MANUFACTURE OF AMYL m-CRESOL

Field of the Invention

The present invention relates to novel processes to a therapeutically active agent and to novel forms of the crude active agent pertaining thereto and to novel processes for preparing intermediates thereto.

More particularly, the invention relates to novel processes for the preparation of amyl- m-cresol and to intermediates for use in the preparation of amyl-w-cresol.

Background to the Invention

Amyl-zw-cresol (AMC) is 5-methyl-2-pentyl phenol of formula I:

AMC is useful as an antiseptic, germicide and a mould preventative. It is best known as an active ingredient in preparations for the treatment of fungal infections of the throat and/or oral cavity, for example, in lozenges, such as Strepsils® where it is used in conjunction with other active ingredients such as 2, 4-dichlorobenzyl alcohol.

The synthesis of AMC was first reported in 1930. UK Patent No. 330,333 (Boots Pure Drug Company Limited) describes the preparation of AMC by the rearrangement of /w-cresol valerate (MCV) in the presence of aluminium chloride, to form valeryl m- cresol. The valeryl m-cresol is then reduced with zinc amalgam to form AMC.

The current process used for the large scale manufacture of AMC follows a similar general synthetic route and comprises the formation of the intermediate valeryl-m- cresol (VMC) followed by reduction of VMC to form AMC. However, it has been found that operation of the prior art process uses reagents which are undesirable, especially in a large scale manufacturing process, such as, phosphoryl chloride. Furthermore, the prior art process requires several complex multi-stage fractional distillations as part of the process in order to be able to manufacture AMC capable of meeting the purity level needed for pharmaceutical use.

VMC

In the existing method, synthesis of VMC is followed by a complex fractional distillation procedure in order to purify the VMC prior to further processing, i.e. conversion into AMC.

The purified VMC is then reduced using zinc amalgam to form AMC: AMC

We have found that in order to meet the purity requirement for pharmaceutical use, AMC made by the prior art method requires further processing including fractional distillation.

Thus, overall the existing process used for large scale manufacture of VMC and AMC is complex and inefficient since, inter alia,

the VMC process uses A1C1 ₃ or POCl ₃ , high temperatures and lengthy reaction times; the VMC process requires complex fractional distillations;

the AMC process requires long reaction times, provides poor conversion of VMC to AMC and produces significant levels of impurity in the product; and

the AMC process requires numerous fractional distillations.

A major disadvantage of the existing process is the use of aluminium chloride (AICI3) or phosphoryl chloride (POCl ₃ ), since these are hazardous chemicals. It will be understood by the person skilled in the art that the destruction of aluminium chloride or phosphoryl chloride is a particular problem since the hydrolysis is highly exothermic and generates hydrogen chloride gas. In the case of phosphoryl chloride there can be a considerable delay in the onset of the exothermic hydrolysis reaction, which may lead to a hazardous situation when carried out at large scale. A layer of dense and cold liquid (POCI3) may survive for several minutes under water before violent, almost instantaneous, hydrolysis occurs when disturbed.

A further disadvantage of the use of aluminium chloride or phosphoryl chloride is the production of large volumes of hazardous waste, which presents a significant environmental challenge in its disposal.

Thus, there is a long felt need for an improved process that, inter alia, avoids the use of particularly hazardous chemicals, produces a less impure product and/or intermediate, requires less onerous purification in order to meet the purity required for pharmaceutical use, and is generally more efficient.

Summary of the Invention

We have now found a novel synthetic route to AMC and VMC which overcomes or mitigates the disadvantages of the prior art process.

Thus, according to a first aspect of the invention we provide a process for the preparation of amyl /n-cresol (AMC) which comprises the steps of;

(i) reaction of m-cresol with valeric acid, or a derivative thereof, (to form m- cresol valerate (3-toluoyl valerate));

(ii) rearrangement, optionally in situ, of 3-toluoyl valerate in the presence of an oxoacid, provided that the oxoacid is not a carboxylic acid or nitric acid; and

(iii) reduction of valeryl w-cresol (VMC). The derivative of valeric acid may comprise any derivative suitably reactive towards a phenol, e.g. the -OH of m-cresol, such as, an acid anhydride or an acid halide, for example, the acid chloride. The reaction of /w-cresol with, for example, valeric acid anhydride or valeric acid halide to form 3-toluoyl valerate (step (i)) is desirably carried out at elevated temperature, such as, from 30 to 40°C, e.g. about 35°C. The reaction of step (i) may be carried out in an organic solvent, e.g. an aromatic solvent such as toluene. However, the reaction is desirably carried out in the absence of any solvent.

