U.S. patent application number 17/071973 was filed with the patent office on 2021-02-11 for glyco-modified atrial natriuretic peptide.
This patent application is currently assigned to DAIICHI SANKYO COMPANY, LIMITED. The applicant listed for this patent is DAIICHI SANKYO COMPANY, LIMITED. Invention is credited to Takeshi HONDA, Mitsuhiro IWAMOTO, Yutaka MORI, Takahiro NAGAYAMA, Keiji SAITO, Takahiro YAMAGUCHI.
Application Number | 20210040170 17/071973 |
Document ID | / |
Family ID | 1000005170148 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040170 |
Kind Code |
A1 |
IWAMOTO; Mitsuhiro ; et
al. |
February 11, 2021 |
GLYCO-MODIFIED ATRIAL NATRIURETIC PEPTIDE
Abstract
The present invention provides a modified atrial natriuretic
peptide that exhibits prolonged duration in blood and maintains
cGMP elevating activity. The present invention provides a modified
peptide in which at least one sugar substance is linked directly
through a glycosidic bond or via a linker structure to at least one
hANP peptide, or a pharmaceutically acceptable salt thereof, a
medicament comprising the modified peptide or the salt thereof as
an active ingredient, etc.
Inventors: |
IWAMOTO; Mitsuhiro; (Tokyo,
JP) ; YAMAGUCHI; Takahiro; (Tokyo, JP) ; MORI;
Yutaka; (Tokyo, JP) ; SAITO; Keiji; (Tokyo,
JP) ; HONDA; Takeshi; (Tokyo, JP) ; NAGAYAMA;
Takahiro; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DAIICHI SANKYO COMPANY, LIMITED |
Tokyo |
|
JP |
|
|
Assignee: |
DAIICHI SANKYO COMPANY,
LIMITED
Tokyo
JP
|
Family ID: |
1000005170148 |
Appl. No.: |
17/071973 |
Filed: |
October 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14806487 |
Jul 22, 2015 |
|
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17071973 |
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PCT/JP2014/051357 |
Jan 23, 2014 |
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14806487 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/58 20130101;
A61K 38/00 20130101; A61K 38/2242 20130101; A61K 47/549 20170801;
A61K 47/65 20170801; A61K 47/60 20170801 |
International
Class: |
C07K 14/58 20060101
C07K014/58; A61K 38/00 20060101 A61K038/00; A61K 47/65 20060101
A61K047/65; A61K 47/54 20060101 A61K047/54; A61K 47/60 20060101
A61K047/60; A61K 38/22 20060101 A61K038/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2013 |
JP |
2013-010612 |
Claims
1. A modified peptide in which at least one sugar substance is
linked directly through a glycosidic bond or via a linker structure
to at least one hANP peptide, or a pharmaceutically acceptable salt
thereof.
2. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is linked
directly through a glycosidic bond or via a linker structure to at
least one of the N terminus of the hANP peptide, the C terminus of
the hANP peptide, and the side chain of at least one amino acid
constituting the peptide.
3. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the hANP peptide is hANP(1-28),
hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27).
4. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is selected
from at least one type of monosaccharide, disaccharide,
trisaccharide, and glycochain of 4 or more monosaccharides bonded
through glycosidic bonds, and when a plurality of sugar substances
are contained in one molecule, the sugar substances may be the same
as or different from each other.
5. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain of 4 or more monosaccharides bonded through glycosidic
bonds.
6. The modified peptide according to claim 5 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycoprotein-derived N-linked glycochain or 0-linked glycochain, or
an altered glycochain thereof.
7. The modified peptide according to claim 6 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a N-linked
glycochain comprising a glycochain structure represented by the
following formula, or a glycochain altered at the reducing end
thereof: ##STR00171## wherein Gxx is GlcNAc, Glc, or Man
(hereinafter, glycochains having the above structure are referred
to as "AG(5)", "AG(5-Glc)", and "AG(5-Man)", respectively,
according to the type of GXX), and "O/N-L" represents binding to
the linker structure or the hANP peptide through an O-glycosidic
bond or a N-glycosidic bond.
8. The modified peptide according to claim 7 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain comprising a glycochain structure represented by the
following formula: ##STR00172## wherein Gxx is GlcNAc, Glc, or Man
(hereinafter, glycochains having the above structure are referred
to as "AG(7)", "AG(7-Glc)", and "AG(7-Man)", respectively,
according to the type of GXX), and "O/N-L" represents binding to
the linker structure or the hANP peptide through an O-glycosidic
bond or a N-glycosidic bond.
9. The modified peptide according to claim 8 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain comprising a glycochain structure represented by the
following formula: ##STR00173## wherein Gxx is GlcNAc, Glc, or Man
(hereinafter, glycochains having the above structure are referred
to as "AG(9)", "AG(9-Glc)", and "AG(9-Man)", respectively,
according to the type of GXX), and "O/N-L" represents binding to
the linker structure or the hANP peptide through an O-glycosidic
bond or a N-glycosidic bond.
10. The modified peptide according to claim 9 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain comprising a glycochain structure represented by the
following formula: ##STR00174## wherein Gxx is GlcNAc, Glc, or Man
(hereinafter, glycochains having the above structure are referred
to as "SG", "SG(Glc)", and "SG(Man)", respectively, according to
the type of GXX), and "O/N-L" represents binding to the linker
structure or the hANP peptide through an O-glycosidic bond or a
N-glycosidic bond.
11. The modified peptide according to claim 7 or a pharmaceutically
acceptable salt thereof, wherein in the sugar substance, Gxx is
GlcNAc.
12. The modified peptide according to claim 11 or a
pharmaceutically acceptable salt thereof, wherein the sugar
substance is SG.
13. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein 10 or less sugar substances are
linked to one hANP peptide.
14. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein 1, 2, or 3 sugar substances are
linked to one hANP peptide,
15. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein one molecule contains a divalent
or higher hANP peptide.
16. The modified peptide according to claim 3 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is linked via
a linker structure to the hANP peptide, and the linker structure is
a chemical structure that has a linking chain of 3 or more atoms
and is bonded at at least one site to the reducing end of the sugar
substance through a glycosidic bond and bonded at at least one site
to the hANP peptide.
17. The modified peptide according to claim 16 or a
pharmaceutically acceptable salt thereof, wherein the sugar
substance is linked to either the N terminus or the C terminus, or
both, of the hANP peptide via a linker structure.
18. The modified peptide according to claim 17 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure is a structure having a linking chain of 15 or less
atoms.
19. The modified peptide according to claim 18 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is SG-hANP(1-28) (compound 2-1), hANP(1-28)-SG (compound
2-2), SG-hANP(1-28)-SG (compound 2-7), AG(9)-hANP(1-28) (compound
2-10), SG-triazole-hANP(1-28) (compound 2-12),
SG-thioacetamide-hANP(1-28) (compound 2-25), or AG(5)-hANP(1-28)
(compound 2-26), or is derived from any of these modified peptides
by the replacement of the sugar substance with SG, SG(Glc),
SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man),
AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of
the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28),
hANP(1-27), hANP(2-27), or hANP(3-27).
20. The modified peptide according to claim 16 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one structure selected from a
polyoxyalkylene chain, an amino acid, and an oligopeptide chain
consisting of 2 or more amino acids.
21. The modified peptide according to claim 20 or a
pharmaceutically acceptable salt thereof, wherein the
polyoxyalkylene chain, the amino acid, and/or the oligopeptide
chain contained in the linker structure is bonded through an amide
bond to the N terminus and/or the C terminus of the hANP
peptide.
22. The modified peptide according to claim 21 or a
pharmaceutically acceptable salt thereof, wherein the
polyoxyalkylene chain is PEG.
23. The modified peptide according to claim 21 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is SG-PEG(3)-(SG-)Asn-hANP(1-28) (compound 2-16),
AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28) (compound 2-21),
AG(7)-(AG(7)-)Asn-PEG(3)-hANP(1-28) (compound 2-22),
SG-PEG(3)-hANP(1-28)-PEG(3)-SG (compound 2-24),
SG-(SG-)Asn-PEG(11)-hANP(1-28) (compound 2-27),
SG-(SG-)Asn-PEG(11)-PEG(11)-hANP(1-28) (compound 2-28),
SG-PEG(3)-hANP(1-28) (compound 2-29), SG-PEG(11)-hANP(1-28)
(compound 2-30), SG-*(SG-)Gln-Mal-PEG(3)-hANP(1-28) (compound
2-31), SG-(SG-)Gln-PEG(3)-Mal-hANP(1-28) (compound 2-32),
SG-(SG-)Asn-(Ser-Gly)3-hANP(1-28) (compound 2-36), or
SG-(SG-)Asn-Gly.sub.6-hANP(1-28) (compound 2-37), or is derived
from any of these modified peptides by the replacement of the sugar
substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man),
AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc
and/or the replacement of the hANP peptide with hANP(1-28),
hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27).
24. The modified peptide according to claim 20 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having a functional
group on the side chain selected from an amino acid having an amino
group on the side chain, an amino acid having SH on the side chain,
an amino acid having a carboxyl group on the side chain, an amino
acid having a hydroxy group on the side chain, and an amino acid
having phenol on the side chain and is linked at the side chain of
the amino acid having a functional group on the side chain to the
sugar substance or the hANP peptide.
25. The modified peptide according to claim 24 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having an amino group
on the side chain and has a structure of the following general
formula (C) in which the sugar substance is linked to the side
chain of the amino acid having an amino group on the side chain:
##STR00175## wherein GLY represents the sugar substance; Lg
represents a structure on the glycochain side in the linker
structure and may be linear or have two or more branches; GLY and L
are bonded through an O- or N-glycosidic bond; when Lg is branched,
the same number of GLY as the number of branch ends is capable of
being linked thereto; and N-(AA) represents a nitrogen atom derived
from the side chain amino group of the amino acid having an amino
group on the side chain.
26. The modified peptide according to claim 25 or a
pharmaceutically acceptable salt thereof, wherein the side chain
amino group and the .alpha. amino group of the amino acid having an
amino group on the side chain form amide bonds with the .alpha.
carboxyl groups of other amino acids.
27. The modified peptide according to claim 25 or a
pharmaceutically acceptable salt thereof, wherein the amino acid
having an amino group on the side chain is Lys.
28. The modified peptide according to claim 27 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is SG-(SG-)Lys-Gly-hANP(1-28) (compound 2-14), [(SG-)
Cys-Gly].sub.3-hANP(1-28) (compound 2-15),
SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal)Lys-]Lys-PEG(3)-hANP(1-28)
(compound 2-19),
[SG.sub.2-Mal-(SG.sub.2-Mal-)Lys-[SG.sub.2-Mal-(SG.sub.2-Mal-)-Lys-
-]Lys-PEG(3)-hANP(1-28) (compound 2-20),
SG-Mal-(SG-Mal-)Lys-hANP(1-28) (compound 2-33),
SG-thioacetamide-(SG-thioacetamide-)Lys-PEG-(3)-hANP(1-28)
(compound 2-34), or SG-(SG-)Lys-PEG(3)-hANP(1-28) (compound 2-35),
or is derived from any of these modified peptides by the
replacement of the sugar substance with SG, SG(Glc), SG(Man),
AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9),
AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP
peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27),
hANP(2-27), or hANP(3-27).
29. The modified peptide according to claim 24 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having a SH group on
the side chain and has a structure of the following general formula
in which the sugar substance is linked to the side chain of the
amino acid having an SH group on the side chain: ##STR00176##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, the same number of
GLY as the number of branch ends is capable of being linked
thereto; and S represents a sulfur atom derived from the side chain
SH group of the amino acid having a SH group on the side chain.
30. The modified peptide according to claim 29 or a
pharmaceutically acceptable salt thereof, wherein the amino acid
having a SH group on the side chain is Cys.
31. The modified peptide according to claim 30 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is [(SG-)Cys-Gly].sub.5-hANP(1-28) (compound 2-17),
[(SG.sub.2-) Cys-Gly].sub.5-hANP(1-28) (compound 2-18), or
SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal-)Lys-]Lys-PEG(11)-hANP(1-28)
(compound 2-23), or is derived from any of these modified peptides
by the replacement of the sugar substance with SG, SG(Glc),
SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man),
AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of
the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28),
hANP(1-27), hANP(2-27), or hANP(3-27).
32. The modified peptide according to claim 24 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having a carboxyl group
on the side chain and has a structure of the following general
formula in which the sugar substance is linked to the side chain of
the amino acid having carboxylic acid on the side chain:
##STR00177## wherein GLY represents the sugar substance; Lg
represents a structure on the glycochain side in the linker
structure and may be linear or have two or more branches; GLY and L
are bonded through an O- or N-glycosidic bond; when Lg is branched,
the same number of GLY as the number of branch ends is capable of
being linked thereto; and CO represents CO derived from the side
chain of the amino acid having carboxylic acid on the side
chain.
33. The modified peptide according to claim 32 or a
pharmaceutically acceptable salt thereof, wherein the sugar
substance is bonded through a N-glycosidic bond to both of the side
chain carboxyl group and the .alpha. carboxyl group of the amino
acid having a carboxyl group on the side chain and bonded to
another linker structure or the hANP peptide via the .alpha. amino
group of said amino acid.
34. The modified peptide according to claim 32 or a
pharmaceutically acceptable salt thereof, wherein the amino acid
having a carboxylic acid group on the side chain is Glu, Gln, Asp,
or Asn.
35. The modified peptide according to claim 34 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is (SG-)Asn-hANP(1-28) (compound 2-3), (SG-)Asn-hANP(2-28)
(compound 2-4), (SG-)Asn-hANP(3-28) (compound 2-8),
SG-(SG-)Asn-hANP(1-28) (compound 2-9), or
SG-(SG-)Asn-PEG(3)-hANP(1-28) (compound 2-13), or is derived from
any of these modified peptides by the replacement of the sugar
substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man),
AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc
and/or the replacement of the hANP peptide with hANP(1-28),
hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27).
36. The modified peptide according to claim 24 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having phenol on the
side chain and has a structure of the following general formula in
which the sugar substance is linked to the side chain of the amino
acid having phenol on the side chain: ##STR00178## wherein GLY
represents the sugar substance; Lg represents a structure on the
glycochain side in the linker structure and may be linear or have
two or more branches; GLY and L are bonded through an O- or
N-glycosidic bond; when Lg is branched, the same number of GLY as
the number of branch ends is capable of being linked thereto; and
the phenol group represents a phenol group derived from the side
chain of the amino acid having a phenol group on the side
chain.
37. The modified peptide according to claim 36 or a
pharmaceutically acceptable salt thereof, wherein the amino acid
having a phenol group on the side chain is Tyr.
38. The modified peptide according to claim 37 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is hANP(1-27)-(SG-)Tyr (compound 2-6), or is derived from
the modified peptide by the replacement of the sugar substance with
SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7),
AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or
the replacement of the hANP peptide with hANP(1-28), hANP(2-28),
hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27).
39. The modified peptide according to claim 24 or a
pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having a hydroxy group
on the side chain and has a structure of the following general
formula in which the sugar substance is bonded through an
O-glycosidic bond to the side chain of the amino acid having a
hydroxy group on the side chain: ##STR00179## wherein GLY
represents the sugar substance; and O represents an oxygen atom
derived from the side chain hydroxy group of the amino acid having
a hydroxy group on the side chain.
40. The modified peptide according to claim 39 or a
pharmaceutically acceptable salt thereof, wherein the amino acid
having a hydroxy group on the side chain is Ser.
41. The modified peptide according to claim 40 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is (SG-)Ser-hANP(2-28) (compound 2-5), or is derived from
the modified peptide by the replacement of the sugar substance with
SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7),
AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or
the replacement of the hANP peptide with hANP(1-28), hANP(2-28),
hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27).
42. The modified peptide according to claim 16 or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide has one or two SG molecules as the sugar substance and one
hANP(1-28) (SEQ ID NO: 1) as the hANP peptide, and the SG is linked
to the N terminus of the hANP(1-28) via a linker structure having a
linking chain of 10 or less atoms.
43. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the modified peptide has a
structure represented by the formula of the following compound 2-1,
2-3, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-25, 2-26, 2-27,
2-29, or 2-30: ##STR00180## ##STR00181## ##STR00182## wherein hANP
is hANP(1-28) consisting of the amino acid sequence of SEQ ID NO: 1
and is bonded at the N terminus of the amino acid sequence to the
linker structure through an amide bond.
44. The salt of the modified peptide according to claim 42, wherein
the pharmaceutically acceptable salt is trifluoroacetate or an
acetate.
45. The modified peptide according to claim 3 or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is linked to
the side chain of an amino acid in the hANP peptide, and the linked
amino acid is an amino acid other than amino acids at amino acid
positions 7 to 23 of SEQ ID NO: 1 contained in the hANP
peptide.
46. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the modified peptide or the
pharmaceutically acceptable salt thereof exhibits a prolonged
duration in blood compared with unmodified hANP(1-28) and maintains
cGMP elevating activity.
47. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the modified peptide or the
pharmaceutically acceptable salt thereof has resistance to the
degradation of the hANP peptide by neutral endopeptidase.
48. The modified peptide according to claim 1 or a pharmaceutically
acceptable salt thereof, wherein the modified peptide or the
pharmaceutically acceptable salt thereof exhibits 3 or more times
the water solubility of unmodified hANP(1-28).
49. A medicament comprising a modified peptide according to claim 1
or a pharmaceutically acceptable salt thereof.
50. The medicament according to claim 48, wherein the medicament is
an agent for treating or alleviating a cardiovascular disease.
51. A method for treating or alleviating a cardiovascular disease,
comprising administering an effective amount of a modified peptide
according to claim 1 or a pharmaceutically acceptable salt
thereof.
52. A method for producing a modified peptide according to claim 1,
comprising the step of linking a hANP peptide, a sugar substance,
and, if necessary, a linker molecule and an acceptor compound.
53. The method according to claim 52, further comprising the step
of transferring a glycochain to a GlcNAc compound, a Glc compound,
or a Man compound by use of Endo-M or a mutant enzyme thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional of U.S. patent
application Ser. No. 14/806,487, filed on Jul. 22, 2015, which is a
Bypass Continuation of International Patent Application No.
PCT/JP2014/051357, filed Jan. 23, 2014, which claims priority to
and the benefit of Japanese Patent Application No. 2013-010612,
filed on Jan. 23, 2013. The contents of these applications are
hereby incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, is
named 098065-0281_SL.txt and is 1 kb in size.
TECHNICAL FIELD
[0003] The present invention relates to a glyco-modified atrial
natriuretic peptide that has a glycochain linkage and exhibits an
improved duration time in blood, a medicament comprising the
modified peptide as an active ingredient, etc.
BACKGROUND ART
[0004] Atrial natriuretic peptides are biologically active peptides
having a vasodilatory effect, a diuretic effect, a cell growth
inhibitory effect, a venous return lowering effect, and a
sympathetic activity inhibitory effect. Native hANP loses its
activity upon cleavage by neutral endopeptidase (NEP) in blood and
therefore has a short half-life in blood. For such reasons, the
native hANP needs to be continuously administered by drip infusion
or the like in current clinical practice.
[0005] Examples of attempts to prolong the half-lives in blood of
such biologically active peptides having a short half-life in blood
include various methods such as utilization of sustained-release
formulations, amino acid substitution or modification, fusion
peptides containing linked albumin, an immunoglobulin Fc portion,
or the like, and modified peptides containing an added polymer
(e.g., PEG). When applying the biologically active peptides to
medicaments for reason of their biological activity, it is required
to prolong their half-lives in blood while maintaining the
biological activity possessed by the peptide at pharmacologically
necessary levels. Attempts to apply such biologically active
peptides having a prolonged half-life in blood to medicaments have
been made on many peptides.
[0006] Non Patent Literature 1 (Proc. Natl. Acad. Sci. USA 1994,
91, 12544-12548) and Non Patent Literature 2 (Bioconjugate Chem.
2008, 19, 342-348) disclose a modified peptide in which PEG is
bonded to atrial natriuretic peptide (ANP).
[0007] Patent Literature 1 (WO2006/076471 A2) discloses a modified
peptide in which PEG is bonded to brain natriuretic peptide
(BNP).
[0008] Patent Literature 2 (WO2008/154226 A1) and Non Patent
Literature 3 (Bioconjugate Chem. 2012, 23, 518-526) describe a
fusion protein in which an immunoglobulin Fc fragment is bonded to
ANP.
[0009] Patent Literature 3 (WO2004/047871 (A2,A3) and Patent
Literature 4 (WO2009/142307 A1) disclose a mutant having an altered
amino acid sequence of ANP.
[0010] However, these techniques are not always successful. In
particular, it is not possible to predict whether or not a
sufficient duration time in blood and maintenance of activity
necessary for pharmacological effects can both be attained, unless
a large number of tests are actually conducted.
CITATION LIST
Patent Literature
[0011] Patent Literature 1: International Publication No.
WO2006/076471 [0012] Patent Literature 2: International Publication
No. WO2008/154226 [0013] Patent Literature 3: International
Publication No. WO2004/047871 [0014] Patent Literature 4:
International Publication No. WO2009/142307
Non Patent Literature
[0014] [0015] Non Patent Literature 1: Proc. Natl. Acad. Sci. USA
1994, 91, 12544-12548 [0016] Non Patent Literature 2: Bioconjugate
Chem. 2008, 19, 342-348) [0017] Non Patent Literature 3:
Bioconjugate Chem. 2012, 23, 518-526
SUMMARY OF INVENTION
Technical Problem
[0018] An object of the present invention is to find a modified
peptide that exhibits a prolonged duration time in blood compared
with native human atrial natriuretic peptide (hANP) and maintains
cGMP elevating activity.
Solution to Problem
[0019] The present inventors have conducted diligent studies on the
modification of hANP so as to prolong the duration time in blood
and to maintain the cGMP elevating activity. As a result, the
present inventors have completed the present invention by finding,
for example, that modified peptides, in which a glycochain is
bonded to hANP by various methods, elevated the intracellular cGMP
concentration of GC-A receptor-expressing cells, exhibited a
prolonged duration time in blood when administered to mice, and
persistently elevated the cGMP concentration in blood even 60
minutes or later after the administration of the modified
peptide.
[0020] The present invention provides the following:
(1) A modified peptide in which at least one sugar substance is
linked directly through a glycosidic bond or via a linker structure
to at least one hANP peptide, or a pharmaceutically acceptable salt
thereof. (2) The modified peptide according to (1) or a
pharmaceutically acceptable salt thereof, wherein the sugar
substance is linked directly through a glycosidic bond or via a
linker structure to at least one of the N terminus of the hANP
peptide, the C terminus of the hANP peptide, and the side chain of
at least one amino acid constituting the peptide. (3) The modified
peptide according to (1) or a pharmaceutically acceptable salt
thereof, wherein the hANP peptide is hANP(1-28), hANP(2-28),
hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). (4) The modified
peptide according to (1) or a pharmaceutically acceptable salt
thereof, wherein the sugar substance is selected from at least one
type of monosaccharide, disaccharide, trisaccharide, and glycochain
of 4 or more monosaccharides bonded through glycosidic bonds, and
when a plurality of sugar substances are contained in one molecule,
the sugar substances may be the same as or different from each
other. (5) The modified peptide according to (1) or a
pharmaceutically acceptable salt thereof, wherein the sugar
substance is a glycochain of 4 or more monosaccharides bonded
through glycosidic bonds. (6) The modified peptide according to (5)
or a pharmaceutically acceptable salt thereof, wherein the sugar
substance is a glycoprotein-derived N-linked glycochain or 0-linked
glycochain, or an altered glycochain thereof. (7) The modified
peptide according to (6) or a pharmaceutically acceptable salt
thereof, wherein the sugar substance is an N-linked glycochain
comprising a glycochain structure represented by the following
formula, or a glycochain altered at the reducing end thereof:
##STR00001##
wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having
the above structure are referred to as "AG(5)", "AG(5-Glc)", and
"AG(5-Man)", respectively, according to the type of Gxx), and
"O/N-L" represents binding to the linker structure or the hANP
peptide through an O-glycosidic bond or a N-glycosidic bond. (8)
The modified peptide according to (7) or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain comprising a glycochain structure represented by the
following formula:
##STR00002##
wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having
the above structure are referred to as "AG(7)", "AG(7-Glc)", and
"AG(7-Man)", respectively, according to the type of Gxx), and
"O/N-L" represents binding to the linker structure or the hANP
peptide through an O-glycosidic bond or a N-glycosidic bond. (9)
The modified peptide according to (8) or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain comprising a glycochain structure represented by the
following formula:
##STR00003##
wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having
the above structure are referred to as "AG(9)", "AG(9-Glc)", and
"AG(9-Man)", respectively, according to the type of Gxx), and
"O/N-L" represents binding to the linker structure or the hANP
peptide through an O-glycosidic bond or a N-glycosidic bond. (10)
The modified peptide according to (9) or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is a
glycochain comprising a glycochain structure represented by the
following formula:
##STR00004##
wherein Gxx is GlcNAc, Glc, or Man (hereinafter, glycochains having
the above structure are referred to as "SG", "SG(Glc)", and
"SG(Man)", respectively, according to the type of Gxx), and "O/N-L"
represents binding to the linker structure or the hANP peptide
through an O-glycosidic bond or a N-glycosidic bond. (11) The
modified peptide according to any of (7) to (10) or a
pharmaceutically acceptable salt thereof, wherein in the sugar
substance, Gxx is GlcNAc. (12) The modified peptide according to
(11) or a pharmaceutically acceptable salt thereof, wherein the
sugar substance is SG. (13) The modified peptide according to (1)
or a pharmaceutically acceptable salt thereof, wherein 10 or fewer
sugar substances are linked to one hANP peptide. (14) The modified
peptide according to (1) or a pharmaceutically acceptable salt
thereof, wherein 1, 2, or 3 sugar substances are linked to one hANP
peptide, (15) The modified peptide according to (1) or a
pharmaceutically acceptable salt thereof, wherein each molecule of
the modified peptide contains a divalent or higher hANP peptide.
(16) The modified peptide according to (3) or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is linked via
a linker structure to the hANP peptide, and the linker structure is
a chemical structure that has a linking chain of 3 or more atoms
and is bonded at at least one site to the reducing end of the sugar
substance through a glycosidic bond and bonded at at least one site
to the hANP peptide. (17) The modified peptide according to (16) or
a pharmaceutically acceptable salt thereof, wherein the sugar
substance is linked to either the N terminus or the C terminus, or
both, of the hANP peptide via a linker structure. (18) The modified
peptide according to (17) or a pharmaceutically acceptable salt
thereof, wherein the linker structure is a structure having a
linking chain of 15 or fewer atoms. (19) The modified peptide
according to (18) or a pharmaceutically acceptable salt thereof,
wherein the modified peptide is SG-hANP(1-28) (compound 2-1),
hANP(1-28)-SG (compound 2-2), SG-hANP(1-28)-SG (compound 2-7),
AG(9)-hANP(1-28) (compound 2-10), SG-triazole-hANP(1-28) (compound
2-12), SG-thioacetamide-hANP(1-28) (compound 2-25), or
AG(5)-hANP(1-28) (compound 2-26), or is derived from any of these
modified peptides by the replacement of the sugar substance with
SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7),
AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or
the replacement of the hANP peptide with hANP(1-28), hANP(2-28),
hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). (20) The
modified peptide according to (16) or a pharmaceutically acceptable
salt thereof, wherein the linker structure comprises at least one
structure selected from a polyoxyalkylene chain, an amino acid, and
an oligopeptide chain consisting of 2 or more amino acids. (21) The
modified peptide according to (20) or a pharmaceutically acceptable
salt thereof, wherein the polyoxyalkylene chain, the amino acid,
and/or the oligopeptide chain contained in the linker structure is
bonded through an amide bond to the N terminus and/or the C
terminus of the hANP peptide. (22) The modified peptide according
to (21) or a pharmaceutically acceptable salt thereof, wherein the
polyoxyalkylene chain is PEG. (23) The modified peptide according
to (21) or a pharmaceutically acceptable salt thereof, wherein the
modified peptide is SG-PEG(3)-(SG-)Asn-hANP(1-28) (compound 2-16),
AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28) (compound 2-21),
AG(7)-(AG(7)-)Asn-PEG(3)-hANP(1-28) (compound 2-22),
SG-PEG(3)-hANP(1-28)-PEG(3)-SG (compound 2-24),
SG-(SG-)Asn-PEG(11)-hANP(1-28) (compound 2-27),
SG-(SG-)Asn-PEG(11)-PEG(11)-hANP(1-28) (compound 2-28),
SG-PEG(3)-hANP(1-28) (compound 2-29), SG-PEG(11)-hANP(1-28)
(compound 2-30), SG-*(SG-)Gln-Mal-PEG(3)-hANP(1-28) (compound
2-31), SG-(SG-)Gln-PEG(3)-Mal-hANP(1-28) (compound 2-32),
SG-(SG-)Asn-(Ser-Gly)3-hANP(1-28) (compound 2-36), or
SG-(SG-)Asn-Gly6-hANP(1-28) (compound 2-37), or is derived from any
of these modified peptides by the replacement of the sugar
substance with SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man),
AG(7), AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc
and/or the replacement of the hANP peptide with hANP(1-28),
hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). (24)
The modified peptide according to (20) or a pharmaceutically
acceptable salt thereof, wherein the linker structure comprises at
least one amino acid having a functional group on the side chain
selected from an amino acid having an amino group on the side
chain, an amino acid having SH on the side chain, an amino acid
having a carboxyl group on the side chain, an amino acid having a
hydroxy group on the side chain, and an amino acid having phenol on
the side chain and is linked at the side chain of the amino acid
having a functional group on the side chain to the sugar substance
or the hANP peptide. (25) The modified peptide according to (24) or
a pharmaceutically acceptable salt thereof, wherein the linker
structure comprises at least one amino acid having an amino group
on the side chain and has a structure of the following general
formula (C) in which the sugar substance is linked to the side
chain of the amino acid having an amino group on the side
chain:
##STR00005##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; and N-(AA) represents a nitrogen atom derived
from the side chain amino group of the amino acid having an amino
group on the side chain. (26) The modified peptide according to
(25) or a pharmaceutically acceptable salt thereof, wherein the
side chain amino group and the .alpha. amino group of the amino
acid having an amino group on the side chain form amide bonds with
the .alpha. carboxyl groups of other amino acids. (27) The modified
peptide according to (25) or (26) or a pharmaceutically acceptable
salt thereof, wherein the amino acid having an amino group on the
side chain is Lys. (28) The modified peptide according to (27) or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is SG-(SG-)Lys-Gly-hANP(1-28) (compound 2-14), [(SG-)
Cys-Gly].sub.3-hANP(1-28) (compound 2-15),
SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal)Lys-]Lys-PEG(3)-hANP(1-28)
(compound 2-19),
[SG.sub.2-Mal-(SG.sub.2-Mal-)Lys-[SG.sub.2-Mal-(SG.sub.2-Mal-)-Lys-
-]Lys-PEG(3)-hANP(1-28) (compound 2-20),
SG-Mal-(SG-Mal-)Lys-hANP(1-28) (compound 2-33),
SG-thioacetamide-(SG-thioacetamide-)Lys-PEG-(3)-hANP(1-28)
(compound 2-34), or SG-(SG-)Lys-PEG(3)-hANP(1-28) (compound 2-35),
or is derived from any of these modified peptides by the
replacement of the sugar substance with SG, SG(Glc), SG(Man),
AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9),
AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP
peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27),
hANP(2-27), or hANP(3-27). (29) The modified peptide according to
(24) or a pharmaceutically acceptable salt thereof, wherein the
linker structure comprises at least one amino acid having an SH
group on the side chain and has a structure of the following
general formula in which the sugar substance is linked to the side
chain of the amino acid having an SH group on the side chain:
##STR00006##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; and S represents a sulfur atom derived from
the side chain SH group of the amino acid having a SH group on the
side chain. (30) The modified peptide according to (29) or a
pharmaceutically acceptable salt thereof, wherein the amino acid
having an SH group on the side chain is Cys. (31) The modified
peptide according to (30) or a pharmaceutically acceptable salt
thereof, wherein the modified peptide is
[(SG-)Cys-Gly].sub.5-hANP(1-28) (compound 2-17), [(SG.sub.2-)
Cys-Gly].sub.5-hANP(1-28) (compound 2-18), or
SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal-)Lys-]Lys-PEG(11)-hANP(1-28)
(compound 2-23), or is derived from any of these modified peptides
by the replacement of the sugar substance with SG, SG(Glc),
SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man),
AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of
the hANP peptide with hANP(1-28), hANP(2-28), hANP(3-28),
hANP(1-27), hANP(2-27), or hANP(3-27). (32) The modified peptide
according to (24) or a pharmaceutically acceptable salt thereof,
wherein the linker structure comprises at least one amino acid
having a carboxyl group on the side chain and has a structure of
the following general formula in which the sugar substance is
linked to the side chain of the amino acid having carboxylic acid
on the side chain:
##STR00007##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; and CO represents CO derived from the side
chain of the amino acid having carboxylic acid on the side chain.
(33) The modified peptide according to (32) or a pharmaceutically
acceptable salt thereof, wherein the sugar substance is bonded
through an N-glycosidic bond to both of the side chain carboxyl
group and the .alpha. carboxyl group of the amino acid having a
carboxyl group on the side chain and bonded to another linker
structure or the hANP peptide via the .alpha. amino group. (34) The
modified peptide according to (32) or (33) or a pharmaceutically
acceptable salt thereof, wherein the amino acid having a carboxylic
acid group on the side chain is Glu, Gln, Asp, or Asn. (35) The
modified peptide according to (34) or a pharmaceutically acceptable
salt thereof, wherein the modified peptide is (SG-)Asn-hANP(1-28)
(compound 2-3), (SG-)Asn-hANP(2-28) (compound 2-4),
(SG-)Asn-hANP(3-28) (compound 2-8), SG-(SG-)Asn-hANP(1-28)
(compound 2-9), or SG-(SG-)Asn-PEG(3)-hANP(1-28) (compound 2-13),
or is derived from any of these modified peptides by the
replacement of the sugar substance with SG, SG(Glc), SG(Man),
AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9),
AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP
peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27),
hANP(2-27), or hANP(3-27). (36) The modified peptide according to
(24) or a pharmaceutically acceptable salt thereof, wherein the
linker structure comprises at least one amino acid having phenol on
the side chain and has a structure of the following general formula
in which the sugar substance is linked to the side chain of the
amino acid having phenol on the side chain:
##STR00008##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; and the phenol group represents a phenol
group derived from the side chain of the amino acid having a phenol
group on the side chain. (37) The modified peptide according to
(36) or a pharmaceutically acceptable salt thereof, wherein the
amino acid having a phenol group on the side chain is Tyr. (38) The
modified peptide according to (37) or a pharmaceutically acceptable
salt thereof, wherein the modified peptide is hANP(1-27)-(SG-)Tyr
(compound 2-6), or is derived from the modified peptide by the
replacement of the sugar substance with SG, SG(Glc), SG(Man),
AG(5), AG(5-Glc), AG(5-Man), AG(7), AG(7-Glc), AG(7-Man), AG(9),
AG(9-Glc), AG(9-Man), or GlcNAc and/or the replacement of the hANP
peptide with hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27),
hANP(2-27), or hANP(3-27). (39) The modified peptide according to
(24) or a pharmaceutically acceptable salt thereof, wherein the
linker structure comprises at least one amino acid having a hydroxy
group on the side chain and has a structure of the following
general formula in which the sugar substance is bonded through an
O-glycosidic bond to the side chain of the amino acid having a
hydroxy group on the side chain:
##STR00009##
wherein GLY represents the sugar substance; and O represents an
oxygen atom derived from the side chain hydroxy group of the amino
acid having a hydroxy group on the side chain. (40) The modified
peptide according to (39) or a pharmaceutically acceptable salt
thereof, wherein the amino acid having a hydroxy group on the side
chain is Ser. (41) The modified peptide according to (40) or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide is (SG-)Ser-hANP(2-28) (compound 2-5), or is derived from
the modified peptide by the replacement of the sugar substance with
SG, SG(Glc), SG(Man), AG(5), AG(5-Glc), AG(5-Man), AG(7),
AG(7-Glc), AG(7-Man), AG(9), AG(9-Glc), AG(9-Man), or GlcNAc and/or
the replacement of the hANP peptide with hANP(1-28), hANP(2-28),
hANP(3-28), hANP(1-27), hANP(2-27), or hANP(3-27). (42) The
modified peptide according to any of (16) to (41) or a
pharmaceutically acceptable salt thereof, wherein the modified
peptide has one or two SG molecules as the sugar substance and one
hANP(1-28) (SEQ ID NO: 1) as the hANP peptide, and the SG is linked
to the N terminus of the hANP(1-28) via a linker structure having a
linking chain of 10 or fewer atoms. In this context, the
pharmaceutically acceptable salt of the modified peptide of the
present invention is preferably trifluoroacetate or an acetate.
