Practical preparation of enantiopure 2-methyl-azetidine-2-carboxylic acid; a γ-turn promoter

Tetrahedron: Asymmetry 23 (2012) 690–696

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Practical preparation of enantiopure 2-methyl-azetidine-2-carboxylic acid; a c-turn promoter Bruno Drouillat, Karen Wright, Jérôme Marrot, François Couty ⇑ Institut Lavoisier de Versailles, UMR CNRS 8180, Université de Versailles St-Quentin en Yvelines. 45, av. des Etats-Unis, 78035 Versailles Cedex, France

a r t i c l e

i n f o

Article history: Received 5 April 2012 Accepted 10 May 2012 Available online 8 June 2012

a b s t r a c t A robust and practical synthesis of each enantiomer of 2-methyl-azetidine-2-carboxylic acid, based on the use of (S)-phenylglycinol as resolving agent, is described. This synthesis affords practical quantities of this quaternary amino acid suitably N- and C-protected for use in further peptide coupling. Synthetic highlights include the formation of the azetidine ring by intramolecular alkylation and the facile separation of the diastereoisomeric amides derived from phenylglycinol. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

2. Results and discussion

The search for new amino acids able to induce particular backbone conformations when included in peptides is an active area of research.1 For example, Ca-methyl proline is known to enhance the propensity of the prototype proline residue for b-turn formation, when located at the i+1 position of a peptide sequence.2 Recently, Ca-alkyl azetidine-2-carboxylic acids were reported on by MartínMartínez et al.3 and demonstrated to induce c-turns4 in short peptides, which is in sharp contrast with their higher Ca-methyl proline homologues. Thus, these constrained amino acids are emerging as new tools for conformational control of the peptide backbone and would be good candidates for c-turn scan studies in higher peptides of biological interest. However, the development of these new tools relies on an easy and scalable synthesis, capable of providing workable quantities of each enantiomer of these quaternary amino acids in a suitably protected form for further peptide coupling. While this is well established for Ca-methyl proline derivatives,5 access to enantiomerically pure a-quaternary azetidines, and more specifically to Ca-alkyl azetidine-2-carboxylic acids is almost unreported in the literature.6 Thus, the most practical route at present relies on the stereocontrolled ring closure of the N-chloroacetyl derivative of an amino ester derived from (+)-10-(N,N-dicyclohexylsulfamoyl)-isoborneol as the chiral auxiliary, which also needs an additional step for the chemoselective reduction of the resulting b-lactam into an azetidine.7 Based on our previous experience in the synthesis and reactivity of azetidinic amino acids,8 we herein report a practical access to this particular class of unnatural azetidinic amino acids relying on ring closure of the azetidine through intramolecular C-alkylation.

2.1. Synthesis of racemic azetidines

⇑ Corresponding author. E-mail address: [email protected] (F. Couty). 0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetasy.2012.05.006

Racemic N-Bn-Ca-methyl azetidine-2-carboxylic acid tert-butyl ester 5a9 was conveniently prepared on a multigram scale, following our recently published procedure9 in a four step sequence starting from commercially available 2-bromo-propionyl bromide involving esterification,10 N-alkylation with N-benzylethanolamine, chlorination, and cyclization. With the aim of extending the scope of this strategy, and to allow a variation of the substituent on the azetidine ring, it was applied for the synthesis of N-BnCa-phenyl azetidine-2-carboxylic acid tert-butyl ester 5b which proceeded uneventfully (Scheme 1). 2.2. Resolution via diastereoisomeric amides We next focused on a practical way to resolve compound 5a, and after some experimentation, found that the diastereoisomeric amides 6a and 6b derived from (S)-phenylglycinol could be conveniently separated by flash chromatography and isolated from 5a in 44% and 43% overall yield, respectively, on a gram scale. The absolute configuration of the quaternary center in 6a was determined by X-ray crystallography11 (Scheme 2). Amides derived from phenylglycinol are relatively easy to hydrolyze and this property has already been used for the purpose of carboxylic acid resolution.12 Thus, N-Fmoc protected derivatives (S)-8 and (R)-8 could be accessed by N-Bn hydrogenolysis, and NFmoc protection followed by acidic hydrolysis of the amide moiety with H2SO4 in a dioxane/water mixture, which occurred without detectable cleavage of the sensitive strained quaternary azetidine. The N-Cbz derivative 10 was prepared via the same strategy, but showed a high discrepancy with the reported3a specific rotation

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B. Drouillat et al. / Tetrahedron: Asymmetry 23 (2012) 690–696

t-BuOH pyridine

O Br X

Br OtBu

Et2O

R

OH BnHN NaI (cat.), K2CO3

O

MeCN

R

1a: R = Me, X = Br 1b: R = Ph, X = Cl

2a: R = Me: 84% 2b: R = Ph: 64%

HO R CO 2tBu

N

3a: R = Me: 90% 3b: R = Ph: 74%

Bn

SOCl2, DCM CO2tBu 5a: R = Me: 83% 5b: R = Ph: 65%

N

Cl

tBuOK, THF 5a or NaHMDS, THF 5b

R CO2tBu

N

R

Bn

Bn

4a: R = Me: 96% 4b: R = Ph: 63% Scheme 1. Synthesis of racemic Ca-quaternary azetidinic amino esters.

1) 2) 3) 4)

TFA / DCM HCl (S)-phenylglycinol, HATU, DIPEA Flash chromatography

O

Ph

O

Ph

OH

5a

(S)

87%

N

N H

(S)

Bn 6a: 44%

(R)

+ N

N H

(S)

OH

Bn 6b: 43%

6a

Scheme 2. Synthesis and separation of diastereoisomeric amides 6a,b and ORTEP view of 6a.

(see Section 4). Alternatively, N-Boc derivative 13 was prepared by acidic methanolysis of the amide, N-Bn to N-Boc exchange and saponification. This procedure could not, however, be applied to the 2-phenyl derivative since N-debenzylation of 5b led exclusively to the azetidine ring opening under the hydrogenolysis conditions employed. (Scheme 3).

and (S)-12 or (R)-12. After N-Boc cleavage with 1.6 M HCl in ether, successful coupling was achieved by HATU activation, leading to the diastereoisomeric dipeptides 14a,b in good yields, as displayed in Scheme 4.

