CA1260941A - Bis¬4-(substituted-amino)-1-pyridinium|alkane saccharin salts and process of preparation thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Bis[4-(substituted-amino)-1-pyridinium]alkane saccharin salts are prepared from corresponding relatively soluble salts and saccharin or a soluble salt of saccharin and are useful as antimicrobial agents, particularly as agents for inhibiting, reducing and preventing dental plaque in dentifrices.

Description

227~9-310

-2- D.N. 1056 BACKGROUND OF THE INVENTION

Field of the Invention ~ ~ =.. ....
The invention relates to bis[4-(substituted-amino)-l-pyridinium]alkane saccharin salts which are useful as antimicrobial agents and a process for prepara-tion thereof.

Information Disclosure Statement Sterling Drug Inc. (Denis M. Bailey, inventor) U.S. Pat. 4,206,215, which issued June 3, 1980, describes antimicrobial bis[4-(substituted-amino)-1-pyridinium]-alkane salts generically at columns 2-3, subgenerically at column 7 and specifically in the examples. The generic description includes inorganic and organic salts, but saccharin salts are not described either subgenerically or specifically. The specification states at line 41 of column 7 that bromide and chloride salts are preferred.
The Merck Index (Ninth Edition, 1976) describes lS saccharin (saccharin insoluble) in monograph 8070 and saccharin soluble (saccharin sodium, that is, the sodium salt of saccharin) in monograph 8071.
Chemical Abstracts (vol. 59, p. 11333b, 1963) sets forth an abstract of Japanese Pa~ent 4891 (1963) describing bis(p-chlorophenylbiguanido)hexane saccharinate, that is, the saccharin salt of the antimicrobial agent chlorhexidine, as being "without bitter taste".

-3- D.N. 1056 Parts A and B of Example 10 of above-cited U.S. Pat. 4,206,215 describe 1,10-bis[4-(octylamino)-1-pyridinium]decane dichloride. The same compound can also be described by the name N,N'(l,10-decanediyldi-1(4H)-5 pyridyl-4-ylidene)bis[l-octaneamine] dihyrochloride, whose hypothetical free base form has been given the generic name octenidine. In oral use the bis[4-(substituted-amino)-l-pyridinium]alkane salts subgenerically and specifically described by U.S. Pat. 4,206,215, for example, the dihydro-chloride salt of octenidine, have an extremely bittertas~e and a lingering bitter metallic aftertaste. This problem is overcome by the presently described and claimed invention.

4- D . N . 1056 SUMMARY OF _THE INVENI'I ON

In a composition of matter aspect ~he invention is a bis[4-(R-amino)-l-pyridinium]alkane disaccharin salt having the structural formula ~NH- ~ N-Y-N \~ -NHR T l ~ ¦

Formula I

wherein Y is alkylene containing from 4 to 18 carbon atoms and separating the two 4-(R-amino)-l-pyridinium groups by from 4 to 18 carbon atoms;
R is the same in both occurrences and is alkyl containing from 6 to 18 carbon atoms, cycloalkyl containing from 5 to 7 carbon atoms, benzyl, or phenyl substituted by methylenedioxy or one or two substituents selected from the group consisting of halo, lower alkyl, lower-alkoxy, nitro, cyano and trifluoromethyl; and Rl is the same in both occurrences and is hydrogen or lower-alkyl.
In a process aspect ~he invention is in the process of preparing a bis[4-(R-amino)-l-pyridinium]alkane disaccharin salt of Formula I which comprises reacting in an ~queous solvent a corresponding relatively soluble

-5- D.N. 1056 bis[4-(R-amino)-l-pyridinium]alkane non-saccharin salt with saccharin or a soluble salt of saccharin and separating the relatively insoluble bis[4-(R-amino)-l-pyridinium]-alkane disaccharin salt.
The compounds of Formula I can also be described as N,N'(alkanediyldi-1(4H)-pyridin-4-ylidene)bis[l-R-amino] disaccharin salts having the structural formula ~N= ~ ~ y~ F
Formula II

wherein Y, R and Rl have the same meanings set forth above for Formula I.
The compounds of Formula I or II are useful as anti-microbial agents and are particularly useful as agents for inhibiting, reducing and preventing dental plaque in dentifrices.

