CA2204529A1 - Smooth muscle cell proliferation inhibitors - Google Patents

Abstract

This invention comprises polyanionic benzylglycosides of benzene triacid amides of general formula (I) wherein each of R1, R2, R3 and R4 are, independently, H, SO3M, or (a) and each oligosaccharide group contains 1 to 3 sugar groups; M is lithium, sodium, potassium, or ammonium; n is 1 or 2; X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms; Y is carbonyl or sulfonyl; or the pharmaceutically acceptable salts thereof, as well as their use as smooth muscle cell antiproliferation inhibitors and as therapeutic compositions for treating diseases and conditions which are characterized by excessive smooth muscle proliferation.

Description

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Wo 96/14324 - Pcr/uss5/l4737 ~MOOTH MIJ~Cl,F. CF.~,l. PRO~.IFF.R~TTON Il~H~RITO~2~

This invention relates to novel polyanionic benzylglycosides of benzene triacid amides. More particularly, this invention relates to polyanionic 5 benzylglycosides of benzene triacid amides and their use as smooth muscle cell .
antlprollferatlon lnhlbltors and as therapeutlc composltlons for treatlng dlseases and conditions which are characterized by excessive smooth muscle proliferation, such as restenosis.
R~k~round of the Invention Smooth muscle cell (SMC) proliferation is a critical event in the pathogenesis of atherosclerosis and transplant arteriosclerosis as well as in the response to injury arising from all forms of vascular reconstruction such as angioplasty (Raines E. W.;
Ross R. Br. Heart J. 1993, 69 (Supplement), S 30; Clowes, A. W.; Reidy, M. A. J.Vasc. Surg 1991, 13, 885; Isik, F. F.; McDonald, T. O.; Ferguson, M.; Yamanaka, E.;
Gordon Am. J. Pathol. 1992, 141, 1139). However, clinically effective inhibitors of smooth muscle cell proliferation for use as antirestenotic agents have not been successful to date (Herrrnan, J. P. R.; Hermans, W. R. M.; Vos, J.; SelTuys P. W.
Drugs 1993, 4, 18 and 249).

Reendothelialization of the injured area conculTent with smooth muscle cell proliferation is a major consideration for inhibiting restenosis (Casscells, W.
Circulation 1992, 86, 722; Reidy, M. A.; Lidner, V. in Endothelial Cell Dysfunctions, Simionescu, N. and Simionescu M., Ed. Plenum Press, NY NY, (1992), 31). Thus, any successful approach to inhibiting SMC proliferation should not interfere with endothelial cell repair or the normal function of other cell types (Weissberg, P. L.;
Grainger, D. J.; Sh:~n~h:~n C. M.; Metcalfe, J. C. Cardiovascular Res. 1993, 27,1 191).
The glycosaminoglycans heparin and heparan sulfate are endogenous inhibitors of SMC proliferation, yet are able to promote endothelial cell growth(Castellot, J. J. Jr.; Wright, T. C.; Karnovsky, M. J. Seminars in Thrombosis and Hemostasis 1987, 13, 489; Wight, T. N. Arteriosclerosis 1989, 9, 1). However, the full clinical benefits of heparin, heparin fragm.onts, chemically modified heparin, low CA 02204~29 1997-0~-0~

molec~ r weight heparins, and other heparin mimicL-in~ anionic polysaccharides may be con~ ~ised due to other pharmacological liabilites (excessive bleeding arising from anticoagulation effects, in particular) coupled with heterogenity of the various pn,pa,~tions (Borman, S. Chemcial and Engineering News, 1993, June 28, 27;
S Schmid, K. M.; Preisack, M.; VoeLker, W.; Sujatta M.; Karsch, K. R. Seminars in Thrombosis and Hemostasis 1993, 19 (Suppl. 1), 155; Amann, F. W.;
Neuenschwander, C.; Meyer, B. Seminars in Thrombosis and Hemostasis 1993, 19 (Suppl. 1), 160; Radhakri~hn~ml-rthy, B.; Sharma, C.; Bhandaru, R. R.; Berenson, G.
S.; S~n7~ni, L.; Mastacchi, R. Atherosclerosis, 1986 60, 141; Maffrand, J. P.;
10 Hervert, M. M.; Bernat, A.; Defreyn, G.; Delevassee, D.; Savi, P.; Pinot, J. J.;
Sampol, J. Seminars in Thrombosis and l~emostasis, 1991, 17 (Suppl. 2), 186). Since the anticoagulant effects of many of these agents are independent of SMC
antiproliferative activity, it would be expected that polyanionic agents which are more homogenous in composition and of more defined molecular structure would exhibit a 15 more desirable and balanced profile with fewer side effects associated with aforementioned anionic polysaccharides.

Prior Art WO 92/18546 discloses specific sequences of heparin, obtainable in pure forrn through synthesis or heparin fragment isolation, which exhibit SMC antiproliferation activity. Beta-Cyclodextrin tetr~decaculfate has been described as a smooth muscle cell proliferation inhibitor and as an effective inhibitor of restenosis (Weisz, P. B.;
Hermann, H. C.; Joullie, M. M.; Kumor, K.; Levine, E. M.; Macarak, E. J.; Weiner, D. B. Angiogenesis: Key Principle - Science - Technology - Medicine- Steiner R.,Weisz, P. B.; Langer, R. Eds. Birkh~l.cer Verlag, Basel Switzerland, 1992, pg. 107;
Hermann, H. C.; Okada, S. S.; Hozakowska, E.; LeVeen, R. F.; Golden, M. A.;
Tomaszewski J. E.; Weisz, P. B.; Barnathan E. S. Arleriosclerosis and Throttlbosis 1993, 13, 924; Reilly, C. F.; Fujita, T.; McFall, R. C.; Stabilito, I. I.; Wai-si E.;
Johnson, R. G. Drug Development Research 1993, 29, 137. U.S. Patent No.
5,019,562 discloses anionic derivatives of cyclodextrins for treating pathological conditions associated with undesirable cell or tissue growth. WO 93/09790 discloses antiproliferative polyanionic derivatives of cyclodextrins bearing at least 2 anionic resi~ es per carbohydrate resiclues. EP 312087 A2 and EP 312086 A2 discloses 35 anliLl~olllbo~ic and anticoagulant plo~ ies of sulfated bis-aldonic acid ~mi-les.

wo 96/14324 PCT/US95/14737 U.S. Patents Nos. 4,431,636, 4,431,637, 4,431,638, and 4,435,387 describe polysulphated, thio- and oxy-aryl glycoside derivatives as modulators of the complement system.

The SMC antiproliferative compounds of the present invention differ from all of the prior art in that the SMC antiproliferative compounds (a) are polyanionicbenzylglycosides of benzene triacid amides which bear no structural resemblance to heparin, or sulfated cyclodextrins, (b) contain no more than three contiguous sugar 10 residues (trisaccharides) and (b) are of defined structure.

nescriDtion of the Invention This invention describes the composition and utility of polyanionic 15 benzylglycosides of benzene triacid amides of formula I

Y ~o~oR3 ~ oR2 J n /~4O~ ~H ~O~oR3 20 wherein each of Rl, R2, R3, and R4 are, independently, H, SO3M, or CA 02204~29 1997-0~-0~

wo 96/14324 PCr/USs5/14737 R40~ 0~0 oR2 and each oligosaccharide group contains 1 to 3 sugar groups;

M is lithium, sodium, potassium, or ammonium;

n is 1 or 2;

X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;

Y is carbonyl or sulfonyl;

or a pharmaceutically acceptable salt thereof.
It is understood that each of the several occurrences of Rl in any given molecule of Formula I herein may be the same or different. Similarly, each of the several occurances of R2, R3 or R4 may be the same or different from the other occurences of R2, R3 or R4, respectively. Similarly, each of the several occurrences 20 herein of Y in any given molecule and each of the occurrences herein of n in any given molecule may be the same or different from any other occurrence of Y or n,respectively.

It is also understood that the lower alkyl groups herein may be straight 25 chained or branched and may be, for example, methyl, ethyl, propyl, isopropyl or butyl groups. Similarly, the lower alkoxy groups herein may be straight chained or branched and may be, for example, methoxyy, ethoxy, propoxy, iS-Jpl~ol)oxy or butoxy groups.

A more ple~.l~,d aspect of this invention is the compounds of formula I:

H N-~oR~3R~\
X ~ ~ oR2 / n G40~X~~ R~0~

~ wherein each of Rl, R2, R3, and R4 are, independently, H, SO3M, or oR2 10 and each oligosaccharide group contains 1 or 2 sugar groups;
M is lithium, sodium, potassium, or ammonium;

n is 1 or 2; X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl;

or a pharm~ceu~i~lly acceptable salt thereof.
The most pre~lled coll~poullds of this invention are:

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E~enzene-1,3,5-tricarboxylic acid tris~[2-methyl-5-(tetra-O-sulfato-~-glucopyranosylo~yll,clhyl)phenyl]amide} dodecasodium salt or a ph~lllaceutically acceptable salt thereof;

Benzene-1,3,5-tricarboxylic acid tris{[2-methyl-5-(hepta-O-sulfato-~-cellobiosyloxymethyl)phenyl]amide ) heneicosasodium salt or a pharrnaceutically acceptable salt thereof;

Benzene-1,3,5-tricarboxylic acid tris{[2-chloro-5-(hepta-O-sulfato-~-D-maltosyloxymethyl)phenyl]amide) heneicosasodium salt or a pharmaceutically acceptable salt thereof;

Benzene-1,3,5-tricarboxylic acid tris{[2-chloro-5-(hepta-O-sulfato-~-D-cellobiosyloxymethyl)phenyl]amide) heneicosasodium salt or a pharmaceutically acceptable salt thereof;

Benzene-1,3,5-tricarboxylic acid tris{[2-chloro-5-(hepta-O-sulfato-~D-lactosyloxymethyl)phenyl]amide) heneicosasodium salt or a pharmaceutically acceptable salt thereof;

Benzene-1,3,5-tricarboxylic acid trisl[3,5-bis-(tetra-O-sulfalo-~-D-glucosyloxymethyl)phenyl]amide) tetracosasodium sall or a pharmaceutically acceptable salt thereof;
Benzene-1,3,5-tricarboxylic acid tris~[3,5-bis-(hepta-O-sulfato-~-D-cellobiosyloxymethyl)phenyl]amide } tetratetracontasodium salt or a pharmaceutically acceptable salt thereof.

