Candida antarctica lipase-catalyzed hydrolysis of 4-substituted bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates as the key step in the synthesis of optically active dihydropyridines

Article abstract of DOI:10.1016/S0957-4166(00)00441-9

Prochiral bis(ethoxycarbonylmethyl) substituted 4-aryl-1,4-dihydropyridine-3,5-dicarboxylates were hydrolyzed enantioselectively by Candida antarctica lipase B (Novozym 435). The enantiomeric excesses varied from 68 to 93%, depending on the substituent at

Full text of DOI:10.1016/S0957-4166(00)00441-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 4559–4569
Candida antarctica lipase-catalyzed hydrolysis of
4-substituted bis(ethoxycarbonylmethyl)
1,4-dihydropyridine-3,5-dicarboxylates as the key step in the
synthesis of optically active dihydropyridines
Arkadij Sobolev,a,b Maurice C. R. Franssen,a,* Natalija Makarova,b Gunars Dubursb and
Aede de Groota
aLaboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
bLatvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
Received 12 October 2000; accepted 6 November 2000
Abstract
Prochiral bis(ethoxycarbonylmethyl) substituted 4-aryl-1,4-dihydropyridine-3,5-dicarboxylates were
hydrolyzed enantioselectively by Candida antarctica lipase B (Novozym 435). The enantiomeric excesses
varied from 68 to 93%, depending on the substituent at position 4. In some cases, the e.e. could be
significantly increased by changing the solvent system. © 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
4-Aryl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1,4-DHP) derivatives are widely
used for the treatment of cardiovascular diseases (hypertension, angina pectoris, infarction).1
1,4-DHPs having different ester groups at the 3- and 5-positions possess a stereogenic carbon at
the 4-position in the 1,4-DHP nucleus, and enantiomers often show different biological
activities.2 The first optically active DHP derivative appeared on the market in 1992; however,
at present, almost all chiral DHPs are still sold as racemates.3
Most investigations in the field of chiral dihydropyridines have been devoted to the synthesis
of calcium antagonists.4 We are focusing on novel activities of 1,4-DHPs such as antidiabetic,
nootropic, neuromodulatory, regulatory mode action and neuropeptide mimicking effects.1,5
These activities have been found for some 4-pyridyl and 2-difluoromethoxyphenyl substituted
dihydropyridines.2,5,6 Derivatives of bis(ethoxycarbonylmethyl) substituted 1,4-dihydropyridine-
3,5-dicarboxylates possess antimetastatic properties in combination with a low toxicity.7
* Corresponding author. Tel: +31 (0)317 482976; fax: +31 (0)317 484914; e-mail: maurice.franssen@bio.oc.wau.nl
0957-4166/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00441-9

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Amongst the many chemical methods for preparing enantiopure compounds, the biotechno-
logical approach based on enzyme-catalyzed enantiomeric differentiation has become a promis-
ing approach for the synthesis of enantiopure 1,4-dihydropyridines.8 The use of biocatalysts
shows a number of distinct advantages as compared to other methods: enzymes are active under
mild conditions and they often exhibit high stereoselectivity, combined with a broad substrate
tolerance. In the case of 1,4-DHPs enzymes are not capable of hydrolyzing alkyl esters at the 3-
and 5-positions of 4-aryl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylates.8 Modification of
the alkyl esters to activated groups makes the hydrolysis possible, in some cases with high
stereoselectivity.9 When substituted alkyl ester chains were introduced at the 3- and/or 5-posi-
tions of the dihydropyridine ring, rabbit liver esterase showed the hydrolysis of the more distant
ester group with a moderate degree of selectivity.10 Seaprose S (Aspergillus melleus) catalyzed the
hydrolysis or transesterification of ethoxycarbonylmethyl esters of 1,4-DHP with splitting of
both ‘outer’ and ‘inner’ ester groups to give the corresponding carboxylic acid or methyl
ester.11,12 It has also been reported that the stereoselectivity of lipase AH (Pseudomonas sp.)
toward the same 1,4-DHPs can be changed or even reversed by changing the solvent.13
In this paper, the Candida antarctica B lipase-catalyzed enantioselective hydrolysis of pro-
chiral bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates 3a–f with different sub-
stituents at the position 4 is described.
2. Results and discussion
Bis(ethoxycarbonylmethyl) substituted 1,4-dihydropyridine-3,5-dicarboxylates 3a–f were pre-
pared by Hantzsch cyclization of ethoxycarbonylmethyl acetoacetate 2 with aromatic aldehydes
1a–f in ethanol with 42–67% yields (Scheme 1). In the case of aryl substituted 1,4-DHPs 3a–e,
Scheme 1.

