Synthesis and biological evaluation of new GABA-uptake inhibitors derived from proline and from pyrrolidine-2-acetic acid

European Journal of Medicinal Chemistry 40 (2005) 231–247

www.elsevier.com/locate/ejmech

Original article

Synthesis and biological evaluation of new GABA-uptake inhibitors

derived from proline and from pyrrolidine-2-acetic acid

Xueqing Zhao, Cornelia E. Hoesl, Georg C. Hoefner, Klaus T. Wanner *

Department für Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany

Received 1 September 2004; accepted 9 November 2004

Available online 07 January 2005

Abstract

Several synthetic approaches to N-alkylated derivatives of 4-hydroxypyrrolidine-2-carboxylic acid and 4-hydroxypyrrolidine-2-acetic acid

are described. The ﬁnal compounds have been evaluated as potential inhibitors of the GABA transport proteins GAT-1 and GAT-3. The

biological assays used were based on bovine material or porcine brain. As compared to the corresponding 4-unsubstituted compounds, the

4-hydroxypyrrolidine-2-carboxylic acid and 4-hydroxypyrrolidine-2-acetic acid derivatives showed a signiﬁcant decrease in the inhibitory

potency at both GAT-1 and GAT-3 with only four compounds having reasonable afﬁnity to GAT-1 (IC₅₀: 5.1, 6.6 and 9.4 µM) or GAT-3 (IC₅₀:

19.9 µM), respectively. The biological data of the 4-hydroxypyrrolidine-2-acetic acid derivatives indicates that (2S)-conﬁguration at the

C-2 position for potent inhibition of GAT-1 and (4R)-conﬁguration at the C-4 position for potent inhibition of GAT-3 may be crucial.

Keywords: GABA-uptake inhibitors; Antiepileptic; GAT-1; GAT-3; Pyrrolidines

1. Introduction

regional distribution in the brain and the body and in their

sensitivity to pharmacological agents [8]. GAT-1 and GAT-

3 are high afﬁnity transporters for GABA expressed speciﬁ-

cally in the CNS thereby being valid targets to modulate

GABA-uptake. Potent inhibitors of GAT-1 such as SK&F

89976-A (1), ( )-cis-SK&F 100591-A (2) [9] and tiagabine

(3) [10] (Fig. 1) have been synthesized and their pharmacol-

ogy was intensively investigated. Dhar succeeded in the syn-

thesis of the ﬁrst GAT-3 selective, highly active inhibitor (S)-

SNAP-5114 (4) (Fig. 1 ) exhibiting an IC₅₀value of 5 µM and

a selectivity of 78:1 (GAT-3:GAT-1) [11]. However, for drug

design and further pharmacological studies of GABA neu-

rotransmission, it is still highly desirable to ﬁnd GABA-

uptake inhibitors having high potency and selectivity.

Previously, we reported the synthesis of the pyrrolidine

derivatives 5 and 6 exhibiting potent inhibition of GAT-1 and

GAT-3 and high selectivity (Fig. 2) [12].

Herein, we present the synthesis and biological evaluation

of new pyrrolidine analogues having further structural modi-

ﬁcations. The afﬁnity of ( )-cis-SK&F 100591-A [9] (2)

(Fig. 1) to GAT-1 implies that a hydroxy group in the

N-heterocycle is likely to be acceptable for GABA-uptake

transporters. Thus, we introduced a hydroxy group into the

C-4 position of pyrrolidine-2-carboxylic acid and pyrrolidine-

Dysfunctioning of GABAergic synapses resulting in a

decrease of GABAergic transmission has been invoked for

diseases such as epilepsy [1], Huntington’s chorea [2], and

Parkinson’s disease [3]. Sodium-dependent GABA-uptake

systems have been found to be the principal means by which

GABA in the synaptic cleft is inactivated. In contrast to the

direct enhancement of GABA neurotransmission by GABA_A

agonists and benzodiazepines, inhibition of the GABA trans-

port system palliates GABA deﬁciency in vivo without giv-

ing rise to the development of tolerance [4]. Four different

GABA transporters have been identiﬁed thus far (GAT-1 [5],

GAT-2 [5 b], GAT-3 [6] and BGT-1 [7]) differing in their

Abbreviations: Bn, benzyl; Cbz, benzyl carboxylate; CC, column chro-

matography; DEAD, diethyl azodicarboxylate; DMAP, (4-dimethylamino)-

pyridine; GAT, GABA transport protein; LDA, lithium diisopropylamide;

prep., preparative; r.t., room temperature; TBAF, tetrabutylammonium ﬂuo-

ride; TBDMSCl, (tert-butyl)dimethlylsilyl chloride; TEA, triethylamine;

TMSCl, trimethylsilyl chloride.

* Corresponding author. Tel.: +49-89-2180-77249; fax:

+49-89-2180-77247.

E-mail address: klaus.wanner@cup.uni-muenchen.de (K.T. Wanner).

doi:10.1016/j.ejmech.2004.11.004

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X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

Fig. 1. Structures of representative known GABA-uptake inhibitors.

Fig. 3. The structures of the target compounds.

Fig. 2

ture.

. Structures of GABA-uptake inhibitors having a pyrrolidine core struc-

Fig. 4. The structures of the four key intermediates prepared from (2S,4R)-

10.

2-acetic acid. The nitrogen atom of potent GABA-uptake

inhibitors is generally substituted by appropriate bulky lipo-

philic groups. Therefore, four typical N-substituents a–d were

chosen and two different series of pyrrolidine derivatives 7

and 8 were prepared in enantiomerically pure form as poten-

tial GABA-uptake inhibitors. In addition, the four stereoiso-

mers of 4-hydroxypyrrolidine-2-acetic acid 9 were synthe-

sized (Fig. 3 ). Compounds 7–9 were evaluated for their

selectivity and inhibitory potency at GAT-1 and GAT-3. The

results are expected to contribute to the optimization of struc-

ture–activity relationships.

rangement initiated by AcOAg-TEA in MeOH afforded

(2S,4R)-17 in 81% yield. A simultaneous N,O-deprotection

of (2S,4R)-17 led to (2S,4R)-12 in 90% yield (47% overall

yield).

Compound (2R,4R)-13 was prepared as depicted in Fig. 6 .

The nitrogen atom of (2S,4R)-10 was protected according to

Ref. [15 a]. Subsequent anodic oxidation in methanol gave

the a-methoxy pyrrolidine derivative 18 as a mixture of dias-

tereomers in 97% yield (ds = 41/59) [16]. Protection of the

hydroxy group using TBDMSCl in the presence of imidazole

(yield: 85%) followed by the nucleophilic addition of

1-ethoxy-1-(trimethylsilyloxy)ethene afforded (2R,4R)-20 as

the major product (yield: (2R,4R)-20: 79%, (2S,4R)-20: 9%)

[17]. O-deprotection of (2R,4R)-20 was accomplished in 88%

yield by means of TBAF in THF [18]. Finally, the resulting

product (2R,4R)-21 was subjected to hydrogenation over 10%

Pd-C in conc. HCl/EtOH to give (2R,4R)-13 in 89% yield

(46% overall yield).

