MXPA96006421A - Dendrimeros antivira - Google Patents

Abstract

An antiviral compound comprises a dendrimer, such as a polyamidoamine or polylysine dendrimer having a plurality of end groups, wherein at least one of the end groups has an anionic or cationic containing moiety linked thereto, particularly a moiety which contains sulfonic acid, which contains carboxylic acid, or contains trimethyl ammonium, or the like

Description

ANTIVIRAL DENDRIMEROS FIELD OF THE INVENTION This invention relates to antiviral agents, and in particular it relates to dendrimers that have been found to have significant antiviral activity against the human immunodeficiency virus (HIV) and other enveloped viruses.

BACKGROUND OF THE INVENTION It has been established that certain sulfonated polysaccharide compounds have an antiviral activity when they are screened against the human immunodeficiency virus; however, these compounds are relatively unstable, and according to the same, large amounts of these compounds are required to obtain effective antiviral effects. In addition, many of these compounds, including heparin and dextran sulfate, for example, are potent anticoagulants, and because of this activity, they are not particularly suitable for clinical use as antiviral agents. The present invention provides a new class of antiviral agents based on a particular type of polymer referred to herein, which have substantial antiviral activity against HIV1 and HIV2, CMV and HSV, and which substantially do not have anticoagulant activity. Accordingly, these compounds are suitable for prophylactic and therapeutic use as antiviral agents in humans.

SUMMARY OF THE INVENTION According to the present invention, there is provided an antiviral compound comprising a dendrimer having a plurality of terminal groups, wherein at least one of these terminal groups has an anionic or cationic containing fraction, different from a fraction of 2-thiosalicylic acid, linked to them. That dendrimer is referred to herein as an "anionic or cationic dendrimer", and this term is used throughout all of this specification and in the following claims, to include not only dendrimers by themselves, but also their pharmaceutically salts or veterinarily acceptable, for example, the alkali metal or alkaline earth metal salts, such as the sodium, potassium or calcium salts.

DETAILED DESCRIPTION OF THE INVENTION Dendrimers are highly branched macromolecular compounds formed by repetitive reaction sequences, starting from an initial core molecule with layers or successive stages that are added in successive "generations" to accumulate a highly ordered three-dimensional polymeric compound. In Figure 1 a generalized dendrimer structure is shown. The dendrimers are characterized by the following features: (i) an initiator core (1), which may have one or more reactive sites, and may be pointed or of a significant size to affect the final topology of the dendrimer; (ii) layers of branched repeat units attached to the initiator core; (iii) functional terminal groups (Z) attached to the surface of the dendrimer. The present invention uses dendritic structures as frames for the union of ionic tractions; The invention is not limited to the spherical dendrimers described in detail herein, but can be based on any dendritic structure. The variety of dendrimers, both in form and in constitution, is well known to those skilled in the art. The preparation of dendrimers is well known, and is described by way of example in U.S. Patent Nos. 4,289,872 and 4,410,688 (which disclose dendrimers based on layers of units of Usin), as well as in U.S. Pat. from North America Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329 (which describe dendrimers based on other units, including polyamidoamine or PAMAM dendrimers). The dendrimers described in these patents of the United States of North America are described, suitable for uses such as surface modifying agents, as metal chelating agents, as demulsifiers or as oil-in-water emulsions, as agents for resistance to moisture in the papermaking, and as agents for modifying the viscosity in aqueous formulations, such as paints. It is also suggested in U.S. Patent Nos. 4,289,872 and 4,420,688 that dendrimers based on Usin units can be used as substrates for the preparation of pharmaceutical dosages. International Patent Publications Numbers WO 88/001178, WO 88/01179 and WO 88/01180 describe conjugates wherein a dendrimer is conjugated or associated with another material, such as a pharmaceutical or agricultural carrier material. These patent publications, together with the aforementioned US Patent, contain a broad description of different dendrimers and processes for their preparation, and the description of each of these publications is incorporated herein by reference. The term "dendrimer" as used herein, should be understood in its broadest sense, to include within its scope all forms and compositions of these dendrimers as described in Patent Publication Numbers WO 88/01178, WO 88 / 01179 and WO 88/01180. 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 with at least two dendritic branches, and preferably extending through at least two generations. Particularly preferred dendrimers are polyamidoamine dendrimers (PAMAM), PAMAM dendrimers (EDA) and polylysine dendrimers. In accordance with the present invention, at least one, and preferably a substantial number, of the end groups on the surface of the dendrimer has an anionic or cationic containing moiety covalently bonded thereto. Branches of the dendrimer can terminate into amino groups or other functional reactive groups, such as OH, SH, or the like, which can subsequently be reacted with the cationic and anionic fractions that form the outer layer of the dendrimer. Where the terminal groups of the dendrimer are amine groups, the anionic or cationic containing fraction can be linked to the dendrimer by a variety of functional groups, including amide and thiourea linkages. Preferred anionic or cationic containing fractions which can be linked to the terminal groups of the dendrimer include the fractions containing sulfonic acid, the carboxylic acid-containing fractions different from the 2-thiosalic acid fractions, the trimethyl ammonium containing fractions, and the fractions containing polyamine-macromocycle. Suitable anionic and cationic containing fractions which can be linked to the amino groups or other terminal groups of the dendrimers, include, by way of example, the following groups (wherein n is zero or a positive integer, more particularly n is zero or an integer from 1 to 20): -NH (CH2) pS03 '- (CH2) nS03- • Ar (S03-) n -ArX (CH2) nS03- X - O.S, NH - CH2CH (S03") COOH - CH (S031CH2COOH - (CH2) nNMe3 -A. (N e3) n • Ar (CH2NMe3) n Particular fractions that can be linked to the terminal groups of the dendrimer according to this invention include the alkylsulfonic acid groups; the sulfoacetamide groups; the sulfosuccinamic acid groups; the N-sulfoalkyl succinamide groups, such as the succinamide N- (2-sulfoethyl) groups; aryl or heteroaryl thioureas substituted with one or more sulphonic acid groups, such as 4-sulfophenyl thiourea groups, 3,6-disulfonaphthyl thiourea groups, 4-sulfonaphthyl thiourea groups, thiourea 3,5-disulfoenyl groups, and thiourea 3, 6, 8-trisulfonaphthyl groups; the aryllic or heteroaryl amides substituted with one or more sulfonic acid, sulfoalkyl, sulfoalkoxy, sulfoalkylamino or sulfoalkylthio groups, such as 4- (sulfomethyl) benzamide groups, or 4-sulfobenzamide groups; aryl or heteroaryl alkamides substituted with one or more sulfonic acid groups, such as N- (4-sulfophenyl) propanamide groups; aryl or heteroaryl ureas substituted with one or more sulfonic acid groups, such as 4-sulfophenyl urea groups; N, N, N-trimethyl amino acid derivatives, such as glycinamide N, N, N-trimethyl groups; aryl or heteroaryl amides substituted with one or more trialkylamino, trialkylaminoalkyl, trialkylaminoalkyloxy, trialkylaminoalkylamino, or trialkylaminoalkylthio groups, such as 4-tri-ethyl ammonium benzamide, or the benzamide 4- (tricyclic-methyl ammonium) groups; the carboxamide N- (2-acetoxyethyl) -N, N- (dimethylammonium) methyl groups; the guanidino groups; the 4-carboxy-3-hydroxybenzyl amine groups; or macrocyclic polyamino groups containing one or more macrocyclic rings connected through an alkyl or aryl splitting moiety with the terminal group of the dendrimer, such as the benzamide groups 4 - ([1,4,8,10-tetraazacyclotetradecan ] methyl).