-6- D.N. 1056 DETAILED DESCRIPTION OF THE INVENTION
INCLUSIVE OF I'HE PREFERRED EMBODIME~TS

Definitions of Variables of Formulas I and II
Y is alkylene and is a bivalent saturated aliphatic - hydrocarbon group containing from 4 to 18, preferably ~rom 8 to 12, carbon atoms arranged in a branched or unbranched chain and separating the t~o 4-(R-amino)-l-pyridinium groups by from 4 to 18 J preferably~from 8to 12, carbon atoms, for example, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene, 1,9-nonylene, 1,10-decylene, l,ll-undecylene, 1,12-dodecylene, 1,13-tri-decylene, 1,14-tetradecylene, 1,15-pentadecylene, 1,16-hexa-decylene, 1,17-heptadecylene, 1,18-octadecylene, l-methyl-1,4-butylene, 3-methyl-1,5-pentylene, 2-ethyl-1,4-butylene, 3-methyl-1,6-hexylene, 2,4-dimethyl-1,5-pentylene, l-methyl-1,7-heptylene, 3-ethyl-1,6-hexylene, 3-propyl-1,5-pentylene, 4,4-di~lethyl-1,7-heptylene, 2,6-dimethyl-1,7-heptylene, 2,4,4-trimethyl-1,6-hexylene, 2,7-dimethyl-1,8-octylene, l-methyl-1,10-decylene, 5-ethyl-1,9-nonylene, 3,3,6,6-tetra-methyl-1,8-octylene, 3,8-dimethyl-1,10-decylene, 3-methyl-l,ll-undecylene, 6-methyl-1,12-dodecylene, 2-methyl-1,13-tri-decylene, 4,9-dimethyl-1,12-dodecylene, 4-methyl-1,14-tetra-decylene, 2,13-dimethyl-1,14-tetradecylene, 1,4-dipropyl-1,4-butylene, 3-(3-pentyl)-1,5-pentylene, 2-(4,8-dimethylnonyl)-1,4-butylene or 1-heptyl-1,5-pentylene.
When R is alkyl containing from 6 to 18 carbon atoms, it can be branched or unbranched, preferably contains

-7- D.N. 1056 from 7 to 9 carbon atoms and is, for example, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, l-methylpentyl, 2,2-dimethyl-butyl, 2-methylhexyl, 1,4-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, 2-methylheptyl, l-ethylhexyl, 2-ethylhexyl, 2-propylpentyl, 2-methyl-3-ethylpentyl, 3-ethylheptyl, 1,3,5-trimethylhexyl, 1,5-dimethyl-4-ethylhexyl, 2-propyl-heptyl, 5-methyl-2-butylhexyl, 2-propylnonyl, 2-butyloctyl, l,l-dimethylundecyl, 2-pentylnonyl, 1,2-dimethyltetradecyl or l,l-dimethylpentadecyl.
. When R is cycloalkyl containing from 5 to 7 carbon atoms, it is, for example, cyclopentyl, cyclohexyl or cycloheptyl.
When R is phenyl substituted by methylenedioxy or one or two substituents selected from the group consisting of halo, lower-alkyl, lower-alkoxy, nitro, cyano and trifluoromethyl, it is, for example, p-chlorophenyl, o-chlorophenyl, m-chlorophenyl, p-bromophenyl, m-fluoro-phenyl, p-iodophenyl, 2,4-dichlorophenyl, 2,4-difluoro-phenyl, 2,5-dibromophenyl, 3,5-dichlorophenyl, 3-chloro-4-fluorophenyl, 3,4-methylenedioxyphenyl, p-ethylphenyl, p-methoxyphenyl, m-nitrophenyl, o-cyanophenyl, m-(trifluoro-methyl)phenyl or 2-methoxy-5-methylphenyl.
When R is benzyl the phenyl group can also be substituted by one or two substituents, for example, -,~- D.N. 1056 -halo, hydroxy, lower-alkyl, lower-alkoxy, nitro, cyano or trifluoromethyl.
Halo is fluoro, chloro, bromo or iodo. Lower-alkyl and lower-alkoxy have from one to four carbon atoms.
Lower-alkyl is thus methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl. Lower-alkoxy is thus methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy or tert-butoxy.

