Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2002-02-12
2004-08-10
Rotman, Alan L. (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C548S556000
Reexamination Certificate
active
06774140
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the inhibition of hexoaminidase and glycosidase. More particularly, the present invention relates to the selective inhibition of hexoaminidases and glycosidases using designed iminocylitols.
BACKGROUND
Enzymatic hydrolysis of glycosidic bonds generally takes place via general acid-base catalyses that require two critical residues, a proton donor and a nucleophile. The process is illustrated in FIG.
1
. Five- or six membered iminocyclitols carrying hydroxyl groups with specific orientation and a secondary amine have been used to mimic the shape and charge of the transition state of the reaction and have been shown to be potent inhibitors of such enzymes (T. A. Beyer, et al.,
J. Biol. Chem
. 1979, 254, 12531-12541; H. Paulsen, et al.,
Adv. Carbohydr. Chem. Biochem
. 1968, 23, 115-232; A. B. Hughes, et al.,
J. Nat. Prod. Rep
. 1994, 135-162; C.-H. Wong, et al.,
Angew. Chem., Int. Ed. Engl
., 1995, 34, 412-432 and 521-546; B. Ganem,
Acc. Chem. Res
. 1996, 29, 340-347; S. Picasso,
Chimia
, 1996, 50, 648-649; L. A. G. M. van den Broek, in Carbohydr. Drug Des. 1997, Eds by Z. J. Witczak, et al., Dekker, New York, pp 1-37 and pp471-493; G. W. Fleet, et al.,
Tetrahedron Lett
. 1985, 26, 3127-3130; Y. T Pan, et al.,
J. Biol. Chem
. 1992, 267, 8313-8318; c) T. D. Heightman. et al.,
Helvetica Chim. Acta
1995, 78, 514-532; and Y. Ichikawa, et al.,
J. Am. Chem. Soc
. 1998, 120, 3007-3018). One process for synthesizing iminocyclitols is based on aldolase-catalyzed reactions (R. L. Pederson, et al.,
Tetrahedron Lett
. 1988, 29, 46454648; T. Ziegler, et al.,
Angew. Chem. Int. Ed. Engl
. 1988, 27, 716-717; C. H. von der Osten, et al.,
J. Am. Chem. Soc
. 1989, 111, 2924-3927; T. Kajimoto, et al.,
J. Am. Chem. Soc
. 1991, 113, 6187-6196; K. K.-C. Liu, et al.,
J. Org. Chem
. 1991, 56, 6280-6289; and Y. F. Wang, et al.,
Angew. Chem. Int. Ed. Engl
. 1994, 33, 1242-1244). Another process for synthesizing iminocyclitols is based on multi-step chemical transformations (S. Hiranuma, et al.,
Tetrahedron Lett
. 1995, 36, 8247-8250; and C.-H. Wong, et al.,
J. Org. Chem
. 1995, 60, 1492-1501). A preferred method for assaying inhibition activity without using radioactive isotopes employs electrospray mass spectrometry and capillary zone electrophoresis (CZE) (S. Takayama, et al.,
J. Am. Chem. Soc
. 1997, 119, 8146-8151; J. Wu, et al.,
Chem. Biol
. 1997, 4, 653-657; Y. Kanie, et al.,
Anal. Biochem
. 1998, 263, 240-245; R., Zeleny, et al.,
Anal. Biochem
. 1997, 256, 96-101; K. B. Lee, et al.,
Anal. Biochem
. 1992, 205, 108-114; and K.-B. Lee, et al.,
Electrophoresis
, 1991, 12, 636-640).
Glycosidases and hexoaminidases catalyze a myriad of clinically important processes. For example, cartilage erosion in arthritic subjects results from the over-catabolism of glycosaminoglycans (GAGs) of proteoglycan (PG)-hyaluronate complex, which fills the most part in cartilage tissue. The process is illustrated in
FIG. 8
The cartilage PG consists of a central protein core to which GAG side chains of chondroitin sulfate (CS) and keratan sulfate (KS) are attached together with O-linked and N-linked oligosaccharides. The PGs bind to hyaluronic acid noncovalently. The degradation of GAGs is a very complicated process, involving a multi-enzyme systems and radical reactions. It is known that subjects with arthritis have an abnormal increase of &bgr;-N-acetylhexoaminidases activities (O. Kida,
J. Jap. Orthop. Ass
. 1968, 42(6), 4010; R. W. Stephen, et al.,
Biochim. Biophys. Acta
1975, 399(1), 101; and J. J. Steinberg, et al.,
Biochim. Biophys. Acta
1983, 757(1), 47). The higher &bgr;-N-acetylhexoaminidases activity amplifies that of hyaluronidase and increases the degradation rate of GAG side chains.