To synthesize the lipophilic target structures shown in

Fig. 7, the key intermediates (2S,4R)-11 and (2R,4R)-11 were

N-alkylated with the respective halides of a and c (see Fig. 7 ).

Subsequent saponiﬁcation led to the target compounds

(2S,4R)-7a, c and (2R,4R)-7a, c in moderate yields. Inver-

sion of the stereocenter at C-4 of the pyrrolidine cycle was

achieved via an intramolecular Mitsunobu reaction of (2S,4R)-

2. Chemistry

L-trans-4-Hydroxypyrrolidine [(2S,4R)-10] was chosen as

a precursor for the synthesis of the four key intermediates

(2S,4R)-11, (2R,4R)-11, (2S,4R)-12 and (2R,4R)-13 (Fig. 4)

Compounds (2S,4R)-11 [13] and (2R,4R)-11 [13,14] were

prepared from (2S,4R)-10 according to literature procedures.

The synthesis of the intermediate (2S,4R)-12 is illustrated

in Fig. 5. The amino and the hydroxy functionality of

(2S,4R)-10 were protected with a Cbz and a Bn group, respec-

tively, according to literature procedures [15]. Following trans-

formation into the diazoketone (2S,4R)-16 employing

(COCl)₂and diazomethane at 0 °C (yield: 80%), Wolff rear-

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

233

Fig. 5

. Synthetic route leading to (2S,4R)-12. (a) Ref. [15a]; (b) Ref. [15b]; (c) (COCl)₂, DMF, CH₂Cl₂, 0 °C; (d) CH₂N₂, Et₂O, 0 °C, 80%; (e) AcOAg/TEA,

MeOH, 25–60 °C, 81%; (f) H₂/10% Pd-C, MeOH, 25 °C, 5 d, 90%.

Fig. 6

. Synthetic route leading to (2R,4R)-13. (a) Electrolysis, TEA, MeOH, 10 °C, 97%; (b) TBDMSCl, imidazole, CH₂Cl₂, 25 °C, 85%; (c) BF₃·Et₂O,

1-ethoxy-1-(trimethylsilyloxy)ethene, CH₂Cl₂, –78 °C, 79%; (d) TBAF, THF, 25 °C, 88%; (e) H₂/10% Pd-C, 37% HCl/EtOH, 25 °C, 89%.

Fig. 7. The synthetic routes leading to the proline derivatives 7a, c. (a) K₂CO₃/KI, bromides of a and c, acetone, r.t.; (b) 0.85 M KOH (or 1.0 M NaOH) in EtOH

(or MeOH), r.t.; (c) Ph₃P/DEAD, THF, 0 °C; (d) Ph₃P/DEAD, AcOH, THF, 0 °C.

7a, c providing lactones (1S,4S)-23a, c [19]. From the latter

upon basic hydrolysis the N-substituted target compounds

(2S,4S)-7a, c were obtained. The N-substituted target com-

pounds (2R,4S)-7a, c were made available by subjecting

(2R,4R)-22a, c to an intermolecular Mitsunobu reaction with

acetic acid as nucleophile to transform the conﬁguration of

the stereocenter in 4-position into (4S). Basic hydrolysis of

the resulting compounds (2R,4S)-24a, c eventually led to

(2R,4S)-7a, c. Similarly to the aforementioned syntheses,

starting from (2S,4R)-12 and (2R,4R)-13, the four sets of ste-

reoisomeric pyrrolidine-2-acetic acid derived target com-

pounds (2S,4R)-8a–d, (2R,4R)-8a–d, (2S,4S)-8a–d and

(2R,4S)-8a–d were obtained as shown in Fig. 8.

the N-Cbz-protected derivatives (2S,4R)-28, (2R,4R)-28,

(2S,4S)-28 and (2R,4S)-28 were synthesized in close analogy

to the preparation of the corresponding N-alkyl derivatives as

outlined in Fig. 8. The enantiopure compounds 28 were even-

tually subjected to hydrogenation providing each of the four

stereoisomers of 9 in excellent yields (89–100%; see Fig. 9).

3. Biological evaluation

The ﬁnal compounds 7a, c, 8a–d and 9 were evaluated for

their inhibitory potency regarding GAT-1- and GAT-3-

mediated GABA-uptake. Measurements of inhibitory potency

at GABA-uptake were performed according to a literature

procedure based on subcellular membrane fractions from fron-

To access each of the four stereoisomers of 4-hydroxy-

pyrrolidine-2-acetic acid 9 in enantiomerically pure form, ﬁrst,

234

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

Fig. 8

.

The synthetic routes leading to the homoproline derivatives 8 and 28. (a) K₂CO₃/KI, bromides of a–d, acetone, r.t.; (b) 0.85 M KOH (or 1.0 M NaOH)

in EtOH (or MeOH), r.t.; (c) Ph₃P/DEAD, THF, 0 °C; (d) Ph₃P/DEAD, AcOH or HCOOH, THF, 0 °C.

ﬁguration: IC₅₀= 56 µM, (R)-conﬁguration: IC₅₀= 2.97 µM

[12 b]]. The loss of potency due to the presence of a 4-hydroxy

substituent was even more prevalent for GAT-3 with all com-

pounds actually being devoid of any reasonable activity (IC₅₀

values>100µM).Ascomparedtothecorresponding4-hydroxy-

pyrrolidine-2-carboxylic acid derivatives 7a, the 4-hydroxy-

pyrrolidine-2-acetic acid derivatives (2S,4R)-8a and

(2S,4S)-8a showed higher afﬁnity for GAT-1 having IC₅₀val-

ues of 3.29 and 4.92 µM, respectively. The observed IC₅₀val-

ues of the two (2S)-conﬁgurated stereoisomers of series 8b

were in the same range as the IC₅₀value of (2S,4R)-8a [IC₅₀

of (2S,4R)-8b = 5.14 µM, IC₅₀of (2S,4S)-8b = 6.56 µM].

Interestingly, according to these data, the stereochemistry at

the 2-position plays a key role for the potency with (2S)-

conﬁgurated derivatives being of high potency at GAT-1 (see

(2S,4S)-8a, (2S,4R)-8a, (2S,4S)-8b, (2S,4R)-8b and also

(2S,4R)-7a) as compared to the (2R)-conﬁgurated counter-

parts. This observation is in line with the results found for the

corresponding compounds lacking a 4-hydroxy group [12].