The term "oxoacid" will be understood by the person skilled in the art, for example, IUPAC defines "oxoacid" as a compound which contains oxygen, at least one other element, and at least one hydrogen bound to oxygen, and which produces a conjugate base by loss of positive hydrogen ion(s). See, for example, the IUPAC Gold Book at http://old.iupac.org/goldbook/O04374.pdf.

In a preferred embodiment of the present invention, the oxoacid comprises an acid moiety of formula I:

Y OH

wherein X is any atom capable of forming at least one covalent double bond with oxygen, provided that X is not carbon;

R is bonded to X via a single or double bond and R is alkyl CI to 10, aryl, OH, -OR ¹ or R is a solid support (e.g. an ion exchange resin) connected to X via a single bond or R is O connected to X via a double bond or R is absent;

Y is oxygen, alkyl, aryl or solid support and may be bonded to X via a single or double bond. Y may also be absent;

R ¹ is alkyl CI to 10 or aryl; and

when both R and Y are absent X is nitrogen.

In the structure of the moiety of formula I each of R and Y may be bonded by a single or a double bond. For the avoidance of doubt, the oxoacid moiety of formula I may be represented by any one of the following:

Preferably X is sulphur or phosphorus.

Although the pKa of the oxoacid used in the process of the invention may vary, preferably, the oxoacid has a pKa of from, about +3 to -14, although it will be understood by the person skilled in the art that the pKa of the oxoacid should be non- limiting. Thus, according to a further aspect of the invention there is provided a process for the preparation of AMC which comprises the steps of;

(i) reaction of m-cresol with valeric acid, or a derivative thereof, (to form m- cresol valerate (3-toluoyl valerate));

(ii) rearrangement, optionally in situ, of 3-toluoyl valerate in the presence of an oxoacid, the oxoacid having a pKa of from +3 to -14, to form VMC; and

(iii) reduction of VMC.

The oxoacid facilitates the rearrangement reaction by acting as a catalyst, although it will be understood by the person skilled in the art that the oxoacid may be present in stoichiometric amounts.

In the process of the invention, the 3-toluoyl valerate formed in (step (i)) may desirably be used in situ in the rearrangement reaction (step (ii)). As hereinbefore described, the rearrangement reaction of 3-toluoyl valerate to form VMC (step (ii)) is carried out in the presence of an oxoacid. For example, the oxoacid may desirably be an oxoacid, preferably with a pKa of from about +3 to about -14, preferably of from +3 to -7, more preferably of from +1 to -5, and more preferably of from -1 to -3, e.g. about -2. It will be understood that the pKa is generally provided as the logarithmic measure of the acid dissociation constant (-log ₁₀ Ka) in water or extrapolated for water at 25°C. However, it will also be understood that some organic acids, for example toluene sulphonic acid, is such a strong acid that its pKa is not measurable in water. Nevertheless, the person skilled in the art will understand that the pKa, especially of such strong acids as toluene sulphonic acid, may be measured in solvents such as acetonitrile or dimethylsulphoxide. Thus, such strong oxoacids which may be used in the rearrangement of the present invention include those oxoacids whose pKa are extrapolated for water at 25°C. For the avoidance of doubt, the pKa range of +3 to - 14 is intended to include both "measured" pKa and "extrapolated" pKa. By way of example only, the following acids have "extrapolated" pKa's as shown:

* either measured or extrapolated for water at 25 °C.

A variety of oxoacids as hereinbefore described may be used, and preferably an oxoacid has a pKa of from about +3 to about -7 in water or extrapolated for water at 25°C. Such oxoacids can include, for example, alkyl(Cl to 10)sulphonic acids, aryl- sulphonic acids, / oluenesulphonic acid, and the like. A preferred oxoacid is, for example, a sulphonic acid or a sulphinic acid. Preferably, but not essentially, the oxoacid has a pKa of from about +3 to about -14 in water or extrapolated for water at 25 °C, as hereinbefore defined. It is within the scope of the present invention that a mixture of one or more of oxoacids may be used, for example, a mixture of oxoacids with a pKa of from about +3 to about -14 in water or extrapolated for water at 25°C. A preferred oxoacid is a sulphonic acid. Such sulphonic acids shall include one or more acidic ion exchange resins, such as, one or more of a Dowex® resin, an Amberlite resin or an Amberlyst resin or other similar resins known to the person skilled in the art. Dowex®, Amberlite™ and Amberlyst™ resins are known to the person skilled in the art and are sulphonic acid derivatives of polystyrene used as ion exchange resins. Dowex® resins are commercially available in the UK from a variety of sources, e.g. Sigma Aldrich. Amberlite™ and Amberlyst™ resins are available in the UK from a variety of sources including the Dow Chemical Company. The use of such resins, e.g. a Dowex® resin, is advantageous in that, inter alia, it may facilitate either a batch or a continuous process, i.e. a continuous flow process, and may remove the need for aqueous work-up.

