Sulphated chelating agent
09950998 ยท 2018-04-24
Inventors
Cpc classification
International classification
C07C305/10
CHEMISTRY; METALLURGY
Abstract
This invention refer to a series of substances according to formula I: ##STR00001## The substances according to formula I above, known as sulphate chelating agent have the ability to enlarge/loosen simple cell membrane nor common organic membrane and its synthesis process. Furthermore, these invented substances have the potential as the biocides raw material, with very low toxicity into mammal. These features make the substances of this invention become very useful on many applications.
Claims
1. A compound of formula I below: ##STR00023## wherein X.sub.1 and X.sub.2, are selected independently from hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, and nitrogen derivatives, X.sub.3 is sulphonate or sulphate, Rx, Rz are selected independently from hydroxy, halide, alkyl C.sub.1-20, alkylene C.sub.1-20, alkyl alcohol C.sub.1-20, aryl, cycloalkyl, sulphite, sulphate, sulphonate, phosphorus derivatives, and nitrogen derivatives, M is selected from hydrogen, group I cation, group II cation, and transition group cations that are pharmaceutically acceptable, n is an integer from 0-3, a is an integer.
2. The compound according to claim 1, whereas the compound is selected from Tetra Hydroxy Ethyl Di Sulphate, Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate, Tri Hydroxy Isobutyl 1,3,4-Tri Sulphate, Tetra Hydroxy Isoamyl 1,3,4,5-Tetra Sulphate, and Ethyl hexa sulphate acid.
3. The compound according to claim 1, whereas M is natrium.
4. The compound according to claim 1, whereas the compound is selected from Tetra Hydroxy Ethyl Di Sulphate Di Natrium, Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Tetra Natrium, Tri Hydroxy Isobutyl 1,3,4-Tri Sulphate Tri Natrium, Tetra Hydroxy Isoamyl 1,3,4,5-TetraSulphate Tetra Natrium, and Ethyl Hexa Sulphate-Hexa Natrium (EHS-6Na).
5. The compound according to claim 1, wherein the compound is a chelating agent.
6. The compound according to claim 1, where the compound increases the permeability of a microbial membrane.
7. The compound according to claim 1, wherein the compound is a biocide.
8. A method of synthesis of a compound according to formula I, the method comprising: reacting at least one of sulphuric acid (H.sub.2SO.sub.4,), sulphonic acid (H.sub.2SO.sub.3), and sulfur dioxide (SO.sub.2), with at least one of formaldehyde, acetaldehyde, formic acid, acetic acid, and oxalic acid to form an intermediate; and reacting the intermediate with at least one of a hydrogen, halide, phosphorous derivative, nitrogen derivative, sulphonate, and sulphate to obtain the compound of Formula I ##STR00024## wherein X.sub.1 and X.sub.2 are selected independently from hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, and nitrogen derivatives, X.sub.3 is sulphonate or sulphate, Rx, Rz are selected independently from hydrogen, hydroxy, halide, alkyl C.sub.1-20, alkylene C.sub.1-20, alkyl alcohol C.sub.1-20, aryl, cycloalkyl, sulphite, sulphate, sulphonate, phosphorus derivatives, and nitrogen derivatives, M is selected from hydrogen, group I cation, group II cation, and transition group cations that are pharmaceutically acceptable, n is an integer from 0-3, a is an integer.
9. The method of claim 8 wherein the at least one of sulphuric acid (H.sub.2SO.sub.4,), sulphonic acid (H.sub.2SO.sub.3), and sulfur dioxide (SO.sub.2), is reacted with at least one of formaldehyde, acetaldehide, formic acid, asetic acid, and oxalic acid.
10. A method to lyse a hydroxy-hydroxy bond in a peptidoglycan or an hydroxyl-amide bound on protein that compose a cell wall membrane by a compound of formula I, wherein the method comprises enlarging, by the compound of formula I, the membrane porosity of both of sides of the membrane, ##STR00025## wherein X.sub.1, X.sub.2, X.sub.3, are selected independently from hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, and nitrogen derivatives, Rx, Rz are selected independently from hydrogen, hydroxy, halide, alkyl C.sub.1-20, alkylene C.sub.1-20, alkyl alcohol C.sub.1-20, aryl, cycloalkyl, sulphite, sulphate, sulphonate, phosphorus derivatives, and nitrogen derivatives, M is selected from hydrogen, group I cation, group II cation, and transition group cations that are pharmaceutically acceptable, n is an integer from 0-3, a is an integer.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
DETAIL DESCRIPTION OF THE INVENTION
(8) In such embodiment of substances have the formula
(9) ##STR00007##
wherein:
X.sub.1, X.sub.2, X.sub.3, are selected from: Hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives, where X.sub.1, X.sub.2, X.sub.3, could be similar or different atoms, molecules or groups.
