MXPA01002665A - Inhibition of toxic materials or substances using dendrimers - Google Patents

Abstract

A method of prophylactic or therapeutic inhibition of a toxic material or substance in a human or non-human animal patient, comprises administration to the patient of an effective amount of a dendrimer having a plurality of terminal groups wherein at least one of the terminal groups has an anionic- or cationic-containing moiety bonded or linked thereto.

Description

I NHIBITION OF SUBSTANCES OR TOXIC MATERIALS USING DENDRIMEROS Field of the invention This invention relates to the inhibition of toxins and other toxic materials or substances, and in particular it relates to the use of dendrimers as binding agents for toxic peptides, proteins or polyamines and other toxic materials or substances.

BACKGROUND OF THE INVENTION Dendrimers are three-dimensional polymeric materials of low polydispersity, which are characterized by a large number of surface end groups. In addition, the manner in which these materials are prepared, allows a strong control over the size, shape and number and type of surface groups. Dendritic materials have several characteristics that are useful for use as therapeutic materials: fixed form that presents a large and defined surface with which to interact with biological surfaces and receptors; and the large number of terminal groups that allow multiple interactions with the biological objectives. International patent applications nos. PCT / AU95 / 00350 (WO 95/34595) and PCT / AU97 / 00447 (WO 98/03573) describe dendrimers, such as polylysine or polyamidoamine dendrimers having a plurality of terminal groups, wherein at least one of The thermionic groups have an anionic or cationic containing portion bound or bonded thereto. The contents of these published international patent applications are incorporated herein by reference. The present invention provides the use of dendritic polymers in the inhibition of toxic materials or substances, including but not limited to toxins or toxic peptides, such as venom, scorpion, spider and bees, as well as toxic peptides or other materials or toxic substances released during bacterial or viral infection.

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, there is provided a method of prophylactic or therapeutic inhibition of a toxic substance or material in a human or non-human patient, which comprises administering to the patient an effective amount of a dendrimer having a plurality of terminal groups, wherein at least one of said terminal groups has an anionic or cationic containing portion attached or bonded thereto. Particularly preferred compounds for use in the method of the present invention are dendrimers having sulfonic acid-containing portions, carboxylic acid-containing portions, phosphoric or phosphonic acid-containing portions, boronic acid-containing portions, neuraminic or sialic acid-containing portions, or acid-containing portions. modified neuraminic or sialic; portions containing primary, secondary, tertiary or quaternary groups, portions containing pyridinium; portions containing ganoidinium; servings containing amid inio; portions containing phenol, heterocycles possessing acidic or basic hydrogens; portions containing zwitterionics; or mixtures of the above portions, linked to terminal groups thereof. The compounds used in the method of this invention are referred to herein as poly-ionic dendrimers, and this term is used throughout this specification and the claims that follow, to include not only the dendrimers per se, but also their salts pharmaceutically or veterinarily acceptable, for example, the alkali metal or alkaline earth metal salts, such as the sodium, potassium or calcium salts, as well as pharmaceutically acceptable anions, such as fluoride, chloride, bromide, iodide, citrate, acetate, p-toluene sulfonate and the like.

DETAILED DESCRIPTION OF THE INVENTION The preferred compounds used in accordance with the present invention include polyionic dendrimers of the general formula I: where: I is an initiating core; Z is an indoor branch unit; n is an integer representing the number of generations of the dendrimer; and A is an anionic or cationic containing portion, which can be linked to an internal branching unit Z through an optional linker group X. Dendrimers are highly branched macromolecular compounds formed by repetitive reaction sequences starting from a initial core molecule with layers or successive stages that are added in successive "generations" to form a three-dimensional, highly ordered polymeric compound. The dendrimers are characterized by the following characteristics: i an initiator core (I), ii the layers of repeating units (Z) unit to the initiator core; iii functional terminal groups (such as portions A) attached to the surface of the dendrimer, optionally through linker groups (such as linker groups X). The present invention uses dendritic structures as frameworks for the binding of ionic portions; the invention is not limited to the spherical dendrimers described in detail herein, but which may be based on any dendritic structure. The variety of dendrimers in both form and constitution are well known to persons skilled in the art. The preparation of dendrimers is well known and is described by way of example in state patents. 4289872 and 4410688 (describing dendrimers based on layers of lysine units), as well as US Pat. 4, 507, 466, 4,558, 1 20, 4, 568,737 and 4, 587, 329 (describing dendrimers based on other units including polya-amidoamine or PAMAM dendrimers). The dendrimers described in these U.S. Patents are described as being suitable for uses such as surface modifying agents, such as metal chelating agents, such as demulsifiers or oil / water emulsions, wet strength agents in papermaking, and as modifying agents. the viscosity in aqueous formulations, such as paints. It is also suggested in US Pat. Nos. 4, 289,872 and 4,41 0,688 that the dendrimers based on lysine units can be used as substrates for the preparation of pharmaceutical dosages. The international patent publications nos. WO 88/01 1 78, WO 88/01 1 79 and WO 88/01 1 80 disclose conjugates in which a dendrimer is conjugated or associated with another material, such as a pharmaceutical or agribyl carrier material. In addition, the international patent publication no. WO 95/24221 describes conjugates of dendritic polymers composed of at least one dendrimer in association with a carrier material, which may be a biological response modifier, and optionally a focus sensor. These patent publications together with the aforementioned US patents contain a broad description of various dendrimers and processes for the preparation thereof, and the description of each of these publications is incorporated herein by reference.

The term "dendrimer", as used herein, will be understood in its broadest sense, and will include within its scope all forms and compositions of these dendrimers as described in the patent publications nos. WO 88/01 1 78, WO 01 01 79 and WO 88/01 1 80. The term also includes linked or bridged dendrimers, as described in these patent publications. Preferred dendrimers of the present invention comprise a polyvalent core covalently linked to at least two dendritic branches, and preferably extend through at least two generations. Particularly preferred dendrimers are polyamidoamine dendrimers (PAMAM), PAMAM dendrimers (EDA), poly (propylene glycol) dendrimers (PPI) and polylysine dendrimers. According to the present invention, at least one, and preferably a substantial number, of the terminal groups on the surface of the dendrimer has an anionic containing or ionic cation bound thereto. Branches of the dendrimer may terminate in amino groups or other functional reactive groups, such as, OH, SH or the like, which may be subsequently reacted with the anionic or cationic portions. Where the terminal groups of the dendrimer are amine groups, the anionic or cationic containing portion can be linked to the dendrimer by a variety of functional groups, including amide and thiourea linkages. Preferred nionic or cationic containing portions, which can be passed to the terminal groups of the dendrimer include portions containing acidic ion, carboxylic acid containing portions (including portions containing neural and sialic acid and portions containing neuraminic acid and sialic modified), boronic acid containing portions, phosphonic and phosphonic acid containing portions (including esterified phosphoric and phosphonic acid containing portions) and primary, secondary, tertiary or quaternary amino containing portions, pyridinium containing portions; portions containing guanidinium; portions containing amidinium; portions containing phenol; heterocycles possessing acidic or basic hydrogens; portions containing zwitterionics; or mixtures of the previous portions. Suitable anionic and cationic containing portions, which can be removed or linked to the terminal or terminal groups, include, by way of example, the following groups (in which n is zero or a positive integer, more particularly n is zero or an integer from 1 to 20): - H (CH2) nS03- - (CH2) nS03- -Ar (SOj-> B -CH2CH (S03 ') COOH -CH (S03") CHZCOOH -ArXlCHjJnSOj- X - OS NH - (CH ^ NMeaAr ( NMe3) "- - (CHjN isJn - ArXP (= O) (OR) 2 X = O, CH 2, CHF, CF 2 R = alkyl, aryl, H, Na.

- ArXP (= O) (OR1) (NR2R3) X = O, CH2, CHF, CF2 Realkyl, aryl, H, Na R2, R3 = alkyl, aryl-Ar [P (= O) (OR) 2] n R = alkyl, aryl, H, Na n = 1-3 - Ar [B (OH) 2] nn = 1-3 - Ar [COOH] nn = 1-3 R = alkyl or arylalkyl; R,, R2, R3 (which may be the same or different) = alkyl or arylalkyl I rent In addition to the foregoing, several portions containing neuraminic or sialic acid or portions containing modified neuramic or sialic acid can be attached or linked to the dendrimers according to this invention. These portions include the various N- and O-substituted derivatives of neuraminic acid, particularly N- and O-acyl derivatives, such as, N-acetyl, O-acetyl and N-glycolyl derivatives, as well as portions in which The neuraminic acid g roup is modified. Suitable modified neuramic acid groups include groups that are substituted at the 4-position with an amino, amido, cyano, azido or ganoidino group, as well as unsaturated neuramic acid groups. These portions can be linked to the dendrimers through positions 2-, 7-, 9- or 5-NAc. Preferably, in the polyionic dendrimers of the formula I, n is an integer from 1 to 20 or more, more preferably from 1 to 1 0. In addition, preferably, the dendrimers include at least three or more terminal groups. Optional linker groups X, which may be present to act as a spacer between the dendrimer and portion A, may consist of an alkyl chain (optionally substituted or branched), an alkoxy, polyalkoxy, alkylthio or polyalkylthio chain ( optionally substituted), or an alkenyl, multiple alkenyl, alkenyl or multiple alkyl chain (optionally substituted). Suitable spacer chains include groups of the formula - (CH2) mZ- (CH2) m-, where Z is -CH2-, -CH = CH-, -C = C-, -O- or -S- and m is an integer from 1 to 1 5 The ammonium or cationic dendrimers of this invention can be prepared by standard chemical methods, which are well known to persons skilled in the art. Suitable methods are described by way of example in the Examples below. As previously described, it has been found that the ammonium or cationic dendrimers of the present invention inhibit toxic substances or materials. The term "toxic substances or materials" as used in The present invention relates in particular to toxins of biological origin (animal, plant, microbial or viral), including but not limited to animal toxins or toxic peptides, such as venom pores, scorpions, spiders and bees, polyamids. toxins, and peptides or other toxic materials or substances released during bacterial infection (such as bacterial exotoxins and endotoxins), or during infection with protozoa, fungi, or viruses. The term "i nh ibition" is used in the present in its most broad to include, either the inhibition or partial or complete suppression of the toxic effect of the toxic material or substance in a human patient The term is also used to encompass both prophylactic and therapeutic treatment. In this way, in another aspect, the present invention provides a pharmaceutical or veterinary composition for the prophylactic or therapeutic inhibition of a toxic substance or material in a patient. human or animal, which comprises a dendrope as broadly described above, in association with at least one pharmaceutically or veterinarily acceptable carrier or diluent. The formulation of such compositions is well known to persons skilled in the art. Pharmaceutical carriers and / or diluents Acceptable acceptable include any and all solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic agents and conventional absorption retardants and the like. The use of such media and agents for pharmaceutically active substances is good C known in the art and described, by way of example, in Remington's Pharmaceutical Sciences, 1 8th edition, Mack Publishing Company, Pennsylvania, USA Except for any conventional media or agent that is incompatible With the active ingredient, the use thereof is contemplated in the pharmaceutical compositions of the present invention. Complementary active ingredients can also be incorporated in the compositions. It is especially advantageous to formulate compositions in dosage unit form for ease of administration and its niformity. dosage The dosage unit form, as used herein, refers to a physically discrete unit suitable as unit dosages for the human subjects to be treated.; each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the carrier and / or pharmaceutical diluent required. The specifications for the novel dosage unit forms of the invention are dictated by and are directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art. of composing such an active ingredient for the particular treatment. In still another aspect, this invention provides the use of an effective amount of a dendrimer as described above broadly in the prophylactic or therapeutic treatment of, or in the manufacture of, a medicament for prophylactic or therapeutic treatment of a human or animal patient by inhibition. of a toxic substance or material. A variety of administration routes are available. The particular mode selected will depend, of course, on the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, can be practiced using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention, without causing unacceptable unacceptable side effects. Such modes of administration include oral, rectal, topical, nasal, inhalation, transdermal or parenteral routes (eg, subcutaneous, intramuscular and intravenous). Formulas for oral administration include discrete units, such as capsules, tablets, pills and the like. Other routes include intrathecal administration directly into the spinal fluid, direct introduction such as by various balloon and catheter angioplasty devices well known to those of ordinary skill in the art and intraparenchymal injection in target areas. The compositions can be conveniently presented in the unit dosage form and can be prepared by any of the methods well known in the pharmacy art. Such methods include the step of bringing the active component into association with a carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and if necessary, shaping the product. The compositions of the present invention suitable for oral administration can be presented as discrete units, such as capsules, cachets, tablets or pills, each containing a predetermined amount of the active component, either as a suspension or as a suspension in a aqueous liquor or non-aqueous liquid, such as a syrup, an ixir or an emulsion. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component, which is preferably isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using dispersants or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic, parenterally-acceptable diluent or solvent, for example, as a solution in polyethylene glycol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspension medium. For this purpose, any soft fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectables. The active component can also be formulated for delivery in a system designed to administer the active component intranasally or by inhalation, for example, as a finely dispersed aerosol atomizer containing the active component. Other delivery systems may include sustained release delivery systems. Sustained-release delivery systems are those that can provide a release of the active component of the invention in capsules or sustained-release pellets. Many types of sustained release delivery systems are available. These include, but are not limited to: (a) wear systems in which the active component is contained within a matrix, and (b) diffusion systems in which the active component is filtered at a controlled rate through a polymer. In addition, an equipment delivery system based on a pump can be used, some of which are adapted for implementation.

