CA2204531A1 - Polyanionic benzylglycosides as inhibitors of smooth muscle cell proliferation - Google Patents

Abstract

This invention relates to the use of polyanionic benzylglycosides as smooth muscle cell proliferation inhibitors and as therapeutic compositions for treating diseases and conditions which are characterized by excessive smooth muscle proliferation, such as restenosis. The compounds of this invention are those of 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 hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms; Y is carbonyl or sulfonyl; Z is alkyl having from 1 to 12 carbon atoms, (a), (b), (c), or (d); and X is as defined above; or a pharmaceutically acceptable salt thereof.

Description

CA 02204~31 1997-0~-0~

pOT,y~ NIc P~RN7,YT,(~T,YCOSIl)F,S AS INHIl~ITOl~.
OF SMOOTH MUSCT,T~ CFI,T, PRO~,~FFRATrON

This invention relates to polyanionic benzylglycosides and their use as smooth 5 muscle cell proliferation inhibitors and as therapeutic compositions for treating diseases and conditions which are characterized by excessive smooth muscle proliferation, such as restenosis.
Ba~k~round of the Invention --All forms of vascular reconstruction such as angioplasty and vein bypass procedures effect a response to injury that Illtim~tely leads to smooth muscle cell (SMC) proliferation and, subsequently, depostion of profuse arnounts of extracellular matrix (Clowes, A. W.; Reidy, M. A. J. Vasc. Surg 1991, 13, 885). These events are 15 also central processes in the pathogenesis of atherosclerosis (Raines E. W.; Ross R.
Br. Heart J. 1993, 69 (Supplement), S 30) as well as transplant arteriosclerosis (Isik, F. F.; McDonald, T. O.; Ferguson, M.; Y:~m:~n~k~, E.; Gordon Am. J. Pathol. 1992, 141, 1139). In the case of restenosis following angioplasty, clinically relevantsolutions for controlling SMC proliferation through pharmacological intervention20 have remained elusive to date (HelTman, J. P. R.; Hermans, W. R. M.; Vos, J.;Serruys P. W. Drugs 1993, 4, 18 and 249). Any successful approach to selective SMC proliferation inhibition must not interfere with endothelial cell repair or the normal proliferation and function of other cells (Weissberg, P. L.; Grainger, D. J.;
Sh~n~h~n C. M.; Metcalfe, J. C. Cardiovascular Res. 1993, 27, 1191). Indeed, an 25 important therapeutic consideration is to promote reendotheliaztion of the injured area concurrent with SMC proliferation inhibiton (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).

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 fr~gTnent~, chemically modified heparin, low molecular weight heparins, and other heparin mimicking anionic polysaccharides may be co~ ised due to other pharmacological liabilites (excessive bleeding arising CA 02204~31 1997-0~-0~
WO 96/14323 PCTtUS95/14686 from anticoag~ tion effects, in particular) coupled with heterogeneity of the various preparations (Borman, S. Chemical and Engineering News, 1993, June 28, 27;
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~m--rthy, B.; Sharma, C.; Bhandaru, R. R.; Berenson, G.
S.; St~n~ni, L.; Mastacchi, R. Atherosclerosis, 1986 60, 141; Maffrand, J. P.;
Hervert, M. M.; Bernat, A.; Defreyn, G.; Delevassee, D.; Savi, P.; Pinot, J. J.;Sampol, J. Seminars in Thrombosis and Hemostasis, 1991, 17 (Suppl. 2), 186). Since 10 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 more desirable profile with fewer side effects associated with the aforementioned anionic polysaccharides.
Prior Art WO 92/18546 discloses specific sequences of heparin, obl;~inable in pure forrn through synthesis or heparin fragment isolation, which exhibit Sl\1C antiproliferation 20 activity. Beta-Cyclodextrin tetradecasulfate has been described as a smoo~h muscle cell proliferation inhibitor and as an effective inhibitor of re~i~eno~i~ (Weisz. P. B.;
Hermann, H. C.; Joullie, M. M.; Kumor, K.; Levine~ E 1~ tacar;lk, E J.; Wein~r, D. B. Angiogenesis: Ke~ Principle - Science - Tccllnol(J"~ t~ilC~n~-- S~einer R., Weisz, P. B.; Langer, R. Eds. Birkhauser Verlag. B;l~l S~lt~rl~n~ '. pg 107;
25 Hermann, H. C.; Okada, S. S.; Hozakowska, E.; Le~een, R ~:~ Golden, M. A.;
Tomaszewski J. E.; Weisz, P. B.; Barnathan E. S. Arteriosclerosis and Thrombosis1993, 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 30 conditions associated with undesirable cell or tissue growth. WO 93/09790 discloses antiproliferative polyanionic derivatives of cyclodextrins bearing at least 2 anionic residues per carbohydrate residue. EP 312087 A2 and EP 312086 A2 discloses an~iLl,~ .botic and anticoagulant ~ ties of sulfated bis-aldonic acid amides.

CA 0220453l lss7-05-05 wo 96/14323 PCT~USg5/14686 .

U.S. Patents Nos. 4,431,636, 4,431,637, 4,431,638, and 4,435,387 describe polysulfated, thio- and oxy-aryl glycoside derivatives as modulators of the complement system.

The compounds of the present invention differ from all of the prior art in that the compounds (a) are benzylglycosides which bear no structural resemblance to heparin, sulfated cyclodextrins, or to sulfated lactobionic acid dimers, (b) contain no more than three contiguous sugar residues (trisaccharides) and (b) are of defined structure.
nescriDtion of the Invention This invention describes the composition and utility of sulfated benzylglycosides of forrnula I
~R40 X\~ X ~OR~\

R~~R~) n ~ ~~~ )n I

wherein each of Rl, R2, R3, and R4 are, independentl~, ~1, SO~M, or a sugar group having the structure:
R40~ ~~ ~i oR2 and each oligosaccharide group of the structure ~,0 ~,0 ~

Rlo~OR3 oR2 contains from 1 to 3 sugar groups;

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M is lithium, sodium, pot~sillm, or ammonium;
n is 1 or 2;
X is hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is a group of formula -(CH2)p-, wherein p is an integer from 1 to 12;

,~/X ~, X H H ~X~

X~, H ~ N

and X is as defmed above;
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 herein of Y in any given molecule and each of the occurrences herein of n in any15 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 chained or branched and may be, for example, methyl, ethyl, propyl, isopropyl orbutyl groups. Similarly, the lower alkoxy groups herein may be straight chained or 20 branched and may be, for example, methoxyy, ethoxy, propoxy, isopropoxy or butoxy groups.

Wo 96/14323 PCT/US95/14686 A more prerellGd aspect or embodiment of this invention is the compounds of ~ formula I:
X,~, [~X

R30~oR1 ¦ R10~oR3 oR2 J n ~ oR2 ~ n I

~ wherein each of R1, R2, R3, and R4 are, independently, H, S03M, or R40~ 0~ 0 oR2 and each oligosaccharide group of the structure ~,0 ~,0 ~

( R10~0R3 oR2 ~0 contains 1 or 2 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2; X is hydrogen, halogen, lower alkvl having I ~o 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl, Z is alkyl having from 1 to 12 carbon atoms, ~, H H

H~H

or a pharmaceutically acceptable salt thereof.

CA 02204~31 1997-0~-0~

The most ~crcl~,d compounds of this invention are:
Do l~c~ne~lioic acid bis{t2-methyl-5-(hepta-0-sulfato-13-D-cellobiosyloxymethyl)-phenyl]amide} tetradecasodium salt or a pharmaceutically acceptable salt thereof;
5 N,N'-Bis[5-(hepta-O-sulfato-,B-D-cellobiosyloxymethyl)-2-methylphenyl]-terephth~l~mide tetradecasodium salt or a pharmaceutically acceptable salt thereof;
Biphenyl-4,4'-dicarboxylic acid bis { [5-(hepta-O-sulfato-~-D-cellobiosyloxymethyl)-2-methylphenyl]amide} tetradecasodium salt or a pharmaceu~ically acceptable salt thereof;
N,N'-Bis[S-(hepta-O-sulfato-~-D-cellobiosyloxymethyl)-2-methylphenyl] -isophth~ micle tetradecasodium salt or a pharmaceutically acceptable salt thereof;
Decanedioic acid bis { [3,5-bis(hepta-O-sulfato-,B-D-cellobiosyloxymethyl)-phenyl]amide}octacosasodium salt or a pharmaceulically acceptable salt thereof;
Biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis(hepta-0-sulfalo-,B-D-cellobiosyl-oxymethyl)phenyllamide3 octacosasodium salt or a pharmaceutically acceptable salt thereof;
Biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis-(hepta-O-sulfato-~-D-lactosyloxy-methyl)phenyl]amide} octacosasodium salt or a pharmaceutically acceptable salt thereof;
Biphenyl-4,4'-dicarboxylic acid bis{[3,5-(bis-(hepta-O-sulfato-~-D-maltosyoxy-methyl)phenyl]amide} octacosasodium salt or a pharmaceutically acceptable salt thereof;
Biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis-(tetra-O-sulfato-~-D-glucosyl- oxymethyl)phenyl]amide~ hex~-lec~codium salt or a pharmaceutically acceptable salt thereof;

WO 96/14323 PCT/US9~;/14686 Biphenyl-4,4'-dicarboxylic acid bis { [2-chloro-5-(hepta-O-sulfato-,B-D-cellobiosyloxymethyl)phenyl]amide} tetra-lec~odium salt or a pharm~re~,ti~z-lly acceptable salt thereof;
Biphenyl-4,4'-dicarboxylic acid bis { [4-chloro-2-(hepta-O-sulfato-~-D-cellobiosyloxymethyl)phenyl]amide} tetradecasodium salt or a pharm~elltic~lly acceptable salt thereof;
N,N'-Bisr3,~-bis-(hepta-0-sulfato-13-D-cellobiosyloxymethyl)phenyl]succin~mi~le octacosasodium salt or a pharmaceutically acceptable salt thereof;
N,N'-Bis[3,5-bis(hepta-O-sulfato-~-D-cellobiosyloxymethyl)phenyl]terephth~l~mideoctacosasodium salt or a pharmaceutically acceptable salt thereof;
Biphenyl-4,4'-disulfonic acid bis { [2-methyl-5-(hepta-O-sulfato-~-D-cellobiosyloxymethyl)phenyl]amide} tetradecasodium salt or a pharmaceutically acceptable salt thereof;
N,N'-Bis[2-methyl-5-(2,3,4,6-tetra-O-sulfato-,B-D-glucopyranosyloxy -methyl)phenyl]succin~mide octasodium salt or a pharmaceutically acceptable salt thereof;
3,3'-[N,N'-Ureido]-bis( N-[2-methyl-5-(hepta-O-sulfato-,B-D-m~ltosyloxyme~hyl)-phenyl])benzamide tetradecasodium salt or a pharmaceuticall~ acceptable salt thereof;
3,3'-(N,N'-Ureido)bis-({N-[2-chloro-5-(hepta-O-sulfato-~-D-cellohio~yloxymethyl)-phenyl]}benzamide) tetradecasodium salt or a pharmaceu~ically acceptable salt thereof;
N,N'-Bis { 3-12-methyl-5-(hepta-O-sulfato-~-D-maltosyloxymethyl)phenyl-carbamoyl]phenyl~isophth~l~micle tetradecasodium salt or a pharmaceutically acceptable salt thereof.

Process of the Invention The compounds of the present invention are p,~l)a ed according to the general sequence of reactions outlined in the Scheme below:

R40~ + ( H ~~ N0 oR2 / 2 glycosidation X

N02 x\~, 3 ~ ~ R30~0R~) 1 ) CIY--Z--YCI
2) NaOCH3 3) S03-(NCH3)3~

~R40~o~0~t~ H-Y-Z--Y-H-~--~ ~ ~O R~\

R30~\0R1 ~ R l o~OR3 oR2 / n I \ oR2 / n wherein R, n, X, Y, and Z 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 mercuric bromide, mercuric cyanide, silver triflate, or silver perchlorate in an aprotic solvent such as dichloromethane, ether, toluene, or nitromethane at temperatures ranging from -40~C to ambient temperature to yield glycoside 3. Reduction of the nitro group of 3 can be accomplished with a reducing 15 agent such as stannous chloride in a polar aprotic solvent such as ethyl acetate at CA 02204~31 1997-0~-0~

WO 96/14323 PCTtUS95/14686 ambient Lelllp~.dture to reflux, or by catalytic hydrogenation in the presence of a catalyst such as pa~ m on carbon gives an anilino compound 4. Coupling of 4 with a bis acid chloride or sulfonyl chloride ClY-Z-YCl can be done in the presence of an amine base such as triethylamine or diisopropylethylamine in an aprotic solvent 5 such as dichl~lull,elllane or tetrahydrofuran, hydrolysis of any acetate groups present on the sugars with a base such as sodium methoxide in methanol or aqueous sodiumhydroxide in methanol at ambient Ic;mpeidture to reflux, and sulfation of some or all of the free hydroxyl groups on the sugars can be completed with a reagent such as sulfur trioxide-trimethylamine complex or sulfur trioxide-pyridine complex in a polar 10 aprotic solvent such as dimethylformamide or dimethylsulfoxide at temperatures ranging from 0~C to 100~C yields the target compounds I.

Alternatively, the sulfate groups can be introduced into compound 3 (after hydrolysis of any acetate groups). Sulfated compound 3 is then reduced to give 15 sulfated 4 and coupled with CIY-Z-YCI to yield I.

This invention is also directed to pharmaceutical compositions, and the manufacture thereof, comprised of sulfated benzylglycosides either alone or in combination with excipients (i.e. pharmaceutically accept;~ble materi~ls with no20 pharmacological effect). Such compositions are useful for dise;~ses ~hich arecharacterized by excessive smooth muscle cell prolifer;ltic n. such ;IS restinosis, most frequently arising from vascular reconstructive sur~ery ;Ind tr;~nspl;lnt~tion~ for example, balloon angioplasty, vascular graft surger~. coron;lr! Irl~r! h!~ s surger~, and heart transplantation. Other disease states in ~hlch thcr~ l~ un~;lnted vascular 25 proliferation include hypertension, asthma, and congestl~e h~;lrt f;~ilure. The compounds of this invention are thus useful for treating 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/h over 5 -30 30 days, or by subcutaneous injection at lower dose, by oral ~clminictration at higherdose than intravenous injection. Localized delivery of the compounds Qf this invention may also be achieved by transmembrane, transdermal, or other topical ~rlministrative routes using ap~.vl~liate continuous release devices such as ~ulJpclLillg matrix, where applicable. The compositions of the invention may be formnl~ted with 35 conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent and the like. These are form~ ted in a conventional manner. It is CA 02204~31 1997-0~-0~

understood that the compounds of this invention may be ~Amini~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 determined on an individual basis by the physician or other skilled medical professional treating the 5 recipient.

Fffe(~t~ on Cell Proliferation 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 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 / anlimyCOtiC (Gibco catalog #
600 - 5240 AG). The tissue was then digested in 10 - 15 mL of "Enzyme Mixture"
containing collagenase type 1, 165 U/mL; elastase type 111, 15 U/mL: BSA, ~ mg/mL;
and soybean trypsin inhibitor, 0.375 mg/mL followed by incuh;~tion a~ 37~C under5% C~2 for 10 - 15 min. After this treatment, the outer surf;lce ad~enti~ia ~as easily removed by peeling with forceps The aorta was then longiludinall- cu~ 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 of 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 rnL of fresh me~ lm 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.

