Polyguanidine polymers and methods of use thereof

Abstract

Polyguanidines and methods of use thereof are described. In particular, polyguanidines of formula (I), formula (II), and formula (III) are described. The polyguanidines can be used to treat infection, such as bacterial, viral or fungal infection, and as an ex vivo microbial agent. ##STR00001##

Claims

1. A polyguanidine of formula (I), formula (II), or formula (III): ##STR00028## wherein R.sub.1 represents either an aromatic ring system with at least one aromatic ring, optionally containing one or more heteroatoms selected from O, N and S and optionally being substituted with one or two vinyl groups to which the —CH.sub.2— group(s) is/are bound, or represents ethylene, wherein n is ≥2.

2. The polyguanidine of claim 1, wherein R.sub.1 is a divalent residue selected from the group consisting of optionally substituted benzene, divinylbenzene, furan, pyrrole, thiophene, pyridine, biphenyl, fluorene and ethylene.

3. The polyguanidine of claim 2, wherein R.sub.1 is a divalent residue selected from the group consisting of benzene, divinylbenzene, pyridine, biphenyl and ethylene.

4. A drug or pharmaceutical composition for antagonizing bacterial infections in a human or animal patient comprising a polyguanidine of claim 1 as an antiinfective.

5. The drug or pharmaceutical composition of claim 4, further comprising at least one pharmaceutically acceptable carrier or excipient and optionally one or more adjuvants and/or one or more further active agents.

6. The drug or pharmaceutical composition of claim 5, comprising at least one further active agent that also shows an antiinfective effect.

7. The drug or pharmaceutical composition of claim 5, comprising at least one further active agent that is effective against a condition other than bacterial infections.

8. The polyguanidine of claim 1, a cyclic structure resulting from cyclization upon elimination of a corresponding guanidine, or a salt of said polyguanidine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the proportion of surviving cells of the exposed HaCaT cell line as cell model on the Y axis for polyguanidines according to the present invention.

DISCLOSURE OF THE INVENTION

(2) In a first aspect, the invention achieves this object by providing a method for producing polycondensation products of guanidine, aminoguanidine or diaminoguanidine G with one or more benzyl or allyl derivatives BA according to the following reaction scheme:

(3) ##STR00008##
wherein
each X independently represents a leaving group;
each R.sub.1 independently represents either an aromatic ring system with at least one aromatic ring, optionally containing one or more hetero atoms selected from O, N and S and optionally being substituted with one or two vinyl groups to which the —CH.sub.2—X group(s) is/are bound, or represents ethylene;
Gua represents a guanidindiyl, aminoguanidindiyl or diaminoguanidindiyl residue;
Y represents H-Gua, and
Z represents H; or
Y and Z together represent a chemical bond to obtain a cyclic structure;
wherein at least one benzyl or allyl derivative BA is subjected to a polycondensation reaction with excessive guanidine, aminoguanidine or diaminoguanidine G upon elimination of HX in order to provide a polyguanidine corresponding to the following formula (I), (II) or (III):

(4) ##STR00009##
or having a cyclic structure resulting from cyclization upon elimination of a corresponding guanidine, or a salt of said polyguanidine.

(5) Contrary to the state of the art, polycondensation in this production method occurs not by cleaving ammoniak, but by cleaving the leaving group X, preferably in the form of a hydrogen halide, e.g. HCl or HBr, or a sulfonic acid, e.g. CH.sub.3SO.sub.2OH (MsOH), which forms acid addition salts with the amino or imino groups present in the molecule, which in turn makes the use of an acid scavenger unnecessary.

(6) This also implicates that polycondensation does not necessarily have to be conducted in a molten state, even though, due to procedural economic reasons, melt polymerization is also the preferred reaction route for the present invention. In preferred embodiments, the at least one benzyl or allyl derivate BA is thus reacted with guanidine, aminoguanidine or diaminoguanidine G by heating the reactants to a temperature above their melting points, the polymerization reaction preferably being conducted for a period of at least 2 h, more preferably at least 3 h. In particular, the reaction is—by analogy with an earlier method of the inventors—conducted in two steps at different temperatures, a first lower and a second higher temperature, in order to guarantee a conversion as complete as possible and thus higher chain lengths and at the same time lower residual monomer contents.

(7) Surprisingly, however, the inventors found that the use of benzylic or allylic structures leads to the formation of mixtures of polycondensation products having structures that differ from those known from the state of the art. However, the main products do not correspond to the structures known from earlier applications by the inventors comprising only mono-substituted nitrogen, since such mono-substituted nitrogen appears to preferably react a second time to form the above structures of formulas (I) to (III).