Rx, Rz are selected from: Hydrogen, hydroxy, halide, alkyl C.sub.1-20, alkylene C.sub.1-20, alkyl alcohol C.sub.1-20, aliphatic or branched substituted or unsubstituted, aryl, cycloalkyl substituted or unsubstituted, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives.
M: are Hydrogen, or group I, group II, transition group cation that are pharmaceutically acceptable.
n: integer from 0-3
a: integer
and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate that are pharmaceutically acceptable.
(10) In the other preferred embodiment of substances derived from Formula I where M is hydrogen.
(11) In the most preferred embodiment of substance is
(12) Tetra Hydroxy Ethyl Di Sulphate
(13) ##STR00008##
(14) Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate
(15) ##STR00009##
(16) Tri Hydroxy Isobuthyl 1,3,4-Tri Sulphate
(17) ##STR00010##
(18) Tetra Hydroxy Isoamyl 1,3,4,5-Tetra Sulphate
(19) ##STR00011##
(20) Ethyl Hexa Sulphate acid (EHS)
(21) ##STR00012##
and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate that are pharmaceutically accepted.
(22) In the other preferred embodiment of such substances from Formula I, where M is Natrium.
(23) In the preferred embodiment of the above substances are
(24) Tetra Hydroxy Ethyl Di Sulphate Di Natrium
(25) ##STR00013##
(26) Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate Tetra Natrium
(27) ##STR00014##
(28) Tri Hydroxy Isobuthyl 1,3,4-Tri Sulphate Tri Natrium
(29) ##STR00015##
(30) Tetra Hidroxy Isoamyl 1,3,4,5-Tetra Sulphate Tetra Natrium.
(31) ##STR00016##
(32) Ethyl Hexa Sulphate-Hexa Natrium (EHS-6Na)
(33) ##STR00017##
and/or its isomer, enantiomer, stereotiomer, salt, solvate, hydrate that are pharmaceutically accepted.
(34) Furthermore, the formed alkyl sulphate substances derived from formula I are still enabled to be polymerized to form longer chain.
(35) In the preferred embodiment of this invention covers breaking or loosening the hydroxyl-hydroxyl bound of peptidoglycan or hydroxyl-ammine bound of protein that compose cell wall membranes or organic membranes by mean utilizing some sulphate chelating agent that result on the enlargement of the membrane porosity, therefore the membrane become permeable on both of its sides. The capability of the said substances was proven by opening/enlargement the microorganism simple cell's membrane, such as bacteria, algae, fungi, virus, etc with some concentration level that works on the cell wall membrane modification from semi permeable membrane into more and more permeable, therefore it becomes possible to insert another ligands inside the microorganism or the cell wall membrane become totally permeable that result on the microorganism death and/or microorganism dormant condition. It is postulated the said mechanism works for all types of microorganism that has cell membrane.
Definition
(36) The following terms are used throughout the specification and have the following meanings unless otherwise specified:
(37) Alkyl means carbon atom chains having the designated number of carbon atoms which can be either straight chain or branched. Examples of alkyl include but are not limited to, methyl, ethyl, propyl, butyl, isobutyl, and the like.
(38) Alkenyl means carbon atom chains having the designated number of carbon atoms which can be either straight chain or branched and which contain at least one double bond. The alkenyl compounds may have more than one such double bond and the orientation about each double bond is independently either cis or trans. Examples of alkenyl include, but are not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl, hexenyl and the like.
(39) Alkynyl means carbon atom chains having the designated number of carbon atoms which can be either straight chain or branched and which contain at least one triple bond.
(40) As used herein the term aryl means single, polynucleic conjugated and fused residues of aromatic hydrocarbon or aromatic heterocyclic ring systems, examples of aryl include, but are not limited to phenyl, naphthyl, fluorenyl, pyrinyl, pyridyl, pyrollyl, and the like.
(41) Cycloalkyl termination refers to non-aromatic aliphatic ring moiety of 3-20 mono-cyclic, bicyclic, or polycyclic carbon atoms. Cycloalkyl can be bicycloalkyl, polycycloalkyl, branched, or spiroalkyl. One or more of the rings might have one or more double bond but no such ring has fully conjugated pi-electron system. Example, without limitation, of cycloalkyl group are: cyclopropane, cyclobutane, cyclopentane, cyclopenthenyl, cyclohexane, cyclohexadiene, adamanthane, cycloheptane, cycloheptatriene, and the like.
