CA1307525C - Sulfoamino derivatives of chondroitin sulfates, of dermatan sulfate and hyaluronic acid and their pharmacological properties - Google Patents

Abstract

SULFOAMINO DERIVATIVES OF CHONDROITIN SULFATES, OF DERMATAN SULFATE AND
OF HYALURONIC ACID AND THEIR PHARMACOLOGICAL PROPERTIES.
ABSTRACT
Novel derivatives are disclosed as obtained from chondroitin sulfate, dermatan sulfate and hyaluronic acid, having a molar ratio between sulfate groups and carboxylic groups like that of heparin and furthermore characterized by possessing, as the heparin, the sulfoamino group at the carbon atom at the 6 position of hexosamine.
The novel compounds show a very remarkable clearing activity in comparison with the starting mucopolysaccharides, this activity being similar and for some derivatives comparable with that of heparin.
Moreover, differently from what normally happens upon sulfating the sulfomucopolysaccharides, the anticoagulating activity of the novel compounds is negligible or anyhow very reduced.
The compounds of the present invention find use in the therapy of arteriosclerosis.

Description

_ -2- 1 3~7525 The present invention relateR to novel derivatives of glycosaminoglycans and more particularly of chondroitin sulfates and of hyaluronic acid.
More specifieally, within the family of the chondroitin sulfates, the compounds are described which are obtained frvm chondroitin sulfates A
and C and related mixtures, as well as from dermatan sulfate.
The glycosaminoglycans or sulfomucopolysaccharides or simply mucopoly-saccharides are polymers very widespread in the animal organs and tissues. Among them the polymer which has undoubtedly achieved maximum use in the t~erapeutical field is heparin,-a macromolecule characterized by a high content of sulfate groups and possessing anticoagulating, ~nti~
thrombotic and clearing activity, the latter activity being due to the lipoprotein lipase and to the hepatic lipase.
These peculiar pharmacological activities have stimulated in the past years, (although for a number of aspects these studies are still pend-ing), a great number of investigations in order to clarify the possible rslationship between the structure of the polymer and the above mentioned activities) for recent comprehensive papers see 8. Casu, Advances Carbo-hydr. Chem. Biochem. 43 51 - 134 1985; L.A. ~ransson in "The Polysac-charides", G.O. Aspinali Ed. Vol. 3, pp. 337 - 415 ~Academia Pres~, New York 1985).
It has been thus a~sessed that to the said activitie~ both the ~ul~oamino group (I. Danishefsky, Fed. Proc. Am. Soc. Exp, Biol. 36 pp. 33-35 1977) and the charge density of the polymer ~R.E. Hurst et Alii, in "Chemi~try and Biology of Heparin" ~lsevier North Holl~nd New York, pp. Z9-40 1981;
R.E. Hurst et Alii, J. Clin. Invest. 72 1042-1045 1983; U. Lindhal et Alii, in Annual ReY. Biochem. 47 pp. 401-406 1978) contribute in a relevant ~easure.

With reference to the charge density, it ha~ been found that in the heparin it is on the avPrage equal to 2, if it i8 expres~ed as the ratio between the moles of sulfate groups and the oorresponding moles of carboxylic groups (B. Casu, see above).

. . -~3~ 1 307525 1 It is worth to note that in the pa~t and in the recent times a number of attempt~ has been carried out to obtain from ~everal glycosaminoglycans, through a reaction of direct sulfation, polymers having property like those of heparin.
For example it is known that the chondroitin sulfates directly subjected to sulfation ~K.H. Meyer, HelY. Chim. Acta 75 574~588 1952), or initially N-deacetylated and then sulfated (M.L. Wolfrom, J.A.C.S. 75 1519 1953) acquire anticoagulating properties.
Alternatively, through an initial depolimerisation followed by a sulfa-tion at the hydroxyl groups, starting from the heparin itæelf as well as also from other glycosaminoglycans, it is possible to obtain polymers which are caracterized by an antithrombotic activity which is comparable or even greater than that of heparin. However, it is the worth to note that only the derivati~es of the latter polysaccharide, among those which are described in the present invention, possess a very reduced anti-coagulating activity (EP-A-86401563.1).
It is moreover to be observed that in the literature several methods are reported for the sulfation of glycosaminoglycans, which generally permit the sulfation of the hydroxyl groups only to be carried out.
As a matter of fact, by means of these methods it i8 not possible to oh-tain the selective 3ulfation of the nitrogen atom in the po~ition 2 of hexosamine. 8y adopting particular conditlons it i8 however pos~ible to obtain, as it will be æhown hereina~ter, the ~elective ~ul~ation of the hydroxyl groups occuping the polymer chain the less sterically hindered ~5 po~ition.
However, these reactions normally lead to products with a high content of sulfur, wherein the distribution of the sulfate group~ ~nong the ~everal positions takes place i~ a non specific manner.
Said reactions most of time are carried out in heterogeneous face, usual-ly in anhydrous pyridine and u~ing as the sulfating agent the pyridine--sulfuric anhydride adduct or chloro~ulfonic acidO