(8) Without wishing to be bound by theory, the inventors assume that the reactivity of the educts, which is due to a mesomeric stabilization of the transition state in the course of the nucleophilic substitution at the benzylic or allylic methylene group, together with the increased reactivity of the primarily formed mono-substituted nitrogen-adducts, leads to the given nitrogen double substitution, which leads to the further assumption that a similar effect is to be expected with at least a majority of known benzylic and allylic structures, i.e. with structures having one methylene group attached to an aromatic ring or a double bond, or with combinations thereof, i.e. in the case of cinnamylic structures in which—regarding the benzyl residue—apparently the known vinylogy effect takes place (see Example 8 below). The latter is, of course, also applicable to conjugated double bonds in aliphatic residues, e.g. in the case of butadiene instead of ethylene. Therefore, any further substituents on these aromatic rings and double bonds shall not be specifically limited at the moment, as long as the aromaticity of the respective ring is not eliminated and the electron density in the aromatic ring or at the double bond is not substantially altered, in particular in the case of tautomeric effects such as keto/enol, imine/ene-amine etc.

(9) In the given structures of formulas (I) to (III), the guanidine or aminoguanidine units are positioned outside of the chain via the nitrogen atom doubly integrated into the chain, which, according to spectroscopic evidence, leads to the structural type of the formulas (I), (II) or (III) in the majority of the oligomer species formed.

(10) The obtained linking type of the new polyguanidines was determined by means of HMBC-NMR: For the polyaminoguanidine of Example 1, for example, the corresponding long range couplings of benzylic CH.sub.2 protons bonded via nitrogen (AB system at 3.8 and 4.2 ppm) are detectable by such an N atom integrated into the oligomer chain as well as two benzylic carbon atoms (at 64 ppm). As an indication of higher branched secondary components (˜15% according to .sup.1H-NMR), signals correlating to benzylation of a further guanidine nitrogen beyond imino functionality were found (AB system at 4.3 and 4.5 ppm, HMBC long range signals in the guanidine carbon region at 160 ppm). A further group of NMR signals CH shift at 8 ppm, .sup.13C shift at 150 ppm) of benzylic imino functionalities correlates with oligomer counterparts of the Sommelet oxidation type, which corresponds to the mass spectrometric data (doublets of the m/z type [M−2] for all oligomers).

(11) The inventors expected even better antimicrobial activity from this new structural type than from their earlier polyaminoguanidines, which, indeed, was confirmed, as is shown by the exemplary embodiments of the invention below: biocidal activity is clearly increased, while toxicity is even lower at the same time.

(12) Without wishing to be bound by theory, the inventors assume that the latter might result from a higher average chain length compared to earlier polyaminoguanidines as well as from an even lower residual monomer content.

(13) To optimize reaction conditions and to find the best possible compromise between reaction time, chain length and residual monomer content, the inventors conducted test series with varying ratios of benzyl or allyl derivative BA and guanidines G, varying temperatures, as well as varying reaction times, and they found out that a ratio G/BA just below 2 leads to products showing the best biological results, wherein the reaction mixtures should preferably first be heated to a temperature of approximately 150-170° C. for 2 to 3 h and then to a temperature of 180-190° C. for 1 to 2 h.

(14) In a second aspect, the invention provides new polyguanidines corresponding to the following formulas (I) to (III), namely

(15) a polyguanidine corresponding to the following formula (I):

(16) ##STR00010##
or having a cyclic structure resulting from cyclization upon elimination of a guanidine;
a polyguanidine corresponding to the following formula (II):

(17) ##STR00011##
or having a cyclic structure resulting from cyclization upon elimination of an aminoguanidine, and
a polyguanidine corresponding to the following formula (III):

(18) ##STR00012##
or having a cyclic structure resulting from cyclization upon elimination of a diaminoguanidine;
wherein R.sub.1 represents either an aromatic ring system with at least one aromatic ring, optionally containing one or more heteroatoms selected from O, N and S and optionally being substituted with one or two vinyl groups to which the —CH.sub.2—X group(s) is/are bound, or represents ethylene, and in preferred embodiments is selected from divalent residues of optionally substituted benzene, divinylbenzene, furan, pyrrole, thiophene, pyridine, biphenyl, fluorene or ethylene, more preferably from a divalent residue of benzene, divinylbenzene, pyridine, biphenyl or ethylene, which residues have already provided good results.

