Supplementary Material for Online Publication
Synthesis of two local anesthetics from toluene: An organic multistep synthesis in a project-oriented laboratory course
Patricia Demare and Ignacio Regla* Facultad de Estudios Superiores-Zaragoza, Universidad Nacional Autónoma de México, Batalla del 5 de Mayo esq. Fuerte de Loreto, Ejército de Oriente, 09230. México, D.F., México *
[email protected]
1. GEERAL IFORMATIO The estimated length of this project is about 8 four-hour lab periods. The first part of the synthetic work (Reaction sequence A in Scheme 1) may be performed by two students as a team; then each student will take over either part B or part C independently. Procaine or tetracaine can be prepared instead of benzocaine. Directions for each step in sequences A and B are given below, which may be used as student handouts. They include notes which might be particularly useful to instructors. Bibliographic references for methodologies of reactions in sequence C are provided. HPLC and GC chromatograms are presented as examples of the mono-nitrotoluene (MNT) isomeric mixture analysis. 1H and 13C NMR spectra for compound 4 are included. The lab manual (in Spanish) used in our course is included in a separate zip file (Manual SFMP-1).
2. BACKGROUD IFORMATIO Topical anesthetics Local anesthetics are drugs that disrupt the propagation of the nerve impulses in tissues, in a lasting and reversible manner. The first topical anesthetic used, for eye-surgery, was cocaine, a naturally occurring substance. Most anesthetics in use today are synthetic drugs that have been developed to avoid the stimulant and addictive effects of natural products such as cocaine. The molecules include a lipophilic fragment (an aromatic ring) and a hydrophilic segment (a secondary or tertiary amine) linked by an intermediate carbonyl containing moiety, either a benzoic ester (see Figure 1) or an anilide (see Figure 2) (1).
1
O
OCH3
O
O
O
O
N
N O
O
O
. HCl
N . HCl
O . HCl Cocaine hydrochloride
NH2
HN
NH2 Benzocaine
Tetracaine hydrochloride
Procaine hydrochloride
Figure 1. Some amino-ester local anesthetics.
Carl Koller, a Viennese ophthalmologist, introduced cocaine as a local anesthesic. He and his friend Sigmund Freud, were experimenting on the pharmacology of cocaine, and Koller, who had been interested in finding a way to locally anesthetize the eye for surgery, noted the numbing effect of the alkaloid. Knowing that cocaine is an extract of coca leaves, Freud wrote a dedication on a reprint of a paper on the effects of cocaine on fatigue, which read: "To my dear friend Coca Koller, from Dr. Sigmund Freud." (2). There are important differences between these two groups of local anesthetic agents. Esters are relatively unstable in solution and are rapidly metabolized in the body. One of the main breakdown products is p-amino benzoic acid (PABA), which is associated with hypersensitivity reactions. In contrast, amide class anesthetics are slowly metabolized and they are less likely to produce an allergic reaction. Other side effects reported for both types of local anesthetics are methemoglobinemia and neuronal apoptosis (3-5). Prilocaine has been reported to induce apoptosis in osteoblastic cells. O HN
O
O N
Lidocaine (Xylocaine)
HN
N
Ropivacaine
HN
O
H N
Prilocaine
HN
N
Bupivicaine
Figure 2. Some amide anesthetic drugs.
Benzocaine (ethyl 4-aminobenzoate; ethyl PABA or Anesthesin) C9H11NO2, [94-09-7] is an ester-type, local anesthetic agent derived from aminobenzoic acid that is applied topically on skin or mucous membranes. It is used in many over-the-counter compounds, such as gels, creams, lotions or sprays, for pruritus and pain. It has a low incidence of toxicity, but sensitization to it may result from prolonged or frequent use. Prilocaine ( -(2-methylphenyl)-2-propylamino-propanamide) hydrochloride C13H20N2O.HCl [1786-81-8] is an amide-type local anesthetic, used in dentistry and in dermal anesthesia. It is no longer available in the US as the pure drug, but is present in the topical anesthetic mixture EMLA (see above). It is used as the racemate, although R(-)-isomer is reported to be slightly more potent (and more toxic) (6). 2
CH3
CH3 HNO3 A)
CH3
CH3
+
H2SO4
CH3
CH3
NO2
NH3Cl
1) NH4HCO2 , Pd/C 2) Ac2O 3) HCl extrn.