It is also possible to operate in a homogeneous phase, in apolar ~on ~ ~ -4- 1 307525 1 aqueous 601vents tfor example dimethylformamide) and using as the sulfat-ing agent sulfure trioxyde or the trimethylamine-sulfur trioxide adduct (J.F. Kennedy, Advances Carbohyd. Chem. Biochem. 29 335-337 1974).
.E. Gilber~ "Sulfonation and related reactions" Interscience publisher, New York 1965, pp 354-360; R.~. Schwei~er "Polysaccharides sulfates. I
Cellulose sulfate with a high degree of substitution" Carbohyd. Res. 21 219 1972: K. Nagasawa et Alii "Chemical sulfation of prepa~ations of chondroitin 4 and 6 sulfate and de~natan ~ulfate. Preparations of chondroitin sulfate E-like material~ from chondroitin sulfate" Carbohyd.
10 Res. 8 1983 1~86).
the glycosaminoglycans to which the present invention relates are preci-sely chondroitinsulfate A and C and their mixtures, dermatan sul~ate or chondroitin sulfate B and ialuronic acid, of which the structural fo~nula of the related repeating di~accaride unit is hereinafter reported.
jo De~natan ~ulfate. (2-acetamido, 2 deo-~ xy, 3-0- ~-D-glucopyranidurosyl, o ~ ~coa, 4-sulfatet B-D-galactopyranose).

r Chondroitin 4-sulfate. (2-acetamido, ~~ _\r ~ ~ 2-deoxy, 3-0-~-D-glucopyranurosyl, ~ ~ ~coc~ 4-sulfate, B-D-galactopyrano3e) C H,OSO~
'' ~ ~ ~\r ~ Chondroitin 6-sul~ate. ~2-acetamido, ~ ~ 2-deoxy, 3-0-~-D-glucopyranuro8yl o WhHCOC~, _ ~ 6-sulfate, B-D-galactopyranos~)~
c~-,O~.
Hyaluronic Acid~ (2-acetamido, 2-deoxy, osll, 3-0-B-D-glucopyranurosyl, B-D-glucopy-~ ranose).

A first object of the present invention thu~ con~ist in novel ~ynthetic derivatives of ~ome glycosaminoglgcans and exactly of chondroitin sul~ate ~ 5 1 3 () 7 5 2 5 1 A and C or their mixtures, chondroitin sulfate and hyaluronic acid, ob-tained by selective N-deacetylation and substequent ~ulfation at ~he nitrogen atom (which in the case of the hyaluronic acid i8 preceeded or followed, as it is disclosed in the examples 6 and 71 by the sulfation at the hydroxyls in the position 6) so as to obtain polymers which, besides the charge density which is characteristic of the heparin as above defined, have also the nitrogen atom at the position Z on the hexoszmine ring substituted with a sulfate group.
It has moreover been found and it is another object of the present inven-tion that these~macromolecules possesses a clearing activity~
The novel polymers9 with reference to the above indicated clearing act-vity and to the said absence of a remarkable anticoagulating activity, are useful as drugs in the terapy of arteriosclerosis. Within the above indicated farmacolo~ic~l activity as shown by the present compounds, a relevant increase of the activity due to the hepatic lipase i~ noticed, which in the case of the derivatives of dermatan sulf~te and of the chon-droitins contributes by fifty percent and more to the clearing activity.
As a matter of fact in the pharmacology to the hepatic lipase an important role i attributed as regards the metabolism of lipoproteins and of the$r influence on the cholesterol cataboli~m (T. Kuusi ct A~ii, FEBS lett. 104 384 l979; H. Jansen et Alii, Biochim. Biophys. ~es. Comm.
92 53 1980, A. Van Tol et Alii, Biochim. Biophy~. Res. Comm 94 lO1, l9B0;
M. Bamberger et Alii, J. Lipid Res. 24 869 1983; H. Jan~en et Alii, Trends ~iochem. Sci. 5 285 1980).
Moreover it has been recently asses~ed during clinical pharmacological experiments (Jacque~ D. Barth et Alii, Atherosclerosi 48 235 1~83 and 68 51 1987) that in individuals su~fering from heavy complisations o~
arterio3clerosi~ the serum values ~f the hepatic lipase are lower than those of norm2l patients, differently from those of the lipoprotein lipa-80. These information indicate per s~ the potential and the remarkableimportance which in the terapy of these diseases the u~e m~y have of a drug which, without influencing the haemosta~ys, ~ould b~ able to .. . . . . .. .

