Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase

Primožič, Ines; Bolant, Marijana; Ramić, Alma; Tomić, Srđanka

doi:10.3390/molecules17010786

Open AccessArticle

Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase

by

Ines Primožič

^*,

Marijana Bolant

,

Alma Ramić

and

Srđanka Tomić

Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102A, HR-10 000 Zagreb, Croatia

^*

Author to whom correspondence should be addressed.

Molecules 2012, 17(1), 786-795; https://doi.org/10.3390/molecules17010786

Submission received: 8 December 2011 / Revised: 5 January 2012 / Accepted: 11 January 2012 / Published: 16 January 2012

(This article belongs to the Special Issue ECSOC-15)

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Abstract

:

Since the optically active quinuclidin-3-ol is an important intermediate in the preparation of physiologically or pharmacologically active compounds, a new biocatalytic method for the production of chiral quinuclidin-3-ols was examined. Butyrylcholinesterase (BChE; EC 3.1.1.8) was chosen as a biocatalyst in a preparative kinetic resolution of enantiomers. A series of racemic, (R)- and (S)-esters of quinuclidin-3-ol and acetic, benzoic, phthalic and isonicotinic acids were synthesized, as well as their racemic quaternary N-benzyl, meta- and para-N-bromo and N-methylbenzyl derivatives. After the resolution, all N-benzyl protected groups were successfully removed by catalytic transfer hydrogenation with ammonium formate (10% Pd-C). Hydrolyses studies with BChE confirmed that (R)-enantiomers of the prepared esters are much better substrates for the enzyme than (S)-enantiomers. Introduction of bromine atom or methyl group in the meta or para position of the benzyl moiety resulted in a considerable improvement of the stereoselectivity compared to the non-substituted compounds. Optically pure quinuclidin-3-ols were prepared in high yields and enantiopurity by the usage of various N-benzyl protected groups and BChE as a biocatalyst.

Keywords:

quinuclidin-3-ol; esters, resolution; butyrylcholinesterase; N-benzyl protected groups

1. Introduction

Butyrylcholinesterase (BChE) is a non-specific ester hydrolyzing enzyme with no known endogenous physiological substrate [1]. It can hydrolyze various esters of choline and other compounds [2] (e.g., cocaine, some organophosphorus compounds), and it is important because of several pharmacological and toxicological functions (e.g., as bioscavenger for the protection of humans against organophosphate toxicity [3], biomarker of exposure to toxic organophosphorus compounds in food and environment [4,5]). This enzyme has not been used to a large extent as a biocatalyst in organic chemistry mainly as a result of its affinity towards positively charged substrates [6]. Structural complementarity of choline and quinuclidin-3-ol pointed to racemic quinuclidine derivatives as candidates for kinetic resolution catalyzed by this enzyme [7,8,9]. Compounds which contain quinuclidin-3-ol moiety are valuable intermediates for the synthesis of optically active pharmaceuticals [10], thus, efficient synthesis of chiral quinuclidin-3-ols is necessary for the preparation of biologically active compounds. In our previous work, various chiral quaternary esters of quinuclidin-3-ol were subjected to hydrolysis in the presence of BChE [11], and it was shown that hydrolysis is highly stereoselective. Kinetic studies revealed the preference of BChE toward (R)-enantiomers of quinuclidin-3-ol compounds. Therefore, we decided to synthesized a series of new quaternary esters with meta- and para-bromo and meta- and para-methylbenzyl protecting groups at the quinuclidine nitrogen atom, to determine whether the substituent at the benzyl moiety can further enhance enantioselectivity of the hydrolysis. A series of racemic, (R)- and (S)- esters of quinuclidin-3-ol and acetic, benzoic, phthalic and isonicotinic acids were synthesized as well as their racemic quaternary meta and para N-bromo and N-methylbenzyl derivatives (Figure 1). After the kinetic resolution using BChE as a catalyst, all N-benzyl protected groups were successfully removed by catalytic transfer hydrogenation with ammonium formate (10% Pd-C, Scheme 1). The stereoselectivity of hydrolysis with horse serum BChE was investigated. A method for the preparation of chiral (R)-esters and (S)-quinuclidin-3-ol by the enzymic kinetic resolution on a preparative scale was proposed.

Figure 1. Synthesized quinuclidinium esters.

