WO2000008040A1 - Inhibiteurs de sialyltransferase - Google Patents

Inhibiteurs de sialyltransferase Download PDF

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Publication number
WO2000008040A1
WO2000008040A1 PCT/EP1999/005697 EP9905697W WO0008040A1 WO 2000008040 A1 WO2000008040 A1 WO 2000008040A1 EP 9905697 W EP9905697 W EP 9905697W WO 0008040 A1 WO0008040 A1 WO 0008040A1
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group
alkyl
compound
sialyltransferase
aryl
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PCT/EP1999/005697
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English (en)
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Richard R. Schmidt
Christoph Schaub
Bernd Müller
Franz Amann
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Universität Konstanz
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Priority to AU60791/99A priority Critical patent/AU6079199A/en
Publication of WO2000008040A1 publication Critical patent/WO2000008040A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • This invention pertains to the field of sialyltransferase inhibitors.
  • Compounds that inhibit sialyltransferases are provided. Also provided are methods of inhibiting sialyltransferases and methods of modulating biological processes that involve sialylation.
  • Sialic acid containing epitopes are involved in important biological processes, such as cell adhesion and inflammation (A. Rosenberg, in Biology of Sialic Acids, Plenum, New York 1995; Dall'Olio et al, Int. J. Cancer 1992, 50, 325-330; Morgenthaler et al, Biochem. Biophys. Res. Commun. 1990, 171, 860-866).
  • sialyl content of glycoconjugates There is also a correlation between sialyl content of glycoconjugates and the malignancy of tumor cells (Bernacki, Science 1977, 195, 557-580; Zhou et al, J. Biol. Chem. 1994, 269, 1959-1965; J. W.
  • Sialylglycosides are synthesized by sialyltransferases, which are a group of glycosyltransferases that transfer sialic acid from an activated sugar nucleotide to acceptor oligosaccharides found on glycoproteins, glycolipids or polysaccharides.
  • sialylated oligosaccharide structures The large number of sialylated oligosaccharide structures has lead to the characterization of many different sialyltransferases involved in the synthesis of these structures. Based on the linkage and acceptor specificity of the sialyltransferases studied so far, it has been determined that at least 13 distinct sialyltransferase genes are present in mammalian systems (Tsuji et al. (1996) Glycobiology 6:v-vii).
  • sialyltransferases employ, independent of their source and their acceptor specificity, cytidine monophosphate N-acetylneuraminic acid (CMP- ⁇ eu5Ac, Scheme 1 ( Figure 1)) as sialyl donor (Harduin-Lepers et al, Glycobiology 1995, 5: 741-758).
  • sialyltransferases Because of the importance of sialylation in biological systems, it is highly desirable to develop efficient inhibitors for sialyltransferases. Thus far, only a few donor and acceptor analogues (substrate analogues) have been reported which serve as sialyltransferase inhibitors (Schaub et al, Glycoconjugate J. 1998, 15: 345-354; Schaub and Schmidt, Abstract C 10, Second European Conference on Carbohydrate Mimics, La Garda (Italy), 1996; Amann et al, Chem. Eur. J. 1998, 4: 1106-1115; Muller et al, Tetrahedron Lett.
  • the invention provides a sialyltransferase inhibitor compound having the formula:
  • R 1 and R 2 are independently selected from the group consisting of H, alkyl, aryl, and alkylaryl;
  • R 3 is selected from the group consisting of R and
  • R 4 and R 5 are independently selected from the group consisting of H, COO" alk l , aryl, alkylaryl, heteroaryl, alkyl heteroaryl,
  • R 6 is selected from the group consisting of H, alkyl, aryl, alkyl aryl, heteroaryl, urea, carbamoyl, and
  • R 7 is H, acyl, alkyl, arylacyl, or heteroarylacyl
  • R 8 is H, alkyl, aryl, alkylaryl, or heteroaryl
  • R 9 is selected from the group consisting of R 5 ,
  • R 10 is R 5 , H, or A;
  • a and B are independently selected from the group consisting of H, OH, O- alkyl, NH-R 6 , NH-R 7 , and guanidino;
  • D is selected from the group consisting of A, alkyl, aryl, heteroaryl, acylaryl, acylalkylaryl, and alkylaryl;
  • X 1 is selected from the group consisting of O, NH, S, CH , and CF 2 ;
  • X 2 is selected from the group consisting of O, N-R ;
  • X 3 is independently selected from the group consisting of N, NH, O, C, and
  • X 4 is independently selected from the group consisting of O, S, N-R , NH, CH 2 , and CH-R 10 ;
  • Y is selected from the group consisting of O, H, NH, N-R ;
  • Z is selected from the group consisting of H, acetyl, glycolyl, acyl, acylaryl, acyl alkylaryl, acyl heteroaryl, guanidino, and
  • R 3 is a ring structure selected from the group consisting of:
  • R is a substituted or unsubstituted benzene.
