported Thiols with PGs. Reaction with
8. To a mixture of the reduced form of activated thiopropyl-Sepharose
6B (1 mL, 20 µmol as SH) and phosphate buffer (0.1 M, pH 7.4, 10
mL) was added a PG solution in methanol (57 mM, 176 µL, 10 µmol)
at 25 °C under argon. At certain intervals (indicated in Figure 4), 1
mL samples were withdrawn and filtered through a glass filter (3G).
The filtrate containing unreacted PG was diluted with phosphate buffer
(0.1 M, pH 7.4, 2 mL) and then subjected to UV measurement.
Supporting Information Available: Spectra of 5f (1H and
13C NMR, FAB MS) and 6f (1H NMR, FAB MS) (6 pages).
See any current masthead page for ordering and Internet access
instructions.
JA9628359