U.S. patent application number 09/833327 was filed with the patent office on 2002-01-17 for colon cancer kh-1 and n3 antigens.
Invention is credited to Danishefsky, Samuel J., Deshpande, Prashant P., Kim, Hyun Jin, Kim, In Jong, Livingston, Philip, Park, Tae Kyo, Ragupathi, Govindaswami.
Application Number | 20020006900 09/833327 |
Document ID | / |
Family ID | 21879664 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006900 |
Kind Code |
A1 |
Danishefsky, Samuel J. ; et
al. |
January 17, 2002 |
Colon cancer KH-1 and N3 antigens
Abstract
The present invention provides processes for the preparation of
the KH-1 and N3 antigens, as well as related analgoues thereof,
which are useful as anticancer therapeutics. The present invention
also provides various intermediates useful in the preparation of
KH-1 and N3 and analogues thereof. Additionally, the invention
provides various compositions comprising any of the analogues of
KH-1 and N3 available through the methods of the invention and
pharmaceutical carriers useful in the treatment of subjects
suffering from various forms of epithelial cancer.
Inventors: |
Danishefsky, Samuel J.;
(Englewood, NJ) ; Deshpande, Prashant P.;
(Plaindome, NJ) ; Kim, In Jong; (Seoul, KR)
; Livingston, Philip; (New York, NY) ; Kim, Hyun
Jin; (New York, NY) ; Ragupathi, Govindaswami;
(New York, NY) ; Park, Tae Kyo; (Taejon,
KR) |
Correspondence
Address: |
Choate, Hall & Stewart
Exchange Place
53 State Street
Boston
MA
02109
US
|
Family ID: |
21879664 |
Appl. No.: |
09/833327 |
Filed: |
April 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09833327 |
Apr 12, 2001 |
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09042280 |
Jan 13, 1998 |
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6238668 |
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60034950 |
Jan 13, 1997 |
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Current U.S.
Class: |
424/184.1 ;
514/15.2; 514/19.5; 514/54; 530/322; 530/395; 536/53 |
Current CPC
Class: |
A61K 47/646 20170801;
A61P 35/00 20180101; A61K 47/643 20170801; C07H 15/10 20130101 |
Class at
Publication: |
514/8 ; 536/53;
514/54; 530/322; 530/395 |
International
Class: |
A61K 038/16; A61K
031/726 |
Goverment Interests
[0002] This invention was made with government support under grants
CA-28824-18, GM-15240-02, GM-16291-01, HL-25848-14 and AI-16943
from the National Institutes of Health. Accordingly, the U.S.
Government has certain rights in the invention.
Claims
What is claimed is:
1. A compound having the structure: 66wherein R is H, substituted
or unsubstituted alkyl, aryl or allyl, or an amino acyl moiety, an
amino acyl residue of a peptide, an amino acyl residue of a
protein, which amino acyl moiety or residue bears an .omega.-amino
group or an .omega.-(C.dbd.O)-- group, which group is linked to O
via a polymethylene chain having the structure
--(CH.sub.2).sub.s--, where s is an integer between about 1 and
about 9, or a moiety having the structure: 67and wherein r, m and n
are independently 0, 1, 2 or 3.
2. The compound of claim 1 having the structure: 68
3. The compound of claim 1 wherein the protein is bovine serum
albumin or KLH.
4. A compound having the structure: 69wherein r is 0, 1, 2, 3 or
4.
5. The compound of claim 4 wherein r is 1.
6. A method of preparing a trisaccharide iodosulfonamide having the
structure: 70which comprises: (a) (i) coupling a disaccharide
glycal with an epoxide having the structure: 71under suitable
conditions to form a trisaccharide intermediate; and (ii)
etherifying the trisaccharide intermediate with a suitable
protecting agent to form a trisaccharide glycal having the
structure: 72and (b) reacting the trisaccharide glycal formed in
step (c) with an iodosulfonamidating agent under suitable
conditions to form the trisaccharide iodosulfonamide.
7. The method of claim 6 wherein the disaccharide glycal having the
structure: 73is prepared by a process which comprises: (a)
protecting a glucal having the structure: 74with a silylating agent
under suitable conditions to form a protected glucal having the
structure: 75(b) (i) alkylating the protected glucal formed in step
(a) with a fucosylfluoride having the structure: 76and (ii)
deprotecting under suitable conditions to form the disaccharide
glycal.
8. The method of claim 7 wherein the silylating agent in step (a)
is triphenylsilyl chloride.
9. The method of claim 7 wherein the alkylating step is effected in
the presence of an ionizing salt, and the ionizing salt is
AgClO.sub.4.
10. The method of claim 7 wherein the conditions of the
deprotecting step comprise a base.
11. The method of claim 10 wherein the base is potassium
carbonate.
12. The method of claim 6 wherein the conditions of the coupling
comprise an acid.
13. The method of claim 6 wherein the acid is a Lewis acid.
14. The method of claim 13 wherein the Lewis acid is zinc
dichloride.
15. The method of claim 7 wherein the protecting agent is
TBSOTf.
16. The method of claim 6 wherein the iodosulfonamidating agent of
step (b) comprises I(coll).sub.2ClO.sub.4 and and
PhSO.sub.2NH.sub.2.
17. A method of preparing a disaccharide stannane having the
structure: 77which comprises: (a) (I) deprotecting a disaccharide
glucal having the structure: 78under suitable conditions to form a
deprotected intermediate; and (ii) selectively reprotecting the
deprotected intermediate with levulinic acid under suitable
conditions to form a disaccharide levulinate having the structure:
79and (b) reacting the disaccharide levulinate formed in step (a)
with a distannyl oxide having the formula (R.sub.3Sn).sub.2O,
wherein R is linear or branched chain alkyl or aryl, under suitable
conditions to form the disaccharide stannane.
18. The method of claim 17 wherein the conditions of the
deprotecting step comprise a fluoride salt.
19. The method of claim 18 wherein the fluoride salt is a
tetraalkylammonium fluoride.
20. The method of claim 19 wherein the tetraalkylammonium fluoride
salt is tetra-n-butylammonium fluoride.
21. The method of claim 17 wherein the conditions of the
reprotecting step comprise 2-chloro-1-methylpyridinium iodide.
22. The method of claim 17 wherein R is n-Bu.
23. A method of preparing a disaccharide ethylthioglycoside having
the structure: 80which comprises: (a) (i) protecting a disaccharide
glucal having the structure: 81with a suitable protecting agent to
form a protected disaccharide glucal; and (ii) reacting the
protected disaccharide glucal under suitable conditions with an
iodosulfonamidating agent to form a disaccharide iodosulfonamide
having the structure: 82and (b) treating the disaccharide
iodosulfonamide formed in step (a)(ii) with ethanethiol under
suitable conditions to form the disaccharide
ethylthioglycoside.
24. The method of claim 23 wherein the disaccharide glucal is
prepared by a process which comprises: (a) alkylating a protected
glucal having the structure: 83with a fucosyl fluoride having the
structure: 84under suitable conditions to form the disaccharide
glucal.
25. The method of claim 24 wherein the conditions of the alkylating
step comprise an ionizing salt.
26. The method of claim 25 wherein the ionizing salt is
AgClO.sub.4.
27. The method of claim 23 wherein the protecting agent is
PMBCl.
28. The method of claim 23 wherein the iodosulfonamidating agent in
step (b)(ii) comprises I(coll).sub.2ClO.sub.4 and
PhSO.sub.2NH.sub.2.
29. The method of claim 23 wherein the conditions of the treating
step comprise a base.
30. The method of claim 29 wherein the base is LHMDS.
31. A method of preparing an N3 allyl glycoside having the
structure: 85which comprises: (a) desilylating a protected N3
glycal having the structure: 86under suitable conditions to form a
desilylated N3 glycal; (b) deprotecting the desilylated N3 glycal
formed in step (a) under suitable conditions to form a deprotected
N3 glycal; (c) treating the deprotected N3 glycal formed in step
(b) with acetic anhydride in the presence of a suitable catalyst to
form an N3 glycal acetate; (d) epoxidizing the N3 glycal acetate
formed in step (c) with an oxygen transfer agent under suitable
conditions to form an N3 glycal epoxyacetate; (e) cleaving the N3
glycal epoxyacetate formed in step (d) with allyl alcohol under
suitable conditions to form an N3 glycal allyl ether; and (f)
saponifying the N3 glycal allyl ether under suitable conditions to
form the N3 allyl glycoside.
32. The method of claim 31 wherein the protected N3 glycal is
prepared by a process which comprises coupling an
ethylthioglycoside having the structure: 87with a heptasaccharide
glycal having the structure: 88wherein R.sub.1 and R.sub.2 are Ac
and R.sub.3 is H, in the presence of an alkylating agent under
suitable conditions to form the protected N3 glycal.
33. The method of claim 32 wherein the alkylating agent is
MeOTf.
34. The method of claim 32 wherein the conditions of the
desilylating step comprise a fluoride salt.
35. The method of claim 34 wherein the fluoride salt is a
tetraalkylammonium fluoride.
36. The method of claim 35 wherein the tetraalkylammonium fluoride
is tetra-n-butylammonium fluoride.
37. The method of claim 31 wherein the catalyst in the treating
step is 2-N,N-dimethylaminopyridine.
38. The method of claim 31 wherein the oxygen transfer agent is
3,3-dimethyidioxirane.
39. A method of preparing a heptasaccharide glycal diacetate
intermediate having the structure: 89wherein R.sub.1 and R.sub.2
are Ac and R.sub.3 is H, which comprises: (a) (i) monoacylating a
heptasaccharide glycal having the structure: 90wherein R.sub.1 and
R.sub.2 are H and R.sub.3 is PMB; with acyl anhydride in the
presence of a catalyst under suitable conditions to form a
heptasaccharide glycal monoacetate; (ii) treating the
heptasaccharide glycal monoacetate formed in step (a)(i) with an
acyl anhydride in the presence of a catalyst under conditions
suitable to form a heptasaccharide glycal diacetate; (iii)
deprotecting the heptasaccharide glycal diacetate under suitable
conditions to form the heptasaccharide glycal diacetate
intermediate.
40. The method of claim 39 wherein the heptasaccharide glycal is
prepared by a process which comprises: (a) (i) reacting a
trisaccharide iodosulfonamide having the structure: 91with a
disaccharide stannane having the structure: 92under suitable
conditions; and (ii) deprotecting under suitable conditions to form
a pentasaccharide glycal having the structure: 93and (b) coupling
the pentasaccharide glycal formed in step (a) with an
ethylthioglycoside having the structure: 94under suitable
conditions to form the heptasaccharide glycal.
41. The method of claim 40 wherein the conditions of the reacting
step comprise an ionizing agent.
42. The method of claim 41 wherein the ionizing agent is
AgBF.sub.4.
43. A method of preparing a protected disaccharide having the
structure: 95wherein R.sub.0 is C.sub.1-9 linear or branched chain
alkyl, arylalkyl, trialkylsilyl, aryldialkylsilyl,
diarylalkylsilyl, and triarylsilyl, which comprises: (a) (i)
epoxidizing a galactal carbonate having the structure: 96with an
oxygen transfer agent under suitable conditions to form an epoxide
galactal; and (ii) coupling the epoxide galactal formed in step (a)
(i) with a doubly protected galactal having the structure: 97under
suitable conditions to form a disaccharide carbonate having the
structure: 98and (b) saponifying the disaccharide carbonate formed
in step (a) (ii) under suitable conditions to form the protected
disaccharide.
44. The method of claim 43 wherein the galactal carbonate is
prepared by a process which comprises: (a) protecting a galactal
having the structure: 99with an alkylating agent under suitable
conditions to form a first protected galactal; and (b) treating the
first protected galactal formed in step (a) with a
carbonate-forming reagent under conditions suitable to form the
galactal carbonate.
45. The method of claim 44 wherein the carbonate-forming reagent is
(Im).sub.2CO/DMAP.
46. The method of claim 43 wherein the doubly protected galactal is
prepared by a process which comprises: (a) protecting a second
galactal having the structure: 100with an alkylating agent under
conditions suitable to form a second protected galactal; and (b)
protecting the second protected galactal formed in step (a) with an
alkylating agent which may be the same or different from that of
step (a) under conditions suitable to form the doubly protected
galactal.
47. The method of claim 46 wherein each alkylating agent is
independently an alkyl, arylalkyl, trialkylsilyl, aryldialkylsilyl,
diarylalkylsilyl or triarylsilyl halide or triflate.
48. The method of claim 47 wherein the alkylating agent is benzyl
bromide.
49. The method of claim 47 wherein the alkylating agent is
TES-Cl.
50. The method of claim 43 wherein the oxygen transfer agent is
DMDO.
51. The method of claim 43 wherein the conditions of the coupling
step comprise ZnCl.sub.2 in THF.
52. The method of claim 43 wherein the conditions of the
saponifying step comprise K.sub.2CO.sub.3 in methanol.
53. A method of preparing an ethylthioglycoside having the
structure: 101wherein R is C.sub.1-9 linear or branched chain
alkyl, arylalkyl, trialkylsilyl, aryldialkylsilyl,
diarylalkylsilyl, and triarylsilyl, which comprises: (a) treating a
protected disaccharide carbonate having the structure: 102with an
iodosulfonamidating agent under suitable conditions to form a
disaccharide iodosulfonamidate having the structure: 103and (b)
reacting the disaccharide iodosulfonamidate formed in step (a) with
ethylthiol under suitable conditions to form the
ethylthioglycoside.
54. The method of claim 53 wherein the protected disaccharide
carbonate is prepared by a process which comprises alkylating a
disaccharide carbonate having the structure: 104with an alkylating
agent under suitable conditions to form the protected disaccharide
carbonate.
55. The method of claim 54 wherein the alkylating agent is an
alkyl, arylalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl
or triarylsilyl halide or triflate.
56. The method of claim 55 wherein the alkylating agent is
TES-Cl.
57. The method of claim 53 wherein the iodosulfonamidating agent is
I(coll).sub.2ClO.sub.4 and PhSO.sub.2NH.sub.2.
58. A method of preparing an ethylthioglycoside having the
structure: 105which comprises: (a) acylating a disaccharide
carbonate having the structure: 106under suitable conditions to
form an acylated disaccharide carbonate having the structure:
107(b) treating the acylated disaccharide carbonate formed in step
(a) with an iodosulfonamidating agent under suitable conditions to
form a disaccharide iodosulfonamidate having the structure: 108(c)
reacting the iodosulfonamidate formed in the step (b) with ethyl
thiol under suitable conditions to form the ethylthioglycoside.
59. The method of claim 58 wherein the conditions of the acylating
step comprise acetic anhydride/pyridine.
60. The method of claim 58 wherein the iodosulfonamidating agent is
I(coll).sub.2ClO.sub.4 and PhSO.sub.2NH.sub.2.
61. A method of preparing a protected hexasaccharide having the
structure: 109which comprises: (a) reacting a protected
tetrasaccharide having the structure: 110with an ethylglycoside
having the structure: 111under suitable conditions to form a
hexasaccharide intermediate; and (b) acetylating the hexasaccharide
intermediate formed in step (a) under suitable conditions to form
the protected hexasaccharide.
62. The method of claim 61 wherein the protected tetrasaccharide is
prepared by a process which comprises: (a) coupling an
ethythioglycoside having the structure: 112with a protected
disaccharide having the structure: 113under suitable conditions to
form a protected tetrasaccharide carbonate; and (b) saponifying the
protected tetrasaccharide carbonate formed in step (a) under
suitable conditions to form the protected tetrasaccharide.
63. The method of claim 62 wherein the conditions of the coupling
step comprise MeOTf/MS.
64. The method of claim 62 wherein the conditions of the
saponifying step comprise K.sub.2CO.sub.3 in methanol.
65. A method of preparing a protected nonasaccharide having the
structure: 114which comprises: (a) deprotecting a protected
hexasaccharide having the structure: 115under suitable conditions
to form a partially deprotected hexasaccharide; and (b) coupling
the partially deprotected hexasaccharide formed in step (a) with a
fucosylfluoride having the structure: 116in the presence of an
organometallic reagent under suitable conditions to form the
protected nonasaccharide.
66. The method of claim 65 wherein the conditions of the
deprotecting step comprise a fluoride salt.
67. The method of claim 66 wherein the fluoride salt is a
tetraalkylammonium fluoride.
68. The method of claim 67 wherein the fluoride salt is TBAF.
69. The method of claim 65 wherein the organometallic reagent is
Sn(OTf).sub.2/DTBP.
70. A method of preparing a protected nonasaccharide ceramide
having the structure: 117which comprises: (a) epoxidizing a
protected nonasaccharide having the structure: 118with an oxygen
transfer agent under suitable conditions to form a protected
nonasaccharide epoxide; (b) coupling the protected nonasaccharide
epoxide formed in step (a) with an azide having the structure:
119under suitable conditions to form a nonasaccharide azide
intermediate; (c) reductively acylating the azide intermediate with
palmitic anhydride under suitable conditions to form a protected
nonasaccharide ceramide; (d) reducing the protected nonasaccharide
ceramide formed in step (c) under suitable conditions to form a
deprotected nonasaccharide ceramide; (e) acylating the deprotected
nonasaccharide ceramide under suitable conditions to form an
acylated nonasaccharide ceramide; and (f) saponifying the acylated
nonasaccharide ceramide under suitable conditions to form the
nonasaccharide ceramide.
71. The method of claim 70 wherein the oxygen transfer agent is
DMDO.
72. The method of claim 70 wherein the conditions of the coupling
step comprise ZnCl.sub.2.
73. The method of claim 70 wherein the azide intermediate is
reductively acylated in step (c) in the presence of Lindlar's
catalyst.
74. The method of claim 70 wherein conditions of the saponifying
step comprise MeONa in methanol.
75. A method of inducing antibodies in a subject, wherein the
antibodies are capable of specifically binding with epithelial
tumor cells, which comprises administering to the subject an amount
of a compound which contains a determinant having a structure
selected from the group consisting of: 120which amount is effective
to induce antibodies.
76. The method of claim 75 wherein the compound is bound to a
suitable carrier protein, said compound being bound either directly
or by a cross-linker selected from the group consisting of a
succinimide and an M.sub.2 linker.
77. The method of claim 75 wherein the compound contains a KH-1 or
N3 epitope.
78. The method of claim 76 wherein the carrier protein is bovine
serum albumin, polylysine or KLH.