(43) The modified peptide according to (1) or a pharmaceutically
acceptable salt thereof, wherein the modified peptide has a
structure represented by the formula of the one of following
compounds 2-1, 2-3, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-25,
2-26, 2-27, 2-29, or 2-30:
##STR00010## ##STR00011##
wherein hANP is hANP(1-28) consisting of the amino acid sequence of
SEQ ID NO: 1 and is bonded at the N terminus of the amino acid
sequence to the linker structure through an amide bond. (44) The
salt of the modified peptide according to any of (1) to (43),
preferably (42) or (43), wherein the pharmaceutically acceptable
salt is trifluoroacetate or an acetate. (45) The modified peptide
according to (3) or a pharmaceutically acceptable salt thereof,
wherein the sugar substance is linked to the side chain of an amino
acid in the hANP peptide, and the linked amino acid is an amino
acid other than amino acids at amino acid positions 7 to 23 of SEQ
ID NO: 1 contained in the hANP peptide. (46) The modified peptide
according to (1) or a pharmaceutically acceptable salt thereof,
wherein the modified peptide or the pharmaceutically acceptable
salt thereof exhibits a prolonged duration time in blood compared
with unmodified hANP(1-28) and maintains cGMP elevating activity.
(47) The modified peptide according to (1) or a pharmaceutically
acceptable salt thereof, wherein the modified peptide or the
pharmaceutically acceptable salt thereof has resistance to the
degradation of the hANP peptide by neutral endopeptidase. (48) The
modified peptide according to (1) or a pharmaceutically acceptable
salt thereof, wherein the modified peptide or the pharmaceutically
acceptable salt thereof exhibits 3 or more times the water
solubility of unmodified hANP(1-28). (49) A medicament comprising a
modified peptide according to any of (1) to (48) or a
pharmaceutically acceptable salt thereof. (50) The medicament
according to (49), wherein the medicament is an agent for treating
or alleviating a cardiovascular disease. (51) A method for treating
or alleviating a cardiovascular disease, comprising administering
an effective amount of a modified peptide according to any of (1)
to (48) or a pharmaceutically acceptable salt thereof. (52) A
method for producing a modified peptide according to any of (1) to
(48) or a pharmaceutically acceptable salt thereof, comprising the
step of linking an hANP peptide, a sugar substance, and, if
necessary, a linker molecule and an acceptor compound. (53) The
method according to (52), further comprising the step of
transferring a glycochain to a GlcNAc compound, a Glc compound, or
a Man compound by use of Endo-M or a mutant enzyme thereof.
Advantageous Effects of Invention
[0021] The modified peptide of the present invention exhibits a
prolonged duration time in blood compared with unmodified
hANP(1-28) (hereinafter, also referred to as "native hANP") and
maintains cGMP elevating activity. The modified peptide of the
present invention is therefore clinically capable of exhibiting
efficacy by non-continuous administration and is applicable to
diseases on which native hANP has no therapeutic effect. In
addition, this modified peptide is superior in water solubility to
native hANP and is therefore susceptible to diverse administration
methods based on higher doses, higher concentrations, etc., of
formulations. This modified peptide can therefore meet diverse
medical needs, which cannot be attained by native hANP.
BRIEF DESCRIPTION OF DRAWING
[0022] [The FIGURE] The FIGURE shows the NMR chart of
SG-oxa/compound 1-12A.
DESCRIPTION OF EMBODIMENTS
[0023] Hereinafter, the present invention will be described in
detail.
[0024] The present invention provides a modified peptide in which
at least one sugar substance is linked directly through a
glycosidic bond or via a linker structure to at least one hANP
peptide. The modified peptide of the present invention exhibits a
prolonged duration time in blood compared with unmodified
hANP(1-28) and maintains cGMP elevating activity possessed by
hANP(1-28). The modified peptide of the present invention is a
modified peptide that has been isolated from the natural world and
artificially produced by the control of the production process and
has a substantially homogeneous structure. The modified peptide of
the present invention does not encompass a peptide that may be
found in nature and is biologically produced in vivo or in cultured
cells. Such a naturally occurring substance itself is definitely
excluded from the scope of the present invention.
[0025] In the present invention, the term "linked" described for a
plurality of structural units (e.g., hANP peptide, sugar substance,
and linker structure) means that these structural units are bonded
directly through a covalent bond or indirectly via a linker
structure so that the structural units exist in one molecule. The
chemical structure that links the structural units is not
particularly limited. In the case of linking via a linear
structure, one each of the structural units is contained in one
molecule. In the case of linking via a branched structure, a
plurality of either or both of the structural units may be
contained in one molecule. The binding pattern between the linker
structure and each structural unit is not particularly limited and
is selected according to the type of the structural unit to be
linked.
<hANP Peptide>
[0026] In the present invention, the "hANP peptide" means a peptide
consisting of an amino acid sequence comprising at least amino
acids at the 7- to 27-positions in the amino acid sequence of human
atrial natriuretic peptide (SEQ ID NO: 1; hereinafter, also
referred to as hANP or hANP(1-28)), which is a biologically active
peptide consisting of 28 amino acids. The hANP exhibits its
biological activity by binding to the GC-A receptor (Chinkers M, et
al., Nature 338; 78-83, 1989)) expressed on the cell surface,
activating guanylate cyclase present in the intracellular domain of
the receptor, and elevating the intracellular cGMP concentration.
As for the native hANP, .alpha.-hANP described in Biochem. Biophys.
Res. Commun., vol. 118, p. 131, 1984, has been approved for
manufacture and sale under the generic name of "carperitide" in
Japan and is commercially available (trade name: HANP).
.alpha.-hANP is also generally known as Human pro-ANP[99-126].
[0027] hANP has an intramolecular ring structure formed by Cys
residues at the 7- and 23-positions of SEQ ID NO: 1 through a
disulfide bond. It is known that this ring structure and the
C-terminal amino acids up to the Arg residue at the 27-position are
important for activation of the GC-A receptor by hANP (Silver, M A,
Curr. Opin. Nephrol. Hypertens. (2006), 15, p. 14-21; and A.
Calderone, Minerva Endocrinol. (2004), 29, p. 113-127). hANP(7-27)
consisting of this ring structure is therefore considered as the
minimum unit for activating GC-A. The hANP peptide of the present
invention is a peptide consisting of an amino acid sequence that
may lack 1 to 6 amino acids consecutively from the N-terminal amino
acid and/or an amino acid at the 28-position in SEQ ID NO: 1, and
is preferably a peptide that may lack at least one of the amino
acids at the 1-position, the 1- and 2-positions, and the
28-position of SEQ ID NO: 1, more preferably a peptide (hANP(2-28),
hANP(3-28), etc.) consisting of an amino acid sequence that may
lack an amino acid at the 1-position or amino acids at the 1- and
2-positions of SEQ ID NO: 1, most preferably a peptide (hANP(1-28))
consisting of the amino acid sequence of SEQ ID NO: 1.
[0028] Examples of the modified peptide of the present invention in
which the hANP peptide and the sugar substance are bonded directly
without the medium of the linker structure can include modified
peptides in which any one or two or more of the hydroxy groups on
the side chains of Ser at the 1-, 5-, 6-, and 25-positions and Tyr
at the 28-position of SEQ ID NO: 1 are bonded directly through an
O-glycosidic bond to the sugar substance, and modified peptides in
which an amide group on the side chain of Asn at the 26-position of
SEQ ID NO: 1 is bonded directly through an N-glycosidic bond to the
sugar substance (in production, the amide bond can also be formed
by converting the amino acid at the position to Asp and reacting
the sugar substance with an azidated reducing end).
[0029] In the case of the modified peptide of the present invention
in which the hANP peptide and the sugar substance are linked via a
linker structure, as mentioned below in detail, the sugar substance
can be linked to: a functional group on the side chain of an amino
acid constituting the hANP peptide, the N terminus, and/or the C
terminus by the adoption of diverse linker structures. The site on
the hANP peptide to which the sugar substance is linked is
preferably the N terminus and/or the C terminus, more preferably
the N terminus.
[0030] The modified peptide of the present invention may comprise
one hANP peptide in one molecule or may be a polyvalent modified
peptide of hANP comprising two or more hANP peptides. The
polyvalent modified peptide of hANP can be appropriately produced
by the selection of a linker molecule having a plurality of
functional groups capable of binding to the hANP peptides such that
a plurality of hANP molecules can be linked to the linker
structure.
<Sugar Substance>
[0031] In the present invention, the "sugar substance" means a
structural unit consisting of one monosaccharide or a structural
unit of two or more monosaccharides bonded to each other through a
glycosidic bond. In the present invention, the sugar substance is
also referred to as "GLY". Alternatively, a specific monosaccharide
or glycochain is also indicated by an abbreviation, for example,
"GlcNAc-" or "SG-". The sugar substance represented by a structural
formula with these abbreviations is bonded at the carbon atom at
the 1-position, which is a reducing end, to the linker structure or
the hANP peptide through an O- or N-glycosidic bond, unless
otherwise specified. An oxygen atom or a nitrogen atom belonging to
the glycosidic bond is not included in the abbreviations indicating
the sugar substance, unless otherwise defined.
[0032] In the present specification, the monosaccharide serving as
the basic unit of the sugar substance is indicated in its ring
structure in which a carbon atom bonded to an oxygen atom
constituting the ring and directly bonded to the hydroxy group (or
the oxygen atom belonging to the glycosidic bond) is defined as the
1-position (2-position only for sialic acid) for the sake of
convenience, unless otherwise specified. The compounds described in
the Examples are named in the light of their whole chemical
structures, so that this rule is not necessarily applicable
thereto.
[0033] The monosaccharide contained in the sugar substance is not
particularly limited as long as the monosaccharide has the basic
structure of a sugar. Various monosaccharides such as 6-membered
and 5-membered sugars can be used. The monosaccharide may be a
sugar found in nature or may be an artificially synthesized sugar.
A sugar found in nature is preferred. Examples of the
monosaccharide can include glucose (Glu), fructose (Flu), mannose
(Man), galactose (Gal), glucosamine (Glc), N-acetylglucosamine
(GlcNAc), glucuronic acid (GlucA), neuraminic acid (Neu), sialic
acid/N-acetylneuraminic acid (NeuNAc/Neu5Ac), galactosamine,
N-acetylgalactosamine (GalNAc), xylose (Xyl), iduronic acid (IdoA),
fucose (Fuc), aldotriose, glyceraldehyde, aldotetrose, erythrose,
threose, aldopentose, ribose, lyxose, arabinose, aldohexose,
allose, talose, gulose, aldose, idose, ketotriose,
dihydroxyacetone, ketotetrose, erythrulose, ketopentose, xylulose,
ribulose, ketohexose, psicose, sorbose, and tagatose.
[0034] An oligosaccharide or a polysaccharide composed of a
plurality of monosaccharides bonded through glycosidic bonds may be
used as the sugar substance of the present invention. The
oligosaccharide is not particularly limited as long as a desired
number of monosaccharides are bonded through glycosidic bonds.
Examples thereof can include: disaccharides such as sucrose,
maltose, lactose, and trehalose; trisaccharides such as
maltotriose, melezitose, and raffinose; and tetrasaccharides such
as nystose, nigerotetraose, and stachyose. Examples of the
polysaccharide can include amylose, glycogen, cellulose, chitin,
chitosan, chondroitin, chondroitin sulfate, hyaluronic acid,
dextran, and dextran sulfate.
[0035] The sugar substance of the present invention may be a
glycochain. The "glycochain" may be a natural glycochain that is
produced in vivo or generated by metabolism and is composed of two
or more monosaccharides bonded through a glycosidic bond or may be
an altered glycochain having an artificial alteration added with
reference to the structure of the natural glycochain. The natural
glycochain exists as a glycochain (carbohydrate) or in the form of
a glycoprotein or a glycolipid in animals, plants, microorganisms,
etc., and can be obtained by isolation and purification therefrom.
The altered glycochain is a glycochain artificially altered from
the glycochain structure of the natural glycochain. The alteration
method can be by way of a synthesis chemical or enzyme chemical
addition, substitution, and/or deletion of one or more
monosaccharides in a naturally derived glycochain and is preferably
an alteration to delete a sugar at the non-reducing end by use of a
glycosidase appropriate for the sugar at the non-reducing end. The
number of monosaccharides contained in the glycochain is not
particularly limited as long as the number is two or more. An
arbitrary number of monosaccharides can be selected from, for
example, approximately 50 or fewer, approximately 40 or fewer, and
approximately 30 or fewer monosaccharides. The number of
monosaccharides contained in the glycochain is preferably
approximately 25 or fewer, more preferably approximately 20 or
fewer, even more preferably approximately 15 or fewer, further
preferably 11 or fewer.
[0036] The glycochain of the present invention may be linear or
branched. The linear glycochain is a glycochain in which all the
monosaccharides contained in the glycochain except for the sugar at
the non-reducing end are linked in a linear form such that each
monosaccharide is bonded at one carbon atom other than the carbon
atom at the 1-position in its ring structure, either directly or
via a substituent, to the carbon atom at the 1-position (2-position
for sialic acid) of another monosaccharide through a glycosidic
bond.
[0037] On the other hand, the branched glycochain is a glycochain
in which one or more monosaccharides contained in the glycochain
are linked in a branched form such that at least one monosaccharide
is bonded at two or more carbon atoms other than the carbon atom at
the 1-position in its ring structure, either directly or via a
substituent, to the carbon atoms at the 1-positions (2-positions
for sialic acid) of other monosaccharides through glycosidic bonds.
For both of the linear and branched glycochains, the end (reducing
end) on the 1-position carbon side of the ring structure is
constituted by one monosaccharide, and the carbon atom at the
1-position (2-position for sialic acid) of the sugar at this
reducing end is bonded through an O- or N-glycosidic bond to the
linker structure or the hANP peptide.
[0038] On the other hand, the monosaccharide at the non-reducing
end of the glycochain does not form a glycosidic bond with another
sugar at a site other than the carbon atom at the 1-position
(2-position for sialic acid). The glycochain has the same number of
non-reducing ends as the number of branches and is altered mainly
at this non-reducing end.
[0039] Glycochains contained in natural glycoproteins are broadly
classified into N-linked glycochains attached to asparagine of a
glycoprotein and O-linked glycochains attached to serine or
threonine thereof, both of which have their characteristic basic
structures. Naturally, the N-linked glycochain is bonded through an
N-glycosidic bond to the amino acid side chain of a protein, while
the O-linked glycochain is bonded through an O-glycosidic bond
thereto. Artificial glycochains can be bonded to other compounds
through any glycosidic bond. Thus, the type of glycosidic bond is
not limited by structure of such glycochain. For example, the sugar
substance is azidated at its reducing end, and this azidated sugar
substance can be reacted with a compound having a carboxyl group in
the presence of triphenylphosphine to bond the compound having the
desired structure to the sugar substance through an N-glycosidic
bond. Alternatively, the sugar substance can be reacted with a
compound having a hydroxy group, such as an alcohol, to bond the
sugar substance to the desired compound through an O-glycosidic
bond.
[0040] The basic structure of the N-linked glycochain is
represented by the following formula (structural formula (I) and
sequence (II)). A glycochain having this glycochain structure is
designated as AG(5).
##STR00012##
[0041] In the above formula, "O/N-L" represents binding to the
linker structure through an O-glycosidic bond or an N-glycosidic
bond. In the above formula, glycochains altered at the reducing end
by the replacement of GlcNAc at the reducing end with Glc or Man
are referred to as AG(5-Glc) and AG(5-Man), respectively.
[0042] Most of the N-linked glycochains have this basic structure.
Its non-reducing end or branched sugar may be further bonded to a
glycochain.
[0043] Human glycochains or human-compatible glycochains are
glycochains known to exhibit no antigenicity in the bodies of
humans. For example, high-mannose, complex, and composite forms of
N-linked glycochains are known. The high-mannose form is a
glycochain having a mannose-rich structure composed of a plurality
of mannose molecules at the non-reducing end of the N-linked basic
structure. The complex form is a glycochain having a
Ga1.beta.1-4GlcNAc motif structure at the non-reducing end of the
N-linked basic structure. The composite glycochain is a glycochain
having a Ga11.beta.-4GlcNAc motif structure at the non-reducing end
of the N-linked basic structure and also having a mannose-rich
structure composed of a plurality of mannose molecules.
<SG,AG(n)>
[0044] The N-linked complex glycochain is typically a glycochain
contained in sialylglycopeptide (hereinafter, referred to as "SGP")
contained in chicken egg yolk. Examples thereof can include sialyl
glycan (hereinafter, referred to as "SG") having a structure
represented by the following structural formula (III) and sequence
(IV):
##STR00013##
[0045] In the above formula, "O/N-L" represents binding to the
linker structure through an O-glycosidic bond or a N-glycosidic
bond. In the above formula, glycochains altered at the reducing end
by the replacement of GlcNAc at the reducing end with Glc or Man
are referred to as SG(Glc) and SG(Man), respectively.
[0046] SG can be obtained, as mentioned later, by reacting SGP with
an enzyme (Endo-M, a mutant thereof, etc.) by a method known in the
art, followed by hydrolytic cleavage or transfer to a desired
compound. SGP can be isolated and purified from chicken egg yolk
according to a conventional method, for example, a method described
in WO2011/0278681. Alternatively, a purified product of SGP is
commercially available (Tokyo Chemical Industry Co., Ltd. or
Fushimi Pharmaceutical Co., Ltd.) and can be purchased.
[0047] The glycochain altered at the reducing end by the
replacement of GlcNAc at the reducing end of SG with another sugar
can be prepared by use of the transglycosylation reaction mentioned
later. The glycochain altered at the reducing end by the
replacement of GlcNAc at the reducing end of SG with Glc is
referred to as SG(Glc). The glycochain altered at the reducing end
by the replacement of GlcNAc at the reducing end of SG with Man is
referred to as SG(Man).
[0048] Specific examples of the altered glycochain that may be used
as the sugar substance of the present invention can include AG(9)
(structural formula (V) and sequence (VI) given below) that lacks
two non-reducing end Neu5Ac residues as a result of the
neuraminidase treatment of SG, AG(7) (structural formula (VII) and
sequence (VIII) given below) that lacks two non-reducing end Gal
residues as a result of the galactosidase treatment of AG(9), and
AG(5) (glycochain having the aforementioned N-linked basic
structure) that lacks two non-reducing end GlcNAc residues as a
result of the further treatment of AG(7) with
N-acetylglucosaminidase. Also, glycochains altered at the reducing
end of AG(9), AG(7), and AG(5) (e.g. AG(9-Glc) with GlcNAc at the
reducing end of AG(9) replaced with Glc, and AG(9-Man) with GlcNAc
at the reducing end of AG(9) replaced with Man) can be obtained by
the same treatment as above using the glycochain altered at the
reducing end of SG (e.g., SG(Glc) or SG(Man)) instead of SG and can
be adopted as the sugar substance of the present invention.
##STR00014##
[0049] In the above formula, "O/N-L" represents binding to the
linker structure through an O-glycosidic bond or an N-glycosidic
bond. In the above formula, glycochains altered at the reducing end
by the replacement of GlcNAc at the reducing end with Glc or Man
are referred to as AG(9-Glc) and AG(9-Man), respectively.
##STR00015##
[0050] In the above formula, "O/N-L" represents binding to the
linker structure through an O-glycosidic bond or an N-glycosidic
bond. In the above formula, glycochains altered at the reducing end
by the replacement of GlcNAc at the reducing end with Glc or Man
are referred to as AG(7-Glc) and AG(7-Man), respectively.
[0051] The modified peptide of the present invention is not limited
by the maximum number of sugar substances as long as, in one
molecule, at least one sugar substance is linked to the hANP
peptide. The number of sugar substances is, for example, 20 or
fewer, more preferably 15 or fewer, even more preferably 12 or
fewer, further preferably 10 or fewer, still further preferably 5
or fewer, still further preferably 1, 2, 3, or 4, most preferably 1
or 2. The sugar substances contained in one molecule may have the
same structure or may be a mixture of sugar substances differing in
structure. Preferably, all of these sugar substances are identical
sugar substances.
[0052] In the present invention, in the case of using a
glycoprotein- or glycolipid-derived glycochain found in nature as
the sugar substance, the glycochain can be used after being cleaved
or isolated or transferred to a desired compound (acceptor
compound) through transglycosylation by use of an enzyme. The
enzyme for use in such a reaction can be selected from diverse
enzymes known in the art according to the structure of the
glycochain used (Endo-A: Li, B., et al, J. Am. Chem. Soc. 127
(2005), pp. 9692-9693; Endo-F: Wei Huang., et al, ChemBioChem 12
(2011), pp. 932-941; Endo-D: Shu-Quan Fan, et al, J. Biol. Chem.
287 (2012), pp. 11272-11281; and Endo-S: Wei Huang, et al, J. Am.
Chem. Soc. 134 (2012), pp. 12308-12318.). As an example of such an
enzyme, for example, endo-.beta.-N-acetylglucosaminidases are known
as a series of enzyme families that hydrolyze .beta.-glycosidic
bonds in chitobiose structures and are known as Endo-A, Endo-D,
Endo-F, Endo-M, Endo-S, etc., according to their origins.
[0053] Of them, Endo-M derived from Mucor hiemalis has the activity
of hydrolyzing the glycosidic bond between GlcNAc-GlcNAc on the
reducing end side in a glycochain having an N-linked basic
structure. In addition, this enzyme even has the activity of
transferring and bonding a glycochain on the non-reducing end side
containing the second reducing end GlcNAc cleaved by this
hydrolysis from the N-linked glycochain basic structure, to the
4-position of GlcNAc of another acceptor compound having a GlcNAc
site (see e.g., Y. Tomabechi, et al, Bioorg. Med. Chem., 18 (2010),
pp. 1259-1264). Also, it is known that when a compound having the
structure of a different sugar unit (e.g., Glc or Man) instead of
GlcNAc is used as an acceptor compound in similar
transglycosylation reactions using Endo-M, the similar transfer
reaction proceeds at a position in the sugar unit corresponding to
the 4-position of GlcNAc (Endoglycosidases--Biochemistry,
Biotechnology, Application, Masahiko Endo et al. Kodansha, Tokyo
(2006)).
[0054] The glycochain structure serving as a substrate of Endo-M
can be any glycochain structure having an N-linked basic structure,
and diverse glycochains such as high-mannose, complex, and
composite forms can be used as the substrate. AG(5), AG(7), AG(9),
and SG also serve as substrates of Endo-M. Endo-M N175Q, which is a
mutant of Endo-M, is a mutant that exhibits reduced hydrolysis
activity while maintaining the substrate specificity and
transglycosylation activity of Endo-M. Endo-M N175Q is particularly
preferred for bonding a glycochain to a desired compound through a
transglycosylation reaction. In the case of using Endo-M N175Q, for
example, an excised glycochain moiety such as SG-Oxa can be used as
a glycochain donor, or a glycopeptide or a glycoprotein such as SGP
may be used (Midori Umekawa et al. JOURNAL OF BIOLOGICAL CHEMISTRY,
285, 2010, pp. 511-521 (which also describes reports of other
mutants)). Endo-M and mutant enzymes thereof can be produced by
genetic engineering by methods known in the art. Alternatively,
Endo-M and Endo-M N175Q may be purchased as commercially available
reagents (distributor: Tokyo Chemical Industry Co., Ltd.).
[0055] In the case of using a glycochain excised by use of
hydrolase, the substrate can be reacted with hydrolase at an
appropriate temperature for an appropriate time, and the glycochain
can be isolated from the obtained reaction solution.
[0056] The excised glycochain may be used as it is or may be
modified at its reducing end for use. For example, in the case of a
glycochain having GlcNAc at the reducing end, this glycochain can
be treated with DMC and isolated as GLY(GlcNAc)-oxa (specifically,
SG-Oxa having an oxazoline ring formed between a hydroxy group
bonded to the carbon atom at the 1-position and a N-acetyl group
bonded to the carbon atom at the 2-position in the ring structure
of GlcNAc). The glycochain thus excised can be used as a substrate
for the transfer reaction to the linker molecule (GlcNAc compound)
and thereby bonded to a desired compound.
[0057] In the case of transferring a glycochain to a desired
compound by use of a glycosyltransferase, a substrate (glycochain
donor), an acceptor compound, and a glycosyltransferase are reacted
at an appropriate temperature for an appropriate time, and the
compound of interest is obtained from the resulting reaction
solution by the isolation of the compound made by the transfer and
binding of the glycochain of interest to the acceptor compound. For
example, in the case of using SGP as the substrate, a GlcNAc
compound as the acceptor compound, and Endo-M N175Q as the
glycosyltransferase, an appropriate amount of the GlcNAc compound
and SGP at a dose appropriate for the GlcNAc valence of the GlcNAc
compound are shaken at 20 to 40.degree. C. (preferably 20 to
30.degree. C., more preferably 22 to 27.degree. C., most preferably
25.degree. C.) for 1 to 10 hours (preferably 2 to 8 hours, more
preferably 3 to 6 hours) in the presence of Endo-M-N175Q and, if
desired, an appropriate amount of DMSO, and the resulting reaction
product can be purified by use of reverse-phase HPLC (ODS; which
employs a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents).
[0058] A functional group such as an amino group (SG-NH.sub.2,
etc.), a carboxyl group (SG-A, etc.), an azide group (SG-N.sub.3,
etc.), a maleimide group (SG-M), or an .alpha.-iodoacetylamide
group (SG-I) can be bonded to the reducing end of the glycochain
through such a transfer reaction by use of various GlcNAc compounds
mentioned later. The glycochain thus prepared can be linked to a
desired linker structure.
[0059] The glycochain-bound compound thus obtained may be bonded
directly to the hANP peptide or may be linked to the hANP peptide
via another linker molecule.
[0060] In this context, the "acceptor compound" includes a sugar
that has not undergone a modification except for the glycosidic
bond at the 1-position carbon, and is not particularly limited as
long as the compound functions as a sugar acceptor in the
transglycosylation reaction. Preferably, a "GlcNAc compound" (which
will be mentioned later in detail) containing GlcNAc as the sugar,
a "Glc compound" containing Glc as the sugar, a "Man compound"
containing Man as the sugar, or the like can be used as the
acceptor compound. A GlcNAc compound is most preferred.
<Linker Structure>
[0061] In the present invention, the linker structure means a
chemical structure that mediates the linking between the hANP
peptide and the sugar substance in the modified peptide of the
present invention. The linker structure is bonded at at least one
site to the hANP peptide and bonded at at least one site to the
sugar substance through a glycosidic bond.
[0062] For the link between the hANP peptide and the linker
structure, the linker structure may be bonded to any position
selected from the N-terminal amino group of the hANP peptide, the
C-terminal carboxyl group of the hANP peptide, and at least one
side chain of the constituent amino acids of hANP peptide. The
number of binding positions may be one or two or more. In the case
of binding to an amino acid side chain, a desired compound having a
functional group appropriate for binding to each functional group
which is a hydroxy group in the side chain of Ser at the 1-, 5-,
6-, or 25-position of SEQ ID NO: 1 or Tyr at the 28-position, or
which is an amide group in the side chain of Asn at the
26-position, or the like may be provided.
[0063] In the notation of a modified peptide of the present
invention or a partial structure thereof, the N terminus (amino
group) and the C terminus (carboxyl group) of an amino acid or a
peptide are indicated on the left and on the right, respectively,
unless otherwise specified. An amino acid or a peptide with the
symbol "*" on the right (e.g., Gln*) represents that contrary to
this rule, the C terminus and the N terminus are indicated on the
left and on the right, respectively.
[0064] In the notation of an amino acid, an amino group and a
carboxyl group, which are structures essential to an amino acid,
directly bonded to the central carbon atom (.alpha. carbon) are
referred to as an ".alpha. amino group" and an ".alpha. carboxyl
group", respectively.
[0065] When the sugar substance is linked to at least one of the N
terminus (amino group) and the C terminus (carboxyl group) of the
hANP peptide, the hANP peptide and the linker structure form an
amide bond. A modified peptide in which the sugar substance is
linked to the N terminus of the hANP peptide via a linker structure
is represented as follows:
GLY-L-hANP (A)
[0066] wherein GLY represents the sugar substance; L represents the
linker structure that is linear or has two or more branches; hANP
represents the hANP peptide; L is bonded through an O- or
N-glycosidic bond to GLY; when L is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; and L is bonded through an amide bond to the
N terminus of the hANP peptide.
[0067] In the notation of a modified peptide or a partial structure
thereof in the present specification, when an amino acid or a
peptide is linked at its N-terminal amino group to another linker,
a symbol representing the structural unit to be linked is indicated
with a hyphen and without parentheses on the left side of a symbol
representing this peptide or amino acid. In this case, the hyphen
represents the amide bond formed between the amino group of the
peptide or the amino acid and the carboxyl group carried by the
linker structure. For example, a structure in which SG is linked to
the amino group of Asn is referred to as "SG-Asn".
[0068] A modified peptide in which the sugar substance is linked to
the C terminus of the hANP peptide via a linker structure is
represented as follows:
hANP-L-GLY (B) wherein GLY represents the sugar substance; L
represents the linker structure that is linear or has two or more
branches; hANP represents the hANP peptide; L is bonded through an
O- or N-glycosidic bond to GLY; when L is branched, there are the
same number of GLY as the number of branch ends that are capable of
being linked thereto; and L is bonded through an amide bond to the
C terminus of the hANP peptide.
[0069] Specifically, in the notation of a modified peptide or a
partial structure thereof in the present specification, when an
amino acid or a peptide is linked at its C-terminal carboxyl group
to another structural unit, a symbol representing the structural
unit to be linked is indicated with a hyphen and without
parentheses on the right side of a symbol representing this peptide
or amino acid. In this case, the hyphen represents the amide bond
formed between the C-terminal carboxyl group of the peptide or the
amino acid and the amino group (or azide group) carried by the
linker structure. For example, a structure in which SG is linked to
the carboxyl group of Tyr is referred to as "Tyr-SG".
[0070] In the present invention, a modified peptide in which the
sugar substance is linked to both of the N terminus and the C
terminus of the hANP peptide is represented by the following
formula C:
GLY-L1-hANP-L2-GLY (C)
[0071] wherein GLY represents a sugar substance; L1 and L2 may be
the same or different and each represent the linker structure that
is linear or has two or more branches; hANP represents the hANP
peptide; L1 and L2 are each bonded through an O- or N-glycosidic
bond to GLY; when L1 or L2 is branched, there are the same number
of GLY as the number of branch ends that are capable of being
linked thereto; and L1 and L2 are bonded through amide bonds to
each of the N terminus and the C terminus of the hANP peptide,
respectively.
[0072] In a modified peptide of the present invention or a partial
structure thereof, the partial structure in which the sugar
substance is linked to an amino acid side chain is represented by
the following formula D:
(GLY-)AA (D)
[0073] wherein GLY represents the sugar substance; AA represents an
arbitrary amino acid; and the side chain of the amino acid is
bonded through an O- or N-glycosidic bond, directly or via a linker
structure, to GLY.
[0074] Specifically, in the notation of the modified peptide or a
partial structure thereof in the present specification, when an
amino acid or a peptide is linked at its side chain functional
group to another structural unit, a symbol representing the
structural unit to be linked is indicated with a hyphen and
parentheses on the left side of a symbol representing the linked
amino acid. In this case, the hyphen represents a chemical
structure containing the glycosidic bond at the reducing end of the
sugar substance. When this linking is mediated by a linker
structure, a structural characteristic of the linker structure may
also be described (e.g., (SG-PEG(3)-)Asn). However, when the linker
structure has no such characteristic structure or is not defined,
this may be omitted (e.g., (SG-)Lys). The names of the compounds
described in the Examples represent specific compounds represented
by the structural formulas provided therewith.
[0075] In accordance with such rules, a partial structure in which
SG is linked to both of the side chain amino group and the .alpha.
amino group of Lys is referred to as "SG-(SG-)Lys". Likewise, a
partial structure in which SG is linked to both of the side chain
carboxyl group and the .alpha. carboxyl group of Glu is referred to
as "(SG-)Gln-SG" or "SG-(SG-)Gln*" (wherein Asp and Glu have the
same structure as Asn/Gln when the side chain carboxyl group forms
an amide bond; and Gln* means that the carboxyl group is located on
the left and the amino group is located on the right).
[0076] When one molecule contains a plurality of hANP peptides,
individual notations are adopted. For example, the structure in
which the sugar substance is linked to the .alpha. carboxyl group
of Lys and the N termini of the hANP peptides are respectively
linked to the .alpha. amino group and the side chain amino group
via PEG linkers is referred to as "GLY-Lys*(-PEG-hANP).sub.2".
[0077] The sugar substance and the linker structure are bonded
through an N- or O-glycosidic bond at the carbon atom at the
1-position of the reducing end of the sugar substance. In this
respect, the configuration of the glycosidic bond may be selected
as any of the .alpha.-position and the .beta.-position. Any binding
pattern can be selectively synthesized according to a method known
in the art (Tomoya Ogawa, et al, Agric. Biol. Chem. 47 (1983), pp.
281-285.; and Mamoru Mizuno, et al, 121 (1999), pp. 284-290.). In
the case of using a glycochain derived from a natural glycoprotein,
the same pattern as the naturally occurring binding pattern of this
glycochain is desirably selected. When the sugar substance is, for
example, SG or an altered glycochain thereof, the .beta.-position
is desirably selected for the glycosidic bond with the linker
structure.
[0078] When the sugar substance is indicated by a symbol (e.g.,
GLY, SG, or GlcNAc) in the present specification, this symbol also
includes carbon at the reducing end and excludes N or O belonging
to the N- or O-glycosidic bond, unless otherwise defined. Likewise,
when the hANP peptide is indicated by a symbol (e.g., hANP or
hANP(1-28)), the symbol also includes N-terminal --NH and
C-terminal C.dbd.O as a rule. The N terminus and the C terminus are
indicated on the left and on the right, respectively, unless
otherwise specified. Specifically, an unmodified hANP peptide is
referred to as H-hANP-OH.
[0079] The chemical structure of the linker structure contained in
the modified peptide of the present invention has an oxygen atom or
a nitrogen atom bonded through a glycosidic bond to at least one
sugar substance and a partial structure bonded to at least one hANP
peptide (NH or C.dbd.O for an amide bond or a structure (which will
be mentioned later) appropriate for the structure of each side
chain for linking to a side chain of the hANP peptide). Other
structures are not limited and may be derived from a single
molecule or may be a plurality of partial structures in which a
plurality of molecules are bonded. Such a molecule from which the
linker structure is derived is referred to as a "linker molecule".
When a plurality of partial structures are contained in the linker
structure, these partial structures may be indicated by "Lx"
according to the partial structures. For example, the partial
structure directly bonded to the sugar substance is indicated by
"Lg". This partial structure fully satisfies the definitions
applied to the linker structure except for the structure directly
bonded to the hANP peptide. Also, the partial structure directly
bonded to the hANP peptide is indicated by "Lp". This partial
structure fully satisfies the definitions applied to the linker
structure except for the structure belonging to the glycosidic bond
with the sugar substance.
[0080] In the linker structure of the present invention, the
shortest chain of atoms that links N or O belonging to the
glycosidic bond and the atom directly bonded to the hANP peptide
(in the case of an amide bond, N or C belonging to the amide bond)
is referred to as a "linking chain". The linking chain contains the
atom belonging to each of the aforementioned bonds. For example,
the modified peptide represented by (GLY)--O--CH2-C(.dbd.O)--(N
term hANP) has a linker structure consisting of a linking chain of
3 atoms. Also, the modified peptide represented by
(GLY)--NH--C(.dbd.O)--CH2-CH2-NH--(C term hANP) has a linker
structure consisting of a linking chain of 5 atoms. The linker
structure of the present invention is not particularly limited as
long as the linker structure has a linking chain of 3 or more
atoms. The linking chain can be, for example, of 200 or fewer atoms
and is preferably of approximately 150 or fewer atoms, more
preferably of 100 or fewer atoms, even more preferably of 70 or
fewer atoms, 50 or fewer atoms, or 30 or fewer atoms, most
preferably of 20 or fewer atoms, 15 or fewer atoms, or 10 or fewer
atoms. For forming such a linker structure, a plurality of linker
molecules can be used to create a linker structure having a
complicated and long linking chain. Preferably, 5 or fewer linker
molecules are adopted. A modified peptide containing a linker
structure derived from 4, 3, 2, or 1 linker molecules is also
preferred.
