POSSESSING BIOCIDAL ACTIVITY

Technical Field

The present invention relates to a novel class of dimeric quaternary pyridinium salts, possessing biocidal activity, which, in particular, can be used as antiseptic and disinfectant compounds. The production process that can easily provide said compounds at low cost from readily available pyridine compounds as starting raw materials is provided below as well.

Background of the invention

The spread of microbes and its increasing resistance to the number of known antimicrobial drugs brings potentially high danger to mankind over the world. That is why the search for new chemical substances demonstrating biocidal properties against wide range of conditionally-pathogenic and pathogenic microorganisms is very important. Nowadays, the task of creation of new generation of highly-efficient antiseptics possessing wide spectrum of antibacterial action together with low toxicity and at low cost is highly actual. Such new biocides would expand a number of available raw materials for creation of various antiseptic and disinfecting agents.

Quaternary ammonium salts (QAC) are one of the most used classes of disinfectants with a large applicability in hospital environments, water treatment, textile, paint and food industries due to their relatively low toxicity to human and animals and to their broad specificity of antimicrobial activity [Block, S.S. Disinfection, Sterilization and Preservation, 5th ed. Lippincott, Williams & Wilkins, Philadelphia, U.S.A. 2001, pp.283-319].

Among them, pyridinium and bispyridinium quaternary salts represent an important group of chemicals widely used as biocides, due to their strong antimicrobial effect even at very low concentrations, on a broad range of gram-positive and gram- negative bacteria, fungi, and some viruses [Chanawanno, K., Chantrapromma, S., Anantapong, T., Kanjana-Opas, A., Fun, H.-K. Synthesis, structure and in vitro antibacterial activities of new hybrid disinfectants quaternary ammonium compounds: Pyridinium and quinolinium stilbene benzenesulfonates. European Journal of Medicinal Chemistry, 45, 4199-4208 (2010); Kourai, H., Yabuhara, T., Shirai, A., Maeda, T., Nagamune, H. Syntheses and antimicrobial activities of a series of new bis-quaternary ammonium compounds. European Journal of Medicinal Chemistry, 41 (4), 437-444 (2006); Perles, C.E., Matheus, A., Volpe, P.L.O. The effect of quaternary ammonium surfactants on the inhibition of the aerobic metabolism of Sacharomyce cerevisiae - A calorimetric study. Thermochimica Acta, 479 (1-2), 28-31 (2008); Ng, C. L.L., Singhal, V., Widmer F., Wright, L. C, Sorell, T. C, Jolliffe, K. A. Synthesis, antifungal and hemolytic activity of a series of bis(pyridinium)alkanes. Bioorganic & Medicinal Chemistry, 15 (10), 3422-3429 (2007)].

In regard to recent results on the low cytotoxic effect of some antimicrobial agents from bis-quaternary pyridinium salts group on human cells it may be expected that pyridinium and bis-pyridinium salts are potential agents recommended for use in composition of hygiene products, in food industry (technological environment, equipment, various surfaces), and in catering units, hospitals, etc. [Nagamune, H., Maeda, T., Ohkura, K., Yamamoto, K., Nakajima, M., Kourai, H. Evaluation of cytotoxic effects of bis-quaternary ammonium antimicrobial reagents on human cells. Toxicology in Vitro, 14 (2), 139-147 (2000)].

In patents and scientific literature there can be found some bis-quaternary pyridinium salts, which possess antimicrobial and antifungal activity [US Patent 4206215 (1980), WO/2007/128059 (2007), US Patent 7612097 (2009)]. The molecular structure of those compounds can be represented by A, B and C types as shown below:

Here in structures A and B the spacer Z is a long aliphatic chain having from 6 to

18 carbon atoms or having similar chain with double and triple bonds inside, or atoms of oxygen, nitrogen or sulfur embedded, or ester groups attached to the nitrogen atom of pyridine rings [structure A - US Patent 4206215 (1980); Bailey D.M., DeGrazia C.G., Hoff S.J., Schulenberg P.L., O'Connor J.R., Paris D.A., Slee A.M., J.Med.Chem. 27, pp.1457-1464 (1984); structure B - WO2007/128059 (2007)].

In structure C the spacer Z is also a long aliphatic chain having from 6 to 18 carbon atoms or having similar chain with double and triple bonds inside or atoms of oxygen, nitrogen or sulfur embedded, or ester groups [structure C - US Patent 7612097 (2009)].

However, there is an important difference of structure C as compared to structures A and B namely in structure C the spacer Z is connected to carbons atom of pyridine rings [US Patent 7612097 (2009)].

One of the compounds with structure A - "octenidine dihydrochloride" widely used as the active antibacterial agent in the creation of antiseptic and disinfectant formulations, and commercially available [US patent application 2001/0036963; US patent application 201 1/0217360: EP 2401914].

The closest analogue to the structure of formula (I) of the present invention is bis- quaternary pyridinium salts of structure C. The major drawbacks of the bis-quaternary pyridinium salts of structure C are high toxicity and skin irritation.

Object of the invention

The purpose of the present invention is to provide new dimeric quaternary pyridinium salts, which can be characterized by higher biocidal activity and prolonged effect, lower toxicity and lower production cost, as well as to provide appropriate methods for production thereof. In comparison with the closest analog - the bis- quaternary pyridinium salts (structure C) with compounds of the formula I of the present invention have a comparable or higher biocidal activity and lower toxicity at the same time.

Brief description of the invention

The present invention describes a new class of dimeric quaternary pyridinium salts. The new compounds are obtained by N-alkylation of the pyridine derivatives of pentaerythritol, dipentaerythritol or hydroquinone with alkyl halides.

The invention relates to the following subject matters:

1. A compound with general formula I: (I), wherein

R is a linear or branched alkyl or alkenylene or alkyne group having from 8 to 18 carbon atoms;

Z is a spacer with a bivalent structure having a formula from the following list, connec

X is a halogen atom of chlorine, bromine or iodine.

2. A compound according to claim 1, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

3. A compound according to claim 1, wherein the bivalent structure of formula

> directly connected to two pyridine rings in the fourth position of pyridine ring.

4. A compound according to claim 1, wherein the bivalent structure of formula

s directly connected to two pyridine rings in the third position of pyridine ring.

5. A compound according to claim 1, wherein the bivalent structure of formula

s directly connected to the third position of one pyridine ring and to the fourth of another pyridine ring.

6. A compound according to claim 1 , wherein the bivalent structure of formula

is directly connected to two pyridine rings in third position of pyridine ring.

7. A compound according to claim 1 , wherein the bivalent structure of formula

OH OH

is directly connected to two pyridine rings in the third position of pyridine ring.

8. A compound according to claim 1 , wherein the bivalent structure of formula

is directly connected to two pyridine rings in the fourth position of pyridine ring.

9. A compound according to claim 1, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

10. A compound according to any one of claims 1 to 9 for use as antiseptics or disinfectants.

11. Use of the compound according to any of claims from 1 to 9, as an active biocidal ingredient for production of antiseptics or disinfectants.

12. A method for producing a compound according to claim 1 , comprising a step of reacting an intermediate of formula II (II),

wherein Z is a bivalent spacer connected directly to the position 3 or 4 in pyridine ring selected from the following structures:

with a compound of formula

R-X

wherein R is a linear or branched alkyl or alkenylene or alkyne group having from 8 to 18 carbon atoms, and X is a halogen atom of chlorine, bromine or iodine; in a solvent.

13. A method according to claim 12, wherein the intermediate of formula II has formula III

14. A method according to claim 12, wherein the intermediate of formula II has formula IV

15. A method according to claim 12, wherein the intermediate of formula II has formula IVa

16. A method according to claim 12, wherein the intermediate of formula II has formula IVb

17. A method according to claim 12, wherein the intermediate of formula II has formula V

18. A method according to claim 12, wherein the intermediate of formula II has formula

19. A method according to claim 12, wherein the intermediate of formula II hasformul

20. A method according to claim 12, wherein the intermediate of formula II has formula VII

The claimed compounds of formula I possessing biocidal activity are applicable, in particular, as an antiseptic and disinfectant active compounds.

The compounds of formula I, wherein spacer Z is connected directly to the position 3 or 4 in pyridine ring, and has a bivalent structure represented by formula selected from the following list:

demonstrate similar biocide properties compared to bis-quaternary pyridinium salts applied in up-to-date antiseptics and disinfecting formulations. However, contrary to the known antiseptics, in particular, to compounds of type C described above, the novel compounds possess significantly reduced toxicity. That is why the application of these compounds can be extended on active ingredients for skin antiseptics as well as for other disinfecting formulas.

The compounds of general formula I can be useful for application as antimicrobial agents. They possess biocidal activity, i.e. bacteriostatic and bactericide activity along with low and even extra low toxicity.

More specifically, the compounds of formula I include the following compounds: (i) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

(ii) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the fourth position of pyridine ring.

(iii) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

(iv) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to the third position of one pyridine ring and to the fourth of another pyridine ring. (v) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

(vi) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

(vii) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the fourth position of pyridine ring.

(viii) Compound represented by formula I, wherein the bivalent structure of formula

is directly connected to two pyridine rings in the third position of pyridine ring.

(ix) The compounds represented by formula I including the compounds of the items (i) to (viii) for use in a biocide treatment.

The technical result of present invention (compounds of formula I) is the lower cell toxicity and thus higher biocompatibility of compounds of formula I together with comparable or higher biocidal efficiency as compared to the closest analogue (bis- quaternary pyridinium salts structure C shown above).

The inventors have discovered a class of dimeric quaternary pyridinium salts derived from pyridine derivatives of pentaerythritol, dipentaerythritol or hydroquinone and alkyl halides, which provide superior biocidal activity, in particular, they can be used as an active compounds in antiseptic and disinfectant compositions. The compounds of formula I of the present invention have a comparable or higher biocidal activity and lower toxicity as compared to existing quaternary ammonium salts. At the same time, this invention relates to a method for producing compounds of the formula I, which can easily provide them at low cost from readily-available pyridine and pentaerythritol, dipentaerythritol or hydroquinone compounds as starting materials.

Detailed description of the invention

Definitions

According to the Directive 98/8/EC of the European Parliament and Council of the 16 February 1998 the following definitions of terms takes place in present invention:

Antibacterial - chemical substance that kills or slows down the growth of bacteria, Antimicrobial chemical substance, which, at low concentrations, exerts an action against microorganisms and destroys them or inhibits their growth,

Antiseptic - antimicrobial substance that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction

Antifungal - chemical substance that destroying or inhibiting the growth of fungi

Antiviral - chemical substance that destroying or inhibiting the growth and reproduction of viruses

Bactericide - chemical substance that kills bacteria and, ideally, nothing else.

Bactericides are disinfectants, antiseptics

Bacteriostatic chemical agent that stops bacteria from reproducing, while not necessarily harming them otherwise.

Biocide (biocidal chemical agent intended to destroy, render harmless, prevent the products) - action of, or otherwise exert a controlling effect on any harmful organism by chemical or biological means. Examples include disinfectants, preservatives, antiseptics, pesticides, herbicides, fungicides and insecticides.

Disinfectant - chemical substance that is applied to non-living objects to destroy microorganisms that are living on the objects

(1) Dimeric quaternary pyridinium salt Nowadays, the resistance of bacteria to modern antibacterial means stimulates the research and development works for creation of new improved biocide materials.

