U.S. patent application number 10/518303 was filed with the patent office on 2005-11-17 for process for the manufacture of n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus obtained.
This patent application is currently assigned to GLYCORES 2000 S.r.1.. Invention is credited to Oreste, Pasqua Anna, Zoppetti, Giorgio.
Application Number | 20050256079 10/518303 |
Document ID | / |
Family ID | 29740492 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050256079 |
Kind Code |
A1 |
Oreste, Pasqua Anna ; et
al. |
November 17, 2005 |
Process for the manufacture of
n-acyl-(epi)k5-amine-o-sulfate-derivatives and products thus
obtained
Abstract
A new method is described for the oversulfation of (epi)KS-N
sulfates to obtain (epi)K5-amine-O-oversulfates at extremely high
degree of sulfation and for the transformation of these
intermediates into new Nacyl-(epi)K5-amine-O-oversulfates basically
free of activity on the coagulation parameters and useful in the
cosmetic or pharmaceutical field. Also described are pharmaceutical
compositions containing, as one of their active ingredients, an
(epi)K5-amine-O-oversulfate.
Inventors: |
Oreste, Pasqua Anna; (Milan,
IT) ; Zoppetti, Giorgio; (Milan, IT) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
GLYCORES 2000 S.r.1.
Milan
IT
|
Family ID: |
29740492 |
Appl. No.: |
10/518303 |
Filed: |
May 26, 2005 |
PCT Filed: |
June 17, 2003 |
PCT NO: |
PCT/IB03/02339 |
Current U.S.
Class: |
514/54 ;
536/54 |
Current CPC
Class: |
C07H 1/00 20130101; A61P
17/16 20180101; A61P 3/06 20180101; A61K 31/737 20130101; A61P
17/06 20180101; A61P 3/10 20180101; A61P 35/04 20180101; C08B
37/006 20130101; A61P 7/02 20180101; A61P 39/06 20180101; A61P
19/02 20180101; A61P 27/02 20180101; C08B 37/0078 20130101; A61P
27/06 20180101; A61K 31/726 20130101; A61K 31/727 20130101; A61P
35/00 20180101; C08B 37/0075 20130101; A61P 31/12 20180101; A61P
9/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/054 ;
536/054 |
International
Class: |
A61K 031/737; C08B
037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2002 |
IT |
MI2002 A 001345 |
Jun 18, 2002 |
IT |
MI2002 A 001346 |
Aug 27, 2002 |
IT |
MI2002 A 001854 |
Claims
1-59. (canceled)
60. A process for the preparation of an
N-acyl-(epi)K5-amine-O-oversulfate- -derivative or of its
chemically or pharmaceutically acceptable salts, which comprises
(a) treating an (epi)K5-N-sulfate-derivative, in acidic form, with
a tertiary or quaternary organic base, letting the reaction mixture
to stand for a time period of 30-60 minutes, maintaining the pH of
the solution at a value of approximately 7 and isolating its salt
with said organic base; (b) treating said organic base salt of said
(epi)K5-N-sulfate-derivative with an O-sulfation reagent in the
conditions of O-oversulfation; and (c) treating the
(epi)K5-amine-O-oversulfate-derivative thus obtained with a
functional derivative of a C.sub.2-C.sub.4 carboxylic acid and
isolating the N-acyl-(epi)K5-amine-O-oversulfate-derivative.
61. Process according to claim 60, wherein said
N-acyl-(epi)K5-amine-O-ove- rsulfate is isolated in sodium salt
form and optionally transformed into another chemically or
pharmaceutically acceptable salt.
62. Process according claim 60, wherein, in step (a),
tetrabutylammonium hydroxide is used as an organic base.
63. Process according to claim 60, wherein in step (b) the
O-oversulfation is carried out in dimethylformamide utilizing 2-4
moles of O-sulfation reagent per available OH per disaccharide at a
temperature of 40-60.degree. C. for 15-20 hours.
64. Process according to claim 60, wherein as starting material an
(epi)K5-N-sulfate-derivative is used having a mean molecular weight
from approximately 1,000 to approximately 25,000.
65. Process according to claim 60, wherein said starting material
is 40-60% C5-epimerized.
66. Process according claim 60, wherein said starting material has
a mean molecular weight from approximately 1,500 to approximately
25,000.
67. Process according to claim 66, wherein said starting material
has a mean molecular weight between 10,000 and 25,000.
68. Process according to claim 66, wherein said starting material
has a mean molecular weight from approximately 1,500 to
approximately 12,000.
69. Process according to claim 68, wherein said starting material
has a mean molecular weight from approximately 1,500 to
approximately 8,000.
70. Process according to claim 60, wherein as starting material an
(epi)K5-N-sulfate-derivative is used consisting of a chain mixture
in which at least 90% of said chains have the formula I 28in which
the glucuronic units/iduronic units ratio is from 100/0 to 40/60, n
is an integer from 2 to 100 and the corresponding cation is
chemically or pharmaceutically acceptable.
71. Process according to claim 70, wherein said starting material
consists of a chain mixture in which at least 90% of said chains
have the formula I, in which the uronic units are 40-60% consisting
of iduronic acid.
72. Process according to claim 70, wherein said starting material
is a LMW-(epi)K5-N-sulfate consisting of a chain mixture in which
at least 90% of said chains have the formula I in which the uronic
units are all consisting of glucuronic acid or are 40-60%
consisting of iduronic acid, n is an integer from 3 to 15 and the
corresponding cation is chemically acceptable.
73. Process according to claim 70, wherein said starting material
is a MW-(epi)K5-N-sulfate consisting of a chain mixture in which at
least 90% of said chains have the formula I' 29in which the uronic
units are 100% consisting of glucuronic acid or 60-40% of
glucuronic acid and 40-60% of iduronic acid, q is an integer from 2
to 20 and the corresponding cation is chemically or
pharmaceutically acceptable.
74. Process according to claim 70, wherein said starting material
is a LMW-(epi)K5-N-sulfate consisting of a chain mixture in which
the preponderant species has the formula I'a 30in which the uronic
units are 100% consisting of glucuronic acid or 60-40% glucuronic
and 40% to 60% of iduronic acid, p is an integer from 4 to 8.
75. Process according to claim 64, wherein said starting material
is a LMW-(epi)K5-N-sulfate obtained by nitrous depolymerization of
the corresponding (epi)K5-N-sulfate and subsequent reduction.
76. Process according to claim 75, wherein said starting
LMW-(epi)K5-N-sulfate contains, at the reducing end of the majority
of the chains in said chain mixture, a 2,5-anhydromanno unit of
structure (a) 31in which X represents a hydroxymethyl group.
77. Process according to claim 75, wherein as starting material a
LMW-(epi)K5-N-sulfate is used consisting of chain mixtures in which
the preponderant species is a compound of formula I'b 32in which X
is hydroxymethyl, m is 4, 5 or 6, the corresponding cation is a
chemically or pharmaceutically acceptable ion, the uronic units are
all of glucuronic acid or the glucuronic and iduronic units are
present alternately, starting with a glucuronic or iduronic
unit.
78. Process according to claim 60, wherein said starting
(epi)K5-N-sulfate-derivative is utilized in sodium salt form.
79. A 100% acylated N-acyl-epiK5-amine-O-oversulfate-derivative, in
which acyl is a (C.sub.2-C.sub.4)acyl, having an iduronic acid
content of 20-60%, a mean molecular weight from approximately 2,000
to approximately 45,000 and a sulfation degree of at least 3.4, or
one of its chemically or pharmaceutically acceptable salts.
80. An N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 79, whose mean molecular weight is between approximately
15,000 and approximately 45,000.
81. An N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 79, whose mean molecular weight is between approximately
4,500 and approximately 8,500.
82. An N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 79, wherein said degree of sulfation is from 3.4 to 3.8.
83. A 100% N-acylated N-acyl-epiK5-amine-O-oversulfate-derivative
consisting of chain mixtures in which at least 90% of said chains
have the formula IV 33in which the uronic units are 20-60%
consisting of iduronic acid, n is an integer from 2 to 100, R, R'
and R" are hydrogen or SO.sub.3--, Z is (C.sub.2-C.sub.4)acyl, the
degree of sulfation is at least 3.4 and the corresponding cation is
chemically or pharmaceutically acceptable.
84. A N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 83, consisting of a chain mixture in which at least 90% of
said chains have the formula IV in which the uronic units are
40-60% consisting of iduronic acid, n is a integer from 3 to 100,
with a mean molecular weight from approximately 2,000 to
approximately 45,000, R is at least 40% SO.sub.3--, R' and R" are
both SO.sub.3-- or one is hydrogen and the other is 5-10%
SO.sub.3-- in monosulfate glucuronic acid and 10-15% SO.sub.3-- in
monosulfate iduronic acid and the corresponding cation is
chemically or pharmaceutically acceptable.
85. A N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 83, which is a LMW-N-acyl-epiK5-O-oversulfate consisting of a
chain mixture in which at least 90% of said chains have the formula
IV' 34in which q is an integer from 2 to 20, R, R' and R" represent
hydrogen or an SO.sub.3-- group for a degree of sulfation from 3.55
to 4, Z is (C.sub.2-C.sub.4)acyl, bearing a sulphated
2,5-anhydromannitol unit of structure (a') 35wherein R represent
hydrogen or SO.sub.3--, in the majority of the chains in said chain
mixture, and the corresponding cation is chemically or
pharmaceutically acceptable.
86. A LMW-N-acyl-epiK5-amine-O-oversulfate according to claim 85,
consisting of a chain mixture in which the preponderant species is
a compound of formula IV'a 36in which p is an integer from 4 to 8,
R, R' and R" are hydrogen or an SO.sub.3-- group for a degree of
sulfation from 3.55 to 4, Z is (C.sub.2-C.sub.4)acyl, and the
corresponding cation is chemically or pharmaceutically
acceptable.
87. A LMW-N-acyl-epiK5-amine-O-oversulfate according to claim 86,
wherein said preponderant species is a compound of formula IV'b
37in which R, R' and R" are hydrogen or SO.sub.3--, Z is
(C.sub.2-C.sub.4)acyl, X" is OH or OSO.sub.3--, m is 4, 5 or 6, for
a degree of sulfation from 3.55 to 4, the uronic units are present
alternately, starting with a glucuronic or iduronic unit, and the
corresponding cation is chemically or pharmaceutically
acceptable.
88. A N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 79 in which the substituent (C.sub.2-C.sub.4)acyl is selected
from the group consisting of acetyl, (2-carboxy)acetyl,
(2-methoxycarbonyl)acetyl, (2-ethoxycarbonyl)acetyl, propionyl,
(3-carboxy)propionyl, N-(3-methoxycarbonyl)propionyl and
(3-ethoxycarbonyl)propionyl.
89. An N-acyl-epiK5-amine-O-oversulfate-derivative according to
claim 79, wherein said salt is an alkaline metal or alkaline-earth
metal, ammonium, (C.sub.1-C.sub.4)tetraalkylammonium, aluminum or
zinc salt.
90. A 100% acylated N-acyl-K5-amine-O-oversulfate-derivative
consisting of a chain mixture in which at least 90% of said chains
have the formula V 38in which n is an integer from 2 to 100, Z is
(C.sub.2-C.sub.4)acyl, R, R' and R" are hydrogen or SO.sub.3--, the
degree of sulfation is from 2.2 to 3, and the corresponding cation
is chemically or pharmaceutically acceptable.
91. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
90, which is a LMW-N-acyl-K5-amine-O-oversulfate consisting of a
chain mixture in which at least 90% of said chains have the formula
V' 39in which q is an integer from 2 to 20, Z is
(C.sub.2-C.sub.4)acyl, R, R' and R" represent hydrogen or an
SO.sub.3-- group for a degree of sulfation from 2.2 to 3, the
majority of the chains in said chain mixture bearing a sulphated
2,5-anhydromannitol unit of structure (a') 40wherein R represent
hydrogen or SO.sub.3--, and the corresponding cation is chemically
or pharmaceutically acceptable.
92. A LMW-N-acyl-K5-amine-O-oversulfate according to claim 91,
consisting of a chain mixture in which the preponderant species is
a compound of formula V'a 41in which p is an integer from 4 to 8, Z
is (C.sub.2-C.sub.4)acyl, R, R' and R" represent hydrogen or an
SO.sub.3-- group for a degree of sulfation from 2.2 to 3, and the
corresponding cation is chemically or pharmaceutically
acceptable.
93. A LMW-N-acyl-K5-amine-O-oversulfate according to claim 92,
wherein said preponderant species is a compound of formula V'b 42in
which Z is (C.sub.2-C.sub.4)acyl, R, R' and R" are hydrogen or
SO.sub.3--, X" is OH or OSO.sub.3--, for a degree of sulfation from
2.2 to 3, m is 4, 5 or 6 and the corresponding cation is a
chemically or pharmaceutically acceptable ion.
94. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
90 wherein said degree of sulfation is from 2.3 to 3.
95. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
94, wherein said degree of sulfation is from 2.5 to 3.
96. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
95, wherein said degree of sulfation is from 2.7 to 2.9.
97. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
90, wherein the substituent (C.sub.2-C.sub.4)acyl is different from
acetyl.
98. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
90, in which the substituent (C.sub.2-C.sub.4)acyl is acetyl,
having a degree of sulfation of 2.7-2.9.
99. An N-acyl-K5-amine-O-oversulfate-derivative according to claim
98, having a degree of sulfation of approximately 2.8.
100. A N-acyl-K5-amine-O-oversulfate-derivative according to claim
90, wherein said salt is an alkaline metal, alkaline-earth metal,
ammonium, (C.sub.1-C.sub.4)tetraalkylammonium, aluminum or zinc
salt.
101. A pharmaceutical composition including, as an active
ingredient, an (epi)K5-amine-O-oversulfate-derivative or one of its
pharmaceutically acceptable salts, isolated in sodium salt form and
optionally transformed into another pharmaceutically acceptable
salt, in mixture with a pharmaceutical excipient.
102. Composition according to claim 101, wherein said active
ingredient is an (epi)K5-amine-O-oversulfate-derivative having a
mean molecular weight from approximately 4,500 to approximately
40,000.
103. Pharmaceutical composition according to claim 101, in which
said active ingredient is an (epi)K5-amine-O-oversulfate-derivative
consisting of a chain mixture in which at least 90% of said chains
have the formula II 43in which n is an integer from 2 to 100, R, R'
and R" are hydrogen or SO.sub.3--, the uronic units are all of
glucuronic acid, for a degree of sulfation from 2.2 to 3, or are
20-60% consisting of iduronic acid, for a sulfation degree of at
least 3.4, and the corresponding cation is pharmaceutically
acceptable.
104. Pharmaceutical composition according to claim 103, wherein
said active ingredient is a LMW-epiK5-amine-O-oversulfate
consisting of a chain mixture in which at least 90% of said chains
have the formula II' 44in which q is an integer from 2 to 20, R, R'
and R" are hydrogen or SO.sub.3--, the uronic units are 20-60%
those of iduronic acid, for a degree of sulfation from 3.55 to 4,
and bearing a sulphated 2,5-anhydromannitol unit of structure (a')
45wherein R represent hydrogen or SO.sub.3--, in the majority of
the chains in said chain mixture.
105. Pharmaceutical composition according to claim 104, wherein, in
said chain mixture of formula II', the uronic units are 40-60%
consisting of iduronic acid, R is at least 40% SO.sub.3--, R' and
R" are both SO.sub.3-- or one is hydrogen and the other is 5-10%
SO.sub.3-- in glucuronic acid and 10-15% SO.sub.3-- in iduronic
acid, n is an integer from 3 to 15, with a mean molecular weight
from approximately 4,000 to approximately 8,000.
106. Pharmaceutical composition according to claim 104, wherein
said LMW-epiK5-amine-O-oversulfate is consisting of a chain mixture
in which the preponderant species has the formula II'a 46in which p
is an integer from 4 to 8, R, R' and R" are as defined above, the
degree of sulfation is from 3.55 to 4, said preponderant species
bearing a sulphated 2,5-anhydromannitol unit of structure (a')
47wherein R represent hydrogen or SO.sub.3--, in the majority of
its chains in said chain mixture and the corresponding cation is
pharmaceutically acceptable.
107. Pharmaceutical composition according to claim 106, wherein
said preponderant species is a compound of formula II'b 48in which
R, R' and R" are hydrogen or SO.sub.3--, X" is OH or OSO.sub.3--, m
is 4, 5 or 6, the uronic units are 40-60% consisting of iduronic
acid, for a degree of sulfation from 3.55 to 4, the iduronic units
being present alternately, starting with a glucuronic or iduronic
unit, and the corresponding cation is a pharmaceutically acceptable
ion.
108. Pharmaceutical composition according to claim 101 including,
as an active ingredient, a K5-amine-O-oversulfate-derivative
consisting of a chain mixture in which at least 90% of said chains
have the formula III 49in which n is a integer from 2 to 100, R, R'
and R" are hydrogen or SO.sub.3--, the degree of sulfation is at
least 2.2, and the corresponding cation is pharmaceutically
acceptable.
109. Pharmaceutical composition according to claim 108, wherein
said active ingredient is a LMW-K5-amine-O-oversulfate consisting
of a chain mixture in which at least 90% of said chains have the
formula III' 50in which q is an integer from 2 to 20, R, R' and R"
represent hydrogen or a SO.sub.3-- group for a sulfation degree of
at least 2.2, and at the reducing end of the majority of the chains
in said chain mixture presents a sulphated 2,5-anhydromannitol unit
of structure (a') 51wherein R represents hydrogen or
SO.sub.3--.
110. Pharmaceutical composition according to claim 109, wherein
said LMW-K5-amine-O-oversulfate consists of a chain mixture in
which the preponderant species has the formula III'a 52in which p
is an integer from 4 to 8, R, R' and R" are as defined above, the
degree of sulfation being from 2.2 to 3.
111. Pharmaceutical composition according to claim 106, wherein
said preponderant species is a compound of formula III'b 53in which
R, R' and R" are hydrogen or SO.sub.3--, X" is OH or OSO.sub.3--,
for a degree of sulfation from 2.2 to 3, m is 4, 5 or 6 and the
corresponding cation is a pharmaceutically acceptable ion.
112. Pharmaceutical composition according to claim 101, wherein
said pharmaceutically acceptable salt or cation is sodium,
potassium, calcium, magnesium or zinc.
113. Pharmaceutical composition according to claim 101, which is in
the form of cream, ointment, liniment, gel, foam, balsam, vaginal
pessary, suppository, solution or suspension for local
administration.
114. A pharmaceutical composition containing, as an active
ingredient, a pharmacologically active amount of a
LMW-(epi)K5-N-sulfate basically free of acetyl groups, or of one of
its pharmaceutically acceptable salts, in mixture with a
pharmaceutical excipient.
115. A cosmetic composition containing an effective amount of a
LMW-(epi)K5-N-sulfate basically free of acetyl groups, or of one of
its pharmaceutically acceptable salts, in mixture with a cosmetic
excipient.
Description
SUBJECT MATTER OF THE INVENTION
[0001] The present invention concerns new derivatives of K5
polysaccharide with a high degree of sulfation, a process for their
preparation, new highly O-sulfated intermediates useful in their
synthesis and pharmaceutical compositions containing said
derivatives of K5 polysaccharide as active ingredients basically
free of activity on coagulation.
[0002] In particular, the invention refers to derivatives of K5
polysaccharide obtained starting with a K5 polysaccharide,
previously N-deacetylated, N-sulfated, optionally depolymerized or
optionally at least 20% C5-epimerized and optionally depolymerized,
by O-oversulfation in suitable conditions and subsequent
N-acylation of the free amine.
BACKGROUND OF THE INVENTION
[0003] The glycosaminoglycans such as heparin, heparan sulfate,
dermatan sulfate, chondroitin sulfate and hyaluronic acid are
biopolymers extracted industrially from various animal organs.
[0004] In particular, heparin, mainly obtained by extraction from
the intestinal mucous membrane of pigs or from bovine lung, is a
polydispersed copolymer with a molecular weight distribution from
approximately 3,000 to approximately 30,000 D consisting of a chain
mixture basically consisting of a uronic acid (glucuronic acid or
iduronic acid) and of an amino sugar (glucosamine) linked by bonds
.alpha.-1.fwdarw.4 or .beta.-1.fwdarw.4. In heparin, the uronic
unit can be O-sulfated in position 2 and the glucosamine unit is
N-acetylated or N-sulfated, 6-O-sulfated and 3-O-sulfated in
approximately 0.5% of the glucosamine units present.
[0005] The properties and the natural biosynthesis of heparin in
mammals have been described by Lindahl et al., 1986 in Lane, D. and
Lindahl, U. (Editors) "Heparin. Chemical and Biological Properties;
Clinical Applications", Edward Arnold, London, Pages 159-190, by
Lindahl, U, Feingold D. S. and Rodn L, 1986 TIBS, 11, 221-225 and
by Conrad H. E. "Heparin Binding Proteins", Chapter 2: Structure of
Heparinoids. Academic Press, 1998. The biosynthesis of heparin
occurs starting with its N-acetyl-heparosan precursor consisting of
a chain mixture consisting of the repetitive
glucuronyl-.beta.-1.fwdarw.4-N-acetylglucosamine disaccharide unit.
Said precursor undergoes enzymatic modifications which partially
hydrolyse the N-acetyl group, substituting it with an
SO.sub.3-group, epimerize the carboxy in position 5 of a part of
the glucuronic units transforming them into iduronic units and
introducing O-sulfate groups to get a product which, once extracted
industrially, has approximately double the number of sulfate groups
as regards carboxy groups per disaccharide unit. These enzymatic
modifications lead, i.a. to the formation of the pentasaccharide
region of a bond to antithrombin III (ATIII), called active
pentasaccharide, which is the structure necessary for the high
affinity bond of heparin to ATIII and fundamental for anticoagulant
and antithrombotic activity of the heparin itself. This
pentasaccharide, present inside only some of the chains which form
heparin, contains a sulfated glucosamine unit in position 3 and a
glucuronic acid spaced out between disaccharides containing
iduronic acids.
[0006] In nature, the formation of the active pentasaccharide is
made possible by the epimerization reaction of the carboxy of a
part of the glucuronic units into iduronic units carried out by the
glucuronyl-C5-epimerase (C5-epimerization) and by suitable
sulfation which also leads to the introduction of a sulfate group
on the hydroxyl in position 3 of the glucosamine. More
particularly, in nature the formation of the active pentasaccharide
is made possible by the fact that C5-epimerization occurs in
clusters, i.e. on portions of chains, and extensively which leads
to a product which contains more iduronic units than glucuronic
ones. In fact, commercial heparin contains approximately 70% of
iduronic units and 30% of glucuronic units.
[0007] Alongside the main anticoagulant and antithrombotic
activities, heparin also exercises antilipaemic, antiproliferative,
antiviral, antitumorous and antimetastatic activities, but its use
as a drug is hindered by the side effects due to the anticoagulant
action which can cause bleeding.
PRIOR ART
[0008] It is known that the capsular K5 polysaccharide isolated
from Escherichia coli, described by Vann W. F. et al., in European
Journal of Biochemistry, 1981, 116, 359-364 ("Vann 1981"), is
consisting of a chain mixture consisting of the repetitive
disaccharide unit glucuronyl-.beta.-1.fwdarw.4-N-acetyl glucosamine
and therefore shows the same repetitive sequence (A) 1
[0009] of N-acetyl-heparosan precursor of heparin. The capsular K5
polysaccharide, referred to hereafter as "K5 polysaccharide" or
more simply "K5", was chemically modified by Lormeau et al. as
described in U.S. Pat. No. 5,550,116 and by Casu et al. as
described in Carbohydrate Research, 1994, 263, 271-284.
K5-O-sulfates having antitumorous, antimetastatic, antiviral, in
particular anti-HIV activities are described in EP 333243 and WO
98/34958. The K5 was also chemically and enzymatically modified in
order to obtain products having in vitro biological activity on
coagulation of the some type as that of heparin as extracted from
animal organs (extractive heparin).
[0010] The attainment of the products having an activity on
coagulation of the some type as that of extractive heparin occurs
by processes which imitate that occurring in nature and envisage
the entire key step of C5-epimerization with D-glucuronyl C5
epimerase.
[0011] The processes described in IT 1230785, WO 92/17507, WO
96/14425 and WO 97/43317 utilize K5 as the starting material. The
K5 originating from fermentation is subjected to N-deacetylation
followed by N-sulfation and on the K5-N-sulfate thus obtained
C5-epimerization with C5-epimerase in solution is performed,
obtained either by chromatography of a solution of microsomal
enzymes from mouse mastocytoma (IT 1230 785) or from bovine liver
(WO 92/17507, WO 96/14425 and WO 97/43317).
[0012] The D-glucuronyl C5 epimerase from bovine liver was purified
by Campbell, P. et al. in J. Biol. Chem., 1994, 269/43, 26953-26958
("Campbell 1994") who also provided its composition in amino acids
and described its use in solution for the transformation of a
K5-N-sulfate into the corresponding 30% epimerized product,
demonstrating the formation of iduronic acid by HPLC method
followed by total nitrous depolymerization to disaccharide.
[0013] The document WO 98/48006 describes the DNA sequence which
codes for the D-glucuronyl C5 epimerase and a recombinant
D-glucuronyl C5 epimerase, obtained from a recombinant expression
vector containing said DNA, afterwards purified by Campbell et al.
as shown by Jin-Ping L. et al. in J. Biol. Chem. 2001, 276,
20069-20077 ("Jin-Ping 2001").