79. The method of claim 76 wherein the compound is a KH-1 or N3
epitope.
80. The method of claim 75 which further comprises co-administering
an immunological adjuvant.
81. The method of claim 80 wherein the adjuvant is bacteria or
liposomes.
82. The method of claim 80 wherein the adjuvant is Salmonella
minnesota cells, bacille Calmette-Guerin or QS21.
83. The method of claim 75 wherein the epithelial tumor cells are
gastrointestinal tumor cells.
84. The method of claim 83 wherein the gastrointestinal tumor cells
are are colon tumor cells.
85. The method of claim 75 wherein the epithelial tumor cells are
lung tumor cells.
86. The method of claim 75 wherein the epithelial tumor cells are
prostate tumor cells.
87. A method of treating a subject suffering from an epithelial
cell cancer, which comprises administering to the subject an amount
of a compound which contains a determinant having a structure
selected from the group consisting of: 121which amount is effective
to treat the cancer.
88. The method of claim 87 wherein the compound is bound to a
suitable carrier protein, said compound being bound either directly
or by a cross-linker selected from the group consisting of a
succinimide and an M.sub.2 linker.
89. The method of claim 88 wherein the carrier protein is bovine
serum albumin, polylysine or KLH.
90. The method of claim 87 or 89 wherein the compound is contains a
KH-1 or N3 epitope.
91. The method of claim 87 or 90 which further comprises
co-administering an immunological adjuvant.
92. The method of claim 91 wherein the adjuvant is bacteria or
liposomes.
93. The method of claim 91 wherein the adjuvant is Salmonella
minnesota cells, bacille Calmette-Guerin or QS21.
94. A method of preventing recurrence of an epithelial cell cancer
in a subject which comprises vaccinating the subject with a
compound which contains a determinant having the structure: (a)
122which amount is effective to prevent recurrence of an epithelial
cell cancer.
95. The method of claim 94 wherein the compound is bound to a
suitable carrier protein.
96. The method of claim 94 wherein the carrier protein is bovine
serum albumin, polylysine or KLH.
97. The method of claim 94 which further comprises co-administering
an immunological adjuvant.
98. The method of claim 97 wherein the adjuvant is bacteria or
liposomes.
99. The method of claim 97 wherein the adjuvant is Salmonella
minnesota cells, bacille Calmette-Guerin or QS21.
100. The method of claim 75, 87 or 94 wherein the compound is
selected from the group consisting of: 123wherein R is H,
substituted or unsubstituted alkyl, aryl or allyl, or an amino acyl
moiety, an amino acyl residue of a peptide, an amino acyl residue
of a protein, which amino acyl moiety or residue bears an
.omega.-amino group or an .omega.-(C.dbd.O)-- group, which group is
linked to O via a polymethylene chain having the structure
--(CH.sub.2).sub.s--, where s is an integer between about 1 and
about 9, or a moiety having the structure: 124and wherein r, m and
n are independently 0, 1, 2 or 3.
101. A composition comprising a compound which contains a
determinant having a structure selected from the group consisting
of: 125and optionally an immunological adjuvant and/or a
pharmaceutically acceptable carrier.
102. The composition of claim 101 wherein the compound is bound to
a suitable carrier protein, said compound being bound either
directly or by a cross-linker selected from the group consisting of
a succinimide and an M.sub.2 linker.
103. The composition of claim 102 wherein the carrier protein is
bovine serum albumin, polylysine or KLH.
104. The composition of claim 101 or 103 wherein the compound
contains a KH-1 or N3 epitope.
105. The composition of claim 101 wherein the immunological
adjuvant is bacteria or liposomes.
106. The composition of claim 105 wherein the adjuvant is
Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
107. The composition of claim 106 wherein the compound has the
structure: (a) 126wherein R is H, substituted or unsubstituted
alkyl, aryl or allyl, or an amino acyl moiety, an amino acyl
residue of a peptide, an amino acyl residue of a protein, which
amino acyl moiety or residue bears an .omega.-amino group or an
.omega.-(C.dbd.O)-- group, which group is linked to O via a
polymethylene chain having the structure --(CH.sub.2).sub.s--,
where s is an integer between about 1 and about 9, or a moiety
having the structure: 127and wherein r, m and n are independently
0, 1, 2 or 3.
Description
[0001] This application is based on U.S. Provisional Application
Serial No. 60/034,950, filed Jan. 13, 1997, the contents of which
are hereby incorporated by reference into this application.
[0003] Throughout this application, citations for various
publications are provided. The disclosures of these publications
are hereby incorporated in their entirety by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
FIELD OF THE INVENTION
[0004] The present invention is in the field of tumor-specific
cell-surface antigens. In particular, the present invention relates
to processes for the preparation of KH-1 and N3 antigens and
analogues thereof which are useful as anticancer therapeutics. The
present invention also provides novel compositions of matter which
serve as intermediates for preparing the KH-1 and N3 antigens.
BACKGROUND OF THE INVENTION
[0005] The function of carbohydrates as structural materials and as
energy storage units in biological systems is well recognized. By
contrast, the role of carbohydrates as signaling molecules in the
context of biological processes has only recently been appreciated.
(M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K.
Singhal, S. Hakomori, J. C. Paulson, Science, 1990, 250, 1130; M.
J. Polley, M. L. Phillips, E. Wagner, E. Nudelman, A. K. Singhal,
S. Hakomori, J. C. Paulson, Proc. Natl. Acad. Sci. USA, 1991, 88,
6224; T. Taki, Y. Hirabayashi, H. Ishikawa, S. Kon, Y. Tanaka, M.
Matsumoto, J. Biol. Chem., 1986, 261, 3075; Y. Hirabayashi, A.
Hyogo, T. Nakao, K. Tsuchiya, Y. Suzuki, M. Matsumoto, K. Kon, S.
Ando, ibid., 1990, 265, 8144; O. Hindsgaul, T. Norberg, J. Le
Pendu, R. U. Lemieux, Carbohydr. Res., 1982, 109, 109; U. Spohr, R.
U. Lemieux, ibid., 1988, 174, 211)
[0006] The elucidation of the scope of carbohydrate involvement in
mediating cellular interaction is an important area of inquiry in
contemporary biomedical research. The carbohydrate molecules,
carrying detailed structural information, tend to exist as
glycoconjugates (cf. glycoproteins and glycolipids) rather than as
free entities. Given the complexities often associated with
isolating the conjugates in homogeneous form and the difficulties
in retrieving intact carbohydrates from these naturally occurring
conjugates, the applicability of synthetic approaches is apparent.
(For recent reviews of glycosylation see: Paulsen, H., Angew Chem.
Int. Ed. Engl., 1982, 21, 155; Schmidt, R. R., Angew. Chem. Int.
Ed. Engl., 1986, 25, 212; Schmidt, R. R., Comprehensive Organic
Synthesis, Vol. 6, Chapter 1 (2), Pergamon Press, Oxford, 1991;
Schmidt, R. R., Carbohydrates, Synthetic Methods and Applications
in Medicinal Chemistry, Part I, Chapter 4, VCH Publishers,
Weinheim, New York, 1992. For the use of glycals as glycosyl donors
in glycoside synthesis, see Lemieux, R. U., Can. J. Chem., 1964,
42, 1417; Lemieux, R. U., Faser-Reid, B., Can. J. Chem., 1965,
43:1460; Lemieux, R. U., Morgan, A. R., Can. J. Chem., 1965, 43,
2190; Thiem, J., Karl, H., Schwentner, J., Synthesis, 1978, 696;
Thiem. J. Ossowski, P., Carbohydr. Chem., 1984, 3, 287; Thiem, J.,
Prahst, A., Wendt, T. Liebigs Ann. Chem., 1986, 1044; Thiem, J., in
Trends in Synthetic Carbohydrate Chemistry, Horton, D., Hawkins, L.
D., McGarvey, G. L., eds., ACS Symposium Series #386, American
Chemical Society, Washington, D.C., 1989, Chapter 8.)
[0007] The carbohydrate domains of the blood group substances
contained in both glycoproteins and glycolipids are distributed in
erythrocytes, epithelial cells and various secretions. The early
focus on these systems centered on their central role in
determining blood group specificities. (R. R. Race and R. Sanger,
Blood Groups in Man, 6th ed., Blackwell, Oxford, 1975) However, it
is recognized that such determinants are broadly implicated in cell
adhesion and binding phenomena. (For example, see M. L. Phillips,
E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S. Hakomori,
J. C. Paulson, Science, 1990, 250:1130.) Moreover, ensembles
related to the blood group substances in conjugated form are
encountered as markers for the onset of various tumors. (K. O.
Lloyd, Am. J. Clinical Path., 1987, 87, 129; K. O. Lloyd, Cancer
Biol., 1991, 2:421) Carbohydrate-based tumor antigenic factors
might find applications at the diagnostic level, as resources in
drug delivery or ideally in immunotherapy. (Toyokuni, T., Dean, B.,
Cai, S., Boivin, D., Hakomori, S., and Singhal, A. K., J. Am. Chem
Soc., 1994, 116, 395; Dranoff, G., Jaffee, E., Lazenby, A.,
Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H.,
Paardoll, D., Mulligan, R., Proc. Natl. Acad. Sci. USA, 1993, 90,
3539; Tao, M. H., Levy, R., Nature, 1993, 362, 755; Boon, T., Int.
J. Cancer, 1993, 54, 177; Livingston, P. O., Curr. Opin. Immunol.,
1992, 4, 624; Hakomori, S., Annu. Rev. Immunol., 1984, 2, 103; K.
Shigeta, et al., J. Biol. Chem., 1987, 262, 1358)
[0008] The use of synthetic carbohydrate conjugates to elicit
antibodies was first demonstrated by Goebel and Avery in 1929.
(Goebel, W. F., and Avery, O. T., J. Exp. Med., 1929, 50, 521;
Avery, O. T., and Goebel, W. F., J. Exp. Med., 1929, 50, 533.)
Carbohydrates were linked to carrier proteins via the
benzenediazonium glycosides. Immunization of rabbits with the
synthetic antigens generated polyclonal antibodies. Other workers
(Allen, P. Z., and Goldstein, I. J., Biochemistry, 1967, 6, 029;
Rude, E., and Delius, M. M., Carbohydr. Res., 1968, 8, 219;
Himmelspach, K., et al., Eur. J. Immunol., 1971, 1, 106; Fielder,
R. J., et al., J. Immunol., 1970, 105, 265) developed similar
techniques for conjugation of carbohydrates to protein carriers.
Most of them suffered by introducing an antigenic determinant in
the linker itself, resulting in generation of polyclonal
antibodies. Kabat (Arakatsu, Y., et al., J. Immunol., 1966, 97,
858), and Gray (Gray, G. R., Arch. Biochem. Bioshys., 1974, 163,
426) developed conjugation methods that relied on oxidative or
reductive coupling, respectively, of free reducing
oligosaccharides. The main disadvantage of these techniques,
however, is that the integrity of the reducing end of the
oligosaccharide was compromised. In 1975 Lemieux described the use
an 8-carbomethoxy-1-octanol linker (Lemieux, R. U., et al., J. Am.
Chem. Soc., 1975, 97, 4076) which alleviated the problem of linker
antigenicity and left the entire oligosaccharide intact. Equally
effective in producing glycoconjugates was the allyl glycoside
method described by Bernstein and Hall. (Bernstein, M. A., and
Hall, L. D., Carbohydr. Res., 1980, 78, C1.) In this technique the
allyl glycoside of the deblocked sugar is ozonized followed by a
reductive workup. The resultant aldehyde is then reductively
coupled to a protein carrier with sodium cyanoborohydride.
[0009] In the mid-70's and early 80's Lemieux and his collaborators
made contributions to antibody production stimulated by synthetic
glycoconjugates (Lemieux, R. U., et al., J. Am. Chem. Soc., 1975,
97, 4076) and to conformational issues (Lemieux, R. U., et al.,
Can. J. Chem., 1979, 58, 631; Spohr, U., et al., Can. J. Chem.,
1985, 64, 2644; Vandonselaar, M., et al., J. Biol. Chem., 1987,
262, 0848) important in the interactions of the blood group
determinants (and analogues thereof) with the carbohydrate binding
proteins known as lectins. More recently, workers at Bristol-Myers
Squibb reported the X-ray crystal structure of the Lewis y epitope
complexed with the antibody BR96. (Jeffrey, P. D., et al., Nature
Structural Biol., 1995, 2, 466.) Two main components appear to
govern recognition between carbohydrates and most antibodies. The
first is multiple hydrogen bonding between the sugar hydroxyls and
the amino acid residues of Asp, Asn, Glu, Gln, and Arg. The second
major interaction is stacking between the sugar-ring faces and
aromatic side chains, which occurs most frequently with tryptophan.
In the complex with BR96 the most significant interactions involve
the latter; additional hydrogen bonding occurs between the sugar
hydroxyls and the indole nitrogens. Most antibody binding sites can
support about 6 linear carbohydrate residues in a groove or cavity
shaped binding site.
[0010] Glycoconjugates may be used in direct immunotherapy or the
monoclonal antibodies generated from vaccinations may be used to
specifically target known chemotherapeutic agents to tumor sites.
The immune response to carbohydrates is generally not strong,
resulting mainly in production of IgM type antibodies. IgM
antibodies are capable of complement fixation. Complement is a
family of enzymes that can lyse cells to which antibodies are
bound. The response to carbohydrate antigens normally does not
enlist the use of T-cells which would aid in the body's rejection
of the tumor. While the probability of complete tumor rejection as
a result of vaccination with a conjugate is unlikely, such
treatments will boost immune surveillance and recurrence of new
tumor colonies can be reduced. (Dennis, J., Oxford Glycosystems
Glyconews Second, 1992; Lloyd, K. O., in Specific Immunotherapy of
Cancer with Vaccines, 1993, New York Academy of Sciences, 50-58.)
Toyokuni and Singhal have described a synthetic glycoconjugate
(Toyokuni, T., et al., J. Am. Chem. Soc., 1994, 116, 395) that
stimulated a measurable IgG titer, a result which is significant
since an IgG response is generally associated with enlistment of
helper T cells.
[0011] The use of immunoconjugates has shown promise in the
reduction of large tumor masses. The workers at Bristol-Myers
Squibb (Trail, P. A., et al., Science, 1993, 261, 212) have
described the attachment of the known chemotherapeutic drug
doxorubicin to the antibody BR96. BR96 is an anti-Lewis y antibody
which has been shown to bind to human breast, lung and colon
carcinomas. Athymic mice that have had human cancers (L2987-lung,
RCA-colon, and MCF7-breast carcinomas) xenografted subcutaneously
were treated with the drug-antibody conjugate (BR96-DOX). The
result was complete regression of the tumor mass in 78% of the mice
treated. BR96 is efficiently-internalized by cellular lysosomes and
endosomes following attachment to the cell surface. The change in
pH upon internalization results in cleavage of the labile hydrazone
thereby targeting the drug specifically to the desired site.
[0012] Many of the blood group determinant structures can also
occur in normal tissues. Antigen expression in normal cells and
cancer cells can have subtle distributional differences. In the
case of Le y, which does appear in normal tissues, the expression
of the determinant in tumor cells tends to be in the form of mucins
which are secreted. Mucins are glycoproteins with a high content of
the amino acids serine and threonine. It is through the hydroxyl
functionality of these amino acids that Lewis y is linked. Thus, in
terms of generating competent antibodies against tumor cells
expressing the Le y antigen, it is important that the antibody
recognize the mucin structure.
[0013] Structurally, the blood group determinants fall into two
basic categories known as type I and type II. Type I is
characterized by a backbone comprised of a galactose 1-3 .beta.
linked to N-acetyl glucosamine while type II contains, instead, a
1-4 .beta. linkage between the same building blocks (cf. N-acetyl
lactosamine). The position and extent of a-fucosylation of these
backbone structures gives rise to the Lewis-type and H-type
specificities. Thus, monofucosylation at the C4-hydroxyl of the
N-acetyl glucosamine (Type I series) constitutes the Le a type,
whereas fucosylation of the C3-hydroxyl of this sugar (Type II
series) constitutes the Le x determinant. Additional fucosylation
of Le a and Le x types at the C2' hydroxyl of the galactose sector
specifies the Le b and Le y types, respectively. The Le y
determinant is expressed in human colonic and liver
adenocarcinomas. (Levery, S. B., et al., Carbohydr. Res., 1986,
151, 311; Kim, Y. S., J. Cellular Biochem. Suppl., 16G 1992, 96;
Kaizu, T., et al., J. Biol. Chem., 1986, 261, 11254; Levery, S. B.,
et al., Carbohydr. Res., 1986, 151, 311; Hakomori, S., et al., J.
Biol. Chem., 1984, 259, 4672; Fukushi, Y., et al., ibid., 1984,
259, 4681; Fukushi, Y., et al., ibid., 1984, 259, 10511.)
[0014] The presence of an .alpha.-monofucosyl branch, solely at the
C2'-hydroxyl in the galactose moiety in the backbone, constitutes
the H-type specifity (Types I and II). Further permutation of the
H-types by substitution of .alpha.-linked galactose or
.alpha.-linked N-acetylgalactosamine at its C3'-hydroxyl group
provides the molecular basis of the familiar serological blood
group classifications A, B, and O. (Lowe, J. B., The Molecular
Basis of Blood Diseases, Stamatoyannopoulos, et al., eds., W. B.
Saunders Co., Philadelphia, Pa., 1994, 293.)
[0015] Several issues merit consideration in contemplating the
synthesis of such blood group substances and their
neoglycoconjugates. For purposes of synthetic economy it would be
helpful to gain relief from elaborate protecting group
manipulations common to traditional syntheses of complex branched
carbohydrates. Another issue involves fashioning a determinant
linked to a protein carrier. It is only in the context of such
conjugates that the determinants are able to galvanize B-cell
response and complement fixation. In crafting such constructs, it
is beneficial to incorporate appropriate spacer units between the
carbohydrate determinant and the carrier. (Stroud, M. R., et al.,
Biochemistry, 1994, 33, 0672; Yuen, C. T., et al., J. Biochem.,
1994, 269, 1595; Stroud, M. R., et al., J. Biol. Chem., 1991, 266,
8439.)