The anionic or cationic dendrimers of this invention can be prepared by conventional chemical methods that are well known to those skilled in the art. Suitable methods are described by way of example in Examples 1 to 20 below. As described above, the anionic or cationic dendrimers of the present invention exhibit significant antiviral activity, particularly against HIV. Accordingly, these anionic or cationic dendrimers are useful in the prophylactic and therapeutic treatment of viral infections, for example, HIV1 and HIV2 infections, and other enveloped viruses, including flaviviruses, such as Hepatitis B and Hepatitis C viruses, Bovine Viral Diarrhea Virus, Human Influenza A and B Virus, Rhinovirus, Human Parainfluenza Virus, Respiratory Syncytial Virus (RSV); Varicella Zoster Virus (VZV), Human Cytomegalovirus (CMV), Epstein Bar Virus (EBV). Human Papilloma Virus (HPV), Adenovirus-8, Herpes Simplex Virus (HSV) type 1 and 2, Measles Virus and Vesicular Stomatitis Virus (VSV). Accordingly, in another aspect, the present invention provides a pharmaceutical or veterinary composition for the prophylactic or therapeutic antiviral treatment of a human or non-human animal, which comprises an anionic or cationic dendrimer, as broadly described above, in association with at least one pharmaceutically or veterinarily acceptable vehicle or diluent. The formulation of these compositions is well known to those skilled in the art. Suitable pharmaceutically acceptable carriers and / or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of these media and agents for pharmaceutically active substances is well known in the art, and is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except when any conventional medium or agent is incompatible with the active ingredient, the use thereof in the pharmaceutical compositions of the present invention is contemplated. Complementary active ingredients can also be incorporated into the compositions. It is especially convenient to formulate compositions in a unit dosage form for ease of administration and uniformity of dosage. The unit dosage form as used herein, refers to physically separate units suitable as unitary dosages for the human subjects to be treated; each unit, must contain a predetermined amount of active ingredient, calculated to produce the desired therapeutic effect in association with the vehicle and / or pharmaceutical diluent required. The specifications for the novel unit dosage forms of the invention without dictated by, and directly depend on: (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technique of the composition, such as an active ingredient for the particular treatment. In another aspect, the present invention provides a method for prophylactic or therapeutic treatment of an HIV or other viral infection in a human or in a non-human animal, which comprises administering to this human or animal, an effective antiviral amount of a prophylactic or therapeutic manner, of an anionic or cationic dendrimer, as broadly described in the foregoing. In still another aspect, this invention provides the use of a prophylactic or therapeutic effective antiviral amount of an anionic or cationic dendrimer, as broadly described in the foregoing., in the prophylactic or therapeutic treatment of, or in the manufacture of, a medicament for prophylactic or therapeutic treatment of an HIV or other viral infection in a human being or in a non-human animal. There are a variety of administration routes available. The particular mode selected will depend, of course, on the particular condition being treated, and on the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, can be practiced employing any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention, without causing clinically unacceptable adverse effects. These modes of administration include the oral, rectal, local, nasal, transdermal or parenteral (eg, subcutaneous, intramuscular and intravenous) routes. Formulations for oral administration include separate units, such as capsules, tablets, dragees and the like. Other routes include intrathecal administration directly into the spinal fluid, direct introduction, such as by different balloon and catheter angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection in target areas. The compositions may conveniently be presented in a unit dosage form, and may be prepared by any of the methods well known in the art of pharmacy. These methods include the step of placing the active component in association with a vehicle, 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 then, if necessary, shaping the product. The compositions of the present invention suitable for oral administration can be presented as separate units, such as capsules, lozenges, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or in a non-aqueous liquid, such as a syrup, an elixir, 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 suitable dispersing 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 may be employed 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 may be employed, including synthetic mono- or di-glycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectables. Other application systems may include sustained release application systems. Preferred sustained release application systems are those that can provide a release of the active component of the invention in sustained release granules or capsules. There are many types of sustained release application systems available. These include, but are not limited to: (a) erosional systems where the active component is contained within a matrix, and (b) diffusion systems, where the active component is permeated at a controlled rate through a polymer . In addition, you can use an application system with hardware based on pumping, some of which are adapted for implantation. The active component is administered in prophylactically or therapeutically effective amounts. A prophylactically or therapeutically effective amount means the amount necessary to obtain at least partially the desired effect, or to delay the establishment of, inhibit the progress of, or totally stop, the establishment or progress of the particular condition being treated.

These amounts will, of course, depend on the particular condition being treated, the severity of the condition, and the 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 with no more than routine experimentation. In general it is preferred that a maximum dose be used, that is, the highest safe dose according to good medical judgment. It will be understood by ordinary experts in the field, however, that a lower dose or a tolerable dose may be administered for medical reasons, psychological reasons, or virtually for any other reason. In general, daily oral doses of the active component will be from about 0.10 milligrams / kilogram per day to 1000 milligrams / kilogram per day. Small doses (from 0.01 to 1 milligram) may be administered initially, followed by increasing doses to approximately 1000 milligrams / kilogram per day. In the event that the response of a subject is insufficient with these doses, even higher doses (or higher effective doses through a different, more localized application route) can be used to the extent that the patient's tolerance allows it. . Multiple doses per day are contemplated to achieve appropriate systemic levels of the compounds.

Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", or variations such as "comprises", or "comprising", shall be construed to imply the inclusion of a declared integer or integer group, but not the exclusion of any other integer or group of integers. Other 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, the PAMAM dendrimers refer to polyamidoamine dendrimers based on an ammonia core as detailed in U.S. Patent Nos. 4,507,466, 4558,120, 4,568,737 and 4,587,329: PAMAM dendrimers (EDA ) refer to polyamidoamine dendrimers based on an ethylene diamine core; and the BAHAlys? lysylysz dendrimers, refer to non-symmetrical polylysine dendrimers based on a benzhydryl amine nucleus, and branch units of Usin, as described in U.S. Patent Nos. 4,289,872 and 4,410,688. Dendrimers polyamidoamine 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 BHAlyslys2, BHAlyslys2 Lys4, BHAlyslys2 lys4lys8 and BHAlyslys2lys4lys8lys16, BHAlyslys2lys4lys8lys16lys32, BHAlyslys2lys4lys8lys16lys32lys54 , or higher generations are prepared as described in U.S. Patent Nos. 4,289,872, 4,410,688, 4,507,466, 4,558,120, 4,568,737 and 4,587,329, and in International Patent Publications Numbers WO 88/01178, WO 88/01179 and WO 88/01180 referred to above.

EXAMPLE 1. Reaction of polymers with propan-sulfonic acid of 2-acrylamido-2-p.ethyl, to give dendrimers terminated in sulfonic acid.

A. PAMAM 1.0 Solid sodium carbonate (0.13 grams, 1.0 mmol) was slowly added to a stirred solution of 2-acrylamido-2-methyl propanesulfonic acid (0.41 grams, 2.0 mmol) in water (3 milliliters). After the evolution of gas ceased, the pH of the solution was 8.0. Then a solution of PAMAM 1.0 (0.12 grams, 0.33 millimoles) in water (1 milliliter) was added to the solution, followed by the addition of four drops of a 40 percent aqueous solution of benzyltrimethyl ammonium 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 the sulfonated PAMAM 1.0 dendrimer as a white solid (0.51 grams). The spectra of ""? and 13C showed a mixture of dialkylated and monoalkylated PAMAM 1.0 dendrimer (approximately 70:30). 13C nm (D20): 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 (Compound No. 20) PAMAM 2.0 was reacted with 2-acrylamido-2-methyl propanesulfonic acid as described above. The crude product was purified by gel filtration (Sephadex Gl ?; water), and then freeze dried to give a white solid. The spectra of 1H and 13 C NMR showed a dialkylated and monoalkylated PAMAM 2.0 dendrimer mixture (approximately 65:35). 1JC rmn (D20): 6 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 benzyltrimethyl ammonium hydroxide, a similar result was obtained.

