Presentación de los resultados de acuerdo a los objetivos específicos

The derivatisation of the enkephalin molecule is done to increase stability, activity and hydrophobicity.

The inclusion of the lipidic amino acids on the enkephalin molecule either by substitution or by conjugation increases hydrophobicity. This increase should allow greater crossing of biomembranes and the blood-brain barrier.

It was hoped that the correct substitution position would increase activity and alter enkephalin receptor selectivity from S to n .

Amide and ester or di-ester linkages were used in the enkephalin molecule for several reasons. Reduction of the terminal carbonyl to amides and ester is known to increase enkephalin activity. In addition, esters and amides act as pro-drugs which deliver the drug effectively and selectively to the active site, the subsequent removal of these groups then leaves the free parent molecule. The ester and amide groups were therefore used to take the enkephalin molecule to the required active site and then release the enkephalin by spontaneous or enzymatic transformation. The pro­ drug linkages to the enkephalin molecule should therefore enhance efficacy and reduce the toxicity and unwanted side effects, by controlling their absorption.

6. Elastase Inhibitors

Elastases are proteolytic enzymes which degrade elastin found in elastic fibres and are involved in connective tissue disorders and inflammatory diseases.

The objective of the work described is the solution phase synthesis of elastase inhibitors with increased lipophilicity. The conjugation of lipidic amino acids and their oligomers to the tetrapeptide sequence (143) increases lipophilicity and enhances their ability to cross biomembranes (eg gut, blood brain barrier) and also the ability to attach to the cell membrane. The elastase inhibitors produced could prevent proteolysis and the subsequent damage caused to cartilage and elastic fibres.

One of the substrates of the elastases is elastin. Structurally, elastin has a

repeating amino acid sequence, the amino acids present being mainly nonpolar with aliphatic side chains. The elastase inhibitors synthesised have a common tetrapeptide sequence, which partially mimics the amino acid sequences found in elastin. The recognition of the elastase inhibitor sequence will allow the inhibitor to bind the elastase enzyme preventing the elastin from breaking.

The tetrapeptide sequence synthesised for inclusion in the elastase inhibitors is Ala-Ala-Pro-Val (143). 143 has been synthesized by Homebeck et a l^ and found to inhibit the action of human leucocyte and pancreatic (porcine) elastase.

— N— C— C ~ N — C ~ C ~ N

I

CWj

C H ÿ ) = ç Hi

0

N - C - C -

The tetrapeptide sequence Ala-Ala-Pro-Val (143) alone is not a potent inhibitor but becomes so when attached to a lipophilic moiety. The importance of the lipophilic group attached to the elastin mimicing peptide sequence is the ability to anchor the inhibitor to the cell membrane, so protecting elastin.

The lipophilic groups which have previously been used in the elastase inhibitors include oleic acid (144), myristic acid (145), succinic acid (146) and methoxycinnamoyl-5-amino-hexanoate (147). The lipidic amino acids and their oligomers were attached via carbodiimide coupling to the peptide sequence (143) to act as the lipophilic moiety. The hydrophobicity of the lipidic oligomers can be controlled by increasing or decreasing the number of amino acids constituting the hydrophobic group or by altering the length of the amino acid alkyl chain.

0

C H - ( C H ^ ) - C H = C H - ( C H ^ ) - C - O H

%

y

y

C H — ( C H ^ l^ ^ C -O H (145) H O - C — C H ^ C H ^ C - O H (146)

CHgO—

C H = C H —[ — N — ( C H 2 ) ^ C " 0 H (i4 7 )

The rationale for the synthesis of elastase inhibitors containing lipidic amino acid oligomers was first, the ability to control their hydrophobicity and secondly to standardise the structure of the hydrophobic group.

To date all the lipidic amino acids and their oligomers have passed all biocompatibility and toxicological tests, it is thus hoped that toxicity or antigenicity problems will not occur.

6. 1 Chemistry of the Elastase Inhibitors

The synthesis of the elastase inhibitors was carried out using solution phase peptide synthesis, in a stepwise method.

N-a-tertiarybutoxycarbonyl proline (BocPro) (148) and valine methyl ester hydrochloride (Val OMe HCl) (149) were coupled using dicyclohexylcarbodiimide (DCC) giving the dipeptide Boc-Pro-Val OMe (CXXXV) (Scheme 35). The Boc group was used to protect the terminal amino group of dipeptide (CXXXV) and a methyl ester was used to protect the terminal carboxyl group. Boc and methyl ester protecting groups were chosen due to their ease of synthesis and removal, the Boc group can be removed by acid hydrolysis and the methyl ester by alkali saponification. q

( C H ^ ) ^ C O C - f T

H 0

C f 3 I

3

(1481

OH

CHj

C H ,

0 . ( C H ^ l j C O C - t ^ l ^ Ijl g

C - N - Ç - C - O C H

0

CH

k x x x v )

The synthesis of the elastase inhibitor was continued with the deprotection of the terminal amino group of (CXXXV) and the addition of BocAla (136) giving BocAla-Pro-Val-OMe (CXXXVIIa). Tripeptide (CXXXVIIa) was deprotected at the terminal amino group to yield amine (CXXXXIII), with (CXXXXIII) acting as the core group to which a series of peptides and peptide derivatives were added (scheme

36).

n

H

k x x x v lHCtXMs m

H 0

^CHg^lOC-

' '

n

I 1 11

0

^QH

The second alanine in the elastase mimicing sequence (143) was included in the lipophilic synthon, prior to its combination with amine (CXXXXIII). The synthor method was used to aid stability and solubility.

Oleic acid (144) and alanine ethyl ester hydrochloride (150) were coupled with DCC to give oleoyl-alanine ethyl ester (CXXXI), which was subsequently deprotected at the carbonyl terminus to give the free acid (CXXXII) (scheme 37).

0 H O