FIELD OF THE INVENTION
[0001] This invention relates to antiviral agents, and in particular it relates to dendrimers
which have been found to have significant antiviral activity against human immunodeficiency
virus (HIV) and other enveloped viruses.
BACKGROUND OF THE INVENTION
[0002] It has been established that certain sulfonated polysaccharide compounds have antiviral
activity when screened against HIV, however these compounds are relatively unstable
and accordingly 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 suited for clinical use as antiviral agents.
[0003] The present invention provides a new class of antiviral agents based on a particular
type of polymer referred to herein as a "dendrimer", which have substantial antiviral
activity against HIV1 and HIV2, CMV and HSV, and which have substantially no anticoagulant
activity, These compounds are therefore well suited for prophylactic and therapeutic
use as antiviral agents in humans.
SUMMARY OF THE INVENTION
[0004] According to the present invention, there is provided
1. A compound being a dendrimer having a plurality of terminal groups, characterised in that
(i) said compound has in vitro inhibitory activity against a virus selected from the group consisting of HIV1 or
HIV2, Hepatitis B or C Bovine Viral Diarrhoea Virus, Rhinovirus, Human Parainfluenza
Virus, Respiratory Syncytial Virus (RSV), Varicella Zoster Virus (VZV), Human Cytomegalovirus
(CMV), Epstein Barr Virus (EBV), Human Papilloma Virus (HPV), Adenovirus-8, Herpes
Simplex Virus (HSV) type 1 and 2, Measles Virus, and Vesicular Stomatitis Virus (VSV),
and
(ii) at least one of said terminal groups has an anionic- or cationic-containing moiety
bonded thereto.
[0005] Such a dendrimer is referred to herein as an "anionic or cationic dendrimer", and
this term is used throughout this specification and the claims which follow to include
not only the dendrimers
per se, but also their pharmaceutically or veterinarily acceptable salts, for example the
alkaline metal or alkaline earth metal salts such as the sodium, potassium or calcium
salts.
DETAILED DESCRIPTION OF THE INVENTION
[0006] Dendrimers are macromolecular highly branched compounds formed by reiterative reaction
sequences starting from an initial, core molecule with successive layers or stages
being added in successive "generations" to build up a three-dimensional, highly ordered
polymeric compound. A generalised dendrimer structure is shown in Figure 1. Dendrimers
are characterised by the following features: i an initiator core(I) which may have
one or more reactive sites and be point-like or of significant size so as to effect
the final topology of the dendrimer; ii layers of branched repeating 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 frameworks for the
attachment of ionic moieties; 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 in both shape and constitution are well known to persons skilled in
the art.
[0007] The preparation of dendrimers is well known, and is described by way of example in
U.S. Patents Nos. 4,289,872 and 4,410,688 (describing dendrimers based on layers of
lysine units), as well as U.S. Patents Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329
(describing dendrimers based on other units including polyamidoamine or PAMAM dendrimers).
The dendrimers disclosed in these US patents are described as being suitable for uses
such as surface modifying agents, as metal chelating agents, as demulsifiers or oil/water
emulsions, wet strength agents in the manufacture of paper, and as agents for modifying
viscosity in aqueous formulations such as paints. It is also suggested in U.S. Patents
Nos. 4,289,872 and 4,410,688 that the dendrimers based on lysine units can be used
as substrates for the preparation of pharmaceutical dosages.
[0008] International Patent Publications Nos. WO 88/01178, WO 88/01179 and WO 88/01180 disclose
conjugates in which a dendrimer is conjugated or associated with another material
such as a carried pharmaceutical or agricultural material. These patent publications
together with the U.S. patents mentioned above contain a broad disclosure of various
dendrimers and processes for the preparation thereof.
[0009] EP-A-0328403, EP-A-0339695, WO 93/03766 and US-5,229,490 all describe various multiple
antigen peptide systems, methods of preparing such systems and uses of such systems.
The multiple antigenic peptide systems of these prior art references are capable iliciting
an immune response when administered to a subject. EP-A-0328403 specifically describes
polypeptides which are able to provide for immunological competition with HIV-gp 120
of at least one of the strains of the HIV retrovirus. The synthesis of dentritic sialoside
inhibitors of the Influenza A Virus haemagglutinin is disclosed in Roy et al, J. Chem.
Soc, Chen Commun, 1993, pp. 1869-1993
[0010] The term "dendrimer" as used herein is to be understood in its broadest sense, and
to include within its scope all forms and compositions of these dendrimers as disclosed
in Patent Publication Nos. WO 88/01178, WO 88/01179 and WO 88/01180. The term also
includes linked or bridged dendrimers as disclosed in these patent publications.
[0011] The preferred dendrimers of the present invention comprise a polyvalent core covalently
bonded to at least two dendritic branches, and preferably extend through at least
two generations. Particularly preferred dendrimers are polyamidoamine (PAMAM) dendrimers.
PAMAM (EDA) dendrimers and polylysine dendrimers.
[0012] In accordance with the present invention, at least one, and preferably a substantial
number, of the terminal groups on the surface of the dendrimer has an anionic- or
cationic- containing moiety covalently bonded thereto. The branches of the dendrimer
may terminate in amino groups or other functional reactive groups such as OH, or SH,
which subsequently can be reacted with the cationic and anionic moieties forming the
outer layer of the dendrimer. Where the terminal groups of the dendrimer are amine
groups, the anionic- or cationic-containing moiety may be bonded to the dendrimer
by a variety of functional group including amide and thiourea linkages. Preferred
anionic- or cationic-containing moieties which may be bonded to the terminal groups
of the dendrimer include sulfonic acid-containing moieties, carboxylic acid-containing
moieties other than 2-thiosalic acid moieties, trimethylammonium-containing moieties
and polyamino-macrocycle-containing moieties.
[0013] Suitable anionic- and cationic-containing moieties which may be bonded to the amino
or other terminal groups of dendrimers 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
of from 1 to 20):
―NH(CH
2)
nSO
3- ―(CH
2)
nSO
3- ―Ar(SO
3)
n
―CH
2CH(SO
3-)COOH -CH(SO
3-)CH
2COOH ―ArX(CH
2)
nSO
3- (X=O,S,NH)

[0014] Particular moieties which may be bonded to the terminal groups of the dendrimer in
accordance with this invention include alkyl sulfonic acid groups; sulfoacetamide
groups; sulfosuccinamic acid groups; N-sulfoalkyl succinamide groups, such as N-(2-sulfoethyl)succinamide
groups; aryl or heteroaryl thioureas substituted with one or more sulfonic acid groups,
such as 4-sulfophenylthiourea groups, 3,6-disulfonapthylthiourea groups, 4-sulfonapthylthiourea
groups, 3,5-disulfophenyl thiourea groups and 3,6,8-trisulfonapthylthiourea groups;
aryl 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 alkanamides 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 derivatives of amino acids, such as N,N,N-trimethylglycinamide
groups; aryl or heteroarylamides substituted with one or more trialkylamino, trialkylaminoalkyl,
trialkylaminoalkyloxy, trialkylaminoalkylamino or trialkylaminoalkylthio groups, such
as 4-trimethylammonium benzamide or 4-(trimethylammonium methyl) benzamide groups;
N-(2-acetoxyethyl)-N,N-(dimethylammonium)methylcarboxamide groups; guanidino groups;
4-carboxy-3-hydroxybenzylamine groups; or macrocyclic polyamino groups containing
one or more macrocyclic rings connected through an alkyl or aryl spacer moiety to
the terminal group of the dendrimer, such as 4-([1,4,8,11-tetraazacyclotetradecane]methyl)benzamide
groups.
[0015] The anionic or cationic dendrimers of this invention may be prepared by standard
chemical methods which are well known to persons skilled in this art. Suitable methods
are described by way of example in Examples 1 to 20 below.
[0016] As previously described, the anionic or cationic dendrimers of the present invention
have been found to exhibit significant antiviral activity, particularly against HIV.
Accordingly, these anionic or cationic dendrimers are useful in prophylactic and therapeutic
treatment of viral infections, for example infections by HIV1 and HIV2 and other enveloped
viruses including flaviviruses such as Hepatitis B and Hepatitis C, Bovine Viral Diarrhoea
Virus, Human Influenza Virus A and B, Rhinovirus, Human Parainfluenza Virus, Respiratory
Syncytial Virus (RSV), Varicella Zoster Virus (VZV), Human Cytomegalovirus (CMV),
Epstein Barr Virus (EBV), Human Papilloma Virus (HPV), Adenovirus-8, Herpes Simplex
Virus (HSV) type 1 and 2, Measles Virus, and Vesicular Stomatitis Virus (VSV).
[0017] Thus, in another aspect the present invention provides a pharmaceutical or veterinary
composition for 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 carrier
or diluent.
[0018] The formulation of such compositions is well known to persons skilled in this field.
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 such media and agents for pharmaceutically active substances
is well known in the art, and it is described, by way of example, in
Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional
media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical
compositions of the present invention is contemplated. Supplementary active ingredients
can also be incorporated into the compositions.
