Highly selective chemoenzymatic synthesis of enantiopure orthogonally protected trans-3-amino-4-hydroxypiperidines

Accepted Manuscript Highly selective chemoenzymatic synthesis of enantiopure orthogonally protected trans-3-amino-4-hydroxypiperidines Ángela Villar-Barro, Vicente Gotor, Rosario Brieva PII:

S0040-4020(15)01051-0

DOI:

10.1016/j.tet.2015.07.014

Reference:

TET 26959

To appear in:

Tetrahedron

Received Date: 11 May 2015 Revised Date:

29 June 2015

Accepted Date: 5 July 2015

Please cite this article as: Villar-Barro Á, Gotor V, Brieva R, Highly selective chemoenzymatic synthesis of enantiopure orthogonally protected trans-3-amino-4-hydroxypiperidines, Tetrahedron (2015), doi: 10.1016/j.tet.2015.07.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highly selective chemoenzymatic synthesis of enantiopure orthogonally protected trans-3amino-4-hydroxypiperidines

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Ángela Villar-Barro, Vicente Gotor* and Rosario Brieva* Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006-Oviedo (Asturias), Spain.

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Tetrahedron journal homepage: www.elsevier.com

Ángela Villar-Barro, Vicente Gotor* and Rosario Brieva*

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Highly selective chemoenzymatic synthesis of enantiopure orthogonally protected trans-3-amino-4-hydroxypiperidines

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Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006-Oviedo (Asturias), Spain

ABSTRACT

Article history: Received Received in revised form Accepted Available online

Optically pure orthogonally protected trans-3-amino-4-hydroxypiperidines have been easily prepared from (±)-1-benzyl-3,4-epoxypiperidine. The key steps are a regioselective epoxide ring-opening with diallylamine and the enzymatic resolution of the resulting aminoalcohol. It is remarkable the high enantioselectivity obtained with Candida antarctica lipase B in the acetylation of (±)-trans-1-benzyl-3-(diallylamino)-4-hydroxypiperidine. The versatility of the protecting groups is showed in the subsequent transformations performed in relation to the assignment of the absolute configuration of the products.

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ARTICLE INFO

1. Introduction

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Keywords: 3-Amino-4-hydroxypiperidines Heterocyclic amino alcohols Regioselective epoxide opening Enzymatic resolution Biocatalysis

The synthesis of 4-amino-3-hydroxypiperidines by a regioselective ring opening of 1-benzyl-3,4-epoxypiperidine, (±)4 with secondary cyclic amines in presence of a Lewis acid, has been described by Ganina et al.7 Later, Tokuda et al.8 and Sheunemann et al.9 investigated the C4 or C3 regioselective ring opening with amines. More recently, in relation with the large scale preparation of BMS-690514, 3, Young et al.10 selected, among various strategies, the regioselective ring opening of the piperidine epoxide followed by a classical resolution, for the large-scale preparation of the optically pure (−)-4-amino-3hydroxypiperidine regioisomer.

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Functionalized piperidines are common components of natural products and essential structural units of biologically active compounds.1 In particular, piperidines substituted at C3 and C4 by a vicinal amino alcohol moiety are found widespread in pharmaceutical related research, significant examples of both regioisomers are showed in figure 1. The trans-3-amino-4hydroxypiperidine structure is present in compound 1, that inhibits various non-receptor tyrosine kinases, including Bruton’s tyrosine kinase; these inhibitors are targets of interest for the treatment of autoimmune disorders.2 Different radiolabeled derivatives of trozamicol, 2 has been evaluated as potential radiotracers for single photon and positron emission tomography (SPECT and PET).3 A representative example of trans-4-amino3-hydroxypiperidines is the valuable BMS-690514, 3 a new drug candidate for the treatment of lung cancer.4 Moreover, the inversion of the hydroxyl group at the trans-isomers provides an easy access to the structure of the cis-amino alcohol or cisdiaminopiperidine, also present in a number of bioactive compounds such as the PAD4 inhibitors,5 or the factor Xa inhibitors.6

2009 Elsevier Ltd. All rights reserved.

The presence of the amino hydroxypiperidine moiety and their absolute (as well as relative) stereochemistry is essential for the biological activity of these molecules. Therefore, development of practical and selective methods for its synthesis is required.

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Figure 1.

The results of these authors and our own experience in the application of biocatalytic methods to the enantiselective

Corresponding author. Tel.: +34-985-102-994; fax: +34-985-103-448; e-mail: [email protected], [email protected].

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In the first set of experiments, all the tested lipases catalysed the transesterification reaction of substrate (±)-trans-6 (Table 1, entries 1-4). Also, all the enzymes showed the same stereochemical preference; in the case of CAL-B, this was the expected preference according with the Kazlauskas’ rule12 (the assignation of absolute configurations of the products and the remaining substrate is described later).

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groups. Secondly, the high sterical demand of the nucleophile could improve the regioselectivity of the epoxide opening, especially in absence of the Li salt, and finally, the presence of a bulky substituent adjacent to the secondary carbon bearing the hydroxyl group could favor the enantioselectivity of the enzymatic resolution.