C. PAMAM 3.0 The PAMAM 3.0 was reacted with 2-acrylamido-2-methyl propanesulfonic acid as above, except that a slight excess of sodium carbonate was used, and the benzyltrimethyl ammonium hydroxide was omitted.

The "" -H and 13C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 3.0 dendrimer (approximately 50:50). 13C rmn (D20): 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, 179.9, 179.0, 179.8.

D. PAMAM 4.0 PAMAM 4.0 was reacted with 2-acrylamido-2-methyl propanesulfonic 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 (approximately 35:65). 13C rmn (D20): 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 grams, 1.5 millimoles) in dry dimethyl formamide (1 milliliter) was added to a stirred solution of PAMAM 1.0 (0.18 grams, 0.5 millimoles) in dimethyl formamide (3 milliliters) ). The resulting yellow solution was stirred for 20 hours at room temperature, when a 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 (2 times), and finally with ethyl acetate. The aqueous solution was concentrated (35 ° C / 25 mmHg), to give the milliliter) of bromoacetylated PAMAM 1.0 as a yellow oil (0.36 grams, 100 percent). 13C nm (D20): d 32.8, 33. 3, 43.0, 43.5, 54.4, 174.5, 176.4. A solution of sodium sulfite (0.2 grams, 1.6 millimoles) in water (1 milliliter) was added to a solution of milliliters) of bromoacetylated PAMAM 1.0 described above (0.36 grams, 0.5 millimoles) in water (5 milliliters), and the solution it was allowed to stand at room temperature for eleven days. The yellow solution was concentrated to give a yellowish solid (0.60 grams). 13C nm (D20): 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, simply by 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-nitrophenyl bromoacetate (0.18 grams, 0.7 mmol) in dry dimethyl formamide (1 milliliter) was added to a stirred solution of PAMAM 2.0 (0.10 grams, 0.1 mmol) in dimethyl formamide (3 mL). milliliters). The resulting yellow solution was stirred for 20 hours at room temperature, when the ninhydrin test was negative. Then the solution was swirling with water (150 milliliters), and the mixture was extracted with chloroform (3 times) and ethyl acetate. A solution of sodium sulfite (0.1 grams, 0.8 mmol) in water (1 milliliter) 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 PAMAM 2.0 dendrimer finished in sodium sulphoacetamide (103 milligrams). 13C nm (D20): d 33.0, 35.7, 36.0, 37.7, 40.3, 43.0, 43.2, 53.4, 53.7, 56.0, 61.6, 171.2, 174.6, 178.5.

Method 2: Solid succinimidyl acetylthioacetate was added (67 milligrams; 0.05 millimoles) to a PAMAM 2.0 solution (52 milligrams; 0.05 millimoles) in dry dimethyl formamide (2 milliliters), and the resulting solution was stirred at room temperature for two days. Then the mixture was concentrated (30 ° C / 10 ~ 3 mmHg) to give an oily residue. The residue was partitioned between water and chloroform, and the water layer was separated and concentrated to give a viscous oil (117 milligrams). ? H and 13C nmr showed that the oil was a mixture of the asylated dendrimer and N-hydroxysuccinimide. Gel filtration (Sephadex G10, water) provided a pure sample of the PAMAM 2.0 dendrimer terminated in acetyl thioacetamide (29 milligrams). 13C nm (D20): 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 percent aqueous formic acid (7 milliliters) was then added to a freshly prepared ice solution of performic acid (1.6 mmol) in formic acid (2 milliliters). The mixture was stirred for one hour at 0 ° C, and then for 20 hours at room temperature. Then a small amount of activated carbon was added to decompose any excess of peracid, 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 acidic sodium bicarbonate, and the material was desalted by passing it through a Sephadex G10 column. A white solid (20 milligrams) was obtained after the leofilization, which was spectroscopically essentially the same as the material obtained by method one. 13C nm (D20): 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 sulfosuccinamic acid.

A. PAMAM 1.0 Solid maleic anhydride (0.11 grams, 1.1 mmol) was added to a stirred solution of PAMAM 1.0 (0.12 grams, 0.33 mmol) in dry dimethyl formamide (3 milliliters). The mixture became a little hot and brown as the anhydride dissolved, and the resulting solution was stirred overnight at room temperature. Then the solution was concentrated (30 ° C / 10 ~ 4 mmHg) to give a viscous oil. and 13C nmr (D20) showed a complete conversion of PAMAM 1.0 into trisamide, along with some maleic acid. 13C nm (D20): 5 33.1, 42.8, 43.1, 54.3, 135.0, 137.1, 169.1, 171.9, 173.3. The crude trisamide was then dissolved in water (4 milliliters), and solid sodium sulfite (0.20 grams, 1.6 millimoles) was added. The resulting solution was allowed to stand at room temperature for four days, and then concentrated. and 13 C nmr (D20) showed a 1: 1 mixture of PAMAM 1.0 dendrimers terminated in regioisomeric sodium sulfosuccinic acid, together with some sulfosuccinic acid. The crude product was purified by gel filtration (Sephadex G10, water) to give a sample of the PAMAM 1.0 dendrimers terminated in sodium sulfosuccinamic acid (107 milligrams). 13C nm (D20): d 33.3, 39.6, 40.0, 42.9, 43.1, 54.0, 67.9, 173.8, 176.3, 177.6, 181.8.

B. PAMAM 2.0 A mixture of the PAMAM 2.0 dendrimers terminated in regioisomeric sodium sulfosuccinnamic acid was prepared as described above. C rmn thin of maleamic acid of PAMAM 2.0 (D20): d 32.8, 33.0, 38.7, 42.9, 53.8, 54.3, 56.5, 135.2, 136.8, 169.2, 171.9, 173.5, 174.6. 13C NMR of sodium sulfosuccinamic acid derivatives of PAMAM 2.0 (D20): 37.0, 40.1, 41.1, 43.0, 43.2, 43.9, 53.0, 53.3, 55.5, 68.0, 69.4, 173.8, 177.6, 179.1, 179.5, 179.8, 182.3 .