A. Sobole6 et al. / Tetrahedron: Asymmetry 11 (2000) 4559–4569
4561
gradual addition of ammonia to the reaction mixture increases the yield by compensating for the
loss of ammonia due to evaporation.
The first enzymatic asymmetrizations of the substrates 3a–f were performed in phosphate
buffer pH 7.5, modified with 15% of acetonitrile, at 45°C using protease P6 (Aspergillus melleus)
and acylase 30,000 (Aspergillus sp.). Both enzymes readily hydrolyzed the ‘outer’ ester group on
both sides of the substrates, eventually leading to the diacids 5a–f. This contrasts the earlier
reports where these enzymes and Seaprose S hydrolyzed the ‘inner’ ester.11,12 Furthermore,
alkaline hydrolysis only takes place at the ‘outer’ ester groups7 because of steric and electronic
factors.
Long reaction times and low enantioselectivity of protease P6 (Aspergillus melleus) and
acylase 30,000 (Aspergillus sp.) in the given reaction conditions led us to investigate other
possibilities of conversion of substrates 3a–f. Initial studies of the enantioselectivity of Candida
rugosa lipase and Rhizomucor miehei lipase towards the substrates 3a–f showed that these
enzymes have also low enantioselectivity under the given reaction conditions.
Candida antarctica lipase B (CAL B) is a very efficient catalyst for the enantioselective
transformations of different kinds of substrates and is widely used in practice.14 When Candida
antarctica B lipase was used for the asymmetrization of 3a–f in phosphate buffer pH 7.5
(modified with acetonitrile, at 45°C; Table 1), a rather high enantioselectivity was reached
together with shorter reaction times, so this enzyme was used for all subsequent experiments.
The reaction should be carefully monitored by HPLC because of the subsequent hydrolysis to
the diacid. The limiting factor of the hydrolysis of the aryl substituted 3a–e is their insolubility
in aqueous medium. Mixing the phosphate buffer with acetonitrile enhances the solubility of the
substrates 3a–e, but the reaction mixture remains heterogenous at the start. The fact that the
substrates dissolve during the reaction makes it difficult to directly compare reaction rates for
the different substrates. Only in the case of 4-pyridyl substituted 1,4-DHP 3f was good solubility
in buffer with just 5% of acetonitrile observed.
Table 1
CAL B-catalyzed hydrolysis of 3a–f in phosphate buffer, pH 7.5 modified with acetonitrile at 45°C
Entry Substrate
Reaction
mediuma
Time (h)
Chemical
yield (%)
Enantiomeric excess
(e.e., %)
Optical rotation,
[h]2D0
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
A
A
A
A
A
B
19
48
20
96
48
18
87
44
66
31
29
58
93
79
77
68
72
82
+49.9
+44.7
+52.5
+5.5
+15.1
+23.7
a (A) 15% solution of acetonitrile in 20 mM K-phosphate buffer, pH 7.5; (B) 5% solution of acetonitrile in 20 mM
K-phosphate buffer, pH 7.5.
The above mentioned solubility difficulties of the substrates such as 3d,e and insufficient
enantioselectivity of CAL B under the given reaction conditions towards substrates 3b–f forced
us to search for more suitable reaction conditions. It is known that the stereoselectivity of an
enzyme can change considerably and sometimes even can reverse on transition from one solvent
to another.13,15

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Altering the reaction media led in some cases to a higher enantioselectivity of CAL B towards
substrates 3; some examples are given in Table 2. From this table it is clear that the e.e. of 4b
was remarkably improved when water-saturated isopropylether (IPE) was used, albeit at the
expense of the reaction rate. However, in other cases changing the reaction medium to IPE did
not have a significant influence on the enantioselectivity of enzyme. Other organic solvents such
as acetone, tetrahydrofuran, t-butanol and dimethylsulfoxide were used instead of acetonitrile,
but in all cases (including acetonitrile) an increase of the amount of organic solvent in reaction
mixture led to longer reaction times and a decreased e.e. of the product.
Table 2
Some examples of CAL B-catalyzed hydrolysis of 3a–f in different reaction media at 45°C
Entry Substrate
Reaction
mediuma
Time (h)
Chemical yield of 4 (%) Enantiomeric excess (e.e., %)
1
2
3
4
5
6
7
8
9
3a
3a
3b
3c
3d
3d
3e
3e
3e
3e
C
D
C
C
D
E
D
E
F
48
22
282
168
48
48
200
24
21b
49b
47
88
55
97
67
68
69
1
70
92
93
15b
27b
35b
40
39b
55
39
28
138
10
G
a Reaction medium: (C) IPE/water; (D) 15% solution of t-butyl alcohol in 20 mM phosphate buffer pH 7.5; (E)
15% solution of acetone in 20 mM phosphate buffer pH 7.5; (F) 1% DMSO solution in 20 mM phosphate buffer pH
7.5; (G) 20% of DMSO in 20 mM phosphate buffer pH 7.5.
b Chemical yields were determined by HPLC.