2.3. Peptide coupling

In conclusion, we have reported on a practical route to both enantiomers of Ca-methyl azetidine-2-carboxylic acid on a multigram scale, a particularly strained amino acid which is a promising new tool for the induction of c-turns in peptidic backbones. This synthesis relies on a very efficient intramolecular alkylation to

The Ca-quaternary amino acids may be slow to undergo N-acylation in peptide coupling reactions, due to their hindered nature. We therefore tested the peptide coupling between Boc-L-Ala-OH

3. Conclusion

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OH

2) FmocCl, DIPEA 6a

Ph

O

1) H2, Pd/C(cat.), EtOH

N H

79%

N

H2SO4 water/dioxane

7a

66%

Ph

O

OH

2) FmocCl, DIPEA 6b

N H

59%

N

H2SO4 water/dioxane

7b

70%

6a

Ph

O

OH

2) CbzCl, DIPEA

N H

78%

6a or 6b

N

CO2Me

COOH N

H2SO4 water/dioxane

9

69%

(R)-8

COOH 10

N Cbz

Cbz

H2SO4 MeOH

(S)-8

Fmoc

Fmoc 1) H2, Pd/C(cat.), EtOH

N Fmoc

Fmoc 1) H2, Pd/C(cat.), EtOH

COOH

H2 , Pd(OH)2 Boc2O

CO2Me

93%

N

N

Bn

Boc

(S)-11: 71% (R)-11: 66%

1) NaOH 2) HCl

COOH N Boc

(S)-12: 94% (R)-12: 76%

13

Scheme 3. Synthesis of suitably N-protected derivatives of Ca-methyl-azetidine-2-carboxylic acid.

N

(S)-12 or (R)-12

1) 1.7 M HCl in Et2O 2) Boc-L-Ala-OH HATU, DIPEA, DMF

CO2Me

O

14a: 70% from (S)-12 NHBoc

N

CO2Me

O

14b: 80% from (R)-12 NHBoc

Scheme 4. Dipeptide synthesis from (S)-12 and (R)-12.

build the azetidine ring and on a facile resolution step via the diastereoisomeric amides derived from phenylglycinol. These amides can be hydrolyzed using particularly mild conditions, which are compatible with the strained four-membered ring. A generalization of this strategy for other quaternary azetidinic amino acids is currently under investigation.

4. Experimental 4.1. General The 1H and 13C spectra were recorded on a Bruker Avance spectrometer at 200 or 300 and 75 or 50 MHz respectively; chemical shifts are reported in ppm from TMS. Optical rotations were determined with a Perkin Elmer 141 instrument. All reactions were carried out under argon. Column chromatography was performed on a silica gel 230–400 mesh by using various mixtures of solvents. TLCs were run on Merck Kieselgel 60F254 plates. Melting points are uncorrected. Infrared spectra were recorded on a Nicolet iS

10 (SMART iTR diamond ATR) spectrophotometer. High resolution mass spectra (HR-MS) were obtained on a Water Micromass Q-Tof Micro instrument. 4.2. Synthesis of azetidines 4.2.1. [Benzyl-(2-hydroxyethyl)-amino]-phenyl-acetic acid tertbutyl ester 3b To a solution of N-benzylethanolamine (0.66 mL, 4.58 mmol) in acetonitrile (20 mL) were added potassium carbonate (0.887 g, 6.42 mmol), bromophenylacetic acid tert-butyl ester (1.5 g, 5.5 mmol), and sodium iodide (0.138 g, 0.92 mmol). The reaction mixture was heated at reflux overnight, cooled to room temperature, and concentrated under reduced pressure. The residue was partitioned between water (70 mL) and ether (70 mL), and the organic layer was successively washed with 5 wt-% aqueous sodium thiosulfate (40 mL) and brine (40 mL). The organic layer was dried over magnesium sulfate, evaporated to dryness, and the residue was purified by chromatography (petroleum ether/ ethyl acetate 9/1). Compound 3b was obtained as a colorless oil

