Enzyme-catalysed improved resolution of (RS)-4-cyano-4-(3,4- dimethoxyphenyl)-4-isopropyl-1-butanol

Article abstract of DOI:10.1016/S0957-4166(99)00389-4

Resolutions of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1- butanol 1 using various enzymes were performed. Among them, Pseudomonas fluorescens resolved it with moderate stereoselectivity (E=13) and reacted faster with the (S)-enantiomer. To optimize enzyme-catalysed reaction conditions for the resolution, the effect of solvents and additives was studied. In n-hexane:ethyl acetate (9:1), both reaction rate and selectivity were high. When pyridine, potassium carbonate and molecular sieves were used as additives, the enantiomeric excess of the (R)-enantiomer was 99, 99 and 98% at 52-60% conversion, respectively. However, in diisopropyl ether, the enantiomeric excess of unreacted alcohol (R)-1 was up to 99% at 70% conversion without additives.

Full text of DOI:10.1016/S0957-4166(99)00389-4

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3759–3767
Enzyme-catalysed improved resolution of (RS)-4-cyano-
4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol
Dai Sig Im,a Chan Seong Cheong,b,∗ So Ha Lee,b Hokoon Park b and Byoung Hee Youn a
aDepartment of Chemistry, Yonsei University, Shinchon, Seoul 120-749, South Korea
bLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, South Korea
Received 23 July 1999; accepted 23 August 1999
Abstract
Resolutions of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1 using various enzymes were
performed. Among them, Pseudomonas fluorescens resolved it with moderate stereoselectivity (E=13) and reacted
faster with the (S)-enantiomer. To optimize enzyme-catalysed reaction conditions for the resolution, the effect of
solvents and additives was studied. In n-hexane:ethyl acetate (9:1), both reaction rate and selectivity were high.
When pyridine, potassium carbonate and molecular sieves were used as additives, the enantiomeric excess of
the (R)-enantiomer was 99, 99 and 98% at 52–60% conversion, respectively. However, in diisopropyl ether, the
enantiomeric excess of unreacted alcohol (R)-1 was up to 99% at 70% conversion without additives. © 1999
Elsevier Science Ltd. All rights reserved.
1. Introduction
4-Cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1 is a valuable compound as the interme-
diate for manufacturing not only Ca++ antagonists but also α-blockers.1 Verapamil, which is used
extensively for the treatment of hypertension as a racemate, has one stereogenic center and can be
synthesized from the compound 1 (Fig. 1). Recently, the pharmacological properties of (R)- and (S)-
enantiomers of verapamil have been shown to be quite different. The (S)-enantiomer has greater effects on
the slow calcium ion current and the (R)-enantiomer is useful in reversal of multidrug resistance in cancer
chemotherapy.2 Thus, attention has focused on methods to obtain the individual enantiomers. Among
the various results concerning the synthesis of compound 2, one is an asymmetric synthesis3 using the
synthon, (S)-1,2-propandiol, and there is a process via classical resolution of verapamil itself. An im-
proved method4 was reported using quinine and 4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropylbutanoic
∗
Corresponding author. E-mail: c2496@kistmail.kist.re.kr
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00389-4