+ NO2
+ NHCOCH3
NO2
CH3
CH3 NH3Cl
B)
1) AcONa, H2O
CH3
H N
Cl O
2) CH3CHClCOCl 2
1) PrNH2 2) HCl (g)
COOH H3O
3
COOEt EtOH
MgSO4 NHCOCH3
N H H
5
COOH KMnO4
Cl
H N O
4
CH3 C)
3
2
1
H NHCOCH3 6
NH2
NH2
7
8
Scheme 1. Divergent synthesis of prilocaine hydrochloride (5) and benzocaine (8)
3. DISCUSSIO 3.A. Reaction sequence A. 3.A.1. Toluene nitration Aromatic nitration is a reaction of great synthetic and industrial importance, as aromatic nitro compounds are widely used as starting materials for the production of pharmaceuticals, dyes, pesticides and plastics. Classic aromatic nitration makes use of the mixed acid system, in which nitric acid is used in conjunction with an excess of concentrated sulfuric acid as catalyst. Although it has the advantage of using cheap and readily available reactants, this method gives rise to problems pertaining to regioselectivity, toxic waste, safety and oxidation. In order to overcome these problems, a number of alternative methods for aromatic nitration have been developed, which include the use of transition metals (7) and solid acid catalysts (8). Toluene nitration affords a mixture of isomeric ortho-, meta- and para-mononitrotoluenes (MNT) (1) in varying ratios depending on the method. Polynitration is usually not a problem because the nitro group introduced in the first stage is powerfully deactivating, but it is important to exert care in controlling the temperature, especially on a large scale. In this reaction, regioselectivity is often a goal, as both o- and p- attack of the nitrating species is favored due to stabilization of specific resonance structures via the electrondonating inductive effect of the methyl group. The nitric-sulfuric acid method described below affords a MNT mixture in a typical ratio of about 58:3.5:38.5 o-/m-/p-. The isomer 3
percentage in MNT mixture may be established either by GC or HPLC (9). If not analyzed, a 38 % p-nitrotoluene proportion can be assumed for the purposes of this project.
3.A.2. MT reduction Reduction of aromatic nitro compounds can be performed by a wide variety of methods, including iron and acid (10), and catalytic hydrogenation. This last reduction strategy is usually a clean reaction, as water is usually the only by-product, and the reaction product is easily isolated by filtration and evaporation of the filtrate. The big disadvantage of this alternative is the inherent risk of explosion of hydrogen gas. Catalytic transfer hydrogenations (11) use a hydrogen donor such as ammonium formate or cyclohexene and a metal catalyst (12). In the presence of Pd/C, ammonium formate decomposes to carbon dioxide, ammonia and hydrogen, all gaseous substances; hydrogen gas is absorbed onto the surface of the catalyst, where it can react with nitrocompounds, among other functional groups. These methods allow a safer practice and the use of ordinary laboratory glassware. Students in our lab have reduced MNT mixtures either by catalytic hydrogen transfer with ammonium formate or by catalytic hydrogenation (with the balloon strategy described by Williamson) (13). The former method has proven to yield the toluidine mixture in excelent yields and it has great safety advantages. Both reactions can be performed in ethyl acetate as solvent and 10% Pd/C as catalyst.
