Use of alpha-hydroxy carbonyl compounds as reducing agents

Abstract

There is provided the use as reducing agents of alpha-hydroxycarbonyl compounds capable of forming cyclic dimers. There is also provided corresponding methods of reducing reducible compounds, particularly reduction-activated prodrugs. Examples of the alpha-hydroxycarbonyl compounds used are dihydroxyacetone, glycolaldehyde, glyceraldehyde, erythrose, xylulose, erythrulose or 3-hydroxy-2-butanone.

Claims

1. A topical pharmaceutical composition suitable for use in medicine comprising 5-(aziridine-1-yl)-4-hydroxylamino-2-nitrobenzamide and a topically-acceptable adjuvant, a topically-acceptable diluent or a topically-acceptable carrier.

2. The topical pharmaceutical composition according to claim 1, wherein the composition is a solution or suspension.

3. The topical pharmaceutical composition according to claim 2, wherein the solution or suspension is sprayable.

4. The topical pharmaceutical composition according to claim 1, wherein the composition is a cream, lotion or ointment.

5. A dry powder aerosol composition comprising 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide.

6. A therapeutic system comprising: (a) a dry powder inhalation device, and (b) one or more discrete doses of the dry powder aerosol composition according to claim 5.

7. The therapeutic system according to claim 6, wherein the dry powder inhalation device contains a source of propellant gas.

8. A device that is: (a) a mechanical sprayer having a reservoir loaded with a solution or suspension comprising 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide and a pharmaceutically-acceptable adjuvant, diluent or carrier; or (b) an aerosol device comprising a solution or suspension comprising one or more propellant gases and 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide and a pharmaceutically-acceptable adjuvant, diluent or carrier.

Description

(1) The invention will now be described in more detail by reference to the following Figures and Examples wherein

(2) FIG. 1 shows the structure of tretazicar (5-(aziridin-1-yl)-2,4-dinitrobenzamide).

(3) FIG. 2 shows the bioactivation of tretazicar.

(4) FIG. 3 shows the structure of dihydroxyacetone (DHA; CAS No: 62147-49-3, Beil. 8, 266, Merck Index 13, 3166). The normal form is the dimer, but this rapidly reverts to the monomer in solution.

(5) FIG. 4 shows the reduction of tretazicar by DHA.

(6) FIG. 5 shows the reduction of tretazicar by DHA in an E45 based cream (as described in Example 2 below). In each of A, B and C of FIG. 5, the upper of the two traces is that measured at 325 nm, whereas the lower is that measured at 260 nm.

(7) FIG. 6 shows the spectral matching of the peak seen with a retention time of 5.0 minute in FIG. 5B, with that obtained with a synthetic standard of 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. The continuous line is the standard and the dashed line is the sample. The spectra are normalised for absorbance at 260 nm.

(8) FIG. 7 shows the percentage survival of Chinese Hamster V79 cells over the course of 2 hours at pH 7.5 and 37 C. in a 10 mM phosphate buffer containing 140 mM NaCl and the following: A: control (i.e. no additional substances); B: 10 mM DHA; C: 10 M E09; B+C: a combination of 10 mM DHA and 10 M E09; D: 100 M E09; and B+D: a combination of 10 mM DHA and 100 M E09.

(9) The survival of V79 cells was determined using the following method: Volumes (1 mL, 210.sup.5 cells/mL) of V79 cells in 10 mM phosphate buffer (pH 7.5) containing 140 mM NaCl were incubated at 37 C. and then reagents (as indicated above, at the concentrations stipulated) were added (except for the control experiment). After a 2 hour incubation, the cells were harvested by centrifugation, diluted out serially (410-fold) and the cells plated into growth medium and assayed for their colony forming ability after growth for 1 week in a humidified 5% CO.sub.2 atmosphere.

(10) As the chart of FIG. 7 demonstrates, the cytotoxic effect of E09 is greatly enhanced by the addition of DHA, which promotes formation of the active form of E09.

(11) In relation to the examples below, tretazicar is commercially available for research purposes from Morvus Technology Limited and Sigma Chemical Company.