CA 02204~31 1997-0~-0~

B. Effects of c~ -ds on cell proliferation using 3H Thymidine inco ~ tion Cells were assayed in early passage (generally passage 3 - 7~ at sub-confluent 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 merlillm (AIM-V; Gibco) for 24 - 48 h prior to initi~ting the experimental protocol.
Although compounds were found to be more effective with longer pre-incubations, in general, experiments were initi~ted 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 ~lL / 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 inH20 and serially diluted into media. Compounds were routinely assayed at 1, 10, and 100 ~IM. 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 ~lg/mL.

At the completion of the experiment, plates were placed on ice, washed three 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 HCI (500 ~L / vial to neutralize NaOH) and each well was rinsed two times with water (500 IlL) for a total volume of 2 mL / vial.
Data was obtained, in triplicate, for both control and experimental samples.
Control (100%) data was obtained from maximally stim~ tçA cells, as the result of growth factor or serum stimulation. Experimental data was obtained from cells maximally stim-ll~t~d with growth factor or serum and treated with compound. Data was expressed as a percent of control from which ICsos could be determined. The compounds of the present invention are effective inhibitors of smooth muscle cell CA 02204~31 1997-0~-0~

proliferation as ~u,~ ;ed in Table I. Furthermore, the compounds of the present invention 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 Upstate Biotechnology Inc., Lake Placid, NY). For example, the sulfated compoundof Example 18 inhibits HAOSMC proliferation driven by FBS (ICso 200 nM) as well as by 5 ng/mL of PDGF (ICso 380 nM).

C. Effect on Endothelial Cell Growth vs. Smooth ~uscle Cell Proliferation The promotion of endothelial cell proliferation concurrent with inhibition of smooth muscle 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 inhibitory towards human smooth muscle cell proliferation driven by 10% FBS
as represented by the sulfated compound of Example 18 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 Mrr (3~ 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Briefly, cells were ag;lin grown in 24 well plates to 70 - 80 % confluency and, as before, serum deprived for 24 - 48 h prior to initiation of the experimental protocol. To insure that the MTT assay monitored toxicity rather than proliferation, cells were incubated with 250 ~LM drug in fresh medium without serum for 24 h at 37~C in a humidified CO2 incubator. Upon completion of the compound treatment, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) indicator dye was added for 4 h at 37~C. Cells werethen 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. Results are reported as percent viable using no drug (100% viable) and pre-solubilization (0% viable) standards. No toxicity wasobserved up to 250 ~M with sulfated compounds of Examples 10 - 25 and Example 26 (Step 5).

WO 96/14323 PCT/US9~i/14686 Anti~n~ nt Activitv ~ The anticlotting activity of the compounds of this invention was evaluated in S a partial thromboplastin time (APTT) assay using normal human plasma collectedfrom 5 donors using the procedure 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 experiments. This reagent was added to human citrated plasma equilibrated at 37~C
10 in a plastic well in the clot timer. Calcium at 37~C was added, the clot timer was started and the time for ~lbrin 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 1~ 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 >1 indicates weaker activity relative to heparin on a ~lg/mL comparison.
Table I

Porcine Smooth Muscle SULFATEDCell Antiproliferation ICs() Antlcoa~ulation Activity COMPOUND OF or (APl~) Relative to Heparin EXAMPLE (% Inhibition at x concentration ) EXAMPLE 26, STEP 5 1 18 ~M nt EXAMPLE 2~(8% inhibition at 50 ~L~/mL) nt EXAMPLE10 161,uM nt Table I Co~ Pd Porcine Smooth Muscle SULFATEDCellAntiproliferation ICso AnticoagulationActivity COMPOUND OF or (AP~) Relative to Heparin EXAMPLE (% Inhibition at x concentration) EXAMPLE24 58 ~LM nt EXAMPLE 11 117 ~M nt EXAMPLE 12 72 ~LM nt EXAMPLE 13 40.5 - 80 ~M nt EXAMPLE 14 3 ~IM 6.7 EXAMPLE 15 4 .7 ~M 2.0 - 2.3 EXAMPLE20 45 ~M nt EXAMPLE 21 43 ~M nt EXAMPLE 22 5.2 - 22 ~M 2.0 EXAMPLE 16 3.6 ~M 2.2 insignificant activity at EXAMPLE 17 23 - 50.8 IlM 50 ~g/rnL

EXAMPLE 18 0.85 IlM 2.1 A~ g4027 Tsble I Con~in~
~orcine Smooth Muscle SUL~ATI~:D Cell Antiproliferarion rC5~) Anticoa~ulation Activity COMPOUNDOFor (APrr) Relative toHepar~n EXA~LE(% Inhibitian at x conce:~ation~
(lO - 50~a inhibition at E~A~PI 1~. 19 ~Q ~l!vI) nt EXAMP~E 23 9.5 - 40 ~I 2.0 (45 - 83% }nhibition at hepann (~I-700 nt = not tested Spe~ific pr~edures are descri~cd in the followin,~, examples. l'hese e,YaT~ples are given to illus~ate the invention ~d shoul~ not be constn~ed as limitin~ the invention set fo~th in the appended claims. The terrns "Sephadex G-~0", "Duwex","Amberlite IR-20"~ "Sephadex LH-2~-100" and "Sephadex SP-C25" a~ used her~in ~re Trade Marks.
~X~MPLE 1 5-~Iept~.O.~cetyl. B~D-~l~syloxy~e~ 2-m~ y1-l-nitro~en~ene To 4.0 ;~, ~4 mmol) of 4-methyl-3-I~itroben7yl alcohol alld 20.0 g (~9 mmol) o~cetobromo-a-m~lto~,e in ~0 mL of CH3NO2 w~s added 6.15 g (24 mmol) of H~(CN),. ~ .91 g ~1~ mmol~ of EgBr2. After stirrin~ at ambient temper~ re overnight, the reaction was quenched with sal. NaCl and w~s stirred for 20 Inin. Thc reaction mixtu~e was ex~acted into CH2C12. The org~c phase was washed ~ith gat.
NaC], dcied (~lgSO4) and flash ¢hromato~raphed ( 1: 2 an~ I: 1 EtOAc I pe~roleum~0 ether). Rechrom~tagraphy using ether ~ petroleum ether ~3: 1, then 4: 1, then 100:
O) gaYe 7.97 g of the title compound ~ ~ colc~rless ~olid: IH-NMR (CDC1~, 300 M~) ~ 7.92 (s, l E~), 7.41 (d, 1 H), 7.32 ~d, 1 H~, 5.42 (d, 1 H), S.36 (t, 1 H)~ 5.25 (t, lH),5.0~(t,1H),4.8-5.0(m,3~),4.65~d,~H~,4,62(d,1H),4.S4(dd,}H),4.~-4.3~m,2H),3.9-4.1~m,3H)13.~-3.71(m, lH~,~.60(s,3H~,2.1~(s,3H),~.11 (s, 3 H), 2.04 (s~ 3 H), 2.03 (s, 6 H), 2.01 (s, 3 El~, and ~.~0 ppm (s, 3 H~.

~ S~&&~

TOTRL P . 05 WO 96tl4323 PCTIUS95/14686 ~te~ 2 5-(Heut~O-acetvl-~-D-m~ltosyloxvmethvl)-2-methylphenvlamine Proce~lure A: A ~ Lulci of 4.0 g (5.09 mrnol) of 5-(hepta-O-acetyl-,B-D-maltosyl-oxymethyl)-2-methyl-1-ni~obenzene prepared in Example 1 and 8.00 g (35 mmol) of SnCl2-H2O in 100 mL EtOAc was heated at reflux for 2 h. The reaction mixture wascooled to room tem~ dture and quenched with sat. NaHC03. After stirring for 15 min, the Illi~UlC; was diluted with CH2C12 (200 mL) and filtered through solka floc (CH2Cl2). The organic phase was dried (MgSO4) and concentrated. Flash 10 cl"~,lllalography (EtOAc / CH2C12, 1: 5, then 1: 4, then 1: 2, then 1: 1) gave 3.42 g (89% yield) of the title compound as a colorless foam: IH-NMR (CDC13, 300 MHz) ~ 7.01 (d, 1 H), 6.63 (s, 1 H), 6.62 (d, 1 H), 5.41 (d, 1 H), 5.39 (t, 1 H), 5.20 (t, 1 H), 5.05 (t, 1 H), 4.82 - 4.90 (m, 2 H), 4.75 (d, 1 H), 4.54 (d, 1 H), 4.51 (d, 1 H), 4.26 (dd, 2 H), 3.95 - 4.01 (m, 3 H), 3.63 - 3.67 (m, 1 H), 2.17 (s, 6 H), 2.11 (s, 3 H), 2.03 (s, 6 15 H), 2.00 (s, 3 H), and 1.99 ppm (s, 6 H).

Procedure B: A solution of 31.1 g (39.6 mmol) of 5-(hepta-O-acetyl-,B-D-maltosyl-oxymethyl)-2-methyl- 1 -nitrobenzene prepared in step 1 of Example I was hydrogenated at 50 psi over 10% Pd/C (10.0 g) for 1 h. The catalyst was removed by 20 filtration and the filtrate was concentrated to give a white foam. Trituration with water gave 28.0 g (94%) of the title compound as a white solid, mp 15~ - 156 ~C.
F~AMPI,F 2 step 1 5-(He~ta-O-acetyl-~-l)-cellobiosyloxvrnethvl~-2-Tnethvl-l -nitroben~ene 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.
Purification was achieved by flash chromatography (EtOAc / petroleum ether, 1: 2 to 1: 1 to 2: 1): IH-NMR (CDC13, 300 MHz) ~ 7.91 (s, 1 H), 7.40 (d, 1 H), 7.32 (d, 1 H), 4.80 - 5.20 (m, 6 H), 4.40 - 4.80 (m, 4 H), 4.36 (dd, 1 H), 4.02 - 4.13 (m, 2 H), 3.81 (t, 1 H), 3.60 - 3.68 (m, 2 H), 2.60 (s, 3 H), 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).

SteD 2 5-fHe~ta-O-~cetyl-~ llobiosvloxvmethvl)-2-methvlDhenvla~nine The title compound, mp 180 - 182~C, was prepared in 62% yield from 5-(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-2-methyl-1-nitrobenzene using ProcdureA 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 - 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, 3 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.98 (s, 3 H), 1.96 (s, 3 H), 1.95 (s, 3 H), 1.94 (s, 3 H), and 1.91 ppm (s, 1 H); mass spectrum (+FAB) m/z 778. Anal. Calcd. for C34H46NOI8: C, 53.97; H, 6.13; N, 1.85. Found: C, 53.67; H, 5.92; N, 1.62.

FXAMP~ ,F 3 ~tee I
5-(He,Dta-O-acetyl-,~-D-cellobiosvloxvmethvl)-2-chloro-1-nitroben~ene The title compound was prepared in 45~ yield by the p~ocedure described in step 1 of Example 1 using 4-chloro-3-nitrobenzvl alcohol In~ ~celohromocellobiose:
IH-NMR (CDC13, 300 MHz) o 7.82 (d, 1 H), 7.53 (d, I ~1), 7.~ (d~ 1). 4.~ 5.20 (m, S H), 4.73 (d, 1 H), 4.51 - 4.66 (m, 4 H), 4.3() - ~ ~1() (nl. I 11). ~ ()3- ~.11 (m. 2 H), 3.81 (t, 1 H), 3.60 - 3.68 (m, 2 H), 2.13 (s, 3 H), ()~ . ' ()h (s~ ~ }3), 2.0 (s, 3 H), 2.01 (s, 3 H), 2.01 (s, 3 H), 1.99 (s, 3 H), and 1.57 ppm (~. ~ Il).

steD ~
5-(HeDta-O-acetvl-,B-D-cellobiosyloxvmethvl)-2-chlorol~henvlamine The title compound was plepalcd from 5-(hepta-O-acetyl-,B-D-cellobiosyl-~ oxymethyl)-2-chloro-1-nitrobenzene in 61% yield as a solid by trituration with ether using Procedure A of step 1 of Example 1: lH-NMR (CDCl3, 300 MHz) ~ 7.20 (d, 1 H), 6.75 (s, 1 H), 6.60 (d, 1 H), 4.89 - 5.19 (m, 5 H), 4.73 (d, 1 H), 4.47 - 4.60 (m, 4 H),4.36(dd,1H),4.02-4.13(m,2H),3.80(t,1H),3.55-3.67(m,2H),2.14(s,3 H), 2.08 (s, 3 H), 2.03 (s, 3 H), 2.02 (s, 6 H), 2.01 (s, 3 H), and 2.00 ppm (s, 3 H).

FX~l\IP~ 4 Ste~ 1 2-(HeDt~-O-~cetyl-~-n-- ~llobjosyloxy~ ,vl)-4-chloro-1-njtrobenzene s The title compound was prepared in 47% yield by the procedure of step 1 of Example 1 using S-chloro-2-nitrobenzyl alcohol and acetobromocellobiose. Partialpurifieation was achieved using flash chromatography (CH2C12 / EtOAc, 6: 1) and the product used as is in the next reaction: IH-NMR (CDC13, 300 MHz) o 8.09 (d, 1 H), 7.71 (d, 1 H), 7.43 (dd, 1 H), 4.90 - 5.25 (m, 8 H), 4.63 (d, 1 H), 4.52 (d, 1 H), 4.49 (d, 1 H), 4.38 (dd, 1 H), 4.03 - 4.16 (m, 3 H), 3.79 (t, I H), 3.60 - 3.68 (m, 2 H), 2.13 (s, 3 H), 2.10 (s, 6 H), 2.04 (s, 6 H), 2.01 (s, 3 H), and 1.98 ppm (s, 3 H).

SteD 2 2-(HeDta-O-aeetvl-~-D-eellobiosvloxYmethvl)-4-chloroDhenylarnine The title compound was prepared in 55% yield from 2-(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-S-chloro-1-nitrobenzene by Procedure A of step 2 of Example 1. Purification was achieved by trituration with ether: IH-NMR (CDC13, 300 MHz) ~7.11 (dd, 1 H), 7.04 (d, 1 H), 6.65 (d, 1 H), 5.03 - 5.18 (m 3 H), 4.88 - 4.97 (m, 2 H), 4.45 - 4.80 (m, 5 H), 4.36 (dd, I H), 4.02 - 4.13 (m, ~ Hh 3.60 - 3.81 (m, 3 H), 2.16 (s, 3 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 2.01 (s, 6 H), 2.00 (~ and 1.98 ppm (s, 3 H)-F.XAMPT ,F 5 steD 1 S-(Tetra-O-acetvl-~-D-~luco~Dvranosvloxvmethyl~-2-methvl-1-nitroben~ene The title compound was prepared in 54% yield by the procedure described in step 1 of Example 1 using a-D-glucopyranosyl bromide tetraacetate and 4-methyl-3-nitrobenzyl alcohol: lH-NMR (CDC13, 300 MHz) ~ 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, 1 H), 2.60 (s, 3 H), 2.11 (s, 3 H), 2.07 (s, 3 H), 2.03 (s, 3 H), and 2.01 ppm (s, 3 H).

5-(T~tra-O-~cetyl-~-n-pl~-co~yr~nosYloxy~netlllyl)-2-methylphenvlamine The title compound was prepared in 63% yield by Procedure A of step 2 of S Example 1 from 5-(tetra-O-acetyl-,B-D-glucopyranosyloxymethyl)-2-methyl-ni~obenzene and punfied by tnturation of the crude reaction mixture with ether: lH-NMR (CDC13, 300 MHz) ~ 7.02 (d, 1 H), 6.64 (s, 1 H), 6.63 (d, 1 H), 5.02 - 5.17 (m, 3 H), 4.79 (d, 1 H), 4.50 - 4.55 (m, 2 H), 4.29 (dd, 1 H), 4.17 (dd, 1 H), 3.64 - 3.68 (m, 1 H), 2.18 (s, 3 H), 2.11 (s, 3 H), 2.02 (s, 3 H), 2.01 (s, 3 H), and 2.00 ppm (s, 3 10 H).