Only (2R,4R)-7a represents an exemption not following this

general trend. The inhibitory potency of the stereoisomers of

8c at GAT-1 with IC₅₀values greater than 100 µM was very

low if not negligible at all. Actually, it is a common ﬁnding,

that N-residues like R = a or R = b are more beneﬁcial for a

high activity at GAT-1 but not at GAT-3, whereas for R = c

the opposite is observed. This is especially well illustrated by

the parent compound 9, which are devoid of any signiﬁcant

activity at GAT-1 or GAT-3. For some stereoisomers of series

8 provided with the residues R = a or R = b reasonable potency

at GAT-1 are seen. Analogously, the presence of R = c in

series 8c leads to a distinct increase in potency at GAT-3 as

compared to the parent compound 9. It is interesting to note,

that the lower homologues 10 do not gain potency at GAT-

3 upon N-substitution with R = c with all compounds 7c being

devoid of a reasonable activity (IC₅₀> 100 µM). Within the

series of compounds 8c, stereoisomer (2R,4S)-8c appears to

be the most potent substance (IC₅₀19.9 µM). However, as

referred to the corresponding 4-unsubstituted compound

Fig. 9. The synthetic route leading to 9.

tal cortex (bfcP2B) and brain stem (bbsP2C), which had been

modiﬁed to allow a higher throughput [20]. Due to the emer-

gence of bovine spongiform encephalitis in Germany, calf

brain (cfcP2B, cbsP2C) was temporarily used instead of the

bovine brain material for the biological tests and ﬁnally por-

cine brain material (pfcP2B, pbsP2C) was applied. But spe-

cies differences seemed to be negligible, as no signiﬁcant dif-

ferences could be detected for a set of standard GABA-

uptake inhibitors with respect to their potency evaluated when

membrane fractions from different species were used.

4. Results and discussion

The stereoisomers of pyrrolidine-2-carboxylic acid deriva-

tives 7a and 7c showed a signiﬁcant decrease of inhibitory

potency at both GAT-1 and GAT-3 (Tables 1 and 2 ) as com-

pared with the corresponding compounds lacking a hydroxy

group at the 4-position of the pyrrolidine moiety. The latter

displayed IC₅₀values up to 2.56 µM for GAT-1 when R = a

and 18.5 µM for GAT-3 when R = c was present [12 b]. In

case of compounds 7, only (2R,4R)-7a showed a reasonable

potency at GAT-1. However, it was still approximately three

times less potent at GAT-1 (IC₅₀= 9.4 µM) than the corre-

sponding 4-unsubstituted compounds [R = a, (S)-con-

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

235

Table 1

The uptake inhibition [IC₅₀(µM)] at GAT-1 or GAT-3 exhibited by 4-hydroxyproline-2-carboxylic acid derivatives^{I, II, III}

IC₅₀S.E.M. (µM)

GAT-1 GAT-3

4328 708^a

GAT-1

GAT-3

–

GAT-1

GAT-3

>10 mM^b

GAT-1

GAT-3

–

R = H

R =

–

>10 mM^a

–

1340 290^b

(2R,4R)-10¹⁴

(2S,4R)-10¹³

>100^a

>100^b

9.4 0.4^a

313 39^b

>100^a

>100^b

79.6^a

>100^b

(2S,4S)-7a

(2S,4S)-7c

(2R,4R)-7a

(2R,4S)-7a

(2S,4R)-7a

>100^a

>100^b

>100^a

>100^b

(2R,4R)-7c

>100^a

>100^b

(2R,4S)-7c

>100^a

>100^b

(2S,4R)-7c

R =

^IThe abbreviations a–b specify the used biological material: ^abfcP2B, ^bbbsP2C.

^IIAll results were processed and evaluated in triplicate.

^IIIEach IC₅₀was given as mean S.E.M. Whenever preliminary experiments indicated that the IC₅₀value is larger than 100 µM, no exact value was esta-

blished.

Table 2

The uptake inhibition [IC₅₀(µM)] at GAT-1 or GAT-3 exhibited by 4-hydroxypyrrolidine-2-acetic acid derivatives^{I, II, III}

IC₅₀S.E.M. (µM)

GAT-1

>100^c

GAT-3

>100^d

GAT-1

>100^c

GAT-3

>100^d

GAT-1

>1.0 mM^a

(2R,4S)-9

GAT-3

GAT-1

>1.0 mM^c

(2S,4R)-9

3.29 0.54^a

GAT-3

R = H

R =

>1.0 mM^d

(2S,4S)-9

4.92 0.42^a

(2R,4R)-9

40.7 8^a

>100^b

28.9 11.6^a

6.56 0.7^a

>100^f

>100^b

(2S,4S)-8a

5.14 2.8^c

(2R,4R)-8a

(2R,4S)-8a

(2S,4R)-8a

3.15 0.30^a

(2S,4R)-8b

>100^b

96.6^an = 1

>100^b

R =

(2S,4S)-8b

(2R,4R)-8b

(2R,4S)-8b

>100^c

>100^a

58.6 17.1^d

>100^a

126 7.0^b

>100^c

>100^a

19.9 1.1^d

>100^a

(2S,4S)-8c

(2S,4S)-8d

(2R,4R)-8c

(2R,4S)-8c

(2S,4R)-8c

>100^f

>100^b

(2R,4R)-8d

>100^b

>100^e

>100^b

(2S,4R)-8d

R =

(2R,4S)-8d

^IThe abbreviations a–f specify the used biological material: ^abfcP2B, ^bbbsP2C, ^cpfcP2B, ^dpbsP2C, ^ecfcP2B, ^fcbsP2C.

^IIAll results were processed and evaluated in triplicate.

^IIIEach IC₅₀was given as mean S.E.M. Whenever preliminary experiments indicated that the IC₅₀value is larger than 100 µM, no exact value was esta-

blished.

(GAT-1: IC₅₀= 67.8 µM; GAT-3: IC₅₀= 3.0 µM [12]), the

introduction of the 4-hydroxy substituent leading to

(2R,4S)-8c caused a severe decrease in inhibitory potency at

both GAT-1 (IC₅₀= 100 µM) and GAT-3 (IC₅₀= 19.9 µM).

The amino acids series 8d bearing a carbazole moiety on the

pyrrolidine nitrogen atom showed no inhibition at GAT-1 as

well as GAT-3 when tested at a concentration of 100 µM.

Thus far, residue d has been reported only for GAT inhibi-

236

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

tions based on a 4-hydroxypiperidine skeleton. According to

our results, the carbazole moiety d seems not to be beneﬁcial

in compounds having skeletons derived from amino acids.

tive (1.0 equiv. hydrochloride) and K₂CO₃(5.0 equiv.) in

acetone (4–8 ml mmol^–1). The reaction mixture was stirred at

r.t. for 72–144 h. Inorganic salts were removed by ﬁltration

and the ﬁltrate was evaporated in vacuo. The residue was puri-

ﬁed by CC.

5. Conclusion

6.1.3. General procedure 2 (GP2)

The biological results presented herein indicate that, in gen-

eral, a 4-hydroxy group at C-4 position of the pyrrolidine ring

of proline and pyrrolidine-2-acetic acid derivatives decreases

the potency of GABA-uptake inhibitors at both GAT-1 and

GAT-3. Furthermore, it was found that, as referred to the

pyrrolidine-2-acetic acids derivatives, the (2S)-conﬁguration

for potent inhibition at GAT-1 or (2R)-conﬁguration for potent

inhibition at GAT-3 may be crucial.

