Bioactive polymers

Abstract

Polycondensation products of aminoguanidine and/or 1,3-diaminoguanidine with one or more diamines are provided including polyguanidine derivatives of the following formula (I) or a salt thereof: ##STR00001##
wherein X is selected from NH.sub.2, aminoguanidino and 1,3-diaminoguanidino; Y is selected from H and R.sub.1NH.sub.2; or X and Y together represent a chemical bond to give a cyclic structure; R.sub.1 is selected from divalent organic radicals having 2 to 20 carbon atoms, in which optionally one or more carbon atoms are replaced by O or N; a and b are each 0 or 1, wherein a+b2 if no 1,3-diaminoguanidine units are included; R.sub.2 is selected from H and NH.sub.2, wherein R.sub.2 is NH.sub.2 if a+b=0, R.sub.2 is H or NH.sub.2 if a+b=1, and R.sub.2 is H if a+b=2; and n2. Production methods and uses of the polyguanidine derivatives are also provided.

Claims

1. A polyguanidine derivative comprising a polycondensation product of aminoguanidine and/or 1,3-diaminoguanidine with at least one diamine and having the following formula (I) or a salt thereof: ##STR00009## wherein X is selected from NH2, aminoguanidino, and 1,3-diaminoguanidino; Y is selected from H and R.sub.1NH.sub.2; or X and Y together represent a chemical bond to give a cyclic structure; R.sub.1 is selected from divalent organic radicals having 2 to 20 carbon atoms, in which optionally at least one carbon atom is replaced by O or N; a and b are each 0 or 1, wherein a+b2 if no 1,3-diaminoguanidine units are contained; R.sub.2 is selected from H and NH2, wherein R.sub.2 is NH.sub.2 if a+b=0, R.sub.2 is H or NH.sub.2 if a+b=1, and R.sub.2 is H if a+b=2; and n>2.

2. The polyguanidine derivative according to claim 1, wherein R.sub.1 is selected from alkylene radicals, in which optionally at least one carbon atom is replaced by O or N.

3. The polyguanidine derivative according to claim 2, wherein R.sub.1 is selected from radicals of the following general formulas (II) to (V):
(CH.sub.2).sub.cZ.sub.1(CH.sub.2).sub.d(II),
(CH.sub.2).sub.cZ.sub.1(CH.sub.2).sub.dZ.sub.2(CH.sub.2).sub.e(III),
(CH.sub.2).sub.cZ.sub.1(CH.sub.2).sub.dZ.sub.2(CH.sub.2).sub.eZ.sub.3(CH.sub.2).sub.f(IV), and
(CH.sub.2).sub.cZ.sub.1(CH.sub.2).sub.dZ.sub.2(CH.sub.2).sub.eZ.sub.3(CH.sub.2).sub.fZ.sub.4(CH.sub.2).sub.g(V), wherein Z.sub.1 to Z.sub.4 are each independently a heteroatom selected from O and N, and indexes c to g are each independently integers in a range of 1 to 12, such that a total number of atoms of radical R.sub.1 does not exceed 20.

4. The polyguanidine derivative according to claim 3, wherein all heteroatoms Z within one radical R.sub.1 are either O or N.

5. The polyguanidine derivative according to claim 4, wherein R.sub.1 represents a divalent radical of a polyether diamine.

6. The polyguanidine derivative according to claims 1, wherein n=2 to 6.

7. The polyguanidine derivative according to claim 1, wherein the salt is an acid addition salt in a form of a hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfate, carbonate, borate, cyanate, thiocyanate, phosphate, mesylate, nitrate, acetate, benzoate, lactate, tartrate, citrate, maleate, fumarate, a partial ester of one of these acids, in case they are difunctional or higher, or as a mixture of at least two of these salts and/or partial esters.

8. A method for producing a polyguanidine derivative according to claim 1, the method comprising polycondensing a guanidine derivative selected from aminoguanidine, 1,3-diaminoguanidine and an acid addition salt thereof with at least one diamine of the formula H.sub.2NRNH.sub.2 by heating.

9. The method according to claim 8, wherein the at least one diamine is used at an excess of 3 to 5 molar % in relation to the guanidine derivative.