[0019] It is especially advantageous to formulate compositions in dosage unit form for ease
of administration and uniformity of dosage. Dosage unit form as used herein refers
to physically discrete units suited as unitary dosages for the human subjects to be
treated; each unit containing a predetermined quantity of active ingredient calculated
to produce the desired therapeutic effect in association with the required pharmaceutical
carrier and/or diluent. The specifications for the novel dosage unit forms of the
invention are dictated by and 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 compounding such an active ingredient for
the particular treatment.
[0020] 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 non-human animal, which
comprises administering to said human or animal a prophylactic- or therapeutic-antiviral-effective
amount of an anionic or cationic dendrimer as broadly described above.
[0021] In yet another aspect, this invention provides the use of a prophylactic- or therapeutic-antiviral-effective
amount of an anionic or cationic dendrimer as broadly described above 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 or non-human
animal.
[0022] A variety of administration routes are available. The particular mode selected will
depend, of course, upon the particular condition being treated and the dosage required
for therapeutic efficacy. The methods of this invention, generally speaking, may be
practised 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 clinically unacceptable adverse effects. Such modes of administration include
oral, rectal, topical, nasal, transdermal or parenteral (e.g. subcutaneous, intramuscular
and intravenous) routes. Formulations for oral administration include discrete units
such as capsules, tablets, lozenges and the like. Other routes include intrathecal
administration directly into spinal fluid, direct introduction such as by various
catheter and balloon angioplasty devices well known to those of ordinary skill in
the art, and intraparenchymal injection into targeted areas.
[0023] The compositions may conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art of pharmacy. 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 then, if necessary, shaping the product.
[0024] Compositions of the present invention suitable for oral administration may be presented
as discrete units such as capsules, cachets, tablets or lozenges, each containing
a predetermined amount of the active component, in liposomes or as a suspension in
an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
[0025] 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 may : be formulated according to
known methods using those suitable dispersing or wetting agents and suspending agents.
The sterile injectable preparation may 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 suspending medium. For this
purpose, any bland 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.
[0026] Other delivery systems can include sustained release delivery systems. Preferred
sustained release delivery systems are those which can provide for release of the
active component of the invention in sustained release pellets or capsules. Many types
of sustained release delivery systems are available. These include, but are not limited
to: (a) erosional systems in which the active component is contained within a matrix,
and (b) diffusional systems in which the active component permeates at a controlled
rate through a polymer. In addition, a pump-based hardware delivery system can be
used, some of which are adapted for implantation.
[0027] The active component is administered in prophylactically or therapeutically effective
amounts. A prophylactically or therapeutically effective amount means that amount
necessary at least partly to attain the desired effect, or to delay the onset of,
inhibit the progression of, or halt altogether, the onset or progression of the particular
condition being treated. Such amounts will depend, of course, on the particular condition
being treated, the severity of the condition and individual patient parameters 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 addressed with no more
than routine experimentation. It is preferred generally that a maximum dose be used,
that is, the highest safe dose according to sound medical judgement. It will be understood
by those of ordinary skill in the art, however, that a lower dose or tolerable dose
may be administered for medical reasons, psychological reasons or for virtually any
other reasons.
[0028] Generally, 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) may be administered initially,
followed by increasing doses up to about 1000 mg/kg per day. In the event that the
response in a subject is insufficient at such doses, even higher doses (or effective
higher doses by a different, more localised delivery route) may be employed to the
extent patient tolerance permits. Multiple doses per day are contemplated to achieve
appropriate systemic levels of compounds.
[0029] Throughout this specification and the claims which follow, unless the context requires
otherwise, the word "comprise", or variations such as "comprises" or "comprising",
will be understood to imply the inclusion of a stated integer or group of integers
but not the exclusion of any other integer or group of integers.
[0030] Further features of the present invention will be apparent from the following Examples
which are included by way of illustration, not limitation of the invention. In the
following Examples, PAMAM dendrimers refer to polyamidoamine dendrimers based on an
ammonia core as detailed in US Patents Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329;
PAMAM (EDA) dendrimers refer to polyamidoamine dendrimers based on an ethylene diamine
core; and BHAlys
xlys
ylys
z dendrimers refer to polylysine unsymmetrical dendrimers based on a benzhydrylamine
core and lysine branching units as described in US Patents Nos. 4,289,872 and 4,410,688.
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 BHAlyslys
2, BHAlyslys
2lys
4, BHAlyslys
2lys
4lys
8 and BHAlyslys
2lys
4lys
8lys
16, BHAlyslys
2lys
4lys
8lys
16lys
32, BHAlyslys
2lys
4lys
8lys
16lys
64, or higher generations are prepared as described in U.S. Patents Nos. 4,289,872,
4,410,688, 4,507,466, 4,558,120, 4,568,737 and 4,587,329 and International Patent
Publications Nos. WO 88/01178, WO 88/01179 and WO 88/01180 referred to above.
EXAMPLE 1 Reaction of polymers with 2-acrylamido-2-methyl propane sulfonic acid to
give sulfonic acid terminated dendrimers.
A. PAMAM 1.0
[0031] Solid sodium carbonate (0.13g; 1.0mmol) was added slowly to a stirred solution of
2-acrylamido-2-methyl propane sulfonic acid (0.41g; 2.0mmol) in water (3ml). After
the evolution of gas had ceased, the pH of the solution was 8.0. A solution of PAMAM
1.0 (0.12g; 0.33mmol) in water (1ml) was then added to the solution followed by the
addition of four drops of a 40% aq. solution of benzyl trimethylammonium hydroxide.
The solution was then heated under nitrogen at 60° 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 an off-white solid (0.51g).
1H and
13C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 1.0 dendrimer
(ca. 70:30).
13C nmr (D
2O): δ 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)
[0032] 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.
1H and
13C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 2.0 dendrimer
(ca. 65:35).
13C nmr (D
2O): δ 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
[0033] PAMAM 3.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as above except
that a slight excess of sodium carbonate was used and the benzyltrimethylammonium
hydroxide was omitted.
1H and
13C nmr spectra showed a mixture of diallrylated and monoalkylated PAMAM 3.0 dendrimer
(ca. 50:50).
13C nmr (D
2O): δ 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
[0034] PAMAM 4.0 was reacted with 2-acrylamido-2-methyl propane sulfonic acid as described
for PAMAM 3.0.
1H and
13C nmr spectra showed a mixture of dialkylated and monoalkylated PAMAM 4.0 dendrimer
(ca. 35:65).
13C nmr (D
2O): δ 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 sodium sulfoacetamide terminated dendrimers.
A. PAMAM 1.0
[0035] A solution of 4-nitrophenyl bromoacetate (0.40g; 1.5mmol) in dry DMF (1ml) was added
to a stirred solution of PAMAM 1.0 (0.18g; 0.5mmol) in DMF (3ml). The resulting yellow
solution was stirred for 20 hours at room temperature, when a ninhydrin test was negative.
The solution was concentrated (30°/ 0.1mmHg) to give a yellow oil. This oil was partitioned
between water and chloroform and the aqueous layer separated and washed with chloroform
(2X) and finally with ethyl acetate. The aqueous solution was concentrated (35°/ 25mmHg)
to give the bromoacetylated PAMAM 1.0 dendrimer as a yellow oil (0.36g;100%).
13C nmr (D
2O): δ 32.8, 33.3, 43.0, 43.5, 54.4, 174.5, 176.4.
[0036] A solution of sodium sulfite (0.2g; 1.6mmol) in water (1ml) was added to a solution
of the bromoacetylated PAMAM 1.0 dendrimer described above (0.36g; 0.5mmol) in water
(5ml) and the solution left to stand at room temperature for eleven days. The yellow
solution was concentrated to give a yellowish solid (0.60g).
13C nmr (D
2O): δ 34.4, 43.1, 43.4, 54.0, 61.7, 171.3, 177.2.
[0037] The above reaction sequence could be carried out 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:
[0038] A solution of 4-nitrophenyl bromoacetate (0.18g; 0.7mmol) in dry DMF (1ml) was added
to a stirred solution of PAMAM 2.0 (0.10g; 0.1mmol) in DMF (3ml). The resulting yellow
solution was stirred for 20 hours at room temperature, when a ninhydrin test was negative.
The solution was then added with swirling to water (150ml) and the mixture extracted
with chloroform (3X) and ethyl acetate. A solution of sodium sulfite (0.1g; 0.8mmol)
in water (1ml) was added to the crude bromoacetylated dendrimer solution and the mixture
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 sodium sulfoacetamide terminated PAMAM 2.0 dendrimer
(103mg).
13C nmr (D
2O): δ 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:
[0039] Solid succinimidyl acetylthioacetate (67mg; 0.33mmol) was added to a solution of
PAMAM 2.0 (52mg; 0.05mmol) in dry DMF (2ml) and the resulting solution stirred at
room temperature for two days. The mixture was then concentrated (30°/10
-3mmHg) to give an oily residue. The residue was partitioned between water and chloroform,
and the water layer separated and concentrated to give a viscous oil (117mg).
1H and
13C nmr showed the oil to be a mixture of the acylated dendrimer and N-hydroxy succinimide.
Gel filtration (Sephadex G10; water) provided a pure sample of the acetylthioacetamide
terminated PAMAM 2.0 dendrimer (29mg).
13C nmr(D
2O): δ 34.0, 34.2, 37.3, 43.0, 43.1, 43.3, 53.5, 54.0, 56.3, 175.4, 177.2, 177.5.