The active component is administered in prophylactically or therapeutically effective amounts. A prophylactically or therapeutically effective amount means that amount necessary to at least partially achieve the desired effect, or to delay the onset of, inhibit the progression of, or completely stop, the onset or progression of the particular condition being treated. Such amounts will, of course, depend on the particular condition being treated, the severity of the condition and parameters of the individual patient including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be solved without further routine experimentation. In general, it is preferred that a maximum dose be used, that is, the highest safe dose according to the correct medical judgment. However, those of ordinary skill in the art will understand that a lower dose or tolerable dose can be admired for medical reasons, psychological reasons or virtually for any other reason. In general, daily oral doses of active component will be from about 0.01 mg / kg per day to 1000 mg / kg per day. Small doses (0.01-1 mg) can be administered initially, followed by increasing doses to approximately 1000 mg / kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or higher effective doses for a different, more localized delivery route) can be employed to the tolerance range allowed by the patient. Multiple doses per day are contemplated to achieve systemic levels of appropriate compounds.

The active component according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for: (a) oral administration, external application, e.g., purgative doses (e.g., aqueous or non-aqueous solutions or suspensions); tablets or bowls; powders, granules or pellets for mixing with forges, pastes for application to the tongue; (b) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection, for example, as a sterile solution or suspension; or (when appropriate) by injection into a nonmamma where a suspension or solution is introduced into the udder via the nipple; (c) topical application, for example, as a cream, ointment or atomizer applied to the skin; or (d) intravaginally, for example, as a pessary, cream or foam. Throughout this specification and the claims that follow, unless the context otherwise requires it, the word "understand", or variations such as "understand" or "understanding", will be understood to imply the inclusion of an integer or group of integers declared, but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE DIAMETERS In the accompanying drawings: Figures 1 to 4 show the effects of various concentrations of BRI 2923 on the inhibition of the P3 fraction of HIV toxic Vpr peptide. Additional features of the present invention will be apparent from the following Examples, which are included by way of illustration and not limitation of the invention. In the following Examples, PAMAM dendrimers refer to polyamidoamine dendrimers based on ammonia core, as detailed in U.S. Pat. 4,507,466, 4,558, 1 20, 4, 568,737 and 4, 587,329; PAMAM dendrimers (EDA) refer to polyamidoamine dendrimers based on an ethylene diamine core; and BHAIysxlysylysz dendrimers refer to asymmetric polylysine dendrimers based on a benzhydrylamine core and lysine branching units as described in US Pat. 4, 289,872 and 4,41 0,688. The polyamidoamine dendrimers PAMAM 1 .0, PAMAM 2.0, PAMAM 3.0, PAMAM 4.0, PAMAM 5.0 or higher generation, PAMAM 4.0 (EDA), and the polylysine dendrimers BHAIyslys, BHAIyslys2lys, BHAIyslys2lys lys8 and BHAIyslys2lys4lys8lys16, BHAIyslys2lys4lys8lys16lys32, BHAIyslys2lys4lys8lys16lys32lys6, or higher prepared generations as described in US Pat. Nos. 4289872, 4410688, 4507466, 45581 20, 4568737 and 4578239 and international patent publications nos. WO 88/01 178, WO 88/01 1 79, WO 88/01 1 80 and WO 95/24221 referred to above.

EXAMPLE 1 Reaction of dendritic polymers with 2-acrylamido-2-methyl propane sulfonic acid to give dendrimers terminated in sulfonic acid A PAMAM 1.0 Solid sodium carbonate (0.13 g, 1.0 mmol) was slowly added to a stirred solution of 2-acrylamido-2-methyl propane sulfonic acid (0.41 g, 2.0 mmol) in water (3 mL). After the gas emission had ceased, the pH of the solution was 8.0. Then a solution of PAMAM 1.0 (0.12 g, 0.33 mmol) in water (1 ml) was added to the solution, followed by the addition of four drops of a 40% aqueous solution of benzyl trimethylammonium hydroxide. The solution was then heated under nitrogen at 60 ° C for three days and then concentrated. The residue was purified by gel filtration (Sephadex G10, water) and then freeze-dried to give sulfonated PAMAM 1.0 dendrimer as an off-white solid (0.51 g). The 1 H and 3 C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 1.0 dendrimer (ca 70:30). 13C nmr (D2O): d 31.0, 31.1, 37.1, 37.7, 41.3, 48.6, 51.5, 53.1, 53.4, 55.6, 56.2, 61.2, 61.5, 178.3, 179.0, 179.8.

B PAMAM 2.0 PAMAM 2.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as described above. The crude product was purified by gel filtration (Sephadex G10, water) and then freeze-dried to give an off-white solid. The 1 H and 13 C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 2.0 dendrimer (ca. 65:35). 3C nmr (D2O): d 31.0, 31.1, 37.1, 37.7, 41.3, 48.7, 51.5, 53.4, 55.6, 56.2, 61.2, 61.5, 178.4, 179.0, 179.1, 179.6. When the above reaction was repeated omitting the benzyltrimethylammonium hydroxide, a similar result was obtained.

C PAMAM 3.0 BRI2783 PAMAM 3.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as before, except that a slight excess of sodium carbonate was used and the benzyltrimethylammonium hydroxide was omitted. The 1 H and 13 C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 3.0 dendrimer (ca. 50:50). 3C nmr (D2O): d 31.0, 31.1, 36.9, 37.4, 41.1, 48.6, 51.5, 53.4, 55.7, 56.2, 61.1, 61.5, 178.2, 178.9, 179.0, 179.8.

D PAMAM 4.0 BRI2784 PAMAM 4.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as described for PAMAM 3.0. The 1 H and 13 C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 4.0 dendrimer (ca. 35:65). 13C nmr (D2O): d 31.0, 31.1, 36.9, 37.3, 41.1, 48.5, 51.5, 53.5, 55.7, 56.2, 61.1, 61.5, 178.1, 178.9, 179.0, 179.8 EXAMPLE 2 Preparation of finished dendrimers in sodium sulfoacetamide A PAMAM 1.0 A solution of 4-nitrophenyl bromoacetate (0.40 g, 1.5 mmol) in dry DMF (1 ml) was added to a stirred solution of PAMAM 1.0 (0.18 g, 0.5 mmol) in DMF (3 ml). The resulting yellow solution was stirred for 20 hours at room temperature, when the ninhydrin test was negative. The solution was concentrated (30 ° C / 0.1 mmHg) to give a yellow oil. This oil was divided between water and chloroform and the aqueous layer was separated and washed with chloroform (2X) and finally with ethyl acetate. The aqueous solution was concentrated (35 ° C / 25 mmHg) to give the bromoacetylated PAMAM 1.0 dendrimer as a yellow oil (0.36 g, 100%). 13C nmr (D2O): d 32.8, 33.3, 43.0, 43.5, 54.4, 174.5, 176.4. A solution of sodium sulfite (0.2 g, 1.6 mmol) in water (1 ml) was added to a solution of the bromoacetylated PAMAM 1.0 dendrimer described above (0.36 g, 0.5 mmol) in water (5 ml) and the solution was left remain at room temperature for eleven days. The yellow solution was concentrated to give a yellowish solid (0.60 g). 13C nmr (D2O): d 34.4, 43.1, 43.4, 54.0, 61.7, 171.3, 177.2. The above reaction sequence could be performed without isolating the bromoacetylated dendrimer by simply adding the sodium sulfite solution to the crude aqueous extract obtained from the first reaction.

B PAMAM 2.0 Method 1: A solution of 4-n-tropholene bromoacetate (018 g) was added, 07 mmol) in dry DMF (1 ml) was added to a stirred solution of PAMAM 20 (019 g, 01 mmol) in DMF (3 ml) The resulting yellow solution was stirred for 20 hours at room temperature, when the test was negative. ninhydpna The solution was then added with turbulence to water (150 ml) and the mixture was extracted with chloroform (3X) and ethyl acetate. A solution of sodium sulfite (01 g, 08 mmol) in water (1 ml) was added to the crude bromoacetylated dendrimer solution and the mixture was allowed to stand for three days at room temperature. The yellowish solution was then concentrated to give a yellow solid residue, which was purified by gel filtration (Sephadex LH20, water) to give the dendrimer PAMAM 20 finished in sodium sulphoacetamide (103 mg) 13C nmr (D2O) d 330, 357, 360, 377, 403, 430, 432, 534, 537, 560, 61 6, 171 2 1746, 1785 Method 2 Solid succymlmidyl acetylthioacetate (67 mg, 033 mmol) was added to a solution of PAMAM 20852 mg, 005 mmol) in dry DMF (2 ml) and the resulting solution was stirred at room temperature for two days. Then the mixture was concentrated (30 ° C / 103 mmHg) to give an oily residue The residue was partitioned between water and chloroform, and the aqueous layer was separated and concentrated to give a viscous oil (117 mg) 1H and 13C nmr showed that the oil was a mixture of the acylated dendimer and N-hydroxy succinimide The gel filtration (Sephadex G10, water) provided a pure sample of the PAMAM dendrimer 20 terminated in acetylthioacetamide (29 mg). 13C nmr (D2O): d 34.0, 34.2, 37.3, 43.0, 43.1, 43.3, 53.5, 54.0, 56.3, 175.4, 177.2, 177.5. A solution of the above functionalized dendrimer in 40% aqueous formic acid (7 ml) was then added to a freshly prepared ice solution of performic acid (1.6 mmol) in formic acid (2 ml). The mixture was stirred for one hour at 0 ° C and then for twenty hours at room temperature. A small amount of activated carbon was then added to decompose any excess of permeation, the mixture was stirred for 30 minutes, then filtered and concentrated to give a viscous oil. The crude product was dissolved in water, the pH was adjusted to 9.0 with aqueous sodium bicarbonate and the material was desalted by passing through a Sephadex G10 column. A white solid (20 mg) was obtained after lyophilization, which was essentially spectroscopically equal to the material obtained by method 1. 13C nmr (D2O): d 33.0, 38.7, 42.9, 43.0, 43.1, 53.9, 54.3, 56.5, 61.6, 171.2, 176.4, 177.0.

EXAMPLE 3 Preparation of dendrimers terminated in sodium sulfuccinic acid A PAMAM 1.0 Solid maleic anhydride (0.11 g, 1.1 mmol) was added to a stirred solution of PAMAM 1.0 (0.12 g, 0.33 mmol) in dry DMF (3 mL).

The mixture became a little brown and a little hot as the anhydride dissolved and the resulting solution was stirred overnight at room temperature. Then the solution was concentrated (30 ° C / 104 mmG) to give a viscous oil 1H and 13C nmr (D2O) showed a complete conversion of PAMAM 10 to tpsamide together with some maleic acid 3C nmr (D2O) d 331, 428, 431, 543, 1350, 1371, 1691, 171 9, 1733 The tpsamide curda was dissolved then in water (4 ml) and solid sodium sulfite (020 g, 16 mmol) was added. The resulting solution was allowed to stand at room temperature for four days and then concentrated 1H and 3C nmr (D2O) showed a mixture of 1 1 of the PAMAM 1 0 dendrimers terminated in sodium sulphosuccinic acid regioisomeccos together with some sulfosuccinic acid The crude product was purified by gel filtration (Sephadex G10, water) to give a sample of the finished PAMAM dendrimers in á sodium sulfosuccinnamic acid (107 mg) 13C nmr (D2O) d 333, 396, 400, 429, 431, 540, 679, 694, 1738, 1763, 1776, 181 8 B PAMAM 20 A mixture of PAMAM dendrimers 20 terminated in sodium sulfosuccinamic acid regioisomepcos was prepared as described above 13C nmr of maleamic acid derivative of PAMAM 20 (D2O) d 328 330, 387, 429, 538, 543, 565 , 1352, 1368, 1692, 171 9 1735 1746 3C nmr of sodium sulfosuccinic acid derivatives of PAMAM 20 (D20) d 370, 401 41 1 430 432 439 530, 533 555 680 694, 1738 1776 179 1 1795 1798 1823 C PAMAM 4.0 BRI6038 Solid maleic anhydride (60 mg, 0.6 mmol) was added to a stirred solution of PAMAM 4.0 (51 mg, 0.01 mmol) in dry DMF (2 mL). The mixture turned turbid initially but soon gave a clear solution which was stirred overnight at room temperature. The solution was then concentrated (35 ° C / 10"4 mmH) to give a viscous oil, 1H and 13C nmr (D2O) showed complete conversion of PAMAM 4.0 to the polyamide together with some maleic acid. Then it was dissolved in water (2 ml) and a solution of sodium sulphite (126 mg) was added.; 1.0 mmol) in water (2 ml). The resulting solution was allowed to stand at room temperature for two days and then concentrated. H and 13C nmr (D2O) showed a mixture of PAMAM 4.0 dendrimers terminated in regioisomeric sodium sulfosuccinic acid together with some sulfosuccinic acid. The crude product was purified by gel filtration (Sephadex LH20, water) to give a PAMAM 4.0 sample terminated with 24 regioisomeric sulfosuccinnamic acid groups (90 mg). 1 H nmr (D 2 O): d 2.4-2.6; 2.7-3.1; 3.2-3.4; 3.9-4.0. 13C nmr (D2O): d 36.2; 39.8; 40.5; 43.0; 43.2; 53.5; 55.8; 68.1; 69.5; 173.8; 177.4; 177.6; 178.7; 182.3.