B. Drouillat et al. / Tetrahedron: Asymmetry 23 (2012) 690–696

(1.16 g, 74%). Rf: 0.17 (petroleum ether/ethyl acetate 90/10), 1H NMR (300 MHz, CDCl3): d = 1.51 (s, 9H, tBu), 2,65 (s, 1H, OH), 2.79–2.85 (m, 1H, NCHHCH2OH), 2.94–3.01 (m, 1H, NCHHCH2OH), 3.39–3.49 (m, 1H, CHHOH), 3.50–3.63 (m, 1H, CHHOH), 3.83 (s, 2H, NCH2Ph), 4,52 (s, 1H, CHa), 7.28–7.41 (m, 10H, Ar) ppm. 13C NMR (75 MHz, CDCl3): d = 28.0 (tBu), 52.2, 55.4, 59.6 (CH2), 67.5 (Ca), 81.7 (CqtBu), 127.3, 127.9, 128.4, 128.5, 128.7, 128.9 (CHAr), 136.6, 139.2 (Cq Ar), 171.7 (CO) ppm. IR (ATR) mmax 3505, 3448, 3084, 3027, 2974, 2933, 2843, 1722, 1698, 1495, 1450, 1366, 1139, 1057, 735, 694 cm1; ESIHRMS (positive mode) m/z calcd for C21H28NO3 [M+H]+: 342.2069, found 342.2078. 4.2.2. [Benzyl-(2-chloroethyl)-amino]-phenyl-acetic acid tertbutyl ester 4b To a solution of 3b (1.16 g, 3.40 mmol) in DCM (20 mL) was added thionyl chloride (0.5 mL, 6.8 mmol). The reaction mixture was heated at reflux for 2 h, cooled to 0 °C, and neutralized by the careful addition of a saturated aqueous solution of sodium hydrogen carbonate (35 mL). The aqueous layer was extracted with DCM (2  20 mL) and the organic layer was washed with brine (20 mL), dried over magnesium sulfate, and evaporated to dryness. Compound 4b was obtained as a white solid (0.70 g, 63%). Rf: 0.80 (petroleum ether/ethyl acetate 90/10), Mp: 58 °C, 1H NMR (300 MHz, CDCl3): d = 1.43 (s, 9H, tBu), 2.92–2.99 (m, 2H, NCH2CH2Cl), 3.11–3.22 (m, 2H, NCH2CH2Cl), 3.71 (d, part of a AB syst., J = 13.9 Hz, 1H, NCHHPh), 3.81 (d, part of a AB syst., J = 13.9 Hz, 1H, NCHHPh), 4,43 (s, 1H, CHa), 7.16–7.28 (m, 10H, Ar) ppm. 13C NMR (75 MHz, CDCl3): d = 28.2 (tBu), 42.6, 46.8, 56.5 (CH2), 68.4 (Ca), 81.7 (CqtBu), 127.3, 127.6, 127.9, 128.4, 128.5, 128.8, 128.9 (CHAr), 137.2, 139.5 (Cq Ar), 171.5 (CO) ppm. IR (ATR) mmax 3084, 3061, 3025, 2978, 2938, 2863, 1717, 1491, 1452, 1366, 1117, 743, 694 cm1; ESIHRMS (positive mode) m/z calcd for C21H27NO2Cl [M+H]+: 360.1730, found 360.1731. 4.2.3. 1-Benzyl-2-phenyl-azetidine-2-carboxylic acid tert-butyl ester 5b Compound 4b (0.70 g, 2.14 mmol) was dissolved in dry THF (32 mL) at 78 °C after which a 2 M solution of sodium bis(trimethylsilyl)amide in THF (1.4 mL, 2.8 mmol) was added dropwise. The reaction mixture was warmed up to room temperature over 2 h and hydrolyzed by the addition of a saturated aqueous solution of ammonium chloride (30 mL). The aqueous layer was extracted with ethyl acetate (2  20 mL) and the organic layer was washed with brine (20 mL), dried over magnesium sulfate, and evaporated to dryness. The residue was purified by flash chromatography (petroleum ether/diethyl ether 95/5). Compound 5b was obtained as a pale yellow oil (0.447 g, 65%). Rf: 0.63 (petroleum ether/ethyl acetate 95/5), 1H NMR (300 MHz, CDCl3): d = 1.54 (s, 9H, tBu), 2.29–2.38 (m, 1H, H-3), 2.92–2.99 (m, 1H, H-30 ), 3.23–3.35 (m, 2H, H-4, H-40 ), 3.61 (d, part of AB syst., J = 13.0 Hz, 1H, NCHHPh), 4.03 (d, part of AB syst., J = 13.0 Hz, 1H, NCHHPh), 7.28–7.49 (m, 10H, Ar) ppm. 13C NMR (75 MHz, CDCl3): d = 28.3 (tBu), 30.1, 50.1, 57.6 (CH2), 81.8 (CqtBu), 125.3, 127.0, 127.3, 128.2, 128.4, 128.6 (CHAr), 138.5, 143.2 (Cq Ar), 171.8 (CO) ppm. IR (ATR) mmax 3060, 3026, 2974, 2928, 2832, 1715, 1600, 1493, 1446, 1391, 1366, 1251, 1163, 1136, 1102, 844, 696 cm1; ESIHRMS (positive mode) m/z calcd for C21H26NO3 [M+H]+: 324.1964, found 324.1967. 4.3. Resolution via amides 4.3.1. (1S,2S)- and (1S,2R)-1-Benzyl-N-(2-hydroxy-1-phenylethyl)-2-methylazetidine-2-carboxamide 6a and 6b To a solution of 5a (3.57 g, 13.6 mmol) in DCM (25 mL) cooled to 0 °C was added trifluoroacetic acid (25 mL). The reaction mixture was stirred overnight at 25 °C, concentrated under reduced