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11 preparation of diverse functionalized heterocycles, have MANUSCRIPT After obtaining the substrates (±)-trans-6 and (±)-trans-7 their ACCEPTED prompted us to prepare orthogonally protected 3,4-substituted ester derivatives (±)-trans-8a-b and (±)-trans-9a-b were prepared in good yields by conventional acylation methods. amino hydroxyl piperidines in order to study them as substrates for a biocatalytic resolution. 2.2. Enzymatic resolutions 2. Results and discussion Initially, the enzymatic transesterification reaction of (±)trans-N-benzyl-3-(diallylamino)-4-hydroxypiperidine, (±)-trans2.1. Regioselective synthesis of substrates 6 using vinyl acetate as the acyl donor was studied. Lipases from Initially, we were interested in test as substrates for the Candida antarctica (CAL-A and CAL-B), Burkholderia cepacia enzymatic processes the two regioisomers (±)-trans-N(PSL-IM), Pseudomonas fluorescens (AK) and Thermomyces benzyl-3-(diallylamino)-4-hydroxypiperidine, (±)-trans-6 and lanuginosus (TL-IM) were tested as catalyst. The reactions were (±)-trans-N-benzyl-4-(diallylamino)-3-hydroxypiperidine, carried out at 30 ºC in organic solvent (tBuOMe) and using 5 (±)-trans-7 obtained by the regioselective opening the epoxide equivalents of vinyl acetate. All the processes were carried out on (±)-4 with diallylamine, (Scheme 1). The diallylamine was a 50 mg scale monitoring the progress of each reaction by chiralchosen to incorporate the amine function at the 3- or 4HPLC; the reactions were stopped when the conversion was near position of the piridine ring for several reasons. First, the allyl 50% or when hardly further progress was observed. (Scheme 2, groups are easily removed in the presence of other protecting Table 1).

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The reaction rate was moderate, in the best case, the reaction catalyzed by CAL-B, 4 days of reaction are needed to achieved a 31% of conversion (entry 2), and only a slightly increment of conversion was observed after another additional 3 days.

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Scheme 1. Synthesis of (±)-trans-6 and (±)-trans-7 and their esterified derivatives.

The epoxide (±)-4 was synthesized in high yields following the optimized procedure described by Young et al.10b

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The product of the nucleophilic attack on C3, the (±)-trans-3(diallylamino)-4-hydroxypiperidine, (±)-trans-6 was obtained, as expected, in absence of the metal salt. Only a 50% of conversion was achieved at room temperature and the remaining epoxide (±)-4 was recovered unaltered. The yield of the process was significantly improved when the reaction was carried out at 80ºC in a closed tube; in these conditions, a yield higher to 80% of the isolate (±)-trans-6 can be achieved after the separation of the remaining epoxide. Under all the reaction conditions, the opposite regioisomer (±)-trans-7 was no detected. The regioselectivity was switched from C3 to C4 in the LiClO4-assited epoxide ring opening with diallylamine and the reactivity was significantly increased. The reaction was completed after 12 hour at room temperature yielding predominantly (±)-trans-7 in a ratio C4/C3 up to 9:1 (similar results were obtained using LiCl). The regioisomer (±)-trans-7 can be isolated in a 85% yield after chromatographic purification.

Scheme 2. Lipase catalysed acetylation of (±)-trans-6.

Table 1. Lipase catalyzed acylation of (±)-trans-6 in organic solvent, using 5 eq. of vinyl acetate at 30 ºC. Entry Enzyme 1 2 3 4 5 6 7 8 9

CAL-A CAL-B AK PSL-IM CAL-B CAL-A CAL-A CAL-B CAL-B

Solvent t

t ees eep c T (ºC) (days) (%)[a] (%)[a] (%)[b] 30 >99 5 40 27 30 >99 4 46 31 30 >99 6 28 22 30 >99 6 13 11 47 30 4 58 55 30 >99 4 64 40 >99 45 4 16 14 45 >99 4 62 38 45 >99 7 87 47

BuOMe BuOMe t BuOMe t BuOMe VAc VAc VAc t BuOMe t BuOMe t BuOMe/Et3N >99 10 CAL-B 45 3 81 (10:1) a Enantiomeric excess of substrate (ees) and product (eep) were determined by chiral HPLC. t

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E[b] >200 >200 >200 >200 5 >200 >200 >200 >200 >200

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Conversion, c = ees/(ees + eep), enantiomeric ratio, E = ln[(1 – c)(1 – ees)]/ln [(1 – c)((1 + ees)].13

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Vinyl acetate (VA) is used as solvent.

In order to improve the conversion the influence of different reaction parameters in the processes catalyzed by lipases CAL-A and CAL-B was studied. The influence of the solvent was tested