EXAMPLE 4 Preparation of dendrimers terminated in sodium N- (2-sulfoethyl) succinamide a Preparation of N- (2-sulfoethyl) succinnamic acid of tetrabutylammonium Solid succinic anhydride (0.5 g, 5.0 mmol) was added to a stirred solution of sodium hydroxide. tetrabutylammonium amine-ethylsulfonic acid (1.83 g, 5.0 mmol) in dry dichloromethane (30 ml). The succinic anhydride was slowly dissolved and the resulting cloudy solution was stirred overnight at room temperature. The mixture was filtered and the filtrate was concentrated to give a viscous oil (2.41 g). 13C nmr showed a complete conversion to the desired monoamide together with a small amount of succinic acid. Repeated precipitation of the product by droplet addition of a dichloromethane solution to a large excess of diethyl ether gave tetrabutylammonium N- (2-sulfoethyl) succinamic acid as a white solid (1762 g, 76%), mp 125 -127 ° C. 1 H nmr (CDCl 3): d 0.86 (t, 12 h, 4 x CH 3), 1.28 (m, 8 H, 4 x CH 2), 1.50 (m, 8 H, 4 x CH 2), 2.33 (m, 2 H, CH-COOH), 2.44 (m, 2 H , CH2CONH), 2.76 (m, 2H, CH2NHCO), 3.12 (m, 8H, 4xCH2N), 3.50 (m, 2H, CH2SO3), 7.53 (br t, 1H, NH). 13C nmr (CDCl 3): d 13.5, 19.5, 23.8, 30.1, 30.9, 35.6, 50.0, 58.5, 172.0, 174.1.

Preparation of 4-nitrophenyl tetrabutylammonium N- (2-sulfoethyl) succinamate A solution of dicyclohexylcarbodimide (45 mg, 0.22 mmol) in dry dichloromethane (1 ml) was added to a stirred solution of N- (2-sulfoethyl) succinamic acid. of tetrabutylammonium (94 mg, 0.20 mmol) in dichloromethane (2 ml), and the mixture was stirred overnight at room temperature. The resulting suspension was filtered and the filtrate was concentrated to give the crude active ester, which was used without further purification.

A Preparation of PAMAM dendrimers terminated in N- (2-sulfoethyl) succinamide sodium PAMAM 4.0 BRI2786 A solution of crude tetrabutylammonium 4- (2-sulfoethyl) succinamate succinamate (0.30 mmol) in dry DMF (1 ml) was added to a stirred solution of PAMAM 4.0 (51.5 mg, 0.01 mmol) dissolved in 50% aqueous DMF (3 ml) and the resulting yellow solution was stirred overnight at room temperature. The mixture was then concentrated (35 ° C / 105 mmHg) and the yellow residue was partitioned between water and chloroform. The aqueous layer was separated, washed with chloroform (2X) and ethyl acetate, and then concentrated to give the yellow oil (134 mg). The crude product was converted to the sodium salt by passing through a column of Amberlite IR 120 (Na) to produce 85 mg of "material.This material was further purified by gel filtration (Sephadex LH20, water) to give the PAMAM 4.0 dendrimer terminated in sodium N- (2-sulfoethyl) succinamide (45 mg) 13C nmr (D2O): d 33.2, 33.6, 35.5, 390, 395, 428, 432, 53.8, 54.1, 54.4, 56.6 , 1765, 176.9, 177.2, 178.9, 179.4 The corresponding PAMAM 1.0 and PAMAM 3.0 (BRI2785) dendrimers terminated with sodium N- (2-sulfoetyl) succ-namide groups were similarly prepared 13C nmr derivative PANAM 3.0 (D2O): d 33.4, 35.5, 39.0, 39.5, 42.9, 43.2, 53.8, 54.1, 54.3, 56.5, 176.4, 176.9, 177.4, 178.9, 179.4, 13C nmr of PANAM derivative 1.0 (D2O): d 34.9 , 35.5, 39.5, 42.9, 43.1, 53.7, 54.1, 179.0, 179.1, 179.3.

B Preparation of polylysine dendrimers terminated in N- (2-sulfoethyl) succinamide sodium BHAIyslys2lys4lys8lys16 BRI2789 Trifluoroacetic acid (1 ml) was added to a suspension of BHAIyslys2lys4lys8DBLi6 (36.5 mg, 5.0 μmol) in dry dichloromethane (1 ml) and the resulting solution was stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in dry DMSO (2 ml) and the pH adjusted to 8.5 with triethylamine. A solution of the crude tetrabutylammonium 4-nitrophenyl-succinamate succinamate (ca. 0.2 mmol) in DMSO (1 ml) was then added in the form of drops and the mixture was stirred overnight at room temperature. The yellow solution was then concentrated (50 ° C / 10"5 mmHg) and the yellow residue was partitioned between water and chloroform.The aqueous layer was separated, washed with chloroform (3X) and ethyl acetate, and then concentrated to give an oil (99 mg) The crude product was converted to the sodium salt by passing through a column of Amberlite IR 120 (Na) to produce 81 mg of material.This material was further purified by gel filtration ( Sephadex LH20; water) to give the dendrimer of BHAIyslys2lys4lys8lys16 terminated in N- (2-sulfoethyl) succinamide sodium (39 mg). 13C nmr (D2O): d 27.0, 32.3, 35.2, 35.3, 35.6, 35.7, 39.5, 43.5, 54.1, 58.5, 131.5, 132.0, 133.3, 145.1, 177.8, 178.0, 178.4, 178.8, 178.9, 179. 2, 179.7, 179.8. The BHAIyslys2, BHAIyslys2lys4 (BRI2787) and BHAIyslys2lys4lys8 (BRI2788) terminated with corresponding N- (2-sulfoethyl) succinamide sodium groups, were prepared in a similar manner. 13C nmr of derivative of BHAIyslys2lys4lys8 (D2O): d 26.9, 32.3, 35.1, . 3, 35.6, 35.7, 39.5, 43.5, 54.1, 58.5, 131.6, 131.9, 132.2, 132.3, 133.2, 133. 3, 145.0, 145.2, 177.2, 177.8, 177.9, 178.0, 178.2, 178.3, 178.6, 178.6, 178.7, 178.8, 178.9, 179.2, 179.3, 179.7, 179.8. 13C nmr of derivative of BHAIyslys2lys4 (D2O): d 26.9, 32.3, 35.1, . 4, 35.7, 35.8, 39.5, 43.5, 54.1, 58.5, 61.8, 131.7, 132.0, 132.2, 132.3, 133. 2, 133.3, 145.0, 145.1, 177.3, 178.0, 178.3, 178.4, 178.7, 178.9, 179.0, 179.3, 179.7, 179.8. 13C nmr of derivative of BHAIyslys2 (D20); d 26.9, 27.1, 32.2, 32.3, 34. 7, 34.8, 35.1, 35.3, 35.6, 35.7, 39.5, 43.4, 54.1, 58.6, 61.8, 131.7, 131.9, 132.2, 132.3, 133.3, 144.9, 145.0, 177.7, 178.4, 178.8, 179.0, 179. 3, 180.0.

EXAMPLE 5 Preparation of Dendrimers Terminated in Sodium 4-Sulfophenylthiourea A PAMAM 4.0 BRI2791 Solid sodium 4-sulphophene? -sun? Octanate sodium hydroxide (500 mg, 1 96 mmol) was added to a solution of PAMAM 40 (300 mg, 00582 mmol) in water (10 ml) and the resulting solution was heated under nitrogen at 53 ° C for two hours and then cooled. The solution was concentrated and the yellow solid residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and freeze dried to give the PAMAM 40 dendrimer terminated in sodium 4-sulphophenylthiourea as a fluffy white solid (370 mg) 1H nmr (D2O) d 228, 252, 269, 315, 327, 360, 732 (d, J = 9Hz), 772 (d, J = 9Hz) 13C nmr (D2O) d 369, 41 1, 431, 483, 536, 558, 1290, 131 1, 1444, 1785, 1791, 1844 PAMAM 1 0 dendrimers, PAMAM 20 (BRI2790), PAMAM 30 and PAMAM 50 (BRI2991) correspondingly terminated with 3, 6, 12 and 48 groups of 2-sulfofen? Lt? Ourea de sodium respectively, were prepared in a similar manner B PAMAM 40 (EDA) BRI6045 Sodium monohydrate 4-sulfofen? L? Sot? Oc? Otate (130 mg, 05 mmol) was added to a solution of PAMAM 40 (EDA) (69 mg, 001 mmol) in Water (4 ml) and the resulting solution was heated under nitrogen at 53 ° C for two hours and then cooled. The solution was concentrated and the solid residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and were freeze-dried to give PAMAM 40 terminated with 32 groups of sodium 4-sulphophenolitrea as a fluffy white solid (136 mg) 1H nmr (D20) d 230 250, 270, 318, 362, 735, (d , J = 9Hz), 772 (d, J = 9Hz) 13C nmr (D2O) d 368, 410, 431, 484, 536, 557, 1289, 1310, 1443, 1785, 1790, 1845 C BHAIyslys2lys4lys8lys16 BRI2792 Tpfluoroacetic acid (4 ml) was added to a suspension of BHAIyslys2lys4lys8DBL16 (073 g, 01 mmol) in dry dichloromethane (4 ml) under nitrogen. A vigorous gas emission was observed for a short time and the resulting solution was stirred at room temperature for two hours and then concentrated. The residual syrup was dissolved in water (5 ml), the solution was passed through a column of Amberlite IRA-401 (OH) and the filtrate was concentrated to give BHAIyslys2lys4lys8lys16 as a viscous oil (049 g) The oil was redissolved in water (5 ml) and N, Nd? met? lN-al? lam? na buffer (pH 95, 3 ml) was added. Solid sodium 4-sulphophenol? sot? octanate monohydrate (1 30 g, 5 1) was then added. mmol) and the resulting solution was heated under nitrogen at 53 ° C for two hours and then cooled. The solution was concentrated and the brownish brown solid was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined, passed through through a column of Amerlite IR 120 (Na) and dry on by freezing to give the dendrimer of BHAIyslys2lys4lys8lys16 terminated in 4-sulphophenolitrea of sodium as a fluffy white solid (374 mg) 1H nmr (D2O) d 1 40, 1 72, 308, 342, 424, 460, 730 , 740 (d, J = 9Hz), 778 (d, J = 9Hz) 13C nmr (D20) d 273 325, 359, 437, 489, 586 633, 1288, 131 0, 1437 1447, 1451 1777 1781 1838, 1852 The corresponding BHAIyslys2lys4lys8, BHAIyslys2lys4lys8lys16lys32 (BRI2992) and BHAIyslys2lys4lys8lys16lys32lys64 (BRI2993) dendrimers, terminated with 16, 64 and 128 sodium 4-sulfophenylthiourea groups respectively, were prepared in a similar manner.

EXAMPLE 6 Preparation of Dendrimers Terminated in 3,6-Disulfonaphthylthiourea Sodium A PAMAM 4.0 BRI2923 Solid sodium 3,6-disulfonaphthylisothiocyanate (160 mg, 0.41 mmol) was added to a solution of PAMAM 4.0 (51 mg, 0.01 mmol) in water (3 mL) and the resulting solution was heated under nitrogen to 53 ° C for two hours and then cooled. The solution was concentrated and the solid coffee residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and concentrated to give the PAMAM 4.0 dendrimer terminated in sodium 3,6-disulfonaphthyl thiorea as a brownish brown solid (73 mg). 1 H nmr (D 2 O): d 2.30; 2.60; 2.74; 3.20; 3.57; 7.75; 7.86; 8.28. 13C nmr (D2O): d 35.0; 39.9; 43.1; 48.1; 53.8; 56.1; 128.4; 128.6; 129.3; 131.0; 131.3; 136.0; 136.8; 138.2; 145.5; 146.0; 177.2; 177.8; 188.5. The corresponding PAMAM 2.0 dendrimer terminated with sodium 3,6-disulfonaphthyl thiourea groups was prepared in a similar manner.

B PAMAM 40 (EDA) BRI6046 Solid 3,6-d? Sulfonaft? L? Sot? Oc? Otate sodium (220 mg, 057 mmol) was added to a solution of PAMAM 40 (EDA) (74 mg, 001 mmol). in water (4 ml) and the resulting solution was heated under nitrogen at 53 ° C for two hours and then cooled. The solution was concentrated and the brownish brown solid residue was purified by gel filtration (Sephadex LH20, water). Pure fractions were combined and concentrated to give PAMAM 40 terminated with 32 sodium 3,6-disulfonaphthyl thiourea groups as a tan solid (148 mg) 1 H nmr (D 2 O) d 230, 280, 320, 354, 774, 785, 825 , 13C nmr (D2O) d 360, 408, 431, 483, 536, 559, 1285, 1294, 131 0, 131 3, 1360, 1368, 1383, 1455, 1460, 1782, 1856 C BHAIyslys2lys4lys8lys16 BRI2999 Tffluoroacetic acid (2 ml) was added to a suspension of BHAIyslys2lys4lys8DBL16 (73 mg, 001 mmol) in dry dichloromethane (2 ml) under nitrogen. A vigorous gas emission was observed for a short time and the resulting solution was stirred at room temperature for two hours and then concentrated The residual syrup was dissolved in water (5 ml), the solution was passed through a column of Amberlite IRA-401 (OH) and the filtrate was concentrated to give BHAIyslys2lys4lys8lys16 as a viscous oil The oil was redissolved in water (5 ml) and a N, Nd? Met? N-al? Lam? Na buffer (pH 95, 3 ml) was added. Then 3,6-d? Sulfonaft? L? Sot was added. Sodium oateate (234 mg, 060 mmol), the resulting solution was heated under nitrogen at 53 ° C for two hours and then cooled. The solution was concentrated and the brownish brown solid residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined, passed through a column of Amberlite IR 120 (Na) and freeze dried to give BHAIyslys2lys4lys8lys16 terminated with 32 sodium 3,6-disulfonaphthylthiourea groups as a white fluffy solid (119 mg). 1H nmr (D2O): d 1.0-2.0; 3.18; 3.43; 4.31; 7.22; 7.80; 7.89; 8.25. 13C nmr (D2O) d 27.2; 32.4; 35.3; 43.7; 49.0; 58.5; 63.6; 128.4; 129.1; 131.4; 136.1; 138.6; 139.0; 145.1; 145.6; 178.4; 184.8; 186.7.