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D. S. Im et al. / Tetrahedron: Asymmetry 10 (1999) 3759–3767
Figure 1.
acid. These methods appear to be tedious and involve long synthetic processes. To our best knowledge,
there are no reports concerning the resolution of the compound 1 using enzymes.
Lipases as chiral catalysts play an important role in the synthesis of enantiomerically pure compounds
by the resolution of racemates and have been used to catalyse the hydrolysis and esterification on a wide
variety of substrates.5 Although their usefulness is stressed, their application is limited to narrow fields
because of their specificities. Many studies have been carried out in order to develop the stereospecific
rules between lipases and substrates.6 A simple rule comes out from the relative sizes of the substituents
and some rules also include polarity or specific size restrictions for the two substituents. These rules seem
to work very well for secondary alcohols, but not for primary or tertiary alcohols. Our target molecule is
an uncommon structure for using enzymes and it cannot be predicted which enantiomer will react faster.
In the previous work,7 we used several lipases for resolving the similar structure to the compound 1. As
part of our continuing interest in the resolution of the quaternary chiral primary alcohol, we tried to get
the compound 1 in enantiomerically pure form as the single isomers (R) or (S) using various enzymes
and optimized resolution condition.
2. Results and discussion
Compound 1 was prepared, as shown in Scheme 1, to submit to the enzyme reactions. We used it and
its acetate 1-1 to obtain the pure enantiomers.
Scheme 1. (a) NaH, DMF, 2-bromopropane, 0°C→rt; (b) NaH, DMF, 3-tetrahydropyranyloxypropyl bromide, 0°C→rt; (c) 1N
HCl, MeOH
We investigated the enzyme-catalysed transesterification between the alcohol 1 and an acyl donor
(Scheme 2). As acyl donor, vinyl acetate and isopropenyl acetate were used to maintain irreversible
conditions and they produced similar results. Thus, we used the reactions of the alcohol with vinyl

D. S. Im et al. / Tetrahedron: Asymmetry 10 (1999) 3759–3767
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acetate in the presence of different commercially available enzymes. All of the reactions were carried
out at 32–34°C and under anhydrous conditions. These results were obtained simply to screen for the
appropriate enzymes when the used enzyme was 10% mass ratio to the substrate and the reaction time was
within 24 h. Actually, we presumed Pig liver esterase (PLE), Porcine pancreas lipase (PPL), or Candida
rugosa lipase (CRL) as the candidate enzymes for resolving compound 1 because the bulky stereogenic
centers should be resolved by them. However, as shown in Table 1, only Pseudomonas fluorescens (LAK)
and Pseudomonas cepacia (LPS) resolved it moderately and showed the same enantiomeric preference
as the (S)-enantiomer.
Scheme 2.
Although none of the lipases gave a high enantioselectivity (E8=6.5 at best), LAK was selected as the
best candidate for resolving the target molecule among the various enzymes. When it was used as mass
equivalent, the conversion was 50% and the selectivity of a resolution (E) was a moderate 13. In the
previous reports about the active site model for LAK, the primary and secondary alcohols were analysed
by virture of the shapes and sizes of the hydrophobic binding sites and the predictive stereospecificity
of their reactions worked very well. Also, the existence of a hydrophobic cleft in the active center of
Table 1
Lipase-catalysed resolutions of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1