  • the benzene is substituted at the 4' position with, for example, an acetyl amine group. In other embodiments, the benzene is substituted at the 5' position with
  • the compounds of the invention also include those in which R is
  • R 3 is a substituted or an unsubstituted aryl group.
  • the ;ssee ccoompounds include, for example, those in which R 3 is selected from the group consisting of:
  • R 11 is selected from the group consisting of alkyl, aryl, heteroaryl, acylalkyl, Cl, NO 2 , Br, I, CF 3 , S alkyl, H, OH, O alkyl, SH, COO-R 6 , and
  • R 3 is a heteroaryl or an alkylheteroaryl group.
  • R 3 can be selected from, among other moieties, the group consisting of:
  • X 5 is selected from the group consisting of N and C;
  • X 6 is selected from the group consisting of O, N, C, and S;
  • X 7 is selected from the group consisting of N, O, NH, and S; and when X 5 or X 6 is C, R 11 is selected from the group consisting of alkyl, aryl, heteroaryl, acylalkyl, Cl, NO 2 , Br, I, CF 3 , S alkyl, H, OH, O alkyl, SH, COO-R 6 , and
  • sialyltransferase inhibitor compounds of the invention include inhibitors of 2,3 sialyltransferases, 2,6 sialyltransferases, and ⁇ 2,8 sialyltransferases.
  • pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a sialyltransferase inhibitor compound as described herein in a unit dosage capable of inhibiting activity of a sialyltransferase in a mammal.
  • the invention provides methods of inhibiting sialyltransferase-catalyzed synthesis of a sialylglycoside, the method comprising contacting a sialyltransferase with a sialyltransferase inhibitor compound of the invention.
  • the methods are useful, for example, to inhibit the synthesis of a sialylgalactoside such as, for example, a structure selected from the group consisting of Sia ⁇ 2-6Gal ⁇ l-4GlcNAc, Sia ⁇ 2,3Gal ⁇ l- 3GalNAc, Sia ⁇ 2,3Gal ⁇ l-4GlcNAc.
  • the invention also provides a method of inhibiting intercellular adhesion mediated by a mammalian cell surface receptor which comprises a sialic acid linked to an acceptor moiety, the method comprising contacting a sialyltransferase which can link the sialic acid to the acceptor moiety with an effective amount of a sialyltransferase inhibitor compound as described above and elsewhere herein.
  • the receptor can comprise a SLe x moiety or Sia ⁇ 2-6Gal ⁇ l-4GlcNAc or Sia ⁇ 2,3Gal ⁇ l-3GalNAc, among many others.
  • the invention also provides a method of treating an inflammatory disease process mediated by a selectin cell surface receptor in a mammal, the method comprising administering to the mammal an effective amount of a sialyltransferase inhibitor compound as described above.
  • sialyltransferase inhibitor compounds of the invention have the formula
  • R and R are independently selected from the group consisting of H, alkyl, aryl, and alkylaryl;
  • R is selected from the group consisting of
  • R > 5 is selected from the group consisting of H, COO " , alkyl, aryl, alkylaryl, heteroaryl, alkyl heteroaryl,
  • R 6 is selected from the group consisting of H, alkyl, aryl, alkyl aryl, heteroaryl, urea, carbamoyl, and
  • R 8 is H, alkyl, aryl, alkylaryl, or heteroaryl; R 9 is selected from the group consisting of R 5 ,
  • R 10 is R 5 , H, or A
  • R ⁇ is selected from the group consisting of alkyl, aryl, heteroaryl, acylalkyl,
  • R and R are independently selected from the group consisting of R 3.' R D 4 ,
  • X 2 is O or N-R 6 ; and A and B are independently selected from the group consisting of H, OH, O alkyl, NH-R 6 , NH-R 7 , and guanidino.
  • Figure 1 shows a reaction scheme for sialyltransferase-catalyzed synthesis of an ⁇ -sialoside.
  • the transition state is shown as CMP-Neu5Ac*.
  • FIG. 2 shows Compounds 1 and (R)-2.
  • (R)-2 was found to be a very potent sialyltransferase inhibitor.
  • Figure 3 shows Compounds 3h and 31, and 4h and 41, along with a diagram of the method for synthesis of these compounds.