(42) Sulphite structure could be described with three equal resonance structures. In every resonance structure, the sulphur atom has a double bond with one oxygen atom with zero/neutral charge, and the single sulphur atom is bound into two other oxygen atoms that each has formal charge of 1. It is also found free electrons pair on Sulphur atom, therefore the predicted structure by VSEPR theory is a pyramid trigonal similar to amonia (NH3)
(43) ##STR00018##
(44) Sulphate termination refers to sulphur as the centered atom surrounded by four equal oxygen atoms in tetrahedral structure. Sulphur atom in its oxidation state +6 while the four oxygen atoms, each in its oxidation state 2. Sulphate ion carry negative charge and two of them are the alkaline conjugate of bisulphate (or hydrogen sulphate), HSO.sub.4.sup., as the alkaline conjugate of H.sub.2SO.sub.4, sulphuric acid.
(45) ##STR00019##
(46) Sulphonate termination refers to an ester of sulphonic acid. An ester with the common formula ROSO.sub.2R is a sulphonic ester. The common structures of sulphonic ester shown by the structures below:
(47) ##STR00020##
(48) Nitrogen derivatives termination refers to carbon substances that contain nitrogen atom, cyclical or aliphatic, straight or branched chain, if it has cyclical structure, then it could be in fusion or non fusion form, could be aromatic or non aromatic.
(49) Phosphorous derivatives termination refers to carbon substances that contain phosphorous atom, cyclical or aliphatic, straight or branched chain, if it has cyclical structure, then it could be in fusion or non fusion form, could be aromatic or non aromatic.
(50) Pharmaceutically accepted salts refer to alkaline addition types of salts synthesized by adding some alkaline substances to the said substances in this invention. The common pharmaceutically accepted salts are: natrium, kalium, calcium or zink.
(51) In the specification, the termination of substituted means that a group may be further substituted with one or more groups selected from alkyl, alkenyl, alkunyl, aryl, fluoro, chloro, bromo, hydroxyl, alkoxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl or two or more substituents may, together with the carbon atoms to which they are attached from a 5 or 6 membered aromatic or non aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen and sulphur.
(52) Main selection of the preferred substances of this invention are: a. Tetra Hydroxy Ethyl Di Sulphate b. Tetra Hydroxy Ethyl Di Sulphate Di Natrium c. Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate d. Tetra Hydroxy Buthyl 1,2,3,4 Tetra Sulphate Tetra Natrium. e. Tri Hydroxy Isobuthyl 1,3,4-Tri Sulphate f. Tri Hydroxy Isobuthyl 1,3,4-Tri Sulphate Tri Natrium g. Tetra Hydroxy Isoamyl 1,3,4,5-Tetra Sulphate h. Tetra Hydroxy Isoamyl 1,3,4,5-Tetra Sulphate Tetra Natrium i. Ethyl hexa sulphate acid j. Ethyl hexa sulphate-hexa sodium (EHS-6Na)
Synthesis of the Substances of Formula (1) or (2) are of Below:
(53) reacting keton/aldehyde/carboxylate compound in formula III below
(54) ##STR00021##
wherein:
X.sub.1, X.sub.2, X.sub.3, are selected from: Hydrogen, hydroxy, halide, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives, where X.sub.1, X.sub.2, X.sub.3, could be similar or different atoms, molecules or groups.
Rx, Rz are selected from: Hydrogen, hydroxy, halide, alkyl C.sub.1-20, alkylen C.sub.1-20, alkyl alcohol C.sub.1-20, aliphatic or branched substituted or unsubstituted, aryl, cycloalkyl substituted or unsubstituted, sulphite, sulphate, sulphonate, phosphorus derivatives, nitrogen derivatives;
a: integer;
with sulphuric acid (H.sub.2SO.sub.4) and/or sulphonic acid (H.sub.2SO.sub.3) and/or sulphur trioxide (SO.sub.3) and/or sulphur dioxide (SO.sub.2),
EXAMPLES OF THE SYNTHESIS OF SOME SUBSTANCES WITHIN THIS INVENTION
Example 1: Synthesis of Tetra Hydroxy Ethyl Di Sulphate (THES)
(55) 225 g of oxalic acid crystal was diluted in 150 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 250 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation until the next 180 minutes with the installation of vertical pipe condenser for reflux purposes to maintain the volume of the liquid until a very clear solution is achieved.