EXAMPLE 1: CHEMICAL ACTIVATION OF TRETAZICAR

(12) Reported chemical methods of producing the active 4-hydroxylamino derivative from tretazicar use harsh reducing conditions in organic solvents with yields less than 30% (Knox et al, 1993; Knox et al, 1988). I have discovered that dihydroxyacetone (DHA) can reduce tretazicar to the required hydroxylamine in aqueous solution under mildly alkaline conditions. At pH 9 the yield is >85% and the only other product of tretazicar reduction detected is 5-(aziridin-1-yl)-2-hydroxylamino-4-nitrobenzamide.

(13) Dihydroxyacetone (DHA; 1,3-dihydroxy-2-propanone; CAS No: 62147-49-3, Beil. 8, 266, Merck Index 13, 3166; FIG. 3) is the active ingredient in sunless or self-tanning lotions and has received approval by the FDA. DHA is a colourless sugar that darkens the skin by staining. It interacts with the dead surface cells found in the epidermis producing a color change. As the dead skin cells are naturally sloughed off, the color gradually fades, typically within 5 to 7 days of application. Sunless tanning products contain dihydroxyacetone concentrations of up to 5%. The higher the concentration, the darker is the tan that will follow. As a tanning agent it stable in pH conditions of between 4 and 6. Too alkali or too acidic results in brown compounds forming, reducing the solutions effectiveness as a tanner. Erythrulose (1,3,4-trihydroxy-2-butanone) is similar in action to DHA, but it does not produce as deep or fast a tan. DHA is produced through the fermentation of glycerine and is a simple three-carbon sugar, non-toxic in nature and comes in the form of a white powder. The normal form is the dimer (C.sub.6H.sub.12O.sub.6) but this rapidly reverts to the monomer in solution. The FDA has approved DHA only for external use, and recommends that users should take protective measures to avoid contact with eyes, nose and mucous membranes.

(14) The only adverse effect reported is allergic contact dermatitis. This is reported rarely and most causes of sensitivity in tanning creams are due to other ingredients such as preservatives in the preparation. See the Code of Federal Regulations, page 376:

(15) TITLE 21FOOD AND DRUGS

(16) CHAPTER IFOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES

(17) PART 73LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATIONTable of Contents

(18) Subpart CCosmetics

(19) Sec. 73.2150 Dihydroxyacetone.

(20) DHA has never been reported as a reducing agent. Reducing sugars are known but these are aldoses and have an aldehyde in one end. The aldehyde behaves as a reducing agent and in the presence of mild oxidizing agent, such as Cu.sup.2+ or Fe.sup.3+, there is oxidation of the aldehyde to a carboxylic acid. As a ketol an equivalent reaction with DHA is not possible. However, dihydroxyacetone has been shown to promote the formation of hydroxyl radicals in the presence of iron (III) chelates (Malisza & Hasinoff, 1995). This type of reaction may be related to its reducing ability and its alkali instability that results in the formation of brown compounds.

(21) Under alkali conditions and using an excess of DHA there is a linear rate of reduction of tretazicar with time (FIG. 4).

(22) The assay was started by addition of 100 L of 100 mM DHA in water to a mixture of tretazicar (100 M) in 0.1M sodium bicarbonate buffer, pH 9 or pH10, to give a final volume of 1 ml. The mixture was incubated at 37 C. and aliquots (10 L) were taken every 6 min and assayed immediately by HPLC [Partisil 10 SCX (42150 mm) (Whatman, Maidstone, Kent, U.K.] eluted isocratically with 0.13 M sodium phosphate (pH 5) at 1.5 mL/min). The concentration of tretazicar was determined in each sample by reference of the corresponding peak area with an external standard, quantified by absorbance at 325 nm. Initial rates were calculated by curve fitting (FigP, Biosoft, Cambridge, U.K.). Reduction products were identified by retention time relative to an authentic standard.

(23) Only two products of tretazicar reduction were observed the 2- and 4-hydroxylamino derivatives. The rate of tretazicar reduction and the proportion of the hydroxylamine products was dependent on pH. Reduction at pH 10 (0.69 nmol/min) was slower than at pH 9 (0.92 nmol/min). After 30 min the ratio of the 4- to the 2-hydroxylamines was 2.7:1 at pH 10 and 9.7:1 at pH 9. Thus, at pH 9, reduction is faster and gives mostly the preferred 4-hydroxylamine product in very high yield (FIG. 4).