3.A.3. Selective acetylation of p-toluidine Since toluidine isomers have similar boiling points, distillation is not a practical method to separate them, but as a student found out some time ago (by hypothesis, testing and confirmation), the weak steric hindrance induced by the methyl group influences toluidine isomers’ reactivity as nucleophiles, affecting the rate of acetylation. A one-pot reduction-acetylation sequence can be performed by using ethyl acetate or toluene as a solvent. Reaction of the toluidine isomeric mixture with the appropriate amount (one molar equivalent relative to p-toluidine in the mixture) of acetic anhydride at low temperature affords an easily separable mixture of o-toluidine and p-methylacetanilide (3). The approximately 4 % m-isomer is probably acetylated along with the p-isomer, and then removed during recrystallization of 3. The acetyl group on nitrogen serves also as a protecting group for the subsequent reaction in the first step of the synthesis of benzocaine (8) (see reaction sequence C). A flow diagram like that in Fig. 3, with blanks left to fill in, will stress the importance of having a clear understanding of the main point on this synthetic strategy: calculating the proper amount of acetic anhydride.
4
CH3
CH3
CH3 NO2 NO2
____ g (___mmol)
NO2
____ g (____ mmol)
NH3, CO2
NH4HCO2, Pd/C, AcOEt
CH3
CH3
CH3
____ g (____ mmol)
NH2
AcOEt NH2
_____ mmol
_____ mol
Pd/C
NH4HCO3
NH2 _____ mol
1) Ac2O ( ____ mmol), 0 °C 2) 10 % HCl (acid/base extraction)
Aqueous phase
Organic phase CH3
CH3
CH3 NH3 Cl
H2O
HCl
AcOEt
AcOH
NHAc NHAc
____ mmol O Cl CH3
H N
Isolation and purification Cl CH3
Cl O
NHAc 1) H2N
C
2) HCl (g) COOEt CH3
H N O
N H
Prilocaine hydrochloride
. HCl NH2 Benzocaine
Figure 3. Flow diagram for the divergent synthesis of Prilocaine hydrochloride and Benzocaine
5
3.B. Prilocaine hydrochloride synthesis
3.B.1. o-Toluidine acylation Acylation of o-toluidine to prilocaine precursor 4 was carried out by adding αchloropropionyl chloride to the buffered aqueous hydrochloride solution (2). This otoluidine selective attack at the acyl carbon atom of the bifunctional reagent is illustrative of the significantly greater reactivity of nucleophiles toward acylation, compared to alkylation. By lowering the reaction temperature, this reaction can take place in the presence of water, which is another example of selectivity, as amines are better nucleophiles than water. Chloroamide 4 was isolated and purified after a four step telescoped (no intermediates isolated) reaction sequence.
3.B.2. Halogen displacement Reaction of an alkyl halide with a large excess of a primary amine yields the corresponding secondary amine, presumably by a SN2 mechanism. A big excess of the amine is necessary in order to preclude further alkylation of the secondary amine product. In the patented methodologies (14) displacement of halide ion with n-propylamine is carried out by heating the reaction mixture in benzene for 8 h in an autoclave at 80 °C. We achieved similar results by dissolving 4 in n-propylamine and allowing this solution to stand for two days in a sealed container. After a simple semi-workup, a prilocaine solution was obtained, which was used in the last step of the synthesis, the preparation of the hydrochloride.