Scheme 1. Syntheses of esters and the method for resolution with butyrylcholinesterase.

2. Results and Discussion

Twelve quaternary racemic esters of quinuclidin-3-ol and acetic, benzoic, isonicotinic and phthalic acids were synthesized, and a method for their resolution with butyrylcholinesterase was explored (Scheme 1). All esters were synthesized in good yields by the esterification reaction of quinuclidin-3-ol with an appropriate anhydride. Quaternization of esters with substituted meta and para- bromo and methylbenzyl bromides followed. The purity and structure of all compounds were determined by IR, MS, one- and two-dimensional ¹H- and ¹³C-NMR. Quaternary racemic esters 1–12 were then hydrolyzed using horse serum BChE as a catalyst. The hydrolysis was stopped when 50% of the ester was hydrolyzed, Table 1. Acetates, benzoates, isonicotinates and phthalates were hydrolyzed with different rates. Changes to the acyl moiety of the substrate affected the activity of the enzyme: The fastest reactions were those of acetates and isonicotinates, while benzoates and phthalates were hydrolyzed much slower. The best substrate for the enzyme was isonicotinate 10, while benzoate 8 was the worst substrate. All reactions proceeded slower than the reactions of the appropriate tested non-substituted N-benzyl derivatives indicated that meta and para substituent at the N-benzyl protecting group generally lowered the affinity of the enzyme toward the substrates.

After the kinetic resolution, the obtained mixtures of quaternary N-benzyl esters and 3-hydroxyquinuclidinium derivatives were subjected to the catalytic transfer hydrogenation, since it was not possible either to monitor optical purity of the reaction directly (e.g., chiral HPLC) or successfully separate quaternary ammonium compounds. Accordingly, chiral (S)-quinuclidine esters and (R)-quinuclidin-3-ol were isolated after the column chromatography. Specific optical rotation values were determined with an Optical Activity LTD automatic polarimeter and are presented in Table 1. Enantiopurity of the obtained esters or quinuclidin-3-ol for compounds 2, 6 and 8–10 were higher than 95% while the optical purity for other compounds varied from 62–89%. This data imply that BChE is adequate biocatalyst for the kinetic resolution of N-benzyl esters: by varying the reaction time, both enantiomers of quinuclidin-3-ol can be obtained in very high optical purity. The enantiopurest (S)-esters were benzoates 6 and 8, compounds with the para-bromo and para-methyl N-benzyl protecting groups. At the same time, the reaction stereoselectivity of derivatives 5 and 7 with meta substituents at the benzyl moiety were much lower.

Table 1. Hydrolysis of compound 1–12 catalyzed with butyrylcholinesterase: optical purities of products were determined after the catalytic transfer hydrogenation.

**Table 1.** Hydrolysis of compound 1–12 catalyzed with butyrylcholinesterase: optical purities of products were determined after the catalytic transfer hydrogenation.
Compound	Time (BChE hydrolysis) /min	[α]_D²⁶ ester	[α]_D²⁶ quinuclidin-3-ol	Optical purity (S)-ester /%	Optical purity (R)- quinuclidin-3-ol /%
1	80	−140	−80	88	89
2	60	−140	−90	88	100
3	60	−140	−80	88	89
4	60	−140	−80	88	89
5	185	−82.3	−60	82	67
6	258	−100	−60	100	67
7	360	−62	−70	62	78
8	1320	−100	−70	100	78
9	60	−200	−70	95	78
10	45	−200	−70	95	78
11	170	110	−70	85	78
12	75	100	−80	77	89

To identify the binding interactions of the enzyme with the benzoate esters, a molecular docking study was performed using Autodock 4.0.2. The flexible ligands 5–8 were docked to the active site of BChE by using the default settings of Lamarckian genetic algorithm. A general trend in binding of all benzoates was observed: Due to the bulkiness of the molecules, productive binding of (R)- and (S)-esters are not the one with the lowest energies: The carbonyl group of the compounds is pointed far away from Ser200 oxygen atom and the catalytic process cannot occur (Figure 2).