Process of the Invention The compounds of the present invention are prepared according to the general sequence of reactions outlined in the Scheme below:

R40~ + n( ~ NO2 9lycosidation 1 oR2 2 ~40~X ~\~ NO2 ~40 ~ ~~ NH2 ORZ ~ R~o~ n Y ~%R3 ~) Y~Y--Cl(R O~OR ~~~

2) NaOCH3 or NaOH oR2 n OR n 3) SO3 (NCH3)3 wherein R, n, and X are as defined above.

Thus, a glycosyl bromide 1 is coupled with a benzylic alcohol 2 in the presence of a catalyst such as a ll~el.;ul,c bromide, mercuric cyanide, silver triflate, or silver perchlorate in an aprotic solvent such as dichloromethane, ether, toluene, or nillu~l~ethane at temperatures ranging from -40~C to ambient temperature to yield 10 glycoside 3.

Reduction of the nitro group of 3 with a reducing agent such as stannous chloride is accomplished in a polar aprotic solvent such as ethyl acetate at ambient lempel~ture to reflux, or by catalytic hydrogenation in the presence of a catalyst such 15 as p~ll~linm on carbon gives an anilino compound 4.

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Coupling of 4 with a benzene triacid chloride or benzene trisulfonyl chloride is completed in the presence of an amine base such as triethylamine or diisopropylethylamine in an aprotic solvent such as dichloromethane or tetrahyd~oru,all. Hydrolysis of any acetate groups present on the sugars with a base such as sodium methoxide in methanol or aqueous sodium hydroxide in methanol at ambient ~ per~ture to reflux, and sulfation of some or all of the free hydroxyl groups on the sugars with a reagent such as sulfur trioxide-trimethylamine complex or sulfur trioxide-pyridine complex in a polar aprotic solvent such as dimethylro~ ide or dimethylsulfoxide at temperatures ranging from 0~C to 10(PC
yields the target compounds I.

The invention is also directed to pharmaceutical compositions comprised of an effective amount of one or more of the polyanionic benzylglycosides of benzene triacid amides of this invention either alone or in combination with excipients (i.e.
pharmaceutically accpetable materials with no pharmacological effect). Such compositions are useful for diseases and conditions which are characterized by excessive smooth muscle cell proliferation most frequently arising from vascularreconstructive surgery and transplantation, for example, balloon angioplasty, vascular graft surgery, coronary artery bypass surgery, and heart transplantation. Other disease states in which there is unwanted vascular proliferation include hypertension, asthma, and congestive heart failure. The compounds of ~he invention are thus useful fortreating these diseases and states.

The compounds of this invention may be administered systemically, for example by intravenous injection, typically ranging from 0.1 to 10 mg / kg/ hr over 5 - 30 days, or by subcutaneous injection at lower dose, by oral ~tlmini~tration at higher dose than intravenous injection. Localized delivery of the compounds of this invention may also be achieved by transmembrane, transdermal, or other topical ~(lrninistrative routes using ap~l~Jpliate continuous release devices such as ~u~ol~ g matrix, where applicable. The compositions of the invention may be forrn~ t~d with conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent and the like. These are forrnul:lted in a conventional manner. It is understood that the compounds of this invention may be ~lmini~tered in any manner and at any concentration that is efficacious to the particular recipient. The manner of delivery and composition and concentration of the dose will be detellllined on an CA 02204~29 1997-0~-0~
Wo 96/14324 PCT/USg5/14737 individual basis by the physician or other skilled medical professional treating the F.ff~ on Cell Prolifer~tion A. Cell Sources The ability of the compounds of the present invention to inhibit smooth muscle cell proliferation and modulate endothelial cell growth was established using 10 isolated aortic cells obtained from commercial sources or, for certain species, prepared in-house. Cell lines used in this study include human and porcine aortic smooth muscle cells and human aortic endothelial cells. Human aortic cell lines were obtained from Clonetics Corporation (San Diego).

Porcine aortas were received from a local slaughterhouse. The material was iced during transit. The aorta was scrupulously cleansed of fatty tissue and rinsed in sterile phosphate-buffered saline with 2% antibiotic/antimycotic (Gibco catalog # 600 - 5240 AG). The tissue was then digested in 10 - 15 mL of "Enzyme Mixture"
containing collagenase type I, 165 U/mL; elastase type rII, 15 U/mL; BSA, 2 mg/mL;
and soybean trypsin inhibitor, 0.375 mg/mL followed by incubation at 37~C under 5% C~2 for 10 - 15 min. After this treatment, the outer surface adventitia was easily removed by peeling with forceps. The aorta was then longitudinally cut and laid open and the endothelial layer was removed by scraping.

The medial layer of cells was rinsed in enzyme solution, and placed in a new 100 mm dish with 10 mL enzyme solution. The aorta was minced using a fine pair of scissors and digested for 2 - 3 h at 37~C in 30 mL of fresh enzyme solution. After digestion, the tissue was homogenized using a sterile Pasteur pipette with a fire polished tip or an eppendorf pipetter with a 200 - 1000 mL sterile pipette tip. The suspension was then centrifuged for 10 min at 8000 rpm and the pellet was suspended in 4 - 6 mL of fresh medium and plated onto 4 - 6 100 mm flasks with vented caps.
Cells were allowed to grow to confluence and split using 0.25 % trypsin. Cells were evaluated for purity and overall quality using antibody to SMC actin.

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B. Effects of compounds on cell proliferation using 3H Thymidine inc D. I ~dtiOn Cells were assayed in early passage (generally passage 3 - 7) at sub-confluent 5 conditions. Cultures were grown in 16 mm (24 well) multi-well culture dishes in media 199 supplemented with 10% fetal bovine serum and 2% antibiotic/
antimycotic. At sub-confluence, the cells were placed in a defined serum free medium (AIM-V; Gibco) for 24 - 48 h prior to initiating the experimental protocol.

Although compounds were found to be more effective with longer pre-incubations, in general, experiments were initiated with the addition of compound, 3H
thymidine and serum / growth factor to serum deprived synchronized cells and results are reported in this invention accordingly. Growth factor and serum stimul~tionswere optimized for each cell type.
Compounds were added to each well at 50 fold dilution (20 ~L / well) and the plates were incubated for 24 - 36 h at 37~C in 5% CO2. In this series, all compounds were found to be H2O soluble and hence, test compounds were initially diluted inH2O and serially diluted into media. Compounds were routinely assayed at 1, 10, and 20 100 IlM. As a control, grade II porcine intestinal mucosal heparin (sodium salt) from Sigma (H-7005) was routinely assayed in all cell preparations at concentrations from 0.1 to 100 ~g/mL.

At the completion of the experiment, plates were placed on ice, washed three 25 times with ice cold PBS and incubated in ice cold 10% trichloroacetic acid (TCA) for 30 min to remove acid soluble proteins. Solution was transferred to scintillation vials containing 0.4N HCl (500 ~lL / vial to neutralize NaOH) and each well was rinsedtwo times with water (500 ~L) for a total volume of 2 mL / vial.

Data was obtained, in triplicate, for both control and experiment~l samples.
Control (100%j data was obtained from maximally stim~ ted cells, as the result of growth factor or serum stimlll~tion. Experimental data was obtained from cells maximally stimlll~t~-A with growth factor or serum and treated with compound. Data was expressed as a percent of control from which ICsos could be determined.
Compounds of the present invention are effective inhibitors of smooth muscle cell .

proliferation as s~ . ized in Table I. Furthermore, the compounds of the presentinvention exhibit human smooth muscle cell (HAOSMC) antiprolierative activity inthe case where proliferation is driven by either 10% fetal bovine serum (FBS) orplatelet derived growth factor (PDGF; human recombinant PDGF-AB purchased from S Upstate Biotechnology Inc., Lake Placid, NY). For example, the sulfated compound of Example 11 inhibits HAOSMC proliferation driven by F~3S (ICso 100 nM) as wellas by 5 ng/mL of PDGF (ICso 100 nM).

C. Effect on Endothelial Cell Growth vs. Smooth Muscle Cell Proliferation.
The promotion of endothelial cell proliferation concurrent with inhibition of smooth cell proliferation is an important consideration for inhibiting the exaggerated response to injury arising from vascular reconstruction. The compounds of the present invention enhance human endothelial cell growth driven by 2% FBS at doses 15 inhibitory towards human smooth muscle cell proliferation driven by 10% FBS as represented by the sulfated compound of Example 11 as shown in Figure 1.