3.C. Synthesis of benzocaine Benzocaine synthesis starting from p-methylacetanilide was adapted from different sources to suit small scale: p-Methylacetanilide (3) oxidation to p-acetamidobenzoic acid (6): (15, 16, 18) Hydrolysis of 6 to p-aminobenzoic acid (7): (15, 18) Esterification of 7 to obtain benzocaine (8): (17,18, 19, 20)
6
4. EXPERIMETAL PROCEDURE Reaction sequence A. 4.A.1. Mononitrotoluene isomeric mixture (1) CH3
CH3
CH3
CH3
NO2
HNO3
+
+
H2SO4, H2O, 15 °C
NO2 58.5 %
3.5 %
NO2 38 %
1
To a stirred solution of 3 mL (44.7 mmoles) 67 % nitric acid in 3.75 mL of water, sulfuric acid (9 mL) was slowly added at 10-15 °C. This nitrating solution was added dropwise with vigorous stirring (ote 1) to 4 mL (37.5 mmoles) of toluene while cooling in icewater in order to maintain the reaction temperature between 15 and 25 °C (ote 2). Stirring was continued for 20 minutes at room temperature and the reaction mixture carefully quenched over 15 g of crushed ice (ote 3). The organic layer was separated (ote 4), and the aqueous layer extracted with diethyl ether (2 X 10 mL) (ote 5). The organic extracts were combined and washed with 10 % NaOH (3 X 5 mL) (ote 6) and brine (5 mL), dried with anhydrous sodium sulfate and evaporated until constant in weight. A sample was analyzed by gas chromatography in order to determine MNT isomer ratio (otes 7 and 8). otes: 1. Efficient stirring (i.e., an evident vortex) is essential at this stage, as it is a biphasic reaction. 2. Careful temperature monitoring is important in order to avoid poly-nitration, as well as other undesired reactions. 3. This operation must be supervised, as quenching is highly exothermic. 4. Check which is the organic layer. This could be a good occasion to discuss solute effect on density of layers. 5. Extractions may be omitted, with about 10 % decrease in yield. 6. NaOH washings remove nitrophenol by-products as yellow water-soluble phenoxides. Students must repeat this step if a yellow color continues to appear in aqueous washings. 7. Typical yields for the MNT mixture (2) are around 60 to 80 %. A typical reaction sample analyzed by GC showed the following ratio: 58.5 % ortho-, 3.8 % metaand 37.7 % para- nitrotoluene. (GC and HPLC analysis for two different MNT
7
mixture samples are shown in pages 13 and 14). If not analyzed, a 38 % pnitrotoluene proportion can be assumed for the purposes of this project.
Safety considerations: Eye protection, gloves and a lab coat should be worn. Care should be exercised in the handling of the following chemicals: sulfuric acid and nitric acids, which are extremely corrosive; contact with skin or eyes may cause severe burns and permanent damage. Toluene and diethyl ether are flammable and toxic; keep away from heat, sparks, and open flame. Avoid skin contact and inhalation. Nitrotoluenes and 10 % NaOH are toxic and skin irritants. Waste disposal: Students must: a) neutralize aqueous acid solutions prior to pouring into drain and b) collect solvent waste in the organic waste container.
4.A.2. Preparation of o-toluidine hydrochloride solution (2) and p-methyl acetanilide (3)
CH3
CH3 +
NH3Cl
1) NH4HCO2 , Pd/C
+
2) Ac2O 3) HCl extrn.
NO2
+ NHCOCH3
NO2
1
CH3
CH3
CH3
NO2
2
3
A mixture of 2.4 g (17.5 mmoles) of the quantified MNT (1) mixture, 3.6 g (57.1 mmoles) of ammonium formate and 100 mg of 10% Pd/C in 12 mL ethyl acetate, was stirred for 15 min and then refluxed with stirring for 1 h (otes 1 and 2). After verifying completion of reaction by TLC (silica gel; 93:7 hexane-MTBE), the reaction mixture was dried over anhydrous sodium sulfate and the mixed solids were filtered through a pad of filter-aid and rinsed with ethyl acetate (2 x 5 mL) (ote 3). The toluidine isomeric mixture solution was cooled to 0 oC (ote 4) and one equivalent of acetic anhydride, as to the quantity of p-toluidine theoretically present in the solution (ote 5), was added dropwise with stirring. The stirring was continued for 15 minutes while the reaction mixture warmed to room temperature. TLC analysis (ote 6) confirmed the selective acetylation of p-toluidine (Fig. 4). The reaction mixture was extracted twice with 5 mL of a 10 % hydrochloric acid solution. The aqueous layer (o-toluidine hydrochloride solution (2)), was saved for prilocaine synthesis and the organic layer was washed with 5 % sodium bicarbonate solution (CAUTIO: CO2 release), dried with anhydrous sodium sulfate and evaporated to afford p-methylacetanilide (3), which was recrystallized with ethyl alcohol (ote 7). 8