(19) Due to the high antimicrobial effectiveness of the new structures, the invention provides in a third aspect a new polyguanidine as defined above for use as an antibiotic or antiinfective, preferably for antagonizing bacterial, viral or fungal infections in a human or animal patient. The polyguanidine may serve for topical or systemic administration, preferably for administration in the form of a drug or a pharmaceutical composition.

(20) Alternatively, the new polyguanidines may also be used ex vivo as antimicrobial agents, preferably as active components of antimicrobial paints, coatings, foils, or membranes, or the like.

(21) In a fourth aspect, the invention thus provides a drug or a pharmaceutical composition for antagonizing bacterial, viral or fungal infections in a human or animal patient, comprising at least one of the new polyguanidines as an antiinfective and preferably also at least one pharmaceutically acceptable carrier or excipient and optionally one or more adjuvants and/or one or more further active agents.

(22) Preferably, the drug or pharmaceutical composition contains at least one further active agent that also shows an antimicrobial effect in order to increase the effect and utilize possible synergetic effects. The at least one further active agent may also be effective against another conditions than bacterial infections. Examples include antidiarrheals and so-called gastroprotective agents.

(23) Below, the invention will be described in further details by means of non-limiting examples.

EXAMPLES

Example 1

Preparation of Polyaminoguanidine (1)

(24) ##STR00013##

(25) α,α′-Dichloro-p-xylene (880 mg, 5.03 mmol) and 1.95 equivalents of aminoguanidine hydrochloride (1083 mg, 9.80 mmol) were heated with stirring to 160° C. for 3 h in an open reaction vessel, followed by heating to 180° C. for 2 h. After the reaction mixture had cooled to below 80° C., the tenfold amount of water was added to the reaction product, and after thoroughly mixing by means of stirring or ultrasound treatment, a clear, light yellow solution with traces of solid contents was obtained. It was filtered through a 0.2 μm PFTE membrane and then evaporated to obtain polyguanidine (1) as a yellow, amorphous solid.

(26) For analysis, a sample was dissolved in a tenfold amount of D.sub.2O. When recording the .sup.1H and the .sup.13C NMR spectra, DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) was added for reference as an internal standard:

(27) .sup.1H NMR (D.sub.2O), δ (ppm): 3.72-3.91 (ad, CH.sub.2A—N(Gua)-CH.sub.2A, J.sub.A,B=12.4 Hz, CH.sub.2A chain), 3.93-4.05 (as, CH.sub.2—NH-Gua, CH.sub.2 terminal), 4.10-4.23 (ad, CH.sub.2B—N(Gua)-CH.sub.2B, J.sub.A,B=12.4 Hz, CH.sub.2B chain), 4.29-4.39 (m, CH.sub.2A α-Gua), 4.45-4.52 (m, CH.sub.2B α-Gua), 7.30-7.83 (m, ═CH Ar), 8.08 (as, N═CH).

(28) .sup.13C NMR (D.sub.2O), δ (ppm): 46.25, 46.56, 46.94 (CH.sub.2 α-Gua), 56.90, 56.97, 57.03 (CH.sub.2 terminal), 63.87, 64.02 (CH.sub.2—N(Gua)-CH.sub.2 chain), 128.93, 129.04, 129.57, 129.63, 129.78, 129.84, 130.20, 130.32, 130.49, 130.66, 132.10, 132.17, 132.30, 132.40, 132.62, 132.67, 132, 75, 132.83, 132.92, 133.20 (CH Ar), 135.02, 135.19, 137.54, 137.92, 138.13, 138.50, 139.07, 139.23, 141.31, 142.53 (C.sub.q Ar), 150.21, 151.05, 151.12 (N═CH), 157.60, 159.67, 159.73, 160.85 (C.sub.q Gua).

(29) The NMR signals in the ranges of 3.72-3.91 ppm and 4.10-4.23 ppm (.sup.1H axis) and at 64.02 ppm (.sup.13C axis) confirm the presence of a doubly substituted nitrogen atom of the aminoguanidine.

(30) MALDI-MS—MALDI-TOF (in the positive ion mode (matrix suppression off)); scan 20-3000 m/z (deflection off); matrix: ACH (α-cyano-4-hydroxy cinnamic acid); (m/z): 247.3, 249.3, 251.4, 425.3, 427.3, 601.4, 603.4, 777.5, 779.5, 953.7, 955.7, 1129.8, 1131.9, 1306.0, 1308.0, 1482.1, 1484.1, 1658.0, 1660.0, 1834.1, 1836.1, 2010.2, 2012.2, 2186.3, 2188.3, 2362.4.