C. PAMAM 4.0 (Compound No. 14) Solid maleic anhydride (60 milligrams, 0.6 mmol) was added to a stirred solution of PAMAM 4.0 (51 milligrams, 0.01 millimole) in dimethyl formamide sesea (2 milliliters). The mixture initially became cloudy, but soon gave a clear solution, which was stirred overnight at room temperature. Then the solution was concentrated (35 ° C / 10 ~ 4 mmHg) to give a viscous oil. 1H and 13C nmr (D 0) showed a complete conversion of PAMAM 4.0 into the polyamide, together with some maleic acid. Then the crude polyamide was dissolved in water (2 milliliters), and a solution of sodium sulfite (126 milligrams) was added.; 1.0 millimoles) in water (2 milliliters). The resulting solution was allowed to stand at the temperature for two days, and then concentrated. 1H and 13C nmr (D20) 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 provide a finished PAMAM 4.0 sample with 24 regioisomeric sulfosuccinnamic acid groups (90 milligrams). 1H nm (D20): d 2.4-2.6; 2.7-3.1; 3.2-3.4; 3.9-4.0. 13C nm (D20): 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. to. Preparation of N- (2-sulfoethyl) succinic acid tetrabutyl ammonium acid. Solid succinic anhydride (0.50 grams, 5.0 mmol) was added to a stirred solution of 2-aminoethylsulfonic acid tetrabutyl ammonium (1.83 grams, 5.0 mmol) in dry dichloromethane (30 milliliters). The succinic anhydride was slowly dissolved, and the resulting nebulized solution was stirred overnight at room temperature. The mixture was filtered, and the filtrate was concentrated to give a viscous oil (2.41 grams). The 13C nmr showed a complete conversion to the desired monoamide, together with a small amount of succinic acid. Repeated precipitation of the product by dropwise addition of a dichloromethane solution to a large excess of diethyl ether gave N- (2-sulfoethyl) succinnamic ammonium tetrabutylic acid as a white solid (1762 grams, 76 percent), mp. . 125-127 ° C. 1H NMR (CDC13): d 0.86 (t, 12h, 4xCH3), 1.28 (, 8H, 4XCH2), 1.50 (m, 8H, 4 CH2), 2.33 (m, 2H, CH2C00H), 2.44 (m, 2H, CH2C0NH ), 2.76 (m, 2H, CH2NHC0), 3.12 (m, 8H, 4xCH2N), 3.50 (m, 2H, CH2S03_), 7.53 (br t, 1H, NH). 13C nm (CDC13): d 13.5, 19. 5, 23.8, 30.1, 30.9, 35.6, 50.0, 58.5, 112. O, 174.1 b. Preparation of N- (2-sulfoethyl) -succinamate 4- nitrophenyl ammonium tetrabutyl. A solution of dicyclohexyl carbodiimide (45 milligrams, 0.22 millimoles) in dry dichloromethane (1 milliliter) was added to a stirred solution of N- (2-sulfoethyl) succinnamic ammonium tetrabutyl acid (94 milligrams, 0.20 millimoles) and 4-nitrophenol ( 28 milligrams; 0.20 mmol) in dichloromethane (2 milliliters), 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 sodium N- (2-sulfoethyl) succinamide.

PAMAM 4.0 A solution of the crude tetrabutyl ammonium N- (2-sulfoethyl) succinamate succinamate 4-nitrophenyl (0.30 mmol) in dry dimethyl formamide (1 milliliter) was added to a stirred solution of PAMAM 4.0 (51.5 milligrams, 0.01 millimole) dissolved in 50 percent aqueous dimethyl formamide (3 milliliters), and the resulting yellow solution was stirred overnight at room temperature. Then the mixture was concentrated (35 ° C / 10 ~ 5 mmHg), and the yellow residue was divided between water and chloroform. The water layer was separated, washed with chloroform (2 times) and ethyl acetate, and then concentrated to give a yellow oil (134 milligrams). The crude product was converted to the sodium salt by passing it through a column of Amberlite IR-120 (Na) to give 85 milligrams of the material. This material was further purified by gel filtration (Sephadex LH20, water) to give the finished PAMAM 4.0 dendrimer in succinamide N- (2-sulfoethyl sodium (45 milligrams) 13C nmr (D20): d 33.2, 33.6, 35.5, 39.0, 39.5, 42.8, 43.2, 53.8, 54.1, 54.4, 56.6, 176.5, 176.9, 177.2, 178.9, 179.4.

The corresponding PAMAM 1.0 and PAMAM 3.0 dendrimers terminated with sodium N- (2-sulfoethyl) succinamide groups were prepared in a similar manner. 13CMN of the derivative of PAMAM 3.0 (D20): 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. 13CMN of the derivative of PAMAM 1.0 (D20): 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) sodium succinamide. BHAlyslys2lys4lys8lys16 Trifluoroacetic acid (1 milliliter) was added to a suspension of BHAlyslys2lys4lys8DBL16 (36.5 milligrams, 5.0 micromoles) in dry dichloromethane (1 milliliter), and the resulting solution was stirred at room temperature under nitrogen for two hours, and then concentrated. The residue was dissolved in dry dimethyl sulfoxide (2 milliliters), and the pH was adjusted to 8.5 with triethyl amine. Then a solution of the crude tetrabutyl ammonium 4-nitrophenyl) succinamate 4-nitrophenyl (about 0.2 mmol) in dimethyl sulfoxide (1 milliliter) was added dropwise, and the mixture was stirred overnight at room temperature. Then the yellow solution was concentrated (50 ° C / 10 ~ 5 mmHg), and the yellow residue was divided between water and chloroform. The aqueous layer was separated, washed with chloroform (3 times) and ethyl acetate, and then concentrated to give an oil (99 milligrams). The crude product was converted to the sodium salt by svi passage through a column of Amberlite IR 120 (Na) to give 81 milligrams of the material. This material was further purified by gel filtration (Sephadex LH20; water) to give the. dendrimer of BHAlyslys2lys4lys8lys16 terminated in sodium N- (2-sulfoethyl) succinamide (39 milligrams). 13C rmn (D20): 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 corresponding BHAlyslys2, BHAlyslys2lys and BHAlyslys2lys4lys8, terminated with sodium N- (2-sulfoethyl) succinamide groups, were similarly prepared. 13C nmr of the derivative of BHAlyslys2lys4lys8 (D20): d 26. 9, 32.3, 35.1, 35.3, 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, 178.2, 178.3, 178.6, 178.6, 178.7, 178.8, 178.8, 178.2, 179.2, 179.3, 179.7, 179.8. 13Cm of the derivative of BHAlyslys2lys4 (D20): d 26.9, 32. 3, 35.1, 35.4, 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. 13Cm of the derivative of BHAlyslys2 (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 finished thiourea 4-sulfophenyl sodium dendrimers.

A. PAMAM 4.0 (Compound No. 1) Solid sodium 4-sulfophenyl isothiocyanate monohydrate (500 milligrams, 1.96 millimoles) was added to a solution of PAMAM 4.0 (300 milligrams, 0.0582 millimoles) in water (10 milliliters), and the 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 4.0 dendrimer terminated in sodium 4-sulfophenyl thiourea as a fluffy white solid (370 milligrams). rmn (D20): d 2.28; 2.52; 2.69; 3.15; 3.27; 3.60; 7.32 (d, J = 9Hz); 7.72 (d, J = 9Hz). 13C nm (D20); d 36.9; 41.1; 43.1; 48.3; 53.6; 55.8; 129.0; 131.1; 144.4; 178.5; 179.1; 184.4. The corresponding dendrimers of PAMAM 1.0 and PAMAM 2.0, PAMAM 3.0 and PAMAM 5.0 (Compound No. 2) terminated with 3, 6, 12 and 48 sodium thiourea 4-sulfophenyl groups, respectively, were prepared in a similar manner.

B. PAMAM 4.0 (EDA) (Compound No. 3) Sodium 4-sulfophenyl isothiocyanate monohydrate (130 milligrams, 0.5 mmol) was added to a solution of PAMAM 4.0 (EDA) (69 milligrams, 0.01 mmol) in water (4 milliliters), 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 freeze dried to give the finished PAMAM 4.0 with 32 sodium thiourea 4-sulfophenyl groups as a fluffy white solid (136 milligrams). 1Hrmn (D20): d 2.30; 2.50; 2.70; 3.18; 3.62; 7.35 (d, J = 9Hz); 7.72 (d, J = 9Hz). 13C nm (D20): d 36.8; 41.0; 43.1; 48.4; 53.6; 55.7; 128.9; 131.0; 144.3; 178.5; 179.0; 184.5.