EXAMPLE 7 Preparation of dendrimers terminated in sodium 4-sulfonaphthylthiourea PAMAM 4.0 BRI2997 Solid sodium 4-sulfonaphthysothiocyanate (180 mg, 0.5 mmol) was added to a solution of PAMAM 4.0 (51 mg, 0.01 mmol) in water (5 ml) and the mixture was heated under nitrogen at 53 ° C for two hours. hours and then it cooled down. The water was distilled off under reduced pressure from the resulting suspension and the off-white solid residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and freeze dried to give the PAMAM 4.0 dendrimer terminated in sodium 4-sulfonaphthylthiourea as a fluffy white solid (60 mg). 1 H nmr (D20): d 2.20; 2.60; 3.14; 3.48; 7.23; 7.47; 7.56; 7.77; 7.93 (d, J = 6Hz); 8.56 (d, J = 6Hz). 13C nmr (D20): d 35.8; 40. 5; 43.1; 48.4; 53.6; 55.9; 127.6; 128.6; 130.3; 131.9; 132.5; 133.5; 134.7; 140.5; 142.7; 177.8; 178.0; 185.4.

EXAMPLE 8 Preparation of dendrimers terminated in sodium 3,5-disulfophenyl thiourea PAMAM 4.0 BRI6039 Solid sodium 3,5-disulfophenylisothiocyanate (110 mg, 0.32 mmol) was added to a solution of PAMAM 4.0 (63 mg, 0.012 mmol) in water (3 ml) and the resulting solution was heated under nitrogen at 53 ° C. C for two hours and then cooled. The solution was concentrated and the brownish brown solid residue was purified by gel filtration (Sephadex G25, water). The pure fractions were combined and concentrated to give PAMAM 4.0 terminated with 24 sodium 3,5-disulfophenylthiourea groups as an off-white solid (110 mg). 1 H nmr (D 2 O): d 2.53; 3.08; 3.36; 3. 66; 7.90; 7.95. 13C nmr (D2O): d 34.8; 41.0; 43.1; 48.0; 53.7; 56.2; 124.1; 128. 6; 143.5; 148.8; 177.6; 185.0.

EXAMPLE 9 Preparation of finished dendrimers in sodium 3,6,8-trisulfonaphthylthiourea PAMAM 4.0 BRI2998 Solid sodium 3,6,8-trisulfonaphthisothiocyanate (250 mg, 0.5 mmol) was added to a solution of PAMAM 4.0 (51 mg, 0.01 mmol) and N, N-dimethyl-N-allylamine buffer (pH 9.5 1 ml) in water (2 ml), and the mixture was calcined under nitrogen at 53 ° C for two hours, then cooled. The mixture was concentrated under reduced pressure to give an orange solid. The residual solid was dissolved in water (2 ml) and passed through a short column of Amberlite IR-120 (Na). The filtrate was then concentrated and the residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and freeze dried to give the PAMAM 4.0 dendrimer terminated in sodium 3,6,8-trisulfonaphthyl thiorea as an off-white solid (102 mg). 1 H nmr (D 2 O): d 2.65; 3.02; 3.30; 3.66; 8.05; 8.42; '8.59; 8.67. 13C nmr (D2O): d 33.2; 38.7; 43.2; 43.7; 47.8; 54.0; 54.3; 56.7; 131.0; 131.3; 131.9; 135.9; 138.0; 139.6; 143.8; 144.1; 145.6; 176.2; 176.5; 186.0. The dendrimer terminated in the corresponding sodium 3,6,8-trisulfonaphthylthiourea BHAIys.lys2lys lys8lys16 BRI7011 was prepared in a similar manner.

EXAMPLE 10 Preparation of dendrimers terminated in sodium 4- (sulfomethyl) benzamide PAMAM BRI6040 Solid 4-nitrophenyl 4- (chloromethyl) benzoate (200 mg, 0.68 mmol) was added to a stirred solution of PAMAM 4.0 (70 mg, 0.014 mmol) in dry DMSO (4 mL) and the resulting yellow solution it was stirred at room temperature for two hours. The solution was then concentrated (10"4 mmHg, 40 ° C) and the residue was extracted with a mixture of water and dichloromethane (1: 1) The remaining solid material was dissolved in DMSO (5 ml) and a solution was added of sodium sulfite (130 mg, 1 mmol) in water (3 ml) The resulting slightly turbid mixture was allowed to remain for four days, after which the addition of more water (2 ml) resulted in the formation of a clear yellow homogenous solution The solution was then concentrated, first at 25 mmHg and 40 ° C, then at 10"4 mmHg and 50 ° C, to give the crude product. The crude product was purified by gel filtration (Sephadex G25, water) to give PAMAM 4.0 terminated with 24 groups of sodium 4- (sulfomethyl) benzamide (24 mg). 1 H nmr (D 2 O): d 2.25; 2.66; 3.08; 3.20; 3.33; 3.38; 4.01; 7.40 (br d); 7.62 (br d). 13C nmr (D2O): d 36.7; 40.9; 43.0; 43.6; 53.5; 55.6; 61.0; 131.6; 135.0; 137.2; 140.4; 174.5; 178.6; 179.2.

EXAMPLE 11 Preparation of dendrimers terminated in 4-sulfobenzamide PAMAM 4.0 (EDA) BRI6116 Solid potassium N-hydroxysuccinimidyl 4-sulfobenzoate (100 mg, 0.3 mmol) was added to a solution of PAMAM 4.0 (EDA) (35 mg, 0.005 mmol) in 0.1 M borate buffer pH 8.5 (5 mg). ml) and the solution was stirred at room temperature for two hours. The milky solution resulting in this step had a pH of 4.5. Then a 1M sodium carbonate solution (1 ml) was added to give a clear solution which was concentrated to give the crude product as a white solid. The crude product was purified by gel filtration (Sephadex G25, water) to give PAMAM 4.0 (EDA) terminated with 32 groups of sodium 4-sulfobenzamide (47 mg). 1 H nmr (D 2 O): d 2.25; 2.42; 2.63; 3.05; 3.18; 3.31; 3.38; 7.72 (d, J = 8Hz); 7.78 (d, J = 8Hz). 3C nmr (D2O): d 36.0; 40.4; 43.0; 43.7; 53.7; 55.8; 130.2; 132.2; 140.4; 150.1; 173.6; 178.0; 178.5.

EXAMPLE 12 Preparation of dendrimers terminated in sodium N- (4-sulfophenyl) propanamide PAMAM 4.0 (EDA) BRI6117 Solid sodium N- (4-sulfophenyl) acrylamide (250 mg, 1 mmol) and solid sodium carbonate (106 mg, 1 mmol) were added successively to a stirred solution of PAMAM 4.0 (EDA) ( 78 mg, 0.011 mmol) in water (4 ml). The resulting solution was stirred under nitrogen for four days and then dried by freezing to give a fluffy white solid. The crude product was purified by gel filtration (Sephadex LH20, water) to give PAMAM 4.0 (EDA) terminated with 64 groups of sodium N- (4-sulfophenyl) propanamide (206 mg). 13C nmr showed faint traces of what was taken as mono-alkylated terminal amino groups. 1 H nmr (D 2 O): d 2.10; 2.48; 2.58; 2.79; 3.20; 7.42 (d, J = 7Hz); 7.65 (d, J = 7Hz). 13C nmr (D2O): d 36.5; 37.9; 41.1; 53.4; 55.6; 124.8; 130.9; 143.0; 144.2; 1774. 178.5.

EXAMPLE 13 Preparation of dendrimers terminated in sodium 4-sulfophenylurea PAMAM 4.0 (EDA) BRI6115 A solution of sodium sulphanilic acid (195 mg, 1 mmol) in dry DMSO (3 mL) was added dropwise to a solution of N, N'-disuccinimidyl carbonate (530 mg; mmol) in dry DMSO (4 ml) and the resulting brownish brown solution was stirred at room temperature for 20 hours. A solution of PAMAM 4.0 (EDA) (75 mg, 0.011 mmol) in dry DMSO (1 ml) was added and the solution was stirred for an additional 18 hours. The solution was then concentrated under high vacuum (105 mmHg, 35 ° C) to give a yellowish semi-solid. The crude product was dissolved in DMSO (4 ml) and the solution was added to 200 ml of well-stirred ethyl acetate. The precipitated white solid was collected by filtration and washed with ethyl acetate (2X) and ether (2X), then dried to give a white powder (275 mg). This material was further purified by gel filtration (Sephadex LH20, water) to give PAMAM 4.0 (EDA) terminated with 32 sodium 4-sulfophenylurea groups (106 mg). 1 H nmr (D20): d 2.31; 2.55; 2.75; 3.19; 7.32 (d, J = 9Hz); 7.63 (d, J = 9Hz). 13C nmr (D20): d 36.3; 40.7; 43.3; 43.8; 53.7; 55.7; 123.3; 130.9; 140.9; 146.0; 161.4; 178.2; 178.6.

EXAMPLE 14 Preparation of N-N, N-Trimethylglycinamide Chloride-Finished Dendrimers BHAIyslys2lys4lys8lys16 BRI2992 Trifluoroacetic acid (4 ml) was added to a suspension of BHAIyslys2lys lys8DBL16 (220 mg, 30 μmol) in dry dichloromethane (2 ml) and the resulting solution was stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in dry DMSO (5 ml) and the pH adjusted to 8.5 with triethylamine. Then solid 4-nitrophenyl N, N, N-trimethylglycinate chloride (0.50 g, 1.8 mmol) was added and the mixture was stirred overnight at room temperature. The cloudy solution was then concentrated (50 ° C / 10"5 mmHg) and the residue was partitioned between water and dichloromethane.The aqueous layer was separated, washed with dichloromethane (3X) and ethyl acetate, and then concentrated to give an oil (1128 g) The crude product was purified by gel filtration (Sephadex LH20, water) to give the dendrimer of BHAIyslys2lys4lys8lys16 terminated in N, N, N-trimethylglycinamide (116 mg), 13C nmr (D2O): d 25.5 , 30.5, 30.8, 33.4, 42.1, 56.5, 57.1, 67.5, 68.1, 166.7, 167.0, 167.1, 176.0, 176.2.

EXAMPLE 15 Preparation of dendrimers terminated in 4-trimethylammoniobenzamide PAMAM 4.0 BRI6043 1, 1 '-carbonyldnidazole (85 mg, 052 mmol) was added to a solution of 4-tpmet-lamonyl-obenzoic acid iodide (154 mg, 05 mmol) in dry DMF (4 mL) and the The mixture was stirred at room temperature under argon for two hours. During this time, a white solid was prepared from the solution. Then a solution of PAMAM 40 (58 mg, 0.011 mmol) in dry DMF (2 ml) and the mixture were added. it was stirred overnight at room temperature. After this time, most of the precipitate had dissolved and a ninhydpna test of the solution was negative. The mixture was concentrated (104 mmHg.; 30 ° C) to give a white solid residue. The crude product was purified by gel filtration (Sephadex LH20, 10% AcOH) to give PAMAM 40 terminated with 24 groups of 4-t-methylammoniobenzamide as the acetic acid salt (89 mg). 1 H nmr (D 2 O): d 1 96; 265-285, 325-3.55; 3.64; 792 13C nmr (D2O) d 258, 33.1, 335, 38.7; 431; 435, 53.5; 54.1; 56.4; 61.2, 124.8; 1336; 139.9; 1532; 173.2; 176.3; 176.8; 1826. The corresponding PAMAM 2.0 dendrimer terminated with 6 groups of 4-trimethamione obenzamide was prepared in a similar manner.

EXAMPLE 16 Preparation of dendrimers terminated in 4- (trimethylammoniomethyl) benzamide PAMAM 4.0 BRI6044 4- (Chloromethyl) benzoate of solid 4-n-trophonol (150 mg, 05 mmol) was added to a stirred solution of PAMAM 4.0 (52 mg, 0.01 mmol) in dry DMSO (3 mL). The resulting yellow solution was stirred at room temperature for 20 hours, when a ninhydrin test was negative (pH ca.8.5). The solution was then concentrated (105 mmHg, 40 ° C) and the residue was stirred with a mixture of water and dichloromethane (1: 1). The insoluble gel-like material was collected by filtration, washed with water (2X) and dichloromethane (2X), and then dried with air. The dendrimer ending in crude 4- (chloromethyl) benzamide was dissolved in 25% aqueous trimethylamine (20 ml) and the yellow solution allowed to remain overnight. The solution was then concentrated, the residue was dissolved in water (5 ml) and the solution was passed through a column of Amberlite IRA-401 (OH). The colorless filtrate was concentrated to give a viscous oil, which was purified by gel filtration (Sephadex G10; 10% AcOH) to give PAMAM 4.0 finished with 24 groups of 4- (trimethylammoniomethyl) benzamide (90 mg). 1 H nmr (D 2 O): d 1.88; 2.65-2.80; 2.98; 3.10-3.60; 7.52 (br d, J = 9 Hz); 7.72 (br d, J = 9 Hz). 13C nmr (D2O): d 26.6; 33.4; 38.8; 43.2; 43.5; 53.6; 53.6; 54.1; 56.8; 62.8; 73.0; 132.1; 135.3; 137.5; 140.0; 176.4; 176.9; 183.6.