The present invention proves the fact that the dimeric quaternary pyridinium salts having general formula I, and being the derivatives of pentaerythritol, dipentaerythritol or hydroquinone, combine the biocidal efficiency in wide antimicrobial spectrum together with low toxicity, and can be used as active ingredient for creation of new antiseptics and disinfectants generation, possessing low toxicity as compared to known agents applied in practice, i.e., for medicine purposes and home use.

According to modern scientific views onto the principle of antiseptic action, the biocide action of the compounds of general formula I can be defined by, but not limited to, its ability to stick to the cell wall and the bacteria membrane as well as to intrude inside of the cell core and then to inhibit the cell.

The sequence of the cell destruction by biocide can be described as follows:

1. Biocide adsorption on the cell surface.

2. Biocide molecule diffusion through cell wall inside of the cell.

3. Connection of the diffused biocide molecule to the cytoplasmic membrane.

4. Destabilization and destruction of cytoplasmic membrane.

5. Cytoplasm compounds extraction from the cell.

6. The destruction of the cell.

The ability to connect to the membranes is mostly determined, on one hand, by presence of positively charged groups in compounds of formula I, in particular, of two quaternary pyridinium groups, and, on the other hand, by presence of negative charge on the cell surface, which is typical for phosphate groups of lipids and acids.

The mechanism for interaction of the compounds of formula I with microorganisms' membranes can be described as below. After the contact of the cell and the biocide molecule, the electrostatic interaction of negatively charged groups at cell surface and the biocide molecule occurs. This leads to the reorientation of the biocide molecule and intrusion of its charged fragments inside of the lipid monolayer (membrane). The compound of formula I cooperatively may connect to a large number of phospholipids in membrane and cause its negative charge neutralization. The created complex is stabilized by strong hydrophobic interaction with alkyl chains of fatty acids of phospholipids. This leads to strong misbalance in electrostatic and hydrophobic interactions and reduces the lipid-lipid interaction. At the same time, another aspect of the biocide adsorption on the cell surface is the inhibition of barrier and transport functions of membrane. Further intrusion of hydrophobic fragment of biocide molecule inside of non-polar part of the cell membrane leads to its expansion and reduction of Van der Waals forces between lipid molecules. As consequence, firstly, the membrane permittivity is changed, secondly, the membrane integrity is disrupted, and finally the membrane is fragmented and destroyed.

The positive input into high antibacterial activity accompanied with low toxicity level of compounds of formula I may be brought by spacer Z. The presence of sufficient number of oxygen atoms inside of the spacer structure evidently secures the good balance between hydrophobic and hydrophilic components of the molecule. This leads to high antibacterial activity, and low toxicity.

In formula I, the R is a linear or branched alkyl or alkenylene or alkyne group having from 8 to 18 carbon atoms.

Terms of the "alkyl" in the compounds of the present invention include typically

C8-18, preferably C8-16, more preferably C8-12. Examples of such alkyl groups include n-octyl, tert-octyl, 2-ethlylhexyl, nonyl, decyl, iso-decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl etc. Preferable groups are C8-18 linear alkyl groups such as n-octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl; more preferable are n-octyl, decyl, dodecyl.

Terms of the "alkenylene" in the compound of the present invention include typically C8-18, preferably C8-16, more preferably C8-12, and at least one double bond. Examples of such alkenylene groups include octenyl, nonenyl, decenyl, iso- decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl and octadecenyl, etc. Preferable groups are C8-18 linear alkenylene groups such as octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl and octadecenyl; more preferable are octenyl, decenyl, dodecenyl.

Terms of the "alkyne" in the compound of the present invention include typically C8-18, preferably C8-16, more preferably C8-12, and at least one triple bond. Examples of such alkyne groups include octynyl, nonynyl, decynyl, isodecynyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadecynyl and octadecynyl, etc. Preferable groups are C8-18 linear alkyne groups such as octynyl, decynyl, dodecynyl, tetradecynyl, hexadecynyl and octadecynyl; more preferable are octynyl, decynyl, dodecynyl.

Overwhelming majority of the compounds represented by formula I was evaluated and screened for possible antibacterial and antifungal activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, Enterococcus faecalis and Candida albicans microorganisms using the paper disk diffusion method [Bauer A.W.; Kirby W.M.; Sherds J.C.; Turck M. Am. J. Clinic. Pathol., 45, 493-496 (1966)]. These strains are recommended for testing of bactericidal activities of disinfectant [European Committee for Standardization. EN 1040, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - Test method and requirements. (Phase 1) Brussels, Belgium, (2005)].

Some of the compounds represented by formula I, in particular, the compounds (Ic, Ih, Ik, In, Ip, Is, It and Ix) were superior to the control compound - benzalkonium chloride, demonstrating high bacteriostatic, bactericidal and antifungal activity against all of the microorganisms mentioned above.

Benzalkonium chloride (BAC) was used as reference drug for control. Although the BAC is benzyl ammonium compound, which does not have bis-quaternary ammonium groups, it was chosen as standard because currently it is a common widely used bactericidal agent [Murguia M.C., Vaillard V.A., Sanchez V.G., Conza J.D., Grau R.J. Synthesis, surface-active properties, and antimicrobial activities of new double- chain gemini surfactants. J Oleo Sci. 57 (5), 301-308 (2008); US Patent 7612097 (2009)].

Most of the compounds represented by formula I have low toxicity, in particular, the compounds (Ic, Ih, Ik, In, Ip, Is, It and Ix) possess lower cytotoxicity than benzalkonium chloride and other bis-quaternary pyridinium salts, for example compound of structure C [US Patent 7612097 (2009)] and commercially available bis- quaternary pyridinium salt - "octenidine dihydrochloride" [Muller G., Kramer A. Biocompatibility index of antiseptic agents byparallel assessment of antimicrobial activity and cellular cytotoxicity. J. Antimicrob Chemother 61 , pp. 1281-7 (2008)].

The compounds of formula I may be used as antiseptics or disinfectants. They may be dissolved, suspended or emulsified in a co-solvent, e.g. an alcohol (for example: methanol, ethanol or isopropanol, as well as a mixture thereof). The solvent may be a polar organic solvent. It may be aqueous alcohol. It may be water. The compounds may be applied by spraying, wiping etc. They may be incorporated (e.g. impregnated) into disinfectant wipes. The dimeric quaternary pyridinium compounds described herein may be combined with one of more acceptable adjuvants and/or carriers to form a formulation. The formulation may also comprise other (non- bispyridinium) antimicrobial compounds. The formulation may comprise one or more than one (e.g. 2, 3, 4 or 5) different dimeric quaternary pyridinium compounds as described in the present invention. The formulation may be a solution, a suspension, an emulsion or a dispersion. It may be a liquid formulation, e.g., washing liquid. It may be a solid, e.g. a powder or soap. It may be, for example, in the form of a cream, paste, salve, lotion, foam, ointment, balm or a spray. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension, in an aqueous or non-aqueous fluid, encapsulated, with a greasy or non-greasy basis.

(2) Method for Producing Dimeric quaternary pyridinium salt

Another part of current invention includes methods for producing the compounds of general formula I according to schemes 1-8, including step 1), 2) or 3) and step 4) as follows:

1) Production of pentaerythritol derivatives represented by formulas II, III, Ilia, or Illb:

Production of dipentaerythritol derivatives represented by formulas IV, V, or

3) Production of h droquinone derivatives represented by formulas VI:

4) Quaternization of dipyridine derivatives of pentaerythritol II, III, Ilia, Illb, dipentaerythritol IV, V, Va or hydroquinone VI with haloidalkanes of formula Vlla-c:

R-X (Vlla-c),

(wherein R is a linear or branched alkyl or alkenylene or alkyne group having from 8 to 18 carbon atoms; X is a halogen atom of chlorine (a), bromine (b) or iodine (c) by refluxing in such solvents as butanol, 4-methylpentane-2-on, or dimethylformamide.

Compounds of formula I(a-e) can be obtained via five-step synthesis according to the Scheme 1 with following steps:

- Acetal 1 is obtained by reaction of pentaerythritol with benzaldehyde using the known method [Org. Synth. 4, 679, 1963.] with 75% yield.

- Ditosylate 2 is obtained using known technique [P. Pecquet, F. Huet, M. Legraverend, E. Bisagni Heterocycles 34 p. 739-745 (1992)] with 97% yield.

- The alkylation of 3-hydroxypyridine with ditosylate 2 in DMF in the presence of potassium carbonate at temperature of 120 °C results in formation of compound 3 with 88% yield.

- The hydrolysis of compound 3 in water solution of hydrochloric acid gives rise to compound II with 78% yield.

- The quaternization of the compound II with alkylbromide Vllb is conducted by refluxing in 4-methylpentane-2-on media and leads to compounds I (a-e) with 80-85% yield.

e 1

R = CgH, ₇ (a), C ₁₀ H _{2 l} (b), C ₁₂ H ₂₅ (c), C, ₄ H ₂₉ (d), C^e); X = Br

Compounds of formula I(f-h) can be obtained via two-step synthesis according to the Scheme 2 with following steps:

- Diacetal III is obtained by reaction of pentaerythritol with 4- pyridinecarboxaldehyde in the presence of toluenesulfonic acid refluxing in toluene with Dean-Stark trap in 70% yield.

- The quaternization of compound III by alkylbromide of formula Vllb is performed by refluxing in 4-methylpentane-2-on and leads to compounds I(f-h) in 80- 90% yield.

Scheme 2

l(f-h)

R= C ₈ H ₁₇ (f) _> C ₁₀ B ₂₁ (g), C ₁₂ H ₂₅ (h); X = Br

The compounds of formula I(i-k) are obtained via two-step synthesis according to the Scheme 3 with following steps:

- Diacetal Ilia is obtained by reaction of pentaerythritol with 3- pyridinecarboxaldehyde in the presence of toluenesulfonic acid by refluxing in toluene with Dean-Stark trap in 84% yield.

- The quaternization of compound Ilia by alkylbromide of formula Vllb is accomplished by refluxing in 4-methylpentane-2-on media and leads to compounds I (i- k) in 70-75% yield.

Scheme 3

l(i-k)

R = C ₈ H ₁₇ (i), C _lo H ₂₁ 0), C ₁₂ H ₂₅ (k); X = Br

Compounds of formula I(l-n) are obtained via three-step synthesis according to the Scheme 4 with following steps:

- Acetal 4 is obtained by reaction of pentaerythritol with 4- pyridinecarboxaldehyde in the presence of toluenesulfonic acid by refluxing in toluene with Dean-Stark trap in 74% yield.

- Diacetal Illb is obtained by reaction of acetal 4 with 3 -pyridinecarboxaldehyde in the presence of toluenesulfonic acid refluxing intoluene with Dean-Stark trap in 83% yield.

- The quaternization of compound Illb by alkylbromide of formula Vllb is performed by refluxing in 4-methylpentane-2-on and leads to compounds I(l-n) in 70- 75% yield. Scheme 4

l(l-n)

R = C ₈ H ₁₇ G), C ₁₀ H ₂₁ (m), C ₁₂ H ₂₅ (n); X = Br

Compounds of formula I(o-q) are obtained via five-step synthesis according to the Scheme 5 with following steps:

- Acetal 5 is obtained by reaction of dipentaerythritol with benzaldehyde in the presence of toluenesulfonic acid by refluxing in toluene with Dean-Stark trap in 95% yield.

- The reaction of acetal 5 with toluenesulfonyl chloride leads to ditosylate 6 product in 75% yield.

- The alkylation of 3-hydroxypyridine by ditosylate 6 in DMF in the presence of potassium carbonate at 100 °C results in formation of compound 7 in 74% yield.