FXAMPT,F. 6 ,~teu 1 3. 5-Bi~hepta-O-acetvl-,B-D-cellobiosvloxvmethvl~-l-nitroben~ene The title compound was prepared in 42% yield by the procedure described in step 1 of Example 1 using one equivalent of 5-nitro-m-xylene-a,a-diol, two equivalents of acetobromocellobiose, and two equivalents of all other reagents.
Purification was achieved by flash chromatography (EtOAc / CH2CI2, 3: 1) and trituration with ether: IH-NMR (CDCl3, 300 MHz) ~ 8.10 (s, 2 H), 7.48 (s, 1 H), 4.88 - 5.30 (m, 12 H), 4.68 (d, 2 H), 4.52 - 4.59 (m, 6 H), 4.38 (dd, 2 H), 4.00 - 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).

3. 5~ (heDta-O-acetyl-,B-D-cellobiosyloxvmethvl)phenvlamine The title compound was prepared in 54% yield by Procedure A of step 2 of Example 1 using 3,5-bis(hepta-O-acetyl-,B-D-cellobiosyloxymethyl)-l-nitrobenzene.
pllrific~tion was achieved by flash chromatography (EtOAc / petroleum ether, 1: 1):
lH-NMR (CDC13, 300 MHz) o 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 (m, 6 H), 4.37 (dd, 2 H), 4.02 -4.13 (m, 6 H), 3.81 (t, 2 H), 3.57 - 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).

CA 02204~31 1997-0~-0~

FX~MPT,F. 7 ~teD I
3, 5 Ri~(he~t~ O ~cetyl-~-D-I~ctosvloxymethvl)-l-nitrobenzene s The title compound was prepared by the procedure described in step I of Example 1 using 1 equivalent of 5-nitro-m-xylene-oc,a-diol, two equivalents of acetobromolactose, and two equivalents of all other reagents. Purification was achieved initially by flash chromatography (EtOAc / CH2C12 (3: 1)) and then rechromatography first with toluene / EtOAc (1: 1) and then with EtOAc / CH2C12 (1 : 4 to 1: 2) to give partially pure product which was used directly in the next reaction.

~2 3. 5-Bi~(heDta-O-acetyl-,B-D-lactosvloxvmethvl)Dhenvlamine The title compound was prepared by procedure A of step 2 of Example 1 using 3,5-bis(hepta-O-acetyl-,B-D-lactosyloxymethyl)- I -nitrobenzene. Partial purification was achieved by flash chromatography (CH2CI~ / EtOAc (': I to 1: 2)) and then rechromatography (CH2CI2 / EtOAc (3: I to 1: 1 )) to ~i~e the title compound.

FXAI~IPI,F 8 ~L~
3.5-Bi~(tetra-O-acetvl-,B-D-~lucosvlo~;-meth-lt-l-ni~r~ en~.ene The title compound was prepared by the procedure described in step 1 of Example 1 using 1 equivalent of 5-nitro-m-xylene-o~,a-diol, Iwo equivalents of acetobromoglucose, and two equivalents of all other reagents. Purification 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.

CA 0220453l lss7-05-05 Wo 96/14323 PcrluS9S/14686 Step 2 3, 5-Bis(tetra-O-acetyl-,B-D-glucosyl(~y~,-elllyl)phenylamine The title compound was prepared by procedure A of step 2 of Example 1 5 using 6.27 g of 3, 5-bis-(tetra-O-acetyl-~-D-glucosyloxymethyl)- l-nitrobenzene.
Puriflcation was achieved by flash chromatography (CH2C12 / EtOAc (3: 2)) to give 2.00 g (39% yield) of the title compound as a yellow oil: partial lH-NMR (CDCl3,300 MHz) â 6.56 (s, 3 H), 5.0 - 5.2 (m, 6 H), 4.8 (d, 2 H), 4.4 - 4.6 (m, 4 H), 3.7 ppm (brd, 2 H).
F.XAMP~ ~ 9 3. ~ he~ta-O-:~cetyl-B-D-maltosvloxvmethvl)-l-nitroben~ene The title compound was prepared by the procedure described in step 1 of Example 1 using 2.57 g (14.02 mmol; 1 equivalent) of 5-nitro-m-xylene-oc,a-diol,two equivalents of acetobromomaltose, and two equivalents of all other reagents, all in CH3NO2 (200 mL). Purification was achieved by flash chromatography (EtOAc /
CH2Cl2 (1: 2 to 1: 1). Rechromatography (Et2O) gave 11.4 g (57% yield) of the title compound which was used in the next reaction: partial IH-NMR (CDC13, 300 MHz) ~ 8.11 (s, 2 H), 7.49 (s, 1 H), 5.42 (d, 2 H), 5.36 (t, 2 H), 5.26 (t, 2 H), 5.06 (t, 2 H), 4.21 -4.29(m,4H),and3.69-3.72ppm(m,2H).

SteD 2 3. 5~i~(heDta-O-acetvl-,B-D-maltosvloxvmethvl)Dhenvlamine The title compound was prepared by procedure A of step 2 of Example 1 using 3,5-bis(hepta-O-acetyl-,B-D-maltosyloxymethyl)-l-nitrobenzene. Purification was achieved by flash chromatography (CH2C12 / EtOAc (1: 1)) to give 4.43 g (40%yield) of the title compound: partial lH-NMR (CDC13, 300 MHz) o 6.57 (s, 3 H), 5.41 (d, 2 H), 5.36 (t, 2 H), 5.23 (t, 2 H), 5.50 (t, 2 H), 4.75 (d, 2 H), 4.26 (m, 4 H), and 3.65 - 3.69 ppm (m, 2 H).

CA 02204~3l lss7-o~-o~
Wo 96/14323 PCT/US95/14686 ~,X~MP~,F, 10 Step 1 nodecanedioic A~id Ri~r5-(B-n-cellobiosvl~xvl"~lhvl)-2-methvl~henvllamide~

To a solution of 5-(hepta-O-acetyl-13-D-cellobiosyloxymethyl)-2-methyl-phenylamine (1.03 g, 1.36 mmol) and triethylamine (189 ~L, 1.36 mmol) in THF (15mL) was added dodecanedioyl dichloride (170 ~LL, 0.681 mmol). After stirring at room temperature for 2 h, the reaction mixture was quenched with MeOH, diluted with CH2C12, and then washed with water. The organic phase was dried (MgSO4), filtered, and concentrated to give 1.157 g of a colorless solid. This product was dissolved in MeOH (25 mL) and was treated with 11.9 mL (11.9 mmol) of 1 N
NaOH. After stirring at 50~C for 5 h, the reaction mixture was cooled to room temperature and treated with 9.5 mL (9.5 mmol) of 1 N HCI. Collection of the colorless solid provided the title compound (566 mg, 75% yield), mp >200~C: partial lH (DMSO-d6, 400 MHz) o 9.25 (s, 2 H), 7.32 (s, 2 H), 7.16 (d, 2 H), 7.10 (d, 1 H), 4.77 (d, 2 H), 4.50 (d, 2 H), 4.24 - 4.30 (two doublets, 2 H), 3.78 (d, 2 H), 3.69 (d, 2 H), 3.63 (br d, 2 H), 2.99 (t, 2 H), 2.30 (t, 4 H), 1.58 (br t, 4 H), and 1.29 ppm (br s, 12 H); 13C-NMR (DMSO-d6, 100 MHz) o 171.1, 136.2, 135.5, 131.1, 130.0, 124.8, 103.2, 101.7, 80.6, 76.8, 76.4, 75.0, 74.9, 73.3, 73.1, 70.0, 69.5, 61.0, 60.4, 35.7, 28.9, 28.8, 28.7, 25.3, and 17.6 ppm; mass spectrum (-FAB) m/z 1115 (M-H); IR (KBr) 3430 and 1660 cm-l. Anal. Calcd. for Cs2H8oN2o24-4 H2O: C, 52.51; H, 7.46; N, 2.36. Found: C, 52.53; H, 7.28; N, 2.27.

Ste~ 2 nodecanedioic Acid Bi~r2-methvl-5-(heDta-O-sulfato-,~-l)-cellobiosvloxvmethyl)~henYllamide~ Tetradecasodium Salt A solution of dodecanedioic acid bis ~ [5-(~-D-cellobiosyloxymethyl)-2-methyl-phenyl]amide} (391 mg, 0.350 mmol) and sulfur trioxide trimethylamine complex (3.66 g, 26.3 mmol) in DMF (40 mL) was heated a 70~C for 3 days. The reaction mixture was concentrated in vacuo and passed through a Sephadex G-10 column (twice). Cation exchange was effected using a Dowex 50 x 8 strongly acidic (Na form) column to give 433 mg of the title compound: partial IH-NMR (D20, 400 MHz)~7.39(d,2H),7.36(d,2H),7.29(s,2H),4.94(d,2H),4.80-4.90(m,4H), 4.59 (dd, 2 H), 2.49 (t, 4 H), 2.23 (s, 3 H), 2.30 - 2.40 (m, 4 H), and 1.30 - 1.50 ppm CA 0220453l lss7-05-05 WO 96/14323 pcTtuss~ll4686 (m, 12 H); 13C-NMR (D2O, 100 MHz) ~ 176.8, 135.2, 134.9, 134.4, 130.9, 127.8, 127.1, 100.0, 99.1, 77.7, 77.4, 77.1, 74.4, 73.7, 73.5, 73.1, 70.7, 67.7, 66.6, 35.8, 28.5, 28.3, 25.5, and 16.9 ppm; mass spectrum (negative electrospray) (m-~Na)lz 825.6 (M-3Na)3-, 613.5 (M-4Na)4-, and 486.2 (M-5Na)5-. Anal. Calcd. for Cs2H66N2O66S14Nal4-14 H2O: C, 17.83; H, 2.70; N, 0.80; S, 17.40. Found: C, 17.96; H, 2.57; N, 0.72; S, 17.11.

F.XAMPI,F 11 steD I
N.N'-Bi~r5-~,B-cellobiosvloxvmethvl)-2-methvlphenvlltereDhthalamide To 813 mg (1.08 mmol) of 5-(hepta-O-acetyl-,13-D-cellobiosyloxymethyl)-2-methylphenylamine in THF (20 mL) containing triethylamine (148 ~LL, 1.08 mmol) was added terephthaloyl chloride (109 mg, 0.538 mmol). After stirring at ambienttemperature for 2 h, the reaction mixture was quenched with MeOH, diluted with CH2C12 and washed with water. Drying (MgSO4) and concentration gave crude product, which was diluted with MeOH (25 mL) and treated with 8 7 mL (8 7 mmol) of 1 N NaOH. After stirring at 50~C for 3 h, the suspension was filtered tO provide 512 mg (89%) yield of the title compound as a colorless solid, mp >210~C: IH
(DMSO-d6; 400 MHz) o 10.07 (s, 2 H), 8.10 (s, 4 H), 7.35 (s. 2 H), 7.26 (d, 2 H), 7.22(d,2H),5.22-5.24(m,4H),5.01 (d,2H), 4.99(d,2H),4.~2(d,2H),4.68(d, 2 H), 4.54 - 4.64 (m, 6 H), 4.32 (d, 2 H), 4.25 (d, 2 H), 3.75 - 3.80 (d, 2 H), 3.63 -3.77 (m, 4 H), 3.37 - 3.41 (m, 2 H), 2.97 - 3.19 (m, 10 H), and 2.23 ppm (s, 6 H); 13C-NMR (DMSO-d6; 100 MHz) o 164.6, 137.0, 136.0, 135.9, 133.0, 130.1, 127.7, 126.0,125.6, 103.2, 101.8, 80.6, 76.8, 76.4, 75.1, 74.9, 73.3, 73.2, 70.0, 69.3, 61.0, 60.4, and 17.7 ppm; mass spectrum m/z 1052, 889, and 725. Anal. Calcd. for C48H64N2~24:
C, 54.75; H, 6.13; N, 2.66. Found: C, 55.54, H, 6.33; N, 2.57.

CA 02204~31 1997-0~-0~

.

~n 2 N.N'-Ri~r5-(heDt~-O-sulfato-~-D-cellobiosvloxymethvl)-2-m~tl~ henylltereDhthalamide TetradecaSodium Salt S A mixture of N,N'-bis[5-(,~-cellobiosyloxymethyl)-2-methylphenyl]-terephth~l~micle (321 mg, 0.305 mmol) and sulfur trioxide trimethylamine complex(3.06 g, 22.0 mmol) in DMF (25 mL) was stirred at 70~C for 4 days. The reaction mixture was concentrated and passed through a Sephadex G-10 column. IH-NMR
analysis showed incomplete sulfation. The product was resubmitted to sulfation with 3.06 g (22.0 mmol) of sulfur trioxide trimethylamine complex in 25 mL of DMF at 70~C for 5 days. The reaction mixture was cooled to ambient temperature, concentrated in vacuo, and passed through a Sephadex G-10 column. Cation exchange using a Dowex column (50 x 8; strongly acidic; Na form) provided 469 mg(62% yield) of the title compound, mp 178 ~C (dec): partial lH-NMR (D2O; 400 MHz)o8.11 (s,4H),7.40-7.50(m,6H),4.98(d,2H),4.95(d,2H),and2.32ppm (s, 6 H); 13C-NMR (D2O; 100 MHz) o 169.5, 136.9, 135.4, 135.2, 134.3, 131.0, 128.1, 128.0, 127.0, 100.0, 99.1, 77.7, 77.4, 77.3, 77.1, 74.4, 73.7, 73.4, 73.1, 70.7, 67.7, 66.7, and 16.8 ppm. Anal. Calcd. for C4gHsoN2o66s l4Nal4- 10 H20: C, 20.58;
H, 2.51; N, 1.00; S, 17.17. Found: C, 20.21; H, 2.84; N, 0.96; S, 17.34.
FXAMPl ~ 12 ~tee 1 Bi~henvl-4.4'-dicarboxvlic Acid Bi~r~-(B-D-cellobios~ meth~1)-2-methvlDhenYllamide~

To a solution of 887 mg (1.18 mmol) of 5-(hepta-O-acetyl-,B-D-cellobiosyl-oxymethyl)-2-methylphenylamine and 162 ~L (1.18 mmol) of triethylamine in THF
(20 mL) was added 164 mg (0.588 mmol) of biphenyl 4,4'-dicarboxylic acid chloride.
After stirring at room temperature for 4 h, the reaction mixture was quenched with MeOH, diluted with CH2C12 and washed with water. The organic phase was dried (MgSO4), filtered, and concentrated to a colorless solid (1.01 g). A solution of the crude material (985 mg, 0.574 mmol) in MeOH (25 mL) containing 9.18 mL (9.18 mmol) of 1 N NaOH was stirred for 3 h at 50~C. The reaction mixture was cooled, and title compound was collected as a white powder (444 mg, 69% yield): partial lH-NMR (DMSO-d6; 300 MHz) ~ 10.01 (s, 2 H), 8.13 (d, 4 H), 7.94 (d, 4 H), 7.37 (s, 2 CA 02204531 lss7-os-05 wo 96/14323 PCT/USg5l14686 H), 7.28 (d, 2 H), 7.23 (d, 2 H), 5.24 (d, 2 H), 5.00 (q, 2 H), 4.84 (d, 2 H), 4.56 - 4.69 (m, 4 H), 4.33 (d, 2 H), 4.27 (d, 2 H), and 2.25 ppm (s, 3 H).

