DK141982B - Antimicrobial preservative, especially for use in cosmetic creams. - Google Patents

Description

(11) PUBLICATION 141982

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\ Ra / DENMARK (61) lntcl · 3 Α 01 μ ^ 3/32 // c 07 d 319/06 § (21) Application No. 801/74 (22) Filed on 15 · f © b. 1974 (23) Race day 15 · Feb. 1974 (44) The application presented and. _ the petition published on ^ · SUS · 19 © 0

DIRECTORATE OF

PATENT AND TRADE MARKET (30) Priority requested from it

Feb 16 1973, 333418, US Jul 26 1973, 382992, US

(71) ELI LILLY AND COMPANY, 3-7 East McCarty Street, Indianapolis, Indiana, US. ~ (72) Inventors Lewis Christopher Lappas, 5331 Hawthorne Drive, Indianapolis, Indiana, US: Clarence August ~ Hirsch, 6737 Dover Road, Indianapolis, Ine diana, US.

(74) Plenipotentiary in the proceedings:

Hofman-Bang & Boutard engineering company .___ (64) Antimicrobial preservative, Issar for use 1 cosmetic creams.

The present invention relates to an antimicrobial preservative, especially for use in cosmetic creams. The preservative is characterized in that it contains as an active ingredient a 5-bromo-5-nitro-2-alkyl-substituted -1,3-dioxane of the general formula set forth in claim 1, such as 5-bromo-2-methyl-5-nitro -1,3-dioxane, with effect against both bacteria and fungi.

A wide variety of antimicrobial agents are known. A large number of antibiotics and synthetic and organic chemicals are used as bacteriostats, bacteriocides, fungistates, fungicides etc. Many of these effective antimicrobial agents have an important role in controlling microorganisms that infect humans. Some have a broad spectrum of activity, while others are used within a relatively narrow range. A few are even limited to a single use.

2 141982

Antimicrobial preservatives are known as important ingredients in cosmetic creams that are used as a base for face makeup. These creams generally consist of an oil-in-water emulsion. The ingredients provide a favorable substrate for microorganisms such as bacteria and fungi. Furthermore, since both oil and water are present, the choice of an appropriate preservative requires attention to the need for antimicrobial action in both systems. Since an effective antimicrobial agent is likely to work best when it is soluble in the substrate to be protected, it is important that the preservative for such a cosmetic cream has an effective solubility in both oil and water.

A preservative that has been used successfully in cosmetic creams is 2-bromo-2-nitropropane-1,3-diol. However, this compound has the disadvantage of being chemically unstable to alkali.

A recently introduced compound that can be used to preserve cosmetic creams is the 5-bromo-5-nitro-1,3-dioxane known from German Publication No. 1,920,298. This compound is chemically stable to alkali. However, although 5-bromo-5-nitro-1,3-dioxane has a relatively broad spectrum of action, there are important microorganisms against which this compound is inactive or exhibits little activity at relatively high concentrations. Therefore, the investigation for an alkali-stable compound, which is both soluble in oil and water, and which has an even wider spectrum of action against microorganisms at low concentration, has continued.

It is, therefore, an object of the present invention to provide an alkali-stable compound which has an appropriate oil and water solubility, and which is effective in relatively small concentrations against a wide variety of bacteria and fungi.

From Roczniki Chemii 46, 1559-1565 (1972), the 2-methyl and 2-isopropyl derivatives of the above-mentioned 5-bromo-5-nitro-1,3-dioxane are known.

Surprisingly, it has now been found that these and other 2-alkyl-substituted 5-nitro-5-bromo-1,3-dioxanes are effective in low concentrations against a wide range of bacteria and fungi.

Such compounds here provide excellent chemical stability at pH

5-9 and a solubility in both water and oil (mineral oil) of up to 1.1-1.55X5. Cosmetic creams can be effectively preserved by incorporating as little as 0.01% of these compounds.