The respective ester (1 equiv.) was hydrolyzed in EtOH or

MeOH (10–50 ml mmol^–1) at r.t. with aq. 0.85 M KOH or

1.0 M NaOH (3, 6 equiv. for derivatives having two ester

groups) and for the time given. Following neutralization with

aq. 1.0 M HCl to pH 7, a buffer (pH 5.5–6.6) was added and

the mixture was evaporated. The residue was puriﬁed by CC

and subsequent recrystallization.

6.1.4. General procedure 3 (GP3)

The respective ester (1.00 equiv.) was hydrolyzed with

1.0 M NaOH (2.05–4.10 equiv.) in MeOH (10–50 ml mmol^–1)

at r.t. for the time given. After MeOH had been removed in

vacuo, the residue was dissolved in a small amount of water

and acidiﬁed to pH 1–2 with aq. 1.0 M HCl. The mixture was

extracted with CH₂Cl₂and the combined extracts were dried

(Na₂SO₄) and evaporated to yield the respective carboxylic

acid.

6. Experimental section

6.1. Chemistry

6.1.1. General

Tetrahydrofuran and diisopropylamine were distilled from

sodium under nitrogen. Triethylamine was reﬂuxed with ben-

zoyl chloride and then distilled under nitrogen. Other com-

mon solvents for recrystallization, column chromatography,

analytical HPLC and preparative HPLC were distilled before

use. Purchased chemical reagents were used without further

puriﬁcation. Aqueous buffer (pH 5.5, 0.42 M and pH 6.6,

0.42 M) was prepared from KH₂PO₄and KOH. TLC

plates were made from silica gel 60 F₂₅₄on aluminum

sheets (Merck). Compounds were stained with 5%

(NH₄)₆Mo₇O₂₄·4H₂O, 0.2% Ce(SO₄)₂·4H₂O and 5% conc.

H₂SO₄. If nothing else is stated, Merck silica gel (mesh 230–

400) was used as stationary phase for ﬂash chromatography

(CC). Analytical HPLC: Column LiChrospher Si 60 (5 µm,

250 × 4 mm with precolumn 4 × 4 mm). Preparative HPLC:

Column LiChrospher Si 60 (7 µm, 250 × 25 mm). Optical

rotations: Polarimeter 241 MC at k 589 cm^–1. Melting points:

m.p. (uncorrected) were determined with a Büchi 510 Melt-

ing Point apparatus. Elementary analysis: Elementaranalysa-

tor Rapid (Heraeus). IR spectroscopy: FT-IR Spectrometer

1600 and Paragon 1000 (Perkin Elmer), oils were measured

as ﬁlm, solid samples as KBr-pellets for measurements. Mass

spectrometry: Mass Spectrometer 5989A with 59,980 B par-

ticle beam LC/MS interface (Hewlett Packard), method:

6.1.5. General procedure 4 (GP4)

DEAD (1.2–1.5 equiv.) was added at 0 °C to a solution of

the N-substituted or N-protected 4-hydroxypyrrolidine deriva-

tive (1.0 equiv.) and Ph₃P (1.0–1.5 equiv.) in THF (10–

50 ml mmol^–1). The resulting yellow solution was stirred for

10 min followed by the addition of AcOH or HCOOH (1.05–

1.50 equiv.) at 0 °C (for intramolecular Mitsunobu reaction

no additional acid was used). Stirring was continued at 0 °C

for the time given. The solvent was evaporated in vacuo and

the residual oil was puriﬁed by CC. The puriﬁcation of some

compounds required prep. HPLC.

6.1.6. General procedure 5 (GP5)

To a solution of the respective N-protected pyrrolidine

(1 equiv.) in MeOH or EtOAc (40–50 ml mmol^–1) 10% Pd-C

(0.5–1.0 w/w) was added. This mixture was hydrogenated at

r.t. and 1 bar for the time given. The reaction mixture was

ﬁltrated and evaporated to give the N-deprotected amine.

6.1.7. (2S,4R)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-carboxylic acid [(2S,4R)-7a]

+

chemical ionization (CH₅) unless otherwise stated. NMR

According to GP2 from 185 mg (0.527 mmol) of (2S,4R)-

22a, 1.85 ml (1.58 mmol) of aq. 0.85 M KOH in 7 ml of

EtOH; reaction time: 1.5 h. Yield: 170 mg (96%); colorless

crystals, m.p. 162–165 °C (acetone). [␣]_D²²= −49.2 (c = 0.85,

spectroscopy: NMR spectra were recorded on JNMR-GX

(JEOL, 400 and 500 MHz) with TMS as internal standard

and integrated with the program of NMR-software Nuts (2D

Version 5.097, Acorn NMR, 1995). If nothing else is stated,

measurements were performed at 400 MHz at room tempera-

ture.

1

EtOH). H NMR (CD₃OD): d = 2.09 (ddd, J = 13.7, 10.6,

4.4 Hz, 1H, NCHCH₂), 2.39 (ddt, J = 13.7, 7.6, 1.9 Hz, 1H,

NCHCH₂), 2.55 (q, J = 7.6 Hz, 2H, NCH₂CH₂), 3.00 (dt,

J = 12.3, 1.9 Hz, 1H, NCH₂CHOH), 3.35 (dt, J = 12.4, 7.6 Hz,

1H, NCH₂CH₂), 3.46 (dt, J = 12.4, 7.6 Hz, 1H, NCH₂CH₂),

3.64 (dd, J = 12.3, 4.4 Hz, 1H, NCH₂CHOH), 4.05 (dd,

6.1.2. General procedure 1 (GP1)

The respective alkyl halide RX (1.0 equiv.) and KI

(0.2 equiv.) were added to a mixture of the pyrrolidine deriva-

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

237

J = 10.6, 7.6 Hz, 1H, NCH), 4.43–4.45 (m, 1H, CHOH), 6.09

(t, J = 7.6 Hz, 1H, = CHCH₂,) 7.17–7.43 (m, 10H, H_aromat).

IR:

v = 3424 cm^–1, 1628. MS; m/z (%): 338 (100) [M + 1]⁺.

6.1.12. (2R,4S)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-carboxylic acid [(2R,4S)-

7c]

˜

According to GP2 from 80 mg (0.14 mmol) of (2R,4S)-

24c, 0.85 ml (0.85 mmol) of aq. 1.0 M NaOH in 3.5 ml of

MeOH; reaction time: 3 d.Yield: 68 mg (94%); colorless crys-

tals, m.p. 135–137 °C (C₆H₆/iPr₂O). [␣]_D²⁰= +20.6 (c = 0.90,

EtOH). The spectra (¹H NMR, MS and IR) were identical

with those of (2S,4R)-7c. C₂₉H₃₃NO₇·1.5 H₂O (534.61).

C₂₁H₂₃NO₃(337.42).

6.1.8. (2R,4S)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-carboxylic acid [(2R,4S)-7a]

According to GP2 from 85 mg (0.21 mmol) of (2R,4S)-

24a, 1.26 ml (1.26 mmol) of aq. 1.0 M NaOH in 6 ml of

EtOH; reaction time: 3 d.Yield: 65 mg (92%); colorless crys-

tals, m.p. 181–184 °C (EtOH/iPr₂O). [␣]_D²⁰= +50.6 (c = 0.93,

EtOH). The spectra (¹H NMR, IR, and MS) were identical

with those of (2S,4R)-7a. C₂₁H₂₃NO₃·H₂O (355.44).