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carrying out the acylation in toluene, acetonitrile, 1,4-dioxane MANUSCRIPT resolution of trans-3,4-dihydroxypiperidines.11c In that case, ACCEPTED and vinyl acetate. The results obtained in the CAL-B catalyzed the hydrolysis catlyzed by CAL-B showed a total regioselectivity towards the hydroxyl group at the C4 position of transesterifications were similar but do not improved those the piperidine, the process was also highly enantioselective. The obtained in tBuOMe (entry 2) except in vinyl acetate where the enantioselectivity decreased considerably (entry 5). However, in acylation of the trans-diol afforded also very good regioselectivity towards the hydroxyl group at C4. Then, we the case of CAL-A, the reaction was accelerated using vinyl conclude that the C4 position is more accessible into the catalytic acetate as a solvent, a 40% of conversion was achieved after four days of reaction (entry 6). active center of the enzyme, so further research work on enzymatic aminolysis of this regioisomer is in progress. Raising the temperature to 45 ºC caused a drastic fall in 2.3. Assignment of the absolute configurations. activity of CAL-A in vinyl acetate as solvent (entry 7). On the contrary, the increment of the temperature accelerated slightly the For the assignment of the chemical configuration of (+)-transprocess catalyzed by CAL-B in tBuOMe, a 38% of conversion 8a and the remaining (+)-trans-6 obtained from the lipase was achieved after four days of reaction (entry 8) and with a catalysed the transesterification, a series of chemical longer incubation time of 7 days a 47% of conversion can be transformations were carried out to convert the optically pure achieved (entry 9). (+)-trans-N-benzyl-3-(diallylamino)-4-acetoxypiperidine, (+)As an additional fine-tuning of the reaction conditions, the trans-8a into the previously described (3R,4R)-Neffect of a basic additive in the reaction media was studied. It has benzyloxycarbonyl-3-tert-butoxycarbonylamino-4been demonstrated that the use of certain additives in the reaction hydroxypiperidine (3R,4R)-12 (Scheme 4). These mixture may have a beneficial influence on the reactivity and transformations have also allowed us to test the versatility of the selectivity of the enzymatic processes.14 Effectively, by the protecting groups on (+)-trans-8a. addition of a small amount of triethyl amine in the best found conditions for the CAL-B catalysed process the reaction rate was O O O O significantly increased in an early stage, after three days of N N reaction 45% of conversion was achieved (entry 10). The a b, c reaction was slowed down near the 50% of conversion and the N N results obtained after 7 days of reaction were the same as in Bn Cbz absence of the amine. (3R,4R)-(+)-8a

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In contrast to the good results obtained in the transesterification of substrate (±)-trans-6, no satisfactory results were achieved in the hydrolysis of its ester derivatives. The reaction conditions are showed in scheme 3. In the case of the acetylated derivative (±)-trans-8a, no conversion was observed after 4 days of reaction. When the activated ester (±)-trans-8b was used as substrate for the enzymatic hydrolysis only a conversion between 2 and 5%, were detected after 10 hours of reaction. Even though the formed product was optically pure at the first stage of the process, no further advance of the reaction was observed at longer times.

Scheme 3. Lipase catalysed hydrolysis of (±)-trans-8a-b.

The resolution of the regioisomer (±)-trans-7 was studied using the same procedure as for substrate (±)-trans-6, the enzymatic acylation was studied using the lipases (CAL-A, CALB, PSL-IM, AK and TL-IM) at 30 ºC in organic solvent (tBuOMe) and 5 equivalents of vinyl acetate as acylating agent, but no reaction was observed in any of the tested processes. Also no reaction was observed when different reaction parameters were modified (temperature, solvent or the ratio of acylating agent). The hydrolysis of the ester derivatives (±)-trans-9a and 9b was also carried out in the reaction conditions detailed for (±)trans-8a-b with the same negative results. On the other hand, these results were in accordance with the previous described

O

(3R,4R)-(+)-10

O

OH

NHBoc

OH NHBoc

d

NHBoc e

N

N

N

Cbz

Cbz

Cbz

(3R,4R)-(+)-11

(3R,4R)-(+)-12

(3R,4S)-(+)-12

Scheme 4. (a) CbzCl, CH3CN, rt, 1 h, 85%. (b) Pd(PPh3)4, NDMBA, PhCH3, 110 ºC, 48 h. (c) Boc2O, CH2Cl2, rt, 12 h, 90%. (d) K2CO3, MeOH/H2O, rt, 3 h, 95 %. (e) ref. 5.

Debenzylation and subsequent protection of the pyperidine nitrogen was carried out in one step by the treatment of (+)-8a with benzylcloroformate15 to obtain in high yields the corresponding N-benzyloxycarbonyl protected derivative (+)10. Removal of the allyl groups of (+)-10 in presence Pd(0) and N,N’-dimethylbarbituric acid (NDMBA) as allyl group scavenger,16 afforded the corresponding crude amine which was next treated with tert-butoxycarbonyl anhydride to achieve the Boc protected derivative (+)-11, in high yield (90%, calculated from (+)-10). A hydrolysis of (+)-11 afforded (+)-12, which has the (3R,4R) configuration, as was established by quiral-HPLC analysis in the same conditions as the reported for (3R,4R)-12.17 Thus, the absolute configuration (3R,4R) was assigned to the enzymatically produced (+)-8a. The inversion of the hydroxyl group at C4 in the (3R,4R)-12 was also carried out to yield the cis-isomer (+)-(3R,4S)-12, that provide us an additional confirmation of the assigned configuration by quiral-HPLC analysis in the same conditions as the reported by Atkinson et al.5 3. Conclusions

This paper describes an easy methodology for the preparation of orthogonally protected (3R,4R)-3-amino-4hidroxipiperidine with control over the relative and absolute

