N-Hydroxyurea 5-Lipoxygenase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 1967
yield by metalation of thieno[2,3-b]pyridine31 with n-butyl-
lithium (1.1 equiv, 2.5 M in hexanes) in THF at -78 °C and
reaction with acetaldehyde. 2-(1-Hydroxyethyl)thieno[2,3-b]-
pyridine was converted to N,O-bis(tert-butyloxycarbonyl)-N-
[1-(thieno[2,3-b]pyrid-2-yl)ethyl]hydroxylamine using N,O-bis-
(tert-butyloxycarbonyl)hydroxylamine and the procedure
described in method 5. The final chromatographic purification
was done using a gradient elution of 10-25% ethyl acetate
and gave a 33% yield of the desired hydroxylamine derivative
as an oil.
To a stirred room temperature solution of N,O-bis(tert-
butyloxycarbonyl)-N-(1-(thieno[2,3-b]pyrid-2-yl)ethyl)hy-
droxylamine (2.75 g, 6.97 mmol) in CH2Cl2 (3 mL) was added
TFA (3 mL). The mixture was stirred for 0.5 h at room
temperature and evaporated to dryness. The residue was
dissolved in ethyl acetate, washed with saturated NaHCO3,
dried (MgSO4), and concentrated to give 1.11 g (82%) of N-[1-
(thieno[2,3-b]pyrid-2-yl)ethyl]hydroxylamine.
porting departments at Abbott Laboratories for techni-
cal assistance. We also thank Dr. L. Dube´, Head of the
Immunoscience Venture and members of the Clinical
Study Group, for the clinical results.
Su p p or tin g In for m a tion Ava ila ble: Spectral data and
experimental procedures for all compounds not reported in the
general methods (39 pages). Ordering information is given
on any current masthead page.
Refer en ces
(1) (a) Israel, E. Moderating the inflammation of asthma: inhibiting
the production or action of products of the 5-lipoxygenase
pathway. Ann. Allergy 1994, 72, 279-284. (b) Brooks, D. W.
Progress with investigational drugs for the treatment of pul-
monary and inflammatory diseases. Expert Opin. Invest. Drugs
1994, 3, 185-190.
(2) Samuelsson, B. Leukotrienes: mediators of immediate hyper-
sensitivity reactions and inflammation. Science 1983, 220, 568-
575.
(3) (a) Corey, E. J .; Niwa, H.; Falck, J . R.; Mioskowski, C.; Arai, Y.;
Marfat, A. Recent studies on the chemical synthesis of eicosanoids.
Adv. Prostaglandin Thromboxane Res. 1980, 6, 19-25. (b)
Borgeat, P.; Sirois, P. Leukotrienes: a major step in understand-
ing immediate hypersensitivity reactions. J . Med. Chem. 1981,
24, 121-126.
(4) Brooks, C. D. W.; Summers, J . B. Modulators of Lekotriene
Biosynthesis and Receptor Activation. J . Med. Chem. 1996, 39,
2629-2654.
The hydroxylamine derivative was converted to 4j using
trimethylsilyl isocyanate (42%) as described in method 1a: mp
1
181-182 °C; H NMR (300 MHz, DMSO-d6) δ 1.52 (d, J ) 7
Hz, 3H) 5.59 (q, J ) 7 Hz, 1H), 6.51 (bs, 2H), 7.72 (d, J ) 1
Hz, 1H), 7.48 (dd, J ) 4 Hz, 1H), 8.17 (dd, J ) 1.5 Hz, J ) 7.5
Hz, 1H), 8.49 (dd, J ) 1.5 Hz, J ) 5 Hz, 1H), 9.31 (s, 1H);
MS(DCI-NH3) m/z 255 (M + NH4)+, 238 (M + H)+. Anal.
(C10H11N3O2S) C, H, N.