The structure of the products was proven by mass spectrometry. Since mass spectra of the
enzymatic products 4a–f did not show molecular ions, the acids were converted into the
corresponding esters 6a–f by esterification with MeI in DMF (Scheme 2). Other ester derivatives
of compound 3b which could be suitable for the determination of the absolute configuration by
X-ray analysis were synthesized using different approaches. Attempts to obtain suitable crystals
failed, despite the fact that racemic analogues of the synthesized esters are often crystalline
materials, as for example in the case of 6d,e. It is already known16 that problems with
crystallization of stereoisomers may occur even if the corresponding racemate is a crystalline
substance.
Scheme 2.

A. Sobole6 et al. / Tetrahedron: Asymmetry 11 (2000) 4559–4569
4563
3. Conclusions
In conclusion, the Candida antarctica lipase catalyzed asymmetrization of 4-substituted
bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates has been developed.
Although complete stereoselectivity of Candida antarctica lipase towards all substrates was not
achieved in all cases, the results obtained have shown that a change of the reaction conditions
can improve the stereoselectivity of the process. Our investigations of lipase-catalyzed hydrolysis
of 1,4-DHPs show the way for the synthesis of various chiral biologically active dihydropyri-
dines containing different aryl and heterocyclic substituents in position 4.
4. Experimental
4.1. General
Flash column chromatography was performed on Merck silica gel 60 (230–400 or 70–230
1
mesh). H NMR spectra were recorded on a Bruker WH 90/DC (90 MHz) or a Bruker AC-E
200 (200 MHz) or a Bruker Avance DPX 400 (400 MHz) spectrometer. 13C NMR spectra were
recorded on a Bruker AC-E 200 (50 MHz) or a Bruker Avance DPX 400 (100 MHz)
spectrometer. Chemical shifts are reported in parts per million (ppm) relative to trimethylsilane
(l 0.00). Mass spectral data and accurate mass measurements were determined on a Finnigan
MAT 95 mass spectrometer. Melting points were determined on a Boetius apparatus and are
uncorrected. Optical rotation values were measured with a Perkin–Elmer 241 digital polarimeter.
Elemental analyses were determined on a Carlo Erba elemental analyzer. The reaction mixtures
were analyzed by HPLC on a 4.6×250 mm column packed with 5 mm Spherisorb ODS-2 (Phase
Separations) using a Gynkotek 480 pump and Applied Biosystems 758A programmable
absorbance detector at 254 nm. The solvent system acetonitrile/water/acetic acid (60:40:0.1) was
used as mobile phase at a flow rate of 1.0 ml/min. Determination of enantiomeric excesses of the
products 4a–f was performed by direct analysis on a chiral column Chirex 3011, 4.6×250 mm,
5 mm (Phenomenex) using a Ginkotek 580A pump and an Applied Biosystems 759A absorbance
detector at 254 nm. Other Pirkle and cavity-type columns were useful for the analysis of e.e. of
products of the reactions but this was the most universal one. The eluent was 0.05 M
ammonium acetate in methanol at a flow rate of 1.0 ml/min for 4a–c,d,f or dichloromethane/
methanol/acetic acid (80:20:0.5) for compound 4e. Peak areas were determined electronically
with the Chromeleon chromatography data system, Dionex Softron GmbH (Germering, Ger-
many). Enzymatic reactions were carried out in a New Brunswick Scientific Innova 4080
incubatory orbital shaker. Immobilized Candida antarctica lipase B, Novozym 435, was a gift
from Novo Nordisk A/S (Bagsvaerd, Denmark).
4.2. General procedure of the synthesis of bis(ethoxycarbonylmethyl)
4-aryl-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates 3a–e
Ethoxycarbonylmethyl acetoacetate 2 (29.0 g, 0.15 mol), 0.077 mol of benzaldehyde 1a or the
corresponding substituted benzaldehyde 1b–e and 5 ml (0.065 mol) of 25% aqueous ammonia
were dissolved in 50 ml of ethanol and were heated under reflux. After 2 h of refluxing, 5 ml of
25% aqueous ammonia was added in two to three portions with 0.5 h intervals. The usual

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A. Sobole6 et al. / Tetrahedron: Asymmetry 11 (2000) 4559–4569
quantity of added ammonia was 10 ml (0.13 mol). Usual time of refluxing was 6 h. After cooling
until −5°C the precipitated product was filtered and recrystallized from ethanol.