. -6- 1 3 0 7 5 ~ 5 1 stimulate an effective activity of the hepatic lipase in the blood circuit.
Coming back to the chemical structure of these co~pounds, it iæ worth to note that the reactions of N-deacetylation and subsequent selective N-sulfation, which by the way are carried out ~ith per sé knawn method, do not change the amount and distribution of the sulfate groups are init ially present in the polymer.
It is fur~hermore to be mentioned, as it will be demonstrated hereinaf-ter, that by other methods already described in the literature for the sulfation of glycosaminoglycans, even in the presence of compaunds which are partially or totally N deacetylated, it is not possible to obtain polymers which, although having the same content of sulfur, have the same distribution of æulfate groups.
The structures which are obtained by carrying out the above reactions on the abo~e said the macromolecules can be syntheti~ally represented with the structural formulas which are hereinafter reported:

/

.. /

C~,-R

I ~
I~H SOJ -. ~I) . n CHl OSO~ l ~ U~

¦(UH ~0 ~¦ /
-O `L_( N~lsO~, .
01~ . n 1 I X ~

'' :

.~ -8- 1 307525 1 The formula I relate~ to the derivat~ves which are obtained starting from chondroitin sulfate A and C respectively, or from their mixtures, and from dermatan sulfate or chondroitin ~ulfate B; more precisely:
- chondroitin 6-sul~ate N-sulfate (dimeric unit: 2-~ulfoaminol 2-deoxy 3-0-~-D-glucopyranurosyl, 6-sulfate, B-D~galactopyranose):
R = S03 Rl = H, Y = H, X = COO .
chondroitin 4-sulfate N-sulfate (dimeric unit: 2-sulfoamino, 2-deoxy, 3-0-B-D-glucopyranurosyl, 4-sulfate, B-D-galactopyranose):
R = OH~ R1 = S03, Y = H, X = COO .
- dermatan sulfate N-sulfate (dimeric unit: 2-sulfoamino, 2-deoxy, 3-0-~-D-glucopyraniduronosyl, 4-sulfate, B-D-galactopyranose):
R = OH, R1 = S03, Y = COO , X = H
The formula II on the contrary relates to the compound obtained from hyaluronic acid (dimeric unit: 2-sulfoamino, 2-deoxy, 3-0-B-D-glucopy-raniduronosyl, 6-sulfate, ~-D-glucopyranose).
The aoove dimeric units are repeated within the polymer for a number of times varying between 4 and 52, respectively equivalent, in the case of the corresponding sodium salt, to a molecolar ~eight of about 2,300 and 30,000.
The reactions through which the synthesis of the ab~ve compounds has been ~arried out are per se known in the literature and consist, as regards the chondroitin sulfate, in an initial N-deacetylation carried out by heating in a carious pipe to the temperature of 105C the sub~tance with an excess of hydrazine sulfate and anhydrou~ hydra~ine ~or a time of 6 hours or le~s (B.A. Dimitrev et Alii, "Selecti~e cleavaye of glyc~sidic lir~ages" Carbohyd, Res. 29 451 1973; "Selective cleavage of glycosidic linkages, studies with the model compound ber~yl-Z-acetamido-deoxy 6-0 -D-mannopyranosyl-D-glucopyran 08 ide" Carbohyd. Res. 30 45 1973; "Selec-tive ~leavages of glycosidic linkages, Btudies with the O-specific poly-~accharide from Shigella Di6enteriae Type 3" Carbohyd. Re~. 40 365 1975).
The subsequent reaction of ~elective N-~ulfation is carried out in aqueou~ phase at pH 9, using an excess of the ~ulfating agent trimethy-.....

.; ' ' , ' '' , .