That can explain why the rate of hydrolysis is slower compared to N-methyl and non-substituted N-benzyl derivatives [11]. Thus, both enantiomers of compounds have to rotate inside the active site for hydrolyses to occur. There is a difference in accommodation of bromo and methyl groups as a result of their different size and electron features. Comparison of meta- and para-derivatives reveals that position of the substituent determines the modes of binding of substrates: only in the case of 5-R and 6-R (Figure 2a) the position of the methyl substituent does not influence significantly the geometry of the substrate in the most stable complex, while in all other complexes there is a radical change in the position of carbonyl group as well as two aromatic moieties.

Figure 2. Substrate-BChE complexes derived from docking studies (a) 5-R (carbon atoms dark grey) and 6-R (carbon atoms light grey); (b) 7-R (carbon atoms dark grey) and 8-R (carbon atoms light grey); (c) 5-S (carbon atoms dark grey) and 6-S (carbon atoms light grey); (d) 7-S (carbon atoms dark grey) and 8-S (carbon atoms light grey). BChE active site is represented by three structurally important amino acids: Ser200 (part of the catalytic triad), Glu 199 and Trp84 (part of the choline binding site). Hydrogen atoms are omitted for clarity, with the exception of the polar ones.

3. Experimental

3.1. General

All reagents and solvents were analytical grade or purified by standard procedures as described in the literature. All melting points were determined on a Melting Point B-540 apparatus (Büchi, Germany) and are uncorrected. IR spectra were recorded with a Perkin-Elmer FTIR 1725 X spectrometer. ¹H- and ¹³C- 1D and 2D NMR spectra were recorded with a Varian XL-GEM 600 spectrometer at room temperature. Chemical shifts are given in ppm downfield from TMS as internal standard. The reactions with enzyme were carried out in Heidolph UNIMAX 1100 Shaker. BChE (EC 3.1.1.8), type IV-S lyophilized powder from horse serum (Sigma Chemical Co.) was used without further purification. Optical rotations were measured on an Optical Activity AA-10 automatic polarimeter at ambient temperature. Acetate, benzoate, phthalate and isonicotinate esters were synthesized as described previously [12]. Chiral (R)- and (S)-quinuclidin-3-ols were obtained by the resolution of racemic quinuclidin-3-yl acetates with D-and L-tartaric acid [13]. Standards of chiral alcohol and esters were prepared: (R)-quinuclidin-3-ol: [α]_D²⁶ −90° (c = 0.02, 1 M HCl); (S)-Quinuclidin-3-yl acetate: [α]_D²⁶ −160° (c = 0.3, CHCl₃); (S)-Quinuclidin-3-yl benzoate: [α]_D²⁶ −100° (c = 0.34, EtOH); (S)-Quinuclidin-3-yl isonicotinate: [α]_D²⁶ −211° (c = 0.3, CHCl₃); (S)-Quinuclidin-3-yl phthalate: [α]_D²⁶ 130° (c = 1.0, EtOH).

3.2. General Procedure for Quaternization of Esters

The appropriate ester (1.5 mmol) was dissolved in dry acetone. Substituted N-benzyl bromide (1.7 mmol) was slowly added to the solution. The mixture was left at room temperature and after 24 h the crystals were formed. They were washed several times with dry ether and dried under reduced pressure to give the title compounds as white crystals (unless stated otherwise).

3-Acetoyloxy-1-(3-methylbenzyl)quinuclidinium bromide (1). Yield: 41%; m.p. 196.3–197.8 °C; IR (cm⁻¹): 2969 (C-H), 2890 (C-H), 1275 (C=O), 1254 (C-O), 1042 (C-N); ¹H-NMR δ: 1.89–2.05 (m, 2H, H₅), 2.07 (s, 3H, CH₃C=O), 2.15–2.23 (m, 2H, H₈), 2.32–2.35 (s, 3H, CH₃Bz), 2.38–2.40 (m, 1H, H₄),3.62–3.78 (m, 1H, H₇), 3.82–3.89 (m, 2H, H₆), 3.93–4.04 (m, 1H, H_2b), 4.24–4.32 (dd, 1H, H_2a), 5.00–5.10 (m, 3H, H₃ and CH₂Bz), 7.23–7.44 (m, 4H, H₂H₄H₅H₆Bz); ¹³C-NMR δ: 18.39 (C₅), 21.28 (C₄), 21.29 (CH₃Bz), 21,37 (C₈), 24.69 (CH₃C=O), 52.87 (C₆), 53.69 (C₇), 59.90 (C₂), 66.70 (CH₂Bz), 67.16 (C₃), 126.63 (C₁Bz), 129.06 (C₅Bz), 130.45 (C₆Bz), 131.33 (C₂Bz), 133.75 (C₄Bz), 139.13 (C₃Bz), 170.02 (C=O). ESMS: m/z (calcd for C₁₇H₂₄NO₂⁺ 274.18) found 274.2.