The active compounds in the preservative according to the invention have the general formula according to claim 1. The compounds can be prepared by using 2-bromo-2-nitropropane-1,3-diol as starting material. The compound 2-bromo-2-nitropropane-1,3-diol is commercially available under the trademark MBronopoln and can be prepared in various ways. An effective method involves the use of nitromethane as a reactant and a reaction with formaldehyde to form 2-nitropropane-1,3-diol which is converted to sodium salt and brominated to give 2-bromo-2-nitropropane-1,3-diol. Preparation 1 below exemplifies the preparation of 2-bromo-2-nitropropane-1,3-diol from nitromethane.

Preparation 1 20 g (0.5 mole) of sodium hydroxide was dissolved in 400 ml of water and the solution was cooled to 10 ° C. To 74 ml of 35% formaldehyde (1.0 mol) was added 76 ml of the previously prepared sodium hydroxide solution. To the combined formaldehyde sodium hydroxide solution was added dropwise 27 ml (0.5 mole) of nitromethane while maintaining the temperature of the reaction mixture at 25-30 ° C by dipping the reaction vessel into an ice-water bath. The reaction between nitromethane and formaldehyde was catalyzed by sodium hydroxide and 2-nitropropane-1,3-diol was formed, but this was not isolated. The diol was converted to the sodium salt by adding the rest of the previously prepared sodium hydroxide solution to the reaction vessel at a temperature of 20 ° C and the mixture was stirred at room temperature and atmospheric pressure for half an hour.

300 ml of ethyl acetate was cooled to -5 ° C using an ice-acetone bath. To the cold ethyl acetate was added 25.5 ml (0.5 mole) of bromine in a slow steady stream, maintaining the temperature of 5 ° C or below. The previously prepared sodium-2-nitropropane-1,3-diol reaction mixture was slowly added to the ethyl acetate-bromine mixture, keeping the temperature at 5-10 ° C. The reaction mixture was stirred for 5 minutes at room temperature and atmospheric pressure, then the pH was adjusted to 2.0 with 6N hydrochloric acid. 200 g of sodium chloride was added to the reaction mixture, whereby the 2-bromo-2-nitropropane-1,3-diol was transferred to the ethyl acetate phase.

This was separated and dried over magnesium sulfate and filtered. Ethyl acetate was removed from the filtrate under vacuum and the resulting solid mass was washed with methyl cyclohexane and evaporated to dryness. 30.1 g (83.1% yield) of 2-bromo-2-nitropropane-1,3-diol were collected. This had a melting point of 108-114 ° C.

The active compounds can be prepared by reacting an appropriate aldehyde with 2-bromo-2-nitropropane-1,3-diol in an acidic medium. The preparation of 5-bromo-2-methyl-5-nitro-1,3-dioxane is exemplified in Example 1.

EXAMPLE 1 40 g (0.2 mole) of 2-bromo-2-nitropropane-1,3-diol was suspended in 200 ml of benzene and 0.3 g of p-toluenesulfonic acid was added. The mixture was cooled in an ice-water bath and a reflux was put on the reaction vessel. Ca. 13.5 g (0.3 mole) of acetaldehyde was slowly added through the back condenser and the reaction mixture was stirred for 30 minutes. A Dean-Stark water trap was applied and the mixture was slowly heated and refluxed until no water developed. (About 4 ml of water appeared over 1.5 hours). The solution was filtered and evaporated on a rotary evaporator at 45 ° C. A dark oil residue was obtained which was vacuum distilled at 0.25 mm. There was thus obtained 36.5 g (75% yield) of 5-bromo-2-methyl-5-nitro-1,3-dioxane having a boiling range at 0.25 mmHg of 72-74 ° C. The NMR and IR spectra were consistent with the structure and the elemental analysis had the following percentages:

Carbon: calculated 26.57, found 26.85;

Hydrogen: calculated 3.57, found 3.85;

Nitrogen: calculated 6.20, found 6.04;

Bromine: calculated 35.35, found 35.21.

The compound 5-bromo-2-methyl-5-nitro-1,3-dioxane is a water-clear, colorless liquid at room temperature and first solidifies at -10 ° C.