693

pressure, and the residue was coevaporated with toluene to remove trace amounts of trifluoroacetic acid. This residue was taken up in toluene (10 mL) and a 2 M HCl aqueous solution (10 mL) was added. The mixture was concentrated under reduced pressure. To a solution of the resulting hydrochloride salt in THF (120 mL) were added (S)-phenylglycinol (2.14 g, 16.3 mmol), DIPEA (8.4 mL, 50.4 mmol) and HATU (5.93 g, 16.3 mmol). The reaction mixture was stirred overnight at 25 °C and concentrated under reduced pressure. The residue was taken up in DCM (400 mL) and the organic layer was successively washed with a saturated aqueous solution of NH4Cl (100 mL), a saturated aqueous solution of NaCl (100 mL) and a saturated aqueous solution of NaHCO3 (100 mL). The organic phase was dried over magnesium sulfate, the solvent was evaporated to dryness, and the residue was purified by chromatography (200 g of silica for 5 g of crude residue) using gradient elution (petroleum ether/EtOAc/15% aqueous NH3: 40/60/0.38 to 20/80/0.38, Rf: 0.4). Compound 6a was first eluted as a white solid (1.94 g, 44% overall) and compound 6b was obtained as a white solid (1.89 g, 43% overall). 6a: Rf: 0.40 (DCM/MeOH: 8/2), Mp: 88– 1 90 °C, ½a20 H NMR (300 MHz, CDCl3): D ¼ 37:9 (c 1.1, CH2Cl2), d = 1.61 (s, 3H, Me), 1.88–1.96 (m, 1H, H-3), 2.18–2.27 (m, 1H, H30 ), 3.06–3.15 (m, 2H, OH, H-4), 3.28–3.34 (m, 1H, H-40 ), 3.48 (d, part of AB syst., J = 12.9 Hz, 1H, NCHHPh), 3.71 (ad, J = 5.6 Hz, 2H, CH2OH), 3.76 (d, part of a AB syst., J = 12.9 Hz, 1H, NCHHPh), 4.89–4.95 (m, 1H, Ha), 7.26–7.39 (m, 10H, Ar), 8.46 (d, J = 6.5 Hz, 1H, NH) ppm. 13C NMR (75 MHz, CDCl3) d = 14.8 (CH3), 30.3, 48.9, 55.2 (CH2), 56.2, (CHa), 67.2 (CH2), 67.8 (Cq), 126.6, 127.2, 127.8, 128.5, 128.7, 128.8 (CHAr), 137.9, 139.1 (CqAr), 176.2 (CO) ppm. IR (ATR) mmax 3373, 3314, 2950, 2865, 1647, 1508, 1494, 1455, 1334, 1247, 1082, 941, 738, 695 cm1; ESIHRMS (positive mode) m/z calcd for C20H25N2O2 [M+H]+: 325.1913, found 325.1916. 6b: Rf: 0.38 (DCM/MeOH: 8/2), Mp: 103 °C, ½a20 D ¼ þ36:5 (c 1.9, CH2Cl2), 1H NMR (300 MHz, CDCl3): d = 1.49 (s, 3H, Me), 1.89– 1.96 (m, 1H, H-3), 2.24–2.34 (m, 1H, H-30 ), 3.02–3.10 (m, 1H, H4), 3.28–3.34 (m, 2H, OH, H-40 ), 3.39 (d, part of AB syst., J = 12.9 Hz, 1H, NCHHPh), 3.59 (d, part of a AB syst., J = 12.9 Hz, 1H, NCHHPh), 3.81 (ad, J = 5.4 Hz, 2H, CH2OH), 4.83–4.89 (m, 1H, Ha), 7.07–7.30 (m, 10H, Ar), 8.47 (d, J = 6.7 Hz, 1H, NH) ppm. 13C NMR (75 MHz, CDCl3): d = 14.6 (CH3), 30.7, 49.0, 55.4 (CH2), 56.3, (CHa), 67.8 (CH2), 67.9 (Cq), 126.6, 127.2, 127.9, 128.5, 128.6, 128.9 (CHAr), 137.7, 138.8 (CqAr), 176.6 (CO) ppm. IR (ATR) mmax 3449, 3364, 2911, 2860, 1648, 1505, 1455, 1434, 1374, 1195, 1091, 938, 760, 702 cm1; ESIHRMS (positive mode) m/z calcd for C20H25N2O2 [M+H]+: 325.1913, found 325.1916. 4.4. Hydrolysis and protection 4.4.1. (1S,2S)-(9H-Fluoren-9-yl)methyl 2-(2-hydroxy-1-phenylethylcarbamoyl)-2-methylazetidine-1-carboxylate 7a To a solution of 6a (0.825 g, 2.54 mmol) in 95% ethanol (44 mL) and 2 M HCl aqueous solution (2.55 mL) was added 10% wt Pd on carbon (0.4 g). The reaction mixture was hydrogenated overnight at atmospheric pressure at 25 °C. After filtration of the catalyst, sodium carbonate (0.54 g, 5.08 mmol) was added to the filtrate and the solvent was evaporated to dryness. To a solution of the resulting residue in acetonitrile (25 mL) cooled to 0 °C were added DIPEA (0.89 mL, 5.08 mmol) and FmocCl (0.92 g, 3.55 mmol). The reaction mixture was stirred overnight at 25 °C and concentrated under reduced pressure. The residue was taken up in ethyl acetate (20 mL) and a saturated aqueous solution of NaCl (20 mL) was added. The aqueous layer was extracted with ethyl acetate (2  20 mL), the organic layer was dried over magnesium sulfate, and the solvent was evaporated to dryness to give a residue, which was purified by chromatography (petroleum ether/EtOAc 1/1). Compound 7a was obtained as a white foam (0.683 g, 79% overall). Rf: 0.18 (petroleum 1 ether/EtOAc: 1/1), Mp: 61 °C, ½a20 D ¼ 86 (c 1.4, CH2Cl2), H NMR