EXAMPLE 17 Preparation of dendrimers terminated in N- (2-acetoxyethyl) -N, N- (dimethylammonium) methyl carboxamide PAMAM 4.0 Solid 1,1'-carbonyldiimidazole (85 mg, 0.52 mmol) was added to a solution of N- (2-acetoxyethyl) -N- (carboxymethyl) -N, N-dimethylammonium bromide (135 mg, 05 mmol) in Dry DMF (3 ml) and the resulting solution was stirred under nitrogen for two hours. Then PAMAM 40 (60 mg, 0012 mmol) in DMF (2 ml) was added, which caused the immediate formation of a flocculent precipitate, which was redissolved slowly The mixture was stirred for two days and then concentrated (104 mmHg 40 ° C) to give a viscous oil The crude product was purified by gel filtration (Sephadex G10, 10% AcOH) to give PAMAM 40 finished with 24 N- (2-acetox? et? l) -N, N- (d? met? lamon? o) met? lcarboxam? da (64 mg) 1 H nmr (D20) d 1 93, 205, 270, 310- groups 360, 328, 393 (m), 414, 448 (m) 13C nmr (D2O) d 246, 262, 332, 387, 428, 429, 539, 574, 626, 673, 675, 1689, 1764, 1768, 1773 , 1832 EXAMPLE 18 Preparation of guanidino-terminated dendrimers PAMAM 40 BRI6042 A solution of PAMAM 40 (63 mg, 0012 mmol) and methylthiopseudourea sulfate (170 mg, 061 mmol) in water (5 ml) (pH 105) was heated under nitrogen at 80 ° C for two hours then concentrated the solution and the residue was purified by gel filtration (Sephadex G10, 10% AcOH) to give PAMAM 40 terminated with 24 guanidm groups such as the acetate salt (107 mg) 1 H nmr (D 2 O) d 200, 280 (br t ), 309 (br t), 332, 345 (br t), 360 (br t) 13 C nmr (D 2 O) d 252, 332, 334, 387, 41 2, 426, 434, 447, 535, 540, 563, 1765, 1767, 1769, 181 6 The corresponding PAMAM 2.0 dendrimer terminated with 6 guanidino groups was prepared in a similar manner.

EXAMPLE 19 Preparation of dendrimers terminated in 4 - ([1,4,8,11-tetraazacyclotetradecane] methyl) benzamide PAMAM 4.0 BRI6041 A solution of 1- (4-carboxyphenyl) methyl-1,4,8,11-tetraazacyclotetradecane tetrahydrochloride (120 mg, 0.25 mmol), N-hydroxysuccinimide (60 mg, 0.52 mmol) and hydrochloride 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (250 mg, 1.3 mmol) in phosphate buffer of pH 7 (10 ml), it remained at room temperature for one hour and then a solution of PAMAM 4.0 (32 mg; 0.006 mmol) in pH 7 phosphate buffer (10 ml). The mixture was allowed to remain for two days and then concentrated. The residue was purified by gel filtration (Sephadex LH20; 10% AcOH) to give PAMAM 4.0 finished with ca. 12 groups of 4 - ([1, 4,8,11-tetraazacyclotetradecane] methyl) -benzamide as determined by 1H and 13C nmr (80 mg). The product was then dissolved in water and passed through a column of Amberlite resin IRA-401 (C19 and then concentrated.The residue was dissolved in water (1 ml), concentrated HCl (1 ml) was added and the solution was diluted with ethanol (30 ml) to precipitate a white solid. The solid was collected by filtration (68 mg). Once again, the 1H and 13C nmr showed ca. 50% of the functionalization of the terminal amino groups 1H nmr (D20) d 2 17, 236 250, 278, 285, 325, 340, 350, 360, 362 449 763 (br d), 778 (br d) 13 C nmr ( D20) d 227 231 332 388 399 402 403 41 0, 41 2, 420, 429, 432, 436, 455, 46 1, 491, 522, 539 543 566, 627, 1325, 1357, 137 1, 1397, 1743, 1762, 1763, 1767, 1770 1782, 1785 EXAMPLE 20 Preparation of dendrimers terminated in 4-carboxy-3-h idroxy benzylamine PAMAM 40 (EDA) BRI6119 Sodium cyanoborohydride (32 mg, 05 mmol) was added to a mixture of PAMAM 40 (EDA) (69 mg, 001 mmol), 4-formyl-2-hydroxybenzoic acid (83 mg, 05 mmol) and sodium hydrogen carbonate (42 mg, 05 mmol) in water (4 ml) The inhomogeneous orange mixture was stirred for four hours at room temperature, during which time it became homogeneous. The orange solution was then concentrated and the residue it was purified by gel filtration (Sephadex LH20, water) to give PAMAM 40 (EDA) terminated with ca 32 groups of 4-carbox? -3-hydrox? benc? lam? na (91 mg) 1H and 13C nmr ( D2O) show mostly monoalkylation, but with some signs of dialkylation of the terminal ammo groups, both spectra show broad peaks 13C nmr (D2O) d 370, 41 1, 509, 534, 555, 558, 61 5, 1209, 1222 , 1224, 1323, 1327, 1350, 1358, 1635, 1637, 1690, 1786, 1793 1H nmr (D2O) d 220, 235, 260, 315, 330, 335, 425, 668, 7 12, 755 EXAMPLE 21 Preparation of finished dendrimers in 4-carboxyphenylamide PAMAM 40 (EDA) Solid 4-carbox? Phenolite was added (86 mg, 048 mmol) to a solution of PAMAM 40 (EDA) (69 mg, 001 mmol) in water (20 ml). The pH of the resulting cloudy solution was adjusted to 9 with saturated NaHCO3 solution and allowed to stir at room temperature for 24 hours. The reaction mixture was then filtered and the filtrate was concentrated to give a white solid residue, which was purified by gel filtration (Sephadex LH20, water) and then freeze-dried to give the product as a fluffy white solid (68). mg) EXAMPLE 22 Preparation of dendrimers terminated in 3,5-dicarboxyphenylamide PAMAM 40 (EDA) Solid 3,5-d? Carbox? Phenol? Sot? Octoate (112 mg, 05 mmol) was added to a solution of PAMAM 40 (EDA) (70 mg, 001 mmol) in water (5 ml) The pH of the resulting cloudy solution was adjusted to 10 with Na2CO31M solution and heated under nitrogen at 53 ° C for 2 hours. The reaction mixture was then filtered and the filtrate was concentrated to give a brownish brown solid residue. , which was purified by gel filtration (Sephadex LH20; ag ua) and then dried by freezing to give the product as a pale brown solid (11.2 mg).

EX EMPLOY 23 Preparation of dendrimers terminated in 4-phosphonooxyfeni lthiourea of sodium PAMAM 4.0 (EDA) Solid 4-phosphonoxyphenylisothiocyanate (251 mg) was added to a solution of PAMAM 4.0 (EDA) (69 mg, 0.01 mmol) in water (20 ml). The resulting solution (pH 9) was stirred for 24 hours at room temperature under nitrogen. The reaction mixture was then concentrated to give a white solid residue, which was purified by gel filtration (Sephadex LH20, water) and then freeze-dried to give the product as a fluffy white solid (86 mg).

EXAMPLE 24 Preparation of dendrimers terminated in 4- (phosphonomethyl) phenylthiourea sodium PAMAM 4.0 (EDA) Solid sodium 4- (phosphonomethyl) phenylisothiocyanate (97 mg) was added to a solution of PAMAM 4.0 (EDA) (69 mg, 0.01 mmol) in water (30 ml).

The resulting solution was stirred for 3 days at room temperature under nitrogen, maintaining the pH at 8 with periodic addition of saturated NaHCO 3 solution. The reaction mixture was concentrated to give a white solid residue, which was purified by gel filtration (Sephadex LH20, water) and then freeze-dried to give the product as a fluffy white solid (10 mg). .

EXAMPLE 25 Preparation of finished dendrimers in sodium 4- (phosphonomethyl) phenylthiourea sodium PAMAM 4.0 (EDA) Solid sodium 4- (phosphonomethyl) phenylisothiocyanate (1.09 mg) was added to a solution of PAMAM 4.0 (EDA) (69 mg, 0.01 mmol) in DMF (30 ml). The resulting solution was stirred for 1 7 hours at room temperature under nitrogen, maintaining the pH at 8 with periodic addition of saturated NaHCO3 solution. The reaction mixture was then concentrated to give a white solid residue, which was purified by gel filtration (Sephadex LH20, water) and then freeze-dried to give the product as a fluffy white solid (30 mg).

