Pregunta Específica

Variance from the published procedure occurred in the second and third steps o f the synthetic scheme. The first step, a Knoevenagel condensation between 4-

chlorobenzaldehyde 59 and diethyl malonate 60, catalysed by piperidine, was

monitored by the volume o f water byproduct formed, and the reaction terminated when water production ceased (its volume roughly agreed with that calculated from a literature yield; Pratt and Werble 1950). The pure product 61 was distilled in good yield.

In the second step, yields o f the cyano compound 62 decreased dramatically on scale-up, with the formation o f the cyano-acid 63 and many byproducts. The hydrocyanation reaction (Bredt and Kallen 1896) is believed to form a p-cyano-ketone intermediate 64 which is unstable in base. The strongly alkaline reaction medium- hydroxide ions are liberated as cyanide is consumed, promotes hydrolysis and subsequent decarboxylation (Nagata and Yoshioka 1985) to give product 62. It was assumed that prolonged reaction at elevated temperature permitted additional attack by hydroxide on species 62, resulting in the free acid 63 (after work-up), and promoted possible hydrolysis o f the newly introduced nitrile group to yield byproducts. Therefore, the temperature was reduced by 10°C, and the reaction progress carefully monitored by thin layer chromatography to observe when compound 63 and byproducts began to form, and followed by IR spectrofcoj>y. Periodically, aliquots o f the reaction mixture were extracted into ether and spectra obtained. The course o f the reaction was thus followed by comparing the increase in peak area o f the nitrile group to the decrease in area o f the carbonyl group, and terminating the reaction when a favourable ratio was reached.

C O O H Cl I b C N 'OH C O O E t I . la H ■c = I O E t 6 3 64

By varying the reaction conditions a yield o f 80% was obtained, which compares favourably with the literature yield o f 72%, and with 40-50% for the same compound (Allen and Johnson 1963).

In the third step it was found that catalytic hydrogenation at atmospheric pressure o f cyano intermediate 62 produced the hydrochlorides o f three compounds, the desired amino acid ester 65 and baclofen 38 as major products, with the lactam 4- (4-chlorophenyl)-pyrrolidine-2-one 66 as a minor constituent. Scale-up o f twenty-fold required a Parr Hydrogenator to decrease reaction times (from days to hours). This

resulted in a higher ratio o f lactam to the other two species. The reaction progress was again followed by IR spectrometry- by observing the ratio o f decrease in peak area o f the nitrile group with respect to peak area o f a carbonyl group at 1730 wavenumbers. The reaction was stopped when the absorption band o f the nitrile group was very weak. At this stage, the theoretical volume o f hydrogen taken up had been exceeded. The synthesis was continued using the lactam (the amino acid ester being stored for use as a precursor for lipidic amino acid derivatives). The long reaction period (33 hours) may have enabled acidolysis o f the ester group o f 65 to form baclofen, which may then have undergone dehydration due to high pressure (30psi) to give the lactam.

Kung et al (1983) obtained the corresponding lactam o f 4-amino-3-(3-bromo-4-

chlorophenyl)-butanoic acid by repeated sublimation in a bulb tube under reduced pressure and elevated temperature, whilst Blade-Font (1980) cyclodehydrated baclofen using an alumina support in boiling toluene. With hindsight and knowledge o f the results o f the third step, it may have been possible to alter the conditions o f the second step so that compound 63 was formed without too many byproducts. Then following hydrogenation, baclofen (as the major product) and lactam, may have been formed exclusively.

In the final step, acid hydrolysis of a sample o f the ester intermediate 65 produced baclofen, however, the amide bond o f the lactam 66 resisted boiling aqueous HCl, but was easily converted to the title compound by saponification with refluxing sodium hydroxide.

2.1.3 Svnthesis o f baclofen-lipidic amino acid analogues.

Baclofen was protected as its Boc derivative or its ethyl ester hydrochloride, then LA A ’s were reacted with one or both terminal groups, using amide or ester (giving a modified peptide bond) linkages, to furnish dimeric or trimeric peptides 71, 72, 75, 77, 79 (Table 2) and intermediates 74 and 78. It was envisaged that enzymic hydrolysis would release the parent compound, following peptidase activity in brain vasculature, hence the conjugates may act as pro-drugs.

X — (HÏT—Ç H —CO)p — HN GHa O I I C —Y — (CH—C ) — Z I l I r O ^CHg)^ CH 3 71, 72, 74, 75, 77, 78, 79

Table 2. Baclofen conjugates.

Cpd. X _P Y r n Z

71a Boc 0 -0 - 1 5 OCH3

71b Boc 0 - 0 - 1 11 OCH3 71c Boc 0 - 0 - 1 13 OCH3 72a H 0 - 0 - 1 5 OCH3 72b H 0 - 0 - 1 11 OCH3 72c H 0 -0- 1 13 OCH3 75a H 0 -NH 1 7 OCH3 75b H 0 -NH 1 9 OCH3 75c H 0 -NH 1 11 OCH3 75d H 0 -NH 1 13 OCH3

77a Boc 1 -NH l 7 OCH3

77b Boc 1 -NH 1 9 OCH3 77c Boc 1 -NH 1 11 OCH3 77d Boc 1 -NH 1 13 OCH3 79a Boc 1 0 0 7 O H 79b Boc 1 0 0 9 OH 79c Boc 1 0 0 11 OH 79d Boc 1 0 0 13 OH

Compounds 71a-71c (Scheme 2) were obtained by reacting the potassium crown ether salt o f protected baclofen with the appropriate a-brom oalkanoate. Baclofen was suspended in a mixture o f tert-butano 1/water (2:3) and sodium hydroxide added to form the corresponding sodium salt, then reacted with ditertbutyl-dicarbonate. After acidification o f the sodium salt with citric acid (mineral acid such as dilute hydrochloric acid may hydrolyse the newly introduced Boc group), Boc-protected

baclofen 67 was formed in good yield. Compound 67 was dissolved in ethanol, reacted

with aqueous potassium hydroxide and crown ether (18-crown-6), then solvent was evaporated and the residue lyophilized to give the potassium crown ether complex 68.

Bromoalkanoic acids (2-bromooctanoic, 2-bromotetradecanoic, and 2-

bromohexadecanoic acid) 69a-69c were esterified using methanol/thionyl chloride to

yield their methyl esters 70a-70c, and reacted immediately with the unstable lyophilate

68 to form products 71a-71c containing an ester (modified peptide) bond at the

Cl c l Cl HN COOH 6 9 , 7 0 , 7 1 B r - C H — C O O C H j 7 o a , b , c 6 9 a , b , c Cl dry DMF (CH ■7 la, b, c