- The hydrolysis of compound 7 in hydrochloric acid water solution leads to compound IV in 80% yield.

- The quaternization of compound IV by alkylbromide of formula Vllb is performed by refluxing in 4-methylpentane-2-on media and leads to compounds I(o-q) in 75-80% yield. e 5

Compounds of formula I(r,s) are obtained via two-step synthesis according to the Scheme 6 with following steps:

- Diacetal V is obtained by reaction of dipentaerythritol with 4- pyridinecarboxaldehyde in the presence of toluenesulfonic acid by refluxing in toluene with Dean-Stark trap in 57% yield.

- The quaternization of compound V by alkylbromide of formula Vllb is performed by refluxing in 4-methylpentane-2-on media and gives compounds I(r,s) in 70-75% yield.

eme 6

l(r,s)

R= C _I(l H ₂₁ (r),C, ₂ H _!s (s); X= Br

Compounds of formula I(t,u) are obtained via two-step synthesis according to the Scheme 7 with following steps:

- Diacetal Va is obtained by reaction of dipentaerythritol with 4- pyridinecarboxaldehyde in the presence of toluenesulfonic acid by refluxing in toluene with Dean-Stark trap in 64% yield.

- The quaternization of compound Va by alkylbromide of formula Vllb is performed by refluxing in 4-methylpentane-2-on media and leads to compounds I(t,u) in 73-75% yield.

e 7

l(t,u)

R = C ₁₂ H ₂₅ (t), C ₁₆ H ₃ 3(u); X = Br

The compounds of formula I(v-z) are obtained via two-step synthesis according to the Scheme 8 with following steps:

- Diacetal VI is obtained by alkylation of 3-hydroxypyridine with 1 ,4- dibromobenzene in boiling DMF in the presence of potassium carbonate and copper within 48 hours in 70% yield. Note: Previously the compound VI has been obtained only in 17% yield via alkylation of 3-hydroxypiridine with 1 ,4-dibromobenzene in boiling dimethylacetamide in presence of potassium carbonate and copper within 18 hours [D.A. McMorran, P.J. Steel, Acta Cryst. C54, pp.1132-1 133 (1998)].

- The quaternization of compound VI by alkylbromide of formula Vllb is performed by refluxing in 4-methylpentane-2-on media and leads to compounds I(v-z) in 63-69% yield. Scheme 8

l(v-z)

R = C ₈ H ₁₇ (v), CioH ₂₁ (w), C ₁₂ H ₂₅ (x),C ₁₄ H ₂₉ (y), C ₁₆ H ₃₃ (z); X = Br

Other objects and advantages of the present invention will become apparent upon detailed description reading of the examples and the appended claims provided below.

In order that the invention should be more readily understood, reference is made to the following examples, which are intended to be illustrative for the invention, but are not intended to be limiting in scope.

(3) Chemistry

General methods

Melting points were determined using a Stuart ESTSMP3 melting point apparatus and are uncorrected. Ή nuclear magnetic resonance spectra were recorded using a Bruker Avance II 300 spectrometers at a frequency of 300.13 MHz and chemical shifts are reported as parts per million (ppm) with deuterochloroform (CDC1 ₃ ; 5 _H 7.26), deuteromethanol (CD ₃ OD-d ₄ ; δ _Η 3.31) or deuterodimethylsulfoxide (DMSO-d ₆ ; 6H 2.50) as internal reference. The data are reported as chemical shift (δ), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (J Hz), relative integral assignment. Analytical thin layer chromatography (TLC) was perfomed using precoated silica gel plates (Merck Kieselgel 60 F254) and visualized using UV-light apparatus with wavelength of λ=254 nm. Preparative column chromatography was carried out using Merck Kieselgel 60 silica gel (Si0 ₂ , 0.04-0.065 mm) with the indicated solvent systems. Rations of solvents for TLC and column chromatography are expressed in (v/v) as specified. All solvents were distilled before use. Mass-spectra were obtained directly using Finningan MAT INCOS 50 spectrometer (EI, 70 eV). Examples 1-5 [Scheme 1 ]

General synthetic procedure for the synthesis dimerlc compounds (Ia-e)

A mixture of 2,2-bis[(pyridin-3-yloxy)methyl]propane-l,3-diol (0.4 g, 1.38 mmol) and alkylbromide (2.8 mmol) in 1.5 ml of dry 4-methyIpentan-2-one was refluxed for 15 hours. The resulting solution was cooled to room temperature, the solvent was decanted, the residue was washed with hexane and ether and then was dissolved in acetonitrile. Afterwards charcoal was added and the mixture was refluxed for 30 min. Filtration from the charcoal, evaporation of the solution and recrystallization from acetonitrile/ether or acetone/acetonitrile leads to pure product as a white solid.

3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(l-octylpyrldlnlum-3- yl)oxy]methyl}propo -l-octylpyrldlnlum dlbromlde (la)

Yield 80% (0.75 g). M = 676.6. C ₃ iH ₅₂ Br ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): δ = 0.91 (t, J = 6.7 Hz, 6H, CH ₃ ), 1.22-1.50 (m, 20H, CH ₂ ), 2.00-2.16 (m, 4H, CH ₂ ), 3.84 (s, 4H, O- CH ₂ ), 4.43 (s, 4H, Py-0-CH ₂ ), 4.66 (t, J = 7.5 Hz, 4H, f-CH ₂ ), 8.03 (dd, J, = 8.6 Hz, J ₂ = 5.8 Hz, 2H, CH, Py), 8.28 (dd, J, = 8.6 Hz, J ₂ = 1.8 Hz, 2H, CH, Py), 8.64 (d, J = 5.8 Hz, 2H, N-CH, Py), 8.95-9.05 (m, 2H, N-CH, Py).

3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(l-decylpyrldlnlum-3- yl)oxy]methyl}pro xy)-l-decylpyrldlnlum dlbromlde (lb)

Yield 83% (0.84 g). M = 732.7. C ₃₅ H ₆ oBr ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): δ = 0.91 (t, J = 6.7 Hz, 6H, CH ₃ ), 1.18-1.50 (m, 28H, CH ₂ ), 2.00-2.16 (m, 4H, CH ₂ ), 3.84 (s, 4H ₃ O- CH ₂ ), 4.43 (s, 4H, Py-0-CH ₂ ), 4.65 (t, J = 7.7 Hz, 4H, N ⁺ -CH ₂ ), 8!03 (dd, J, = 8.8 Hz, J ₂ = 5.9 Hz, 2H, CH, Py), 8.27 (dd, J, = 8.8 Hz, J ₂ = 1.9 Hz, 2H, CH, Py), 8.64 (d, J = 5.9 Hz, 2H, N-CH, Py), 8.95-9.05 (m, 2H, N-CH, Py).

3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(l-dodecylpyridinium-3- yl)oxy]methyl}pro oxy)-l-dodecylpyridinium dibromide (Ic)

Yield 85% (0.93 g). M = 788.8. C ₃ 9H ₆₈ Br2N ₂ 04. Ή NMR (CD ₃ OD): δ = 0.91 (t, J = 6.7 Hz, 6H, CH ₃ ), 1.18-1.50 (m, 36H, CH ₂ ), 2.00-2.16 (m, 4H, CH ₂ ), 3.82 (s, 4H, O- CH ₂ ), 4.41 (s, 4H, Py-0-CH ₂ ), 4.66 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 8.02 (dd, J, = 8.5 Hz, h = 5.8 Hz, 2H, CH, Py), 8.27 (dd, Jj = 8.5 Hz, J ₂ = 2.0 Hz, 2H, CH, Py), 8.64 (d, J = 5.8 Hz, 2H, N-CH, Py), 8.96-9.06 (m, 2H, N-CH, Py).

3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(l-tetradecylpyridinium-3 - yl)oxy]methyl ropoxy)-l-tetradecylpyridinium dibromide (Id)

Yield 85% (0.99 g). M = 844.9. C ₄₃ H ₇₆ Br ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): 5 = 0.91 (t, J = 6.7 Hz, 6H, CH ₃ ), 1.18-1.50 (m, 44H, CH ₂ ), 2.00-2.16 (m, 4H, CH ₂ ), 3.82 (s, 4H, O- CH ₂ ), 4.41 (s, 4H, Py-0-CH ₂ ), 4.66 (t, J = 7.4 Hz, 4H, N ⁺ -CH ), 8.02 (dd, J, = 8.5 Hz, = 5.8 Hz, 2H, CH, Py), 8.27 (dd, Ji = 8.5 Hz, J ₂ = 2.0 Hz, 2H, CH, Py), 8.64 (d, J = 5.8 Hz, 2H, N-CH, Py), 8.96-9.06 (m, 2H, N-CH, Py).

3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(l-hexadecylpyridinium-3- yl)oxy]methyl}propoxy)-l-hexadecylpyridinium dibromide (Ie)

Yield 85% (1.06 g). M = 901.0. C ₄₇ H84Br ₂ N ₂ 04. Ή NMR (CD ₃ OD): δ = 0.91 (t, J = 6.7 Hz, 6H, CH ₃ ), 1.18-1.50 (m, 52H, CH ₂ ), 2.00-2.16 (m, 4H, CH ₂ ), 3.82 (s, 4H, O- CH ₂ ), 4.41 (s, 4H, Py-0-CH ₂ ), 4.66 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 8.02 (dd, J, = 8.5 Hz, J ₂ = 5.8 Hz, 2H, CH, Py), 8.27 (dd, J, = 8.5 Hz, J ₂ = 2.0 Hz, 2H, CH, Py), 8.64 (d, J = 5.8 Hz, 2H, N-CH, Py), 8.96-9.06 (m, 2H, N-CH, Py).

Examples 6-8 [Scheme 2]

General synthetic procedure for the synthesis dimeric compounds (If-h)

Mixture of diacetal III (1.57 g, 5.0 mmol) and alkylbromide (15.0 mmol) in 5 ml of dry 4-methylpentan-2-one was refluxed for 6 hours. The resulting solution was cooled to room temperature, the solvent was decanted, the residue was washed with ether and dissolved in methanol. Afterwards charcoal was added, and the mixture was boiled for 30 min. The charcoal was removed by filtration, and evaporation of methanol under reduced pressure afforded crude reaction product. Recrystallization of the crude product from acetone gives rise to desired compound obtained in a pure form as a white solid.

N-Octyl{4,4 ^, -(2,4,8,10-tetraoxaspiro[5.5]-undecan-3,9-diyl)dipyrid inium dibromid

Yield 80% (2.79 g). M = 700.6. C ₃ 3H ₅₂ Br ₂ N ₂ 0 ₄ . Ή NMR (DMSO-d ₆ ): δ = 0.84

(t, J = 7.0 Hz, 6H, CH ₃ ), 1.18-1.32 (m, 20H, CH ₂ ), 1.84-1.96 (m, 4H, CH ₂ ), 3.81-4.13 (m, 6H, CH ₂ ), 4.52-4.60 (m, 2H, CH ₂ ), 4.66 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 5.88 (s, 2H, CH), 8.18 (d, J = 6.7 Hz, 4H, CH, Py), 9.21 (d, J = 6.7 Hz, 4H, N-CH, Py).