9- 1 3n75~s 1 lamine-sulfuric anhydride (L. Levy et Alii, "Chemical and pharmasological studies on N-resul~ated heparin" Proc~ Soc. Exp. Biol. Med~ 109 901 1982; Y. Ynoue et Alii, "Selective N-desulfation of heparin with dimethyl sulfoxide containing water and methanol" Carbohyd. Res. 46 87 1976)o It is noticeable that the N-deacetylation reaction can be repeated a second time so as to lower the content of acetyl groups to the requested level when it has not been possible in the first step.
It is now to be pointed out that the C NMR spectra herein enclosed and particularly tho6e of the figures 1, 2, 3 and 6, clearly show that under the adopted conditions it takes place in an almost complete manner not only the N-deacetylation but also the subsequent N-sulfation7 as it is shown by the peak relating to the sulfoamino group (about 55 ppm) and from the small importance of the peak corresponding to the acetylamino group (25 ppm).
It is furthermore to be observed that as regards the preparation of compounds having low molecolar weight, the glycosamunoglycans may, i~
necessary, undergo a preliminary reaction of the depolimeri~ation which can be carried out with per se known methods, either of chemical or of enzymatic nature (L.A. Fransson "Mammalian glycosaminoglycans" in GØ
Aspinall "The polysaccharides", Academic Press vol. 3 337 1985).
Lastly, as regards the hyaluronic acid, the ~ulfation at the oxygen atom can be carried out be~ore the depolimerisation or, a~ it is p~efer~ble, after thi~ step. The sulration reaction is carried out selectively at the hydroxyl of the carbon atom at fi, u~ing methods known in the prior art, such as for example the reaction in dimethyl formamide us~ng the triethy-lamine-sulfurtrioxide adduct (example 4) of the sulfation with a mixture of sulf~ric acid-chlorosulfonic acid (Ao Naggi et Alii7 "Supersulfated heparin fragments, a new type of low-molecular weight heparin". ~iochem.

~harmacol. 36 17 1985 1987).

~he chemical parameters which characterise the polymers obtained through the above reaction are the following ~the data have been calcula~ed on dry basi~ and relate to the corresponding sodium ~alt of these s~bstan-0- 1 3()7525 ce s ) - Sulfoni~ sulfur: 8.0-12% preferably 9.5~ 4%
- Sulfates/carboxyls: 1.5-2.~ preferably 1.8-2.1 - Residual N-acetyl groups: 3% preferably 1%
- ~ Molecular weight: 2,3-30 X 10 preferably 3~-15 X 10 (E.A. Johnson et Alii, Carbohyd, Res. 51 119 1976).
In order to demonstrate that by the methods used for the sulfation of the hydroxyls it is not possible to obtain, starting from the above sulfomucopoly accharides, compounds having the chemical properties described in the present invention, a sulfation has been carried out on a 10 preparation of dermatan sulfate which previously had been selectively N-deacetylated, by reaction with trimethylamine-sulfur trioxyde adduct in dimethylformamide, according to the disclosure of the EP-A-86401563.1O
The po1ymer, as it is required by the process described in the above patent application, has been previously con~erted into the corresponding salt with a quaternary ammonium cation (triethylammonium).
15 m e experiment and the obtained results are descri~ed in the example 4.
It has been assessed that in the range (105-2.~) of the ratio between the sulfate groups/carboxylic groups of interest for the present in~ention the sulfation reaction takes place under the above condition~ almost ex-clusively at the oxygen atom Or the alcohoic group which in the polymer chain has the less sterically hindered position, namely that bound to the carbon atom positioned at po~ition 6 in the galacto3a~ ne ring.
It has been moreover assessed (example 5) that al80 adopting the aulfa-tion conditions reported by M.L. Wolfrom in J.A.C.S. 75 1519 1953 (pyr-idine + chlorosul~onic acid) and even starting from a preparation of dermatan sulfate previously N-deacetylated, it is not possible to have is the reaction occurring at the ammonium group of the galactosamine.
As a matter of fact, al80 in this case it has been demonstrated that the reaction takes place at the hydroxyl groups; ~ore precis~ly the sulfation takes place~ at the hydroxyl at position 6 of galacto3amine and that at the position 2 of the hyaluronic acld (figure 5).

1 From the preceeding infor~ation it i~ eYident that the sulfated product of the present invention are obtained only under partioular sulfation conditions.
As regards the method which have been used to evaluate the pharmacological properties of the novel derivatives of the in~ntion, the anticoagulating activit~ has been assessed in vitro acoording to the m~thod USP XX, using aæ the reference standard the third international standard of heparin.
The clearing activi~y induced both by lipoprotein lipase and by hepatic lipase has been assessed according to the method described in the paper by R. Pescador et Alii, Biochem. Pharmacol. 36 4 253 1987.
For the reasons which are hereinafter stated with reference to table I, the results obtained for the activity of the lipoprotein lipase and of the hepatic lipase (expressed in micromoles of fre~ fatty acids) relating to the lowest and to the highest among the administered doses have been reported.
The data which have been obtained in the previous experiments, are reported in table I in Yhich a comparison is carried out with those relating to the starting glycosaminoglycans.
For these substances ho~ever no statistically slgnific~nt relationship between the administered do~e and the corresponding activity has been as~e~sed. For the~e reasons in the table I the re~ults have bsen reported corresponding to the lo~est dose and to the highest dose among those ad-mdnistered, in order to render anyhow possible a comparison.
From the table I it is çvident the relevant increase of the clearing ac-tivity of the novel çompounds in comparison with that of the starting ~lfomucopolysaccarides.
~oreover, it i~ observed that in the ca~e of ~he derivatives of chondroitins ~nd of the dermatan sulphats the hepatic lipa~e contribu~es to th~ total clearing activity by fifty percent and more.
It is worth to note that, with reference to the data reported in the in-dicated table, at the do~e of 1.25 mgJkg, the activi~y ~f the hepatic ~ .. ., . . ~, . . . . . . . . . .