(24) DHA or DHA-associated products from the reaction were not detected with the above HPLC method.

EXAMPLE 2: ACTIVATION OF TRETAZICAR IN A CREAM FORMULATED FOR TOPICAL APPLICATION

(25) Two creams designated A and B have been made. For use, these are mixed in equal amounts. Cream A consists of an E45 base (white soft paraffin BP 14.5% w/w, light liquid paraffin Ph Eur 12.6% w/w, hypoallergenic anhydrous lanolin (Medilan) 1.0% w/w, Crookes Healthcare Ltd, Nottingham, UK) containing 10 mg tretazicar, 10 mg NaHCO.sub.3 & 90 mg Na.sub.2CO.sub.3 per g. Cream B contains E45 with 100 mg DHA dimer per g. Mixing the two components, A and B, produced a pale yellow cream. This turned brown over a few hours and continued to darken for about 24 hours. Suspension of 200 g of cream in 1 mL of water with vigorous agitation produced a solution with a pH of about 10 as shown by pH indicating papers. Preliminary experiments with creams containing 10% of the above amounts of buffer salts gave a solution with the same initial pH. However, after 4 hours a solution, prepared as above, was neutral and the cream no longer darkened. This would suggest that by varying the initial buffer concentrations both the duration and extent of the reaction can be controlled.

(26) After mixing and at various times, 200 g of cream was extracted into 1 mL of DMSO. The extract was then diluted 1/100 with 50 mM ammonium bicarbonate buffer (pH 10) and analysed by reversed-phase HPLC. At the starting time, analysis of the extract at 325 nm showed only a single major peak and this corresponded to tretazicar as shown by the same retention time and the spectral matching of the UV absorbance spectrum relative to an authentic standard. After 4 hours, analysis of the extract showed many more peaks on both the 260 nm and 325 nm traces (FIG. 5).

(27) Cream A (consisting of an E45 base (white soft paraffin BP 14.5% w/w, light liquid paraffin Ph Eur 12.6% w/w, hypoallergenic anhydrous lanolin (Medilan) 1.0% w/w, Crookes Healthcare Ltd, Nottingham, UK) containing 10 mg tretazicar, 10 mg NaHCO.sub.3 & 90 mg Na.sub.2CO.sub.3 per g and cream B (containing E45 with 100 mg DHA dimer per g) were mixed and at various times 200 g was extracted with 1 mL of DMSO with vigorous agitation. The extract was cleared by centrifugation and then diluted 1/100 with 50 mM ammonium bicarbonate buffer (pH 10) and analysed by HPLC. A sample (10 L) was injected onto a Waters Symmetry Shield RP18 column (1503.9 mm) and eluted with a linear gradient (1-40% over 30 minutes) of acetonitrile in 10 mM ammonium formate (pH 4.5). The eluate was continually monitored for UV absorbance between 230 and 400 nm using a TSP UV3000 scanning detector. A.) An extract prepared immediately after the mixing of the cream. B.) An extract from the cream 4 hours after it had been mixed. C.) a synthetic standard of 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. The blue line (the lower line in each pair of traces) is the trace obtained at 260 nm and the red line (the upper line in each pair of traces) the trace obtained at 325 nm. The peak at 13 minutes is CB 1954 and the large 260 nm peak at 19 minutes comes from the E45. It is not possible to measure the efficiency of the extraction method.

(28) Tretazicar was still detected and a peak with a retention time of 5.0 minutes was identified as the 4-hydroxylamine of tretazicar as indicated by the same retention time and the spectral matching of the UV absorbance spectrum relative to an authentic standard (FIG. 6). The other peaks did not correspond to known tretazicar reduction products and may have arisen from the oxidation of DHA or reaction of reduced tretazicar products with either cream or DHA.

(29) The active form of tretazicar, (5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide), is formed in the E45 based cream.

EXAMPLE 3: TOPICAL APPLICATION OF A TRETAZICAR CREAM

(30) A cream formulated above was mixed and about 0.1 g applied to a wart (growing, dome 1.5 mm high) located on the finger of a healthy human volunteer and covered with a plaster. An initial warmth was reported from the applied cream. After about 4 hours the plaster was removed and the wart was found to have sloughed off and left a pit of 1 mm depth. The immediate surrounding tissue had a yellow colouration. This gradually turned white over a few days and no re-growth of the wart was reported after 6 weeks. No adverse affects were apparent or reported.