  • Figure 4 shows Compounds (R)-6, (S)-6, (R)-7, and (S)-7, and a method for their synthesis.
  • Figure 5 shows Compounds (R)-6 and (S)-6 in which the phosphonate group is replaced with a carboxylate group.
  • Figure 6 shows the compounds (E)-8 and (Z)-9, and a scheme for synthesis of these compounds.
  • Figure 7 shows the structures of the compounds (R)-13, (S)-13, and (E)-14.
  • Ara arabinose
  • GalNAc N-acetylgalactosamine
  • GlcNAc N-acetylglucosamine
  • Man mannose
  • oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non- reducing end on the left and the reducing end on the right.
  • oligosaccharides described herein are described with the name or abbreviation for the non-reducing saccharide (e.g., Gal), followed by the configuration of the glycosidic bond ( ⁇ or ⁇ ), the ring bond, the ring position of the reducing saccharide involved in the bond, and then the name or abbreviation of the reducing saccharide (e.g., GlcNAc).
  • the linkage between two sugars may be expressed, for example, as 2,3, 2 ⁇ 3, or (2,3).
  • Each saccharide is a pyranose.
  • sialic acid refers to any member of a family of nine-carbon carboxylated sugars.
  • the most common member of the sialic acid family is N-acetyl- neuraminic acid (2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-l- onic acid (often abbreviated as Neu5Ac, NeuAc, or NANA).
  • a second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated.
  • a third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al. (1990) J. Biol. Chem. 265: 21811-21819. Also included are 9-substituted sialic acids such as a 9-O- C1-C6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-
  • the invention provides compositions and methods for inhibiting inflammatory and other disease responses that are mediated by sialyl glycosides.
  • the present invention relates to sialyltransferase inhibitors that can to inhibit the activity of sialyltransferases involved in the synthesis of ligands recognized by cell surface receptors, such as selectin receptors.
  • Methods are also disclosed for preparing the • sialyltransferase inhibitors as well as various screening assays to identify suitable candidates. Therapeutic and other uses for these compounds are also provided.
  • the biology and biochemistry of enzymes involved in the biosynthesis of sialylated glycosides, including Sia ⁇ 2,6 galactosides and Sia ⁇ 2,3 galactosides, have been extensively studied.
  • the invention provides novel sialyltransferase inhibitors.
  • the inhibitor compounds of the invention are generally of high potency against sialyltransferases. Often, the compounds are modifications of CMP-NeuAc, which is a natural inhibitor of sialyltransferases.
  • the inhibitor compounds have the formula I:
  • R and R are independently selected from the group consisting of H, alkyl, aryl, and alkylaryl. Where R 1 and R 2 are both H, the compound includes a cytidine nucleotide attached to a ribose sugar.
  • R group of Formula I is generally selected so as to be a relatively flat ring structure.
  • R 3 can be, for example, the moieties specified for R 4 , or
  • the compounds of the invention generally have one or more of the following features which distinguish them from CMP -NeuAc.
  • the compounds often include a methylene carbon to which are attached one or all of the following: a CMP residue, a phosphonate or other negatively charged functional group, and a flat or nearly flat pyranosyl or furanosyl ring.
  • the inhibitor compounds have two negatively charged moieties which are separated by about 2-8 chemical bonds, more preferably by agout4-6 bonds, and most preferably the negative charges are separated by about four bonds.
  • R is a planar or nearly planar benzene ring structure, either substituted or unsubstituted.
  • the following groups are suitable at the R position of the sialyltransferase inhibitor compounds:
  • Example 1 provides protocols for the synthesis of these compounds. Results of sialyltransferase inhibition assays are also provided in this Example.
  • R and R > 5 i • n these formulae are independently selected from the group consisting of H, COO " , alkyl, aryl, alkylaryl, heteroaryl, alkyl heteroaryl,
  • R 6 is selected from the group consisting of H, alkyl, aryl, alkyl aryl, heteroaryl, urea, carbamoyl, and
  • R 7 is H, acyl, alkyl, arylacyl, or heteroarylacyl
  • R 8 is H, alkyl, aryl, alkylaryl, or heteroaryl
  • R 9 is selected from the group consisting of R ,
  • R 10 is R 5 , H, or A.
  • a and B in these formulae are independently selected from the group consisting of H, OH, O-alkyl, NH-R 6 , NH-R 7 , and guanidino
  • D is selected from the group consisting of A, alkyl, aryl, heteroaryl, acylaryl, acylalkylaryl, and alkylaryl.