[0040] A solution of the above functionalised dendrimer in 40% aqueous formic acid (7ml)
was then added to an ice cold freshly prepared solution of performic acid (1.6mmol)
in formic acid (2ml). The mixture was stirred for one hour at 0° and then for twenty
hours at room temperature. A small amount of activated charcoal was then added to
decompose any excess peracid, the mixture stirred for 30 minutes then filtered and
concentrated to give a viscous oil. The crude product was dissolved in water, the
pH adjusted to 9.0 with aqueous sodium bicarbonate and the material desalted by passage
through a column of Sephadex G10. A white solid (20mg;) was obtained after lyophylisation
which was spectroscopically essentially the same as the material obtained by method
1.
13C nmr (D
2): δ 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 sodium sulfosuccinamic acid terminated dendrimers
A. PAMAM 1.0
[0041] Solid maleic anhydride (0.1 1g; 1. 1mmol) was added to a stirred solution of PAMAM
1.0 (0.12g; 0.33mmol) in dry DMF (3ml). The mixture became a little warm and brownish
as the anhydride dissolved and the resulting solution was stirred overnight at room
temperature. The solution was then concentrated (30°/10
-4mmHg) to give a viscous oil.
1H and
13C nmr (D
2O) showed complete conversion of the PAMAM 1.0 to the trisamide together with some
maleic acid.
13C nmr (D
2O): δ 33.1, 42.8, 43.1, 54.3, 135.0, 137.1, 169.1, 171.9, 173.3.
[0042] The crude trisamide was then dissolved in water (4ml) and solid sodium sulfite (0.20g;
1.6mmol) added. The resulting solution was allowed to stand at room temperature for
four days and then concentrated.
1H and
13C nmr (D
2O) showed a 1:1 mixture of the regioisomeric sodium sulfosuccinamic acid terminated
PAMAM 1.0 dendrimers together with some sulfosuccinic acid. The crude product was
purified by gel filtration (Sephadex G10; water) to afford a sample of the sodium
sulfosuccinamic acid terminated PAMAM 1.0 dendrimers (107mg).
13C nmr (D
2O): δ 33.3, 39.6, 40.0, 42.9, 43.1, 54.0, 67.9, 69.4, 173.8, 176.3, 177.6, 181.8.
B. PAMAM 2.0
[0043] A mixture of the regioisomeric sodium sulfosuccinamic acid terminated PAMAM 2.0 dendrimers
was prepared as described above.
13C mnr PAMAM 2.0 maleamic acid derivative (D
2O): δ 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 PAMAM 2.0 sodium sulfosuccinamic acid derivatives (D
2O): δ 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)
[0044] Solid maleic anhydride (60mg; 0.6mmol) was added to a stirred solution of PAMAM 4.0
(51mg; 0.01 mmol) in dry DMF (2ml). The mixture initially became cloudy but soon gave
a clear solution which was stirred overnight at room temperature. The solution was
then concentrated (35°/10
-4 mmHg) to give a viscous oil.
1H and
13C nmr (D
2O) showed complete conversion of the PAMAM 4.0 to the polyamide together with some
maleic acid. The crude polyamide was then dissolved in water (2ml) and a solution
of sodium sulfite (126mg; 1.0mmol) in water (2ml) added. The resulting solution was
allowed to stand at room temperature for two days and then concentrated.
1H and
13C nmr (D
2O) showed a mixture of the regioisomeric sodium sulfosuccinamic acid terminated PAMAM
4.0 dendrimers together with some sulfosuccinic acid. The crude product was purified
by gel filtration (Sephadex LH20; water) to afford a sample of PAMAM 4.0 terminated
with 24 regioisomeric sulfosuccinamic acid groups (90mg).
1H nmr (D
1O): δ 2.4-2.6; 2.7-3.1; 3.2-3.4; 3.9-4.0.
13C nmr (D
2O): δ 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 sodium N-(2-sulfoethyl)succinamide terminated dendrimers
a. Preparation of tetrabutylammonium N-(2-sulfoethyl)succinamic acid
[0045] Solid succinic anhydride (0.50g; 5.0mmol) was added to a stirred solution of tetrabutylammonium
2-aminoethylsulfonic acid (1.83g; 5.0mmol) in dry dichloromethane (30ml). The succinic
anhydride slowly dissolved and the resulting cloudy solution was stirred overnight
at room temperature. The mixture was filtered and the filtrate concentrated to give
a viscous oil (2.41 g).
13C nmr showed 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 tetrabutylammonium
N-(2-sulfoethyl)succinamic acid as a white solid (1.762g; 76%), mp 125-127°C. 1 H
nmr (CDCl
3): δ 0.86 (t, 12h, 4xCH
3), 1.28 (m, 8H, 4xCH)
2, 1.50 (m, 8H, 4xCH
2), 2.33 (m, 2H, CH
2COOH), 2.44 (m, 2H, CH
2CONH), 2.76 (m, 2H, CH
2NHCO), 3.12 (m, 8H, 4xCH
2N), 3.50 (m, 2H, CH
2SO
3-), 7.53 (br t, 1H, NH).
13C nmr (CDCl
3): δ 13.5, 19.5, 23.8, 30.1, 30.9, 35.6, 50.0, 58.5, 172.0, 174.1.
b. Preparation of tetrabutylammonium 4-nitrophenyl N-(2-sulfoethyl)-succinamate
[0046] A solution of dicyclohexylcarbodiimide (45mg; 0.22mmol) in dry dichloromethane (1ml)
was added to a stirred solution of tetrabutylammonium N-(2-sulfoethyl)succinamic acid
(94mg; 0.20mmol) and 4-nitrophenol (28mg; 0.20mmol) in dichloromethane (2ml), and
the mixture stirred overnight at room temperature. The resulting suspension was filtered
and the filtrate concentrated to give the crude active ester, which was used without
further purification.
A. Preparation of sodium N-(2-sulfoethyl)succinamide terminated PAMAM dendrimers
PAMAM 4.0
[0047] A solution of the crude tetrabutylammonium 4-nitrophenyl N-(2-sulfoethyl)succinamate
(0.30mmol) in dry DMF (1ml) was added to a stirred solution of PAMAM 4.0 (51.5mg;
0.01mmol) dissolved in 50% aqueous DMF (3ml) and the resulting yellow solution stirred
overnight at room temperature. The mixture was then concentrated (35°/ 10
-5mmHg) and the yellow residue partitioned between water and chloroform. The water layer
was separated, washed with chloroform (2X) and ethyl acetate, and then concentrated
to give a yellow oil (134mg). The crude product was converted to the sodium salt by
passage through a column of Amberlite IR- 120(Na) to yield 85mg of material. This
material was further purified by gel filtration (Sephadex LH20; water) to give the
sodium N-(2-sulfoethyl)succinamide terminated PAMAM 4.0 dendrimer (45mg).
13C nmr (D
2O): δ 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.
[0048] The corresponding PAMAM 1.0 and PAMAM 3.0 dendrimers terminated with sodium N-(2-sulfoethyl)succinamide
groups were similarly prepared.
13C nmr PAMAM 3.0 derivative (D
2O): δ 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 PAMAM 1.0 derivative (D
2O):δ 34.9,35.5,39.5,42.9,43.1,53.7,54.1, 179.0, 179.1, 179.3.
B. Preparation of sodium N-(2-sulfoethyl)succinamide terminated polylysine dendrimers.
BHAlyslys2lys4lys8lys16
[0049] Trifluoroacetic acid (1ml) was added to a suspension of BHAlysly
2lys
4lys
8DBL
16 (36.5mg; 5.0µmol) in dry dichloromethane (1ml) and the resulting solution stirred
at room temperature under nitrogen for two hours and then concentrated. The residue
was dissolved in dry DMSO (2ml) and the pH adjusted to 8.5 with triethylamine. A solution
of the crude tetrabutylammonium 4-nitrophenyl N-(2-sulfoethyl)succinamate (ca. 0.2mmol)
in DMSO (1ml) was then added dropwise and the mixture stirred overnight at room temperature.
The yellow solution was then concentrated (50°/10
-5mmHg) and the yellow residue partitioned between water and chloroform. The aqueous
layer was separated, washed with chloroform (3X) and ethyl acetate, and then concentrated
to give an oil (99mg). The crude product was converted to the sodium salt by passage
through a column of Amberlite IR 120(Na) to yield 81mg of material. This material
was further purified by gel filtration (Sephadex LH20; water) to give the sodium N-(2-sulfoethyl)succinamideterminatedBHAlyslys
2lys
4lys
8lys
16 dendrimer (39mg).
13C nmr (D
2O): δ 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.
[0050] The corresponding BHAlyslys
2, BHAlysly
2lys
4 and BHAlysly
2lys
4lys
8 terminated with sodium N-(2-sulfoethyl)succinamide groups were similarly prepared.
13C nmr BHAlysly
2lys
4lys
8 derivative (D
2O): δ 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, 178.3, 179.6,
178.7, 178.8, 178.9, 179.2, 179.3, 179.7, 179.8.
13C nmr BHAlyslys
2lys
4 derivative (D
2O): δ 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.