(56) Then the liquid temperature was raised to 90-100 C.
(57) Evaporation of the solvent followed by crystallization result in formation of the desired substance as an off white powder with the following properties: melting point >200 C., boiling point >400 C., specific gravity=1.86, water solubility 48%.
Example 2: Synthesis of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-2Na)
(58) 90 g of oxalic acid crystal was diluted in 150 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 100 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry heated with agitation until the next 180 minutes with the installation of vertical pipe condenser for reflux was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation until the next 180 minutes with the installation of vertical pipe condenser for reflux purposes to maintain the volume of the liquid until a very clear solution is achieved.
(59) To this clear solution was added dropwise a solution of 73 g NaOH flakes in 75 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 5020 C. Upon completion of addition, the temperature was adjusted slowly to ambient and the solution was held agitated in the ambient temperature for the next 60 minutes.
(60) Evaporation of the solvent followed by crystallization result in formation of the desired substance as an off white powder with the following properties: melting point >200 C., boiling point >400 C., specific gravity=2.2, water solubility 31%.
Example 3: Synthesis of Tetra Hydroxy Butyl 1,2,3,4-Tetra Sulphate
(61) 184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 230 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Then the temperature was adjusted slowly to ambient and the solution was held agitated in the ambient temperature for the next 60 minutes.
(62) Upon 60 minutes agitation completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35-40% of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into 10 C. by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate acid crystals, the separated crystals then oven dried at 70-80 C. until the constant weight reached.
Example 4: Synthesis of Tetra Hidroxy Butyl 1,2,3,4-Tetra Sulphate-Tetra Natrium
(63) 184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 230 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. To this clear solution was added dropwise a solution of 170 g NaOH flakes in 200 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 5020 C. Upon completion of addition, the temperature was adjusted slowly to ambient and the solution was held agitated in the ambient temperature for the next 60 minutes.
(64) Upon 60 minutes agitation completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35-40% of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into 10 C. by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Tetra Natrium crystals, the separated crystals then oven dried at +70-80 C. until the constant weight reached.
Example 5: Synthesis of Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate
(65) 138 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 173 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Next, prepare methanol solution of 34 g methanol 96% technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly.
(66) Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate.
(67) Upon the filtration completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35-40% of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into 10 C. by ice water bath. The resulting suspension then is filtrated to separate the Tri Hydroxy Butyl 1,2,3 Tri Sulphate acid; the separated crystals then oven dried at 70-80 C. until the constant weight reached.
Example 6: Synthesis of Tri Hidroxy Isobutyl 1,2,3-Tri Sulphate-3Natrium
(68) 138 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 173 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Next, prepare methanol solution of 34 g methanol 96% technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly.
(69) Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate.
(70) To this clear solution was added dropwise a solution of 130 g NaOH flakes in 200 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 5020 C.
(71) Upon completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35-40% of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into 10 C. by ice water bath. The resulting suspension then is filtrated to separate the Tri Hydroxy Butyl 1,2,3 Tri Sulphate-3Natrium crystals; the separated crystals then oven dried at 70-80 C. until the constant weight reached.
Example 7: Synthesis of Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulphate
(72) 184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 230 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved. Next, prepare methanol solution of 34 g methanol 96% technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly.
(73) Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate.
(74) Upon the filtration completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35-40% of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into 10 C. by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulphate acid; the separated crystals then oven dried at 70-80 C. until the constant weight reached.
Example 8: Synthesis of Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulfat-4Natrium
(75) 184 g formic acid crystal was diluted in 300 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 230 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation for the next 60 minutes, then 8.6 gram of natrium peracetate was added as the catalyst for polymerization process with the installation of vertical pipe condenser during the next 120 minutes reflux until a very clear solution is achieved.
(76) Next, prepare methanol solution of 34 g methanol 96% technical grade in 60 mL aquadess; into this solution, 43 g of Caustic soda flakes was added following by 30 minutes agitation, until the salt precipitated perfectly.
(77) Then the said hot liquid was cooling slowly into ambient temperature while still agitated for the next 60 minutes. Separate the precipitated salt through filter to get the clear filtrate.
(78) To this clear solution was added dropwise a solution of 170 g NaOH flakes in 200 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 5020 C.