[0016] The present invention provides new strategies and protocols
for oligosaccharide synthesis. The object is to simplify such
constructions such that relatively complex domains can be assembled
with high stereo-specifity. Major advances in glycoconjugate
synthesis require the attainment of a high degree of convergence
and relief from the burdens associated with the manipulation of
blocking groups. Another requirement is that of delivering the
carbohydrate determinant with appropriate provision for conjugation
to carrier proteins or lipids. (Bernstein, M. A., and Hall, L. D.,
Carbohydr. Res., 1980, 78, Cl; Lemieux, R. U., Chem. Soc. Rev.,
1978, 7, 423; R. U. Lemieux, et al., J. Am. Chem. Soc., 1975, 97,
4076.) This is a critical condition if the synthetically derived
carbohydrates are to be incorporated into carriers suitable for
biological application.
[0017] Antigens which are selective or ideally specific for cancer
cells could prove useful in fostering active immunity. (Hakomori,
S., Cancer Res., 1985, 45, 2405-2414; Feizi, T., Cancer Surveys,
1985, 4, 245-269) Novel carbohydrate patterns are often presented
by transformed cells as either cell surface glycoproteins or as
membrane-anchored glycolipids. In principle, well chosen synthetic
glycoconjugates which stimulate antibody production could confer
active immunity against cancers which present equivalent structure
types on their cell surfaces. (Dennis, J., Oxford GlycOsystems
Glyconews Second, 1992; Lloyd, K. O., in Specific Immunotherapy of
Cancer with vaccines, 1993, New York Academy of Sciences pp. 50-58)
Chances for successful therapy improve with increasing restriction
of the antigen to the target cell. A glycosphingolipid was isolated
by Hakomori and collaborators from the breast cancer cell line
MCF-7 and immunocharacterized by monoclonal antibody MBrl. (Bremer,
E. G., et al., J. Biol. Chem., 1984, 259, 14773-14777; Menard, S.,
et al., Cancer Res., 1983, 43, 1295-1300).
[0018] The compounds prepared by processes described herein are
antigens useful in adjuvant therapies as vaccines capable of
inducing antibodies immunoreactive with epithelial carcinomas, for
example, human colon, lung and ovarian tumors. Such adjuvant
therapies have potential to reduce the rate of recurrence of cancer
and increase survival rates after surgery. Clinical trials on 122
patents surgically treated for AJCC stage III melanoma who were
treated with vaccines prepared from melanoma differentiation
antigen GM2 (another tumor antigen which like MBr1 is a cell
surface carbohydrate) demonstrated in patients (lacking the
antibody prior to immunization) a highly significant increase in
disease-free interval (P. O. Livingston, et al., J. Clin Oncol.,
12, 1036 (1994)).
[0019] The effectiveness of a vaccine derived from a
tumor-associated antigens increases with the greater specificity of
the carbohydrate domain of the antigen. One such antigen is the
glycolipid KH-1, immunocharacterized by Hakomori et al. who have
proposed its structure as 1. (Nudelman, E.; Levery, S. B.; Kaizu,
T; Hakomori, S. -I., J. Biol. Chem., 1986, 261, 11247. Kaizu, T.;
Levery, S. B.; Nudelman, E; Stenkamp, R. E.; Hakomori, S. -I, J.
Biol. Chem., 1986, 261, 11254; Kim, S. Y.; Yuan, M.; Itzkowitz, S.
H.: Sun, Q.; Kaizu, T.; Palekar, A; Trump, B. F.; Hakamori, S. -I,
Cancer Res., 1986, 46, 5985.)
[0020] This antigen has been claimed to be a highly specific marker
for malignancy and pre-malignancies involving colonic
adenocarcinoma. The nonasaccharide character of 1 (FIG. 1) is
unique from a structural standpoint. The crystallographically
derived presentation of the monoclonal antibody BR 96 bound to a
Le.sup.y tetrasaccharide glycoside has been reported. (Jeffery, P.
D.; Bajorath, J.; Chang, C. Y.; Dale, Y.; Hellstrom, I.; Hellstrom,
E. K.; Sheriff, S., Nature Structural Biology, 1995, 2, 456.) The
structure of the BR96:Le.sup.y complex suggested that this antibody
might also have the capacity to recognize higher order fucosylated
arrays.
[0021] Accordingly, the present invention relates to the total
synthesis not only of 1 itself, but of congeners (cf. structure 2)
which are suitable for conjugation to appropriate bioactive carrier
systems.
SUMMARY OF THE INVENTION
[0022] Therefore, one object of the present invention is to provide
processes for the preparation of the KH-1 and N3 antigens, as well
as related analgoues thereof, useful as anticancer
therapeutics.
[0023] Another object of the present invention is to provide
various compounds useful as intermediates in the preparation of
KH-1 and N3 and analogues thereof. A further object of the present
invention is to provide methods of preparing such
intermediates.
[0024] An additional object of the invention is to provide
compositions comprising any of the analogues of KH-1 and N3
available through the preparative methods of the invention and
pharmaceutical carriers useful in the treatment of subjects
suffering from cancer. A further object of the invention is to
provide methods of treatment of cancer using any of the analogues
of KH-1 and N3 alone or conjugated to suitable carriers as
disclosed herein available through the preparative methods of the
invention, optionally in combination with pharmaceutical
carriers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 show the structure of the cell surface antigen KH-1
ceramide and its bioconjugateable O-allyl ether form.
[0026] FIG. 2 provides synthetic Scheme 1. Reagents: (a) (i)
3,3-dimethyldioxirane, CH.sub.2Cl.sub.2; (ii) 4 or 5, ZnCl.sub.2,
THF 65% for 6 & 55% for 7; (b) (i) TESOTf, Et.sub.3N, DMAP,
CH.sub.2Cl.sub.2, 92%, (ii) I(coll).sub.2ClO.sub.4,
PhSO.sub.2NH.sub.2, 4 .ANG. molecular sieves, CH.sub.2Cl.sub.2,
>90%; (iii) LHMDS, EtSH, DMF>90%; (c) (i) Ac.sub.2O,
Et.sub.3N, DMAP, CH.sub.2Cl.sub.2, 95%; (ii)
I(coll).sub.2ClO.sub.4, PhSO.sub.2NH.sub.2, 4 .ANG. molec-ular
sieves, CH.sub.2Cl.sub.2, >90%; (iii) LHMDS, EtSH, DMF (iv)
Ac.sub.2O, Et.sub.3N, DMAP, CH.sub.2Cl.sub.2, 85%; (d)
K.sub.2CO.sub.3, MeOH 80%; (e) (i) MeOTf, di-t-butylpyridine,
Et.sub.2O:CH.sub.2Cl.sub.2 (2:1), 4 .ANG. MS (55%), (ii)
K.sub.2CO.sub.3, MeOH (85%); (f) (i) MeOTf, di-t-butylpyridine,
Et.sub.2O:CH.sub.2Cl.sub.2 (2:1), 4 .ANG. MS (60%); (ii) Ac.sub.2O,
Py, DMAP, CH.sub.2Cl.sub.2 (95%); (g) TBAF:AcOH (93%).
[0027] FIG. 3 provides synthetic Scheme 2. Reagents: (a) 14,
Sn(OTf).sub.2, Tol:THF (10:1), 4 .ANG. MS (60%); (b) (i)
3,3-dimethyldioxirane, CH.sub.2Cl.sub.2; (ii) EtSH,
CH.sub.2Cl.sub.2, H.sup.+ (cat); (iii) Ac.sub.2O, Py,
CH.sub.2Cl.sub.2 60% (3 steps) (c) 17, MeOTf,
Et.sub.2O:CH.sub.2Cl.sub.2 (2:1), 4 .ANG. MS (55%); (d) (i)
Lindlar's catalyst, H.sub.2, palmitic anhydride, EtOAc, 85% (ii)
Na, NH.sub.3, THF; (MeOH quench); (iii) Ac.sub.2O, Et.sub.2N, DMAP,
CH.sub.2Cl.sub.2 (iv) MeONa, MeOH, 70% (3 steps); (e) (i) Na,
NH.sub.3, THF; (MeOH quench); (ii) AC.sub.2O, Et.sub.3N, DMAP,
CH.sub.2Cl.sub.2; (iii) 3,3-dimethyldioxirane, CH.sub.2Cl.sub.2;
(iv) Allyl Alcohol (v) MeONa, MeOH, 60%.
[0028] FIG. 4 provides a synthetic strategy for N3 antigen.
[0029] FIG. 5 provides a synthetic stratety for the Le x donor
portion.
[0030] FIG. 6 provides a synthetic stratety for the Le a donor
portion.
[0031] FIG. 7 provides a synthetic stratety for the N3 acceptor
portion.
[0032] FIG. 8 provides a 2+2 coupling for the major N3 antigen.
[0033] FIG. 9 provides a 2+4 and 1+1 coupling for the N3
antigen.
[0034] FIG. 10 provides a pathway for deprotection of the major N3
epitope.
[0035] FIG. 11 provides a synthetic stratety for the KH-1
tetrasaccharide and hexasaccharide intermediates.
[0036] FIG. 12 illustrates the direct coupling of KH-1 to KLH.
[0037] FIG. 13 illustrates the coupling of KH-1 to KLH via a
M.sub.2 cross-linker.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The subject invention provides a compound having the
structure: 1
[0039] wherein R is H, substituted or unsubstituted alkyl, aryl or
allyl, or an amino acyl moiety, an amino acyl residue of a peptide,
an amino acyl residue of a protein, which amino acyl moiety or
residue bears an .omega.-amino group or an .omega.-(C.dbd.O)--
group, which group is linked to O via a polymethylene chain having
the structure --(CH.sub.2).sub.s--, where s is an integer between
about 1 and about 9, or a moiety having the structure: 2
[0040] and wherein r, m and n are independently 0, 1, 2 or 3.
[0041] The present invention also provides a compound having the
structure: 3
[0042] In one embodiment, the invention provides a compound wherein
the protein is bovine serum albumin or KLH.
[0043] The invention also provides a compound having the structure:
4
[0044] wherein r is 0, 1, 2, 3 or 4. In one embodiment, the
invention provides the compound wherein r is 1.
[0045] The invention further provides a method of preparing a
trisaccharide iodosulfonamide having the structure: 5
[0046] which comprises:
[0047] (a) (i) coupling a disaccharide glycal with an epoxide
having the structure: 6
[0048] under suitable conditions to form a trisaccharide
intermediate; and
[0049] (ii) etherifying the trisaccharide intermediate with a
suitable protecting agent to form a trisaccharide glycal having the
structure: 7
[0050] and
[0051] (b) reacting the trisaccharide glycal formed in step (c)
with an iodosulfonamidating agent under suitable conditions to form
the trisaccharide iodosulfonamide. In one embodiment, the invention
provides the method wherein the disaccharide glycal has the
structure: 8
[0052] is prepared by a process which comprises:
[0053] (a) protecting a glucal having the structure: 9
[0054] with a silylating agent under suitable conditions to form a
protected glucal having the structure: 10
[0055] (b)(i) alkylating the protected glucal formed in step (a)
with a fucosylfluoride having the structure: 11
[0056] and
[0057] (ii) deprotecting under suitable conditions to form the
disaccharide glycal. In one embodiment, the invention provides the
method wherein the silylating agent in step (a) is triphenylsilyl
chloride. In another embodiment, the invention provides the method
wherein the alkylating step is effected in the presence of an
ionizing salt, and the ionizing salt is AgClO.sub.4. In an
additional embodiment, the invention provides the method wherein
the conditions of the deprotecting step comprise a base. In yet
another embodiment, the invention provides the method wherein the
base is potassium carbonate. The method also encompasses the
embodiment wherein the conditions of the coupling comprise an acid.
The method further encompasses the embodiment wherein the acid is a
Lewis acid. One example of the Lewis acid is zinc dichloride. One
example of the silylating agent used is TBSOTf. The
iodosulfonamidating agent of step (b) above may comprise
I(coll).sub.2ClO.sub.4 and and PhSO.sub.2NH.sub.2.
[0058] The present invention also provides a method of preparing a
disaccharide stannane having the structure: 12
[0059] which comprises:
[0060] (a) (i) deprotecting a disaccharide glucal having the
structure: 13
[0061] under suitable conditions to form a deprotected
intermediate; and
[0062] (ii) selectively reprotecting the deprotected intermediate
with levulinic acid under suitable conditions to form a
disaccharide levulinate having the structure: 14
[0063] and
[0064] (b) reacting the disaccharide levulinate formed in step (a)
with a distannyl oxide having the formula (R.sub.3Sn).sub.2O,
wherein R is linear or branched chain alkyl or aryl, under suitable
conditions to form the disaccharide stannane. The invention
encompasses the method wherein the conditions of the deprotecting
step comprise a fluoride salt. The invention further encompasses
the method wherein the fluoride salt is a tetraalkylammonium
fluoride. The method additionally encompasses the method wherein
the tetraalkylammonium fluoride salt is tetra-n-butylammonium
fluoride. The invention also encompasses the method wherein the
conditions of the reprotecting step comprise
2-chloro-1-methylpyridinium iodide. In one embodiment, the
invention provides the method wherein R is n-Bu.
[0065] The present invention further provides a method of preparing
a disaccharide ethylthioglycoside having the structure: 15
[0066] which comprises:
[0067] (a)(i) protecting a disaccharide glucal having the
structure: 16
[0068] with a suitable protecting agent to form a protected
disaccharide glucal; and
[0069] (ii) reacting the protected disaccharide glucal under
suitable conditions with an iodosulfonamidating agent to form a
disaccharide iodosulfonamide having the structure: 17
[0070] and
[0071] (b) treating the disaccharide iodosulfonamide formed in step
(a)(ii) with ethanethiol under suitable conditions to form the
disaccharide ethylthioglycoside. The method encompasses the
embodiment wherein the disaccharide glucal is prepared by a process
which comprises:
[0072] (a) alkylating a protected glucal having the structure:
18
[0073] with a fucosyl fluoride having the structure: 19
[0074] under suitable conditions to form the disaccharide
glucal.
[0075] The method encompasses the embodiment wherein the conditions
of the alkylating step comprise an ionizing salt. In addition, the
method encompasses the example wherein the ionizing salt is
AgClO.sub.4. The method also includes the example wherein the
protecting agent is PMBCl. The method further encompasses the
embodiment wherein the iodosulfonamidating agent in step (b) (ii)
comprises I(coll).sub.2ClO.sub.4 and PhSO.sub.2NH.sub.2. The method
also encompasses the embodiment wherein the conditions of the
treating step comprise a base. The method also includes the
instance wherein the base is LHMDS.
[0076] The invention also provides a method of preparing an N3
allyl glycoside having the structure: 20
[0077] which comprises:
[0078] (a) desilylating a protected N3 glycal having the structure:
21
[0079] under suitable conditions to form a desilylated N3
glycal;
[0080] (b) deprotecting the desilylated N3 glycal formed in step
(a) under suitable conditions to form a deprotected N3 glycal;
[0081] (c) treating the deprotected N3 glycal formed in step (b)
with acetic anhydride in the presence of a suitable catalyst to
form an N3 glycal acetate;
[0082] (d) epoxidizing the N3 glycal acetate formed in step (c)
with an oxygen transfer agent under suitable conditions to form an
N3 glycal epoxyacetate;
[0083] (e) cleaving the N3 glycal epoxyacetate formed in step (d)
with allyl alcohol under suitable conditions to form an N3 glycal
allyl ether; and
[0084] (f) saponifying the N3 glycal allyl ether under suitable
conditions to form the N3 allyl glycoside.
[0085] The invention also encompasses the method wherein the
protected N3 glycal is prepared by a process which comprises
coupling an ethylthioglycoside having the structure: 22
[0086] heptasaccharide glycal having the structure: 23
[0087] wherein R.sub.1 and R.sub.2 are Ac and R.sub.3 is H, in the
presence of an alkylating agent under suitable conditions to form
the protected N3 glycal. The invention encompasses the method
wherein the alkylating agent is MeOTf. The invention also
encompasses the method wherein the conditions of the desilylating
step comprise a fluoride salt. The invention also encompasses the
method wherein the fluoride salt is a tetraalkylammonium fluoride.
The invention also encompasses the method wherein the
tetraalkylammonium fluoride is tetra-n-butylammonium fluoride. The
invention further includes the method wherein the catalyst in the
treating step is 2-N,N-dimethylaminopyridine. The invention also
encompasses the method wherein the oxygen transfer agent is
3,3-dimethyldioxirane.
[0088] The present invention encompasses a method of preparing a
heptasaccharide glycal diacetate intermediate having the structure:
24
[0089] wherein R.sub.1 and R.sub.2 are Ac and R.sub.3 is H, which
comprises:
[0090] (a)(i) monoacylating a heptasaccharide glycal having the
structure: 25
[0091] wherein R.sub.1 and R.sub.2 are H and R.sub.3 is PMB; with
acyl anhydride in the presence of a catalyst under suitable
conditions to form a heptasaccharide glycal monoacetate;
[0092] (ii) treating the heptasaccharide glycal monoacetate formed
in step (a) (i) with a n acyl anhydride in the presence of a
catalyst under conditions suitable to form a heptasaccharide glycal
diacetate;
[0093] (iii) deprotecting the heptasaccharide glycal diacetate
under suitable conditions to form the heptasaccharide glycal
diacetate intermediate.
[0094] The invention encompasses the method wherein the
heptasaccharide glycal is prepared by a process which
comprises:
[0095] (a) (i) reacting a trisaccharide iodosulfonamide having the
structure: 26
[0096] with a disaccharide stannane having the structure: 27
[0097] under suitable conditions; and (ii) deprotecting under
suitable conditions to form a pentasaccharide glycal having the
structure: 28
[0098] and
[0099] (b) coupling the pentasaccharide glycal formed in step (a)
with an ethylthioglycoside having the structure: 29
[0100] under suitable conditions to form the heptasaccharide
glycal. The invention encompasses the method wherein the conditions
of the reacting step comprise an ionizing agent.
[0101] The invention also encompasses the method wherein the
ionizing agent is AgBF.sub.4.
[0102] The invention further encompassses a method of preparing a
protected disaccharide having the structure: 30
[0103] wherein R.sub.0 is C.sub.1-9 linear or branched chain alkyl,
arylalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl, and
triarylsilyl, which comprises:
[0104] (a)(i) epoxidizing a galactal carbonate having the
structure: 31
[0105] with an oxygen transfer agent under suitable conditions to
form an epoxide galactal; and
[0106] (ii) coupling the epoxide galactal formed in step (a) (i)
with a doubly protected galactal having the structure: 32
[0107] under suitable conditions to form a disaccharide carbonate
having the structure: 33
[0108] and (b) saponifying the disaccharide carbonate formed in
step (a) (ii) under suitable conditions to form the protected
disaccharide.