otes: 1. Warm gently, as the reaction is mildly exothermic. 2. Because of the evolution of gases (CO2 and NH3), foaming occurs; a 100 mL
round bottomed flask is advised. Some of these gases react in vapor phase and deposit ammonium carbonate in the condenser tube. Also, a gas bubbler connected to the condenser through a rubber hose may be used to monitor reaction progress. 3. Avoid allowing the catalyst to become dry, as the reactive hydrogen adsorbed on
its surface may spontaneously ignite. After filtering, they should use water to dissolve salts and to wash off the catalyst into a special container. 4. Don’t forget to save a drop of this solution in order to use it later in a TLC. 5. If the MNT isomer proportion was not determined, 38 % p-toluidine content may
be assumed. 6. TLC (Fig. 4) is run on silica gel plates. All spots can be observed under a UV lamp
at 254 nm. 7. After recrystallization and drying, typical masses of product 3 are around 0.8 g. 8. Ammonium formate may be prepared by bubbling ammonium gas into a cold
solution of 20 mL 88% formic acid in 80 mL 2-propanol. Ammonium gas can be generated by slowly adding 40 mL 28 % ammonium hydroxide to 30 g sodium hydroxide, in a similar manner as the HCl generation (21).
1
2
80:20 Hexane-AcOEt
3 2 1
1:0.85:0.15 AcOEt-Hexane-MTBE (run twice)
Figure 4. TLC plates for experiment 4.A.2 in two different mobile phases. Sample 1: Toluidine isomer mixture; Sample 2: Acetylation reaction mixture; Sample 3: o-Methylacetanilide
Safety considerations: The use of eye-protection and gloves when performing this experiment is recommended. Care should be exercised with the following chemicals: 9
Palladium on carbon is pyrophoric. Toluidine isomers and their hydrochlorides are skin irritants and suspected carcinogens. Acetic anhydride is a lachrymator. MTBE and ethyl acetate are volatile, toxic and flammable liquids. Hydrochloric acid is corrosive to eyes, skin, and mucous membranes; exposure may cause severe burns. Reflux and workup should be done in a hood. Waste Disposal: Collect moist Pd/C catalyst in a special container, as it may be recycled. Collect organic solvents in the appropriate waste container. Collect silica gel plates in a special solid waste container.
4.B. Prilocaine hydrochloride synthesis 4.B.1. 2-chloro--o-tolilpropanamide (4) CH3
1) NaOAc, H2O NH3Cl
CH3
H N
Cl O
2) Cl
Cl O
2
4
The o-toluidine hydrochloride solution (2, about 10 mmoles) from part A was placed in a cooling bath and 8 mL of a 10 % NaOH solution added (ote 1) with stirring. Sodium acetate trihydrate (2 g) was added in order to buffer the solution up to pH=5-6. After cooling again to 0 °C, 2-chloropropionyl chloride (1 mL; 1.4 g; 11 mmol) was added with stirring and the reaction mixture stirred for 30 minutes, filtered and washed with water. The tan-colored precipitate was recrystallized from methanol to afford chloroamide 4 as white crystals, m.p. 111 – 112 °C (otes 2 and 3). otes: 1. The NaOH will neutralize most of the excess HCl in the hydrochloride solution. Students can make this calculation using their own data. Resulting pH should be around 2 or 3. 2. Overall yields of 4 from toluene (no isolated intermediates) are typically in the range from 15 to 30 %. 3. Analogous bromoamide (m.p. 117-8 °C) may be prepared with the same methodology, using bromopropionyl chloride or bromide. 4. 1H and 13C NMR spectra of compound 4 are displayed below.
Safety considerations: All procedures must be conducted in a fume hood. Eye protection and gloves must be worn at all times. Take special care when dispensing αchloropropionyl chloride, as it produces hydrogen chloride by contact with water vapor in the air and is extremely corrosive to the eyes, skin, and mucous membranes. Sodium hydroxide is corrosive. Avoid skin and eye contact. Methanol is volatile and flammable. 10
Waste disposal: Students must: a) neutralize aqueous acid solutions prior to pouring into drain and b) collect solvent waste in the organic waste container.