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(300 MHz, CDCl3): d = 1.64 (s, 3H, CH3), 1.89–1.97 (m, 1H, H-3), 2.59–2.65 (m, 1H, H-30 ), 3.65–3.81 (m, 4H, CH2OH, H-4, H-40 ), 4.11 (t, J = 6.1 Hz, 1H, NCOOCH2CH), 4.30 (d, J = 6.1 Hz, 2H, NCOOCH2CH), 4.97–4.99 (m, 1H, Ha), 7.12–7.23 (m, 7H, Ar), 7.28–7.33 (m, 2H, Ar), 7.43–7.48 (m, 2H, Ar), 7.66–7.68 (m, 2H, Ar), 8.23 (d, J = 7.3 Hz, 1H, NH) ppm. 13C NMR (75 MHz, CDCl3): d = 22.7 (CH3), 25.5, 44.5 (CH2), 47.1 (CH), 55.9 (CHa), 66.9, 67.2 (CH2), 70.4 (Cq), 120.1, 124.9, 126.7, 127.1, 127.8, 128.8 (CHAr), 138.7, 141.3, 143.5, 143.6 (CqAr), 156.3, 174.7 (CO) ppm. IR (ATR) mmax 3404, 3306, 3065, 2966, 2928, 1674, 1526, 1450, 1411, 1342, 1167, 1078, 738, 698 cm1; ESIHRMS (positive mode) m/z calcd for C28H28N2O4Na [M+Na]+: 479.1947, found 479.1947. 4.4.2. (1S,2R)-(9H-fluoren-9-yl)methyl 2-(2-hydroxy-1-phenylethylcarbamoyl)-2-methylazetidine-1-carboxylate 7b Following the above procedure for 7a, and starting with 6b (0.800 g, 2.46 mmol), compound 7b was obtained as a white solid (0.557 g, 59% overall). Rf: 0.18 (petroleum ether/EtOAc: 1/1), Mp: 1 68 °C, ½a20 H NMR (300 MHz, CDCl3): D ¼ þ29 (c 1.1, CH2Cl2); d = 1.66 (s, 3H, Me), 2.00–2.09 (m, 1H, H-3), 2.50 (s, 1H, OH), 2.77–2.86 (m, 1H, H-30 ), 3.75–3.94 (m, 4H, CH2OH, H-4, H-40 ), 4.25 (t, J = 6.7 Hz, 1H, NCOOCH2CH), 4.37–4.49 (m, 2H, NCOOCH2CH), 5.04–5.10 (m, 1H, Ha), 7.27–7.36 (m, 7H, Ar), 7.40–7.45 (m, 2H, Ar), 7.58–7.61 (m, 2H, Ar), 7.77–7.80 (m, 2H, Ar), 8.37 (d, J = 7.3 Hz, 1H, NH) ppm. 13C NMR (75 MHz, CDCl3): d = 22.5 (CH3), 27.5, 44.5 (CH2), 47.1 (CH), 55.9 (CHa), 66.9, 67.2 (CH2), 70.3 (Cq), 120.0, 125.0, 125.1, 126.6, 127.1, 127.8, 128.8 (CHAr), 138.8, 141.3, 143.6, 143.6 (CqAr), 156.4, 174.5 (CO) ppm. IR (ATR) mmax 3400, 3307, 2926, 1674, 1526, 1450, 1411, 1342, 1167, 1078, 738, 698 cm1; ESIHRMS (positive mode) m/z calcd for C28H28N2O4Na [M+Na]+: 479.1947, found 479.1947. 4.4.3. (S)- and (R)-2-Methyl-azetidine-1,2-dicarboxylic acid 1(9H-fluoren-9-ylmethyl) ester 8 A solution of 7a (0.658 g, 1.44 mmol) in dioxane (40 mL) and 3 M H2SO4 aqueous solution (40 mL) was stirred at reflux for 1 h. After cooling, the aqueous layer was extracted with ethyl acetate (2  70 mL). The organic layer was washed with water (3  70 mL) and dried over magnesium sulfate. The solvent was evaporated to dryness and the residue was purified by chromatography (dichloromethane/methanol 98/2). Compound (S)-8 was obtained as a white solid (0.320 g, 66%). Rf: 0.30 (dichloromethane/ 1 methanol 90/10), Mp: 126 °C, ½a20 D ¼ 82 (c 1.0, CH2Cl2), H NMR (300 MHz, CDCl3): Two rotamers (75:25 ratio), d = 1.43 (minor rotamer) and 1.73 (major rotamer) (s, 3H, Me), 2.12–2.21 (m, 1H, H3), 2.29–2.46 (minor rotamer) and 2.71–2.74 (major rotamer) (m, 1H, H-30 ), 3.89–3.97 (m, 2H, H-4, H-40 ), 4.17–4.27 (m, 1H, OCH2CH), 4.40–4.52 (m, 2H, OCH2), 7.30–7.36 (m, 4H, Ar), 7.40–7.45 (m, 2H, Ar), 7.56–7.58 (m, 2H, Ar) ppm. 13C NMR (75 MHz, CDCl3): Major rotamer d = 22.2 (CH3), 28.3, 28.9, 44.6, 45.0 (CH2), 47.1 (CH), 66.9, 67.6 (CH2), 68.0, 68.7 (Cq), 120.0, 125.0, 127.1, 127.6, 127.9 (CHAr), 141.3, 143.4, 143.5, 143.8 (CqAr), 155.8, 156.5 (NCOO), 175.5, 177.6 (COOH) ppm. IR (ATR) mmax 3065, 2964, 2887, 1743, 1705, 1661, 1448, 1417, 1345, 1156, 1078, 757, 738 cm1; ESIHRMS (positive mode) m/z calcd for C20H19NO4Na [M+Na]+: 360.1212, found 360.1203. Following the same procedure, and starting from 7b (0.591 g, 1.29 mmol), (R)-8 was obtained as a white solid (0.305 g, 70%): Mp: 122 °C, ½a20 D ¼ þ76 (c 1.1, CH2Cl2); ESIHRMS (positive mode) m/z calcd for C20H19NO4 [M]+: 337.1314 found 337.1306. 4.4.4. (1S,2S)-2-(2-Hydroxy-1-phenyl-ethylcarbamoyl)-2-methylazetidine-1-carboxylic acid benzyl ester 9 To a solution of 6a (0.130 g, 0.401 mmol) in 95% ethanol (7 mL) and 2 M HCl aqueous solution (0.4 mL) was added 10%wt Pd on carbon (63 mg). The reaction mixture was hydrogenated overnight