  • X 1 is selected from the group consisting of O, NH, S, CH 2 , and CF 2 ;
  • X is selected from the group consisting of O, N-R ;
  • X 3 is independently selected from the group consisting of N, NH, O, C, and CH;
  • X 4 is independently selected from the group consisting of O, S, N-R 6 ,
  • Y is selected from the group consisting of O, H, NH, N-R ;
  • Z is selected from the group consisting of H, acetyl, glycolyl, acyl, acylaryl, acyl alkylaryl, acyl heteroaryl, guanidino, and
  • the sialyltransferase inhibitor compounds of Formula I that are encompassed by the invention specifically exclude the compound (R)-2 and other compounds that are described in Amann et al. (1998) Eur. J. Biochem. 4: 1108-1115.
  • sialyltransferase inhibitors of the invention are shown in Table 1, together with their K j for ⁇ 2,6-sialyltransferase inhibition.
  • Each of the compounds is also inhibitor of o2,3 sialyltransferases except for compound (Z)-9, which is specif i c for ⁇ 2 , 6 sialyltransferases.
  • This compound , (Z)-9 has a K, of 0.04 ⁇ M for ⁇ 2,6 sialyltransferase, for which enzyme the inhibitor is specific.
  • the compound (Z)-9 is specific for ⁇ 2,6 sialyltransferase, as it is not an inhibitor of ⁇ 2,3 sialyltransferase.
  • Preferred compounds of the invention are potent inhibitors of one or more sialyltransferases.
  • preferred compounds have a K t for one or more sialyltransferases that is less than or equal to about 300 nM. More preferably the preferred compounds have a K m of about 200 nM or less; still more preferably less than about 50 nM.
  • the invention provides sialyltransferases that are specific for one or more sialyltransferases
  • sialyltransferases that are capable of inhibiting more than one s i alyltransferase , e.g., an ⁇ 2,3 sialyltransferase and an 2,8 sialyltransferase.
  • alkyl as used herein means a branched or unbranched , saturated or unsaturated, monovalent or divalent, hydrocarbon radical having from 1 to 20 carbons, including lower alkyls of 1-8 carbons such as methyl, ethyl, n-propyl, butyl, n- hexyl, and the like, cycloalkyls (3-7 carbons), cycloalkylmethyls (4-8 carbons), and arylalkyls.
  • alkoxy refers to alkyl radicals attached to the remainder of the molecule by an oxygen, e.g., ethoxy, methoxy, or n-propoxy.
  • alkylthio refers to alkyl radicals attached to the remainder of the molecule by a sulfur.
  • acyl refers to a radical derived from an organic acid by the removal of the hydroxyl group. Examples include acetyl, propionyl, oleoyl, myristoyl.
  • aryl refers to an aromatic monovalent carbocyclic radical having a single ring (e.g., phenyl) or two condensed rings (e.g., naphthyl), which can optionally be mono-, di-, or tri-substituted, independently, with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, lower- alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl, lower- alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-
  • R , n' ' is selected from the group consisting of alkyl, aryl, heteroaryl, acylalkyl, Cl, N0 2 , Br, I, CF 3 , S alkyl, H, OH, O alkyl, SH, COO-R 6 , and R 6
  • heteroaryl includes the following groups:
  • X 5 is selected from the group consisting of N and C;
  • X 6 is selected from the group consisting of O, N, C, and S;
  • X 7 is selected from the group consisting of N, O, NH, and S; and when X 5 or X 6 is C, R u is selected from the group consisting of alkyl, aryl, heteroaryl, acylalkyl, Cl, NO 2 , Br, I, CF 3 , S alkyl, H, OH, O alkyl, SH, COO-R 6 , and
  • the phosphonate group in the sialyltransferase inhibitors of Formula I can be replaced with, for example, CO 2 2' and other groups that provide a similar spacing between negative charges.
  • Other suitable negatively charged groups include, for example, phosphate, phosphonate, sulfamide, sulfonate, and the like.
  • sialyltransferase inhibitor compounds provided by the present invention include those having Formula HI:
  • R 1 and R 2 are independently selected from the group consisting of H, alkyl, aryl, and alkylaryl;
  • R 3 is selected from the group consisting of
  • R 5 is selected from the group consisting of H, COO " , alkyl, aryl, alkylaryl, heteroaryl, alkyl heteroaryl,
  • R 6 is selected from the group consisting of H, alkyl, aryl, alkyl aryl, heteroaryl, urea, carbamoyl, and
  • R 7 is H, acyl, alkyl, arylacyl, heteroacyl
  • R 8 is H, alkyl, aryl, alkylaryl, or heteroaryl
  • R 9 is selected from the group consisting of R ,
  • R 11 is selected from the group consisting of alkyl, aryl, heteroaryl, acylalkyl, Cl, NO 2 , Br, I, CF 3 , S alkyl, H, OH, O alkyl, SH, COO-R 6 , and
  • R 14 and R 15 are each independently selected from the group consisting of R 3 '
  • X 2 is O or N-R 6 ;
  • a and B are independently selected from the group consisting of H, OH, O alkyl, NH-R 6 , NH-R 7 , and guanidino.