Preparation of ~he Compounds The process aspect of the invention is carried out using as the aqueous solvent water alone or a mixture of water and one or more water-miscible organic solvents selected from the alcohols, ketones, acids, amides, nitriles and sulfoxides and multifunctional and mixed functional solvents. Water alone is the preferred solvent.
The saccharin salts of Formula I or II are insoluble or only slightly soluble in water. The relatively soluble bis[4-(R-amino)-l-pyridinium]alkane non-saccharin salt or N,N'-(alkanediyldi-1(4H)-pyridin-4-ylidene)bis[l-R-amino] non-saccharin salt can be any more soluble salt described by above-cited U.S. Patent 4,206,215. Chloride and bromide salts~are preferred.
Saccharin is only slightly soluble in water.
The process aspect of the invention is therefore preferably carried out using a soluble salt of saccharin, for example, an alkali metal salt. The sodium salt (saccharin soluble) is most preferred.

-9- D.N. 1056 The process aspect of the invention is carried out with or without heating or cooling, preferably at a temperature in the range o 0-100C., most preferably at a temperature in the range of 20-60C.
Structures of products are inferred from structures of sta-ting ma~erials and the expected course of the preparative reaction. The methods and measurements used to confirm structures and estimate purities of starting materials and products include melting temperature range (m.r.), elemental analysis, infrared (IR~ spectral analysis, ultraviolet (UV) spectral analysis, mass spectral (MS) analysis, nuclear magnetic resonance (NMR) spectral analysis, gas chromatography (GC), high pressure liquid chromato-graphy (HPLC) and thin layer chromatography (TLC).

-10- D.N. 1056 EXAMPLE

A warm (50C.) solution of N,N~ ,lO-decanediyldi-1(4H)-pyridin-4-ylidene)bis[l-octanamine](octenidine) dihydrochloride salt (623 g., 1.00 mole) in deionized water (6 l.) was added with stirring during 15 minutes to a warm (50C.) solution of saccharin sodium salt (520 g., 2.00 mole) in deionized water (2 1.). Stirring was continued for two hours, then the mixture was filtered. The solid was washed with hot deionized water (3 l.) and dried at 60C. under vacuum, affording N,N'-(l,lO-decanediyldi-1(4H)-pyridin-4-ylidene)bis[l-octanamine](octenidine) disaccharin (saccharin) salt (molecular weight 917.1, 890 g., 97% yield, m.r. 82-85C.).