D. Cytotoxicity:

Visually, all cells were found to tolerate high levels of all compounds quite well, however to insure that no toxicity was present, cytotoxicity of compounds was examined using a commercial modification of the Ml~ assay. Briefly, cells were again grown in 24 well plates to 70 - 80 % confluency and, as before, serum deprived for 24 - 48 hours prior to initiation of the experimental protocol. To insure that the MTT assay monitored toxicity rather than proliferation, cells were incubated with 250 ~M drug in fresh medium without serum for 24 hrs. at 37~C in a humidifled CO2 incubator. Upon completion of the compound treatment, MTT indicator dye was added for 4 hours at 37~C. Cells were then lysed and aliquots from each well were transferred to a 96-well plate for analysis. Absorbance at 570 nm wavelength with a reference wavelength of 630 nm was recorded using an ELISA plate reader. Resultsare reported as percent viable using no drug (100% viable) and pre-solubilization (0%
viable) standards. Sulfated compounds of Examples 8-14 exhibited no toxicity at 250 ~M.

WO 96114324 - PCTIUS95tl4737 E. Antic~ snt Activity The andclotting activity of the compounds of this invention were evaluated in a partial thromboplastin time (APTT) assay using normal human plasma collected S from 5 donors using the ~ ce~lulG of Fenichel et. al. (Clin. Chem. 1964, 10, 69). A
BBL Fibrometer automatic precesion coagulation timer utilizing a 0.3 ml probe was employed. An Ellagic acid activated partial thromboplastin was used for these exp~ el~ts. This reagent is added to human citrated plasma equilibrated at 37~C in a plastic well in the clot timer. Calcium at 37~C is added, the clot timer is started and the time for fibrin clot formation (in seconds) was recorded. The effect of the compounds, added to plasma, over a concentration of 12.5 - 200 ~g/mL was determined. Any plasma which did not clot after 240 seconds was assigned a clotting time of 240 seconds. An unfractionated heparin comparator was used over the concentration range of 1.25 - 10 ~g/mL. Clotting tests at all concentrations were run in triplicate. Analysis of variance for a randomized block design was used to determine the significance of differences observed in the clotting times. The potency is reported relative to heparin wherein ratio >I indicates weaker activity relative to heparin on a ~g/rnL basis.

Table I

Porcine Smooth Muscle SULFATEDCellAntiproliferation ICso Anticoagulation Activity COMPOUND OF or (APTT) Relative to Heparin EXAMPLE (% Inhibition at x concentration) 8 247 ',lM nt 9 12.7 ~M no signifcant activity at 25 -200 ~g/mL
11.8 ~lM nt 11 2.6 ~M 72 12 2.0 IlM nt 13 (23 - 48% at 20 ~lM) nt 14 3.5 ~lM 3.1 nt = not tested Wo 96/14324 PCr/USss/14737 F. Effect in E~ l Model of Restenosis Male Sprague-Dawley Rats weighing between 0.3 - 0.4 kg were housed in S polycall~nate cages (2 per box) with corn cob bedding and provided with Purina5001 rat chow and water (ad. lib. A 12 hr / 12 hr light: dark cycle was m~int~ined with light on at 7:00 A.M). After acclimation, rats were randomly assigned to treatment groups (12 per group). The rats were anesthetized with Ketamine, Aceplu,llazone, and Xylazine. The anterior portion of the neck was shaved, a 3 cm 10 incision was made along the midline, and the trachea was exposed by gently parting the underlying tissues and muscle. A retractor was inserted to hold the muscles and tissues aside and the common carotid artery was isolated from the surrounding tissue.

A 2 cm segment of the right common carotid artery was lifted such that an 11 15 mm appro~i,l,ator was placed on the vessel at about 2 and 7 cm before the bifurcation of the internal and extemal branches of the carotid artery. The 5 mm segment of the common carotid artery between the clamps of the approximator was punctured at about 0.5 mm from the edge of each clamp with a 30 G needle and a gentle stream of isotonic saline was injected through the isolated segment to flush out any blood A
20 steady stream of air, at a flow rate of 30 - 35 cc/min, was then directed through the artery for 5 minutes to desiccate the vessel. The approxima~or ~ ~s loosened initially to remove air from the segment and then removed to restore blo{)d flOw. Gentle pressure was applied to the neck until bleeding stopped The neck incision was sutured closed and the animal retumed to individual housing (polycarbonate boxes25 with com cob bedding).

A sham operation was performed on a control group of rabbits in which the carotid artery was exposed but not subjected to air drying.

E~ elltal drugs were ~ministered i.v. from ALZA 2ml 2 osmotic pumps for 2 weeks starting 2 days before air drying. A 12 mm incision was made along the posterior rnidline of the rat's neck. To insert the pumps, a subcutaneous pocket was formed through the incision along the animal's back and the pump was implanted in the pocket and the attached cannula was pulled subcutaneously to the anterior neck where it was inserted into the jugular vein contralateral to the injured arterial vessel.

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Two weeks following surgery, the rats were sacrificed via anesthetized exsanquination. The injured arterial tissue was fixed in situ by perfusion with Zn2+
Formalin, then removed for histological processing and evaluation. Arterial tissue was cut in 5 micron sections at 200 to 500 micron intervals. The cross sections were photographed and the arterial media and intima were digitized and measured. These values were used to determine the intima / media (I/M) ratio; group differences were compared by ANOVA. The compounds of the present invention have been found to be effective inhibitors of intimal thickening in this experimental model of restenosis.
For example the sulfated compound of Example 11 inhibited intimal thickening by 10 50% in comparison to untreated ~nim~l~ at a dose of 125 llg/mL according to the procedure described above.

Specific procedures are described in the following examples. These exarnples are given to illustrate the invention and should not be construed as limiting the 15 invention set forth in the appended claims. The terms "Sephadex G-10" and "Dowex", as used herein are Trade Marks.

Step 1 5-(Tetra-O-acetyl~ lucopvranosyloxyrne~hyl)-2-methyl-1-nitrobenzene A solution of 10.3 g (61.5 mmol) of 4-methyl-3-nitrobenzyl alcohol, 30.3 g (73.7 mmol) of acetobromoglucose, 13.3 g (51.7 mmol) of Hg(CN)2, and 18.6 g (52.0 mmol) of HgBr2 in nitromethane (150 mL) was stirred at room temperature for 20 h.
25 The reaction mixture was quenched with 250 mL of 2.0 M KBr and then extractedinto CH2C12. The organic phase was washed with sat. NaHCO3 and brine. The organic phase was dried over MgSO4, concentrated to an oil and semi-purified by flash chromatography using petroleum ether / ethyl acetate (2: 1). Solvent removal provided a total of 16.4 g (54% yield) of the title compound as a colorless solid: lH-30 NMR (CDCl3, 300 MHz) o 7.92 (d, 1 H), 7.43 (dd, 1 H), 7.33 (d, 1 H), 5.06 - 5.24 (m, 3 H), 4.92 (d, 1 H), 4.66 (d, 1 H), 4.59 (d, 1 H), 4.28 (dd, 1 H), 4.18 (dd, 1 H), 3.68-3.72(m,1H),2.60(s,3H),2.11(s,3H),2.07(s,3H),2.03(s,3H),and2.01 ppm (s, 3 H).

Wo 96/14324 - PcrluS9S/14737 ,Ste,D 2 5-(TP~r~-O ~cetvl-~-pl-~co~yr~nosvlu,-vlllethvl)-2-rn~tll~yl~DhPnvl~mine A solution of 5-(tetra-O-acetyl-~-glucopyranosyl-oxymethyl)-2-methyl-S nillubenzene (6.73 g, 13.5 mmol) in EtOAc (150 mL) containing 21.4 g (94.8 mmol) of SnC12 dihydrate was stirred at 75~C for 3 h. The reaction mixture was cooled and quenched with ca. 450 mL of sat. NaHCO3, diluted with CH2C12, and filtered through solka floc. The organic phase of the filtrate was dried (MgSO4), and concentrated to give yellow solid which was washed with a little Et2O to provide 3.85 (63% yield) of the title co~ )ound: lH-NMR (CDC13, 300 MHz) o 7.02 (d, 1 H), 6.64 (s, 1 H), 6.63 (d,lH),5.02-5.17(m,3H),4.79(d,1H),4.50-4.55(m,2H),4.29(dd,1H),4.17 (dd,lH),3.64-3.68(m,1H),2.18(s,3H),2.11(s,3H),2.02(s,3H),2.01(s,3H), and 2.00 ppm (s, 3 H).

FX~MPLF 2 ste,D 1 5-(He~t~-O-acetvl-B-D-cellobiosyloxymethvl)-2-methyl-1-nitrobemene The title compound was prepared in 47% yield by the procedure of step 1 of Example 1 using 4-methyl-3-nitrobenzyl alcohol and acetobromocellobiose as well as sat. NaCl in the quenching step. Purification was achieved by flash chromato~raphy (EtOAc / petroleum ether, 1: 2 to 1: l to 2: l ): lH-NMR (CDC13. 300 MHz) o 7.91(s, 1 H), 7.40 (d, 1 H), 7.32 (d, 1 H), 4.80 - 5.20 (m, 6 H), 4.4() - 4.80 (m, 4 H), 4.36 (dd,lH),4.02-4.13(m,2H),3.81(t,1H),3.60-3.68(m,2H),2.60(s,3H),2.14 (s, 3 H), 2.08 (s, 3 H), 2.06 (s, 3 H), 2.03 (s, 3 H), 2.02 (s, 3 H), 2.01 (s, 3 H), and 1.98 ppm (s, 3 H).