Example 2

Preparation of Polyaminoguanidine (2)

(31) ##STR00014##

(32) In analogy to Example 1, polyguanidine (2) was prepared from α,α′-dichloro-m-xylene and aminoguanidine hydrochloride, yielding a yellowish, amorphous, completely water-soluble solid.

(33) .sup.1H NMR (D.sub.2O), δ (ppm): 3.73-3.92 (ad, CH.sub.2A—N(Gua)-CH.sub.2A, J.sub.A,B=12.7 Hz, CH.sub.2A chain), 3.94-4.05 (as, CH.sub.2—NH-Gua, CH.sub.2 terminal), 4.10-4.23 (ad, CH.sub.2B—N(Gua)-CH.sub.2B, J.sub.A,B=12.7 Hz, CH.sub.2B chain), 4.29-4.38 (m, CH.sub.2A α-Gua), 4.45-4.53 (m, CH.sub.2B α-Gua), 7.23-7.85 (m, ═CH Ar), 8.10 (as, N═CH).

(34) .sup.13C NMR (D.sub.2O), δ (ppm): 46.36, 46.66, 47.01 (CH.sub.2 α-Gua), 57.01, 57.04, 57.12, 57.14 (CH.sub.2 terminal), 63.94 (CH.sub.2—N(Gua)-CH.sub.2 chain), 129.63, 129.75, 130.09, 130.20, 130.83, 131.38, 131.44, 131.53, 131.57, 131.67, 131.82, 131.89, 132.18, 132.34, 132.73, 133.52, 134.23, 134.52, 135.29 (CH Ar), 135.72, 135.81, 136.12, 138.59, 138.69, 138.73, 139.13, 139.77, 139.90, 140.30 (C.sub.q Ar), 151.24 (N═CH), 157.67, 159.78, 159.81, 160.86 (C.sub.q Gua).

(35) The NMR signals in the ranges of 3.73-3.92 ppm and 4.10-4.23 ppm (.sup.1H axis) and at 63.94 ppm (.sup.13C axis) again confirm the presence of a doubly substituted nitrogen atom of the aminoguanidine.

(36) MALDI-MS—MALDI-TOF (m/z): 247.3, 249.3, 251.4, 425.3, 427.3, 601.4, 603.4, 777.5, 779.5, 953.7, 955.7, 1129.8, 1131.9, 1306.0, 1308.0, 1482.1, 1484.1, 1658.0, 1660.0, 1834.1, 1836.1, 2010.2, 2012.2, 2186.3, 2188.3.

Example 3

Preparation of Polyaminoguanidine (3)

(37) ##STR00015##

(38) In analogy to Example 2, polyguanidine (3) was prepared from 132 mg (0.5 mmol) of α,α′-dibromo-m-xylene (instead of the dichloro derivative) as well as 1.75 equivalents of aminoguanidine hydrochloride (97 mg, 0.88 mmol), yielding a brownish, amorphous, water-soluble solid.

(39) .sup.1H NMR (D.sub.2O), δ (ppm): 3.63-3.95 (m, CH.sub.2A—N(Gua)-CH.sub.2A, CH.sub.2A chain), 3.95-4.08 (as, CH.sub.2—NH-Gua, CH.sub.2 terminal), 4.13-4.24 (ad, CH.sub.2B—N(Gua)-CH.sub.2B, J.sub.A,B=12.5 Hz, CH.sub.2B chain), 4.31-4.40 (m, CH.sub.2A α-Gua), 4.47-4.55 (m, CH.sub.2B α-Gua), 7.17-7.86 (m, ═CH Ar), 8.12 (as, N═CH).

(40) .sup.13C NMR (D.sub.2O), δ (ppm): 46.38, 46.64, 46.99 (CH.sub.2 α-Gua), 56.98, 57.11, 57.48 (CH.sub.2 terminal), 63.90 (CH.sub.2—N(Gua)-CH.sub.2 chain), 128.58, 129.08, 129.64, 129.76, 130.05, 130.20, 130.81, 130.98, 131.35, 131.41, 131.51, 131.71, 131, 80, 131.87, 132.16, 132.33, 132.69, 133.49, 134.21, 134.51, 135.29 (CH Ar), 135.66, 135.76, 136.06, 138.68, 138.98, 139.07, 139.25, 139.72, 139.85, 140.25 (C.sub.q Ar), 150.46, 151.29 (N═CH), 159.73, 160.84 (C.sub.q Gua).