6.1.13. (2R,4R)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-carboxylic acid [(2R,4R)-

7c]

According to GP2 from 66 mg (0.13 mmol) of (2R,4R)-

22c, 0.38 ml (0.38 mmol) of aq. 1.0 M NaOH in 2.5 ml of

EtOH; reaction time: 2 h; puriﬁcation by CC (Al₂O₃, pH 7

0.5; EtOH/H₂O, 4:1).Yield: 43 mg (67%); colorless crystals,

6.1.9. (2R,4R)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-carboxylic acid [(2R,4R)-7a]

20

m.p. 126–130 °C (petrol ether). [␣]_D= +12.1 (c = 0.7,

According to GP2 from 68 mg (0.19 mmol) of (2R,4R)-

22a, 0.58 ml (0.58 mmol) of aq. 1.0 M NaOH in 3 ml of

EtOH; reaction time: 3 h.Yield: 58 mg (92%); colorless crys-

tals, m.p. 112–116 °C (C₆H₆/iPr₂O). [␣]_D²⁰= +43.1 (c = 0.90,

EtOH). ¹H NMR (CD₃OD, 500 MHz): d = 2.15–2.21 (m, 1H,

NCHCH₂), 2.48–2.63 (m, 3H, NCHCH₂and NCH₂CH₂), 3.03

(dd, J = 11.5, 3.8 Hz, 1H, NCH₂CHOH), 3.18–3.24 (m, 1H,

NCH₂CH₂), 3.32–3.41 (m, 2H, NCH₂CH₂and NCH₂CHOH),

3.79 (dd, J = 10.6, 4.4 Hz, 1H, NCH), 4.36–4.39 (m, 1H,

CHOH), 6.07 (dd, J = 7.9, 6.9 Hz, 1H, =CHCH₂), 7.17–7.44

EtOH). ¹H NMR (CD₃OD): d = 2.23–2.29 (m, 1H, NCHCH₂),

2.54 (ddd, J = 13.9, 11.0, 4.4 Hz, 1H, NCHCH₂), 3.06 (dd,

J = 11.6, 3.6 Hz, 1H, NCH₂CHOH), 3.23–3.37 (m, 3H,

NCH₂CHOH and NCH₂CH₂), 3.51–3.61 (m, 2H, NCH₂CH₂),

3.77 (s, 9H, OCH₃), 3.98 (dd, J = 11.0, 4.0 Hz, 1H, NCH),

4.38–4.41 (m, 1H, CHOH), 6.86–6.90 (m, 6H, H_aromat), 7.34–

7.38 (m, 6H, H_aromat). IR:

C₂₉H₃₃NO₇·H₂O (525.60).

˜

v = 3414 cm^–1, 1638.

6.1.14. (2S,4S)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-carboxylic acid [(2S,4S)-

7c]

(m, 10H, H_aromat). IR: ˜

v = 3274 cm^–1, 1633. MS; m/z (%):

338 (36) [M + 1]⁺. C₂₁H₂₃NO₃·1.2 H₂O (362.88).

According to GP4 from 151 mg (0.298 mmol) of

(2S,4R)-7c and 161 mg (0.596 mmol) of PPh₃(67 mg,

0.39 mmol) of DEAD in 6.5 ml of THF; reaction time: 20 h.

Puriﬁcation by CC (Al₂O₃, pH 7.5 0.5, gradient elution,

n-heptane/acetone = 2:1 → EtOH/H₂O = 4:1) directly yielded

(2S,4S)-7c.Yield: 115 mg (76%); colorless crystals, m.p. 147–

149 °C (EtOH/H₂O). [␣]_D²⁴= –11.9 (c = 0.75, EtOH). The

spectra (¹H NMR, and IR) were identical with those of

(2R,4R)-7c. C₂₉H₃₃NO₇(507.58).

6.1.10. (2S,4S)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-carboxylic acid [(2S,4S)-7a]

According to GP2 from 157 mg (0.491 mmol) of (1S,4S)-

23a, 2.1 ml (1.800 mmol) of aq. 0.85 M KOH in 12 ml of

EtOH; reaction time: 40 min.Yield: 158 mg (95%); colorless

crystals, m.p. 138–140 °C (acetone). [␣]_D²⁴= –42.8 (c = 1.00,

EtOH). The spectra (¹H NMR, IR, and MS) were identical

with those of (2R,4R)-7a. C₂₁H₂₃NO₃(337.42).

6.1.11. (2S,4R)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-carboxylic acid [(2S,4R)-

7c]

6.1.15. (2R,4R)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-acetic acid [(2R,4R)-8a]

According to GP2 from 53 mg (0.14 mmol) of (2R,4R)-

30a, 0.42 ml (0.42 mmol) of aq. 1.0 M NaOH in 2 ml of

MeOH; reaction time: 41 h. Yield: 41 mg (84%); colorless

According to GP2 from 299 mg (0.574 mmol) of (2S,4R)-

22c, 1.7 ml (1.500 mmol) of aq. 0.85 M KOH in 23 ml of

EtOH; reaction time: 4 h.Yield: 243 mg (85%); colorless crys-

20

crystals, m.p. 70–76 °C (MeOH/iPr₂O). [␣]_D= +52.2

20

1

tals, m.p. 142–144 °C (acetone). [␣]_D= –24.8 (c = 1.12,

(c = 0.50, MeOH). H NMR (CD₃OD): d = 1.71–1.77 (m,

1

EtOH). H NMR (CD₃OD): d = 2.15 (ddd, J = 13.8, 10.1,

1H, CH₂CHCH₂COO), 2.46–2.57 (m, 4H, CH₂COO,

CH₂CHCH₂COO and 2H of NCH₂CH₂), 2.63 (dd, J = 16.7,

6.3 Hz, 1H, CH₂COO), 2.92–3.01 (m, 2H, NCH₂CH₂and

NCH₂CHO), 3.17–3.29 (m, 1H, NCH₂CHO), 3.46–3.57 (m,

2H, NCH₂CH₂and NCH), 4.35–4.40 (m, 1H, CHOH), 6.02

(t, J = 7.3 Hz, 1H, =CHCH₂), 7.14–7.40 (m, 10H, H_aromat).

4.7 Hz, 1H, NCHCH₂), 2.41 (ddt, J = 13.8, 7.8, 1.9 Hz, 1H,

NCHCH₂), 3.08 (br. d, J = 12.4 Hz, 1H, NCH₂CHOH), 3.29–

3.35 (m, 1H, NCH₂CH₂), 3.40–3.61 (m, 4H, NCH₂CH₂,

NCH₂CHOH and NCH₂CH₂), 3.76 (s, 9H, ArOCH₃), 4.19

(dd, J = 10.1, 7.8 Hz, 1H, NCH), 4.43–4.46 (m, 1H, CHOH),

6.85–6.88 (m, 6H, H_aromat), 7.32–7.36 (m, 6H, H_aromat). IR:

IR:

C₂₂H₂₅NO₃(351.45)·1.0 H₂O.