  • Enzyme inhibition generally involves the interaction of a substance with an enzyme so as to decrease the rate of the reaction catalyzed by that enzyme.
  • Inhibitors can be classified according a number of criteria. For example, they may be reversible or irreversible. An irreversible inhibitor dissociates very slowly, if at all, from its target enzyme because it becomes very tightly bound to the enzyme, either covalently or noncovalently. Reversible inhibition, in contrast, involves an enzyme-inhibitor complex which may dissociate. Inhibitors can also be classified according to whether they are competitive, noncompetitive or uncompetitive inhibitors.
  • the enzyme can bind either the substrate or the inhibitor, but not both.
  • competitive inhibitors resemble the substrate or the product(s) and bind the active site of the enzyme, thus blocking the substrate from binding the active site.
  • a competitive inhibitor diminishes the rate of catalysis by effectively reducing the affinity of the substrate for the enzyme.
  • an enzyme may be competitively inhibited by its own product because of equilibrium considerations. Since the enzyme is a catalyst, it is in principle capable of accelerating a reaction in the forward or reverse direction.
  • Noncompetitive inhibitors allow the enzyme to bind the substrate at the same time it binds the inhibitor.
  • a noncompetitive inhibitor acts by decreasing the turnover number of an enzyme rather than diminishing the proportion of free enzyme.
  • Another possible category of inhibition is mixed or uncompetitive inhibition, in which the inhibitor affects the binding site and also alters the turnover number of the enzyme.
  • Enzyme inhibition of kinetically complex systems involving more than one substrate as is the case for glycosyltransferases, are described in Segel, Enzyme Kinetics, (Wiley, N.Y. 1975), which is incorporated herein by reference.
  • Sialyltransferase activity and its inhibition is typically assayed according to standard methods for determining enzyme activity.
  • enzyme assays see, Rossomando, "Measurement of Enzyme Activity" in Guide to Protein
  • An assay for sialyltransferase activity typically contains a buffered solution adjusted to physiological pH, a source of divalent cations, a donor substrate (usually labeled CMP-sialic acid), an acceptor substrate (e.g., LacNAc or Gal ⁇ l,3GalNAc), sialyltransferase (typically ST6Gal or ST3Gal), and the sample or fraction of a sample whose inhibitory activity is to be tested.
  • a donor substrate usually labeled CMP-sialic acid
  • an acceptor substrate e.g., LacNAc or Gal ⁇ l,3GalNAc
  • sialyltransferase typically ST6Gal or ST3Gal
  • sialylated product is isolated and measured according to standard methods (e.g., in a scintillation counter).
  • Sialyltransferase assays which use a UV-labeled acceptor and lead to a UV-labeled product that can be readily separated by reverse phase HPLC and quantitated by UV spectroscopy are described in Schaub et al. (1998) Glycoconjugate J. 15: 345-354. See also, Kajihara et al, Carbohydr. Res. 1994, 264, C1-C5; J. Org. Chem. 1995, 60, 5732-
  • Inhibition of sialyltransferase activity in an assay as defined herein refers to a decrease in enzyme specific activity of at least about 70%, more preferably at least about 90%.
  • Sialyltransferase inhibitors of the invention can be further tested for their ability to inhibit inflammatory and other disease responses in laboratory animals.
  • Animals can be treated with pharmacological doses of the blocking agent to block addition of sialic acid to cell surface carbohydrates of leukocytes and other cells.
  • the target condition involves SLe x -mediated cell adhesion
  • animals can then be tested for susceptibility for neutrophil infiltration into sites of irLflammation (Mourshargh et al, J. Immunol. 142:3193-98 (1989), reperfusion injury (Vedder et al, Proc. Natl Acad. Sci. USA 87:2643-46) and other animal models suitable for assay of leukocyte induced tissue damage.
  • Assays that are suitable for testing the effect of a sialyltransferase inhibitor on other types of immune responses include, for example, B cell proliferation assays, CTL activation assays, and the like. Such assays are described in, for example, Hennet et al. (1998) Proc. Nat 7. Acad. Sci. USA 95: 4504-4509. Uses for Sialyltransferase Inhibitors
  • the invention also provides methods of inhibiting sialyltransferase-catalyzed synthesis of a sialylglycoside by contacting a sialyltransferase with a compound of the invention.