(31) Cream A (consisting of an E45 base (white soft paraffin bp 14.5% w/w, light liquid paraffin Ph Eur 12.6% w/w, hypoallergenic anhydrous lanolin (Medilan) 1.0% w/w, Crookes Healthcare Ltd, Nottingham, UK) containing 10 mg tretazicar, 10 mg NaHCO.sub.3 & 90 mg Na.sub.2CO.sub.3 per g and cream B (containing E45 with 100 mg DHA dimer per g) were mixed and applied (100 g) to a wart (growing, dome 1.5 mm high) located on the finger of a healthy human volunteer and covered with a plaster. After about 4 hours the plaster was removed and the wart was found to have sloughed off and left a pit of 1 mm depth. The immediate surrounding tissue had a yellow colouration. This gradually turned white over a few days and no re-growth of the wart was reported after 6 weeks. No adverse affects were apparent or reported. The photograph was taken after 1 week.

(32) Activation of tretazicar in a topical application has a marked effect on a wart with minimal effects on normal surrounding tissue.

EXAMPLE 4: TREATMENT OF BLADDER CANCER

(33) Bladder chemotherapy instillations, or intravesical chemotherapy, are given to people who have superficial bladder cancer by filling the bladder with medication to fight the cancer cells. Although superficial bladder cancers are an early form of cancer, many will recur after initial removal. However, by using treatment which puts medication directly in contact with the bladder wall, it may well be possible to prevent recurrence or lengthen the time until recurrence. Intravesical chemotherapy is a brief procedure. A catheter is put into the bladder through the urethra. Tretazicar in bicarbonate buffer (pH 9) is instilled over 2 to 3 minutes, followed by an infusion of DHA in water over a similar time. The catheter is removed and after 2 hours the medication removed by urination.

EXAMPLE 5: DHA REDUCTION OF PRODRUGS AT NEAR NEUTRAL PH

(34) Assay: HPLC

(35) The assay mixture contained the compound under test (100 M) and DHA (10 mM) in a final reaction volume of 1 mL of sodium phosphate buffer (of the required pH). The reaction was started by addition of DHA and the mixture is incubated at 37 C. Aliquots (10 L) were taken every 20 min and assayed immediately by HPLC on a Partisphere 5 C18 (4.2150 mm) column (Whatman, Maidstone, Kent, U.K.], eluted with a gradient of acetonitrile in water (1-95% over 10 minutes) at 2.0 mL per minute. The eluate was continuously monitored for absorbance using a photodiode array UV-VIS detector. The concentration of drug was determined in each sample by reference of the corresponding peak area with an external standard and quantified by absorbance at a suitable wavelength determined from the PDA scan. Initial rates were calculated by curve fitting (FigP software).

(36) TABLE-US-00001 INITIAL RATE (nmoles/min/mL) Compound Structure 0.1 M PO.sub.4, pH7.5 0.1 M PO.sub.4, pH8 Tretazicar <0.01 0.158 Metronidazole 0.131 0.369 Misonidazole 0.221 1.922 Nitrofurazone 0 0.177 0.203 RSU-1069/RB-1645 <0.01 2.57 Tirapazamine 0.523 0.512 Mitomycin C 1.524 (PBS) 0.052.sup.a E09 0.272 0.605 Notes .sup.a1 mM DHA employed (instead of 10 mM DHA)

EXAMPLE 6: DHA REDUCTION OF NITRO COMPOUNDS AND PRODRUGS AT VARIOUS PH VALUES

(37) Samples were assayed as described above for Example 5.