~n2 S Rj~hel~,yl-4,4'-djc~rboxvlic Acid Bi~r5-(heDta-O-sulfato-~-D-cellobiosyl~y...~ll,yl)-2-1n~thvlDhenvllamide~ Tetradecasodium Salt A solution of 437 mg (0.387 mmol) of biphenyl-4,4'-dicarboxylic acid bis{ [5-,B-D-cellobiosyloxymethyl)-2-methylphenyl]amide} and 3.85 g (27.7 mmol) of sulfur ~ioxide trimethylamine complex in DMF (25 mL) was stirred at 70~C for 5 days.
The reaction mixture was quenched with water and concentrated in vacuo. The residue was dissolved in a small amount of water and passed through a Sephadex G-10 column. Cation exchange was accomplished using Dowex 50 x 8 strongly acidic (Na form) resin to provide, after azeotropic drying with toluene, 742 mg (75% yield) of the title compound, mp 170~C (dec): partial IH-NMR (D20; 400 MHz) o 8.09 (d, 4 H), 7.97 (d, 4 H), 7.40 (m, 6 H), 4.98 (d, 2 H), 4.94 (d, 2 H), 4.63 - 4.68 (m, 4 H), 4.56 (dd, 2 H), 4.41 (dd, 2 H), 4.30 - 4.37 ( m, 6 H), 4.17 - 4.23 (m, 4 H), 3.99 - 4.05 (m, 4 H), and 2.31 ppm (s, 6 H); 13C-NMR (D~O; 100 MHz) â 169.7, 143.2, 135.3, 135.2, 134.4, 132.8, 130.9, 128.1, 128.0, 127.4, 127.0, 99.9, 98.9, 77.6, 77.3, 77.2, 77.0, 74.2, 73.6, 73.3, 73.0, 70.6, 67.6, 66.6, and 16.7 ppm Anal. Calcd. for CssHs4N2O66S14Nal4-14 H20: C, 21.97; H, Z.80; ~'. 0.95; S, 16.~9. Found: C, 21.77; H, 2.90; N, 0.95; S, 13.66.

F.XAMPL,F. 13 steD 1 N.N'-Bi~r5-(~-D-cellobiosyloxvmethvl)-2-methvl~henvlli~oDhthalamide To a solution of 887 mg (1.18 mmol) of 5-(hepta-O-acetyl-,B-D-cellobiosyl-3Q oxymethyl)-2-methylphenylamine in THF (20 mL) containing triethylamine (162 ~LL, 1.18 mmol) was added isophthaloyl dichloride (119 mg, 0.587 mmol). After stirring at ambient tenl~elatulc for 2 h, the reaction mixture was quenched with MeOH, diluted with CH2C12, and washed with water. The organic phase was dried (MgSO4) and concentrated to give 1.00 g of a colorless solid. A solution of the crude product in MeOH (25 mL) containing 9.76 mL (9.76 mmol) of 1 N NaOH was stirred at 50~C

CA 02204~31 1997-0~-0~

for 3 h. The resulting solid was collected and azeotropically dried over toluene to give 355 mg (55% yield) of the title compound as a colorless powder, mp 200-203~C:
partial lH-NMR (DMSO-d6; 400 MHz) o 10.09 (s, 2 H), 8.54 (s, 1 H), 8.15 (d, 2 H), 7.68 (t, 1 H), 7.34 (s, 2 H), 7.26 (d, 2 H), 7.21 (d, 2 H), 5.24 (t, 4 H), 5.01 (dd, 2 H), 4.82 (d, 2 H), 4.69 (s, 2 H), 4.54 - 4.69 (m, 4 H), 4.32 (d, 2 H), 4.25 (d, 2 H), 3.76 -3.80 (m, 2 H), 3.63 - 3.69 (m, 4 H), and 2.24 ppm (s, 6 H); 13C-NMR (DMSO-d6;
100 MHz) o 164.9, 136.1, 135.9, 134.8, 133.0, 130.5, 130.1, 128.6, 127.1, 125.9,125.6, 103.2, 101.8, 80.6, 76.8, 76.4, 75.1, 74.9, 73.3, 73.2, 70.0, 69.4, 61.0, 60.4, and 17.7 ppm; mass spectrum (-FAB) m/z 1051, 727, and 889. Anal. Calcd. for C4gH64N2O24-3 H20: C, 52.08; H, 6.37; N, 2.53. Found: C, 51.89; H, 6.31; N, 2.26.

Ste~ ~
N.N'-Ri~r5-(heDta-O-sulfato-,B-D-cellobiosvloxvmethvl)-2-lnethvlDhenylli~oDhthalamide Tetradecasodiuln Salt A solution of N,N'-bis[5-(~-D-cellobiosyloxymethyl)-2-methylphenyl]-isophth~l~mide (180 mg, 0.171 mmol) and 1.79 g (11.98 mmol) of sulfur trioxide trimethylamine complex in DMF (25 mL) was stirred at 70~C for 4 days. The reaction mixture was concentrated and then purifled using a Sephadex G-10 columnwith water elution. Cation exchange was effected with a column of Dowex 50 x 8 strongly acidic (Na form) resin with water elution. Removal of water in vacu~ and azeotropic drying with toluene gave 358 mg (84% yield) of the ~itle compound as a colorless solid, mp 171 ~C (dec): partial lH-NMR (D20; 40() 1~1Hz) o 8.43 (s, 2 H), 8.21 (dd, 2 H), 7.78 (t, 1 H), 7.40 - 7.50 (m, 6 H), 4.97 (d, 2 H), 4.94 (d, 2 H), 4.56 (dd, 2 H), and 2.30 ppm (s, 6 H); 13C-NMR (D2O; 100 MHz) o 169.2, 135.3, 135.1, 134.3, 134.0, 131.0, 130.9, 129.3, 128.0, 126.9, 126.5, 99.9, 99.0, 135.5, 77.3, 77.2, 77.0, 74.3, 73.6, 73.3, 73.0, 70.6, 67.6, 66.6, and 16.7 ppm. Anal. Calcd. for C48HsoN2o66sl4Nal4-l4 H20-Na2S04: C, 19.12, H, 2.61; N, 0.93; S, 17.01.
Found: C, 18.97; H, 2.45; N, 0.94; S, 15.68.

CA 0220453l lss7-05-05 WO 96/14323 pcTruss5ll4686 ~ F,XAMP~,F, 14 ~!~D 1 necane~lioic Acid Ri~r~ ~-bi~ llobiosvloxvmethvl)Dhenvllamide~
s To a solution of 3, 5-bis(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-phenylamine (817 mg, 0.588 mmol) and triethylamine (82 ~LL, 0.59 mmol) in THF
(25 mL) was added 74 ~L (0.295 mmol) of dodecanedioyl dichloride. The reaction mixture was stirred at ambient temperature for 90 min, quenched with MeOH, diluted with CH2Cl2, and washed with water. The organic phase was dried (MgSO4) and concentrated to a colorless solid (834 mg, 95% yield). The crude product was dissolved in MeOH (25 mL) and was treated with 8.4 mL (8.4 mmol) of 1 N NaOH.
After stirring at 50~C for 2 h, the reaction mixture was quenched at room temperature with 7.84 mL of 1 N HCI, concentrated, and purified by reverse phase column chromatography (RP silica 60) using MeOH / H2O (1: 1) elution. Rechromatography using MeOH / H2O (2: 3) provided 438 mg (87% yield) of the title compound as a colorless solid: partial IH-NMR (D2O; 400 MHz) ,o 7.4~ (s, 4 H), 7.26 (s, 2 H), 4.64 (d,4H),4.47(t,8H),3.80(dd,4H),3.71 (dd,4H),2.30(brt,4H), 1.55(t,4H), and 1.17 ppm (brm, 12 H); 13C-NMR (D2O; 100 MHz) ~ 175.1, 138 1, 137.7, 124.4, 120.3, 10~.5, 101.3, 78.7, 75.9, 75.5, 74.7, 74.3, 73.1, 72.8. 70 8. 69 4. ~0.~, 60.0, 36.6, 28.7, 28.6, 28.5, and 25.3 ppm. Anal. Calcd. for C7t,~ 3~0~ l() H2O:
C, 46.15; H, 7.13; N, 1.42. Found: C, 45.91; H, 6.82; l~i, I .~ I

ste~ 2 Decanedioic Acid E~ r3.5-bis~he~ta-O-sulf.~tu-13-1)-cellobiosvloxvmethvl)Dhenvllamide~ Octacosasvdium Salt A solution of decanedioic acid bis{[3,5-bis(~-cellobiosyloxymethyl)phenyl]-amide (177 mg, 98.4 mmol) and sulfur trioxide trimethylamine complex (1.97 g, 14.2 mmol) in DMF (25 mL) was stirred at 70~C for 25 h. The reaction mixture was concentrated and purified by chromatography with Sephadex G-10 using water elution. Cation exchange using a column of Dowex 50 x 8 strongly acidic resin (Na form) provided 292 mg (64% yield) after azeotropic drying with toluene: partial lH-NMR (D2O; 400 MHz) o 7.55 (s, 4 H), 7.35 (s, 2 H), 4.96 - 4.98 (m, 8 H), 2.45 (br t, 4 H), 1.40 - 1.60 (br t, 4 H), and 1.36 - 1.41 ppm (br m, 12 H); 13C-NMR (D2O; 100 MHz) o 176.1, 138.0, 137.1, 124.5, 121.0, 99.9, 99.6, 77.6, 77.43, 77.39, 77.0, 74.3, 73.4, 72.9, 70.9, 67.6, 66.3, 36.5, 28.7, 28.5, and 25.3 ppm. Anal. Calcd. for C76Hg2N2O30-3 Na2S04-28 H20: C, 16.34; H, 2.67; N. 0.50; S, 17.8. Found:
C, 16.25; H, 2.63; H, 0.59; S, 17.07.

5FX~MP~,F 15 Step 1 RiDhenvl 4.4'-dicarboxylic Acid Bisrr3~5-bis(,B-D-cellobiosyloxvmethyl)Dhenvllarnide~
To a solution of 3,5-bis(hepta-O-acetyl-~-D-cellobiosyloxymethyl)-phenylamine (817 mg, 0.588 mmol) and triethylamine (86 ~LL, 0.62 mmol) in THF
(25 mL) was added 4,4-biphenyldicarboxylic acid dichloride (86 mg, 0.309 mmol).
After stirring at room temperature for 2 h, the reaction mixture was quenched with 15MeOH, diluted with CH2CI2, and washed with H20. The reaction mixture was dried(MgSO4) and concentrated to give 921 mg of crude product, which was dissolved inMeOH (20 mL) and treated with 9.3 mL of 1 N NaOH. After sthTing ~t 50~C for 4 h,8.6 mL (8.6 mrnol) of 1 N HCI was added to the cooled reaction mix~ure. Collection of the solid provided 482 mg (86% yield) of the title compound: pani;ll lH-NMR
20(DMSO-d6; 400 MHz) o 10.40 (s, 2 H), 8.11 (d, 4 H), 7.93 (Lh 4 H), 7.74 (s, 4 H), 7.18 (s, 2 H), 4.86 (d, 4 H), 4.56 (d, 4 H), 4.35 (d, 4 H), 4.27 (d. ~ X0 (d, 4 H), and 2.97 - 3.07 ppm (m, 4 H); 13C-NMR (DMSO-d~" 10() 1~1}1z) ~ 165.0, 142.0, 138.9, 138.2, 134.1, 128.5, 126.9, 122.8, 119.3, 103.', 1()' (). ~ . 7~ . 76.5. 75.1, 75.0, 73.3, 73.2, 70.02, 69.96, 61.0, and 60.4 ppm; IR (~Br) 1~ -nl l Anal Calcd for C78HI08N2O46-l0 H2O: C, 47.08; H, 6.48; 1~ 1. Found C. ~6.6~; H, 6.22;
N, 1.6 SteD 2 BiDhenvl-4.4'-dicarboxvIic Acid ~is~r3 ~-bis(heDta-O-sulfato-,B-D-30f ~IIobiosvloxymethvI)Dhenvllamide~ Octacosasodium Salt A solution of 338 mg (0.187 mmol) of biphenyl-4,4'-dicarboxylic acid bis{[3,5-bist,B-D-cellobiosylo~ylIlelhyl)phenyl]amide} and 3.64 g (26.1 mmol) ofsulfur trioxide trimethylamine complex in DMF (25 mL) was stirred at 70~C for 2 35 days. The reaction llli~lure was cooled to ambient temperature, quenched with water, and concentrated. Purification was achieved with a Sephadex G-10 column (H2O
elution). Cation exchange was accomplished by passing an aqueous solution of theproduct through a column of Dowex 50 x 8 strongly acidic (Na form) resin. Removal of solvent and azeotropic drying with toluene gave 765 mg (88% yield) of the title compound as an off-white solid, mp 180~C (dec): partial lH-NMR (D2O; 400 MHz) ~ 8.08 (d, 4 H), 7.97 (d, 4 H), 7.70 (s, 4 H), 7.39 (s, 2 H), 5.02 (d, 4 H), 4.98 (d, 4 H), 4.15 - 4.23 (m, 8H)t and 4.00 ppm (m, 8 H); 13C-NMR (D2O, 100 MHz) â 169.2, 143.3, 138.2, 137.3, 133.5, 128.3, 127.5, 125.0, 121.8, 100.0, 99.7, 77.7, 77.54, 77.51, 77.1, 74.4, 73.5, 73.0, 71.0, 67.7, and 66.4 ppm; mass spectrum (electrospray) (m-0 zNa)lz 401.3 (m - llNa)ll-, 443.7 (m - 10Na)l0-, 495.6 (m - 9Na)9-, 560.4 (m -8Na)8-, and 643.7 (m - 7Na)7~ .
Anal. Calcd. for C7gHgoN2ol3os28Na28-2 Na2so4-28 H2O:
C, 17.17, H, 2.51; N, 0.51; S, 16.46. Found: C, 17.06; H, 211; N, 0.55; S, 12.04.

FXAMPT,F 16 steD 1 BiDhenvl-4.4'-dicarboxvlic Acid Bis~r3.5-Bis(he~ta-O-~cetvl-,B-D-lactosyloxvmethvl)Dhenvllamide~
To a solution of 3, 5-bis(hepta-O-acetyl-~-D-lac~osyloxvmethyl)phenylamine (1.05 g, 0.757 mmol) and triethylamine (105 ~L, 0.757 mmol) in THF (20 mL) was added 4,4-biphenyldicarboxylic acid dichloride (106 mg, ().~7X mmol). After stining at room temperature for 2 h, the reaction mixture ~s quenched ~ith MeOH, dilutedwith CH2C12, and washed with H20. The reaction mixture W;lS dried (MgSO4), concentrated, and purified by trituration from CH2C12 / Et2O to give 984 mg (87%yield) of the title compound as a colorless solid, mp 163 - 167~C: lH (CDC13; 400 MHz) o 8.62 (s, 2 H). 8.05 (d, 4 H), 7.74 (d, 4 H), 7.66 (s, 4 H), 6.93 (s, 2 H), 5.33 (d, 2 H), 5.17 (t, 2 H), 5.07 - 5.12 (m, 2 H), 4.91 - 4.97 (m, 4 H), 4.79 (d, 2 H), 4.62 -4.66 (m, 4 H), 4.57 (d, 2 H), 4.51 (d, 2 H), 4.03 - 4.15 (m, 6 H), 3.80 - 3.88 (m, 4 H), 3.62 (dq, 2 H), 2.13 (s, 12 H), 2.10 (s, 12 H), 2.03 (s, 24 H), 2.02 (12 H), and 1.95 ppm (s, 12 H); mass spectrum (electrospray, Ca2+ adduct) m/z 1513.2 (m + Ca)2+.
~ Anal. Calcd. for Cl34H164N2O74 1 H2O: C, 53.56; H, 5.57; N, 0.93. Found: C, 53.25; H, 5.55; N, 1.06.