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3054, 2986 cm-1; HRMS-ESI+ requires [C18H27N2O]+ (M+H)+ stereochemistry of the 3,4-amino alcoholACCEPTED moiety. These MANUSCRIPT optically pure compounds could be useful intermediates in the 287,2118 m/z, found 287.2125 synthesis of a number of bioactive compounds. 4.3. Synthesis of (±)-trans-1-benzyl-4-(diallylamino)-3After the regioselective preparation of the racemic (±)-transhydroxypiperidine, (±)-trans-7. N-benzyl-3-(diallylamino)-4-hydroxypiperidine, (±)-trans-NTo a solution of 3,4-epoxypiperidine (±)-4 (0.32 g, 1.69 benzyl-4-(diallylamino)-3-hydroxypiperidine and their ester mmol) synthesized following the procedure described by Young derivatives, different lipases, solvents and reaction conditions et al.10b in acetonitrile (15 mL), LiClO4 (538 mg, 5.07 mmol) and were tested. The resolution of the 3-(diallylamino)-4-hydroxy diallylamine (243 mL, 2.5 mmol) was added. The mixture was substituted piperidine was accomplished by a successful stirred at at room temperature for 12 h. After this time, the transesterification catalyzed by CAL-B. In contrast, these lipases solvent was removed under reduced pressure to afford the were ineffective in the resolution of the opposite regioisomer. corresponding crude residue that was purified by flash Furthermore, the selective transformations performed in order chromatography on silica gel (CH2Cl2/EtOH 10:0.1) to afford the to assign the absolute configuration of the optically pure (3R,4R)product (±)-trans-7 as a colourless oil (85% yield); 1H-NMR N-benzyl-3-(diallylamino)-4-acetoxypiperidine, showed the (CDCl3, 300.13 MHz): δ 7.39 – 7.24 (m, 5H), 5.87 – 5.73 (m, versatility of the protecting groups at this key structural unit. 2H, 2 x CH=CH2), 5.22 – 5.09 (m, 4H, 2 x CH=CH2), 3.64 – 3.57 (m, 3H, H-3, CH2), 3.40 – 3.37 (m, 2H, 2 x CH-CH=CH2), 3.23 4. Experimental section. (ddd, J = 10.4, 4.6, 2.1 Hz, 1H, H-2), 2.97 – 2.90 (m, 3H, H-6 and 2 x CH=CH2), 2.44 (ddd, J = 12.0, 9.8, 4.0 Hz, 1H, H-4), 4.1. General Remarks 1.99 (dt, J = 12.0, 2.6 Hz, 1H, H-6), 1.89 (t, J = 10.0 Hz, 1H, H2), 1.78 – 1.63 (m, 1H, H-5), 1.53 (dddd, J = 12.0, 12.0, 12.0, 4.0 Enzymatic reactions were carried out in a Gallenkamp Hz, 1H, H-5). 13C-NMR (CDCl3, 75.5 MHz): δ 137.1 (C), 129.5 incubatory orbital shaker. Immobilized Candida antarctica (CH), 128.6 (CH), 127.5 (CH), 117.6 (CH2), 67.0 (C-3), 64.6 (Clipase B, CAL-B (Novozym 435, 7300 PLU/g), was a gift 4), 63.1 (CH2), 59.2 (C-2), 53.3 (C-6), 53.0 (CH), 22.5 (C-5). IR from Novo Nordisk co., immobilized CAL-A (lipase NZL(neat, NaCl): νmax 3463, 2988, 2087, 1644, 1802; HRMS-ESI+ 101, 6,2 U/g) is commercialized by Codexis, Pseudomonas requires [C18H27N2O]+ (M+H)+ 287.2118 m/z, found 287.2121. fluorescens (AK, 22100 U/g), and Thermomyces lanuginosus

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(TL IM, 560 TBU/g) is commercialized by Novo Nordisk. Chemical reagents were commercialized by Aldrich, Merck, ACROS organics or Alfa Aesar. Solvents were distilled over an appropriate desiccant under nitrogen. Flash chromatography was performed using Merck silica gel 60 (230-400 mesh). Optical rotations were measured using a Perkin-Elmer 343 polarimeter and are quoted in units of 10-1 deg cm2 g-1.1H-NMR, 13C-NMR and DEPT spectra were recorded in a Bruker AC-300, Bruker AC-300 DPX or Bruker AV-400 spectrometer using CDCl3 as solvent. The chemical shift values (δ) are given in ppm. HRMS were measured in ESI+ mode with a Bruker micrOTOF spectrometer. IR spectra were recorded in a UNICAM Mattson 3000 FT. The enantiomeric excesses were determined by chiral HPLC analysis on a Hewlett-Packard 1100, LC liquid chromatograph, using a CHIRALPAK IA column (4.6 × 250 mm) and CHIRALPAK AD-H column (4.6 × 250 mm).