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Pseudomonas cepacia has recently been postulated.9 According to that, we predicted the (R)-enantiomer
of the compound as the faster reacting one. In the other work, we had obtained (S)-2-butyl-2-cyano-2-
phenylethanol using Pseudomonas fluorescens by the three-dimensional model,6d as shown in Fig. 2.
However, in the resolution of compound 1 we obtained the opposite result as the (S)-enantiomer reacted
faster. This result seems to obey the flat or non-flat hydrophobic pocket model.6f Thus, we suggest that
this difference results from the molecular structure of the compound 1 as follows: (i) the reactive site is
far from the stereogenic center (three bonds), (ii) the stereogenic carbon is quaternary, not secondary.
Figure 2. Two models suggested by the experimental results
Next, we tried to establish reaction conditions to increase the stereoselectivity. To do this, such factors
as temperature effects,10 water level,11 solvent effect,12 variation of the co-substrate and the additive13
such as bases and metal salts in the irreversible transesterification were studied. We chose the two
variables, solvent and additive. At first, we investigated the proper choice of the solvent. The effect
of the solvent on substrate specificity and enantioselectivity of enzymes was well studied. In the enzyme
reaction under the organic medium, when a substrate interacts with the enzyme’s active center, water must
be excluded.14 Therefore, this phenomenon will affect reaction rate and reaction specificity in relation to
solvent polarity.
As shown in Table 2, using ethyl acetate, 1,4-dioxane and n-hexane, the reaction proceeded smoothly
within 10 h and the selectivities (E) were between 4.9 and 8.3. In polar solvents such as methanol, dichlo-
roethane and acetonitrile, the reaction rate was slow and, especially in dichloroethane and methanol, the
reaction did not proceed over 70 h. Among the above solvents, n-hexane as a non-polar solvent showed
good results. In the case of diisopropyl ether, unreacted alcohol R-1 was resolved in 99% ee. When n-
hexane:ethyl acetate as co-solvent was used, the reaction rate increased and the selectivity (E) was 13. We
assumed that this resulted from the solubility of the alcohol 1. Thus, we chose the co-solvent system to
increase the stereoselectivity in the range of 10–50% ethyl acetate:n-hexane solution. Finally, we studied
the effect of the additives on the stereoselectivity. These results are shown in Table 3.
One of the additive effects on stereoselectivity is deactivation of the enzyme. In the irreversible reaction
using an acyl-donor, the acetaldehyde seems to be detrimental to the reaction rate and stereospecificity.13
The mechanism of enzyme deactivation by aldehydes involves Schiff-base formation on lysine residues.
Another additive effect is modification of the lipase local conformation as mentioned by Itoh et al.15
These two aspects would explain the results as shown in Table 3. Although Pseudomonas lipases are less
sensitive to aldehyde than the other lipases,13 we investigated the aldehyde effect on LAK in the above
reaction. Among the various additives, pyridine and potassium carbonate gave the (R)-enantiomer in up
to 99% ee. This result is independent of solubility. In particular, it is interesting that molecular sieves