Ste~ 2 henvl-4.4'-dicarboxvlic Acid Ri.~r3 ~-bi~hepta-O-sulfato-,B-D-l~ctosyl~v-..etl~vl)Dh-onvllamide~ Octacos~odium S~lt A solution of 871 mg (0.292 mmol) of biphenyl-4,4'-dicarboxylic acid bis~[3,5-bis(hepta-O-acetyl-,13-D-lactosyloxymethyl)phenyl]amide} in MeOH (15 mL) containing 8.75 mL of 1 N NaOH (8.75 mmol) was stirred at 50~C for 3 h. The reaction mixture was cooled to ambient temperature and was quenched with 8.16 mL(8.16 mmol) of 1 N HCI. The resulting solid was collected and azeotropically dried with toluene: 13C-NMR (DMSO-d6; 100 MHz) o 164.9, 141.9, 138.9, 138.2, 134.1, 128.4, 126.8, 119.2, 103.8, 101.9, 80.7, 75.5, 74.99, 74.96, 73.3, 73.2, 70.6, 69.9, 68.0, 60.5, and 60.3 ppm.

A solution of 396 mg (0.219 mmol) of biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis-(,B-D-lactosyloxymethyl)phenyl]amide3 and 4.26 g (30.6 mmol) of sulfur trioxide timethylamine complex in DMF (20 mL) was stirred at 70~C for 3 days. The reaction mixture was cooled to ambient temperature, quenched with distilled H2O,and concentrated. The residue was purified by passing throuL~h ~ Sephadex G-10 column (H2O elution). Cation exchange was accomplished by passing an aqueous solution of the product through a column of Dowex 5() x R stronrly acidic ('.~'a form) resin. Removal of solvent and azeotropic drying with toluene ~ e ~1 m~ (fi5%) ofa shiny white solid, mp 149~C (dec): 13C-NMR (D~O: 1()0 ;'~l~lz) ~ 1~9.1, 143.1,138.1, 137.3, 133.4, 128.2, 127.4, 124.9, 121.7, 1()().'~ . 77.(). 75 X. 75.3, 75.05, 75.00, 73.0, 71.1, and 66.34 ppm; mass spcctrum ~lcc~ro~r;l~ ~ /m :Na)l.
643.7 (m - 7 Na)7-, 754.8 (m - 6 Na)6- and 910.4 (m - 5 I~ . An;ll. Calcd. for C78H80N2O130S28Na28 28 H2O: C, 18.12; H, 2.65; N, 0.54; S, 17.36. Found:
C, 18.06; H, 2.67; N, 0.50; S, 14.75. (NMR and capillary electrophoresis show one major component and several minor componen~s; mass spec~rum analysis indicates products from 20 - 28 sulfates).

l~,X~MP~,F, 17 Step 1 Bipher~vl-4.4'~ carbo~ylic Acid Bi.~rr~ ~-bi~(tetra-O-acetvl-~-D-pll-cosyluAv.-.ethvl)Dhenvllamide~

To a solution of 3, 5-bis(tetra-O-acetyl-,B-D-glucosyloxymethyl)phenylamine (996 mg, 1.22 mmol) and triethylamine (160 ~LL, 1.22 mmol) in THF (15 mL) was added 4,4'-biphenyldicarboxylic acid dichloride (171 mg, 0.612 mmol). After stirring at room temperature for 3 h, the reaction mixture was quenched with MeOH, diluted with CH2C12, and washed with H2O. The reaction mixture was dried (MgSO4), concentrated, and purified by trituration from CH2C12 / Et20 to give 984 mg (87%yield) of the title compound as a colorless solid, mp 125 - 130 ~C: IH (CDC13; 400 MHz)~8.42(s, 12H),8.04(d,4H),7.75 (d,4H),7.66(s,4H),6.98 (s,2H),5.03-5.22 (m, 12 H), 4.85 (d, 4 H), 4.67 (d, 4 H), 4.61 (d, 4 H), 4.22 - 4.32 (m, 8 H), 3.72 (dq, 4 H), 2.06 (s, 12 H), 2.024 (s, 12 H), 2.019 s, 12 H), and 1.99 ppm (s, 12 H);
mass spec ((-)-FAB), m/z 1159 (M-H). Anal. Calcd. for Cs4H68N2o26 6H2~: C, 51.10; H, 6.35; N, 2.21. Found: C, 51.01; H, 6.09; N, 2.25.

Stee 2 Ri~henvl-4.4'-dicarboxvlic Acid Bis~3 -~-bis(~
~lucosvloxvmethvl)~hen~ llalTIide~

A solution of 575 mg (0.495 mmol) of biphenyl-4.~-dic;lrboxylic acid bis { [3,5-bis-(tetra-O-acetyl-~-D-glucosyloxymethyl)phenyl lamide ) and 8.91 mL (8.9 mmol) of 1 N NaOH in MeOH (15 mL) was stirred at 50~C for 4 h. The reaction mixture was quenched with 1 N HCI (7.9 mL) and purified by Sephadex G-10 chromatography (H20 elution). Removal of H20 in vacuo gave 144 mg (25% yield) of the title compound, mp 160~C (dec): IH (D2O; 400 MHz) ~ 7.38 (d, 4 H), 7.20 (s, 4H),7.16(d,4H),7.07 (s,2H),4.72(d,4H),4.47 (d,4H),4.38 (d,4H),3.88 (d,4 H), 3.69 (dd, 4 H), and 3.27 - 3.44 ppm (m, 16 H); 13C-NMR (D2O; 100 MHz) ~
166.3, 141.6, 137.6, 137.2, 131.7, 127.5, 126.3, 124.0, 120.1, 101.5, 75.7, 75.6, 73.0, 70.7, 69.5, and 60.6 ppm; mass spectrum ((-)-FAB) m/z 1159.4 (M - H), 997.3, 981.3, and 699.2. Anal. Calcd. for Cs4H68N2o26-6H2o: C, 51.10; H, 6.35; N, 2.21. Found:C,51.01;H,6.09;N,2.25.

CA 02204~31 1997-0~-0~

Step 3 Bil~her~vl-4.4'~dicarboxvlic Acid Bi~rr3.'.-bi~(tetra -O-sulfato-B-D-cosvlo~vnlethvl)Dhenvllalnide~ Hexade~odium Salt A solution of 92 mg (0.079 mmol) of biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis(,B-D-glucosyloxymethyl)phenyl]amide} and 450 mg (6.83 mmol) of sulfur trioxide trimethylamine complex in DMF (15 mL) was stirred at 70~C for 3 days.
The reaction mixture was cooled to ambient temperature, quenched with distilled H2O, and concentrated. The residue was purified by passing through a Sephadex G-10 column (H2O elution). Cation exchange was accomplished by passing an aqueous solution of the product through a column of Dowex 50 x 8 strongly acidic (Na form) resin. Removal of solvent and azeotropic drying with toluene gave 161mg (73%) of a shiny white solid, mp 172~C (dec): IH-NMR (D2O; 400 MHz) ~ 8.10 (dd, 4 H), 7.97 (dd, 4 H), 7.71 (s, 4 H), 7.40 (s, 2 H), 5.05 (d, 4 H), 5.01 (dd, 4 H), 4.89 (d, 4 H), 4.47 - 4.60 (m, 12 H), 4.28 (dt, 4 H), and 4.19 - 4.22 ppm (m, 4 H); 13C-NMR (D2O; 100 MHz) o 169.1, 143.1, 138.0, 137.3, 133.4, 128.2, 127.3, 125.0, 121.8, 99.3, 76.0, 75.7, 73.4, 72.5, 70.8, and 67.7 ppm. Anal. Calcd. for Cs4Hs2N2o74sl6Nl6 l6-H2o:C, 21.01; H, 2.75; N, 0.91; S, 16.65. Found: C, 20.93; H, 2.78; N, 0 77; S, 16.86.
Capillary electrophoresis shows purity in excess of 91 %.
FXAMPI.F lX

Step I
Biphenvl-4.4'-dicarboxvlic Acid Bis~. 5-bis(he~ta-O-ace~YI-~-D-~n~ltosvloxvmethyl)~henvllamide~

To a solution of 3,5-bis(hepta-O-acetyl-,~-D-maltosyloxymethyl)phenylamine (1.07 g, 0.769 mmol) and triethylamine (101 ~L, 0.769 mmol) in THF (20 mL) was added 4,4'-biphenyldicarboxylic acid dichloride (107 mg, 0.385 mmol). After stirring at room temperature for 3 h, the reaction mixture was quenched with MeOH, diluted with CH2Cl2, and washed with H2O. The reaction mixture was dried (MgSO4) and concentrated to give 1.10 g (96% yield) of the title compound as a colorless solid, mp 139 - 144~C: 1H-NMR (CDCI3, 400 MHz) o 8.66 (bs, 2 H), 8.03 (d, 4 H), 7.72 (d, 4H), 7.67 (s, 4 H), 6.93 (s, 2 H), 5.39 (d, 4 H), 5.32 (t, 4 H), 5.20 (t, 4 H), 5.02 (t, 4 H), 4.76 - 4.86 (m, 12 H), 4.57 - 4.66 (m, 12 H), 4.20 - 4.25 (m, 8 H), 3.93 - 4.04 (m, 12 H), 3.6 - 3.7 (m, 4 H), 2.09 (s, 12 H), 2.06 (s, 12 H), 1.99 (s, 12 H), 1.98 (s, 12 H), CA 0220453l lss7-05-05 WO 96/14323 pcT/uss5ll4686 1.97 (s, 12 H), 1.96 (s, 12 H), and 1.95 ppm (s, 12 H); IR (KBr)1745 em~l. Anal.Caled. for C134H164N2074: C, 53.89; H, 5.53; N, 0.94. Found: C, 53.49; H, 5.55; N, 0.94.
.

~itep 2 RiDhenvl.4.4'-diearboxylie Aeid Bi~rr3. 5-bis(,~
~n~ltosyloxymet't~ henyll~mide~

A solution of 973 mg (0.326 mmol) of biphenyl-4,4'-diearboxylie aeid bis{[3,5-bis(hepta-O-aeetyl-,B-D-maltosyloxymethyl)phenyl]amide} and 9.77 mL
(9.77 mmol) of 1 N NaOH in MeOH (15 mL) was stirred at 50~C for 4 h. The reaetion l~lixlure was eooled to room temperature, quenehed with 1 N HCI (9.12 mL), concentrated, and purified by Sephadex G-10 chromatography (H2O elution).
Removal of H2O in vacuo gave 494 mg (75% yield) of the title eompound, mp >200~C: IH-NMR (D2O, 400 MHz) o 7.54 (s, 4 H), 7.33 (s, 4 H), 7.23 - 7.27 (br, 4H), 7.15 (s, 2 H), 5.32 (s, 4 H), 4.51 - 4.20 (brd 4 H), 4.39 (d, 4 H), 3.50 - 3.90 (m, 36 H), and 3.30 - 3.45 ppm (m, 12 H); 13C-NMR (D20; 100 MHz) ~ 166.3, 141/7, 137.8, 137.3, 132.0, 127.7, 126.5, 124.1, 120.1, 101.3, 9g.9, 77.4, 75.9, 74.4, 72.8, 72.6, 71.6, 70.6, 69.1, 60.6, and 60.3 ppm. Anal. Calcd. for C7~Hlo8N2o46-8H2o:
C, 48.85; H, 6.31; N, 1.46. Found: C, 48.48; H, 6.28; N, 1.58.

Ste~ 3 BiDhenvl-4.4'-dicarboxylic Acid Bis~ bis(heDta-O-sulf~ ,B-D-ln~ltosvloxymethvl~vhenyllamide~ Oetaeosasodium Salt A solution of 315 mg (0.174 mmol) of biphenyl-4,4'-dicarboxylic acid bis([3,5-bis(~-D-maltosyloxymethyl)phenyl]amide} and 3.58 g, (25.7 mmol) of sulfur trioxide trimethylamine complex in DMF (15 mL) was stirred at 70~C for 3 days. The reaction mixture was cooled to ambient temperature, quenched with distilled H2O, and eoncentrated. The residue was purified by passing through a Sephadex G-10 column (H2O elution). Cation exchange was accomplished by passing an aqueous solution of the product through a column of Dowex 50 x 8 strongly aeidie (Na form) resin. Removal of solvent and azeotropie drying with toluene gave 609mg (75%) of an off-white solid, mp 178~C (dec): lH-NMR (D~O;
400 MHz) ~ 8.06 (d, 4 H), 7.94 (d, 4 H), 7.69 (s, 4 H), 7.36 (s, 2 H), 5.58 (d, 4 H), CA 02204~31 1997-0~-0~
WO 96tl4323 PCT/US95/14686 5.06 (d, 4 H), 5.02 (d, 4 H), 4.7 - 4.9 (m, 12 H), 4.58 - 4.61 (m, 8 H), 4.49 (dd, 4 H), 4.12 - 4.46 ppm (m, 28 H); 13C-NMR (D2O; 100 MHz) ~ 169.1, 143.1, 138.0, 137.3, 133.4, 128.2, 127.4, 124.9, 121.7, 99.3, 94.1, 77.1, 76.0, 74.8, 73.2, 73.1, 72.3, 71.8, 70.5, 69.9, 67.5, and 66.0 ppm; mass spectrum (ele~ y) (m-zNa)tz 1143.7 (m-4 Na)4-, 910.4 (m-5 Na)5-, 754.8 (m-6 Na6~), 643.7 (m - 7 Na7~), 560.4 (m- 8 Na)8-.
Calcd- for C78H80N2O130S28Na2g 28H2O: C, 18.12; H, 2.65; N, 0.54; S, 17.36 Found: C, 18.33; H, 2.73; N, 0.46; S, 17.72.

F~xAMpT~F~ 19 ~tep 1 3.3'-(N.N'-Ureido~bi~rN-r5-(he~ta-0-acetvl-13-D-cellobiosvloxvmethyl)-2-chloroDhenvllbe~7~mide~

To a solution of 1.10 g (1.42 mmol) of 5-(hepta-O-acetyl-,~-D-cellobiosyloxymethyl)-2-chlorophenylamine in THF (20 mL) containing 198 ~L
(1.42 mmol) of triethylamine was added 3-nitrobenzoyl chloride (316 mg, 1.70 mmol). After 3 h, the reaction mixture was quenched with MeO~ and diluted with CH2C12. The reaction mixture was washed with H2O, dried (MgSO4), filtered, and concentrated to 1.31 g (1.42 mmol) of crude product which was used directly in the next reaction. The crude material was dissolved in EtOAc and W;15 treated with 2.32 g (10.28 mmol) of SnCI2 H2O. After stirring at reflux for 5 h, Ihe reaction mixture was cooled to room temperature and was quenched with sat. NaHCO~ (300 mL). The reaction mixture was diluted with EtOAc, and filtered. Th~ org~nic phase was separated and the aqueous phase was extracted with CH2C12. The organic phases were combined, dried (MgSO4) and concentrated to give 1.18 g (93% yield) of crude product which was used directly in the next reaction. To the product in THF (20 mL) containing 108 ~L (7.98 mmol) of pyridine was added 66 mg (0.22 mmol) of triphosgene. The mixtu re was stirred at ambient temperature overnight. The reaction llli~LUlG was quenched with H20 and then stirred for another 30 min. The reaction mixture was filtered. The solid was collected and dissolved in CH2C12, dried (MgSO4), concentrated, and then triturated with Et2O to give 995 mg (82% yield) of the title compound, mp 173 - 178~C: IH-NMR (CDC13, 400 MHz) o 8.47 (s, 2 H), 8.41 (d,2H),7.45(d,2H),7.38(t,2H),7.33(d,2H),6.96(dd,2H),5.10-5.16(m, 4 H), 5.03 (t, 2 H), 4.90 - 4.96 (m, 4 H), 4.79 (d, 2 H), 4.59 (t, 2 H), 4.51 - 4.56 (m, 4 H), 4.30 (dd, 1 H), 4.14 (dd, 1 H), 3.99 (dd, 2 H), 3.92 (t, 2 H), 3.65 - 3.70 (dq, 2H), - 2.09 (s, 6 H), 2.04 (s, 6 H), 2.02 (s, 12 H), 1.987 (s, 6 H), 1.985 (s, 6 H), and 1.96 ppm (s, 6 H); 13C-NMR (CDCl3; 100 MHz) ~ 170.6, 170.49, 170.46, 169.83, 169.17, 169.14, 165.22, 152.47, 139.75, 136.78, 134.85, 134.43, 129.68, 129.13, 124.35, 123.08, 122.95, 121.18, 120.57, 117.99, 100.70, 98.92, 73.22, 72.64, 71.89, 71.74, 71.55, 71.49, 69.97, 67.90, 62.04, 61.53, 20.86, 20.72, 20.64, 20.58, 20.53, and 20.45 ppm; mass spectrum ((+)-FAB) m/z 1837.5 (M + Na). Anal. Calcd. for CglHg2N4O3gCl2-H2O: C, 53.03; H, 5.16; N, 3.05. Found: C, 52.74; H, 5.09; N, 3.15.