[0081] When the linker structure of the present invention is
derived from one linker molecule, this linker molecule is a
compound containing, in one molecule, both a functional group
binding through a glycosidic bond to the sugar substance, and a
functional group binding to the hANP peptide, and is preferably,
for example, an amino acid or a peptide because of having an amino
group and a carboxyl group, more preferably an amino acid having an
amino group, a carboxyl group, a hydroxy group, or the like on the
side chain. Specific examples of such a linker molecule can include
HO--CH2-COOH, HO--CH2-CH2-NH2, aspartic acid, glutamic acid,
serine, and lysine.
[0082] When the linker structure of the present invention is
derived from a plurality of linker molecules, at least a compound
containing a functional group capable of binding through a
glycosidic bond to the sugar substance, and a compound having a
functional group capable of binding to the hANP peptide are used as
these linker molecules. These two compounds also have functional
groups that permit the compounds to be bonded to each other
directly. Alternatively, these two compounds may be linked to each
other via an additional compound. A method known in the art can be
applied to the binding between such linker molecules. Examples
thereof can include, but are not particularly limited to, an amide
bond between an amino group and a carboxyl group, a bond between an
SH group and a maleimide group, a bond between an SH group and an
iodoacetyl group, a bond between a phenol group and a triazoledione
group (Hitoshi Ban, et al, 132 (2010), 1523-1525), an ester bond
between an alcohol and a carboxyl group, and a bond between an
azide group and an acetylene group through Huisgen reaction. The
linker molecules used in the present invention can be appropriately
selected from those having functional groups appropriate for these
binding patterns and used in the formation of the linker
structure.
[0083] The modified peptide of the present invention needs to
exhibit a certain degree of hydrophilicity as a whole. It is
therefore preferred to adopt a highly hydrophilic structure when
the linker structure has a size above a certain level. Examples of
such a structure include a polyoxyalkylene and a polyamide or other
biologically applicable repeat structures.
[0084] Examples of the polyoxyalkylene can include polyethylene
glycol (PEG), polypropylene glycol, polybutylene glycol, and
polyvinyl alcohol (PVA). PEG is preferred. In the notation of the
linker structure containing PEG in the present specification, the
number of ethoxy repeats is represented by, for example, PEG(3) or
PEG(11). Examples of a modified peptide having such a structure can
include compounds 2-13 and 2-27 to 2-34 prepared in the working
examples.
[0085] Alternatively, the linker structure of the present invention
may contain an amino acid or an oligopeptide chain of two or more
amino acids bonded through a peptide bond. Such an amino acid or
oligopeptide chain can assume a structure that is directly bonded
through an amide bond to the N terminus or the C terminus of the
hANP peptide to extend the peptide chain of the hANP peptide.
Alternatively, this structure may be linked to the hANP peptide via
a non-peptide structure or may have both of these structures.
[0086] The amino acid contained in the linker structure of the
present invention is not particularly limited as long as the amino
acid has an amino acid structure in which a hydrogen atom, an amino
group, and a carboxyl group are bonded to the same carbon atom. The
amino acid may be a naturally occurring amino acid or may be an
artificial amino acid. The artificial amino acid may be a
synthetically produced amino acid such as a D amino acid or may be
an altered amino acid having an artificially modified side chain of
a naturally occurring amino acid. The amino acid is preferably a
naturally occurring amino acid or an altered amino acid, more
preferably a naturally occurring amino acid. When the modified
peptide of the present invention is used as an active ingredient
for a medicament, it is preferred that the amino acid contained in
the linker structure should not have its own biological activity,
and it is also preferred to adopt Gly or an amino acid whose side
chain is bonded to another structure. Examples of the linker
structure containing such an amino acid can include a linker
structure that contains Gly or oligo-Gly consisting of two or more
Gly residues and is linked at the N terminus and the C terminus to
the hANP peptide and the sugar substance. Specific examples of such
a modified peptide can include compound 2-40 in which SG(Glc) is
used as the sugar substance and Gly is contained as the linker.
[0087] For example, an "amino acid having an amino group on the
side chain", such as Lys, which has a side chain amino group, "an
amino acid having an SH group on the side chain", such as Cys,
which has a side chain SH group, "an amino acid having a carboxyl
group on the side chain", such as aspartic acid or glutamic acid,
which have a side chain carboxyl group, "an amino acid having a
hydroxy group on the side chain", such as serine, which has a side
chain hydroxy group, or "an amino acid having phenol on the side
chain", such as tyrosine, which has a side chain p-phenol group,
can be adopted as the amino acid having such a side chain
structure. This amino acid having the side chain structure can
attain linking at three sites, i.e., the amino group and the
carboxyl group bonded to the .alpha. carbon and the side chain
functional group. For forming a branched linker structure, it is
therefore preferred to contain at least one of these amino acids
having the side chain structure.
[0088] The amino acid having an amino group on the side chain is
not particularly limited as long as the amino acid has at least one
amino group (except for an amide group) on the side chain. The
natural amino acid having an amino group on the side chain is Lys.
Examples of the artificial amino acids having an amino group on the
side chain can include altered amino acids obtained by the reaction
of a divalent amine such as 1,2-diaminoethane with the side chain
carboxyl group of Glu or Asp.
[0089] When the linker structure contains at least one amino acid
having an amino group on the side chain and the sugar substance is
linked to the side chain of the amino acid having an amino group on
the side chain, a partial structure of the following general
formula can be taken:
##STR00016##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; N-(AA) represents a nitrogen atom derived
from the side chain amino group of the amino acid having an amino
group on the side chain; and (AA) contains the basic structure of
the amino acid and a structural moiety linked to the side chain
amino group.
[0090] The linker structure having such a partial structure can be
produced by introducing a protective group to the carboxyl group of
the amino acid having an amino group on the side chain or a linker
molecule containing this amino acid, subsequently reacting
therewith a linker molecule represented by "GLY-Lg-COOH",
deprotecting the carboxyl group, and bonding the resulting compound
to the hANP peptide or Lp. In this reaction, when the amino acid
having an amino group on the side chain has a free .alpha. amino
group, Lg can form a linker structure having a branched structure
through amide bonds to both of the side chain amino group and the
.alpha. amino group. The modified peptide thus constituted is, for
example, a modified peptide having a partial structure such as
SG-(SG-)Lys. Specific examples of such a compound can include
compounds 2-14 and 2-35 prepared in the working examples.
[0091] In similar production, a modified peptide having a partial
structure in which the hANP peptide is linked to the side chain of
the amino acid having an amino group on the side chain can be
produced by the replacement of Lg(-sugar substance) with Lp(-hANP
peptide). Such a partial structure can be produced by introducing a
protective group to the carboxyl group of the amino acid having an
amino group on the side chain or a linker molecule containing this
amino acid, subsequently reacting therewith a linker molecule
represented by "Lp-COOH", deprotecting the carboxyl group, and
bonding Lg or the sugar substance to the carboxyl group. In this
reaction, when the amino acid having an amino group on the side
chain has a free .alpha. amino group, Lp can form a linker
structure having a branched structure through amide bonds to both
of the side chain amino group and the .alpha. amino group. The
modified peptide thus constituted is, for example, a modified
peptide having a partial structure such as Lp-(Lp-)Lys-Lg. Specific
examples of such a compound can include compounds 2-38 and 2-39. In
addition, a modified peptide containing a plurality of sugar
substances and/or a plurality of hANP peptides in one molecule can
be appropriately produced by the combined use with other linker
molecules having diverse structures.
[0092] Alternatively, a structure having a large number of amino
groups may be formed by repeatedly forming respective amide bonds
from the side chain amino group and the .alpha. amino group of each
amino acid having an amino group on the side chain with the .alpha.
carboxyl groups of identical amino acids having an amino group on
the side chain, and this structure can be bonded to Lg to form a
linker structure linked to the same number of sugar substances as
the number of amino groups. For example, a plurality of amino
groups in such a branched peptide can be reacted with glycochains
having carboxyl groups, for example, by use of a condensing agent
such as HATU, to introduce a plurality of glycochains via the
branched peptide. Examples of the modified peptide thus produced
include linker structures such as "SG-[SG-(SG-)Lys-]Lys" and
"SG-(SG-)Lys-[SG-(SG-)Lys-]Lys". Specific examples of such a
modified peptide can include compounds 2-14 and 2-35.
[0093] The amino acid having an SH group on the side chain is not
particularly limited as long as the amino acid has at least one SH
group on the side chain. The natural amino acid having an SH group
on the side chain is Cys. Examples of artificial amino acids having
an SH group on the side chain can include SH group-containing
altered amino acids obtained by the reaction of a compound having,
for example, a HOOC--R-STr (trityl sulfide) structure with the side
chain amino group of Lys or by the reaction of a compound having,
for example, a H.sub.2N--R-STr structure with the side chain
carboxyl group of Glu or Asp.
[0094] When the linker structure contains at least one amino acid
having an SH group on the side chain and the sugar substance is
linked to the side chain of the amino acid having the SH group on
the side chain, a partial structure of the following general
formula can be taken:
##STR00017##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; S represents a sulfur atom derived from the
side chain SH group of the amino acid having the SH group on the
side chain; and (AA) contains the basic structure of the amino acid
and a structural moiety linked to the side chain SH group.
[0095] The linker structure having such a partial structure can be
produced by reacting the amino acid having the SH group on the side
chain or a linker molecule containing this amino acid with a linker
molecule represented by (GLY)-Lg-N maleimide. In this reaction, the
amino acid having the SH group on the side chain can be linked at
its .alpha. amino group and/or a carboxyl group to another sugar
substance, thereby forming a linker structure having a branched
structure. For example, a plurality of glycochains can be
introduced via such a branched peptide by: reacting a plurality of
amino groups in the branched peptide with 3-mercaptopropionic acid
having a protected SH group; deprotecting the C terminus of the
peptide; after binding to the hANP peptide, deprotecting the SH
group; and reacting therewith glycochains having maleimide groups
in a 0.2 M phosphate buffer solution (pH 6.75). Specific examples
of such a compound include compounds 2-19, 2-20, and 2-23.
[0096] In similar production, a modified peptide having a partial
structure in which the hANP peptide is linked to the side chain of
the amino acid having the SH group on the side chain can be
produced by the replacement of Lg(-sugar substance) with Lp(-hANP
peptide). In addition, a modified peptide containing a plurality of
sugar substances and/or a plurality of hANP peptides in one
molecule can be appropriately produced by the combined use with
other linker molecules having diverse structures.
[0097] The amino acid having a carboxyl group on the side chain is
not particularly limited as long as the amino acid has at least one
carboxyl group on the side chain. The natural amino acids having a
carboxyl group on the side chain are Asp or Glu. Examples of an
artificial amino acid having a carboxyl group on the side chain can
include altered amino acids obtained by the reaction of a divalent
carboxylic acid such as maleic acid with the side chain amino group
of Lys.
[0098] When the linker structure contains at least one amino acid
having a carboxylic acid group on the side chain and the sugar
substance is linked to the side chain of the amino acid having the
carboxylic acid group on the side chain, a partial structure of the
following general formula can be taken:
##STR00018##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; CO represents CO derived from the side chain
of the amino acid having carboxylic acid on the side chain; and
(AA) contains the basic structure of the amino acid and a
structural moiety linked to the side chain carboxylic acid
group.
[0099] The linker structure having such a partial structure can be
produced by introducing, if necessary, a protective group to the
amino group of the amino acid having the carboxyl group on the side
chain or a linker molecule containing this amino acid, subsequently
reacting therewith a linker molecule represented by "Lg-NH.sub.2",
then deprotecting the amino group, and bonding the resulting
compound to Lg or the C terminus of the hANP peptide. In this
reaction, when the amino acid having the carboxyl group on the side
chain has a free .alpha. carboxyl group, Lg can form a linker
structure having a branched structure through amide bonds to both
of the side chain carboxyl group and the .alpha. carboxyl group.
For example, a plurality of glycochains can be introduced via such
a branched peptide by activating the carboxylic acids of the
branched peptide having a plurality of carboxyl groups by use of
trifluoroacetic anhydride and N-hydroxysuccinimide and reacting
therewith glycochains (SG-NH.sub.2, etc.) having amino groups. Such
a partial structure in which Glu is used as the amino acid having
the carboxyl group on the side chain and is linked to SG is
represented by, for example, "-(SG(Lg)-)Gln-(Lg) SG" (wherein Glu
and Asp have the same structure as Gln/Asn because their side
chains form amide bonds). Specific examples of such a modified
peptide can include compounds 2-31 and 2-32.
[0100] In similar production, a modified peptide having a partial
structure in which the hANP peptide is linked to the side chain of
the amino acid having the carboxyl group on the side chain can be
produced by the replacement of Lg(sugar substance) with Lp(hANP
peptide). The linker structure having such a partial structure can
be produced by introducing, if necessary, a protective group to the
amino group of the amino acid having the carboxyl group on the side
chain or a linker molecule containing this amino acid, subsequently
reacting therewith a linker molecule represented by
"(hANP)-Lp-NH.sub.2", then deprotecting the amino group, and
bonding the resulting compound to Lg having an appropriate
functional group. In this reaction, when the amino acid having a
carboxyl group on the side chain has a free .alpha. carboxyl group,
Lp can form a linker structure having a branched structure through
amide bonds to both of the side chain carboxyl group and the
.alpha. carboxyl group. For example, a polyvalent modified peptide
of hANP in which a plurality of hANP peptides are introduced via
such a branched peptide can be produced by activating the
carboxylic acids of the branched peptide having a plurality of
carboxyl groups by use of trifluoroacetic anhydride and
N-hydroxysuccinimide and reacting therewith Lp having amino groups
or the N-terminal amino groups of the hANP peptides. Such a partial
structure in which Glu is used as the amino acid having a carboxyl
group on the side chain and is linked to hANP is represented by,
for example, "SG Gln-(Lp-hANP).sub.2" (wherein Glu and Asp have the
same structure as Gln/Asn because their side chains form amide
bonds). In addition, a modified peptide containing a plurality of
sugar substances and/or a plurality of hANP peptides in one
molecule can be appropriately produced by the combined use with
other linker molecules having diverse structures.
[0101] Alternatively, a structure having a large number of carboxyl
groups may be formed by repeatedly forming respective amide bonds
from the side chain carboxyl group and the .alpha. carboxyl group
of each amino acid having a carboxyl group on the side chain with
the .alpha. amino groups of other amino acids (which may be of the
same type or of different types and is preferably of the same type)
having a carboxyl group on the side chain, and this structure can
be bonded to Lg through the aforementioned reaction to form a
linker structure linked to the same number of Lg as the number of
carboxyl groups. Such a partial structure of the linker structure
is, for example, "H-[(SG-)Gln-SG]Gln-SG".
[0102] The carboxyl group of the amino acid having the carboxyl
group on the side chain can be further linked directly to the sugar
substance through an N-glycosidic bond to form the following
partial structure:
##STR00019##
wherein GLY represents the sugar substance; NH--CO-(AA) represents
an amide structure derived from the side chain of the amino acid
having the carboxyl group on the side chain; and (AA) contains the
basic structure of the amino acid and a structural moiety linked to
the side chain carboxylic acid group.
[0103] Such a partial structure can be produced by reacting the
amino acid having carboxylic acid on the side chain or a linker
molecule containing this amino acid with a sugar substance azidated
at the reducing end in the presence of triphenylphosphine, and
bonding the reducing end of the sugar substance to the side chain
through an N-glycosidic bond. Also, a compound having a structure
in which the sugar substance is linked to the .alpha. carboxyl
group of the amino acid having the carboxyl group on the side chain
can be synthesized by a method based on the Examples. Specific
examples of such a sugar-modified peptide include compounds 2-3,
2-4, 2-8, 2-9, 2-13, 2-21, 2-22, 2-27, 2-28, 2-38, and 2-39.
[0104] The amino acid having a phenol group on the side chain is
not particularly limited as long as the amino acid has at least one
p-phenol group on the side chain. The natural amino acid having a
phenol group on the side chain is Tyr. Examples of an artificial
amino acid having a phenol group on the side chain can include
altered amino acids obtained by the reaction of p-aminophenol with
the side chain carboxyl group of Glu or Asp.
[0105] When the linker structure contains at least one amino acid
having a phenol group on the side chain and the sugar substance is
linked to the side chain of this amino acid, a partial structure of
the following general formula can be taken:
##STR00020##
wherein GLY represents the sugar substance; Lg represents a
structure on the glycochain side in the linker structure and may be
linear or have two or more branches; GLY and L are bonded through
an O- or N-glycosidic bond; when Lg is branched, there are the same
number of GLY as the number of branch ends that are capable of
being linked thereto; the phenol group represents a phenol group
derived from the side chain of the amino acid having phenol on the
side chain; and (AA) contains the basic structure of the amino acid
and a structural moiety linked to the side chain phenol group.
[0106] The linker structure having such a partial structure can be
produced by reacting the amino acid having the phenol group on the
side chain or a linker molecule containing this amino acid with a
linker molecule represented by (GLY)-Lg-N triazoledione(*). In this
reaction, the amino acid having the phenol group on the side chain
can be linked at its .alpha. amino group and/or a carboxyl group to
another sugar substance, thereby forming a linker structure having
a branched structure. For example, a GlcNAc structure can be
selectively introduced to the phenolic side chain of Tyr
corresponding to the 28-position of hANP by activating GlcNAc
having a triazoledione structure by use of N-bromosuccinimide, and
reacting therewith hANP. A transglycosylation reaction can be
carried out with this compound as a starting material. Specific
examples of such a compound include compound 2-6.
[0107] In similar production, a modified peptide having a partial
structure in which the hANP peptide is linked to the side chain of
the amino acid having the phenol group on the side chain can be
produced by the replacement of Lg(sugar substance) with Lp(hANP
peptide). In addition, a modified peptide containing a plurality of
sugar substances and/or a plurality of hANP peptides in one
molecule can be appropriately produced by the combined use with
other linker molecules having diverse structures.
[0108] The amino acid having a hydroxy group on the side chain is
not particularly limited as long as the amino acid has at least one
hydroxy group on the side chain. A natural amino acid having a
hydroxy group on the side chain is Ser or Tyr. Examples of an
artificial amino acid having a hydroxy group on the side chain can
include altered amino acids obtained by the reaction of an
aminoalcohol such as 2-aminoethanol with the side chain carboxylic
acid of Asp or Glu.
[0109] When the linker structure contains at least one amino acid
having a hydroxy group on the side chain and the sugar substance is
linked to the side chain of this amino acid, a partial structure of
the following general formula can be taken:
##STR00021##
wherein GLY represents the sugar substance; O represents an oxygen
atom derived from the side chain hydroxy group of the amino acid
having a hydroxy group on the side chain; and (AA) contains the
basic structure of the amino acid and a structural moiety linked to
the side chain hydroxy group.
[0110] The linker structure having such a partial structure can be
produced by reacting the amino acid having the hydroxy group on the
side chain or a linker molecule containing this amino acid with the
sugar substance under conditions that form an O-glycosidic bond. In
this reaction, the amino acid having the hydroxy group on the side
chain can be linked at its .alpha. amino group and/or a carboxyl
group to another sugar substance, thereby forming a linker
structure having a branched structure. For example, GlcNAc can be
introduced to the side chain of serine via an O-glycosidic bond by
reacting a glucosamine derivative having a trichloroacetimidate
structure with the side chain hydroxy group of serine in the
presence of trimethylsilyl trifluoromethanesulfonate, followed by
several steps. Transglycosylation reactions can be carried out with
this compound as a starting material. Specific examples of such a
modified peptide include compound 2-5.
[0111] In similar production, a modified peptide having a partial
structure in which the hANP peptide is linked to the side chain of
the amino acid having a hydroxy group on the side chain can be
produced by the replacement of Lg(sugar substance) with Lp(hANP
peptide). In addition, a modified peptide containing a plurality of
sugar substances and/or a plurality of hANP peptides in one
molecule can be appropriately produced by the combined use with
other linker molecules having diverse structures.
[0112] At least the same number of sugar substances as the number
of these functional group-containing side chains can be linked
through various reactions mentioned above using a linker molecule
containing an oligopeptide containing a plurality of such amino
acids having the side chain structure or a plurality of linker
molecules each containing one or more each of these amino acids.
Such an oligopeptide is not particularly limited as long as the
oligopeptide comprises the aforementioned amino acids having the
side chain. The oligopeptide may further contain a certain number
of Gly and preferably has a repeat sequence in which these Gly
residues are regularly arranged. When the amino acid having the
side chain is defined as, for example, Xaa, an oligopeptide
represented by (Xaa-Gly.sub.m).sub.n (wherein m and n each
independently represent a natural number of 1 or larger) is
preferred. Although there are no particular upper limits on n and
m, each of n and m is preferably 10 or smaller, more preferably 7
or smaller. Even more preferably, m is 3 or smaller. Specific
examples of the oligopeptide can include, but are not limited to,
(Cys-Gly).sub.3, (Cys-Gly)s, (Lys-Gly-Gly).sub.3, and
(Tyr-Gly-Gly-Gly).sub.3. Specific examples of the modified peptide
having such a structure can include compounds 2-15, 2-17, and
2-18.
[0113] Various linker molecules and GlcNAc compounds as described
above can be bonded by an appropriate combination to synthesize a
linker structure having a structure designed as desired. Such
linker structures can be designed as very diverse structures and
can also control the number of sugar substances to be bonded. Many
variations of modified peptides can be produced by such design and
synthesis.
<Production Method and GlcNAc Compound>
[0114] In the present invention, the modified peptide in which the
sugar substance is bonded through an O-glycosidic bond to the
linker structure can be produced by the reaction of GlcNAc-oxa (or
its related substance, for example, in which three hydroxy groups
contained at the GlcNAc moiety are protected by acetylation), which
is an oxazoline derivative of N-acetylglucosamine (GlcNAc), with a
linker molecule having a hydroxy group (e.g., benzyl glycolate or
an amino acid having a hydroxy group on the side chain, such as
serine or tyrosine). Also, the modified peptide in which the sugar
substance is bonded through an N-glycosidic bond to the linker
structure can be produced by the reaction of a sugar substance
having an azide group with a linker molecule having a carboxyl
group in the presence of triphenylphosphine.
[0115] In the modified peptide of the present invention, in the
case of linking a sugar substance having a glycochain structure as
the sugar substance, the reducing end of the glycochain may be
appropriately modified and bonded to the linker molecule.
Alternatively, an acceptor compound having a particular sugar unit
and a desired structure (e.g., a GlcNAc compound) may be
synthesized, and the modified peptide can also be produced by the
transfer of a glycochain to the sugar unit (e.g., GlcNAc) by use of
a glycosynthase (e.g., Endo-M N175Q).
[0116] In the present invention, a "GlcNAc compound" is a compound
containing GlcNAc that has not undergone a modification except for
the glycosidic bond at the 1-position carbon, and functional groups
capable of binding to other molecules, or a compound linked to the
hANP peptide. The GlcNAc compound may be, for example, a compound
in which GlcNAc as a monosaccharide is bonded through a glycosidic
bond to a desired compound, amino acid, or the like in one
molecule, a glycochain (e.g., AG(5)) having GlcNAc at the
non-reducing end, or a compound bonded thereto. The glycosidic bond
between the compound and GlcNAc may have either of the
.alpha.-position or the .beta.-position, both of which promote the
transfer reaction (Endoglycosidases: Masahiko Endo, et al.,
Biochemistry, Biotechnology, Application). For the glycochain
structure after a transfer with respect to a natural glycochain, it
is preferred to have the same binding pattern as in the naturally
occurring glycochain. For example, when the sugar-bound compound
after the transfer has SG, the .beta.-position is also preferred
for the GlcNAc compound as an acceptor of the SG.
[0117] The GlcNAc compound can be produced according to various
methods known in the art. For example, glucosamine or
4,5-dihydro-2-methyloxazolo[5',4':1,2]-3,4,6-tri-O-acetyl-1,2-dideoxy-.al-
pha.-glucopyranose (the following formula; see Bull. Chem. Soc.
Jpn., 2003, 76, 485-500)):
##STR00022##
[0118] can be used as a starting material to synthesize a compound
having a desired structure and functional group appropriately.
Specific examples of such a GlcNAc compound can include compound
1-2C having a carboxyl group, compound 1-7A having an amino group,
and compound 1-6D having a triazoledione group. Also, the GlcNAc
compound in which an amino acid is linked to GlcNAc can be
synthesized by the selective addition of a protective group to
either the amino group or the carboxyl group. Specific examples
thereof can include Boc-(GlcNAc-)Ser (compound 1-1D),
Boc-(GlcNAc-)Asn (J. Am. Chem. Soc., 1999, 121, 284-290), and
(GlcNAc-)Gln.
The GlcNAc compound as described above can be bonded to another
linker molecule or to the peptide to synthesize GlcNAc compounds
having diverse structures and linking functional groups. Such
diverse GlcNAc compounds can be used in the production of the
modified peptide of the present invention. For example, a GlcNAc
compound having an amino group (or a carboxyl group) can be bonded
to a linker molecule having a carboxyl group (or an amino group)
and a desired structure and functional group in order to synthesize
a GlcNAc compound having the desired functional group (specific
examples: compound 1-7B having a maleimide group).
[0119] For example, a GlcNAc compound having an amino group (or a
carboxyl group) can be bonded to a PEG linker molecule having a
carboxyl group (or an amino group) to synthesize a GlcNAc compound
having a desired length of PEG (specific examples: compounds 1-3A,
1-4B, 1-5A, and 1-19). Alternatively, a polyamide such as polyGly
or poly(Ser-Gly) can be used instead of PEG to synthesize a GlcNAc
compound having a desired length of polyamide linker (specific
examples: compound 1-21B). In this context, an amino acid having a
functional group on the side chain can be adopted as the amino acid
contained in the polyamide to bond an additional GlcNAc compound to
the functional group.
[0120] The GlcNAc compound used in the present invention may be a
compound containing a plurality of individual GlcNAc residues in
one molecule. Examples of such a polyvalent GlcNAc compound include
compounds in which two GlcNAc residues are bonded through
N-glycosidic bonds to the aforementioned amino acid, and compounds
in which a plurality of the aforementioned GlcNAc compounds are
bonded to the linker molecule. For example, an amino acid having an
amino group on the side chain, such as Lys can be reacted as a
linker molecule with 2 equivalents of a GlcNAc compound having a
carboxyl group to synthesize a GlcNAc compound having two GlcNAc
residues and one carboxyl group, such as GlcNAc-(GlcNAc-)Lys-OH.
This GlcNAc-(GlcNAc-)Lys-OH can be further reacted with lysine (an
amino acid having an amino group on the side chain) to synthesize a
tetravalent GlcNAc compound. This step can be repeated to
synthesize a GlcNAc compound containing a large number of GlcNAc
residues. When the GlcNAc compound has a plurality of functional
groups, a polyvalent GlcNAc compound can also be synthesized
(specific examples: compound 1-5B) by introducing a protective
group to a portion of these functional groups, linking GlcNAc
thereto, then deprotecting the functional groups, and bonding
additional GlcNAc compounds to the deprotected functional groups.
These approaches can be appropriately repeated and/or combined
according to conventional methods to synthesize diverse GlcNAc
compounds.
[0121] Compounds derived from the GlcNAc compound by the
replacement of GlcNAc as in the above definition, form, etc., with
Glc or Man are defined as a Glc compound and a Man compound,
respectively. These compounds are also preferably adopted as the
acceptor compound for the production of the modified peptide of the
present invention.
[0122] In the production of the modified peptide of the present
invention, the modified peptide can be produced by the appropriate
binding, transfer, etc. of intermediates (hANP, sugar substance,
linker molecule, acceptor compound, etc.). The order in which these
intermediates are produced is not particularly limited, and the
intermediates can be produced by various methods according to
conventional processes. The functional group carried by each
intermediate is appropriately subjected to activation,
deactivation, addition of a protective group, deprotection, etc.,
according to conventional methods depending on the production
process.
[0123] The sugar substance can be linked by the adoption of various
methods. For example, a glycochain is transferred to a linker
molecule having GlcNAc, Glc, or the like, and this glycochain-bound
linker molecule can be linked to the hANP peptide. Alternatively,
an intermediate in which Glc or GlcNAc is linked to the hANP
peptide may be used as an acceptor compound for the transfer of a
glycochain in the production of the modified peptide. The hydroxy
group carried by the sugar substance can be appropriately subjected
to steps such as acetylation and deacetylation and thereby
prevented from causing unnecessary side reactions.
<Function and Activity>
[0124] The modified peptide of the present invention exhibits a
prolonged duration time in blood and excellent water solubility
compared with unmodified hANP(1-28) and maintains cGMP elevating
activity. hANP(1-28) disappears rapidly from blood and therefore
needs to be continuously administered in clinical practice. By
contrast, the modified peptide of the present invention can exert
pharmacological effects even by non-continuous administration.
Furthermore, the modified peptide of the present invention is
superior in water solubility to native hANP and is therefore
applicable to a formulation containing an active ingredient at a
high concentration. Such characteristics of the modified peptide of
the present invention allow for adoption of administration methods,
administration routes, and formulation techniques, which cannot be
attained by conventional native hANP, and also enable the modified
peptide to be used in the treatment of acute cardiovascular
diseases as well as chronic cardiovascular diseases (hypertension,
chronic heart diseases, etc.). Moreover, the modified peptide of
the present invention is also useful as a biological research tool.
It is unclear how or whether native hANP migrates to a tissue when
residing in blood for a long period. By contrast, such localization
or the influence of the long-term residence of hANP in blood on
living bodies can be examined by the administration of the modified
peptide of the present invention. The duration time of the modified
peptide of the present invention in blood can be tested according
to the method of Test Example 3 by administering the modified
peptide to an animal and then detecting the cGMP concentration in
peripheral blood and/or the modified peptide contained in the
peripheral blood sample. The modified peptide of the present
invention maintains the effect of elevating the cGMP concentration
in peripheral blood even approximately 15 minutes after
intracorporeal administration, more preferably maintains this
effect even approximately 30 minutes after the administration, even
more preferably maintains this effect even approximately 45 minutes
after the administration, and further preferably maintains this
effect even approximately 60 minutes after the administration. As
for the detection of the modified peptide from peripheral blood
after the administration of the modified peptide, this peptide is
preferably detected even approximately 30 minutes later, more
preferably even approximately 45 minutes later, even more
preferably even approximately 60 minutes later, further preferably
even approximately 90 minutes later.
[0125] The modified peptide of the present invention exhibits
excellent water solubility by virtue of the linked sugar substance.
This excellent water solubility is also influenced by the chemical
structure of the linker structure. The amount of native hANP
dissolved per ml of water is 32 mmol at which point the peptide is
gelled. By contrast, 60 mmol or more, preferably 80 mmol or more,
more preferably 100 mmol (e.g., specifically, 112 mmol) of the
modified peptide of the present invention is soluble per ml of
water. Thus, the modified peptide of the present invention has
approximately 2 or more times, preferably approximately 3 or more
times the water solubility of native hANP.
[0126] The duration time in blood of the modified peptide of the
present invention can be measured by administering the modified
peptide to an organism, sampling blood at certain intervals of
time, and detecting the modified peptide contained in the blood
samples. Various methods, for example, detection by LC-MS and ELISA
using an antibody specifically recognizing the ring structure of
hANP, can be used as methods for detecting the modified peptide. In
the case of administering the modified peptide of the present
invention at a dose that produces its cGMP elevating activity, the
cGMP levels of the blood samples are measured by use of a
commercially available measurement kit and compared with the cGMP
level in blood determined before the start of the administration.
In this way, the duration time of the modified peptide in blood can
be measured as biological activity. Alternatively, the modified
peptide may be labeled with a radioisotope and detected by
separating blood samples by SDS-PAGE or the like and detecting the
radioactive signals.
[0127] In the present invention, the "prolonged duration time in
blood" means that the modified peptide exhibits a longer duration
time in blood than that of native hANP. Native hANP subcutaneously
administered to a monkey is no longer detected in blood 30 minutes
after the administration. Hence, if the modified peptide can be
detected 30 minutes after the administration, its duration time in
blood can be regarded as prolonged. Also, the elevation of the cGMP
level in blood by the native hANP subcutaneously administered to a
monkey returns, 60 minutes after the administration, to the same
level as that before the administration. Hence, if the modified
peptide exhibits, 60 minutes after the administration, a higher
cGMP level than that before the administration, its duration time
in blood can be regarded as prolonged.
[0128] The modified peptide of the present invention also has
resistance to the degradation of the hANP peptide by NEP. This is
probably responsible in part for the prolonged duration time. Such
resistance to the NEP degradation can be measured by a method known
in the art.
[0129] The cGMP elevating activity of the modified peptide of the
present invention can be measured by stimulating GC-A
receptor-expressing cells with a test substance prepared in
concentration gradient up to a sufficient amount to provide the
maximum activity, then lysing the cells, measuring cGMP
concentrations in the cell lysates, and identifying the maximum
cGMP concentration (Emax). The phrase "maintaining cGMP elevating
activity" described for the modified peptide of the present
invention means that the maximum cGMP concentration exhibited by
the modified peptide is approximately 30% or more compared with the
maximum cGMP concentration of native hANP. The maximum cGMP
concentration exhibited by the modified peptide is preferably
approximately 50% or more, more preferably approximately 70% or
more. The modified peptide of the present invention can be
formulated at a high concentration compared with native hANP and
exhibits a prolonged duration time in blood. In this respect, it is
not appropriate to define the activity of the modified peptide of
the present invention on the basis of an index such as so-called
EC50 values. If the maximum activity of a modified peptide at the
elevated concentration can be activity equal to or greater than a
certain level of activity of native hANP, the modified peptide can
display sufficient efficacy when administered continuously and/or
at a high concentration in clinical practice.
[0130] The present invention provides a medicament comprising the
modified peptide of the present invention as an active
ingredient.
<Medicament>
[0131] The substance that may be used as an active ingredient for
the medicament according to the present invention may be a
pharmaceutically acceptable salt of the modified peptide mentioned
above. Specifically, in the present invention, an acid (an
inorganic acid, for example, hydrochloric acid, sulfuric acid, or
phosphoric acid, or an organic acid, for example, formic acid,
acetic acid, butyric acid, trifluoroacetic acid (TFA), succinic
acid, or citric acid)-addition salt of the substance may be used as
the active ingredient. Alternatively, in the present invention, a
metal (e.g., sodium, potassium, lithium, or calcium) salt of the
substance or a salt form based on an organic base thereof may be
used as the active ingredient. Such a salt of the modified peptide
of the present invention may be a salt based on the hANP peptide
moiety or may be a salt formed in the structure of the sugar
substance. The salt of the modified peptide of the present
invention is preferably a pharmaceutically acceptable salt formed
at the hANP peptide moiety, more preferably trifluoroacetate or an
acetate formed at the hANP peptide moiety. The pharmaceutical
composition according to the present invention may contain a free
form of the substance related to the active ingredient or a
pharmaceutically acceptable salt thereof.
[0132] The substance that may be used as an active ingredient for
the medicament according to the present invention, or the
pharmaceutically acceptable salt thereof is preferably mixed with a
pharmaceutically acceptable carrier, excipient, diluent, or the
like known in the art and administered to an individual by an
administration method generally used for medicaments, i.e., an oral
administration method or a parenteral administration method such as
transmucosal administration, intravenous administration,
intramuscular administration, or subcutaneous administration.
[0133] The dose of the substance that may be used as an active
ingredient for the medicament according to the present invention
differs depending on the type of disease, the age, body weight, and
severity of the individual's (patient's) condition, and the
administration route, etc. In general, the upper limit of the daily
dose is, for example, approximately 100 mg/kg or lower, preferably
approximately 50 mg/kg or lower, more preferably 1 mg/kg or lower.
The lower limit of the daily dose is, for example, approximately
0.1 .mu.g/kg or higher, preferably 0.5 .mu.g/kg or higher, more
preferably 1 .mu.g/kg or higher.
[0134] The frequency of administration of the medicament according
to the present invention varies depending on the active ingredient
used, the administration route, and the particular disease to be
treated. In the case of orally administering, for example, a
peptidic substance, this substance is preferably prescribed such
that the number of doses per day is 4 or fewer. In the case of
parenteral administration, for example, intravenous administration,
the medicament can be injected by use of a normal syringe or may be
continuously administered by use of an infusion pump, a catheter,
or the like. Alternatively, administration through a route such as
subcutaneous injection or intramuscular injection is also
preferred. In this case, various administration devices that are
usually used can be adopted.