Specific oxoacids which may be mentioned include, but shall not be limited to, one or more of methanesulphonic acid, sulphuric acid, phosphoric acid, camphorsulphonic acid, benzenesulphonic acid, p-toluenesulphonic acid, trifluoromethanesulphonic acid and Dowex® Amberlyst 15-Wet.

An especially preferred oxoacid is an alkyl (CI to 10) sulphonic acid, preferably an alkyl (CI to 6) sulphonic acid, and especially methanesulphonic acid.

As used herein, the term "alkyl" refers to a hydrocarbon moiety, a straight chain or branched chain, i.e. primary, secondary or tertiary alkyl. Where not otherwise identified, preferably the alkyl comprises 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, wo-propyl, n-butyl, sec-butyl, wo-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, «-octyl, n-nonyl, «-decyl and the like. Where appropriate the term "alkyl" may refer to cycloalkyl or alkyl substituted by cycloalkyl or alkyl substituted by aryl.

As used herein, the term "aryl" refers to 6-carbon monocyclic, 10-carbon bicyclic, 14- carbon tricyclic aromatic ring system. Examples of "aryl" are phenyl, and naphthyl. As used herein, the term "aryl" preferably refers to phenyl.

The amount of oxoacid present may vary, depending upon, inter alia, the nature of the oxoacid used, etc. However, a preferred amount of oxoacid, e.g. when the oxoacid is a sulpho ic acid as hereinbefore described, is from 0.2 to 2 molar equivalents (relative to the 3-toluoyl valerate), preferably from 0.2 to 1 molar equivalents, more preferably from 0.2 to 0.6 molar equivalents, especially about 0.4 molar equivalents.

The rearrangement is also desirably carried out at elevated temperature.

One advantage of the process of the invention is that the preparation of VMC may be conducted at a temperature lower than the prior art process(es) which are generally carried out at about 160°C. Therefore, in the process of the present invention for the preparation of VMC which comprises the rearrangement of 3-toluoyl valerate, the rearrangement may desirably be carried out at a temperature of below 160°C, preferably, at a temperature of from 100 to 130°C, more preferably at a temperature of about 110°C.

The process for the preparation of AMC is novel per se and advantageous since, inter alia, it produces AMC with an improved impurity profile, i.e. AMC which requires a much more simplified, two stage, fractional distillation to remove undesirable impurities. It is a particular aspect of the invention that the AMC with an improved impurity profile may be prepared with a reduced number of fractional distillations. Whilst in prior art methods the impurity profile of AMC is generally improved by repeated fractional distillations, the process of the present invention requires reduced number of fractional distillations to obtain AMC to a medicinal standard. Thus, according to the present invention we provide a process as hereinbefore described for the preparation of AMC with an improved impurity profile, i.e. prior to fractional distillation and/or hydroxylamine treatment as hereinafter described.

A major impurity in AMC is a ketone impurity, 5-methyl-2-pentylcyclohexanone;

Thus, the term "improved impurity profile" may mean AMC which has less than 4% w/w of 5-methyl-2-pentylcyclohexanone impurity, preferably less than 3% w/w of 5- methyl-2-pentylcyclohexanone impurity, more preferably less than 2% w/w of 5- methyl-2-pentylcyclohexanone impurity, i.e. from 1.5 to less than 2% w/w of 5- methyl-2-pentylcyclohexanone impurity. This compares to an average level of methyl-2-pentylcyclohexanone impurity in the current large scale manufacturing process of more than 4.5% w/w. The amount of 5-methyl-2-pentylcyclohexanone present in the AMC prepared by the process of the invention can generally be determined by gas chromatography techniques as defined by Example 3 herein.

The ketone impurity, 5-methyl-2-pentylcyclohexanone, may be removed from the crude AMC by formation of an oxime, e.g. by treatment of the crude reaction mixture with hydroxylamine and subsequent fractional distillation. The residual 5-methyl-2- pentylcyclohexanone oxime remains after the desired AMC is distilled off. Removal of the ketone impurity by oxime formation has not been previously disclosed and therefore forms a novel aspect of the present invention. Therefore, according to a further aspect of the invention any ketone impurity is substantially converted to an oxime, e.g. by treatment with hydroxylamine.