~Q 2 S-(HeDta~Q acetyl-~-n-(~Pllobiosylo~vll-ethyl)-2-methyl~henylamine The title compound, mp 180 - 182 ~C, was pl~l)aled in 62% yield from 5-(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-2-methyl- 1 -nitrobenzene using the procedure of step 2 of Example 1. Purification was achieved by flash chromatography (EtOAc / petroleum ether, 1: 1 to 2: 1): lH-NMR (DMSO-d6, 400 MHz) ~ 6.86 (d, 1 H), 6.48 (d, 1 H), 6.36 (dd, 1 H), 5.24 (t, 1 H), 5.10 (m, 1 H), 4.80 -CA 02204~29 1997-0~-0~
Wo 96tl4324 - PCT/USg5/14737 4.90 (m, 3 H), 4.61 - 4.72 (m, 2 H), 4.56 (d, 1 H), 4.30 - 4.31 (m, 2 H), 4.22 (dd, 1 H), 4.08 (dd, 1 H), 3.99 - 4.03 (m, 1 H), 3.94 (dd, 1 H), 3.73 - 3.81 (m, 2 H), 2.10 (s, 3H),2.10(s,3H),2.00(s,3H), 1.98(s,3H), 1.96(s,3H), 1.95(s,3H), 1.94(s,3 H), and 1.91 ppm (s, 1 H); mass spectrum (+FAB) m/z 778. Anal. Calcd. for S C34H46NOlg: C, 53.97; H, 6.13; N, 1.85. Found: C, 53.67; H, 5.92; N, 1.62.

FX~MPI,F 3 5-(HeDt~-O-~cetyl-B-D-cellobiosvloxymethvl)-2-chloro-1-nitrobenzene The title compound was ~ ;paled in 45% yield by the procedure described in step 1 of Example 1 using 4-chloro-3-nitrobenzyl alcohol and acetobromocellobiose as well as sat. NaCl in the quenching step: IH-NMR (CDC13, 300 MHz) ~ 7.82 (d 1 H),7.53(d, 1 H),7.42(dd, 1 H),4.8 -5.2(m,5H),4.73 (d, I H),4.51 -4.66(m,4 H), 4.3 - 4. 4. (m, 1 H), 4.03- 4.11 (m, 2 H), 3.81 (t, I H), 3.60 - 3.68 (m, 2 H), 2.13 (s,3H),2.09(s,3H),2.06(s,3H),2.03(s,3H),2.01 (s,3H),2.01 (s,3H), I.99(s, 3 H), and 1.57 ppm (s, 3 H).

~te~ 2 5-(Hevt~-O-~cetyl-B-n-cellobiosvlo~vmeth~l)-2-chloro-vhenylamine The title compound was prepared from 5-(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-2-chloro-1-nitrobenzene in 61~ yield as a solid by trituration with ether using the procedure of step 2 of Example 1: IH-NMR (CDC13, 300 MHz) ~ 7.20 (d, 1 H), 6.75 (s, 1 H), 6.60 (d, I H), 4.89 - 5.19 (m, 5 H), 4.73 (d, I H), 4.47 -4.60(m,4H),4.36(dd,1H),4.02-4.13(m,2H),3.80(t,1H),3.55-3.67(m,2H), 2.14(s,3H),2.08(s,3H),2.03(s,3H),2.02(s,6H),2.01 (s,3H),and2.00ppm(s, 3H).

CA 02204~29 1997-0~-0~

WO 96tl4324 PCT/US95/14737 F.X~MPI,F, 4 Step 1 5-(He~ta-O-acetYl-13-mqltosylo~methvl)-2-chloro-~ r~ f~ r s The title colllpound was p~e~ ed in 50% yield by the procedure described in step 1 of Example 1 using 4-chloro-3-nitrobenzyl alcohol and acetoblu~ .lllaltose as well as sat. NaCl in the quenching step. Purification was achieved by flash chromatography (CH2C12: EtOAc (5: 1) to provide the title compound: partial IH-NMR (CD13; 300 MHz) ~ 7.83 (d, 1 H), 7.53 (d, 1 H), 7.42 (dd, 1 H), 5.42 (d, 1 H), 5.36 (t, 1 H), 5.06 (t, 1 H), 2.15 (s, 3 H), 2.11 (s, 3 H), 2.04 (s, 3 H), 2.03 (s, 3 H), 2.02 (s, 3 H), and 2.01 ppm (s, 3 H).

Step 2 5-(HeDt~-O-~cetyl-~ ltosyloxymethyl)-2-chloro-~henvl~mine The title compound was prepared from 5-(hepta-O-acetyl-~-maltosyloxymethyl)-2-chloro-1-nitrobenzene in 96% yield by the procedure of step 2 of Example 1. The crude product was obtained as a solid and was used without further purification: partial 1H-NMR (CD13; 300 MHz) ~ 7.20 (d, I H), 6.73 (d, 1 H), 6.60 (dd, 1 H), 5.41 (d, I H), 5,30 - 5.39 (m, 2 H), 5.23 (t, 1 H), 5.05 (t, 1 H), 4.83 -4.89 (m, 2 H), 4.74 (d, I H), 2.16 (s, 3 H), 2.11 (s, 3 H), 2.032 (s, 3 H), 2.027 (s, 3 H), and2.00ppm(s,6H).

FX~MP~,F. S
,stee I
S-(Hept~-O-acetvl-~-lactosylo~,,vmethvl)-2-chloro-l-nitroben7~ne The title compound was prepared in 51% yield by the procedure described in step 1 of Example 1 using 4-chloro-3-nitrobenzyl alcohol and acetobromolactose as well as sat. NaCl in the quenching step. Purification was achieved by an intial flash chromatography (CH2C12: EtOAc (4: 1) and then re-flash rechromatography (CH2C12: EtOAc (9: 1)) to provide the title compound as a solid: partial lH-NMR
(CD13; 300 MHz) ~ 7.82 (d, 1 H), 7.53 (d, 1 H), 7.42 (dd, 1 H), 5.35 (d, 1 H), 5.21 (s, CA 02204~29 1997-0~-0~

lH),4.87(d,1H),4.66(d,1H),2.15(s,3H),2.13(s,3H),2.06(s,6H),2.05(s,6 H), and 1.97 ppm (s, 3 H).

Ste~ 2 5-(HeDt~-O-acetvl-,B-lactosylo~ nethyl)-2-chloro-Dhenvl~ ne The title compound was prepared from S-(hepta-O-acetyl-~-lactosyl-oxymethyl)-2-chloro-1-nitrobenzene in 94% yield by the procedure 1 of step 2 of Example 1. The crude product was obtained as a solid and was used without further purification: partial lH-NMR (CDl3; 300 MHz) o 7.19 (d, 1 H), 6.72 (d, 1 H), 6.59 (dd,lH),5.35(d,1H),5.08-5.20(m,2H),4.73(d,1H),2.15(s,3H),2.14(s,3 H), 2.06 (s, 3 H), 2.05 (s, 3 H), 2.02 (s, 3 H), and 1.97 ppm (s, 3 H).

FX~MPI,F 6 Ste~ 1 3. 5-Ri~-(heDta-o-~cetyl-B-n-cellobiosvloxymethyl)-l-nitroben~ne The title compound was prepared in 42% yield by the procedure described in step 1 of Example 1 using 1 equivalent of S-ni~o-m-xylene-a,ot-diol, two equivalents of acetobromocellobiose, and two equivalents of all other reagents as well as sat.
NaCI in the quenching step. Purification was achieved initially by flash chromatography (EtOAc / CH2C12 (3: 1)) and then by trituration from ether IH-NMR(CDCl3,300MHz)~8.10(s,2H),7.48(s, l H),4.88-5.30(m, 12H),4.68(d, 2 H), 4.52 - 4.59 (m, 6 H), 4.38 (dd, 2 H), 4.0 - 4.13 (m, 4 H), 3.82 (t, 2 H), 3.58 -3.68 (m, 4 H), 2.13 (s, 6 H), 2.08 (s, 6 H), 2.07 (s, 6 H), 2.03 (s, 12 H), 2.01 (s, 6 H), and 1.98 ppm (s, 6 H).

Ste~ 2 3. S-Ri~ el)h-O-~cetyl-B-D-cellobiosyloxvmethyl~henyl~mine The title compound was ple~ ed in 54% yield by procedure of step 2 of Example 1 using 3, 5-bis-(~-D-cellobiosyloxymethyl)-1-nitrobenzene. Purificationwas achieved using EtOAc / petroleum ether (1 ~ H-NMR (CDC13, 300 MHz) 6.73 (bs, 2 H), 6.67 (bs, 1 H), 5.03 - 5.30 (m, 6 H), 4.97 (d, 2 H), 4.91 (d, 2 H), 4.75 (d, 2 H), 4.49 - 4.61 (6 H), 4.37 (dd, 2 H), 4.02 - 4.13 (m, 6 H), 3.81 (t, 2 H), 3.57 -wo 96/14324 Pcr/uss5/14737 3.69 (m, 4 H), 2.15 (s, 6 H), 2.08 (s, 6 H), 2.03 (s, 6 H), 2.01 (s, 18 H), and 1.98 ppm - (s, 6 H).