10. The method according to claim 8, wherein the acid addition salt of amino-guanidine or 1,3-diaminoguanidine is heated together with the at least one diamine, initially to a first, lower temperature and then to a second, higher temperature.

11. The method according to claim 10, wherein the acid addition salt of aminoguanidine or 1,3-diaminoguanidine is heated together with the at least one diamine, initially to 110-130 C. and then to 160-180 C.

12. The method according to claim 10, wherein a reaction mixture the guanidine derivative and diamine is kept at the first temperature for 1 to 3 hours and at the second temperature for 1 to 8 hours.

13. The method according to claim 8, wherein the polyguanidine derivative is purified by dissolution in approximately a 3-to 10-fold amount of water.

14. A method of using the polyguanidine derivative according to claim 1 for in human and veterinary medical fields for antagonizing bacterial, fungal and viral infections and their aftereffects.

15. A method of using the polyguanidine derivative according to claim 1 as a pesticide and disinfectant in the agricultural and environmental fields for reducing and eliminating germs.

16. A method of using the polyguanidine derivative according to claim 1 as an antiparasitic.

17. A method of using the polyguanidine derivative according to claim 1 as a supplement for stabilizing or sterilizing products.

18. A method of using the polyguanidine derivative according to claim 1 as a nebulization substance, wherein the polyguanidine derivative is present in a dissolved form for cold/wet nebulization, micronization and vapor sterilization.

19. A composition comprising the polyguanidine derivative according to claim 1, wherein an effective amount of the polyguanidine derivative is present as a solution in a 3- to 10-fold amount of water.

Description

EXAMPLES

Examples 1 to 6 & Comparative Examples 1 and 2

Production of the Polymers

Example 1

(1) 23 mmol of 1,3-diaminoguanidinium hydrochloride and 24 mmol of 4,9-dioxadodecane-1,12-diamine were heated in a reaction vessel closed with a drying tube at 120 C. for 90 min with stirring, then the temperature was increased to 180 C. for 100 min, at the end of this reaction time under reduced pressure (50 mbar) for 45 min. After the reaction mixture had cooled off to below 80 C., 25 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

(2) The water was evaporated from a sample of the aqueous solution obtained, and the residue obtained was dried in vacuum, which resulted in a reddish, viscous liquid. It was dissolved in 2 ml of D.sub.2O (with a deuterization degree >99,5%), and a .sup.1H nuclear resonance (.sup.1H-NMR-) spectrum was obtained. The positions of methylene proton groups of the radical R.sub.1 in the product distinguishable in this way are as follows:

(3) .sup.1H-NMR (D.sub.2O), (ppm): 1.54-1.67 (m, OCH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.80-1.95 (m, NCH.sub.2CH.sub.2), 3.23-3.38 ppm (m, NCH.sub.2), 3.42-3.65 ppm (m, CH.sub.2CH.sub.2OCH.sub.2CH.sub.2).

(4) This confirms the structure of the diamine component used, 4,9-dioxadodecane-1,12-diamine.

Example 2

(5) 4.6 mmol of 1,3-diaminoguanidinium hydrochloride and 4.8 mmol of 4,9-dioxadodecane-1,12-diamine were heated in a reaction vessel closed with a drying tube at 120 C. for 90 min with stirring, then the temperature was increased to 180 C. for 8 h, at the end of this reaction time under reduced pressure (50 mbar) for 45 min. After the reaction mixture had cooled off to below 80 C., 16 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

Example 3

(6) 4.6 mmol of N-aminoguanidinium hydrochloride and 4.8 mmol of 4,9-dioxadodecane-1,12-diamine were heated in a reaction vessel closed with a drying tube at 120 C. for 90 min with stirring, then the temperature was increased to 180 C. for 3.5 h, at the end of this reaction time under reduced pressure (50 mbar) for 60 min. After the reaction mixture had cooled off to below 80 C., 16 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

Example 4

(7) 1.16 mmol of 1,3-diaminoguanidinium hydrochloride and 1.21 mmol of tris(2-aminoethyl)amine were heated in a reaction vessel closed with a drying tube at 120 C. for 150 min with stirring, then the temperature was increased to 160 C. for 2.5 h, at the end of this reaction time under reduced pressure (50 mbar) for 45 min. After the reaction mixture had cooled off to below 80 C., 4 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