(41) The NMR signals in the ranges of 3.63-3.95 ppm and 4.13-4.24 ppm (.sup.1H axis) and at 63.90 ppm (.sup.13C axis) again confirm the presence of a doubly substituted nitrogen atom of the aminoguanidine.

(42) MALDI-MS—MALDI-TOF (m/z): 247.3, 249.3, 251.4, 425.3, 427.3, 601.4, 603.4, 777.5, 779.5, 953.7, 955.7, 1129.8, 1131.9, 1306.0, 1308.0, 1482.1, 1484.1, 1658.0, 1660.0, 1834.1, 1836.1, 2010.2, 2012.2, 2186.3, 2188.3.

Example 4

Preparation of Polydiamino Guanidine (4)

(43) ##STR00016##

(44) In analogy to Example 2, polyguanidine (4) was prepared from 88 mg (0.5 mmnol) of α,α′-dichloro-m-xylene and 1 equivalent of diaminoguanidine hydrochloride (68 mg, 0.5 mmol), yielding a yellowish, amorphous, water-soluble solid.

(45) The structural determination by means of .sup.1H and .sup.13C NMR shows, in addition to the product species found in the Examples 1 to 3, also the presence of larger proportions of highly branched components in which another guanidine nitrogen is benzylized beyond the imino functionality.

Example 5

Preparation of Polyguanidine (5)

(46) ##STR00017##

(47) In analogy to Example 3, polyguanidine (5) was prepared from 132 mg (0.5 mmol) of α,α′dibromo-p-xylene and 1.75 equivalents of guanidine hydrochloride (83 mg, 0.88 mmol), yielding a water-soluble, reddish, amorphous solid.

(48) The structural determination by means of and .sup.13C NMR shows, in addition to the product species found in the Examples 1 to 3, also the presence of larger proportions of highly branched components in which another guanidine nitrogen is benzylized beyond the imino functionality.

(49) MALDI-MS—MALDI-TOF (m/z): 355.3, 382.3, 516.4, 543.4, 677.3, 704.5, 838.5, 865.5, 999.6, 1026.6, 1160.7, 1187.7, 1321.8, 1348.8, 1483.9, 1510.9, 1672.0, 1833.1, 1995.2.

Example 6

Preparation of Polyaminoguanidine (6)

(50) ##STR00018##

(51) 2,6-Bis(bromomethyl)pyridine (265 mg, 1 mmol) and 1.95 equivalents of aminoguanidine hydrochloride (216 mg, 1.95 mmol) were heated with stirring to 160° C. for 1.5 h in an open reaction vessel, followed by heating to 180° C. for 1.5 h. After the reaction mixture had cooled to below 80° C., water (4.81 ml) was added to the reaction product, and after thoroughly mixing by means of stirring or ultrasound treatment as well as filtration through a 0.2 μm PFTE membrane, a clear, dark brown solution was obtained.

(52) MALDI-MS—MALDI-TOF (m/z): 248.4, 250.4, 252.4, 421.4, 423.4, 425.4, 427.4, 429.4, 598.4, 600.4, 602.4, 604.4.

Example 7

Preparation of Polyaminoguanidine (7)

(53) ##STR00019##

(54) In analogy to Example 2, polyguanidine (7) was prepared from 4,4′-bis(chloromethyl)biphenyl (251 mg, 1 mmol) as well as 1.95 equivalents of aminoguanidine hydrochloride (216 mg, 1.95 mmol), yielding a yellowish, amorphous solid, which is, apart from small amounts of a solid residue, easily soluble in water.

(55) MALDI-MS—MALDI-TOF (m/z): 323.4, 325.4, 327.4, 575.4, 577.4, 579.4, 827.6, 829.6, 831.6.

Example 8

Preparation of Polyaminoguanidine (8)

8.1 Preparation of dimethyl-3,3′-(1,3-phenylene)-(2E,2′E)-diacrylate

(56) ##STR00020##

(57) To a solution of 0.75 mmol of isophthalic aldehyde in 10 ml THF, a solution of 2.05 equivalents of (methoxycarbonylmethylene)triphenylphosphorane (1.54 mmol) in 15 ml THF was added with the exclusion of air. The reaction mixture was stirred for 12 h at 50° C. and then concentrated. A chromatographic purification (silica, dichloromethane) yielded 0.62 mmol (83% o.th.) of a white solid.