'~ -12- l 3 0 7 5 ~ 5 1 lipase iR similar and in the case of the compounds obtained rrom dermatan sulfate and from hyaluronic acid is comparable with the corre~ponding one of heparin at the do~e of 1.4 mg/kg.
In the case of the derivative of hyaluronic acid the activity of lipoprotein lipase is comparable with that of heparin.
In the table II it is demonstrated that by introducing in a N-deacetylat-ed and subsequently N~sulfated derivative of chondroitin sulfate (in the s~bject case chondroitin sulfate A ~ C) a sulfate group at a hydroxyl (with reference to the relating unit, see the previous structural formu-la) a relevant increase of anticoagulating activity is obtained which isthree times greater than that of the starting polymer.
In the table III the data are illustrated relating to the anticoagulating ~CtiYity of a sample of dermatan sulfate having sulfate groups substitut-ed for some hydroxyl functions (GAG 938) and furthermore characterized by havir~ a ratio between sulfate groups and carboxylic groups of 2, and consequently formally falling within the range foreseen by the present invention.
The above said ~able demostrates that the s~lfation at the oxygeatom does not produce the same effect, as regards the ~nticoagulating activity, as the like reaction at the nitrogen atom of galactosamine.
As a matter of fa~t in the case of the GAG 938 derivative thi~ activity i8 at least double with respect to the starting polymer and is ~nyhow greater than that of the corresponding d~rivatiYc wherein the sulfation has on the contrary o~ occurred at the nitrogen atom of the galactosamine (GAG 944 IV, table I).
These examples confirm that for the terapeutical use as drugs ha~ing clearir~ acti~ity of sulfate derivatives of chondroitin sulfates, of dermatan sulfate and of hyaluronic acid, ~hen these compounds have no site effects of haemostasis, two structural requirements are e ~ential, namely the selective sulfation at the nitrDgen atom in the position 2 of the pyranose ring of hexo amine and the ratio between moles of sul~at~

grOUp8 and of carboxylic groups, which must be within the range for seen ... . ~ ~ ..... . . . .. ...... .... .. . . . . . .. . . .
' ' ', :

~ 13- l 307S25 1 by the present invention.
As a confirmation of the above structural requirements which as stated are critical in order to obtain compounds having an evident clearing ac-tivity without undesired effects on the coagulation, it is worth to note 5 that by acting on a polymer which does not contain sulfate groups at the hydroxyls, as for example hyaluronic acid, a N-sulfation reaction at the nitrogen atom in the position 2 of hexosamine according to the process described in the example 6, a substance is obtained ~GAG 1045) having a very reduced clearing activity with respect to the corresp~nding loderivatives in which also the hydroxyl in the position 6 is ~ub~tituted for with a group -OS03.

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1 The substances according to the presente invention can be administered in form o~ their pharmacologically acceptable salts, such as, as a non limiting example, the corresponding ~alts with alkali and earth-alcali metals, for example sodium and calcium salts.
The foreseen pharmaceutical forms are the standard one~: ~terile and ap-irogenic solutions in vials and lyophilized preparations packaged in sterile bottle~ to be dissolved upon administering in the solvent for the administration by parenteral and subcutaneous route; tablets, gelatin or gastroresistant capsules, granulates for the oral administration.
The solutions for parenteral administration may contain 1 to 250 mg/ml of active principle, preferably 1 to 150 mg/~l, whereas the co~positions ~or oral use have a content of active principle as a unitary dose of 1-500 mg.
The daily posology may vary from 1 to 2,000 mg, preferably from 1 to 500 mg.
Example 1 1 g of chondroitin 4-sulfate (ratio sulfate groups/ carboxylic groups 1.07; identification n. GAG 920; m.w. 12,000) is admixed with 0,325 g of anhydrous hydrazine9 directly in a carius pipe which is immediatly after tightly sealed.
The N-deacetylation reaction is carried out by heating the pipe to 105C
for 5 hour 8 .
At the end the liquid pha~e i8 evaporated under reduced pressure making use of subaequent addition of toluene to promote the removal of hyd-razine.