[0014] The complete sequence of the C5-epimerase was described by
Crawford B. E. et al. in J. Biol. Chem., 2001, 276(24), 21538-21543
(Crawford 2001)
[0015] The document WO 01/72848 describes a method for the
preparation of N-deacetylated N-sulfate derivatives of K5
polysaccharide, at least 40% epimerized of iduronic acid as regards
the total of the uronic acids, having a molecular weight from 2000
to 30,000, containing from 25 to 50% of high affinity chains for
ATIII and having an anticoagulant and antithrombotic activity
expressed as HClI/antiXa ratio from 1.5 to 4. Said document
describes the oversulfation of a 40-60% epimerized K5-N-sulfate and
shows that the product obtained, whose .sup.13C-RMN is illustrated,
has a sulfate group content per disaccharide unit of 2-3.5.
Repeating the aforesaid oversulfation in the conditions described
and examining the .sup.13C-RMN it is ascertained that the product
obtained is actually a free amine whose content of 6-O-sulfate is
80-95%, that of 3-O-sulfate on the amino sugar is 30%, but whose
sulfation degree is 3.2. It is also ascertained that in the
conditions of oversulfation described in WO 01/72848 a sulfation
degree of more than 3.2 is not obtained.
[0016] The patent application M12001A/00397 (WO 02/068477),
describes K5-N,O-oversulfated having a sulfation degree of more
than 3.2, obtained starting with a K5 free of lipophilic substances
or one of its moieties of molecular weight of approximately 5,000
by (a) N-deacetylation/N-sulfa- tion, (b) O-oversulfation and,
optionally, (c) N-resulfation.
[0017] None of the aforesaid documents describes LMW-K5-N-sulfates,
optionally 40-60% epimerized, in which NH.sub.2 or acetyl groups
are virtually absent.
[0018] In order to standardize the terminology and render the text
more comprehensible, in the present description conventional terms
or expressions will be used, in the singular or plural. In
particular:
[0019] by "K5" or "K5 polysaccharide" is meant the capsular
polysaccharide from Escherichia coli obtained by fermentation, i.e.
a chain mixture consisting of disaccharide units (A) optionally
containing a double bond at the non-reducing end as shown above, in
any case prepared and purified according to the methods described
in literature, in particular according to Vann 1981, according to
Manzoni M. et al., Journal of Bioactive Compatible Polymers, 1996,
11, 301-311 ("Manzoni 1996") or according to the method described
in WO 01/72848 and in WO 02/068447; it is obvious for a person
skilled in the art that what is shown hereafter can be applied to
any N-acetylheparosan;
[0020] by "C5-epimerase" is meant the D-glucuronyl C-5 epimerase,
extractive or recombinant, in any case prepared, isolated and
purified, in particular as described in Campbell 1994, in WO
98/48006, in Jin-Ping L. et al. in J. Biol. Chem. 2001, 276,
20069-20077 (Jin-Ping 2001") or in Crawford 2001;
[0021] by K5-amine is meant at least 95% N-deacetylated K5, but
generally in which acetyl groups are undetectable by a current NMR
apparatus;
[0022] by "K5-N-sulfate" is meant at least 95% N-deacetylated and
N-sulfate K5 as described hereafter, but in which acetyl groups are
undetectable with a normal NMR apparatus;
[0023] by "epiK5" is meant the K5 and its derivatives in which
20-60% of the glucuronic units are C5-epimerized to iduronic
units;
[0024] by "epiK5-N-sulfate" is meant the K5-N-sulfate in which
20-60% of the glucuronic units is C5-epimerized to iduronic units
of the type described in WO 92/17507 or WO 01/72848;
[0025] by "epiK5-amine-O-oversulfate" is meant an O-sulfated
epiK5-amine with a sulfation degree of at least 3.4;
[0026] by "N-acyl-epiK5-amine-O-oversulfate" is meant an N-acylated
epiK5-amine O-oversulfate, with a sulfation degree of at least
3.4;
[0027] by "K5-amine-O-oversulfate" is meant an O-sulfated K5-amine
with a sulfation degree of at least 2.2;
[0028] by "N-acyl-K5-amine-O-oversulfate" is meant an N-acylated
N-acyl-K5-amine-O-oversulfate with a sulfation degree of at least
2.2;
[0029] In addition:
[0030] the conventional terms and expressions herein defined above
refer to K5 as isolated after fermentation, generally with a
molecular weight distribution from approximately 1,500 to
approximately 50,000 with a mean molecular weight of 10,000-25,000,
advantageously of 15,000-25,000;
[0031] excepting specific designation of the molecular weight, the
conventional terms and expressions herein defined above, when
preceded by the acronym "LMW" (low molecular weight), for example
LMW-K5-N-sulfate, LMW-epiK5-N-sulfate indicate low molecular weight
products, having a mean molecular weight of up to 12,000;
[0032] the conventional terms and expressions as herein defined
above, when followed by "-derivative" indicate as a whole both the
derivatives from native K5 and those of a low molecular weight;
[0033] the prefix "(epi)", which precedes "K5" in conventional
terms and expressions as herein defined above, indicates as a whole
both the products from native K5 and those 20-60% epimerized as
defined above, i.e. chain mixtures all consisting of glucuronic
acid or glucuronic acid and, 20-60%, of iduronic acid and is
denoted in the formulae by the symbol "";
[0034] by the term "approximately" referring to the molecular
weight is meant the molecular weight [.+-. the theoretical weight
of a disaccharide unit, including the weight of the sodium,
calculated as 461 in the case of an (epi)K5-N-sulfate-derivative]
measured by viscometry according to Johnson et al. Carb. Res. 1976,
51, 119-127 utilizing samples whose molecular weight was calculated
by HPLC as the standard;
[0035] by the expression "preponderant species", is meant the
compound which, in the mixture constituting a
lmw-(epi)K5-N-sulfate, a lmw-(epi)K5-amine-oversulfate or a
lmw-N-acyl-(epi)K5-amine-O-oversulfate- , is the most represented
type, determined by the peak of the curve of the molecular weight
measured by HPLC;
[0036] unless otherwise specifically stated, by "degree of
sulfation" is meant the SO.sub.3--/COO.sup.- ratio, expressible
also as the number of sulfate groups per disaccharide unit,
measured with the conductometric method described by Casu B. et al.
in Carbohydrate Research, 1975, 39, 168-176 (Casu 1975), the same
utilized in WO 01/72848;
[0037] by "conditions of O-oversulfation" is meant an extreme
O-sulfation performed for example according to the method C
described by B. Casu et al. in Carbohydrate Research, 1994, 263,
271-284 (Casu 1994);
[0038] by the term "alkyl" is meant a linear or branched alkyl,
whereas the term "tetrabutylammonium" indicates the
tetra-n-butylammonium.
SUMMARY OF THE INVENTION
[0039] It has now been found that, starting with an
epiK5-N-sulfate, it is possible to obtain an epiK5-amine-O-sulfate
with a degree of sulfation greater than that of every other
epiK5-amine-O-sulfate described in literature, for example in WO
01/72848, preparing the salt with a tertiary or quaternary organic
base of said epiK5-N-sulfate-derivative taking care to let the
reaction mixture to stand for a time period of 30-60 minutes
maintaining the pH at approximately 7 with the same organic base
and then treating the salt obtained with an O-sulfation reagent in
the conditions of O-oversulfation. Also a lmw-epiK5-N-sulfate, when
subjected to the same method of salification and of O-oversulfation
gives a lmw-epiK5-amine-O-sulfate with an extremely high degree of
sulfation. In the case of such epi-K5-amine-oversulfate-derivatives
the degree of sulfation is more than 3.2.
[0040] Similarly, it was found that, applying the conditions of
O-oversulfation described above to a lmw-K5-N-sulfate a
lmw-K5-amine-O-sulfate is obtained at a high degree of sulfation.
Also a K5-N-sulfate, when subjected to the same method of
salification and of O-oversulfation, gives a K5-amine-O-sulfate
with a high degree of sulfation. In the case of such
K5-amine-oversulfate-derivatives the degree of sulfation is more
than 2.2.
[0041] In fact, it was surprisingly found that, preparing the
tetrabutylammonium salt of an (epi)K5-N-sulfate-derivative in the
aforesaid conditions, the salification becomes complete and, during
the subsequent O-oversulfation, favours the total displacement of
the sulfate group from the nitrogen atom in position 2 of the
glucosamine to the oxygen atoms of the glucosamine itself, in
particular to the oxygen atom in position 3, thus giving rise to
(epi)K5-amine-O-sulfate-derivatives with a high degree of sulfation
in which the NH.sub.2 group is strictly free. The quantitative
difference in sulfation degree of the derivatives epiK5 and K5
depends on their configuration which favours the sulfation of the
epimerized derivatives.
[0042] It has also been found that, subjecting an
(epi)K5-amine-O-oversulf- ate-derivotive thus obtained to
N-acylation, new N-acyl-(epi)K5-amine-O-su- lfate-derivatives are
obtained free of activity on coagulation and useful for the
preparation of pharmaceutical or cosmetic compositions. By this new
and versatile process it is thus possible to prepare in a
reproducible manner epiK5-O-sulfate-derivatives, their N-acyl
analogues, a K5-O-sulfate having a precise degree of sulfation and,
above all, its derivatives of low molecular weight and their
N-acyl-analogues.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 shows the NMR proton spectrum of K5-NH.sub.2
O-sulfate with a sulfate/carboxy ratio of 2.87 (obtained according
to Example 1);
[0044] FIG. 2 shows the NMR proton spectrum of epiK5-N-acetylate,
O-sulfate with a sulfate/carboxy ratio of 4.26 (obtained according
to Example 2);
[0045] FIG. 3 shows the NMR proton spectrum of K5-N-acetylate,
O-sulfate with a sulfate/carboxy ratio of 2.87 (obtained according
to Example 3).
DETAILED DESCRIPTION
[0046] Thus, according to one of its aspects, the present invention
provides a process for the preparation of
N-acyl-(epi)K5-amine-O-oversulf- ate-derivatives, characterized in
that
[0047] (a) an (epi)K5-N-sulfate-derivative, in acidic form, is
treated with a tertiary or quaternary organic base, letting the
reaction mixture to stand for a time period of 30-60 minutes,
maintaining the pH of the solution at a value of approximately 7
and its salt is isolated with said organic base;
[0048] (b) said salt of organic base of said
(epi)K5-N-sulfate-derivative is treated with an O-sulfation reagent
in the conditions of O-oversulfation;
[0049] (c) the product thus obtained is treated with a functional
derivative of a (C2-C4) carboxylic acid, the
N-acyl-(epi)K5-amine-O-overs- ulfate-derivative thus obtained is
isolated.
[0050] Generally the N-acyl-(epi)K5-amine-O-oversulfate-derivative
is isolated in sodium salt form and optionally said sodium salt is
transformed into another chemically or pharmaceutically acceptable
salt.
[0051] In this context, the term "chemically acceptable" refers to
a cation usable in chemical synthesis, such as the ions sodium,
ammonium, (C.sub.1-C.sub.4)tetraalkylammonium, or for the
purification of the product, whereas "pharmaceutically acceptable"
is self-explanatory.
[0052] Advantageous cations are those derived from alkaline metals,
alkaline-earth metals, ammonium,
(C.sub.1-C.sub.4)tetraalkylammonium, aluminum and zinc. Preferred
cations are the sodium, calcium and tetrabutylammonium ions.
[0053] According to an advantageous procedure, the step (a) is
carried out by passing a solution of the sodium salt of the
(epi)K5-N-sulfate-derivat- ive, i.e. of K5 polysaccharide,
previously N-deacetylated, N-sulfated preferably 100%, optionally
20-60% C5-epimerized and optionally depolymerized with nitrous
acid, having a mean molecular weight from approximately 1,000 to
approximately 25,000, advantageously from approximately 1,500 to
approximately 25,000, through an acid ionic exchange resin, for
example of the type IR-120H.sup.+, collecting the eluate including
also the washing water of the resin and neutralizing the eluate
with tertiary or quaternary organic base, preferably with an
aqueous solution of tetrabutylammonium hydroxide. The solution is
let to stand for 1 hour, maintaining its pH at approximately 7
(i.e. trying to maintain neutrality) by addition of the same
tertiary or quaternary organic base, preferably an aqueous solution
of tetrabutylammonium hydroxide, and the salt thus obtained is
recovered by lyophilization.
[0054] In step (b), the O-oversulfation occurs utilizing an excess
of O-sulfating agent and working at a temperature from 20 to
70.degree. C. for a time period of up to 24 hours in an aprotic
polar solvent.
[0055] Advantageously, the salt with a tertiary or quaternary
organic base of the (epi)K5-N-sulfate-derivative, i.e. of K5
polysaccharide, previously N-deacetylated, N-sulfated preferably
100%, optionally 20-60% C5epimerized and optionally depolymerized
with nitrous acid, having a mean molecular weight from
approximately 1,500 to approximately 25,000 as isolated in step
(a), is dissolved in dimethylformamide and treated with 2-10 moles
of an O-sulfation reagent for every free hydroxyl at a temperature
of 40-60.degree. C. for 10-20 hours. As the O-sulfation reagent is
advantageously used the pyridine.SO.sub.3 adduct in a quantity of
2.5-5 moles, preferably 2.5-4 moles per free hydroxyl per
disaccharide and the reaction is advantageously carried out at
50-60.degree. C., preferably at 55.degree. C. overnight. The
product obtained upon termination of the reaction is isolated by
the addition of 0.1-1 volume of water and neutralization,
preferably with sodium hydroxide, precipitation with a saturated
sodium chloride solution in acetone, filtration and possible
ultrafiltration.