Antimicrobial Properties of the Compounds As stated above the compounds of Formula I
or II are Jseful as antimicrobial agents. This has been shown by determining the antibacterial and antifungal properties in vitro of a sample of the compound of the foregoing example prepared as described above using distilled water instead of deionized water and without heating (m.r. 71.5-73C.).
A solution of the compound in propylene glycol (6.4 ~M./ml.) was prepared and tested against one strain each of Staphylococcus aur~eus and Streptococcus pyogenes representing the gram-positive bacteria, Escherichia coli, -11- D.N. 1056 Klebsiella pneumoniae, Proteus mirabilis, Proteus vul~aris and Pseudomonas aeruginosa representing the gram-negative bacteria, and Aspergillis niger, Candida albicans and Trichophyton mentagrophytes representing the fungi.
The cultures of S. aureus, the five gram-negative strains and C. albicans were grown in ~ryptose phosphate broth.
S. pyogenes was grown in brain-heart infusion broth with lQ% normal horse serum. These eight cultures were adjusted to an optical density of 0.10 (650 n~., B&L Spectronic 20), then diluted in double-strength culture medium to 2x105 cells/ml. A. niger and T. menta~rophytes were maintained as spore suspensions and diluted in double-strength maltose peptone ~3 broth to 2x105 spores/ml. as determined previously by plate count.
Serial twofold dilutions of the propylene glycol solution of the test compound were prepared in sterile distilled water in test tubes. Each dilution was inoculated with an equal volume of each of the diluted microbial cultures and incubated, the bacteria for 18-20 hours at 37C. and the fungi for Eive days at 25C. The lowest concentration of the test compound which prevented visible growth of the microorganism is the minimum inhibitory concentration, whose values are shown in the following table.

-12- D.N. 1056 Minimum Inhibitory Microor~anism Concentration (~M./ml.) S. aureus Smith 1.56 S. pyogenes C203 1.56 E. coli Vogel 1.56 K. pneumoniae 39545 6.25 P. mirabilis MGH-l 12.5 P. vulgaris 9920 3.13 P. aerug,_osa MGH-2 6.25 A. ni~er 16404 12.5 C. albicans 10231 3.13 T. mentagr~ytes 9129 3.13 Comparative Taste and Aftertaste As shown by above-cited U.S. Pat. 4,206,215 octenidine dihydrochloride salt is useful for preventing dental plaque. Octenidine disaccharin salt is intended to be used, and dentiErice formulations thereof have been shown to be useful, for the same purpose. Octenidine dihydrochloride salt and a sample of octenidine disaccharin salt, which was prepared as described in the example above using distilled water instead of deionized water and without heating (m.r. 71.5-73C.), were therefore compared for taste and aftertaste. Octenidine dihydro-chloride salt had an extremely bitter taste and a lingering bitter metallic aftertaste. Octenidine disaccharin salt had a sweetish taste and a mild, slightly bitter (transient) aftertaste.

-13- D.N. 1056 Dentifrice Compositions and Antiplaque Properties Thereof The compounds of Formula I or II are preferably formulated in dentifrice compositions comprising an effective dental plaque inhibiting, reducing or preventing amount thereof; a polishing agent or thickening agent selected from the group consisting of silicon dioxide, silicic acid, silica, amorphous silica, precipitated silica, h~drated silica, silica hydrate, silica gel, silica xerogel and hydrous silica gel; and a foaming agent selected from the group consisting of N-alkanoylsarcosine or N-alkenoyl-sarcosine or an alkali metal, ammonium or alkanolaminesalt thereof, wherein alkanoyl or alkenoyl has from 8 to 18 carbon atoms and alkanolamine has from 2 to 9 carbon atoms.
A preferred compound of Formula I or II is octenidine saccharin salt of the foregoing example. The effec~ive dental plaque inhibiting, reducing or preventing amount of the compound of Formula I or II is generally in the ran~e of 0.1-10% and most generally of the order of 1% by weight of the dentifrice.
In N-alkanoylsarcosine and N-alkenoylsarcosine alkanoyl and alkenoyl having from 8 to 18 carbon atoms can be branched or unbranched. Unbranched alkanoyl having an even number of carbon atoms is preferred, especially lauroyl having 12 carbon atoms. Preferred alkanolamine salts include the monoethanolamine, diethanolamine and triethanolamine salts. The alkali metal salts include -14- D.N. 1056 the lithium, sodium and potassium salts. Sodium salts are preferred. The most preferred foaming agent is sodium N-lauroyl sarcosinate. The amount of foaming agent is generally in the range or 0.1-10% and most generally of the order of 1% by weight of the dentifrice.
In addition to the above-described components the dentifrices of the invention generally also contain one or more humectants, for example, sorbitol, glycerin - or a polyethylene-glycol, for example, PEG-6-32, which is an adopted name for a mixture of the polyethylene glycol having 6 ethylene oxide units and the polyethylene glycol having 32 ethylene oxide units; caries reducing, inhibiting or preventing fluoride salts, for example, sodium fluoride, sodium monofluorophosphate or stannous fluoride, sweeteners, for example, free saccharin in addition to the bound saccharin of the above-described saccharin salts, cyclamate or aspartame; flavors; colorants;
acids or bases to adjust the pH (desirably about 7), for example, hydrochloric acid or sodium hydroxide; and water.
To ensure maximum possible antiplaque effect the saccharin salt of Formula I or II is added as the last ingredient of the dentifrice formulations. A typical order of addition of the remaining ingredients of a typical formulation is: water, ~luoride salt, humectants, sweetener, foaming agent, flavors, colorant, acid or base to adjust -15- D.N. 1056 pH, and polishing and thickening aents. Accordingly the following composition was prepared.