In addition to the removal of the ketone impurity, residual w-cresol valerate (3-toluoyl valerate) may be substantially removed by treatment with a base, such as sodium carbonate. The base treatment will generally comprise heating the crude AMC with a base, such as sodium carbonate. It will be understood by the person skilled in the art that this base treatment may be conducted during or prior to fractional distillation. However, it is preferred to conduct the base treatment in situ with the fractional distillation. The amount of base used may vary depending upon, inter alia, the nature of the base, the anticipated amount of residual m-cresol valerate, etc.

Although, as hereinbefore described, the AMC with an improved impurity profile prepared by the process of the present invention can be prepared by the use of fractional distillation techniques, crude AMC with an improved impurity profile as hereinbefore defined, i.e. prior to hydroxylamine treatment and/or fractional distillation, is novel per se. Therefore, according to a further aspect of the invention there is provided a form of crude AMC which has less than 4% w/w of 5-methyl-2- pentylcyclohexanone impurity, i.e. prior to hydroxylamine treatment and/or fractional distillation. The invention particularly provides crude AMC which has less than 3% w/w of 5-methyl-2-pentylcyclohexanone impurity, more preferably less than 2% w/w of 5-methyl-2-pentylcyclohexanone impurity, i.e. from 1.5 to less than 2% w/w of 5- methyl-2-pentylcyclohexanone impurity, as hereinbefore defined. A further impurity found in AMC is the alcohol, 2-(l-hydroxypentyl)-5- methylphenol. This impurity is especially undesirable since it may dehydrate to form the corresponding alkene during the distillation step.

In addition the process of the present invention is also advantageous in that the reduction of VMC to AMC (step (iii)) comprises a hydrogenation which is, inter alia, more efficient than prior art reduction methods.

Thus, the hydrogenation step of the process of the present invention (step (iii)) preferably comprises the use of a palladium catalyst, such as palladium on charcoal. Advantageously the hydrogenation step according to this aspect of the invention utilises commercial grades of palladium on charcoal, such as 5% or 10% w/w palladium on charcoal, preferably 5% w/w palladium on charcoal, more preferably Johnson Matthey® palladium on charcoal catalysts of type 39, most preferably Johnson Matthey® palladium on charcoal type 394, 5% w/w, 5% water wet.

Hydrogenation of VMC, e.g. using a palladium on charcoal catalyst, has not been previously disclosed and therefore forms a novel aspect of the present invention. Therefore, according to a further aspect of the invention the process provides the reduction of VMC, e.g. VMC prepared according to the process hereinbefore described, to AMC, by hydrogenation. The hydrogenation according to this aspect of the invention may be carried out in a variety of solvents. A non-basic reaction environment is preferred, thus the solvent may be neutral or acidic, for example a protic solvent may be used, such as water or, preferably, an alcohol, e.g. an alkyl (CI to CIO) alcohol, such as methanol, ethanol or isopropyl alcohol, preferably isopropyl alcohol. However, it will be understood by the person skilled in the art that mixtures of solvents maybe used, for example, aqueous alcohols, mixtures of different protic solvents, e.g. alcohols, one or more protic solvents, e.g. alcohols, mixed with one or more other organic solvents, such as toluene, etc.

As hereinbefore described the hydrogenation process should be carried out in a pH neutral or an acidic environment. The use of a pH neutral or an acidic environment is advantageous, in that, inter alia, it reduces the level of ketone impurity in the final AMC product. One or more acidic additives, such as, citric acid and the like, may be included in the hydrogenation reaction. Although a variety of acidic additives may be used in the hydrogenation process of the invention, desirably, the hydrogenation may be carried out using an acidic additive that gives a resulting reaction mixture of from about pH 0 to about pH 7, preferably from about pH 1 to about pH 6, preferably from about pH 1 to about pH 5, preferably from about pH 2 to about pH 4, preferably from about pH 2.5 to about pH 3.5, e.g. about pH 3, when measured at room temperature. A preferred acidic additive is an organic carboxylic acid, for example, citric acid. The solvent being protic facilitates the hydrogenolysis reaction whilst the acidic additive helps prevent the ketone formation.

The process for the preparation of the intermediate VMC is also novel per se and advantageous since, inter alia, it is useful in the preparation of AMC as hereinbefore described. Thus, according to an alternative aspect of the invention we provide a process for the preparation of VMC which comprises the steps of;

(i) reaction of w-cresol with valeric acid, or a derivative thereof, (to form 3- toluoyl valerate fm-cresol valerate)); and

(ii) rearrangement, optionally in situ, of 3-toluoyl valerate to form VMC in the presence of an oxoacid, provided that the oxoacid is not a carboxylic acid or nitric acid.