[0135] When the active ingredient for the medicament of the present
invention is prepared in a solution, the modified peptide of the
present invention or the pharmaceutically acceptable salt thereof
can be dissolved in an aqueous solvent and supplemented, if
necessary, with a stabilizer, a pH adjuster, a surfactant, and the
like to prepare the solution. When the active ingredient is
prepared in a lyophilized formulation, the solution thus prepared
can be lyophilized and dissolved in physiological saline,
injectable water, or a glucose solution in use.
[0136] The medicament of the present invention is administered to a
patient with a disease that is treatable by the activation on GC-A
and the resulting elevation of the cGMP level, and is thereby
effective for treating such a disease. In this context, the
"treatment" of the disease or its symptoms means that the
progression of a pathological condition expected to be normalized
by the activation of GC-A is delayed, alleviated, reduced, and/or
suppressed, thereby making the condition closer to a normal state.
The medicament of the present invention is expected to be effective
for preventing the aggravation or onset of a disease by starting
its administration at an early stage of the disease or to an
individual at a high risk of the disease. Although a patient who
has developed the disease in the past is at risk of recurrence or
chronicity, the medicament of the present invention can be expected
to reduce the risk of recurrence or chronicity by continuous
administration to such a patient. These effects are also included
in the scope of the treatment.
[0137] Examples of such a disease include hypertension, acute heart
failure (including the management of a medical condition after the
onset of acute heart failure), chronic heart failure, ischemic
heart diseases, acute nephritis (including the management of a
medical condition after the onset of acute nephritis), chronic
nephritis, acute renal failure (including the management of a
medical condition after the onset of acute renal failure), chronic
renal failure, ischemic heart diseases (myocardial infarction,
etc.), metastasis of malignant tumor, hepatic fibrosis, hepatic
cirrhosis, tissue adhesion caused by dialysis, and fibrosis.
EXAMPLES
[0138] Hereinafter, the present invention will be specifically
described with reference to Examples. The embodiments of the
present invention described in the Examples are given merely for
illustrative purposes, and the present invention is not intended to
be limited by these examples.
[0139] Example 1 is a production example of a linker molecule, a
GlcNAc compound, a sugar substance, or a derivative thereof, which
is an intermediate for the production of the modified peptide of
the present invention. Example 2 is a production example of the
modified peptide using these intermediates. Test Examples are
examples of tests on the characteristics or effects of the modified
peptide of the present invention.
Example 1
Example 1-1
[0140] (1-1A) Synthesis of
[(2R,3S,4R,5R,6R)-3,4-diacetoxy-6-hydroxy-5-(2,2,2-trichloroethoxycarbony-
lamino)tetrahydropyran-2-yl]methyl acetate (compound 1-1A: compound
of the following formula)
##STR00023##
[0141] Glucosamine hydrochloride (10.0 g, 46.38 mmol) and sodium
bicarbonate (11.7 g, 139 mmol) were dissolved in water (100 mL). To
the solution, 2,2,2-trichloroethyl chloroformate (7.66 mL, 55.7
mmol) was added dropwise at room temperature, and the mixture was
stirred for 1 hour. The reaction solution was neutralized by the
addition of 1 N hydrochloric acid, and the resulting precipitates
were collected by filtration. The solid was washed with water and
then dried in a vacuum pump. The obtained solid was dissolved in
pyridine (50 mL). To the solution, acetic anhydride (24.1 mL, 255
mmol) was added at room temperature, and the mixture was stirred
overnight. The solvent was distilled off under reduced pressure to
obtain a crude product. This product was purified by silica gel
column chromatography (hexane:ethyl acetate=80:20-33:67, v/v) to
obtain a crude product of the intermediate as a colorless oil (19.4
g).
[0142] The obtained crude product (19.4 g) of the intermediate was
dissolved in N,N-dimethylformamide (200 mL). To the solution,
hydrazine acetate (4.01 g, 44.5 mmol) was added at room
temperature, and the mixture was stirred for 1 hour. The reaction
solution was diluted with ethyl acetate and washed with 10% saline
twice and saturated saline once. After drying over anhydrous sodium
sulfate and filtering, the solvent was distilled off under reduced
pressure to obtain a crude product. This product was purified by
silica gel column chromatography (hexane:ethyl acetate=80:20-25:75,
v/v) to obtain the title compound 1-1A as a white solid (11.6 g,
yield through 2 steps: 52%).
[0143] .sup.1H-NMR (CDCl.sub.3) .delta.: 5.42 (1H, d, J=9.8 Hz),
5.37-5.32 (2H, m), 5.16-5.10 (1H, m), 4.80 (1H, d, J=11.7 Hz), 4.64
(1H, d, J=12.1 Hz), 4.26-4.22 (2H, m), 4.17-4.12 (1H, m), 4.09-4.03
(1H, m), 3.46-3.43 (1H, m), 2.10 (3H, s), 2.05 (3H, s), 2.02 (3H,
s).
[0144] (1-1B) Synthesis of
[(2R,3S,4R,5R,6S)-3,4-diacetoxy-6-(2,2,2-trichloroethanimidoyl)oxy-5-(2,2-
,2-trichloroethoxycarbonylamino)tetrahydropyran-2-yl]methyl acetate
(compound 1-1B: compound of the following formula)
##STR00024##
[0145] The compound 1-1A (5.00 g, 10.4 mmol) was dissolved in
dichloromethane (35 mL). To the solution, trichloroacetonitrile
(10.4 mL, 104 mmol) and diazabicycloundecene (0.467 mL, 3.12 mmol)
were added at 0.degree. C. The reaction solution was heated to room
temperature and stirred for 40 minutes. The solvent was distilled
off under reduced pressure to obtain a crude product. This product
was purified by silica gel column chromatography (hexane:ethyl
acetate=75:25-50:50, v/v) to obtain the title compound 1-1B as a
colorless oil (3.70 g, yield: 57%).
[0146] .sup.1H-NMR (CDCl.sub.3) .delta.: 8.81 (1H, s), 6.43 (1H, d,
J=3.9 Hz), 5.37-5.34 (1H, m), 5.27-5.22 (2H, m), 4.72 (2H, dd,
J=16.2, 11.9 Hz), 4.32-4.26 (2H, m), 4.16-4.10 (2H, m), 2.09 (3H,
s), 2.06 (6H, s).
[0147] (1-1C) Synthesis of benzyl
(25)-3-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetra-
hydropyran-2-yl]oxy-2-(tert-butoxycarbonylamino)propanoate
(compound 1-1C: compound of the following formula)
##STR00025##
[0148] The compound 1-1B (2.77 g, 4.44 mmol) was dissolved in
dichloromethane (50 mL). To the solution, benzyl
(2S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanoate (1.31 g, 4.44
mmol) and trimethylsilyl trifluoromethanesulfonate (8.0 .mu.L,
0.0444 mmol) were added at room temperature, and the mixture was
stirred for 1 hour. Triethylamine (0.1 mL) was added thereto, and
the solvent was distilled off under reduced pressure to obtain a
crude product. This product was purified by silica gel column
chromatography (hexane:ethyl acetate=75:25-33:67, v/v) to obtain a
crude product of the intermediate as a white solid (2.01 g).
[0149] The obtained crude product (2.01 g) of the intermediate was
dissolved in acetic anhydride (50 mL). To the solution, zinc (1.5
g, 22.9 mmol), washed with 0.1 N hydrochloric acid, methanol, and
diethyl ether in this order and dried, was added at room
temperature, and the mixture was stirred for 6 hours. The reaction
solution was filtered through celite, and the solvent was distilled
off under reduced pressure to obtain a crude product. This product
was purified twice by silica gel column chromatography
(hexane:ethyl acetate=50:50-0:100, v/v) to obtain the title
compound 1-1C as a colorless solid (0.846 g, yield through 2 steps:
31%).
[0150] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.40-7.33 (5H, m),
5.52-5.43 (2H, m), 5.29-5.15 (3H, m), 5.04 (1H, t, J=9.6 Hz), 4.70
(1H, d, J=8.2 Hz), 4.49-4.46 (1H, m), 4.27-4.23 (2H, m), 4.11-4.07
(1H, m), 3.84 (1H, dd, J=10.6, 3.5 Hz), 3.75-3.73 (1H, m),
3.64-3.62 (1H, m), 2.07 (3H, s), 2.03 (3H, s), 2.02 (3H, s), 1.94
(3H, s), 1.46 (9H, s).
[0151] (1-1D) Synthesis of
(2S)-3-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetra-
hydropyran-2-yl]oxy-2-(tert-butoxycarbonylamino)propanoic acid
(compound 1-1D: compound of the following formula)
##STR00026##
[0152] To the compound 1-1C (846 mg, 1.35 mmol), 10%
palladium-carbon (approximately 50% water-wetted product) (150 mg),
ethyl acetate (5 mL), and ethanol (5 mL) were added, and the
mixture was stirred at room temperature for 3 hours under hydrogen
atmosphere. The reaction solution was filtered, and the solvent was
distilled off under reduced pressure to obtain a crude product of
the intermediate as a colorless oil (740 mg).
[0153] The obtained crude product (740 mg) of the intermediate was
dissolved in methanol (10 mL). To the solution, a 0.5 M solution of
sodium methoxide in methanol (14 mL, 7.0 mmol) was added, and the
mixture was stirred at room temperature for 20 hours. Dowex-50 was
added to the reaction solution until the reaction solution became
weakly acidic. Then, the mixture was filtered, and the solvent was
distilled off under reduced pressure to obtain the title compound
1-1D as a light brown solid (543 mg, yield through 2 steps:
98%).
[0154] .sup.1H-NMR (CD.sub.3OD) .delta.: 4.44 (1H, d, J=8.8 Hz),
4.28-4.25 (1H, m), 4.16 (1H, dd, J=10.4, 4.4 Hz), 3.87 (1H, dd,
J=12.0, 2.0 Hz), 3.80 (1H, dd, J=10.4, 4.4 Hz), 3.68 (1H, dd,
J=12.0, 5.6 Hz), 3.62-3.58 (1H, m), 3.45 (1H, dd, J=10.5, 8.5 Hz),
3.29-3.24 (2H, m), 2.00 (3H, s), 1.44 (9H, s).
[0155] FAB-MS: Calcd for C.sub.16H.sub.28N.sub.2O.sub.10:
[M+H].sup.+ 409, Found 409.
Example 1-2
[0156] (1-2A) Synthesis of benzyl
2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-
pyran-2-yl]oxyacetate (compound 1-2A: compound of the following
formula)
##STR00027##
[0157]
4',5'-Dihydro-2'-methyloxazolo[5',4':1,2]-3,4,6-tri-O-acetyl-1,2-di-
deoxy-.alpha.-glucopyranose (4.30 g, 13.1 mmol) produced according
to the description of Bull. Chem. Soc. Jpn., 2003, 76, 485-500 was
dissolved in dichloroethane (50 ml). To the solution, benzyl
glycolate (5.56 ml, 39.1 mmol) and pyridinium p-toluenesulfonate
(1.64 g, 6.53 mmol) were added at room temperature, and the mixture
was heated to reflux for 3 hours. The reaction solution was cooled
and then added to a saturated aqueous solution of sodium
bicarbonate under ice cooling, and the organic matter was extracted
with dichloromethane. The organic layer was washed with a 1 N
aqueous hydrochloric acid solution, a saturated aqueous solution of
sodium bicarbonate, and saturated saline, then dried over anhydrous
sodium sulfate, and filtered, and the solvent was distilled off
under reduced pressure to obtain a crude product. This product was
purified by silica gel column chromatography (hexane:ethyl
acetate=60:40-0:100, v/v) to obtain the title compound 1-2A as a
colorless solid (3.45 g, yield: 53%).
[0158] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.37-7.34 (5H, m), 5.85
(1H, d, J=8.8 Hz), 5.16-5.08 (4H, m), 4.66 (1H, d, J=8.3 Hz), 4.33
(2H, s), 4.22 (1H, dd, J=12.2, 4.4 Hz), 4.09 (1H, dd, J=12.2, 2.4
Hz), 4.07-4.02 (1H, m), 3.64-3.62 (1H, m), 2.05 (3H, s), 2.01 (3H,
s), 1.99 (3H, s), 1.91 (3H, s).
[0159] (1-2B) Synthesis of
2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-
pyran-2-yl]oxyacetatic acid (compound 1-2B: compound of the
following formula)
##STR00028##
[0160] The compound 1-2A (3.45 g, 6.96 mmol) was dissolved in
methanol (54 ml). To the solution, 20% palladium hydroxide-carbon
(690 mg) was added, and the mixture was stirred at room temperature
for 3.0 hours under hydrogen atmosphere. The reaction mixture was
filtered through celite, and the solvent was distilled off under
reduced pressure. The solid obtained by the addition of diisopropyl
ether was collected by filtration to obtain the title compound 1-2B
as a colorless solid (2.72 g, yield: 96%).
[0161] .sup.1H-NMR (CDCl.sub.3) .delta.: 6.36 (1H, d, J=8.8 Hz),
5.21-5.10 (2H, m), 4.70 (1H, d, J=8.8 Hz), 4.39 (1H, d, J=16.9 Hz),
4.32 (1H, d, J=16.9 Hz), 4.28 (1H, dd, J=12.2, 4.9 Hz), 4.15 (1H,
dd, J=12.2, 2.4 Hz), 4.11-4.05 (1H, m) 3.72-3.70 (1H, m), 2.10 (3H,
s), 2.07 (3H, s), 2.04 (3H, s), 1.97 (3H, s).
[0162] (1-2C) Synthesis of
2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyacetic acid (compound 1-2C: compound of the following
formula)
##STR00029##
[0163] The compound 1-2B (2.72 g, 6.73 mmol) was dissolved in
methanol (42 ml). To the solution, a 5 mol/L solution of sodium
methoxide in methanol (2 ml, 10 mmol) was added at room
temperature, and then the mixture was stirred overnight at room
temperature. After the completion of the reaction, distilled water
(4 ml) was added thereto, and then an ion-exchange resin (Dowex
50wx8) was added to the mixture to adjust its pH to 3. The reaction
solution was filtered, and the solvent was distilled off under
reduced pressure. The solid obtained by the addition of diisopropyl
ether was collected by filtration to obtain the title compound 1-2C
as a colorless solid (2.72 g, yield: 96%).
[0164] .sup.1H-NMR (D.sub.2O, TMSP) .delta.: 4.57 (1H, d, J=8.6
Hz), 4.17 (2H, s), 3.91 (1H, dd, J=12.5, 1.6 Hz), 3.77-3.72 (2H,
m), 3.56-3.41 (3H, m), 2.05 (3H, s).
[0165] ESI-LC-MS: Calcd for C.sub.10H.sub.27NO.sub.8: [M+H].sup.+
280, Found 280.
Example 1-3
[0166] (1-3A) Synthesis of
3-[2[2[2[2[[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethy-
l)tetrahydropyran-2-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propano-
ic acid (compound 1-3A: compound of the following formula)
##STR00030##
[0167] A 1.20 mmol/g 2-chlorotrityl chloride resin (694 mg, 0.833
mmol) was placed in a column for solid-phase synthesis.
Dichloromethane (5 mL) was added thereto, and the mixture was
shaken for 5 minutes. After filtration, a solution of
3-[2[2[2[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]ethoxy]etho-
xy]propanoic acid (488 mg, 1 mmol) and N,N-diisopropylethylamine
(730 .mu.L, 4.17 mmol) in dichloromethane (5 mL) was added thereto,
and the mixture was stirred at room temperature for 2 hours. After
filtration, the resin was washed with a dichloromethane mixed
solution
(dichloromethane:methanol:N,N-diisopropylethylamine=85:10:5, v/v)
three times, dichloromethane three times, and N,N-dimethylformamide
three times. A 20% solution of piperidine in N,N-dimethylformamide
(5 mL) was added thereto, and the mixture was shaken for 5 minutes,
followed by filtration. This operation was carried out 4 times. The
resin was washed with N,N-dimethylformamide three times,
dichloromethane three times, and diethyl ether three times and
dried in a vacuum pump. An aliquot (200 mg) of the obtained resin
(800 mg) was placed in a column for solid-phase synthesis, and
N,N-dimethylformamide (2.5 mL), triethylamine (406 .mu.L, 2.92
mmol), and water (0.5 mL) were added thereto. A solution obtained
by the stirring of the compound 1-2C (174 mg, 0.625 mmol),
N,N-dimethylformamide (3 mL), triethylamine (174 .mu.L, 1.25 mmol),
and dimethylthiophosphonoyl chloride (80 mg, 0.625 mmol) at room
temperature for 1 hour was added thereto. The mixture was stirred
at room temperature for 2 hours and then filtered, and the resin
was washed with N,N-dimethylformamide three times and
dichloromethane three times. A 1% solution of trifluoroacetic acid
in dichloromethane (2 mL) was added thereto, and the mixture was
shaken for 2 minutes, followed by the recovery of the filtrate.
This operation was carried out 10 times. The solvent in the
recovered solution was distilled off under reduced pressure to
obtain a crude product. The crude product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents to
obtain the title compound 1-3A as a white solid (25 mg).
[0168] ESI-LC-MS: Calcd for C.sub.21H.sub.38N.sub.2O.sub.13:
[M+H].sup.+ 526, Found 526.
Example 1-4
[0169] (1-4A) Synthesis of
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]et-
hoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic
acid (compound 1-4A: compound of the following formula)
##STR00031##
[0170]
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(9H-Fluoren-9-ylmethoxycarbon-
ylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth-
oxy]ethoxy]ethoxy]propanoic acid (320 mg, 0.38 mmol) was dissolved
in methanol (2 ml) and distilled water (2 ml). To the solution, a 1
N aqueous sodium hydroxide solution (1 ml) was added at 0.degree.
C., and the mixture was stirred at room temperature for 1.5 hours.
1 N hydrochloric acid (1 ml) was added thereto at 0.degree. C., and
the organic solvent was distilled off under reduced pressure. To
the residue, distilled water (10 ml) was added, and the resulting
product was washed with dichloromethane, then separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents, and
lyophilized to obtain the title compound 1-4A as a colorless oil
(233.5 mg, 99%).
[0171] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.63 (2H, br s), 3.79 (2H,
t, J=5.1 Hz), 3.75 (2H, t, J=5.9 Hz), 3.70-3.68 (2H, m), 3.65-3.61
(42H, m), 3.21-3.17 (2H, m), 2.58 (2H, t, J=5.9 Hz).
[0172] (1-4B) Synthesis of
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-[(2R,3R,4R,5S,6R)-3-acetamido-4-
,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]ethoxy]-
ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho-
xy]propanoic acid (compound 1-4B: compound of the following
formula)
##STR00032##
[0173] The compound 1-2C (36.6 mg, 0.13 mmol) was dissolved in
dimethylformamide (500 .mu.l). To the solution, triethylamine (45.7
.mu.l, 0.33 mmol) was added at room temperature, then a solution of
dimethylthiophosphinoyl chloride (16.9 mg, 0.13 mmol) in
dimethylformamide (500 .mu.l) was added at 0.degree. C., and the
mixture was stirred at 0.degree. C. for 0.5 hours.
[0174] The compound 1-4A (67.5 mg, 0.11 mmol) was dissolved in
dimethylformamide (500 .mu.l). To the solution, prepared active
ester was added at 0.degree. C., and the mixture was stirred at
room temperature for 6 hours. Distilled water (3 ml) and acetic
acid (100 .mu.l) were added thereto, and the resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound 1-4B as a
colorless oil (31.0 mg, 32%).
[0175] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.62 (1H, d, J=6.8 Hz),
7.40 (1H, t, J=5.4 Hz), 4.47 (1H, d, J=8.3 Hz), 4.27 (1H, d, J=16.1
Hz), 4.22 (1H, d, J=16.1 Hz), 3.90 (1H, dd, J=12.0, 3.2 Hz), 3.81
(1H, dd, J=12.2, 4.9 Hz), 3.77 (2H, t, J=6.1 Hz), 3.74-3.52 (54H,
m), 3.39-3.34 (2H, m), 2.59 (2H, t, J=6.1 Hz), 2.08 (3H, s).
[0176] MALDI-TOF-MS: Calcd for C.sub.37H.sub.70N.sub.2O.sub.21:
[M+Na].sup.+ 901, Found 901.
Example 1-5
[0177] (1-5A) Synthesis of
3-[2-[2-[2-[2-[[(2S)-4-[[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hy-
droxymethyl)tetrahydropyran-2-yl]amino-2-(tert-butoxycarbonylamino)-4-oxob-
utanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (compound
1-5A: compound of the following formula)
##STR00033##
[0178]
3-[2-[2-[2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy-
]propanoic acid (200 mg, 0.547 mmol) was dissolved in a solution of
4 N hydrochloric acid in dioxane (2 mL), and the solution was
stirred at room temperature for 1 hour. The solvent was distilled
off under reduced pressure, and the residue was dried in a vacuum
pump to obtain a crude product of the intermediate as a light brown
oil (165 mg).
[0179]
(2S)-4-[[(2R,3R,4R,5S,6R)-3-Acetamido-4,5-dihydroxy-6-(hydroxymethy-
l)tetrahydropyran-2-yl]amino]-2-(tert-butoxycarbonylamino)-4-oxobutanoic
acid (158 mg, 0.364 mmol) produced according to the approach of J.
Am. Chem. Soc., 1999, 121, 284-290 and HATU (138 mg, 0.364 mmol)
were dissolved in N,N-dimethylformamide (3 mL). To the solution,
N,N-diisopropylethylamine (128 .mu.L, 0.728 mmol) was added, and
the mixture was stirred at room temperature for 1 minute. This
solution was added to the obtained crude product (54.9 mg) of the
intermediate, further N,N-diisopropylethylamine (128 .mu.L, 0.728
mmol) was added, and the mixture was stirred at room temperature
for 0.5 hours. The reaction solution was diluted with water, and
the resulting product was separated and purified by reverse-phase
HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents to obtain the title
compound 1-5A as a white solid (53 mg, yield through 2 steps:
43%).
[0180] ESI-LC-MS: Calcd for C.sub.28H.sub.50N.sub.4O.sub.15:
[M+H].sup.+ 683, Found 683.
[0181] (1-5B) Synthesis of
3-[2-[2-[2-[2-[[(2S)-4-[[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hy-
droxymethyl)tetrahydropyran-2-yl]amino]-2-[[2-[(2R,3R,4R,5S,6R)-3-acetamid-
o-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-4-o-
xobutanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid
(compound 1-5B: compound of the following formula)
##STR00034##
[0182] To the compound 1-5A (53 mg, 0.0777 mmol), a 30% aqueous
trifluoroacetic acid solution was added, and the mixture was
stirred at room temperature for 7 hours. The reaction solution was
diluted with water and lyophilized to obtain a crude product of the
intermediate as a light brown oil (45 mg).
[0183] The compound 1-2C (65.1 mg, 0.233 mmol) and triethylamine
(65 .mu.L, 0.466 mmol) were dissolved in N,N-dimethylformamide (0.5
mL). To the solution, a solution of dimethylthiophosphonoyl
chloride (30 mg, 0.233 mmol) in N,N-dimethylformamide (0.5 mL) was
added at 0.degree. C. The mixture was heated to room temperature
and stirred for 1 hour. This solution was cooled to 0.degree. C.,
and a mixed solution of the obtained crude product (45 mg) of the
intermediate, triethylamine (152 .mu.L, 1.088 mmol),
N,N-dimethylformamide (2.5 mL), and water (0.5 mL) was added
thereto. The mixture was heated to room temperature and stirred for
8 hours. The reaction solution was diluted with water, and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents to obtain the title
compound 1-5B as a white solid (9.25 mg, yield through 2 steps:
14%).
[0184] MALDI-TOF-MS: Calcd for C.sub.33H.sub.57N.sub.5O.sub.20:
[M+H].sup.+ 844, Found 844.
Example 1-6
[0185] (1-6A) Synthesis of
[(2R,3S,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[2-(4-nitrophenyl)ethoxy]te-
trahydropyran-2-yl]methyl acetate (compound 1-6A: compound of the
following formula)
##STR00035##
[0186]
4,5-Dihydro-2-methyloxazolo[5',4':1,2]-3,4,6-tri-O-acetyl-1,2-dideo-
xy-.alpha.-glucopyranose (1.00 g, 3.04 mmol) produced according to
the description of Bull. Chem. Soc. Jpn., 2003, 76, 485-500 was
dissolved in dichloroethane (10 ml). To the solution, molecular
sieve 4A (312 mg), 2-(4-nitrophenyl)-ethanol (2.54 g, 15.2 mmol),
and (+)-camphorsulfonic acid (0.78 g, 3.34 mmol) were added at room
temperature, and the mixture was stirred at 60.degree. C. for 3
hours. The reaction solution was added to a saturated aqueous
solution of sodium bicarbonate, and the organic matter was
extracted with ethyl acetate. The organic layer was washed with
saturated saline, then dried over anhydrous sodium sulfate, and
filtered, and the solvent was distilled off under reduced pressure
to obtain a crude product. This product was purified by silica gel
column chromatography (hexane:ethyl acetate=67:33-0:100, v/v) to
obtain the title compound 1-6A as a colorless solid (1.51 g, yield:
65%).
[0187] .sup.1H-NMR (CDCl.sub.3) .delta.: 8.14 (2H, d, J=8.9 Hz),
7.38 (2H, d, J=9.0 Hz), 5.32 (1H, d, J=8.6 Hz), 5.21 (1H, dd,
J=10.6, 9.4 Hz), 5.07 (1H, t, J=9.6 Hz), 4.63 (1H, d, J=8.2 Hz),
4.25 (1H, dd, J=12.1, 4.7 Hz), 4.20-4.12 (2H, m), 3.88 (1H, dt,
J=10.6, 8.6 Hz), 3.72-3.64 (2H, m), 3.06-2.93 (2H, m), 2.09 (3H,
s), 2.03 (3H, s), 2.03 (3H, s), 1.84 (3H, s).
[0188] (1-6B) Synthesis of
[(2R,3S,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[2-(4-aminophenyl)ethoxy]te-
trahydropyran-2-yl]methyl acetate (compound 1-6B: compound of the
following formula)
##STR00036##
[0189] The compound 1-6A (982.0 mg, 1.98 mmol) was dissolved in
ethyl acetate (12 ml) and ethanol (12 ml). To the solution, 10%
palladium-carbon (250 mg) was added, and the mixture was stirred at
room temperature for 1.5 hours under hydrogen atmosphere. The
reaction mixture was filtered through celite, and the solvent was
distilled off under reduced pressure to obtain the title compound
1-6B as a colorless solid (758 mg, yield: 82%).
[0190] .sup.1H-NMR (CDCl.sub.3) .delta.: 6.99 (2H, d, J=8.6 Hz),
6.61 (2H, d, J=8.6 Hz), 5.32 (1H, d, J=9.0 Hz), 5.24 (1H, dd,
J=10.6, 9.4 Hz), 5.06 (1H, t, J=9.6 Hz), 4.60 (1H, d, J=8.2 Hz),
4.26 (1H, dd, J=12.1, 4.7 Hz), 4.15-4.04 (3H, m), 3.84 (1H, dt,
J=10.6, 8.4 Hz), 3.68-3.58 (4H, m), 2.78-2.77 (2H, m), 2.09 (3H,
s), 2.02 (3H, s), 2.02 (3H, s), 1.88 (3H, s).
[0191] (1-6C) Synthesis of
[(2R,3S,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[2-[4-[(ethoxycarbonylamino-
)carbamoylamino]phenyl]ethoxy]tetrahydropyran-2-yl]methyl acetate
(compound 1-6C: compound of the following formula)
##STR00037##
[0192] The compound 1-6B (568.0 mg, 1.22 mmol) was dissolved in
tetrahydrofuran (22 ml). To the solution, triethylamine (424 .mu.l,
3.04 mmol) was added at room temperature, then 4-nitrophenyl
chloroformate (441.8 mg, 2.19 mmol) was added at -10.degree. C.,
and the mixture was stirred at room temperature for 1.5 hours.
Triethylamine (424 .mu.l, 3.04 mmol) was added thereto at room
temperature, then ethyl carbazate (228.2 mg, 2.19 mmol) was added,
and the mixture was stirred at room temperature for 30 minutes. The
reaction solution was added to water, and the organic matter was
extracted with ethyl acetate twice. The organic layer was washed
with saturated saline, then dried over anhydrous sodium sulfate,
and filtered, and the solvent was distilled off under reduced
pressure to obtain a crude product. This product was purified by
silica gel column chromatography
(dichloromethane:methanol=100:0-90:10, v/v) to obtain the title
compound 1-6C as a colorless foam (650.4 mg, yield: 90%).
[0193] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.88 (1H, br s), 7.24 (2H,
s), 7.02 (2H, d, J=8.2 Hz), 6.58 (1H, br s), 5.27 (1H, t, J=10.0
Hz), 5.04 (1H, t, J=10.0 Hz), 4.64 (1H, d, J=8.6 Hz), 4.27-4.05
(5H, m), 3.90-3.81 (1H, m), 3.73-3.68 (1H, m), 3.61-3.55 (1H, m),
3.49 (2H, d, J=4.3 Hz), 2.85-2.70 (2H, m), 2.07 (3H, s), 2.00 (6H,
s), 1.82 (3H, s), 1.27 (3H, t, J=9.4 Hz).
[0194] (1-6D) Synthesis of
N-[(2R,3S,4R,5R,6R)-3-acetamido-2-[2-[4-(3,5-dioxo-1,2,4-triazolidin-4-yl-
)phenyl]ethoxy]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetam-
ide (compound 1-6D: compound of the following formula)
##STR00038##
[0195] The compound 1-6C (650.0 mg, 1.09 mmol) was dissolved in
methanol (30 ml). To the solution, potassium carbonate (451.8 mg,
3.27 mmol) was added at room temperature, and the mixture was
stirred at 60.degree. C. for 9.5 hours. After cooling to room
temperature, the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound 1-6D as a colorless solid (462.4 mg,
85%).
[0196] .sup.1H-NMR (CD.sub.3OD) .delta.: 7.35 (4H, dd, J=13.1, 8.8
Hz), 4.36 (1H, d, J=8.2 Hz), 4.17 (1H, dt, J=11.0, 4.8 Hz), 3.87
(1H, dd, J=11.9, 2.2 Hz), 3.70-3.62 (3H, m), 3.39 (1H, dd, J=10.2,
8.6 Hz), 3.28-3.22 (2H, m), 2.90 (2H, t, J=6.3 Hz).
[0197] ESI-TOF-MS: Calcd for C.sub.18H.sub.24N.sub.4O.sub.8:
[M+H].sup.+ 425, Found 425.
Example 1-7
[0198] (1-7A) Synthesis of
2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-
pyran-2-yl]oxyethylamine (compound 1-7A: compound of the following
formula)
##STR00039##
[0199]
N-[(2R,3R,4R,5S,6R)-3-Acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetr-
ahydropyran-2-yl]oxyethylazide (500 mg, 1.72 mmol) was dissolved in
ethanol (20 ml). To the solution, 10% palladium-carbon (200 mg) was
added, and the mixture was stirred at room temperature for 3 hours
under hydrogen atmosphere. The reaction mixture was filtered
through celite, and the solvent was distilled off under reduced
pressure to obtain the title compound 1-7A as a colorless solid
(460 mg, yield: quant).
[0200] .sup.1H-NMR (CD.sub.3OD) .delta.: 4.38 (1H, d, J=8.3 Hz),
3.90-3.82 (2H, m), 3.69-3.55 (2H, m), 3.46-3.40 (1H, m), 2.80-2.73
(2H, m), 1.98 (3H, s).
[0201] (1-7B) Synthesis of
N-[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahy-
dropyran-2-yl]oxyethyl]-3-(2,5-dioxopyrrol-1-yl)propanamide
(compound 1-7B: compound of the following formula)
##STR00040##
[0202] The compound 1-7A (100 mg, 0.378 mmol) was dissolved in
N,N-dimethylformamide. To the solution, 2,5-dioxopyrrolidin-1-yl
3-(2,5-dioxopyrrol-1-yl)propanoate (0.126 mg, 0.473 mmol) was
added, and the mixture was stirred at room temperature for 3 hours.
The reaction solution was diluted with water, and the resulting
product was separated and purified by reverse-phase HPLC (GL
Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic
acid solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents to obtain the title compound 1-7B as a
white solid (93 mg, yield: 59%).
[0203] .sup.1H-NMR (CD.sub.3OD) .delta.: 6.82 (2H, s), 4.38 (1H, d,
J=8.6 Hz), 3.89-3.87 (1H, m), 3.78-3.75 (3H, m), 3.66-3.62 (3H, m),
3.44-3.38 (1H, m), 2.67 (4H, s), 2.46 (2H, t, J=7.0 Hz), 1.98 (3H,
s).
[0204] ESI-LC-MS: Calcd for C.sub.17H.sub.25N.sub.3O.sub.9:
[M+H].sup.+ 416, Found 416.
Example 1-8
[0205] (1-8A) Synthesis of
2-[[(2R)-2-[[2-[[(2R)-2-[[2-[[(2R)-2-[[2-[[(2R)-2-[[2-[[(2R)-2-acetamido--
3-tritylsulfanylpropanoyl]amino]acetyl]amino]-3-tritylsulfanylpropanoyl]am-
ino]acetyl]amino]-3-tritylsulfanylpropanoyl]amino]acetyl]amino]-3-tritylsu-
lfanylpropanoyl]amino]acetyl]amino]-3-tritylsulfanylpropanoyl]amino]acetat-
ic acid (compound 1-8A: compound of the following formula)
##STR00041##
[0206] A 1.20 mmol/g 2-chlorotrityl chloride resin (833 mg, 1.00
mmol) was placed in a column for solid-phase synthesis.
[0207] Dichloromethane (7.5 mL) was added thereto, and the mixture
was shaken for 10 minutes. After filtration, a solution of
2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (594 mg, 2 mmol)
and N,N-diisopropylethylamine (0.86 mL, 5 mmol) in dichloromethane
(7.5 mL) was added thereto, and the mixture was stirred at room
temperature for 2 hours. After filtration, the resin was washed
with a dichloromethane mixed solution
(dichloromethane:methanol:N,N-diisopropylethylamine=85:10:5, v/v)
three times, dichloromethane three times, and diethyl ether three
times. The resin was dried in a vacuum pump and recovered (1.83 g).
An aliquot (1.37 g) of the recovered resin was placed in a column
for solid-phase synthesis. A 20% solution of piperidine in
N,N-dimethylformamide (20 mL) was added thereto, and the mixture
was shaken for 5 minutes, followed by the filtration of the
reaction solution. This operation was carried out 4 times. After
washing with N,N-dimethylformamide 4 times, a solution of
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid (1.32 g, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (669
mg, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid (1.32 g, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (669
mg, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid (1.32 g, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (669
mg, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added, and the mixture was shaken
at room temperature for 30 minutes. After filtration, the resin was
washed with N,N-dimethylformamide 4 times. A 20% solution of
piperidine in N,N-dimethylformamide (20 mL) was added thereto, and
the mixture was shaken for 5 minutes, followed by filtration of the
reaction solution. This operation was carried out 4 times. After
washing with N,N-dimethylformamide 4 times, a solution of
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid (1.32 g, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (669
mg, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. After washing with N,N-dimethylformamide 4 times, a
solution of
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid (1.32 g, 2.25 mmol), HATU (856 mg, 2.25 mmol), and
N,N-diisopropylethylamine (582 .mu.L, 4.50 mmol) in
N,N-dimethylformamide (15 mL) was added thereto, and the mixture
was shaken at room temperature for 30 minutes. After filtration,
the resin was washed with N,N-dimethylformamide 4 times. A 20%
solution of piperidine in N,N-dimethylformamide (20 mL) was added
thereto, and the mixture was shaken for 5 minutes, followed by
filtration of the reaction solution. This operation was carried out
4 times. The resin was washed with N,N-dimethylformamide 4 times
and washed with dichloromethane 4 times and diethyl ether 4 times.
The resin was dried in a vacuum pump and recovered (1.83 g). An
aliquot (360 mg) of the recovered resin was placed in a column for
solid-phase synthesis. A solution of acetic acid (27 mg, 0.45
mmol), HATU (171 mg, 0.45 mmol), and N,N-diisopropylethylamine (154
.mu.L, 0.90 mmol) in N,N-dimethylformamide (5 mL) was added
thereto, and the mixture was shaken at room temperature for 30
minutes. After filtration, the resin was washed with
N,N-dimethylformamide 4 times and dichloromethane 4 times. A mixed
solution of 1,1,1,3,3,3-hexafluoro-2-propanol (1 mL) and
dichloromethane (3 mL) was added thereto, and the mixture was
shaken at room temperature for 1.5 hours. The resin was filtered
off, and the filtrate was concentrated under reduced pressure. The
concentrate was subjected to azeotropy with dichloromethane three
times and dried in a vacuum pump to obtain the title compound 1-8A
as a brown solid (176 mg).