N-decyl{4,4'-(2,4,8,10-tetraoxaspiro[5.5]-undecan-3,9-diyl)d ipyridinium dibromide (Ig)

2Bf

Yield 82% (3.10 g). M = 756.7. C ₃₇ H ₆ oBr ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): 6 = 0.91 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.22-1.48 (m, 28H, CH ₂ ), 1.96-2.12 (m, 4H, CH ₂ ), 3.84-4.16 (m, 6H, CH ₂ ), 4.62-4.78 (m, 6H, 2 N ⁺ -CH ₂ , CH ₂ ), 5.88 (s, 2H, CH), 8.21 (d, J = 6.4 Hz, 4H, CH, Py), 9.08 (d, J = 6.4 Hz, 4H, N-CH, Py).

N-dodecyl{4,4 '-(2,4, 8,10-tetraoxaspirofS.5J-undecan-3, 9-diyl)dipyridinium dibromide (Ih)

Yield 90% (3.66 g). M = 812.8. C ₄ iH ₆₈ Br ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): δ = 0.91 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.24-1.48 (m, 36H, CH ₂ ), 1.96-2.12 (m, 4H, CH ₂ ), 3.86-4.16 (m, 6H, CH ₂ ), 4.62-4.80 (m, 6H, 2N ⁺ -CH ₂ , CH ₂ ), 5.88 (s, 2H, CH), 8.20 (d, J = 6.4 Hz, 4H, CH, Py), 9.08 (d, J = 6.4 Hz, 4H, N-CH, Py).

Examples 9-11 [Scheme 3]

General synthetic procedure for the synthesis dimeric compounds (Ii-k)

A mixture of diacetal Ilia (0.628 g, 2.0 mmol) and alkylbromide (6.0 mmol) was refluxed in 2 ml of dry 4-methylpentan-2-one for 6 hours. The resulting solution was cooled to room temperature, the solvent was decanted, the residue was washed with ether and dissolved in methanol/acetonitrile mixture (1/5 by volume). Afterwards charcoal was added and the mixture was refluxed for 30 min until complete decolorization. The charcoal was removed by filtration; solution was evaporated under reduced pressure. Recrystallisation from acetone affords pure product as a white solid.

N-octyl{3,3'-(2,4,8,10-tetraoxaspiro[5.5]-undecan-3,9-diyl)} dipyridinium dibromide Π)

2Br ^"

Yield 70% (2.45 g). M = 700.6. C ₃₃ H ₅₂ Br ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): δ = 7.0 Hz, 6H, CH ₃ ), 1.18-1.38 (m, 28H, CH ₂ ), 1.90-2.04 (m, 4H, CH ₂ ), 3.78-4.08 (m, 6H, CH ₂ ), 4.58-4.76 (m, 6H, 2N ⁺ -CH ₂ , CH ₂ ), 5.82 (s, 2H, CH), 8.11 (dd, J, = 7.7 Hz, J ₂ = 6.2 Hz, 2H, CH, Py), 8.54-8.64 (m, 2H, CH, Py), 8.96-9.04 (m, 2H, N-CH, Py), 9.08- 9.12 (m, 2H, N-CH, Py).

N-decyl{3,3 '-(2,4, 8,10-tetraoxaspiro[5.5]-undecan-3, 9-diyl) dipyridinium dibromide (Ij)

2Br ^"

Yield 74% (2.80 g). M = 756.7. C ₃₇ H ₆₀ Br ₂ N ₂ O ₄ . Ή NMR (CD ₃ OD): δ = 0.89 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.14-1.40 (m, 28H, CH ₂ ), 1.88-2.06 (m, 4H, CH ₂ ), 3.78-4.10 (m, 6H, CH ₂ ), 4.57-4.77 (m, 6H, 2^-CH^ CH ₂ ), 5.83 (s, 2H, CH), 8.1 1 (dd, J, = 8.0 Hz, J ₂ = 6.6 Hz, 2H, CH, Py), 8.56-8.66 (m, 2H, CH, Py), 8.96-9.04 (m, 2H, N-CH, Py), 9.07- 9.14 (m, 2H, N-CH, Py).

N-dodecyl{3,3'-(2,4,8,10-tetraoxaspiro[5.5]-undecan-3,9-diyl )}dipyridinium dibromide (Ik)

Yield 80% (3.25 g). M = 812.8. C ₄ iH ₆₈ Br ₂ N ₂ 0 ₄ . Ή NMR (CD ₃ OD): δ = 0.88 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.13-1.41 (m, 36H, CH ₂ ), 1.88-2.06 (m, 4H, CH ₂ ), 3.78-4.10 (rn, 6H, CH ₂ ), 4.58-4.80 (m, 6H, 2N ⁺ -CH ₂ , CH ₂ ), 5.82 (s, 2H, CH), 8.10 (dd, J, = 7.7 Hz, J ₂ - 6.2 Hz, 2H, CH, Py), 8.56-8.66 (m, 2H, CH, Py), 8.96-9.04 (m, 2H, N-CH, Py), 9.07- 9.14 (m, 2H, N-CH, Py).

Examples 12-14 [Scheme 4]

General synthetic procedure for the synthesis dimeric compounds (Il-n)

A mixture of diacetal Illb (0.943 g, 3.0 mmol) and alkylbromide (6.0 mmol) was refluxed in 15 ml of dry 4-methylpentan-2-one for 10 hours. The resulting solution was cooled to room temperature, the solvent was decanted, the residue was washed with hexane and dissolved in methanol (30 ml). Afterwards charcoal was added and the mixture was refluxed for 30 min until complete decolorization. The charcoal was removed by filtration; solution was evaporated under reduced pressure to give pure product as a white solid.

N-Octyl{3,4'-(2,4,8,10-tetraoxaspiro[5.5]-undecan-3,9-diyl)} dipyridinium dibromid

2Br ^"

Yield is 70% (1.47 g). M = 700.6. C33H ₅₂ Br ₂ N ₂ 0 ₄ . Ή NMR (300 MHz, CD ₃ OD): δ = 0.88 (t, J - 7.0 Hz, 6H, CH ₃ ), 1.18-1.40 (m, 20H, CH ₂ ), 1.90-2.06 (m, 4H, CH ₂ ), 3.80-4.12 (m, 6H, CH ₂ ), 4.57-4.73 (m, 6H, CH ₂ ), 5.83 (s, 1H, CH), 5.84 (s, 1H, CH), 8.07-8.17 (m, 3H, CH, Py), 8.58-8.66 (m, 1H, CH, Py), 8.98-9.15 (m, 4H, CH, Py).

N-Decyl{3,4 '-(2,4, 8,10-tetraoxaspiro[5.5]-undecan-3, 9-diyl)}dipyridinium dibromide (Im)

2Br ^"

Yield is 71% (1.61 g). M = 756.7. C ₃₇ H ₆ oBr ₂ N ₂ 0 ₄ . ¾ NMR (300 MHz, CD ₃ OD): δ = 0.88 (t, J = 7.0 Γιι, 6H, CH ₃ ), 1.14-1.40 (m, 28H, CH ₂ ), 1.90-2.06 (m, 4H, CH ₂ ), 3.80-4.12 (m, 6H, CH ₂ ), 4.57-4.77 (m, 6H, CH ₂ ), 5.84 (s, 1H, CH), 5.85 (s, 1H, CH), 8.07-8.17 (m, 3H, CH, Py), 8.58-8.66 (m, 1H, CH, Py), 9.00-9.16 (m, 4H, CH, Py).

N-Dodecyl{3,4'-(2,4,8,10-tetraoxaspiro(5.5]-undecan-3,9-diyl )}dipyridinium dibromide (In)

2Br

Yield 75% (1.83 g). M = 812.8. C ₄₁ H ₆₈ Br ₂ N ₂ 0 ₄ . Ή NMR (300 MHz, CD ₃ OD): δ

= 0.86 (t, J = 7.0 ΓΧ 6H, CH ₃ ), 1.18-1.40 (m, 36H, CH ₂ ), 1.90-2.06 (m, 4H, CH ₂ ), 3.80-4.10 (m, 6H, CH ₂ ), 4.57-4.73 (m, 6H, CH ₂ ), 5.81 (s, 1H, CH), 5.82 (s, 1H, CH), 8.06-8.16 (m, 3H, CH, Py), 8.57-8.65 (m, 1H, CH, Py), 8.97-9.13 (m, 4H, CH, Py).

Examples 15-17 [Scheme 5]

General synthetic procedure for the synthesis dimeric compounds (lo-q) A mixture of compound IV (85.8 mg, 0.21 mmol) and alkylbromide (0.44 mmol) was refluxed in 3 ml of acetonitrile for 24 h. The resulting solution was cooled to room temperature and diluted with 15 ml of ether. The solution was decanted and the product was washed twice with ether until complete crystallization. The product was dissolved in EtOH and stirred with charcoal at room temperature for 30 min. Filtration from charcoal and evaporation under vacuo affords pure white crystalline compound.

l-Octyl-3-[3-[3-[(l-octylpyridinium-3-yl)oxy]-2,2-bis(hydrox methy

2,2-bis(hy roxymethyl)propoxy]pyridinium dibromide (Io)

Yield 75% (0.125 g). M = 794.7. C ₃₆ H ₆₂ Br ₂ N ₂ 0 ₇ . Ή NMR (CD ₃ OD-d4): δ = 0.90

(t, J = 6.6 Hz, 6H, CH ₃ ), 1.23-1.45 (m, 20H, CH ₂ ), 1.95-2.10 (m, 4H, CH ₂ ), 3.54 (s, 4H, OCH ₂ ), 3.66 (s, 8H, OCH ₂ ), 4.24 (s, 4H, OCH ₂ ), 4.57 (t, J = 8.0 Hz, 4H, N ⁺ CH ₂ ), 7.94 (dd, J| = 8.8 Hz, = 5.9 Hz, 2H, Py), 8.16 (dd, J, = 8.8 Hz, J ₂ = 1.5 Hz, 2H, Py), 8.55 (d, J = 5.1 Hz, 2H, Py), 8.85 (br s, 2H, Py).

l-Decyl-3-f3-f3-f(l-decylpyridinium-3-yl)oxyJ-2,2-bis(hydrox ymethyl)propoxyJ- 2,2-bis(hydroxymethyl)propoxy]pyridinium dibromide (Ip)

Yield 80% (0.143 g). M = 850.8. C ₄₀ H ₇₀ Br ₂ N ₂ O ₇ . Ή NMR (CD ₃ OD-d ₄ ): δ = 0.90 (t, J = 6.6 Hz, 6H, CH ₃ ), 1.23-1.45 (m, 28H, CH ₂ ), 1.95-2.10 (m, 4H, CH ₂ ), 3.54 (s, 4H, OCH ₂ ), 3.66 (s, 8H, OCH ₂ ), 4.24 (s, 4H, OCH ₂ ), 4.57 (t, J = 8.0 Hz, 4H, N ⁺ CH ₂ ), 7.94 (dd, J, = 8.8 Hz, h = 5.9 Hz, 2H, Py), 8.16 (dd, J, = 8.8 Hz, J ₂ = 1.6 Hz, 2H, Py), 8.55 (d, J = 5.0 Hz, 2H, Py), 8.85 (br s, 2H, Py).

l-Dodecyl-3-[3-[3-[(l-dodecylpyridinium-3-yl)oxy]-2,2- bis(hydroxymethyl)propoxy]-2,2-bis(hydroxymethyl)propoxy]pyr idinium dibromide (Iq)

Yield 77% (0.146 g). M = 906.9. C44H ₇₈ Br ₂ N ₂ 0 ₇ . Ή NMR (CD3OD-CI4): δ = 0.90 (t, J = 6.6 Hz, 6H, CH ₃ ), 1.23-1.45 (m, 36H, CH ₂ ), 1.95-2.10 (m, 4H, CH ₂ ), 3.54 (s, 4H, OCH ₂ ), 3.66 (s, 8H, OCH ₂ ), 4.24 (s, 4H, OCH ₂ ), 4.57 (t, J = 8.0 Hz, 4H, Ν¾Η ₂ ), 7.94 (dd, J, - 8.8 Hz, = 5.9 Hz, 2H, Py), 8.16 (dd, J, = 8.8 Hz, J ₂ = 1.6 Hz, 2H, Py), 8.55 (d, J = 5.0 Hz, 2H, Py), 8.85 (br s, 2H, Py).