13C nmr BHAlyslys
2 derivative (D
2O): δ 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 sodium 4-sulfophenylthiourea terminated dendrimers
A. PAMAM 4.0 (Compound No. 1)
[0051] Solid sodium 4-sulfophenylisothiocyanate monohydrate (500mg; 1.96mmol) was added
to a solution of PAMAM 4.0 (300mg; 0.0582mmol) in water (10ml) and the resulting solution
heated under nitrogen at 53° for two hours and then cooled. The solution was concentrated
and the yellow solid residue purified by gel filtration (Sephadex LH20; water). The
pure fractions were combined and freeze dried to give the sodium 4-sulfophenylthiourea
terminated PAMAM 4.0 dendrimer as a fluffy white solid (370mg).
1H nmr (D
2O) : δ 2.28; 2.52; 2.69; 3.15; 3.27; 3.60; 7.32 (d, J=9Hz); 7.72 (d, J=9Hz).
13C nmr (D
2O) : δ 36.9; 41.1; 43.1; 48.3; 53.6; 55.8; 129.0; 131.1; 144.4; 178.5; 179.1; 184.4.
[0052] The corresponding PAMAM 1.0 and PAMAM 2.0, PAMAM 3.0 and PAMAM 5.0 (Compound No.
2) dendrimers terminated with 3, 6, 12 and 48 sodium 4-sulfophenylthiourea groups
respectively were similarly prepared.
B. PAMAM 4.0 (EDA) (Compound No. 3)
[0053] Solid sodium 4-sulfophenylisothiocyanate monohydrate (130mg; 0.5mmol) was added to
a solution of PAMAM 4.0 (EDA) (69mg; 0.01mmol) in water (4ml) and the resulting solution
heated under nitrogen at 53° for two hours and then cooled. The solution was concentrated
and the solid residue purified by gel filtration (Sephadex LH20; water). The pure
fractions were combined and freeze dried to give PAMAM 4.0 terminated with 32 sodium
4-sulfophenylthiourea groups as a fluffy white solid (136mg).
1H nmr (D
2O) : δ 2.30; 2.50; 2.70; 3.18; 3.62; 7.35 (d, J=9Hz); 7.72 (d, J=9Hz).
13C nmr (D
2O): δ 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)
[0054] Trifluoroacetic acid (4ml) was added to a suspension of BHAlyslys
2lys
4lys
8DBL
16 (0.73g; 0.1mmol) in dry dichloromethane (4ml) 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 (5ml), the solution passed through a column of Amberlite IRA-401(OH) and
the filtrate concentrated to give BHAlyslys
2lys
4lys
8lys
16 as a viscous oil (0.49g). The oil was redissolved in water (5ml) and N,N-dimethyl-N-allylamine
buffer (pH 9.5; 3ml) added. Solid sodium 4-sulfophenylisothiocyanate monohydrate (1.30g;
5.1mmol) was then added and the resulting solution heated under nitrogen at 53° for
two hours and then cooled. The solution was concentrated and the brownish solid residue
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 sodium
4-sulfophenylthiourea terminated BHAlysly
2lys
4lys
8lys
16 dendrimer as a fluffy white solid (374mg).
1H nmr (D
2O) : δ 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 nmr (D
2O) : δ 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.
[0055] The corresponding BHAlyslys
2lys
4lys
8, BHAlysly
2lys
4lys
8lys
16lys
32 (Compound No. 5), and BHAlyslys
2lys
4lys
8lys
16lys
32lys
64 (Compound No. 6) dendrimers terminated with 16, 64, and 128 sodium 4-sulfophenylthiourea
groups respectively were similarly prepared.
EXAMPLE 6 Preparation of sodium 3,6-disulfonapthylthionrea terminated dendrimers
A. PAMAM 4.0 (Compound No. 9)
[0056] Solid sodium 3,6-disulfonapthylisothiocyanate (160mg; 0.41mmol) was added to a solution
of PAMAM 4.0 (51mg; 0.01mmol) in water (3ml) and the resulting solution heated under
nitrogen at 53° for two hours and then cooled. The solution was concentrated and the
brown solid residue purified by gel filtration (Sephadex LH20; water). The pure fractions
were combined and concentrated to give the sodium 3,6-disulfonapthylthiourea terminated
PAMAM 4.0 dendrimer as a brownish solid (73mg).
1H nmr (D
2O): δ 2.30; 2.60; 2.74; 3.20; 3.57; 7.75; 7.86; 8.28.
13C nmr (D
2O): δ 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.
[0057] The corresponding PAMAM 2.0 dendrimer terminated with sodium 3,6-disulfonapthylthiourea
groups was similarly prepared.
B. PAMAM 4.0 (EDA) (Compound No. 11)
[0058] Solid sodium 3,6-disulfonapthylisothiocyanate (220mg; 0.57mmol) was added to a solution
of PAMAM 4.0 (EDA) (74mg; 0.01mmol) in water (4ml) and the resulting solution heated
under nitrogen at 53° for two hours and then cooled. The solution was concentrated
and the brownish solid residue purified by gel filtration (Sephadex LH20; water).
The pure fractions were combined and concentrated to give PAMAM 4.0 terminated with
32 sodium 3,6-disulfonapthylthiourea groups as a tan solid (148mg).
1H nmr (D
2O) : δ 2:30; 2.80; 3.20; 3.54; 7.74; 7.85; 8.25.
13C nmr (D
2O) : δ 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)
[0059] Trifluoroacetic acid (2ml) was added to a suspension of BHAlyslys
2lys
4lys
8DBL
16 (73mg; 0.01mmol) in dry dichloromethane (2ml) 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 (5ml), the solution passed through a column of Amberlite IRA-401(OH) and
the filtrate concentrated to give BHAlyslys
2lys
4lys
8lys
16 as a viscous oil. The oil was redissolved in water (5ml) and N,N-dimethyl-N-allylamine
buffer (pH 9.5; 3ml) added Solid sodium 3,6-disulfonapthylisothiocyanate (234mg; 0.60mmol)
was then added and the resulting solution heated under nitrogen at 53° for two hours
and then cooled. The solution was concentrated and the brownish solid residue 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 BHAlyslys
2lys
4lys
8lys
16 terminated with 32 sodium 3,6-disulfonapthylthiourea groups as a fluffy off-white
solid (119mg).
1H nmr (D
2O) : δ 1.0-2.0; 3.18; 3.43; 4.31; 7.22; 7.80; 7.89; 8.25.
13C nmr (D
2O) : δ 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 sodium 4-sulfonapthylthiourea terminated dendrimers
PAMAM 4.0 (Compound No. 8)
[0060] Solid sodium 4-sulfonapthylisothiocyanate (180mg; 0.5mmol) was added to a solution
of PAMAM 4.0 (51mg; 0.01mmol) in water (5ml) and the mixture heated under nitrogen
at 53° for two hours and then cooled. The water was distilled under reduced pressure
from the resulting suspension and the off white solid residue purified by gel filtration
(Sephadex LH20; water). The pure fractions were combined and freeze dried to give
the sodium 4-sulfonapthylthiourea terminated PAMAM 4.0 dendrimer as a fluffy white
solid (60mg).
1H nmr (D
2O) : δ 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 (D
2O) : δ 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 sodium 3,5-disulfophenylthiourea terminated dendrimers
PAMAM 4.0 (Compound No. 7)
[0061] Solid sodium 3,5-disulfophenylisothiocyanate (110mg; 0.32mmol) was added to a solution
of PAMAM 4.0 (63mg; 0.012mmol) in water (3ml) and the resulting solution heated under
nitrogen at 53° for two hours and then cooled. The solution was concentrated and the
brownish solid residue 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 (110mg).
1H nmr (D
2O) : δ 2.53; 3.08; 3.36; 3.66; 7.90; 7.95.
13C nmr (D
2O) : δ 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 sodium 3, 6, 8-trisulfonaphthylthiourea terminated dendrimers
PAMAM 4.0 (Compound No. 10)
[0062] Solid sodium 3, 6, 8-trisulfonaphthylisothiocyanate (250mg; 0.5mmol) was added to
a solution of PAMAM 4.0 (51mg; 0.01 mmol) and N,N-dimethyl-N-allylamine buffer (pH
9.5; 1ml) in water (2ml) and the mixture heated under nitrogen at 53° 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 (2ml) and passed through a short
column of Amberlite IR-120(Na). The filtrate was then concentrated and the residue
purified by gel filtration (Sephadex LH20; water). The pure fractions were combined
and freeze dried to give the sodium 3, 6, 8-trisulfonaphthylthiourea terminated PAMAM
4.0 dendrimer as an off-white solid (102mg).
1H nmr (D
2O) : δ 2.65; 3.02; 3.30; 3.66; 8.05; 8.42; 8.59; 8.67.
13C nmr (D
2O) : δ 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 sodium 4-(sulfomethyl)benzamide terminated dendrimers
PAMAM 4.0 (Compound No. 13)
[0063] Solid 4-nitrophenyl 4-(chloromethyl)benzoate (200mg; 0.68mmol) was added to a stirred
solution of PAMAM 4.0 (70mg; 0.014mmol) in dry DMSO (4ml) and the resulting yellow
solution stirred at room temperature for two hours. The solution was then concentrated
(10
-4mmHg; 40°) and the residue extracted with a mixture of water and dichloromethane (1:1).