Example 5

(8) 8.12 mmol of 1,3-diaminoguanidinium hydrochloride and 8.47 mmol of tris(2-aminoethyl)amine were heated in a reaction vessel closed with a drying tube at 130 C. for 120 min with stirring, then the temperature was increased to 180 C. for 8 h, at the end of this reaction time under reduced pressure (50 mbar) for 90 min. After the reaction mixture had cooled off to below 80 C., 28 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

Example 6

(9) 2.32 mmol of 1,3-diaminoguanidinium hydrochloride and 2.43 mmol of 3,6-dioxaoctane-1,8-diamine were heated in a reaction vessel closed with a drying tube at 120 C. for 60 min with stirring, then the temperature was increased to 170 C. for 4 h, at the end of this reaction time under reduced pressure (50 mbar) for 60 min. After the reaction mixture had cooled off to below 80 C., 7 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

Comparative Example 1

(10) 23.2 mmol of guanidinium hydrochloride, 5.4 mmol of 3,6-dioxaoctane-1,8-diamine and 18.1 mmol of 1,6-diaminohexane were heated in a reaction vessel closed with a drying tube at 120 C. for 90 min with stirring, then the temperature was increased to 170 C. for 8 h, at the end of this reaction time under reduced pressure (50 mbar) for 90 min. After the reaction mixture had cooled off to below 80 C., 60 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

(11) The structure of the polymer obtained corresponds to that disclosed in WO 2006/047800 A1.

Comparative Example 2

(12) 2.00 mmol of guanidinium hydrochloride, 1.70 mmol of 1,6-hexamethylene diamine and 0.3 mmol of hydrazine hydrate were heated in a reaction vessel closed with a drying tube at 160 C. for 90 min with stirring, then the temperature was increased to 180 C. for 3.5 h, at the end of this reaction time under reduced pressure (50 mbar) for 60 min. After the reaction mixture had cooled off to below 80 C., 4 ml of water were added to the gelatinous reaction product. After several hours, a clear solution was obtained.

(13) The structure of the polymer obtained corresponds to that disclosed in WO 2011/043690 A1.

Example 7

Determination of Activity: Antimicrobial/Antifungal/Antiviral Effects

(14) The activities of the new compounds were tested in screening systems in multiplicate. The antibacterial and antifungal activities were tested in a MIC assay. MIC refers to minimal inhibitory concentration and is the lowest concentration of a substance that will inhibit the growth of microorganisms discernible with the naked eye. The MIC is determined using a so-called titer method, where the substance is diluted and then the pathogen is added.

(15) Usually this allows for the determination of the concentration of an antibiotic that is just high enough to inhibit growth of a bacterial strain. The MIC is specified in micrograms per milliliter (g/ml) or in % per volume, and the dilutions are generally conducted in log 2 steps. Herein, an initial concentration of 1% each was 2-fold diluted, which consequently resulted in test concentrations of 0.5%, 0.25%, 0.125%, etc. Lower values thus reflect better activity as anti-infective.

(16) The assays were conducted according to the standards required by EUCAST (European Committee for Antimicrobial Susceptibility Testing) and according to the AFST (Antifungal Susceptibility Testing) regulations of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).

(17) The screening system for viruses is an infection system in which host cells are infected in vitro, and the test substance is added before or after the infection and its activity determined. All these assays were conducted according to internal standard regulations of SeaLife Pharma for drug screening, wherein analogous serial dilutions were used like in the antibacterial/antifungal assay.

(18) The following tables 1 to 3 summarize the test results regarding the anti-infective effect of the inventive new compounds of Examples 1, 3, 4 and 5 against multiresistant bacteria and fungi as well as viruses. The data are mean values of multiple determinations.