[0208] MALDI-TOF-MS: Calcd for
C.sub.122H.sub.114N.sub.10O.sub.12S.sub.5: [M+Na].sup.+ 2094, Found
2094.
Example 1-9
[0209] (1-9A) Synthesis of benzyl
2-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]acetate
(compound 1-9A: compound of the following formula)
##STR00042##
[0210] To a solution of
(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid (1.70 g, 5.00
mmol), benzyl 2-aminoacetate (1.00 g, 5.00 mmol), and HATU (2.90 g,
7.50 mmol) in N,N-dimethylformamide (25 mL),
N,N-diisopropylethylamine (2.60 mL, 15.0 mmol) was added, and the
mixture was stirred at room temperature for 20 hours. The reaction
solution was added to water, and the organic matter was extracted
with ethyl acetate. The organic layer was washed with saturated
saline, then dried over anhydrous sodium sulfate, and filtered, and
the solvent was distilled off under reduced pressure to obtain a
crude product. This product was purified by silica gel column
chromatography (hexane:ethyl acetate=90:10-0:100, v/v) to obtain
the title compound 1-9A as a pale yellow oil (2.30 g, yield:
93%).
[0211] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.40-7.33 (5H, m), 6.63
(1H, s), 5.18 (2H, s), 5.11 (1H, s), 4.63 (1H, s), 4.15-4.03 (3H,
m), 3.16-3.06 (2H, m), 1.91-1.81 (1H, m), 1.70-1.59 (1H, m),
1.53-1.24 (22H, m).
[0212] MS (ESI): Calcd for C.sub.25H.sub.40N.sub.3O.sub.7:
[M+H].sup.+ 494, Found 494.
[0213] (1-9B) Synthesis of benzyl
2-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]acetate
(compound 1-9B: compound of the following formula)
##STR00043##
[0214] The compound 1-9A (250 mg, 0.507 mmol) was dissolved in
dichloromethane (3.0 ml). To the solution, trifluoroacetic acid
(1.0 mL) was added, and the mixture was stirred at room temperature
for 1 hour. The solvent was distilled off under reduced pressure to
obtain the title compound 1-9B as a pale yellow oil (247 mg, yield:
100%).
[0215] .sup.1H-NMR (DMSO-d.sub.6) .delta.: 8.95 (1H, t, J=5.9 Hz),
8.18 (2H, s), 7.71 (2H, s), 7.42-7.34 (5H, m), 5.16 (2H, d, J=12.5
Hz), 4.12-3.96 (2H, m), 3.87-3.78 (1H, m), 2.78-2.66 (2H, m),
1.76-1.66 (2H, m), 1.54-1.47 (2H, m), 1.40-1.31 (2H, m).
[0216] (1-9C) Synthesis of benzyl
2-[[(2S)-2,6-bis[[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydrox-
ymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]hexanoyl]amino]acetate
(compound 1-9C: compound of the following formula)
##STR00044##
[0217] The title compound 1-9C was obtained as a colorless foam
(180 mg, yield: 88%) according to the same method as in (1-5B)
using the compounds 1-9B (93.0 mg, 0.190 mmol) and 1-2C (160 mg,
0.573 mmol).
[0218] .sup.1H-NMR (CD.sub.3OD) .delta.: 7.39-7.29 (5H, m), 5.16
(2H, s), 4.94-4.84 (2H, m), 4.46-4.39 (3H, m), 4.33-4.27 (2H, m),
4.13-4.00 (3H, m), 3.94 (1H, d, J=17.6 Hz), 3.91-3.84 (2H, m),
3.78-3.66 (4H, m), 3.50-3.42 (2H, m), 3.38-3.28 (2H, m), 3.22 (2H,
t, J=6.6 Hz), 2.03 (3H, s), 2.01 (3H, s), 1.88-1.81 (1H, m),
1.78-1.67 (1H, m), 1.58-1.50 (2H, m), 1.48-1.35 (2H, m).
[0219] (1-9D) Synthesis of
2-[[(2S)-2,6-bis[[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydrox-
ymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]hexanoyl]amino]acetic
acid (compound 1-9D: compound of the following formula)
##STR00045##
[0220] The compound 1-9C (180 mg, 0.221 mmol) was dissolved in
methanol (50 ml). To the solution, 10% palladium-carbon (180 mg)
was added, and the mixture was stirred at room temperature for 2
hours under hydrogen atmosphere. The reaction mixture was filtered
through celite, and the solvent was distilled off under reduced
pressure to obtain the title compound 1-9D as a pale yellow foam
(160 mg, yield: 100%).
[0221] MS (ESI): Calcd for C.sub.28H.sub.48N.sub.5O.sub.17:
[M+H].sup.+ 726, Found 726.
Example 1-10
[0222] (1-10A) Synthesis of
2-[[(2R)-2-[[2-[[(2R)-2-[[2-[[(2R)-2-acetamido-3-sulfanylpropanoyl]amino]-
acetyl]amino]-3-sulfanylpropanoyl]amino]acetyl]amino]-3-sulfanylpropanoyl]-
amino]acetic acid (compound 1-10A: compound of the following
formula)
##STR00046##
[0223] A 1.20 mmol/g 2-chlorotrityl chloride resin (208 mg, 0.25
mmol) was placed in a column for solid-phase synthesis.
Dichloromethane (2.5 mL) was added thereto, and the mixture was
shaken for 10 minutes. After filtration, a solution of
2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (149 mg, 0.5
mmol) and N,N-diisopropylethylamine (219 .mu.L, 1.25 mmol) in
dichloromethane (2.5 mL) was added thereto, and the mixture was
stirred at room temperature for 2 hours. After filtration, the
resin was washed with a dichloromethane mixed solution
(dichloromethane:methanol:N,N-diisopropylethylamine=85:10:5, v/v)
three times, dichloromethane three times, and N,N-dimethylformamide
three times. The obtained resin was loaded in a peptide synthesizer
(433A Peptide Synthesizer manufactured by Applied Biosystems, Inc.)
and subjected to deprotection, condensation, deprotection,
condensation, deprotection, condensation, deprotection,
condensation, deprotection, condensation, deprotection, and
condensation in the synthesizer to elongate the peptide chain. For
the deprotection, piperidine and N-methylpyrrolidone were used. For
the condensation reactions, HATU, N,N-diisopropylethylamine,
N-methylpyrrolidone, and various carboxylic acids were used. The
carboxylic acids were used in each condensation reaction in the
order of
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid, 2-(9H-fluoren-9-ylmethoxycarbonylamino) acetic acid,
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid, 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid,
(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl]propanoic
acid, and acetic acid. A half (450 mg) of the amount of the
obtained resin (900 mg) was placed in a column for solid-phase
synthesis, and a mixed solution of trifluoroacetic acid (2.64 mL),
water (0.27 mL), phenol (0.06 g), and triisopropylsilane (0.03 mL)
was added thereto. The mixture was shaken at room temperature for 2
hours, and trifluoroacetic acid was distilled off. The reaction
solution was diluted with water, and the resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents to obtain the title compound 1-10A as a white solid (11 mg,
yield: 16%).
[0224] ESI-LC-MS: Calcd for C.sub.17H.sub.28N.sub.6O.sub.8S.sub.3:
[M+H].sup.+ 541, Found 541.
[0225] (1-10B) Synthesis of
2-[[(2R)-2-[[2-[[(2R)-2-[[2-[[(2R)-2-acetamido-3-[1-[3-[2-[(2R,3R,4R,5S,6-
R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethy-
lamino]-3-oxopropyl]-2,5-dioxopyrrolidin-3-yl]sulfanylpropanoyl]amino]acet-
yl]amino]-3-[1-[3-[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydrox-
ymethyl)tetrahydropyran-2-yl]oxyethylamino]-3-oxopropyl]-2,5-dioxopyrrolid-
in-3-yl]sulfanylpropanoyl]amino]acetyl]amino]-3-[1-[3-[2-[(2R,3R,4R,5S,6R)-
-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxyethyla-
mino]-3-oxopropyl]-2,5-dioxopyrrolidin-3-yl]sulfanylpropanoyl]amino]acetic
acid (compound 1-10B: compound of the following formula)
##STR00047##
[0226] The compounds 1-10B (11 mg, 0.0203 mmol) and 1-7B (33 mg,
0.0794 mmol) were dissolved in a mixed solution of acetonitrile (1
mL) and a 0.2 M phosphate buffer of pH 6.75 (1 mL), and the
solution was stirred at room temperature for 2 hours. The reaction
solution was diluted with water, and the resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents to obtain the title compound 1-10B as a white solid (27 mg,
yield: 74%).
[0227] MALDI-TOF-MS: Calcd for
C.sub.68H.sub.103N.sub.15O.sub.35S.sub.3: [M-H].sup.+ 1784, Found
1784.
Example 1-11
[0228] (1-11A) Synthesis of
N-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyethylamine trifluoroacetate (compound 1-11A: compound
of the following formula)
##STR00048##
[0229] The compound 1-7A (120 mg) was dissolved in distilled water
(6 ml). To the solution, trifluoroacetic acid (48 .mu.l) was then
added, and the mixture was lyophilized. The obtained amorphous
solid 1-11A was used without being purified.
[0230] (1-11B) Synthesis of SG-NH.sub.2 (compound 1-11B: compound
of the following formula)
##STR00049##
[0231] Sialylglycopeptide (60 mg) was dissolved in a 0.2 M
phosphate buffer solution (pH 6.25) (260 .mu.l). To the solution,
an aqueous solution (100 .mu.l) of glycosynthase (Endo-M-N175Q,
Tokyo Chemical Industry Co., Ltd., 1 U/ml) was then added. The
compound 1-11A (28 mg) in a 0.2 M phosphate buffer solution (pH
6.25) (160 .mu.l) was further added thereto, and the mixture was
reacted at 28.degree. C. for 72 hours. The reaction was terminated
by the addition of a 0.2% aqueous trifluoroacetic acid solution
(2480 .mu.l) to the reaction solution, and the resulting product
was separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound SG-NH.sub.2
(28.5 mg).
[0232] ESI-TOF-MS: Calcd for C.sub.86H.sub.143N.sub.7O.sub.62:
[M-H].sup.- 2264.8, Found 2264.8.
[0233] (1-11C) Synthesis of SG-I (compound 1-11C: compound of the
following formula)
##STR00050##
[0234] The compound SG-NH.sub.2 (15.0 mg) produced in (1-11B) was
dissolved in a 43 mM aqueous sodium bicarbonate solution (750
.mu.l). To the solution, a 30 mM solution of iodoacetic acid
N-hydroxysuccinimide ester in acetone (250 .mu.l) was added under
ice cooling, and the mixture was stirred at room temperature for 1
hour. The reaction was terminated by the addition of acetic acid
(1.8 .mu.l) to the reaction solution, and the organic solvent was
removed under reduced pressure. The resulting product was separated
and purified by reverse-phase HPLC (GL Sciences Inc., Inertsil
ODS-3) using a 0.1% aqueous trifluoroacetic acid solution and a
0.1% solution of trifluoroacetic acid in acetonitrile as eluents
and lyophilized to obtain the title compound SG-I (13.4 mg).
[0235] ESI-TOF-MS: Calcd for C.sub.88H.sub.144IN.sub.7O.sub.63:
[M+2H].sup.2+ 1218.5 (ave.), Found 1218.3.
Example 1-12
[0236] (1-12A) Synthesis of SG-Oxa (compound 1-12A: compound of the
following formula)
##STR00051##
[0237] Disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.,
26.0 mg, 12.8 .mu.mol) was dissolved in distilled water (210
.mu.l). To the solution, triethylamine (80.7 .mu.l, 579 .mu.mol)
was added at room temperature. An aqueous solution (52 l) of
2-chloro-1,3-dimethylimidazolium chloride (32.6 mg, 192 .mu.mol)
was added thereto at 0.degree. C., and the mixture was stirred at
0.degree. C. for 2 hours. The resulting product was purified with
Sephadex G15 (0.03% aqueous NH.sub.3 solution). A 0.1 N aqueous
sodium hydroxide solution (100 .mu.l) was added thereto, and the
mixture was lyophilized to obtain the title compound SG-Oxa as a
colorless solid (24.6 mg, 95%).
[0238] NMR (in D2O) (chart of The FIGURE).
Example 1-13
[0239] (1-13A) Synthesis of SG-M (compound 1-13A: compound of the
following formula)
##STR00052##
[0240] The compound SG-NH.sub.2 (30.0 mg) produced in (1-11B) was
dissolved in a 43 mM aqueous sodium bicarbonate solution (1500 ul).
To the solution, a 13.9 mM solution of
3-(2,5-dioxopyrrol-1-yl)butyric acid N-hydroxysuccinimide ester in
acetone (500 ul) was added under ice cooling, and the mixture was
stirred at room temperature for 1 hour. The reaction was terminated
by the addition of acetic acid (3.6 ul) to the reaction solution,
and the organic solvent was removed under reduced pressure. The
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound SG-M (29 mg).
[0241] ESI-TOF-MS: Calcd for C.sub.93H.sub.148N.sub.8O.sub.65:
[M+2H].sup.2+ 1209.4, Found 1209.4.
Example 1-14
[0242] (1-14A) Synthesis of
2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyacetatic acid ammonium salt (compound 1-14A: compound
of the following formula)
##STR00053##
[0243] The compound 1-2C (200 mg) was dissolved in distilled water
(2 ml). To the solution, 28 to 30% ammonia water (100 .mu.l) was
then added, and the mixture was lyophilized. The obtained amorphous
solid 1-14A was used without being purified.
[0244] (1-14B) Synthesis of SG-A (compound 1-14B: compound of the
following formula)
##STR00054##
[0245] Sialylglycopeptide (58 mg) was dissolved in a 0.2 M
phosphate buffer solution (pH 6.25) (254 .mu.l). To the solution,
an aqueous solution (100 .mu.l) of glycosynthase (Endo-M-N175Q,
Tokyo Chemical Industry Co., Ltd., 1 U/ml) was then added. The
compound 1-14A (24 mg) in a 0.2 M phosphate buffer solution (pH
6.25) (152 .mu.l) was further added thereto, and the mixture was
reacted at 28.degree. C. for 72 hours. The reaction was terminated
by the addition of a 0.2% aqueous trifluoroacetic acid solution
(3000 .mu.l) to the reaction solution, and the resulting product
was separated and purified by reverse-phase HPLC (Shiseido Co.,
Ltd., Proteonavi) using a 0.1% aqueous trifluoroacetic acid
solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain the title
compound SG-A (19.5 mg).
[0246] ESI-TOF-MS: Calcd for C.sub.86H.sub.140N.sub.6O.sub.64:
[M+2H].sup.2+ 1142.0 (ave.), Found 1141.4.
Example 1-15
[0247] (1-15A) Synthesis of di-tert-butyl
(25)-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]pentanedicarboxylate
(compound 1-15A: compound of the following formula)
##STR00055##
[0248] A solution of di-tert-butyl (2S)-2-aminopentanedicarboxylate
hydrochloride (295 mg, 1.00 mmol) in N,N-dimethylformamide (5.0 mL)
was cooled to 0.degree. C. N,N-Diisopropylethylamine (0.510 mL,
3.00 mmol) and (2,5-dioxopyrrolidin-1-yl)
3-(2,5-dioxopyrrol-1-yl)propanoate (293 mg, 1.10 mmol) were added
thereto in this order, and the mixture was stirred at 0.degree. C.
for 1 hour, further heated to room temperature, and stirred at room
temperature for 18 hours. Ethyl acetate was added to the reaction
solution. The organic layer was washed with 1 M hydrochloric acid
and saturated saline in this order, then dried over anhydrous
sodium sulfate, and filtered, and the solvent was distilled off
under reduced pressure to obtain a crude product. This product was
purified by silica gel column chromatography (hexane:ethyl
acetate=95:5-0:100, v/v) to obtain the title compound 1-15A as a
pale yellow oil (400 mg, yield: 98%).
[0249] .sup.1H-NMR (CDCl.sub.3) .delta.: 6.70 (2H, s), 6.22 (1H, d,
J=7.8 Hz), 4.48-4.42 (1H, m), 3.91-3.79 (2H, m), 2.60-2.52 (2H, m),
2.35-2.18 (2H, m), 2.13-2.04 (1H, m), 1.93-1.84 (1H, m), 1.46 (9H,
s), 1.44 (9H, s).
[0250] (1-15B) Synthesis of
(25)-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]pentanedicarboxylic
acid (compound 1-15B: compound of the following formula)
##STR00056##
[0251] The title compound 1-15B (260 mg, yield: 89%) was obtained
according to the same approach as in (1-9B) using the compound
1-15A (400 mg, 0.976 mmol).
[0252] MS (ESI): Calcd for C.sub.12H.sub.15N.sub.2O.sub.7:
[M+H].sup.+ 299, Found 299.
[0253] (1-15C) Synthesis of SG-(SG-Gln*)-Mal (compound 1-15C:
compound of the following formula)
##STR00057##
[0254] A solution of the compound 1-15B (30.0 mg, 0.101 mmol) and
N-hydroxysuccinimide (58.0 mg, 0.504 mmol) in dichloromethane
(0.400 mL) was cooled to 0.degree. C. Pyridine (0.200 mL, 2.48
mmol) and trifluoroacetic anhydride (70.0 .mu.L, 0.500 mmol) were
added thereto in this order, and the mixture was stirred at
0.degree. C. for 10 minutes. The reaction mixture was heated to
room temperature and further stirred at room temperature for 30
minutes. Dichloromethane was added to the reaction solution. The
organic layer was washed with 1 M hydrochloric acid, then dried
over anhydrous sodium sulfate, and filtered, and the solvent was
distilled off under reduced pressure to obtain a crude product.
This product was purified by silica gel column chromatography
(dichloromethane:ethyl acetate=25:75-0:100, v/v) to obtain a pale
yellow foam (25 mg).
[0255] Subsequently, an aliquot (2.00 mg) of the obtained product
was dissolved in N,N-dimethylformamide (200 .mu.L). The solution
was added to a solution of the compound SG-NH.sub.2 (20.0 mg, 8.41
.mu.mol) produced in (1-11B) and N,N-diisopropylethylamine (15
.mu.L, 88.0 .mu.mol) in N,N-dimethylformamide (600 .mu.L), and the
mixture was stirred at room temperature for 2 hours. A 0.2% aqueous
trifluoroacetic acid solution (2.0 mL) was added to the reaction
solution, and the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound SG-(SG-Gln*)-Mal (15.0 mg, yield:
76%).
[0256] ESI-TOF-MS: Calcd for C.sub.184H.sub.299N.sub.16O.sub.129:
[M+3H].sup.3+ 1599.7 (ave.), Found 1599.6.
Example 1-16
[0257] (1-16A) Synthesis of di-tert-butyl
(25)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]etho-
xy]ethoxy]ethoxy]propanoylamino]p entanedicarboxylate (compound
1-16A: compound of the following formula)
##STR00058##
[0258] A solution of di-tert-butyl (2S)-2-aminopentanedicarboxylate
hydrochloride (58.0 mg, 0.196 mmol) in N,N-dimethylformamide (1.0
mL) was cooled to 0.degree. C. N,N-Diisopropylethylamine (0.100 mL,
0.588 mmol) and (2,5-dioxopyrrolidin-1-yl)
3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethox-
y]ethoxy]propanoate (100 mg, 0.195 mmol) were added thereto in this
order, and the mixture was stirred at 0.degree. C. for 1 hour,
further heated to room temperature, and stirred at room temperature
for 20 hours. Ethyl acetate was added to the reaction solution. The
organic layer was washed with 1 M hydrochloric acid and saturated
saline in this order, then dried over anhydrous sodium sulfate, and
filtered, and the solvent was distilled off under reduced pressure
to obtain a crude product. This product was purified by silica gel
column chromatography (dichloromethane:methanol=98:2-90:10, v/v) to
obtain the title compound 1-16A as a pale yellow oil (100 mg,
yield: 78%).
[0259] .sup.1H-NMR (CDCl.sub.3) .delta.: 6.84 (1H, br s), 6.70 (2H,
s), 6.46 (1H, br s), 4.53-4.45 (1H, m), 3.85 (2H, t, J=7.2 Hz),
3.80-3.71 (2H, m), 3.68-3.60 (12H, m), 3.54 (2H, t, J=5.1 Hz), 3.42
(2H, q, J=5.1 Hz), 2.54-2.49 (4H, m), 2.37-2.21 (2H, m), 2.16-2.07
(1H, m), 1.92-1.83 (1H, m), 1.46 (9H, s), 1.43 (9H, s).
[0260] MS (ESI): Calcd for C.sub.31H.sub.52N.sub.3O.sub.12:
[M+H].sup.+ 658, Found 658.
[0261] (1-16B) Synthesis of
(2S)-2-[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]etho-
xy]ethoxy]ethoxy]propanoylamino]pentanedioic acid (compound 1-16B:
compound of the following formula)
##STR00059##
[0262] The title compound 1-16B (83 mg, yield: 100%) was obtained
according to the same approach as in (1-9B) using the compound 1-16
(100 mg, 0.152 mmol).
[0263] MS (ESI): Calcd for C.sub.23H.sub.36N.sub.3O.sub.12:
[M+H].sup.+ 546, Found 546.
[0264] (1-16C) Synthesis of SG-(SG-Gln*)-PEG(3)-Mal (compound
1-16C: compound of the following formula)
##STR00060##
[0265] A solution of the compound 1-16B (32.0 mg, 58.7 .mu.mol) and
N-hydroxysuccinimide (34.0 mg, 0.295 mmol) in dichloromethane
(0.400 mL) was cooled to 0.degree. C. Pyridine (0.200 mL, 2.48
mmol) and trifluoroacetic anhydride (42.0 .mu.L, 0.300 mmol) were
added thereto in this order, and the mixture was stirred at
0.degree. C. for 30 minutes. The reaction mixture was heated to
room temperature and further stirred at room temperature for 2
hours. Dichloromethane was added to the reaction solution. The
organic layer was washed with 1 M hydrochloric acid, then dried
over anhydrous sodium sulfate, and filtered, and the solvent was
distilled off under reduced pressure to obtain a crude product (35
mg).
[0266] Subsequently, an aliquot (0.31 mg) of the obtained crude
product was dissolved in N,N-dimethylformamide (20 .mu.L). The
solution was added to a solution of the compound SG-NH.sub.2 (2.0
mg, 0.88 .mu.mol) produced in (1-11B) and N,N-diisopropylethylamine
(1.5 .mu.L, 8.8 .mu.mol) in N,N-dimethylformamide (30 .mu.L), and
the mixture was stirred at room temperature for 18 hours. A 0.2%
aqueous trifluoroacetic acid solution (2.0 mL) was added to the
reaction solution, and the resulting product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents and
lyophilized to obtain the title compound SG-(SG-Gln*)-PEG(3)-Mal
(0.60 mg, yield: 28%).
[0267] ESI-TOF-MS: Calcd for C.sub.195H.sub.320N.sub.17O.sub.134:
[M+3H].sup.3+ 1682.2 (ave.), Found 1682.2.
Example 1-17
[0268] (1-17A) Synthesis of AG(9)-P (compound 1-17A: compound 1 of
the following formula)
##STR00061##
[0269] Sialylglycopeptide (200 mg) was dissolved in a 0.2 M acetate
buffer solution (pH 5.0) (1000 .mu.l). To the solution, an aqueous
solution (1000 .mu.l) of neuraminidase ([E.C.3.2.1.18], Nacalai
Tesque, Inc., 1 U/ml) was then added, and the mixture was reacted
at 37.degree. C. for 17 hours. After the completion of the
reaction, a 0.2% aqueous trifluoroacetic acid solution (2000 .mu.l)
was added thereto. Two lots of this reaction were combined, and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound AG(9)-P (307 mg).
[0270] ESI-TOF-MS: Calcd for C.sub.90H.sub.155N13054: [M+2H].sup.2+
1142.6 (ave.), Found 1142.0.
[0271] (1-17B) Synthesis of AG(7)-P (compound 1-17B: compound of
the following formula)
##STR00062##
[0272] The compound AG(9)-P (100 mg) produced in (1-17A), magnesium
sulfate (0.48 mg), and .beta.-D-galactosidase (Wako Pure Chemical
Industries, Ltd., 600 U/mg) (2 mg) were dissolved in a 0.2 M
phosphate buffer solution (pH 7.0) (2000 .mu.l), and the solution
was reacted at 37.degree. C. for 24 hours. .beta.-D-galactosidase
(Wako Pure Chemical Industries, Ltd., 600 U/mg) (1 mg) was added
thereto, and the mixture was further reacted for 24 hours. After
the completion of the reaction, a 0.2% aqueous trifluoroacetic acid
solution (2000 .mu.l) was added thereto. Two lots of this reaction
were combined, and the resulting product was separated and purified
by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a
0.1% aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound AG(7)-P (158 mg).
[0273] ESI-TOF-MS: Calcd for [M+H].sup.+
C.sub.78H.sub.135N.sub.13O.sub.44 1959.0 (ave.), Found 1958.9.
[0274] (1-17C) Synthesis of AG(5)-P (compound 1-17C: compound of
the following formula)
##STR00063##
[0275] The compound AG(7)-P (100 mg) produced in (1-17B) was
dissolved in a 0.2 M phosphate buffer solution (pH 6.25) (3150
.mu.l). To the solution, 100.times.BSA (New England BioLabs Japan
Inc.) (43 .mu.l) and .beta.-N-acetylglucosaminidase (New England
BioLabs Japan Inc., 4000 U/ml) (100 .mu.l) were added, and the
mixture was reacted at 37.degree. C. for 20 hours. After the
completion of the reaction, a 0.2% aqueous trifluoroacetic acid
solution (1000 .mu.l) was added thereto. The resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound AG(5)-P (73.8
mg).
[0276] ESI-TOF-MS: Calcd for [M+2H].sup.2+
C.sub.62H.sub.109N.sub.11O.sub.34 777.3 (ave.), Found 777.3.
Example 1-18
[0277] (1-18A) Synthesis of SG-N3 (compound 1-18A: compound of the
following formula)
##STR00064##
[0278] Sialylglycopeptide (76 mg) was dissolved in a 0.2 M
phosphate buffer solution (pH 6.25) (330 .mu.l). To the solution,
an aqueous solution (100 .mu.l) of glycosynthase (Endo-M-N175Q,
Tokyo Chemical Industry Co., Ltd., 1 U/ml) was then added.
N-[(2R,3R,4R,5S,6R)-3-Acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-
pyran-2-yl]oxyethylazide (23 mg) in a 0.2 M phosphate buffer
solution (pH 6.25) (230 .mu.l) was further added thereto, and the
mixture was reacted at 28.degree. C. for 96 hours. The reaction was
terminated by the addition of a 0.2% aqueous trifluoroacetic acid
solution (3000 .mu.l) to the reaction solution, and the resulting
product was separated and purified by reverse-phase HPLC (Shiseido
Co., Ltd., Proteonavi) using a 0.1% aqueous trifluoroacetic acid
solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain a solid composed
mainly of the title compound. Subsequently, the obtained solid was
dissolved in distilled water (3000 .mu.l), and the resulting
product was separated and purified by reverse-phase HPLC (GL
Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic
acid solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain the title
compound SG-N.sub.3 (34.4 mg).
[0279] ESI-TOF-MS: Calcd for C.sub.86H.sub.141N.sub.9O.sub.62:
[M+2H].sup.2+ 1147.5, Found 1147.4.
Example 1-19
[0280] (1-19A) Synthesis of tert-butyl
N-[2-[2-[2-[2-[3-[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydrox-
ymethyl)tetrahydropyran-2-yl]oxyethylamino]-3-oxo-propoxy]ethoxy]ethoxy]et-
hoxy]ethyl]carbamate (compound 1-19A: compound of the following
formula)
##STR00065##
[0281] The title compound 1-19A was obtained as a pale yellow foam
(150 mg, yield: 90%) according to the same method as in (1-9A)
using
3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propa-
noic acid (100 mg, 0.274 mmol) and the compound 1-7A (110 mg, 0.291
mmol).
[0282] MS (ESI): Calcd for C.sub.26H.sub.49N.sub.3O.sub.13:
[M+H].sup.+ 612, Found 612.
[0283] (1-19B) Synthesis of
N-[2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahy-
dropyran-2-yl]oxyethyl]-3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]pro-
panamide (compound 1-19B: compound of the following formula)
##STR00066##
[0284] The compound 1-19A (150 mg, 0.245 mmol) was dissolved in
dichloromethane (2.0 ml). To the solution, trifluoroacetic acid
(2.0 mL) was added, and the mixture was stirred at room temperature
for 2 hours. The solvent was distilled off under reduced pressure,
and the obtained residue was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound 1-19B (70 mg, 55%) as a colorless
oil.
[0285] MS (ESI): Calcd for C.sub.21H.sub.41N.sub.3O.sub.11:
[M+H].sup.+ 512, Found 512.
Example 1-20
[0286] (1-20A) Synthesis of
3-[2-[2-[2-[2-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]etho-
xy]ethoxy]ethoxy]ethoxy]propanoic acid (compound 1-20A: compound of
the following formula)
##STR00067##
[0287] To a solution of
(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid (210 mg, 607
.mu.mol) and HATU (220 mg, 579 .mu.mol) in N,N-dimethylformamide
(2.0 mL), N,N-diisopropylethylamine (0.410 mL, 2.41 mmol) was
added, and the mixture was stirred at room temperature for 2
minutes. The obtained reaction solution was added to a solution of
3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoic acid (150
mg, 497 .mu.mol) produced according to the approach of (1-5A) and
N,N-diisopropylethylamine (0.260 mL, 1.53 mmol) in
N,N-dimethylformamide (0.50 mL), and the mixture was stirred at
room temperature for 2 hours. The solvent was distilled off under
reduced pressure, and the obtained residue was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents and
lyophilized to obtain the title compound 1-20A (240 mg, 80%) as a
pale yellow oil.
[0288] MS (ESI): Calcd for C.sub.27H.sub.52N.sub.3O.sub.11:
[M+H].sup.+ 594, Found 594.
Example 1-21
[0289] (1-21A) Synthesis of
2-[[(2S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-amino-3-tert-butoxy-propanoyl]amino]-
acetyl]amino]-3-tert-butoxy-propanoyl]amino]acetyl]amino]-3-tert-butoxy-pr-
opanoyl]amino]acetic acid (compound 1-21A: compound of the
following formula)
##STR00068##
[0290] A 1.20 mmol/g 2-chlorotrityl chloride resin (166 mg, 0.200
mmol) was placed in a column for solid-phase synthesis.
Dichloromethane (3 mL) was added thereto, and the mixture was
shaken for 10 minutes. After filtration, a solution of
2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (119 mg, 0.400
mmol) and N,N-diisopropylethylamine (171 .mu.L, 1.00 mmol) in
dichloromethane (3 mL) was added thereto, and the mixture was
stirred at room temperature for 2 hours. After filtration, the
resin was washed with a dichloromethane mixed solution
(dichloromethane:methanol:N,N-diisopropylethylamine=85:10:5, v/v)
three times, dichloromethane three times, and N,N-dimethylformamide
three times. The obtained resin was loaded in a peptide synthesizer
(433A Peptide Synthesizer manufactured by Applied Biosystems, Inc.)
and subjected to deprotection, condensation, deprotection,
condensation, deprotection, condensation, deprotection,
condensation, deprotection, condensation, and deprotection in the
synthesizer to elongate the peptide chain. For the deprotection,
piperidine and N-methylpyrrolidone were used. For the condensation
reactions, HATU, N,N-diisopropylethylamine, N-methylpyrrolidone,
and various carboxylic acids were used. The carboxylic acids were
used in each condensation reaction in the order of
(2S)-3-tert-butoxy-2-(tert-butoxycarbonylamino)propanoic acid,
2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid,
(2S)-3-tert-butoxy-2-(tert-butoxycarbonylamino)propanoic acid,
2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid, and
(2S)-3-tert-butoxy-2-(tert-butoxycarbonylamino)propanoic acid. The
obtained resin was placed in a column for solid-phase synthesis. A
mixed solution of hexafluoroisopropanol (1 mL) and dichloromethane
(3 mL) was added thereto, and the mixture was shaken at room
temperature for 2 hours. The resin was filtered off, and the
obtained filtrate was concentrated under reduced pressure. The
concentrate was subjected to azeotropy with dichloromethane 6 times
and dried in a vacuum pump to obtain the title compound 1-21A as a
white solid (120 mg, yield: 97%).
[0291] MALDI-TOF-MS: Calcd for C.sub.27H.sub.50N.sub.6O.sub.10:
[M+H].sup.+ 619.4, Found 619.4.
[0292] (1-21B) Synthesis of
2-[[(2S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-4-[[(2R,3R,4R,5S,6R)-3-acetam-
ido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]amino]-2-[[2-[[(2R-
,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-
-yl]amino]acetyl]amino]-4-oxobutanoyl]amino]-3-tert-butoxy-propanoyl]amino-
]acetyl]amino]-3-tert-butoxy-propanoyl]amino]acetyl]amino]-3-tert-butoxy-p-
ropanoyl]amino]acetic acid (compound 1-21B: compound of the
following formula)
##STR00069##
[0293]
(2S)-4-[[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethy-
l)tetrahydropyran-2-yl]amino]-2-(tert-butoxycarbonylamino)-4-oxobutanoic
acid (84.5 mg, 0.194 mmol) produced according to the approach of J.
Am. Chem. Soc., 1999, 121, 284-290 and HATU (73.8 mg, 0.194 mmol)
were dissolved in N,N-dimethylformamide (5 mL). To the solution,
N,N-diisopropylethylamine (66 .mu.L, 0.388 mmol) was added, and the
mixture was stirred at room temperature for 3 minutes. This
solution was added to the compound 1-21A (100 mg, 0.162 mmol), and
the mixture was stirred at room temperature for 0.5 hours. The
reaction solution was diluted with water, and the resulting product
was separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents. To the obtained compound, a mixed solution of
trifluoroacetic acid (0.1 mL) and water (0.9 mL) was added, and the
mixture was stirred overnight. The reaction solution was diluted
with water, and the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents to obtain the
intermediate as a white solid (14.7 mg, 10%).
[0294] The compound 1-2C (5.91 mg, 0.0212 mmol) and HATU (8.04 mg,
0.0212 mmol) were dissolved in N,N-dimethylformamide (1 mL). To the
solution, N,N-diisopropylethylamine (9.05 .mu.L, 0.0529 mmol) was
added, and the mixture was stirred at room temperature for 3
minutes. This solution was added to a solution of the obtained
intermediate (16.5 mg, 0.0176 mmol) in N,N-dimethylformamide (1
mL), and the mixture was stirred at room temperature for 0.5 hours.
The reaction solution was diluted with water, and the resulting
product was separated and purified by reverse-phase HPLC (GL
Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic
acid solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents to obtain the title compound 1-21B as a
white solid (14.0 mg, yield: 66%).
[0295] MALDI-TOF-MS: Calcd for C.sub.49H.sub.85N.sub.11O.sub.23:
[M+H].sup.+ 1197.6, Found 1197.5.
Example 1-22
[0296] (1-22A) Synthesis of
(25)-2,6-bis[3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy-
]ethoxy]ethoxy]ethoxy]propanoylamino]hexanoic acid (compound 1-22A:
compound of the following formula)
##STR00070##
[0297] Lysine (26.0 mg, 0.178 mmol) was dissolved in a 0.10 M
phosphate buffer (pH 7.0) (0.40 ml). To the solution, a solution of
(2,5-dioxopyrrolidin-1-yl)
3-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethox-
y]ethoxy]propanoate (200 mg, 0.390 mmol) in N,N-dimethylformamide
(0.40 mL) was added, and the mixture was stirred at room
temperature for 3 hours. A 0.2% aqueous trifluoroacetic acid
solution (2.0 mL) was added to the reaction solution, and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound 1-22A (106 mg, yield: 63%).
[0298] MS (ESI): Calcd for C.sub.42H.sub.65N.sub.6O.sub.18:
[M-H].sup.- 941, Found 941.
[0299] (1-22B) Synthesis of SG-Lys*-[PEG(3)-Mal].sub.2 (compound
1-22B: compound of the following formula)
##STR00071##
[0300] To a solution of the compound 1-22A (10.0 mg, 10.6 .mu.mol)
thus produced, the compound SG-NH.sub.2 (19.4 mg, 8.13 .mu.mol)
produced in (1-11B), and HATU (4.00 mg, 10.6 .mu.mol) in
N,N-dimethylformamide (1.0 mL), N,N-diisopropylethylamine (8.80
.mu.L, 51.7 .mu.mol) was added, and the mixture was stirred at room
temperature for 1 hour. The reaction solution was added to a 0.5%
aqueous trifluoroacetic acid solution (6.0 mL), and the resulting
product was separated and purified by reverse-phase HPLC (GL
Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic
acid solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain the title
compound SG-Lys*-[PEG(3)-Mal].sub.2 (10.0 mg, yield: 25%).