4.2.1. Bis(ethoxycarbonylmethyl) 1,4-dihydro-2,6-dimethyl-4-phenylpyridine-3,5-dicarboxylate 3a
1
Yield: 42%, mp 84–85°C; H NMR (CDCl3, 90 MHz): l 1.23 (6H, t, J=7.0 Hz, CH2CH3),
2.33 (6H, s, 2×CH3), 4.15 (4H, q, J=7.0 Hz, CH2CH3), 4.55 (4H, s, 2×COOCH2COO), 5.09
(1H, s, CH), 6.00 (1H, br s, NH), 7.05–7.30 (5H, m, C6H5); anal. calcd for C23H27NO8: C, 62.01;
H, 6.11; N, 3.14; found: C, 62.06; H, 6.12; N, 3.14.
4.2.2. Bis(ethoxycarbonylmethyl) 4-(2%-difluoromethoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine-
3,5-dicarboxylate 3b
1
Yield: 55%, mp 101–102°C; H NMR (CDCl3, 90 MHz): l 1.20 (6H, t, J=7.0 Hz, CH2CH3),
2.30 (6H, s, 2×CH3), 4.10 (4H, q, J=7.0 Hz, CH2CH3), 4.50 (4H, s, 2×COOCH2COO), 5.33
(1H, s, CH), 6.25 (1H, br s, NH), 6.45 (1H, t, J=75 Hz, OCHF2), 6.87–7.40 (4H, m, C6H4);
anal. calcd for C24H27F2NO9: C, 56.36; H, 5.32; N, 2.74; found: C, 56.42; H, 5.33; N, 2.71.
4.2.3. Bis(ethoxycarbonylmethyl) 1,4-dihydro-2,6-dimethyl-4-(3%-nitrophenyl)-pyridine-3,5-
dicarboxylate 3c
1
Yield: 67%, mp 157–159°C; H NMR (CDCl3, 90 MHz): l 1.18 (6H, t, J=7.0 Hz, CH2CH3),
2.33 (6H, s, 2×CH3), 4.09 (4H, q, J=7.0 Hz, CH2CH3), 4.50 (4H, s, 2×COOCH2COO), 5.77
(1H, s, CH), 6.57 (1H, br s, NH), 7.10–7.67 (4H, m, C6H4); anal. calcd for C23H26N2O10: C,
56.32; H, 5.34; N, 5.71; found: C, 56.38; H, 5.32; N, 5.70.
4.2.4. Bis(ethoxycarbonylmethyl) 4-(4%-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate 3d
1
Yield: 69%, mp 130–132°C; H NMR (CDCl3, 90 MHz): l 1.22 (6H, t, J=7.0 Hz, CH2CH3),
2.33 (6H, s, 2×CH3), 4.15 (4H, q, J=7.0 Hz, CH2CH3), 4.54 (4H, s, COOCH2COO), 5.05 (1H,
s, CH), 6.07 (1H, br s, NH), 7.08 and 7.23 (4H, two d, J=9 Hz, C6H4); anal. calcd for
C23H26ClNO8: C, 57.56; H, 5.46; N, 2.92; found: C, 57.56; H, 5.47; N, 2.90.
4.2.5. Bis(ethoxycarbonylmethyl) 4-(2%-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine-3,5-
dicarboxylate 3e
1
Yield: 38%, mp 115–117°C; H NMR (CDCl3, 90 MHz): l 1.16 (6H, t, J=7.0 Hz, CH2CH3),
2.30 (6H, s, 2×CH3), 4.10 (4H, q, J=7.0 Hz, CH2CH3), 4.50 (4H, s, COOCH2COO), 5.44 (1H,
s, CH), 6.41 (1H, br s, NH), 6.88–7.41 (4H, m, C6H4); anal. calcd for C23H26ClNO8: C, 57.56;
H, 5.46; N, 2.92; found: C, 57.56; H, 5.45; N, 2.91.
4.3. Bis(ethoxycarbonylmethyl) 1,4-dihydro-2,6-dimethyl-4-(3%-pyridyl)-pyridine-3,5-
dicarboxylate 3f
Ethoxycarbonylmethyl acetoacetate 2 (37.6 g, 90.2 mol), 9.4 ml (10.7 g, 0.1 mol) of
pyridine-3-carboxaldehyde 1f and 10 ml (0.13 mol) of 25% aqueous ammonia solution were
dissolved in 50 ml of ethanol and were heated under reflux for 3 h. After cooling until −5°C the
precipitate was filtered off and recrystallized from ethanol to give 25.4 g (57%) of crystalline
1
product, mp 143–145°C; H NMR (CDCl3, 90 MHz): l 1.21 (6H, t, J=7.0 Hz, CH2CH3), 2.34
(6H, s, 2×CH3), 4.17 (4H, q, J=7.0 Hz, CH2CH3), 4.54 (4H, s, 2×COOCH2COO), 5.09 (1H, s,

A. Sobole6 et al. / Tetrahedron: Asymmetry 11 (2000) 4559–4569
4565
CH), 7.06 (1H, br s, NH), 7.17 (1H, dd, J5,4=8 Hz, J5,6=4 Hz, H5 Py), 7.65 (1H, dt, J4,5=8 Hz,
J4,2=J4,6=2 Hz, H4 Py), 8.32 (1H, dd, J6,5=4 Hz, J6,4=2 Hz, H6 Py), 8.50 (1H, d, J2,4=2 Hz,
H2 Py); anal. calcd for C22H26N2O8: C, 59.19; H, 5.87; N, 6.27; found: C, 59.04; H, 5.86; N, 6.17.