EXAMPLE 26 Preparation of dendrimers terminated in 2-thiosiolate bound to Cn-alkyl [(N-hydroxysuccimide ester of 8-octanoic acid) 5-acetamido-4,7,8,8-tetra-O-acetyl-3,5-dideoxy -2-thio-D-glycero-aD-galacto-2-methyl nonulopyranosidjonate, was prepared by the following procedure. To a solution of 5-acetamido-4,7,8,9-tetra-O-acet-l-2-S-acetyl-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2 Methyl nonulopyranosonate (Hasegawa et al, 1986) (1000 mg) in dry dimethylformamide (1 ml), 8-bromooctanoic acid (41 mg) and diethylamine (280 mg) were added, and the solution was stirred at 20 °. C for 1 7 hours. The solvent was removed under vacuum and the residue was partitioned between ethyl acetate and 5% ice cold hydrochloric acid. The organic layer was washed with water, dried over sodium sulfate and evaporated to give a residue (1 30 mg). This was dissolved in ethyl acetate (5 ml) and N-hydroxysuccinimide (26 mg) and dicyclohexylcarbodiimide (46 mg) were added. The mixture was stirred at 20 ° C for 17 hours and then the white precipitate was filtered. The filtrate was concentrated and purified by flash chromatography on silica gel by levigating with ethyl acetate. The product containing fractions were combined and evaporated to give a 97 mg white foam. 71%. They were prepared in a similar manner: [(N-hydroxysuccinimide ester of 1 1 -undecanoic acid) 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero - methyl D-galacto-2-nonulpyranosidejonate. [(N-hydroxysuccinimide acetic acid ester) 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranoside] methyl onate [(Nh? Drox? Succ? N? Mido ester of 4-butane acid? Co) 5-acetamido-4, 7 8, 9-tetra-O-acet? L-3, 5-d? Deox ? -2-t? OD-gl? Zero-aD-galacto-2-nonulopyranosidjonate methyl [(N? Όx? Succ? N? M? Ester of 4-met? Lbenzo? Co acid) 5-acetam? Do -4, 7, 8, 9-tetra-O-acetyl-3, 5-d? Deox? -2-t? OD-gl? Zero-aD-galacto-2-nonulopyranosidjonate of methyl A PAMAM [EDA] 4 0 [(8-octanamido) -5-acetamido-3, 5-d? Deox? -2-t? OD-gl? Cero-aD-galacto-2-nonulop acid? ranos? do? co] 32 BRI61 1 2 To a solution of PAMAM [EDA] 4 0 (50 mg) in dry dimethyl sulfoxide (4 ml) under an inert atmosphere, [(Nh? drox? succ? n? m? octanoic acid ester) 5-acetamido-4,7,8,9-tetra-O-acet? l-3,5-d? deox? -2-t? oDg l? zero-ad- galacto-2-nonulop? anes? d] methyl methasone (300 mg) and the solution was stirred for 60 hours at 20 ° C. The solvent was removed under vacuum and the residue was dissolved in methanol (2 ml). The solvent was removed under vacuum and the residue was dissolved in methanol (2 ml) This solution was subjected to size exclusion chromatography on Sephadex LH20 by levigating with methanol On evaporation of the solvent, the product, PAMAM [EDA] 4 0 [[(8-octanam ? do) 5-acetamido-4,7,8, 9-tetra-O-acet? l-3,5-d? deox? -2-t? oD-gl? cero-aD-galacto-2- nonulop? ions? d] methionate] 32 was obtained as a white powder 1 82 mg 93% This was converted to the free sialoside by the following method To a solution of PAMAM [EDA] 4 0 [[(8-octanamido) 5-acetamido-4, 7, 8,9-tetra-O-acet-l-3, 5-d? Deox? -2-t? OD-gl? Zero-aD-galacto-2-nonulop? Anes? D] methionate] 32 (1 82 mg) in dry methanol (3 ml) under argon at 20 ° C, a freshly prepared 0.1 9 M solution of sodium methoxide in methanol (7 ml) was added and the mixture was stirred for 2.5 hours. The solvent was evaporated, the residue was dissolved in water (10 ml) and stirred for 3 hours. This solution was subjected to size excision chromatography on Sephadex LH20 by levigating with water. On lyophilization, the product, PAMAM [EDA] 4.0 [(8-octanamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-ad-galacto-2-nonulopyranosidoic acid] 32 was obtained as a pale lemon powder 1 1 0 mg. 77%. Using a similar procedure, the following were prepared: PAMAM [EDA] 4.0 [(1 1 -undecanamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranosidoic acid] 32 BRI 6147 PAMAM [EDA] 4.0 [acid (acetamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranoside] 32 BRI 6121 PAMAM [EDA] 4.0 [acid (4- methylbenzamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranosidoic acid] 32 BRI 61 20 B BHAIyslys2lys4lys8lys16 [(8-octanamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranosidoic acid] 32 BRI 61 69 A solution of BHAIyslys2lys lys8lys16 ( t-Boc) 32 (20.3 mg) in a mixture of trifluoroacetic acid (2 ml) and dichloromethane (2 ml) at 20 ° C for 2 hours, then the solvent was removed under vacuum. The residue was dissolved in dry dimethyl sulfoxide (1 ml) and diisopropylethylamine (25 mg) and [(N-hydroxysuccinimide 8-octanoic acid ester) 5-acetamido-4,7,8,8-tetra-O- were added. methyl acetyl-3, 5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranoside] onate (78 mg). The mixture was stirred under argon at 20 ° C for 60 hours, then the solvent was removed under vacuum. The residue was dissolved in a freshly prepared 0.1M solution of sodium methoxide in methanol (2.5 ml) and the mixture was stirred for 3 hours under argon at 20 ° C. The solvent was evaporated and the residue was dissolved in water (1 ml) and stirred for 1 7 hours. This solution was subjected to size exclusion chromatography on Sephadex LH20 by levigating with water. After lyophilization, the product, BHAIyslys2lys4lys8lys16 [(8-octanamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-aD-galacto-2-nonulopyranosidoic acid] 32 was obtained as a white powder 44 mg. 86% EXAMPLE 27 Preparation of dendritic sialosides modified in position 4 of sialic acid. 4-Azido-5-acetamido-7,8,9-trio-O-acetyl-2-S-acetyl-3,4,5-trideoxy was prepared. -2-thio-D-glycero-aD-galacto-2-nonulopyranosonate methyl by the following procedure. To a solution of 4-azido-5-acetamido-7,8,9-tri-O-acetyl-2-chloro-3,4,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate from methyl (Sabesan, 1994) (5 g) in dichloromethane (150 ml) was added powdered fine potassium thiolacetate (5.8 g) and the suspension was stirred vigorously at 20 ° C for 48 hours. The mixture was filtered and evaporated to give a clear brown foam (5.2 g). The required product was isolated by preparative reverse phase HPLC [C 8, 30% acetonitrile / water] as a white foam 3.9 g. 72%. [(N-hydroxysuccinimide 8-octanoic acid ester) 4-azido-5-acetamido-7,8,9-tri-O-acetyl-3,4,5-trideoxy-2-thio-D-glycero-aD- galacto-2-nonulopylans-methyl] ate was prepared by the following procedure to a solution of 4-azido-5-acetamido-7, 8 9-tr? oO-acet-l-2 -S-acetyl-3, 4, 5-tr? Deox? -2-t? OD-gl? Zero-aD-galacto-2-nonu lop? Ranosonate methyl (300 mg) in dry dimethylformamide (3 5 ml ), 8-bromooctanoic acid (1 55 mg) and diethylamine (26 ml) were added, and the solution was stirred at 20 ° C for 1.7 hours. The solvent was removed under vacuum and the residue was partitioned between ethyl acetate and 1 0% ice cold hydrochloric acid The organic layer was washed with water, dried over sodium sulfate and evaporated to give a yellow foam (385 mg). This was dissolved in ethyl acetate (20 ml) and N-hydroxysuccinimide was added. (95 mg) and dicyclohexylcarbodnmide (175 mg) The mixture was stirred at 20 ° C for 1.7 hours, then the white precipitate was filtered. This was concentrated and purified by preparative reverse phase H PLC [C18, 30% acetonitoplo / water] to give a white foam 340 mg 83% A PAMAM [EDA] 4 0 ((8-octanamido) -4-azido-5-acetamido-3,4,5-tr? Deox? -2-t? OD-gl? Zero- aD-galacto-2-nonulop? ions? do? co] 32 BRI 6146 To a solution of PAMAM [EDA] 4 0 (72 mg) in dry dimethyl sulfoxide (5 ml) under an inert atmosphere, [(Nh) drox? succ? n? 8-octane? co) 4-azido-5-acetamido-7,8,9-tr? -O-acet? l-3,4 acid ester, 5-tr? Deox? -2-t? OD-gl? Zero-aD-galacto-2-nonulop? Anes? D] methyl oate (31 8 mg) and the solution was stirred for 60 hours at 20? C The solvent was removed under vacuum and the residue was dissolved in methanol (2 ml). This solution was subjected to size exclusion chromatography on Sephadex LH 20, levigating with methanol. On evaporation of the solvent, the product was obtained, PAMAM [E DA] 4 0 [[(3-octanamido) 4-azido-5-acetamido-7, 8, 9-tr? -O-acet? L-3, 4, 5-tr? Deox? -2-t? OD-gl? Zero-aD-galacto-2-nonu lop? Ranos? D] methionate] 32, as a white foam 225 mg 81% The free sialoside was obtained by the following method To a solution of PAMAM [E DA] 4 0 [[(8-octanamido) 4-azido-5-acetamido-7, 8, 9-tr? OO -acetyl-3,4, 5-tr? deox? -2-t? oD-gl? zero-aD-galacto-2-nonulop? anes? d] methionate] 32 (21 5 mg) in dry methanol (1 ml) under argon at 20 ° C, a freshly prepared 1M solution of sodium methoxide in methanol (1 ml) was added and the mixture was stirred for 3 hours. The solvent was evaporated and the residue was dissolved in ag ua (2 ml) and stirred for 1 7 hours This solution was subjected to size exclusion chromatography on Sephadex LH20, levigating with water On the hofilization, the product, PAMAM [EDA] 4 0 [8-octanam? do) -4-azido-5-acetamido-3,4, 5-tr? deox? -2-t? oD-gl? cero-aD-galacto-2-nonulop? ranos? do? co] it was obtained as a spongy white powder 160 mg 90% B PAMAM [EDA] 4 0 [(8-octannamic acid) -4-am? No-5-acetam? Do-3,4,5-tr? Deox? -2-t? OD-gl? Zero- aD-galacto-2-nonulop? ions? do? co] 32 BRI 6149 A slow steam of hydrogen sulphide gas was passed to a solution of PAMAM [EDA] 4 0 [(8-octanamide) -4- az-do-5-acetamido-3,4, 5-tr? deox? -2-t? oD-gl? zero-aD-galacto-2-nonulop? canos? do? co] 32 (25 mg) in a mixture of pipdin (40 ml) and water (20 ml) at 20 ° C for 5 days The solution was then bubbled with nitrogen for 2 hours -.-. to remove excess hydrogen sulfide. The solution was evaporated to dryness and the residue was taken up in water (5 ml) and filtered through a 0.45 μm membrane filter to remove the sulfur. On freeze drying, the product was obtained, PAMAM [EDA] 4.0 [(8-octanamido) -4-am ino-5-acetamido-3,4,5-trideoxy-2-thio-D-glycero-aD-galacto acid -2-nonulopyranosidoic acid] 32, as a spongy white powder. 23 mg. 96% EXAMPLE 28 Preparation of Dendrimers Finished in Boronic Acid N-hydroxysuccinimide ester of 4-carboxyphenylboronic acid To a solution of 4-carboxyphenylboronic acid (500 mg) in dried formamide dimethyl (5 ml) were added N-hydroxysuccinimide (380 mg) and dicyclohexylcarbodiimide (680 mg). The mixture was stirred at 20 ° C for 64 hours and then the white precipitate was filtered. The solvent was removed under vacuum and the residue was dissolved in ethyl acetate (1000 μl). This solution was washed with water, dried over sodium sulfate and evaporated to give a white solid, which was crystallized from acetonitrile / water as fine needles. 730 mg. 92% PAMAM [EDA] 4.0 [4-benzamidoboronic acid] 32 BRI 6160 To a solution of PAMAM [EDA] 4.0 (69 mg) in dry dimethyl sulfoxide (5 ml) under an inert atmosphere, N-hydroxysuccinimide acid ester was added. -carbophenyloboronic acid (1 30 mg), and the solution was stirred for 65 hours at 20 ° C, the thick paste was added a solution of 1 M sodium carbonate (1 ml) and the clear solution was stirred an additional 24 hours. The solvent was removed under vacuum and the residue was dissolved in a 10% ammonia solution (5 ml). This solution was subjected to size exclusion chromatography on Sephadex LH20, levigating with a 10% ammonia solution. evaporation of the solvent, the product was obtained, PAMAM [EDA] 4 0 [4-benzam? doboron? co] 32 acid as a white fluffy solid 1 1 0 mg 94% EXAMPLE 29 Preparation of 3,6-disu-terminated sodium dinofluorophthylthiourea BHAIyslys2lys4lys8lys16lys32 Tpfluoroacetic acid (2 ml) was added to a stirred suspension of BHAIyslys2lys lys8lys16DBL32 (147 mg) in dry dichloromethane (2 ml) and the resulting solution was stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in N, Nd? met? N-al? lam? na buffer (pH 9.5 5 ml) and then 3.6 isothiocyanate was added. solid sulfone (400 mg) The pH of the mixture was then adjusted to 9 5 by the addition of 1 M sodium carbonate and the solution was heated at 53 ° C for three hours under nitrogen. The residue was concentrated and the residue redissolved in water and the solution was passed through a column of Amberlite IR 1 20 (Na) The filtrate was concentrated to give the crude product, which was purified by gel filtration (Sephadex LH20, water) to give BHAIyslys2 lys4lys8lys1 6lys32 with 64 groups of 3,6-disulfonaphthylurea sodium as a fluffy white solid (1575 mg).

EXAMPLE 30 Preparation of dendrimers terminated in 3,5-disulfofen sodium luthou BHAIyslys2lys4lys8lys16lys32 Trifluoroacetic acid (3 ml) was added to a stirred suspension of BHAIyslys2lys lys8lys16DBL32 (300 mg, 0.02 mmol) in dry dichloromethane (3 ml), The resulting solution was stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in water and the solution was passed through a column of Amberlite I RA 401 (OH) and the filtrate was concentrated to give a viscous oil (187 mg). The oil was dissolved in a 1: 1 mixture of pyridine / water (8 ml) and solid sodium 3,5-disulfophenyl isothiocyanate (680 mg) was added.; 2 mmol). The resulting solution was heated at 53 ° C for three hours under nitrogen. The solution was then concentrated to give a white solid residue. The crude product was purified by gel filtration (Sephadex LH20, water) to give BHAIyslys2lys4lys8lys16lys32 with 64 sodium 3,6-disulfophenylurea groups as a fluffy white solid.

EXAMPLE 31 Preparation of dendrimers terminated in 3,5-dicarboxyphenethylthiourea sodium BHAIyslys2lys4lys8lyS? 6lys32 BRI 6741 Trifluoroacetic acid (3 ml) was added to a stirred suspension of BHAIyslys2lys4lys8lys -? 6DBL32 (300 mg, 0.02 mmol) in dry dichloromethane (3 ml), the resulting solution was stirred at room temperature under nitrogen for two hours and then concentrated. The residue was dissolved in water and the solution was passed through a column of Amberlite I RA 401 (OH) and the filtrate was concentrated to give a viscous oil (86 mg). The oil was dissolved in a 1: 1 mixture of pyridine / water (8 ml) and 3, 5-dicarboxyphenyl isothiocyanate sodium (450 mg, 2 mmol) was added. The resulting solution was heated at 53 ° C for 1 3 hours under nitrogen. The solution was then concentrated to give a residue of white solid. The crude product was purified by gel filtration (Sephadex LH20, water) to give BHAIyslys2lys4lys8lys16lys32 with 64 sodium 3,6-dicarboxyphenylurea groups as a fluffy white solid. The PAMAM 4.0 (EDA) BRI 6195 dendrimer terminated in the corresponding 3, 5-dicarboxyphenylthiourea sodium was prepared in a similar manner.

EXAMPLE 32 Preparation of dendrimers terminated in sodium 4-phosphonooxyphenylthiourea BHAIyslys2lys4lys8lys16lys32 BRI 6181 Trifluoroacetic acid (2ml) was added to a stirred suspension of BHAIyslys2lys4lys8lys16DBL32 (147 mg, 0.01 mmol) in dry dichloromethane (2 ml) and the resulting solution was stirred at room temperature under nitrogen for two hours, and then concentrated to give a viscous oil. The oil was dissolved in N, N-dimethyl-N-allylamine buffer (pH 9.5, 5 ml) and solid 4-phosphonooxyphenyl isothiocyanate (250 mg) was added. The pH of the resulting solution was adjusted to 100 with 1 M sodium carbonate and the mixture was heated at 53 ° C for three hours under nitrogen. The solution was then concentrated to give a white solid residue. The residue was redissolved in water, the solution was passed through a column of Amberlite I R 1 (Na) and the filtrate was concentrated. The residue was then purified by gel filtration (Sephadex LH20, water) to give BHAIyslys2lys4lys8lys16lys32 with 64 sodium 4-phosphonooxyphenylurea groups as a fluffy white solid (150 mg).

EXAMPLE 33 Preparation of thermionized dendrimers in sodium 4-phosphonophenylthiourea BHAIyslys2lys4lys8lyS? 6lys32 Trifluoroacetic acid (2 ml) was added to a stirred suspension of BHAIyslys2lys4lys8lys16DBL32 (147 mg, 0.01 mmol) in dry dichloromethane (2 ml), the resulting solution was stirred at room temperature under nitrogen for two hours and then concentrated to give a viscous oil. The oil was dissolved in N, N-dimethyl-N-allylamine buffer (pH 9.5, 5 ml) and solid 4-phosphonophenyl isothiocyanate (250 mg) was added. The pH of the resulting solution was adjusted to 9 with saturated sodium bicarbonate solution and the mixture was heated at 53 ° C for three hours under nitrogen. The solution was then concentrated to give a white solid residue. The residue was redissolved in water, the solution was passed through a column of Amberlite I R 1 (Na) and the filtrate was concentrated. The residue was then purified by gel filtration (Sephadex LH20; ag ua) to give BHAIyslys2lys4lys8lys16lys32 with 64 groups of sodium 4-phosphonophenylurea urea BRI 61 96 as a fluffy white solid (1 52 mg) after freeze drying.

EXAMPLE 34 Preparation of dendrimers terminated in sodium 4,6-diphosphonaphonaphthyl thiourea PAMAM 4.0 A solution of sodium 4,6-diphosphonaphthyl isothiocyanate (165 mg) in water (2 ml) was added to a solution of PAMAM 4.0 (51 mg; 0.01 mmol) in water (2 ml). The pH of the mixture was adjusted to 9.5 with saturated sodium bicarbonate solution and the mixture was stirred vigorously for one hour at room temperature, then heated at 53 ° C for three hours under nitrogen. The mixture was then filtered and the filtrate was concentrated to give a brown solid residue. The crude product was purified by gel filtration (Sephadex G25, water) to give PAMAM 4.0 terminated with 24 sodium 4,6-diphosphonaphonaphthylthiourea groups as a brown solid (81 mg) after freeze drying.