[0056] The product thus obtained is generally the sodium salt of an
(epi)K5-amine-O-oversulfate-derivative having a glucuronic and
iduronic acids content as shown above. When the oversulfation
reaction is performed on the salt of the tertiary or quaternary
base, preferably on the tetrabutylammonium salt of an
epiK5-N-sulfate-derivative having a mean molecular weight from
approximately 1,000 to approximately 25,000, advantageously from
approximately 1,500 to approximately 25,000 in which the iduronic
acid is 20-60% of the total of the uronic acids, an
epiK5-amine-O-oversulfate-derivative is obtained having a mean
molecular weight from approximately 3,500 to approximately 40,000,
advantageously from approximately 4,500 to approximately 40,000,
and a sulfation degree of at least 3.4, advantageously of at least
3.5, more advantageously from 3.55 to 4, preferably from 3.55 to
3.8.
[0057] When the oversulfation reaction is performed on the salt of
the tertiary or quaternary base, preferably on the
tetrabutylammonium salt of a K5-N-sulfate-derivative having a mean
molecular weight from approximately 1,500 to approximately 25,000,
a K5-amine-O-oversulfate-der- ivative is obtained having a mean
molecular weight from approximately 3,500 to 40,000, advantageously
from approximately 4,500 to approximately 40,000 and a sulfation
degree of at least 2.2, advantageously from 2.2 to 3 or from 2.3 to
3, more advantageously from 2.5 to 3, preferably from 2.7 to 2.9.
Preferably, the product thus obtained comes from a lmw-K5-N-sulfate
and is preferably the sodium salt of a lmw-K5-amine-O-oversulfated
having a degree of sulfation from 2.3 to 3. The mean molecular
weight of such product can be from approximately 3,500 to
approximately 11,000.
[0058] The sodium salt thus obtained can be converted into another
salt. By way of example, calcium ionic exchange can be performed
working with an ultrafiltration membrane.
[0059] In step (c), the (epi)K5-amine-O-oversulfate-derivative with
a high degree of sulfation is N-acylated utilizing the known
methods in literature.
[0060] The N-acylation is performed by making the
(epi)K5-amine-O-oversulf- ate-derivative originating from step (b)
react with a functional derivative of a mono or dicarboxylic acid
containing from 2 to 4 carbon atoms in hydroalcoholic solution at a
temperature of approximately 4.degree. C. As functional derivatives
of said (C.sub.2-C.sub.4) carboxylic acids, preferably of acetic,
propionic, malonic, or succinic acid or of mono esters, in
particular methylic or ethylic, of the latter, one can use the
anhydride, the chloride, a mixed anhydride or an active ester. The
product obtained, an N-(C.sub.2-C.sub.4)acyl-(epi)K5-amine-O-o-
versulfate-derivative, is neutralized with a base, preferably
sodium hydroxide, and then isolated by ultrafiltration and
precipitation with a saturated sodium chloride solution in acetone.
Optionally the step of N-acylation is repeated until obtaining
total substitution or however more than 95%. Are thus obtained the
N-acyl-(epi)K5-amine-O-oversulfates, in particular the N-acetyl-,
N-(2-carboxy)acetyl-, N-(2-methoxycarbonyl)acetyl-,
N-(2-ethoxycarbonyl)acetyl-, N-propionyl-, N-(3-carboxy)
propionyl-, N-(3-methoxycarbonyl)propionyl-, N-(3-ethoxycarbonyl)
propionyl-(epi) K5-amine-O-oversulfates.
[0061] The new N-acyl-(epi)K5-amine-O-oversulfate-derivatives thus
obtained are generally in their sodium salt forms. Said sodium salt
can be converted into another chemically or pharmaceutically
acceptable salt. Particularly advantageous salts are those of
alkaline metals, alkaline-earth metals, of ammonium,
(C.sub.1-C.sub.4)tetraalkylammonium, aluminum and zinc. The salts
of sodium, calcium and tetrabutylammonium are preferred.
[0062] Unlike the process described in WO 98/34958, which consists
in a sulfation in heterogeneous phase of the K5 sodium salt to
obtain K5-O-sulfates (according to the terminology used herein
"N-acetyl-K5-amine-O-sulfates") with a degree of sulfation from 0.5
to 4 and which has to resort to the use of chlorosulfonic acid to
obtain derivatives of low molecular weight in an uncontrolled
manner, the process of the present invention is extremely
versatile. In fact, it allows the preparation of
N-acyl-K5-amine-O-oversulfates of every type in very high yields,
with easily controllable reactions. In particular, the process of
the present invention allows the preparation of
N-acyl-K5-amine-O-oversulfates otherwise unobtainable, like the
epimerized C-5 derivatives, the N-acylated derivatives with a
different acyl from the acetyl present in the native K5 and the
derivatives of low molecular weight. Besides, unlike the method
described in WO 98/34958, the process of the present invention
allows the attainment of LMW-K5-O-sulfates in controlled manner in
order to obtain the mean molecular weight desired and, above all, a
well defined sulfation degree which can range from 2.3 to 3 but
which can regularly be 2.7-2.9. Finally, all the products of the
process of the present invention, in particular the free
intermediate amines and the N-acyl-K5-amine-O-oversul- fates are
useful active ingredients for pharmaceutical or cosmetic
compositions.
[0063] The starting materials of step (a) are
(epi)K5-N-sulfate-derivative- s known in literature or their
moieties, or LMW-(epi)K5-N-sulfates prepared by nitrous
depolymerization of the corresponding (epi)K5-sulfates.
[0064] The starting (epi)K5-N-sulfate-derivatives have a mean
molecular weight from approximately 1,000 to approximately 25,000,
advantageously from approximately 1.500 to approximately 25,000.
Preferably, as starting material is used an (epi)K5-N-sulfate
having a mean molecular weight between 10,000 and 25,000 or
LMW-(epi)K5-N-sulfate having a mean molecular weight from
approximately 1,500 to approximately 12,000, advantageously from
approximately 1,500 to approximately 8,000, preferably from
approximately 1,500 and approximately 7,500.
[0065] A lmwepiK5-N-sulfate having an iduronic units content of
approximately 20%, obtained by N-deacetylotion, N-sulfation and
C5-epimerization of a moiety of K5 having a mean molecular weight
of 5,000 is described in WO 92/17507, but this product contains a
high percentage of acetyl groups.
[0066] An epiK5-N-sulfate particularly advantageous as starting
material is that obtained by epimerization of a K5-N-sulfate
virtually free of acetyl groups in turn prepared from particularly
pure K5, in particular not containing lipophilic substances,
described in WO 02/068477.
[0067] The low molecular weight C5-epimerized K5-N-sulfates having
a higher content of iduronic units, in particular 40-60%,
preferably 50-55%, are instead particularly advantageous new
products as starting materials in the preparation of the
N-substituted epiK5-amine-O-oversulfa- ted-derivatives of the
present invention.
[0068] The LMW-epiK5-N-sulfates con also be obtained by
C5-epimerization of the corresponding LMW-K5-N-sulfates, preferably
when their mean molecular weight is more than 4,000. The
LMW-K5-N-sulfates virtually free of acetyl groups are all new
products preparable by fractionation or, preferably, by
depolymerization of a K5-N-sulfate free of acetyl groups. The
K5-N-sulfate is well known in literature and is described in
documents cited herein above to illustrate the state of the art.
The aforesaid starting material is invariably obtained by
N-deacetylation of K5 and subsequent N-sulfation of the K5-amine
thus obtained. However, it was ascertained that the preparation of
a K5-N-sulfate virtually free of acetyl or NH.sub.2 groups is
helped if the K5 from which it is prepared is particularly pure, in
particular if it does not contain lipophilic substances. It is
therefore preferred, according to the present invention, to use a
starting K5-N-sulfate prepared from a purified K5 as described in
WO 02/068477. Said K5-N-sulfate, whose .sup.13C-RMN spectrum does
not show traces of N-acetyl or NH.sub.2 groups is also described in
WO 02/068477.
[0069] The new LMW-epiK5-N-sulfates as shown above are prepared by
a process characterized in that a K5-N-sulfate is subjected, in any
one order,
[0070] (i) to C5-epimerization with a D-glucuronyl C5-epimerase
that is isolated, purified and in solution or immobilized on a
solid support, at a pH of approximately 7, at a temperature of
approximately 30.degree. C. and for a time period of 12-24 hours in
the presence of at least one bivalent ion selected among calcium,
magnesium, barium and manganese; and
[0071] (ii) to nitrous depolymerization optionally followed by
reduction, normally with sodium borohydride.
[0072] The expression "in any order" shows that the process can be
indifferently carried out both in the direction (i)-(ii), i.e. in
the sequence above indicated, and in the reverse direction, i.e.
also in the direction (ii)-(i), subjecting the K5-N-sulfate at
first to the nitrous depolymerization reaction, optionally followed
by reduction with sodium borohydride, and afterwards to the
C5-epimerization in the aforesaid conditions. The preferred order
is in the direction (i).fwdarw.(ii). The sequence (ii)-(i) is
preferably utilized starting with LMW-K5-N-sulfates having a mean
molecular weight of more than 4000, preferably starting with
approximately 6,000. For example, one can determine the amounts of
sodium nitrite which, starting with 1 g of epiK5-N-sulfate, allow
the attainment of a lmw-epiK5-N-sulfate with a molecular weight of
more than 4,000, in particular of at least 6,000, so as to obtain
useful intermediates for the preparation of
LMWepiK5-N,O-oversulfates. In fact, in this case, in step (ii) the
percentage of optimum epimerization is obtained.
[0073] According to a preferential aspect of the invention, the
C5-epimerase is immobilized on an inert solid support.
[0074] The C5-epimerase, preferably recombinant, isolated and
purified for example according to Campbell 1994, WO 98/48006,
Jin-Ping 2001 or Crawford 2001, is immobilized on an inert support
in the presence of the substrate, i.e. in the presence of starting
K5-N-sulfate-derivative or in the presence of LMW-K5-N-sulfate,
advantageously having a mean molecular weight of more than 4,000,
preferably of at least 6,000. The immobilization is performed
according to conventional methods, for example as described in WO
01/72848.
[0075] The C-5epimerization reaction is carried out by
recirculating 20-1,000 ml of a solution of 25 mM HEPES at a pH of
approximately 7 containing 0.001-10 g of substrate (K5-N-sulfate or
LMW-K5-N-sulfate, preferably with a molecular weight of more than
4,000, in particular of at least 6,000) and a cation selected among
calcium, magnesium, barium and manganese at a concentration between
10 and 60 mM through a column containing from 1.2.times.10.sup.7 to
3.times.10.sup.11 cpm of the immobilized enzyme, maintaining the pH
at approximately 7 at approximately 30.degree. C., at a flow of
30-220 ml/hour for a time period of 12-24 hours, advantageously of
15-24 hours.
[0076] Preferably the said solution is recirculated at a flow of
approximately 200 ml/hour overnight (15-20 hours). The product
obtained is purified and separated according to known methods, for
example by ultrafiltration and precipitation with ethanol. The
product thus obtained is comprised either of epiK5-N-sulfate (and
in such case is dissolved in water and subjected to
depolymerization) or of LMW-epiK5-N-sulfate (in such case it
constitutes the end product). The percentage of epimerization, in
practice the amount of iduronic units as regards the glucuronic
ones, is calculated with .sup.1H-RMN according to the method
described in WO 96/4425.
[0077] The nitrous depolymerization reaction is carried out
according to known methods by the depolymerization of heparin, for
example according to the method described in EP 37319, in WO
82/03627 or according to the method by depolymerization of a
K5-N-sulfate described in EP 544592, but starting with a
K5-N-sulfate or an epiK5-N-sulfate containing from 0 to no more
than 10%, preferably no more than 5%, of acetyl groups. Preferably,
the depolymerization, performed with sodium nitrite and
hydrochloric acid on an epiK5-N-sulfate virtually free of acetyl
groups, is followed by in situ reduction with sodium
borohydride.