3-Acetoyloxy-1-(4-methylbenzyl)quinuclidinium bromide (2). Yield: 63%; m.p 174.9–175.6 °C; IR (cm⁻¹): 2945 (C-H), 1720 (C=O), 1241 (C-O), 1025 (C-N); ¹H-NMR δ: 1.89–2.00 (m, 2H, H₅), 2.08 (s, 3H, CH₃C=O), 2.14–2.19 (m, 2H, H₈), 2.33 (s, 3H, CH₃Bz), 2.35–2.39 (m, 1H, H₄), 3.62–3.67 (m, 2H, H₇), 3.83–3.86 (m, 2H, H₆), 3.93–3.95 (m, 1H, H_2b), 4.22–4.29 (m, 1H, H_2a), 4.97–5.07 (m, 3H, H₃ and CH₂Bz), 7.15–7.18 (m, 2H, H₃H₅Bz), 7.37–7.44 (m, 2H, H₂H₆Bz); ¹³C-NMR δ: 17.78 (C₅), 20.47 (C₄), 20.76 (CH₃Bz), 21.37 (C₈), 24.23 (CH₃C=O), 52.50 (C₆), 53.14 (C₇), 59.36 (C₂), 65.95 (CH₂Bz), 66.69 (C₃), 123.25 (C₁Bz), 129.25 (C₃C₅Bz), 132.71 (C₂C₆Bz), 140.13 (C₄Bz), 169.76 (C=O). ESMS: m/z (calcd for C₁₇H₂₄NO₂⁺ 274.18) found 274.2.

3-Acetoyloxy-1-(3-bromobenzyl)quinuclidinium bromide (3). Yield: 63%; m.p. 194–196 °C; IR (cm⁻¹): 3050, 2955, 2870, 1720, 1430, 1371, 1244, 1026; ¹H-NMR δ: 1.92–2.03 (m, 2H, H₅), 2.04–2.09 (s, 3H, CH₃C=O), 2.18–2.20 (m, 2H, H₈), 2.39–2.40 (m, 1H, H₄), 3.70–3.93 (m, 5H, H₆, H₇ and H_2b), 4.33–4.34 (dd, 1H, H_2a), 5.08–5.21 (m, 3H, H₃ and CH₂Bz), 7.36–7.41 (m, 4H, H₂H₄ H₅H₆Bz); ¹³C-NMR δ: 17.89 (C₅), 20.51 (C₄), 20.90 (C₈), 24.15 (CH₃C=O), 52.89 (C₆), 53.34 (C₇), 59.56 (C₂), 64.75 (CH₂Bz), 66.57 (C₃), 122.58 (C₁Bz), 128.70 (C₃Bz), 130.30 (C₆Bz), 131.80 (C₅Bz), 133.25 (C₄Bz), 135.25 (C₂Bz), 169.67 (C=O). ESMS: m/z (calcd for C₁₆H₂₁BrNO₂⁺ 338.08) found 338.2.

3-Acetoyloxy-1-(4-bromobenzyl)quinuclidinium bromide (4). Yield: 60%; m.p. 239–241 °C; IR (cm⁻¹): 3050, 2956, 2870 (v_s C-H); 1735, 1488,1422, 1363, 1239, 1029; ¹H-NMR δ: 1.89–2.02 (m, 2H, H₅), 2.04–2.10 (s, 3H, CH₃C=O), 2.17–2.21 (m, 2H, H₈), 2.38–2.39 (m, 1H, H₄), 3.64–3.72 (m, 2H, H₇), 3.77–3.95 (m, 3H, H₆ and H_2b), 4.28–4.29 (dd, 1H, H_2a), 5.06–5.07 (m, 3H, H₃), 5.14–5.21 (m, 2H, CH₂Bz), 7.50–7.52 (m, 2H, H₂H₆Bz), 7.56–7.58 (m, 2H, H₃H₅Bz); ¹³C-NMR δ: 17.87 (C₅), 20.47 (C₄), 20.88 (C₈); 24.15 (CH₃C=O), 52.80 (C₆), 53.21 (C₇), 59.51 (C₂), 64.79 (CH₂Bz), 66.57 (C₃); 124.91(C₁Bz), 125.36 (C₄Bz), 131.96 (C₃C₅Bz), 134.47 (C₂C₆Bz), 169.65 (C=O). ESMS: m/z (calcd for C₁₆H₂₁BrNO₂⁺ 338.08) found 338.2.