FX~MPI,F, 7 S ste,D 1 3. 5~Ri~(tetra~o~cetv~ D~lucosyloxymethyl)~l~nitrob~n7~ne The title compound was prepared by the procedure described in step I of Example 1 using 1 equivalent of 5-nitro-m-xylene-a,a-diol, tWO equivalents of 10 acetobromoglucose, and two equivalents of all other reagents as well as sat. NaCl in the quenching step. Purif1cation was achieved by flash chromatography (EtOAc /
CH2C12 (1: 4 to 1: 2) to give a 29% yield of nearly pure title compound which was used without further purification.

Step 2 3. 5.Ri~(t~tra.O.~cetvl-B-D-plucosvloxymethyl)~Dhenyl~mine The title compound was prepared by the procedure of step 2 of Example l using 5.29 g 3, 5-bis-(tetra-O-acetyl-~-D-glucosyloxymethyl)-l-nitrobenzene.
Purification was achieved by flash chromatography (CH2C12 / EtOAc (3: 2)) tO give 2.00 g (39% yield) of the title compound as a yellow oil: panial IH-NMR (CDC13, 300MHz)o6.56(s,3H),5.0-5.2(m,6H),4.8(d,2H),4.4-4.6(m,4H),3.7ppm (brd, 2 H).

stee 1 Rr..,r..~ -tril~rboxylic Acid Tris~r2-methyl-5-(~-~II-coDvr~nosyloxy~ thyl)Phenvllamide~

To a solution of 5-(tetra-O-acetyl-~-glucopyranosyloxymethyl)-2-methylphenylamine (1.51 g, 3.22 mmol) and 3.22 mmol of triethylamine in THF (35 mL) was added 285 mg (1.01 mmol) of benzene-1,3,5-tricarboxylic acid chloride.
The reaction ~ ure was stirred for 4 h, quenched with MeOH, diluted with CH2C12,and washed with H2O. The organic phase was dried (MgSO4) and concel-t,~ted to anoil which was purified by flash chromatography (CH2Cl2: EtOAc (1: 1) to give CA 02204~29 1997-0~-0~

crude product. (1.17 g, 70% yield). To a solution of 983 mg (0.631 mmol) of the crude m~t~ l in MeOH (25 mL) was added 1 N NaOH (9.47mL, 9.47 mmol). After stirring at 50 ~C for 3 h, the reaction mixture was quenched with 1 N HCI (7.58 mL, 7.58 mmol) and the product i~ol~ted by filtration. Drying in vacuo provided 700 mg 5 (100% yield) of crude title co~ )oulld. The solid was suspended in H20 to remove additional salts to provide pure title compound, mp > 200 ~C: lH-NMR (DMSO-d6, 400 MHz) o 10.3 (s, 3 H), 8.73 (s, 3 H), 7.37 (s, 3 H), 7.28 (d, 3 H), 7.23 (d, 3 H), 5.10 (bs, 3 H), 4.84 (d, 3 H), 4.57 (d, 3 H), 4.25 (d, 3 H), 3.69 (dd, 3 H), 3.44 - 3.49 (m, 3 H), 3.0 - 3.2 (m, 6 H), and 2.28 ppm (s, 9 H); 13C-NMR (DMSO-d6; 100 MHz) 10 ~ 164.6, 136.1, 135.3, 133.0, 130.3, 129.8, 126.0, 125.7, 102.1, 77.0, 76.8, 73.6, 70.2, 69.2, 61.2, and 17.8 ppm; mass spectrum (-FAB) mle 1052.3, 890.3; IR (KBr) 1650 cm-1. Anal. Calcd. for CslH63N3O2l 4 H2O: C, 54.40; H, 6.35; N, 3.73. Found: C, 54.44; H, 6.15; N, 3.68.

Step 2 R~on7~ne~ -Trir~rboxvlic Acid Tri~rr2-methyl-~-(tetra-o-slllfat ~lllco~vr~nosyloxv~nethvl)Dhenyllamide~ Doder~odium S~lt A solution of benzene-1,3,5-tricarboxylic acid tris-~l2-methyl-5-(~-glucopyranosyloxymethyl)phenyll-amide) (761 mg~ 0.748 mmol) and sulfur trioxide (6.24 g, 44.9 mmol) in DMF (30 mL) was stirred at 70~C for 4 days. The reaction mixture was quenched at room tempeldture with H2O and concentrated. Purificationwas achieved by Sephadex G-10 chromatography (H2O elution) followed by cation ion exchange using a column of Dowex 50 x 8 strongly acidic resin (Na form) to provide 825 mg (48% yield) of the title compound as colorless solid, mp > 200~C:IH-NMR (D2O, 400 MHz) ~ 8.73 (s, 3 H), 7.46 - 7.51 ( bm, 9 H), 4.97 - 5.00 (m, 6H), 4.87 (d, 3 H), 4.76 (t, 3 H), 4.47 - 4.53 (m, 6H), 4.22 - 4.27 (m, 3 H), 4.15 - 4.19 (m, 3 H), and 2.37 ppm (s, 9 H); 13C-NMR (D2O; 100 MHz) ~ 168.3, 135.3, 135.2, 135.0, 134.3, 131.1,. 130.0, 128.1, 127.0, 99.1, 76.2, 75.9, 73.4, 72.6, 70.7, 67.8, and 16.8 ppm; mass ~e~ (ele~;llosplay) (m - zNa)lz 736.5 (m - 3 Na)3-, 546.2 (m - 4 Na)4-, and 433.4 (m- 5 Na)5~. Anal. Calcd. for CslH5lN3Nal2oslsl2 12 H2O: C, 24.55; H, 3.01; N, 1.68; S, 15.40 Found: C, 24.29; H, 2.78; N, 2.69; S, 15.84.

FX~ P! F 9 ~tep 1 R~..7. ..e~ r~ rboxvlic Acid Tri~rr2 rne~ 5-f~-lobiosvlo,~v~ hvl)phenvllAm~

To a solution of 5-(tetra-O-acetyl-~-cellobiosyloxymethyl)-2-methylphenyl-amine (1.49 g, 1.97 mmol) and 217 ~lL (1.97 mmol) of tnethylarnine in THF (35 rnL) was added 174 mg (0.66 mmol) of benzene-1,3,5-tricarboxylic acid chloride. The reaction mixture was sti~red for 4 h, quenched with MeOH, diluted with CH2C12, and washed with H2O. The organic phase was dried (MgSO4) and concentrated to an oil which was purified by trituration with ether to give 873 mg (55% yield) of an off-white solid. To a solution of the crude material in MeOH (25 mL) was added 1 N
NaOH (9.1 mL, 9.1 mmol). After stining at 50~C for 3 h, the reaction mixture wasquenched with 1 N HCl (7.64 mL, 7.64 mmol) and the product isolated by filtra~ion.
Drying in vacuo provided 408 mg (80% yield) of title compund. mp > 200 ~C: partial IH-NMR (DMSO-d6, 400 MHz) o 10.33 (s, 3 H), 8.75 (s, 3 H), 7.36 (s, 3 H), 7.27 (d, 3 H), 7.23 (d, 3 H), 5.2 - 5.3 (br, 6 H), 5.00 (br, 6 H), 4.84 (d, 3 H), 4.70 (s, 3 H), 4.56 -4.64(m,9H),4.33(d,3H),4.26(d,3H),3.77 (dd.3H),3.64- 3.70(m,6H),and 2.27 ppm (s, 9 H): 13C-NMR (DMSO-d6; 100 MHz) ~ I ~ 5. 136.0, 135.9, 135.1, 133.0, 130.2, 129.8, 125.9, 125.6, 103.2, 101.8. R0 5. 7~ X. 76 1. 75 0, 74 9, 73.3, 73.2, 70.0, 69.4, 61.0, 60.4, and 17.8 ppm; m~ss speclrum ~ AB) 15~9 2 (m) ~tep 2 R.on7~ne~ -tri~rboxylic Acid Tris~2-meth~l-5-(heDta-O-sulfato-~
cellobiosylo?~.ymetllyl)Dh~nvll~mide~ Heneicos~sodillm S~lt A solution of 279 mg (149 mmol) of benzene-1,3,5-tricarboxylic acid tris-(12-methyl-5-(~-cellobiosyloxymethyl)phenyl]amide} and sulfur trioxide trimethylamine - complex (3.19 g) in DMF (30 mL) was stirred at 70~C for 4 days. The reaction ~ was quenched at room tempel ~ture with H20 and concentrated in vacuo. The residue was purified by Sephadex G-10 chromatography (H2O elution). Cation exchange was effected using a column of Dowex 50 x 8 strongly acidic resin (Na form) to provide 357 mg of the title compound as a tan solid, mp > 200 ~C: partial lH-NMR (D20, 400 MHz) ~ 8.70 (s, 3 H), 7.44 - 7.52 (m, 9 H), 4.99 (d, 3 H), 4.97(d,3H),4.86(d,3H),4.83(d,3H),4.68(d,3H),4.64(d,3H),4.58(dd,3H),4.01 CA 02204~29 1997-0~-0~

- 4.04 (m, 6 H), and 2.34 ppm (s, 9 H); 13C-NMR (D2O; 100 MHz) ~ 168.17, 135.4, 135.0, 134.9, 134.2, 131.0, 129.9, 127.9, 126.7, 99.9, 99.2, 77.6, 77.34, 77.31, 77.02, 74.3, 73.6, 73.4, 73.0, 70.6, 67.6, 66.5, and 16.7 ppm; mass ~ecl-ulll (electrospray) (m - z Na)/z 897.8 (m - 4 Na)4-, 713.2 (m - 5 Na)5-, and 590.8 (m - 6 Na)6~. Anal.
Calcd. for C6gH72N3Na21OggS21 ~ 18 H2O: C, 20.67; H, 2.70; N, 1.05; S, 16.78.
Found: C, 20.35; H, 2.78 N, 1.00; S, 14.19.