˜

v = 3388 cm^–1, 1608. MS; m/z (%): 352 (23) [M + 1]⁺.

˜

v= 3464 cm^–1, 1607. C₂₉H₃₃NO₇(507.58).

238

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

6.1.16. (2S,4S)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-acetic acid [(2S,4S)-8a]

6.1.20. (2S,4S)-1-[4,4-Di(3-methylthien-2-yl)but-3-en-1-

yl]-4-hydroxypyrrolidine-2-acetic acid [(2S,4S)-8b]

According to GP2 from 67 mg (0.18 mmol) of (1S,5S)-

26b, 0.54 ml (0.54 mmol) of aq. 1.0 M NaOH in 4.5 ml of

MeOH; reaction time: 25 h. Yield: 59 mg (84%); colorless

According to GP2 from 60 mg (0.18 mmol) of (1S,5S)-

26a, 0.54 ml (0.54 mmol) of aq. 1.0 M NaOH in 3 ml of

MeOH; reaction time: 20 h. Yield: 49 mg (78%); colorless

20

powder, m.p. 72–78 °C (C₆H₅/iPr₂O). [␣]_D= –34.4

crystals, m.p. 95–97 °C (C₆H₆). [␣]_D= –51.5 (c = 0.47,

(c = 0.52, MeOH). The spectra (¹H NMR, IR, and MS) were

identical with those of (2R,4R)-8b. C₂₀H₂₅NO₃S₂·1.1 H₂O.

MeOH). The spectra (¹H NMR, IR, and MS) were identical

with those of (2R,4R)-8a. HRMS (CI) Calc. for C₂₂H₂₅NO₃:

352.1913. Found: 352.1895.

6.1.21. (2R,4S)-1-[4,4-Di(3-methylthien-2-yl)but-3-en-1-

yl]-4-hydroxypyrrolidine-2-acetic acid [(2R,4S)-8b]

According to GP2 from 70 mg (0.15 mmol) of (2R,4S)-

31b, 0.91 ml (0.91 mmol) of aq. 1.0 M NaOH in 4 ml of

MeOH; reaction time: 3 d. Yield: 56 mg (94%); pale yellow

6.1.17. (2R,4S)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-acetic acid [(2R,4S)-8a]

According to GP2 from 36 mg (0.085 mmol) of (2R,4S)-

31a, 0.51 ml (0.510 mmol) of aq. 1.0 M NaOH in 4.5 ml of

MeOH; reaction time: 3 d.Yield: 28 mg (93%); colorless crys-

20

crystals, m.p. 58–62 °C (C₆H₆/iPr₂O). [␣]_D= +57.5

1

(c = 0.52, MeOH). H NMR (CD₃OD): d = 1.87–1.94 (m,

20

tals, m.p. 57–60 °C (C₆H₆/iPr₂O). [␣]_D= +65.6 (c = 0.5,

1H, CH₂CHCH₂COO), 1.99 (s, 3H, thienyl-CH₃), 2.04 (s,

3H, thienyl-CH₃), 2.13 (dd, J = 13.6, 6.5 Hz, 1H,

CH₂CHCH₂COO), 2.49 (dd, J = 16.6, 3.2 Hz, 1H, CH₂COO),

2.50–2.61 (m, 2H, NCH₂CH₂), 2.63 (dd, J = 16.6, 6.0 Hz,

1H, CH₂COO), 2.85 (br. d, J = 12.1 Hz, 1H, NCH₂CHO),

2.99–3.05 (m, 1H, NCH₂CH₂), 3.42 (dt, J = 12.4, 8.2 Hz, 1H,

NCH₂CH₂), 3.60 (dd, J = 12.1, 4.9 Hz, 1H, NCH₂CHO),

3.68–3.76 (m, 1H, NCH), 4.40–4.43 (m, 1H, CHOH), 6.05

(t, J = 7.4 Hz, 1H, =CHCH₂), 6.77 (d, J = 5.2 Hz, 1H,

SC=CH), 6.91 (d, J = 5.2 Hz, 1H, SC=CH), 7.15 (d,

J = 5.2 Hz, 1H, SCH=), 7.36 (d, J = 5.2 Hz, 1H, SCH=). IR:

MeOH). ¹H NMR (CD₃OD): d = 1.93 (ddd, J = 13.6, 11.4,

5.1 Hz, 1H, CH₂CHCH₂COO), 2.13 (ddt, J = 13.6, 6.5, 1.7 Hz,

1H, CH₂CHCH₂COO), 2.46 (dd, J = 16.5, 3.0 Hz, 1H,

CH₂COO), 2.56 (q, J = 7.4 Hz, 2H, NCH₂CH₂), 2.70 (dd,

J = 16.5, 5.9 Hz, 1H, CH₂COO), 2.82 (d, J = 12.5 Hz, 1H,

NCH₂CHO), 3.03–3.10 (m, 1H, NCH₂CH₂), 3.46–3.53 (m,

2H, NCH₂CH₂and NCH₂CHO), 3.74–3.80 (m, 1H, NCH),

4.38–4.41 (m, 1H, CHOH), 6.09 (t, J = 7.4 Hz, 1H, =CHCH₂),

7.21–7.46 (m, 10H, H_aromat). IR: ˜

v = 3295 cm^–1, 1624. MS;

m/z (%): 352 (19) [M + 1]⁺. C₂₂H₂₅NO₃(351.45)·1.3 H₂O.

˜

v = 3380 cm^–1, 1588. MS; m/z (%): 392 (45) [M + 1]⁺.

C₂₂H₂₅NO₃S₂(391.54)·1.0 H₂O.

6.1.18. (2S,4R)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-

pyrrolidine-2-acetic acid [(2S,4R)-8a]

6.1.22. (2S,4R)-1-[4,4-Di(3-methylthien-2-yl)but-3-en-1-

yl]-4-hydroxypyrrolidine-2-acetic acid [(2S,4R)-8b]

According to GP2 from 90 mg (0.22 mmol) of (2S,4R)-

25b, 0.67 ml (0.67 mmol) of aq. 1.0 M NaOH in 3.5 ml of

MeOH; reaction time: 3 h.Yield: 80 mg (92%); yellow foam

According to GP2 from 50 mg (0.14 mmol) of (2S,4R)-

25a, 0.42 ml (0.42 mmol) of aq. 1.0 M NaOH in 3 ml of

EtOH; reaction time: 3 h. Yield: 40 mg (83%); colorless oil.

[␣]_D²⁰= –69.4 (c = 0.95, MeOH). The spectra (¹H NMR, IR,

and MS) were identical with those of (2R,4S)-8a. C₂₂H₂₅NO₃

(351.45).

20

(MeOH), m.p. 74–80 °C (C₆H₆/iPr₂O). [␣]_D= –58.3

(c = 1.00, MeOH). The spectra (¹H NMR, IR, and MS) were

identical with those of (2R,4S)-8b. C₂₀H₂₅NO₃S₂·1.0 H₂O

(391.54).