at atmospheric pressure at 25 °C. After filtration of the catalyst, sodium carbonate (85 mg, 0.80 mmol) was added to the filtrate and the solvent was evaporated to dryness. To a solution of the resulting residue in acetonitrile (6 mL) cooled to 0 °C were added DIPEA (0.170 mL, 0.80 mmol) and CbzCl (80 lL, 0.56 mmol). The reaction mixture was stirred overnight at 25 °C and concentrated under reduced pressure. The residue was taken up in ethyl acetate (10 mL) and a saturated aqueous solution of NaCl (10 mL) was added. The aqueous layer was extracted with ethyl acetate (2  10 mL). The organic layer was dried over magnesium sulfate. The solvent was evaporated to dryness and the residue was purified by chromatography (petroleum ether/EtOAc 25/75). Compound 9 was obtained as a white solid (0.115 g, 78% overall). Rf: 0.5 (EtOAc), Mp: 84 °C, 1 ½a20 D ¼ þ95:5 (c 0.62, CHCl3), H NMR (300 MHz, CDCl3): Two rotamers (9:1 ratio), d = 1.61 (minor rotamer) and 1.68 (major rotamer) (s, 3H, Me), 1.91–2.01 (m, 1H, H-3), 2.57–2.66 (m, 1H, H-30 ), 2.94 (br s, 1H, OH), 3.54–3.98 (m, 4H, H-4, H-40 , CH2OH), 4.83–5.12 (m, 3H, CHPh, PhCH2O), 7.06–7.25 (m, 5H, Ar), 8.31 (d, J = 6.0 Hz, 1H, NH) ppm. 13C NMR (75 MHz, CDCl3): Major rotamer d = 22.8 (CH3), 27.6, 44.5 (CH2), 55.9, (CHa), 66.9, 67.0 (CH2), 70.5 (Cq), 126.8, 127.7, 128.0, 128.3, 128.6, 128.8 (CHAr), 136.1, 139.0 (CqAr), 156.4, 174.8 (CO) ppm; ESIHRMS (positive mode) m/z calcd for C21H24N2O4Na (M+Na)+: 391.1634, found 391.1641. 4.4.5. (S)-2-Methyl-azetidine-1,2-dicarboxylic acid 1-benzyl ester 103a A solution of 9 (0.103 g, 0.279 mmol) in dioxane (6 mL) and aqueous 3 N H2SO4 (6 mL) was refluxed for 1 h, then cooled to rt, diluted with water (15 mL) and extracted with EtOAc (3  15 mL). The organic layer was washed with water (20 mL), brine (20 mL) and dried over MgSO4. Concentration under reduced pressure gave 10 as a clear oil (48 mg, 69%). Rf: 0.3 (EtOAc/EtOH: 20 3a 95/5), ½a20 D ¼ 88 (c 1.2, CHCl3), Lit : ½aD ¼ 7:6 (c 0.09, CHCl3); 1 H NMR (300 MHz, CDCl3): Two rotamers (7:3 ratio), d = 1.67 (minor rotamer) and 1.74 (major rotamer) (s, 3H, Me), 1.98–2.09 (m, 1H, H-3), 2.30–2.42 (m, 1H, H-30 minor rotamer), 2.55–2.68 (br s, 1H, H-30 minor rotamer), 3.58–3.98 (m, 2H, H-4, H-40 ), 5.06 (br s, 2H, PhCH2O), 7.09–7.28 (m, 5H, Ar), 8.41 (br s, 1H, COOH) ppm; 13 C NMR (75 MHz, CDCl3): Major rotamer d = 22.2 (CH3), 28.3, 44.8, 67.4, (CH2), 70.5 (Cq), 128.1, 128.4, 128.6, 128.8 (CHAr), 135.8 (CqAr), 156.7, 175.3 (CO) ppm; ESIHRMS (positive mode) m/z calcd for C13H15NO4Na (M+Na)+: 272.0899, found 272.0912. 4.4.6. (S)- and (R)-1-Benzyl-2-methyl-azetidine-2-carboxylic acid methyl ester 11 A solution of 6b (3.065 g, 9.46 mmol) in MeOH (50 mL) and H2SO4 (9.7 mL) was stirred under reflux for 2 days. After cooling to 0 °C, the reaction mixture was poured into a saturated aqueous solution of sodium hydrogen carbonate (500 mL). The aqueous layer was extracted with ethyl acetate (2  250 mL). The organic layer was dried over magnesium sulfate, the solvent was evaporated to dryness and the residue was purified by chromatography (pentane/ethyl acetate 75/25). Compound (R)-11 was obtained as a colorless oil (1.36 g, 66%). Rf: 0.70 (pentane/ethyl acetate 25/ 1 75), ½a20 H NMR (300 MHz, CDCl3): D ¼ þ47 (c 1.1, CH2Cl2), d = 1.41 (s, 3H, Me), 1.80–1.88 (m, 1H, H-3), 2.46–2.55 (m, 1H, H30 ), 2.98–3.05 (m, 1H, H-4), 3.13–3.20 (m, 1H, H-40 ), 3.49 (d, part of AB syst., J = 12.7 Hz, 1H, NCHHPh), 3.66 (s, 3H, MeO), 3.68 (d, part of a AB syst., J = 12.7 Hz, 1H, NCHHPh), 7.11–7.24 (m, 5H, Ar) ppm. 13C NMR (75 MHz, CDCl3): d = 18.7 (CH3), 28.6, 49.1 (CH2), 51.7, (CH3), 55.7 (CH2), 67.7 (Cq), 126.9, 128.2, 128.7 (CHAr), 138.2 (CqAr), 175.2 (CO) ppm. IR (ATR) mmax 3448, 3364, 2959, 2912, 2860, 1723, 1649, 1495, 1456, 1373, 1270, 1175, 1107, 1091, 1027, 832, 697 cm1; ESIHRMS (positive mode) m/z calcd for C13H18NO2 [M+H]+: 220.1338, found 220.1336. Following the same procedure, and starting from 6a (1.487 g, 4.59 mmol),