F.XAMPI E 10 Step 1 Rf~n~ne~ -tri~rboxvlic Acid Trisfr5-(heDta-O-acetyl-~-n-ln~ltosylo~ ,..ethvl~-2-chloroDhenvllamide~

To a solution of 962 mg (1.24 mmol) of 5-(hepta-O-acetyl-~-maltosyloxymethyl)-2-chlorophenylamine in THF (20 mL) containing 173 ~LL (1.24 mmol) of triethylamine was added 110 mg (0.413 mmol) of benzene-1,3,5-tricarboxylic acid chloride. The reaction mixture was stirred for 90 min, quenched with MeOH, diluted with CH2C12, and washed with H2O. The organic phase was dried (MgSO4) and concentrated to a light yellow solid. Purification by flash chromatography (CH2C12: EtOAc (2: 1 to 1: l) gave 607 mg (59% yield) of the title compound as a colorless solid, mp 132~C (dec): partial IH-NMR (CDC13, 400 MHz) ~ 8.66 (s, 3 H), 8.65 (s, 3 H), 8.48 (d, 3 H), 7.42 (d, 3 H), 7.08 (dd, 3 H), 5.40 (d, 3 H), 5.33 (t, 3 H), 5.23 (t, 3 H), 5.03 (t, 3 H), 4.82 - 4.93 (m, 9 H), 4.66 (d, 3 H), 4.62 (d, 3 H), 4.53 (dd, 3 H), 3.67 - 3. 70 (m, 3 H), 2.14 (s, 3 H), 2.10 (s, 3 H), 2.04 (s, 3 H), 2.01 (s, 3 H), 1.99 (s, 3 H), 1.995 (s, 3 H), 1.986 (s, 3 H), and 1.97 ppm (s, 3 H):
13C-NMR (CDC13; 100 MHz) ~ 170.5; 170.2, 169.9, 169.7, 169.4, 162.9, 137.0, 136.1, 134.1, 129.2, 128.9, 124.2, 123.1, 121.1, 99.1, 95.5, 75.4, 72.6, 72.2, 72.0, 70.1, 70.0, 69.3, 68.5, 68.0, 62.8, 61.5, 20.9, 20.7, 20.6, and 20.5 ppm; mass spectrum (positive (Ca2+) electrospray) 1262 (m + Ca2 +)/2. Anal. Calcd. for C108H126C13N3~57 1 H20: C, 51.83; H, 5.16; N, 1.68. Found: C, 51.64; H, 5.11;
N, 1.53.

CA 02204~29 1997-0~-0~

WO 96/14324 PCrlUS95/14737 ~2 n~ ..e-l ~, C,-trir~rboxvlic A-~id Tri~rr2-chloro-5-(~-D-lnqltosyl(),.
~h~orull~mide~

To a solution of 983 mg (0.194 mmol) of benzene-1,3,5-tricarboxylic acid tris ( [5-(hepta-O-acetyl-~-D-maltosyloxymethyl)-2-chlorophenyl]amide } in MeOH
(15 mL) was added 1 N NaOH (4.67 mL, 4.67 mmol). After stirring at 50~C for 4 h,the reaction mixture was quenched with 1 N HCI (4.08 mL, 4.08 mmol) and the product isolated by filtration. Drying in vacuo provided 309 mg (99% yield) of title co.llpulld. mp > 200 ~C: partial IH-NMR (DMSO-d6, 400 MHz) ~ 10.56 (s, 3 H), 8.78(s,3H),7.60(d,3H),7.56(d,3H),7.37(dd,3H),5.02(d,3H),4.89(d,3H), and 4.63 ppm (d, 3 H); 13C-NMR (DMSO-d6; 100 MHz) ~ 164.57, 137.83, 134.73, 134.61, 130.13, 129.36, 128.67, 127.53, 126.85, 102.12, 100.75, 79.54, 76.37, 75.28, 73.45, 73.24, 73.02, 72.42, 69.87, 68.72, 60.76, and 60.64 ppm; mass spectrum ((-)-FAB) m/z 1598.4 (M - H), 1436.3, and 1274.3. Anal. Calcd. f~rC~6H84C13N3~36 ~9H2O: C, 44.94; H, 5.83; N, 2.38. Found: C, 44.48; H, 5.46; N, 2.78.

Step 3 Renzene~ tri~rboxvlic Acid Tri~2-chloro-~-(heDta-O-sulfato-B-D-m~ltosvloxymethyl)Dhenyllamide~ Heneicos~odium Salt A solution of benzene-1,3,5-tricarboxylic acid Iris-ll2-chloro-5-(~-D-maltosyloxymethyl)phenyl]amide ) (208 mg, (130 mmol ) and sulfur trioxide trimethylamine complex (1.90 g) in DMF (20 mL) was stirred at 70~C for 2.5 days.The reaction mixture was quenched at room temperature with H2O and concentrated in vacuo. The residue was purified by Sephadex G-10 chromatography (H20 elution). Cation exchange was effected using a column of Dowex 50 x 8 strongly acidic resin (Na form) to provide 400 mg (82% yield) of the title compound as a tan solid, mp > 178~C (dec): partial lH-NMR (D2O, 400 MHz) ~ 8.74 (s, 3 H), 7.67 -7.70(m,6H),7.57(d,3H),5.62(d,3H),5.09(d,3H),5.04(d,3H),4.66(t,3H), 4.61 (dd, 3 H), and 4.52 ppm (t, 3 H); 13C-NMR (D2O; 100 MHz) ~ 168.1, 136.9, 134.7, 132.9, 130.3, 130.1, 129.7, 128.6, 127.6, 98.8, 94.1, 77.4, 76.0, 74.8, 73.4, 73.2, 72.3, 71.8, 69.9, 69.7, 67.6, and 66.1 ppm; mass spectrum (elec~ y) (m - zNa)lz 1225.0 (m - 3 Na)3-, 912.8 (m - 4 Na)4- 725.6 (m - 5 Na)5-, and 601 (m - 6 Na)~- Anal. Calcd. for C66H63C13N3Na21OggS21 4 Na2SO4 21 H2O: C, 16.90; H, 2.25; N, 0.90, S, 17.09. Found: C, 16.77; H, 2.29; N, 0.97; S, 16.90.

F,XAl~PT,F 11 Step 1 n~ .- . --e~ Ç~rj~rboxylic Acid Tri~r5-(heDta -O-~cetvl-~-n-~llobjos~ yl)-2-( hloro~henyll~lnide~

To a solution of 1.43 g (1.85 mmol) of 5-(hepta-O-acetyl-,B-cellobiosyloxymethyl)-2-chlorophenylamine in THF (25 rnL) containing 254 ~L
(1.85 mmol) of triethylamine was added 163 mg (0.615 mmol) of benzene-1,3,5-tricarboxylic acid chloride. The reaction mixture was stirred for 90 min, quenched with MeOH, diluted with CH2Cl2, and washed with H2O. The organic phase was dried (MgSO4) and concentrated to an off-white solid. Purification by flash chromatography (CH2Cl2: EtOAc (1: 1)) gave 937 mg (62% yield) of product. An analytical sample, obtained as a white crystal (mp >200~C), was prepared from a separate run: 13C-NMR (CDC13; 100 MHz) ~ 170.5, 170.4, 170.2, 169.8, 169.6, 169.3, 169.0, 163.0, 137.0, 136.1, 134.0, 129.2. 128 9, 12~.7. 123.2. 121.2, 100.75, 99.4, 76.37, 72.90, 72.8, 72.47, 7l.93, 71.59, 71.~. 7() 1. ~7 X, ~ . 61.5, 20.87, 20.66, 20.60, and 20.50 ppm; mass spectrum (posinve (C~'~) electrospray) (m +
Ca)2+12 1262. Anal. Calcd. for C108Hl26cll!~3o~l 'H~O C. ~l ~6; H. 5.'(); ~.
1.67. Found: C, 51.44; H, 4.88; N, 1.71.