C. BHAlyslys2lys4lys8lys16 (Compound No. 4) Trifluoroacetic acid (4 milliliters) was added to a suspension of BHAlyslys2lys4lys8DBL16 (0.73 grams, 0.1 mmol) in dry dichloromethane (4 milliliters) under nitrogen. A vigorous evolution of gas 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 milliliters), the solution was passed through a column of Amberlite IRA-401 (OH), and the filtrate was concentrated to give BHAlyslys2lys4lys8lys16 as a viscous oil (0.49 grams). The oil was redissolved in water (5 milliliters), and an N, N-dimethyl-N-allylic amine pH buffer (pH 9.5, 3 milliliters) was added. Then solid sodium 4-sulfophenyl isothiocyanate monohydrate (1.30 grams, 5.1 mmol) was added, and the resulting solution was heated under nitrogen at 53 ° C for 2 hours, and then cooled. The solution was concentrated, and the chestnut 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 the dendrimer of BHAlyslys2lys4lys8lys16 terminated in 4-sulfonale sodium thiourea as a fluffy white solid (374 milligrams). 1Hrmn (D20): d 1.40; 1.72; 3.08; 3.42; 4.24; 4.60; 7.30; 7.40 (d, J = 9Hz); 7.78 (d, J = 9Hz). 13C nm (D20): d 27.3; 32.5; 35.9; 43.7; 48.9; 58.6; 63.3; 128.8; 131.0; 143.7; 144.7; 145.1; 177.7; 178.1; 183.8; 185.2. The corresponding dendrimers of BHAlyslys2lys4lys8lys16, BHAlyslys2lys4lys8lys16lys32 (Compound No. 5), and BHAlyslys2lys4lys8lys16lys32lys64 (Compound No. 6) terminated with 16, 64 and 128 sodium thiourea 4-sulfophenyl groups, respectively, were prepared in a similar manner.

EXAMPLE 6. Preparation of Dendrimers Terminated in Thiourea 3,6-Disulfonaphthyl Sodium.

A. PAMAM 4.0 (Compound No. 9) Solid sodium 3,6-disulfonaphthyl isothiocyanate (160 milligrams, 0.41 millimole) was added to a solution of PAMAM 4.0 (51 milligrams, 0.01 millimole) in water (3 milliliters), and the The resulting solution was heated under nitrogen at 53 ° C for two hours, and then cooled. The solution was concentrated, and the chestnut solid residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and concentrated to give the PAMAM 4.0 dendrimer finished in thiourea 3,6-disulfonaphthyl sodium as a tan solid (73 milligrams). 1Hrmn (D20): d 2.30; 2.60; 2.74; 3.20; 3.57; 7.75; 7.86; 8.28. 13C nm (D20): 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; 185.5. The corresponding PAMAM 2 dendrimer terminated with thiourea 3,6-disulfonaphthyl sodium groups was prepared in a similar manner.

B. PAMAM 4.0 (EDA) (Compound No. 11) Solid sodium 3,6-disulfonaphtyl isothiocyanate (220 milligrams, 0.57 millimole) was added to a solution of PAMAM 4.0 (EDA) (74 milligrams, 0.01 millimole) in water ( 4 milliliters) and the resulting solution was heated under nitrogen at 53 ° C for two hours, and then cooled. The solution was concentrated, and the chestnut solid residue was purified by gel filtration (Sephadex LH20, water). The pure fractions were combined and concentrated to give the finished PAMAM 4.0 with 32 thiourea 3,6-disulfonaphthyl sodium groups as a tan solid (148 milligrams). ""? rmn (D20): d 2.30; 2.80; 3.20; 3.54; 7.74; 7.85; 8.25. 13C nm (D20): d 36.0; 40.8; 43.1; 48.3; 53.6; 55.9; 128.5; 129.4; 131.0; 131.3; 136.0; 136.8; 138.3; 145.5; 146.0; 178.2; 185.6.

C. BHAlyslys2lys4lys8lys16 (Compound No. 12) Trifluoroacetic acid (2 milliliters) was added to a suspension of BHAlysly? 2ly? 4lys8DBL16 (73 milligrams; 0.01 mmol) in dry dichloromethane (2 milliliters) under nitrogen. A vigorous gas evolution 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 milliliters), the solution was passed through a column of Amberlite IRA-401 (OH), and the filtrate was concentrated to give BHAlyslys2lys4lys8DBL16 as a viscous oil. The oil was redissolved in water (5 milliliters), and an N, N-dimethyl-N-allyl amine pH buffer (pH 9.5, 3 milliliters) was added. Then, solid sodium 3,6-disulfonaphtyl isothiocyanate (234 milligrams, 0.60 millimoles) was added, and the resulting solution was heated under nitrogen at 53 ° C for two hours, and then cooled. The solution was concentrated, and the chestnut 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 BHAlyslys2lys4lys8DBL15 terminated with 32 thiourea 3,6-disulfonaphthyl sodium groups as a fluffy white solid (119 milligrams) . "" "H rmn (D20): d 1.0-2.0, 3.18, 3.43, 4.31, 7.22, 7.80, 7.89, 8.25, 13C nm (D20): d 27.2, 32.4, 35.3, 43.7, 49.0, 58.5, 63.6, 128.4; 129.1; 131.4; 136.1; 136.6; 138.6; 139.0; 145.1; 145.6; 178.4; 184.8; 186.7.

EXAMPLE 7. Preparation of finished dendrimers in 4-sulfonaphthyl sodium thiourea.

PAMAM 4.0 (Compound No. 8) Solid sodium 4-sulfonaphthyl isothiocyanate (180 milligrams, 0.5 millimole) was added to a solution of PAMAM 4.0 (51 milligrams, 0.01 millimole) in water (5 milliliters), and the mixture was heated under nitrogen at 53 ° C for two hours, and then cooled. The water was distilled under reduced pressure from the resulting suspension, and the 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 4-sulfonaphthyl sodium thiourea as a fluffy white solid (60 milligrams). 1H nm (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 nm (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 3, 5-disulfophenyl sodium thiourea.

PAMAM 4.0 (Compound No. 7) Solid sodium 3,5-disulfophenyl isothiocyanate (110 milligrams, 0.322 millimoles) was added to a solution of PAMAM 4.0 (63 milligrams, 0.012 millimole) in water (3 milliliters) and the resulting solution was added. heated under nitrogen at 53 ° C for two hours, and then cooled. The solution was concentrated, and the chestnut solid residue was purified by gel filtration (Sephadex G25, water). The pure fractions were combined and concentrated to give PAMAM 4.0 finished with 24 thiourea 3, 5-disulfophenyl sodium groups as a white solid (110 milligrams). 1Hrmn (D20): or "2.53; 3.08; 3.36; 3.66; 7.90; 7.95. 13Crmn (D20): 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 thiourea 3,6,8-trisulfonaphthyl sodium.

PAMAM 4.0 (Compound No. 10) Solid sodium 3, 6, 8-trisulfonaphthyl isothiocyanate (250 milligrams, 0.5 millimole) was added to a PAMAM 4.0 solution (51 milligrams, 0.01 millimole) and a pH regulator of amine N, N-dimethyl-allylic (pH of 9.5; 1 milliliter) in water (2 milliliters), and the mixture was heated under nitrogen at 53 ° C for two hours and then cooled. The mixture was concentrated under reduced pressure to give an orange solid. The residual solid was dissolved in water (2 milliliters), and passed through a short column of Aberlite 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 thiourea 3,6,8-trisulfonaphthyl sodium as a white solid (102 milligrams). 1H nm (D20): d 2.65; 3.02; 3.30; 3.66; 8.05; 8.42; 8.59; 8.67. 13C nm (D20): 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.

EXAMPLE 10. Preparation of dendrimers terminated in 4- (sodium sulfomethyl) benzamide.