Meth od 7. Preparation of N-substituted N-hydroxyureas
by Mitsunobu alkylation of N,O-bis(carbophenoxy)hydroxy-
lamine and reaction with ammonia. This procedure was done
as previously described.22,23
Biologica l Meth od s. Percent inhibition was computed by
comparing individual values in treatment groups to the mean
value of the control group. Statistical significance was deter-
mined using one way analysis of variance and Tukeys multiple
comparison procedure. Linear regression was used to estimate
IC50 and ED50 values.
Glu cu r on osyltr a n sfer a se Activity in Mon k ey Liver
Micr osom es. Glucuronidation rates were obtained using
microsomal pellets prepared from frozen cynomolgus monkey
livers as previous described by Bell et al.29 Data shown are
from rates derived from duplicate incubations at the times
noted.
(5) (a) Rouzer, C. A.; Samuelsson, B. Leukocyte arachidonate
5-lipoxygenase isolation and characterization. Methods Enzymol.
1990, 187, 312-319. (b) Steinhilber, D. 5-Lipoxygenase: enzyme
expression and regulation of activity. Pharm. Acta Helv. 1994,
69, 3-14. (c) Radmark, O. Arachidonate 5-lipoxygenase. J . Lipid
Med. Cell Signalling 1995, 12, 171-184.
(6) Percival, M. D. Human 5-lipoxygenase contains an essential iron.
J . Biol. Chem. 1991, 266, 10058-10061.
(7) (a) Musser, J . H.; Kreft, A. F. 5-Lipoxygenase: properties,
pharmacology and the quinolinyl(bridged)aryl class of inhibitors.
J . Med. Chem. 1992, 35, 2502-2524. (b) Garland, L. G.; Salmon,
J . A. Hydroxamic acids and hydroxyureas as inhibitors of
arachidonate 5-lipoxygenase. Drugs Future 1991, 16, 547-558.
(c) Batt, D. G. 5-Lipoxygenase inhibitors and their anti-inflam-
matory activities. In Progress in Medicinal Chemistry; Ellis, G.
P., Luscombe, D. K., Eds.; Elsevier Science Publishers: New
York, 1992; Vol. 29, pp 1-63. (d) McMillan, R. M.; Walker, E.
R. H. Designing therapeutically effective 5-lipoxygenase inhibi-
tors. Trends Pharmacol. Sci. 1992, 13, 323-330.
(8) Corey, E. J .; Cashman, J . R.; Kanter, S. S.; Corey, D. R.
Rationally designed, potent competitive inhibitors of leukotriene
biosynthesis. J . Am. Chem. Soc. 1984, 106, 1503-1504.
(9) Summers, J . B.; Gunn, B. P.; Mazdiyasni, H.; Goetze, A. M.;
Young, P. R.; Bouska, J . B.; Dyer, R. D.; Brooks, D. W.; Carter,
G. W. In vivo characterization of hydroxamic acid inhibitor of
5-lipoxygenase. J . Med. Chem. 1987, 30, 2121-2126.
Deter m in a tion of Dr u g P la sm a Con cen tr a tion s. Com-
pounds for oral administration were suspended in 0.2% HPMC
with a Potter-Elvehjem homogenizer equipped with a Teflon-
coated pestle and administered orally to cynomolgus monkeys
and or rats as described by Bell et al.29 Data presented are
means from at least three animals.
(10) (a) Summers, J . B.; Gunn, B. P.; Martin, J . G.; Mazdiyasni, H.;
Stewart, A. O.; Young, P. R.; Goetze, A. M.; Bouska, J . B.; Dyer,
R. D.; Brooks, D. W.; Carter, G. W. Orally active hydroxamic
acid inhibitors of leukotriene biosynthesis. J . Med. Chem. 1988,
31, 3-5. (b) Summers, J . B.; Gunn, B. P.; Martin, J . G.; Martin,
M. B.; Mazdiyasni, H.; Stewart , A. O.; Young, P. R.; Bouska, J .
B.; Goetze, A. M.; Dyer, R. D.; Brooks, D. W.; Carter, G. W.