3-Benzoyloxy-1-(3-methylbenzyl)quinuclidinium bromide (5). Yield: 89%, m.p. 145.8–146.9 °C; IR (cm⁻¹): 2969 (C-H), 1710 (C=O), 1453 (C=C), 1278 (C-O), 1024 (C-N); ¹H-NMR δ: 1.95–2.29 (m, 4H, H₅ and H₈), 2.32 (s, 3H, CH₃Bz), 2.54–2.57 (m, 1H, H₄), 3.76–4.13 (m, 5H, H₆, H₇ and H_2b), 4.37–4.47 (m, 1H, H_2a), 5.10 (s, 2H, CH₂Bz), 5.33–5.36 (m, 1H, H₃), 7.19–7.29 (m, 2H, H₂H₆Bn), 7.39–7.46 (m, 2H, H₂H₆Bz), 7.53–7.59 (m, 3H, H₃H₄H₅Bn), 8.01–8.04 (m, 2H, H₄H₅Bz); ¹³C-NMR δ: 18. 54 (C₅), 21.28 (CH₃Bz), 21.37 (C₈), 24.94 (C₄), 53.31 (C₆), 53.98 (C₇), 59.97 (C₂), 66.81 (CH₂Bz), 67.79 (C₃), 126.69 (C₁Bz), 128.81 (C₃Bz), 129.08 (C₃C₅Bn), 128.38 (C₁₀), 129.86 (C₂C₆Bz), 130.43 (C₂C₆Bn), 130.43 (C₅Bz), 131.29 (C₄Bz), 133.82 (C₄Bn), 139.31 (C₃Bz), 165.40 (C=O). ESMS: m/z (calcd for C₂₂H₂₆NO₂⁺ 336.20) found 336.2.

3-Benzoyloxy-1-(4-methylbenzyl)quinuclidinium bromide (6). Yield: 81%, m.p. 209.6–210.4 °C; IR (cm⁻¹): 2968 (C-H), 2879 (C-H), 1721 (C=O), 1449 (C=C), 1271 (C-O), 1022 (C-N); ¹H-NMR δ: 1.95–2.28 (m, 4H, H₅ and H₈), 2.56 (s, 3H, CH₃Ph), 2.52–2.61 (m, 1H, H₄), 3.68–4.16 (m, 5H, H₆, H₇ and H₂), 4.32–4.41 (m, 1H, H₂), 5.11 (s, 2H, H₁₆), 5.32–5.35 (m, 1H, H₃), 7.18–7.23 (m, 2H, H₁₁ and H₁₅), 7.43–7.48 (m, 2H, H₁₈ and H₂₂), 7.51–7.62 (m, 3H, H₁₂, H₁₃ and H₁₄), 8.01–8.04 (m, 2H, H₁₉ and H₂₁); ¹³C-NMR δ: 18.85 (C₅), 21.30 (CH₃Bz), 21.38 (C₈), 24.98 (C₄), 53.35 (C₆), 53.93 (C₇), 59.99 (C₂), 66.68 (CH₂Bz), 67.77 (C₃), 123.64 (C₄Bz), 128.61 (C₃C₅Bn), 128.76 (C₁Bz), 129.87 (C₂C₆Bz), 129.94 (C₂C₆Bn), 133.22 (C₃C₅Bz), 133.73 (C₄Bn), 140.92 (C₁Bn), 165.51 (C=O). ESMS: m/z (calcd for C₂₂H₂₆NO₂⁺ 336.20) found 336.2.