(19) It is obvious that the new compounds of the invention show excellent activity against Gram-positive as well as Gram-negative pathogens:

(20) TABLE-US-00001 TABLE 1 MIC assay Staphylococcus streptococcus Enterococcus Propionibacter results MRSA epidermis pneumoniae faecalis acne E. coli Example 1 0.001% 0.001% 0.004% 0.008% 0.001% 0.016% Example 3 0.001% 0.001% 0.001% 0.008% 0.001% 0.02% Example 4 0.001% 0.001% 0.001% 0.008% 0.001% 0.016% Example 5 0.001% 0.001% 0.002% 0.002% 0.001% 0.020% MIC assay Klebsiella Pseudominas Acinetobacter Enterobacter Salmonella results pneumoniae aeruginosa baumanii cloace enterica Example 1 0.02% 0.02% 0.06% 0.03% 0.03% Example 3 0.02% 0.02% 0.06% 0.2% 0.03% Example 4 0.016% 0.030% 0.02% 0.016% 0.030% Example 5 0.02% 0.04% 0.04% 0.13% 0.03%

(21) Also against fungi and yeasts:

(22) TABLE-US-00002 TABLE 2 MIC assay Candida Candida Candida Candida Aspergillus Aspergillus Fusarium Trichophyton Alternarria Microsporum Dematiacea results albicans papillosis glabrata kruzei terreus fumigatus rosei sp. alternarria canis sp. Example 1 0.008% 0.03% 0.02% 0.02% 0.02% 0.03% 0.03% 0.02% 0.02% 0.03% 0.02% Example 3 0.02% 0.02% 0.02% 0.02% 0.03% 0.03% 0.03% 0.02% 0.02% 0.02% 0.02% Example 4 0.008% 0.016% 0.016% 0.008% 0.125% 0.125% n.t. n.t. n.t. n.t. n.t. Example 5 0.02% 0.02% 0.02% 0.020% 0.016% 0.016% n.t. n.t. n.t. n.t. n.t.

(23) As well as against viruses:

(24) TABLE-US-00003 TABLE 3 Virological Influenza Human Parainfluenza Herpes assay results A and B rhinovirus virus simplex virus Example 1 0.008% 0.008% 0.008% 0.02% Example 3 0.02% 0.02% 0.02% 0.02% Example 4 0.04% 0.02% 0.04% 0.02% Example 5 0.04% 0.04% 0.04% 0.02%

(25) Thus, all new compounds tested show very good to excellent activity against various pathogenswith significantly lower toxicity than the polyguanidine derivatives known from prior art, as is shown by the following toxicity assays.

Example 8

Toxicity Assays

(26) AlamarBlue Assays as described below were used to study 4 polymers with regard to their toxicological potential (including proliferation, cell death, cell metabolism), and the IC.sub.50 value and the non-toxic concentration were determined with primary keratinocytes (HKER) and primary endothelial cells (HUVEC). FIG. 1 shows the toxic effect of the various polymers depending on their concentration.

(27) AlamarBlue Assay: 20,000 human keratinocytes (HKER) or endothelial cells (HUVEC) were plated in 96 well plates and incubated for 24 h, before different concentrations (5% to 0.005%) of the new polymers of Examples 1 and 3 as well as of the comparative substances of Comparative Examples 1 and 2 were added. After 24 hours, 10 l AlamarBlue were added to each well (100 l medium), and after 3 hours of incubation, the color reaction was detected using a multiplate reader (ex: 530 nm; em: 590 nm). HKER: human primary keratinocytes; HUVEC: human umbilical vein endothelial cells.

(28) The polymers of Comparative Examples 1 and 2 show significant toxic effects against HKER as well as HUVEC at already very low concentrations, i.e. an IC.sub.50 of approximately 0.01% or below. In comparison, the new polymers produced by the inventors of Examples 1 and 3 show toxic effects at significantly higher concentrations: for Example 1, the IC.sub.50 for both cell types is approximately 1%, and for Example 3, it ranges between 0.05% and 0.1%. The toxicity produced by the comparative examples is reached by the polymer of Example 3 only at the 5-fold concentration, and by that of Example 1 only at the at least 100-fold concentration. The DAG derivative thus showed much better results in this assay than the MAG polymer.

(29) Consequently, the new compounds show very good to excellent activity against various pathogenswith significantly lower toxicity than polyguanidine derivatives known from prior art.