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4.2. Synthesis of (±)-trans-1-benzyl-3-(diallylamino)-4hydroxypiperidine, (±)-trans-6. To a solution of 3,4-epoxypiperidine (±)-4 (0.3 g, 1.59 mmol) synthesized following the procedure described by Young et al.10b in ethanol (10 mL), diallylamine (231 mL, 2.38 mmol) was added. The mixture was stirred at 80 ºC, for 48 h, in a sealed tube. After this time, the solvent was removed under reduced pressure to afford the corresponding crude residue that was purified by flash chromatography on silica gel (CH2Cl2/EtOH 10:0.2) to afford the product (±)-trans-6 as a pale yellow oil (80 % yield); 1H-NMR (CDCl3, 300.13 MHz): δ 7.42-7.22 (m, 5H), 5.77 (m, 2H, 2 x CH=CH2), 5.27-5.03 (m, 4H, 2 x CH=CH2), 3.54 (AB, J = 13.5 Hz, 2H, CH2), 3.49-3.40 (m, 1H, H-4), 3.373.30 (m, 2H, 2 x CH-CH=CH2), 2.95-2.80 (m, 4H, H-2 and H-6, 2 x CH-CH=CH2), 2.75 (td, J = 10.8, 3.6 Hz, 1H, H-3), 2.16-1.93 (m, 2H, H-2 or H-6, H-5), 1.85 (t, J = 10.8 Hz, 1H, H-2 or H-6), 1.72-1.53 (m, 1H, H-5). 13C-NMR (CDCl3, 75.5 MHz): δ 138.4 (C), 136.6 (CH), 129.0 (CH), 128.8 (CH), 128.2 (CH), 127.0 (CH), 117.3 (CH2), 68.0 (C-4), 63.1 (C-3), 62.7 (CH2), 52.7 (CH2), 51.7 (CH2), 50.3 (CH2), 32.3 (C-5). IR (neat, NaCl): νmax

4.4. General procedure for the acylation of (±)-trans-6 and (±)-trans-7.

To a solution of the corresponding (diallylamino)alcohol (1.0 mmol) in dichloromethane (10 mL), DMAP (6.4 mg, 0.05 mmol), Et3N (217,6 µL, 1.56 mmol) and the corresponding acyl chloride (2.08 mmol) were added under inert atmosphere. The mixture was stirred at room temperature for 12 h. Then, the mixture was washed with water (3 x 15 mL) and the organic phase was dried over Na2SO4. The solvent was removed under reduced pressure and the crude residue was purified by flash chromatography on silica gel (CH2Cl2/EtOH 10:0.1). 4.4.1. (±)-trans-4-acetoxy-1-benzyl-3-(diallylamino)piperidine, (±)-trans-8a.

Colourless oil, yield 85%; 1H-NMR (CDCl3, 300.13 MHz): δ 7.32 (m, 5H), 5.71 (m, 2H, 2 x CH=CH2), 5.19 – 5.02 (m, 4H, 2 x CH=CH2), 4.98 – 4.85 (dt, J = 10.2, 4.9 Hz, 1H, H-4), 3.55 (AB, J = 12.8 Hz, 2H, CH2), 3.32 – 3.21 (m, 2H, 2 x CH-CH=CH2), 3.15 – 2.89 (m, 3H, H-2 or H-6, 2 x CH-CH=CH2), 2.81 (m, 1H, H-3), 2.14 – 2.07 (s and m, 7H, CH3, H-5, H-2 and H-6), 1.77 – 1.55 (m, 1H, H-5). 13C-NMR (CDCl3, 75.5 MHz): δ 170.5 (CO), 138.3 (C), 137.5 (CH), 128.9 (CH), 128.2 (CH), 127.1 (CH), 116.2 (CH2), 71.0 (C-4), 62.6 (CH2), 59.7 (C-3), 53.2 (CH2), 53.2 (CH2), 51.4 (CH2), 30.7 (C-5), 21.4 (CH3). IR (neat, NaCl): νmax 2088, 1644; HRMS-ESI+ requires [C20H29N2O2]+ (M+H)+ 329.2224 m/z, found 329.2205. 4.4.2. (±)-trans-1-benzyl-3-(diallylamino)-4-methoxyacetoxypiperidine, (±)-trans-8b. Colourless oil, yield 88%; 1H-NMR (CDCl3, 300.13 MHz): δ 7.31 (m, 5H), 5.84 – 5.61 (m, 2H, 2 x CH=CH2), 5.20 – 4.91 (m, 5H, H-4, 2 x CH=CH2), 4.06 (s, 2H, CH2), 3.54 (m, 2H, CH2), 3.48 (s, 3H, CH3), 3.28 (dd, J = 14.2, 5.1 Hz, 2H, 2 x CHCH=CH2), 2.98 (m, 3H, H-2 or H-6, 2 x CH-CH=CH2), 2.83 (d, J = 11.3, 1H, H-2 or H-6), 2.09 – 1.93 (m, 3H, H-5, H-2 and H-6), 1.77-1.67 (m, 1H, H-5). 13C-NMR (CDCl3, 75.5 MHz): δ 169.8 (CO), 137.2 (C), 128.9 (CH), 128.4 (CH), 128.3 (CH), 127.1 (CH), 116.5 (CH2), 71.3 (C-4), 70.0 (CH2), 62.5 (CH2), 59.7 (C3), 59.3 (CH3), 53.1 (CH2), 52.5 (CH2), 51.3 (CH2), 30.6 (C-5).

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4.4.4. (±)-trans-1-benzyl-4-(diallylamino)-3-methoxyacetoxypiperidine, (±)-trans-9b.

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4.5. General procedure for the enzymatic acylation.