(79) Upon completion, the condenser was released, then the solution is boiled for around 2 hours to evaporate the water until 35-40% of its original volume evaporated. The resulting solution then undergoes slow cooling into ambient temperature and cooling further into 10 C. by ice water bath. The resulting suspension then is filtrated to separate the Tetra Hydroxy Isoamyl 1,2,3,4 Tetra Sulphate-4Natrium; the separated crystals then oven dried at +70-80 C. until the constant weight reached.
Example 9: Synthesis of Ethyl Hexa Sulphate (EHS)
(80) 75 g of oxalic acid crystal was diluted in 75 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 250 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry heated with agitation until the next 180 minutes with the installation of vertical pipe condenser for reflux was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation until the next 120 minutes with the installation of vertical pipe condenser for reflux purposes to maintain the volume of the liquid until a very clear solution is achieved; then the vertical pipe condenser was released, the resulting solution is still heated with another 30 minutes agitation to ensure complete and homogenous sulphatation reaction.
(81) Then the liquid temperature was raised to 100-105 C. Evaporate half of the solvent volume followed by crystallization result in formation of the desired substance as an off white powder with the following properties: melting point >210 C., boiling point >400 C., specific gravity=1.87, water solubility 48% and very higroscopic.
Example 10: Synthesis of Ethyl Hexa Sulphate-Hexa Natrium (EHS-6Na)
(82) 75 g of oxalic acid crystal was diluted in 75 ml of water and the resulted slurry heated to 40-50 C. To this slurry was added dropwise 250 ml of 98% H.sub.2SO.sub.4. The rate of acid addition was controlled to ensure that the temperature of the slurry heated with agitation until the next 180 minutes with the installation of vertical pipe condenser for reflux was in the range of 90-100 C. at atmospheric pressure. Upon completion of H.sub.2SO.sub.4 acid addition, the solution is still heated with agitation until the next 120 minutes with the installation of vertical pipe condenser for reflux purposes to maintain the volume of the liquid until a very clear solution is achieved; then to this clear solution was added dropwise a solution of 185 g NaOH flakes in 190 ml of water with the rate of addition being controlled so that the temperature of the solution remained at 5020 C. within one hours time. Then the vertical pipe condenser was released, the resulting solution is still heated with another 30 minutes agitation to ensure complete and homogenous neutralization reaction.
(83) Then the liquid temperature was raised to 100-105 C. Evaporate half of the solvent volume followed by crystallization result in formation of the desired substance as an off white powder with the following properties: melting point >360 C., boiling point >400 C., specific gravity=2.21, water solubility 29% and higroscopic.
(84) Analytical Method:
(85) Analytical Method to Determine Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-2Na), Utilizing Iron's Chelating Agent
(86) Test Principle:
(87) Active matter of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-2Na) was determined by chelating reaction of residual metallic ion with several anionic surfactants that prevent this anionic surfactants dissolved in the solution result in the turbid solution measured by its turbidity and/or absorbance spectrophotometer.
(88) Materials and Equipments:
(89) Materials:
(90) Reagent grade NH.sub.3FeSO.sub.4.2H.sub.2O, reagent grade Natrium Laurier Ethoxylate sulphate (Na-SLES), Caustic Soda flakes, aquadess.
(91) Equipments:
(92) Visible Spectrofotometer, cuvet, erlen meyer, beaker glass, pipete, scale weight, magnetic stirrer.
(93) Method:
(94) I. Absorbance:
(95) 1) Add 2 gr Na-SLES into 100 cc aquades, stir with magnetic stirrer for 15 minutes until clear solution (1) reached. 2) Add on 1.0 cc of tested chelating agent (THES-2Na), stir with magnetic stirrer for 15 minutes until homogenous solution (2). 3) Adjust the pH of the solution above into 7 with Caustic Soda flakes. 4) Prepare 50 cc 0.1 M NH3FeSO4 solution in the 100 cc beaker glass. 5) Fill the cuvet with the solution (2), record the absorbance at 450 nm. 6) Titration starts with the addition of 1.0 cc 0.1 M NH3FeSO4 solution into solution (2), stir with magnetic stirrer for 2 minutes until homogenous solution (3). 7) Fill the cuvet with the solution (3), record the absorbance at 450 nm. 8) Repeat step 5 and 6 until end point of 0.1 M NH3FeSO4 reached.