[0078] In practice, a cold aqueous solution of epiK5-N-sulfate is
brought to acid pH (approximately 2) with hydrochloric acid and,
still cold, treated with sodium nitrite maintaining the temperature
(approximately 4.degree. C.) and the pH (approximately 2) constant
and, upon termination of depolymerization (approximately 15-30
minutes) the solution is neutralized with sodium hydroxide and
treated, still at approximately 4.degree. C., with an aqueous
solution of sodium borohydride. Upon termination of the reduction
(approximately 4 hours) the excess sodium borohydride is destroyed
with hydrochloric acid, the solution is neutralized with sodium
hydroxide and the depolymerized (and reduced) product is isolated
according to known methods, for example by straightforward
precipitation with ethanol or acetone. The product obtained upon
termination of the depolymerization can be either a
lmw-epiK5-N-sulfate (in such case it constitutes the end product)
or a lmw-K5-N-sulfate (and in such case is directly subjected to
C5-epimerization as shown herein above, after isolation or also in
solution without being previously isolated), in particular when it
has a mean molecular weight of more than 4,000, preferably of at
least 6,000, or is utilized to prepare LMW-K5-N,O-oversulfated of
antiangiogenetic and antiviral activity. By appropriately
controlling the depolymerization reaction, in particular utilizing
different amounts of sodium nitrite/hydrochloric acid,
LMW-K5-N-sulfates or LMW-epiK5-N-sulfates are obtained having a
mean molecular weight in the entire interval from approximately
1,500 to approximately 12,000, advantageously from approximately
1,500 to approximately 10,000, preferably from approximately 1,500
to approximately 7,500, calculated at the .sup.13C-RMN spectrum
through the integration of the signal attributed to the C2 of the
2,5-anhydromannitol with that of the anomeric carbon of the
glucosamine inside the polysaccharide chain. According to a general
procedure starting for example with 1 g of K5-N-sulfate or of
epiK5-N-sulfate, the starting product is dissolved in 100-200 ml of
deionized water and thermostated at 4.degree. C. Then an amount of
sodium nitrite is added so as to obtain the mean molecular weight
desired, for example from approximately 2,000 to approximately
4,000. Therefore, starting with an epiK5-N-sulfate having a
molecular weight of 20,000 measured with HPLC method equipped with
a BioRad BioSil 250 column and utilizing a heparin standard of
known molecular weight, will require the addition of from 330 to
480 mg of sodium nitrite dissolved in a 0.2% aqueous solution. The
solution containing the epiK5-N-sulfate and the sodium nitrite,
kept at 4.degree. C., is brought to pH 2 through addition of 0.1 N
HCl cooled to 4.degree. C. It is reacted under slow agitation for
20-40 minutes, then is neutralized with 0.1 N NaOH. The product
obtained is brought to room temperature and treated with reducing
agent such as for example sodium borohydride (250-500 mg dissolved
in 50-100 ml of water) and reacted for 4-8 hours. The excess sodium
borohydride is eliminated bringing the pH to 5-5.5 with 0.1 N HCl
and let to stand for a further 2-4 hours. In the end it is
neutralized with 0.1 N NaOH and the product is recovered by
precipitation with acetone or ethanol after having concentrated the
product through evaporation at reduced pressure.
[0079] Similarly, the amounts of sodium nitrite can be determined
which, starting with 1 g of K5-N-sulfate or of epiK5-N-sulfate,
allow the attainment of a lmw-K5-N-sulfate or a lmw-epiK5-N-sulfate
with a mean molecular weight from approximately 4,000 to
approximately 12,000, advantageously from approximately 4,000 to
approximately 7,500, in particular of 6,000-7,500.
[0080] The LMW-epiK5-N-sulfates thus obtained, with an iduronic
acid content from 20 to 60%, advantageously from 40 to 60%,
preferably of 50-55% and virtually free of NH.sub.2 and N-acetyl
groups, having a mean molecular weight from approximately 1,500 to
approximately 12,000, advantageously from approximately 1,500 to
approximately 10,000, preferably from approximately 1,500 to
approximately 7,500 and their chemically or pharmaceutically
acceptable salts constitute new products useful as starting
materials in the preparation of particularly interesting
LMW-epiK5-N,O-oversulfates, but also themselves useful as active
ingredients of pharmaceutical or cosmetic compositions and
constitute an additional aspect of the present invention.
[0081] Advantageously, the starting materials in the preparation of
the N-acyl-(epi)K5-amine-O-oversulfate-derivatives of the present
invention are (epi)K5-N-sulfate-derivatives consisting of a chain
mixture in which at least 90% of said chains have the formula I
2
[0082] in which the glucuronic units/iduronic units ratio is from
100/0 to 40/60, n is a integer from 2 to 100, advantageously from 3
to 100, and the corresponding cation is chemically or
pharmaceutically acceptable.
[0083] In particular, said starting (epi)K5-N-sulfate-derivatives
are consisting of a chain mixture in which at least 90% of said
chains have the aforesaid formula I, with a glucuronic units
content from 100 to 40%, advantageously with a glucuronic acid
content of 100% or 80-40% of glucuronic acid and 20-60% of iduronic
acid, n is a integer from 2 to 100, advantageously from 3 to 100
and the corresponding cation is chemically acceptable. Preferably,
the glucuronic acid content is 100% or out of the total of uronic
acids 40-60% is glucuronic acid and 60-40% is iduronic acid.
[0084] Preferred starting materials are LMW-(epi)K5-N-sulfates
consisting of a chain mixture in which at least 90% of said chains
have the formula I in which the uronic units are all consisting of
glucuronic acid or are 40-60% comprised, preferably 50-55%, of
iduronic acid, n is a integer from 2 to 20, advantageously from 3
to 15 and the corresponding cation is chemically acceptable. The
epiK5-N-sulfates, prepared by C5-epimerization of K5-N-sulfates,
are well known and widely described in literature. Their
preparation by C-5 epimerization of the glucuronic unit of
K5-N-sulfate with a D-glucuronyl C5 epimerase was described in
documents cited herein above to illustrate the state of the art,
for example in WO 92/17507, WO 98/14425, WO 97/43317, WO 01/72848
and U.S. 2002/0062019. However, according to a preferential manner
of procedure, by epimerization a K5-N-sulfate is used obtained from
a K5 free of lipophilic substances as described in WO 02/068477 and
the C5 epimerization is performed with a D-glucuronyl C5-epimerase
isolated, purified and immobilized on a solid support, at a pH of
approximately 7, at a temperature of approximately 30.degree. C.
and for a time period of 12-24 hours in the presence of at least
one bivalent ion selected among calcium, magnesium, barium and
manganese as shown above. Also the K5-N-sulfate was shown herein
above.
[0085] The completely N-sulfated (epi)K5-N-sulfates of low
molecular weight, in particular those in which the uronic units are
all in glucuronic acid and those having an iduronic units content
of 40-60%, preferably 50-55%, are instead all particularly
advantageous new products as starting materials in the preparation
of the N-acyl-(epi)K5-amine-O-ov- ersulfate-derivatives according
to the present invention. Said starting materials are
LMW-(epi)K5-N-sulfates having an iduronic acid content of 0% or
40-60% as regards the total of the uronic acids and a mean
molecular weight from approximately 1,500 to approximately 12,000,
advantageously from approximately 1,500 to approximately 8,000,
preferably from approximately 1,500 to approximately 7,500; and
their chemically or pharmaceutically acceptable salts. The
distribution can range from approximately 1,000 to approximately
10,000.
[0086] Said LMW-(epi)K5-N-sulfates are advantageously consisting of
a chain mixture in which at least 90% of said chains have the
formula I' 3
[0087] in which the uronic units are 100% consisting of glucuronic
acid or 60-40% of glucuronic acid and 40-60% of iduronic acid, q is
a integer from 2 to 20 and the corresponding cation is chemically
or pharmaceutically acceptable.
[0088] In this context, the term "chemically" refers to a cation
usable in chemical synthesis, such as the ions sodium, ammonium,
(C.sub.1-C.sub.4)tetraalkylammonium, or for the purification of the
product.
[0089] Advantageous cations are those derived from alkaline metals,
alkaline-earth metals, ammonium,
(C.sub.1-C.sub.4)tetraalkylammonium, aluminum and zinc. Preferred
cations are the sodium, calcium and tetrabutylammonium ions.
[0090] Preferred starting materials are the new
LMW-(epi)K5-N-sulfates consisting of a chain mixture in which the
preponderant species has the formula I'a 4
[0091] in which the uronic units are 100% consisting of glucuronic
acid or 60-40% glucuronic and 40% to 60% of iduronic acid, p is a
integer from 4 to 8. The mean molecular weight of these products is
from approximately 2000 to approximately 4000 and the corresponding
cation is chemically or pharmaceutically acceptable.
[0092] In particular the new LMW-(epi)K5-N-sulfates are useful
starting materials consisting of a chain mixture in which at least
90% of said chains have the formula I' herein above, obtained by
nitrous depolymerization of the corresponding (epi) K5-N-sulfates
shown above and subsequent possible reduction for example with
sodium borohydride.
[0093] The origin of these (epi)K5-N-sulfates from a step of
nitrous depolymerization involves, at the reducing end of the
majority of the chains in said chain mixture, the presence of a
2,5-anhydromannose unit or, in case of reduction with for example
sodiumborohydride, of 2,5-anhydromannitol of structure (a) 5
[0094] in which X represents a formyl group or a hydroxymethyl
group. Therefore, the reducing end of the majority (60-70%) of the
chains is actually represented by the structure (b) 6
[0095] in which X is as defined above.
[0096] The presence of the structure (a) does not have any
influence on the chemical characteristics of the (epi)K5-N-sulfates
and their derivatives since any sulfations would involve a possible
introduction of one or two sulfate groups which would not
significantly change however the sulfation degree of the O-sulfated
derivatives. Besides, the presence of the structure (a) does not
influence biological activity of the products, as demonstrated by
.O slashed.stergaard P. B. et al. in Thrombosis Research, 1987, 45,
739-749 (.O slashed.stergaard 1987) for heparins of low molecular
weight.
[0097] Particularly advantageous LMW-(epi)K5-N-sulfates according
to the present invention are consisting of chain mixtures in which
the preponderant species is a compound of formula I'b 7
[0098] in which X is formyl or hydroxymethyl, m is 4, 5 or 6, the
corresponding cation is one chemically or pharmaceutically
acceptable ion, the uronic units are all of glucuronic acid or the
glucuronic and iduronic units are present alternately, starting
with a glucuronic or iduronic unit. In such case the
Glucuronic/iduronic ratio is from 45/55 to 55/45, i.e.
approximately 50/50.
[0099] All the LMW-(epi)K5-N-sulfates herein shown above are new
products which constitute an additional object of the present
invention.
[0100] The LMW-(epi)K5-N-sulfates, i.e. the LMW-K5-N-sulfates shown
above and, unlike other epiK5-N-sulfates, for example as indicated
in WO 92/17507, also the LMW-epiK5-N-sulfates of the present
invention, being virtually free of N-acetyl groups, do not have
anticoagulant activity and have an interesting activity against the
free radicals. Thanks to their low toxicity, the new
LMW-K5-N-sulfates and LMW-epiK5-N-sulfates are active ingredients
for the preparation of pharmaceutical compositions utilizable as
adjuvants in the treatment of ischemic cardiopathy and for the
treatment of radiation dermatitis or of cosmetic compositions
useful as anti-ageing of the skin.
[0101] When the LMW-(epi)K5-N-sulfates shown above are used as
starting materials for the O-oversulfation reaction, it is in any
case preferred that, in their preparation by nitrous
depolymerization as shown above, said depolymerization is followed
by the reduction for example with sodium borohydride to give
LMW-K5-N-sulfates characterized by terminal units (a) in which X is
hydroxymethyl, since according to the process of the present
invention said LMW-(epi)K5-N-sulfates are subjected to reactions of
sulfation and acylation whose influence, of the 2,5-anhydromannose
radical of structure (a) is unknown on the formyl group, in which X
represents formyl.
[0102] Said starting materials are preferably used in sodium salt
form, unless a salt is already available with a tertiary or
quaternary organic base prepared according to step (a) shown above,
preferably the tetrabutylammonium salt.
[0103] According to the present invention, the starting
(epi)-K5-N-sulfate-derivatives, preferably 100% N-sulfated, are
subjected to the aforesaid steps (a) and (b), upon termination of
which are isolated the corresponding, new
(epi)K5-amine-O-oversulfate-derivatives, in which the amine is not
substituted, normally in sodium salt form, which can be transformed
into another chemically or pharmaceutically acceptable salt.
[0104] Thus, according to another of its aspects, the present
invention refers to new (epi)K5-amine-O-oversulfate-derivatives and
their chemically or pharmaceutically acceptable salts, obtainable
by a process characterized in that
[0105] (a) an (epi)K5-N-sulfate-derivative, in acidic form, is
treated with a tertiary or quaternary organic base, letting the
reaction mixture to stand for a time period of 30-60 minutes,
maintaining the pH of the solution at a value of approximately 7 by
addition of said tertiary or quaternary organic base and its salt
is isolated with said organic base;
[0106] (b) said salt of organic base of said
(epi)K5-N-sulfate-derivative is treated with an O-sulfation reagent
in the conditions of O-oversulfation and the
(epi)K5-amine-O-oversulfate-derivative is isolated.
[0107] The derivative thus obtained is generally an
(epi)K5-amine-O-oversulfate-derivative having a mean molecular
weight from approximately 4,500 to approximately 40,000, isolated
in sodium salt form which can be transformed into another
chemically or pharmaceutically acceptable salt. Particularly
advantageous salts are those of alkaline metals, alkaline-earth
metals, of ammonium, (C.sub.1-C.sub.4)tetraalkylam- monium,
aluminum and zinc and, among these, the salts of sodium, calcium
and tetrabutylammonium are preferred.