3-Benzoyloxy-1-(3-bromobenzyl)quinuclidinium bromide (7). Yield: 78%; m.p. 140–142 °C; IR (cm⁻¹): 3050, 2966, 2885, 1716, 1450,1409, 1214, 1012; ¹H-NMR δ: 1.94–2.27 (m, 4H, H₅ and H₈), 2.53–2.55 (m, 1H, H₄), 3.76–4.09 (m, 5H, H₆, H₇ and H_2b), 4.37–4.45 (dd, 1H, H_2a), 5.10 (s, 2H, CH₂Bz), 5.33–5.36 (q, 1H, H₃), 7.24–7.29 (m, 1H, H₄Bn), 7.38–7.78 (m, 6H, H₂H₃H₅H₆Bn and H₅H₆Bz), 7.99–8.01 (d, 2H, H₂H₄Bz); ¹³C-NMR δ: 19.95 (C₅), 20.10 (C₈), 24.90 (C₄), 53.07 (C₆), 53.58 (C₇), 55.21 (C₂), 65.68 (CH₂Bzl), 67.68 (C₃), 123.12 (C₁Bz), 128.60 (C₁Bn), 129.07 (C₃C₅Bn), 129.09 (C₃Bz), 129.89 (C₂C₆Bn), 130.82 (C₄Bn), 132.30 (C₆Bz), 133.70 (C₅Bz), 133.81 (C₄Bz), 135.72 (C₂Bz), 165.56 (C=O). ESMS: m/z (calcd for C₂₁H₂₃BrNO₂⁺ 400.09) found 400.2.

3-Benzoyloxy-1-(4-bromobenzyl)quinuclidinium bromide (8). Yield: 67%; m.p. 84–86 °C; IR (cm⁻¹): 3050, 2963, 2885, 1716, 1409, 1214, 1012; ¹H-NMR δ: 2.03–2.27 (m, 4H, H₅ and H₈), 2.55–2.56 (m, 1H, H₄), 3.72–4.05 (m, 5H, H₆, H₇ and H_2b), 4.35–4.45 (dd, 1H, H_2a), 5.18 (s, 2H, CH₂Bz), 5.33–5.36 (q, 1H, H₃), 7.27–7.29 (m, 1H, H₄Bn), 7.41–7.59 (m, 6H, H₂H₃H₅H₆Bn and H₂H₆Bz), 7.99–8.01 (d, 2H, H₃H₅Bz); ¹³C-NMR δ: 19.39 (C₅), 20.10 (C₈), 24.89 (C₄), 53.07 (C₆), 53.74 (C₇), 57.53 (C₂), 62.16 (CH₂Bz), 67.73 (C₃), 125.43 (C₁Bz), 125.61 (C₁Bn), 128.62 (C₃C₅Bn), 128.70 (C₄Bz), 128.73 (C₂C₆Bz), 129.87 (C₂C₆Bn), 131.51 (C₄Bn), 132.51 (C₃C₅Bz), 165.56 (C=O). ESMS: m/z (calcd for C₂₁H₂₃BrNO₂⁺ 400.09) found 400.2.

3-Isonicotinoyloxy-1-(3-bromobenzyl)quinuclidinium bromide (9). Yield: 78%; m.p. 251–255 °C; IR (cm⁻¹): 3050, 2963, 2870, 1720, 1468,1406, 1217, 1039; ¹H-NMR δ: 1.94–1.95 (m, 2H, H₅), 2.10–2.12 (m, 2H, H₈), 2.27–2.31 (m, 1H, H₄), 3.48–4.19 (m, 5H, H₇, H₆ and H_2b), 4.28–4.30 (dd, 1H, H_2a), 4.48–4.51 (m, 2H, CH₂Bz), 5.29–5.30 (q, 1H, H₃), 7.33 (t, 1H, H₅Bz), 7.56–7.70 (m, 2H, H₄H₆Bz), 7.77–7.78 (m, 1H, H₂Bz), 7.89–7.90 (m, 2H, H₂H₆Py), 8.76–8.77 (m, 2H, H₃H₅Py); ¹³C-NMR δ: 17.99 (C₅), 20.82 (C₈), 24.32 (C₄), 52.51 (C₆), 53.52(C₇), 59.40 (C₂), 64.77 (CH₂Bz); 68.10 (C₃), 122.52 (C₂C₆Py), 122.73 (C₁Py), 128.52 (C₁Bz), 130.35 (C₃C₅Py), 130.78 (C₆Bz), 131.33 (C₅Bz), 133.21 (C₃Bz), 133.38 (C₂Bz), 135.23 (C₄Bz), 135.54 (C=O). ESMS: m/z (calcd for C₂₀H₂₂BrN₂O₂⁺ 401.09) found 401.1.