Example Percent by Ingredient Weight Octenidine Saccharin Salt 1.10 Sodium Fluoride 0.220 Sorbitol Solution 46.3 Hydrated Silica, Polishing Grade 17.0 Glycerin 15.0 Hydrated Silica, Thickening Grade 11.1 PEG-6-32 3.00 Sodium Lauroyl Sarcosina~e 2.10 Flavors 1.100 Saccharin 0.100 Colorant 0.00400 Hydrochloric Acid to make pH 7 --Purifi.ed Water to make 100.0 An in vitro test for effect of the foregoing composition against preformed dental plaque was carried out.
Streptococcus mutans NCTC 10449, Streptococcus sanguis ATCC 10558 and Actinomyces viscosus M-100 were used as plaque forming microorganisms and were stored prior to use in a frozen or lyophilized state. Working ~ t~

-16- D.N. 1056 stock cultures were maintained by twice monthly passage in fluid thioglycollate medium containing 20% (w/v) meat extract and excess calcium carbonate. For plaque formation the medium of Jordan et al. (J. Dent. Res., vol. 39, pp. 116-123, 1960) supplemented with 5% (w/v) sucrose was used. All cultures were adopted to this growth medium by at least one growth cycie prior to use in the tests.
The substrates were cleaned, sterilized ceramic hydroxylapatite (durapatite, slabs approximately 1/4"
x 1/2" x 2" in size, which were manipulated during transfer and brushing with sterile clamps (hemostats). Brushing was done with a wetted medium bristle toothbrush and one inch strips of the dentifrice on all sides of the slab for one minute. The slab was then rinsed in running tap water for one minute with manipulation to ensure removal of all visible dentifrice.
Plaques were developed on the slabs by daily passage through the supplemented Jordon et al. medium (20 ml.) in culture tubes. Only the initial culture tube was inoculated with the plaque-forming microorganism (at least 0.3 ml. of a late log-phase culture). Subsequent tubes were not directly inoculated. Thus, only the most adherent microorganisms were carried over, thereby permit-ting formation of strongly adherent plaques. After three successive daily transfers the slabs were brushed with the dentifrice, rinsed and immersed in fresh growth 3~

-17- D.N. 1056 medium containing a pH indicator (bromocresol purple).
The culture tubes were incubated for 24 hours at 37C.
under an anaerobic atmosphere. A once daily brushing regimen was thus simulated. Alternatively the slabs were brushed twice daily and the incubation periods were 6 and 16 hours in duration. Growth and metabolic activity ~acid production) were subsequently assessed, and the treatment and incubation(s) were repeated. The slabs were brushed daily until the cultures failed to yield 0 acid end products and (ideally) yielded viable bacteria.
Metabolic activity (M.A.) was measured 12 and 36 hours after final treatment and scored + (acid produc-tion and turbidity increase), + (no acid shift but notice-able turbidity increase) or + (no acid shift and minimal evidence of turbidity increase). Plaque was measured following staining with erythrosin and scored 0, 1, 2, 3, 4 or Fl (flecks or microcolonies). The following results were obtained from duplicate brushings twice daily for three successive days with only water and no dentifrice, a placebo dentifrice (the composition of the foregoing example wherein the antimicrobial agent was replaced by an equa]. amount of sorbitol solution) and the composition of the foregoing example itself.