To a solution of the optically pure (3R,4R)-8a (250 mg, 0.73 mmol) obtained from the CAL-B catalyzed acetylation of (±)trans-6, in acetonitrile (7.mL), benzyloxycarbonyl hydrochloride (104.0 µL, 0.73 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. Then, the solvent was removed under reduced pressure and ethyl acetate (10 mL) was added to the crude residue; the mixture was washed with water (3 x 10 mL) and the organic phase was dried over Na2SO4. The solvent was removed under reduced pressure and the crude residue was purified by flash chromatography on silica gel (hexane/EtOAc 9:1) to afford the product (3R,4R)-10 as a colourless oil (95% yield); 1H-NMR (CDCl3, 300.13 MHz): δ 7.43 – 7.34 (m, 5H), 5.81 – 5.64 (m, 2H, 2 x CH=CH2), 5.11 (m, 7H, H-4, 2 x CH=CH2, CH2), 4.14 (m, 2H, H-2 and H-6), 3.29 (dd, J = 14.3, 5.0 Hz, 2H, 2 x CH-CH=CH2), 3.08 (m, 2H, 2 x CH-CH=CH2), 2.77 (m, 3H, H-2, H-6 and H-3), 2.08 (s, 3H, CH3), 2.00 (m, 1H, H-5), 1.69 – 1.46 (dd, J = 13.8, 4.6 Hz, 1H, H-5). 13C-NMR (CDCl3, 75.5 MHz): δ 170.3 (CO), 155.1 (CO), 136.8 (CH), 136.6 (C), 128.5 (CH), 128.1 (CH), 127.9 (CH), 116.7 (CH2), 70.3 (C-4), 67.3 (CH2), 59.4 (C-3), 53.0 (CH2), 43.4 (CH2), 42.2 (CH2), 30.7 (C-5), 21.3 (CH3). IR (neat, NaCl): νmax 3054, 2986, 2305, 1724, 1718. HRMS-ESI+ requires [C21H29N2O4]+ (M+ H)+ 373.2122 m/z, found 373.2135. [α]D25 = +5.9 (c = 1, CHCl3), ee >99 %.

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White oil, yield 87%; 1H-NMR (CDCl3, 300.13 MHz): δ 7.36 – 7.21 (m, 5H), 5.72 (m, 2H, 2 x CH=CH2), 5.23 – 5.01 (m, 5H, H-3, 2 x CH=CH2 ), 4.02 (s, 2H, CH2), 3.53 (m, 2H, CH2), 3.51 – 3.44 (s, 3H, CH3), 3.36 – 3.23 (m, 2H, 2 x CH-CH=CH2), 3.06 (ddd, J = 12.0, 5.8, 2.9 Hz, 1H, H-4), 2.92 (m, 3H, H-6 and 2 x CH-CH=CH2), 2.74 – 2.57 (td, J = 10.7, 5.2 Hz, 1H, H-2), 1.95 (m, 2H, H-2 and H-6), 1.82 – 1.70 (m, 1H, H-5), 1.57 (dddd, J = 12.0, 12.0, 12.0, 3.7 Hz, 1H, H-5). 13C-NMR (CDCl3, 75.5 MHz): δ 169.8 (CO), 138.3 (C), 137.9 (CH), 129.3 (CH), 128.7 (CH), 127.6 (CH), 116.7 (CH2), 70.3 (CH2), 69.7 (C-3), 62.8 (CH2), 61.2 (CH3), 59.7 (C-4), 57.2 (C-2), 53.1 (CH2), 52.8 (C-6), 23.8 (C-5). IR (neat, NaCl): νmax 2088, 1644; HRMS-ESI+ requires [C21H31N2O3]+ (M+H)+ 359.2329 m/z, found 359.2333.

4.7.1. Synthesis of (3R,4R)-4-acetoxy-1-benzyloxycarbonyl-3(diallylamino)piperidine, (3R,4R)-10.

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Colourless oil, yield 87%; 1H-NMR (CDCl3, 300.13 MHz): δ 7.38 – 7.22 (m, 5H), 5.83 – 5.63 (m, 2H, 2 x CH=CH2), 5.22 – 5.01 (m, 5H, H-3, 2 x CH=CH2), 3.56 (brs, 2H, CH2), 3.39 – 3.21 (dd, J = 13.8, 5.2 Hz, 2H, 2 x CH-CH=CH2), 3.10 – 2.81 (m, 4H, H-2, H-6 and 2 x CH-CH=CH2), 2.72 – 2.55 (td, J = 12.0, 4.4 Hz, 1H, H-4), 2.06 (s, 3H, CH3), 1.96 (m, 2H, H-2 and H-6), 1.74 (m, 1H, H-5), 1.61 (dddd, J = 12.0, 12.0, 12.0, 3.9 Hz, 1H, H-5). 13 C-NMR (CDCl3, 75.5 MHz): δ 170.5 (CO), 138.3 (C), 138.0 (CH), 129.4 (CH), 128.7 (CH), 127.5 (CH), 116.5 (CH2), 69.4 (C-3), 62.8 (CH2), 61.1 (C-4), 57.3 (C-2), 53.2 (CH2), 52.9 (C-6), 24.2 (C-5), 21.6 (CH3). IR (neat, NaCl): νmax 2088, 1643; HRMSESI+ requires [C20H29N2O2]+ (M+H)+ 329.2224 m/z, found 329.2206.

4.7 Assignment of the absolute configuration.

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4.4.3. (±)-trans-3-acetoxy-1-benzyl-4-(diallylamino)piperidine, (±)-trans-9a.