  • the methods can inhibit the synthesis of many sialic acid-containing compounds.
  • sialyl galactosides which include biologically significant oligosaccharides such as Sia ⁇ 2-6Gal ⁇ l-4Glc ⁇ Ac, Sia ⁇ 2,3Gal ⁇ l-3GalNAc, Siacx2,3Gal ⁇ l-4GlcNAc and the like.
  • Compounds of particular interest for inhibition include, for example, a SLe x or SLe a moiety.
  • the methods involve contacting a sialyltransferase which, in the absence of a sialyltransferase inhibitor, can link the sialic acid to an acceptor moiety with an effective amount of a sialyltransferase inhibitor compound
  • compositions and methods of the present invention find use in both therapeutic and diagnostic applications.
  • the sialyltransferase inhibitors which can act as substrate analogs, are used for in vitro diagnosis of cells, (e.g., cancer cells) that express the particular sialyltransferase of interest.
  • the response of the cells to a biologically effective dose of the agent can then be determined.
  • the sialyltransferase inhibitor compounds of the invention also find use therapeutically to selectively inhibit sialyltransferase activity associated with a variety of immune responses.
  • the inhibitors of the invention can be used to inhibit deleterious immune responses associated with autoimmune disease, graft rejection and allergies. Inappropriate activation of the immune system is a component of a number of immunopathologies, such as autoimmunity, allograft rejection and allergic responses.
  • autoimmune diseases include rheumatoid arthritis, multiple sclerosis, and myasthenia gravis.
  • Allergic responses include allergies to various pollens, dust mites and the like.
  • foreign infectious diseases may cause immunopathology (e.g., lyme disease, hepatitis, LCMV, post-streptococcal endocarditis, or glomerulonephritis).
  • immunopathology e.g., lyme disease, hepatitis, LCMV, post-streptococcal endocarditis, or glomerulonephritis.
  • Food hypersensitivities such as celiac disease and Crohn's disease, as well as other allergic diseases, have been associated with inappropriate immune responses or suspected of having an autoimmune component.
  • compositions of the present invention include, e.g., rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), septic shock, and acute and chronic inflammation, including atopic dermatitis and psoriasis.
  • reperfusion injury the blocking agents are ideally used prophylactically prior to heart surgery to enhance post-surgical recovery.
  • tumor metastasis can be prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.
  • the sialyltransferase inhibitors of the invention are administered to an individual already suffering from an inappropriate or undesirable immune response.
  • Compositions that contain the sialyltransferase inhibitors are administered to a patient in an amount sufficient to suppress the undesirable Immune response and to cure or at least partially arrest symptoms and/or complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the inhibitor composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the inhibitors, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these compositions.
  • the dose of the sialyltransferase inhibitor of the invention for treatment of inflammatory disease will vary according to, e.g., the particular inhibitor, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician.
  • compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
  • topical application non-sprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water are typically used.
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • the compounds are preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of blocking agents are 0.1%- 10% by weight, preferably l%-5%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • esters or partial esters of fatty acids conta ⁇ ig from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arbitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters.
  • an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arbitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure.
  • suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed.
  • a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
  • compositions for intravenous administration which comprise a solution of the sialyltransferase inhibitor dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • concentration of the sialyltransferase inhibitor that can be combined to form a "cocktail" under certain circumstances for increased efficacy in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 1% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intravenous irrfusion could be made up to contain 250 ml of sterile Ringer's solution, and a unit dosage comprising 2-2,000 mg of the compound.
  • Actual methods for preparing parenterally admmistrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remingtons Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA (1990).
  • conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more sialyltransferase inhibitors of the invention, preferably 15%.
  • the sialyltransferase inhibitors of the invention can also be administered via liposomes, which serve to target the conjugates to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • a desired peptide or conjugate of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected sialyltransferase inhibitor compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospho lipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
  • the targeting of liposomes using a variety of targeting agents is well known in the art (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044).
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide or conjugate may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of admiriistration, the conjugate being delivered, and the stage of the disease being treated.
  • compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.
  • compositions are a ⁇ lministered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Unit dosages effective for this use will depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.5 mg to about 10 g of glycosyltransferase inhibitor for a 70 kg patient, usually from about 10 mg to about 5 g, and preferably between about 2 mg and about 1 g.
  • Therapeutic administration may begin at the first sign of disease or the detection or shortly after diagnosis in the case of immune disorder.
  • compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically effective dose.”