PAMAM 4.0 (Compound No. 13) Solid 4-nitrophenyl 4- (chloromethyl) benzoate (200 milligrams, 0.68 millimole) was added to a stirred solution of PAMAM 4.0 (70 milligrams, 0.014 millimole) in dry dimethyl sulfoxide (4 milliliters). ), and the resulting yellow solution 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 dimethyl sulfoxide (5 milliliters), and a solution of sodium sulfite (130 milligrams, 1 millimole) in water (3 milliliters) was added. The resulting slightly cloudy mixture was allowed to stand for four days, after which time, the addition of more water (2 milliliters) resulted in the formation of a homogeneous transparent yellow. The solution was then concentrated, first at 25 mmHg, and at 40 ° C, and then at 10 ~ 4 mmHg and at 50 ° C to give the crude product. The crude product was purified by gel filtration (Sephadex G25, water) to give the finished PAMAM 4.0 with 24 sodium benzamide 4- (sulfomethyl) groups (24 milligrams). ? E rmn (D20): d 2.25; 2.66; 3.08; 3.20; 3.33; 3.38; 4.01; 7.40 (br d); 7.62 (br d). 13C nm (D20): d 36.7; 40.9; 43.0; 43.6; 53.5; 55.5; 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) Solid potassium N-hydroxy-succinimidylic 4-sulfobenzoate (100 milligrams, 0.3 millimole) was added to a PAMAM 4.0 (EDA) solution (35 milligrams, 0.005 millimole) in a 0.1M borate regulator with a pH of 8.5 (5 milliliters), 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 milliliter) 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) finished with 32 groups of sodium 4-sulfobenzamide (47 milligrams). - "- H rmn (D20): 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), 13C nm (D20): d 36.0; 40.4; 43.0; 43.7; 53.7; 55.8; 130.2; 132.2; 1.40.4; 150.1; 173.6; 178.0; 178.5.

EXAMPLE 12. Preparation of dendrimers terminated in sodium N- (4-sulfophenyl) propanamide.

PAMAM 4.0 (EDA) Solid sodium N- (4-sulfophenyl) acrylic amide (250 milligrams, 1 millimole) and solid sodium carbonate (106 milligrams, 1 millimole) were successively added to a stirred solution of PAMAM 4.0 (EDA). (78 milligrams; 0.011 millimoles) in water (4 milliliters). 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 the PAMAM 4.0 (EDA) terminated with 64 N- (4-sulfophenyl) propylene sodium amide groups (206 milligrams). The 13C nmr showed a weak trace of what monoalkylated terminal amino groups were taken. 1Hrmn (D20): d 2.10; 2.48; 2.58; 2.79; 3.20; 7.42 (d, J = 7Hz); 7.65 (d, J = 7Hz). 13C nm (D20): d 36.5; 37.9; 41.1; 53.4; 55.6; 124. 8; 130.9; 143.0; 144.2; 177.4; 178.5.

EXAMPLE 13. Preparation of dendrimers terminated in sodium N- (4-sulfophenyl) propanamide.

PAMAM 4.0 (EDA) A solution of sodium sulphanilic acid (195 milligrams, 1 millimole) in dry dimethyl sulfoxide (3 milliliters) was added dropwise to a solution of N, N'-disuccinimidyl carbonate (530 milligrams, 2 millimoles) ) in dry dimethyl sulfoxide (4 milliliters), and the resulting tan solution was stirred at room temperature for 20 hours. A solution of PAMAM 4.0 (EDA) (75 milligrams; 0.011 mmol) in dry dimethyl sulfoxide (1 milliliter) was added and the solution was stirred for another 18 hours. Then the solution was concentrated under a high vacuum (10 ~ 5 mmHg, 35 ° C) to give a yellowish semi-solid. The crude product was dissolved in dimethyl sulfoxide (4 milliliters), and the solution was added to 200 milliliters of well-stirred ethyl acetate. The precipitated white solid was collected by filtration, and washed with ethyl acetate (2 times) and ether (2 times), and then dried to give a white powder (275 milligrams). This material was further purified by gel filtration (Sephadex LH20, water) to give PANAM 4.0 (EDA) terminated with 32 sodium 4-sulfophenyl urea groups (106 milligrams). ! H ruin (D20): d 2.31; 2.55; 2.75; 3.19; 7.32 (d, J = 9Hz); 7.63 (d, J = 9Hz). 13C nm (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 dendrimers terminated in N, N, N-trimethylic glycinamide chloride).

BHAlyslys2lys4lys8lys16 (Compound No. 15) Trifuluoroacetic acid (4 milliliters) was added to a suspension of BHAlyslys2lys4lys8DBL16 (220 milligrams, 30 micromoles) in dry dichloromethane (2 milliliters), and the resulting solution was stirred at room temperature under nitrogen for two hours , and then concentrated. The residue was dissolved in dry dimethyl sulfoxide (5 milliliters), and the pH was adjusted to 8.5 with triethyl amine. Then, solid 4-nitrophenyl N, N, N-trimethylglycinate chloride (0.50 grams, 1.8 mmol) was added and the mixture was stirred overnight at room temperature. Then the nebulous solution was concentrated (50 ° C / 10 ~ 5 mmHg), and the residue was partitioned between water and dichloromethane. The aqueous layer was separated, washed with dichloromethane (3 times), and ethyl acetate, and then concentrated to give an oil (1128 grams). The crude product was purified by gel filtration (Sephadex LH20, water) to give the dendrimer of BHAlyslys2lys4lys8lys16 terminated in N, N, N-trimethyl glycinamide (116 milligramqs). 13C nm (D20): 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-trimethyl ammonium benzamide.

PANAM 4.0 (Compound No. 16) 1,1-Carbonyldiimidazole (85 milligrams; 0. 52 mmol) was added to a solution of 4-trimethyl ammonium benzoic acid iodide (154 milligrams, 0.5 mmol) in dry dimethyl formamide (4 milliliters), and the mixture was stirred at room temperature under argon for two hours. During this time, a white solid was separated from the solution. Then a solution of PAMAM 4.0 (58 milligrams, 0.011 mmol) in dry dimethyl formamide (2 milliliters) was added and the mixture was stirred overnight at room temperature. After this time, most of the precipitate had dissolved, and a ninhydrin test of the solution was negative. The mixture was concentrated (10 ~ 4 mmHg, 30 ° C) to give a white solid residue. The crude product was purified by gel filtration (Sephadex LH20, 10 percent AcOH) to give the finished PAMAM 4.0 with 24 groups of 4-trimethyl ammonium benzamide as the acetic acid salt (89 milligrams). ^ i rmn (D20): d 1.96; 2.65-2.85; 3.25-3.55; 3.64; 7.92. 13C nm (D20): d 25.8; 33.1; 33.5; 38.7; 43.1; 43.5; 53.5; 54.1; 56.4; 61. 2; 124.8; 133.6; 139.9; 153.2; 173.2; 176.3; 176.8; 182.6. The corresponding PAMAM 2.0 dendrimer terminated with six benzamide 4-trimethyl ammonium groups was prepared in a similar manner.

EXAMPLE 16. Preparation of dendrimers terminated in benzamide 4- (trimethyl-methyl ammonium).

PAMAM 4.0 (Compound No. 17) Solid 4-nitrophenyl 4- (chloromethyl) benzoate (150 milligrams, 0.5 mmol) was added to a stirred solution of PAMAM 4.0 (52 milligrams, 0.01 mmol) in dry dimethyl sulfoxide (3 milliliters). ). The resulting yellow solution was stirred at room temperature for 20 hours, when a ninhydrin test was negative (pH of about 8.5). The solution was then concentrated (10 ~ 5 mmHg, 40 ° C), and the residue was stirred with a mixture of water and dichloromethane (1: 1). The material in the form of insoluble gel was collected by filtration, washed with water (2 times) and dichloromethane (2 times), and then dried in air. The finished dendrimer in crude 4- (chloromethyl) benzamide was dissolved in 25 percent aqueous trimethyl amine (20 milliliters), and the yellow solution was allowed to stand overnight. Then the solution was concentrated, the residue was dissolved in water (5 milliliters), 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 percent AcOH) to give the finished PAMAM 4.0 with 24 benzamide 4- (trimethylammonium ammonium) groups (90 milligrams ). 1Hrmn (D20): 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 nm (D20): d 26.6; 33.4; 38.8; 43.2; 43.5; 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 carboxamide N- (2-acetoxyethyl) -N, N- (dimethyl ammonium) methyl.