5 141982

It is chemically stable at pH 5.0-9.0 and has a smell similar to bromine.

EXAMPLE 2

The compound 5-bromo-2-ethyl-5-nitro-1,3-dioxane was prepared following the procedure described in Example 1 using propiondehyde instead of acetaldehyde. The NMR spectrum was consistent with the structure and an elemental analysis gave the following percentage composition:

Carbon: calculated 30.02; found 30.24

Hydrogen: calculated 4.20; found 4.24

Nitrogen: calculated 5.83; found 5.92

Bromine: calculated 33.29; found 32.99

The white crystalline substance had a melting point of 58 - 59 ° C. The compound is chemically stable at pH 5 - 9 and has the same odor similar to bromine as the methyl derivative.

EXAMPLE 3

The compound 5-bromo-5-nitro-2-n-propyl-1,3-dioxane was prepared following the method described in Example 1, using n-butyraldehyde instead of acetaldehyde. The NMR and IR spectra were consistent with the structure, and an elemental analysis showed the following percentage composition:

Carbon: calculated 33.09; found 33.32;

Hydrogen: calculated 4.76; found 4.63;

Nitrogen: calculated 5.51; found 5.66;

Bromine: calculated 31.45; found 31.64;

The compound was a colorless oil which, when distilled at 0.005 mmHg, had a boiling point of 73 - 75 ° C. The oil solidified to a waxy solid upon standing at room temperature. There was a brominish odor present.

EXAMPLE 4

The compound 5-bromo-2-isopropyl-5-nitro-1,3-dioxane was prepared 6AA1982 using the method described in Example 1 using isobutyraldehyde instead of acetaldehyde. The MR and IR spectra were consistent with the structure, and an elemental analysis yielded the following percentage composition:

Carbon: calculated 33.09; found 33.33

Hydrogen: calculated 4.76; found 4.83 Nitrogen: calculated 5.51; found 5.33 Bromine: calculated 31.45; found 31.24

The compound was a colorless crystalline mass which, after recrystallization from 1: 1 isopropanol: water, had a melting point of 42 - 44 ° C. There was a brominish odor present.

The active compounds of the preservative of the invention exemplified in Examples 1, 2, 3 and 4 may exist as cis and trans isomers. The method described in Example 1 gives a ratio of the isomers of 9: 1. It is intended to include both geometric isomers as chemical units as well as the isomeric mixture. Antimicrobial experiments with isomeric mixtures of 5-bromo-2-methyl-5-nitro-1,3-dioxane, one with a high content of the cis form, and another with a high content of the trans form, show equal activity , indicating that there is no significant difference between the two geometric isomers. As a consequence, the experiments described below land use of the isomeric mixture (about 9: 1) prepared as described in Example 1.

One of the active compounds in the preservative of the invention, namely 5-bromo-2-methyl-5-nitro-1,3-dioxane, is extremely effective in vitro against a wide variety of microbial organisms, both bacteria and fungi. Furthermore, the antimicrobial effect of this compound is up to 20 times that of other compounds such as 5-bromo-5-nitro-1,3-dioxane. Eg. this active compound has a minimal inhibitory concentration of 5 µg / ml against Corynebacterium aenes and Staphylococcus aureus compared to a minimum inhibitory concentration of 100 µg / ml for the compound 5-bromo-5-nitro-1,3-dioxane against the same microorganisms in a conventional dilution test. In this experiment, the inoculated fluid concentration was 10 µm organisms / ml.

In vitro activity of the active compounds, 5-bromo-2- methyl-5-nitro-1,3-dioxane (Compound A), 5-bromo-5-nitro-1,3-dioxane (Compound B) and 2-Bromo-2-nitro-propane-1,3-diol (Compound C) was determined by plating six organisms on agar plates containing the active antimicrobial agents A, B and C.

The minimum inhibitory concentration of compound A was from 1/5 to 1/30 of the minimum inhibitory concentration observed for compound B, and from 1/20 to 1/67 of the minimal inhibitory concentration found for compound C. The tests were conducted simultaneously and there were 10 microorganisms / ml. The microorganisms used as well as the experimental results are shown in Table I below.