[0301] ESI-TOF-MS: Calcd for C.sub.128H.sub.205N.sub.13O.sub.79:
[M-2H].sup.2- 1595.0 (ave.), Found 1595.0.
Example 1-23
[0302] (1-23A) Synthesis of
(25)-2,6-bis[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1--
yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-
ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]hexanoic acid (compound
1-23A: compound of the following formula)
##STR00072##
[0303] The title compound 1-23A was obtained as a colorless oil
(36.0 mg, yield: 41%) according to the same method as in (1-22A)
using lysine (7.70 mg, 52.7 .mu.mol) and (2,5-dioxopyrrolidin-1-yl)
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoyla-
mino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-
]ethoxy]ethoxy]propanoate (100 mg, 115 .mu.mol).
[0304] MS (ESI): Calcd for C.sub.74H.sub.129N.sub.6O.sub.34:
[M-H].sup.- 1645, Found 1645.
[0305] (1-23B) Synthesis of SG-Lys*-[PEG(11)-Mal].sub.2 (compound
1-23B: compound of the following formula)
[Formula 89]
[0306] The title compound SG-Lys*-[PEG(11)-Mal]2 was obtained as a
colorless oil (14.0 mg, yield: 34%) according to the same method as
in (1-22B) using the compound 1-23A (19.0 mg, 11.5 .mu.mol) and the
compound SG-NH.sub.2 (25.0 mg, 10.5 .mu.mol) produced in
(1-11B).
[0307] ESI-TOF-MS: Calcd for C.sub.180H.sub.269N.sub.13O.sub.95:
[M-2H].sup.2- 1947.4 (ave.), Found 1947.3.
Example 1-24
[0308] (1-24A) Synthesis of benzyl
2-[[2-[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-
-2-yl]oxyacetic acid]amino]acetate (compound 1-24A: reaction
product of the following formula)
##STR00073##
[0309] A known compound
2-[[2-[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-
-2-yl]oxyacetic acid (Tetrahedron Asymmetry, 2008, 19, 1919-1933)
(670 mg) was dissolved in DMF (6 ml). To the solution, HATU (630
mg) and DIPEA (0.57 ml) were added, and the mixture was stirred at
room temperature for 4 minutes. Then, glycine benzyl ester
hydrochloride (370 mg) was added thereto, and the mixture was
stirred at room temperature for 1 hour. The reaction solution was
diluted with ethyl acetate and washed with 10% saline twice and 1 N
hydrochloric acid once. After drying over anhydrous sodium sulfate
and filtration, the solvent was distilled off under reduced
pressure to obtain a crude product. This product was purified by
silica gel column chromatography (hexane:ethyl acetate=60:40-20:80,
v/v) to obtain the title compound 1-24A (840 mg, yield: 62%) in an
amorphous form.
[0310] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.38-7.35 (5H, m),
6.99-6.98 (1H, br m), 5.23-5.21 (3H, m), 5.11-5.04 (2H, m), 4.56
(1H, d, J=7.8 Hz), 4.34 (1H, d, J=15.1 Hz), 4.20-4.09 (5H, m),
3.73-3.71 (1H, m), 2.08 (3H, s), 2.07 (3H, s), 2.04 (3H, s), 2.03
(3H, s).
[0311] ESI-LC-MS: Calcd for C.sub.25H.sub.31NO.sub.13: [M+H].sup.+
554, Found 554.
[0312] (1-24B) Synthesis of
2-[[2-[(2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-
-2-yl]oxyacetic acid]amino]acetic acid (compound 1-24B: reaction
product of the following formula)
##STR00074##
[0313] The title compound 1-24B (700 mg, yield: quant.) was
obtained in an amorphous form according to the same approach as in
(1-2B) using the compound 1-24A (840 mg).
[0314] ESI-LC-MS: Calcd for C.sub.18H.sub.25NO.sub.13: [M-H].sup.-
462, Found 462.
[0315] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.05-7.04 (1H, br m), 5.24
(1H, t, J=9.5 Hz), 5.12-5.05 (2H, m), 4.58 (1H, d, J=7.8 Hz), 4.35
(1H, d, J=15.6 Hz), 4.27-4.05 (6H, m), 3.76-3.75 (1H, m), 2.08-2.04
(12H, m).
[0316] (1-24C) Synthesis of
2-[[2-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-
-2-yl]oxyacetic acid]amino]acetic acid (compound 1-24C: reaction
product of the following formula)
##STR00075##
[0317] The title compound 1-24C (286 mg, yield: quant.) was
obtained in an amorphous form according to the same approach as in
(1-2C) using the compound 1-24B (450 mg). This compound was used
directly in the next reaction.
[0318] .sup.1H-NMR (D.sub.2O, TMSP) 6:4.54 (1H, d, J=7.8 Hz), 4.43
(1H, d, J=15.6 Hz), 4.31 (1H, d, J=15.6 Hz), 3.91-3.89 (3H, m),
3.73 (1H, dd, J=12.2, 5.4 Hz), 3.53-3.37 (4H, m).
[0319] ESI-LC-MS: Calcd for C.sub.10H.sub.27NO.sub.9: [M-H].sup.-
294, Found 294.
[0320] (1-24D) Synthesis of
2-[[2-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-
-2-yl]oxyacetic acid]amino]acetic acid ammonium salt (compound
1-24D: compound of the following formula)
##STR00076##
[0321] The title compound 1-24D (325 mg) was obtained in an
amorphous form according to the same approach as in (1-14A) using
the compound 1-24C (286 mg). This compound was used without being
purified.
[0322] (1-24E) Synthesis of SG(Glc)-Gly-A (compound 1-24E: compound
of the following formula)
##STR00077##
[0323] Sialylglycopeptide (191 mg) was dissolved in a 0.2 M
phosphate buffer solution (pH 6.25) (1000 .mu.l). To the solution,
an aqueous solution (300 .mu.l) of glycosynthase (Endo-M-N175Q,
Tokyo Chemical Industry Co., Ltd., 1 U/ml) was then added. The
compound 1-24D (125 mg) in a 0.2 M phosphate buffer solution (pH
6.25) (370 .mu.l) was further added thereto, and the mixture was
reacted at 28.degree. C. for 72 hours. The reaction was terminated
by the addition of a 0.2% aqueous trifluoroacetic acid solution
(3000 .mu.l) to the reaction solution, and the resulting product
was separated and purified by reverse-phase HPLC (Inertsil ODS-3,
GL Sciences Inc.) using a 0.1% aqueous trifluoroacetic acid
solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain the title
compound SG(Glc)-Gly-A (88 mg).
[0324] ESI-TOF-MS: Calcd for C.sub.86H.sub.140N.sub.6O.sub.65:
[M+4H].sup.4+ 1149.4 (ave.), Found 1149.4.
[0325] Also, a compound derived from the compound 1-24D by the
replacement of the sugar structure with a sugar other than Glc
(e.g., Man or Gal) can be synthesized with reference to the
reaction of Example 1-24. A glycochain altered at the reducing end
of SG(Man)-Gly, SG(Gal)-Gly, or the like can be synthesized through
the same transglycosylation reaction as in (1-24E) by use of the
compound thus synthesized as an acceptor compound.
[0326] In addition, a glycochain altered at the reducing end of
SG-NH2, SG-I, SG-oxa, SG-A, or the like can be appropriately
synthesized by the appropriate conversion of the sugar structure of
the starting material (acceptor compound) to a desired one with
reference to the reaction of Example 1-24 in the methods of
Examples 1-11, 1-12, 1-13, and 1-14.
Example 2
[0327] Hereinafter, the simple term "hANP" in a structural formula
represents that the hANP peptide in the modified peptide is
hANP(1-28).
<Example 2-1> Synthesis of SG-hANP(1-28) (compound 2-1)
[0328] (2-1A) Preparation of hANP-TFA salt (trifluoroacetate)
[0329] The hANP-TFA salt used in the reactions given below was
prepared according to the following procedures: Preparation Method
1
[0330] Carperitide acetate (hANP(1-28) acetate) (100 mg) was
dissolved in distilled water (4000 .mu.l), and the resulting
product was separated and purified by reverse-phase HPLC (GL
Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic
acid solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain the title
compound (89.6 mg).
[0331] Preparation Method 2
[0332] Carperitide acetate (hANP(1-28) acetate) (250 mg) was
dissolved in distilled water (30 ml). To the solution,
trifluoroacetic acid (600 .mu.l) was added, and the mixture was
lyophilized. This compound was used directly without being further
purified.
[0333] (2-1B) Synthesis of SG-hANP (1-28) (compound 2-1: compound
of the following formula)
##STR00078##
[0334] (2-1B-1) Synthesis of SG-hANP(1-28) (compound 2-1) TFA
salt
[0335] To a solution of the compound SG-A (97.7 mg) synthesized in
(1-14B) in N,N-dimethylformamide (1000 .mu.l), a solution of HATU
(16.3 mg) in N,N-dimethylformamide (1000 .mu.l) was added, then
diisopropylethylamine (30 .mu.l) was added, and the mixture was
stirred at room temperature for 5 minutes and immediately used in
the next reaction.
[0336] The hANP-TFA salt (100 mg) prepared according to the
procedures of (2-1A) was dissolved in N,N-dimethylformamide (1200
.mu.l) and distilled water (320 .mu.l). To the solution,
diisopropylethylamine (22.5 .mu.l) was added. To this solution, a
solution containing active ester prepared beforehand in
N,N-dimethylformamide (2000 .mu.l) was added, and the mixture was
stirred for 1 hour. After the completion of the reaction, a 0.2%
aqueous trifluoroacetic acid solution (20 ml) was added thereto
under ice cooling. Insoluble matter was dissolved by the addition
of acetic acid (2 ml), and the resulting product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents and
lyophilized to obtain the title compound SG-hANP(1-28) (compound
2-1)-TFA salt (91.0 mg).
[0337] MALDI-TOF-MS: Calcd for
C.sub.213H.sub.341N.sub.51O.sub.102S.sub.3: [M+H].sup.+ 5342.2,
Found 5342.2.
[0338] (2-1B-2) Synthesis of SG-hANP(1-28) (Compound 2-1)
Acetate
[0339] To a solution of the compound SG-A (790 mg) synthesized in
(1-14B) in N,N-dimethylformamide (18 ml), a solution of TSTU
(O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate) (104 mg) in N,N-dimethylformamide (2 ml) was
added, then diisopropylethylamine (241 .mu.l) was added, and the
mixture was stirred at room temperature for 60 minutes and used in
the next reaction.
[0340] Carperitide acetate (hANP(1-28) acetate) (1000 mg) was
dissolved in N,N-dimethylformamide (12 ml) and distilled water (3.2
ml). To the solution, diisopropylethylamine (241 .mu.l) was added.
To this solution, a solution containing active ester prepared
beforehand in N,N-dimethylformamide (20 ml) was added, and the
mixture was stirred for 1 hour. After the completion of the
reaction, acetonitrile (32 ml) was added thereto, and the
precipitates were collected by filtration. After washing with
N,N-dimethylformamide/acetonitrile (1/1) (30 ml) and acetonitrile
(100 ml), the obtained solid matter was dried under reduced
pressure. This solid matter was dissolved in distilled water, and
the resulting product was separated and purified by reverse-phase
HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous acetic
acid solution and a 0.1% solution of acetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound SG-hANP(1-28)
(compound 2-1)-acetate (1056 mg).
[0341] ESI-TOF-MS: Calcd for
C.sub.213H.sub.341N.sub.51O.sub.102S.sub.3: [M+4H].sup.4+ 1337.1
(ave.), Found 1337.0.
[0342] As mentioned above, the modified peptide of interest is also
synthesized as a salt of a type corresponding to the salt of the
hANP peptide used as a starting material. In the Examples below,
the TFA salt of the hANP peptide was adopted, and the modified
peptide of interest was obtained as a TFA salt, unless otherwise
specified. In these cases, the type of the salt is not particularly
described. All title compounds can be synthesized as acetates by
synthesis according to the procedures of (2-1B-2).
<Example 2-2> Synthesis of hANP(1-28)-SG (compound 2-2)
[0343] (2-2A) Synthesis of Boc-hANP(1-28) (compound of the
following formula)
##STR00079##
[0344] The hANP(1-28)-acetate (62.5 mg) was dissolved in distilled
water (1.3 ml). To the solution, a solution of di-t-butyl
dicarbonate (0.9 mg, 324.6 .mu.mol) in t-butyl alcohol (400 .mu.l)
was added at room temperature, then an aqueous solution (200 .mu.l)
of triethylamine (13.6 .mu.l) was added, and the mixture was
stirred at room temperature for 3 hours. Insoluble matter was
dissolved by the addition of distilled water (6 ml), acetonitrile
(2 ml), and acetic acid (1 ml), and the resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound Boc-hANP(1-28)
(59.9 mg).
[0345] MALDI-TOF-MS: Calcd for
C.sub.132H.sub.211N.sub.45O.sub.41S.sub.3: [M+H].sup.+ 3179.5,
Found 3179.7
[0346] (2-2B) Synthesis of Boc-hANP(1-28)-GlcNAc (compound of the
following formula)
##STR00080##
[0347] The Boc-hANP(1-28) (59.9 mg) produced in (2-2A) and the
compound 1-7A (59.7 mg) were dissolved in distilled water (0.2 ml).
To the solution, a solution of HATU (35.8 mg) in dimethylformamide
(2.0 ml) and triethylamine (15.8 .mu.l) were added at room
temperature, and the mixture was stirred at room temperature for 2
hours. The reaction solution was added to an ice-cold aqueous
solution (5 ml) of trifluoroacetic acid (8.7 .mu.l), and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound Boc-hANP(1-28)-GlcNAc (38.7 mg).
[0348] MALDI-TOF-MS: Calcd for
C.sub.142H.sub.229N.sub.47O.sub.46S.sub.3: [M+H].sup.+ 3425.6,
Found 3426.0.
[0349] (2-2C) Synthesis of hANP(1-28)-GlcNAc (compound of the
following formula)
##STR00081##
[0350] The Boc-hANP(1-28)-GlcNAc (38.7 mg) produced in (2-2B) was
dissolved in a 20% aqueous trifluoroacetic acid solution (5 ml) and
acetic acid (1 ml), and the solution was left standing at room
temperature for 7 hours. Distilled water (10 ml) was added thereto,
and the mixture was lyophilized. The resulting crude product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound
hANP(1-28)-GlcNAc (21.8 mg).
[0351] MALDI-TOF-MS: Calcd for
C.sub.137H.sub.221N.sub.47O.sub.44S.sub.3: [M+H].sup.+ 3325.6,
Found 3425.5.
[0352] (2-2D) Synthesis of hANP(1-28)-SG (compound 2-2: compound of
the following formula)
##STR00082##
[0353] To the compound SG-Oxa produced in (1-12A) in a 0.2 M
phosphate buffer solution (60 mM, 120 .mu.l), glycosynthase
(Endo-M-N175Q, Tokyo Chemical Industry Co., Ltd., 1 U/ml, 48 .mu.l)
was added at room temperature, then a solution of the
hANP(1-28)-GlcNAc (6.0 mg, 1.8 .mu.mol) produced in (2-2C) in
dimethyl sulfoxide (72 .mu.l) was added in two portions at an
interval of 15 minutes at room temperature, and the mixture was
shaken at 25.degree. C. for 2 hours. The reaction was terminated by
the addition of a 0.2% aqueous trifluoroacetic acid solution (1.5
ml) at room temperature, and the resulting product was separated
and purified by reverse-phase HPLC (GL Sciences Inc., Inertsil
ODS-3) using a 0.1% aqueous trifluoroacetic acid solution and a
0.1% solution of trifluoroacetic acid in acetonitrile as eluents
and lyophilized to obtain the title compound hANP(1-28)-SG
(compound 2-2) (6.2 mg).
[0354] MALDI-TOF-MS: Calcd for
C.sub.213H.sub.334N.sub.52O.sub.100S.sub.3: [M+H].sup.+ 5327.3,
Found 5326.7.
<Example 2-3> Synthesis of (SG-)Asn-hANP(1-28) (compound
2-3)
[0355] (2-3A) Synthesis of Boc-(GlcNAc-)Asn-hANP(1-28) (compound of
the following formula)
##STR00083##
[0356] The title compound Boc-(GlcNAc-)Asn-hANP(1-28) (13.0 mg) was
obtained according to the same approach as in (2-1B) using
(2S)-4-[[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetr-
ahydropyran-2-yl]amino]-2-(tert-butoxycarbonylamino)-4-oxobutanoic
acid (42.4 mg, 0.0976 mmol) synthesized according to the
description of J. Am. Chem. Soc., 1999, 121, 284-290 and
hANP(1-28)-TFA salt prepared from the hANP(1-28)-acetate (31.3 mg)
by Preparation Method 2 of (2-1A).
[0357] MALDI-TOF-MS: Calcd for
C.sub.144H.sub.230N.sub.48O.sub.48S.sub.3: [M+H].sup.+ 3495.6,
Found 3496.5.
[0358] (2-3B) Synthesis of (GlcNAc-)Asn-hANP(1-28) (compound of the
following formula)
##STR00084##
[0359] The title compound (GlcNAc-)Asn-hANP(1-28) (5.83 mg) was
obtained according to the same approach as in (2-2C) from the
Boc-(GlcNAc-)Asn-hANP(1-28) (13.0 mg) produced in (2-3A).
[0360] MALDI-TOF-MS: Calcd for
C.sub.139H.sub.222N.sub.48O.sub.46S.sub.3: [M+H].sup.+ 3396.6,
Found 3396.6.
[0361] (2-3C) Synthesis of (SG-)Asn-hANP(1-28) (compound of the
following formula: compound 2-3)
##STR00085##
[0362] The title compound (GlcNAc-)Asn-hANP(1-28) (compound 2-3)
(2.17 mg) was obtained according to the same approach as in (2-2D)
from the (GlcNAc-)Asn-hANP(1-28) (4.90 mg) produced in (2-3B).
[0363] MALDI-TOF-MS: Calcd for
C.sub.215H.sub.345N.sub.53O.sub.102S.sub.3: [M+H].sup.+ 5398.3,
Found 5398.4.
<Example 2-4> Synthesis of (SG-)Asn-hANP(2-28) (compound
2-4)
[0364] (2-4A) Preparation of hANP(2-28)
[0365] The hANP(1-28)-acetate (100 mg) was dissolved in a mixed
solution of 1.5% dimethylallylamine, 60% pyridine, and 38.5% water
(10.4 mL). To the solution, phenyl isothiocyanate (1.04 mL) was
added at room temperature, and the mixture was stirred at
50.degree. C. for 30 minutes. The reaction mixture was cooled to
room temperature, and water and acetic acid were added thereto.
After washing with benzene three times, the aqueous layer was
lyophilized. Trifluoroacetic acid (2.6 mL) was added thereto, and
the mixture was stirred at 50.degree. C. for 30 minutes. Then,
trifluoroacetic acid was distilled off. To the residue, water and
acetic acid were added. After washing with benzene three times, the
aqueous layer was lyophilized. The dried product was dissolved in
water and acetic acid, and the resulting product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents to
obtain the title compound ANP(2-28) as a white solid (50 mg).
[0366] MALDI-TOF-MS: Calcd for
C.sub.124H.sub.198N.sub.44O.sub.37S.sub.3: [M+H].sup.+ 2992.4,
Found 2992.0.
[0367] (2-4B) Synthesis of Boc-(GlcNAc-)Asn-hANP(2-28) (compound of
the following formula)
##STR00086##
[0368] The title compound Boc-(GlcNAc-)Asn-hANP(2-28) (13.0 mg) was
obtained according to the same approach as in (2-3A) using the
hANP(2-28) (25.0 mg) produced in (2-4A) instead of the
hANP(1-28).
[0369] MALDI-TOF-MS: Calcd for
C.sub.141H.sub.225N.sub.47O.sub.46S.sub.3: [M+H].sup.+ 3409.6,
Found 3409.5.
[0370] (2-4C) Synthesis of (GlcNAc-)Asn-hANP(2-28) (compound of the
following formula)
##STR00087##
[0371] The title compound (GlcNAc-)Asn-hANP(2-28) (6.86 mg) was
obtained according to the same approach as in (2-2C) from the
Boc-(GlcNAc-)Asn-hANP(2-28) (13.0 mg) produced in (2-4B).
[0372] MALDI-TOF-MS: Calcd for
C.sub.136H.sub.217N.sub.47O.sub.44S.sub.3: [M+H].sup.+ 3309.5,
Found 3309.5.
[0373] (2-4D) Synthesis of (SG-)Asn-hANP(2-28) (compound 2-4:
compound of the following formula)
##STR00088##
[0374] The title compound (SG-)Asn-hANP(2-28) (compound 2-4) (3.80
mg) was obtained according to the same approach as in (2-2D) from
the (GlcNAc-)Asn-hANP(2-28) (5.75 mg) produced in (2-4C).
[0375] MALDI-TOF-MS: Calcd for
C.sub.212H.sub.340N.sub.52O.sub.100S.sub.3: [M+H].sup.+ 5313.4
(ave.), Found 5314.7.
<Example 2-5> Synthesis of (SG-)Ser-hANP(2-28) (compound
2-5)
[0376] (2-5A) Synthesis of Boc-(GlcNAc-)Ser-hANP(2-28) (compound of
the following formula)
##STR00089##
[0377] The title compound Boc-(GlcNAc-)Ser-hANP(2-28) (13.0 mg) was
obtained according to the same approach as in (2-3A) using the
compound 1-1D (40.8 mg) and the hANP(2-28) (25.0 mg) produced in
(2-4A).
[0378] MALDI-TOF-MS: Calcd for
C.sub.140H.sub.224N.sub.46O.sub.46S.sub.3: [M+H].sup.+ 3382.6,
Found 3382.7.
[0379] (2-5B) Synthesis of (GlcNAc-)Ser-hANP(2-28) (compound of the
following formula)
##STR00090##
[0380] The title compound (GlcNAc-)Ser-hANP(2-28) (6.36 mg) was
obtained by the removal of Boc according to the same approach as in
(2-2C) using the Boc-(GlcNAc-)Ser-hANP(2-28) (13.0 mg) produced in
(2-5A).
[0381] MALDI-TOF-MS: Calcd for
C.sub.135H.sub.216N.sub.46O.sub.44S.sub.3: [M+H].sup.+ 3282.5,
Found 3282.6.
[0382] (2-5C) Synthesis of (SG-Ser)-hANP(2-28) (compound 2-5:
compound of the following formula)
##STR00091##
[0383] The title compound (SG-)Ser-hANP(2-28) (compound 2-5) (4.40
mg) was obtained according to the same approach as in (2-2D) using
the (GlcNAc-)Ser-hANP(2-28) (5.47 mg) produced in (2-5B).
[0384] MALDI-TOF-MS: Calcd for
C.sub.211H.sub.339N.sub.51O.sub.100S.sub.3: [M+H].sup.+ 5284.2,
Found 5284.4.
<Example 2-6> Synthesis of hANP(1-27)-(SG-)Tyr (compound
2-6)
[0385] (2-6A) Synthesis of hANP(1-27)-(GlcNAc-)Tyr (compound of the
following formula)
##STR00092##
[0386] The compound 1-6D (30.6 mg) was dissolved in
dimethylformamide (200 .mu.l). To the solution, a solution of
N-bromosuccinimide (11.5 mg) and pyridine (5.2 .mu.l) in
dimethylformamide (125 .mu.l) was added at 0.degree. C., and the
mixture was stirred for 5 minutes.
[0387] The hANP(1-28)-acetate (50.0 mg) was dissolved in a 0.1 M
phosphate buffer solution (pH 7.0, 1 ml). To the solution, a
solution of a triazoledione derivative prepared in advance in
dimethylformamide (325 .mu.l) was added at 0.degree. C. The mixture
was left standing at room temperature for 5 hours, and then, the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound hANP(1-27)-(GlcNAc-) Tyr (9.8 mg).
[0388] MALDI-TOF-MS: Calcd for
C.sub.145H.sub.225N.sub.49O.sub.47S.sub.3: [M+H].sup.+ 3501.6,
Found 3501.6.
[0389] (2-6B) Synthesis of hANP(1-27)-(SG-)Tyr (compound 2-6:
compound of the following formula)
##STR00093##
[0390] The title compound hANP(1-27)-(SG-)Tyr (compound 2-6) (6.3
mg) was obtained according to the same approach as in (2-2D) using
the hANP(1-27)-(GlcNAc-)Tyr (8.3 mg) produced in (2-6A).
[0391] MALDI-TOF-MS: Calcd for
C.sub.221H.sub.348N.sub.54O.sub.103S.sub.3: [M+H].sup.+ 5503.3,
Found 5502.8.
<Example 2-7> Synthesis of SG-hANP(1-28)-SG (compound
2-7)
[0392] (2-7A) Synthesis of GlcNAc-hANP(1-28) (compound of the
following formula)
##STR00094##
[0393] The compound 1-2C (25.0 mg) was dissolved in
dimethylformamide (0.5 ml). To the solution, triethylamine (34 ml)
was added at room temperature, then a solution of
dimethylthiophosphinoyl chloride (12.0 mg) in dimethylformamide
(0.5 ml) was added under ice cooling, and then the mixture was
stirred at room temperature for 1 hour. Meanwhile,
hANP(1-28)-acetate (25 mg) was dissolved in dimethylformamide (1
ml) and distilled water (0.32 ml). To the solution, triethylamine
(25.3 .mu.l) was added at room temperature, then a solution of
activated ester prepared in advance in dimethylformamide (433
.mu.l) was added under ice cooling, and the mixture was stirred at
room temperature for 2 hours. The reaction solution was added to an
ice-cold 0.5% aqueous trifluoroacetic acid solution (5 ml), and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound GlcNAc-hANP(1-28) (17.8 mg).
[0394] MALDI-TOF-MS: Calcd for
C.sub.137H.sub.218N.sub.46O.sub.46S.sub.3: [M+H].sup.+ 3340.5,
Found 3340.5.
[0395] (2-7B) Synthesis of GlcNAc-hANP(1-28)-GlcNAc (compound of
the following formula)
##STR00095##
[0396] The title compound GlcNAc-hANP(1-28)-GlcNAc (10.0 mg) was
obtained by the linking of GlcNAc to the C terminus of hANP
according to the same approach as in (2-2B) using the
GlcNAc-hANP(1-28) (30.0 mg) produced in (2-7A) and the compound
1-7A.
[0397] MALDI-TOF-MS: Calcd for
C.sub.147H.sub.236N.sub.48O.sub.51S.sub.3: [M+H].sup.+ 3586.7,
Found 3586.7.
[0398] (2-7C) Synthesis of SG-hANP(1-28)-SG (compound 2-7: compound
of the following formula)
##STR00096##
[0399] The title compound SG-hANP(1-28)-SG (compound 2-7) (13.0 mg)
was obtained according to the same approach as in (2-2D) using the
GlcNAc-hANP(1-28)-GlcNAc (6.0 mg) produced in (2-7B).
[0400] MALDI-TOF-MS: Calcd for
C.sub.299H.sub.482N.sub.58O.sub.163S.sub.3: [M+H].sup.+ 7590.0,
Found 7589.0.
<Example 2-8> Synthesis of (SG-)Asn-hANP(3-28) (compound
2-8)
[0401] (2-8A) Synthesis of (GlcNAc-)Asn-hANP(3-28) (compound of the
following formula)
##STR00097##
[0402] The N-terminal amino acid of the hANP(2-28) (32.0 mg)
produced in (2-4A) was removed by the method of (2-4A), again, to
obtain hANP(3-28). The title compound (GlcNAc-)Asn-hANP(3-28) (3.5
mg) was obtained according to the same approach as in Example 2-4
using the obtained hANP(3-28) instead of hANP(2-28).
[0403] MALDI-TOF-MS: Calcd for
C.sub.130H.sub.206N.sub.46O.sub.43S.sub.3: [M+H].sup.+ 3196.5,
Found 3196.7.
[0404] (2-8B) Synthesis of (SG-)Asn-hANP(3-28) (compound of the
following formula: compound 2-8)
##STR00098##
[0405] The title compound (SG-)Asn-hANP(3-28) (compound 2-8) ((2.5
mg) was obtained according to the same approach as in (2-2D) using
the GlcNAc-Asn-hANP(3-28) (3.5 mg) produced in (2-8A).
[0406] MALDI-TOF-MS: Calcd for
C.sub.206H.sub.329N.sub.51O.sub.99S.sub.3: [M+H].sup.+ 5198.1,
Found 5198.3.
<Example 2-9> Synthesis of SG-(SG-)Asn-hANP(1-28) (compound
2-9)
[0407] (2-9A) Synthesis of GlcNAc-(GlcNAc-)Asn-hANP(1-28) (compound
of the following formula)
##STR00099##
[0408] The title compound GlcNAc-(GlcNAc-)Asn-hANP(1-28) (6.15 mg)
was obtained by the linking of GlcNAc to the amino group of Asp
according to the same approach as in (1-5B) using the
(GlcNAc-)Asn-hANP(1-28) (16.0 mg) produced in (2-3B).
[0409] MALDI-TOF-MS: Calcd for
C.sub.149H.sub.237N.sub.49O.sub.53S.sub.3: [M+H].sup.+ 3657.7,
Found 3658.1.
[0410] (2-9B) Synthesis of SG-(SG-)Asn-hANP(1-28) (compound 2-9:
compound of the following formula)
##STR00100##
[0411] The title compound SG-(SG-)Asn-hANP(1-28) (compound 2-9)
(6.7 mg) was obtained by the same approach as in 2-2D using the
compound SG-Oxa produced in (1-12A) in a 0.2 M phosphate buffer
solution (60 mM, 168 .mu.l), glycosynthase (Endo-M-N175Q, Tokyo
Chemical Industry Co., Ltd., 1 U/ml, 67 .mu.l), and the
GlcNAc-(GlcNAc-)Asn-hANP(1-28) (6.15 mg) produced in (2-9A).
[0412] MALDI-TOF-MS: Calcd for
C.sub.301H.sub.483N.sub.59O.sub.165S.sub.3: [M+H].sup.+ 7661.0,
Found 7660.7.
<Example 2-10> Synthesis of AG(9)-hANP(1-28) (compound
2-10)
[0413] (2-10A) Synthesis of AG(9)-hANP(1-28) (compound 2-10:
compound of the following formula)
##STR00101##
[0414] The SG-hANP(1-28) (21 mg) synthesized in (2-1B) was
dissolved in a 0.2 M acetate buffer solution (pH 5.0) (1000 .mu.l).
To the solution, an aqueous solution (1000 .mu.l) of neuraminidase
([E.C.3.2.1.18], Nacalai Tesque, Inc., 1 U/ml) was then added, and
the mixture was reacted at 37.degree. C. for 17 hours. After the
completion of the reaction, a 0.2% aqueous trifluoroacetic acid
solution (2000 .mu.l) was added thereto. Insoluble matter was
dissolved by the addition of acetic acid (200 .mu.l). Two lots of
this reaction solution were combined, and the resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound
AG(9)-hANP(1-28) (compound 2-10) (33.8 mg).
[0415] MALDI-TOF-MS: Calcd for
C.sub.191H.sub.307N.sub.49O.sub.86S.sub.3: [M+H].sup.+ 4760.0,
Found 4760.4.
<Example 2-11> Synthesis of AG(7)-hANP(1-28)
[0416] (2-11A) Synthesis of AG(7)-hANP(1-28) (compound 2-11:
compound of the following formula)
##STR00102##
[0417] The AG(9)-hANP(1-28) (16 mg) synthesized in (2-10A) was
dissolved in distilled water (1425 .mu.l). To the solution, a 0.2 M
phosphate buffer solution (pH 6.25) (1500 .mu.l) and an aqueous
solution (75 .mu.l) of .beta.1-4 galactosidase (New England BioLabs
Japan Inc., 8000 U/ml) were then added, and the mixture was reacted
at 37.degree. C. for 24 hours. After the completion of the
reaction, a 0.2% aqueous trifluoroacetic acid solution (3000 ul)
was added thereto. Insoluble matter was dissolved by the addition
of acetic acid (300 .mu.l), and the resulting product was separated
and purified by reverse-phase HPLC (GL Sciences Inc., Inertsil
ODS-3) using a 0.1% aqueous trifluoroacetic acid solution and a
0.1% solution of trifluoroacetic acid in acetonitrile as eluents
and lyophilized to obtain the title compound AG(7)-hANP(1-28)
(compound 2-11) (11.7 mg).
[0418] MALDI-TOF-MS: Calcd for
C.sub.179H.sub.287N.sub.49O.sub.76S.sub.3: [M+H].sup.+ 4435.9,
Found 4435.8.
<Example 2-12> Synthesis of SG-triazole-hANP(1-28) (compound
2-12)
[0419] (2-12A) Synthesis of Pentynoyl-hANP(1-28) (compound of the
following formula)
##STR00103##
[0420] The title compound Pentynoyl-hANP(1-28) (26.0 mg) was
obtained according to the same method as in (2-7A) using the
hANP(1-28)-acetate (50.0 mg) and 4-pentynoic acid (10.0 mg, 101
.mu.mol).
[0421] MALDI-TOF-MS: Calcd for
C.sub.132H.sub.207N.sub.45O.sub.40S3: [M].sup.+ 3158.4, Found
3158.0.
[0422] (2-12B) Synthesis of GlcNAC-triazole-hANP(1-28) (compound of
the following formula)
##STR00104##
[0423] To the Pentynoyl-hANP(1-28) (22.0 mg) produced in (2-12A), a
30 mM aqueous
N-[(2R,3R,4R,5S,6R)-2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymet-
hyl)tetrahydropyran-3-yl]acetamide solution (0.37 mL, 11.1
.mu.mol), a 30 mM aqueous sodium ascorbate solution (0.26 mL, 7.8
.mu.mol), a 10 mM aqueous copper sulfate solution (0.15 mL, 1.5
.mu.mol), and a 0.1 M phosphate buffer (pH 7.0) were added in this
order, and the mixture was stirred at room temperature for 4 hours.
A 0.2% aqueous trifluoroacetic acid solution (12 mL) was added to
the reaction solution, and the resulting product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents and
lyophilized to obtain the title compound GlcNAc-triazole-hANP(1-28)
(15.0 mg).
[0424] MALDI-TOF-MS: Calcd for
C.sub.142H.sub.225N.sub.49O.sub.46S.sub.3: [M+H].sup.+ 3449.6,
Found 3449.6.
[0425] (2-12C) Synthesis of SG-triazole-hANP(1-28) (compound 2-12:
compound of the following formula)
##STR00105##
[0426] The title compound SG-triazole-hANP(1-28) (compound 2-12)
(9.3 mg) was obtained according to the same approach as in (2-2D)
using the GlcNAc-triazole-hANP(1-28) (8.0 mg) produced in
(2-12B).
[0427] MALDI-TOF-MS: Calcd for
C.sub.218H.sub.348N.sub.54O.sub.102S.sub.3: [M+H].sup.+ 5451.3,
Found 5451.1.
<Example 2-13> Synthesis of SG-(SG-)Asn-PEG(3)-hANP(1-28)
(Compound 2-13)
[0428] (2-13A) Synthesis of GlcNAc-(GlcNAc-Asn)-PEG(3)-hANP(1-28)
(compound of the following formula)
##STR00106##
[0429] The title compound GlcNAc-(GlcNAc-)Asn-PEG(3)-hANP(1-28)
(12.0 mg) was obtained according to the same approach as in (2-1B)
from the compound 1-5B (9 mg) and the hANP-TFA salt (16.6 mg)
prepared by Preparation Method 2 of (2-1A).
[0430] MALDI-TOF-MS: Calcd for
C.sub.160H.sub.258N.sub.50O.sub.58S.sub.3: [M+H].sup.+ 3904.8,
Found 3904.5.
[0431] (2-13B) Synthesis of SG-(SG-)Asn-PEG(3)-hANP(1-28) (compound
2-13: compound of the following formula)
##STR00107##
[0432] The title compound SG-(SG-)Asn-PEG(3)-hANP(1-28) (compound
2-13) (6.40 mg) was obtained according to the same approach as in
(2-9B) using the GlcNAc-(GlcNAc-)Asn-PEG(3)-hANP(1-28) (6.00 mg)
produced in (2-13A).