  • the inhibitor compounds are administered to risk groups. In this use, the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.5 mg to about 10 g of glycosyltransferase inhibitor for a 70 kg patient, usually from about 10 mg to about 5 g, and preferably between about 2 mg and about 1 g.
  • Single or multiple adrninistrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of inhibitor of this invention sufficient to effectively treat the patient.
  • the effect of administration of the sialyltransferase inhibitors of the invention can be monitored by detecting the levels of sialylgalactosides in a sample from a patient. This can be performed according to standard methods for detection of desired carbohydrate structures. For instance, specific lectins or antibodies raised against the ligand can be used.
  • CD22 or a moiety having the Sia6LacNAc binding activity of CD22 can also be used to detect the presence or absence of Sia ⁇ LacNAc.
  • Immune disorders that involve CTLs can be detected using an agent that binds to ⁇ 2,3 sialylgalactosides.
  • a suitable binding agent for ⁇ 2,3 is the MAL II lectin, which can be isolated from Maackia amurensis seeds.
  • Glycosyltransferases themselves, in particular the acceptor binding domain of a glycosyltransferase, are also useful as binding moieties in the diagnostic assays of the invention.
  • concentration of acceptor moieties tends to increase.
  • ST6Gal sialyltransferase causes a dramatic increase in terminal galactose residues (i.e., Gal ⁇ l,4GlcNAc-) on B cells.
  • ST6Gal sialyltransferase as a detection moiety to determine whether ST6Gal is deficient in the cells.
  • An ST3Gal transferase can be used similarly as a detection moiety.
  • the detection moieties are labeled with a detectable label.
  • the detectable labels can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, as is common in immunological labeling).
  • Primary and secondary labels can include undetected elements as well as detected elements.
  • Useful primary and secondary labels in the present invention can include spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyesTM, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, 32 P, 33 P, etc.), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc.), spectral colorimetric labels such as colloidal gold or colored glass or plastic (e.g.
  • fluorescent dyes e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM
  • the label may be coupled directly or indirectly to a component of the detection assay (e.g., the detection reagent) according to methods well known in the art.
  • a component of the detection assay e.g., the detection reagent
  • a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Preferred labels include those that use: 1) cher luminescence (using horseradish peroxidase or luciferase) with substrates that produce photons as breakdown products as described above) with kits being available, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim, and Life Technologies/ Gibco BRL; 2) color production (using both horseradish peroxidase and/or alkaline phosphatase with substrates that produce a colored precipitate [kits available from Life Technologies/Gibco BRL, and Boehringer-Mannheim]); 3) hemifluorescence using, e.g., alkaline phosphatase and the substrate AttoPhos [Amersham] or other substrates that produce fluorescent products, 4) fluorescence (e.g., using Cy-5 [Amersham]), fluorescein, and other fluorescent tags]; 5) radioactivity.
  • kits being available, e.g., from Molecular Probes, Amersham, Boehringer-Mann
  • Preferred enzymes that can be conjugated to detection reagents of the invention include, e.g., luciferase, and horse radish peroxidase.
  • the chemiluminescent substrate for luciferase is luciferin.
  • Embodiments of alkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP), which is detected with a spectrophotometer; 5-bromo-4- chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) and fast red napthol AS-TR phosphate, which are detected visually; and 4-methoxy-4-(3-phosphonophenyl) spiro[l,2- dioxetane-3,2'-adamantane], which is detected with a luminometer.
  • Embodiments of horse radish peroxidase substrates include 2,2'azino-bis(3-emylbenzthiazoline-6 sulfonic acid)
  • ABTS 5-aminosalicylic acid
  • 5AS 5-aminosalicylic acid
  • OPD o-dianisidine
  • TMB 3,3,5,5'-tetramethylbenzidine
  • DAB 3,3'diaminobenzidine
  • AEC 3-amino-9-ethylcarbazole
  • 4C1N 4-chloro-l-naphthol
  • Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
  • detection moieties that are suitable for use in the methods of the invention include SNA-fluorescein isothiocyanate (FITC) lectin (FL-1301, Vector Laboratories, Burlingame CA) and biotinylated SNA lectin (B-1305, Vector Laboratories) for cx2,3 sialyl galactosides.
  • FITC fluorescein isothiocyanate
  • B-1305 Biotinylated SNA lectin
  • MAL II- FITC lectin and biotinylated MAL II lectin B-1265, Vector Laboratories
  • Efficacy of a treatment regime is indicated by a substantial reduction of ⁇ 2,3- or ⁇ .2,6-sialylgalactosides in a sample, e.g., lymphocytes, obtained from the patient
  • a sample e.g., lymphocytes
  • methods for detecting levels of specific glycosyltransferase activities can be used.