B. Drouillat et al. / Tetrahedron: Asymmetry 23 (2012) 690–696

compound (S)-11 was obtained as an oil (0.718 g, 71%): ½a20 D ¼ 42 (c 2.0, CH2Cl2); ESIHRMS (positive mode) m/z calcd for C13H18NO2 [M+H]+: 220.1338, found 220.1337. 4.4.7. (S)- and (R)-2-Methyl-azetidine-1,2-dicarboxylic acid 1tert-butyl ester 2-methyl ester 12 To a solution of (R)-11 (1.36 g, 6.21 mmol) in 95% EtOH (50 mL) were added Pd(OH)2 on carbon, 50% wt in water (150 mg) and Boc2O (2.71 g, 12.42 mmol). The reaction mixture was stirred under a hydrogen pressure of 8 bars for 16 h. After filtration over Celite, the solvent was evaporated to dryness and the residue was purified by chromatography (pentane/ethyl acetate 90/10). Compound (R)-12 was obtained as a colorless oil (1.085 g, 76%). Rf: 1 0.70 (pentane/ethyl acetate 25/75), ½a20 D ¼ þ36 (c 3.3, CH2Cl2), H NMR (300 MHz, CDCl3): d = 1.39 (s, 9H, tBu), 1.60 (s, 3H, Me), 2.06–2.15 (m, 1H, H-3), 2.26 (m, 1H, H-30 ), 3.76 (s, 3H, MeO), 3.77–3.82 (m, 1H, H-4), 3.93–3.99 (m, 1H, H-40 ) ppm. 13C NMR (75 MHz, CDCl3): d = 22.8 (CH3), 28.2 (CH2), 28.3 (tBu), 44.6 (CH2), 52.2, (CH3), 67.9, 79.6 (Cq), 155.2, 173.4 (CO) ppm. IR (ATR) mmax 2975, 2933, 2895, 1740, 1701, 1451, 1382, 1364, 1285, 1252, 1194, 1145, 1128, 1070, 876, 859, 778, 542 cm1; ESIHRMS m/z calcd for C11H19NO4Na [M+Na]+: 252.1212, found 252.1211. Following the same procedure, and starting from (S)11 (637 mg, 2.9 mmol), compound (S)-12 was obtained as an oil (627 mg, 94%): ½a20 D ¼ 41 (c 2.4, CH2Cl2); ESIHRMS (positive mode) m/z calcd for C11H19NO4Na [M+Na]+: 252.1212, found 252.1210. 4.4.8. (S)-2-Methyl-azetidine-1,2-dicarboxylic acid 1-tert-butyl ester 13 To a solution of (S)-12 (0.250 g, 1.09 mmol) in THF (13 mL) and methanol (7 mL) was added a solution of sodium hydroxide (0.087 g, 2.17 mmol) in water (1.3 mL). After stirring at 60 °C for 1 h, water (50 mL) was added to the reaction mixture and the organic solvents were evaporated. The aqueous layer was washed with ether (4  10 mL) and acidified to pH 1 with a 0.5 M HCl aqueous solution (5 mL). The aqueous layer was extracted with ether (4  25 mL). The organic layer was dried over magnesium sulfate and evaporated to dryness. Compound 13 was obtained as a white solid (0.209 g, 93%). Rf: 0.40 (DCM/methanol 90/10), Mp: 1 99 °C, ½a20 H NMR (300 MHz, CDCl3): D ¼ 126 (c 0.5, CH2Cl2), d = 1.48 (s, 9H, tBu), 1.72 (s, 3H, CH3), 2.02–2.11 (m, 1H, H-3), 2.71–2.80 (m, 1H, H-30 ), 3.75–3.90 (m, 2H, H-4, H-40 ), 8.51–10.56 (s, 1H, COOH) ppm. 13C NMR (75 MHz, CDCl3): d = 22.2 (CH3), 27.6 (C3), 28.3 (tBu), 44.5 (C-4), 69.1 (C-2), 82.6 (Cq), 157.3, 174.8 (CO) ppm. IR (ATR) mmax 2973, 2931, 2896, 2653, 2552, 1723, 1622, 1455, 1431, 1366, 1282, 1252, 1160, 1085, 860, 769, 778, 716 cm1; ESIHRMS (positive mode) m/z calcd for C12H17NO4Na [M+Na]+: 238.1055, found 238.1059. 4.5. Peptide coupling 4.5.1. (1S,2S)-1-(2-tert-Butoxycarbonylamino-propionyl)-2-methylazetidine-2-carboxylic acid methyl ester 14a Compound (S)-12 (0.627 g, 2.73 mmol) was dissolved in a 1.7 M HCl solution in ether (10 mL). After overnight stirring, a precipitate was formed. The solvent was removed in vacuo. To the salt were added THF at 0 °C (20 mL), Boc-L-Ala-OH (0.622 g, 3.28 mmol), HATU (1.25 g, 3.28 mmol), and DIPEA (1.76 mL, 10.10 mmol). After stirring overnight, the THF was removed by evaporation and the residue was taken up in dichloromethane (100 mL). The organic layer was successively washed with a saturated aqueous solution of sodium hydrogen carbonate (40 mL) and brine (40 mL). The solvent was dried over magnesium sulfate and evaporated to dryness. The residue was purified by chromatography (pentane/ethyl acetate 70/30). Compound 14a was obtained as a colorless oil