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The reaction mixture containing the corresponding (diallylamino)piperidine (50 mg), the lipase (100 mg), and vinyl acetate (5 eq) in the corresponding organic solvent (1.5 mL), was shaken at 45 oC and 250 rpm in an orbital shaker. In the case of substrate (±)-trans-6, for a faster process, 150 µL of Et3N were added to the reaction media. The progress of the reaction was monitored by chiral HPLC until achievement of the required conversion. The enzyme was then removed by filtration and washed with tBuOMe. The solvent was evaporated under reduced pressure and the crude residue was purified by flash chromatography on silica gel (hexane/EtOAc 8:2). 4.5.1. (3S,4S)-1-benzyl-3-(diallylamino)-4-hydroxypiperidine, (3S,4S)-6. Yield (26.0 mg, 52%); [α]D25 = +5.2 (c = 1, CHCl3), ee >87 %. 4.5.2. (3R,4R)-4-acetoxy-1-benzyl-3-(diallylamino)piperidine, (3R,4R)-8a. Yield (25.8 mg, 45%); [α]D25 = +1.8 (c = 1, CHCl3), ee >99 %. 4.5 Determination of the ee by HPLC analysis: Chiralpak IA, 30 ºC, hexane/2-propanol (95:5), UV 210 nm, 0.8 mL min-1, tR 4.78 min (3R,4R)-8a, tR 5.32 min (3S,4S)-8a, 6.53 min (3S,4S)6, tR 6.96 min (3R,4R)-6. 4.6. General procedure for the enzymatic hydrolysis. The reaction mixture, containing the corresponding acylated (diallylamino)piperidine (50 mg), the lipase (100 mg) and H2O (5 equiv.) in tBuOMe (1,5 mL), was shaken at 30 oC and 250 rpm in an orbital shaker. The progress of the reaction was monitored by

4.7.2. Synthesis of (3R,4R)-4-acetoxy-1-benzyloxycarbonyl-3tert-butoxycarbonylaminopiperidine, (3R,4R)-11. A solution of Pd(OAc)2 (5 mg, 0.023 mmol) and PPh3 (23 mg, 0.087 mmol) in anhydrous CH2Cl2 (2.0 mL) was added drop wise under nitrogen atmosphere to a solution of (3R,4R)-10 (216.0 mg, 0.58 mmol) in anhydrous CH2Cl2 (2.0 mL). (For the in situ formation of the catalysts, it is convenient to maintain the solution in the addition funnel over five minutes prior to addition). N,N’-dimethylbarbituric acid (272 mg, 1.74 mmol) was then added and the resulting mixture was refluxed under nitrogen atmosphere for 48 h. After this time, the solution was washed with NaOH 3N (4 x 10 mL), the organic phase was dried over Na2SO4 and the solvent was removed under reduced pressure. The crude residue was filtered through silica gel (CH2Cl2/MeOH 20:1) to afford the crude (3R,4R)-1benzyloxycarbonyl-4-acetoxy-3-aminopiperidine, which was dissolved newly in CH2Cl2 (2.5 mL) and treated with di-tertbutyl pyrocarbonate (15.6 mg, 0.71 mmol). The resulting mixture was stirred at room temperature for 12 h. After this time, the solvent was removed under reduced pressure and the crude residue that was purified by flash chromatography on silica gel (CH2Cl2/MeOH 30:1) to afford the product (3R,4R)-11 as a yellow oil (90% yield); 1H-NMR (CDCl3, 400 MHz): δ 7.38 (m, 5H), 5.15 (m, 2H, CH2), 4.84 (m, 1H, H-4), 4.63 (m, 1H, H-3), 4.05 (m, 1H, H-2 or H-6), 3.77 (m, 2H, H-2 and H-6), 3.16 (m, 2H, H-5, H-2 or H-6), 2.09 (s, 3H, CH3), 1.93 (s, 1H, H-5), 1.44 (s, 9H, CH3). 13C-NMR (CDCl3, 101 MHz): δ 170.7 (CO), 155.0 (CO), 154.9 (CO), 136.4 (C), 128.5 (CH), 128.1 (CH), 127.95 (CH), 79.9 (C), 71.3 (C-4), 67.5 (CH2), 50.2 (C-3), 46.3 (CH2), 41.1 (CH2), 28.5 (C-5), 28.3 (CH3), 21.1 (CH3). IR (neat, NaCl): νmax 3055, 2986, 1701, 1697, 1673. [α]D25 = +1.5 (c = 1, CHCl3), ee >99 %.

4.7.3. Synthesis of (3R,4R)-1-benzyloxycarbonyl-4-hydroxy-3tert-butoxycarbonylaminopiperidine, (3R,4R)-12.

6

Tetrahedron

Yield > 99 %; [α]D25 = +3.1 (c = 1, CHCl3), ee >99 % 4.7.4. Synthesis of (3R,4S)-1-benzyloxycarbonyl-4-hydroxy-3tert-butoxycarbonylaminopiperidine, (3R,4S)-12.

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The synthesis was carried out following the procedure described in ref. 5. The spectroscopic data and tR for the obtained (+)-cis-12 analyzed by quiral-HPLC was in accordance with that reported for (3R,4S)-isomer. [α]D25 = +1.9 (c = 1, CHCl3), ee >99 %

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Acknowledgments This work was supported by the Ministerio de Ciencia e Innovation of Spain (Project CTQ 2011-24237 and CTQ 201455015) and Principado de Asturias (FC-15-GRUPIN14-002). A. V.-B. thanks also Principado de Asturias for a predoctoral fellowship. References and notes

5.