[0108] As already herein shown above, the degree of sulfation of
the (epi)K5-amine-O-oversulfate-derivatives as obtained above
depends on the configuration of the starting
(epi)K5-N-sulfate-derivative, since the volume in the disaccharide
chain of K5-N-sulfate-derivative, in which the uronic units are
comprised exclusively of glucuronic acid, allows a oversulfation
which is less than that occurring with the
epiK5-N-sulfate-derivative, especially if the epimerization degree
of the latter is 40-60% of glucuronic acid and 60-40% of iduronic
acid.
[0109] In fact it has been ascertained that, subjecting a
K5-N-sulfate-derivative to the aforesaid steps (a) and (b) a
K5-amine-O-oversulfate-derivative is obtained having a sulfation
degree from 2.2 to 3, advantageously from 2.5 to 3, preferably from
2.7 to 2.9.
[0110] Subjecting an epiK5-N-sulfate-derivative to the aforesaid
steps (a) and (b) in which the iduronic acid content is 20-60%,
preferably 40-60% of the total of the uronic acids, an
epiK5-amine-O-oversulfate-derivative is obtained having a sulfation
degree of at least 3.4, advantageously of at least 3.5, more
advantageously from 3.55 to 4, preferably from 3.55 to 3.8.
[0111] These (epi)K5-amine-O-oversulfated-derivatives are useful
products as intermediates in the preparation of their
N-(C.sub.2-C.sub.4)acylated derivatives and as active ingredients
basically free of activity on the coagulation parameters but having
other interesting pharmacobiological properties, useful for the
preparation of pharmaceutical compositions. For use of the
epiK5-amine-O-oversulfate-derivatives of the present invention as
active ingredients of pharmaceutical compositions, it is
advantageous to prepare both derivatives of low molecular weight,
with a mean molecular weight from approximately 3,000 to
approximately 11,500, preferably from approximately 4,500 to
approximately 8,500, with a molecular weight distribution of
between approximately 1,000 and approximately 15,000, preferably
between approximately 2,000 and approximately 10,000 and
derivatives of high molecular weight, originating from the
unfractionated K5, with a mean molecular weight from approximately
15,000 to approximately 45,000, preferably between approximately
20,000 and approximately 45,000, with a molecular weight
distribution from approximately 2,000 to approximately 70,000.
[0112] Thus, according to another of its aspects the invention
provides new active ingredients of pharmaceutical compositions
consisting of (epi)K5-amine-O-oversulfate-derivatives consisting of
chain mixtures in which at least 90% of said chains have the
formula II 8
[0113] in which n is a integer from 2 to 100, preferably from 3 to
100, R, R' and R" are hydrogen or SO.sub.3--, the uronic units are
all of glucuronic acid, for a sulfation degree of at least 2.2,
advantageously from 2.2 to 3 or from 2.3 to 3, more advantageously
from 2.5 to 3, preferably from 2.7 to 2.9, or are 20-60% consisting
of iduronic acid, for a sulfation degree of at least 3.4,
advantageously of at least 3.5, more advantageously from 3.55 to 4,
preferably from 3.55 to 3.8 and the corresponding cation is
chemically or pharmaceutically acceptable.
[0114] Advantageous epiK5-amine-O-oversulfated-derivatives of
extremely high degree of sulfation are consisting of a chain
mixture in which at least 90% of said chains have the formula II in
which the uronic units are 40-60% consisting of iduronic acid, n is
a integer from 2 to 100, preferably from 3 to 100, with a mean
molecular weight from approximately 2,000 to approximately 40,000,
advantageously from approximately 4,500 to approximately 40,000, R
is at least 40%, preferably 50-80% SO.sub.3--, R' and R" are both
SO.sub.3-- or one is hydrogen and the other is 5-10% SO.sub.3-- in
monosulfate glucuronic acid and 10-15% SO.sub.3-- in monosulfate
iduronic acid, the degree of sulfation is more than 3.4 and the
corresponding cation is chemically or pharmaceutically
acceptable.
[0115] Preferred epiK5-amine-O-oversulfate-derivatives of very high
degree of sulfation are the LMW-epiK5-amine-O-oversulfates
consisting of a chain mixture in which at least 90% of said chains
have the formula II' 9
[0116] in which q is a integer from 2 to 20, R, R' and R" are
hydrogen or SO.sub.3--, the uronic units are 20-60% comprised,
preferably 40-60%, of iduronic acid, for a degree of sulfation from
3.55 to 4, and the corresponding cation is one chemically or
pharmaceutically acceptable ion.
[0117] Are particularly interesting the chain mixtures of formula
II' in which the uronic units are 40-60% comprised, preferably
50-55%, of iduronic acid, R is at least 40%, advantageously 50-80%,
preferably approximately 65% SO.sub.3--, R' and R" are both
SO.sub.3-- or one is hydrogen and the other is 5-10% SO.sub.3-- in
glucuronic acid and 10-15% SO.sub.3-- in iduronic acid, n is a
integer from 3 to 15, with a mean molecular weight from
approximately 4,000 to approximately 8,000 and the corresponding
cation is chemically or pharmaceutically acceptable.
[0118] Among these LMW-epiK5-amine-O-oversulfates are preferred
those consisting of a chain mixture in which the preponderant
species has the formula II'a 10
[0119] in which p is a integer from 4 to 8, R, R' and R" are as
defined above, the degree of sulfation is from 3.55 to 4 and the
corresponding cation is chemically or pharmaceutically
acceptable.
[0120] The origin of the new LMW-epiK5-amine-O-oversulfated from
LMW-epiK5-N-sulfates obtained by nitrous depolymerization and
subsequent reduction with, for example, sodium borohydride,
involves, at the reducing end of the majority of the chains in said
chain mixture, the presence of a sulfated 2,5-anhydromannitol unit
of structure (a') 11
[0121] in which R" represents hydrogen or SO.sub.3--.
[0122] Thus, the reducing end of the majority of the chains in said
chain mixture is represented by the structure (b') 12
[0123] in which the uronic unit can be glucuronic or iduronic.
[0124] Among the aforesaid new LMW-epiK5-amine-O-oversulfates, are
preferred those consisting of mixtures in which the preponderant
species is a compound of formula II'b 13
[0125] in which R, R' and R" are hydrogen or SO.sub.3--, X" is OH
or OSO.sub.3--, m is 4, 5 or 6, the uronic units are 40-60%
consisting of iduronic acid, for a degree of sulfation from 3.55 to
4, the iduronic units being present alternately, starting with a
glucuronic or iduronic unit, and the corresponding cation is one
chemically or pharmaceutically acceptable ion.
[0126] According to another of its aspects the invention provides
new K5-amine-O-oversulfate-derivatives consisting of chain mixtures
in which at least 90% of said chains have the formula III 14
[0127] in which n is a integer from 2 to 100, preferably from 3 to
100, R, R' and R" are hydrogen or SO.sub.3--, the degree of
sulfation is at least 2.2, advantageously from 2.2 to 3 or from 2.3
to 3, more advantageously from 2.5 to 3, preferably from 2.7 to 2.9
and the corresponding cation is chemically or pharmaceutically
acceptable.
[0128] Preferred K5-amine-O-oversulfates are the
LMW-K5-amine-O-oversulfat- es consisting of chain mixtures in which
at least 90% of said chains have the formula III' 15
[0129] in which q is a integer from 2 to 20, R, R' and R" represent
hydrogen or an SO.sub.3-- group, for a sulfation degree of at least
2.2, advantageously from 2.2 to 3 or from 2.3 to 3, more
advantageously from 2.5 to 3, preferably from 2.7 to 2.9 and the
corresponding cation is one chemically or pharmaceutically
acceptable ion.
[0130] Among these LMW-K5-amine-O-oversulfates are preferred those
consisting of a chain mixture in which the preponderant species has
the formula III'a 16
[0131] in which p is a integer from 4 to 8, R, R' and R" are as
defined above, the degree of sulfation is at least 2.2,
advantageously from 2.2 to 3 or from 2.3 to 3, more advantageously
from 2.5 to 3, preferably from 2.7 to 2.9 and the corresponding
cation is chemically or pharmaceutically acceptable.
[0132] The origin of the new LMW-K5-amine-O-oversulfated from
LMW-K5-sulfates obtained by nitrous depolymerization and subsequent
reduction with, for example, sodium borohydride, involves, at the
reducing end of the majority of the chains in said chain mixture,
the presence of a sulfated 2,5-anhydromannitol unit of structure
(a') as shown above, in which R" represents hydrogen or
SO.sub.3--.
[0133] Thus, the reducing end of the majority of the chains in said
chain mixture is represented by the structure (b') as shown above,
in which the uronic unit is still of glucuronic acid.
[0134] Among the aforesaid new LMW-K5-amine-O-oversulfates, are
preferred those consisting of mixtures in which the preponderant
species is a compound of formula III'b 17
[0135] in which R, R' and R" are hydrogen or SO.sub.3--, X" is OH
or OSO.sub.3--, for a sulfation degree of at least 2.2,
advantageously from 2.2 to 3 or from 2.3 to 3, more advantageously
from 2.5 to 3, preferably from 2.7 to 2.9, m is 4, 5 or 6 and the
corresponding cation is one chemically or pharmaceutically
acceptable ion.
[0136] These LMW-K5-amine-O-oversulfated are new products useful as
intermediates in the preparation of their N-sulfated or N-acylated
derivatives but themselves have interesting pharmacological
properties, in particular as anti-free radicals and microbicides
and, as such, constitute new active ingredients of pharmaceutical
compositions which constitute therefore an additional aspect of the
present invention.
[0137] In particular, according to another of its aspects, the
invention concerns the use of the aforesaid
(epi)K5-amine-O-oversulfate-derivatives with a high degree of
sulfation for the preparation of new N-substituted
epiK5-amine-O-oversulfate-derivatives.
[0138] The N-acyl-(epi)K5-amine-O-oversulfate-derivatives according
to the present invention are obtained by subjecting the
(epi)K5-amine-O-oversulf- ate-derivatives to the step (c) of the
process of the present invention.
[0139] Thus, according to one of its additional aspects, the
present invention provides new N-acyl (epi)
K5-amine-O-oversulfate-derivatives obtainable by a process
characterized in that
[0140] (a) an (epi)K5-N-sulfate-derivative, in acidic form, is
treated with a tertiary or quaternary organic base, letting the
reaction mixture to stand for a time period of 30-60 minutes,
maintaining the pH of the solution at a value of 7 by addition of
said tertiary or quaternary organic base and its salt is isolated
with said organic base;
[0141] (b) said salt of organic base of said
(epi)K5-N-sulfate-derivative is treated with an O-sulfation reagent
in the conditions of O-oversulfation;
[0142] (c) the product thus obtained is treated with a functional
derivative of a (C.sub.2-C.sub.4) carboxylic acid, the
N-acyl-(epi)K5-amine-O-oversulfate-derivative thus obtained is
isolated. Generally the
N-acyl-(epi)K5-amine-O-oversulfate-derivative is isolated in sodium
salt form and optionally said sodium salt is transformed into
another chemically or pharmaceutically acceptable salt.
[0143] As occurs for the (epi)K5-amine-O-oversulfate-derivatives,
also the degree of sulfation of the N-acyl-(epi)
K5-amine-O-oversulfate-derivative- s as obtained above depends on
the configuration of the starting (epi)K5-N-sulfate-derivative,
since the volume in the disaccharide chain of
K5-N-sulfate-derivative, in which the uronic units are comprised
exclusively of glucuronic acid, allows a oversulfation which is
less than that occurring with the epiK5-N-sulfate-derivative,
especially if the epimerization degree of the latter is 40-60% of
glucuronic acid and 60-40% of iduronic acid.
[0144] In fact, it has been ascertained that, subjecting a
K5-N-sulfate-derivative to the aforesaid steps (a)-(c) an
N-acyl-K5-amine-O-oversulfate-derivative is obtained having a
degree of sulfation from 2.2 to 3, advantageously from 2.5 to 3,
preferably from 2.7 to 2.9.
[0145] Subjecting an epiK5-N-sulfate-derivative to the aforesaid
steps (a), (b) and (c) in which the iduronic acid content is 20-60%
of the total of the uronic acids,
N-acyl-epiK5-amine-O-oversulfate-derivatives are obtained having a
sulfation degree of at least 3.4, advantageously of at least 3.5,
more advantageously from 3.55 to 4, preferably from 3.55 to
3.8.
[0146] Thus, according to another of its aspects, the present
invention provides new
N-acyl-(epi)K5-amine-O-oversulfate-derivatives in which the acyl is
a (C.sub.2-C.sub.4) carboxylic acid, having a mean molecular weight
from approximately 2,000 to approximately 45,000, preferably
between approximately 4,500 and approximately 40,000, a degree of
sulfation as indicated above, said derivatives being basically
inactive on the coagulation parameters but useful active
ingredients of pharmaceutical or cosmetic compositions.