[0109] The invention encompasses the method wherein the galactal
carbonate is prepared by a process which comprises:
[0110] (a) protecting a galactal having the structure: 34
[0111] with an alkylating agent under suitable conditions to form a
first protected galactal; and
[0112] (b) treating the first protected galactal formed in step (a)
with a carbonate-forming reagent under conditions suitable to form
the galactal carbonate. The invention further provides the method
wherein the carbonate-forming reagent is (Im).sub.2CO/DMAP.
[0113] The invention also provides a method wherein the doubly
protected galactal is prepared by a process which comprises:
[0114] (a) protecting a second galactal having the structure:
35
[0115] with an alkylating agent under conditions suitable to form a
second protected galactal; and
[0116] (b) protecting the second protected galactal formed in step
(a) with an alkylating agent which may be the same or different
from that of step (a) under conditions suitable to form the doubly
protected galactal. The invention encompasses the method wherein
each alkylating agent is independently an alkyl, arylalkyl,
trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl or triarylsilyl
halide or triflate. The invention further encompasses the method
wherein the alkylating agent is benzyl bromide. In one example, the
alkylating agent is TES-Cl. The method also encompasses the method
wherein the oxygen transfer agent is DMDO. The method further
encompasses conditions for the coupling step comprising ZnCl.sub.2
in THF. The additionally encompasses conditions for the saponifying
step comprising K.sub.2CO.sub.3 in methanol.
[0117] The present invention further provides a method of preparing
an ethylthioglycoside having the structure: 36
[0118] wherein R is C.sub.1-9 linear or branched chain alkyl,
arylalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl, and
triarylsilyl, which comprises:
[0119] (a) treating a protected disaccharide carbonate having the
structure: 37
[0120] with an iodosulfonamidating agent under suitable conditions
to form a disaccharide iodosulfonamidate having the structure:
38
[0121] and
[0122] (b) reacting the disaccharide iodosulfonamidate formed in
step (a) with ethylthiol under suitable conditions to form the
ethylthioglycoside.
[0123] The invention also provides a method wherein the protected
disaccharide carbonate is prepared by a process which comprises
alkylating a disaccharide carbonate having the structure: 39
[0124] with an alkylating agent under suitable conditions to form
the protected disaccharide carbonate. The method encompasses within
the scope of the method any alkylating agent selected from the
group including an alkyl, arylalkyl, trialkylsilyl,
aryldialkylsilyl, diarylalkylsilyl or triarylsilyl halide or
triflate. An example of the alkylating agent is TES-Cl. An example
of the the iodosulfonamidating agent is I(coll).sub.2ClO.sub.4 and
PhSO.sub.2NH.sub.2.
[0125] The present invention provides a method of preparing an
ethylthioglycoside having the structure: 40
[0126] which comprises:
[0127] (a) acylating a disaccharide carbonate having the structure:
41
[0128] under suitable conditions to form an acylated disaccharide
carbonate having the structure: 42
[0129] (b) treating the acylated disaccharide carbonate formed in
step (a) with an iodosulfonamidating agent under suitable
conditions to form a disaccharide iodosulfonamidate having the
structure: 43
[0130] and
[0131] (c) reacting the iodosulfonamidate formed in the step (b)
with ethyl thiol under suitable conditions to form the
ethylthioglycoside. The invention encompasses the method wherein
the conditions of the acylating step comprise acetic
anhydride/pyridine. An example of the iodosulfonamidating agent is
I(coll).sub.2ClO.sub.4 and PhSO.sub.2NH.sub.2.
[0132] The present invention also provides a method of preparing a
protected hexasaccharide having the structure: 44
[0133] which comprises:
[0134] (a) reacting a protected tetrasaccharide having the
structure: 45
[0135] with an ethylglycoside having the structure: 46
[0136] under suitable conditions to form a hexasaccharide
intermediate; and
[0137] (b) acetylating the hexasaccharide intermediate formed in
step (a) under suitable conditions to form the protected
hexasaccharide.
[0138] The invention provides a method wherein the protected
tetrasaccharide is prepared by a process which comprises:
[0139] (a) coupling an ethythioglycoside having the structure:
47
[0140] with a protected disaccharide having the structure: 48
[0141] under suitable conditions to form a protected
tetrasaccharide carbonate; and
[0142] (b) saponifying the protected tetrasaccharide carbonate
formed in step (a) under suitable conditions to form the protected
tetrasaccharide. The invention encompasses the method wherein the
conditions of the coupling step comprise MeOTf/MS. The invention
also encompasses the method wherein the conditions of the
saponifying step comprise K.sub.2CO.sub.3 in methanol.
[0143] The present invention provides a method of preparing a
protected nonasaccharide having the structure: 49
[0144] which comprises:
[0145] (a) deprotecting a protected hexasaccharide having the
structure: 50
[0146] under suitable conditions to form a partially deprotected
hexasaccharide; and
[0147] (b) coupling the partially deprotected hexasaccharide formed
in step (a) with a fucosylfluoride having the structure: 51
[0148] in the presence of an organometallic reagent under suitable
conditions to form the protected nonasaccharide. The method
encompasses conditions of the deprotecting step comprising a
fluoride salt. The fluoride salt may be a tetraalkylammonium
fluoride. Specifically, the fluoride salt may be TBAF. The
invention encompasses the method wherein the organometallic reagent
is Sn(OTf).sub.2/DTBP.
[0149] The present invention also provides a method of preparing a
protected nonasaccharide ceramide having the structure: 52
[0150] which comprises:
[0151] (a) epoxidizing a protected nonasaccharide having the
structure: 53
[0152] with an oxygen transfer agent under suitable conditions to
form a protected nonasaccharide epoxide;
[0153] (b) coupling the protected nonasaccharide epoxide formed in
step (a) with an azide having the structure: 54
[0154] under suitable conditions to form a nonasaccharide azide
intermediate;
[0155] (c) reductively acylating the azide intermediate with
palmitic anhydride under suitable conditions to form a protected
nonasaccharide ceramide;
[0156] (d) reducing the protected nonasaccharide ceramide formed in
step (c) under suitable conditions to form a deprotected
nonasaccharide ceramide;
[0157] (e) acylating the deprotected nonasaccharide ceramide under
suitable conditions to form an acylated nonasaccharide ceramide;
and
[0158] (f) saponifying the acylated nonasaccharide ceramide under
suitable conditions to form the nonasaccharide ceramide. The
invention encompasses the method wherein the oxygen transfer agent
is DMDO. The invention also encompasses the method wherein the
conditions of the coupling step comprise ZnCl.sub.2. The method
further encompasses use of an azide intermediate which is
reductively acylated in step (c) in the presence of Lindlar's
catalyst. The invention further encompasses the method wherein
conditions of the saponifying step comprise MeONa in methanol.
[0159] The present invention provides a method of inducing
antibodies in a subject, wherein the antibodies are capable of
specifically binding with epithelial tumor cells cells, which
comprises administering to the subject an amount of a compound
which contains a determinant having a structure selected from the
group consisting of: 55
[0160] which amount is effective to induce antibodies. In one
embodiment, the invention encompasses a method wherein the compound
is bound to a suitable carrier protein, said compound being bound
either directly or by a cross-linker selected from the group
consisting of a succinimide and an M.sub.2 linker. Preferably, the
compound contains a KH-1 or N3 epitope. The method specifically
encompasses use of the carrier protein selected from the group
consisting of bovine serum albumin, polylysine or KLH. The method
also encompassses the method disclosed which further comprises
co-administering an immunological adjuvant. In particular, the
adjuvant may include bacteria or liposomes. Specifically, the
adjuvant may be Salmonella minnesota cells, bacille Calmette-Guerin
or QS21. In a certain embodiment, the method includes use of the
compound having the structure: 56
[0161] wherein R is H, substituted or unsubstituted alkyl, aryl or
allyl, or an amino acyl moiety, an amino acyl residue of a peptide,
an amino acyl residue of a protein, which amino acyl moiety or
residue bears an .omega.-amino group or an .omega.-(C.dbd.O)--
group, which group is linked to O via a polymethylene chain having
the structure --(CH.sub.2).sub.s--, where s is an integer between
about 1 and about 9, or a moiety having the structure: 57
[0162] and wherein r, m and n are independently 0, 1, 2 or 3. The
invention encompasses the method wherein the subject is in clinical
remission or, where the subject has been treated by surgery, has
limited unresected disease.
[0163] In the practice of the invention, the method encompasses the
induction of antibodies capable of specifically binding with
gastrointestinal tumor cells, colon tumor cells, lung tumor cells,
prostate tumor cells.
[0164] In addition, the invention provides a method of treating a
subject suffering from an epithelial cell cancer, which comprises
administering to the subject an amount of a compound which contains
a determinant having a structure selected from the group consisting
of: 58
[0165] which amount is effective to treat the cancer. The method
may be practiced wherein the compound is bound to a suitable
carrier protein, said compound being bound either directly or by a
cross-linker selected from the group consisting of a succinimide
and an M.sub.2 linker. Faborably, the carrier protein is bovine
serum albumin, polylysine or KLH, and the compound contains a KH-1
or N3 epitope. The method may further comprise co-administering an
immunological adjuvant. The adjuvant is bacteria or liposomes. In
particular, the adjuvant is Salmonella minnesota cells, bacille
Calmette-Guerin or QS21.
[0166] The invention further provides a method of preventing
recurrence of epithelial cell cancer in a subject which comprises
vaccinating the subject with a compound which contains a
determinant having the structure: 59
[0167] which amount is effective to induce the antibodies. The
invention encompasses the method wherein the compound is bound to a
suitable carrier protein. The method specifically encompasses use
of any effective carrier protein including bovine serum albumin,
polylysine or KLH. In addition, the method may further comprises
co-administering an immunological adjuvant. The adjuvant may be
bacteria or liposomes. In particular, the adjuvant may be
Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
[0168] The method may carried out using a compound selected from
the group consisting of: 60
[0169] wherein R is H, substituted or unsubstituted alkyl, aryl or
allyl, or an amino acyl moiety, an amino acyl residue of a peptide,
an amino acyl residue of a protein, which amino acyl moiety or
residue bears an .omega.-amino group or an .omega.-(C.dbd.O)--
group, which group is linked to O via a polymethylene chain having
the structure --(CH.sub.2).sub.s--, where s is an integer between
about 1 and about 9, or a moiety having the structure: 61
[0170] and wherein r, m and n are independently 0, 1, 2 or 3.
[0171] The processes of the present invention for preparing KH-1
and N3 anitgens and analogues thereof and intermediates thereto
encompass the use of various alternate protecting groups known in
the art. Those protecting groups used in the disclosure including
the Examples below are merely illustrative. One of ordinary skill
would understand how to substitute equivalent protecting groups for
those illustrated.
[0172] The subject invention also provides pharmaceutical
compositions for treating cancer comprising any of the analogues of
KH-1 or N3 antigens as disclosed herein, optionally in combination
with a pharmaceutically suitable carrier.
[0173] The subject invention further provides a method of treating
cancer in a subject suffering therefrom comprising administering to
the subject a therapeutically effective amount of any of the
analogues of KH-1 or N3 antigens disclosed herein and a
pharmaceutically suitable carrier.
[0174] The invention provides a method of preventing recurrence of
an epithelial cell cancer in a subject which comprises vaccinating
the subject with a compound which contains a determinant having the
structure: 62
[0175] which amount is effective to prevent recurrence of an
epithelial cell cancer.
[0176] The invention provides a composition comprising a compound
which contains a determinant having a structure selected from the
group consisting of: 63
[0177] and optionally an immunological adjuvant and/or a
pharmaceutically acceptable carrier.
[0178] The invention also provides the composition wherein the
compound is bound to a suitable carrier protein, said compound
being bound either directly or by a cross-linker selected from the
group consisting of a succinimide and an M.sub.2 linker. The
composition is also provided wherein the carrier protein is bovine
serum albumin, polylysine or KLH. In particular, the composition is
characterized wherein the compound contains a KH-1 or N3
epitope.
[0179] Additionally, the composition is provided wherein the
immunological adjuvant is bacteria or liposomes. The adjuvant may
be Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
[0180] Favorably, the composition is provided wherein the compound
has the structure: 64
[0181] wherein R is H, substituted or unsubstituted alkyl, aryl or
allyl, or an amino acyl moiety, an amino acyl residue of a peptide,
an amino acyl residue of a protein, which amino acyl moiety or
residue bears an .omega.-amino group or an .omega.-(C.dbd.O)--
group, which group is linked to O via a polymethylene chain having
the structure --(CH.sub.2).sub.s--, where s is an integer between
about 1 and about 9, or a moiety having the structure: 65
[0182] and wherein r, m and n are independently 0, 1, 2 or 3.
[0183] Utilities
[0184] The compounds taught above which are related to KH-1 and N3
cell-surface antigens are capable of preventing recurrence of
various types of epithelial cancer in a subject, including lung,
gastrointestinal, prostate and colon cancers, and inducing
antibodies useful as a vaccine in the treatment of such types of
cancer, both in vivo and in vitro. Thus, these antigens and
analogues thereof are useful to treat, prevent or ameliorate such
cancers in subjects suffering therefrom.
[0185] The magnitude of the therapeutic dose of the compounds of
the invention will vary with the nature and severity of the
condition to be treated and with the particular compound and its
route of administration. In general, the daily dose range for
anticancer activity or antibody induction lies in the range of
0.001 to 25 mg/kg of body weight in a mammal, preferably 0.001 to
10 mg/kg, and most preferably 0.001 to 1.0 mg/kg, in single or
multiple doses. In unusual cases, it may be necessary to administer
doses above 25 mg/kg.
[0186] Any suitable route of administration may be employed for
providing a mammal, especially a human, with an effective dosage of
a compound disclosed herein. For example, oral, rectal, topical,
parenteral, ocular, pulmonary, nasal, etc., routes may be employed.
Dosage forms include tablets, troches, dispersions, suspensions,
solutions, capsules, creams, ointments, aerosols, etc.
[0187] The pharmaceutical compositions of the present invention
comprise a compound containing any of the KH-1 and N3 antigens of
the subject invention, as an active ingredient, and may also
contain a pharmaceutically acceptable carrier and, optionally,
other therapeutically active ingredients.
[0188] The compositions include compositions suitable for oral,
rectal, topical (including transdermal devices, aerosols, creams,
ointments, lotions and dusting powders), parenteral (including
subcutaneous, intramuscular and intravenous), ocular (ophthalmic),
pulmonary (nasal or buccal inhalation) or nasal administration.
Although the most suitable route in any given case will depend
largely on the nature and severity of the condition being treated
and on the nature of the active ingredient. They may be
conveniently presented in unit dosage form and prepared by any of
the methods well known in the art of pharmacy.
[0189] In preparing oral dosage forms, any of the unusual
pharmaceutical media may be used, such as water, glycols, oils,
alcohols, flavoring agents, preservatives, coloring agents, and the
like in the case of oral liquid preparations (e.g., suspensions,
elixers and solutions); or carriers such as starches, sugars,
microcrystalline cellulose, diluents, granulating agents,
lubricants, binders, disintegrating agents, etc., in the case of
oral solid preparations are preferred over liquid oral preparations
such as powders, capsules and tablets. If desired, capsules may be
coated by standard aqueous or non-aqueous techniques. In addition
to the dosage forms described above, the compounds of the invention
may be administered by controlled release means and devices.
[0190] Pharmaceutical compositions of the present invention
suitable for oral administration may be prepared as discrete units
such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient in powder or granular
form or as a solution or suspension in an aqueous or nonaqueous
liquid or in an oil-in-water or water-in-oil emulsion. Such
compositions may be prepared by any of the methods known in the art
of pharmacy. In general compositions are prepared by uniformly and
intimately admixing the active ingredient with liquid carriers,
finely divided solid carriers, or both and then, if necessary,
shaping the product into the desired form. For example, a tablet
may be prepared by compression or molding, optionally with one or
more accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredient in a
free-flowing form such as powder or granule optionally mixed with a
binder, lubricant, inert diluent or surface active or dispersing
agent. Molded tablets may be made by molding in a suitable machine,
a mixture of the powdered compound moistened with an inert liquid
diluent.
[0191] The present invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described in
the claims which follow thereafter.
EXAMPLE 1
[0192] 6-O-Benzyl-3,4-O-carbonate-galactal (3): To a solution of
3,4-carbonate-galactal (5.36 g, 34.37 mmol) in dry DMF (50 mL) at
0.degree. C. was added benzyl bromide (12.26 mL, 103.0 mmol),
followed by NaH (60% oil dispersion, 1.5 gm. 1.1 eq). The reaction
was stirred for 1 hr, diluted with CHCl.sub.3 (50 mL) and then
brine solution (20 mL) was added and stirred for 5 min. The organic
layer was separated, dried (MgSO.sub.4), concentrated, and
subjected to chromatographic purification (1:1, Hex:EA) to obtain
compound 3 (85%) as a syrup: [.alpha.].sup.23.sub.D=-92.0 (c 1.0,
CHCl.sub.3); FTIR (thin film) 3030, 2875, 1797, 1647, 1496, 1453,
1371, 1244, 1164, 1110, 1010, 837, 699 cm.sup.-1; .sup.1H NMR (400
MHz, CDCl.sub.3 .delta. 3.7-3.9 (m, 2H, H-6), 4.08 (bt, 1H, J=7.36
Hz, H-5), 4.58 (s, 2H, --CH.sub.2Ar), 4.90 (d, 1H, J=7.76 Hz, H-4),
4.93 (bm, 1H, H-3), 5.14 (dd, 1H, J=3.16 Hz, J=7.72 Hz, H-2), 6.66
(d, 1H, J=6.24 Hz, H-1), 7.28-7.45 (m, 5H, Ar--H); .sup.13C NMR
(400 MHz, CDCl.sub.3 .delta. 67.97, 68.74, 72.41, 73.14, 73.66,
97.97, 127.77, 127.93, 128.44, 137.18, 149.06, 153.98.
EXAMPLE 2
[0193] 6-O-Benzylglucal (3'): To a solution of a glucal (10 g,
68.42 mmol) in a dry DMF (200 mL) was added at -40 .degree. C.