6. 7. 8. 9. 10.

11.

12. 13. 14.

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Källström, S.; Leino, R. Bioorg. Med Chem. 2008, 16, 601-635. Hopkins, B. T.; Cai, X.; Chan, T. R.; Conlon, P.; Humora, M.; Jenkins, T. J.; MacPhee, J. M.; Shi, X.; Miller, R. A.; Thompson, A. WO 2013185082 A2 2013, CAN, 160:69958. (a) Khare, A. B.; Langason, R. B.; Parsons, S. M.; Mach, R. H.; Efange, S. M. N. Nucl. Med. Biol. 1999, 26, 609-617. (b) Efange, S. M. N.; Mach, R. H.; Khare, A.; Michelson, R. H.; Nowak, P. A.; Evora, P. H. App. Radiat. Isot. 1994, 45, 465-72. Fink, B. E.; Gavai, A. V.; Vite, G. D.; Chen, P.; Mastalerz, H.; Norris, D. J.; Tokarski, J. S.; Zhao, Y.; Han, W.-C. WO 2005066176 A1 2005. CAN, 143:153408. Atkinson, S. J.; Barker, M. D.; Campbell, M.; Diallo, H.; Douault, C.; Garton, N. S.; Liddle, J.; Sheppard, R. J.; Walker, A. L.; Wellaway, C.; Wilson, D. M. WO 2014015905 2014, CAN, 160:278922. Mochizuki, A.; Nakamoto, Y.; Naito, H.; Uoto, K.; Ohta, T. Bioor. Med.Chem. Lett.2008, 18, 782-787. Ganina, O. G.; Veselov, I. S.; Grishina, G. V.; Fedorov, A. Yu.; Beletskaya, I. P. Russ. Chem. Bull. 2006, 55, 1642-1647. Tokuda, O.; Aikawa, T.; Ikemoto, T. Kurimoto, I. Tetrahedron Lett. 2010, 51, 2832-2834. Sheunemann, M.; Hennig, L.; Funke, U. Steinbach, J. Tetrahedron 2011, 67, 3448-3456. (a) Young, I. S.; Ortiz, A.; Sawyer, J. R.; Conlon, D. A.; Buono, F. G.; Leung, S. W.; Burt, J. L.; Sortore, E. W. Org. Process Res. Dev. 2012, 16, 1558-1565. (b) Ortiz, A.; Young, I. S.; Sawyer, J. R.; Hsiao. Y.; Singh, A.; Sugiyama, M.; Corbett, R. M.; Chau, M.; Shi, Z. Conlon, D. A. Org. Biomol. Chem. 2012, 10, 5253-5257. (a) Villar-Barro, A.; Gotor, V.; Brieva, R.; Bioorg. Med Chem. 2014, 22, 5563-5568 and references cited therein. (b) RodríguezRodríguez, J. A.; Brieva, R.; Gotor, V. Tetrahedron 2010, 66, 6789–6796; (c) Solares, L. F.; Lavandera, I.; Gotor-Fernández, V.; Brieva, R.; Gotor, V. Tetrahedron 2006, 62, 3284-3291. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. J. Org. Chem. 1991, 56, 2656–2665. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J., J. Am. Chem. Soc., 1982, 104, 7294-7299. Theil, F., Tetrahedron, 2000, 56, 2905-2919.

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1. 2.

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15. Rawal, V. H.; Jones, R. J.; Cava, M. P. J. Org. Chem. 1987, 52, A solution of (+)-trans-11 (100 mg, 0.25 ACCEPTED mmol) and K2CO3 MANUSCRIPT 19-28. (35 mg) in methanol/water 2:1 (1.5 mL) was stirred at room 16. Garro-Helion, F.; Merzouk, A. Guibé, F. J. Org. Chem. 1993, 58, temperature for 3h. Then, the solvent was removed under reduced 6109-6613. pressure the crude was dissolved newly in ethyl acetate (10 mL). 17. HPLC data is described in the supporting information of: Burger, The resulting solution was washed with water (3 x 15mL) and the M. T.; Han, W.; Lan, J.; Nishiguchi, G.; Bellamacina, C.; Lindval, M.; Atallah, G.; Ding, Y.; Mathur, M.; McBride, C.: Beans, E. L.; organic phase was dried over Na2SO4. The solvent was removed Muller, K.; Tamez, V.; Zhang , Y.; Huh, K.; Feucht, P.; under reduced pressure afford quantitatively the product (+)Zavorotinskaya, T.; Dai, Y.; Holash, J.; Castillo, J.; Langowski, J.; trans-12 as a colorless oil. No further purification was necessary. Wang, Y.; Chen, M. Y.; García, P. D. J. Med. Lett. 2013, 4, 1193The spectroscopic data and tR for the obtained (+)-trans-12 1197. analyzed by quiral-HPLC was in accordance with that reported for (3R,4R)-1217 Thus, the absolute configuration (3R,4R) was assigned to the enzymatically produced (+)-8a.

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Figure 1. Significant examples of piperidines substituted at C3 and C4 by a vicinal amino alcohol moiety.

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ACCEPTED MANUSCRIPT