(58) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.82 (s, 6H), 6.47 (d, J=16 Hz, 2H), 7.42 (dd, J=7.7+7.7 Hz, 1H), 7.54 (dd, J=7.7+1.7 Hz, 2H), 7.64 (t, J=1.7 Hz, 1H), 7.69 (d, J=16 Hz, 2H).

8.2 Preparation of (2E,2′E)-3,3′-(1,3-phenylene)-bis(prop-2-en-1-ole)

(59) ##STR00021##

(60) In a Schienk vessel, 1.50 mmol of dimethyl-3,3′-(1,3-phenylene)-(2E,2′E)-diacrylate were dissolved in 30 ml anhydrous dichloromethane. At −78° C., 4.5 equivalents of diisobutyl-aluminum hydride were slowly added dropwise as a 1 M solution in toluene (6.75 ml). The reaction mixture was stirred for 2 h at −78° C. and subsequently hydrolyzed at 0° C. with methanol. The resulting white precipitate was filtered, the filtrate was concentrated and chromatographically purified (silica, DCM:EE 1:1), with 1.05 mmol (70% o.th.) of a white solid being isolated.

(61) .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm): 4.26 (m. 4H), 6.33 (dm. J=16 Hz, 2H), 6.56 (br,d, J=16 Hz, 2H), 7.22 (m, 3H), 7.34 (br s, 1H).

8.3 Preparation of 1,3-bis((E)-3-chloroprop-1-en-1-yl)benzene

(62) ##STR00022##

(63) In a Schienk vessel, 0.95 mmol of dimethyl-3,3′-(1,3-phenylene)-(2E,2′E)-diacrylate and 3 equivalents of diisopropylethylamine (DIPEA, 2.85 mmol) in 20 ml dichloromethane were provided and cooled to −40° C., followed by the addition of 2.38 mmol of methanesulfonyl chloride and stirring of the reaction mixture at room temperature for 12 h. After withdrawal of the solvent, the crude product was purified chromatographically (silica, DCM), with 0.57 mmol (60% o.th.) of a white, crystalline solid being isolated.

(64) .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm): 4.25 (dd, J=7.1+1.2 Hz, 4H), 6.34 (dt, J=15.7+7.1 Hz, 2H), 6.65 (dt, J=15.7+1.2 Hz, 2H), 7.30 (m, 3H), 7.40 (m, 1H).

8.4 Preparation of Polyaminoguanidine (8)

(65) ##STR00023##

(66) In analogy to Example 2, polyguanidine (8) was prepared from 1,3-bis((E)-2-chlorovinyl)-benzene (200 mg, 1 mmol) and 1.95 equivalents of aminoguanidine hydrochloride (216 mg, 1.95 mmol), yielding a yellowish, translucent, water-soluble gel.

(67) MALDI-MS—MALDI-TOF (m/z): 303.3, 531.4, 759.6, 833.7, 987.8, 1061.9, 1216.0.

Example 9

Preparation of Polyaminoguanidine (9)

9.1 Preparation of cis-1,4-bis(methylsulfonyloxy)but-2-ene

(68) ##STR00024##

(69) 10 g of cis-but-2-ene-1,4-diol (113 mmol) and 3.0 equivalents of diisopropylethylamine (44 g, 340 mmol, 60 ml) were dissolved in 250 ml of dichloromethane and cooled in an argon atmosphere to −40° C., after which 2.4 equivalents of methanesulfonyl chloride (30.9 g, 270 mmol, 20.9 ml) were added portionwise and the reaction mixture was allowed to warm up to +10° C. for 1 h. The clear, yellow solution was poured into 500 ml ice-cold water and the organic phase was washed with further 500 ml of cold water, then with 200 ml of 2 N HCl, then twice with 200 ml each of saturated NaHCO.sub.3 solution, and finally again twice with 200 ml each of water. The dichloromethane solution of the product was dried over Na.sub.2SO.sub.4 and the solvent was withdrawn in vacuo until a white precipitate appeared, whereafter the minimum amount of dichloromethane was added in order to again obtain a clear solution. After the addition of 25 ml of diethylether, the product was left to crystallize from the solution at −20° C., whereafter 10 g of cis-1,4-bis(methylsulfonyloxy)-but-2-ene were isolated as a crystalline, white solid.

(70) .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm): 3.04 (s, 3H), 4.84 (m, 2H), 5.95 (m, 1H).