LHMDS (1.0 M soln in THF, 75.26 mL, 1.1 eq) dropwise, followed by
BnBr (8.18 mL, 68.42 mmol). The solution was stirred mechanically
for 6 hrs allowing the temperature to rise to 0.degree. C. At room
temperature, a sat'd solution of ammonium chloride (50 mL) was
added, followed by EtOAc (200 mL). The organic layer was separated;
the aqueous layer was extracted with EtOAc (3.times.50 mL).
Combined organic layers were washed with brine (50 mL), water (50
mL), dried with (MgSO.sub.4), filtered, concentrated and purified
by column chromatgraphy (1:1 Hex: EtOAc) to obtain compound 3' as a
syrup: [.alpha.].sup.23.sub.D=+11.0 (c 1.0, CHCl.sub.3); FTIR (thin
film): 3342, 2871, 1642, 1656, 1231, 1101, 1027, 851, 738
cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.6-3.85 (m,
5H,), 4.06 (d, 1H, J=4.0 Hz, --OH), 4.11 (bt, 1H, H-3), 4.46 (d,
1H, J=12.0 Hz, --CH.sub.2Ar), 4.52 (d, 1H, J=12.0 Hz,
--CH.sub.2Ar), 4.57 (dd, 1H, J=1.84 Hz, J=5.96 Hz, H-2). 6.21 (d,
1H, J=5.96 Hz, H-1), 7.15-7.35 (m, 5H, Ar--H); .sup.13C NMR (400
MHz, CDCl.sub.3) .delta. 69.06, 69.63, 70.28, 73.427, 76.95,
102.72, 127.29, 127.55, 128.21, 128.24, 137.60, 137.75, 143.86.
EXAMPLE 3
[0194] 6-O-Benzyl-3-O-triethylsilylglucal (4): To a solution of
compound 3 (5 g, 21.16 mmol) in dry CH.sub.2Cl.sub.2 (50 mL) was
added imidazole (1.72 g, 25.39 mmol), DMAP (10 mg). At 0.degree. C.
TESCl (3.90 mL, 23.27 mmol) was added dropwise. The reaction
mixture was stirred for 9 hrs, washed with water (2.times.10 mL)
and brine (10 mL). The organic layer was separated and dried
(MgSO.sub.4), concentrated and purified by column chromatography
(20% EA in hexane) to obtain 4 (5.47 mg, 73%) as a syrup:
[.alpha.].sup.22.sub.D+44.0 (c 1.0, CHCl.sub.3); FTIR (thin film)
3468, 3030, 2953, 2875, 1644, 1453, 1237, 1086, 871, 737 cm.sup.-1;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.55 (q, 6H, J=7.90 Hz,
--SiCH.sub.2CH3), 0.88 (t, 9H, J=7.90 Hz, --SiCH2CH.sub.3), 2.47
(d, 1H, J=4.12 Hz, --OH), 3.6-3.75 (m, 3H, 2H-6, H-4), 4.13 (bd,
1H, J=6.4 Hz, H-3), 4.47 & 4.52 (2d, 2H, J=12.00 Hz,
--CH.sub.2Ar), 4.55 (dd, 1H, J=2.24 Hz, J=6.16 Hz, H-2), 6.21 (d,
1H, J=5.96 Hz, H-1), 7.10-7.40 (m, 5H, Ar--H); .sup.13C NMR (400
MHz, CDCl.sub.3) .delta. 4.84, 6.66, 69.05, 69.64, 70.56, 73.47,
76.97, 103.44, 127.59, 127.64, 128.27, 137.78, 143.33.
EXAMPLE 4
[0195] 3,6,6'-Tri-O-benzyl-4',5'-carbonate-lactal (7): To a
solution of compound 3 (3.00 gm, 11.43 mmol) in a dry
CH.sub.2Cl.sub.2 (20 mL) at 0.degree. C. was added
3,3-dimethyldioxirine (300 mL, 0.08 M solution in acetone). The
reaction was stirred at 0.degree. C. for 1 h. The organic solvent
was evaporated in a stream of N.sub.2 gas. The residue was dried in
vacuum for 10 minutes. The resulting anhydro sugar was dissolved in
a solution of the compound 3,6-dibenzylglucal (5.29 gm, 17.15 mmol)
in a dry THF (30 mL). At 0.degree. C. a 1.0 M solution of
ZnCl.sub.2 in ether (5.71 mL, 0.5 eq) was added. The reaction was
stirred at room temperature for 24 h, diluted with EtOAc (50 mL),
washed with a sat'd solution of NaHCO.sub.3 (2.times.10 mL). The
organic layer was separated, dried (MgSO.sub.4) and purified by
chromatography using EA:Hexane (1:1) to obtain compound 7, 3.3 g
(48%) (60% wrt recovered SM) as a syrup:
[.alpha.].sup.22.sub.D-38.0 (c 1.0, CHCl.sub.3); FTIR (thin film)
3437, 3029, 2871, 1804, 1648, 1453, 1367, 1166, 1097, 1027, 739,
697 cm.sup.-1; .sup.1H NMR (400 Mhz, CDCl.sub.3) .delta. 3.55-3.62
(m, 2H), 3.62-3.70 (m, 2H), 3.70-3.78 (m, 2H), 3.95-4.11 (m, 2H),
3.95-4.11 (m, 2H), 4.17 (dd, 1H, J=5.36 Hz, J=7.04), 4.27 (ddd, 1H,
J=1.12 Hz, J=1.73 Hz, J=5.29 Hz), 4.44 (s, 2H, --CH2Ar), 4.77 (dd,
1H, J=2.48 Hz, J=6.12 Hz, H-2), 6.28 (d, 1H, J=6.04 Hz, H-1),
7.10-7.40 (m, 15H, Ar--H); .sup.13C NMR (400 MHz, CDCl.sub.3)
.delta. 68.00, 68.09, 70.55, 70.63, 72.20, 73.58, 73.81, 74.58,
74.82, 75.26, 76.18, 78.47, 100.17, 101.32, 127.43 (2C), 127.56,
127.72 (2C), 127.83, 127.90, 128.00 (2C), 128.31 (2C), 128.37 (2C),
128.44 (2C), 137.28, 137.43, 138.29, 144.59, 153.97.
EXAMPLE 5
[0196] 3,6,6'-Tri-O-benzyl-lactal (10): To a solution of compound 7
(3.00 g, 4.96 mmol) in MeOH (100 mL) was added dropwise a solution
of sodium methoxide (1 mL, 25% by wt in MeOH). The reaction was
stirred for 1 h, and the solvent was evaporated. The syrup obtained
was rapidly purified by column chromatography (2.5% MeOH in EtOAc)
to obtain 2.68 g (91%) of 10 as syrup: [.alpha.].sup.22.sub.D-14.0
(c 1.0, CHCl3) ; FTIR (thin film) 3415, 3029, 2867, 1647, 1453,
1246, 1068, 735 cm.sup.-1; .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 3.48-3.56 (m, 2H), 3.62 (dd, 1H, J=4.80 Hz, J=8.0 Hz),
3.66-3.78 (m, 3H), 3.91 (d, 1H, J=4.4 Hz), 3.97 (dd, 1H, J=4.0 Hz,
J=8.8 Hz), 4.18-4.28 (m, 4H), 4.47 (s, 2H, --CH2Ar), 4.52 (d, 1H,
J=8.0 Hz), 5.59 (s, 1H, --CH2Ar), 4.57-4.65 (m, 2H, --CH2Ar), 4.85
(dd, 1H, J=2.4 Hz, J=4.8 Hz, H-2), 6.41 (d, 1H, J=4.8 Hz, H-1),
7.20-7.45 (m, 15H, Ar--H); C NMR (400 MHz, CDCl.sub.3) .delta.
67.92, 68.86, 69.19, 69.82, 71.53, 73.35 (2C), 73.39, 73.42, 73.87,
76.26, 100.01, 103.30, 127.35, 127.42, 127.59, 127.74, 128.19,
128.29, 137.72, 137.81, 138.52, 144.57.
EXAMPLE 6
[0197] Monosilylated lactal (6): To a solution of compound 3 (3.00
gm, 11.43 mmol) in a dry CH.sub.2Cl.sub.2 (20 mL) at 0.degree. C.
was added 3,3-dimethyldioxirine (300 mL, 0.08 M solution in
acetone). The reaction was stirred at 0.degree. C. for 1 h, and the
organic solvents were evaporated in a N.sub.2 gas stream. The
residue was dried in vacuum for 10 minutes. The resulting anhydro
sugar was dissoved in a solution of compound 4 (6 gm, 9.2 mmol) in
a dry THF (30 mL), at 0.degree. C. was added a 1.0 M solution of
ZnCl.sub.2 in ether (6 g, 0.5 eq). Reaction was stirred at room
temperature for 24 h. Diluted with EtOAc (50 mL), washed with sat.
solution of NaHCO.sub.3 (2.times.10 mL), organic olayer was
separated, dried (MgSO.sub.4) submitted for chromatography
EA:Hexane (2:3) to obtain compound 6 (4.8 g 66%) (81% wrt recovered
7) as a syrup: [.alpha.].sup.22.sub.D -25.0 (c 1.0, CHCl3); IR
(thin film) 3439, 3030, 2910, 1804, 1725, 1647, 1453, 1371, 1243,
1074, 847, 741 cm.sup.-1; 1H NMR (CDCl3, 400 MHz) .delta. 0.58 (q,
6H, J=8.0 Hz, --SiCH2CH3), 0.92 (t, 9H, J=8.0 Hz, --SiCH2CH3), 3.51
(d, 1H, J=2.8 Hz, --OH), 3.62 (ddd, 1H, J=2.8 Hz, J=7.2 Hz, J=7.2
Hz, H-2'), 3.65.3.75 (m, 3H), 3.85 (m, 1H), 3.93 (dd, 1H, J=4.92
Hz, J=11.24 Hz), 3.99 (bt, 1H, J=5.32 Hz, J=6.48 Hz), 4.09 (bm,
1H), 4.27 (bt, 1H, J=4.16 Hz), 4.48-4.68 (m, 6H, --CH2Ar), 4.70
(dd, 1H, J=3.36 Hz, J=6.16 Hz, H-2), 4.74 (dd, 1H, J=1.8 Hz, J=7.16
Hz, H-4), 6.32 (d, 1H, J=6.04, H-1), 7.2-7.4 (m, 10H, Ar--H); C
(500 MHz, CDCl.sub.3) .delta. 4.75, 6.67, 65.65, 67.79, 67.93,
70.42, 71.49, 73.43, 73.58, 74.46, 75.27, 75.42, 78.05, 99.94,
102.61, 127.79, 127.85, 128.14, 128.33, 137.36, 137.53, 143.00,
153.96.
EXAMPLE 7
[0198] Acetylated silyl lactal: To a solution of compound 6 (3.5 g,
5.50 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added pyridine (3 mL),
Ac.sub.2O (3 mL) and DMAP (cat). The reaction was stirred
overnight, and then diluted with EtOAc (50 mL), washed with a sat'd
solution of CuSO.sub.4 (3.times.10 mL), water (1.times.10 mL),
NaHCO.sub.3 (2.times.10 mL), and brine (1.times.10 mL). The organic
layer was separated, dried, and concentrated. The residue was
purified by chromatography (1:1, Hex:EA) to obtain 6' in
quantitative yield; [U].sup.23.sub.D -42.0 (c 1.0, CHCl.sub.3); IR
(film) 2954, 2875, 1809, 1755, 1646, 1454, 1222, 1060, 743
cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.58 (q, 6H, J=7.92 Hz,
--SiCH.sub.2CH.sub.3), 0.92 (t, 9H, J=7.92 Hz,
--SiCH.sub.2CH.sub.3), 2.06 (s, 3H, --COCH.sub.3), 3.63 (dd, 1H,
J=2.92 Hz, J=10.92 Hz, H-5), 3.70 (bd, 2H, J=10.52 Hz, 2H-6), 3.85
(dd, 1H, J=6.08 Hz, J=10.92, H-5s), 3.9-4.0 (m, 2H, 2H-6'),
4.10-4.2 (m, 2H), 4.5-4.6 (m, 4H, 2--CH.sub.2Ar), 4.64 (dd, 1H,
J=3.96 Hz, 8.0 Hz, H-3'), 4.71 (dd, 1H, J=4.24 Hz, J=5.84 Hz, H-2),
4.84 (dd, 1H, J=1.04 Hz, J=8.08 Hz, H-4'), 4.90 (d, 1H, J=4.60 Hz,
H-1'), 4.99 (t, 1H, J=4.20 Hz, H-4'), 6.30 (d, 1H, J=6.16 Hz, H-1),
7.15-7.40 (m, 10H, Ar--H); .sup.13C NMR (CDCl.sub.3) .delta. 4.78,
6.73, 20.58, 64.78, 67.94, 67.99, 69.38, 69.50, 73.19, 73.35,
73.79, 73.92, 74.56, 74.86, 96.82, 102.27, 127.60, 127.75, 127.78,
127.93, 128.29, 128.44, 137.35, 138.01, 142.99, 153.27, 168.54.
EXAMPLE 8
[0199] Iodosulfonamide (6"): To a solution of compound 6' (2.5 gm,
3.72 mmol) (suspended with 4 A MS (3.00 g)) and benzenesulfonamide
(2.92 g, 18.57 mmol) at 0.degree. C., was added (via cannula) a
solution of I.sup.+(coll).sub.2ClO.sub.4.sup.- (freshly prepared
from Ag(coll)ClO.sub.4 (8.36 g, 18.59 mmol) and I.sub.2 (4.53 g,
18.53 mmol)) in CH.sub.2Cl.sub.2 (40 mL). The reaction mixture was
allowed to warm to r.t. and stirred for 1 hr. The mixture was
filtered through a pad of silica gel. The filtrate was washed with
a sat'd solution of Na.sub.2S.sub.2O.sub.3 (3.times.25 mL),
followed by a sat'd solution of CuSO.sub.4 (5.times.25 mL), and
H.sub.20 (2.times.10 mL). The organic layer was separated and dried
(MgSO.sub.4), concentrated and purified by column chromatogrphy (5%
EA in CH.sub.2Cl.sub.2, in a gradient elution) to obtain 6", 2.9 g
(81%) as a syrup; [.alpha.].sup.23.sub.D -30.0 (c 1.0, CHCl.sub.3);
IR (film) 3267, 2954, 1806, 1755, 1495, 1458, 1370, 1342, 1090,
813, 750 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.66 (m, 6H,
J=7.8 Hz, --SiCH.sub.2CH.sub.3) 0.95 (t, 9H, J=7.8 Hz,
--SiCH.sub.2CH.sub.3), 2.04 (s, 3H, --COCH.sub.3), 3.44 (dd, 1H,
J=5.56 Hz, J=10.16 Hz, H-5), 3.55-3.72 (m, 4H), 3.86 (bs, 1H), 4.11
(t, 1H, J=6.96 Hz), 4.23 (bs, 1H), 4.35 (dd, 1H, J=2.28 Hz, J=9.98
Hz), 4.44 & 4.50 (2d, 2H, 11.88 Hz, --CH.sub.2Ar), 4.57 (s, 2H,
--CH.sub.2 Ar), 4.70 (bd, 1H, J=8.32 Hz), 4.89 (bs, 1H), 4.95-5.0
(m, 2H), 5.25 (t, 1H, J=9.64 Hz), 5.60 (d, 1H, J=9.92 Hz), 7.2-7.5
(m, 13H, Ar--H), 7.88 (d, 2H, J=7.72 Hz, Ar--H); .sup.13C NMR
(CDCl.sub.3) .delta. 4.93, 6.95, 20.64, 67.49, 67.86, 68.40, 68.46,
71.91, 72.57, 73.33, 73.94, 75.20, 79.30, 126.39, 127.35, 127.67,
127.85, 127.98, 128.09, 128.36, 128.54, 128.58, 129.10, 132.35,
132.68, 137.14, 137.91, 141.36, 153.60, 168.69.
EXAMPLE 9
[0200] Thiodonor (9): To a solution of iodosulfonamide 6" (2.8 g,
2.93 mmol) in dry DMF (40 mL) at -40.degree. C. was added EtSH
(1.08 mL, 14.65 mmol), followed by dropwise addition of a solution
of LHMDS (1.0 M solution in THF, 8.80 mL). The reaction mixture was
stirred for 1 hr while allowing it to warm up to r.t., and then
neutralized with a saturated solution of NH.sub.4Cl (10 mL), and
extracted with EtOAc (5.times.20 mL). The organic layer was washed
with brine (15 mL), separated, dried (MgSO.sub.4), and
concentrated. The resulting residue was acetylated in
CH.sub.2Cl.sub.2 (50 mL) with pyridine (1.0 mL), .sub.2Ac O (1.0
mL) overnight. The organic layer was washed with a sat'd solution
of CuSO.sub.4 (3.times.15 mL), water (1.times.10 mL), a and sat'd
solution of NaHCO.sub.3 (2.times.15 mL). The organic layer was
separated, dried (MgSO.sub.4) and concentrated. The residue was
purified by chromatography (1:1, Hex: EA) to obtain 9 (2.38 g, 91%)
as syrup; [.alpha.].sup.23.sub.D -4.0 (c 1.0, CHCl.sub.3); IR
(film) 3316, 2955, 2875, 1815, 1745, 1448, 1371, 1330, 1227, 1092,
897, 740 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) 67 0.51 (q, 6H, J=8.0
Hz, --SiCH.sub.2CH.sub.3), 0.88 (t, 9H, J=7.92 Hz,
--SiCH.sub.2CH.sub.3), 1.09 (t, 3H, 7.20 Hz, --SCH.sub.2CH.sub.3),
2.09 (s, 3H, --COCH.sub.3), 2.44 (m, 2H, --SCH.sub.2CH.sub.3), 3.48
(bm, 1H, H-2), 3.83-3.70 (m, 7H), 3.89 (bt, 1H), 3.95 (bs, 1H),
4.43 (d, 1H, J=5.44 Hz, H-1), 4.48 (bd, 2H, --CH.sub.2Ar), 4.53 (d,
1H, J=6.32 Hz, H-1'), 4.57 (s, 2H, --CH.sub.2Ar), 4.75 (bt, 1H,
J=5.72 Hz, H-2'), 4.84 (bd, 1H, 9.88 Hz, --NHSO.sub.2Ph), 7.20-7.40
& 7.40-7.60 (m, 13H, Ar--H), 7.97 (d, 2H, J=7.16 Hz, Ar--H);
.sup.13C NMR (CDCl.sub.3) .delta. 4.28, 6.65, 14.56, 20.64, 20.89,
56.96, 67.64, 70.49, 70.52, 70.57, 71.14, 73.26, 73.72, 73.96,
74.99, 75.02, 76.88, 82.48, 97.83, 126.21, 127.30, 127.61, 127.78,
127.87, 128.29, 128.38, 128.62, 128.94, 132.17, 137.32, 137.94,
141.38, 153.27, 170.99.