8 141982

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The antimicrobial activity of 5-bromo-2-methyl-9-nitro-1,3-dioxane (compound A), 5-bromo-5-nitro-1,3-dioxane (compound B) and 5-bromo-2 Ethyl 5-nitro-1,3-dioxane (Compound D) was simultaneously tested against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli in a conventional dilution test, and against Aspergillus flavus, Aspergillus niger and Candida albicans using agar plates. The concentrations of microorganisms in the dilution experiments were 104 organisms per ml. and the concentration expressed on the agar plates was 10 µ microorganisms per ml. ml of agar. The figures in Table II show the lowest concentrations in µg / ml of active compound in the liquid or on the agar where no growth was observed. This was used as the minimum inhibitory concentration of the active compound against each microorganism.

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In a third experiment, the antimicrobial activity of Compound A, Compound B and 5-bromo-5-nitro-2-n-propyl-1,3-dioxane (Compound E) was determined simultaneously against the same six microorganisms as shown in Figs. Table I. Compound A was clearly superior to Compound B against four of the six microorganisms, and Compound E showed a clear antimicrobial action. The experiments were in vitro assays using agar plates containing the active compound at a concentration of 10, 25 and 50 µg per ml. ml of agar. The organisms were used at a concentration of 10b organisms per day. ml and the seven were smeared on the solid agar. The plates were incubated for 24 hours and the presence or absence of growth was noted. The minimum inhibitory concentration was determined as the lowest concentration of active compound in the agar where no growth was observed. Table III below shows the results of this experiment.

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The active compounds in the preservative of the invention also show exceptionally good antifungal effect. This effect was determined against 16 common fungi, and against 14 of the 16 organisms, 5-bromo-2-methyl-5-nitro-1,3-dioxane had a lower mininal inhibition concentration than that observed when 5- bromo-5-nitro-1,3-dioxane was used in a concurrent experiment. The experiments were carried out by adding the active compound to molten agar at concentrations of 200, 100, 50, 25 and 10 µg / ml, solidifying the agar and stretching the organisms thereon. The results of the experiments are shown in Table V.

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The antifungal effect of compounds A and B was determined for 7 fungi and a yeast fungus in another experiment. Aqueous concentrations of 0.1, 0.05 and 0.025% by weight of each compound A and B were seeded at ca. 10 ^ organisms per ml. Viable organisms were counted after 24 hours, as shown in Table VI. Compound A was observed to be significantly better than Compound B against yeast, Saccharamyces pastorianus, at a concentration of 0.025 percent. Furthermore, Compound A had an improved effect on Trichophyton menta-grophytes relative to Compound B.

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o CpsramoBPi 19 1A19 82 In Table VI, a number of organisms of less than 100 are considered indicative of complete extinction, the 2-methyl derivative of 5-bromo-5-nitro-1,3-dioxane being a particularly preferred compound as active component of the preservative of the invention.

Experiments were performed to assess the skin irritation and skin sensitivity properties of the compound 5-bromo-5-nitro-1,3-dioxane and the preferred active compound 5-bromo-2-methyl-5-nitro-1,3-dioxane. The following procedure was used:

200 people were used for each study. A repeated irritation technique was used in the beauty spot test. A different group of people was used for each assessment.

The technique used in the stain test requires that a series of 9 stains of each active compound be applied to each person. The series was followed 2 weeks later with a single spot of each active compound to test skin sensitivity.

The 9-spot series was performed on Monday, Wednesday and Thursday, and contact with the skin was maintained for 24 hours, after which the stain was removed and the site of the skin inspected for irritation. The Thursday spots were placed immediately after removing and assessing Wednesday's applications. After the ninth applied stain was applied, a period of 2 weeks went by where no stains were applied, and then a stain was applied to determine the sensitivity reactions. A new spot on the skin was used for this new 24 hour spot. This location was always selected next to a treated site, i.e. a place where repeated additions were performed during the series with the 9 spots applied. The last stain was assessed 24 and 72 hours after application.