Ste~ 2 Ren7~ne~ -tri~rboxylic Acid Tris~2-chloro-5-(,B-D-cellobiosyloxymethyl)phenyllamide~

A solution of 917 mg (0.375 mmol) of benzene-1,3,5-tricarboxylic acid tris { [5-(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-2-chlorophenyl]amide ) in MeOH
(25 mL) was treated with 9.0 mL (9.0 mmol) of 1 N NaOH. After stirring for 3 h at 50~C, the reaction Illi~lUle was quenched at room le~ erature with 1 N HCl (7.9 mL, 7.9 mmol) and stirred for 10 min. The solid was collected and dried in vcauo to provide 579 mg (96% yield) of the title compound as colorless solid, mp > 200~C:partial lH-NMR (DMSO-d6, 400 MHz) ~ 10.57, (s, 3 H) 8.78 (s, 3 H), 7.58 (s, 3 H), CA 02204~29 1997-0~-0~
Wo 96/14324 PCT/USs5/14737 7.55(d,3H),7.36(s,3H),4.87(d,3H),4.34(d,3H),4.25(d,3H),3.77(d,3H), and 3.68 ppm (d, 3 H); 13C-NMR (DMSO-d6; 100 MHz) ~ 164.6, 137.9, 134.7, 134.6, 130.2, 129.4, 128.7, 127.5, 126.9, 103.2, 102.0, 80.5, 76.8, 76.5, 75.0, 73.3, 73.2, 70.0, 68.8, 61.0, and 60.4 ppm; IR (KBr) 1655 cm-l; mass spectrum (+FAB) m/z 1622.2 (M + Na). Anal. Calcd. for C66Hg4CI3N3O36-5 H20: C, 46.86; H, 5.60;
N, 2.48. Found: C, 46.84; H, 5.46; N, 2.37.

~ten 3 R~n~ne.l ~ ~-trir~rboxylic Acid Tri~r2-chloro-5-(heDta-O-sulfo-B-O-cellobiosvloxyl,.ethyl)phenvllamide) Heneiso~codium Salt A soluiton of 403 mg (0.252 mmol) of benzene-1,3,5-tricarboxylic acid tris{[2-chloro-5-(~-D-cellobiosyloxymethyl)phenyl]amide} and sulfur trioxide trimethylamine complex (3.89 g, 28 mmol) in DMF (25 mL) was stirred at 70~C for 5 days. The reaction mixture was quenched at room temperature with H2O and concenL,~ted in vacuo. The residue was purified by Sephadex G-10 chromatography (H2O elution). Cation exchange was effected using a column of Dowex 50 x B
strongly acidic resin (Na form) to provide 949 mg of the title compound as a tan solid, mp > 178~C (dec): partial IH-NMR (D20, 400 MHz) o 8 73 (s, 3 H), 7.70 (s, 3 H), 7.67(d,3H),7.57(d,3H),4.99-5.03(m,6H),4.68(t,3H),4.64(t,3H),4.60(dd, 3 H), 4.24 (d, 3 H), 4.20 (t, 3 H), and 4.02 - 4.05 ppm (m, 3 H); 13C-NMR (D2O; lO0 MHz) ~ 168.1, 139.0, 134.7, 132.8, 130.3, 130.0, 129.6, 128.5, 127.4, 99.9, 99.4, 77.5, 77.4, 77.3, 77.0, 74.3, 73.7, 73.3, 73.1, 70.1, 67.6, and 66.6 ppm. Anal. Calcd.
for C66H63C13N3Na21~99-33 H20: C, 18.28; H, 2.93; N, 0.97; S, 15.53. Found: C, 18.20; H, 2.67; N, 0.76; S, 15.51. Capillary electrophoresis showed purity in excess of 98~o.

FXAMPT F. 12 ~e~? 1 Rr.~i~ .. e~ Tri- ~rboxvlic Acid Tri~r2-chloro-5-(~-1)-o~luAv~ Dhenvlamide~

To a solution of 993 mg (1.28 mmol) of 5-(hepta-O-acetyl-~-lactosyloxymethyl)-2-chloro-phenylamine in THF (20 mL) containing 178 ~L (1.28 mmol) of triethylamine was added 113 mg (0.427 mmol) of benzene-1,3,5-CA 02204~29 1997-0~-0~

tricarboxylic acid chloride. The reaction lllixLule was stirred for 90 min, quenched with MeOH, diluted with CH2C12, and washed with H20. The organic phase was dried (MgSO4) and concentrated to an off-white solid. Purification by flash chromatography (CH2C12: EtOAc (2: 1 to 1: 1)) gave 431 mg (41% yield) of product. A solution of 431 mg (0.174 mmol) of the compound in MeOH (15 mL) was treated with 4.2 mL (4.2 mmol) of 1 N NaOH. After stirring for 4 h at 50~C, the reaction mixture was quenched at room temperature with 1 N HCl (3.6 mL, 3.6 mmol). The solid was collected and dried in vacuo to provide 193 mg (69% yield) of the title compound as colorless solid, mp > 200~C: partial IH-NMR (DMSO-d6, 400 MHz)olO.58(s,3H),8.79(s,3H),7.59(d,3H),7.56(d,3H),7.36(dd,3H),4.88 (d, 3 H), 4.63 (d, 3 H), 4.35 (d, 3 H), 4.20 (d, 3 H), 3.79 (d, 3 H), and 3.11 (s, 3 H) 3C-NMR (DMSO-d6; 100 MHz) o 164.6, 137.8, 134.7, 134.6, 130.2, 129.4, 128.7, 127.5, 126.8, 103.8, 102.0, 80.7, 75.5, 75.0, 74.9, 73.22, 73.19, 70.5, 68.8, 68.1, 60.5, and 60.3 ppm; mass spectrum ((-)-FAB) m/z 1598.8 and 1600.8 (both M-H). Anal.
Calcd- for C66H84C13N3O36 9 H2O: C, 44.94; H, 5.83; N, 2.38. Found: C, 44.24;
H, 5.38; N, 2.29.

~teD 2 R.on7~ne l.~ tricarboxylic Acid Tri~-r~2-chloro-5-(heDta-O-sulfato-B-n-l~ctosyloxv~ lhyl)Dhenyllamide~ Heneicos~sodium S~lt A soluiton of 126 mg (0.079 mmol) of benzene-1,3,5-~ricarboxylic acid tris{[2-chloro-5-(~-D-lactosyloxymethyl)phenyl]amide I and sulfur trioxide trimethylamine complex (1.5 g, 8.26 mmol) in DMF (20 mL) was stirred at 70~C for2.5 days. The reaction mixture was quenched at room temperature with H2O and concentrated in vacuo. The residue was purified by Sephadex G-10 chromatography (H2O elution). Cation exchange was effected using a column of Dowex 50 x 8 strongly acidic resin (Na form) to provide 207 mg (70% yield) of the title compound as a tan solid, mp > 171~C (dec): partial lH-NMR (D2O; 400 MHz) ~ 8.73 (s, 3 H),7.68 (s, 3 H), 7.67 (d, 3 H), 7.58 (d, 3 H), 5.13 (d, 3 H), 5.08 (d, 3 H), 5.01 (d, 3 H), 4.43 (t, 3 H), and 4.24 ppm (t, 3 H); 13C-NMR (D2O; 100 MHz) ~ 168.1, 137.0, 134.7, 132.8, 130.3, 130.1, 130.0, 129.6, 128.5, 127.5, 100.9, 99.2, 77.6, 77.0, 75.6, 75.3, 75.04, 74.97, 73.2, 71.7, 70.0, 66.5, and 66.3 ppm; mass ~l~CCI~ (electrospray (m-zNa)lz 913 (M-4Na)4- and 725.9 (M-5 Na)5- Anal. Calcd. for CA 02204~29 1997-0~-0~

C66H63Cl3N3Na21OggS21 21 H20: C, 19.23; H, 2.57; N, 1.02; S, 16.33. Found: C, - 19.51; H, 2.61; N, 1.11; S, 15.97.

FXAMP~,F 13 ~tep 1 R~..,.r.,e 1.~ ~ trjr~rboxylic Acid Trisrr3--~-bis-(tetr~-O-acetyl-b-n-copyranosyloxymethyl)phenyllamide~

To a solution of 861 mg (1.06 mmol) of 3, 5-bis(tetra-O-acetyl-~-D-glucosyloxymethyl)phenylamine in THF (20 mL) containing 147 ~L (1.06 mmol) of triethylamine was added 93.6 mg (0.353 mmol) of benzene-1,3,5-tricarboxylic acidchloride. The reaction ll~ixlu~e was stirred for 90 min, quenched with MeOH, diluted with CH2C12, and washed with H2O. The organic phase was dried (MgSO4) and concentrated to a light yellow solid. Purification was achieved by trituration with ether to provide 849 mg (0.327 mmol) of the title compound as colorless crystals, mp 120-129~C: IH-NMR (CDCl3; 400 MHz) ~ 9.1 (bs, 3 H), 8.73 (s, 3 H), 7.67 (s, 6 H), 6.99(s,3H),5.18(t,6H),5.10(t,6H),5.03(dd,6H),4.84(d,6H),4.65(d,6H), 4.61 (d, 6 H), 4.27 (br s, 12 H), 3.72 - 3.74 (m, 6 H), 2.06 (s, 18 H), 2.01 (s, 36 (H), and 1.97 ppm (s, 18 H); 13C-NMR (CDC13; 100 MHz) ~ 171.2, 170.2, 169.4, 163.9, 20 138.4, 138.2, 135.7, 129.2, 123.1, 119.5, 100.1, 72.8, 71.9, 71.3, 71.1, 68.4, 62.0, 20.8, 20.64, and 20.56 ppm. Anal. Calcd. for Cll7HI4lN3O63 2H2O: C, 53.36; H, 5.55; N, 1.60. Found: C, 53.29; H, 5.29; N, 1.61.