The remaining solid material was dissolved in DMSO (5ml) and a solution of sodium
sulfite (130mg; 1mmol) in water (3ml) added. The slightly cloudy mixture that resulted
was left to stand for four days, after which time the addition of more water (2ml)
resulted in the formation of a clear homogeneous yellow solution. The solution was
then concentrated, first at 25mmHg and 40° then at 10
-4 mmHg and 50° to give the crude product. The crude product was purified by gel filtration
(Sephadex G25; water) to give PAMAM 4.0 terminated with 24 sodium 4-(sulfomethyl)benzamide
groups (24mg).
1H nmr (D
2O) : δ 2.25; 2.66; 3.08; 3.20; 3.33; 3.38; 4.01; 7.40 (br d); 7.62 (br d).
13C nmr (D
2O) : δ 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 4-sulfobenzamide terminated dendrimers
PAMAM 4.0 (EDA)
[0064] Solid potassium N-hydroxysuccinimidyl 4-sulfobenzoate (100mg; 0.3mmol) was added
to a solution of PAMAM 4.0 (EDA) (35mg; 0.005mmol) in 0.1M pH 8.5 borate buffer (5ml)
and the solution stirred at room temperature for two hours. The resulting milky solution
at this stage had a pH of 4.5. 1M Sodium carbonate solution (1ml) was then 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 sodium 4-sulfobenzamide groups (47mg).
1H nmr (D
2O) : δ 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 nmr (D
2O) : δ 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 Sodium N-(4-sulfophenyl)propanamide terminated dendrimers
PAMAM 4.0 (EDA)
[0065] Solid sodium N-(4-sulfophenyl)acrylamide (250mg; 1mmol) and solid sodium carbonate
(106mg; 1mmol) were added successively to a stirred solution of PAMAM 4.0 (EDA) (78mg;
0.011mmol) in water (4ml). The resulting solution was stirred under nitrogen for four
days and then freeze dried 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
sodium N-(4-sulfophenyl)propanamide groups (206mg).
13C nmr showed a faint trace of what was taken to be mono alkylated terminal amino groups.
1H nmr (D
2O) : δ 2.10; 2.48; 2.58; 2.79; 3.20; 7.42 (d, J=7Hz); 7.65 (d, J=7Hz).
13C nmr (D
2O) : δ 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 Sodium 4-sulfophenylurea terminated dendrimers
PAMAM 4.0 (EDA)
[0066] A solution of sodium sulfanilic acid (195mg; 1mmol) in dry DMSO (3ml) was added dropwise
to a solution ofN,N'- disuccinimidyl carbonate (530mg; 2mmol) in dry DMSO (4ml) and
the resulting brownish solution stirred at room temperature for 20 hours. A solution
of PAMAM 4.0 (EDA) (75mg; 0.011mmol) in dry DMSO (1ml) added and the solution stirred
for a further 18 hours. The solution was then concentrated under high vacuum (10
-5mmHg; 35°) to give a yellowish semi solid. The crude product was dissolved in DMSO
(4ml) and the solution added to 200ml 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 (275mg). This material was further purified
by gel filtration (Sephadex LH20; water) to give PAMAM 4.0 (EDA) terminated with 32
sodium 4-sulfophenylurea groups (106mg).
1H nmr (D
2O) :
δ 2.31; 2.55; 2.75; 3.19; 7.32 (d, J=9Hz); 7.63 (d, J=9Hz).
13C nmr (D
2O) : δ 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-trimethylglycinamidechloride terminated dendrimers.
BHAlyslys2lys4lys8lys16 (Compound No. 15)
[0067] Trifluoroacetic acid (4ml) was added to a suspension of BHAlyslys
2lys
4DBL
8DBL
16 (220mg; 30µmol) in dry dichloromethane (2ml) and the resulting solution stirred at
room temperature under nitrogen for two hours and then concentrated. The residue was
dissolved in dry DMSO (5ml) and the pH adjusted to 8.5 with triethylamine. Solid 4-nitrophenyl
N,N,N-trimethylglycinate chloride (0.50g; 1.8mmol) was then added and the mixture
stirred overnight at room temperature. The cloudy solution was then concentrated (50°/10
-5 mmHg) and the residue partitioned between water and dichloromethane. The aqueous
layer was separated, washed with dichloromethane (3X) and ethyl acetate, and then
concentrated to give an oil (1.128g). The crude product was purified by gel filtration
(Sephadex LH20; water) to give the N,N,N-trimethylglycinamide terminated BHAlyslys
2lys
4lys
8lys
16 dendrimer (116mg). "C nmr (D
2O): δ 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 4-Trimethylammoniumbenzamide terminated dendrimers
PAMAM 4.0 (Compound No. 16)
[0068] 1,1'-Carbonyldiimidazole (85mg; 0.52mmol) was added to a solution of 4-trimethylammoniumbenzoic
acid iodide (154mg; 0.5mmol) in dry DMF (4ml) and the mixture stirred at room temperature
under argon for two hours. During this time a white solid separated from the solution.
A solution of PAMAM 4.0 (58mg; 0.011 mmol) in dry DMF (2ml) was then added and the
mixture 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°) to give a white solid residue. The crude product was purified by gel filtration
(Sephadex LH20; 10% AcOH) to give PAMAM 4.0 terminated with 24 4-trimethylammoniumbenTamide
groups as the acetic acid salt (89mg).
1H nmr (D
2O) : δ 1.96; 2.65-2.85; 3.25-3.55; 3.64; 7.92.
13C nmr (D
2O) :
δ 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.
[0069] The corresponding PAMAM 2.0 dendrimer terminated with 6 4-trimethylammonium benzamide
groups was similarly prepared.
EXAMPLE 16 Preparation of 4-(Trimethylammoniummethyl)benzamide terminated dendrimers
PAMAM 4.0 (Compound No. 17)
[0070] Solid 4-nitrophenyl 4-(chloromethyl)benzoate (150mg; 0.5mmol) was added to a stirred
solution of PAMAM 4.0 (52mg; 0.01mmol) in dry DMSO (3ml). 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 (10
-5mmHg; 40°) and the residue shaken 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 air dried. The crude 4-(chloromethyl)benzamide
terminated dendrimer was dissolved in 25% aq. trimethylamine (20ml) and the yellow
solution left to stand overnight. The solution was then concentrated, the residue
dissolved in water (5ml) and the solution passed through a column of Amberlite IRA-401
(OH). The colourless filtrate was concentrated to give a viscous oil which was purified
by gel filtration (Sephadex G10; 10% AcOH) to give PAMAM 4.0 terminated with 24 4-(trimethylammoniummethyl)benzamide
groups (90mg).
1H nmr (D
2O) : δ 1.88; 2.65-2.80; 2.98; 3.10-3.60; 7.52 (br d, J=9Hz); 7.72 (br d, J=9Hz).
13C nmr (D
2O) : δ 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 N-(2-Acetozyethyl)-N,N-(dimethylammonium)methylcarbozamide
terminated dendrimers
PAMAM 4.0
[0071] Solid 1,1'-carbonyldiimidaz,ole (85mg; 0.52mmol) was added to a solution of N-(2-acetoxyethyl)-N-(carboxymethyl)-N,N-dimethylammonium
bromide (135mg; 0.5mmol) in dry DMF (3ml) and the resulting solution stirred under
nitrogen for two hours. A solution of PAMAM 4.0 (60mg; 0.012mmol) in DMF (2ml) was
then added, which caused the immediate formation of a flocculant precipitate which
slowly redissolved. The mixture was stirred for two days and then concentrated (10
-4 mmHg; 40°) to give a viscous oil. The crude product was purified by gel filtration
(Sephadex G10; 10% AcOH) to give PAMAM 4.0 terminated with 24 N-(2-Acetoxyethyl)-N,N-(dimethylammonium)methylcarboxamide
groups (64mg).
1H nmr (D
2O) : δ 1.93; 2.05; 2.70; 3.10-3.60; 3.28; 3.93 (m); 4.14; 4.48 (m).
13C nmr (D
2O) : δ 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 Guanidino terminated dendrimers
PAMAM 4.0 (Compound No. 18)
[0072] A solution of PAMAM 4.0 (63mg; 0.012mmol) and methylthiopseudourea sulfate (170mg;
0.61mmol) in water (5ml) (pH 10.5) was heated under nitrogen at 80° for two hours.
The solution was then concentrated and the residue purified by gel filtration (Sephadex
G10; 10% AcOH) to give PAMAM 4.0 terminated with 24 guanidino groups as the acetate
salt (107mg).
1H nmr (D
2O): δ 2.00; 2.80 (br t); 3.09 (br t); 3.32; 3.45 (br t); 3.60 (br t).
13C nmr (D
2O): δ 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.
[0073] The corresponding PAMAM 2.0 dendrimer terminated with 6 guanidino groups was similarly
prepared.