[0147] For use of the N-acyl-epiK5-amine-O-oversulfate-derivatives
of the present invention as pharmaceutical or cosmetic products it
is advantageous to prepare both derivatives of low molecular
weight, with a mean molecular weight from approximately 3,000 to
approximately 11,500, preferably from approximately 4,500 to
approximately 8,500, with a molecular weight distribution of
between approximately 1,000 and approximately 15,000, preferably
between approximately 2,000 and approximately 10,000 and
derivatives of high molecular weight, originating from the
unfractionated K5, with a mean molecular weight from approximately
15,000 to approximately 45,000, preferably between approximately
20,000 and approximately 45,000, with a molecular weight
distribution from approximately 2,000 to approximately 70,000.
[0148] In the N-acyl-epiK5-amine-O-oversulfate-derivatives of the
present invention, the degree of sulfation is very high, since on
the 4 free hydroxyls available per disaccharide unit, at least 3.4,
preferably from 3.5 to 3.8, are sulfated, whereas the nitrogen of
the glucosamine is virtually 100% acylated. Besides, the
N-acyl-epiK5-amine-O-oversulfate-de- rivatives are 100%
6-O-sulfated and 50-80% 3-O-sulfated in their glucosamine units,
5-10% 3-O-monosulfated in glucuronic units, 10-15% O-monosulfated
in iduronic units and 2.3-di-O-sulfated in the remaining uronic
units, considering that the degree of sulfation is at least
3.4.
[0149] The N-acyl-K5-amine-O-oversulfate-derivatives which, as
indicated above, have a degree of sulfation from 2.2 to 3, are at
least 90% 6-O-sulfated.
[0150] According to another of its aspects, the invention provides
new N-acyl-epiK5-amine-O-oversulfate-derivatives consisting of
chain mixtures in which at least 90% of said chains have the
formula IV 18
[0151] in which the uronic units are 20-60% consisting of iduronic
acid, n is a integer from 2 to 100, preferably from 3 to 100, R, R'
and R" are hydrogen or SO.sub.3--, Z is (C.sub.2-C.sub.4)acyl, the
degree of sulfation is at least 3.4, advantageously of at least
3.5, more advantageously from 3.55 to 4, preferably from 3.55 to
3.8 and the corresponding cation is chemically or pharmaceutically
acceptable.
[0152] Advantageous N-acyl-epiK5-amine-O-oversulfate-derivatives of
extremely high degree of sulfation are consisting of a chain
mixture in which at least 90% of said chains have the formula IV in
which the uronic units are 40-60% consisting of iduronic acid, n is
a integer from 2 to 100, preferably from 3 to 100, with a mean
molecular weight from approximately 2,000 to approximately 45,000,
advantageously from approximately 4,500 to approximately 45,000, R
is at least 40%, preferably 50-80% SO.sub.3--, R' and R" are both
SO.sub.3-- or one is hydrogen and the other is 5-10% SO.sub.3-- in
monosulfate glucuronic acid and 10-15% SO.sub.3-- in monosulfate
iduronic acid, the degree of sulfation is more than 3.4 and the
corresponding cation is chemically or pharmaceutically
acceptable.
[0153] The N-acyl-epiK5-amine-O-oversulfate-derivatives of
extremely high degree of sulfation of particular interest are
N-acyl-LMW-epiK5-amine-O-o- versulfates consisting of a chain
mixture in which at least 90% of said chains have the formula IV'
19
[0154] in which q is a integer from 2 to 20, R, R' and R" represent
hydrogen or an SO.sub.3-- group for a degree of sulfation from 3.55
to 4, Z is (C.sub.2-C.sub.4)acyl, and the corresponding cation is
one chemically or pharmaceutically acceptable ion.
[0155] Chain mixtures of formula IV' in which the uronic units are
40-60% comprised, preferably 50-55%, of iduronic acid, R is at
least 40%, advantageously 50-80%, preferably approximately 65%
SO.sub.3--, R' and R" are both SO.sub.3-- or one is hydrogen and
the other is 5-10% SO.sub.3-- in glucuronic acid and 10-15%
SO.sub.3-- in iduronic acid, q is a integer from 3 to 15, with a
mean molecular weight from approximately 4,500 to approximately
9,000 and the corresponding cation is chemically or
pharmaceutically acceptable are preferred.
[0156] Other particularly interesting
N-acyl-LMW-epiK5-amine-O-oversulfate- s are those of very low
molecular weight, consisting of a chain mixture in which the
preponderant species is a compound of formula IV'a 20
[0157] in which p is a integer from 4 to 8, R, R' and R" are
hydrogen or an SO.sub.3-- group for a degree of sulfation from 3.55
to 4, Z is (C.sub.2-C.sub.4)acyl, and the corresponding cation is
one chemically or pharmaceutically acceptable ion.
[0158] The origin of the new N-acyl-LMW-epiK5-amine-O-oversulfates
from LMW-epiK5-sulfates obtained by nitrous depolymerization and
subsequent reduction with, for example, sodium borohydride,
involves, at the reducing end of the majority of the chains in said
chain mixture, the presence of a sulfated 2,5-anhydromannitol unit
of structure (a') defined above, in which R" represents hydrogen or
SO.sub.3--.
[0159] Thus, the reducing end of the majority of the chains in said
chain mixture is represented by the structure (b") 21
[0160] in which Z represents (C.sub.2-C.sub.4)acyl and the uronic
unit can be glucuronic or iduronic.
[0161] Among the aforesaid new
N-acyl-LMW-epiK5-amine-O-oversulfates, are preferred those
consisting of mixtures in which the preponderant species is a
compound of formula IV'b 22
[0162] in which R, R' and R" are hydrogen or SO.sub.3--, Z is
(C.sub.2-C.sub.4)acyl, X" is OH or OSO.sub.3--, m is 4, 5 or 6, for
a degree of sulfation from 3.55 to 4, the uronic units are present
alternately, starting with a glucuronic or iduronic unit, and the
corresponding cation is one chemically or pharmaceutically
acceptable ion.
[0163] Said cations are advantageously those of alkaline metals,
alkaline-earth metals, of ammonium,
(C.sub.1-C.sub.4)tetraalkylammonium, aluminum and zinc and, among
these, preferably the salts of sodium, calcium and
tetrabutylammonium.
[0164] In all the N-acyl-epiK5-amine-O-oversulfates shown above, in
particular those of formula IV, IV', IV'a, IV'b, as also in the
structure b", the acyl group (Z) is preferably a
(C.sub.2-C.sub.4)acyl selected among the group consisting of
acetyl, (2-carboxy)acetyl, (2-methoxycarbonyl)acetyl,
(2-ethoxycarbonyl)acetyl, propionyl, (3-carboxy) propionyl,
N-(3-methoxycarbonyl) propionyl and (3-ethoxycarbonyl)
propionyl.
[0165] According to another of its aspects, the invention provides
new N-acyl-K5-amine-O-oversulfate-derivatives consisting of chain
mixtures in which at least 90% of said chains have the formula V
23
[0166] in which n is a integer from 2 to 100, preferably from 3 to
100, Z is (C.sub.2-C.sub.4)acyl, R, R' and R" are hydrogen or
SO.sub.3--, the degree of sulfation is at least 2.2, advantageously
from 2.2 to 3 or from 2.3 to 3, more advantageously from 2.5 to 3,
preferably from 2.7 to 2.9 and the corresponding cation is
chemically or pharmaceutically acceptable. Particularly interesting
are new N-acyl-LMW-K5-amine-O-oversu- lfates consisting of chain
mixtures in which at least 90% of said chains have the formula V'
24
[0167] in which q is a integer from 2 to 20, Z is
(C.sub.2-C.sub.4)acyl, R, R' and R" represent hydrogen or an
SO.sub.3-- group for a sulfation degree of at least 2.2,
advantageously from 2.2 to 3 or from 2.3 to 3, more advantageously
from 2.5 to 3, preferably from 2.7 to 2.9 and the corresponding
cation is one chemically or pharmaceutically acceptable ion.
[0168] Among these N-acyl-LMW-K5-amine-O-oversulfates are preferred
those consisting of a chain mixture in which the preponderant
species has the formula V'a 25
[0169] in which p is a integer from 4 to 8, Z is
(C.sub.2-C.sub.4)acyl, R, R' and R" are as defined above, the
degree of sulfation is at least 2.2, advantageously from 2.2 to 3
or from 2.3 to 3, more advantageously from 2.5 to 3, preferably
from 2.7 to 2.9 and the corresponding cation is chemically or
pharmaceutically acceptable.
[0170] The origin of the new N-acyl-LMW-K5-amine-O-oversulfates
from LMW-K5-sulfates obtained by nitrous depolymerization and
subsequent reduction with, for example, sodium borohydride,
involves, at the reducing end of the majority of the chains in said
chain mixture, the presence of a sulfated 2,5-anhydromannitol unit
of structure (a') as shown above, in which R" represents hydrogen
or SO.sub.3--.
[0171] Thus, the reducing end of the majority of the chains in said
chain mixture is represented by the structure (b'") 26
[0172] in which Z, R, R' and R" are as defined above.
[0173] Among the aforesaid new N-acyl-LMW-K5-amine-O-oversulfates,
are preferred those consisting of mixtures in which the
preponderant species is a compound of formula V'b 27
[0174] in which Z is (C.sub.2-C.sub.4)acyl, R, R' and R" are
hydrogen or SO.sub.3--, X" is OH or OSO.sub.3--, for a sulfation
degree of at least 2.2, advantageously from 2.2 to 3 or from 2.3 to
3, more advantageously from 2.5 to 3, preferably from 2.7 to 2.9, m
is 4, 5 or 6 and the corresponding cation is one chemically or
pharmaceutically acceptable ion.
[0175] In all the N-acyl-K5-amine-O-oversulfates shown above, in
particular those of formula V, V', V'a, V'b, as also in the
structure b'", the acyl group (Z) is preferably a
(C.sub.2-C.sub.4)acyl selected among the group consisting of
acetyl, (2-carboxy)acetyl, (2-methoxycarbonyl)acetyl,
(2-ethoxycorbonyl)acetyl, propionyl, (3-carboxy) propionyl,
N-(3-methoxycarbonyl)proponyl and (3-ethoxycarbonyl)proponyl.
[0176] Particularly interesting are the
N-acyl-K5-amine-O-oversulfates shown above in which Z is a
(C.sub.2-C.sub.4)acyl different from acetyl. Also interesting and
particularly active are the N-acyl-K5-amine-O-oversu- lfates shown
above, in which Z is acetyl, having a degree of sulfation of
2.7-2.9, preferably of approximately 2.8.
[0177] The new N-acyl-(epi)K5-amine-O-oversulfate-derivatives,
especially in their salts form, are highly anionic products able to
capture the free radicals. These can be used in the cosmetics
industry as coadjuvants against hair loss or to prepare
"anti-ageing" creams, but are above all useful in the
pharmaceutical industry, as products for the treatment of
dermatitis and as microbicides.
[0178] Thus, according to one of its additional aspects, the
present invention provides pharmaceutical compositions including,
as one of their active ingredients, a pharmacologically active
amount of an N-acyl-(epi)K5-amine-O-oversulfate-derivative as shown
above or of one of its pharmaceutically acceptable salts, in
mixture with a pharmaceutical excipient.
[0179] In the pharmaceutical compositions of the present invention
for oral, subcutaneous, intravenous, transdermal or topical
administration, the active ingredients are preferably administered
in the form of dosage units, in mixture with the classic
pharmaceutical excipients or vehicles. The dose regimen can vary
widely depending on the age, the weight and health condition of the
patient. This dose regimen includes the administration of a dose
from 1 to 1000 mg, advantageously from 10 to 750 mg, preferably 250
to 500 mg from one to three times a day by intravenous,
subcutaneous, oral, transdermal or topical administration.
[0180] The present invention also provides a cosmetic composition
including an effective amount of an
N-acyl-(epi)K5-amine-O-oversulfated-d- erivative or one of its
pharmaceutically acceptable salts, in mixture with a cosmetic
excipient.
[0181] Besides, the present invention provides a pharmaceutical
composition including, as one of its active ingredients, an
(epi)K5-amine-O-oversulfate-derivative obtainable according to
steps (a) and (b) of the process described above, or one of its
pharmaceutically acceptable salts, in mixture with a pharmaceutical
excipient. Advantageously, said
(epi)K5-amine-O-oversulfate-derivative is consisting of a chain
mixture in which at least 90% of said chains have the formula II,
II', III or III' or in which the preponderant species is a compound
of formula IIa, II'a, III'a or III'b. Preferred active ingredient
is a lmw-K5-amine-O-oversulfate having a degree of sulfation from
2.2 to 3, advantageously having a mean molecular weight from
approximately 3,500 to approximately 11,000, more advantageously
from approximately 3,500 to approximately 5,200 and basically free
of N-acetyl and N-sulfate groups.