3-Isonicotinoyloxy-1-(4-bromobenzyl)quinuclidinium bromide (10). Yield: 78%; m.p. 119–121 °C; IR (cm⁻¹): 3050, 2953, 2870, 1731, 1462,1409, 1214, 1012; ¹H-NMR δ: 1.92–2.33 (m, 4H, H₅ and H₈), 2.57–2.58 (m, 1H, H₄), 3.50–4.24 (m, 5H, H₆, H₇ and H_2b), 4.32–4.51 (dd, 1H, H_2a), 5.25 (s, 2H, CH₂Bz), 5.29–5.41 (q, 1H, H₃), 7.47–7.66 (m, 4H, H₂H₃H₄H₅Bz), 7.89–7.91 (m, 2H, H₂H₆Py), 8.78–8.80 (m, 2H, H₃H₅Py); ¹³C-NMR δ: 18.47 (C₅), 21.30 (C₈), 24.05 (C₄), 53.06 (C₆), 53.61 (C₇), 59.78 (C₂), 65.33 (CH₂Bz), 68.33 (C₃), 119.46 (C₂C₆Py), 125.69 (C₁Py), 131.95 (C₂C₆Bz), 134.49 (C₃C₅Bz), 136.03 (C₁Bz); 144.13 (C₄Bz), 151.03 (C₃C₅Py), 164.38 (C=O). ESMS: m/z (calcd for C₂₀H₂₂BrN₂O₂⁺ 401.09) found 401.1.

3-Phthaloyloxy-1-(3-methylbenzyl)quinuclidinium bromide (11). Yield: 49%, yellow viscous oil; IR (cm⁻¹): 3344 (O-H), 2923 (C-H), 1721 (C=O), 1488 (C=C), 1284 (C-O), 1039 (C-N); ¹H-NMR δ: 0.88–0.99 (m, 2H, H₅), 1.20–1.48 (m, 2H, H₈), 1.80–2.15 (m, 1H, H₄), 2.32–2.40 (CH₃Bz), 3.38–3.97 (m, 5H, H₆, H₇ and H_2b), 4.65–4.71(m, 1H, H_2a), 5.17–5.24 (s, 2H, CH₂Bz), 5.29–5.36 (s, 1H, H₃), 7.08–7.31 (m, 4H, H₃H₄H₅H₆Pht), 7.56–8.25 (m, 4H, H₂H₄H₅H₆Bz); ¹³C-NMR δ: 13.36 (CH₃Bz), 18.05 (C₅), 20.62 (C₈), 21.23 (C₄), 26.22 (C₆), 29.98 (C₇), 64.05 (C₃), 67.53 (CH₂Bz), 67.83 (C₂), 125.49 (C₂Bz), 128.41–129.29 (C₂C₄C₅C₆Bz), 130.55 (C₁Pht), 130.89–131.97 (C₃C₄C₅C₆Pht), 132.75 (C₁Bz), 135.17 (C₂Pht), 138.35 (C₃Bz), 168.09 (COOH), 171.03 (C=O). ESMS: m/z (calcd for C₂₃H₂₆NO₄⁺ 380.19) found 380.3.

3-Phthaloyloxy-1-(4-methylbenzyl)quinuclidinium bromide (12). Yield: 81%, yellow viscous oil; IR (cm⁻¹): 3419 (O-H), 2957 (C-H), 1722 (C=O), 1458 (C=C), 1285 (C-O), 1039 (C-N); ¹H-NMR δ: 1.77–1.87 (m, 4H, H₅ and H₈), 2.27–2.35 (m, 1H, H₄), 2.28 (s, 3H, CH₃Bz), 3.45–3.52 (m, 5H, H₆, H₇and H_2b), 3.94–3.90 (m, 1H, H_2a), 4.84 (s, 2H, CH₂Bz), 4.51 (q, 1H, H₃); 7.11–7.16 (m, 4H, H₂H₃H₅H₆Bz), 7.27–7.36 (m, 2H, H₄H₅Pht), 7.45–7.52 (m, 2H, H₃H₆Pht), 8.19–8.24 (m, 1H, COOH); ¹³C-NMR δ: 17.97 (C₅), 21.23 (C₄), 21.57 (C₈), 26.92 (CH₃Bz), 53.27 (C₇), 54.36 (C₆), 63.77 (C₂), 64.09 (C₃), 67.63 (CH₂Bz), 123.44 (C₁Bz), 129.93 (C₃C₆Pht), 131.04 (C₂C₃C₅C₆Bz), 132.43 (C₄Pht), 132.88 (C₅Pht), 134.11 (C₁C₂Pht), 140.89 (C₄Bz), 170.95 (C=O, COOH). ESMS: m/z (calcd for C₂₃H₂₆NO₄⁺ 380.19) found 380.3.