[0433] ESI-TOF-MS: Calcd for
C.sub.312H.sub.504N.sub.60O.sub.170S.sub.3: [M+4H].sup.4+ 1979.0
(ave.), Found 1978.5.
<Example 2-14> Synthesis of SG-(SG-)Lys-Gly-hANP(1-28)
(Compound 2-14)
[0434] (2-14A) Synthesis of GlcNAc-(GlcNAc-)Lys-Gly-hANP(1-28)
(compound of the following formula)
##STR00108##
[0435] The title compound GlcNAc-(GlcNAc-)Lys-Gly-hANP(1-28) (14.9
mg) was obtained according to the same method as in (2-7A) using
the hANP(1-28)-acetate (50.0 mg) and the compound 1-9D (29.0 mg,
40.0 .mu.mol).
[0436] MALDI-TOF-MS: Calcd for
C.sub.155H.sub.249N.sub.50O.sub.55S.sub.3: [M+H].sup.+ 3786.7,
Found 3786.8.
[0437] (2-14B) Synthesis of SG-(SG-)Lys-Gly-hANP(1-28) (compound
2-14: compound of the following formula)
##STR00109##
[0438] The title compound SG-(SG-)Lys-Gly-hANP(1-28) (compound
2-14) (7.6 mg) was obtained according to the same approach as in
(2-2D) using the GlcNAc-(GlcNAc-)Lys-Gly-hANP(1-28) (6.0 mg)
produced in (2-14A).
[0439] ESI-TOF-MS: Calcd for
C.sub.307H.sub.498N.sub.60O.sub.167S.sub.3: [M+4H].sup.4+ 1949.4
(ave.), Found 1949.2.
<Example 2-15> Synthesis of [(SG-)Cys-Gly].sub.3-hANP(1-28)
(Compound 2-15)
[0440] (2-15A) Synthesis of [(GlcNAc-)Cys-Gly]3-hANP(1-28)
(compound of the following formula)
##STR00110##
[0441] The title compound [(GlcNAc-)Cys-Gly].sub.3-hANP(1-28) (18.4
mg) was obtained according to the same approach as in (2-1B) using
the compound 1-10B (27 mg).
[0442] MALDI-TOF-MS: Calcd for
C.sub.195H.sub.304N.sub.60O.sub.73S.sub.6: [M+H].sup.+ 4847.0,
Found 4847.2.
[0443] (2-15B) Synthesis of [(SG-)Cys-Gly]3-hANP(1-28) (compound
2-15: compound of the following formula)
##STR00111##
[0444] The title compound [(SG-)Cys-Gly]3-hANP(1-28) (compound
2-15) (5.23 mg) was obtained by the same approach as in 2-2D using
the compound SG-Oxa produced in (1-12A) in a 0.2 M phosphate buffer
solution (60 mM, 118 .mu.l), glycosynthase (Endo-M-N175Q, Tokyo
Chemical Industry Co., Ltd., 1 U/ml, 47 .mu.l), and the
[(GlcNAc-)Cys-Gly]3-hANP(1-28) produced in (2-15A).
[0445] ESI-TOF-MS: Calcd for
C.sub.423H.sub.673N.sub.75O.sub.241S.sub.6: [M+5H].sup.5+2172.5
(ave.), Found 2172.4.
<Example 2-16> Synthesis of SG-PEG(3)-(SG-)Asn-hANP(1-28)
(Compound 2-16)
[0446] (2-16A) Synthesis of GlcNAc-PEG(3)-(GlcNAc-)Asn-hANP(1-28)
(compound of the following formula)
##STR00112##
[0447] The title compound GlcNAc-PEG(3)-(GlcNAc-)Asn-hANP(1-28)
(9.10 mg) was obtained according to the same approach as in (2-7A)
from the (GlcNAc-)Asn-hANP (19.0 mg) produced in (2-3B) and the
compound 1-3A (8.8 mg).
[0448] MALDI-TOF-MS: Calcd for
C.sub.160H.sub.258N.sub.50O.sub.58S.sub.3: [M+H].sup.+ 3904.8,
Found 3904.4.
[0449] (2-16B) Synthesis of SG-PEG(3)-(SG-)Asn-hANP(1-28) (compound
2-16: compound of the following formula)
##STR00113##
[0450] The title compound SG-PEG(3)-(SG-)Asn-hANP(1-28) (compound
2-16) (3.10 mg) was obtained according to the same approach as in
(2-9B) using the GlcNAc-PEG(3)-(GlcNAc-)Asn-hANP(1-28) (3.50 mg)
produced in (2-16A) at room temperature.
[0451] ESI-TOF-MS: Calcd for
C.sub.312H.sub.504N.sub.60O.sub.170S.sub.3: [M+4H].sup.4+ 1978.1
(ave.), Found 1978.8.
<Example 2-17> Synthesis of [(SG-)Cys-Gly].sub.5-hANP(1-28)
(Compound 2-17)
[0452] (2-17A) Synthesis of [Cys-Gly].sub.5-hANP(1-28) (compound of
the following formula)
##STR00114##
[0453] The intermediate (13.0 mg) was obtained according to the
same approach as in (2-1B) from the compound 1-8A (50.6 mg) and
hANP-TFA salt prepared from the hANP-acetate (47.0 mg) by
Preparation Method 2 of (2-1A).
[0454] To the obtained intermediate (13.0 mg), a mixed solution of
trifluoroacetic acid (1.9 mL), water (0.05 mL), and
triisopropylsilane (0.05 mL) was added. The mixture was shaken at
room temperature for 1 hour. The reaction solution was diluted with
water, and the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents to obtain the title
compound [Cys-Gly].sub.5-hANP(1-28) as a white solid (3.2 mg).
[0455] MALDI-TOF-MS: Calcd for
C.sub.154H.sub.245N.sub.55O.sub.50S.sub.8: [M+H].sup.+ 3921.6,
Found 3921.9.
[0456] (2-17B) Synthesis of [(SG-)Cys-Gly].sub.5-hANP(1-28)
(compound 2-17: compound of the following formula)
##STR00115##
[0457] The [Cys-Gly].sub.5-hANP(1-28) (3.00 mg) produced in (2-17A)
and the compound SG-M (9.94 mg) produced in (1-13A) were dissolved
in a mixed solution of acetonitrile (0.25 mL) and a 0.2 M phosphate
buffer of pH 6.75 (0.25 mL), and the solution was stirred at room
temperature for 2 hours. The reaction solution was diluted with
water, and the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents to obtain the title
compound [(SG-) Cys-Gly]5-hANP(1-28) (compound 2-17) (8.19 mg).
[0458] ESI-TOF-MS: Calcd for
C.sub.619H.sub.985N.sub.95O.sub.375S.sub.8: [M+6H].sup.6+ 2670.1
(ave.), Found 2670.1.
<Example 2-18> Synthesis of [(SG.sub.2-)Cys-Gly]5-hANP(1-28)
(Compound 2-18)
[0459] (2-18A) Synthesis of tert-butyl
N-[(1R)-1-(prop-2-ynylcarbamoyl)but-3-ynyl]carbamate (compound of
the following formula)
##STR00116##
[0460] The title compound was obtained as a pale yellow solid (480
mg, yield: 96%) according to the same method as in (1-9A) using
(2R)-2-(tert-butoxycarbonylamino)-4-pentynoic acid (430 mg, 2.02
mmol) and propargylamine (140 mg, 2.54 mmol).
[0461] MS (ESI): Calcd for C.sub.13H.sub.19N.sub.2O.sub.3:
[M+H].sup.+ 251, Found 251.
[0462] (2-18B) Synthesis of
(2R)-2-amino-N-prop-2-ynyl-pent-4-enamide trifluoroacetate
(compound of the following formula)
##STR00117##
[0463] The title compound was obtained as a pale yellow oil (270
mg, yield: 100%) according to the same method as in (1-9B) using
the title compound (250 mg, 1.00 mmol) obtained in (2-18A).
[0464] .sup.1H-NMR (CD.sub.3OD) .delta.: 4.11-3.96 (3H, m),
2.87-2.74 (2H, m), 2.68-2.65 (2H, m).
[0465] (2-18C) Synthesis of
(2R)-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]-N-prop-2-ynyl-pent-4-enam-
ide (compound of the following formula)
##STR00118##
[0466] The title compound was obtained as a pale yellow solid (220
mg, yield: 73%) according to the same method as in (1-15A) using
the title compound (270 mg, 1.00 mmol) obtained in (2-18B).
[0467] MS (ESI): Calcd for C.sub.15H.sub.14N.sub.3O.sub.4:
[M-H].sup.- 300, Found 300.
[0468] (2-18D) Synthesis of [(SG.sub.2-)Cys-Gly].sub.5-hANP(1-28)
(compound 2-18: compound of the following formula)
##STR00119##
[0469] The compound SG-N3 (20.0 mg, 8.73 .mu.mol) produced in
(1-18A) was dissolved in a 0.10 M phosphate buffer (pH 8.0) (0.840
ml). To the solution, a 3.3 mM solution of the title compound
obtained in (2-18C) in tert-butanol (1.06 mL, 3.50 .mu.mol), a 50
mM aqueous sodium ascorbate solution (0.420 mL, 21.0 .mu.mol), and
a 10 mM aqueous copper sulfate solution (0.420 mL, 4.20 .mu.mol)
were added in this order, and the mixture was stirred at room
temperature for 1 hour and 30 minutes. A 0.2% aqueous
trifluoroacetic acid solution (12 mL) was added to the reaction
solution, and the resulting product was separated and purified by
reverse-phase HPLC (Shiseido Co., Ltd., Proteonavi) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the intermediate (6.20 mg, 36%).
[0470] The title compound [(SG.sub.2-)Cys-Gly].sub.5-hANP(1-28)
(compound 2-18) (8.42 mg) was obtained according to the same
approach as in (2-17B) from the (Cys-Gly).sub.5-hANP(1-28) (1.76
mg) produced in (2-17A) and the synthesized intermediate (10.3
mg).
[0471] ESI-TOF-MS: Calcd for
C.sub.1089H.sub.1730N.sub.160O.sub.690S.sub.8: [M-10H].sup.10-
2835.1 (ave.), Found 2834.9.
<Example 2-19> Synthesis of
SG-(SG-)Lys-[SG-(SG-)Lys-]Lys-PEG(3)-hANP(1-28) (Compound 2-19)
[0472] (2-19A) Synthesis of
TrS-(TrS-)Lys-[TrS-(TrS-)Lys-]Lys-PEG(3) (compound of the following
formula)
##STR00120##
[0473] A 1.20 mmol/g 2-chlorotrityl chloride resin (250 mg, 0.300
mmol) was placed in a column for solid-phase synthesis.
Dichloromethane (5 mL) was added thereto, and the mixture was
shaken for 10 minutes. After filtration, a solution of
3-[2[2[2[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]ethoxy]etho-
xy]propionic acid (175 mg, 0.360 mmol) and
N,N-diisopropylethylamine (257 .mu.L, 1.50 mmol) in dichloromethane
(5 mL) was added thereto, and the mixture was stirred at room
temperature for 2 hours. After filtration, the resin was washed
with a dichloromethane mixed solution
(dichloromethane:methanol:N,N-diisopropylethylamine=85:10:5, v/v)
three times, dichloromethane three times, and N,N-dimethylformamide
three times. A 20% solution of piperidine in N,N-dimethylformamide
(10 mL) was added thereto, and the mixture was shaken for 5
minutes, followed by filtration. This operation was carried out 4
times. The resin was washed with N,N-dimethylformamide 4 times. A
solution of
(2S)-2,6-bis(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid (532
mg, 0.900 mmol), HATU (342 mg, 0.900 mmol), and
N,N-diisopropylethylamine (308 .mu.L, 1.80 mmol) in
N,N-dimethylformamide (10 mL) was added to the resin, and the
mixture was shaken at room temperature for 30 minutes. After
filtration, the resin was washed with N,N-dimethylformamide three
times. A 20% solution of piperidine in N,N-dimethylformamide (10
mL) was added thereto, and the mixture was shaken for 5 minutes,
followed by filtration. This operation was carried out 4 times. The
resin was washed with N,N-dimethylformamide 4 times. A solution of
(2S)-2,6-bis(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid
(1060 mg, 1.80 mmol), HATU (684 mg, 1.80 mmol), and
N,N-diisopropylethylamine (616 .mu.L, 3.60 mmol) in
N,N-dimethylformamide (10 mL) was added to the resin, and the
mixture was shaken at room temperature for 1 hour. After
filtration, the resin was washed with N,N-dimethylformamide 4
times. A 20% solution of piperidine in N,N-dimethylformamide (10
mL) was added thereto, and the mixture was shaken for 5 minutes,
followed by filtration. This operation was carried out 5 times. The
resin was washed with N,N-dimethylformamide 4 times. A 1/3 amount
(corresponding to 0.100 mmol) of the obtained resin was placed in a
column for solid-phase synthesis. A solution of
3-tritylsulfanylpropionic acid (418 mg, 1.20 mmol), HATU (456 mg,
1.20 mmol), and N,N-diisopropylethylamine (411 .mu.L, 2.40 mmol) in
N,N-dimethylformamide (10 mL) was added thereto, and the mixture
was shaken at room temperature for 1 hour. After filtration, a
solution of 3-tritylsulfanylpropionic acid (418 mg, 1.20 mmol),
HATU (456 mg, 1.20 mmol), and N,N-diisopropylethylamine (411 .mu.L,
2.40 mmol) in N,N-dimethylformamide (10 mL) was added again to the
resin, and the mixture was shaken at room temperature for 1 hour.
After filtration, the resin was washed with N,N-dimethylformamide 4
times and dichloromethane three times. A mixed solution of
1,1,1,3,3,3-hexafluoro-2-propanol (2.5 mL) and dichloromethane (7.5
mL) was added thereto, and the mixture was shaken at room
temperature for 1.5 hours. The resin was filtered off, and the
filtrate was concentrated under reduced pressure. The concentrate
was subjected to azeotropy with dichloromethane 6 times and dried
in a vacuum pump to obtain the title compound
TrS-(TrS-)Lys-[TrS-(TrS-) Lys-]Lys-PEG(3) as a brown solid (170
mg).
[0474] (2-19B) Synthesis of
HS-(HS-)Lys[HS-(HS-)Lys-]Lys-PEG(3)-hANP(1-28) (compound of the
following formula)
##STR00121##
[0475] The intermediate (40 mg) was obtained according to the same
approach as in (2-17A) from the compound (66.1 mg) produced in
(2-19A).
[0476] The title compound
HS-(HS-)Lys[HS-(HS-)Lys-]Lys-PEG(3)-hANP(1-28) (2.3 mg) was
obtained according to the same approach as in (2-17A) from the
obtained intermediate (7.0 mg).
[0477] MALDI-TOF-MS: Calcd for
C.sub.168H.sub.276N.sub.52O.sub.51S.sub.7: [M+H].sup.+ 4062.9,
Found 4062.8.
[0478] (2-19C) Synthesis of
SG-(SG-)Lys-[SG-(SG-)-Lys-]Lys-PEG(3)-hANP(1-28) (compound of the
following formula: compound 2-19)
##STR00122##
[0479] The title compound
[SG-(SG-)Lys-[SG-(SG-)-Lys-]Lys-PEG(3)-hANP(1-28) (compound 2-19)
(5.31 mg) was obtained according to the same approach as in (2-17B)
from HS(HS-)-Lys-[HS-(HS-)Lys-]Lys-PEG(3)-hANP(1-28) (2.90 mg)
produced by the approach of (2-19B).
[0480] ESI-TOF-MS: Calcd for
C.sub.540H.sub.868N.sub.84O.sub.311S.sub.7: [M+7H].sup.7+ 1963.4
(ave.), Found 1963.4.
<Example 2-20> Synthesis of
[SG.sub.2-(SG.sub.2-)Lys-[SG.sub.2-(SG.sub.2-)-Lys-]Lys-PEG(3)-hANP(1-28)
(Compound 2-20)
[0481] (2-20A) Synthesis of
SG.sub.2-(SG.sub.2-)Lys-[SG.sub.2-(SG.sub.2-)-Lys-]Lys-PEG(3)-hANP(1-28)
(compound 2-20: compound of the following formula)
##STR00123##
[0482] The title compound
SG.sub.2-(SG.sub.2-)Lys-[SG.sub.2-(SG.sub.2-)-Lys-]Lys-PEG(3)-hANP(1-28)
(compound 2-20) (4.79 mg) was obtained according to the same
approach as in (2-17B) from the
HS(HS-)-Lys-[HS-(HS-)Lys-]Lys-PEG(3)-hANP(1-28) (2.20 mg) produced
in (2-19B) and the compound SG-(SG-)Gln*-Mal (12.1 mg) produced in
(1-15C).
[0483] ESI-TOF-MS: Calcd for
C.sub.904H.sub.1460N.sub.116O.sub.567S.sub.7: [M+8H].sup.8+2907.3
(ave.), Found 2907.
<Example 2-21> Synthesis of
AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28) (Compound 2-21)
[0484] (2-21A) Synthesis of AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28)
(compound 2-21: compound of the following formula)
##STR00124##
[0485] The title compound AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28)
(compound 2-21) (8.2 mg) was obtained according to the same
approach as in (2-10A) using the SG-(SG-Asn)-PEG-hANP(1-28) (16 mg)
synthesized in (2-13B).
[0486] ESI-TOF-MS: Calcd for
C.sub.268H.sub.436N.sub.56O.sub.138S.sub.3: [M+4H].sup.4+ 1687.7,
Found 1687.4.
<Example 2-22> Synthesis of
AG(7)-(AG(7)-)Asn-PEG(3)-hANP(1-28) (Compound 2-22)
[0487] (2-22A) Synthesis of AG(7)-(AG(7)-)Asn-PEG(3)-hANP(1-28)
(compound 2-22: compound of the following formula)
##STR00125##
[0488] The title compound AG(7)-(AG(7)-)Asn-PEG(3)-hANP(1-28)
(compound 2-22) (6.6 mg) was obtained according to the same
approach as in (2-11A) using the
AG(9)-(AG(9)-)Asn-PEG(3)-hANP(1-28) (8 mg) synthesized in
(2-21A).
[0489] ESI-TOF-MS: Calcd for
C.sub.244H.sub.396N.sub.56O.sub.118S.sub.3: [M+4H].sup.4+ 1525.6,
Found 1525.4.
<Example 2-23> Synthesis of
SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal-)Lys-]Lys-PEG(11)-hANP(1-28)
(Compound 2-23)
[0490] (2-23A) Synthesis of
TrS-(TrS-)Lys-[TrS-(TrS-)Lys-]Lys-PEG(11)-CO.sub.2H (compound of
the following formula)
##STR00126##
[0491] The title compound
TrS-(TrS-)Lys-[TrS-(TrS-)Lys-]Lys-PEG(11)-CO.sub.2H (135 mg) was
obtained according to the same approach as in (2-19A) from
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(9H-fluoren-9-ylmethoxycarbonylamin-
o)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]et-
hoxy]ethoxy]propionic acid (202 mg).
[0492] (2-23B) Synthesis of
HS-(HS-)Lys-[HS-(HS-)Lys-]Lys-PEG(11)-hANP(1-28) (compound of the
following formula)
##STR00127##
[0493] The title compound
HS-(HS-)Lys-[HS-(HS-)Lys-]Lys-PEG(11)-hANP(1-28) (12.0 mg) was
obtained according to the same approach as in (2-17A) from the
TrS-(TrS-)Lys-[TrS-(TrS-)Lys-]Lys-PEG(11)-CO.sub.2H (45.3 mg)
produced in (2-23A).
[0494] MALDI-TOF-MS: Calcd for
C.sub.184H.sub.308N.sub.52O.sub.59S.sub.7: [M+H].sup.+ 4415.1,
Found 4416.1.
[0495] (2-23C) Synthesis of SG.sub.4-Lys.sub.3-PEG(11)-hANP(1-28)
(compound 2-23: compound of the following formula)
##STR00128##
[0496] The title compound SG-Mal-(SG-Mal-)Lys-[SG-Mal-(SG-Mal-)
Lys-]Lys-PEG(11)-hANP(1-28) (compound 2-23) (4.40 mg) was obtained
according to the same approach as in (2-17B) from the
HS-(HS-)Lys-[HS-(HS-)Lys-]Lys-PEG(11)-hANP(1-28) (3.16 mg) produced
in (2-23B).
[0497] ESI-TOF-MS: Calcd for
C.sub.556H.sub.900N.sub.84O.sub.319S.sub.7: [M+6H].sup.6+ 2349.3
(ave.), Found 2349.2.
<Example 2-24> Synthesis of SG-PEG(3)-hANP(1-28)-PEG(3)-SG
(Compound 2-24)
[0498] (2-24A) Synthesis of GlcNAc-PEG(3)-hANP(1-28) (compound of
the following formula)
##STR00129##
[0499] The title compound GlcNAc-PEG(3)-hANP(1-28) (25.0 mg) was
obtained according to the same method as in (2-7A) using the
hANP(1-28)-TFA salt (33.0 mg) produced in (2-1A) and the compound
1-3A(13.0 mg, 24.7 .mu.mol).
[0500] MALDI-TOF-MS: Calcd for
C.sub.148H.sub.240N.sub.47O.sub.51S.sub.3: [M+H].sup.+ 3587.7,
Found 3587.6.
[0501] (2-24B) Synthesis of GlcNAc-PEG(3)-hANP(1-28)-PEG(3)-GlcNAc
(compound of the following formula)
##STR00130##
[0502] The title compound GlcNAc-PEG(3)-hANP(1-28)-PEG(3)-GlcNAc
(3.0 mg) was obtained according to the same method as in (2-2B)
using the GlcNAc-PEG(3)-hANP(1-28) (21.0 mg) produced in (2-24A)
and the compound 1-19B (46 mg, 73.6 .mu.mol).
[0503] MALDI-TOF-MS: Calcd for
C.sub.169H.sub.278N.sub.50O.sub.61S.sub.3: [M+H].sup.+ 4080.9,
Found. 4080.9.
[0504] (2-24C) Synthesis of SG-PEG(3)-hANP(1-28)-PEG(3)-SG
(compound 2-24: compound of the following formula)
##STR00131##
[0505] The title compound SG-PEG(3)-hANP(1-28)-PEG(3)-SG (compound
2-24) (5.5 mg) was obtained according to the same approach as in
(2-2D) using the GlcNAc-PEG(3)-hANP(1-28)-PEG(3)-GlcNAc (4.0 mg)
produced in (2-24B).
[0506] ESI-TOF-MS: Calcd for
C.sub.321H.sub.528N.sub.60O.sub.173S.sub.3: [M+4H].sup.4+ 2023.0
(ave.), Found 2022.8.
<Example 2-25> Synthesis of SG-thioacetamide-hANP(1-28)
(Compound 2-25)
[0507] (2-25A) Synthesis of TrS-hANP(1-28) (compound of the
following formula)
##STR00132##
[0508] 3-Tritylsulfanylpropionic acid (2.24 mg, 6.43 .mu.mol) was
dissolved in dimethylformamide (100 .mu.l). To the solution,
triethylamine (1.79 .mu.l, 12.8 .mu.mol) was added at room
temperature, then a solution of dimethylthiophosphinoyl chloride
(0.83 mg, 6.46 .mu.mol) in dimethylformamide (60 .mu.l) was added
under ice cooling, and then the mixture was stirred at room
temperature for 1 hour. Meanwhile, the hANP(1-28)-TFA salt (10 mg)
was dissolved in dimethylformamide (200 .mu.l) and distilled water
(60 .mu.l). To the solution, triethylamine (4.2 .mu.l) was added at
room temperature, then a solution of active ester prepared in
advance in dimethylformamide (160 .mu.l) was added under ice
cooling, and the mixture was stirred at room temperature for 5
hours. The reaction solution was added to an ice-cold 0.5% aqueous
trifluoroacetic acid solution (2 ml), and the resulting product was
separated and purified by reverse-phase HPLC (GL Sciences Inc.,
Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic acid solution
and a 0.1% solution of trifluoroacetic acid in acetonitrile as
eluents and lyophilized to obtain the title compound TrS-hANP(1-28)
(5.77 mg).
[0509] ESI-TOF-MS: Calcd for
C.sub.149H.sub.221N.sub.45O.sub.40S.sub.4: [M+3H].sup.3+ 1138.0
(ave.), Found 1137.8.
[0510] (2-25B) Synthesis of HS-hANP(1-28) (compound of the
following formula)
##STR00133##
[0511] The TrS-hANP(1-28) (5.77 mg) synthesized in (2-25A) was
dissolved in a trifluoroacetic acid/distilled
water/triisopropylsilane (90/5/5) solution, and the solution was
stirred at room temperature for 1 hour. After the completion of the
reaction, insoluble matter was dissolved by the addition of a
distilled water/acetic acid (10/1) solution (3 ml), and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound HS-hANP(1-28) (2.88 mg).
[0512] MALDI-TOF-MS: Calcd for
C.sub.130H.sub.207N.sub.45O.sub.40S.sub.4: [M+H].sup.+ 3167.4,
Found 3167.7.
[0513] (2-25C) Synthesis of SG-thioacetamide-hANP(1-28) (compound
2-25: compound of the following formula)
##STR00134##
[0514] The HS-hANP(1-28) (2.88 mg) synthesized in (2-25B) and the
compound SG-I (2.66 mg) synthesized in (1-11C) were dissolved in
dimethylformamide (300 .mu.l). To the solution,
diisopropylethylamine (0.77 .mu.l) was added, and the mixture was
stirred at room temperature for 1 hour. After the completion of the
reaction, insoluble matter was dissolved by the addition of a
distilled water/acetic acid (10/1) solution (3 ml), and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound SG-thioacetamide-hANP(1-28) (compound
2-25) (2.90 mg).
[0515] ESI-TOF-MS: Calcd for
C.sub.218H.sub.350N.sub.52O.sub.103S.sub.4: [M+4H].sup.4+ 1369.9
(ave.), Found 1369.6.
<Example 2-26> Synthesis of AG(5)-hANP(1-28) (Compound
2-26)
[0516] (2-26A) Synthesis of AG(5)-hANP(1-28) (compound 2-26:
compound of the following formula)
##STR00135##
[0517] Trifluoroacetate of the GlcNAc-hANP synthesized in (2-7A)
was replaced with another salt by use of an ion-exchange resin
(Dowex 1.times.8), and the resulting GlcNAc-hANP acetate was used
in the next reaction.
[0518] The compound AG(5)-P (18 mg) synthesized in (1-17C) was
dissolved in a 0.2 M phosphate buffer solution (pH 6.75, 160
.mu.l). To the solution, a solution of glycosynthase (Endo-M-N175Q,
Tokyo Chemical Industry Co., Ltd., 1 U/ml, 64 .mu.l) and acetate
(8.0 mg) of the hANP-GlcNAc synthesized in (2-7A) in dimethyl
sulfoxide (96 .mu.l) was then added, and the mixture was reacted at
25.degree. C. for 3 hours. The reaction was terminated by the
addition of a 0.2% aqueous trifluoroacetic acid solution (1.5 ml)
at room temperature, and the resulting product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents and
lyophilized to obtain the title compound AG(5)-hANP(1-28) (compound
2-26) (5.6 mg).
[0519] ESI-TOF-MS: Calcd for
C.sub.163H.sub.261N.sub.47O.sub.66S.sub.3: [M+3H].sup.3+1345.1
(ave.), Found 1344.6.
<Example 2-27> Synthesis of SG-(SG-)Asn-PEG(11)-hANP(1-28)
(Compound 2-27)
[0520] (2-27A) Synthesis of Boc-(GlcNAc-)Asn-PEG(11)-CO.sub.2H
(compound of the following formula)
##STR00136##
[0521]
(2S)-4-[[(2R,3R,4R,5S,6R)-3-Acetamido-4,5-dihydroxy-6-(hydroxymethy-
l)tetrahydropyran-2-yl]amino]-2-(tert-butoxycarbonylamino)-4-oxobutanoic
acid (187 mg, 0.43 mmol) produced according to the description of
J. Am. Chem. Soc., 1999, 121, 284-290 and HATU (163 mg, 0.43 mmol)
were dissolved in N,N-dimethylformamide (3.0 ml). To the solution,
diisopropylethylamine (150 .mu.l, 0.86 mmol) was added at room
temperature, and the mixture was stirred for 3 minutes. This
reaction solution was added to the
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]et-
hoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic
acid (0.22 g, 0.36 mmol) produced in (1-4A), and the mixture was
stirred at room temperature for 3 hours. This reaction mixture was
added dropwise to an ice-cold mixed solvent of distilled water (3
ml) and acetic acid (100 .mu.l) and dissolved therein, and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound Boc-(GlcNAc-Asn)-PEG(11)-CO.sub.2H (210
mg).
[0522] MALDI-TOF-MS: Calcd for C.sub.44H.sub.82N.sub.4O.sub.23:
[M+K].sup.+ 1073.6, Found 1073.5.
[0523] (2-27B) Synthesis of Boc-(GlcNAc-)Asn-PEG(11)-hANP(1-28)
(compound of the following formula)
##STR00137##
[0524] The Boc-(GlcNAc-)Asn-PEG(11)-CO.sub.2H (7.3 mg, 7.1 .mu.mol)
produced in (2-27A) was dissolved in N,N-dimethylformamide (150
.mu.l). To the solution, a solution of triethylamine (5.9 .mu.l, 43
.mu.mol) and dimethylthiophosphinoyl chloride (1.7 mg, 21 .mu.mol)
in N,N-dimethylformamide (50 .mu.l) was added under ice cooling.
This reaction solution was heated to room temperature while stirred
for 1.5 hours. This reaction solution was added under ice cooling
to a solution of the hANP-TFA salt (36 mg, 60 w/w %, 7.1 .mu.mol)
prepared according to the procedures of (2-1A) and triethylamine
(14 .mu.l, 99 .mu.mol) dissolved in a mixed solvent of
N,N-dimethylformamide (1500 .mu.l) and distilled water (300 .mu.l),
and the mixture was heated to room temperature while stirred for 1
day. This reaction solution was added to an ice-cold 0.2 v/v %
aqueous trifluoroacetic acid solution (8.3 ml), and the resulting
product was separated and purified by reverse-phase HPLC (GL
Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous trifluoroacetic
acid solution and a 0.1% solution of trifluoroacetic acid in
acetonitrile as eluents and lyophilized to obtain the title
compound GlcNAc-(GlcNAc-)Asn-PEG (11)-CO.sub.2H (14.8 mg).
[0525] ESI-TOF-MS: Calcd for
C.sub.171H.sub.283N.sub.49O.sub.61S.sub.3: [M+2H].sup.2+ 2049.8
(ave.), Found 2049.5.
[0526] (2-27C) Synthesis of GlcNAc-(GlcNAc-)Asn-PEG(11)-hANP(1-28)
(compound of the following formula)
##STR00138##
[0527] The Boc-(GlcNAc-)Asn-PEG(11)-hANP(1-28) (14.8 mg) produced
in (2-27B) was dissolved in a 33 v/v % aqueous trifluoroacetic acid
solution (1.0 ml), and the solution was left standing at room
temperature for 3 hours. This reaction solution was added to an
ice-cold mixed solvent of distilled water (9.5 ml) and acetic acid
(0.5 ml), and the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the intermediate (10.2 mg).
[0528] The
2-[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)-
tetrahydropyran-2-yl]oxyacetic acid (1.4 mg, 5.1 .mu.mol) produced
in (1-2C) was dissolved in N,N-dimethylformamide (150 .mu.l). To
the solution, triethylamine (2.1 .mu.l, 15 .mu.mol) and
dimethylthiophosphinoyl chloride (0.98 mg, 7.7 .mu.mol) were added
under ice cooling. This reaction solution was heated to room
temperature while stirred for 1.5 hours. This reaction solution was
added under ice cooling to a solution of the obtained intermediate
(10 mg, 2.6 .mu.mol) and triethylamine (5.0 .mu.l, 36 .mu.mol)
dissolved in a mixed solvent of N,N-dimethylformamide (1500 .mu.l)
and distilled water (3 00 .mu.l), and the mixture was heated to
room temperature while stirred for 3 days. This reaction solution
was added to an ice-cold 0.2 v/v % aqueous trifluoroacetic acid
solution (8.5 ml), and the resulting product was separated and
purified by reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3)
using a 0.1% aqueous trifluoroacetic acid solution and a 0.1%
solution of trifluoroacetic acid in acetonitrile as eluents and
lyophilized to obtain the title compound
GlcNAc-(GlcNAc-)Asn-PEG(11)-hANP(1-28) (9.0 mg).
[0529] ESI-TOF-MS: Calcd for
C.sub.176H.sub.290N.sub.50O.sub.66S.sub.3: [M-2H].sup.2- 2128.3
(ave.), Found 2128.0.
[0530] (2-27D) Synthesis of SG-(SG-Asn)-PEG(11)-hANP(1-28)
(compound 2-27: compound of the following formula)
##STR00139##
[0531] To the compound SG-Oxa produced in (1-12A) in a 0.2 M
phosphate buffer solution (60 mM, 188 .mu.l), Endo-M-N175Q (1 U/ml,
100 .mu.l) was added at room temperature, then a solution of the
hANP-GlcNAc (8.0 mg, 1.9 .mu.mol) produced in (2-27D) in dimethyl
sulfoxide (120 .mu.l) was added in two portions at an interval of
15 minutes at room temperature, and the mixture was shaken at
25.degree. C. for 1 day. The reaction was terminated by the
addition of a mixed solvent of a 0.2% aqueous trifluoroacetic acid
solution (4.5 ml) and acetic acid (0.5 ml) at room temperature, and
the resulting product was separated and purified by reverse-phase
HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound SG-(SG-)Asn-PEG(11)-hANP(1-28) (compound
2-27) (3.4 mg).
[0532] ESI-TOF-MS: Calcd for
C.sub.328H.sub.536N.sub.60O.sub.178S.sub.3: [M+5H].sup.5+ 1653.8
(ave.), Found 1653.7.
<Example 2-28> Synthesis of
SG-(SG-)Asn-PEG(11)-PEG(11)-hANP(1-28) (compound 2-28)
[0533] (2-28A) Synthesis of Fmoc-PEG(11)-PEG(11)-CO.sub.2H
(compound of the following formula)
##STR00140##
[0534]
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(9H-Fluoren-9-ylmethoxycarbon-
ylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth-
oxy]ethoxy]ethoxy]propanoic acid (350 mg, 0.42 mmol) and HATU (192
mg, 0.50 mmol) were dissolved in N,N-dimethylformamide (3.0 ml). To
the solution, diisopropylethylamine (176 .mu.l, 1.01 mmol) was
added at room temperature, and the mixture was stirred for 3
minutes. This reaction solution was added to the
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]et-
hoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic
acid (259 mg, 0.42 mmol) produced in (1-4A), and the mixture was
stirred at room temperature for 3 hours. This reaction mixture was
added dropwise to an ice-cold mixed solvent of distilled water (3
ml) and acetic acid (117 .mu.l) and dissolved therein, and the
solution was further diluted with a mixed solvent of
N,N-dimethylformamide (3.0 ml) and distilled water (15 ml). The
resulting product was separated and purified from the solution by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound Fmoc-PEG(11)-PEG(11)-CO.sub.2H (399
mg).
[0535] ESI-TOF-MS: Calcd for C.sub.69H.sub.118N.sub.2O.sub.29:
[M-H].sup.- 1437.8, Found 1437.8.
[0536] (2-28B) Synthesis of H.sub.2N-PEG(11)-PEG(11)-CO.sub.2H
(compound of the following formula)
##STR00141##
[0537] The title compound H.sub.2N-PEG(11)-PEG(11)-CO.sub.2H (77
mg) was obtained according to the same approach as in (1-4A) from
the Fmoc-PEG(11)-PEG(11)-CO.sub.2H (250 mg) produced in
(2-28A).
[0538] MALDI-TOF-MS: Calcd for C.sub.54H.sub.108N.sub.2O.sub.27:
[M+H].sup.+ 1217.7, Found 1217.9.
[0539] (2-28C) Synthesis of
Boc-(GlcNAc-)Asn-PEG(11)-PEG(11)-CO.sub.2H (compound of the
following formula)
##STR00142##
[0540] The title compound
Boc-(GlcNAc-)Asn-PEG(11)-PEG(11)-CO.sub.2H (58 mg) was obtained
according to the same approach as in (2-27A) from
(25)-4-[[(2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetr-
ahydropyran-2-yl]amino]-2-(tert-butoxycarbonylamino)-4-oxobutanoic
acid (32 mg, 74 .mu.mol) produced according to the description of
J. Am. Chem. Soc., 1999, 121, 284-290 and the
H.sub.2N-PEG(11)-PEG(11)-CO.sub.2H (76 mg) produced in (2-28B).