~tep 2 Rr.. 7~ -tri~rboxvlic Acid Tri~r3.5-bis-(tetr~-0-s~lf~to-B-n-cosyloxynl~tllyl)~h~ yllamide~ tetracos~co-lium ~lt A solution of 780 mg (0.300 mmol) of benzene-1,3,5-tricarboxylic acid tris { [3,5-bis-(tetra-O-acetyl-~-D-glucopyranosyloxymethyl)phenyl]amide) in MeOH
(15 mL) con~ ing 8.1 mL (8.1 mmol) of 1 N NaOH was stirred at 50~C for 5 h. The reaction ~ ul~ was cooled to room temperature and quenched with 1 N HCl (7.2 mL, 7.2 mmol). The product, benzene-1,3,5-tricarboxylic acid tris([3,5-bis-(,B-D-glucosyloxymethyl)phenyl]amide}, was collected (338 mg, 81% yield) and used directly in the next reaction: partial lH-NMR (DMSO-d6; 400 MHz) o 10.73 (s, 3 H),8.74(s,3H),7.78(s,6H),7.22(s,3H),4.88(d,6H),4.57(d,6H),4.29(d,6 CA 02204~29 1997-0~-0~

WO 96tl4324 PCT/US95/14737 H), 3.71 (d, 6 H), and 3.48 (dd, 6 H); 13C-NMR (DMSO-d6; 100 MHz) ~ 164.5, 138.7, 138.4, 135.3, 129.8, 123.0, 119.1, 102.2, 77.0, 76.8, 73.5, 70.1, 70.0, and 61.1 ppm.

A solution of 317 mg (0.200 mmol) of benzene-1,3,5-tricarboxylic acid tris~[3,5-bis-(~-D-glucosyloxymethyl)phenyl]amide } and sulfur trioxide trimethylamine complex (3.59 g, 25.8 mmol) in DMF (25 mL) was stirred at 70~C for 3 days. The reaction mixture was quenched at room temperature with H2O and concenl-aled in vacuo. The residue was purified by Sephadex G- 10 chromatography(H2O elution) to give a yellow solid, which by NMR contained some residual trimethylall~l,onium sulfate. Cation exchange was effected using a column of Dowex 50 x 8 strongly acidic resin (Na form) to provide 771 mg (ca. 95% yield) of the title co~ ound, mp 174~C, as coloress solid: lH-NMR (D20; 400 MHz) o 8.63 (s, 3 H), 7.68 (br s, 6 H), 7.44 (br s, 3 H), 5.02 (d, 6 H), 4.98 (d, 6 H), 4.85 (d, 6 H), 4.54 (t, 6 H), 4.46 - 4.50 (m, 12 H), 4.24 (dd, 6 H), and 4.16 ppm (dt, 6 H); 13C-NMR (D2O;100 MHz) o 167.7, 138.0, 137.1, 135.3, 129.9, 125.3, 122.0, 99.5, 76.2, 75.9" 73.4, 72.6, 70.9, and 67.6 ppm. Anal. Calcd. for C6gH6gN3Na24OIllS24 24H2O 4Na2SO4 C, 16.45; H, 2.34; N, 0.83; S, 17.82. Found: C. 16.51; H, 2.17; N, 0.84, S, 18.17.

F.XAMPI F 14 ~teD I
Ren7~ne.1.~.~-tri~rboxylic Acid Tris~3 ~-bis-(,B-D-cellobiosvloxymethvl)Dhen~llamide~

To a solution of 802 mg (0.577 mmol) of 3, 5-bis-(hepta-O-acetyl-~-D-cellobiosyloxymethyl)phenylamine in THF (25 mL) containing 80.6 ~lL (1.28 mmol) of triethylamine was added 51.2 mg (0.193 mmol) of benzene-1,3,5-tricarboxylic acid chloride. The reaction mixture was stirred for 90 min, quenched with MeOH, diluted with CH2C12, and washed with H2O. The organic phase was dried (MgSO4) and concellllaled to a light yellow solid. Purification was achieved by trituration from CH2C12 / petroleum ether to provide 777 mg (94% yield) of product. A solution of777 mg (0.180 mmol) of the compound in MeOH (25 mL) was treated with 8.1 mL
(8.1 mmol) of 1 N NaOH. After stirring for 2.5 h at 50~C, the reaction Illi~lUI~; was quenched at room telllpelature with 1 N HCl (7.6 mL, 7.6 mmol). The solid was collected and purified by reverse phase chromatography (RP silica 60) using elution of MeOH / H2O (3: 7) and then by Sephadex G- 10 chromatography (H2O elution) to provide 262 mg (57% yield) of the title compound as a colorless solid, mp >171~C(dec): 13C-NMR (D2O, 100 Hz) ~ 165.3, 138.4, 137.2, 134.0, 130.0, 123.9, 119.9, 102.6, 101.7, 78.7, 75.9, 75.5, 74.6, 74.2, 73.1, 72.8, 70.6, 69.4, 60.5, and 60.0 ppm.

Step 2 RPn~Pne.l ~ ~-tri~rboxvlic Acid Tri~r3.5-bis-~he~ta-0-sulf~to-~-n-lobiosyloxylne~lvl)phenyll~mide~ Tetratetracont~codium ~lt A solution of 139 mg (0.0543 mmol) of benzene-1,3,5-tricarboxylic acid tris([3,5-bis-(~-D-lactosyloxymethyl)phenyl]amide ) and sulfur trioxide trimethylarnine complex (1.68 g, 12.07 mmol) in DMF (25 mL) was stirred at 70~C
for 3 days. The reaction mixture was quenched at room temperature with H2O and concentrated in vacuo. The residue was purified by Sephadex G-10 chromatography 15 (H2O elution). Cation exchange was effected using a column of Dowex 50 x 8 strongly acidic resin (Na form) to provide 317 mg (85% yield) of the title compound as a tan solid, mp > 180~C (dec): partial IH-NMR (D2O; 400 MHz) ~ 8.64 (s, 3 H),7.67 (s, 6 H), 7.46 (s, 3 H), 5.02 (d, 6 H), 4.96 (d, 6 H), 4.64 (t, 6 H), and 3.88 - 4.01 (m, 12 H); 13C-NMR (D2O; 100 MHz) ~ 167.7, 138.0, 137.0, 135.3, 129.9, 125.1, 121.8, 99.8, 77.7, 77.5, 77.4, 77.0, 74.2, 73.5, 73.4, 72.9, 71.0, 67.6, 66.1 ppm; mass spectrum (electrospray) (m-zNa)lz 883.4 (m - 8 Na)~- 738.2 (m - 9 Na)9-, 662.0 (m -10 Na)10-, 599.5 (m - ll Na)ll~ Anal. Calcd. for Cl05HIIIN3Na42Ol95S42 42 H2O: C, 16.59; H, 2.58; N, 0.55; S, 17.71. Found: C, 16.47; H, 2.86; N, 0.45; S,14.72.

Claims (13)

What is claimed:

1. A compound of formula I

I

wherein each of R1, R2, R3, and R4 are, independently, H, SO3M, or ;

and each oligosaccharide group contains 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 of formula I:
I

wherein each of R1, R2, R3, and R4 are, independently, H,SO3M, or and each oligosaccharide group contains 1 or 2 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl;
or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris{[2-methyl-5-(tetra-O-sulfato-.beta.-glucopyranosyloxy-methyl)phenyl]amide} dodecasodium salt or a pharmaceutically acceptable salt thereof.

4. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris{[2-methyl-5-(hepta-O-sulfato-.beta.-cellobiosyloxy-methyl)phenyl]amide} heneicosasodium salt or a pharmaceutically acceptable salt thereof.

5. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris{[2-chloro-5-(hepta-O-sulfato-.beta.-D-maltosyloxy-methyl)phenyl]amide) heneicosasodium salt or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris[2-chloro-5-(hepta-O-sulfato-.beta.-D-cellobiosyloxy-methyl)phenyl]amide} heneicosasodium salt or a pharmaceutically acceptable salt thereof.

7. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris{[2-chloro-5-(hepta-O-sulfato-.beta.-D-lactosyloxymethyl)-phenyl]amide} heneicosasodium salt or a pharmaceutically acceptable salt thereof.

8. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris([3,5-bis-(tetra-O-sulfato-.beta.-D-glucosyloxymethyl)-phenyl]amide} tetracosasodium salt or a pharmaceutically acceptable salt thereof.

9. A compound according to claim 2 which is benzene-1,3,5-tricarboxylic acid tris {[3,5-bis-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)-phenyl]amide} tetratetracontasodium salt or a pharmaceutically acceptable salt thereof.

10. A method of treating a human suffering from a condition which is characterized by excessive smooth muscle proliferation, the method comprising administering to the human an effective amount of the compound of the formula I

I

wherein each of R1, R2, R3, and R4 are, independently, H,SO3M, or ;

and each oligosaccharide group contains 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising an effective amount of a compound of formula I

I

wherein each of R1, R2, R3, and R4 are, independently, H,SO3M, or ;

and each oligosaccharide group contains 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
or a pharmaceutically acceptable salt thereof.

12. The use, to treat a human suffering from a condition which is characterized by excessive smooth muscle proliferation, of an effective amount of the compound of formula I

I

wherein each of R1, R2, R3, and R4 are, independently, H,SO3M, or ;

and each oligosaccharide group contains 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
or a pharmaceutically acceptable salt thereof.

13. The use, in the manufacture of a medicament to treat a human suffering from a condition which is characterized by excessive smooth muscle proliferation, of an effective amount of the compound of formula I

I

wherein each of R1, R2, R3, and R4 are, independently, H,SO3M, or ;

and each oligosaccharide group contains 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
or a pharmaceutically acceptable salt thereof.