4.4. General procedure for enzymatic hydrolysis of 1,4-dihydropyridine-3,5-dicarboxylates 3a–e
A solution of 0.5 mmol of 3a,c,d or 0.4 mmol of 3b,e in 60 ml of acetonitrile was added to
340 ml of 20 mM K2HPO4/KH2PO4 buffer (pH 7.5) and heated to 45°C, after which 600 mg of
Novozym 435 was added. The resulting mixture was shaken at 350 rpm and heated at 45°C.
Reactions were monitored by HPLC (see Tables 1 and 2 for further details about reaction
times). After removal of the enzyme by filtration, the filtrate was adjusted to pH 5.0 by adding
1 M HCl and extracted with chloroform (3×100 ml). The combined organic layers were
concentrated under reduced pressure. The residue was flash chromatographed on silica gel using
solvent system chloroform/isopropyl alcohol/acetic acid (100:20:0.1) to give the following
monoacids.
4.4.1. (+)-1,4-Dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxycarbonyl-4-phenyl-3-pyridine-
carboxylic acid 4a
Yield: 181 mg (87%) as a precipitate from hexane, mp 169–170°C; [h]2D0 +49.9 (c 1.0, MeOH);
1H NMR (DMSO-d6, 200 MHz): l 1.17 (3H, t, J=7.1 Hz, CH2CH3), 2.31 (6H, s, 2×CH3), 4.10
(2H, q, J=8.0 Hz, CH2CH3), 4.32 (2H, ABq, COOCH2COO), 4.60 (2H, s, COOCH2COO), 4.98
(1H, s, CH), 7.12–7.21 (5H, m, C6H5), 9.03 (1H, s, NH); 13C NMR (CD3OD, 50 MHz): l 14.41
(CH3), 18.86 (CH3), 18.91 (CH3), 40.20 (CH), 60.41 (CH2), 62.17 (CH2), 63.26 (CH2), 102.97,
104.10, 127.04 (CH), 128.69 (CH), 128.90 (CH), 147.51, 148.47, 148.92, 168.78, 169.48, 170.10,
176.51.
4.4.2. (+)-4-(2%-Difluoromethoxyphenyl)-1,4-dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxy-
carbonyl-3-pyridinecarboxylic acid 4b
Yield: 85 mg (44%) as a precipitate from hexane, mp 151–153°C; [h]2D0 +44.7 (c 0.855, MeOH);
1H NMR (DMSO-d6, 400 MHz): l 1.13 (3H, t, J=7.1 Hz, CH2CH3), 2.26 (3H, s, CH3), 2.29
(3H, s, CH3), 4.08 (2H, q, J=7.1 Hz, CH2CH3), 4.24 (2H, ABq, COOCH2COO), 4.50 (2H, ABq,
COOCH2COO), 5.25 (1H, s, CH), 6.95–7.13 (3H, m, C6H4), 7.00 (1H, t, J=74.9 Hz, OCHF2),
7.29 (1H, dd, C6H4), 9.03 (1H, s, NH); 13C NMR (DMSO-d6, 100 MHz): l 14.76 (CH3), 19.10
(CH3), 19.19 (CH3), 34.91 (CH), 60.82 (CH2), 61.33 (CH2), 63.35 (CH2), 100.73, 103.16, 117.94
(CH, t, J=252.9 Hz, OCHF2), 118.29 (CH), 125.89 (CH), 128.26 (CH), 131.70 (CH), 139.81,
145.71, 148.10, 148.98, 167.27, 167.60, 169.00, 173.06.