~2 3..~ fN.N'-Ureido~b;~N-r5-(~ cellobiosvloxvmethvl)-2-.~hlorol~henvllb~n7~mide~

A solution of 908 mg (0.50 mmol) of 3,3'-(N,N'-ureido)bis{N-[5-(hepta-O-acetyl-,B-cellobiosyloxymethyl)-2-chlorophenyl]benzamide } in MeOH (20 mL) containing 7.5 mL (7.5 mmol) of 1 N NaOH was stirred at 50~C under N2 for 4 h.
The reaction mixture was cooled to room temperature and was quenched with l N
HCl (7.0 mL, 7.0 mmol). The reaction mixture was sti~;red for 15 min and filtered.
The solid was collected and azeotropically dried with toluene to provide 566 mg (92% yield) of the title compound as an off-white solid, mp 183~C: partial ~ NMR(DMSO-d6, 400 MHz) o 10.06 (s, 2 H), 9.28 (s, 2 H), (s, I r{) ' .~ 7.7~ (d. I H), 7.60 (d, 2 H), 7.59 (s, 2 H), 7.50 - 7.55 (q, 4 H), 7.44 (d. H), 7.33 (dd, 2 ~i), 5.30 (s, 2 H), 5.25 (d, 2 H), 4.86 (d, 2 H), 4.70 (s, 2 H), 4.33 (d. ~ r-3). ~ ~. (d. ~ H), 3.75 -3.80 (dd, 2 H), 3.60 - 3.71 (m, 4 H), and 3.0 - 3.2 ppm (m. 8 is); ~ IR (DMSO-d6; 100 MHz) o 165.54, 152.70, 140.05, 137.74, 134.90, 13~.7~i. 129.'7, 128.95, 128.48, 127.36, 126.53, 121.58, 120.91, 117.80, 103.21, 101.99, 80.49, 76.79, 76.46, 75.01, 73.29, 73.19, 70.04, 68.82, 61.02, and 60.43 ppm; mass spec~,rum ((+)-FAB) m/z 1249.2 (M + Na). Anal. Calcd. for Cs3H64N4O2scl2 4H2O: C, 48.97; H, 5.58;
N, 4.31. Found: C, 49.25; H, 5.58; N, 4.28.

CA 02204~31 1997-0~-0~
WO 96/14323 PCT/US9~;/14686 te~! 3 (N.N'-Ureido)bi~(rN-r2-- hloro-5-(heDta-O-sulfato-,B-D-cellobiosvlo~...~ yl~Dhenvll~b~n7:-mide) Tetr~decasodium Salt S To a solution of 3,3'-(N,N'-ureido)bis{N-[5-(,1~-cellobiosyloxymethyl)-2-chlorophenyl]ben7~mide} (475 mg, 0.387 mmol) in DMF (25 mL) containing 3.77 g (27.08 mmol) of sulfur trioxide trimethylamine complex was stirred at 70~C for 3days. The reaction ll~i~lulG was cooled to room temperature, quenched with distilled H2O, and concentrated. The residue was purified by passing through a Sephadex G-10 column (H2O elution). Cation exchange was accomplished by passing an aqueous solution of the product through a column of Dowex 50 x 8 strongly acidic (Na form) resin. Removal of solvent and azeotropic drying with toluene gave 926 mg (0.349 mmol) of the title compound as an off-white solid, mp 168~C (dec): l3C-NMR (D2O;100 MHz) ~ 169.46, 155.29, 138.29, 136.85, 133.89, 133.06, 129.90, 129.63, 129.56, 128.43, 127.62, 124.74, 122.79, 119.59, 99.91, 99.14, 77.61, 77.32, 77.03, 74.28, 73.63, 73.34, 70.0, 67.60, and 66.60 ppm. Anal. Calcd. for C53H50N4O67S14Nal4-3Na2SO4-18H2O C, 19.08; H, 2.60; N, 1.68; S, 16.34.
Found: C, 19.01; H, 2.21; N, 1.74; S, 16.82. Capillary zone electrophoresis shows >90% purity.
FXAMPI,F. 20 ~teD I
RiDhenvl-4.4'-dicarboxylic Acid ~is~2-chloro~
cellobiosvloxvmethyl)~henYllamide~

To a solution of 5-(hepta-O-acetyl-,B-D-cellobiosyloxymethyl)-2-chlorophenylamine (995 mg, 1.28 mmol) and triethylamine (148 ~L, 1.28 mmol) in THF (25 mL) was added 4,4'-biphenyldicarboxylic acid dichloride (179 mg, 0.642 mmol). After stirring at room tc;nlpe-~lule for 2 h, the reaction mixture was quenched with MeOH, diluted with CH2C12, and washed with H2O. The reaction mixture was dried (MgSO4) and concenLI~Led to an oil which was triturated with ether to provide 1.09 g of crude product (99% yield). The material was dissolved in MeOH (20 mL) and treated with 9.5 mL of 1 N NaOH. After stirring at 50~C for 4 h, the reaction mixture was cooled to room le~ ule and treated with 8.9 mL (8.9 mmol) of 1 N
HCl. Collection of the solid provided 670 mg (91% yield) of the title compound, mp WO 96/14323 PCTtUS95/14686 , >200~C, after azeotropic drying with toluene: partial lH-NMR (DMSO-d6; 400 MHz)~;10.21(s,2H),8.13(d,4H),7.95(d,4H),7.61(d,2H),7.55(d,2H),7.35 (dd, 2 H), 5.29 (d, 2 H), 5.25 - 5.26 (br, 2 H), 4.87 (d, 2 H), 4.71 (s, 2 H), 4.34 (d, 2 H), 4.26 (d, 2 H), 3.78 (dd, 2 H), 3.15 (br d, 2 H), and 3.05 ppm (br t, 2 H); partial 13C-NMR (DMSO-d6; 100 MHz) ~ 165.0, 128.5, 127.0, 103.2, 102.0, 80.5, 76.8, 76.4, 75.0, 73.3, 73.2, 70.0, 68.8, 61.0, and 60.4 ppm.; mass spectrum (-FAB) m/z 1166.9, 1004.9, and 842.9. Anal. Calcd. for Cs2H62N2O24C12-5 H2O: C, 49.57; H, 5.76; N, 2.22. Found: C, 49.81; H, 5.36; N, 2.14.

~
henvl-4~4'-dicarboxvlic Acid Bi~rr2-chloro-~-(heDta-()-sulfato-,~-D-cellobiosyloxy~ thvl~l~henvllamide~ Tetradecasodi~lm Salt A solution of 531 mg (0.454 mmol) of biphenyl-4,4'-dicarboxylic ~cid bis~ [2-chloro-5-(13-D-cellobiosyloxymethyl)phenyl]amide ~ and 4.42 g (31.8 mmol) of sulfur trioxide trimethylamine complex in DMF (25 mL) was stirred at 70~C for 2 days.
The reaction mixture was concentrated and purified b~ Seph;ldex G-10 chromatography using H20 elution. Cation exchange ~ as effecled usin~ a column of Dowex 50 x 8 strongly acidic (Na form) with H2O elution Remo~al of water in vacuo and azeotropic drying with toluene gave 705 m~ (h()~ ) of tht~ e compound as a white solid, mp 165~C (dec): partial lH-NMR (D~SO-d" ~()() .'~l~lz) ~ X 10 (d, 4 H), 7.97 (d, 4 H), 7.66 (d, 2 H), 7.65 (d, 2 H), 7.5 (dd. ' ~ 3 ~ 'J (d. ' ~ 97 (d, 2 H), 4.58 (d, 2 H), and 4.00 - 4.06 ppm (m, 4 ~ D~() 1()() ~l~lz) 169.8, 143.5, 137.0, 133.2, 132.6, 130.04, 130.01, 1~; 7. 1'~ (). 1_7.~, 9~
99.2, 77.7, 77.4, 77.1, 74.3, 73.6, 73.4, 73.2, 67.7, and ~6.h PF"~ m:l~s spectrum (electrospray) (m-zNa)/z 496.7 (m - SNa)5-, 626.6 (m-4Na)4-, ~nd 843.2 (m-3Na)3-Anal. Calcd. for Cs2H48Cl2N2Nal4O66SI4-16 H20: C, 21.63; ~1, '.79; N, 0.97; S, 15.55. Found: C, 21.24; H, 2.31; N, 0.93; S, 12.25.

CA 02204~31 1997-0~-0~

F,X~MP~,~ 21 ~tep 1 Ri~h~nyl 4.4'-di~rboxylic Arid l~is~r2-(~-D-cellobiosyloxvmethvl)-4-l~hloro~henyll~mide~

To a solution of 861 mg (1.11 mmol) of 2-(hepta-O-acetyl-,l~-D-cellobiosyloxymethyl)-4-chlorophenylamine and triethylamine (155 ~L, 1.11 mmol) in THF (20 mL) was added 4,4'-biphenyldicarboxylic acid dichloride (155 mg, 0.555 mmol). After stirring at ambient temperature for 2 h, the reaction mixture was quenched with MeOH, diluted with CH2C12 and washed with H2O. The organic phase was dried (MgS04), filtered, and concentrated to give 975 mg of crude product.
This material was dissolved in MeOH (15 mL) and was treated with 8.3 mL (8.3 rnmol) of 1 N NaOH. After stirring for 4 h at ambient temperature, the reaction mixture was quenched with 7.8 mL of 1 N HCI and the solid was collected to provide 590 mg (91% yield) of the title compound as an off-white solid, mp >200~C: partial lH-NMR (DMSO-do; 400 MHz) o 9.95 (s, 2 H), 8.11 (d, 4 H), 7.93 (d, 4 H), 7.67 (d, 2 H), 7.62 (d, 2 H), 7.42 (dd, 2 H), 5.45 (d, 2 H), 4.91 (d, 2 H), 4.34 (d, 2 H), and 4.25 ppm (d, 2 H); partial 13C-NMR (DMSO-d6; 100 MHz) ~ 164.9, l42.~, 134.6, 134.5, 133.4, 128.5, 127.0, 103.2, 101.7, 80.4, 80.2, 76.8, 76.4, 75Ø 74.9, 73.~, 70.0, 66.3, 61.0, and 60.3 ppm; IR (KBr) 1650 cm-l; mass spectrum ~-FAB) ~tl ' 1 l67.3, 843.3, and 704.2. Anal. Calcd. for Cs2H62CI2N2O24-4 H~O: C, 50.~ h 5.~8; N, 2.26.
Found: C, 50.37; H, 5.38; N, 2.32.

~teD 2 Biphenyl-4,4'-dicarboxylic Acid Bis~14-chloro-~-(heDta-O-sulfato-~-D-cellobiosYIoxvmethvl)~henvllamide~ Tetrade(~ odium Salt A solution of 447 mg (0.382 mmol) of biphenyl-4,4'-dicarboxylic acid bis{[2-(~-D-cellobiosyloxymethyl)-4-chlorophenyl]amide} and 3.85 g (27.7 mmol) of sulfur trioxide trimethylamine complex in DMF (25 mL) was stirred at 70~C for 6 days.
The reaction mixture was quenched at room temperature with H2O, concentrated, and passed through a Sephadex G-10 column (H2O elution). Cation exchange was effected by passing an aqueous solution of the compound through a Dowex 50 x 8 strongly acidic (Na form) cation exchange column (H2O elution). Removal of solvent and azeotropic drying with toluene gave 666 mg (67% yield) of the title ~ cc,~ vund as an off-white solid, mp 172~C, which was ca. 70% pure as determined - by capillary elec~ophoresis: partial IH-NMR (D2O; 400 MHz) ~ 8.10 (d, 4 H), 7.99 (d, 4 H), 7.74 (s, 2 H), 7.46 - 7.53 (br, 4 H), 4.95 (d, 2 H), and 4.90 ppm (d, 2 H);
13C-NMR (D2O; 100 MHz) o 1~9.9, 143.4, 134.5, 133.4, 132.4, 129.7, 129.0, 128.7,128.3, 127.6, 99.9, 98.8, 77.6, 77.4, 77.0, 74.3, 73.5, 73.4, 73.0, 67.6, 66.9, and 66.3 ppm. Anal. Calcd. for Cs2H48cl2N2Nal4o66sl4-l4 H20: C, 21.92; H, 2.69; N, 0.98; S, 15.75. Found: C, 21.98; H, 2.63; N, 1.08; S, 16.05.

FX~MPI,F 22 steD 1 N.N'-R~i~r~ bi~(,B-D-cellobiosvloxvmethvl~vhenvll~uccinamide To a solution of 840 mg (0.604 mmol) 3, 5-bis(hepta-O-acetyl-~-D-cellobiosyloxymethyl)phenylamine and 86.4 ~L (0.31 mmol) of triethylamine in THF(20 mL) was added succinyl chloride (34.2 IlL, 0.31 mmol). After stirring at room temperature for 1 h, incomplete reaction was observed. Another 10 ,uL (0.09 mmol) of succinyl chloride was added. The reaction was stirred for another 15 min, quenched with MeOH, diluted with CH2C12, washed with H2O, dried (MgSO4), filtered, and concentrated. Trituration with Et2O gave a green solid which was semi-purified by flash chromatography (EtOAc) to give a yellow solid (633 mg, 75%
yield). The compound was dissolved in MeOH and was treated with 6.6 mL of I N
NaOH (6.6 mmol). After stirring at 50~C for 4 h, the reaction rnixture was cooled to room temperature and to it was added strongly acidic Amberlite resin until neutral pH
was obtained. After stirring for 5 min, the reaction mixture was filtered and concentrated to a yellow solid. Purification by reverse phase chromatography (RP-60 silica gel) using elution of MeOH: H2O (3: 7) and then by Sephadex G-10 chromatography (H2O elution) gave 278 mg (94%) of the title compound as a yellowsolid: partial IH-NMR ~D2O; 400 MHz) ~ 7.48 (s, 4 H), 7.31 (s, 2 H), 4.91 (d, 4 H), 4.71 (d, 4 H), 4.52 (t, 8 H), 3.82 (dd, 4 H), 3.74 (dd, 4 H), 3.43 (d, 4 H), and 2.81 ppm (s, 4 H); l3C-NMR (D2O; 100 MHz) ~ 173.3, 137.9, 137.3, 124.9, 120.9, 102.4, 101.0, 78.5, 75.8, 75.3, 74.6, 74.1, 73.0, 72.7, 70.7, 69.3, 60.4, 59.9, and 31.4 ppm.