also gave pure enantiomer. They are assumed to act by adsorption of the aldehyde or capturing water

D. S. Im et al. / Tetrahedron: Asymmetry 10 (1999) 3759–3767
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Table 2
Solvent effects on the resolution of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1
using Pseudomonas fluorescens
Table 3
Additive effects on the resolution of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1
using Pseudomonas fluorescens
molecules. As additive, when 18-crown-6 was used, the stereoselectivity (E) was best at 49. Additionally,
the acetate 1-1 was hydrolysed in buffer solution using various enzymes, as shown in Scheme 3.
From screening enzymes for the resolution of 1-1, Pseudomonas fluorescens was best and the fast
reacting enantiomer was the (S) one. Its enantiomeric purity was up to 86% at 26% conversion when the
enzyme was used in mass equivalent to the substrate 1-1. When the second resolution of (S)-enantiomer
was performed, its purity rose to 99%.

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Scheme 3.
3. Conclusions
We resolved the (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1 to the (S)- and (R)-
enantiomers using Pseudomonas fluorescens (LAK) among the various lipases. These enantiomers will
be used to synthesize enantiopure (S)- and (R)-verapamil. This resolving efficiency on primary alcohol
containing a quaternary stereogenic carbon can be extended to other similar structures and we will
continue to study the reason for the discrimination in such a molecule.
4. Experimental
4.1. General
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Varian Gemini 300 MHz
spectrometer with TMS as internal reference. IR spectra were recorded on a MIDAC 101025 FT-IR
spectrometer and optical rotation was measured on an Autopol® III polarimeter (Rudolph Research Co.).
Low EI resolution mass spectra were determined on an HP GC 5972 and HP MS 5988A system at 70
eV. High pressure liquid chromatography for the determination of enantiomeric purity was performed
on a Varian 9010 system with Varian 9050 UV detector at 254 nm and Chiralcel OD. The pH stat used
was Metrohm 718 STAT Titrino with Metrohm electrode and Metrohm 722 stirrer. All reactions were
carried out under an atmosphere of nitrogen or argon. Column chromatography was performed on Merck
silica gel 60 (230–400 mesh). TLC was carried out using glass sheets pre-coated with silica gel 60 F254
prepared by E. Merck. All the commercially available reagent chemicals were obtained from Aldrich,
Fluka and Tokyo Kasei Chemical Company and generally used without further purification. Solvents
were distilled over appropriate drying materials before use.
4.2. Synthesis of 2-(3,4-dimethoxyphenyl)-3-methylbutyronitrile 2
To a stirred suspension of sodium hydride (2.26 g, 54.6 mmol, 60% dispersion in mineral oil) in dried
DMF (70 ml) was added dropwise (3,4-dimethoxyphenyl)acetonitrile (9.16 g, 51.7 mmol) in DMF (10
ml) over a period of 30 min at 0°C. After the mixture was stirred for another 30 min, 2-bromopropane
(5.78 g, 47.0 mmol) was added at 15°C. The mixture was stirred at room temperature for 15 h. The
reaction medium was quenched with ice cold water (200 ml) and extracted with diethyl ether (3×50
ml). The combined organic extracts were washed with saturated NaHCO3, brine, dried over MgSO4
and concentrated to furnish 14 g of crude 2-(3,4-dimethoxyphenyl)-3-methylbutyronitrile 2. Silica gel