4.4.3. (+)-1,4-Dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxycarbonyl-4-(3%-nitrophenyl)-
3-pyridinecarboxylic acid 4c
Yield: 153 mg (66.0%) as crystals from ether/petroleum ether, mp 172–174°C; [h]2D0 +52.5 (c
1
1.0, MeOH); H NMR (DMSO-d6, 200 MHz): l 1.10 (3H, t, J=7.0 Hz, CH2CH3), 2.32 (6H, s,
2×CH3), 4.04 (2H, q, J=7.1 Hz, CH2CH3), 4.25 (2H, ABq, COOCH2COO), 4.59 (2H, s,
COOCH2COO), 5.07 (1H, s, CH), 7.50 (1H, t, C6H4), 7.73 (1H, d, C6H4), 7.96 (2H, d+s, C6H4),
9.33 (1H, s, NH); 13C NMR (CD3OD, 50 MHz): l 14.37 (CH3), 18.85 (CH3), 18.93 (CH3), 40.55
(CH), 61.44 (CH2), 62.20 (CH2), 63.47 (CH2), 102.21, 103.35, 122.06 (CH), 123.53 (CH), 130.08
(CH), 135.51 (CH), 148.36, 149.34, 149.38, 151.21, 168.18, 168.93, 169.89, 176.83.

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4.4.4. (+)-4-(4%-Chlorophenyl)-1,4-dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxycarbonyl-3-
pyridinecarboxylic acid 4d
Yield: 70 mg (31%) as a viscous oil; [h]2D0 +5.5 (c 1.0, MeOH); H NMR (CDCl3, 200 MHz):
1
l 1.24 (3H, t, J=7.1 Hz, CH2CH3), 2.33 (6H, s, 2×CH3), 4.19 (2H, q, J=7.1 Hz, CH2CH3), 4.59
(2H, ABq, COOCH2COO), 4.60 (2H, s, COOCH2COO), 5.06 (1H, s, CH), 6.45 (1H, s, NH),
7.16 and 7.25 (4H, two d, J=9 Hz, C6H4); 13C NMR (CDCl3, 50 MHz): l 14.07 (CH3), 19.10
(CH3), 38.61 (CH), 60.06 (CH2), 60.53 (CH2), 61.49 (CH2), 102.50, 102.59, 128.14 (CH), 129.24
(CH), 131.92, 145.58, 146.19, 146.39, 166.68, 168.70, 173.10.
4.4.5. (+)-4-(2%-Chlorophenyl)-1,4-dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxycarbonyl-
3-pyridinecarboxylic acid 4e
Yield: 53 mg (29%) as a precipitate from ether/petroleum ether, mp 175–177°C; [h]2D0 +15.1 (c
1
1.0, MeOH); H NMR (CDCl3, 200 MHz): l 1.22 (3H, t, J=7.2 Hz, CH2CH3), 2.34 (3H, s,
CH3), 2.36 (3H, s, CH3), 4.16 (2H, q, J=7.1 Hz, CH2CH3), 4.54 (2H, ABq, COOCH2COO),
4.60 (2H, s, COOCH2COO), 5.48 (1H, s, CH), 6.28 (1H, s, NH), 7.00–7.23 (3H, m, C6H4), 7.39
(1H, dd, 2-Cl-C6H4); 13C NMR (CDCl3, 50 MHz): l 14.04 (CH3), 19.20 (CH3), 19.31 (CH3),
37.05 (CH), 59.76 (CH2), 60.12 (CH2), 61.40 (CH2), 102.60, 126.98 (CH), 127.41 (CH), 129.20
(CH), 131.60 (CH), 132.40, 145.66, 146.18, 146.32, 166.74, 166.78, 168.95, 172.97.
4.5. (+)-1,4-Dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxycarbonyl-4-(3%-pyridyl)-3-pyridine-
carboxylic acid 4f
A solution of 223 mg (0.5 mmol) of 3f in 20 ml of acetonitrile was added to 380 ml of 20 mM
K2HPO4/KH2PO4 buffer solution and heated until 45°C after which 600 mg of Novozym 435
was added. The resulting mixture was shaken at 350 rpm for 17 h at 45°C. After removal of
enzyme by filtration, the filtrate was extracted with chloroform (2×100 ml). The filtrate was
acidified to pH 5.0 by adding 1 M HCl and concentrated in vacuum. The residue was flash
chromatographed on silica gel using chloroform/isopropyl alcohol/acetic acid (100:20:0.1) to
(50:50:0.1) to give 4f. Crystallization from ethyl acetate gave 122 mg (58%) of 4f as a powder,
mp 187–192°C (dec.); [h]2D0 +23.7 (c 1.0, MeOH); 1H NMR (DMSO-d6): l 1.13 (3H, t, J=7.0 Hz,
CH2CH3), 2.30 (6H, s, 2×CH3), 4.06 (2H, q, J=7.1 Hz, CH2CH3), 4.21 (2H, ABq,
COOCH2COO), 4.59 (2H, s, COOCH2COO), 4.95 (1H, s, CH), 7.23 (1H, dd, Py), 7.57 (1H, dt,
Py), 8.29 (1H, d, Py), 8.39 (1H, br s, Py), 9.13 (1H, s, NH).