EXAMPLE 10
[0201] Disilylated lactal (6'"): To a solution of lactal 6 (3 gm,
4.77 mmol) in dry CH.sub.2Cl.sub.2 (50 mL) at 0.degree. C., was
added Et.sub.3N (3.34 mL), followed by the dropwise addition of
TESOTf (1.61 mL, 7.15 mL). The reaction mixture was stirred for 3
h, and washed with a sat'd solution of NaHCO.sub.3 (2.times.15 mL).
The organic layer was separated, dried (MgSO.sub.4), and
concentrated. The residue was purified by chromatography (4:1,
Hex:EA) to obtain 6'" (3.27 g, 92%) as a syrup; [.alpha.].sub.23D
-38.0 (c 1.0, CHCl3) ; IR (thin film) .delta. 3087, 2953, 2875,
1819, 1647, 1647, 1454, 1365, 1240, 1101, 854, 739 cm.sup.-1; 1H
NMR (CDCl3, 400 MHZ) 0.57 & 0.617 (2q, 12H, J=8.0 Hz,
--SiCH2CH3), 0.92 & 0.94 (2t, 18H, J=8.0 Hz, --SiCH2CH3),
3.5-3.75 (m, 4H), 3.8-4.0 (m, 3H), 4.05-4.20 (m, 2H), 4.49 (dd, 1H,
J=4.36 Hz, J=7.24 Hz), 4.50-4.62 (m, 4H, --CH2Ar), 4.64 (d, 1H,
J=5.2 Hz, H-1'), 4.70 (dd, 1H, J=4.0 Hz, J=5.60 Hz, H-4'), 4.76
(bd, 1H, J=7.5 Hz, H-2), 6.32 (d, 1H, J=6.0 Hz, H-1); 13C NMR
(CDCl3, 400 MHz) .delta. 4.56, 4.79, 6.58, 6.76, 65.24, 67.99,
68.02, 69.48, 71.06, 73.37, 73.76, 74.24, 74.37, 75.10, 78.21,
99.21, 99.34, 102.56, 127.63, 127.77, 127.79, 127.88, 128.32,
128.43, 137.53, 138.09, 143.08, 153.87.
EXAMPLE 11
[0202] Disilylated Iodosulfonamide (6""): To a solution of lactal
6'" (2.5 g, 3.36 mmol) (suspended with 4 .ANG. MS (3 g)) and
benzenesulfonamide (2.64 g, 3.36 mmol) at 0.degree. C., was added a
freshly prepared solution of I(sym-coll).sub.2ClO.sub.4 (Seq) in
CH.sub.2Cl.sub.2 The reaction mixture was stirred at r.t. for 1 hr,
filtered through a pad of silica gel, washed with a sat'd solution
of Na.sub.2S.sub.2O.sub.3 (3.times.25 mL), CuSO.sub.4 (5.times.25
mL), and water (2.times.10 mL). The organic layer was separated,
dried (MgSO.sub.4), and concentrated. The resulting residue was
purified by chromatography (5% EA in CH.sub.2Cl.sub.2) to obtain
6"" (3.20 g, 92%) as a syrup; [.alpha.].sup.23.sub.D -19.0 (c 1.0,
CHCl.sub.3); IR (thin film) 3258, 2953, 2875, 1806, 1788, 1453,
1331, 1105, 849, 745 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. -0.57 & 0.64 (2q, 12H, J=8.0 Hz, --SiCH.sub.2CH.sub.3),
0.90 & 0.95 (2t, 18H, J=8.0 Hz, --SiCH.sub.2CH.sub.3), 3.39
(bm, 1H, H-2), 3.60-3.70 (m, 4H), 3.78-3.83 (bm, 2H), 4.05-4.17 (m,
3H), 4.34 (dd, 1H, J=2.40 Hz, J=8.68 Hz), 4.45 & 4.52 (2d, 2H,
J=12.0 Hz, --CH.sub.2Ar), 4.55 (s, 2H, --CH.sub.2Ar), 4.68 (d, 1H,
J=2.96 Hz), 4.89 (d, 1H, J=8.56 Hz), 5.29 (t, 1H, J=8.36 Hz), 5.47
(d, 1H, J=9.64 Hz, --NHSO.sub.2Ph), 7.2-7.5 (m, 13H, Ar--H), 7.89
(d, 2H, J=7.6 Hz, Ar--H); .sup.13H NMR (CDCl.sub.3, 400 MHz)
.delta. 4.54, 4.94, 6.59, 6.95, 67.94, 68.12, 68.39, 68.63, 73.12,
73.31, 73.36, 73.90, 75.26, 75.33, 76.86, 79.66, 100.04, 127.40,
127.67, 127.76, 127.93, 128.01, 128.36, 128.51, 128.60, 132.39,
137.34, 138.02, 141.31, 154.01.
EXAMPLE 12
[0203] Disilylated thiodoner (8): To a solution of iodosulfonamide
6"" (2.7 g, 2.63 mmol) in dry DMF (40 mL) at -40.degree. C., was
added EtSH (0.584 mL, 7.89 mmol), followed by the dropwise addition
of a solution of LHMDS (1.0 M solution in THF, 7.89 mL). The
reaction mixture was stirred for 1 hr while allowing it to warm up
to r.t., and then neutralized with a saturated solution of
NH.sub.4Cl (10 mL). EtOAc was added (50 mL). The organic layer was
washed with brine (5 mL), separated, dried (MgSO.sub.4), and
concentrated. The residue was purified by chromatography (7:3,
Hex:EA) to obtain 8 (2.3 g, 91%) as syrup; [.alpha.].sup.23.sub.D
-64.0 (c 1.0, CHCl.sub.3); IR (thin film) 3314, 2954, 2875, 1807,
1453, 1330, 1181, 1104, 739 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400
MHz) .delta. 0.50 (q, 6H, J=7.88 Hz, --SiCH.sub.2CH.sub.3), 0.624
(q, 6H, J=7.6 Hz, --SiCH.sub.2CH.sub.3), 0.87 (t, 9H, J=7.88 Hz,
--SiCH.sub.2CH.sub.3), 0.94 (t, 9H, J=7.96 Hz, --SiCH2CH), 1.11 (t,
3H, J=7.44 Hz, --SCH.sub.2CH.sub.3), 2.48 (m, 2H,
--SCH.sub.2CH.sub.3), 3.35 (m, 1H, H-2), 3.85-3.68 (m, 6H), 3.86
(bm, 1H), 3.97 (bt, 1H), 4.06 (bt, 1H, J=6.56 Hz), 4.49 (s, 2H,
--CH.sub.2Ar), 4.57 (s, 2H, --CH.sub.2Ar), 4.55 (m, 1H), 4.61 (d,
1H, J=6.28 Hz, H-1), 4.67 (d, 1H, J=4.0 Hz, H'-4), 4.87 (d, 1H,
J=8.84 Hz, H-1), 5.50 (d, 1H, J=8.84 Hz, --NHSO.sub.2Ph), 7.2-7.4
(m, 10H, Ar--H), 7.45-7.55 (m, 3H, Ar--H), 7.94 (d, 2H, J=7.2 Hz,
Ar--H); .sup.13C NMR (CDCl.sub.3) .delta. 4.28, 4.45, 6.56, 6.71,
14.58, 25.64, 57.42, 67.82, 69.05, 69.68, 70.37, 71.80, 73.13,
73.66, 73.75, 76.45, 76.64, 77.05, 82.68, 100.94, 127.52, 127.57,
127.79, 127.83, 128.22, 128.35, 128.84, 132.25, 137.49, 138.01,
140.70.
EXAMPLE 13
[0204] Tetrasaccharide diol (9'): To a solution of disaccharide 10
(100 mg, 0.173 mmol) and thiodonor 9 (308 mg, 0.34 mmol) in dry
CH.sub.2Cl.sub.2 (8 mL), suspended with 4 .ANG. MS (1.0 g) was
added di-t-butylpyridine (0.311 mL, 1.36 mmol), cooled to
-10.degree. C. Then, MeOTf (0.156 mL, 1.36 mmol) was added. The
reaction mixture was stirred for 2 h, then at 0.degree. C. for 24
h. After neutralizing with Et.sub.3N (0.1 ml), the mixture was
diluted with EtOAc (25 mL), and filtered through a pad of silica
gel. The filtrate was washed with a sat'd solution of NaHCO.sub.3
(2.times.10 mL). The organic layer was separated, dried
(MgSO.sub.4), and concentrated. The residue was purified by
chromatography to obtain tetrasaccharide 9' in 55% as syrup;
[.alpha.].sup.23.sub.D -28.0 (c 1.0, CHCl.sub.3); I.R. (film) 3491,
3029, 3874, 1815, 1753, 1647, 1453, 1370, 1221, 1160, 1064, 738
cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.38 (q, 6H, J=7.96 Hz,
--SiCH.sub.2CH.sub.3), 0.76 (t, 9H, J=7.96 Hz,
--SiCH.sub.2CH.sub.3), 1.97 (s, 3H, --COCH.sub.3), 3.2-3.32 (m,
2H), 3.35-3.55 (m, 5H), 3.55-3.7 (m, 7H), 3.7-3.8 (m, 4H), 3.95
(dd, 1H, J=4.64 Hz, J=11.28 Hz), 4.0-4.12 (m, 2H), 4.18 (bs, 1H),
4.3-4.65 (m, 15H), 4.7-4.8 (m, 2H), 4.89 (t, 1H, J=5.24 Hz), 5.31
(d, 1H, J=8.4 Hz), 6.32 (d, 1H, J=6.04 Hz, H-1), 7.1- 7.5 (m, 28H,
Ar--H), 7.85 (d, 2H, J=7.4 Hz, Ar--H); .sup.13C NMR (CDCl.sub.3)
.delta. 4.47, 6.74, 20.62, 58.45, 67.89, 68.16, 68.91, 69.63,
70.01, 70.23, 70.70, 70.74, 72.95, 73.26, 73.32, 73.40, 73.43,
73.79, 74.38, 74.75, 74.81, 75.34, 76.60, 77.19, 82.21, 97.41,
100.41, 102.53, 102.84, 127.30, 127.44, 127.50, 127.56, 127.59,
127.62, 127.76, 127.82, 127.84, 127.96, 128.18, 128.24, 128.33,
128.38, 128.46, 128.83, 132.49, 137.32, 137.89, 138.70, 140.76,
144.51, 153.43, 168.94.
EXAMPLE 14
[0205] Tetrasaccharide pentaol (11): To a solution of
tetrasaccharide 9' (370 mg, 0.26 mmol) in MeOH (5 mL) was added
K.sub.2CO.sub.3 (370 mg). The reaction mixture was stirred for 15
min, diluted with CH.sub.2Cl.sub.2 (100 mL), and filtered through a
pad of silica gel, followed by washing with EtOAc (100 mL). The
filtrates were combined, and concentrated to obtain 11 (295 mg,
85%) as a syrup; [.alpha.].sup.23.sub.D -18.0 (c 1.0, CHCl.sub.3);
IR (film) 3469, 3030, 2873, 1648, 1496, 1452, 1328, 1092, 909, 737
cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.31 (q, 6H, J=6.38 Hz,
--SiCH.sub.2CH.sub.3), 0.70 (t, 9H, J=6.38 Hz,
--SiCH.sub.2CH.sub.3), 2.49 (bs, 1H, --OH), 2.82 (bs, 1H, --OH),
3.16 (m, 1H, --CHNHSO.sub.2Ph), 3.3-3.6 (m, 12H), 3.6-3.78 (m, 6H),
3.79 (bs, 2H), 3.8-3.85 (m, 3H), 3.92 (bd, 1H, J=4.23 Hz), 4.0 (bt,
1H), 4.05-4.10 (m, 2H), 4.10-4.25 (m, 3H), 4.30-4.40 (m, 6H),
4.4-4.55 (m, 7H), 4.75 (dd, 1H, J=2.73 Hz, J=4.94 Hz), 4.9 (d, 1H,
J=4.20 Hz), 6.17 (d, 1H, 6.63 Hz, --HNSO.sub.2Ph), 6.31 (d, 1H,
J=4.9 Hz, H-1), 7.0-7.4 (m, 23H, Ar--H), 7.80 (d, 2H, J=6.00 Hz,
Ar--H); .sup.13C NMR (CDCl.sub.3) .delta. 4.30, 6.72, 57.88, 68.00,
68.78, 68.84, 69.20, 70.46, 70.86, 71.39, 71.99, 73.04, 73.14,
73.31, 73.40, 73.54, 73.79, 75.72, 76.01, 76.16, 81.44, 100.15,
101.85, 102.32, 102.60, 127.30, 127.45, 127.58, 127.62, 127.65,
127.73, 128.17, 128.21, 128.30, 128.33, 128.90, 132.52, 137.71,
137.87, 137.91, 138.10, 138.62, 140.18, 144.27.
EXAMPLE 15
[0206] Hexasaccharide tetrol (15'): To a solution of disaccharide 8
(197 mg, 0.20 mmol) and tetrasaccharide 15 (275 mg, 0.20 mmol) in
CH.sub.2Cl.sub.2:Et.sub.2O (1:2, 15 mL) (suspended with 4 .ANG.
molecular sieves (1.20 g)) and di-t-butylpyridine (0.184 mL, 0.80
mmol) at -10.degree. C. was added MeOTf (0.092 mL, 0.80 mmol. The
reaction mixture was stirred for 2 h, allowed to warm up to
0.degree. C. After stirring for 24 h, the mixture was diluted with
EtOAc (15 mL), filtered through a pad of silica gel, and washed
with a sat'd solution of NaHCO.sub.3 (2.times.10 mL). The organic
layer was separated, dried (MgSO.sub.4), and concentreted. The
resdidue was purified by chromatography (1:1, Hex:EA) to obtain 15'
(276 mg, 60%) as a syrup; [.alpha.].sup.23.sub.D -23.0 (c, 1.0,
CHCl.sub.3); I.R. (film) 3490, 3030, 2875, 1807, 1649, 1453, 1330,
1093, 909, 743 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.25 (m,
6H, --SiCH.sub.2CH.sub.3), 0.37 (q, 6H, J=7.92 Hz,
--SiCH.sub.2CH.sub.3), 0.71 (t, 9H, J=7.88 Hz,
--SiCH.sub.2CH.sub.3), 0.76 (t, 9H, J=7.92 Hz,
--SiCH.sub.2CH.sub.3), 0.86 (t, 9H, J=7.88 Hz,
--SiCH.sub.2CH.sub.3), 2.46 (s, 1H, --OH), 2.52 (s, 1H, --OH), 3.15
(m, 1H, --CHNHSO.sub.2Ph), 3.21 (m, 1H, --CHNHSO.sub.2Ph), 3.28
(dd, 1H, J=3.04, J=9.24 Hz), 3.37-3.55 (m, 7H), 3.55-3.79 (m, 14H),
3.82 (bs, 2H), 3.89 (bs, 1H), 3.94-4.11 (m, 4H), 4.18 (bs, 1H),
4.28 (m, 1H), 4.33-4.40 (m, 3H), 4.41 (s, 2H, --CH.sub.2Ar),
4.44-4.47 (m, 3H), 4.49 (s, 2H, --CH.sub.2Ar), 4.52 (m, 1H), 4.54
(s, 2H, --CH.sub.2Ar), 4.55-4.63 (m, 2H), 4.66 (dd, 2H, J=3.88 Hz,
J=6.04 Hz), 4.74 (dd, 1H, J=2.76 Hz, J=6.08 Hz), 5.28 (d, 1H,
J=7.52 Hz, --NHSO.sub.2Ph), 5.51 (d, 1H, J=8.32 Hz,
--NHSO.sub.2Ph), 6.32 (d, 1H, J=6.04 Hz, H-1), 7.10-7.55 (m, 41H,
Ar--H), 7.83 (d, 2H, J=7.36 Hz, Ar--H), 7.89 (d, 2H, J=7.48 Hz,
Ar--H); .sup.13C NMR (CDCl.sub.3) .delta. 4.36, 4.44 (2C), 6.55,
6.67, 6.87, 58.52, 58.82, 67.61, 67.76, 67.82, 68.11, 68.71, 68.94,
69.07, 69.49, 69.75, 69.78, 69.92, 70.47, 70.73, 72.51, 72.92,
73.26, 73.31, 73.34, 73.37, 73.68, 73.85, 74.26, 74.61, 75.21,
75.27, 75.75, 75.90, 76.40, 77.10, 82.97, 83.60, 99.93, 100.49,
101.64, 102.74, 102.82, 103.08, 127.20, 127.38, 127.42, 127.49,
127.54, 127.62, 127.72, 127.76, 127.88, 128.10, 128.17, 128.26,
128.30, 128.40, 128.84, 128.97, 132.38, 132.71, 137.37, 137.57,
137.87, 137.90, 138.12, 138.18, 138.76, 139.9, 140.62, 144.45,
154.16.
EXAMPLE 16
[0207] Fully protected Hexasaccharide (12): To a solution of
hexasaccharide 15' (175 mg, 0.078 mmol) in dry CH.sub.2Cl.sub.2 (20
mL) was added pyridine (2 mL), Ac.sub.2O (2 mL) and DMAP (cat). The
reaction mixture was stirred for 24 h, washed with CuSO.sub.4
solution (3.times.10 mL), and sat'd NaHCO.sub.3 (3.times.10 mL).
The organic layer was separated, dried (MgSO.sub.4), and
concentrated. The residue was purified by chromatography to obtain
12 (175 mg, 95) as a syrup; [.alpha.].sup.23.sub.D; I.R.