4.B.2. Prilocaine hydrochloride (5)
CH3
CH3
H N
Cl O
4
1) PrNH2 2) HCl (g)
Cl
H N O
N H H
5
In an airtight vial, a solution of 250 mg (1.26 mmoles) of chloroamide 4 in 2 mL (23.7 mmoles) of n-propylamine was kept at room temperature for three days (ote 1). After verifying the end of the reaction by TLC (hexane:AcOEt 1:1), the excess n-propylamine was removed in a water bath and the residue blown with nitrogen in the foomhood (ote 2). The residue was then dissolved in 5 mL AcOEt, washed with 2 mL of a 5 % aqueous NaHCO3 solution and dried with anh. sodium sulfate. Prilocaine hydrochloride was obtained by adding 3 mL of saturated hydrogen chloride solution in isopropyl alcohol (ote 3) and stirring it for half an hour in a cooling bath (ote 4). White crystals, m.p. 163-164, were obtained by vacuum filtration. otes: 1. Longer reaction times do not affect the outcome of the reaction. 2. It is very important to evaporate n-propylamine as completely as posible (i.e., no odor detected). 3. A 0.5 N hydrogen chloride solution in isopropyl alcohol (VWR®) is commercially available. Alternatively, gaseous HCl may be safely generated in the laboratory (21). 4. This is a mixed-solvent crystallizing system. Students might want to read chapter 13 (‘Recrystallization’) of Zubrick’s book (22).
Safety considerations: Safety glasses, gloves and a fume hood are recommended when performing this experiment. Propylamine is highly flammable, toxic and irritating to the skin; avoid skin contact and inhalation. Hydrogen chloride is corrosive and irritating to the eyes, skin, and mucous membranes; inhalation may result in pulmonary edema. Waste disposal: Students must: a) neutralize aqueous acid solutions prior to pouring into drain and b) collect solvent waste in the organic waste container. 11
List of substances used:
NFPA RATINGS Compound
CAS #
Health
Flammability
Reactivity
Acetic anhydride
108-24-7
2
2
1
Ammonium formate
540-69-2 90
1
0
0
2-Chloropropionyl chloride
7623-09-8
3
3
2
Ethyl acetate
141-78-6
1
3
0
Hydrochloric acid
7647-01-0
3
0
0
Hydrogen chloride (gas)
7647-01-0
3
0
1
Isopropyl alcohol
67-63-0
1
3
0
Methyl t-butyl ether
1634-4-4
2
3
0
Nitric acid
7697-37-2
3
0
0
n-Propylamine
107-10-8
3
3
0
Paladium 5% on carbon
7440-05-3
1
3
0
Sodium acetate trihydrate
6131-90-4
1
1
0
Sodium hydroxide
144-55-8
3
0
1
Sodium sulfate
7757-82-6
1
0
0
Sulfuric acid
7664-93-9
4
0
2
Toluene
108-88-3
2
3
0
Special Hazards
W
Oxidizer
W
12
GC analysis of the MT mixture
Instrument: Hewlett Packard 5880
Tinj = 260 °C
Detector: flame ionization
Tdet = 260 °C
Column: Zebron ZB-5 capillary column
Carrier gas: H2
(5 % phenyl, 95 % dimethylpolisiloxane). L=30 m
ID= 0.25 mm df = 0.25 µm
Ti = 100 °C
µ = 55 cm / sec
ti = 0 min
Flow rate = 1.6 mL / min
vp = 10 °C / min
ΦCol = 1.6 mL / min
Tf = 150 °C
γsplit = 50
tf = 0 min
ΦSplit = 80 mL / min
Retention times: ortho: 3.29 min; meta: 3.61; para: 3.82 min.