4.6. (+)-4-(2%-Difluoromethoxyphenyl)-1,4-dihydro-2,6-dimethyl-5-ethoxycarbonylmethoxy-
carbonyl-3-pyridinecarboxylic acid 4b by hydrolysis in wet isopropyl ether
To a solution of 204 mg (0.40 mmol) of 3b in 50 ml of water-saturated isopropyl ether, 600
mg of Novozym 435 was added and the resulting mixture was shaken at 350 rpm for 12 days
(282 h) at 45°C. The enzyme was removed by filtration and washed additionally with methanol
and the filtrate was concentrated under reduced pressure. The residue was flash chro-
matographed on silica gel with chloroform/isopropyl alcohol/acetic acid (100:20:0.1) to give 86
mg (45%) of 4b as a solid and 95 mg (47%) of unreacted 3b. Data for 4b: [h]2D0 +72.6 (c 1.0,
1
MeOH); H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) are identical to
those for 4b described in Section 4.4.2.

A. Sobole6 et al. / Tetrahedron: Asymmetry 11 (2000) 4559–4569
4567
4.7. General procedure for the preparation of 6a–f
All derivatives were prepared in the same manner as described below for compound 6c.
4.7.1. Ethoxycarbonylmethyl methoxycarbonylmethyl 1,4-dihydro-2,6-dimethyl-4-(3%-nitro-
phenyl)-pyridine-3,5-dicarboxylate 6c
To a solution of 65 mg (0.14 mmol) of 4c in 1 ml DMF 29 mg (0.21 mmol) of K2CO3 was
added at rt and the resulting mixture was stirred for 2 h after which 0.017 ml (0.28 mmol) of
MeI was added. The reaction mixture was stirred for additional 2 h. The mixture was poured
into water and extracted with CHCl3. The extract was washed successively with water and brine,
then dried over MgSO4. After removal of solvent in vacuum the residue was flash chro-
matographed on silica gel with petroleum ether (bp 40–60°C)/chloroform/isopropyl alcohol
1
(8:2:2) to give 46 mg (69%) of 6c, mp 85–88°C (triturated with hexane–ether); H NMR (CDCl3,
200 MHz): l 1.24 (3H, t, J=7.2 Hz, CH2CH3), 2.42 (6H, s, 2×CH3), 3.72 (3H, s, CH3), 4.19 (2H,
q, J=7.2 Hz, CH2CH3), 4.60 (2H, s, COOCH2COO), 4.61 (2H, s, COOCH2COO), 5.24 (1H, s,
CH), 6.03 (1H, s, NH), 7.39 (1H, t, C6H4), 7.73 (1H, dd, C6H4), 8.02 (1H, dt, C6H4), 8.14 (1H,
t, C6H4); MS m/z (rel. abund.): 476 (M+, 4), 387 (3), 373 (4), 356 (3), 355 (16), 354 (100), 345
(3), 326 (3), 296 (10), 268 (6); HRMS calcd for C22H24N2O10: 476.1431; found: 476.1428.
4.7.2. Ethoxycarbonylmethyl methoxycarbonylmethyl 1,4-dihydro-2,6-dimethyl-4-phenyl-
pyridine-3,5-dicarboxylate 6a
1
Yield: 65% as a powder triturated with hexane, mp 112–114°C; H NMR (CDCl3, 200 MHz):
l 1.25 (3H, t, J=7.1 Hz, CH2CH3), 2.38 (6H, s, 2×CH3), 3.72 (3H, s, CH3), 4.19 (2H, q, J=7.2
Hz, CH2CH3), 4.59 (2H, ABq, COOCH2COO), 4.61 (2H, s, COOCH2COO), 5.14 (1H, s, CH),
5.89 (1H, s, NH), 7.10–7.36 (5H, m, C6H5); MS m/z (rel. abund.): 431 (M+, 4), 355 (11), 354
(100), 238 (3); HRMS calcd for C22H25NO8: 431.1580; found: 431.1578.
4.7.3. Ethoxycarbonylmethyl methoxycarbonylmethyl 4-(2%-difluoromethoxyphenyl)-1,4-
dihydro-2,6-dimethylpyridine-3,5-dicarboxylate 6b
1
Yield: 80% as a powder triturated with hexane, mp 83–85°C; H NMR (CDCl3, 200 MHz):
l 1.22 (3H, t, J=7.1 Hz, CH2CH3), 2.35 (6H, s, 2×CH3), 3.67 (3H, s, CH3), 4.15 (2H, q, J=7.1
Hz, CH2CH3), 4.55 (2H, ABq, COOCH2COO), 4.57 (2H, s, COOCH2COO), 5.40 (1H, s, CH),
5.96 (1H, s, NH), 6.49 (1H, t, J=75.3 Hz, OCHF2), 6.97–7.42 (4H, m, C6H4); MS m/z (rel.
abund.): 497 (M+, 6), 495 (4), 408 (3), 394 (4), 391 (4), 368 (9), 366 (5), 355 (16), 354 (100), 296
(5); HRMS calcd for C23H25F2NO9: 497.1497; found: 497.1501.