D. S. Im et al. / Tetrahedron: Asymmetry 10 (1999) 3759–3767
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column chromatography (n-hexane:ethyl acetate 10:1) provided the compound 2 (7.08 g, 69%) as clear
liquid. Compound 2: GC/MSD (m/e) 51, 63, 76, 90, 103, 115, 131, 146, 162, 176 (100), 189, 203, 219
1
(m+); H NMR (300 MHz, CDCl3, ppm) 1.04 (d, J=6.7 Hz, 6H, -CH3×2), 2.07–2.14 (m, 1H), 3.88 (s,
3H, -OCH3), 3.90 (s, 3H, -OCH3), 6.79–6.84 (m, 3H, aromatic CH).
4.3. Synthesis of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropylbutanol 1
3-Tetrahydropyranyloxypropyl bromide was prepared from the commercially available 3-bromo-1-
propanol (6.95 g, 50 mmol), 3,4-dihydro-2H-pyran (5.04 g, 5.47 ml, 60 mmol) and pyridinium p-toluene
sulfonate as catalyst in CH2Cl2 (50 ml) for 3 h at room temperature. A solution was diluted with diethyl
ether (50 ml), washed once with half-satd brine to remove the catalyst and concentrated to furnish 12.0
g of the crude compound 3-tetrahydropyranyloxypropyl bromide. Silica gel column chromatography (n-
hexane:ethyl acetate 20:1) provided 10.5 g (94%) as clear liquid. GC/MSD (m/e) 223 (m+), 196, 167,
151, 137, 123, 107, 93, 85 (100), 67, 56; 1H NMR (300 MHz, CDCl3, ppm) 1.15–1.63 (m, 6H), 2.02–2.10
(m, 2H), 3.40–3.48 (m, 4H, -OCH2×2), 3.76–3.81 (m, 2H), 4.53 (t, J=3.0 and 3.7 Hz, 1H, chiral CH);
13C NMR (75 MHz, CDCl3, ppm) 19.8, 25.7, 30.9, 30.9, 33.2, 62.5, 65.1, 99.1.
To a stirred suspension of sodium hydride (1.20 g, 25.0 mmol, 60% dispersion in mineral oil) in dried
DMF (50 ml) was added dropwise the compound 2 (5.48 g, 25.0 mmol) in DMF (5 ml) at 0°C. After the
mixture was stirred for 30 min, the above 3-tetrahydropyranyloxypropyl bromide (5.5 g, 25 mmol) was
added at 15°C. The mixture was stirred at room temperature for 15 h. The reaction medium was quenched
with ice cold water (200 ml) and extracted with diethyl ether (3×50 ml). The combined organic extracts
were washed with saturated NaHCO3, brine and H2O, dried over MgSO4 and concentrated to furnish 11
1
g of the crude compound 3. H NMR (300 MHz, CDCl3, ppm) 0.79 (d, J=6.7 Hz, 3H, -CH3), 1.17 (d,
J=6.6 Hz, 3H, -CH3), 1.19–2.09 (m, 11H), 3.36–3.86 (m, 4H), 3.86 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3),
4.42–4.55 (m, 1H, chiral CH), 6.81–6.99 (m, 3H, aromatic CH).
To a solution of crude (RS)-3 (11 g) in methanol (10 ml) was added 1N methanolic HCl (20 ml) at
room temperature and stirred for 3 h. After evaporation of methanol, the residue was neutralized with
satd aqueous NaHCO3 and extracted with diethyl ether (3×50 ml). The combined organic extracts were
washed with satd NaHCO3, brine, dried over MgSO4 and concentrated to furnish 8.1 g of the crude (RS)-
1. Silica gel column chromatography (n-hexane:ethyl acetate 10:1) provided 5.80 g (84%) as a clear
liquid. Compound (RS)-1: GC/MSD (m/e) 277 (m+), 234, 216 (100), 189, 185, 170, 146, 138, 115, 103,
1
77, 65, 51; H NMR (300 MHz, CDCl3, ppm) 0.81 (d, J=6.8 Hz, 3H, -CH3), 1.20 (d, J=6.6 Hz, 3H,
-CH3), 1.22–1.30 (m, 1H), 1.32 (s, 1H, -OH), 1.55–1.69 (m, 1H), 1.87–1.99 (m, 1H), 2.05–2.16 (m, 1H),
2.18–2.30 (m, 1H), 3.40–3.44 (m, 2H, -CH2OH), 3.87 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 6.79–6.84
(m, 3H, aromatic CH); 13C NMR (75 MHz, CDCl3, ppm) 18.8, 19.2, 29.1, 34.5, 38.1, 53.5, 56.1, 56.3,
62.2, 109.8, 111.4, 119.1, 121.8, 130.7, 148.5, 149.3; IR (neat, cm−1) 3520 (OH), 3150 (aromatic CH),
2964 (aliphatic CH), 2234 (CN), 1592, 1520, 1465, 1456, 1375, 1258, 1152, 1026, 806, 768; HPLC
analysis [column: Chiralcel OD (cellulose carbamate derivative), eluent: n-hexane:IPA 9:1, flow rate:
1.0 ml/min; detector: UV 254 nm]; retention time (min): 18.30 for (R)-enantiomer and 32.46 for (S)-
enantiomer.
4.4. Synthesis of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropylbutyl acetate 1-1
To a stirred solution of alcohol (ꢀ)-1 (1.7 g, 6.14 mmol) in dried CH2Cl2 (10 ml) was added 4-N,N-
dimethylaminopyridine (100 mg), pyridine (1.5 ml, 18.4 mmol) and acetic anhydride (1.74 ml, 18.4
mmol) at 0°C. After the mixture was stirred for 3 h at room temperature, the reaction mixture was poured