(cm.sup.-1); .sup.1H NMR (CDCl.sub.3) .delta. -0.24 (m, 12H,
--SiCH.sub.2CH.sub.3), 0.54 (q, 6H, J=8.08 Hz,
--SiCH.sub.2CH.sub.3), 0.68 (bt, 9H, J=7.70 Hz,
--SiCH.sub.2CH.sub.3), 0.70 (bt, 9H, J=7.8 Hz,
--SiCH.sub.2CH.sub.3), 0.87 (t, 9H, J=7.9 Hz,
--SiCH.sub.2CH.sub.3), 1.86 (s, 3H, --COCH.sub.3), 1.90 (s, 3H,
--COCH.sub.3), 2.08 (s, 2H, --COCH.sub.3), 2.15 (s, 3H,
--COCH.sub.3), 3.03 (bd, 1H, J=7.68 Hz, --CHNHSO.sub.2Ph), 3.2-3.4
(m, 8H), 3.4-3.85 (m, 30H), 3.85-4.2 (m, 8H). 4.20-4.6 (m, 29H),
4.75 (q, 1H, J=3.12 Hz, 6.0 Hz), 4.8 (bd, 1H, J=8.2 Hz, 4.88 (d,
1H, J=3.48 Hz), 5.10 (m, 2H, J=8.76 Hz), 5.26 (d, 1H, J 2.52 Hz),
5.33 (d, 1H, J -8.68 Hz, --NHSO.sub.2Ph), 5.42 (d, 1H, J=2.64 Hz),
5.90 (d, 1H, J=10.84 Hz, --NHSO.sub.2Ph), 6.31 (d, 1H, 6.0 Hz,
H-1), 7.1-7.5 (m, 41H, Ar--H), 7.82 & 7.89 (2bm, 4H, Ar--H);
.sup.13H NMR (CDCl.sub.3) .delta. 4.10, 4.14, 4.49, 6.52, 6.60,
6.64, 20.75, 20.81, 20.09, 21.46, 55.97, 56.73, 67.83, 68.41,
68.63, 68.80, 69.35, 69.82, 69.88, 70.12, 70.49, 71.09, 71.20,
71.71, 72.84, 72.95, 73.11, 73.38, 73.53, 73.60, 73.67, 73.74,
73.79, 74.10, 74.33, 74.40, 75.32, 75.78, 75.89, 76.18, 76.77,
77.20, 99.75, 100.15, 100.38, 100.53, 101.55, 102.17, 127.26,
127.34, 127.42, 127.47, 127.52, 127.58, 127.61, 127.62, 127.66,
127.73, 127.73, 127.80, 127.85, 128.14, 128.21, 128.26, 128.39,
128.41, 128.66, 128.99, 131.93, 132.60, 137.47, 137.66, 137.77,
137.92, 138.31, 138.43, 138.77, 139.96, 141.74, 144.48, 154.07,
169.44, 169.60, 169.64, 171.34.
EXAMPLE 17
[0208] Hexasaccharide triol (13): To a solution of hexasaccharide
12 (175 mg, 0.0725 mmol) in dry THF (5 mL) was added a solution of
TBAF (1.0 M in THF): AcOH (0.725 mL, 10 eq). The reaction mixture
was stirred at 35.degree. C. for 24 h, diluted with EtOAc (10 mL),
and washed with a saturated solution of NaHCO.sub.3 (2.times.5 mL).
The organic layer was separated, dried (MgSO.sub.4), and
concentrated. The residue was purified by chromatography (1:4,
Hex:EA) to obtain 13 (143 mg, 93%) as a white glassy substance;
.sup.1H NMR (CDCl.sub.3) .delta. 1.88, 1.92, 2.01, 2.02 (4s, 3H
each, --COCH.sub.3), 2.85 (bt, 1H, J=8.24 Hz, --CHNHSO.sub.2Ph),
3.02 (bq, 1H, J=7.0 Hz, --CHNHSO.sub.2Ph), 3.20 (dd, 1H, J=7.56 Hz,
J=8.0 Hz), 3.27 (dd, 2H, J=4.72 Hz, J=10.00 Hz), 3.3-3.8 (m, 36H),
3.87 (bs, 2H), 4.03 (bd, 3H), 4.10 (bs, 1H), 4.2-4.65 (m, 33H),
4.66 (d, 1H, 5.1 Hz), 4.77 (q, 1H, J=3.2 Hz), 5.01 (dd, 1H, J=8.32
Hz, J=9.68 Hz), 5.12 (dd, 1H, J=8.2 Hz, J=9.84 Hz), 5.25 (d, 1H,
J=3.16 Hz), 5.39 (d, 1H, J=3.08), 6.32 (d, 1H, J=6.12 Hz, H-1),
7.10-7.45 (m, 41H, Ar--H), 7.78 (m, 4H, Ar--H);
EXAMPLE 18
[0209] Nonasaccharide (15): To a solution of hexasaccharide 13 (140
mg, 0.067 mmol) and 13-flourofucose 14 (241 mg, 0.53 mmol) in dry
toluene (10 mL) (suspended with 4 .ANG. molecular sieves) at
0.degree. C., was added di-t-butylpyridine (0.152 mL, 0,67 mmol)
followed by a solution of Sn(OTf).sub.2 (0.223 mg, 0.53 mmol) in
THF (1 mL). The reaction mixture was stirred for 36 h, and then
diluted with EtOAc (25 mL), and filtered through a pad of silica
gel. The organic layer was washed with a sat'd solution of
NaHCO.sub.3 (2.times.10 mL). The organic layer was separated, dried
(MgSO.sub.4), and concentrated. The residue was purified by
chromatography (1:1, Hex:EA) to obtain 15 (135 mg, 60%) as a syrup;
.sup.1H NMR (CDCl.sub.3) .delta. 0.87 (d, 3H, J=6.24, --CH.sub.3),
), 0.93 (d, 3H, J=6.32 Hz, --CH.sub.3), 1.07 9d, 3H, J=6.36 Hz,
--CH.sub.3), 1.61 9s, 3H, --COCH.sub.3), 1.80 (s, 3H,
--COCH.sub.3), 1.93 (s, 3H, --COCH.sub.3), 1.98 (s, 3H,
--COCH.sub.3), 3.2-3.4 (m, 6H), 3.4-3.9 (m, 27H), 3.9-4.0 (m, 2H),
4.0-4.2 (m, 8H), 4.2-4.65 (m, 34H), 4.65-4.8 (m, 7H), 4.84 (d, 1H,
J=5.3 Hz), 4.89 (bt, 1H, J=8.4 Hz), 5.05-5.15 (m, 2H), 5.28 (bs,
1H), 5.35 (d, 1H, J=2.76 Hz), 5.40 (bs, 1H), 5.53 (bs, 3H), 5.65
(d, 1H, J=5.60 Hz), 6.33 (d, 1H, J=6.04 Hz, H-1), 7.)-7.3 (m, 68H,
Ar--H), 7.3-7.45 (m, 9H, Ar--H), 7.45-7.57 (m, 3H), 7.65 (d, 2H,
J=7.56 Hz, Ar--H), 7.75 (d, 2H, J=7.48 Hz, Ar--H), 7.99, 7.96, 7.94
(3d, 6H, J=7.52 Hz, Ar--H.
EXAMPLE 19
[0210] Thioglycoside of Nonasaccharide (16): To a solution of a
nonasaccharide 15 (50 mg, 0.0149 mmol) in dry CH.sub.2Cl.sub.2 (1
mL) (suspended with 4 .ANG. molecular sieves (100 mg)), was added a
solution of dimethyldioxirane in acetone (ca 0.08 M, 3 mL). The
reaction mixture was stirred for 45 min, and then solvents were
evaporated under a stream of N.sub.2 gas. The residue was dried in
vacuum (10 min), and then dissolved in CH.sub.2Cl.sub.2 (1 mL), and
after cooling to -78.degree. C., was reacted with EtSH (1 mL) and
TFAA (5 .mu.L). After 30 min, the mixture was evaporated under a
stream of N.sub.2 gas, and the residue was dried in vaccume. The
crude product was dissolved in CH.sub.2Cl.sub.2 (1 mL) and then
reacted with acetic anhydride (0.5 mL) and pyridine (0.5 mL). After
drying for 24 hrs under reduced presure, the residue was
chromatographed (3:2, Hex:EA) to obtain thioglycoside 16 (60%) as a
syrup; .sup.1H NMR (CDCl.sub.3) .delta. 0.94 (d, 3H, --CH.sub.3),
1.02 (d, 3H, --CH.sub.3), 1.16 (d, 3H, --CH.sub.3), 1.28 (t, 3H,
--CH.sub.3), 1.93 (s, 3H, --COCH.sub.3), 2.0 (s, 3H, --COCH.sub.3),
2.04 (s, 3H, --COCH.sub.3), 2.07 (s, 3H, --COCH.sub.3), 2.14 (s,
3H, --COCH.sub.3), 2.71 (m, 2H, --SCH.sub.2CH.sub.3), 3.1-4.0 (m,
several protons), 4.1-5.0 (m, several protons), 4.82 (d, 1H), 4.89
(t, 1H), 5.20 (d&m, 2H), 5.35 (d, 1H), 5.45 (d, 1H), 5.50 (bs,
1H), 5.63 (bs, 2H), 5.74 (m, 1H), 7.0-8.2 (m, 90H, Ar--H);
EXAMPLE 20
[0211] Sphingosine glycoside (18): To a solution of thioglycoside
16 (30 mg, 0.0086 mmol) and azidohydrin 17 at 0.degree. C. in dry
CH.sub.2Cl.sub.2: Ether (1:2, 1.5 mL) (suspended with 4 .ANG.
molecular sieves (100 mg)) was added MeOTf (0.0038 mL, 4 eq). The
reaction mixture was allowed to warm up to room temperature. After
24 hrs, the mixture was diluted with EtOAc (5 mL), filtered through
a pad of silica gel, and washed with a sat'd solution of
NaHCO.sub.3 (2.times.5 mL). The organic layer was separated, dried
(MgSO.sub.4), and concentrated. The residue was purified by column
chromatogrphy (1:1 Hex:EA) to obtain Sphingosine glycoside 18 (55%)
as a syrup; .sup.1H NMR (CDCl.sub.3) .delta. 0.80 (m, 9H, 0.85 (d,
3H, --CH.sub.3), 0.93 (d, 3H, --CH.sub.3), 1.07 (d, 3H,
--CH.sub.3), 1.18 (bm, 23H, aliphatic --CH.sub.2), 1.33 (bs, 2H)
1.5 (bd, 4H), 1.81 (s, 3H, --COCH.sub.3), 1.94 (s, 3H,
--COCH.sub.3), 1.97 (s, 3H, --COCH.sub.3), 2.0 (s, 6H,
--COCH.sub.3), 3.1-3.7 (m, several protons), 3.7-4.1 (m, several
protons), 4.2-4.8 (m, several protons), 4.82 (d, 1H), 4.89 (t, 1H),
4.97 (d, 1H), 5.1 (m, 2H), 5.37 (m, 4H), 5.47 (d, 1H), 5.53 (bs,
2H), 5.60 (d, 1H), 5.7 (m, 1H), 7.0-8.1 (m, 95H, Ar--H).
EXAMPLE 21
[0212] Amide (protected KH-1 antigen) (18'): To a solution of azide
18 (15 mg, 0.0039 mmol) in EtOAc (3 mL) was added Lindlar's
catalyst (50 mg) and Palmitic anhydride (10 mg, 0.020 mmol). The
reaction mixture was stirred at room temperature under a H.sub.2
atmosphere for 24 h, and then filtered through a pad of silica gel,
rinsed with EtOAc (20 mL), and concentrated. The residue was
purified by chromatography (1:1 EA:Hex) to give amide 18' (85%) as
a syrup: .sup.1H NMR (CDCl.sub.3) .delta. 0.79 (m, 9H, 0.84 (d, 3H,
--CH.sub.3), 0.92 (d, 3H, --CH.sub.3), 1.06 (d, 3H, --CH.sub.3),
1.17 (bm, 45H, aliphatic --CH.sub.2), 1.48 (bs, 9H), 1.77 (s, 3H,
--COCH.sub.3), 1.90 (s, 3H, --COCH.sub.3), 1.95 (s, 3H,
--COCH.sub.3), 1.97 (s, 6H, --COCH.sub.3), 3.0-3.9 (m, several
protons), 4.0-5.0 (m, several protons) 5.51 (bs, 1H), 5.2-5.4 (m,
3H), 5.5 (bs, 1H), 5.6-5.8 (m, 2H); 7.0-8.1 (m, 95H, Ar--H).
EXAMPLE 22
[0213] KH-1 antigen (1): To a solution of liquid ammonia (5 mL)
under N.sub.2 at -78.degree. C. was added sodium (18 mg). To the
resulting blue solution was added a solution of protected KH-1
derivative 18' (20 mg, 0.005 mmol) in dry THF (1 mL). After 45 min
at -78.degree. C., the reaction mixture was quenched with absolute
MeOH (5 mL). Most of the ammonia was removed in a stream of
nitrogen gas. The resulting solution was diluted with MeOH (5 mL)
and stirred overnight, and then neutralized with Et.sub.3N.HCl.
After stirring for 15 min, the mixture was dried under nitrogen.
The crude product was then suspended in DMF (1.0 mL), THF (1.0 mL),
and Et.sub.3N (1.0 mL) and treated with Ac.sub.2O (1 mL) and DMAP
(cat). After stirring overnight, the reaction mixture was
concentrated, passed through a plug of silica gel using EtOAc as an
elutant and concentrated. The syrup obtained was dissolved in MeOH
(5 mL) and treated with MeONa (5 mg) for 24 h, and then neutralized
with Dowex 50-X8. Filtration and concentration gave the KH-1
antigen (70%). An analytical sample was prepared by RP column
chromatography, eluting with water-5% methanolic water, followed by
lyophilization to obtain 1 as a white powder; .sup.1H NMR (DMSO)
.delta. 0.95 (m, 3H), 1.1-1.35 (3d, 9H, --CH.sub.3), 1.38 (bm,
multipleple protons, alphatic --CH.sub.2), 1.5 (m, 9H), 1.85 (s,
6H, NHCOCH.sub.3), 1.9 (m, 2H), 2.0-2.20 (m, 6H), 3.0-4.0 (m,
Multiple protons), 4.1 (q, 1H), 4.17 (d, 1H, H-1), 4.27 (m, 1H),
4.34 (bm, 1H), 4.41 (d, 1H), 4.6 (q, 1H), 4.67 (m), 4.75 (t, 2H),
4.88 (d, 2-3H), 4.97 (d, 1H), 5.36 (m, 1H), 5.56 (m, 1H).
EXAMPLE 23
[0214] Allyl glycoside (2): To a solution of liquid ammonia (5 mL)
under N.sub.2 at -78.degree. C. was added sodium (94 mg). To the
resulting blue solution was added a solution of nonasacchride
glycal 15 (75 mg, 0.022 mmol) in a dry THF (3 mL). After 45 min at
-78.degree. C., the reaction was quenched with absolute MeOH (5
mL). Most of the ammonia was removed with a stream of nitrogen. The
solution was diluted with MeOH (5 mL), stirred overnight, and
neutralized with Dowex 50-X8 (846 mg). The resulting mixture was
stirred for 15 min, and filtered. The resins were washed with
NH.sub.3--MeOH sloution (3.times.20 ml). The filtrates were
combined , and dried under a stream of nitrogen gas. The crude
product was then suspended in DMF (1.0 mL), THF (1.0 mL), and
Et.sub.3N (1.0 mL) and then treated with Ac.sub.2O (1 mL) and DMAP
(cat). The reaction mixture was stirred for 24 h, concentrated,
passed through a plug of silica gel in EtOAc, and again
concentrated. The syrup obtained was dissolved in CH.sub.2Cl.sub.2,
then treated with dimethyldioxirane solution in acetone (ca. 0.08
M, 5 mL) at 0.degree. C. under N.sub.2. The mixture was stirred for
45 min, and concentrated under a stream of N.sub.2 gas. The syrup
obtained was reacted with allyl alcohol (5 mL). After 24 h, excess
allyl alcohol was evaporated and the crude syrup was dissolved in
MeOH and treated with MeONa (25% in MeOH, 60 .mu.L). After 24 h,
the mixture was neutralized with Dowex 50-X8, filtered and
concentrated to give allylated nonasaccharide 2 (60%). An
analytical sample was prepared by RP column chromatography, eluting
with water-5% methanolic water, followed by lyophilization to
obtain white powder; .sup.1H NMR (D.sub.2O) .delta. 1.0-1.35 (3d,
9H, --CH.sub.3), 2.0 (s, 6H, --COCH.sub.3), 3.3 (bm, 1H, --CHNHAc),
3.4-4.0 (m, multiple protons), 4.08 (bs, 1H), 4.12 (bs, 1H), 4.22
(m, 1H), 4.42 (t, 2H), 4.5 (t, 2H), 4.7 (d, 2H), 4.86 (d, 1H), 5.1
(bs, 2H), 5.26 (bs, 1H), 5.39 (d, 1H) 5.95 (m, 1H,
--CHCH.dbd.CH.sub.2).
PREPARATION OF HEPTA AND KH-1-KLH CONJUGATES BY DIRECT AND
CROSS-LINKER METHOD
[0215] The allyl glycoside of KH-1 was conjugated to KLH (Keyhole
Lympet Hemocyanin) protein via two different methods. The first was
the direct coupling method which utilized the reductive amination
reaction between the lysines of KLH protein and the aldehyde moiety
obtained by ozonolisis of KH-1 allyl glycoside. This method
typically provides the glycoprotein with around 141 carbohydrate
units (KH-1) per KLH.
[0216] The other conjugation method utilized a cross linker known
as M.sub.2. The same aldehyde of KH-1 antigen utilized in direct
coupling was further derivatized to a suitable conjugatable form
containing M.sub.2 linker. Then the resulting compound was coupled
to thiolated KLH protein. This crosslinker method was highly
efficient, providing the glycoprotein conjugate with around 492
carbohydrate units (KH-1) per KLH. FIGS. 12 and 13 describe two
coupling methods.
[0217] Groups of mice were immunized with both types of
glycoprotein conjugates (KH1-KLH and KH1-M.sub.2-KLH). An
immunological adjuvant QS-21 was co-administered in the
immunization. The antibodies thus elicited were assayed by ELISA
and FACS method. The cross-linked conjugate (KH1-M.sub.2-KLH)
showed increased immune response from the mice, though both types
of conjugate effectively elicited antibodies. FIG. 11 describes an
alternative synthesis of KH-1 tetrasaccharide and
hexasaccharide.