EXAMPLE 19 Preparation of 4-([1,4,8,11-tetraazacyclotetradecane]methyl)benzamide terminated
dendrimers
PAMAM 4.0 (Compound No. 19)
[0074] A solution of 1-(4-carboxyphenyl)methyl-1,4,8,1]-tetraazacyclotetradecane tetra hydrochloride
(120mg; 0.25mmol), N-hydroxysuccinimide (60mg; 0.52mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (250mg; 1.3mmol) in pH 7 phosphate buffer (10ml) was allowed to stand
a room temperature for one hour and then a solution of PAMAM 4.0 (32mg; 0.006mmol)
in pH 7 phosphate buffer (10ml) added. The mixture was allowed to stand for two days
and then concentrated. The residue was purified by gel filtration (Sephadex LH20;
10% AcOH) to give PAMAM 4.0 terminated with ca. 12 4-([1,4,8,11-tetraazacyclotetradecane)methyl)benzamide
groups as determined by
1H and
13C nmr (80mg). 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 (1ml), concentrated HCl (1ml) added, and the solution diluted with ethanol (30ml)
to precipitate a white solid. The solid was collected by filtration (68mg). Once again
1H and
13C nmr showed ca. 50% functionalisation of the terminal amino groups.
1H nmr (D
2O) : δ 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 nmr (D
2O) : δ 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; 4b.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 4-Carbory-3-hydrorybenzylamine terminated dendrimers
PAMAM 4.0 (EDA)
[0075] Sodium cyanoborohydride (32mg; 0.5mmol) was added to a mixture of PAMAM 4.0 (EDA)
(69mg; 0.01mmol), 4-formyl-2-hydroxybenzoic acid (83mg; 0.5mmol), and sodium hydrogen
carbonate (42mg; 0.5mmol) in water (4ml). 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 purified by gel filtration (Sephadex
LH20; water) to give PAMAM 4.0 (EDA) terminated with ca. 32 4-carboxy-3-hydroxybenzylamine
groups (91mg).
1H and
13C nmr (D
2O) shows mostly mono alkylation but with some signs of dialkylation of the terminal
amino groups, both spectra show broad peaks.
13C nmr (D
2O):δ 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 nmr (D
2O): δ 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
[0076] Bovine blood was collected from the abattoir, where an animal was bled into a bucket
containing sodium citrate at a concentration of 3.5g per litre of fresh blood. This
blood was returned to the laboratory where it was kept in a 37°C water bath.
[0077] Aliquots of the whole blood were then centrifuged at 3000rpm 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 later testing.
[0078] The procedure actually tests the recalcification time of the citrated blood at 37°C.
All glassware was washed, dried and silated with 'Coatasil' before re-washing and
drying. Each 12 X 75mm culture tube contained 0.1ml of plasma, 0.1ml of saline solution
(0.9% NaCl) followed by 0.1ml of 0.025M CaCl
2 at which time the stop watch was started. Every 15 sec the tube was tilted to one
side and clotting was assessed. When a firm clot had formed, the watch was stopped
and the time recorded. In the case of testing anti-coagulants 0.1 ml of the test substance
replaced the saline. The times for a range of concentrations for the test compounds
are recorded in Table 1. Heparin, sodium citrate and test compouond were made up in
water as 10mg/ml solutions. These solutions were diluted to give a range of concentrations.
The final concentrations in the test tubes are given in the table. The figures in
the table represent average times for, up to ten replicates.
TABLE 1
|
Time for plasma coagulation at 37°C for following concentrations of anticoagulant |
Compound |
Controls |
0.0003 mg/ml |
0.003 mg/ml |
0.03 mg/ml |
0.33 mg/ml |
3.33 mg/ml |
Heparin |
2.29min |
2.30min |
>30min |
>30min |
>30min |
- |
Sodium citrate |
2.29min |
- |
- |
2.30min |
2.30min |
>30min |
Compound No.1 |
2.11min |
- |
- |
2.29min |
4.00min |
>30min |
Compound No.4 |
2.08min |
- |
- |
1.72min |
10.00min* |
>30min |
Compound No. 15 |
2.20min |
- |
2.34min |
7min* |
8min* |
- |
Compound No.9 |
2.27min |
- |
2.19min |
4min |
5.40min* |
- |
EXAMPLE 23 Test for antiviral activity.
1. A compound being a dendrimer having a plurality of terminal groups,
characterised in that
(i) said compound has in vitro inhibitory activity against a virus selected from the group consisting of HIV1 or
HIV2, Hepatitis B or C Bovine Viral Diarrhoea Virus, Rhinovirus, Human Parainfluenza
Virus, Respiratory Syncytial Virus (RSV), Varicella Zoster Virus (VZV), Human Cytomegalovirus
(CMV), Epstein Barr Virus (EBV), Human Papilloma Virus (HPV), Adenovirus-8, Herpes
Simplex Virus (HSV) type 1 and 2, Measles Virus, and Vesicular Stomatitis Virus (VSV),
and
(ii) at least one of said terminal goups has an anionic- or cationic-containing moiety
bonded thereto.
2. A compound according to claim 1, wherein said dendrimer, comprises a polyvalent core
covalently bonded to at least two dendritic branches, and extends through at least
two generations.
3. A compound according to claim 1 or claim 2, wherein said dendrimer is a polyamidoamine
dendrimer based on an ammonia core.
4. A compound according to claim 1 or claim 2, wherein said dendrimer is a polyamidoamine
dendrimer based on an ethylene diamine core.
5. A compound according to claim 1 or claim 2, wherein said dendrimer is a polylysine
dendrimer based on a benzhydrylamine.
6. A compound according to any of claims 1 to 5 wherein said anionic- or cationic-containing
moiety or moieties are bonded to terminal amine, sulfhydryl, hydroxy or other reactive
functional terminal groups of said dendrimer by amide or thiourea linkages.
7. A compound according to any of claims 1 to 6 wherein said anionic-containing moiety
or moieties are sulfonic acid-containing moieties or carboxylic acid-containing moieties
other than 2-thiosialic acid moieties.
8. A compound according to any of claims 1 to 6 wherein said cationic-containing moiety
or moieties are trimethylammonium-containing moieties or polyamino-macrocyclic-containing
moieties.
9. A compound according to any of claims 1 to 6, wherein the moiety or moieties which
are bonded to amino of other reactive functional terminal groups of the dendrimer
are selected from the following groups:
―NH(CH
2)
nSO3
- ―(CH
2)
nSO
3 ―Ar(SO
3)
n
―CH
2CH(SO
3)COOH ―CH(SO
3)CH
2COOH ―ArX(CH
2)
nSO
3 X = O, S,NH

in which n is zero or a positive integer.
10. A compound according to any of claims 1 to 6 which is an alkylsulfonic acid terminated
dendrimer.
11. A compound according to any of claims 1 to 6 which is a sulfoacetamide terminated
dendrimer.
12. A compound according to any of claims 1 to 6 which is a sulfosuccinamic acid terminated
dendrimer.
13. A compound according to any of claims 1 to 6 which is a N-(2-sulfoethyl) succinamide
terminated dendrimer.
14. A compound according to any of claims 1 to 6 wherein the moiety bonded to the terminal
groups of the dendrimer is an aryl or heteroarylthiourea substituted with one or more
sulfonic acid groups.
15. A compound according to claim 14 which is a 4-sulfophenylthiourea terminated dendrimer.
16. A compound according to claim 14 which is a 3,6-di-sulfonapthylthiourea terminated
deadrimer.
17. A compound according to claim 14 which is a 4-sulfonapthylthiourea terminated dendrimer.
18. A compound according to claim 14 which is a 3,5-di-sulfophenylthiourea terminated
dendrimer.
19. A compound according to claim 14 which is a 3,6,8-tri-sulfonapthylthiourea terminated
dendrimer.
20. A compound according to any of claims 1 to 6 wherein the moiety bonded 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 claim 20 which is a 4-(sulfomethyl) benzamide terminated dendrimer.
22. A compound according to claim 20 which is a 4-sulfobenzannide terminated dendrimer.
23. A compound according to any of claims 1 to 6 wherein the moiety bonded to the terminal
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 moiety bonded 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 4-sulfophenylurea terminated dendrimer.
27. A compound according to any of claims 1 to 6 wherein the moiety bonded to the terminal
groups of the dendrimer is an N,N,N-trimethyl derivative of an amino acid.
28. A compound according to claim 27 which is a N,N,N-tri-methylglycinamide terminated
dendrimer.
29. A compound according to any of claims 1 to 6 wherein the moiety bonded to the terminal
groups of the dendrimer is an aryl or heteroaryl amide substituted with one or more
trialkylamino, trialkylaminoalkyl, trialkylaminoalkyloxy, trialkylaminoalkylamino
or trialkylaminoalkylthio groups.
30. A compound according to claim 29 which is a 4-trimethylammonium benzamide terminated
dendrimer.
31. A compound according to claim 29 which is a 4-(trimethylammonium methyl)benzamide
terminated dendrimer.
32. A compound according to any of claims 1 to 6 which is a N-(2-acetoxyethyl)-N,N-(dimethylammonium)methylcarboxamide
terminated dendrimer.
33. A compound according to any of claims 1 to 6 which is a guanidino terminated dendrimer.
34. A compound according to any of claims 1 to 6 wherein the dendrimer is a macrocyclic
polyamino group containing one or more macrocyclic rings connected through an alkyl
or aryl spacer moiety to the terminal group of the dendrimer.
35. A compound according to claim 34 which is a 4-([1,4,8,11-tetraazacyclotetradecane]methyl)benzamide
terminated dendrimer.