(38) TABLE-US-00002 Initial Rate (nmoles/ Name Structure pH min/mL) Tretazi- car 8 9 10 10.6 0.158 0.933 1.248 5.801 5-chloro- 2,4- dinitro- toluene 8 9 10 10.6 0.132 0.499 0.690 1.703 4-nitro- benzyl alcohol 8 9 10 10.6 <0.01 <0.01 0.105 0.087 4-chloro- 3,5- dinitro- benzoic acid 8 9 10 10.6 0.195 1.293 1.727 2.442 Misoni- dazole 8 9 10 10.6 1.922 1.851 3.180 Metroni- dazole 0 8 9 10 10.6 0.369 0.890 1.239 0.807 Mitomy- cin C 7 8 8.5 9 1.019 0.052.sup.a 0.103.sup.a Too fast to measure Nitro- furazone 7.5 8 8.5 9 10 0.177 0.203 0.260 2.390 3.789 Tirapa- zamine 7.5 8 8.5 9 10 0.523 0.512 0.639 3.503 4.209 Notes .sup.a1 mM DHA employed (instead of 10 mM DHA)

EXAMPLE 7: REDUCTION OF NADP+ BY -HYDROXYCARBONYL COMPOUNDS

(39) A 10 L aliquot of test compound (at a concentration of 100 mM in water) is added to a 200 M NADP.sup.+ aqueous solution (approximately 990 L), buffered to pH 10 (1 mM NaHCO.sub.3 buffer). The final concentration of the test compound in the assay solution was 1 mM. Reduction of NADP.sup.+ was then monitored by measuring the increase, over the course of 2 minutes, of absorption at 350 nM on a spectrophotometer. Initial rates were recorded as the change in A350 per minute.

(40) TABLE-US-00003 Compound Structure Rate Dihydroxyacetone (dimer).sup.1 0.063 Hydroxyacetone (acetol).sup.2 0.003 D,L- Glyceraldehyde.sup.3 0.015 Glycolaldehyde dimer.sup.4 0.029 D()-Erthrose.sup.5 0.004 L-Xylulose 0.003 Notes .sup.1Supposed to form monomer in solution. .sup.2Technical grade (>90%) used. Lag of about 60 s before any increase in absorption at 350 nm observed. .sup.3Concentration calculated for monomer. .sup.4Mix of stereoisomers. No effect on rate observed after addition of 1 mM EDTA to the reaction mixture. .sup.5Only ~50% pure. Lag of about 90 s before any increase in absorption at 350 nm observed.

(41) Without wishing to be bound by theory, it is believed that the compounds showing reducing activity in the above assay are -hydroxycarbonyl compounds that are capable of forming cyclic dimers of the type depicted in Formula Ia.

(42) In contrast, compounds for which rate in the above assay determined to be below 0.001 (i.e. for which no reducing activity detected) include the following: glycerol; glyoxal; D-glucose; diglycolic anhydride; ()-tetrahydrofurfuryl alcohol; 1,4-dioxane-2,3-diol; and 2-(hydroxymethyl)tetrahydropyran.

EXAMPLE 8: REDUCTION OF TRETAZICAR BY -HYDROXYCARBONYL COMPOUNDS

(43) The assay was started by addition of 100 L of 100 mM test compound (compound of formula I) in water to a mixture of tretazicar (100 M) in 0.1 M sodium bicarbonate buffer, pH 9 or pH 10, to give a final volume of 1 mL. The mixture was incubated at 37 C. and aliquots (10 L) were taken every 6 min and assayed immediately by HPLC [Partisil 10 SCX (4.2150 mm) (Whatman, Maidstone, Kent, U.K.] eluted isocratically with 0.13 M sodium phosphate (pH 5) at 1.5 ml/min). The concentration of tretazicar was determined in each sample by reference of the corresponding peak area with an external standard, quantified by absorbance at 325 nm. Initial rates were calculated by curve fitting (FigP, Biosoft, Cambridge, U.K.). Reduction products were identified by retention time relative to an authentic standard.

(44) TABLE-US-00004 Initial Rate (nmoles/ Compound Structure pH min/mL) Dihydroxy- acetone (used in dimeric 0 9 10 0.92 0.69 form) D,L-Glyce- raldehyde.sup.3 9 10 0.53 0.62 Glycolal- dehyde (used in dimeric 9 10 0.18 0.14 form).sup.4 D()- Erthrose.sup.5 9 10 <0.01 0.28 L- Xylulose 9 10 <0.01 0.28 3-hydroxy- 2-butanone 9 10 0.034 0.184

(45) In contrast, compounds for which rate in the above assay determined to be below 0.001 (i.e. for which no reducing activity detected) include the following: glycerol; glyoxal; D-glucose; diglycolic anhydride; ()-tetrahydrofurfuryl alcohol; 2-(hydroxymethyl)tetrahydropyran; 4-hydroxy-2-butanone; dichloroacetone; 1,4-dioxane-2,3-diol; and dichloroacetyl chloride.