Examples 18-21 [Schemes 6, 7]

General synthetic procedure for the synthesis dimeric compounds (Ir-u)

A mixture of diacetal V or Va (1.03 g, 2.38 mmol) and alkyibromide (9.5 mmol) in 15 ml of dry 4-methylpentan-2-one was refluxed for 8 hours. The resulting solution was cooled to room temperature, the solvent was decanted, the residue was washed with hexane and dissolved in acetonitrile/methanol (9/1 volume) mixture. Afterwards charcoal was added and the mixture was boiled for 15 min until complete decolorization. The charcoal was removed by filtration. Evaporation of solvent under reduced pressure afforded crude product as a white solid.

N-Decyl-4,4'-(oxybis{methylene[5-(hydroxymethyl)-l,3-dioxane -5,2- diyl]})dipyridinium dibromide (Ir)

Mixture of isomers. Yield 70% (1.45 g). M = 874.8. C ₄₂ H ₇ oN ₂ 0 ₇ . Ή NMR (CD ₃ OD): δ = 0.87-0.98 (m, 6H, CH ₃ ), 1.24-1.50 (m, 32H, CH ₂ ), 1.99-2.14 (m, 4H, CH ₂ ), 3.28-3.40 (m, 4H, C¾), 3.49 (br. s, 2H, OH), 3.70-3.80 (m, 2H, CH ₂ ), 3.84-3.90 (m, 2H, CH ₂ ), 3.98-4.12 (m, 4H, CH ₂ ), 4.69 (t, J = 7.3 Hz, 4H, N^CH^, 5.77-5.87 (m, 2H, CH from different isomers), 8.14-8.24 (m, 4H, CH, Py), 9.04-9.12 (m, 4H, N-CH, Py).

N-Dodecyl-4,4 '-(oxybis{methylene[5-(hydroxymethyl)-l,3-dioxane-5,2- diyl]})dipyridinium dibromide (Is)

2Br ^"

Mixture of isomers. Yield 75% (1.66 g). M = 930.9. C ₄₆ H ₇₈ Br ₂ N ₂ 0 ₇ . Ή NMR (CD ₃ OD): δ = 0.87-0.98 (m, 6H, CH ₃ ), 1.24-1.50 (m, 36H, CH ₂ ), 2.00-2.14 (m, 4H, CH ₂ ), 3.28-3.38 (m, 4H, CH ₂ ), 3.73 (d, J = 14.6 Hz, 2H, CH ₂ ), 3.87 (d, J = 2.2 Hz, 2H, CH ₂ ), 4.05 (t, J - 11.0 Hz, 4H, CH ₂ ), 4.15- 4.27 (m, 4H, CH ₂ ), 4.69 (t, J = 7.3 Hz, 4H, N ⁺ -CH ₂ ), 5.77-5.87 (m, 2H, CH from different isomers), 8.13-8.24 (m, 4H, CH, Py), 9.07-9.14 (m, 4H, N-CH, Py).

N-Dodecyl-3,3'-(oxybis{methylene[5-(hydroxymethyl)-l,3-dioxa ne-5,2- diyl]})dipyridinium dibromide (It)

2Br ^"

Mixture of isomers. Yield 73% (1.62 g). M = 930.9. C ₄₆ H ₇₈ Br ₂ N ₂ 0 ₇ . Ή NMR (CD ₃ OD): δ = 0.87-0.98 (m, 6H, CH ₃ ), 1.26-1.48 (m, 36H, CH ₂ ), 1.98-2.14 (m, 4H, CH ₂ ), 3.30-3.36 (m, 4H, CH ₂ ), 3.73 (d, J = 14.6 Hz, 2H, CH ₂ ), 3.86-3.92 (m, 2H, CH ₂ ), 4.05 (t, J = 1 1.0 Hz, 4H, CH ₂ ), 4.15- 4.27 (m, 4H, CH ₂ ), 4.71 (t, J = 7.3 Hz, 4H, N ⁺ - CH ₂ ), 5.78-5.88 (m, 2H, CH from different isomers), 8.13-8.22 (m, 2H, CH, Py), 8.64- 8.72 (m, 2H, CH, Py), 9.03-9.10 (m, 2H, N-CH, Py) 9.13-9.18 (m, 2H, N-CH, Py).

N-Hexadecyl-3,3'-(oxybis{methylene[5-(hydroxymethyl)-l,3-dio xane-5,2- diyl]})dipyridinium dibromide (Iu)

Mixture of isomers. Yield 75% (1.86 g). Ή NMR (CD ₃ OD): δ = 0.87-0.98 (m, 6H, CH ₃ ), 1.22-1.50 (m, 52H, CH ₂ ), 1.98-2.14 (m, 4H, CH ₂ ), 3.30-3.36 (m, 4H, CH ₂ ), 3.73 (d, J = 14.7 Hz, 2H, CH ₂ ), 3.86-3.92 (m, 2H, CH ₂ ), 4.05 (t, J = 10.8 Hz, 4H, CH ₂ ), 4.15- 4.27 (m, 4H, CH ₂ ), 4.69 (t, J = 7.3 Hz, 4H, N ⁺ -CH ₂ ), 5.78-5.88 (m, 2H, CH from different isomers), 8.13-8.22 (m, 2H, CH, Py), 8.64-8.72 (m, 2H, CH, Py), 9.03-9.10 (m, 2H, N-CH, Py) 9.13-9.18 (m, 2H, N-CH, Py).

Examples 22-26 [Scheme 8]

General synthetic procedure for the synthesis dimeric compounds (Iv-z)

A mixture of 3,3'-[l,4-phenylenebis(oxy)]dipyridine (0.314 g, 1.19 mmol) and alkylbromide (2.4 mmol) in 3 ml of dry 4-methylpentan-2-one was refluxed for 10 hours. The resulting solution was cooled to room temperature, the solvent was decanted. The residue was washed with hexane, acetone and filtered off to give pure product as a white solid.

3,3 '-[l 4-Phenylenebis(oxy)]bis(l-octylpyridinium) dibromide (Iv)

2Br ^'

Yield 63% (0.48 g). M = 650.5. C ₃ 2H4 ₆ Br ₂ N ₂ 0 ₂ . Ή NMR (CD ₃ OD): δ = 0.88 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.26-1.56 (m, 20H, CH ₂ ), 2.02-2.16 (m, 4H, CH ₂ ), 4.75 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 7.48 (s, 4H, Ar), 8.13 (dd, J, = 8.8 Hz, J ₂ = 6.0 Hz, 2H, CH, Py), 8.34 (dd, J, = 8.8 Hz, J ₂ = 2.2 Hz, 2H, CH, Py), 8.83 (d, J = 6.0 Hz, 2H, N-CH, Py), 9.13 (d, J = 2.2 Hz, 2H, N-CH, Py).

3, '-[l,4-Phenylenebis(oxy)]bis(l-decylpyridinium) dibromide (Iw)

2Br ^"

Yield 65% (0.55 g). M - 706.6. C ₃₆ H ₅₄ Br ₂ N ₂ 0 ₂ . Ή NMR (CD ₃ OD): δ = 0.88 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.24-1.54 (m, 28H, CH ₂ ), 2.00-2.14 (m, 4H, CH ₂ ), 4.74 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 7.47 (s, 4H, Ar), 8.07-8.17 (m, 2H, CH, Py), 8.29-8.35 (m, 2H, CH, Py), 8.79-8.86 (m, 2H, N-CH, Py), 9.10-9.16 (m, 2H, N-CH, Py).

3,3 '-[l,4-Phenylenebis(oxy)]bis(l-dodecylpyridinium) dibromide βχ) Yield 69% (0.63 g). M = 762.7. C ₄ oH6 ₂ Br ₂ N ₂ 0 ₂ . Ή NMR (CD ₃ OD): δ = 0.87 (t, J = 7.0 Hz, 6H, CH ₃ ), 1.24-1.56 (m, 36H, CH ₂ ), 1.98-2.10 (m, 4H, CH ₂ ), 4.73 (t, J = 7.4 Hz, 4H, N ⁺ -CH;.), 7.46 (s, 4H, Ar), 8.11 (dd, Ji = 8.6 Hz, J ₂ = 6.0 Hz, 2H, CH, Py), 8.32 (dd, J, = 8.6 Hz, J ₂ = 1.7 Hz, 2H, CH, Py), 8.80 (d, J = 6.0 Hz, 2H, N-CH, Py), 9.10 (d, J = 1.7 Hz, 2H, N-CH, Py).

,-[l,4-Phenylenebis(oxy)]bis(l-tetradecylpyridinium) dibromide (Iy)

2Br ^"

Yield 69% (0.67 g). M = 818.8. C ₄ 4H ₇ oBr ₂ N ₂ 0 ₂ . Ή NMR (CD ₃ OD): δ = 0.87 (t, 6H, J = 7.0 Hz, CH ₃ ), 1.24-1.56 (m, 44H, CH ₂ ), 1.96-2.10 (m, 4H, CH ₂ ), 4.73 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 7.46 (s, 4H, Ar), 8.12 (dd, J, = 8.6 Hz, J ₂ = 6.0 Hz, 2H, CH, Py), 8.34 (dd, J, = 8.6 Hz, = 2.2 Hz, 2H, CH, Py), 8.83 (d, J = 6.0 Hz, 2H, N-CH, Py), 9.1 1 (d, J = 2.2 Hz, 2H, N-CH, Py).

3 '-[l,4-Phenylenebis(oxy)]bis(l-hexadecylpyridinium) dibromide (Iz)

2Br

Yield 65% (0.68 g). M = 875.0. C ₄₈ H ₇₈ Br ₂ N ₂ 0 ₂ . Ή NMR (CD ₃ OD): δ = 0.87 (t, 6

H, J = 7.0 Hz, CH ₃ ), 1.22-1.48 (m, 52H, CH ₂ ), 1.96-2.12 (m, 4H, CH ₂ ), 4.74 (t, J = 7.4 Hz, 4H, N ⁺ -CH ₂ ), 7.45 (s, 4H, Ar), 8.12 (dd, Ji = 8.8 Hz, J ₂ = 6.0 Hz, 2H, CH, Py), 8.34 (dd, J, = 8.8 Hz, = 2.3 Hz, 2H, CH, Py), 8.83 (d, J = 6.0 Hz, 2H, N-CH, Py), 9.12 (d, J = 2.2 Hz, 2H, N-CH, Py).

Starting materials preparation

The starting materials applied in examples above are the compounds obtained using standard methods from known materials. Some of the starting materials fall under the coverage of the present invention.

For example, the methods A-N below are shown only for illustration and do not limit the synthesis ways for those starting materials.

Methods for scheme 1

Method A. (2-Phenyl-l,3-dioxane-5,5-diyl)dimethanol (1)

Acetal 1 is obtained using known technique [Org. Synth. 4, 679, 1963]. M.p. 134- 136°C.