CA 02204~31 1997-0~-0~

N.N'-13i~r~ ~-bi~(he~ta-O-s--lf~to-~-n-cellobiosvloxymethvl)~h~nvll~uccinamide O-~t~cos~odium S~lt S A solution of 278 mg (0.208 mmol) of N,N'-bis[3,5-bis(,B-D-cellobiosyl-oxyrriethyl)phenyl]succin:~mide and sulfur trioxide trimethylamine complex (4.23 g, 29.0 mmol) in DMF (20 mL) was stirred at 70~C for 3 days. The reaction mixture was quenched at room temperature with H2O and then concentrated. Purification bySephadex G-10 chromatography (H2O elution) and then cation exchange using Dowex 50 x 8 strongly acidic (Na form) resin gave 606 mg (64% yield) of the title compound as a tan solid, mp 168~C (dec): partial lH-NMR (D2O; 400 MHz) o 7.58 (s, 4 H), 7.36 (s, 2 H), 4.00 - 5.00 (m, 64 H), and 2.87 ppm (s, 4 H); 13C-NMR (D2O;
100 MHz) ~ 173.3, 138.0, 137.1, 124.4, 99.9, 99.7, 77.6, 77.4, 77.0, 74.3, 73.4, 72.9, 70.9, 67.6, 66.2, and 37.1 ppm; mass spectrum (electrospray) (m-zNa)lz 734.1 (m -6Na)6-, 885.5 (m-SNa)5-, and 1112.7 (m-4Na)4- Anal. Calcd. for C68H76N2Ol30S28Na28-38 H2O: C, 15.62; H, 2.93; N, 0.54; S, 17.18. Found: C, 15.24; H, 2.43; N, 0.75; S, 17.67.

FXAI~PT F. 23 SteD 1 N~N'-Bi~3 ~-bis(~-D-cellobiosYloxymethyl)DhenvlltereDhthalamide To a solution of 3, 5-bis(hepta-O-acetyl-~-D-cellobiosvloxvmethyl)-phenylamine (823 mg, 0.592 mmol) and triethylamine (82.5 IlL, 0.59~ mmol) in THF(15 mL) was added terephthaloyl chloride (60.1 mg, 0.296 mmol). After stirring at ambient temperature for 2 h, the reaction mixture was quenched with MeOH, diluted with CH2CI2 and washed with H2O. The organic phase was dried (MgSO~), filtered, concentrated, and triturated with Et2O to provide 845 mg (98%) of crude product.This was dissolved in MeOH (20 mL) and treated with 8.7 mL (8.7 mmol) of 1 N
NaOH. After stirring at 50~C for 3 h, the reaction mixture was cooled to ambienttemperature and 8.1 mL (8.1 mmol) of 1 N HCI was added. The solid was collected and dried in vacuo to provide 295 mg (59% yield) of the title compound as a white solid, mp 185~C (dec): partial IH-NMR (DMSO-d6 with 5 drops D2O; 400 MHz) 8.01 (s, 4 H), 7.64 (s, 4 H), 7.18 (s, 2 H), 4.81 (d, 2 H), 4.56 (d, 2 H), 4.33 (d, 2 H), 4.26 (d, 2 H), and 3.77 ppm (d, 2 H); 13C-NMR (DMSO-d6; 100 MHz) o 164.7, 138.7, 138.2, 137.3, 127.7, 122.9, 119.3, 103.2, 101.9, 80.5, 76.8, 76.4, 75.1, 75.0, ~ 73.3, 73.2, 70.0, 69.9, 61.0, and 60.4 ppm; IR (KBr) 1650 cm~l. Anal. Calcd. for C72Hlo4N6O46-12 H20: C, 44.35; H, 6.62; N, 1.44. Found: C, 44.05; N, 6.30; N, 1.41.

S

N~ ni~r3--~-bi~fhevta-o-sulfato-~
(~llobiosylo~ P~ h~-lylltereDhth~l~mide Octacos~odium Salt A solution of 178 mg (0.103 mmol) of N,N'-bis[3,5-bis(,B-D-cellobiosyloxymethyl)phenyl]terephth~l,.mid~ and sulfur trioxide trimethylamine complex (2.0 g, 14.4 mmol) in DMF (20 mL) was stirred at 70~C for 3 days. The reaction mixture was cooled, quenched with water, and concentrated. The residue was purified by Sephadex G-10 chromatography (H20 elution). Cation exchange was 15 effected using a column of Dowex 50 x 8 strongly acidic (Na fonn) resin to afford 402 mg (85% yield) of the title compound as an off-white solid, mp 163~C (dec):
partial IH-NMR (D2O; 400 MHz) o 8.09 (s, 4 H), 7.71 (s, 4 H), 7.43 (s, 2 H), 5.03 (d, 2 H), and 5.00 ppm (d, 2 H); 13C-NMR (DMSO-d6; 100 MHz) ~ 168.7, 138.1, 137.2, 137.1, 127.8, 125.0, 121.7, 99.9, 99.7, 77.6, 77.4, 77.0, 74.3, 73.4, 7~.9, 70.9, 67.6, 20 and 66.3 ppm; mass spectrum (electrospray) (m-zNa)/z 632.8 (m - 7Na)7~, 742.1 (m-6Na)6~, and 895.2 (m-SNa)5-, and 1124.7 tm- 4)4- An;ll. Calcd. for C72H76N2Na28Ol30S28-28 H2O: C, 16.97; H, 2.61; N, 0.55, S, 17.62. Found: C, 16.77; N, 2.41; N, 0.59; S, 17.42.

FX~MP~,F 24 Step 1 BiDhenvl-4.4'--1iculfonic Acid Bi~rr5-(,B-D-cellobiosvloxvmethYI)-2-methvl~henyllamide~
To 970 mg (1.28 mmol) of 5-(hepta-O-acetyl-,B-D-cellobiosyloxymethyl)-2-methylphenylamine in CH2Cl2 (20 mL) cont,.ining 1.0 mL (12.4 mmol) of pyridine ~ was added 225 mg (0.641 mmol) of biphenyl-4,4'-disulfonyl chloride. After stirring at room temperature for 90 min, the reaction mixture was quenched with sat.
35 NaHCO3 and extracted into EtOAc. The bright yellow organic phase was washed CA 02204~31 1997-0~-0~

with pH 7 buffer, dried (MgSO4), and flash chromatographed (EtOAc / Et2O /
CH2C12, 1: 1: 1) to give 504 mg (58% yield) of an orange powder. This material (a total of 675 mg (0.378 mmol) from two separate runs) was dissolved in MeOH (10 mL) and THF (10 mL) and was treated with 6 rnL of 1 N NaOH. After stirring at room IC~ 1dlU1C for 12 h, the reaction mixture was quenched with 6 mL of 1 N HCl, and was concentrated to ca. 6 - 8 mL. The resulting precipitate was collected and was washed with 15 mL of H2O and then Et2O. Azeotropic drying with toluene gave 335 mg (74% yield) of a pale yellow powder, mp 230~C (dec): IH-NMR (DMSO, 400 MHz)~9.69(s,2H),7.91 (d,4H),7.75(d,4H),7.16(d,2H),7.11 (s,2H),7.10(d, 2H),4.71 (d,2H),4.46(d,2H),4.24-4.28,3.77(d,2H),3.70(d,2H),3.58-3.64 (m, 2 H), 2.97 - 3.43 (m, 18 H), and 1.91 ppm (s, 6 H); 13C-NMR (DMSO, 100 MHz) o 142.2, 140.5, 136.1, 134.4, 132.9, 130.5, 127.8, 127.2, 126.0, 103.2, 101.6, 80.5, 76.7, 76.4, 75.1, 74.9, 73.2, 73.1, 70.0, 69.1, 61.0, 60.4, and 17.2 ppm; mass spectrum ((-)-FAB), m/z 1199.3, 750.2, 514.2. Anal. Calcd. for Cs2H68N2o26s2~H2o: C, 51.23; H, 5.79; N, 2.30. Found: C, 51.04; H, 5.64; N, 2.29.

~teD 2 E~iDhenvl-4.4'~ ulfonic Acid Bis~r2-methvl-5-(heDta-O-sulfato-,B-I)-cellobiosvloxy~n~thyl)~henvllamide~ Tetradeca~odium Salt To 214 mg (0.178 mmol) of biphenyl-4,4'-disulfonic acid bis{[5-(~-D-cellobiosyloxymethyl)-2-methylphenyl]amide) in DMF (10 mL) was added 1.8 g (12.9 mmol) of sulfur trioxide trimethylamine complex. After stilTing for 3 days at 70~C, the reaction mixture was cooled to room temperature, quenched with 10 mL of water, and stirred for 20 min. This was concentrated in vacuo and purified by gel filtration (Sephadex G-10) to give 419 mg of pale tan flakes. The compound was dissolved in a minim~31 amount of water and passed through an ion exchange column (Dowex 50 x 8 strongly acidic (Na-form)) to give 350 mg of pale tan flakes afterazeotropic drying with toluene: 1H-NMR (D2O, 400 MHz) o 7.89 (d, 4 H), 7.81 (d, 4 H), 7.39 (d, 2 H), 7.32 (s, 2 H), 7.25 (d, 2 H), 4.94 (d, 2 H), 4.65 - 4.87 (m, 8 H), 4.57 (dd, 2 H), 4.18 - 4.51 (m, 16 H), 3.98 - 4.05 (m, 4 H), and 1.90 ppm (s, 6 H); 13C-NMR (D20, 100 MHz) ~ 143.8, 138.0, 135.4, 134.9, 131.1, 128.1, 128.0, 127.8, 127.3, 99.9, 98.7, 77.6, 77.3, 77.2, 76.9, 74.3, 73.7, 73.3, 72.93, 70.3, 67.6, 66.6., and 16.4 ppm. Anal. Calcd. for Cs2Hs4N2Nal4O6gS16-18 H2O: C, 21.14; H, 3.05; N, 0.95. Found: C, 21.17; H, 2.71; N, 0.96.

Wo 96114323 Pcr/uS9S/14686 F.XAl~IPI .~ 25 steD 1 N.N'-Bi~r2-methvl-5-(B-l)-~ coDyranosyl~xv".elhvl)Dhenvllsuccinamide To a solution of 879 mg (1.88 mmol) of 5-(tetra-O-acetyl-~-D-glucopyranosyloxymethyl)-2-methylphenylamine and 262 ~L (1.88 mmol) of triethylamine in THF (10 mL) was added succinyl chloride (104 ~LL, 0.94 mmol).
10 After stirring at room temperature for 3 h, the reaction mixture was quenched with MeOH (20 mL), diluted with CH2CI2, washed with H2O, dried (MgSO4), and concentrated. Crystallization from Et2O gave 741 mg (78%) of a green solid whichwas used directly in the next reaction. To a solution of 360 mg (0.354 mmol) of the crude product in MeOH (7.5 mL) was added 1 N NaOH (3.54 mL, 3.54 mmol). After 15 stirring at 50~C for 90 min, the reaction mixture was quenched at room temperature with 1 N HCl (2.85 mL, 2.50 mmol). After stirring for 1 h at room temperature, the solid was collected to provide 229 mg of the title compound: partial IH-NMR
(DMSO-d6,300MHz)~9.41 (s,2H),7.36(s,2H),7.17 (d,2H),7.12 (d,2H),4.78 (d,2H),4.50(d,2H),4.22(d,2H),3.69(d,2H),2.67(s,4H),and2.18ppm(s,6 20 H).

Stev 2 N.N'-Bi~r2-methyl-~-(2 ~.4.6-tetra-O-sulfat(>~
~luco~vranosvloxvmethvl~Dhenyll~uccinamide Octasodium Salt A solution of 229 mg (0.337 mmol) of N,N'-bis[2-methyl-5-(,B-D-glucopyranosyloxymethyl)phenyl]succinamide and sulfur trioxide trimethylamine complex (2.11 g, 15.2 mmol) in DMF (30 mL) was stirred at 50~C overnight. The reaction mixture was quenched with H2O (10 mL) and concentrated to an oily solid.
30 The solid was removed and rinsed with a very small amount of H2O. The combined - filtrates were concentrated and partially purified by repeated Sephadex G-10 chromatogrphy (H2O elution) and then by dialysis using a 500 MW cut off bag. A
small amount of trimethylammonium sulfate was still seen by NMR. Cation ~ exchange was effected with a column of Dowex 50 x 8 strongly acidic (Na form) 35 resin (H2O eluton) to provide 249 mg (49% yield) of the title compound as an off-white solid, mp >200~C: partial lH-NMR (D2O, 400 MHz) ~ 7.39 (d, 2 H), 7.37 (d, CA 02204~31 1997-0~-0~

2 H), and 4.55 ppm (t, 2 H); l3C-NMR (D2O, 100 MHz) ~ 173.9, 134.82, 134.80, 134.3, 130.8, 127.6, 126.7, 98.8, 7~.9, 75.5, 73.4, 72.4, 70.5, 67.8, 31.0, and 16.7 ppm; mass ~.lJC~ ll ((-) FAB) mz 1472.6 (M-Na)-, 1370.7 (m-Na-NaS03+H)-, 765.9, and 564.8. Anal. Calcd. for C32H36N2NagO3gSg-2 Na2SO4-6 H2O: C, 20.34; H, 2.54; N, 1.48. Found: C, 20.19; H, 2.22; N, 1.39.

li.X~MP~,F, 26 Step 1 N-r5-(HeDt~-O-acetyl-~-D-m~ltosyloxvmethvl)-2-methylDhenY11-3-n;trobenzamide To a stirred mixture of 5-(hepta-O-acetyl-,B-maltosyloxymethyl)-2-methylphenylamine (1.90 g, 2.52 mmol) and pyridine (0.22 g, 2.77 mmol) in CH2Cl2(10 mL) was added 3-nitrobenzoyl chloride (0.51 g, 2.77 mmol). After 18 h, the mixture was diluted with EtOAc, washed with sat. NaHCO3, H2O, and b;ine, dried (MgSO4), and concentrated. Purification by flash chromatography (1: 1 EtOAc /
hexane) gave 2.00 g (88%) of the title compound as a white foam. lH-NMR (DMSO-d6, 400 MHz) ~ 10.28 (s, 1 H), 8.80 (s, 1 H), 8.45 (dd, 1 H), 8 42 (d, I H), 7.84 (m, I
H), 7.28 (d, 1 H), 7.25 (s, 1 H), 7.10 (d, 1 H), 5.30 (m, 2 H), 5.20 (t, I H), 5.05 (t, I
H), 4.85 (d, 1 H), 4.80 (m, 1 H), 4.75 (m, 2 H), 4.56 (d, I H), 4.40 (dd, l H), 4.19 (m, 2 H), 4.0 (m, 4 H), 2.20 (s, 3 H), and 2.00 ppm (m, 21 H). An~l C~lcd. for C4lH4gN2O2l: C, 54.42; H, 5.35; N, 3.10. Found: C, 54.30; H, 5.'7; N, 3.10.

C.tep 2 N-r5-(B-D-M~ltosyloxnnethyl)-2-methYl~henvll-3-nitroben~,~mide To a stirred solution of N-[S-(hepta-O-acetyl-,B-D-maltosyloxy)-2-methylphenyl]-3-nitrobenzamide (2.00 g, 2.21 mmol) in MeOH (20 mL) was added 25 wt% NaOMe in MeOH (5.10 mL, 22.10 mmol). After 3 h, 10 g of Amberlite IR-120 (H+) ion exchange resin was added. The mixture was filtered and the filtrate was concentrated to give 1.30 g (96%) of the title compound as a white solid, mp 166-168~C: lH-NMR (DMSO-d6, 400 MHz) ~ 10.37 (s, 1 H), 8.80 (t, 1 H?, 8.43 (m, 2 H), 7.85 (m, 1 H), 7.35 (s, 1 H), 7.25 (m, 2 H), 5.00 (d, 1 H), 4.85 (d, 1 H), 4.57 (d, 1 WO 96/14323 Pcr/uss5114686 H), 4.30 (d, 1 H), 3.75 (m, 2 H), 3.60 (m, 2 H), 3.40 (m, 6 H), 3.10 (m, 2 H), and 2.21 - ppm (s, 3 H); mass ~e~ mle 609 (M-H).

