Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

A. Sobolev

Research output: Thesisinternal PhD, WU


Chirality is important for the activity of many biologically active compounds, since differences in biological properties of stereoisomers occur frequently. The exact stereochemical composition of each new chiral compound as well as toxicological and pharmacological data for racemic and enantiomerically pure compounds are required for their approval as new chiral medicines in the EU.

The research described in this thesis deals with the chemoenzymatic synthesis of 1,4-dihydropyridine derivatives (1,4-DHPs) in enantiopure form as the key intermediates for chiral analogues of symmetrical biologically active compounds. The use of enzymes is an advantageous alternative to classical chemical methods, as enzymes are efficient catalysts with high chemo-, regio- and stereoselectivity under mild conditions.

In Chapter 1, a literature review is given about the synthesis of 1,4-dihydropyridine derivatives, and their biological activities. Synthesis of 1,4-DHPs by cyclocondensation reactions and reduction of pyridines are described whereby special attention is paid to stereoselective chemical and biotechnological methods for the synthesis of enantiopure 1,4-DHPs.

Derivatives of bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates have shown antimetastatic activities as well as activities against the Herpes simplex virus. The first objective of the current research is the enzyme-catalysed hydrolysis of these compounds as described in Chapter 2.

Prochiral bis(ethoxycarbonylmethyl) substituted 4-aryl-1,4-dihydropyridine-3,5-dicarboxylates 1a-f are hydrolysed enantioselectively by Candida antarctica lipase B (Novozym 435 ®) (Scheme 1). The enantiomeric excesses range from 68 to 97%, depending on the substituent at position 4. In some cases, the e.e. can be significantly increased by changing the solvent system.

Scheme 1

Cerebrocrast (2,6-dimethyl-3,5-bis[2-(propoxy)ethoxycarbonyl]-4-[2-(difluoromethoxy)phenyl]-1,4-dihydropyridine) is a highly active neuroprotector. This compound has been found active in the treatment of diabetes and various inflammatory disorders. Chapter 3 is devoted to the synthesis of chiral analogues of cerebrocrast in enantiopure form via enzyme-catalysed kinetic resolution of 2,6-dimethyl-4-[2-(difluoromethoxy)phenyl]-1,4-dihydropyridine 3,5-diesters.

Alkyl esters at the 3- and 5-positions of 2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxylates are not hydrolysed by commercially available hydrolases, and 4-substituted bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates can be cleaved by lipases only at the 'outer' ester group. Therefore, derivatives have been prepared which contain a spacer that spontaneously detaches after enzymatic hydrolysis of the 'outer' ester group (Chapter 3). Seven acyloxymethyl esters of 5-methyl- and 5-(2-propoxyethyl) 4-[2-(difluoromethoxy)phenyl]-2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate 3 have been synthesised and subjected to Candida rugosa lipase (CRL) catalysed hydrolysis in wet diisopropyl ether (Scheme 2). A methyl ester at the 5-position and a long or branched acyl chain at C(3) give the highest enantiomeric ratio ( E value). The most stereoselective reaction ( E =21) is obtained with 3-[(isobutyryloxy)methyl] 5-methyl 4-(2-difluoromethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 3 , and this compound is used to prepare both enantiomers of 3-methyl 5-(2-propoxyethyl) 4-[2-(difluoromethoxy)phenyl]-2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate 5 .

Scheme 2

The absolute configuration of the enzymatically-produced carboxylic acid has been established to be 4 R by X-ray crystallographic analysis of its 1-( R )-phenylethyl amide.

In Chapter 4, an efficient chemoenzymatic synthesis of (—)-( R )- 5 is described (Scheme 3). The key step in this approach is the asymmetrisation of a symmetrical bifunctional substrate. The enantioselectivity of Candida rugosa lipase-mediated asymmetrisation of the prochiral bis[(isobutyryloxy)methyl] 4-[2-(difluoromethoxy)phenyl]-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate is excellent (≥99%).

Scheme 3

The disodium salt of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)-glutaric acid (glutapyrone) possesses an unusually broad spectrum of biological activities at low concentrations such as neuromodulatory and neuroregulatory action. It is an anticonvulsant, stress-protective, antiarrhythmic, cognition and memory enhancing compound.

3,4,5-Trialkyl 2,6-dimethyl-1,4-dihydro-3,4,5-pyridinetricarboxylates like glutapyrone are inert to the attack of hydrolytic enzymes. The exchange of at least one alkyl group to an enzymatically labile moiety ( e.g. acyloxymethyl or ethoxycarbonylmethyl) turns these 1,4-dihydroisonicotinic acid derivatives into substrates for hydrolytic enzymes. Since acyloxymethyl and ethoxycarbonylmethyl derivatives of 2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxylates have been recognised as being susceptible to lipases (see Chapters 2-4), the corresponding derivatives of 1,4-dihydroisonicotinic acid 8 and 10 have been prepared (see Scheme 4). The lipase-catalysed kinetic resolution of five derivatives of 4-[(acyloxy)methyl] and 4-ethoxycarbonylmethyl 3-methyl 5-propyl 2,6-dimethyl-1,4-dihydro-3,4,5-pyridinetricarboxylates 8 and 10 has been investigated, and the results are described in Chapter 5. Whereas the enantioselectivity of lipases towards the acyloxymethyl derivatives 8 is rather low, the Candida antarctica lipase B (Novozym 435 ®) catalysed hydrolysis of the ethoxycarbonylmethyl ester of 1,4-dihydroisonicotinic acid 10 is enantioselective. In water-saturated diisopropyl ether at 45°C the enantioselectivity of Novozym 435 ®toward the ethoxycarbonylmethyl ester 10 is rather moderate ( E =13.8), but it is enhanced at rt and +4°C ( E =21.5 and E =28.9, respectively). A high enantiomeric ratio ( E =45.3) is reached at subzero temperatures, although at the expense of the reaction rate.

Scheme 4

Polycyclic 1,4-DHPs in enantiopure form are desired for extended pharmacological studies, since racemic 1,4-dihydrobenzothieno[3,2- b ]pyridine-5,5-dioxides and 5-oxo-4,5-dihydro-1,4-indeno[1,2- b ]pyridines possess various biological activities such as coronary dilating and anticancer; they have also been found active as glutathione S-transferase inhibitors.

The lipase-catalysed kinetic resolution of enzymatically labile 3-(isobutyryloxy)methyl 4-[2-(difluoromethoxy)phenyl]-2-methyl-5,5-dioxo-1,4-dihydrobenzothieno[3,2- b ]pyridine-3-carboxylate rac -12 is described in Chapter 6 (Scheme 5). The most enantioselective reaction ( E =28) is a CRL-mediated transesterification with n -butanol in water-saturated toluene, at 45°C.

Scheme 5

The main results of this study are evaluated in Chapter 7. The approaches to overcome inactivity of hydrolytic enzymes toward simple esters of 1,4-DHPs, with their advantages, disadvantages and limitations, are discussed. The perspectives of the applications of chemoenzymatic approaches to the synthesis of enantiopure pharmacologically important novel dihydropyridine derivatives are also outlined.

It can be concluded that the use of hydrolytic enzymes, acting on hydrolysable groups on spacers, is a useful and widely applicable method for the enantioselective synthesis of hydrogenated pyridines.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • de Groot, Aede, Promotor
  • Franssen, Maurice, Co-promotor
Award date17 Jun 2003
Place of Publication[S.I.]
Print ISBNs9789058088222
Publication statusPublished - 2003


  • synthesis
  • enzyme activity
  • enantiomers
  • pyridines
  • derivatives

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