Method B. (2-Phenyl-l,3-dioxane-5,5-diyl)di(methylene) bis(4- methylbenzenesulfonate) 2)

Ditosylate 2 is obtained using known technique [P. Pecquet, F. Huet, M. Legraverend, E. Bisagni Heterocycles 34 p. 739-745 (1992)]. M.p. 173-175°C.

Method C. 3,3 '-[(2-Phenyl-l,3-dioxane-5,S-diyl)bis(methyleneoxy)]dipyridi ne (3)

In a 3 L three-necked round bottom flask equipped with a powerful mechanical stirrer and thermometer, (47.5 g, 0.5 mol) of 3-hydroxypyridine, 278 g (2.00 mol) potassium carbonate and 133.1 g (0.25 mol) of ditosylate 2 are placed in 2 L of dry DMF and then heated at 120°C during 84 h. After cooling to room temperature, the reaction mixture was poured into 1 L of water and extracted with 3x400 ml of ether. The ether layer was washed with water and dried over anhydrous sodium sulfate. The solvent was removed in a rotary evaporator giving 83 g (88%) of 3 as a yellowish viscous liquid. M = 532.6. C ₂₆ H ₂₈ 0 ₈ S ₂ . Ή NMR (CDC1 ₃ ): δ = 4.02 (s, 2H, PyO-CH ₂ ),

4.12 (d, J = 1 1.9 Hz, 2H, 0-CH ₂ ), 4.42 (d, J = 11.9 Hz, 2H, 0-CH ₂ ), 4.52 (s, 2H, PyO- CH ₂ ), 5.57 (s, 1H, CH), 7.15-7.55 (m, 9H, Ar), 8.22-8.40 (m, 4H, Ar). MS (EI, 70 eV): m/z (%) = 378 [M ⁺ ]. Method D. 2,2-Bis((pyridi -3-yloxy)methyl]propane-l,3-diol (II)

In a 1 L three-necked round-bottomed flask equipped with a powerful mechanical stirrer a solution 100.0 ml of concentrated hydrochloric acid in 470 ml of water and 76 g (0.2 mol) of compound 3 were placed. The resulting mixture was stirred at rt for 48 h. The reaction mixture was extracted with 2x50 ml of ether. Solution of 36 g sodium hydroxide in 600 ml water was added to aqueous layer until pH of mixture became 10. The formed precipitate was filtered off, washed with 100 ml of water, 100 ml of acetone and recrystallized from acetonitrile to give 46 g (78%) of compound 7. M.p. 138-140°C. M = 290.3. C, ₅ Hi _g N ₂ 0 ₄ . Ή NMR (CDC1 ₃ ): δ = 3.02 (br s, 2H, OH), 3.98 (s, 4H, 0-CH ₂ ), 4.16 (s, 4H, 0-CH ₂ ), 7.15-7.21 (m, 2H, Py), 7.23-7.281 (m, 2H, Py), 8.16-8.21 (m, 2H, Py), 8.26-8.31 (m, 2H, Py). MS (EI, 70 eV): m/z (%) = 290. [M ⁺ ].

Methods for scheme 2

Method E. {4,4'-(2, -tetraoxaspiro[5.5]-undecan-3,9-diyl)dipyridine (III)

A mixture of 4-pyridinecarboxaldehyde (2.14 g, 0.02 mol), pentaerythritol (1.36 g, 0.01 mol) and p-toluenesulfonic acid (6.92 g, 0.04 mol) in 50 ml of dry toluene was refluxed for 6 hours. The precipitate formed during the process was filtered out and washed with toluene and with aqueous acetone. The residue was recrystallized from ethanol to give pure product as a white solid. Yield 70% (2.2 g). M.p. 162-165°C. M = 314.3. Ci ₇ Hi ₈ N ₂ 0 ₄ . Ή NMR (DMSO-d ₆ ): δ = 3.78-4.60 (m, 8H, CH ₂ ), 5.88 (s, 2H, CH), 7.42 (d, J = 6.6 Hz, 4H, CH, Py), 8.6 (d, J = '6.6 Hz, 4H, N-CH, Py).

Methods for scheme 3

Method F. {3,3 '-(2,4,8,10-tetraoxaspiro[5.5]-undecan-3,9-diyl)}dipyridine (Ilia)

A mixture of 3-pyridinecarboxaldehyde (10.71 g, 0.1 mol), pentaerythritol (6.81 g, 0.05 mol,) and p-toluenesulfonic acid (20.66 g, 0.12 mol) was refluxed for 6 hours in 400 ml of dry toluene. After reflux the toluene was decanted, a solid phase was dissolved in 200 ml of chloroform and mixed with 200 ml of 20% NaOH. The mixture was stirred vigorously for 2h at rt. Organic layer was separated, washed with water, dried over magnesium sulfate, and the chloroform was evaporated. The residue was recrystallized from ethanol to give diacetal as a white solid. Yield 84% (13.2 g). M.p. 230-232°C. M = 314.3. Ci ₇ Hi ₈ N ₂ 0 ₄ . Ή NMR (CDC1 ₃ ): δ = 3.62-3.90 (m, 6H, CH ₂ ), 4.78-4.90 (m, 2H, CH ₂ ), 5.51 (s, 2H, CH), 7.32 (dd, J, = 7.7 Hz, J ₂ = 4.8 Hz, 2H, CH, Py), 7.79-7.85 (m, 2H, CH, Py), 8.62 (dd, J, = 5.1 Hz, J ₂ = 1.5 Hz, 2H, N-CH, Py), 8.72 (d, J = 1.5 Hz, 2H, N-CH, Py).

Methods for scheme 4

Method G. (2-pyridin-4-yl-l,3-dioxane-5,5-diyl)dimethanol (4)

A mixture of 4-pyridinecarboxaldehyde (5.35 g, 0.05 mol), pentaerythritol (6.81 g, 0.05 mol) and p-toluenesulfonic acid (9.88 g, 0.052 mol) in 150 ml of dry toluene was refluxed for 6 hours. Toluene was decanted; solid phase was mixed with 20 ml of 20% NaOH and stirred vigorously for 2h at rt. The precipitate was filtered out, dried and recrystallised twice from water to give pure 4. Additional amount of product was isolated as follows. Water phase (containing NaOH) was evaporated to dryness and the residue was extracted in 100 ml of chloroform. Solvent was evaporated and the mixture was purified by column chromatography (Si02, eluent chloroform:methanol = 8: 1). Overall yield is 74% (8.33 g). M.p. 148-150°C. M = 225.2. C1 1H15NO4. Ή NMR

(CD ₃ OD): δ = 3.56 (s, 2H, OH), 3.78-3.88 (m, 4H, CH ₂ ), 3.99-4.07 (m, 2H, CH ₂ ), 4.75- 4.90 (m, 2H, CH ₂ ), 5.43 (s, 1H, CH), 7.45 (d, J = 5.9 Hz, 2H, CH, Py), 8.48 (d, J = 5.9 Hz, 2H, CH, Py). MS (EI, 70 eV): m/z (%) = 225 [M ⁺ ].

Method H. 3-(9-pyridin-4-yl-l,5,8,10-tetraoxaspiro[5.5]undec-3-yl)pyri dine

(Mb)

A mixture of 3-pyridinecarboxaldehyde (1.61 g, 15 mmol), 2-pyridin-4-yl-l ,3- dioxane-5,5-diyl)dimethanol (3.38 g, 15 mmol) and p-toluenesulfonic acid (5.83 g, 30.7 mmol) in 130 ml of dry toluene was refluxed for 6 hours. Toluene was decanted; solid phase was diluted with 30 ml of 20% NaOH and stirred vigorously for 30 min at rt. The precipitate was filtered out, dried and recrystallised from ethanol to give pure Illb. Yield 83% (3.91 g). M.p. 172 °C. M = 314.3. C ₁₇ Hi ₈ N ₂ 0 ₄ . Ή NMR (CDC1 ₃ ): δ = 3.67 (d, J = 1 1.7 Hz, 2H, CH ₂ ), 3.78-3.90 (m, 4H, CH ₂ ), 4.75-4.87 (m, 2H, CH ₂ ), 5.44 (s, 1H, CH), 5.51 (s, 1H, CH), 7.29 (dd, J, = 7.7 Hz, J ₂ = 3.3 Hz, 1H, CH, Py), 7.40 (d, J = 5.9 Hz, 2H, CH, Py), 7.77-7.87 (m, 1H, CH, Py), 8.58-8.75 (m, 4H, CH, Py). MS (EI, 70 eV): m/z (%) = 314 [M ⁺ ].

Methods for scheme 5

Method I. {Oxybis[methylene(2-phenyl-l,3-dioxane-5,5-diyl)]}dimeth nol (mixture of isomers)

Dipentaerythritol (lOg, 0.039 mol) was suspended in 100ml of toluene. Benzaldehyde (9.12 g, 0.086 mol) and several crystals of p-toluenesulfonic acid were added and the mixture was refluxed with Dean-Stark nozzle for 5 h until separation of water through the nozzle is finished. The solution was filtered from insoluble impurity, the filtrate was evaporated and the residual product was crystallized using small amount of ether. Ether was diluted twice with hexane, the crystalline product was filtered off and washed with hexane. Yield is 95% (15.9 g). M = 430.5. C ₂₄ H ₃₀ O ₇ . Ή

NMR (CDCI3): δ = 2.66 (br. s, 2H, OH), 3.20-3.48 (m, 4H, OCH ₂ ), 3.64-4.20 (m, 12H,

OCH ₂ ), 5.40, 5.41 (2 s, 2H, CH-protons from cis- and trans- isomers), 7.30-7.54 (m, 10H, Ph).

Method J. Oxybis[methylene(2-phenyl-l,3-dioxane-5,S-diyl)methylene] bis (4- methylbenzene sulfonate) (mixture of isomers) (6)

Compound 5 (16.5 g, 0.0384 mol) was dissolved in 130 ml of acetonitrile and 16 ml (0.1 16 mol) of triethylamine was added consequently. The solution was cooled to 20°C and 15.4 g (0.0768 mol) of p-toluenesulfonyl chloride was added by small portions. The mixture was stirred for 4 days at ambient temperature, then the solution was poured into mild alkaline water (600 ml) giving separate oil fraction. Water fraction was decanted and the residual oil fraction was washed again with alkaline water. After second wash the oil has crystallized and the crystalline product was filtered off. The precipitate was washed with ether, filtered and dried yielding 1 1 g of the pure product. The ether filtrate was concentrated and crystallized from alcohol (methanol or ethanol) giving rise to additional 10 g of the product. Total yield is 75% (21.2 g). M = 738.9. C-sgH^O, ^. Ή NMR (CDC1 ₃ ): δ = 2.41, 2.43, 2.44 (6 s, CH ₃ - from different isomers), 3.25 (d, J = 10.3 Hz, 2H, OCH ₂ ), 3.64-4.08 (m, 12H, OCH ₂ ), ), 4.34 (d, J = 10.3 Hz, 2H, OCH ₂ ), 5.30, 5.35, 5.41 (3 s, 2H, CH-protons from different isomers), 7.22-7.52 (m, 14H, Ar), 7.77-7.90 (m, 4H, Ar).