3.3. Kinetic Resolution with BChE

The appropriate quaternary ester (40 mg) was dissolved in a minimal volume of 0.1 M phosphate buffer. The solution was placed in shaker for 10 min and BChE (100 μL, 10 mg/mL) was added to a reaction mixture. Reaction was stopped when 50% of the ester was hydrolyzed by adding ethanol (10 mL). The reaction mixture was dried under the reduced pressure and extracted with chloroform. The extract was dried over Na₂SO₄ and evaporated under reduced pressure.

3.4. Catalytic Transfer Hydrogenation

Dry methanol (3 mL) was added to the residue. This methanol solution was placed in a 2-bottom flask and 10% Pd-C (40 mg) was added. Ammonium formate (30 mg) was added in a single portion. The reaction mixture was stirred at the reflux temperature. After the completion of the reaction, it was filtered through a Celite pad, and washed with dry methanol (5 mL). The filtrate was dried under the reduced pressure, made alkaline with saturated aqueous solution of K₂CO₃ and extracted with chloroform. The extract was dried and evaporated under reduced pressure. Products were separated by column chromatography (aluminum oxide 90 active neutral (70–230 mesh ASTM), (Merck, Darmstadt, Germany) with chloroform-methanol solution (9:1) as eluent.

4. Conclusions

A series of novel meta- and para-substituted N-benzyl protected quinuclidinium esters were prepared and stereoselectivity of hydrolysis catalyzed with horse serum BChE was investigated. The kinetic resolutions were performed at 37 °C in 0.1 M phosphate buffer, pH 7.4. The hydrolyses of (R)-esters proceeded much faster than those of (S)-enantiomers. Introduction of N-benzyl para- and meta- bromine atoms or methyl groups resulted in a significant improvement of the stereoselectivity compared to non-substituted N-benzyl protected groups. Thus, optically pure quinuclidin-3-ol derivatives were prepared in high yields and enantiopurity by the esterification, quaternization with derivatives of benzyl bromide, BChE-catalysed hydrolysis and finally catalytic transfer hydrogenation.

Acknowledgements

The authors wish to thank Tomica Hrenar for his valuable assistance in docking studies. This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, Research Project No. 119-1191344-3121.

Sample Availability: Samples of the compounds are available from the authors.

References and Notes

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Primožič, I.; Bolant, M.; Ramić, A.; Tomić, S. Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase. Molecules 2012, 17, 786-795. https://doi.org/10.3390/molecules17010786

AMA Style

Primožič I, Bolant M, Ramić A, Tomić S. Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase. Molecules. 2012; 17(1):786-795. https://doi.org/10.3390/molecules17010786

Chicago/Turabian Style

Primožič, Ines, Marijana Bolant, Alma Ramić, and Srđanka Tomić. 2012. "Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase" Molecules 17, no. 1: 786-795. https://doi.org/10.3390/molecules17010786

APA Style

Primožič, I., Bolant, M., Ramić, A., & Tomić, S. (2012). Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase. Molecules, 17(1), 786-795. https://doi.org/10.3390/molecules17010786

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Preparation of Novel meta- and para-Substituted N-Benzyl Protected Quinuclidine Esters and Their Resolution with Butyrylcholinesterase

Abstract

1. Introduction

2. Results and Discussion

3. Experimental

3.1. General

3.2. General Procedure for Quaternization of Esters

3.3. Kinetic Resolution with BChE

3.4. Catalytic Transfer Hydrogenation

4. Conclusions

Acknowledgements

References and Notes

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