The application to the skin for each active compound was performed using oval adhesive spots (1 "x 1 1/4") with circular gauze centers. The gaseous centers were covered with approximately 0.03 ml of the tested material just prior to application. used in the skin irritation reactions and in assessing the sensitivity were as follows:

TABLE VII

Experiments with repeated application of irrigation patches to humans

Skin system scoring system Erythema and eschar formation

No reaction 0

Very poor erythema (almost not visible) 1

Mild, well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (red as beetroot) for eschara formation (damage in depth) 4

Total possible Erythema point 4 edema formation

No reaction 0

Very low edema (just visible) 1

Slight edema (the edge of the area is well defined by a definite elevation) 2

Moderate edema (area elevated approximately 1 mm) 3

Severe edema (area elevated more than 1 mm and · extending beyond the area directly affected) 4

Total possible edema point 4

Total possible primary irritation point 8

Four separate formulations of each compound were tested together with two formulations of parabens. These formulations were all cosmetic creams with different concentrations of the active compounds, as shown in Table VIII.

TABLE VIII

Formulation Active compound Concentration% Cream type A 5-bromo-5-nitro-0.05 Nonionic B 1,3-dioxane 0.1 Nonionic C 0.05 Anionic D 0.1 Anionic E 5-bromo-2-methyl-5- 0.01 Nonionic F nitro-1,3-dioxane 0.1 Nonionic G 0.01 Anionic

Table VIII (continued) 21 141982

Formulation Active compound Concentrations% Cream type H 0.1 Anionic K Parabens 0.6 Nonionic L 0.6 Anionic

Table IX below shows a summary of the points obtained in all the experiments:

TABLE IX

Comparison of 5-bromo-5-nitro-1,3-dioxane and 5-bromo-2-methyl-1,3-dioxane in terms of irritation and sensitivity in a human experiment.

5-Bromo-5-aitro-l, 5-dioxane

Assessment of the irritation

Total reactions with points on:

Tested Number of positive formulation reactions 012345678 A 25 1766 18 3 3 1 1100 B 41 1735 29 12 9 3 3 0 0 2 C 21 1768 18 6 1 0 1 1 0 0 D 56 1693 59 23 6 6 2 4 0 0 K 19 1771 21 5 0 0 0 0 01 0 L 20 1767 21 5 2 1 1000

Assessment of the sensitivity

Total reactions with points on:

Tested Number of positive formulation reactions 012345678 A 3 197 00110 11 00 B 11 189 2 2 2 1 11 21 0 11 C 2 198 0 0 1 0 0 11 0 0 D 20 180 1 5 2 5 11 31 11 21 K 2 198 11000000 L 1 199 00010000

Indicates skin sensitivity 22 5-Bromo-2-methyl-5-nitro-1,3-dioxane

Assessment of the irritaion

Total reactions with points on:

Tested Number of positive formulation reactions 0 1 2 3 45 678 E 13 1787 13 0 0 00000 F 26 1774 23 2 1 00000 G 25 1775 22 1 2 00000 H 25 1775 21 2 2 0 0 0 0 0 K 20 1777 19 4 0 0 0 0 0 0 L 24 1776 22 2 0 00000

Assessment of the sensitivity

Total number of reactions with points on:

Tested Number of positive formulation reactions 0 l 2 3 4 5678 E 0 200 0 0 0 0 0 0 0 0 F 0 200 0 0 0 0 0 0 0 0 G 0 200 0 0 0 0 0 0 0 0 H 2 198 2 0 0 0 0 00 0 K 1 199 10000000 L 2 198 2 0-0 0 000-0

From the figures in Table IX, it is clear that 5-bromo-5-nitro-1,3-dioxane has a distinct sensitivity effect, with 11 out of 200 people being assessed as being sensitive to a concentration of 0.1 percent of the compound at the sensitivity test, while none of the 200 subjects were sensitive to a 0.1% concentration of 5-bromo-2-methyl-5-nitro-1,3-dioxane. A score of 5 to 8 was judged to be indicative of the development of skin sensitivity to the tested compound. Furthermore, the irritation points of the compound 5-bromo-5-nitro-1,3-dioxane were more severe than those listed for the 2-methyl derivative.