EXAMPLE 35 Preparation of dendrimers terminated in fluoresceinourea PAMAM 4.0 (EDA) Solid fluorescein isothiocyanate (1 88 mg) was added to a solution of PAMAM 4 0 (EDA) (74 mg, 0 01 m mol) in water (3%). ml) A solution of saturated sodium bicarbonate was added to adjust the pH to 9, the homogeneous solution was stirred overnight at room temperature and then concentrated. The orange residue was purified by gel filtration (Sephadex LH20, ua) to give PAMAM 4 0 (EDA) finished with 21 fluoresceintiourea groups as a fluffy orange solid (1 93 mg) after freeze drying EXAMPLE 36 Preparation of dendrimers terminated in (Phen-3-boronic acid) - sodium thiourea PAMAM 4 0 (EDA) Isothiocyanate was added (phenol-3-boron-co-solid) (1 00 mg, 5 μmol) was added to a solution of PAMAM 4 0 (EDA) (69 mg, 0.01 mmol) in water (5 ml). 1 M sodium carbonate was added to the dissolved isothiocyanate (pH ca 10). The mixture was then heated at 53 ° C for two hours under nitrogen, then filtered and the filtrate was concentrated to give a brownish brown solid residue The crude product was purified by gel filtration (Sephadex LH20; water) to give PAMAM 4 0 (EDA) terminated with 32 groups of (phenol-3-boronic acid) t-ourea as a fluffy white solid (87 mg) after freeze drying EXAMPLE 37 Preparation of dendrimers terminated in pipdinium dodecyl carboxamide Dendpimer PAMAM 20 BRI-6807 The PAMAM generation 20 core (0479 mmol, 50 mg) was evaporated from 05 ml of solution in MeOH and then redissolved in 10 ml of water. to the solution 1-Np? r? d? n? 12-dodecanoic acid bromide (014 g, 0384 mmol), N-hydroxybenzotpazole hydrate [HOBT] (52 mg, 0384 mmol), tetylamine (53? l, 0384) mmol) and 1- (3-d? et? lam? noprop? l-3-et? l) carbod? m HCl [EDC] (74 mg, 0384 mmol) This reaction mixture was stirred overnight at room temperature The volume was reduced to one third under reduced pressure and the solution was subjected to chromatography on an LH20 column using water as the levigant. Fractions containing the product were collected and pipdinium dodecylcarboxamide bromide 20 was isolated as a white solid. spongy by freeze-drying 1H nmr (D2O) d 1 15, 1 45, 1 9, 215, 275, 28, 315, 335, 35, 455, 805, 85, 88 PAMAM 40 BRI-6809 Dendrimer The PAMAM generation 40 nucleus (005 mmol, 69 mg) of 10 ml of solution in MeOH was evaporated and then redissolved in 15 ml of water. 1-Np? Bromide was added to the solution. r? d? n? 12-dodecanoic acid (0143 g, 04 mmol), N-hydroxybenzotpazole hydrate [HOBT] (77 mg, 04 mmol), tetylamine (56 μl, 04 mmol) and 1- (3-d? et? lam? nopropyl-3-ethyl carbodnide HCl [EDC] (77 mg, 04 mmol) This reaction mixture was stirred overnight at room temperature The volume was reduced to one third under reduced pressure and the solution was subjected to chromatography on a LH20 column using 1% tpetilam ina in water as the levigant. Fractions containing the product were collected and the PAMAM bromide 4 0 dodecylcarboxam ida of pipdimo was isolated as a fluffy white solid by freeze drying. react a small amount of the product with acetic anhydride to confirm the complete cover of the fine NH2 groups of the dendrimer nucleus 1 H nmr (D2O) d 1 1 0, 1 45, 1 9, 2 1, 2 30, 2 5, 2 7, 3 2, 4 5, 8 00, 8 45, 8 80 EXAM PLO 38 Preparation of saccharin-terminated dendrimers PAMAM Dendrimer 4 0 BRI-6157 To a solution of PAMAM 4 0 dendrimer nucleus of ethylenediamine core (275 mg, 39 8 uM) in dry dimethylformamide (25 ml) was added isothiocyanate. 6- (benzosulf? Mido) (400 mg, 1 67 mM) and the mixture was stirred at room temperature for 24 h. The cloudy solution was clarified by adjusting the pH with sodium carbonate solution to pH 1 0-1. 0 5 This solution was stirred for a further 24 h and the volatiles were removed on a rotary evaporator. The solution was subjected to chromatography on a large Sephadex LH20 column and the frontal fraction was collected. The remaining fractions were collected and re-subjected to chromatography. In a smaller column, the combined front fractions were evaporated and freeze-dried to yield the finished dendrimer product in saccharin (450 mg).; 78%) as a white fluffy solid. 1 H nmr (D2O): d 2.20, 2.50, 3.23, 3.46, 3.63, 7.52, 7.87. The dendrimer BHAIyslys2lys lys8lys16lys32 ... terminated in corresponding saccharin BRI-6189 was prepared in a similar manner.

EXAMPLE 39 Inhibition of cobra venom and bee venom toxin.

A Materials and methods Cytosensor microphysiometer protocol The cytosensor microphysiometer (Molecular Devices Inc., CA) is a device based on a light-weight, potentiometric sensor that can be used to indirectly measure the metabolic rate of cells in vitro (Parce et al. 1989, McConnell et al., 1992). Metabolism is determined by measuring the rate of production of acid metabolites from immobilized cells within a microvolume flow chamber. Human CEM cells were centrifuged and resuspended in serum free / bicarbonate free RPMI 1640 medium with low buffering (Molecular Devices, hereinafter referred to as "modified medium"). The cells were seeded at a density of 60,000-75,000 cells / capsule on the polycarbonate membrane (porosity of 3 μm) of cups of cellular capsules (Molecular Devices) The cells were immobilized using an agarose trap media (Molecu). lar Devices) Capsu cups sown were transferred to sensor chambers containing the silicon sensor, which detects changes in pH (and thus cellular metabolism). The cytosensor system used for this set of experiments contained eight separate chambers. for the measurement of acidification rates The modified media were pumped through the cells at a rate of 1 00-120 μl / mm Each cell chamber was served by fluid from either of two tanks To measure the rate of acidification, the flow of the modified medium was periodically interrupted, allowing the accumulation of excreted acid metabolites (lactic acid and CO2). In this set of experiments, the flow was stopped for 30s, during which time, a tilt adjustment of least squares for the change in the voltage signal over time, the acidification rate (measured as μ V / s) These velocity data were normalized (using the 4-5 speed points before the addition of the compound) to allow a direct comparison of the signals of the four chambers Acidification rate measurements were made every 2 min The chamber was maintained at 37 ° C The basal acidification rates were monitored (in the absence of any treatment) during at least 30 min. After this time, the poisons / peptides were exposed to the cells at a range of concentrations during periods of h up to 4 h A toxin concentration showing a pronounced effect on the cells, but less than the maximum, was selected to test the inhibition of this toxicity by a range of concentrations of BRI2923 In all the experiments, at least one chamber was not exposed to none of the compounds, providing a negative control BRI 2923 was dissolved in water and the solution was adjusted to a pH of 7 2.

Concentrations ranging from 1000 μM to 1 nM were added to the venom / medium solutions and incubated for periods ranging from 6 min (the lowest possible incubation period using this equipment) to 1 h, and then introduced into the cells All the experiments were repeated in triplicate B Results Inhibition of BRI2923 from cobra venom Crude venom from the forest cobra (Naja malenoleuca) was tested. The cobra venom added to the CEM cells caused an initial increase in the cellular metabolism followed by cell death (cell lysis). The cobra venom was particularly harmful to the cells, causing an initial increase in the metabolism of approximately 80%, followed rapidly by 100% cell death within the first 10 minutes. The poisons were tested imediately at 10, 50 and 100 μg / ml. The maximum response of 50 μg / μl was selected as the test dose in most of the experiments Two concentrations of BRI2923 (1 0 ~ 4 M and 1 0"5 M) were used in all incubation periods (6, 30 and 60 mm), BRI2923 10"5 M incubated with 50 μg / ml of venom, reduced the initial increase in metabolic rate from approximately 80% to approximately 5%, and delayed the onset of cell death by approximately 1 5-20 minutes BRI2923 1 0"4 M, incubated with 50 μg / ml of venom, blocked both the initial increase in metabolic rate, as well as the rapid subsequent cell death seen at this concentration of venom alone. BRI2923 1 0"4 M, incubated with 1 00 μg / ml of venom, also eliminated the initial increase in metabolic rate and the cells proceeded to cell death after the venom / dendrimer solution had been washed. vipers consist of many toxic components, each of which has different modes of toxicity.Cobra venoms contain a cytotoxic peptide, which causes cell lysis in a manner similar to the bee venom toxin, melittin. Amino acid sequence of the cytaxine isolated from Naja malenoleuca indicates that this toxin is highly basic (cationic) and thus, would be rapidly inactivated by polyanionic compounds, such as BRI2923.This electrostatic inactivation as a basis for reduced toxicity, is supported by the experimental finding that an incubation period of 6 minutes gives the same result as the longer incubation periods of 30-60 m inuto s used.

Inhibition of BRI2923 of melitin (main toxin of bee venom) Melitin was added to the CEM cells in average logarithmic doses ranging from 1 0"5 M to 1 0" 7 M. The two highest doses (1 0"5 and 5 x 1 0" 6 M) caused total cell death within 15 minutes without initial activation of the cells. 10"6 M caused a transient increase in cellular metabolic rate followed by complete cell lysis after approximately 1 h 5 x 10" 7 M caused the lysis curve to shift further to the right and 107 M had no effect. This determination of dose response was repeated and 10"6 M melitin was selected as the submaximal concentration to be used with BRI2923. A range of BRI2923 concentrations were incubated with 10 ~ 6 M melitin for 20 minutes, BRI2923 10" 4 M and 10.5 M completely inhibited the toxic effects of melittin. BRI292310 ~ 6 M completely blocked the melitin toxicity for approximately 30 minutes and during the final 30 minutes of exposure, the metabolic rate only fell by approximately 10-15% less than the control cells. Concentrations 10"7 M, 5 x 10" 7 M, and 10"8 M of BRI2923 reduced, but did not prevent, the toxicity, BRI292310" 9 M had no effect on melitin 10"6 M.

EXAMPLE 40 Inhibition of HIV toxin (AIDS) Introduction Vpr is a protein of 96 amino acids, 14 kDa, accessory gene product of the human immunodeficiency virus (HIV-1). The vpr gene is highly conserved in HIV-1, HIV-2 and the Vpr gene product optimizes HIV infection and disease progression (reviewed by Cullen, 1998; Emerman &Malim, 1998). Among its actions, Vpr induces apoptosis and cytopathic effects in both human cells and yeast (Stewart et al, 1997, Zhao et al, 1996, Macreadie et al, 1996). 6 The Vpr protein has been fractionated and the peptide sequence, which causes apoptosis, has been isolated and designed Vpr P3 It has been contrasted that Vpr P3 to permeabilize CD4 + T lymphocytes (such as CEM cells) and causes their death by apoptosis The following experiments use this P3 fraction of toxic Vpr peptide (Arunag iri et al, 1 997) Method using the cytosensor microphysiometer The human CEM cells were centrifuged at 1400 RPM for 7 minutes and resuspended in modified RPM I 1640 medium. The cells were then immobilized using agarose trap media and stained in the center of the polycarbonate membrane of the capsule cup (Molecular Devices Ltd, CA.). The cells were seeded at a density of about 43,000-75,000 cells per cup of capsule. The cups of seeded capsules were transferred to sensor chambers containing the silicon sensor and placed in the microphysiometer. The modified medium was pumped through the cells at a rate of 1 20 μl / min. Each cell chamber was served by fluid from either of two tanks, which could be alternated using a computer program command. The cells were allowed to equilibrate inside the chambers for 30-60 minutes, or until a stable metabolic rate was reached. In this set of experiments, the flow was stopped for 30 s, during which time, a minimum square fit tilt was calculated for the change in the voltage signal with time, the rate of acidification (measured as μV). / s) These velocity data were normalized (using the 4-5 speed points before the addition of the compound) to allow direct comparison of the signals from the four chambers. Acidification rate measurements were made every 2 m in. chamber was maintained at 37 ° C. For all experiments, CEM cells were used at 1 3 x 1 05 to 1 3 x 1 06 cells / ml, corresponding to an early logarithmic phase at the mean growth of these cells. The cells were established. in the microphysiometer and Vpr P3 peptide was diffused through the cell chambers after an equilibrium period of at least 30 minutes. The concentration range used for Vpr P3 was 1 0 5 M - 2 x 1 0 5 M Vpr P3 needed to be in contact with the cells for up to 2 hours so that the full effect of toxicity was evident For experiments that prove the inhibition of Vpr peptide toxicity by BRI2923, solutions of Vpr P3 peptide medium were made at the appropriate concentration and BRI2923 was added, the solution was mixed and the equilibrium was allowed to reach for 20-30 minutes before placing it in the cytosensor. With each inhibition experiment, it was also positive control channel containing the same concentration of Vpr P3 used to test the inhibition of BRI2923 for the same period, as well as a time control channel The peptide solution Vpr P3 and the solution of Vpr P3 / BRI2923 were in contact with cells for a minimum period of 2 h 30 min at a maximum period of 4 h In addition, after washing the compounds, the metabolic rate of the cells was monitored for a short period (minimum of 6 min).