In the process of the present invention the 3-toluoyl valerate formed by the reaction of w-cresol with valeric acid, or a derivative thereof, e.g. a valeric acid anhydride or valeric acid halide, may be used in situ in the rearrangement of 3-toluoyl valerate to form VMC.

Therefore, according to a further aspect of the invention we provide a one stage process for the preparation of VMC with an improved impurity profile which comprises the steps of;

(i) reaction of τη-cresol with valeric acid, or a derivative thereof, such as, a valeric acid anhydride or valeric acid halide, to form 3-toluoyl valerate; and

(ii) rearrangement of 3-toluoyl valerate to form VMC in the presence of an oxoacid, provided that the oxoacid is not a carboxylic acid or nitric acid.

A further advantage of the process of the invention is that the preparation of VMC may be conducted at a temperature lower than the prior art process(es) which are generally carried out at about 160°C. Therefore, in the process of the present invention for the preparation of VMC which comprises the rearrangement of 3-toluoyl valerate, the rearrangement may desirably be carried out at a temperature of below 160°C, preferably, at a temperature of from 100 to 130°C, more preferably at a temperature of about 110°C.

The AMC with an improved impurity profile prepared by the method as hereinbefore described is advantageous in that, inter alia, it may be used in the preparation of AMC which can be included in a composition for the treatment of mild bacterial or fungal infections of the throat and/or oral cavity. Thus, according to this aspect of the invention there is provided a composition comprising AMC prepared by a process as hereinbefore described in admixture with a suitable adjuvant, diluent or carrier.

The invention further provides a composition comprising AMC prepared via crude AMC which has less than 4% w/w of 5-methyl-2-pentylcyclohexanone impurity prior to hydroxylamine treatment and/or fractional distillation in admixture with a suitable, e.g. a pharmaceutically acceptable, adjuvant, diluent or carrier.

Such a composition may, for example, be in the form of a lozenge, gel, spray, capsule, pastille, gum or tablet. The composition according to this aspect of the invention may also include one or more other antiseptic, antibacterial and analgesic therapeutic agents. Such agents include but shall not be limited to, one or more of, analgesics, such as flurbiprofen and oxycodone; antiseptics, such as, 2,4-dichlorobenzyl alcohol (DCBA) and hexylresorcinol; cetylpyridinium chloride, dequalinium chloride and combined therapies, such as oxyprofen; local anaesthetics, such as, lidocaine, benzocaine and menthol; mucolytic agents, such as, ambroxol hydrochloride; antitussive agents, such as, dextromethorphan hydrobromide; expectorants, such as, guaifenesin; and combinations thereof.

According to a yet further aspect of the invention there is provided the use of AMC prepared by a process as hereinbefore described in the treatment of a mild bacterial or fungal infection of the throat and/or oral cavity.

There is therefore also provided a method of treatment of mild bacterial or fungal infections of the throat and/or oral cavity which comprises administering AMC prepared by a process as hereinbefore described or a composition thereof.

Examples:

The following Examples serve to illustrate the invention without limiting its scope. Abbreviations used are those conventional in the art.

Abbreviations

AMC amyl-m-cresol

DI deionised

IPA wo-propanol

MCV m-cresol valerate

VMC valeryl w-cresol Example 1 Preparation of A my I m-CresoI

1.1. VMC Stage 1: Valeryl m-Cresol (Standard Process)

etor-cresol (157.4 g, 1.46 mol) was charged to a 1 litre 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (184.3 g, 1.53 mol) was added from a dropping funnel slowly over 30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then methanesulphonic acid was added (56.0 g, 0.58 mol) over 5 minutes. The reaction was stirred at 110°C for 7 hours, then cooled to between 40 and 50°C. Toluene (115.2 g) and deionised water (453.8 g) were charged to a second 1 litre three-necked flask. The reaction mixture from the first flask was added slowly to the second flask (toluene/water) with stirring, and then the mixture was stirred for 15 minutes. The contents of the flask were transferred to a 1 litre separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (115.2 g), and the layers separated as before. The organic layers were combined and washed with a solution of deionised water (190.6 g), sodium carbonate (16.0 g), and sodium chloride (18.8 g), and then a solution of deionised water (190.6 g) and sodium chloride (18.8 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product as a dark brown oil containing -5% toluene. Expected yield ~ 290 g

Purity data:

Data quoted by GC normalisation. VMC Stage 1:

1.2. VMC Stage 2: Distillation of Valeryl /w-Cresol

The crude valeryl /w-cresol from Example 1.1 was charged to a 1 litre three-necked flask and set up for fractional distillation with a. The pressure of the system was reduced to 50 mbar, and the contents heated to 110°C. Toluene was distilled off, then the pressure dropped to 35 mbar and the remaining toluene was distilled. The pressure was further reduced to 5 mbar and the temperature increased to 118°C. The system was left to equilibrate under these conditions for around 1 hour, until the first fraction (w-cresol) began to distil. Once no further material distilled over at this temperature, the temperature was increased stepwise to 125°C, 130°C and 140°C and further fractions collected at each temperature. Then the temperature was increased to 145°C and the main fraction collected.

Purity data:

Data quoted by GC normalisation. VMC Stage 2:

1.3. AMC Stage 1 Standard Process

VMC stage 2 (182.1g) was charged as a slurry in isopropyl alcohol (IP A) (440ml) to a 2 litre jacketed hydrogenator vessel. Palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (11.5g) was charged to the vessel as a slurry in EPA (220ml). EPA (220ml) was charged to the vessel and the jacket temperature set point was set to 17.5°C (operating temperature range 15 -20°C). The vessel was pressurised to 0.7bar with hydrogen and the vessel contents were hydrogenated for 16 hours. The reaction mixture was sampled and analysed for reaction completion by gas chromatography (GC). Once reaction completion had been achieved the reaction mixture was filtered at between 15 and 20°C to remove the catalyst via a sinter funnel. The catalyst was then washed with EPA (37 ml, 29.2 g) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation until the residue temperature reached 95°C.

Expected yield 218g Purity data:

The average level of 5-methyl-2-pentylcyclohexanone in the batches made via this process was 1.73% w/w, (this compares to about an average ketone level of 4.63% w/w when made by prior art processes).

1.4. AMC Stage 2/3

The charges for the oxime treatment to remove the ketone impurity were scaled based upon the quantity of the impurity present in the starting material.

Crude amyl mera-cresol stage 1 (218 g) was charged to a 1 litre three-necked flask, set up for distillation. Iso-propyl alcohol (EPA) (5.8g) was charged to the reaction flask followed by sodium carbonate (1 l.Og). The flask contents were heated to reflux for 30 minutes then the IP A removed by distillation until the internal temperature reached 140°C. The residue was cooled to less than 30°C and water (250ml) was charged. The flask contents were stirred for 30 minutes before being transferred to a separating funnel and the phases allowed to separate. The aqueous phase was removed and the product washed with water (150ml).

To a clean 1 litre three-necked flask hydroxylamine (3.962 x mass ketone), sodium acetate trihydrate (1.124 x mass ketone) and water ((12.081 x mass ketone) - (mass sodium acetate x 0.397)) were charged. The flask contents were warmed to 35-40°C and held at this temperature for 30 minutes. The crude AMC product was charged to the flask and the contents stirred at 35-40°C for 3 hours before being transferred to a separating funnel and the phases allowed to separate. The aqueous phase was removed and the product phase washed three times with water (5.055 x mass ketone). The crude AMC stage 3 was then purified via fractional distillation at 5mbar. A foreruns fraction (~5% total mass charged) was taken followed by a main fraction (~70% total mass charged) and two tail fractions (-8% each of the total mass charged).

Purity data:

The data given is for final product AMC.

Example 2

VMC Stage 1: Valeryl m-cresol

A eta-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeric anhydride (28.2g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour.

The temperature was raised to 110°C, then methanesulphonic acid was added (5.4 g,

0.056 mol). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C.

Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a

100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (19.1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 27.2 g. Composition: 2.47% m-cresol, 13.80% MCV, 82.57% VMC.

Example 3

VMC Stage 1: Valeryl m-cresol

Meta-cresol valerate (3.0 g, 15.6 mmol) was charged to a 25 ml flask. Then methanesulphonic acid was added (1 ml, 15.6 mmol), with stirring. The reaction was stirred at room temperature for -42 hours. Toluene (10 g) was charged to the flask followed cautiously by DI water (20 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 50ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (10 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (15 g), sodium carbonate (3.0 g), and sodium chloride (1.5 g), and then a solution of DI water (15 g) and sodium chloride (1.5 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 2.94 g. Composition: 8.7% m-cresol, 79.8% MCV, 9.4% VMC. Example 4

VMC Stage 1; Valeryl m-cresol

e/a-cresol valerate (1.0 g, 5.2 mmol) was charged to a 25 ml flask. The reaction was heated to 130°C with stirring, then methanesulphonic acid was added (169 μΐ, 2.6 mmol). The reaction was stirred at 130°C for 30 min, then cooled to <50°C. Toluene (5 g) was charged to the flask followed cautiously by DI water (15 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 50ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (10 g), sodium carbonate (2.0 g), and sodium chloride (1.0 g), and then a solution of DI water (10 g) and sodium chloride (1.0 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 1.02 g. Composition: 8.2% m-cresol, 13.6% MCV, 75.3% VMC.