PAMAM 4.0 Solid 1, 1'-carbonyldimidazole (85 milligrams, 0.52 millimole) was added to a solution of ammonium bromide N- (2-acetoxyethyl) -N- (carboxymethyl) -N, N-dimethyl (135 milligrams; 0.5 millimoles ) in dry dimethyl formamide (3 milliliters), and the resulting solution was stirred under nitrogen for two hours. Then a solution of PAMAM 4.0 (60 milligrams, 0.012 millimoles) in dimethyl formamide (2 milliliters) was added, which caused the immediate formation of a flocculent precipitate that slowly dissolved again. The mixture was stirred for two days, and then concentrated (10 ~ 4 mmHg; 40 ° C) to give a viscous oil. The crude product was purified by gel filtration (Sephadex G10, 10 percent AcOH) to give the finished PAMAM 4.0 with 24 carboxamide N- (2-acetoxyethyl) -N, N- (dimethyl ammonium) -methyl carboxamide groups (64 milligrams ). - "" H rmn (D20): d 1.93; 2.05; 2.70; 3.10-3.60; 3.28; 3.93 (m); 4.14; 4.48 (m). 13C nm (D20): d 24.6; 26.2; 33.2; 38.7; 42.8; 42.9; 53.9; 57.4; 62.6; 67.3; 67.5; 168.9; 176.4; 176.8; 177.3; 183.2.

EXAMPLE 18. Preparation of dendrimers terminated in guanidino.

PAMAM 4.0 (Compound No. 18) A solution of PAMAM 4.0 (63 milligrams, 0.012 millimole) and methylthioseudourea sulfate (170 milligrams, 0.061 millimole) in water (5 milliliters) (pH of 10.5) was heated under nitrogen at 80 ° C. during two hours. The solution was then concentrated, and the residue was purified by gel filtration (Sephadex G10, 10 percent AcOH) to give the finished PAMAM 4.0 with 24 guanidino groups as the acetate salt (107 milligrams). 1Hrmn (D20): d 2.00; 2.80 (br t); 3.09 (br t); 3.32; 3.45 (br t), 3.60 (br t). 13C nm (D20): d 25.2; 33.2; 33.4; 38.7; 41.2; 42.6; 43.4; 44.7; 53.5; 54.0; 56.3; 176.5; 176.7; 176.9; 181.6. The corresponding PAMAM 2.0 dendrimer terminated with six guanidino groups was prepared in a similar manner.

EXAMPLE 19. Preparation of dendrimers terminated in b e n z a m i d a 4 - ([1, 4 8 1 1 - tetraazasiclotetradecan] methyl).

PAMAM 4.0 (Compound No. 19) A solution of l- (4-carboxyphenyl) methyl-1,4,8,1-tetraazacyclotetradecane tetraclohydrate (120 milligrams, 0.25 millimoles), N-hydroxysuccinimide (60 milligrams, 0.52 millimoles), and carbodiimide hydrochloride 1- (3-dimethylaminopropyl) -3-ethyl (250 milligrams, 1.3 millimoles) in a phosphate buffer of a pH of 7 (10 milliliters), allowed to stand at room temperature for one hour, and then a solution of PAMAM 4.0 (32 milligrams, 0.006 millimoles) in a phosphate buffer of a pH of 7 (10 milliliters) was added. The mixture was allowed to stand for two days, and then concentrated. The residue was purified by gel filtration (Sephadex LH20, 10 percent AcOH) to give the finished PAMAM 4.0 with approximately 12 benzamide groups 4- ([1, 4,8, 11-tetraazaciclotetradecan] methyl), determined by 1H and 13 C nmr (80 milligrams). The product was then dissolved in water and passed through a column of Amberlite IRA-401 (Cl) resin, and then concentrated. The residue was dissolved in water (1 milliliter), concentrated, HCl (1 milliliter) was added, and the solution was diluted with ethanol (30 milliliters) to precipitate a white solid. The solid was collected by filtration (68 milligrams). Once again, the 1H and 13C nmr showed functionalization of approximately 50 percent of the terminal amino groups. 1H nm (D20): d 2.17; 2.36; 2.50; 2.78; 2.85; 3.25; 3.40; 3.50; 3.60; 3.62; 4.49; 7.63 (br d); 7.78 (br d). 13C nm (D20): < S 22.7; 23.1; 33.2; 38.8; 39.9; 40.2; 40.3; 41.0; 41.2; 42.0; 42.9; 43.2; 43.6; 45.5; 46.1; 49.1; 52.2; 53.9; 54.3; 56.6; 62.7; 132.5; 135.7; 137.1; 139.7; 174.3; 176.2; 176.3; 176.7; 177.0; 178.2; 178.5.

EXAMPLE 20. Preparation of dendrimers terminated in 4-carboxy-3-hydroxybenzyl amine.

PAMAM 4.0 (EDA) Sodium cyanoborohydride (32 milligrams; 0. 5 millimoles) to a mixture of PAMAM 4.0 (EDA) (69 milligrams, 0.01 millimole), 4-formyl-2-hydroxybenzoic acid (83 milligrams, 0.5 millimole), and sodium acid carbonate (42 milligrams, 0.5 millimole) in water (4 milliliters). The inhomogeneous orange mixture was stirred for four hours at room temperature, during which time, it became homogeneous. Then the orange solution was concentrated, and the residue was purified by gel filtration (Sephadex LH20, water) to give the PAMAM 4.0 (EDA) terminated with about 32 4-carboxy-3-hydroxybenzyl amine groups (91 milligrams). The ""? and 13C nmr (D20) shows mostly monoalkylation, but with some signs of alkylation of the terminal amino groups, and both spectra show broad peaks. 13C nm (D20): d 37.0; 41.1; 50.9; 53.4; 55.5; 55.8; 61.5; 120.9; 122.2; 122.4; 132.3; 132.7; 135.0; 135.8; 163.5; 163.7; 169.0; 178.6; 179.3. 1H nm (D20): d 2.20; 2.35; 2.60; 3.15; 3.30; 3.55; 4.25; 6.68; 7.12; 7.55.

EXAMPLE 21. Test for anticoagulant activity. Bovine blood was collected from the slaughterhouse, where an animal was bled into a bucket containing sodium citrate at a concentration of 3.5 grams per liter of fresh blood. This blood was returned to the laboratory, where it was kept in a water bath at 37 ° C. Aliquots of the whole blood were then centrifuged at 3,000 rpm for 5 minutes to separate the plasma. This was collected and returned to the water bath. Extra plasma was also prepared, and stored in liquid nitrogen for a subsequent test. The procedure actually tests the recalcification time of the citrated blood at 37 ° C. All the glassware was washed, dried and silo with "Coatasil" before being washed and dried again. Each 12 x 75 millimeter culture tube contained 0.1 milliliter of plasma, 0.1 milliliter of whey solution (0.9 percent NaCl), followed by 0.1 milliliter of 0.025M CaCl2, at which time the timer was started. Every 15 seconds, the tube was tilted to one side, and coagulation was evaluated. When a firm clot had formed, the stopwatch was stopped, and time was recorded. In the case of the anticoagulant test, 0.1 milliliter of the test substance replaced the serum. The times for a concentration scale is for the test compounds recorded in Table 1. Heparin, sodium citrate and the test compound were formed in water, solutions of 10 milligrams / milliliter. These solutions were diluted to give a scale of concentrations. The final concentrations of the test tubes are given in the Table. The figures in the Table represent average times for up to ten replicates.

TABLE 1 EXAMPLE 23. Test for antiviral activity.