4.7.4. Ethoxycarbonylmethyl methoxycarbonylmethyl 4-(4%-chlorophenyl)-1,4-dihydro-2,6-
dimethylpyridine-3,5-dicarboxylate 6d
1
Yield: 71% as a precipitate from hexane–ether, mp 131–132°C; H NMR (CDCl3, 200 MHz):
l 1.25 (3H, t, J=7.2 Hz, CH2CH3), 2.39 (6H, s, 2×CH3), 3.72 (3H, s, CH3), 4.20 (2H, q, J=7.2
Hz, CH2CH3), 4.60 (2H, s, COOCH2COO), 4.61 (2H, s, COOCH2COO), 5.11 (1H, s, CH), 5.83
(1H, s, NH), 7.19 and 7.28 (4H, two d, J=9 Hz, C6H4); MS m/z (rel. abund.): 465 (M+, 4), 378
(3), 376 (3), 362 (3), 356 (3), 355 (17), 354 (100), 348 (3), 334 (4), 268 (3); HRMS calcd for
C22H24ClNO8: 465.1190; found: 465.1185.

4568
A. Sobole6 et al. / Tetrahedron: Asymmetry 11 (2000) 4559–4569
4.7.5. Ethoxycarbonylmethyl methoxycarbonylmethyl 4-(2%-chlorophenyl)-1,4-dihydro-
2,6-dimethylpyridine-3,5-dicarboxylate 6e
1
Yield: 40% as a precipitate from hexane–ether, mp 91–93°C; H NMR (CDCl3, 200 MHz): l
1.21 (3H, t, J=7.1 Hz, CH2CH3), 2.35 (3H, s, CH3), 2.36 (3H, s, CH3), 3.64 (3H, s, CH3), 4.15
(2H, q, J=7.2 Hz, CH2CH3), 4.57 (2H, ABq, COOCH2COO), 4.58 (2H, s, COOCH2COO), 5.50
(1H, s, CH), 6.23 (1H, s, NH), 7.00–7.24 (3H, m, C6H4), 7.40 (1H, dd, C6H4); MS m/z (rel.
abund.): 465 (M+, 3), 429 (3), 428 (13), 370 (4), 362 (3), 355 (14), 354 (100), 334 (3), 296 (5), 268
(4); HRMS calcd for C22H24ClNO8: 465.1190; found: 465.1186.
4.7.6. Ethoxycarbonylmethyl methoxycarbonylmethyl 1,4-dihydro-2,6-dimethyl-4-(3%-pyridyl)-
pyridine-3,5-dicarboxylate 6f
1
Yield: 60% as a powder triturated with hexane, mp 124–125°C; H NMR (CDCl3, 200 MHz):
l 1.24 (6H, t, J=7.2 Hz, CH2CH3), 2.39 (6H, s, 2×CH3), 3.71 (3H, s, CH3), 4.19 (4H, q, J=7.2
Hz, CH2CH3), 4.59 (2H, s, COOCH2COO), 4.61 (2H, s, COOCH2COO), 5.14 (1H, s, CH), 6.28
(1H, br s, NH), 7.18 (1H, dd, Py), 7.68 (1H, dt, Py), 8.40 (1H, br d, Py), 8.50 (1H, br s, Py);
MS m/z (rel. abund.): 432 (M+, 5), 355 (18), 354 (100), 343 (5), 329 (6), 301 (5), 297 (3), 296 (23),
268 (8), 211 (3); HRMS calcd for C21H24N2O8: 432.1533; found: 432.1532.
Acknowledgements
Financial support was provided by Copernicus programme (ERB-CIPA-CT 94 0121), NATO
(Linkage grant LST.CLG 974948), the Netherlands Ministry of Agriculture, Nature Manage-
ment and Fisheries (IAC grant), the Ph.D. programme of Wageningen University and a Ph.D.
Grant of the Latvian Council of Science. We are grateful to Novo Nordisk A/S (Bagsvaerd,
Denmark) for the donation of Candida antarctica lipase B, Novozym 435. We are indebted to
Mr. A. van Veldhuizen for recording the NMR spectra and to Mr. C. J. Teunis and Mr. H.
Jongejan for the mass spectral analyses.
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