The active compounds have the important property that they are soluble in water up to 1.5% and in mineral oil up to 1.1%. This valuable property makes these binderies particularly useful as active ingredients in antimicrobial agents for preserving cosmetic creams. Effective preservation of a cosmetic cream can be accomplished by incorporating from 0.01 to 1.5%, preferably 0.1 to 1.0%, of such a compound. The preparation of a typical cosmetic cream using 5-bromo-2-methyl-5-nitro-1,3-dioxane at a concentration of 0.1% as active ingredient in a preservative is exemplified in Example 5.

Example 5 7.5 kg of a nonionic surfactant cosmetic based cream is prepared as follows:

Add to a first suitable container Phase I:

Heavy mineral oil 1,875 g

Cetyl alcohol 300 g

White wax 450 g

Anhydrous lanolin 225 g

Sorbitan monosterate 300 g

Polyoxyethylene (20) sorbitan monosterate 450 g

Heat phase I to 80 ° C with continuous stirring and add to another suitable container phase II:

Purified water 3,750 ml

Heat phase II to 80 ° C.

Add Phase I to Phase II with continuous stirring.

Remove the heat and allow the mixture to cool to 45 ° C with continuous stirring.

Add to a third suitable container phase III:

Polyethylene Glycol 200 93.7 g 5-Bromo-2-methyl-5-nitro-1,3 dioxane 7.5 g

Heat to 45 ° C to dissolve the compounds with stirring. Add phase III to the reaction mixture between phase I and phase II at 45 ° C and mix well with continuous stirring.

Make up with water to 7.5 kg, i.e. 200 ml of water ·

Cool to 37 ° C with continuous stirring.

24 141982

The active compounds of the preservative of the invention are effective antimicrobial agents for inhibiting the growth of microorganisms selected from the group consisting of gram positive and gram negative bacteria as well as fungi. The growth of such microorganisms can be hindered by contacting them with an amount of such compound which is effective in inhibiting this growth. Effective amounts of the compounds vary from as little as 0.01% by weight. to 2.0% by weight of this mixture in which the compound is incorporated, preferably 0.1-1.0% by weight.

Typical compositions of such agents, and the concentration of the active compound therein, are set forth in Examples 6-12.

EXAMPLES 6-12

Antimicrobial solution: 95% SD3A Alcohol 50 parts

Ion-exchanged water 49.5 parts 5-Bromo-2-methyl-5-nitro-1,3-dioxane 0.5 parts

Antimicrobial ointment:

Polyethylene glycol 300 49.5 parts

Polyethylene Glycol 1500 49.5 parts 5-Bromo-5-nitro-2-n-propyl-1,3-dioxane 1.0 parts

Antimicrobial powder:

Talkum, U.S.P. 99 parts 5-Bromo-2-methyl-5-nitro-1,3-dioxane 1 part

Shampoo:

Sodium lauryl sulfate 40 parts

Coconut fatty acid diethanolamide 6 parts

Ion-exchanged water 53 parts 5-Bromo-2-ethyl-5-nitro-1,3-dioxane 1 part

Antimicrobial Soap:

Coconut oil glycerides, sodium salt 60 parts

Beef Glycerides, Sodium Salt 39 parts 5-Bromo-2-methyl-5-nitro-1,3-dioxane 1 part

Antimicrobial Cleaner:

Fatty alcohol sulfate (sodium sulfate) 25 parts

Sodium carbonate 7 parts

Sodium sulphate 15 parts

Trisodium phosphate 40 parts

Pentan sodium aminotrimethylene phosphate 10 parts

Carboxymethyl cellulose 1 part 5-Bromo-2-methyl-5-nitro-1,3-dioxane 2 parts