E. P. Schreiner et al. /Bioorg. Med. Chem. Lett. 13 (2003) 4313–4316
4315
mixture was refluxed with TFA in toluene for 7 h. At
this stage purification was achieved by crystallisation of
the trifluoroacetic acid salt of 6 from ethanol to give
pure 7 (75%).19 Finally, heating of 6 with 7 in DMA at
50 ꢁC for 1 h and crystallization after aqueous workup
cleanly provided 1 in 87% yield. Within this sequence
the most expensive synthone 2 is introduced late in the
synthesis and no chromatographic purification is
needed. Overall yield amounted to 45%.
In conclusion, we identified 1 as the first potent non-
estrogenic irreversible inhibitor of human steroid sulfa-
tase featuring a 5,6-bicyclic ring system as a mimicry for
the steroidal A- and B-ring. 1 is a candidate for further
evaluation of its potential as a drug in the treatment of
estrogen- and androgen-dependent diseases.
References and Notes
The target compound 1 inactivated purified human STS
irreversibly. From the kinetics of inactivation, followed
by measuring residual enzyme activity after incubation
with various concentrations of the inhibitor,11,20 an
inhibition constant (Ki) of 6 mM and a rate constant of
inactivation (kinact) of 0.097 msꢀ1 was obtained. These
values may be compared with Ki=0.47 mM and
kinact=0.009 sꢀ1 reported for EMATE as a reference
compound.20 While 1 shows weaker binding to STS
than EMATE, the efficiency of inactivation, as expres-
1. (a) Pasqualini, J. R.; Gelly, C.; Nguyen, B. L.; Vella, C. J.
Steroid Biochem. 1989, 34, 155. (b) Reed, M. J.; Purohit, A.
Rev. Endocrine Related Cancer 1993, 45, 51.
2. Utsumi, T.; Yoshimura, N.; Takeuchi, S.; Maruta, M.;
Maeda, K.; Harada, N. J. Steroid Biochem. Mol. Biol. 2000,
73, 141.
3. Voigt, W.; Sawaya, M. E.; Hsia, S. L.; Amthor, C. IRSC
Med. Sci. 1982, 10, 529.
4. (a) Hoffmann, R.; Rot, A.; Niiyama, S.; Billich, A. J.
Invest. Dermatol. 2001, 117, 1342. (b) Billich, A.; Rot, A.;
Lam, C.; Schmidt, J. B.; Schuster, I. Horm. Res. 2000, 532, 92.
5. (a) Jagannathan, S.; Johnson, D. A. Cognitive Brain Res.
1995, 2, 251. (b) Rhodes, M. E.; Li, P. K.; Burke, A. M.;
Johnson, D. A. Brain Res. 1997, 773, 28. (c) Li, P. K.; Rhodes,
M. E.; Johnson, D. A.; Wu, T.; Li, P.; Maher, T. J. Brain Res.
2000, 865, 286.
6. Howarth, N. M.; Purohit, A.; Reed, M. J.; Potter, B. V. L.
J. Med. Chem. 1994, 37, 219.
7. Poirier, D.; Ciobanu, L. C.; Maltais, R. Exp. Opin. Ther.
Pat. 1999, 9, 1083.
sed by the ratio of kinact/Kꢀi 1, is about equal (1.6 vs
4
1.9 10 sꢀ1 Mꢀ1) for the two compounds.
.
To assess the selectivity of 1 for STS over human aryl-
sulfatases A (ASA) and B (ASB), both enzymes were
isolated from human placenta,21 and assayed in pre-
sence of 1.22 Whereas ASA was not inhibited by 1 u p to
the highest test concentration of 10 mM, mASB was
weakly inhibited (by 25% at 10 mM) in a time-indepen-
dent fashion, suggesting reversible binding to the active
site of this enzyme.
8. Nussbaumer, P.; Billich, A. Exp. Opin. Ther. Pat. 2003, 13,
605.
9. Elger, W.; Schwarz, S.; Hedden, A.; Reddersen, G.;
Schneider, B. J. Steroid Biochem. Mol. Biol. 1995, 55, 395.
10. (a) Ciobanu, L. C.; Boivin, R. P.; Luu-The, V.; Poirier, D.