13
HPLC analysis of the MT mixture (3)
Instrument: Waters 600E Column: Symmetry C18 (4.6 X 250 mm) Data acquisition system: Chrom Perfect Flow rate: 1.0 mL/min Injection volume: 5 µL (1 mg/mL) Detector: Waters 486 tunable absorbance detector Detector wavelength: 254 nm Eluent: 60% methanol / 40% water Retention times: ortho: 11.40 min; para: 12.07 min; meta: 12.91. 14
1
H NMR and 13C NMR spectra for compound 4
CH3
H N
Cl O
15
5. LITERATURE CITED 1. Daryl L. Ostercamp, D. L. and Brunswold, R. J. Chem. Educ. 2006, 83 (12), 1816-1820. 2. Hall, M. Anesthesia Progress, 1972, 65-67. 3.Werdehausen, R.; Fazeli, S.; Braun, S.; Hermanns, H.; F. Essmann, F.; Hollmann, M. W.; I. Bauer, I. and Stevens, M. F. British J. Anesthaesia 2009, 103 (5), 711-718 . 4. Guay, J. Anesthesia and Analgesia 2009, 108 (3), 837–845 5. Nakamura, K. et al. Can. J. Anesth 1999, 46 (5), 476-482. 6. a) Akermann, B.; Persson, H. and Tegner, C. Acta Pharmacol. et Toxicol. 1967, 25, 233-241. b) Akerman, B.; Ross, S. Acta Pharmacologica et Toxicologica 1970, 28 (6), 445–453. 7. Waller, F. J.; Barrett, A. G. M.; D. Braddockb, C. and Ramprasada, D. Chem. Commun., 1997, 613-614. 8. a) Gigante, B.; Prazeres, A. O.; Marcelo-Curto, M. J.; Cornelis, A. and Laszlo, P. J. Org. Chem. 1995, 60 (11), 3445-7. b) Kogelbauer, A.; Vassena, D.; Prins, R. and Armor, J. N. Catalysis Today 2000, 55, 151–160. 9. Blankespoor, R. L.; Hogendoorn, S. and Pearson, A. J. Chem. Ed. 2007, 84, 697-698. 10. Owsley, D.C. and Bloomfield, J. J. Synthesis 1977, 2, 118-120. 11. Johnstone, R. A.; Anna H. Wilby, A. H.; Entwistle, I. D. Chem. Rev. 1985, 85 (2), 129-170. 12. Gowda, D. C. and Mahesh, B. Synthetic Communications 2000 30 (20), 3639-3644. 13. Williamson, K. L. Macroscale and microscale organic experiments, 3rd. ed., Houghton Mifflin: New York, 1999. 14. a) Lofgren, N. and Tegner, C. P. U.S. Pat. 3,160,662 (1964). b) Brown, C. L., U.S. Pat. 3,646,137 (1972). 15. Kremer, C. B. J. Chem. Educ. 1956, 33 (2), 71-72. 16. Organic Syntheses, Coll. Vol. I, Wiley: New York, 1941; pp 111-113. (http://www.orgsyn.org/). 17. Pavia, D.L., Lampman, G. M., Kriz, G. S. and Engel, R. G., Introduction to Organic Laboratory Techniques. A Microscale Approach. 3° ed., Saunders: Fort Worth, 1999; pp 362364. 18. Wilcox, F.; Wilcox, M. Experimental Organic Chemistry, A Small-Scale Approach. PrenticeHall:New Jersey, 1995; pp 489-491. 19. Vogel´s Text-book of Practical Organic Chemistry. 3th Ed. Longman: London, 1957; p 1000. 20. Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchell, A. R. Vogel’s Textbook of Practical Organic Chemistry, 5th ed., Longman Group: London, 1989 p 897. 21. Arnáiz, F. J. J. Chem. Educ., 1995, 72 (12), 1139. 22. Zubrick, J. The Organic Chem Lab Survival Manual. J. Wiley and Sons, 4th. Ed., 1997, 132135.
16