The residue is taken with water and the alcalinity is neutrali~e by ~d-ding 1 M HCl and by cooling in water and ice bath.
The solution is then dialized with distilled water throught dialysis membranes of 3500 D (Thomas dialy~er tubing 3787-H47~.

The solution is then evaporated under reduced pressur~ thu~ recovering 7DO mg of chondroitin 4-sulfate N-deacetylated (identification NoO G~G
~57).

,.

1 1 g of the product i6 di~solved in 20 ml of distilled water, the pH of the resulting solution being adjusted to 9 by means of few drop~ of 2N
NaOH.
The solution is heated to 55~C and under stirring 1 g of the trimethy-lamine-sulfur trioxide complex is added together with about 900 mg of sodium bicarbonate, in order to restore the alckaline pH. Subsequently, at 4-hour intervals (at the fourth and eight hour) the addition of 1 g of reactant and of gOO mg of the salt is repeated.
After 16 hours the reaction is stopped by cooling the reaction mixture to room temperature.
Then the solution is dialized, by using the same above indicated membrane but substituting ~or the distilled water a 0.5M solution of sodium chloride. By evaporation under reduced pressure there are obtained 1.1 g of chondroitin sulfate N-sulfate with a ratio sulfate/carboxylic groups =

2.2; m.w. 8000; identification number: GAG 973). The C N~R speckrum (figure 1) shows an attenuated signal at 25ppm (group-CH3, letter A), a signal at about 55 ppm (letter E) corresponding to the carbon atom at the position 2 of the galactosamine bearing the group NH-S03. There are furthermore seen, as in the starting polymer, a ~ignal at about 60 ppm (letter C) correspondlng to the carbon atom in the position 6 of the galacto~amine bearing a hydroxyl group, and at about 70 ppm ~letter D) relating to the carbon atom in the position 4 o~ ~he galacto~amine bear~
ing a ~ul~ate group.

The signal indicated in figure 1 with the letter B corresponds to the carbon atom in the position 2 Df the galacto~amine bearing as ~ubstituent the group -NH-CH3.
Example 2 1 g of chondroitin 6-sulfate ( ratio of sulfate/carboxylic group = 1 9 id-entification No. GAG 921, m.w. 18.000) i8 admixed ~ith the same amount of hydrazine sulfate indicated in the above example. The N-deacetylation re~ction is carried out a~ above descrlbed~
Then the N-~ulfation is carried out obtaining 1.2 g o~ final product ~id--20- l 307525 1 entificatlon N. GAG 974~ having a ratio between sulfate groups ~nd carboxylic group~ of 2.1, m.wO 15.000.
The spectrum, reported in figure 2, apart from the 8ignals common to that showed in figure 1 as regards the almost total absence of N-acetyl groups and the appearance of the signal corresponding to the sulfoamino group, shows at about 70 ppm the signal (letter C) of the carbon atom in the position 6 of the galactosamine bearing the sulfate group.
Example 3 1 g of dermatan solfate ( ratio between sulfate groups and carboxylic groups of 1.1; identification n. GAG 968; m.w. 9000~ is subjected to the N~deacetylation reaction and subsequent N-sulfation.
There are obtained 695 mg of the corresponding derivative. The substance (identification n. GAG 944 IV) has a sulfate groups/carboxylic groups ratio = 1.8 , m.w. 6000.
The C NMR spect~um (figure 33 shows9 as in the starting substance, one peak at 60 ppm (letter C) corresponding to the carbon a*om in the po~i-tion 6 of the galactosamine bearing a hydroxyl group, one a peak at 5S pm (letter E) relating to the carbon atom in the position 2 of the galac-tosamine bearing a group NH-S03 and lastly, likewise the dermatan sul~a~
te, one peack at 77 ppm (letter D) corresponding to the carbon atom in the position 4 of the same ring bearing a sulfate group.
Example 4 ~hi8 example relate to the sul~onation carried out one sample of N-deacetylated dermatan sulfate, prepared according to the previous ex-ample, according to the E.P. Application 86401563.1.
The N-deacetylated sample (lg) i converted into the corresponding acidic ~orm by dissolving the substance in 10 ml of distilled water and admixing with Amberlite IR 120 (form H ). The resin is removed by centrifugation and the pH of the solution i8 brougth to the value of 5 by means of a lOX
~/v solution of triethyla~ine in eth~lol. The solution is extracted with ether.
The aqueou~ fase i~ lastly lyophilized7 1 g of the obtained salt (salt of ~ /~c~e ~h~k ~ 21- 1 ~ 0 75~S