9.2 Preparation of Polyaminoguanidine (9)

(71) ##STR00025##

(72) Cis-1,4-bis(methylsulfonyloxy)but-2-ene (246 mg, 1 mmol) and 1.95 equivalents of aminoguanidine hydrochloride (216 mg, 1.95 mmol) were heated in a closed vessel in an argon atmosphere with stirring to 160° C. for 3 h, then to 180° C. for 2 h. After the reaction mixture had cooled to below 80° C., water (4.67 ml) was added to the reaction product to obtain a clear, yellow-red solution.

(73) MALDI-MS—MALDI-TOF (m/z): 201.3, 251.3, 253.3, 297.2, 325.3, 327.3, 349.2, 377.3, 423.3, 451.3, 453.3, 519.3.

Example 10

Preparation of Polyaminoguanidine (10)

(74) ##STR00026##

(75) 1,4-Dichloro-2-butene (262 mg, 1.3 mmol) and 1.95 equivalents of aminoguanidine hydrochloride (216 mg, 1.95 mmol) were heated in a closed reaction vessel in an argon atmosphere with stirring and repeated (three times per hour) replacement of the atmosphere with fresh argon to 150° C. for 2 h, then to 170° C. for 1 h. After the reaction mixture had cooled to below 80° C., water (4.67 ml) was added to the reaction product to obtain a clear, yellow-red solution.

(76) MALDI-MS—MALDI-TOF (m/z): 201.3, 251.3, 253.3, 297.2, 325.3, 327.3, 377.3, 423.3, 451.3, 453.3.

Comparative Example 1

Preparation of a polyaminoguanidine from diamine and aminoguanidine

(77) ##STR00027##

(78) 23 mmol of 1,3-diaminoguanidinium hydrochloride and 24 mmol of 4,9-dioxadodecane-1,12-diamine were heated to 120° C. in a reaction vessel closed with a drying tube for 90 min with stirring, whereafter the temperature was increased to 180° C. for 100 min, at the end of which reaction time the pressure was reduced for 45 min (50 mbar). After the reaction mixture had cooled to below 80° C., 25 ml of water were added to the gel-like reaction product. After a few hours, a clear solution was obtained.

(79) The water was evaporated from a sample of the resulting aqueous solution and the obtained residue was dried in vacuum, yielding a reddish, viscous liquid. It was dissolved in 2 ml of D.sub.2O (with a deuteration degree >99.5%) and a .sup.1H nuclear resonance CH NMR) spectrum was recorded. The position of the groups of methylene protons of the R.sub.1 residues thus distinguishable is as follows:

(80) .sup.1H NMR (D.sub.2O), δ (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 (in, CH.sub.2CH.sub.2OCH.sub.2CH.sub.2).

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

Example 11

Activity Measurements: Antimicrobial/Antifungal/Antiviral Effect

(82) The activity of the new compounds was tested in screening systems performed several times each. The antibacterial and antifungal activities were examined by means of an MIC test. MIC means “minimal inhibitory concentration” and refers to the lowest concentration of a substance at which no propagation of microorganisms is perceivable with the naked eye. The MIC is determined with a so-called titer process in which the substance is diluted and then the pathogen is added.

(83) This normally serves to determine the concentration of an antibiotic that just inhibits the growth of a bacterial strain. Die MIC is expressed in micrograms per milliliter (μg/ml) or in % by volume, and the dilutions are normally conducted in log 2 steps. Herein, a starting concentration of 1% was diluted twofold each time, leading to test concentrations of 0.5%, 0.25%, 0.125% etc. Lower values thus reflect better activity as antiinfective.

(84) The tests were conducted according to the standards required by EUCAST (European Committee for Antimicrobial Susceptibility Testing) and according to the AFST (“Antifungal Susceptibility Testing”) provisions of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).

(85) 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, followed by the determination of its activity. All these tests were conducted according to internal standard rules of SeaLife Pharma for drug screening using analogous dilution series as in the antibacterial/antifungal test.

(86) In the following Tables 1 to 3, test results regarding the antiinfective effects of the inventive new compounds from the Examples 1, 3, 4 and 5 and from Comparative Example 1 against several multiresistant bacteria and fungi as well as viruses are shown. The data are mean values of multiple determinations.

(87) It is obvious that the new compounds of the invention show excellent activity against gram-positive as well as gram-negative pathogens.