Structure-activity analysis of a class of orally active hydroxamic
acid inhibitors of leukotriene biosynthesis. J . Med. Chem. 1988,
31, 1960-1964
Ra t Ba sop h il Leu k em ia (RBL) Cell Lysa te 5-Lip oxy-
gen a se In h ibitor P oten cy. Adherent rat basophilic leuke-
mia (RBL-1) cells (2H3 subline) lysate was centrifuged at
20000g for 20 min and the supernatant containing 5-LO
activity stored frozen until used. Compounds were evaluated
for 5-lipoxygenase inhibitory activity according to the method
described by Carter et al.11d Data are from duplicate incuba-
tions.
(11) (a) Summers, J . B.; Gunn, B. P.; Brooks, D. W. Indole, benzo-
furan, benzothiophene containing lipoxygenase inhibiting com-
pounds. U.S. Patent 4873259, Oct. 10, 1989. (b) Carter, G. W.;
Young, P. R.; Albert, D. H.; Bouska, J .; Dyer, R. D.; Bell, R. L.;
Summers, J . B.; Brooks, D. W.; Gunn, B. P.; Rubin, P.; Kester-
son, J . A-64077, a new orally active 5-lipoxygenase inhibitor. In
Leukotrienes and Prostanoids in Health and Disease, New
Trends in Lipid Mediators Research; Zor, U., Naor, Z., Danon,
A., Eds.; Karger: Basel, 1989; pp 50-55. (c) Brooks, D. W.;
Summers, J . B.; Gunn, B. P.; Rodriques, K. E.; Martin, J . G.;
Martin, M. B.; Mazdiyasni, H.; Holms, J . H.; Stewart, A. O.;
Moore, J . L.; Young, P. R.; Albert, D. H.; Bouska, J . B.; Malo, P.
E.; Dyer, R. D.; Bell, R. L.; Rubin, P.; Kesterson, J .; Carter, G.
W. The discovery of A-64077, a clinical candidate for treating
diseases involving leukotriene mediators. Abstracts of the
International Chemical Congress of Pacific Basin Societies,
Honolulu, 1989, Abstract BIOS 34. (d) Carter, G. W.; Young, P.
R.; Albert, D. H.; Bouska, J .; Dyer, R.; Bell, R. L.; Summers, J .
B.; Brooks, D. W. 5-Lipoxygenase inhibitory activity of zileuton.
J . Pharmacol. Exp. Ther. 1991, 256, 929-937.
Hu m a n Wh ole Blood Eicosa n oid F or m a tion . Aliquots
of heparinized (20 USP units/mL) human blood (0.3 mL) were
preincubated with drug or vehicle for 15 min at 37 °C, and
ecosanoid biosynthesis was initiated by adding calcium iono-
phore A23187 according to the method described by Bell et
al.29 The amount of LTB4 in aliquots of the extracts was
analyzed by enzyme immunoassay (EIA). All results are
means of at least duplicate and in most case triplicate
determinations.
Ra t P er iton ea l An a p h yla xis Mod el. This in vivo leu-
25
kotriene assay was conducted as described by Young et al.
Samples were analyzed for leukotrienes by enzyme immu-
noassay (EIA reagents for LTE4, LTB4 and thromboxane:
Cayman Chemical CO., Ann Arbor, MI, and LTB4 antibody
from Advance Magnetics, Cambridge, MA, and LTE4 tracer
was prepared at Abbott).
(12) Brooks, D. W.; Carter, G. W. The discovery of zileuton. In The
Search for Anti-Inflammatory Drugs; Merluzzi, V. J ., Adams,
J ., Eds.; Birkhauser: Boston, 1995; Chapter 5, pp 129-160.
Ack n ow led gm en t. We thank the members of the
Leukotriene Biosynthesis Regulators Project and sup-