S N-r5-fHeDt~-O-slllf~to-B-n-nl~ltosvloxy)-2-methvlDhenvll-3-nitroben~amide HeD~codil-m S~lt A mixture of N-[5-(~-D-maltosyloxymethyl)-2-methylphenyl]-3-nitroben7~micle (1.27 g, 2.08 mmol) and sulfur trioxide trimethylamine complex 10 (10.13 g, 72.79 mmol) in DMF (10 mL) was heated at 70 - 75~C for 4 days. The tUl~; was cooled, MeOH was added, and the mixture was filtered. The filtrate wasconcentrated, taken up in MeOH, and passed through a Sephadex LH-20-100 column (1: 1 MeOH/CHC13; 0.1% Et3N). The fractions were concentrated and passed through a Sephadex G-10 gel filtration column (H2O) to give a brown oil. Further15 puri~lcation by reverse phase chromatography (C- 18; H2O), flash chromatography (5:
0.1: 0.5 CH3CN/H2O/Et3N), and ion exchange on Sephadex SP C-25 (Na+) gave 1.10 g (40%) of the title compound as a white solid, mp 168 - 170~C: IH-NMR
tD2O, 400 MHz) ~ 8.82 (t, 1 H), 8.52 (m, 1 H), 8.38 (m, I H), 7.86 (m, 1 H), 7.45, (s, 2 H), 5.57 (d, 1 H), 4.99 (d, 1 H), 4.95 (d, I H), 4.80 (m, 2 H), 4.6~ (~, I H), 4.55 (s, 1 20 H), 4.46 (m, 1 H), 4.30 (m, 4 H), 4.18 (m, 1 H), 4.05 (m, I H), and '.~ I ppm (s, 3 H);
mass spectrum m/z 1323. Anal. Calcd. for C27H 7N~N;~7O3~sS7-~}~O: C, '~.5~; H, 2.41; N, 2.03. Found: C, 22.70; H, 2.69; N, 1.72.

~teD 4 3-rN-r5-(Hel~ta-O-sulfato-,B-D-maltosvlo~ 2-met h ~ 1 Dhen ~ I I phen ~ 1~ mi ne HeDt~odium Salt A solution of N-[5-(hepta-O-sulfato-~-D-maltosyloxy)-2-methylphenyl]-3-nitroben7~micle heptasodium salt (0.60 g, 0.45 mmol) in MeOH, H20, and pyridine 30 was hydrogenated at atmospheric pressure over 10% Pd/C for 6 h. The catalyst was removed by filtration, and the filtrate was concentrated and passed through a Sephadex SP-C25 (Na+) ion exchange column to give 0.40 g (68%) of the title ~ compound as a white solid, mp 232 - 234~C (dec): IH-NMR (I~2O, 300 MHz) ~ 7.20 (m, 6 H), 6.93 (m, 1 H), 5.42 (d, 1 H), 4.75 (m, 2 H), 4.60 (m, 3 H), 4.48 (m, 2 H), CA 02204~31 1997-0~-0~

4.40 (dd, 2 H), 4.30 (t, 1 H), 4.15 (m, 2 H), 4.05 (m, 3 H), 3.95 (m, 2 H), and 2.15 ppm (s, 3 H).

Ste~ 5 3 ~'-rN.N'-Ureidolbi~rrN-r2-me~h~yl-5-(he~ta-0-sulfato-,~
~n~losvl~ thyl)Dh~nvll~b-on7~mideTetrade~ odium Salt To a stirred mixture of 3-[N-[5-(hepta-O-sulfato-,13-D-maltosyloxy)-2-methylphenyl]]phenylamine heptasodium salt (440 mg, 0.34 mmol) and pyridine (5 mL) was added triphosgene (20 mg, 0.057 mmol). Stirring was continued at room temperature for 18 h. The mixture was concentrated, passed through a Sephadex G-10 gel filtration column, and a Sephadex SP-C25 (Na+) ion exchange column. The fractions were concentrated and triturated with MeOH to give 350 mg (79%) of thetitle compound as an off-white solid, mp 220~C (dec): IH-NMR (D20, 400 MHz) ~
7.61(d,2H),7.59(s,2H),7.50(m,2H),7.43(m,8H),7.29(d,2H),5.54(d,2H), 4.95 (t, 4 H), 4.80 (m, 2 H), 4.61 (t, 2 H), 4.52 (dd, 2 H), 4.44 (t, 4 H), 4.30 (m, 12 H), 4.20 (m, 2 H), 3.98 (t, 2 H), and 2.28 ppm (s, 6 H); 13C-NMR (D2O, 100 MHz) o 169.8, 135.3, 135.1, 134.8, 134.4, 131.1, 130.0, 128.1, 126.9, 12~.3, 121.7, 117.6, 94.7, 77.6, 76.6, 75.0, 73.7, 73.3, 73.1, 72.5, 69.8, 67.8, 66.0, and 16.8 ppm; mass spectrum m/z 1797. Anal. Calcd. for CssHs6N4Nal4o67s~ 8H2o: C, 22 47; H, 3.15; N, 1.91. Found: C, 22.11; H, 2.81; N, 1.59.

FXAMPI.F 27 Step 1 N.N'-Bi~3-r2-methvl-5-(he~ta-0-acetvl-~-D-m~ltosYloxvrnethyl)DhenvlcarbamoyllDhenvl~isoDhth~lamide To a stirred mixture of 3-amino-[5-(hepta-O-acetyl-~-D-maltosyloxymethyl)-2-methylphenyl]benzamide (1.0 g, 1.143 mmol) and triethylamine (0.12 g, 1.143 mmol) in CH2C12 (5 mL) was added isophthaloyl dichloride (0.12 g, 0.572 mmol).
After 18 h, water was added and the layers were separated. The organic phase waswashed with saturated aqueous NaHCO3 and brine, dried (MgSO4), and concentrated.~ Purification by flash ch~o",atography gave 0.57 g (53%) of product as a white solid, mp 178 - 180 ~C: lH-NMR (DMSO-d6, 300 MHz) ~ 10.60 (s, 2 H), 9.90 (s, 2 H), ~ 8.60 (s, 1 H), 8.37 (s, 2 H), 8.21 (dd, J = 1.5, 7.7 Hz, 2 H), 8.05 (d, J = 7.7 Hz, 2 H), ~ 7.75(m,3H),7.52(t,J=7.9Hz,2H),7.25(m,4H),7.10td,J=7.8Hz,2H),5.30 (m,SH),5.03(m,3H),4.80(m,4H),4.70(m,4H),4.50(d,J= 12.5Hz,2H),4.40 (d, J = 12.5 Hz, 2 H), 4.20 (m, 4 H), 3.95 (m, 8 H), 2.20 (s, 6 H), and 1.90 - 2.00 ppm (m, 42 H)-Ste~l 2 N.N'-Ri~3-r2~ thvl-5-(,B-D-maltosylo~ymethyl3~henvlcarbamovllphenvl~i~ophthalamide To a warmed solution of N,N'-bis (3-[2-methyl-5-(hepta-O-acetyl-,B-D-maltosyloxymethyl)phenylcarbamoyl]phenyl}isophth~l~mide (0.572 g, 0.304 mmol) in MeOH (10 mL) was added 25 wt% NaOMe in MeOH (0.5 mL, 2.13 mmol). After 2 h, Amberlite IR-120 (H+) resin was added. The mixture was filtered and concentrated to give 0.300 g (77%) of product as a white solid, mp 232-234~C: IH-NMR (DMSO-d6, 300 MHz) ~ 10.65 (s, 2 H), 9.90 (s, 2 H) 8.60 (s, 1 H), 8.35 (s, 2H),8.21 (dd,J= 1.5,7.7Hz,2H),8.05(d,J=7.7Hz,2H),7.70(m,2H),7.50(t,J
= 7.9 Hz, 2 H), 7.35 (s, 2 H), 7.20 (m, 5 H), 5.01 (d, J = 3.9 Hz, 2 H), 4.80 (d, J = 12.5 Hz,2H),4.50(d,J= 12.5Hz,2H),4.30(d,J=7.9Hz,2H),3.20- 3.75(m,26H), 3.10 (m, 4 H), 2.20 (s, 6 H).
Stev ?S
N.N'~ r3-r2-methvl-5-(he~ta-0-sulfato-,B-n-maltos~ lox~-méthvl)phenylcarbamoYIlDhenYl~iso~hthalamide Tetradecasodillm Salt A mixture of N,N'-bis{3-~2-methyl-5-(,B-D-maltosyloxymethyl)-phenyl-carbamoyl]phenyl}isophth~l~mic~e (0.30 g, 0.232 mmol) and sulfur trioxide pyridine complex (2.60 g, 16.267 mmol) in DMF (10 mL) was stirred at room temperature for2 days. The mixture was concentrated, purified on a Sephadex LH-20-100 column (0.5% Et3N/DMF), and converted to a sodium salt with Sephadex SP-C-25 ion exchange resin to give 0.200 g (31%) of product as an off white solid, mp 222~C
(dec): lH-NMR (D2O, 400 MHz) ~ 8.45 (s, 1 H), 8.35 (brs, 2 H), 8.10 (brs, 2 H), 7.95 (d, J = 7.9 Hz, 2 H), 7.85 (d, 7.9 Hz, 2 H), 7.75 (brs, 2 H), 7.57 (brs, 2 H), 7.35 (brs, 5 H), 4.75 (m, 4 H), 4.45 (m, 4 H), 4.30 (m, 4 H), 4.10 (m, 4 H), 3.80 (m, 4 H), 3.57 (m, 4 H), 3.28 (m, 8 H), and 2.20 ppm (s, 6 H); 13C-NMR (D2O, 100 MHz) 160, 137, 134, 130, 128, 120, 102, 96, 75, 74, 72, 62, 56, 49, 18 ppm.

l t~ t ~ "15 ~, i

Claims (25)

We claim:

1. A compound of formula I

I

wherein each of R1, R2, R3, and R4 are, independently, H, SO3M, or a sugar group having the structure:

;

and each oligosaccharide group of the structure contains from 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is alkyl having from 1 to 12 carbon atoms, , , , or ;

and X is as defined above;
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 of the structure contains 1 or 2 sugar groups;

M is lithium, sodium, potassium or ammonium;
n is 1 or 2;
X is hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is alkyl having from 1 to 12 carbon atoms, , , , or ;

or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 2 which is dodecanedioic acid bis{[2-methyl-5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]amide}
tetradecasodium salt or a pharmaceutically acceptable salt thereof.

4. A compound according to claim 2 which is 2 N,N'-bis[5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)-2-methylphenyl]-terephthalamide tetradecasodium salt or a pharmaceutically acceptable salt thereof.

5. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis{[5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)-2-methylphenyl]amide}
tetradecasodium salt or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 2 which is N,N'-bis[5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)-2-methylphenyl]isophthalamide tetradecasodium salt; or a pharmaceutically acceptable salt thereof.

7. A compound according to claim 2 which is decanedioic acid bis{[3,5-bis(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]amide octacosasodium salt or a pharmaceutically acceptable salt thereof.

8. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]amide}
octacosasodium salt or a pharmaceutically acceptable salt thereof.

9. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis-(hepta-O-sulfato-.beta.-D-lactosyloxymethyl)phenyl]amide}
octacosasodium salt or a pharmaceutically acceptable salt thereof.

10. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis {[3,5-(bis-(hepta-O-sulfato-.beta.-D-maltosyloxymethyl)phenyl]amide}
octacosasodium salt or a pharmaceutically acceptable salt thereof.

11. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis{[3,5-bis-(tetra-O-sulfato-.beta.-D-glucosyloxymethyl)phenyl]amide}
hexadecasodium salt or a pharmaceutically acceptable salt thereof.

12. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis{[2-chloro-5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]amide}
tetradecasodium salt or a pharmaceutically acceptable salt thereof.

13. A compound according to claim 2 which is biphenyl-4,4'-dicarboxylic acid bis{[4-chloro-2-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]amide}
tetradecasodium salt or a pharmaceutically acceptable salt thereof.

14. A compound according to claim 2 which is N,N'-bis[3,5-bis(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]succinamide octacosasodium salt or a pharmaceutically acceptable salt thereof.

15. A compound according to claim 2 which is N,N'-bis[3,5-bis(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]terephthalamide octacosasodium salt or a pharmaceutically acceptable salt thereof.

16. A compound according to claim 2 which is biphenyl-4,4'-disulfonic acid bis{[2-methyl-5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]amide}
tetradecasodium salt or a pharmaceutically acceptable salt thereof.

17. A compound according to claim 2 which is N,N'-bis[2-methyl-5-(2,3,4,6-tetra-O-sulfato-.beta.-D-glucopyranosyloxymethyl)phenyl]succinamide octasodium salt or a pharmaceutically acceptable salt thereof.

18. A compound according to claim 2 which is 3,3'-[1,3-ureido]-bis{N-[2-methyl-5-(hepta-O-sulfato-.beta.-D-maltosyloxymethyl)phenyl]}benzamide tetradecasodium salt or a pharmaceutically acceptable salt thereof.

19. A compound according to claim 2 which is 3,3'-(N,N'-ureido)bis-({N-[2-chloro-5-(hepta-O-sulfato-.beta.-D-cellobiosyloxymethyl)phenyl]} benzamide) tetradecasodium salt or a pharmaceutically acceptable salt thereof.

20. A compound according to claim 2 which is N,N'-bis{3-[2-methyl-5-(hepta-O-sulfato-.beta.-D-maltosyloxymethyl)phenylcarbamoyl]phenyl} isophthalamide tetradecasodium salt or a pharmaceutically acceptable salt thereof.

21. 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 a sugar group having the structure:

;

and each oligosaccharide group of the structure contains from 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
is a hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is alkyl having from 1 to 12 carbon atoms, , , , or ;

and X is as defined above;
or a pharmaceutically acceptable salt thereof.

22. The use of Claim 21 wherein the disease or condition which is characterized by excessive smooth muscle proliferation is restenosis

23. 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 a sugar group having the structure:

;

and each oligosaccharide group of the structure contains from 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is alkyl having from 1 to 12 carbon atoms, , , , or ;

and X is as defined above;
or a pharmaceutically acceptable salt thereof.

24. 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

wherein each of Rl, R2, R3, and R4 are, independently, H, S03M, or a sugar group having the structure:

and each oligosaccharide group of the structure contains from 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium.
n is 1 or 2;
X is a hydrogen, halogen, lower alkyl having I to t) c;3rh)n ;310rll~. or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is alkyl having from 1 to 12 carbon atoms, or and X is as defined above;
or a pharmaceutically acceptable salt thereof.

25. 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 a sugar group having the structure:

;

and each oligosaccharide group of the structure contains from 1 to 3 sugar groups;
M is lithium, sodium, potassium, or ammonium;
n is 1 or 2;
X is a hydrogen, halogen, lower alkyl having 1 to 6 carbon atoms, or lower alkoxy having 1 to 6 carbon atoms;
Y is carbonyl or sulfonyl;
Z is alkyl having from 1 to 12 carbon atoms, , , , or ;

and X is as defined above;
or a pharmaceutically acceptable salt thereof.