What are needed are iminocyclitols having inhibitory activities against hexoaminidases and glycosidases.
SUMMARY
One aspect of the invention is directed to designed iminocyclitols and their use for the inhibition of glycosidases. A series of five membered iminocyclitols are synthesized starting from a single starting material through Wittig reaction, Sharpless epoxidation, and double inversion reactions using the chloromethanesulfonyl group as a leaving group. This versitile synthetic strategy provides a useful route to heterocycles having activity as inhibitors of glycosidases.
It is disclosed herein that the differences in the conformation and the orientation of side chains and OH groups might be the main reason for the observed higher inhibitory activity of the 2(R),5(R)-isomer 2 compared to the 2(S),5(R)-isomer 1. Using 2 as a starting material, a number of iminocyclitols were synthesized and tested as glycosidase inhibitors using capillary electrophoresis, and the results showed remarkable specificities toward several glycosidases. Among such compounds, 6 and 3~5 were shown to be potent inhibitors of &bgr;-glucosidase and &bgr;-N-acetylglucosaminidase, respectively.
It is also disclosed herein that the amine function of compound 6 or the OH function of compound 24 may be used to make conjugates with various aglycon groups to prepare inhibitors with improved specificities. In addition, N-methylation of compound 3 is disclosed to enhance its inhibition activity with respect to specific enzymes.
Another aspect of the invention is directed to designed iminocyclitols and their use as inhibitors with respect to hexoaminidases, including the treatment for arthritis. Several forms of arthritis are characterized by abnormally high &bgr;-N-acetylhexoaminidase activity. It is disclosed herein that such forms of arthritis are treatable with designed iminocyclitols having inhibitory activity with respect to &bgr;-N-acetylhexoaminidase. Inhibition of &bgr;-N-acetylhexoaminidase activity is disclosed herein to delay and/or reduce the degradation of PG side chains and the followed cross-reactive immune response.
Two proposed mechanisms for the catalytic mechanism of the &bgr;-N-acetylhexoaminidases are illustrated in FIG.
9
. The first proposed mechanism employs an oxonium ion transition state (D. E. Koshland,
Biol. Rev
. 1953, 28, 416). According to this first mechanism, there is partial positive charge on the ring oxygen atom, which is stabilized by the depronatated carboxyl group from the enzyme. A second mechanism involving the participation of the neighboring C-2 acetamido group has also been proposed (S. Knapp, et al.,
J. Am. Chem. Soc
. 1996, 119, 6804). Transition states analogs corresponding to both mechanisms were synthesized and evaluated, as illustrated in FIG.
10
.
Because all the GAG chains except hyaluronic acid are heavily sulfated, the 6-sulfate inhibitors would mimic the molecular size and charge distribution of the mostly sulfated GAG chain substrates better than 6-OH compounds and thus should show better inhibition activity against hexoaminidases and consequentially the degradation of the GAG side chains. Accordingly, the 6-sulfate, 6-sulfate methyl ester 5-membered iminocyclitols 207, 212 and 215 were also synthesized and assayed for bioactivity.
Another aspect of the invention is directed to an inhibitor of hexoaminidase or glycosidase represented by the following structure:
In the above structure, R
1
may be hydrogen, sulfate, or methyl sulfate; R
2
may be hydrogen, methyl, ethyl, or a branched or unbranched hydrocarbon having between 3 and 8 carbons; and R
2
may be a hydrocarbon having between 1 and 50 carbon atoms. In a first preferred embodiment, R
1
is hydrogen; R
2
is selected from the group consisting of hydrogen, methyl, ethyl, and a branched or unbranched hydrocarbon of between 3 and 8 carbon atoms; and R
3
is a hydrocarbon having between 1 and 20 carbon atoms. In an alternative to this first preferred embodiment, R
3
is a hydrocarbon having between 1 and 8 carbon atoms or is methyl. In a second preferred embodiment, R
1
is a sulfate group; R
2
is hydrogen methyl, ethyl or any branched or unbranched hydrocarbon of between 3 and 8 carbon atoms; R
3
is a hydrocarbon group that has between 1 and 20 carbon atoms. In an alt
Liu Jungie
Wong Chi-Huey
Lewis Donald G.
Rotman Alan L.
The Scripps Research Institute
Wright Sonya
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