[0541] MALDI-TOF-MS: Calcd for C.sub.71H.sub.135N.sub.5O.sub.36:
[M+K].sup.+ 1673.0, Found 1672.9.
[0542] (2-28D) Synthesis of
Boc-(GlcNAc)-Asn-PEG(11)-PEG(11)-hANP(1-28) (compound of the
following formula)
##STR00143##
[0543] The title compound
Boc-(GlcNAc-)Asn-PEG(11)-PEG(11)-hANP(1-28) (35 mg) was obtained
according to the same approach as in (2-27B) using the
Boc-(GlcNAc-Asn)-PEG(11)-PEG(11)-COOH (29 mg, 18 .mu.mol) produced
in (2-28C) and the hANP(1-28)-TFA salt (50 mg, 60 w/w %, 9.7
.mu.mol) produced in (2-1A).
[0544] MALDI-TOF-MS: Calcd for
C.sub.198H.sub.336N.sub.50O.sub.74S.sub.3: [M+H].sup.+ 4695.3,
Found 4697.5.
[0545] (2-28E) Synthesis of
GlcNAc-(GlcNAc-Asn)-PEG(11)-PEG(11)-hANP(1-28) (compound of the
following formula)
##STR00144##
[0546] The title compound
GlcNAc-(GlcNAc-)Asn-PEG(11)-PEG(11)-hANP(1-28) (16 mg) was obtained
according to the same approach as in (2-27C) from the
Boc-(GlcNAc-Asn)-PEG(11)-PEG(11)-hANP(1-28) (35 mg, 7.4 .mu.mol)
produced in (2-28D).
[0547] MALDI-TOF-MS: Calcd for
C.sub.203H.sub.343N.sub.51O.sub.79S.sub.3: [M+H].sup.+ 4859.4
(ave.), Found 4858.4.
[0548] -TOF-MS: Calcd for
C.sub.203H.sub.343N.sub.51O.sub.79S.sub.3: [M+H].sup.+ 4859.4
(ave.), Found 4858.4.
[0549] (2-28F) Synthesis of SG-(SG-Asn)-PEG(11)-PEG(11)-hANP(1-28)
(compound 2-28: compound of the following formula)
##STR00145##
[0550] The title compound SG-(SG-Asn)-PEG(11)-PEG(11)-hANP(1-28)
(compound 2-28) (13 mg) was obtained according to the same approach
as in (2-27D) from the compound SG-Oxa produced in (1-12A) in a 0.2
M phosphate buffer solution (60 mM, 190 .mu.l) and the
GlcNAc-(GlcNAc)-Asn-PEG(11)-PEG(11)-hANP(1-28) (16 mg, 3.2 .mu.mol)
produced in (2-28F).
[0551] ESI-TOF-MS: Calcd for
C.sub.355H.sub.589N.sub.61O.sub.191S.sub.3: [M+4H].sup.4+ 2217.0
(ave.), Found 2216.9.
<Example 2-29> Synthesis of SG-PEG(3)-hANP(1-28) (Compound
2-29)
[0552] (2-29A) Synthesis of SG-PEG(3)-hANP(1-28) (compound 2-29:
compound of the following formula)
##STR00146##
[0553] The title compound SG-PEG(3)-hANP(1-28) (compound 2-29)
(12.32 mg) was obtained according to the same approach as in
(1-14B) from the GlcNAc-PEG(3)-hANP(1-28) (15.0 mg) produced in
(2-24A).
[0554] ESI-TOF-MS: Calcd for
C.sub.224H.sub.362N.sub.52O.sub.107S.sub.3: [M+4H].sup.4+ 1399.0
(ave.), Found 1398.3.
<Example 2-30> Synthesis of SG-PEG(11)-hANP(1-28) (Compound
2-30)
[0555] (2-30A) Synthesis of GlcNAc-PEG(11)-hANP(1-28) (compound of
the following formula)
##STR00147##
[0556] The title compound GlcNAc-PEG(11)-hANP(1-28) (33.7 mg) was
obtained according to the same approach as in (2-7A) using the
hANP(1-28)-TFA salt (43.9 mg) and the compound 1-4B (15.0 mg).
[0557] MALDI-TOF-MS: Calcd for
C.sub.164H.sub.271N.sub.47O.sub.59S.sub.3: [M+H].sup.+ 3939.9,
Found 3939.8.
[0558] (2-30B) Synthesis of SG-PEG(11)-hANP(1-28) (compound 2-30;
compound of the following formula)
##STR00148##
[0559] The title compound SG-PEG(11)-hANP(1-28) (compound 2-30)
(11.52 mg) was obtained according to the same approach as in
(1-14B) using the GlcNAc-PEG(11)-hANP(1-28) (15.0 mg) produced in
(2-30A).
[0560] ESI-TOF-MS: Calcd for
C.sub.240H.sub.394N.sub.52O.sub.115S.sub.3: [M+5H].sup.5+ 1189.8
(ave.), Found 1189.3.
<Example 2-31> Synthesis of
SG-(SG-)Gln*-Mal-PEG(3)-hANP(1-28) (Compound 2-31)
[0561] (2-31A) Synthesis of H.sub.2N-PEG(3)-hANP(1-28) (compound of
the following formula)
##STR00149##
[0562] Boc-PEG(3)-hANP(1-28) (12.7 mg) was obtained according to
the same approach as in (2-1B) using
3-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]propi-
onic acid (11.7 mg) and hANP(1-28)-TFA salt prepared from the
hANP(1-28)-acetate (41.0 mg) by Preparation Method 2 of (2-1A).
[0563] The title compound H.sub.2N-PEG(3)-hANP(1-28) (12.0 mg) was
obtained according to the same approach as in (2-2C) using the
obtained Boc-PEG(3)-hANP(1-28) (12.7 mg).
[0564] MALDI-TOF-MS: Calcd for
C.sub.138H.sub.224N.sub.46O.sub.44S.sub.3: [M+H].sup.+ 3326.6,
Found 3326.6.
[0565] (2-31B) Synthesis of HS-PEG(3)-hANP(1-28) (compound of the
following formula)
##STR00150##
[0566] The title compound HS-PEG(3)-hANP(1-28) (5.00 mg) was
obtained according to the same approach as in (2-25A) and (2-25B)
using the H.sub.2N-PEG(3)-hANP(1-28) (12.0 mg) produced in
(2-31A).
[0567] MALDI-TOF-MS: Calcd for
C.sub.242H.sub.228N.sub.46O.sub.45S.sub.4: [M+H].sup.+ 3414.6,
Found 3414.7.
[0568] (2-31C) Synthesis of SG-(SG-)Gln*-Mal-PEG(3)-hANP(1-28)
(compound of the following formula: compound 3-31)
##STR00151##
[0569] The title compound SG-(SG-)Gln*-Mal-PEG(3)-hANP(1-28)
(compound 2-31) (2.09 mg) was obtained according to the same
approach as in (2-17B) using the HS-PEG(3)-hANP(1-28) (4.00 mg)
produced in (2-31C) and the SG-(SG-)Gln*-Mal (7.43 mg) produced in
(1-15C).
[0570] ESI-TOF-MS: Calcd for
C.sub.325H.sub.524N.sub.62O.sub.174S.sub.4: [M+5H].sup.5+ 1643.4
(ave.), Found 1643.2.
<Example 2-32> Synthesis of
SG-(SG-)Gln*-PEG(3)-Mal-hANP(1-28) (Compound 2-32: Compound of the
Following Formula)
##STR00152##
[0572] The HS-hANP(1-28) (2.3 mg) produced in (2-25B) and the
SG-(SG-)Gln*-PEG(3)-Mal (4.0 mg) produced in (1-16C) were dissolved
in a mixed solvent of a 0.2 M acetate buffer (pH 5.0) (0.10 mL) and
dimethyl sulfoxide (0.10 mL), and the solution was stirred at room
temperature for 5 hours. A 0.2% aqueous trifluoroacetic acid
solution (2.0 mL) was added to the reaction solution, and the
resulting product was separated and purified by reverse-phase HPLC
(GL Sciences Inc., Inertsil ODS-3) using a 0.1% aqueous
trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain the title compound SG-(SG-) Gln*-PEG(3)-Mal-hANP(1-28)
(compound 2-32) (3.5 mg).
[0573] ESI-TOF-MS: Calcd for
C.sub.325H.sub.528N.sub.62O.sub.174S.sub.4: [M+4H].sup.4+ 2054.0
(ave.), Found 2053.8.
<Example 2-33> Synthesis of SG-Mal-(SG-Mal-)Lys-hANP(1-28)
(Compound 2-33)
[0574] (2-33A) Synthesis of TrS-(TrS-)Lys-PEG(3)-CO.sub.2H
(compound of the following formula)
##STR00153##
[0575] A 1.20 mmol/g 2-chlorotrityl chloride resin (83 mg, 0.100
mmol) was placed in a column for solid-phase synthesis.
Dichloromethane (2 mL) was added thereto, and the mixture was
shaken for 10 minutes. After filtration, a solution of
3-[2[2[2[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]ethoxy]etho-
xy]propionic acid (97.5 mg, 0.200 mmol) and
N,N-diisopropylethylamine (85.6 .mu.L, 0.500 mmol) in
dichloromethane (2 mL) was added thereto, and the mixture was
shaken at room temperature for 2 hours. After filtration, the resin
was washed with a dichloromethane mixed solution
(dichloromethane:methanol:N,N-diisopropylethylamine=85:10:5, v/v)
three times, dichloromethane three times, and N,N-dimethylformamide
three times. A 20% solution of piperidine in N,N-dimethylformamide
(2 mL) was added thereto, and the mixture was shaken for 5 minutes,
followed by filtration. This operation was carried out 4 times. The
resin was washed with N,N-dimethylformamide 4 times. A solution of
(2S)-2,6-bis(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid (177
mg, 0.300 mmol), HATU (114 mg, 0.300 mmol), and
N,N-diisopropylethylamine (103 .mu.L, 0.600 mmol) in
N,N-dimethylformamide (2 mL) was added to the resin, and the
mixture was shaken at room temperature for 30 minutes. After
filtration, the resin was washed with N,N-dimethylformamide 4
times. A 20% solution of piperidine in N,N-dimethylformamide (2 mL)
was added thereto, and the mixture was shaken for 5 minutes,
followed by filtration. This operation was carried out 4 times. The
resin was washed with N,N-dimethylformamide 4 times. A solution of
3-tritylsulfanylpropionic acid (209 mg, 0.600 mmol), HATU (228 mg,
0.600 mmol), and N,N-diisopropylethylamine (205 .mu.L, 1.20 mmol)
in N,N-dimethylformamide (2 mL) was added thereto, and the mixture
was shaken at room temperature for 1 hour. After filtration, the
resin was washed with N,N-dimethylformamide 4 times and
dichloromethane 4 times. A mixed solution of
1,1,1,3,3,3-hexafluoro-2-propanol (0.5 mL) and dichloromethane (1.5
mL) was added thereto, and the mixture was shaken at room
temperature for 2 hours. The resin was filtered off, and the
filtrate was concentrated under reduced pressure. The concentrate
was subjected to azeotropy with dichloromethane 6 times and dried
in a vacuum pump to obtain the title compound
TrS-(TrS-)Lys-PEG(3)-CO.sub.2H as a brown solid (105 mg).
[0576] (2-33B) Synthesis of TrS-(TrS-)Lys-PEG-(3)-hANP(1-28)
(compound of the following formula)
##STR00154##
[0577] The title compound TrS-(TrS-)Lys-PEG-(3)-hANP(1-28) (30 mg)
was obtained according to the same approach as in (2-17A) from the
TrS-(TrS-)Lys-PEG(3)-CO.sub.2H (27.4 mg) produced in (2-33A).
[0578] MALDI-TOF-MS: Calcd for
C.sub.188H.sub.272N.sub.48O.sub.47S.sub.5: [M+H].sup.+ 4114.9,
Found 4115.1.
[0579] (2-33C) Synthesis of HS-(HS-)Lys-PEG-(3)-hANP(1-28)
(compound of the following formula)
##STR00155##
[0580] The title compound HS-(HS-)Lys-PEG-(3)-hANP(1-28) (15.3 mg)
was obtained according to the same approach as in (2-17B) from the
TrS-(TrS-)Lys-PEG-(3)-hANP(1-28) (30.0 mg) produced in (2-33B).
[0581] MALDI-TOF-MS: Calcd for
C.sub.150H.sub.244N.sub.48O.sub.47S.sub.5: [M+H].sup.+ 3630.7,
Found 3631.0.
[0582] (2-33D) Synthesis of SG-Mal-(SG-Mal-)Lys-PEG-(3)-hANP(1-28)
(compound 2-33: compound of the following formula)
##STR00156##
[0583] The title compound SG-Mal-(SG-Mal-)Lys-PEG-(3)-hANP(1-28)
(compound 2-33) (8.55 mg) was obtained according to the same
approach as in (2-17C) from the HS-(HS-)Lys-PEG-(3)-hANP(1-28)
(5.00 mg) produced by the approach of (2-33C).
[0584] ESI-TOF-MS: Calcd for
C.sub.336H.sub.540N.sub.64O.sub.177S.sub.5: [M+5H].sup.5+ 1694.7
(ave.), Found 1694.5.
<Example 2-34> Synthesis of
SG-thioacetamide-(SG-Thioacetamide-)Lys-PEG-(3)-hANP(1-28)
(Compound 2-34)
[0585] (2-34A) Synthesis of
SG-thioacetamide-(SG-thioacetamide-)Lys-PEG-(3)-hANP(1-28)
(compound of the following formula)
##STR00157##
[0586] The title compound SG-thioacetamide-(SG-thioacetamide-)
Lys-PEG-(3)-hANP(1-28) (compound 2-34) (4.74 mg) was obtained
according to the same approach as in (2-25C) using the HS-(HS-)
Lys-PEG-(3)-hANP(1-28) (4.14 mg) produced in (2-33C) and the
compound SG-I (7.40 mg) produced in (1-19D).
[0587] ESI-TOF-MS: Calcd for
C.sub.326H.sub.530N.sub.62O.sub.173S.sub.5: [M+5H].sup.5+ 1650.3
(ave.), Found 1650.2.
<Example 2-35> Synthesis of SG-(SG-)Lys-PEG(3)-hANP(1-28)
(Compound 2-35)
[0588] (2-35A) Synthesis of Lys-PEG(3)-hANP(1-28) (compound of the
following formula)
##STR00158##
[0589] To a solution of the compound 1-20A (4.40 mg, 7.42 .mu.mol)
and HATU (2.6 mg, 6.84 .mu.mol) in N,N-dimethylformamide (94
.mu.L), N,N-diisopropylethylamine (5.0 .mu.L, 29.4 .mu.mol) was
added, and the mixture was stirred at room temperature for 3
minutes. The obtained reaction solution was added to a mixed
solution of hANP(1-28)-TFA salt (25 mg) and
N,N-diisopropylethylamine (13 .mu.L, 76.4 .mu.mol) in
N,N-dimethylformamide/water (5:1, v/v) (0.60 mL), and the mixture
was stirred at room temperature for 2 hours. A 0.2% aqueous
trifluoroacetic acid solution (3 mL) was added to the reaction
solution, and the resulting product was separated and purified by
reverse-phase HPLC (GL Sciences Inc., Inertsil ODS-3) using a 0.1%
aqueous trifluoroacetic acid solution and a 0.1% solution of
trifluoroacetic acid in acetonitrile as eluents and lyophilized to
obtain Boc-(Boc-) Lys-PEG(3)-hANP(1-28) (18.0 mg).
[0590] The title compound Lys-PEG(3)-hANP(1-28) (12.0 mg) was
obtained according to the same approach as in (2-2C) using the
obtained Boc-(Boc-)Lys-PEG(3)-hANP(1-28) (18.0 mg).
[0591] MALDI-TOF-MS: Calcd for
C.sub.144H.sub.237N.sub.48O.sub.45S.sub.3: [M+H].sup.+ 3454.7,
Found 3454.7.
[0592] (2-35C) Synthesis of SG-(SG-)Lys-PEG(3)-hANP(1-28) (compound
2-35: compound of the following formula)
##STR00159##
[0593] The title compound SG-(SG-)Lys-PEG(3)-hANP(1-28) (compound
2-35) (6.4 mg) was obtained according to the same approach as in
(2-1B) using the Lys-PEG(3)-hANP(1-28) (6.0 mg) produced in
(2-35B).
[0594] ESI-TOF-MS: Calcd for
C.sub.316H.sub.507N.sub.60O.sub.171S.sub.3: [M-5H].sup.5- 1595.7
(ave.), Found 1595.6.
<Example 2-36> Synthesis of
SG-(SG-)Asn-(Ser-Gly).sub.3-hANP(1-28) (Compound 2-36)
[0595] (2-36A) Synthesis of
GlcNAc-(GlcNAc-)Asn-(tBuSer-Gly).sub.3-hANP(1-28) (compound of the
following formula)
##STR00160##
[0596] The title compound
GlcNAc-(GlcNAc-)Asn-(tBuSer-Gly).sub.3-hANP(1-28) (27.0 mg) was
obtained according to the same approach as in (2-1B) using the
compound 1-21B (13.8 mg) and hANP(1-28)-TFA salt prepared from the
hANP-acetate (37.1 mg) by Preparation Method 2 of (2-1A).
[0597] MALDI-TOF-MS: Calcd for
C.sub.176H.sub.286N.sub.56O.sub.61S.sub.3: [M+H].sup.+ 4258.0,
Found 4257.8.
[0598] (2-36B) Synthesis of
GlcNAc-(GlcNAc-Asn)-(Ser-Gly).sub.3-hANP(1-28) (compound of the
following formula)
##STR00161##
[0599] The title compound
GlcNAc-(GlcNAc-Asn)-(Ser-Gly).sub.3-hANP(1-28) (4.66 mg) was
obtained according to the same approach as in (2-17A) from the
GlcNAc-(GlcNAc-Asn)-(tBuSer-Gly).sub.3-hANP(1-28) (27.0 mg)
produced in (2-36A).
[0600] MALDI-TOF-MS: Calcd for
C.sub.164H.sub.262N.sub.56O.sub.61S.sub.3: [M+H].sup.+ 4089.8,
Found 4090.1.
[0601] (2-36C) Synthesis of SG-(SG-Asn)-(Ser-Gly).sub.3-hANP(1-28)
(compound 2-36: compound of the following formula)
##STR00162##
[0602] The title compound SG-(SG-)Asn-(Ser-Gly).sub.3-hANP(1-28)
(compound 2-36) (3.24 mg) was obtained according to the same
approach as in (2-2D) from the
GlcNAc-(GlcNAc-)Asn-(Ser-Gly).sub.3-hANP(1-28) (3.50 mg) produced
in (2-36B).
[0603] ESI-TOF-MS: Calcd for
C.sub.316H.sub.508N.sub.66O.sub.173S.sub.3: [M+4H].sup.4+ 2025.0
(ave.), Found 2025.0.
<Example 2-37> Synthesis of SG-(SG-)Asn-Gly6-hANP(1-28)
(Compound 2-37)
[0604] (2-37A) Synthesis of Gly.sub.3-hANP(1-28) (compound of the
following formula)
##STR00163##
[0605] Boc-Gly.sub.3-hANP(1-28) (196 mg) was obtained according to
the same approach as in (2-1B) using
2-[[2-[[2-(tert-butoxycarbonylamino)acetyl]amino]acetyl]amino]acetic
acid (27.7 mg) and hANP-TFA salt prepared from hANP-acetate (246
mg) by Preparation Method 2 of (2-1A). The title compound
Gly.sub.3-hANP(1-28) (190 mg) was obtained according to the same
approach as in (2-2C) from the obtained Boc-Gly.sub.3-hANP(1-28)
(196 mg).
[0606] MALDI-TOF-MS: Calcd for
C.sub.133H.sub.212N.sub.48O.sub.42S.sub.3: [M+H].sup.+ 3250.5,
Found 3250.6.
[0607] (2-37B) Synthesis of Gly.sub.6-hANP(1-28) (compound of the
following formula)
##STR00164##
[0608] Boc-Gly.sub.6-hANP(1-28) (115 mg) was obtained according to
the same approach as in (2-1B) from the Gly.sub.3-hANP(1-28) (190
mg) produced in (2-37A) and
2-[[2-[[2-(tert-butoxycarbonylamino)acetyl]amino]acetyl]amino]acetic
acid (13.4 mg).
[0609] The title compound Gly.sub.6-hANP(1-28) (115 mg) was
obtained according to the same approach as in (2-2C) from the
obtained Boc-Gly.sub.6-hANP(1-28) (115 mg).
[0610] MALDI-TOF-MS: Calcd for
C.sub.139H.sub.221N.sub.51O.sub.45S.sub.3: [M+H].sup.+ 3421.6,
Found 3421.5.
[0611] (2-37C) Synthesis of Boc-(GlcNAc-)Asn-Gly.sub.6-hANP(1-28)
(compound of the following formula)
##STR00165##
[0612] The title compound Boc-(GlcNAc-)Asn-Gly.sub.6-hANP(1-28)
(23.0 mg) was obtained according to the same approach as in (1-5A)
from the Gly.sub.6-hANP(1-28) (60.0 mg) produced in (2-37B).
[0613] MALDI-TOF-MS: Calcd for
C.sub.156H.sub.248N.sub.54O.sub.54S.sub.3: [M+H].sup.+ 3838.7,
Found 3839.0.
[0614] (2-37D) Synthesis of
GlcNAc-(GlcNAc-)Asn-Gly.sub.6-hANP(1-28) (compound of the following
formula)
##STR00166##
[0615] The title compound GlcNAc-(GlcNAc-)Asn-Gly.sub.6-hANP(1-28)
was obtained as a white solid (6.77 mg) according to the same
approach as in (1-5B) from the
Boc-(GlcNAc-Asn)-Gly.sub.6-hANP(1-28) (23.0 mg) produced in
(2-37C).
[0616] MALDI-TOF-MS: Calcd for
C.sub.161H.sub.255N.sub.55O.sub.59S.sub.3: [M+H].sup.+ 3999.8,
Found 4000.1.
[0617] (2-37E) Synthesis of SG-(SG-)Asn-Gly.sub.6-hANP(1-28)
(compound 2-37: compound of the following formula)
##STR00167##
[0618] The title compound SG-(SG-)Asn-Gly.sub.6-hANP(1-28)
(compound 2-37) (4.32 mg) was obtained according to the same
approach as in (2-2D) from the GlcNAc-(GlcNAc-)Asn-Gly.sub.6-hANP
(1-28) (3.40 mg) produced in (2-37D).
[0619] ESI-TOF-MS: Calcd for
C.sub.313H.sub.501N.sub.65O.sub.171S.sub.3: [M+4H].sup.4+ 2002.7
(ave.), Found 2002.5.
<Example 2-38> Synthesis of
SG-Lys*-[PEG(3)-Mal-hANP(1-28)].sub.2
[0620] (compound 2-38: compound of the following formula)
##STR00168##
[0621] the Title Compound SG-Lys*-[PEG(3)-Mal-hANP(1-28)].sub.2
(compound 2-38) (7.0 mg) was obtained according to the same method
as in (2-32A) using the SG-Lys-[PEG(3)-Mal]2 (3.0 mg, 0.94 .mu.mol)
produced in (1-22B).
[0622] ESI-TOF-MS: Calcd for
C.sub.388H.sub.616N.sub.103O.sub.159S.sub.8: [M-5H].sup.5- 1904.8
(ave.), Found 1904.8.
<Example 2-39> Synthesis of
SG-Lys*-[PEG(11)-Mal-hANP(1-28)].sub.2 (Compound 2-39: Compound of
the Following Formula)
##STR00169##
[0624] The title compound SG-Lys*-[PEG(11)-Mal-hANP(1-28)].sub.2
(compound 2-39) (7.0 mg) was obtained according to the same method
as in (2-32A) using the SG-Lys*-[PEG(11)-Mal]2 (3.7 mg, 0.95
.mu.mol) produced in (1-23B).
[0625] ESI-TOF-MS: Calcd for
C.sub.420H.sub.680N.sub.103O.sub.175S.sub.8: [M-5H].sup.5- 2045.8
(ave.), Found 2045.8.
<Example 2-40> Synthesis of SG(Glc)-Gly-A-hANP(1-28)
(Compound 2-40: Compound of the Following Formula)
##STR00170##
[0627] The title compound SG(Glc)-Gly-A-hANP(1-28) (compound 2-40)
(34.2 mg) was obtained according to the same approach as in (2-1B)
using the SG(Glc)-Gly-A (30 mg) synthesized in (1-24E).
[0628] ESI-TOF-MS: Calcd for
C.sub.213H.sub.341N.sub.51O.sub.103S.sub.3: [M+4H].sup.4+ 1341.1
(ave.), Found 1341.0.
[0629] Also, modified hANP containing a glycochain altered at the
reducing end as the glycochain can be appropriately produced by use
of various glycochains altered at the reducing end synthesized
according to the methods of Example 1-11, 1-12, 1-13, or 1-14 and
Example 1-24 in the production of each modified hANP of Example
2.
TEST EXAMPLES
<Test Example 1> Test on cGMP Elevating Activity of
Glyco-Modified Peptide
[0630] The cGMP elevating activity of each modified peptide
prepared in Example 2 was measured by the following method:
[0631] CHO/human GC-A cells, which are CHO cells constitutively
expressing human GC-A, were suspended at 2.times.10.sup.5 cells/ml
in .alpha.-MEM, 10% FBS, and 1% penicillin-streptomycin, inoculated
at 20 .mu.l/well (4.times.10.sup.3 cells/well) onto a 384-well
plate (Corning, 3826), and cultured overnight in a CO.sub.2
incubator. On the next day, the medium was removed from this plate,
and then, 1.6 mM IBMX/KRB buffer was added thereto at 10
.mu.l/well. The mixture was stirred on a plate shaker and then
incubated at room temperature for 10 minutes. Next, a test
substance (each modified peptide and native hANP(1-28) (Peptide
Institute, Inc.); dilution series were prepared such that the final
concentration range involved 0.01, 0.1, 1, 10, and 100 nM) prepared
at a concentration 3 times the final concentration by dissolution
in water was added thereto at 5 .mu.l/well. The mixture was stirred
on a plate shaker and then incubated for 15 minutes in a CO.sub.2
incubator. Thereafter, a lysis buffer (50 mM phosphate buffer, pH
7.0, and 1% Triton X-100) was added thereto at 5 .mu.l/well. The
cells were lysed by stirring for 10 minutes on a plate shaker.
Subsequently, the cGMP levels in the cell lysates were measured by
use of a cGMP kit (manufactured by Cisbio Bioassays). Specifically,
to a 384-well plate (Greiner, 784076), 5 .mu.l/well of a diluent
attached to the kit, 5 .mu.l/well of the cell lysate, 5 .mu.l/well
of cGMP-d2, and 5 .mu.l/well of anti cGMP-Cryptate were added. The
mixture was stirred on a plate shaker and then incubated overnight
at 4.degree. C. in the dark, followed by the measurement of
homogeneous time-resolved fluorescence using RubyStar (manufactured
by BMG LABTECH JAPAN Ltd.). The activity value (T/C) of the test
substance at each concentration was calculated when the activity
value of a well supplemented with only a solvent was defined as 0
and the activity value of a well supplemented with 1 nM ANP was
defined as 1. T/C at each concentration was plotted, and the
maximum T/C value in the measurement concentration range was
determined as E.sub.max from the obtained sigmoid curve with the
value of T/C=0.5 defined as EC.sub.50 (Table 1).
[0632] From the results of Table 1, all of the modified peptides
were shown to exhibit 50% or more cGMP elevating activity compared
with hANP (Emax>0.5) and to maintain cGMP elevating activity.
The compounds 2-17, 2-18, and 2-20 were prone to have low Emax on
the order of 0.6 to 0.7, whereas the other modified peptides had
Emax of 0.95 or higher and maintained a cGMP elevating effect
equivalent to that of the native hANP.
[0633] cGMP elevating activity of test compound
TABLE-US-00001 TABLE 1 Compound No. of test EC50 substance (nM)
Emax Native hANP 0.022 2-1 0.04 1.02 2-2 0.1 1.01 2-3 0.12 1.02 2-4
0.08 1.04 2-5 0.082 1.04 2-6 1.7 1.00 2-7 1.7 0.99 2-8 0.2 1.01 2-9
1.1 1.00 2-10 0.03 1.02 2-11 0.048 1.01 2-12 0.69 1.01 2-13 0.34
1.01 2-14 0.6 0.95 2-15 3.1 0.98 2-16 0.98 1.01 2-17 35 0.70 2-18
48 0.63 2-19 9.8 0.90 2-20 64 0.59 2-21 0.097 1.02 2-22 0.08 1.02
2-23 2.4 0.98 2-24 0.54 1.01 2-25 0.054 1.02 2-26 0.024 1.01 2-27
0.26 1.02 2-28 0.31 1.01 2-29 0.1 1.01 2-30 0.12 1.01 2-31 0.31
1.00 2-32 0.41 1.00 2-33 0.4 1.01 2-34 0.53 1.01 2-35 0.31 1.00
2-36 0.39 1.00 2-37 0.4 1.00 2-38 0.016 1.01 2-39 0.0077 1.00 2-40
0.05 1.03
<Test Example 2> Test on NEP Degradation of Modified
Peptide
[0634] The resistance of each modified peptide prepared in Example
2 to degradation by neutral endopeptidase (generic name:
neprilysin) was examined by the following method:
[0635] Neprilysin (R&D systems, Inc.) was added at 1 .mu.g/ml
into a solution of a test substance (each glyco-modified ANP and
native hANP(1-28)), followed by pretreatment at 37.degree. C. for
30 minutes. The neprilysin-treated solution was used to examine the
cGMP elevating activity of the test substance by the method of Test
Example 1.
[0636] As a result, the native hANP lost its activity by the NEP
treatment, whereas the modified peptide of the present invention
maintained cGMP elevating activity at the same level as in Test
Example 1 even after the NEP treatment, demonstrating that the
modified peptide is insusceptible to degradation by NEP.
[0637] The main mechanism underlying the rapid disappearance of the
naturally occurring ANP from the blood of animals is considered to
be the degradation by NEP. The modified peptide of the present
invention maintained cGMP elevating activity even after the NEP
treatment, demonstrating that the modified peptide is insusceptible
to degradation by NEP even in the bodies of animals and, when
administered in an effective amount, can exert cGMP elevating
activity over a long time after the administration.
<Test Example 3> Test on duration time of modified peptide in
blood of rat
[0638] The duration time (the effect of persistently elevating cGMP
in blood and the time for which a test substance was detectable in
blood) of each modified peptide prepared in Example 2 in the blood
of rats was examined by the following method:
(1) Preparation of Plasma Sample
[0639] Isoflurane: Japanese pharmacopoeia isoflurane Needle and
syringe for blood collection: Terumo Syringe 25G.times.1 SR for
Tuberculin Tube for blood collection: CAPIJECT Micro Collection
Tube EDTA-2Na 500 .mu.L Tube for sample storage: MTARIX 4170 Sample
Tracking Tube 0.75 mL
[0640] Each 8-week-old male Slc:SD rat was subjected to isoflurane
inhalation anesthesia ((inhalation of an Escain inhalation
anesthetic kept at a concentration of 1 to 2%). A solution of a
test substance (each modified peptide and native hANP(1-28)
(Peptide Institute, Inc.)) prepared at a concentration of 100 .mu.M
by dissolution in water was rapidly intravenously injected at a
dose of 100 nmol/kg (1 mL/kg) into the jugular vein of the rat.
Before the administration and 15, 30, 60, 90, 120, 180, and 240
minutes after the administration, blood was sampled (200
.mu.L/sampling) over time from the jugular vein. The blood samples
were immediately left on ice.
[0641] The collected blood samples were centrifuged at 5000 rpm at
4.degree. C. for 5 minutes by use of a centrifuge (Sigma 4K15,
rotor: Nr12130-H). The separated plasma samples were divided into
two types (samples for PK measurement and for cGMP measurement) and
stored at -80.degree. C. until measurement.
(2) Measurement of cGMP Concentration in Plasma
[0642] The cGMP concentration in plasma was measured using Amersham
cGMP Enzyme Immunoassay Biotrak.TM. (EIA) System (dual range)
according to the protocol attached thereto. The results were
plotted with the cGMP concentration on the ordinate vs. the elapsed
time (min) after the administration on the abscissa to calculate
AUC of 0 minutes to 240 minutes (AUC0-240) and AUC of 60 to 240
minutes after the administration (AUC60-240) (Table 2).
(3) Detection of Test Substance in Plasma Sample
[0643] An internal standard (20 .mu.L (500 nM) of a stable isotope
of hANP) and an acetic acid mixed solvent (AcOH/distilled
water/DMSO=5/3/2, v/v/v) were added to 50 .mu.L of each rat plasma
sample prepared in (1) and then mixed therewith. The mixture was
transferred to Amicon Ultra-0.5 50K (Millipore Corp., MA) and
centrifuged at 14000 rpm at 15.degree. C. for 30 minutes. The
obtained filtrate was transferred to Amicon Ultra-0.5 3K (Millipore
Corp., MA) and centrifuged again under the aforementioned
conditions. The solution remaining on the filter was recovered and
transferred to a 96-well deep well plate. The content of the test
substance was measured by LC-MS/MS (LC: Shimadzu LC-10ADVP
(Shimadzu Corp.), MS/MS: API 4000 QTrap (AB SCIEX)) to calculate
the concentration in plasma. The time at which the test substance
was finally detected is shown in the rightmost column of Table
2.
Evaluation of Duration Time in Blood of Rat
TABLE-US-00002 [0644] TABLE 2 Maximum Com- Pre-value and time of
pound All values All values higher values detection No. of
integrated * integrated * integrated ** after test AUC AUC AUC
adminis- sub- [(pmol/ml)*h] [(pmol/ml)*h] [(pmol/ml)*h] tration
stance 0-240 60-240 60-240 (hr) Native 13.90 -33.86 0.00 0 hANP 2-1
488.98 188.59 191.43 3 2-3 340.97 116.61 136.00 2 2-10 735.39
323.59 323.59 1.5 2-11 874.06 397.08 399.13 2 2-12 581.28 193.48
198.16 1.5 2-13 268.64 72.88 75.73 3 2-14 179.61 35.76 43.03 1.5
2-15 47.20 7.14 36.06 1.5 2-16 173.71 33.08 8.66 2 2-25 524.33
205.83 207.18 2 2-26 245.50 13.72 60.43 2 2-27 208.58 30.24 41.71 2
2-29 430.38 143.46 156.49 2 2-30 365.22 136.33 138.87 4 * AUC value
obtained by using Pre-value (value at 0 minutes) as a baseline and
integrating differences at all points on the curve from the
baseline. Points on the curve under the baseline were calculated as
negative values. ** AUC value obtained by using Pre-value (value at
0 minutes) as a baseline and integrating differences at only points
on the curve above the baseline, from the baseline. Points on the
curve under the baseline were excluded from the calculation.
[0645] Although a transient upsurge in cGMP caused by the
administration was observed in the native hANP, this cGMP level
decreased 30 minutes after the administration to a level close to
that before the start of the administration. The elevation of cGMP
disappeared completely at 60 minutes or later. The native hANP
therefore had AUC60-240 of 0 or lower and was thus confirmed to
have no duration time in blood. In the detection of this test
substance in plasma, the native hANP was no longer detected in the
plasma sample even 15 minutes after the administration.
[0646] By contrast, the modified peptides of Example 2 exhibited a
high value of AUC60-240. The cGMP concentration in plasma elevated
by the administration of these test substances maintained a value
higher than that before the start of the administration, even at 60
minutes or later after the administration (180 minutes later for
the compounds 2-1 and 2-10, 120 minutes later for the compounds
2-12, 2-13, 2-14, and 2-16, and 60 minutes later for the compounds
2-11, 2-15, and 2-19). In addition, these test substances
themselves were still detected from the plasma sample 1.5 hours or
later after administration, demonstrating that the modified peptide
stays in blood over a long time without being metabolized in vivo.
From these results, the modified peptide of the present invention
was shown to have a prolonged duration time in blood and to
maintain cGMP elevating activity in this duration.
Sequence CWU 1
1
1128PRTHomo sapiensInventorIwamoto, Mitsuhiro;Yamaguchi,
Takahiro;Mori, Yutaka;Saito, Keiji;Honda, Takeshi; Nagayama,
Takahiro 1Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg
Ile Gly1 5 10 15Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20
25
* * * * *