End Point and Calculation
I. Absorbance Method: 1) Absorbance reading will show significant increases, until some points, then the reading will decrease. 2) When this absorbance reading suddenly jump from the increasing trend into decreasing trend, the end point of the titration has reached. 3) Continue the NH3FeSO4 solution addition by another 2 or 3 cc volume to get the smooth curve and/or representative data for accurate interpolation of the end point volume. 4) Draw the absorbance curve versus titrant volume addition then determine the end point volume by this curve or using all absorbance and NH3FeSO4 volume addition through Lagrance interpolation formula to get the end point volume. 5) Calculate the concentration or active matter of Tetra Hydroxy Ethyl Di Sulphate Di Natrium (THES-2Na) using the formula below:
THES 2Na content=(V epM NH3FeSO4MW THES 2Na3.9)/(V samplesample SG1,000) where: V ep: end point volume in cc M NH3FeSO4: molarity NH3FeSO4: 0.1 M BM THES 2Na: molecule weight THES 2Na: 330 V sample: sample volume of tested chelant in cc
EXAMPLE
(96) Take 1.0 cc of 35% Tetra Hidroksi Etil Di Sulfat Di Natrium (THES-2Na) solution as sample, density of 1.1 g/cc. Then add on Na-SLES, titrate with 0.1 M NH3FeSO4; the absorbance record shown in the Table 1 below:
(97) TABLE-US-00001 TABLE 1 ml titer Absorbance No NH3FeSO4 450 nm 1 0 0.001 2 1 0.011 3 2 0.025 4 3 0.042 5 4 0.156 6 5 0.241 7 6 0.32 8 7 0.355 9 8 0.385 10 9 0.397 11 10 0.404 12 11 0.41 13 12 0.411 14 13 0.414 15 14 0.413 16 15 0.412
(98) From Table 1 above, the absorbance curve versus volume NH3FeSO4 trend is shown in
(99) From
THES-2Na concentration=3.2 cc0.1 M3303.9/(1.01.151,000)=0.358=35.8% of chelating agent.
Characteristics of this New Invention Substances
(100) These substances of this new invention are preferred to have the characteristics as below: a. Tetra Hydroxy Ethyl Di Sulphate (THES)
(101) TABLE-US-00002 Molecular weight 286 Crystal density (g/cuCm) 1.865 Solubility @ 25 C. (%) 48.0 pH of 30% solution <0.5 Melting point ( C.) 218 Boiling point ( C.) >400 Colour Off white Crystal charactheristic Hygroscopic b. Tetra Hydroxy Ethyl Di Sulphate Di Natrium
(102) TABLE-US-00003 Molecular weight 330 Crystal density (g/cuCm) 2.215 Solubility @ 25 C. (%) 31.5 pH of 30% solution 0.5-1.5 Melting point ( C.) 287 Boiling point ( C.) >400 Colour Transparance Crystal charactheristic Slightly hygroscopic c. Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate
(103) TABLE-US-00004 Molecular weight 506 Crystal density (g/cuCm) 2.28 Solubility @ 25 C. (%) 39.6 pH of 30% solution <0.0 Melting point ( C.) 242 Boiling point ( C.) >400 Colour White brownies Crystal charactheristic Very hygroscopic d. Tetra Hydroxy Butyl 1,2,3,4 Tetra Sulphate Tetra Natrium
(104) TABLE-US-00005 Molecular weight 594 Crystal density (g/cuCm) 2.978 Solubility @ 25 C. (%) 27.3 pH of 30% solution 0.0-1.0 Melting point ( C.) 288 Boiling point ( C.) >400 Colour Transparance Crystal charactheristic hygroscopic e. Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate
(105) TABLE-US-00006 Molecular weight 393 Crystal density (g/cuCm) 1.97 Solubility @ 25 C. (%) 56.7 pH of 30% solution <0.0 Melting point ( C.) 191 Boiling point ( C.) >400 Colour White brownies Crystal charactheristic Very hygroscopic f. Tri Hydroxy Isobutyl 1,2,3-Tri Sulphate Tri Natrium
(106) TABLE-US-00007 Molecular weight 459 Crystal density (g/cuCm) 2.35 Solubility @ 25 C. (%) 36.2 pH of 30% solution 0.0-1.0 Melting point ( C.) 242 Boiling point ( C.) >400 Colour Off white Crystal charactheristic hygroscopic g. Tetra Hydroxy Isoamyl 1,2,3,4-Tetra Sulphate
(107) TABLE-US-00008 Molecular weight 519 Crystal density (g/cuCm) 2.07 Solubility @ 25 C. (%) 61.2 pH of 30% solution <0 Melting point ( C.) 211 Boiling point ( C.) >400 Colour White brownies Crystal charactheristic Very hygroscopic h. Tetra Hydroxy Isoamyl 1,2,3,4-Tetra Sulphate Tetra Natrium.