Method K. 3,3 '-{Oxybis[methylene(2-phenyl-l,3-dioxane-5,5- diyl)methyleneoxy]}dipyridine (mixture of isomers) (7)

Compound 6 (10.8 g, 14.6 mmol) was dissolved in 100 ml of abs. DMF, 12. lg (87.6 mmol) of potassium carbonate and 2.92g (0.031 mol) of pyridin-3-ol were consequently added and the mixture was heated at 100°C for 48 h. The reaction mixture was cooled to room temperature and poured into 500 ml of ice water with vigorous stirring. Cream-yellow precipitate was filtered, washed with water and dried. Yield is 74% (6.31 g). M = 584.7. C ₃₄ H ₃₆ N ₂ 0 ₇ . Ή NMR (CDC1 ₃ ): 6 = 3.28 (d, J = 10.4 Hz, 2H, OCH ₂ ), 3.62-4.40 (m, 14H, OCH ₂ ), 5.45, 5.49, 5.55 (3 s, 2H, CH-protons from different isomers), 7.16-7.54 (m, 16H, Ar), 8.18-8.40 (m, 2H, Py).

Method L. 2,2 '-[Oxydi(methylene)]bis{2-[(pyridin-3-yloxy)methyl]propane-l ,3- diol} (IV)

Compound 7 (6.2 g, 10.6 mmol) was suspended in 60ml of aqueous HC1 (20 ml of 36% HC1 + 40 ml of water) and stirred at room temperature for 24 hours. The solution was decanted and the remaining resin was extracted with water. The combined aqueous solutions were washed with ether to separate of benzaldehyde, and the final volume of water fraction was reduced to 120 ml by evaporation. The acid/water components were distilled off under vacuo, and the remaining oily product was diluted with a small amount of water (30-50 ml) and basified with sodium bicarbonate to pH 8- 9. The mixture was evaporated and the product was extracted with hot ethanol. The ethanolic solution was refluxed with charcoal for 20 min, filtered and evaporated under vacuo. The resulting oil was refluxed with acetonitrile (50 ml) for 30 min, the formed precipitate was filtered and crystallized from water. The resulting crystalline product was filtered and washed with acetone. Yield is 80% (3.46). M = 408.4. C ₂₀ H ₂₈ N ₂ O ₇ . Ή NMR (MeOD-cU): δ = 3.55 (s, 4H, OCH ₂ ), 3.72 (s, 8H, OCH ₂ ), 4.02 (s, 4H, PyOCH ₂ ), 7.26-7.42 (m, 4H, Py), 8.10 (dd, Ji = 4.6 Hz, J ₂ - 1.3 Hz, 2H, Py), 8.21 (d, J = 2.2 Hz, 2H, Py).

Methods for Schemes 6-7

Method M. General procedure for the synthesis of diacetals (V) and (Va)

A mixture of 4-pyridinecarboxaldehyde or 3-pyridinecarboxaldehyde (2.14 g, 20 mmol), dipentaerythritol (2.54 g, 10 mmol) and p-toluenesulfonic acid (3.7 g, 21.5 mmol) was refluxed in 100 ml of dry toluene for 8 hours. Toluene was decanted; solid phase was diluted with 1 1 ml of 20% NaOH in water and was stirred for lh. Mixture was extracted with chloroform (3 < 100 ml). Organic layer was washed with water, dried over Na ₂ S0 ₄ and the chloroform was evaporated. The residue was dissolved in acetone (50 ml). Afterwards charcoal was added and the mixture was boiled for 15 min until complete decolorization. The charcoal was removed by filtration, and evaporation of acetone under reduced pressure afforded crude product.

{Oxybis[methylene(2-pyridin-4-yl-l,3-dioxane-5,5-diyl)]}dime thanol (V)

Yield is 57% (2.46). M = 432.5. C ₂₂ H ₂ 8N ₄ . M.p. = 200-205 °C. ¾ NMR (CDC1 ₃ ): δ = 2.28 (br. s, 2H, OH), 3.38 (s, 2H, CH ₂ ), 3.51 (s, 2H, CH ₂ ), 3.77 (s, 2H, CH ₂ ), 3.79- 3.85 (m, 4H, CH ₂ ), 4.00 (s, 2H, CH ₂ ), 4.12-4.21 (m, 4H, CH ₂ ), 5.41 (br. s, 2H, CH), 7.34-7.46 (m, 4H, CH, Py), 8.58-8.68 (m, 4H, N-CH, Py). MS (EI, 70 eV): m/z (%) = 432 [M ⁺ ].

{Oxybis[methylene(2-pyridin-3-yl-l,3-dioxane-5,5-diyl)]}dime thanol (Va)

Yield is 64% (2.76). M = 432.5. C ₂₂ H ₂₈ N ₂ 0 ₇ . M.p. - 174-178 °C. Ή NMR (CD ₃ OD): δ = 3.20 (br. s, 2H, OH), 3.25-3.39 (m, 2H, CH ₂ ), 3.49 (s, 2H, CH ₂ ), 3.79 (d, J = 1 1.1 Hz, 4H, CH ₂ ), 3.83 (s, 2H, CH ₂ ), 3.91 -4.03 (m, 2H, CH ₂ ), 4.17 (dd, J, = 1 1.1 Hz, J ₂ = 3.3 Hz, 4H), 5.47 (s, 2H. CH), 7.25-7.35 (m, 2H, CH, Py), 7.73-7.85 (m, 2H, CH, Py), 8.53-8.63 (m, 2H, CH, Py), 8.67-8.75 (m, 2H, CH, Py). MS (EI, 70 eV): m/z (%) = 432 [M ⁺ ],

Methods for Scheme 8

Method N. 3,3 '-[l,4-Phenylenebis(oxy)]dipyridine (VI)

A mixture of 3-hydroxypyridine (0.316 g, 3.33 mmol), 1 ,4-dibromobenzene (0.39 g, 1.65 mmol), potassium carbonate (1.75 g, 12.7 mmol) and copper (0.8 g, 12.6 mmol) was refluxed in 20 ml of dry DMF under argon atmosphere for 48 h. The solvent was decanted, the solid phase was washed with ether. Ether layer was mixed with DMF solution. Mixture was washed with 10 ml of 10% NaOH, water (2x20 ml), dried with Na ₂ S0 ₄ and the solvent was evaporated under reduced pressure. Purification of the residue by Si0 ₂ chromatography gave final product in a pure form. Yield 70% (0.31 g). M = 264.3. C26Hi ₂ N ₂ 0 ₂ . M.p. 78-80 °C. Ή NMR (CDC1 ₃ ): δ = 7.06 (s, 4H, Ar), 7.23- 7.34 (m, 4H, Py), 8.34-8.45 (m, 4H, Py). MS (EI, 70 eV): m/z (%) = 264 [M*].

Examples 27-32

The novel compounds of formula I possess useful antibacterial and antifungal activity being effective against both gram positive and gram negative bacteria, and several species of fungi. Thus, the present compounds are truly broad spectrum biocide agents. Moreover, these compounds possess low toxicity.

The compounds Ic (obtained according to Example 3), Ih (obtained according to Example 8), Ik (obtained according to Example 11), In (obtained according to Example 14), Ip (obtained according to Example 16), Is (obtained according to Example 19), It (obtained according to Example 20) and Ix (obtained according to Example 24) tested below are the typical representatives of compounds according to formula I. The variation of R in formula I is negligible and influences only the solubility of certain compound in the solution but negligibly influences its biocidal property provided by the molecule backbone (two pyridine rings connected with spacer Z).

The test bacteria strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) used in examples below are the most resistant forms of bacteria. The test microorganisms sample (Enterococcus hirae) used in examples below is one of the most resistant forms of microorganisms. The resistance of said bacteria and microorganisms to disinfectant or antiseptic treatment is the highest among the large range of various classes of simple life forms. The activity of chemical agent against said bacteria and microorganisms is a figure of merit for biocidal effect of the chemical agent in test. For example, if the Escherichia coli bacteria are effectively killed after biocidal treatment, the other representatives of E. coli class will be killed in same or even stronger manner by the same biocidal agent.

The Benzalkonium chloride (BAC) compound is often used as a reference for quantitative comparison of various antiseptics and disinfectants [US Patent 7612097 (2009)]. Thus, the tests below are given in comparison to BAC to demonstrate the high biocidal efficiency of the novel compounds under test as well as their low toxicity.

Comparative test toxicity results for representative dimeric quaternary pyridinium salts prepared according to formula I and commercially available benzalkonium chloride (BAC), bis-quatemary ammonium salts: l,4-bis(3,3'-(l- decylpyridinium)methyloxy)butane-dibromide (C) known as "Hygenia" (by Tama Kagaku Kogyo Co, Ltd.) and also the "Octenidine dihydrochloride" (by Dishman Group).

(4) Biological testing

Biocidal efficacies of samples of novel compounds of formula I obtained according to the European standards procedures were determined by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and cell toxicity towards human keratinocytes [European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Clin. Microbiol. Infect. 9, 1-7 (2003); Gerald Muller and Axel Kramer. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. Journal of Antimicrobial Chemotherapy 61 , 1281-1287. (2008)].

The tested compounds I (Ic, Ih, Ik, In, Ip, Is, It and Ix) are typical of the claimed compounds of formula I and the basic structural elements define the biocidal activity and low toxicity are the two pyridine rings connected with a spacer Z, but not the alkyl groups R, which is confirmed in the case of the closest analogue bis-quaternary ammonium salts (structure C) [US Patent 7612097 (2009)] and in several other bis- quaternary ammonium compounds [US Patent 4206215 (1980), WO2007/128059 (2007)].

Materials and Methods

The testing for the biocidal activity of new compounds of formula I has been conducted according to general broth dilution method.

Compounds under test: Ic (obtained according to Example 3), Ih (obtained according to Example 8), Ik (obtained according to Example 1 1), In (obtained according to Example 14), Ip (obtained according to Example 16), Is (obtained according to Example 19), It (obtained according to Example 20) and Ix (obtained according to Example 24).

Reference compounds: benzalkonium chloride (BAC), bis-quaternary ammonium salts: l ,4-bis(3,3'-(l-decylpyridinium)methyloxy)butane-dibromide (C) and "octenidine dihydrochloride". Standard Test Microorganisms:

Bacteria:

- Escherichia coli ATCC 25922

- Staphylococcus aureus ATCC 6538

- Pseudomonas aeruginosa ATCC 27853

MIC test:

- Test samples were prepared at concentration of 20000 ppm with ethanol.

- 150 ΐ, aliquots were transferred using an Eppendorf tube, and a two-step dilution was performed with bouillon medium (Final concentration: 256 ppm - 0.25 ppm).

- 150 iL aliquots were added to a 96 well plate.

- 150 iL aliquots of the bacteria adjusted to approximately 10 ⁶ CFU/mL were added.

- After incubating for 24 hours at 37°C, the MIC was visually determined based on turbidity.

MBC test:

- Test samples were prepared at concentration of 20000 ppm with ethanol.

- Serial dilutions of samples were performed with sterilized water such that concentrations of the active ingredients become 100, 50, 25, 10, 5, and 2 ppm.

- While maintaining the temperature at 20°C, 4.5 mL of each sample was adjusted to 10 ⁷ CFU/mL with physiological saline, followed by the addition of 0.5 mL of the test bacterial solution.

- After a contact time of 10 minutes, a loopful of this was inoculated to 4.5 mL of neutralizedmedium (Tween80 10% + Lecithin 3%) containing bouillon, which was then incubated for 24 hours at 37°C.

- After incubation, the bouillon agar medium was streaked to detect bacterial viability.

Example 27 - testing bacteriostatic activities of the above-described novel compounds (Ic, Ih, Ik, In, Ip, Is, It and Ix) against various bacteria.