Example 12

Toxicity Tests

(88) Attached FIG. 1 further shows that the inventive new polyguanidines show very low toxicity at those concentrations at which they exhibit excellent antimicrobial activity, as is clearly shown by the proportion of surviving cells of the exposed HaCaT cell line as cell model on the Y axis.

(89) TABLE-US-00001 TABLE 1 Effects against gram-positive and gram-negative pathogens Entero- Streptococcus Staphylococcus Klebsiella Entero- Pseudomonas Clostridium MIC [%] MRSA coccus pneumoniae epidermis E. coli pneumoniae bacter aeroginosa def. Salmonella Example 1 >0.0016 >0.0002 >0.0016 >0.0008 >0.0016 >0.025 >0.003 >0.003 >0.0008 >0.003 Example 2 >0.0008 >0.0008 >0.0008 >0.0002 >0.0008 >0.025 >0.0008 >0.0016 >0.0004 >0.0008 Example 3 >0.0004 >0.0008 >0.0008 >0.0004 >0.0008 n.d. >0.0016 >0.003 >0.0004 >0.0008 Example 4 >0.003 >0.003 >0.003 >0.0016 >0.0063 n.d. >0.0125 >0.0125 >0.003 >0.0125 Example 5 >0.0008 >0.0002 >0.0004 >0.0004 >0.0016 >0.0125 >0.0016 >0.003 >0.0004 >0.0016 Example 6 >0.025 >0.0125 >0.025 >0.025 >0.025 >0.05 >0.025 >0.025 >0.025 >0.05 Example 7 >0.0002 >0.0002 >0.0002 >0.0002 >0.0008 >0.0004 >0.0008 >0.0008 >0.0008 >0.0008 Example 8 >0.0004 >0.0004 >0.0004 >0.0004 >0.0008 >0.0008 >0.0008 >0.0008 >0.0008 >0.0008 Example 9 >0.0125 >0.003 >0.0125 >0.0125 >0.0008 >0.0008 >0.025 >0.025 >0.025 >0.025 Example 10 >0.0125 >0.0125 >0.0125 >0.0125 >0.025 >0.025 >0.025 >0.025 >0.025 >0.025 Comparative >0.001 >0.008 >0.004 >0.001 >0.016 >0.02 >0.008 >0.02 n.d. >0.03 example 1

(90) TABLE-US-00002 TABLE 2 Effects against fungi and yeasts Micro- Candida Candida Candida Candida Aspergillus Aspergilus Fusarium Trichophyton Alternarri sporum Dematiacea MIC [%] albicans papillosis glabrata krusei terreus fumigates rosei sp. a sp. canis sp. Example >0.025 >0.025 >0.025 >0.025 >0.05 >0.05 >0.05 >0.025 >0.025 >0.025 >0.025 1 Example >0.025 >0.025 >0.025 >0.025 >0.05 >0.05 >0.025 >0.025 >0.05 >0.025 >0.05 2 Example >0.025 >0.025 >0.025 >0.025 >0.05 >0.05 >0.025 >0.025 >0.05 >0.025 >0.05 3 Example >0.05 >0.05 >0.05 >0.05 >0.1 >0.1 >0.1 >0.05 >0.05 >0.05 >0.1 4 Example >0.025 >0.025 >0.025 >0.025 >0.05 >0.05 >0.025 >0.025 >0.05 >0.025 >0.05 5 Example >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 6 Example >0.025 >0.025 >0.025 >0.025 >0.05 >0.05 >0.025 7 Example >0.025 >0.025 >0.025 >0.025 >0.05 >0.05 >0.05 8 Example >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 9 Example >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 10 Comparative >0.008 >0.03 >0.02 >0.02 >0.02 >0.03 >0.03 >0.02 >0.02 >0.03 >0.02 example 1

(91) TABLE-US-00003 TABLE 3 Effects against viruses MIC [%] Influenza A Influenza B Human Rhinovirus Example 1 >0.0016 >0.0016 >0.0016 Example 2 >0.0016 >0.0016 >0.0016 Example 3 >0.0016 >0.0016 >0.0016 Example 4 >0.003 >0.003 >0.003 Example 5 >0.0016 >0.0016 >0.0016 Example 6 >0.025 >0.05 >0.025 Example 7 >0.0016 >0.0016 >0.0016 Example 8 >0.0032 >0.0016 >0.0032 Example 9 >0.0125 >0.0125 >0.0125 Example 10 >0.0125 >0.0125 >0.0125 Comparative >0.035 >0.008 >0.008 example 1