36. A compound according to any of claims 1 to 6 which is a 4-carboxy-3-hydroxybenzylamine
terminated dendrimer.
37. A pharmaceutical or veterinary composition for prophylactic or therapeutic antiviral
treatment of a human or non-human animal, which comprises a compound of any of claims
1 to 36, in association with at least one pharmaceutically or veterinarily acceptable
carrier or diluent.
38. Use of a compound according to any of claims 1 to 36 for the manufacture of a medicament
for prophylactic or therapeutic antiviral treatment of a human or non-human animal,
wherein the medicament is to be administrated in a prophylactic- or therapeutic-antiviral-effective
amount,
39. Use according to claim 38, wherein said antiviral treatment is treatment of infection
by HIV 1 or HIV2, Hepatitis B or C, Bovine Viral Diarrhoea Virus, Human Influenza
Virus A and B, Rhinovirus, Human Parainfluenza Virus, Respiratory Syncytial Virus
(RSV), Varicella Zoster Virus (VSV), Human Cytomegalovirus (CMV), Epstein Barr Virus
(EBV), Human Papilloma Virus (HPV), Adenovirus-8. Herpes Simplex Virus (HSV) type
1 and 2, Measles Virus, or Vesicular Stomatitis Virus (VSV).
1. Verbindung, bei der es sich um ein Dendrimer mit einer Menge von Endgruppen handelt,
dadurch gekennzeichnet, dass:
(i) die Verbindung in vitro eine hemmende Wirkung gegen ein Virus aufweist, das aus
der Gruppe ausgewählt ist, die aus Folgenden besteht: HIV1 oder HIV2, Hepatitis-B-
oder -C-Virus, Virus der Bovinen Virus-Diarrhöe, Rhinovirus, Humanes Parainfluenza-Virus,
Respiratory Syncytial Virus (RSV), Varicella-zoster-Virus (VZV), Humanes Cytomegalievirus
(CMV), Epstein-Barr-Virus (EBV), Humanes Papillom-Virus (HPV), Adenovirus-8, Herpes-simplex-Virus
(HSV) Typ 1 und 2, Masernvirus und Vesikuläres Stomatitis-Virus (VSV); und
(ii) wenigstens eine der Endgruppen eine daran gebundene anionische oder kationische
Struktureinheit aufweist.
2. Verbindung gemäß Anspruch 1, wobei das Dendrimer einen mehrwertigen Kern umfasst,
der kovalent an wenigstens zwei dendritische Zweige gebunden ist und sich über wenigstens
zwei Generationen erstreckt.
3. Verbindung gemäß Anspruch 1 oder 2, wobei das Dendrimer ein auf einem Ammoniak-Kern
beruhendes Polyamidoamin-Dendrimer ist.
4. Verbindung gemäß Anspruch 1 oder 2, wobei das Dendrimer ein auf einem Ethylendiamin-Kern
beruhendes Pölyamidoamin-Dendrimer ist.
5. Verbindung gemäß Anspruch 1 oder 2, wobei das Dendrimer ein auf einem Benzhydrylamin
beruhendes Polylysin-Dendrimer ist.
6. Verbindung gemäß einem der Ansprüche 1 bis 5, wobei die anionische oder kationische
Struktureinheit oder die anionischen oder kationischen Struktureinheiten über Amid-
oder Thioharnstoffbindungen an Amin-, Sulfhydryl- oder Hydroxy-Endgruppen oder andere
reaktive funktionelle Endgruppen des Dendrimers gebunden sind.
7. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die anionische Struktureinheit
oder die anionischen Struktureinheiten sulfonsäurehaltige Struktureinheiten oder carbonsäurehaltige
Struktureinheiten, aber keine 2-Thiosialinsäure-Struktureinheiten, sind.
8. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die kationische Struktureinheit
oder die kationischen Struktureinheiten Trimethylammonium enthaltende Struktureinheiten
oder Polyaminomakrocyclen enthaltende Struktureinheiten sind.
9. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die Struktureinheit oder Struktureinheiten,
die an Aminoendgruppen oder andere reaktive funktionelle Endgruppen des Dendrimers
gebunden sind, aus den folgenden Gruppen ausgewählt sind:
―NH(CH
2)
nSO
3- ―(CH
2)
nSO
2- ―Ar(SO
3)
n
―CH
2CH(SO
3)COOW ―CH(SO
3)CH
2COOH ―ArX(CH
2)
nSO
3- X = O, S, NH

wobei n = 0 oder eine positive ganze Zahl ist.
10. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
Alkylsulfonsäure-Endgruppen handelt.
11. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
Sulfoacetamid-Endgruppen handelt.
12. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
Sulfosuccinamsäure-Endgruppen handelt.
13. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
N-(2-Sulfoethyl)succinamid-Endgruppen handelt.
14. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die an die Endgruppen des Dendrimers
gebundene Struktureinheit ein mit einer oder mehreren Sulfonsäuregruppen substituierter
Aryl- oder Heteroarylthioharnstoff ist.
15. Verbindung gemäß Anspruch 14, bei der es sich um ein Dendrimer mit 4-Sulfophenylthioharnstoff-Endgruppen
handelt.
16. Verbindung gemäß Anspruch 14, bei der es sich um ein Dendrimer mit 3,6-Disulfonaphthylthioharnstoff-Endgruppen
handelt.
17. Verbindung gemäß Anspruch 14, bei der es sich um ein Dendrimer mit 4-Sulfonaphthylthioharnstoff-Endgruppen
handelt.
18. Verbindung gemäß Anspruch 14, bei der es sich um ein Dendrimer mit 3,6-Disulfophenylthioharnstoff-Endgruppen
handelt.
19. Verbindung gemäß Anspruch 14, bei der es sich um ein Dendrimer mit 3,6,8-Trisulfonaphthylthioharnstoff-Endgruppen
handelt.
20. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die an die Endgruppen des Dendrimers
gebundene Struktureinheit ein mit einer oder mehreren Sulfonsäure-, Sulfoalkyl-, Sulfoalkoxy-,
Sulfoalkylamino- oder Sulfoalkylthiogruppen substituiertes Aryl- oder Heteroarylamid
ist.
21. Verbindung gemäß Anspruch 20, bei der es sich um ein Dendrimer mit 4-(Sulfomethyl)benzamid-Endgruppen
handelt.
22. Verbindung gemäß Anspruch 20, bei der es sich um ein Dendrimer mit 4-Sulfobenzamid-Endgruppen
handelt.
23. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die an die Endgruppen des Dendrimers
gebundene Struktureinheit ein mit einer oder mehreren Sulfonsäuregruppen substituiertes
Aryl- oder Heteroarylalkanamid ist.
24. Verbindung gemäß Anspruch 23, bei der es sich um ein Dendrimer mit N-(4-Sulfophenyl)propanamid-Endgruppen
handelt.
25. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die an die Endgruppen des Dendrimers
gebundene Struktureinheit ein mit einer oder mehreren Sulfonsäuregruppen substituierter
Aryl- oder Heteroarylharnstoff ist.
26. Verbindung gemäß Anspruch 25, bei der es sich um ein Dendrimer mit 4-Sulfophenylharnstoff-Endgruppen
handelt.
27. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die an die Endgruppen des Dendrimers
gebundene Struktureinheit ein N,N,N-Trimethyl-derivat einer Aminosäure ist.
28. Verbindung gemäß Anspruch 27, bei der es sich um ein Dendrimer mit N,N,N-Trimethylglycinamid-Endgruppen
handelt.
29. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei die an die Endgruppen des Dendrimers
gebundene Struktureinheit ein mit einer oder mehreren Trialkylamino-, Trialkylaminoalkyl-,
Trialkylaminoalkyloxy-, Trialkylaminoalkylamino- oder Trialkylaminoalkylthiogruppen
substituiertes Aryl- oder Heteroarylamid ist.
30. Verbindung gemäß Anspruch 29, bei der es sich um ein Dendrimer mit 4-Trimethylammoniumbenzamid-Endgruppen
handelt.
31. Verbindung gemäß Anspruch 29, bei der es sich um ein Dendrimer mit 4-(Trimethylammoniummethyl)benzamid-Endgruppen
handelt.
32. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
N-(2-Acetoxyethyl)-N,N-(dimethylammonium)methylcarboxamid-Endgruppen handelt.
33. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
Guanidino-Endgruppen handelt.
34. Verbindung gemäß einem der Ansprüche 1 bis 6, wobei das Dendrimer eine makrocyclische
Polyaminogruppe ist, die einen oder mehrere makrocyclische Ringe enthält, die über
eine Alkyl- oder Aryl-Spacer-Struktureinheit mit der Endgruppe des Dendrimers verbunden
sind.
35. Verbindung gemäß Anspruch 34, bei der es sich um ein Dendrimer mit 4-([1,4,8,11-Tetraazacyclotetradecan]methyl)benzamid-Endgruppen
handelt.
36. Verbindung gemäß einem der Ansprüche 1 bis 6, bei der es sich um ein Dendrimer mit
4-Carboxy-3-hydroxybenzylamin-Endgruppen handelt.
37. Pharmazeutische oder veterinärmedizinische Zusammensetzung zur prophylaktischen oder
therapeutischen antiviralen Behandlung eines Menschen oder eines nichthumanen Tiers,
die eine Verbindung gemäß einem der Ansprüche 1 bis 36 in Verbindung mit wenigstens
einem pharmazeutisch oder veterinärmedizinisch annehmbaren Träger oder Verdünnungsmittel
umfasst.