1 N-deacetylated dermatan sulfate with triethylammonium) is di~solved in 140 ml of dimethylformamide. It i~ cooled to 0C and 10 g of the trimethylamine - sulfur trioxide adduct, previously dissolved in 60 ml of dimethylformamide, are added.
The reaction is carried out at the indicated temperature for one hour.
Then water cooled at + 5C is added, the pH is rapidly brought to 9 by adding 5N NaOH nd the product is precipitated by adding 600 ml of ethanol saturated with sodium acetate. During this phase the temperature is constantly maintained at +5C. It is filtered, di6~01ved again in 10 ~ater and dialired against distilled water, using the above mentioned di-alysis membrane.
900 mg of product (identification n. GAG 941) are obtained having a ratio sulfate groups~carboxylic group = 1.5.
The NMR spectrum (figure 4) shows an intense signal at about 50ppm (let-15 ter D) corresponding to the carbon atom in the position 2 of the galac-tosamine and bearing the primary amino group (-NH2), one signal between 60 and 65 ppm (letter C~ corresponding to the carbon atom in the position 6 of the galactosamine bearing a free hydroxyl and lastly a signal bet-yeen 65 and 70 ppm (letter E) corresponding to the carbon atom in the 20 position 6 with a substituent-S03 .
~he peaks indicated by the letters A and B respectiYely maintain the meaning attributed thereto in the figure 1.
Example 5 Sulfation o~ denmatan sulfate previously N-deacetylated according to the 25 ~ethod described by M.L. Wolfrom, J.A.C.S~ 75 1519 1953 (pyridine chlorosulfonic acid).
1 g of N-deacetylated dermatan sulfate are dis301ved in 20 ml of dis-tilled water and then precipitated by adding 60 ~1 of methanol. The precipitate is dried.
30 In a flask maintained in nitrogen atmosfere, provided wi~h condenser and ~ropping funnel, there are initially charged 30 ml of anhydrou~ pyridine at 0C. 8y maintaining said temperature in the liquid there are 81oWly - . , . ' ' . . . . . .

~ 22- 1 3~7525 1 dropwi~0 added 4 ~1 of chlorosulfonic acid. Upon the addition is complet-ed, through the ~ame inlet ~ ~uspension, previously cooled at O~C, of N-deacetylated dermatan sulfate in anhydrou~ pyridine is quickly in-troduced.
The mixture is moderately heated up to room temperature and then rapidly to 100C, the reaction being prosecuted at that temperature for one hour.
The mixture is cooled, the superanatant liquid is decanted and a portion o~ 20ml of distalled water is added. The solution is neutralized with 5 N
NaOH.
Then it is dialized against distilled water. Most of the liquid is eY-aporated under a reduced pressure at 35C by means of methyl alcohol and then lyophilized~
There are obtained 750 mg of product (identification n. GAG 986~, Xaving a ratio between sulfate groups and carboxylic group of 1.3.
The N~R spectrum (figure 5) shows a signal at about 50 ppm (letter A) corresponding to the carbon atom in the position 2 of the galacto~amine bearing the group -NH2, one signal at about 60 ppm (letter B) correspond-ing to the carbonato in the position 6 bearing a hydroxil grvup and one peack at 74 ppm (letter C) corresponding to the carbon atom in the posi-tion 2 Or the ring o~ the iduronic acid bearing a sulfate group.Example 6 Sinthesis of the hyaluronic acid N, 6-di~ulfate.
1 g of the compound previously depolymerized (GAG 1039) and having molecular we1ght of 15000, is subjeeted to the N deacetylation reaction according to the example 1.
Then N-sulfation is carried out as described, leading to 800 mg of N-sulfate compound.
The next reaction of O-sulfation at the carbon atom in the po~ition 6 of the glucosamine is carried out according to the conditions already in-dicated in the previous example 4 aR regards the like re~ction ofN-deacetylated dermatan sulfate leading to 650 mg of the ~oreseen compound (GAG 902), m.w. 7000, having a ratio between sul~ate groups and ~ ... .