[0218] Accordingly, the allyl group in KH-1 or the heptasaccharide
disclosed herein was converted to an aldehyde group by ozonolysis
and linked to --NH.sub.2 groups of KLH by reductive amination
method in the presence of sodium cyanoborohydride as described for
globo H. (Ragupathi G, et al., Angew. Chem. Int. Ed. Engl. 1997,
36, 125-128.) In the case of the cross-linker method, the aldehyde
group obtained through ozonolysis was first reacted with hydrazide
of M.sub.2CH.sub.2 (4-maleimidomethyl) cyclohexane-1-carboxyl
hydrazide) and reacted with thiolated KLH as described in Ragupathi
G., et al., "A novel and efficient method for synthetic
carbohydrate conjugate vaccine preparation: Synthesis of sialyl
Tn-KLH conjugate using an M.sub.2C.sub.2H linker arm"
Glycoconjugate J., in press. For example, 4 mg of KH-1 allyl
glycoside in methanol was stirred at -78.degree. C. in a
dry-ice/ethanol bath and ozone gas was passed through the solution
for 0 min under vigorous stirring. The excess of ozone was then
displaced with nitrogen over a period of 5 min. Methyl sulfide (100
.mu.l) was added and the reaction mixture stirred at room
temperature for 2 hours and distributed equally in two vials. The
solvent was removed under a stream of nitrogen. The resulting white
solid was used directly in the subsequent conjugation steps.
[0219] Direct Conjugation of KH-1-aldehyde with KLH:
[0220] Two mg KH-1 aldehyde was dissolved in 1 ml of 0.1M phosphate
buffered saline (PBS) pH 7.2 and 4 mg of KLH in PBS. Two mg sodium
cyanoborohydride was added and the mixture incubated under gentle
agitation at 37.degree. C. for 48 h. After 16 h, an additional 1.0
mg sodium cyanoborohydride was added and the incubation continued.
The unreacted KH-1 aldehyde was removed completely with multiple
washes using a Amicon Centriprep with molecular weight cut-off
value 30000 dalton, with 6-7 changes of PBS at 4.degree. C.
[0221] Conjugation of KH-1-aldehyde through M.sub.2C.sub.2H to
thiolated KLH:
[0222] Preparation of KH-1-M.sub.2C.sub.2H
[0223] Two mg of KH-1-aldehyde was dissolved in 1 ml of 0.1M sodium
acetate buffer pH 5.5, and 4 mg of M.sub.2C.sub.2H in 100 .mu.l of
dimethyl sulfoxide (DMSO) was added. The reaction mixture was
incubated at room temperature for 15 min with gentle stirring. At
the end of 15 min 2 mg of solid sodium cyanoborohydride was added
and the incubation continued at room temperature for 2 h. Unreacted
M.sub.2C.sub.2H was removed in a Sephadex G10 column equilibrated
previously with 0.1 M sodium phosphate buffer pH 6.0 containing 5
mM EDTA and eluted with the same buffer. The fractions positive for
KH-1 by TLC with orcinol were combined.
[0224] Addition of sulfhydryl groups to KLH
[0225] 2-Iminothiolane (2 mg) dissolved in thiolation buffer (50 mM
triethanolamine, 0.15 M NaCl, 5 mM EDTA, pH 8.0) was added to 4 mg
of KLH and incubated with stirring at room temperature for 2 h.
Unreacted 2-iminothiolane was removed by Sephadex G15 column
equilibrated previously with 0.1 M sodium phosphate buffer pH 7.2
containing 5 mM EDTA and eluted with the same buffer. Fractions
positive for KLH with BioRad protein assay dye reagent were
combined. A small portion was used to estimate sulfhydryl groups in
the thiolated KLH using Ellman's reagents and cysteine as standard.
Riddles P. W., et al., Anal. Biochem. 1979, 94, 75-81. The KLH was
estimated by a dye method using BioRad dye reagent according to the
manufacture's instructions.
[0226] Conjugation of KH-1-M.sub.2C.sub.2H product and thiolated
KLH were mixed and adjusted to pH 7.2 with 0.1M sodium phosphate
buffer pH 8.0. The reaction mixture was then incubated at room
temperature overnight. The content of the KH-1-M.sub.2C.sub.2H-KLH
reaction vial was transferred to a Centriprep concentrator 30
(Amicon: molecular cut-off 30000 Dalton) and unreacted
KH-1-M.sub.2C.sub.2H was removed completely with multiple washes.
The conjugate was checked by HPTLC for the absence of unreacted
KH-1 as mentioned above. The epitope ratios of two batches of
conjugate were determined by estimating protein content by BioRad
dye binding protein assay and carbohydrate by a HPAEC-PAD assay.
The epitope ratio of hepta-KLH and hepta-M.sub.2-KLH was 112/1 and
197/1 respectively. The epitope ratio of KH-1-KLH and
KH-1-M.sub.2-KLH was 141/1 and 492/1, respectively.
1TABLE 1 Antibody Titers by ELISA against KH1-KLH Pre-serum 10 days
post 3rd Group IgM IgG IgM IgG KH-1-KLH 1.1 0 0 100 0 1.2 0 0 100 0
1.3 0 0 100 0 1.4 100 0 300 0 1.5 100 0 100 0 KH-1-M.sub.2-KLH 2.1
0 0 0 0 2.2 0 0 900 300 2.3 0 0 300 300 2.4 0 0 900 900 2.5 0 0 100
0 3.1 0 0 2700 24,300 3.2 0 0 2700 8100 3.3 0 0 300 0 3.4 0 100
2700 2700 3.5 100 0 8100 900 (0.2 ug/well antigen plated)
[0227]
2TABLE 2 Cell Surface reactivity of KH-1 antibodies on MCF-7 cells
by FACS. % of cells positive Group IgM IgG KH1-KLH 1.1 28.4% 14.1%
1.2 16.9% 18.8% 1.3 12.9% 11.0% 1.4 36% 12.3% 1.5 35.56% 30.2%
KH1-M2-KLH 2.1 30.18% 88.1% 2.2 36.59% 76.2% 2.3 18.16% 93.1% 2.4
47.9% 91.9% 2.5 20.03% 97.9% Mouse presera IgM: 1.72%, Mouse preIgG
0.76%, Mab BR96.78.17%
[0228] Serological Analysis:
[0229] ELISA: Enzyme-linked immunosorbent assays (ELISAs) were
performed as described by Livingston, P. O. et al., Cancer Immunol.
Immunother., 1989, 29, 179-184, 1989. Serially diluted antiserum
was added to wells coated with antigen (0.1 .mu.g) and incubated
for 1 h at room temperature. Goat anti-mouse IgM or IgG conjugated
with alkaline phosphatase served as secondary antibodies.
Absorbance was measured at 414 nm. The antibody titer was defined
as the highest serum dilution showing an absorbance 0.1 or greater
above that of normal mouse sera.
[0230] Flow Cytometry:
[0231] Cells from the KH-1-positive breast cancer cell line MCF-7
served as target. Soule, H. D., et al., J. Natl. Cancer Inst.,
1973, 51, 1409-1416. Single cell suspensions of 2.times.10.sup.5
cells/tube were washed in PBS with 3% fetal calf serum and 0.01 M
NaN.sub.3 and incubated with 20 .mu.l of 1:20 diluted antisera or
mAb BR-96 for 30 min on ice. After washing the cells twice with 3%
FCS in PBS, 20 .mu.l of 1:15 goat anti-mouse IgM or IgG-labeled
with fluorescein-isothiocyanate (FITC) was added, mixed and
incubated for 30 min. After wash, the positive population and mean
fluorescence intensity of stained cells were analyzed by flow
cytometry (EPICS Profile II, Coulter, Co., Hialeah, Fla.). Zhang,
S. et al., Cancer Immunol. Immunother., 1995, 40, 88-94.
[0232] Immune Adherence (IA) Assay:
[0233] The IA assay measures resetting of human RBC (blood group O)
with guinea pig complement on target cells mediated by IgM
antibodies, and was performed as described previously. Shiku, H.,
et al., J. Exp't Med., 1976, 144, 873-881. Individual target cells
were scored as positive when 50% or more of the cell perimeter 3
was surrounded by indicator cells.
[0234] Complement Dependent Cytotoxicity (CDC):
[0235] Complement dependent cytotoxicity was assayed at a serum
dilution of 1:10 with MCF-7 cells by a 4 h europium-release assay.
Zhang, S., et al., Cancer Immunol. Immunother., 1995, 40, 88-94.
All assays were performed in triplicate. Controls included cells
incubated only with culture medium, complement, antisera or mAb
BR-96. Spontaneous release was the europium released by target
cells incubated with complement alone. Percent cytolysis was
calculated according to the formula: 1 Specific Release ( % ) =
Experimental release - spontaneous release Maximum release -
spontaneous release .times. 100
[0236] Inhibition Assay:
[0237] Antisera at 1:1500 dilution or mAb BR-96 at 0.1 .mu.g/ml
were mixed with various concentrations of structurally related and
unrelated carbohydrate antigens. The mixture was incubated at room
temperature for 30 min, and transferred to an ELISA plate coated
with KH-1-ceramide. ELISAs were performed as described above.
Percentage inhibition was calculated as the difference in
absorbance between the uninhibited and inhibited serum.
[0238] Immunization of Mice
[0239] Groups of mice (CB6F1 female; 6 weeks of age) obtained from
Jackson Laboratory, Bar Harbor, Me., were immunized subcutaneously
with KH-1-KLH or KH-1-M.sub.2C.sub.2H-KLH containing equivalent to
3 .mu.g KH-1 only (the quantity of KLH varied depending on the
epitope density) mixed with 10 .mu.g of immunological adjuvant
QS-21, a saponin derivative from the bark of the Quillaja saponaria
Molina tree (Aquila,. Worcester, Mass.) at 0, 1 and 2 weeks and
bled 10 days after the third immunization. The presence of antibody
was assayed by an enzyme linked immunosorbent assay (ELISA) as
described in Kensil C. R. et al., J. Immunol., 1993, 146, 431-437,
using KH-1 ceramide as target antigen. The cell surface reactivity
of anti-KH-1 antibodies was tested on KH-1 positive MCF-7 cells by
flow cytometry assays. The mice vaccinated with
KH-1-M.sub.2C.sub.2H are made the high titer antibody against the
synthetic KH-1 and the antibodies were reacted strongly on the
cell's surface that expressed KH-1 antigens.
[0240] Binding of Monoclonal Antibody BR 96 with synthetic KH-1 and
other Carbohydrate by Dot-blot Immune Stain:
[0241] 0.5 .mu.g KH-1 ceramide and other Ley antigen and unrelated
antigens were spotted on nitrocellulose strips. Dot blot Immune
staining was performed monoclonal antibody BR 96 after blocked with
6% bovine serum albumin in PBS for 1 h and incubated with antibody
BR 96 (diluted 1:500 in PBS) overnight at room temperature. The
strips were washed with PBS containing 0.05% Tween 20 and incubated
with anti-mouse IgG antibody conjugated with horseradish peroxidase
at 1:200 dilution for 3 h at room temperature. Then the strips were
washed with PBS-0.05% Tween 20 and developed with
4-chloro-1-naphtol-H.sub.2O.sub.2. The results are summarized in
Table 1. The synthetic KH-1 reacted very strongly when compared
with other Le.sup.y related antigens unrelated antigens were failed
to react with BR 96 antibody.
3TABLE 3 Binding of Monoclonal Antibody Br 96 with KH-1 and other
Carbohydrates by Dot-blot. Monoclonal Antibody BR 96 (Le.sup.y F12
Carbohydrate related) (FucosylGM1) KH-1 ceramide very strong
negative (+++) Le.sup.y-ceramide strong (++) negative Le.sup.y-KLH
strong (++) negative Globo H ceramide negative negative TF-ceramide
negative negative SSEA-ceramide negative negative Le.sup.y/Le.sup.b
(Ovarian cyst Mucins-Tighe)* strong (++) negative Le.sup.a/Le.sup.x
(Ovarian cyst mucins-N1)* weak (+) negative Non fucosylated
precursor of Lewis* negative negative Le.sup.a-PAA negative
negative Le.sup.x-PAA weak (+) negative FucGMI negative very strong
(+++) GD3 negative negative *extracted from patient tissue
[0242] Discussion
[0243] Human tumors are often marked by the presence of unusual
carbohydrate structural motifs. Hakomori, S., Cancer Res., 1985,
45, 2405; Feizi, T., Cancer Surveys, 1985, 4, 245; Lloyd, K. O.,
Am. J. Clin. Pathol., 1987, 87, 129; Lloyd, K. O., Cancer Biol.,
1991, 2, 421. These carbohydrate domains are encountered as
cell-surface bound glycolipids or glycoproteins. Hakomori, S.,
Cancer Cells, 1991, 3, 461. It would be useful for cancer therapy
to achieve some level of immune response by vaccinating cancer
patients with such cell-free carbohydrate domains, obtained through
total synthesis and suitably bioconjugated. Preliminary synthetic
studies have been reported. M. T. Bildoeau, T. K. Park, S. Hu, J.
T. Randolph, S. J. Danishefsky, P. O. Livingston, and S. Zhang, J.
Am. Chem. Soc., 1995, 117, 7840; T. K. Park, I. J. Park, I. J. Kim,
S. Hu, M. T. Bilodeau, J. T. Randolph, O. Kwon and S. J.
Danishefsky, J. Am. Chem. Soc., 1996, 118, 11488. In addition, the
utility of tumor-associated carbohydrate antigens is supported by
the observed establishment of responses to the human cancer lines
by sera of mice immunized with such antigens. G. Ragupathi, et al.,
Angewandte Chemie, In Press.
[0244] In conducting this project, the important issue of
"strategy" in oligosacchcaride synthesis is addressed. Of course,
in this field (as opposed to "conventional" natural product
synthesis) the basic building blocks which are considered to be
rather restricted and tend to bear obvious homology with readily
recognized components of the target system.
[0245] From this perspective a plan was pursued which would build a
hexasaccharide (cf. structure 13) so differentiated in terms of its
protecting patterns (see asterisks) as to allow for the unveiling
of the three free hydroxyls to serve as .alpha.-fucosylation
acceptor sites (see structure 13). In this way, the three
immunologically defining .alpha.-fucose units might be introduced
in one concurrent synthetic operation.
[0246] Assembling the hexasaccharide involved a potentially
forbidding network of hydroxyl group functionality. Regarding this,
advantages were observed in drawing from a few of the basic
principles now well appreciated in the logic of glycal assembly.
Bilodeau, M. T.; Danishefsky, S. J., Angew. Chem. Int. Ed. Engl.,
1996, 35, 1380.
[0247] Thus, differentiated glycals 4 and 5 are derived from
D-glucal by exploiting the reliable reactivity preference of the
C.sub.6, C.sub.3 and C.sub.4 hydroxyls
(C.sub.6>C.sub.3>C.sub.4). Moreover, the fashioning of a
clean .alpha.-epoxide from galactal derivative 3 is known. Also,
known (Halcomb, R. L.; Danishefsky, S. J., J. Am. Chem. Soc., 1989,
111, 6661), is the excellent .beta.-galactosyl donating capacity of
such an epoxide. Coupling of this epoxide to 4 and to 5, under
mediation by a simple reagent (anhydrous zinc chloride), gave 6 and
7, respectively. The C.sub.3' hydroxyl of the lactal derivative 6
was protected as a triethylsilyl derivative. In the resultant
structure 8, two of the three sites destined for eventual
fucoyslation have been distinguished. In a parallel experiment,
compound 6 could be converted by acetylation to its C.sub.3'
acetate, and overall sulfonamido (2.alpha.) ethanethiylation
(Griffith, D. A.; Danishefsky, S. J., J. Am. Chem. Soc., 1990, 112,
5811), (1 .beta.) of its glycal linkage (leads to 9 which carries
the third eventual fucosylation center at the site of its TES
group). Cleavage of the carbonate linkage of 7 generated triol 10.
Here, advantage is taken of another well appreciated preference
wherein the glycosyl accepting site in such a triol tends to be at
its C.sub.3' hydroxyl acetate (see asterisk). Kameyama, A; Ishida,
H.; Kiso, M.; Haegawa, A. J., Carb. Chem., 1991, 5, 337. Coupling
of 10 and 9 afforded, after cleavage of its cyclic carbonate and
acetate, a pentaol (see structure 11).
[0248] At this stage, the proposition was pursued in which the 1,
2, 3 in the terminal ring D, rather than the 1, 3 diol in ring B
would serve as the pre-lactosamine acceptor site with donor 8.
This, in fact proved to be the case. The successful glycosylation
was followed by acetylation of the four remaining hydroxyl groups.
This sequence led to 12 and thence to 13 as shown.
[0249] Thus, it was possible to introduce the three
.alpha.-L-fucose residues in one step via donor 14 (Danishefsky, S.
J.; Gervay, J.; Peterson, J. M.; McDonald, F. E.; Koseki, K.;
Oriyama, T.; Griffith, D. A.; Wong, C. -H.; Dumas, D. P., J. Am.
Chem. Soc., 1992, 114, 8331), thereby affording a 60% yield of the
nonasaccharide. From 15, the sorts of protocols required to reach 1
and 2 were qualitatively well precedented. In the case of 2, the
chemistry followed very closely from the methodology developed for
the globo-H breast tumor, conjugatable allyl glycoside. M. T.
Bildoeau, T. K. Park, S. Hu, J. T. Randolph, S. J. Danishefsky, P.
O. Livingston, and S. Zhang, J. Am. Chem. Soc., 1995, 117, 7840; T.
K. Park, I. J. Park, I. J. Kim, S. Hu, M. T. Bilodeau, J. T.
Randolph, O. Kwon and S. J. Danishefsky, J. Am. Chem. Soc., 1996,
118, 11488. To reach the naturally occurring glycolipid antigen 1,
a small but useful variant was introduced wherein the pre-ceramide
acceptor 17 was coupled to an anomeric thioethyl donor derived from
the glycal epoxide. For a review, see: Fugedi, P.; Garegg, P. J.;
Lnn, H.; Norberg, T.; Gycocnjugate J., 1987, 4, 97; Lnn, H.,
Carbohydr. Res., 1985, 139, (105) 115; Lnn, H., Carbohydr. Chem.,
1987, 6, 301.
[0250] The structures of the final products 1 and 2 were fully
substantiated by mass spectroscopy, self consistent nmr analysis,
and in the case of 1, correspondence with the available published
data. Nudelman J. Biol. Chem., 1986, 261, 11247.
* * * * *