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(0.576 g, 70%). Rf: 0.38 (pentane/ethyl acetate 50/50), ½a20 D ¼ 64 (c 1.55, CH2Cl2), 1H NMR (300 MHz, CDCl3): d = 1.18 (d, J = 6.8 Hz, 3H, CH3), 1.31 (1s, 9H, tBu), 2.06–2.20 (m, 1H, H-3), 2.25–2.40 (m, 1H, H-30 ), 3.64 (s, 3H, MeO), 3.99–4.21 (m, 3H, Ha, H-4, H40 ), 5.27 (d, J = 8.07 Hz, 1 H, NH) ppm. 13C NMR (75 MHz, CDCl3): d = 17.8 (CH3: Ca), 21.8 (CH3: C2), 28.2 (tBu), 28.3 (C-3), 45.8 (Ca), 46.5 (C-4), 52.4 (CO2CH3), 67.4 (Cq), 79.3 (Cq), 154.9, 171.66, 172.2 (CO) ppm. IR (ATR) mmax 3321, 2978, 2933, 2888, 1739, 1708, 1649, 1507, 1452, 1427, 1365, 1287, 1247, 1158, 1131, 1060 cm1; ESIHRMS (positive mode) m/z calcd for C12H24N2O5Na [M+Na]+: 323.1583, found 323.1575. 4.5.2. (1S,2R)-1-(2-tert-Butoxycarbonylamino-propionyl)-2methyl-azetidine-2-carboxylic acid methyl ester 14b Following the above procedure for 14a, and starting from (R)-12 (1.36 g, 6.21 mmol), compound 14b was obtained as a colorless oil (1.143 g, 80%). Rf: 0.70 (pentane/ethyl acetate 25/75), ½a20 D ¼ þ27 (c 1.59, CH2Cl2), 1H NMR (300 MHz, CDCl3): Two rotamers (85:15 ratio), d = 1.26–30 (m, 3H, CH3), 1.70 (minor rotamer), 1.74 (major rotamer) (s, 9H, tBu), 2.14–2.23 (m, 1H, H-3), 2.39–2.48 (major rotamer) and 2.51–2.57 (minor rotamer) (m, 1H, H-30 ), 3.76 (major rotamer) and 3.79 (minor rotamer) (s, 3H, MeO), 3.83–4.11 (m, 1H, H-4), 4.14–4.25 (m, 1H, Ha), 4.28–4.37 (m, 1H, H-40 ), 5.08 (d, J = 9.6 Hz, NH minor rotamer), 5.33 (d, J = 7.7 Hz, NH major rotamer) ppm. 13C NMR (75 MHz, CDCl3): Major rotamer d = 18.6 (CH3Ca), 21.8 (CH3C-2), 28.3 (tBu and C-3), 45.9 (Ca), 46.8 (C-4), 52.6 (CO2CH3), 67.5 (C-2), 79.5 (Cq), 155.0, 172.1, 172.3 (CO) ppm. IR (ATR) mmax 3301, 2975, 2933, 2888, 1739, 1709, 1648, 1451, 1426, 1365, 1364, 1288, 1247, 1159, 1131, 1061, 733 cm1; ESIHRMS (positive mode) m/z calcd for C14H24N2O5Na [M+Na]+: 323.1583, found 323.1582. Acknowledgments CNRS and the University of Versailles St-Quentin-en-Yvelines are acknowledged for financial support. References 1. For recent selected reviews on this topic, see: (a) Martinek, T. A.; Fulop, F. Chem. Soc. Rev. 2012, 41, 687–702; (b) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Chem. Rev. 2011, 111, 657–687; (c) Moriuchi, T.; Hirao, T. Acc. Chem. Res. 2010, 43, 1040–1051; (d) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Acc. Chem. Res. 2009, 42, 1628–1639; (e) Prabhakaran, P.; Kale, S. S.; Puranik, V. G.; Rajamohanan, P. R.; Chetina, O.; Howard, J. A. K.; Hofmann, H. J.; Sanjayan, G. J. J. Am. Chem. Soc. 2008, 130, 17743–17754. 2. De Poli, M.; Moretto, A.; Crisma, M.; Peggion, C.; Formaggio, F.; Kaptein, B.; Broxterman, Q. B.; Toniolo, C. Chem. Eur. J. 2009, 15, 8015–8025. 3. (a) Baeza, J. L.; Gerona-Navarro, G.; Pérez de Vega, J.; García-López, M. T.; González-Muñiz, R.; Martín-Martínez, M. Tetrahedron Lett. 2007, 48, 3689– 3693; (b) Baeza, J. L.; Gerona-Navarro, G.; Pérez de Vega, J.; García-López, M. T.; González-Muñiz, R.; Martín-Martínez, M. J. Org. Chem. 2008, 73, 1704–1715; (c) Baeza, J. L.; Gerona-Navarro, G.; Thompson, K.; Pérez de Vega, J.; Infantes, L.; García-López, M. T.; González-Muñiz, R.; Martín-Martínez, M. J. Org. Chem. 2009, 74, 8203–8211; (d) Pérez-Faginas, P.; Aranda, T.; García-López, M. T.; Snoeck, R.; Andrei, G.; Balzarini, J.; González-Muñiz, R. Bioorg. Med. Chem. 2010, 19, 1155–1161; (e) Baeza, J. L.; Bonache, A.; García-López, M. T.; GonzálezMuñiz, R.; Martín-Martínez, M. Amino Acids 2010, 39, 1299–1307. 4. Némethy, G.; Printz, M. P. Macromolecules 1972, 5, 755–758. 5. Beck, A. K.; Blank, S.; Job, K.; Seebach, D.; Sommerfeld, T. Org. Synth. 1995, 72, 62–73. 6. (a) Cativiela, C.; Díaz-de-Villegas, D. Tetrahedron: Asymmetry 2000, 11, 645– 732; (b) Kawabata, T.; Matsuda, S.; Kawakami, S.; Monguchi, D.; Moriyama, K. J. Am. Chem. Soc. 2006, 128, 15394–15395; (c) Drouillat, B.; Couty, F.; Marrot, J. Synlett 2009, 767–770; (d) Cativiela, C.; Ordoñez, M. Tetrahedron: Asymmetry 2009, 20, 1–63. 7. Gerona-Navarro, G.; García-López, M. T.; Gonzàles-Muñiz, R. J. Org. Chem. 2002, 67, 3953–3956. 8. (a) Couty, F.; Evano, G.; Vargas-Sanchez, M.; Bouzas, G. J. Org. Chem. 2005, 70, 9028–9031; (b) Braüner-Osborne, H.; Bunch, L.; Chopin, N.; Couty, F.; Evano, G.; Jensen, A. A.; Kusk, M.; Nielsen, B.; Rabasso, N. Org. Biomol. Chem. 2005, 3, 3926–3936; (c) Couty, F.; Evano, G. Org. Prep. Proced. Int. 2006, 38, 427; (d) Mangaleshwaran, S.; Couty, F.; Evano, G.; Srinivas, B.; Sridhar, R.; RamaRao, K. Arkivoc 2007(x), 71–93; (e) Sivaprakasam, M.; Hansen, K. B.; David, O.; Nielsen,

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B.; Traynelis, S. F.; Clausen, R. P.; Couty, F.; Bunch, L. Chem. Med. Chem. 2009, 4, 110–117. 9. Couty, F.; Drouillat, B.; Lemée, F. Eur. J. Org. Chem. 2011, 794–801. 10. Vollmarr, A.; Dunn, M. S. J. Org. Chem. 1960, 25, 387–390. 11. Crystal system: space group: Orthorhombic, P2(1)2(1)2(1). Unit cell dimensions: a = 10.6000(6) Å, a = 90°, b = 10.6014(6) Å, b = 90°,

c = 15.8021(9) Å, c = 90°. Volume: 1775.76(17) Å3, Z, Calculated density: 4, 1.213 Mg/m3. F(0 0 0)696. Data have been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 866018. 12. Helmchen, G.; Nill, D.; Flockerzi, D.; Youssef, M. S. K. Angew. Chem., Int. Ed. 1979, 18, 63–65.