Eur. J. Med. Chem. 2001, 36, 659. (b) Purohit, A.; Vernon,
K. A.; Hummelinck, A. E. et al. J. Steroid Biochem. Mol. Biol.
1998, 64, 269. (c) Kolli, A.; Chu, H. H.; Rhodes, M. E.; Inoue,
K.; Selcer, K. W.; Li, P. K. J. Steroid Biochem. Molec. Biol.
1999, 68, 31. (d) Malini, B.; Purohit, A.; Ganeshapillai, D.;
Woo, L. W.; Potter, B. V.; Reed, M. J. J. Steroid Biochem.
Mol. Biol. 2000, 75, 253.
11. Nussbaumer, P.; Lehr, P.; Billich, A. J. Med. Chem. 2002,
73, 4310.
12. Billich, A.; Schreiner, E. P.; Wolff-Winiski, B., WO
0136398 A1, 2001.
13. Masters, A. P.; Sorensen, T. S.; Tran, P. M. Can. J. Chem.
1987, 65, 1499.
Measurement of enzyme inhibition in cellular systems is
one step closer to the in vivo situation. First, we asses-
sed the ability of 1 to inhibit human STS in CHO cells
over-expressing STS:23 an IC50 of ꢂ37.5 nM was
determined (Table 1). Furthermore, we tested for inhi-
bition of STS-catalysed hydrolysis of DHEAS in differ-
ent cell types of the skin, namely keratinocytes,
fibroblasts, and sebocytes (Table 1).24 IC50 values in the
range of 0.15–0.8 nM were obtained. Finally, the IC50
value for inhibition of STS-dependent cleavage of
estrone sulfate by the human breast cancer cell line
MCF-7 was 2.3 nM.25
14. Narayanan, V. L. Ger. Offen. 2,043,380 (CA 75,20180k),
1972 (according to the abstract).
15. Appel, R.; Berger, G. Chem. Ber. 1958, 91, 1339.
These IC50 values roughly follow the amount of enzyme
activity in the various cell types (data not shown), which
is in line with the irreversible action of 1 and hence a
dependence on enzyme concentration.
1
16. For 1: H NMR (250 MHz, DMSO-d6) d 8.02 (br.s, 2H),
7.76 (d, J=8.6 Hz, 1H), 7.62 (d, J=2.2 Hz, 1H), 7.26 (dd,
J=8.6 and 2.2 Hz, 1H), 4.46 (s, 2H), 6.24 (s, 1H), 4.22 (s,
1H), 2.68 (s, 1H), 1.79–2.05 (m, 12H). 13C NMR (250 MHz,
DMSO-d6) d (ppm): 2167.76, 163.44, 148.82, 147.28, 140.02,
119.44, 105.43, 103.60, 40.97, 40.03, 39.01, 36.62, 33.50,
27.45, IR (neat) nmax 1192, 1388 cmꢀ1, MS (APCI) m/z 361.1
[MH]+.
The title compound 1 did not show any measurable
affinity to the human estrogen receptors a and b
(EC50>100 mM) whereas for EMATE EC50 values of
2.6 and 3.7 mM were determined.26
17. Lindemann, H.; Koenitzer, H.; Romanoff, S. Liebigs Ann.
Chem. 1927, 456, 284.
Table 1. Inhibition of human STS in different cell types
18. Fujita, S.; Koyama, K.; Inagaki, Y. Synthesis 1982, 68.
19. Procedure: 60 g (0.4 mol) of 6-hydroxy-2-methyl-ben-
zoxazole (10) were dissolved in 1 L of dry THF, cooled to
0 ꢁC, and 75 mL (0.44 mol) of DIEA and 52 mL (0.42
mol) trimethylsilyl chloride were added. Then the ice bath
was removed and stirring continued for 2 h. For the
deprotonation the reaction mixture was cooled to ꢀ78 ꢁC
Cell type
IC50 (nM)
STS-expressing CHO cells
Keratinocytes
Fibroblasts
Sebocytes
MCF-7 breast cancer cells
31.8ꢃ3.6
0.77ꢃ0.32
0.75ꢃ0.15
0.15ꢃ0.01
2.3ꢃ0.8