Example 5

VMC Stage 1; Valeryl m-cresol

Meta-cresol valerate (1.0 g, 5.2 mmol) was charged to a 25 ml flask. The reaction was heated to 110°C with stirring, then methanesulphonic acid was added (34 μΐ, 0.52 mmol). The reaction was stirred at 110°C for 6 hours, then cooled to <50°C. Toluene (5 g) was charged to the flask followed cautiously by DI water (15 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 50ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (10 g), sodium carbonate (2.0 g), and sodium chloride (1.0 g), and then a solution of DI water (10 g) and sodium chloride (1.0 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 1.04 g. Composition: 4.8% m-cresol, 80.0% MCV, 8.7% VMC.

Example 6

VMC Stage 1: Valeryl m-cresol

Meta-cresol valerate (1.0 g, 5.2 mmol) was charged to a 25 ml flask. The reaction was heated to 110°C with stirring, then methanesulphonic acid was added (0.68 ml, 10.4 mmol). The reaction was stirred at 110°C for 5 hours, then cooled to <50°C. Toluene (5 g) was charged to the flask followed cautiously by DI water (15 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 50ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (10 g), sodium carbonate (2.0 g), and sodium chloride (1.0 g), and then a solution of DI water (10 g) and sodium chloride (1.0 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 0.96 g. Composition: 33.9% m-cresol, 5.2% MCV, 44.6% VMC.

Example 7

VMC Stage 1: Valeryl m-cresol

ero-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then sulphuric acid was added (2.7 g, 0.028 mol). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C. Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (19.1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 20.7 g.

Example 8

VMC Stage 1: Valeryl m-cresol

eta-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then phosphoric acid was added (1.8 g, 0.019 mol). The reaction was stirred at 1 10°C for 7 hours, then cooled to <50°C. Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (19.1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 26.0 g.

Example 9

VMC Stage 1; Valeryl m-cresol

Meta-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then camphorsulphonic acid was added (13.0 g, 0.056 mol). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C. Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (1 .1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 28.2 g. Example 10

VMC Stage 1: Valeryl m-cresol

Meta-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then benzenesulphonic acid was added (8.8 g, 0.056 mol). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C. Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (19.1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 24.0 g.

Example 11

VMC Stage 1; Valeryl m-cresol

M?ta-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then p-toluenesulphonic acid was added (9.6 g, 0.056 mol). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C. Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (19.1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 24.7 g.

Example 12

VMC Stage 1; Valeryl m-cresol

ew-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3-necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45 °C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then trifluoromethanesulphonic acid was added (8.4 g, 0.056 mol). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C. Toluene (11.5 g) was charged to the flask followed cautiously by DI water (45.3 g), and the mixture stirred for 15 minutes. The contents of the flask were transferred to a 100ml separating funnel and allowed to settle. The aqueous layer was removed, and the organic phase retained (product). The aqueous phase was extracted with toluene (11.5 g), and the layers separated as before. The organic layers were combined and washed with a solution of DI water (19.1 g), sodium carbonate (5.0 g), and sodium chloride (1.9 g), and then a solution of DI water (19.1 g) and sodium chloride (1.9 g). The organic layer was separated and the solvent removed in vacuo to leave the crude product. Crude yield 22.7 g.

Example 13

VMC Stage 1: Valeryl m-cresol

eta-cresol (15.6 g, 0.14 mol) was charged to a 100 ml 3 -necked flask. The reaction was heated to 35°C with stirring, then valeryl chloride (18.3g, 0.15 mol) was added slowly over 15-30 minutes. The reaction mixture was stirred for a further 30 minutes at around 35 to 45°C. Then the contents were heated to 80°C and stirred for 1 hour. The temperature was raised to 110°C, then Dowex® Amberlyst™ 15-wet (dried prior to use by flushing with methanol) was added (11.9 g, 0.4 eq). The reaction was stirred at 110°C for 7 hours, then cooled to <50°C. The resin was filtered and washed with toluene (3x 11.5g). The washings were added to the filtrate and the solvent removed in vacuo to leave the crude product. Crude yield 15.6 g.