(108) TABLE-US-00009 Molecular weight 607 Crystal density (g/cuCm) 2.63 Solubility @ 25 C. (%) 40.1 pH of 30% solution 0.0-0.5 Melting point ( C.) 247 Boiling point ( C.) >400 Colour Off white Crystal charactheristic hygroscopic i. Asam Etil Heksa Sulfat (EHS)
(109) TABLE-US-00010 Molecular weight 606 Crystal density (g/cuCm) 1.87 Solubility @ 25 C. (%) 48% pH of 30% solution 0.0 Melting point ( C.) >210 C. Boiling point ( C.) >400 C. Colour Off white Crystal charactheristic hygroscopic j. Etil Heksa Sulfat-Heksa Natrium (EHS-6Na)
(110) TABLE-US-00011 Molecular weight 738 Crystal density (g/cuCm) 1.87 Solubility @ 25 C. (%) 48% pH of 30% solution 0.0-0.5 Melting point ( C.) >210 C. Boiling point ( C.) >400 C. Colour Off white Crystal charactheristic hygroscopic
Ability to Enlarge the Simple Cell Membrane
(111) The substances of this invention were found to have the ability to modify the cell membrane or organic membrane by means enlarging/loosening membrane porosity significantly. By not totally theoretically supported, it was postulated due to the sulphate group electronegativity in the said substances as the chelating agent will loosen the hydroxy-hydroxy bound of a peptidoglycan or hydroxy-ammine bound of a protein that compose the membrane cell nor organic membrane. The ability of these substances to enlarge/loosen the membrane porosity were proven by opening/enlarging the microorganism cell membrane, ie: bacteri, algae, fungi, virus, etc with such concentration level that works on the cell wall membrane modification from the semi permeable into more permeable until it is possible to insert another ligands inside the microorganism or the cell wall membrane becomes totally permeable that result on the microorganism death or its latent position. It has been thought that the said mechanism works for all microorganism that has the cell membrane. The said ability to modify the simple cell membrane permeability will be shown by the mechanism and testing data below:
(112) ##STR00022##
Method:
(113) The ability of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES) as a Sulphate chelating agent to open/loosen the cell membrane was tested against two types of bacterias, ie: Staphylococcus aerus (gram positive) that has two cell membrane (the outer membrane as the outer cell wall and the inner membrane) and E. Colli (gram negative) that has three cell membranes. First, the Minimum Inhibitory Concentration (MIC) of each bacterias was determined, then the bacteria membrane alteration after contacting with THES were observed by Electron Microscope (TEM and SEM) with the THES concentration range below and above MIC. Antibacterial activity was tested using disc-diffusion method by checking the clear zone from the paper disc inhibition that contains THES. The growth curve were made by means examining the bacterial culture on the broth media.
(114) Materials and Equipments:
(115) Materials
(116) Difco brand nutrient broth, aquadistilata, THES (tetra hydroxy ethyl di sulphate di natrium) as the tested substance, glutaraldehyde, paraformaldehyde, albumin serum bovin (BSA), buffer phosphate pH 7, NaCl 0.9%, white LR resin, blue toluidine, Natrium citrate trihydrate, NaOH 0.1N, uranil acetate, lead nitrate (Pb(NO.sub.3).sub.2), parafin, transparan capsule, glass cutter, grid, collodion, gold powder.
(117) Equipments:
(118) Incubator, autoclave, petri dish, ose, spectrophotometer (Bausch&Lomb), vibrating incubator, glassware, ultra mikrotom, knife maker, visible microscope, grid pad, transmission electron microscope (Philips), scanning electron microscope (Hitachi).
(119) TABLE-US-00012 TABLE 2 Result of Biocide effication plate test of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-Di Natrium) Total Plate Count Killing No Biocide dosing (Colony/ml) efficiency Total Plate Count Microorganism 1 Blanco/Control 2400 0 2 0.05% THES-di Natrium 132 94.5 3 0.1% THES-di Natrium 115 95.2 4 0.5% THES-di Natrium 101 95.8 5 1.0% THES-di Natrium 80 96.7 E. Coli 1 Blanco 3900 0 2 0.025% THES-di Natrium 2010 48.5 3 0.05% THES-di Natrium 1150 70.5 4 0.1% THES-di Natrium 1050 73.1 5 0.5% THES-di Natrium 970 75.1 6 1.0% THES-di Natrium 720 81.5 7 5.0% THES-di Natrium 630 83.8 Staphylococcus Aerus 1 Blanco 980 0 2 0.025% THES-di Natrium 296 69.8
Test on Microorganism:
(120) Staphylococcus aerus ATCC 6538, Escherichia coli ATCC 9637 were supplied from Chemotherapy Laboratory of Pharmaceutical Department, Bandung Institute of Technology (ITB).