Results In the cytosensor system, the VPR P3 peptide, tested by itself, causes an initial increase in metabolic rate followed by a decrease and subsequent cell death due to the apoptosis of the EM cells. In the experiment shown in Figure 1, the VPR P3 peptide (at 10"5 M) was preincubated with the BRI2923 compound at 10" 4 M (final volume) in a total volume of 1 ml (modified RPMI medium) for 30 minutes . The solutions were then diluted to final volumes (25 μl) and diffused through the cells. A large initial fall in metabolic rate is seen in the chamber of BRI2923 / Vpr P3, due to a combination of the pH difference of the solution of BRI2923 1 0"4 M (acid) and medium (neutral) and a the intrinsic dampening effect of the BRI2923 dendrimer by itself To answer for this drop in metabolic rate, a correction factor has been applied to all the results using BRI2923. The correction factor used was: a) the initial fall calculated in the first point of data added to all subsequent data points, and b) in the same way, the calculated washing effect was subtracted from all points after washing.The corrected graph is shown in Figure 2. This result shows that BRI2923, at 1 0"4 M, gives full protection against toxic effects (apoptosis) induced by VPR P3 at 10 ~ 5 M. This experiment was repeated in quadruplicate. Additional BRI 2923 concentrations were tested against higher concentrations of Vpr P3, and the results are shown in Figures 3 and 4. BRI 2923 at 10 0"5 M also demonstrated inhibition of Vpr-induced apoptosis P3 2x 1 0" 5 M (experiment in triplicate). BRI2923 1 0"6 M showed a significant reduction in the effects produced by Vpr P3 2x1 0" 5 M (experiment in triplicate). BRI2923 10"7 M in the toxicity of Vpr P3 2x1 0" 5 M, attenuate to a lesser degree the rate of decrease in metabolic rate (experiment in duplicate). Unlike time controls, the cells in the chamber containing BRI2923 did not show any time-related decrease in metabolic rate. Previous cytosensor experiments using BRI2923 have also suggested that the dendrimer can confer a cytoprotective effect in addition to the inhibition of apoptosis caused by VPR P3.

EXAMPLE 41 Calcein Release Assay to Deter Inhibition of Cholera Toxin Materials Cell line • Human ileoceral carcinoma epithelial cells HPC-8 Calcein, AM • Molecular probes, catalog # C-1430 • C46H46N2O23, molecular weight 994.87 • Resuspend the vial of 1 mg in 1Oμl of dimethylsulfoxide anhydrous (DMSO) grade reactive, to give a stock concentration of 1.0 mM.

Toxin • Cholera Toxin (Sig ma Cat. No. C8052) - A subunit surrounded by five subunits B.

Inhibitory dendrimers • Dendrimers test - BRI291 3 and BRI2999.

Method 1 HC P-8 (adherent human ileocecal carcinoma epithelial cell line) in logarithmic growth phase was trypsinized (trypsin EDTA) in 2 ml / 75 cm 2 flasks after 2 washes with PBS (phosphate buffered saline). 2. The cells with trypsin solution were incubated for 5 min. 3. 20 ml of culture medium were added to the flask to inactivate the trypsin. 4. The cell solution was transferred to a 50 ml centrifuge tube and rotated at 1200 RPM for 7 min at room temperature (RT). 5. Cells were washed in PBS and spun at 1200 RPM for 7 min at RT. 6. The cells were resuspended in PBS and counted, then greased at 1200 RPM for 7 min at RT and the supernatant discarded. 7. Cells were resuspended in 1 ml of PBS with 5 μl of calcein added, and incubated at 37 ° C for 45 min. 8. The cells were washed in PBS (approximately 5-20 ml) and resuspended in PBS to give the final numbers of 5x1 05 cells / 100 μl. 9. 1 μl of cellular solution (5x1 05 cells per well) were added in each used cavity of a 96-well plate with 50 μl of cholera toxin (dissolved in H2O) and 50 μl of dendrimer solutions (stock solution made at 10mM in DMSO and diluted in PBS). Each concentration was repeated in triplicate. 1 0. The plate was incubated for 2 h at 37 ° C in a 5% CO2 incubator. eleven . The supernatant was collected and the fluorometric analysis was performed.

Results The preliminary experiment was carried out. All results presented are the average fluorescence intensity of three replicates. % inhibition = 100 - (Fl toxin + dendrimer / Fltoxin x 100) 7 References Arunagiri, C.K., Macreadie, I.G., Hewish, D.R. and Azad, A. (1997) A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4 + lymphocytes. (A C-terminal domain of the accessory Vpr protein of HIV-1 is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4 + lymphocytes). Apoptosis, 2, 69-76 Cullen, B.R. (1998) HIV-1 auxiliary proteins: making connections in dying cells. (Auxiliary proteins of HIV-1: making connections between dying cells). Cell, 93, 685-692. Emerman, M., and Malim, M.H. (1998) HIV-1 regulatory / accesory genes: keys to viral unraveling and host cell biology. (Regulatory genes / accessories of HIV-1: keys to decipher the biology of host and viral cells). Science, 280, 1880-1883. McConnell, H.K., Owicki, J.C., Parce, J.W., Miller, D.L., Baxter, G.T., Wada, H.G. and Pitchford, S. (1992). The cytosensor microphysiometer: biological applications of silicon technology. (The cytosensor microphysiometer: biological applications of silicon technology). Science, 257, 1901-1912. Macreadie, I.G., Arunagiri, C.K., Hewish, D.R., White, J.F., and Azad, A. (1996) Extracellular addition of a domain of HIV-1 Vpr containing the aminoacid sequence motif H (S / F) RIG causes cell membrane permeabilization and death. (The extracellular addition of a Vpr domain of HIV-1 containing the amino acid sequence motif H (S / F) RIG causes cell membrane permeabilization and death). Mol. Microbe!., 19 (6), 1185-1192. Parce, J.W., Owicki, J.C., Kercso, K.M., Sigal, G.B., Wada, H.G., Muir, V.C., Bousse, L.J., Ross, K.L., Sikic, B.l. and McConnell, H.M. (1989). Detection of cell-affecting agents with a silicon biosensor. (Detection of agents that affect cells with a silicon biosensor). Science, 246, 243-247. Stewart, S.A., Poon, B., Jowett, J.B.M. and Chen, I.S. (1997) Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. (Vpr of human immunodeficiency virus type 1 induces apoptosis following the arrest of the cell cycle). J. Virol, 71, 5579-5592.

Zhao, Y., Cao, J., O'Gorman, MRG, Yu, M., and Yogev, R. (1996) Effect of human immunodeficiency virus type 1 protein R (Vpr) gene expression on basic cellular function of fission yeast Schizoasaccharomyces pombe. (Effect of gene expression of protein R (Vpr) of human immunodeficiency virus type 1, on the basic cellular function of the fission yeast Schizoasaccharomyces pombe). J. Virol. 70, 5821-5826.

Claims (11)

1. REVIVAL DIVACTION 1 . A method of prophylactic or therapeutic inhibition of a toxic substance or material in a human or animal patient, which comprises administering to the patient an effective amount of a dendrimer having a plurality of ther nal end groups, wherein at least one of said terminal groups has an anionic or cationic containing portion bound or bonded thereto.
2. 2. A method according to claim 1, wherein said compound is a dendrimer, which comprises a polyvalent core covalently linked to at least two dendritic branches and extending through at least two generations.
3. 3. A method according to claim 2, wherein said dendrimer is a polyamidoamine dendrimer based on an ammonia core.
4. 4. A method according to claim 2, wherein said dendrimer is a polyamidoamine dendrimer based on an ethylene diamine core.
5. 5. A method according to claim 2, wherein said dendrimer is a polylysine dendrimer based on a benzhydrylamine core or other suitable core.
6. 6. A method according to claim 2, wherein said dendrimer is a poly (propylene imine) dendrimer.
7. 7. A method according to claim 2, wherein said compound is a polyionic dendrimer of the general formula I: where: I is an initiating core; Z is an indoor branching unit; n is an integer representing the number of generations of the dendrimer; and A is an anionic or cationic containing portion, which may be linked to an internal branching unit Z through an optional linker group X.
8. A method according to any of claims 1 to 7, wherein said compound , said anionic or cationic containing portion or portions are attached to amine, hydrogen sulfide, hydroxy or other reactive end groups of the dendrimer by amide or thiourea linkages.
9. 9. A method according to any one of claims 1 to 8, wherein in said compound, said anionic or cationic containing portions are selected from the group consisting of sulfonic acid containing portions, carboxylic acid containing portions (including acid-containing portions). single and sialic neuram, and portions containing modified neuramic and sialic acid), portions containing boronic acid, portions containing phosphoric and phosphonic acid (including portions containing esterified phosphoric and phosphonic acid), primary, secondary, tertiary or quaternary amino containing portions , pyridinium containing portions, guanidinium containing portions, amidinium containing portions, phenol containing portions, heterocycles possessing acidic or basic hydrogens and zwitterionic containing portions.
10. 1 0. A method according to any one of claims 1 to 9, wherein in said compound, the portion or portions that are attached to amino or other reactive end groups of the dendrimer are selected from the following groups, wherein n is zero or a positive integer: H (CH2) nS03- (CH ^ SC SC) n -CH2CH (S03 ') COOH CH (S03") CHzCOOH A ^ CH ^ SO," X-OS NH + + - (CH2) "NMe3 Ar (NMe3) "Ar (CHjN e3)" - ArXP (= O) (OR) 2 X = O, CH 2, CHF, CF 2 R = alkyl, aryl, H, Na. - ArXP (= O) (OR) (NR2R3) X = O, CH2, CHF, CF2 Realkyl, aryl, H, Na R2, R3 = alkyl, aryl-Ar [P (= O) (OR) 2] n R = alkyl, aryl, H, Na n = 1-3 - Ar [B (OH) 2] nn = 1-3 - Ar [COOH] nn = 1-3 R = alkyl or arylalkyl; R ,, R2, R3 (which may be the same or different) = alkyl or arylalkyl I rent
11. 11. A method according to any of claims 1 to 10, wherein said compound is selected from the group consisting of: i. dendrimers terminated in alkylsulfonic acid; ii. dendrimers ending in sulfoacetamide; iii. dendrimers terminated in sulfosuccinnamic acid; iv. Dendrimers ending in N- (2-sulfoethyl) succinamide; dendrimers terminated in 4-sulfophenylthiourea; VI dendrimers terminated in 3,6-di-sulfonaphthylthiourea; il dendrimers ending in 4-sulfonphthylthiourea; viii. dendrimers terminated in 3,5-di-sulfophenyl thiourea; ix. dendrimers terminated in 3,6,8-tri-sulfonaphthylthiourea; X. dendrimers terminated in 4- (sulfomethyl) benzamide; xi. dendrimers terminated in 4-sulfobenzamide; XÜ. dendrimers terminated in N- (4-sulfophenyl) propanamide; xiii dendrimers terminated in 4-sulfophenylurea; xiv dendrimers terminated in N, N, N-tri-methylglycinamide; XV dendrimers terminated in 4-trimethylammonium benzamide; xvi Dendrimers terminated in 4- (trimethylammoniomethylene) benzamide; xvii. dendrimers terminated in N- (2-acetoxyethyl) -N, N- (dimethylammonium) methyl carboxamide; xviii. dendrimers finished in guanidino; xix. dendrimers ending in 4 - ([1, 4, 8, 1 1 - tet raazaciclotetradeca no] metí I) benzamide; xx. dendrimers terminated in 4-carboxy-3-hydroxy-benzylamine; xxi. dendrimers terminated in 4-carboxyphenylamide; xxii. dendrimers terminated in 3,5-dicarboxyphenylamide; xxiii. dendrimers ending in 4-phosphonooxyfenylthiourea; xxiv. dendrimers ending in 4- (phosphonomethyl) phenylthiourea; xxv. dendrimers terminated in ethyl-4- (phosphonomethyl) phenylthiourea; xxvi. dendrimers terminated in (8-octanamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxvii. dendrimers terminated in (1 1 -undecanamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxviii. dendrimers terminated in acid (acetamido) -5-acetamido-3, 5-dideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxix. dendrimers terminated in (4-butanamid) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxx dendrimers terminated in (4-methylbenzamido) -5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxxi. dendrimers terminated in (8-octanamido) -4-azido-5-acetamido-3,4,5-trideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxxii. dendrimers terminated in (8-octanamido) -4-amino-5-acetamido-3,4,5-trideoxy-2-thio-D-glycero-a-D-galacto-2-nonulopyranosidoic acid; xxx! 11 dendrimers terminated in 4-benzamidoboron? Co, xxxiv dendrimers terminated in 3, 5-d? Carbox? Phen? Lt? Ourea, xxxv dendrimers terminated in 4-phosphonoox? Fen? Lt? Ourea, xxxvi dendrimers finished in 4 phosphonofen? lt? ourea, XXXV II dendrimers terminated in 4,6-d? fosfononaft? lt? ourea, XXXVI II dendrimers terminated in fluoresceintiourea, xxxix dendrimers terminated in (fen? l-3-boron acid? co) - t? ourea, xl dendrimers terminated in dodecylcarboxamide of pipdinium, and xli dendrines terminated in saccharin 1 2 A method according to any of claims 1 to 11, wherein said treatment comprises the inhibition of toxins and toxic peptides of origin biological or toxins and toxic peptides released during infection with bacteria, protozoa, fungi or viruses 1 3 A pharmaceutical or veterinary composition for prophylactic or therapeutic inhibition of a toxic substance or material in a human or animal, which comprises a dendritic ammonium or cationic ammonium as defined in any of claims 1 to 11, in association with at least one pharmaceutically or severely acceptable carrier or diluent. The use of an ammonium or cationic dendrimer as defined in any of claims 1 to 1. 1, in, or in the manufacture of a medicament for, the prophylactic or therapeutic inhibition of a substance or toxic material in a human or animal RES UMEN A method of prophylactic or therapeutic inhibition of a toxic substance or material in a human or animal patient, comprises administering to the patient an effective amount of a dendrimer having a plurality of terminal groups, wherein at least one of the terminal groups has an anionic or cationic moiety linked or bonded thereto.