-18- D.N. 1056 Score S. Mutans S. Sanguis A. Viscosus Gom~ositionMeasurement 10449 10558_ M-100 Water 12-Hr. M.A. +, + +, ~ ~, +
36-Hr. M.A. +, + -~, + +, +
Plaque 3, 3 4, 4 3, 4 Placebo12-Hr. M.A. +, + +, i +, +
36-Hr. M.A. +, + +, ~ +, +
Plaque 1, 1 1, 1 1, 1/2 - 10Example12-Hr. M.A. +, + +, + +, +
36-Hr. M.A. +, + i, + +, +
Plaque Fl, Fl Fl, FlFl, Fl

Claims (15)

We claim:

1. A bis[4-(R-amino)-1-pyridinium]alkane disaccharin salt having the structural formula wherein Y is alkylene containing from 4 to 18 carbon atoms and separating the two 4-(R-amino)-1-pyridinium groups by from 4 to 18 carbon atoms;
R is the same in both occurrences and is alkyl containing from 6 to 18 carbon atoms, cycloalkyl containing from 5 to 7 carbon atoms, benzyl, or phenyl substituted by methylenedioxy or one or two substituents selected from the group consisting of halo, lower-alkyl, lower-alkoxy, nitro, cyano and trifluormethyl; and Rl is the same in both occurrences and is hydrogen or lower alkyl.

2. A compound according to claim 1 wherein R1 is hydrogen.

3. A compound according to claim 2 wherein R is alkyl containing from 6 to 18 carbon atoms.

4. A compound according to claim 3 wherein R is n-octyl.

5. The compound according to claim 4 wherein Y
is 1,10-decylene.

6. The process of preparing a bis[4-(R-amino)-l-pyridinium]alkane disaccharin salt according to claim 1 which comprises reacting in an aqueous solvent a corres-ponding relatively soluble bis[4-(R-amino)-1-pyridinium]-alkane non-saccharin salt with saccharin or a soluble salt of saccharin and separating the relatively insoluble bis[4-(R-amino)-1-pyridinium]alkane disaccharin salt.

7. The process according to claim 6 wherein R1 is hydrogen.

8. The process according to claim 7 wherein R
is alkyl containing from 6 to 18 carbon atoms.

9. The process according to claim 8 wherein R
is n-octyl.

10. The process according to claim 9 wherein Y
is 1,10-decylene.
ll. N,N'(1,10-Decanediyl-di-1(4H)-pyridin-4-ylidene)-bis[1-octanamine] disaccharin salt.

21

12. The process according to claim 6, 7 or 8, wherein the dihydrochloride of bis[4-(R-amino)-1-pyridinium]alkane and an alkali metal salt of saccharin are employed.

13. A process of preparing N,N'-(1,10-decanediyl-di-1(4H)-pyridin-4-ylidene)-bis[1-octanamine]disaccharin salt, which comprises reacting a relatively soluble N,N'-(1,10-decanediyldi-1(4H)-pyridin-4-ylidene)bis [1-octanamine]alkane non-saccharin salt with saccharin or a soluble salt of saecharin and separating the resulting relatively insoluble salt.

14. A process according to claim 13, wherein the dihydrochloride of the bis[1-octanamine] and an alkali metal salt of saccharin are reacted in water.

15. A process according to claim 14, wherein the sodium salt of saccharin is used as the alkali metal salt.