The results of the activity tests against HIVl, HIV2, CMV and different herpes simplex viruses (HSV) are recorded in Tables 2 to 5, respectively.

TABLE 2 0. 0694 > 20 > 300 (9) 0.0032 > 125 > 39000 (10) 0.0041 > 14 > 3500 (11) 0.0051 > 12.9 > 2537 (12) 0.0088 > 15 > 1700 0. 0088 > 24 > 2737 (14) 0.2849 > 25 > 88 (15) 0.0725 > 125 > 1720 (16) 1.2559 > 25 > twenty 'TABLE 3 (17) PAMAM 4.0 > 7,175 7,175 < 1 24 extreme groups (18) PAMAM 4.0 NH > 10,817 10,817 < 1 24 groups of \ end N "H NH2 (19) PAMAM 4.0 0.2175 2.4245 11 approximately 12 CONH- end groups

Claims (40)

1. An antiviral compound comprising a dendrimer having a plurality of end groups, wherein at least one of the terminal groups has an anionic or cationic containing moiety, different from a 2-thiosalic acid moiety, bonded thereto.

2. A compound according to claim 1, wherein the dendrimer comprises a polyvalent core covalently linked with at least two dendritic branches, and extends through at least generations.

3. A compound according to claim 1 or claim 2, wherein the dendrimer is a polyamidoamine dendrimer based on a core of ammonia.

4. A compound according to claim 1 or claim 2, wherein the dendrimer is a polyamidoamine dendrimer based on an ethylene diamine core.

A compound according to claim 1 or claim 2, wherein the dendrimer is a polylysine dendrimer based on a benzhydryl amine nucleus or other suitable core.

6. A compound according to any of claims 1 to 5, wherein the fraction or fractions containing anionic or cationic, are bonded to the terminal amine, sulfhydryl, hydroxy or other functional reactive end groups of the dendrimer, by linkages of amide or thiourea.

A compound according to any of claims 1 to 6, wherein the fraction or fractions containing anionic, are fractions containing sulfonic acid, or fractions containing carboxylic acid other than fractions of 2-thiosalicylic acid.

8. A compound according to any of claims 1 to 6, wherein the fraction or fractions containing cationic, are fractions containing trimethyl ammonium, or fractions containing polycyclin-macrocyclic.

9. A compound according to any of claims 1 to 6, wherein the fraction or fractions that are linked with amino or other reactive functional groups of the dendrimer, are selected from the following groups: -NHíChy-SOa- (CH2) nS03- Ar (S03")" -CH2CH (S03") COOH CH (S03") CH2COOH ArX (CH2) nS03 'X - O, S, NH + + + - (CH2) -NMe3 A / (NMe3) p Ar (CH2NMe3) n where n is zero or a positive integer.

10. A compound according to any of claims 1 to 6, which is a dendrimer terminated in alkylsulphonic acid.

11. A compound according to any of claims 1 to 6, which is a dendrimer terminated in sulfoaceta ida.

12. A compound according to any of claims 1 to 6, which is a dendrimer terminated in sulfosuccinnamic acid.

13. A compound according to any of claims 1 to 6, which is a dendrimer terminated in

14. A compound according to any of claims 1 to 6, wherein the fraction linked to the terminal groups of the dendrimer is an aryl or heteroaryl thiourea substituted with one or more sulphonic acid groups.

15. A compound according to claim 14, which is a dendrimer terminated in 4-sulfophenyl thiourea.

16. A compound according to claim 14, which is a dendrimer terminated in thiourea 3,6-disulfonaphthyl.

17. A compound according to the claim 14, which is a dendrimer terminated in 4-sulfonaphthyl thiourea.

18. A compound according to claim 14, which is a dendrimer terminated in thiourea 3,5-disulfophenyl.

19. A compound according to claim 14, which is a dendrimer terminated in thiourea 3,6,8-trisulfonaphthyl.

20. A compound according to any of claims 1 to 6, wherein the fraction linked to the terminal groups of the dendrimer is an aryl or heteroaryl amide substituted with one or more sulfonic acid, sulfoalkyl, sulfoalkoxy, sulfoalkylamino or sulfoalkylthio groups. .

21. A compound according to the claim 20, is a dendrimer terminated in 4- (sulfomethyl) benzamide.

22. A compound according to claim 20, which is a dendrimer terminated in 4-sulfobenzamide.

23. A compound according to any of claims 1 to 6, wherein the fraction linked to the end groups of the dendrimer is an aryl or heteroaryl alkanamide substituted with one or more sulfonic acid groups.

24. A compound according to claim 23, which is a N- (4-sulfophenyl) propanamide-terminated dendrimer.

25. A compound according to any of claims 1 to 6, wherein the fraction linked to the terminal groups of the dendrimer is an aryl or heteroaryl urea substituted with one or more sulfonic acid groups.

26. A compound according to claim 25, which is a dendrimer terminated in 4-sulfophenyl urea.

27. A compound according to any of claims 1 to 6, wherein the fraction linked to the terminal groups of the dendrimer is a N, N-N-trimethyl derivative of an amino acid.

28. A compound according to claim .2 > 27, which is dendrimer terminated in glycinamide N, N, N-trimethyl.

29. A compound according to any of claims 1 to 6, wherein the fraction linked to the terminal groups of the dendrimer is an aridic amide or Heteroaryl substituted with one or more trialkylamino, trialkylaminoalkyl, trialkylaminoalkyloxy, trialkylaminoalkylamino, or trialkylaminoalkylthio groups.

30. A compound according to claim 20, which is a dendrimer terminated in 4- trimethyl ammonium benzamide.

31. A compound according to claim 29, which is a dendrimer terminated in benzamide 4- (trimethyl ammonium-methyl).

32. A compound according to any one of claims 1 to 6, which is a dendrimer terminated in carboamide N- (2-acetoxyethyl) -N, N- (di-ethyl ammonium) -methyl.

33. A compound according to any of claims 1 to 6, which is a guanidino-terminated dendrimer.

34. A compound according to any one of claims 1 to 6, wherein the dendrimer is a macrocyclic polyamino group containing 1 or more macrocyclic rings connected through an alkyl or aryl splitter moiety with the terminal group of the dendrimer.

35. A compound according to claim 34, which is a dendrimer terminated in benzamide 4- ([1,4,8, 11-tetraazacyclotetradecan] methyl).

36. A compound according to any of claims 1 to 6, which is a 4-carboxy-3-hydroxybenzyl amine-terminated dendrimer.

37. A pharmaceutical or veterinary composition for a prophylactic or therapeutic antiviral treatment of a human or a non-human animal, which comprises a compound of any of claims 1 to 36, in association with at least one pharmaceutically-derived carrier or diluent or veterinarily acceptable.

38. A method for prophylactic or therapeutic anti-viral treatment of a human or non-human animal, which comprises administering to this human or animal, a prophylactic or therapeutic effective antiviral amount of a compound of any one of claims 1 to 36.

39. A method according to claim 38, wherein the antiviral treatment is a treatment of HIV infection or HIV2, Hepatitis B or C, Bovine Viral Diarrhea Virus, Human Influenza Virus A and B, Rhinovirus, Human Parainfluenza Virus, Respiratory Syncytial Virus (RSV), Varicella Zoster Virus (VSV), Human Cytomegalovirus (CMV), Epstein Bar Virus (EBV), Human Papilloma Virus (HPV), Adenovirus-8, Herpes Simplex Virus (HSV) type 1 and 2, Measles Virus, or Vesicular Stomatitis Virus (VSV).

40. The use of a compound of any of claims 1 to 36, in the prophylactic or therapeutic antiviral treatment of, or in the manufacture of a medicament for prophylactic or therapeutic antiviral treatment of, a human or non-human animal.