~ 23- 1 307525 1 carboxylic groups of 1.9.
The C MMR spectrum shows a signal at about 55 ppm corresponding to the carhon atom in khe position 2 of the glucosamine bearing a group NH-S03 and a 3igr}al at about 70 ppm corresponding to the carbon atom in the S position 6 of ths glucosamine bearing a sulfate group.
Example 7 Synthesis of hyaluronic acid N, 6-disulfate.
The previou~ly example is repeated as regard the N-deacetylation and sub-sequent N-sul~ation. The derivative which is obtained (GA~ lQ45, 0.96 g) has ratio sulfate/carboxyl groups of 0.9. The next reaction of sulfation at the oxygen atom in the position 6 of the glucosamine is on the contra-ry carried out by using a mixture of sulfuric acid and chlorosulfonic acid (A. Naggi et Alii, Biochem. Pharmacol. 36 12 1895 1987).
20 ml of 95% H2S04 and lOml of HClS03 are admixed in a flask at - 4C.
g of N-deacetylated and N-sulfated hyaluronic acid, previously dried, is added to the liquid phase maintained under stirring~ at the same above mentioned te~perature. The reaction is continued for one hour at -4C and for further 60 minutes at 25C.
The sulfation is then terminated by pouring the solution in 500 ml of ethyl ether at -4C. The precipitate is filtered, dissolved in 20 ml of distillated water and the obtained solution is added with 0.5 N NaQH un-til the reaction is noutral.
It is dialized against distilled water and the product is recovered by evaporation under reduced pressl~e followed by lyophilization. Ther~ are obtained 900 mg (GAG 1046) of a compound having a ratio bebween sulfate groups and carboxylic groups of 1.8, m.w. 8000. The C NMR spectrum is not different, as regard the distribution of the sulfate group~, from that reported for GAG 902.
Example 8 Synthesi~ of GAG 952 (chondroitin sulfate A ~ C, N-de~cetylnted and ~ub s0quently sulfated both at the nitrogen atom and at the oxygen ~tom).

1.5 g of chondroitin sulfate A ~ C are treated as described $n the ex-.

~24- 1 307525 1 ample 1, giving 1.3 g of product in which the nitrogen atom bound to the carbon atom in the position 2 of the galactosamine is almost quantitati-vely substituted with a sulfate group.
The next sulfation is carried out as desoribed for the sulfation at the oxygen atom described in the previous exampl~ 7.
There are obtained 1.3 g of product having a ratio between sulfate and carboxylic groups of 2.7.
Example 9 Synthesis of GAG 938 (dermatan sulfate sulfated at the hydroxyl groups of 10 the chain.
1 g of dermatan sulfate, GAG 968 is sulfated under the conditions described in the example 7, with respect to the sulfation at the carbon atom in the position 6 of the glucosamine. There are abtained 0.9 g of a product having a ratio sulfate groups/carboxylic groups = 2~ The C MMR
15 spectrum shows at about 25 ppm an intense signal corresponding to the carbon atom in the position 2 of galactosamine bearing a group NH-CH3 and a weak signal at about 50 ppm corresponding to the same carbon atom bear-ing a primary amino function -NH2.

~5 ~ .
''

Claims (12)

1. Synthetic sulfoamino derivatives of glycosaminoglycans selected in the group comprising chondroitin sulfates A and C, their mixtures, dermatan sulfate and chondroitin sulfate B, the dimeric units of which have the following formula:

wherein R represents OH, SO?, R1 represents H, SO?, Y represents H, COO-, and X represents H, COO-, n is a number between 4 and 52 inclusive.

2. Synthetic sulfoamino derivatives of the hyaluronic acid, the dimeric units of which have the formula:

wherein n is a number between 4 and 52 inclusive.

3. Synthetic derivatives according to claims 1 or 2, characterized by following chemical parameters calculated on dry basis and referred to the corresponding sodium salt:
sulfonic sulfur 8,0-12%
sulfate/carboxylic groups 1,5-2,3 residual N-acetyl groups <3%
molecular weight 2,3-30 x 103

4. Synthetic derivatives according to claim 3, characterized in that said parameters are included in the following ranges:
sulfonic sulfur 9,5-11,4%
sulfate/carboxylic groups 1,8-2,1 residual N-acetyl groups < 1%
molecular weight 3-15 x 103

5. A process for the preparation of the derivatives of claim 1 or 2, characterized in that a first deacetylation step in carried out by heat-ing the starting glycosaminoglycane to the temperature of 105°C with an excess of hydrazine sulfate and anhydrous hydrazine for a time not greater than 6 hours and a subsequent step of selective N-sulfation is carried out in aqueous medium at pH 9 with an excess of sulfating agent.

6. A process according to claim 5, characterized in that said sulfating agent is selected among triethylamine-sulfuric anhydride and clorosulfonic acid.

7. A process according to claim 5, characterized in that said N-deacety-lation reaction is carried out in a number of steps until the content of acetyl groups in brought to the level required for the next N-sulfation step.

8. A process according to claim 5, characterized in that before said N-deacetylation step a preliminary depolymerization step is carried out in a per se known manner.

9. A process according to claim 8, characterized in that said depolymerization step is carried out before the N-sulfation step.

10. Pharmaceutical composition, characterized by containig, as the active ingredient, a glycosaminoglycan derivative according to claims 1 or 2, together with the usual excipients.

11. Pharmaceutical compostion according to claim 10, having clearing ac-tivity,

12. Pharmaceutical composition according to claim 10, characterized in that said active ingredient is contained in a unitary dose of 1-500 mg.