EXAMPLE 9: LARGE-SCALE REDUCTION OF TRETAZICAR TO THE CORRESPONDING HYDROXYLAMINE

(46) A solution of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954, 1.00 g, 3.97 mmol) in methanol (AnalaR-grade, 40 mL) was treated with excess powdered anhydrous K.sub.2CO.sub.3 (10.0 g, 72 mmol; aprox. 18 equiv.) and the mixture was heated to 60 C. with stirring. The suspension was stirred at 60 C. beneath a N.sub.2 blanket stream atmosphere. A solution of 1,3-dihydroxyacetone (1.50 g as DHA dimer, 8.33 mmol; 2.1 mol equiv) in methanol (AnalaR-grade, 40 mL), previously deaerated by flushing with N.sub.2 gas, was then added during 15 min to the reaction mixture with rapid stirring while maintaining the temperature at 60 C. A fast reaction is observed with a colour change from pale yellow-orange to brown. Thin-layer chromatography (TLC; Merck silica gel 60 GF254 on aluminium sheets, 9:1 v/v CH.sub.2Cl.sub.2:MeOH) showed quantitative removal of the CB 1954 starting material (Rf 0.73) and formation of two polar products at Rf 0.53 and Rf 0.47. The two products showed identical TLC behaviour to authentic 5-(aziridin-1-yl)-2-hydroxylamino-4-nitrobenzamide and 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide samples, respectively, prepared by published methods [Knox, R. J., Friedlos, F., Jarman, M., Roberts, J. J. Biochemical Pharmacology 37, 4661-4669 (1988); Knox, R. J., Friedlos, F., Biggs, P. J., Flitter, W. D. Gaskell, M., Goddard, P., Davies, L., Jamnnan, M. Biochemical Pharmacology 46, 797-803 (1993)].

(47) The reaction mixture was filtered while hot and the insoluble material was washed with cold methanol (10 mL). The combined filtrate was cooled and rotary evaporated (30 C., high-vacuum) to give a viscous yellow-brown oil (1.02 g, >100%). TLC examination confirmed that two major products were present. Minor spots (<1-2% total, Rf 0.62, 0.66) corresponding to the 2-nitroso and 4-nitroso oxidation products resulting from O2 oxidation under alkaline conditions during handling and work-up were also evident. (Note: exposure to air should be minimised by flushing all apparatus with N.sub.2 gas to prevent oxidation of the hydroxylamines to nitroso products and unwanted coloured by-products). The 2-hydroxylamine (Rf 0.53) and 4-hydroxylamine (Rf 0.47) reaction products were judged to be formed in 40:60 ratio. Chromatographic separation of the isomers is hampered by their close elution properties using available solvent systems. However, flash chromatographic separation of a crude mixture sample (100 mg) (Merck silica gel, 60-200 mesh, 9:1 v/v CH.sub.2Cl.sub.2-MeOH) gave 5-(aziridin-1-yl)-2-hydroxylamino-4-nitrobenzamide (31 mg) and 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide (49 mg) after solvent removal from fractions. Both products gave NMR spectra consistent with the reported properties for the isomeric hydroxylamines and TLC behaviour that was indistinguishable with the authentic compounds [Knox, R. J., Friedlos, F., Jarman, M., Roberts, J. J. Biochemical Pharmacology 37, 4661-4669 (1988)].

(48) Notes:

(49) (1) The preferred reaction solvent is methanol. 1,3-Dihydroxyacetone (DHA) dimer has limited solubility in many common solvents, including acetone and higher alcohols. (2) Reaction is almost instantaneous at 60 C. but is slower at lower temperatures. The use of higher temperature reaction systems may have an adverse effect on relative product yield.

(50) The product hydroxylamines show greater sensitivity to air oxidation in the presence of alkali, hence it is recommended that the K.sub.2CO.sub.3 reagent is removed from the reaction mixture as soon as possible. Chromatographic separation of the mixture containing the 2- and 4-hydroxylamine products requires minimisation of any exposure for dissolved material to O.sub.2 (air) during handling.

REFERENCES

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(52) All references mentioned herein are incorporated herein by reference.