Using benzalkonium chloride (BAC) and l,4-bis(3,3'-(l- decylpyridinium)methyloxy)-butane-dibromide (Bis-QAC-1) as a reference compounds, the minimum inhibitory concentrations (MICs) were determined. The results are presented in Table 1.

Table 1. Bacteriostatic Spectra

Conclusions: Some of the compounds (Ic, Ih, Ik, In, Ip, Is, It and Ix) in comparison with reference compounds - BAC and Bis-QAC- 1 are demonstrating a comparable or higher bacteriostatic activity against all of the test bacteria strains.

Example 28 - testing bactericidal activities of the above-described novel compounds (Ic, Ih and Ix) against various bacteria.

Using benzalkonium chloride (BAC) and l,4-bis(3,3'-(l- decylpyridinium)methyloxy)-butane dibromide (Bis-QAC- 1) as a reference compounds, minimum bactericidal concentrations (MBCs) were determined. The results are presented in Table 2.

Table 2. Bactericidal Spectra

MBC (ppm)

Compound Test microorganism: bacteria

E. coli S. aureus P. aeruginosa

Ic 17,5 37,5 7,5

Ih 5,0 7,5 8,3

Ix 7,5 1 1 ,0 1 1 ,0

Bis-QAC -1 12,0 10,0 25,0 BAC 25,0 10,0 25,0

Conclusions: The compounds (Ic, Ih, and Ix) in comparison with reference compounds - BAC and closest analog Bis-QAC-1 demonstrate a comparable or higher bactericidal activity against all of the test bacteria.

Additionally the compounds (Ic, Ih and Ix) were tested for antibacterial activity against bacteria - Enterococcus hirae and Enterococcus faecalis, antifungal activity against fungi - Candida albicans, antiviral activity against virus - Herpes.

Example 29 - testing bacteriostatic activities of the above-described novel compounds (Ic, Ih and Ix) against Enterococcus hirae bacteria.

Materials and Methods

Compounds under test: Ic (obtained according to Example 3), Ih (obtained according to Example 8) and Ix (obtained according to Example 24).

Reference compound: benzalkonium chloride (BAC).

Standard Test Microorganisms:

- Enterococcus hirae ATCC 10541

MIC Test (performed on E. hirae) :

- Test samples were prepared at a concentration of 100000 ppm with ethanol.

- They were diluted with nutrient broth to 500, 250, 100, 50, 25, 10, 5, 2, 1 ppm by using an Eppendorf tube.

- 150 μί aliquots were added to a 96-well plate.

- 150 μΙ_, aliquots of each bacterial solution (adjusted to approximately 10 ⁶ CFU/mL with nutrient broth) were added to the wells.

- After a 24-hour incubation at 37°C, the MIC was determined based on the degree of turbidity.

Using benzalkonium chloride as a reference compound the minimum inhibitory concentrations (MICs) were determined. The results are presented in Table 3.

Table 3. MIC test results (ppm)

MIC (ppm)

Test microorganism: bacteria Compound

BAC

Ic Ih Ix

E. hirae 5 5 5 2

Bacterial count (CFU/mL) 5.6x l0 ⁶ Conclusions: Most of the samples (Ic, Ih and Ix) tested demonstrated high bacteriostatic activity against E. hirae.

Example 30 - testing bactericidal activities of the above-described novel compounds (Ic, Ih and Ix) against Enterococcus faecalis bacteria.

Materials and Methods

Compounds under test: Ic (obtained according to Example 3), Ih (obtained according to Example 8) and Ix (obtained according to Example 24).

Reference compound: benzalkonium chloride (BAC).

Standard Test Microorganisms:

- Enterococcus faecalis ATCC 29212

MBC Test (performed on E. faecalis):

- Test samples were prepared at a concentration of 100000 ppm with ethanol.

- They were diluted with sterile distilled water to 1000, 500, 250, 100, 50, 25, 10, 5, 2 ppm.

- While maintaining the temperature at 20°C, 0.5 mL of bacterial solution adjusted to 10 ⁷ CFU/mL with sterile distilled water was added to 4.5 mL of each sample.

- After a 10-minute contact time, a loopful was added to 4.5 mL of neutralising medium (10% Tween80 and 3% Lecithin).

- After a 24-hour incubation at 37°C, each sample was streaked on an agar plate, cultured for 24 hours at 37°C and checked for bacterial growth.

Using benzalkonium chloride as a reference compounds, minimum bactericidal concentrations (MBCs) were determined. The results are presented in Table 4.

Table 4. MBC test results (ppm)

Conclusions: All of the samples (Ic, Ih and Ix) were found to be bactericidal effective against E. faecalis when used at a concentration of 25-50 ppm. The MBC test showed that compounds Ih and Ix of the samples tested were more effective than compound Ic and control sample BAC.

Example 31 - testing antifungal activity of the above-described invention compounds (Ic, Ih and Ix) against Candida albicans fungi.

Compounds: Ic (obtained according to Example 3), Ih (obtained according to Example 8), Ix (obtained according to Example 24).

Reference compound: benzalkonium chloride (BAC)

Test fungi:

- Candida albicans ATCC 10231

Test Method:

- Test samples were prepared at a concentration of 100,000 ppm with ethanol.

- They were diluted with sterile distilled water to 1000, 500, 250, 100, 50, 25, 10, 5, 2 ppm.

- While maintaining the temperature at 20°C, 0.5 mL of bacterial solution adjusted to 10 ⁷ CFU/mL with sterile distilled water was added to 4.5 mL of each sample.

- After a 10-minute contact time, 0.5 mL was transferred to 4.5 mL of neutraliser (an aqueous solution of 10% Tween80 and 3% Lecithin) to stop the reaction.

- 0.2 mL of each solution was plated on potato dextrose agar medium, cultured at 30°C for 2 days (C. albicans) and checked for bacterial growth.

Using benzalkonium chloride as a reference compounds the minimum bactericidal concentrations (MBCs) were determined. The results are presented in Table 5.

Table 5. MBC test results (ppm)

Conclusions: All of the samples (Ic, Ih and Ix), except BAC, have demonstrated good fungicidal activity against C. albicans when used at a concentration of 25 ppm.

Example 32 - testing antiviral activity of the above-described novel compounds (Ic, Ih and Ix) against Herpes virus. Compounds: Ic (obtained according to Example 3), Ih (obtained according to Example 8), Ix (obtained according to Example 24).

Reference compound: benzalkonium chloride (BAC)

Preparation of Test Solutions:

Each of the test samples (compounds) was dissolved in 95 wt% ethanol at a concentration of 10 w/v%. They were then diluted 100-fold with distilled water and further diluted 10-fold to obtain 0.01 w/v% solutions.

Virus, Host Cells and Media:

Test virus: Herpes simplex Typel ATCC VR-733 (Herpes)

Host cell: African green monkey kidney cell JCRB9013 (Vero cell)

Cell culture medium: Eagle's minimum essential medium (MEM)

* The MEM used in this experiment was supplemented with non-essential amino acids.

Test Method:

0.1 mL of virus suspension was added to 0.9 mL of each test solution. After 30 seconds and 1 minute of contact time at room temperature, they were diluted 10-fold with MEM containing 10% fetal bovine serum (FBS) to stop the reaction. They were then serially diluted with MEM containing 2% FBS, and 0.1 mL aliquots of each dilution were pipetted into 4 wells of a 96-well plate containing Vero cells. After the 1- hour incubation at 35°C in a 5% C0 ₂ incubator, the medium of each well was replaced with 0.1 mL of fresh MEM containing 2% FBS, and incubated for 5 days in the incubator under the same conditions. The cells were then checked for infection with the viruses under a microscope, and viral infectivity (TCID50) was calculated by the Behrens-Karber method to determine the log 10 reduction of Herpes.

As a control compound, benzalkonium chloride was used. The results are presented in Table 6.

Table 6. Virucidal activity against Herpes (n=2)

Conclusion: All of the samples (Ic, Ih and Ix) were effective against Herpes virus at concentration of 0.01% within 30 seconds of contact time.

Example 33 - testing cytotoxicity of the above-described novel compounds (Ic, Ih, Ik, In, Ip, Is, It and Ix).

Test samples:

Compounds: Ic, Ih, Ik, In, Ip, Is, It and Ix

Compound for comparison: benzalkonium chloride (BAC), bis-quaternary ammonium salts: l,4-bis(3,3'-(l-decylpyridinium)methyloxy)butane-dibromide (Bis- QAC-1) and "Octenidine dihydrochloride" (Bis-QAC-2).

Cells: NHEK (F) (Kurabo): Normal Human Epidermal Keratinocytes.

Growth medium: EpiLife with Ca (Invitrogen MEPI500CA).

Medium additive: HKGS (Kurabo S-001-5).

MTT assay:

- A 50 μΐ of 10% collagen matrix (CellMatrix) was introduced to each well of a 96-well microplate (Corning Inc.) using a multichannel pipette with sterile tips and kept for 30 minutes. After the excess solution was removed, the plate was dried for 30 minutes.

- NHEK cells with a density of 10 ^s cells/ml were seeded onto the 96-well microplate at 100 μΐ /well and cultured in a C0 ₂ incubator at 37°C for 3 days.

- At 3 days of seeding, the medium was replaced with serial dilutions of samples (1 , 5, 10 ppm) in medium (100 μΐ/well) and kept in a C0 ₂ incubator at 37°C for 48 hours. Triplicate wells per concentration were tested.

- The sample-containing medium of each cell was then removed. MTT (Thiazolyl Blue Tetrazolium, 100 μΐ, of 0.5 mg/ml solution) was added to each cell for 2 hours at 37°C.

- After 2 hours, the media was completely removed from each cell and replaced with an equal amount of isopropyl alcohol (100 μΐ,).

- The optical density of each cell was then measured using an automated plate reader (BIOLISE) at 570 nm wavelength.

- The cell viability is expressed as a percentage of the control (treated with purified water).

- Using benzalkonium chloride (BAC), bis-quaternary ammonium salts: 1,4- bis(3,3'-(l-decylpyridinium)methyloxy)butane-dibromide (Bis-QAC-1) and octenidine dihydrochloride (Bis-QAC-2) as a reference compounds, the cytotoxicity data were determined. The results are presented in Table 7.

Table 7. Cytotoxicity Data

*Bis-QAC-l : l ,4-bis(3,3'-(l-decylpyridinium)methyloxy)butane-dibromide (by Tama Kagaku Kogyo Co, Ltd.)

**Bis-QAC-2: "Octenidine dihydrochloride" (Octenidine - HC1, by Dishman Group)

Conclusions:

In accordance with the results of table 7 the compounds: Ic, Ih, Ik, In, Ip, Is, It and Ix have much lower toxicity as compared to the control compounds - benzalkonium chloride (BAC) and the closest structural analog Bis-QAC-1, and other bis-quaternary ammonium compound - Bis-QAC-2.

Based on the above results, one can conclude that a number of compounds of the formula I demonstrate high antibacterial, antifungal, antiviral activity together with low cytotoxicity performance in comparison with the control compound - benzalkonium chloride (BAC) and the closest structural analog Bis-QAC-1 - and can be used as an effective bactericidal active ingredients in disinfectants or antiseptic compositions, which have low toxicity.

Although the present invention has been described in detail with reference to a particular preferred embodiment, persons possessing ordinary skill in the art to which this invention pertains will appreciate that various modifications and enhancements may be made without departing from the spirit and scope of the claims that follow.