6.1.19. (2R,4R)-1-[4,4-Di(3-methylthien-2-yl)but-3-en-1-

yl]-4-hydroxypyrrolidine-2-acetic acid [(2R,4R)-8b]

According to GP2 from 47 mg (0.12 mmol) of (2R,4R)-

30b, 0.56 ml (0.56 mmol) of aq. 1.0 M NaOH in 2 ml of

MeOH; reaction time: 2 d. Yield: 43 mg (95%); slightly yel-

low powder, m.p. 69–74 °C (C₆H₆/petrol ether).

6.1.23. (2R,4R)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-acetic acid [(2R,4R)-8c]

According to GP2 from 54 mg (0.098 mmol) of (2R,4R)-

30c, 0.30 ml (0.300 mmol) of aq. 1.0 M NaOH in 2 ml of

MeOH; reaction time: 2 d; puriﬁcation by CC (Al₂O₃).Yield:

40 mg (78%); colorless crystals, m.p. 85–88 °C (C₆H₆/iPr₂O).

20

1

[␣]_D= +34.8 (c = 0.46, MeOH). H NMR (CD₃OD):

d = 1.72–1.78 (m, 1H, CH₂CHCH₂COO), 1.97 (s, 3 H,

thienyl-CH₃), 2.03 (s, 3 H, thienyl-CH₃), 2.46–2.60 (m, 5 H,

CH₂CHCH₂COO, CH₂COO and NCH₂CH₂), 2.95–3.04 (m,

1H, NCH₂CH₂), 3.08 (dd, J = 11.9, 5.5 Hz, 1H, NCH₂CHO),

3.30–3.60 (m, 3H, NCH₂CHO, NCH₂CH₂and NCH), 4.38–

4.44 (m, 1H, NCH₂CHO), 6.05 (t, J = 7.3 Hz, 1H, =CHCH₂),

6.78 (d, J = 5.2 Hz, 1 H, SC=CH), 6.91 (d, J = 5.2 Hz, 1H,

SC=CH), 7.16 (d, J = 5.2 Hz, 1H, SCH=), 7.35 (d, J = 5.2 Hz,

20

1

[␣]_D= +17.9 (c = 0.90, MeOH). H NMR (CD₃OD): d

= 1.83 (ddd, J = 13.4, 9.0, 4.3 Hz, 1H, CH₂CHCH₂COO),

2.50–2.62 (m, 2H, CH₂COO and CH₂CHCH₂COO), 2.74 (dd,

J = 16.7, 6.2 Hz, 1H, CH₂COO), 2.98–3.04 (m, 1H,

NCH₂CH₂), 3.14 (dd, J = 11.9, 5.7 Hz, 1H, NCH₂CHO),

3.28–3.34 (m, 1H, NCH₂CHO), 3.39–3.60 (m, 4H,

NCH₂CH₂, NCH and NCH₂CH₂), 3.77 (s, 9H, ArOCH₃),

4.41–4.46 (m, 1H, CHOH), 6.85–6.89 (m, 6H, H_aromat), 7.32–

1H, SCH=). IR: ˜

v= 3396 cm^–1, 1591, 1399, 1097, 713. MS;

m/z (%): 392 (12) [M + 1]⁺, 374 140 (100). C₂₀H₂₅NO₃S₂

7.36 (m, 6H, H_aromat). IR:

˜

v = 3420 cm^–1, 1607. MS (ESI⁺);

(391.54)·0.8 H₂O.

m/z (%): 522 [M + 1]⁺(6). C₃₀H₃₅NO₇(521.61)·1.0 H₂O.

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

239

6.1.24. (2S,4S)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

6.1.28. (2S,4S)-1-[3-(Carbazol-9-yl)-1-propyl]-4-hydroxy-

methoxy]-1-ethyl}pyrrolidine-2-acetic acid [(2S,4S)-8c]

According to GP2 from 112 mg (0.227 mmol) of (1S,5S)-

26c, 0.67 ml (0.670 mmol) of aq. 1.0 M NaOH in 7.3 ml of

MeOH; reaction time: 2 d. Yield: 102 mg (96%); colorless

pyrrolidine-2-acetic acid [(2S,4S)-8d]

According to GP2 from 54 mg (0.16 mmol) of (1S,5S)-

26d, 0.49 ml (0.49 mmol) of aq. 1 M NaOH in 4.5 ml of

MeOH; reaction time: 12 h. Yield: 54 mg (95%); colorless

20

crystals, m.p. 102–108 °C (MeOH/EtOAc). [␣]_D= –31.0

crystals, m.p. 95–103 °C (C₆H₆/iPr₂O). [␣]_D= –16.8

(c = 0.51, MeOH). The spectra (¹H NMR, IR, and MS) were

identical with those of (2R,4R)-8d. HRMS (70 eV) Calc. for

C₂₁H₂₄N₂O₃: 352.1787. Found: 352.1764.

(c = 0.75, MeOH). The spectra (¹H NMR and IR) were iden-

tical with those of (2R,4R)-8c. HRMS (CI) Calc. for

[C₃₀H₃₅NO₇+ Na⁺]: 544.2311. Found: 544.2342.

6.1.29. (2R,4S)-1-[3-(Carbazol-9-yl)-1-propyl]-4-hydroxy-

pyrrolidine-2-acetic acid [(2R,4S)-8d]

6.1.25. (2R,4S)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-acetic acid [(2R,4S)-8c]