  • Standard assays for detecting glycosyltransferases such as the ST6Gal and ST3Gal I are known to those of skill in the art.
  • treatment efficacy is indicated by a substantial reduction in activity of the particular glycosyltransferase.
  • a "substantial reduction" in the appropriate sialylgalactoside levels or glycosyltransferase activity refers to a reduction of at least about 30% in the test sample compared to a non-immunodeficient control.
  • the reduction will be at least about 50%, more preferably at least about 75%o, and most preferably sialylgalactoside or glycosyltransferase levels will be reduced by at least about 90%) in a sample from a treated mammal compared to an untreated control.
  • This Example addresses two questions.
  • the first question is whether the neuraminyl residue as shown in (R)-2 ( Figure 2) is required for sialyltransferase inhibition, or whether it can be replaced by a simple aryl, heteroaryl, or related moiety, thus making these type of compounds more readily available.
  • the second question is whether a different geometry of the substituents around the anomeric center increases binding affinity.
  • This compound exhibited very potent competitive inhibition of (2-6)-sialyltransferase with a K value of 40 nm (Table 2), and thus has three orders of magnitude higher affinity for ⁇ (2-6)-sialyltransferase than the natural substrate CMP-Neu5 Ac. This compound also has a similar affinity for a recombinant rat liver ⁇ (2-3)-sialyltransferase.
  • potent sialyltransferase inhibition can be based on flat pyranosyl ring mimics which possess a methyl or a methylene carbon bearing a phosphonate and a CMP residue, as clearly exhibited for the series 1, (i?)-2,3h and (E)-8,(Z)-9.
  • Selected properties of 3h,l; 4h,l; (R)-6; (5)-6; (R)-7; (S)-7, (Z)-9, (R)-13, (S)-13, and ( ⁇ )-ll are provided in Table 3.
  • Table 3 Selected physical data of 3h,l; 4h,l; (R)-6; (S)-6; (R)-7; (S)-7, (Z)-9, (R)-13, (S)-13, and (E)-14.
  • This Example demonstrates that transition state analogues derived from S ⁇ l- type sialyltransfer with CMP-Neu5Ac as donor substrate are useful as sialyltransferase inhibitors.
  • the synthesized ⁇ -hydroxyphosphonate and ⁇ -hydroxycarboxylate derivatives 3h,l, 4h,l, (R)-, (S)-6, and (R)-, (S)-7 have flat pyranyl ring mimics which are connected to the ⁇ -carbon, and a CMP residue which is attached to the ⁇ -hydroxy group.
  • These type of compounds can be readily synthesized from the corresponding ⁇ -hydroxy acids.

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Abstract

L'invention concerne des inhibiteurs puissants de sialyltransférase. Ces inhibiteurs sont utiles pour inhiber la synthèse de glycosides sialylés. On peut, de ce fait, mettre en application ces inhibiteurs de sialyltransférase afin de moduler des processus biologiques dans lesquels joue un rôle l'adhésion cellulaire provoquée par l'intermédiaire de sialylglycosides.
PCT/EP1999/005697 1998-08-07 1999-08-06 Inhibiteurs de sialyltransferase WO2000008040A1 (fr)

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WO2001039804A2 (fr) * 1999-12-06 2001-06-07 Abbott Gmbh & Co. Kg Inhibiteurs de la gd3-synthase pour le traitement de troubles neuropathologiques
US8940719B2 (en) 2006-07-03 2015-01-27 Academia Sinica Lithocholic acid analogues that inhibit sialyltransferase

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JPH07145191A (ja) * 1993-11-24 1995-06-06 Japan Tobacco Inc ガラクトシルホスフォネート誘導体
WO1998025940A1 (fr) * 1996-12-10 1998-06-18 The Scripps Research Institute Inhibiteurs des glycosyltransferases

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WO1998025940A1 (fr) * 1996-12-10 1998-06-18 The Scripps Research Institute Inhibiteurs des glycosyltransferases

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001039804A2 (fr) * 1999-12-06 2001-06-07 Abbott Gmbh & Co. Kg Inhibiteurs de la gd3-synthase pour le traitement de troubles neuropathologiques
WO2001039804A3 (fr) * 1999-12-06 2002-04-11 Knoll Ag Inhibiteurs de la gd3-synthase pour le traitement de troubles neuropathologiques
US8940719B2 (en) 2006-07-03 2015-01-27 Academia Sinica Lithocholic acid analogues that inhibit sialyltransferase

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