Experimental Results (Examples 1.1, 7-13):

^♦ estimated value Example 14

AMC Stage 1: Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (4.6g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 25°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by gas chromatography (GC) analysis. Once reaction completion had been achieved (after around 1 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with Γ Α (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 49.5g.

Example 15

AMC Stage 1: Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (4.6g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 17.5°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IP A (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 52.8g. Example 16

AMC Stage 1: Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (4.6g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 12.5°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IP A (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 56.5g.

Example 17

AMC Stage 1: Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (4.6g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 7.5°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IPA (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 58.3g. Example 18

AMC Stage 1; Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (2.88g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 17.5°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IPA (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 69.1 g.

Example 19

AMC Stage 1: Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (4.6g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 17.5°C. The vessel was pressurised to 0.56bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 17 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IPA (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 56.2g. Example 20

AMC Stage 1: Amy! m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (2.88g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Methanol (220ml) was charged to the vessel and the jacket temperature was set to 17.5°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with methanol (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 42.5g.

Example 21

AMC Stage 1; Amy, m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (4.6g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.5g). Ethanol/methanol (95:5, 220ml) was charged to the vessel and the jacket temperature was set to 25°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with ethanol/ethanol (95:5, 10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 46.8g. Example 22

AMC Stage 1: Amyl m-cresol

VMC stage 2 (32. lg) and palladium on carbon catalyst (Johnson Matthey® type 39 10% w/w, 54.8% water wet) (3.22g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.35g). Iso-propanol (155ml) was charged to the vessel and the jacket temperature was set to 15 ~20°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IPA (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated rotary evaporation. Crude yield 33.6g.

Example 23

AMC Stage 1; Amyl m-cresol

VMC stage 2 (32. lg) and palladium on carbon catalyst (Johnson Matthey® type 58 5% w/w, 50% water wet) (6.44g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.35g). Iso-propanol (155ml) was charged to the vessel and the jacket temperature was set to 15 -20°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 16 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IPA (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated rotary evaporation. Crude yield 27.8g Example 24

AMC Stage 1: Amyl m-cresol

VMC stage 2 (45.5g) and palladium on carbon catalyst (Johnson Matthey® type 394 5% w/w, 5% water wet) (2.88g) were charged to a jacketed hydrogenator vessel, followed by citric acid (0.25g). Iso-propanol (220ml) was charged to the vessel and the jacket temperature was set to 17.5°C. The vessel was pressurised to 0.7bar with hydrogen and stirred at this pressure until judged complete by GC analysis. Once reaction completion had been achieved (after around 1 hr) the reaction mixture was filtered to remove the catalyst. The catalyst was then washed with IP A (10 ml) and the washings added to the reaction mixture. The reaction mixture was concentrated by distillation or rotary evaporation. Crude yield 46.5g.

Experimental Results:

AMC Stage 1:

MCV = m-cresol valerate; VMC = valeryl m-cresol; AMC = amyl m-cresol; nd = not detected Example 25 The European Pharmacopoeia Compendial Specification for AMC

Test Acceptance Criteria

Description: A clear or almost clear, colourless to slightly yellow liquid or solid crystalline mass

Identification by FTIR: Conforms

Sulphated Ash Not more than 0.1 % ^w / _w

Related Substances by GC. Not more than 0.6% ^w / _w 4-methyl-2-pentylphenol

Not more than 0.15% ^w / _w 5-Methyl-2- pentylcyclohexanone (sum of isomers)

Not more than 0.1% ^W / _W any other single unspecified impurity

Not more than 1.0% ^w / _w total impurities

Residual Solvents by GC: Not more than lOOppm Toluene

Not more than 150ppm Methanol

Not more than 0.1% ^W / _W Ethanol

Assay by GC: 98.0% ^w / _w to 102.0% ^w / _w

Example 26 Analytical conditions

Gas Chromatography Instrument Parameters:

Agilent GC6890 and/or Agilent GC7890

Column: 30m x 0.25mm ZB-WAX plus, 0.25μι ^η film

Oven Program: 100°C then 8°C/min to 240°C (Hold for 15 min)

Injector Temperature: 250°C

Detector Temperature 250°C

Injection Volume: Ι.ΟμΙ

Pressure: lO.Opsi, constant, Nitrogen.

Detector: F.I.D.

Column flow: 0.7ml/min

Spilt Flow 6.7ml/min

Split Ratio: 10: 1

Run Time: 32.5 minutes

Retention Time AMC: 18 minutes

Sample Solvent: Ethanol

Sample Concentration: 5mg/ml in Sample Solvent