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D. S. Im et al. / Tetrahedron: Asymmetry 10 (1999) 3759–3767
into cold 10% aq. hydrochloride and extracted with diethyl ether (3×30 ml). The combined organic
extracts were washed with saturated NaHCO3, brine, dried over MgSO4 and concentrated to furnish the
compound 1-1 as crude 4 g. Silica gel column chromatography (n-hexane:ethyl acetate 4:1) provided the
pure compound 1-1 as a pure liquid (1.80 g, 92%). Compound 1-1: GC/MSD (m/e) 319 (m+), 277, 258,
1
235, 216 (100), 189, 174, 138, 128, 115, 91, 77, 51; H NMR (300 MHz, CDCl3, ppm) 0.81 (d, J=6.8
Hz, 3H, -CH3), 1.20 (d, J=5.0 Hz, 3H, -CH3), 1.29–1.33 (m, 1H), 1.65–1.79 (m, 1H), 1.80–1.91 (m, 1H),
2.03 (s, 3H, -COCH3), 2.06–2.13 (m, 1H), 2.14–2.25 (m, 1H), 3.89 (s, 3H, -OCH3), 3.91 (s, 3H, -OCH3),
4.00 (t, J=6.3 Hz, -CH2CO-), 6.85–6.91 (m, 3H, aromatic CH); 13C NMR (75 MHz, CDCl3, ppm) 18.9,
19.3, 21.2, 25.4, 34.8, 38.2, 53.5, 56.2, 56.3, 64.1, 109.8, 111.5, 119.0, 121.4, 130.5, 148.7, 149.4, 171.3;
IR (neat, cm−1) 3150 (aromatic CH), 2968 (aliphatic CH), 2234 (CN), 1732 (C_O), 1592, 1520, 1465,
1456, 1375, 1258, 806, 768, 636; HPLC analysis [column: Chiralcel OD (cellulose carbamate derivative);
eluent: n-hexane:IPA 9:1; flow rate: 1.0 ml/min; detector: UV 254 nm]; retention time (min): 13.34 for
(R)-isomer and 15.00 (S)-isomer.
4.5. Enzymatic esterfication of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-butanol 1
To a stirred solution of alcohol (RS)-1 (100 mg, 0.36 mmol) in dried n-hexane:ethyl acetate (9:1,
10 ml) was added the enzyme (mass equivalent to substrate) and vinyl acetate (31 mg, 0.36 mmol)
at 32–34°C. When the conversion of the reaction reached around 44%, the enzyme was removed by
filtration and washed with diethyl ether. The combined organic layer was concentrated to afford an oily
residue, which was chromatographed on silica gel column with n-hexane:ethyl acetate (10:1) system to
give the corresponding ester (S)-1 and unreacted (R)-1. The isolated (S)-acetate was hydrolysed with
1.2N methanolic KOH to afford (S)-1. The enantiomeric excess of (S)-1 was 76% while that of (R)-1 was
58%.
When each of the resolved acetate (S)-1 and the unreacted alcohol (R)-1 was re-resolved under the
same conditions at the 40% and 50% conversions, their enantiomeric excesses were 96.6 and >99%,
respectively. (R)-(+)-1: [α]29 +16.92 (c=1.77, CHCl3); >99% ee; (S)-(−)-1: [α]29 −16.26 (c=1.15,
D
D
CHCl3); 96.6% ee.
4.6. Enzymatic hydrolysis of (RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropylbutyl acetate 1-1
To a stirred suspension of acetate (RS)-1-1 (100 mg, 0.31 mmol) in phosphate buffer (8 ml, pH 7) was
added the enzyme (mass equivalent to substrate) at room temperature. The pH of the reaction was kept
at 7.0 by 0.01N aqueous NaOH via a syringe pump interfaced with a pH controller. When the conversion
of the reaction was 26%, the reaction mixture was extracted with ethyl acetate (20 ml×2). The combined
organic layer was concentrated to afford an oily residue, which was chromatographed on silica gel column
with n-hexane:ethyl acetate (10:1) system to give the corresponding alcohol (S)-1-1 and unreacted acetate
(R)-(+)-1-1. The isolated (R)-1-1 was hydrolysed with 1.2N methanolic KOH to afford the alcohol. The
enantiomeric excesses of the alcohol and acetate were 86% and 44%, respectively. When the acetate of
the reacted alcohol (S)-1-1 was hydrolysed again under the same conditions, its optical purity was up to
99%.

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Acknowledgements
This study was supported by a grant from the Korea Institute of Science and Technology and we would
like to express our grateful thanks to Amano Pharm. Co. Ltd., Japan.
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