[0182] In the pharmaceutical compositions of the present invention
for oral, subcutaneous, intravenous, transdermal or topical
administration, the active ingredients (epi)
K5-amine-O-oversulfate-derivatives are preferably administered in
the form of dosage units, in mixture with the classic
pharmaceutical excipients or vehicles. The dose regimen can vary
widely depending on the age, the weight and health condition of the
patient. This dose regimen includes the administration of a dose of
an (epi)K5-amine-O-oversulfate-derivative from 1 to 1000 mg,
advantageously from 10 to 750 mg, preferably 250 to 500 mg from one
to three times a day by intravenous, subcutaneous, oral,
transdermal or topical administration.
[0183] The pharmaceutical compositions including an
(epi)K5-amine-O-oversulfate-derivative such as those shown above
are formulated with the classic excipients suitable for different
ways of administration.
[0184] Particularly advantageous are the formulations in the form
of creams, ointments, liniments, gels, foams, balsams, vaginal
pessaries, suppositories, solutions or suspensions suitable for
local administration.
[0185] Finally, the present invention provides a pharmaceutical
composition containing, as one of its active ingredients, a
pharmacologically active amount of a lmw-(epi)K5-N-sulfate, i.e. of
a lmw-K5-N-sulfate or of a lmw-epiK5-N-sulfate as shown above or of
one of their pharmaceutically acceptable salts, in mixture with a
pharmaceutical excipient.
[0186] In the pharmaceutical compositions of the present invention
for oral, subcutaneous, intravenous, transdermal or topical
administration, the active ingredients are preferably administered
in the form of dosage units, in mixture with the classic
pharmaceutical excipients or vehicles. The dose regimen can vary
widely depending on the age, the weight and health condition of the
patient. This dose regimen includes the administration of a dose
from 1 to 1000 mg, advantageously from 10 to 750 mg, preferably 250
to 500 mg from one to three times a day by intravenous,
subcutaneous, oral, transdermal or topical administration.
[0187] According to another of its aspects, the present invention
also provides a cosmetic composition containing, as one of its
active ingredients, an effective amount of a lmw-K5-N-sulfate or
LMW-epiK5-N-sulfate or of one of its pharmaceutically acceptable
salts, in mixture with a cosmetic excipient.
[0188] Advantageously, said LMW-(epi)K5-N-sulfate-derivative is
consisting of a chain mixture in which at least 90% of said chains
have the formula I, or I', or in which the preponderant species is
a compound of formula I'a, or I'b. Preferred active ingredient is a
lmw-(epi)K5-N-sulfate-deriv- ative having a mean molecular weight
from approximately 1,000 to approximately 12,000, advantageously
from approximately 1,500 to approximately 8,000, preferably from
approximately 1,500 to approximately 7,500 and basically free of
N-acetyl groups.
[0189] A salt selected from the group consisting of salts of
alkaline metals or alkaline-earth metals, of ammonium,
(C.sub.1-C.sub.4)tetraalkyl- ammonium, aluminum or zinc, in
particular the salt of sodium, potassium, calcium, magnesium,
aluminum or zinc constitutes an effective active ingredient of the
compositions of the present invention.
[0190] The following examples illustrate the invention without
however limiting it.
Preparation I
[0191] LMW-epiKS-N-sulfate
[0192] a) Epimerization of K5-N-sulfate
[0193] 2 g of K5 N-sulfate, obtained as described in Example 1,
steps (i) and (ii), of WO 02/068477, are dissolved in 120 ml of 25
mM HEPES buffer, pH 7, containing 50 mM CaCl2. The solution
obtained is made to recirculate through a 50 ml column filled with
the resin containing the immobilized enzyme obtained as described
in WO 96/14425. This operation is carried out at 30.degree. C. with
a flow of 200 ml/h for 24 hours. The product obtained is purified
through ultrafiltration over a 1000 D membrane and passage over an
ionic exchange column IR 120 H.sup.+, neutralizing the eluate with
1 N NaOH. The sample is recovered by precipitation with ethanol or
acetone.
[0194] An epimerized product is obtained with an iduronic
acid/glucuronic acid ratio of 55/45 against a ratio of 0/100 of the
starting product. The percentage of epimerization was calculated
with .sup.1H-RMN according to the method described in WO 96/14425.
The yield, calculated by measuring the content of uronic acids
against a standard with the carbazole method (Bitter and Muir Anal.
Biochem. 39, 88-92-1971) is 90%.
[0195] b) Depolymerization of epi-K5-N-sulfate
[0196] 1 g of product obtained in step (a) is depolymerized by the
degradation method with nitrous acid and subsequent reduction of
the aldehyde which forms. In particular one continues by dissolving
the product in 25 ml of distilled water and adding it with 230 mg
of sodium nitrite dissolved in 115 ml of distilled water. The
solution is then brought to 4.degree. C. and the pH to 2 with 0.1 N
HCl and maintained for 30 minutes. At the end of the reaction the
solution is brought to room temperature and the pH to 7 with 0.1 M
NaOH. The solution is then added with 450 mg. of NaBH.sub.4 and
reacted for 4 hours. The product is recovered by precipitation with
3 volumes of acetone at 4.degree. C., filtration with filtering
funnel and dried at 40.degree. C. in a vacuum oven. 900 mg of
LMW-epiK5-N-sulfate are obtained with a molecular weight
distribution measured with HPLC method which ranges from 1,000 to
4,000 and with a glucuronic unit content of 45% and iduronic unit
content of 55%.
[0197] Preparation II
[0198] LMW-K5-N-sulfate
[0199] The product obtained as described in Example 1, steps (i)
and (ii), of WO 02/068477 is depolymerized by the degradation
method with nitrous acid and subsequent reduction of the aldehyde
which forms. One continues by dissolving 1 g of K5-N-sulfate in 200
ml of distilled water and adding it with 480 mg of sodium nitrite
dissolved in 240 ml of distilled water. The solution is then
brought to 4.degree. C. and the pH to 2 with 0.1 N HCl and
maintained for 30 minutes. At the end of the reaction the solution
is brought to pH 7 with 0.1 M NaOH and then to room temperature.
The solution is then added with 450 mg. of NaBH.sub.4 and reacted
for 4 hours. The excess NaBH.sub.4 is eliminated with HCl bringing
the pH to 5-6. The product, neutralized with 0.1 M NaOH, is
recovered by precipitation with 3 volumes of acetone at 4.degree.
C., filtration with filtering funnel and dried at 40.degree. C. in
a vacuum oven. 900 mg of LMW-K5-N-sulfate are obtained with a mean
molecular weight of approximately 2,000, consisting of a chain
mixture in which the preponderant species is a compound of formula
I'b in which m is 4 and the uronic units are those of glucuronic
acid.
[0200] Preparation III
[0201] (a) Depolymerization of K5-N-sulfate
[0202] 2 g of K5-N-sulfate obtained as described in Example 1,
steps (i) and (ii), of WO 02/068477 is depolymerized as described
in PREPARATION II, utilizing 100 mg of sodium nitrite and 300 mg.
of sodium borohydride. 1.8 g. of LMW-K5-N-sulfate are obtained with
a mean molecular weight of 5.000
[0203] (b) Epimerization of LMW-K5-N-sulfate
[0204] 1 g of LMW-K5 N-sulfate obtained in step (a) is treated as
described in step (a) of the Example 1. An epimerized product is
obtained with an iduronic acid/glucuronic acid ratio of 44/56
against a ratio of 0/100 of the starting product, with a molecular
weight distribution from 2,000 to 10,000 and with a mean molecular
weight of 5,000 D. The yield, calculated by measuring the content
of uronic acids against a standard with the carbazole method
(Bitter and Muir Anal. Biochem. 39, 88-92-1971) is 90%.
[0205] Preparation IV
[0206] Preparation of an epiK5-N-sulfate
[0207] A solution of 10 g of K5-N-sulfate obtained as described in
Example 1, steps (i) and (ii), of WO 02/068477 in 600 ml of 25 mM
HEPES buffer at pH 7, containing CaCl.sub.2 at a concentration of
50 mM is made to recirculate through a 50 ml column filled with
Sepharose 4B resin containing 5 g of recombinant C5-epimerase (WO
96/14425) immobilized as described in WO 01/72848. The reaction is
carried out at 30.degree. C. at pH 7 with a flow of 200 ml/h for 24
hours. The product obtained is purified by ultrafiltration and
precipitation with ethanol. Thus an epiK5-N-sulfate is obtained
whose iduronic acid content is 54%.
[0208] Preparation V
[0209] Preparation of a lmw-epiK5-N-sulfate
[0210] 1 g of product obtained in PREPARATION IV is depolymerized
by the degradation method with nitrous acid and subsequent
reduction of the aldehyde which forms. In particular one continues
by dissolving the product in 25 ml of distilled water and adding it
with 230 mg of sodium nitrite dissolved in 115 ml of distilled
water. The solution is then brought to 4.degree. C. and the pH to 2
with 0.1 N HCl and maintained for 30 minutes. At the end of the
reaction the solution is brought to room temperature and the pH to
7 with 0.1 M NaOH. The solution is then added with 450 mg. of
NaBH.sub.4 and reacted for 4 hours. The product is recovered by
precipitation with 3 volumes of acetone at 4.degree. C., filtration
with filtering funnel and dried at 40.degree. C. in a vacuum oven.
900 mg of LMW-epiK5-N-sulfate are obtained with a molecular weight
distribution measured with HPLC method which ranges from 1,000 to
4,000.
EXAMPLE 1
[0211] EpiK5-amine-O-oversulfate
[0212] (a) Tetrabutylammonium salt of epi K5-N-sulfate
[0213] A solution of 400 mg of epiK5-N-sulfate with an iduronic
acid content of 54% as obtained in PREPARATION IV in 40 ml of water
is thermostated at 4.degree. C., then passed over an ionic exchange
resin IR 120H.sup.+ preconditioned with water at 4.degree. C. The
eluate obtained, consisting of 100 ml of a solution at pH 1.94, is
neutralized with a solution of 15% tetrabutylammonium hydroxide and
left at room temperature for one hour, maintaining the pH at 7 by
addition of 15% tetrabutylammonium hydroxide and finally is
lyophilized. Thus 805 mg of tetrabutylammonium salt of
epiK5-N-sulfate are obtained.
[0214] (b) Epi-K5-amine-O-oversulfate
[0215] To a solution containing the 805 mg of the salt thus
obtained in 30 ml of dimethylformamide is set at 55.degree. C. and
treated with 30 ml of dimethylformamide containing 2.26 g of
pyridine.SO.sub.3 adduct. The reaction at 55.degree. C. is
continued overnight then 60 ml of water are added to the mixture.
After neutralization with 1 N NaOH, the product is precipitated
with 3 volumes of acetone saturated with NaCl and set at 4.degree.
C. overnight. The precipitate is recovered by filtration on guch G4
and then ultrafiltered with 1000 D Millipore TFF system and dried
at reduced pressure. 550 mg of epi-K5-amine-O-oversulfated are
obtained having a content of iduronic acid of 54%, of
glucosamine-6-O-sulfate of 100%, of glucosamine 3-O-sulfate of 60%,
of monosulfate glucuronic acid of 10%, of monosulfate iduronic acid
of 15%, the rest of the uronic units being disulfated, with a
sulfation degree of 3.55 measured with the conductometric method
according to Casu et al. 1975.
EXAMPLE 2
N-Acetyl-epiK5-amine-O-oversulfate
[0216] To a solution of 250 mg of epi-K5-amine-O-oversulfate
obtained in Example 1 in 75 ml of water and cooled to 4.degree. C.
are added 7.5 ml of methanol and 3.75 ml of acetic anhydride. The
reaction is kept at 4.degree. C. for 2 hours maintaining the pH at
a value of 7 with 5M NaOH. After ultrafiltration on 1,000 D
membrane are added 3 volumes of acetone saturated with sodium
chloride and the precipitate is recovered by centrifugation at
5,000 rpm for 5'. Thus 249 mg of
N-acetyl-epiK5-amine-O-oversulfated are obtained with on iduronic
acid content of 54%, glucosamine-6-O-sulfate of 100%, N-acetyl of
100%, glucosamine 3-O-sulfate of 60%, monosulfate glucuronic acid
of 10%, monosulfate iduronic acid of 15%, the rest of the uronic
units being disulfated, and whose sulfation degree is 3.5 measured
with the conductometric method according to Casu et al. 1975.
EXAMPLE 3
N-acetyl-K5-amine-O-oversulfate
[0217] 1 gr of K5 N,O oversulfate obtained in Example 2 of
WO/02068477 is dissolved in 200 ml of water and the solution is
brought to pH 2 with 1 N HCl and thermostated at 50.degree. C. The
reaction is kept at 50.degree. C. for 3 hours and then cooled to
room temperature and neutralized with 1 N NaOH. The sample is then
cooled to 4.degree. C. and 20 ml of MeOH and 10 ml of acetic
anhydride are added to the reaction which is left at 4.degree. C.
for 2 hours maintaining the pH constantly at 7. At the end of the
reaction the sample is purified from salts by ultrafiltration on a
membrane with a 1000 D cut off and then recovered by dried at
reduced pressure. A 100% N-acetylated product is obtained, 100% 6-0
sulfate, 30% monosulfate and 70% disulfate on the glucuronic acid
is obtained.
* * * * *