(121) Method:
(122) 1. Bacteria preparation The testing bacterias were growth on the aqueous medium and incubated for 24 hours at 37 C. 2. Tested Biocide preparation The tested Biocide was diluted in distillate water at various gradually decreased concentration. This gradual concentration variation is necessary to determine MIC (Minimum Inhibitory Concentration) and the bacteriostatic/bactericidal properties of the tested Biocide. 3. Determination of Killing method (bacteriostatic and bactericidal properties) The testing was done on the aqueous medium contains the tested substance with the concentration 5, 10 and 20%. The turbidity of the cultured medium then will be measured every 30 minutes until 270 minutes with spectrophotometer. 4. Preparation of microbe cells for electron microscope examination Staphylococcus aereus and Escherichia coli were growth on the aqueous medium within 24 hours, then mixed with tetra hydroxy ethyl di sulphate di sodium (THES) with the final concentration 0.6; 5 and 10%. This system mixtures then was placed under vibrating incubator for several hours. Microbe cells then were separated and washed, first with NaCl 0.9% then with distilled water. Cells then were utilized for the preparation of electron microscope testing. 5. Preparation for Scanning Electron Microscope (SEM) examination Bacteria cells were suspended into 4% collodion. The said suspensions then were fixed on some metal disc and were coated with the gold powder. Then were examined with electron microscope and pictured. 6. Preparation for Transmittance Electron Microscope (TEM) examination a) The prepared microbes in (5) were fixed into a mixture of formaldehyde 2% and glutaraldehyde 0.5% for 2 hours at 4 C. b) The above mixtures then was three times washed with pH 7 phosphate buffer, then were diluted into bovin serum albumin (BSA) 1/5 for embedded preparation, while leave glutaraldehyde hardening at room temperature. c) After leaving dehydrated in alcohol, the white LR resin then will be infiltrated. d) Embedded process were done at white LR resin following by polymerization process at 60 C. for 48 hours time. The polymerization product as hardened capsules then are ready to be sliced. e) Making of semi thin and ultra thin slices. Slices of microbe sample were made by utilizing automatic ultra microtom. To get the precised and accurate slices of sample, semi thin slice were made and coloured with blue toluidine then were examined under the visible microscope. When the right slices were obtained, ultra thin slices were prepared and were made contrast with uranil acetate and lead citrate. The dried ultra thin slices then can be examined with Transmittance Electron Microscope.
Testing Results
Minimum Inhibitory Concentration (MIC)
(123) MIC of tetra hydroxy ethyl di sulphate di natrium against E. coli and S. aureus are shown in Table 3.
(124) TABLE-US-00013 TABLE 3 Antibacterial activity of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-Di Natrium) Minimum Inhibitory Concentration No Microorganisme (MIC) (%) Gram Positive Bacteria 1 Bacillus Subtilis ATCC 0.234 6633 2 Staphylococcus Aerus ATCC 0.234 6538 3 Staphylococcus Epidermidis 0.234 ATCC 12228 Gram Negative Bacteria 1 Acinetobacter Anitratus 0.115 2 Escherichia Coli ATCC 0.234 25922 3 Pseudomonas Aeruginosa 0.115 ATCC 27853 4 Klebsiella Pneumoniae 0.234 5 Salmonella Typhii 0.115 Fungi 1 Aspergillus Niger 0.937 2 Candida Albicans 0.469
(125) The test done on the pH range of 7-7.5 with the sample of Tetra Hydroxy Ethyl Di Sulphate-Di Natrium (THES-Di Natrium) 30% in aqueous solution.
(126) From the transversal section (TEM) of Staphylococcus aerus cell, can be observed between normal cell that still contains cytoplasm and other organic substances (dark sight) and the partly empty cells that has already lost its cytoplasm liquid due to lysis out of cell wall membrane (transparent sight), in fact the E. Colli cell that has more layers cell wall membranes also experienced the said internal cell liquid lysis out of its cell wall membrane layers, although the MIC of E. Colli cell was slightly higher than Staphylococcus aerus cell.
(127) From the outer surface of bacteria's cell wall observation through Scanning Electron Microscope (SEM), it was shown that the outer cell wall membrane of bacteria looked wrinkled/corrugated due to the wider cell wall surface compared to normal cell wall for both Staphylococcus aerus nor E. Colli.