38. Verwendung einer Verbindung gemäß einem der Ansprüche 1 bis 36 zur Herstellung eines
Medikaments zur prophylaktischen oder therapeutischen antiviralen Behandlung eines
Menschen oder eines nichthumanen Tiers, wobei das Medikament in einer prophylaktisch
oder therapeutisch antiviral wirksamen Menge zu verabreichen ist.
39. Verwendung gemäß Anspruch 38, wobei es sich bei der antiviralen Behandlung um die
Behandlung einer Infektion durch folgende Viren handelt: HIV1 oder HIV2, Hepatitis-B-
oder -C-Virus, Virus der Bovinen Virus-Diarrhöe, Humanes Influenza-Virus A und B,
Rhinovirus, Humanes Parainfluenza-Virus, Respiratory Syncytial Virus (RSV), Varicella-zoster-Virus
(VZV), Humanes Cytomegalievirus (CMV), Epstein-Barr-Virus (EBV), Humanes Papillom-Virus
(HPV), Adenovirus-8, Herpes-simplex-Virus (HSV) Typ 1 und 2, Masernvirus oder Vesikuläres
Stomatitis-Virus (VSV).
1. Composé étant un dendrimère comptant une pluralité de groupes terminaux,
caractérisé en ce que :
(i) ledit composé a in vitro une activité inhibitrice contre un virus choisi dans le groupe se composant des éléments
suivants : HTV-1 ou HTV-2, l'hépatite B ou C, le virus de la diarrhée virale des bovins,
le rhinovirus, le virus parainfluenza humain, le virus respiratoire syncytial (RSV),
le virus varicelle-zona (VZV), le cytomégalovirus humain (CMV), le virus Epstein-Barr
(EBV), le papillomavirus humain, l'adénovirus-8, le virus de l'herpès simplex (HSV)
du type 1 et 2, le virus de la rougeole, et le virus de la stomatite vésiculaire (VSV),
et
(ii) au moins l'un desdits groupes terminaux comporte un groupe caractéristique à
contenu anionique ou cationique lié à celui-ci.
2. Composé selon la revendication 1, dans lequel ledit dendrimère comprend un noyau polyvalent
lié de manière covalente a au moins deux branches dendritiques, et s'étend à travers
au moins deux générations.
3. Composé selon la revendication 1 ou la revendication 2, dans lequel ledit dendrimère
est un dendrimère du type polyamidoamine à base d'un noyau ammoniacal.
4. Composé selon la revendication 1 ou la revendication 2, dans lequel ledit dendrimère
est un dendrimère du type polyamidoamine à base d'un noyau d'éthylènediamine.
5. Composé selon la revendication 1 ou la revendication 2, dans lequel ledit dendrimère
est un dendrimère de type polylysine à base d'une benzhydrylamine.
6. Composé selon l'une quelconque des revendications 1 à 5, dans lequel le(s)dit(s) groupe(s)
caractéristique(s) à contenu anionique ou cationique est/sont lié(s) à des groupes
amines terminaux, sulfhydryl ou hydroxy, ou à un autre groupe terminal fonctionnel
réactif dudit dendrimère par des liaisons par amide ou thiourée.
7. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le(s)dit(s) groupe(s)
caractéristique(s) à contenu anionique sont des groupes caractéristiques contenant
un acide sulfonique ou des groupes caractéristiques contenant un acide carboxylique
autres que les groupes caractéristiques à 2-acide thiosialique.
8. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le(s)dit(s) groupe(s)
caractéristique(s) à contenu cationique sont des groupes caractéristiques contenant
du triméthylammonium ou des groupes caractéristiques à contenu polyamino-macrocyclique.
9. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le(s) groupe(s)
caractéristique(s) qui sont liés à des groupes amines terminaux ou à d'autres groupes
terminaux fonctionnels réactifs du dendrimère sont choisis parmi les groupes suivants
:
―NH(CH
2)
nSO
3- ―(CH
3)
nSO
3- -Ar(SOa)
n
―CH
2CH(SO
3)COOH ―CH(SO
2)CH
2COOH -ArX(CH
2)
nSO
2- X--O, S NH

dans le(s)quel(s) n est 0 ou un entier positif.
10. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par un acide alkylsulfonique.
11. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par un sulfacétamide.
12. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par un acide sulfosuccinamique.
13. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par un N-(2-sulfoéthyl)succinamide.
14. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le groupe caractéristique
lié aux groupes terminaux du dendrimère est un aryle ou une hétéroarylthiourée substitué
par un ou plusieurs groupes d'acide sulfonique.
15. Composé selon la revendication 14, qui est un dendrimère se terminant par une 4-sulfophénylthiourée.
16. Composé selon la revendication 14, qui est un dendrimère se terminant par une 3,6-di-sulfonaphtylthiourée.
17. Composé selon la revendication 14, qui est un dendrimère se terminant par une 4-sulfonaphtylthiourée.
18. Composé selon la revendication 14, qui est un dendrimère se terminant par une 3,5-di-sulfophénylthiourée.
19. Composé selon la revendication 14, qui est un dendrimère se terminant par une 3,6,8-tri-
sulfonaphtylthiourée.
20. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le groupe caractéristique
lié aux groupes terminaux du dendrimère est un aryl ou hétéroaryl amide substitué
par l'un ou plusieurs des groupes d'acide sulfonique, sulfoalkyl, sulfoalkoxy, sulfoallcylamino
ou sulfoalkythio.
21. Composé selon la revendication 20, qui est un dendrimère se terminant par une 4-(sulfométhyl)benzamide.
22. Composé selon la revendication 20, qui est un dendrimère se terminant par une 4-sulfobenzamide.
23. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le groupe caractéristique
lié aux groupes terminaux du dendrimère est un aryl ou hétéroaryl alkanamide substitué
par l'un ou plusieurs des groupes d'acide sulfonique.
24. Composé selon la revendication 23, qui est un dendrimère se terminant par une N-(4-sulfophényl)propanamide.
25. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le groupe caractéristique
lié aux groupes terminaux du dendrimère est un aryl ou hétéroaryl urée substitué par
l'un ou plusieurs des groupes d'acide sulfonique.
26. Composé selon la revendication 25, qui est un dendrimère se terminant par une 4-sulfophénylurée.
27. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le groupe caractéristique
lié aux groupes terminaux du dendrimère est un dérivé N,N,N-triméthyl d'un acide aminé.
28. Composé selon la revendication 27, qui est un dendrimère se terminant par un N,N,N-tri-méthylglycinamide.
29. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le groupe caractéristique
lié aux groupes terminaux du dendrimère est un aryl ou hétéroaryl amide substitué
par l'un ou plusieurs des groupes trialkylamino, trialkylaminoalkyl, trialkylaminoalkyloxy,
trialkylaminoalkylamino ou trialkylaminoalkylthio.
30. Composé selon la revendication 29, qui est un dendrimère se terminant par une 4-triméthylammonium
benzamide.
31. Composé selon la revendication 29, qui est un dendrimère se terminant par une 4-(triméthylammoniumméthyl)benzamide.
32. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par un N-(2-acétoxyéthyl)-N,N-(diméthylammonium)méthylcarboxamide.
33. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par un composé guanidino.
34. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le dendrimère
est un groupe macrocyclique, polyamino contenant un ou plusieurs cycles macrocycliques
reliés par un groupe caractéristique espaceur alkyl ou aryl au groupe terminal du
dendrimère.
35. Composé selon la revendication 34, qui est un dendrimère se terminant par une 4-([1,4,8,11-tétra-azacyclotétradécane]méthyl)benzamide,
36. Composé selon l'une quelconque des revendications 1 à 6, qui est un dendrimère se
terminant par une 4-carboxy-3-hydroxy-benzylamine.
37. Composition pharmaceutique ou vétérinaire pour un traitement antiviral prophylactique
ou thérapeutique d'un être humain ou d'un animal, qui comprend un composé selon l'une
quelconque des revendications 1 à 36, en association avec au moins un support ou un
diluant acceptable du point de vue pharmaceutique ou vétérinaire.
38. Utilisation d'un composé selon l'une quelconque des revendications 1 à 36 pour la
production du médicament destiné au traitement antiviral prophylactique ou thérapeutique
d'un être humain ou d'un animal, dans laquelle le médicament qui doit être administré
est une quantité d'antivirus efficace du point de vue prophylactique ou thérapeutique.
39. Utilisation selon la revendication 38, dans laquelle ledit traitement antiviral est
le traitement d'une infection par HIV-1 ou HIV-2, l'hépatite B ou C, le virus de la
diarrhée virale des bovins, le virus de la grippe humaine du type A et B, le rhinovirus,
le virus parainfluenza humain, le virus respiratoire syncytial (RSV), le virus varicelle-zona
(VZV), le cytomégalovirus humain (CMV), le virus Epstein-Barr (EBV), le papillomavirus
humain (HPV), l'adénovirus-8, le virus de l'herpès simplex (HSV) du type 1 et 2, le
virus de la rougeole, et le virus de la stomatite vésiculaire (VSV)