According to GP2 from 70 mg (0.12 mmol) of (2R,4S)-

31c, 0.71 ml (0.71 mmol) of aq. 1.0 m NaOH in 6.3 ml of

MeOH; reaction time: 48 h. Yield: 52 mg (85%); colorless

According to GP2 from 94 mg (0.22 mmol) of (2R,4S)-

31d, 1.3 ml (1.30 mmol) of aq. 1.0 M NaOH in 6.5 ml of

MeOH; reaction time: 3 d.Yield: 73 mg (93%); colorless crys-

20

tals, m.p. 193–197 °C (MeOH/iPr₂O). [␣]_D= +44.4

20

(c = 0.80, MeOH). ¹H NMR (CD₃OD, 500 MHz): d = 1.93

(ddd, J = 13.7, 11.3, 4.7 Hz, 1H, CH₂CHCH₂COO), 2.13 (ddt,

J = 13.7, 6.4, 1.7 Hz, 1H, CH₂CHCH₂COO), 2.21–2.37 (m,

2H, CHNCH₂CH₂), 2.45 (dd, J = 16.5, 3.0 Hz, 1H, CH₂COO),

2.65 (dd, J = 16.5, 6.0 Hz, 1H, CH₂COO), 2.93 (br. d,

J = 12.4 Hz, 1H, NCH₂CHO), 2.96–3.03 (m, 1H, CH₂NCH),

3.36–3.42 (m, 1H, CH₂NCH), 3.64–3.74 (m, 2H, NCH₂CHO

and NCH), 4.39–4.43 (m, 1H, CHOH), 4.45–4.55 (m, 2H,

CH₂-carbazole), 7.21–7.26 (m, 2H, H_aromat), 7.49–7.55 (m,

2H, H_aromat), 7.62–7.67 (m, 2H, H_aromat), 8.08–8.10 (m, 2H,

crystals, m.p. 98–105 °C (C₆H₆/iPr₂O). [␣]_D= +31.5

(c = 0.60, MeOH). ¹H NMR (CD₃OD): d = 1.93 (ddd, J = 13.5,

11.5, 4.6 Hz, 1H, CH₂CHCH₂COO), 2.12 (ddt, J = 13.5, 6.5,

1.6 Hz, 1H, CH₂CHCH₂COO), 2.39 (dd, J = 16.9, 2.4 Hz,

1H, CH₂COO), 2.72 (dd, J = 16.9, 5.3 Hz, 1H, CH₂COO),

3.00 (dt, J = 12.6, 1.6 Hz, 1H, NCH₂CHO), 3.10–3.16 (m,

1H, NCH₂CH₂), 3.38–3.53 (m, 4H, NCH₂CHO, NCH₂CH₂

and NCH₂CH₂), 3.75 (s, 9H, ArOCH₃), 3.79–3.86 (m, 1H,

NCH), 4.39–4.43 (m, 1H, CHOH), 6.83–6.87 (m, 6H, H_aro

^mat), 7.29–7.33 (m, 6H, H_aromat). IR:

C₃₀H₃₅NO₇(521.61)·0.8 H₂O.

-

˜

v = 3398 cm^–1, 1607.

H

_aromat). IR:˜

v= 3405 cm^–1, 1594. MS; m/z (%): 353 (3) [M +

1]⁺. C₂₁H₂₄N₂O₃·1.0 H₂O (370.46).

6.1.26. (2S,4R)-4-Hydroxy-1-{2-[tris(4-methoxyphenyl)-

methoxy]-1-ethyl}pyrrolidine-2-acetic acid [(2S,4R)-8c]

6.1.30. (2S,4R)-1-[3-(Carbazol-9-yl)-1-propyl]-4-hydroxy-

pyrrolidine-2-acetic acid [(2S,4R)-8d]

According to GP2 from 149 mg (0.279 mmol) of (2S,4R)-

25c, 0.85 ml (0.850 mmol) of aq. 1.0 M NaOH in 5 ml of

MeOH; reaction time: 4 h; puriﬁcation by CC (Al₂O₃).Yield:

120 mg (83%); colorless crystals, m.p. 95–103 °C

According to GP2 from 105 mg (0.286 mmol) of (2S,4R)-

25d, 0.86 ml (0.860 mmol) of aq. 1.0 M NaOH in 4 ml of

MeOH; reaction time: 6 h.Yield: 89 mg (88%); colorless crys-

20

tals, m.p. 207–209 °C (MeOH/iPr₂O). [␣]_D= –43.5

20

(c = 0.93, MeOH). The spectra (¹H NMR, IR, and MS) were

identical with those of (2R,4S)-8d. C₂₁H₂₄N₂O₃(352.44).

(C₆H₆/petrol ether). [␣]_D= –33.2 (c = 1.00, MeOH). The

spectra (¹H NMR and IR) were identical with those of

(2R,4S)-8c. C₃₀H₃₅NO₇(521.61)·0.5 H₂O.

6.1.31. (2S,4R)-4-Hydroxypyrrolidine-2-acetic acid

[(2S,4R)-9]

6.1.27. (2R,4R)-1-[3-(Carbazol-9-yl)-1-propyl]-4-hydroxy-

pyrrolidine-2-acetic acid [(2R,4R)-8d]

According to GP5 from 76 mg (0.270 mmol) of (2S,4R)-28

and 38 mg (0.036 mmol) of 10% Pd-C in 10 ml of MeOH;

reaction time: 1 h. Yield: 35 mg (89%); colorless crystals,

m.p. 238–240 °C (decomp.). [␣]_D²⁰= +1.3 (c = 0.31, H₂O).

¹H NMR (D₂O): d = 1.83 (ddd, J = 14.1, 11.4, 4.4 Hz, 1H,

CH₂CHCH₂COO), 2.14–2.20 (m, 1H, CH₂CHCH₂COO),

2.55 (dd, J = 16.6, 7.7 Hz, 1H, CH₂COO), 2.64 (dd, J = 16.6,

5.5 Hz, 1H, CH₂COO), 3.22 (dt, J = 12.8, 1.5 Hz, 1H, NCH₂),

3.43 (dd, J = 12.8, 4.2 Hz, 1H, NCH₂), 4.02–4.08 (m, 1H,

According to GP2 from 88 mg (0.23 mmol) of (2R,4R)-

30d, 0.70 ml (0.70 mmol) of aq. 1.0 M NaOH in 3 ml of

MeOH; reaction time: 14 h. Yield: 73 mg (90%); colorless

20

crystals, m.p. 93–97 °C (MeOH/EtOAc). [␣]_D= +31.2

1

(c = 0.26, MeOH). H NMR (CD₃OD): d = 1.71–1.78 (m,

1H, CH₂CHCH₂COO), 2.18–2.32 (m, 2H, CHNCH₂CH₂),

2.43–2.51 (m, 1H, CH₂CHCH₂COO), 2.58–2.62 (m, 2H,

CH₂COO), 2.90–3.02 (m, 2H, CH₂NCH and NCH₂CHO),

3.41–3.55 (m, 3H, NCH, CH₂NCH and NCH₂CHO), 4.34–

4.51 (m, 3H, CHOH and 2H of CH₂-carbazole), 7.17–7.21

(m, 2H, H_aromat), 7.46–7.53 (m, 4H, H_aromat), 8.05–8.08 (m,

NCH), 4.56–4.59 (m, 1H, CHOH). IR:˜

v= 3231 cm^–1, 1643.

MS; m/z (%): 146 (100) [M + 1]⁺. C₆H₁₁NO₃(145.16).

6.1.32. (2R,4S)-4-Hydroxypyrrolidine-2-acetic acid

[(2R,4S)-9]

According to GP5 from 50 mg (0.180 mmol) of (2R,4S)-28

and 25 mg (0.024 mmol) of 10% Pd-C in 8 ml of MeOH;

2H, H_aromat). IR:˜

v= 3385 cm^–1, 1595. MS; m/z (%): 353 (2)

[M + 1]⁺. HRMS (CI) Calc. for C₂₁H₂₄N₂O₃: 353.1865.

Found: 353.1875.

240

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

reaction time 1 h.Yield: 24 mg (92%); colorless crystals, m.p.

235–237 °C (MeOH, decomp.). [␣]_D²⁰= –2.6 (c = 0.23, H₂O).

The spectra (¹H NMR, IR, and MS) were identical with those

of (2S,4R)-9. C₆H₁₁NO₃(145.16). HRMS (70 eV) Calc. for

C₆H₁₁NO₃: 145.0738. Found: 145.0771.

(c = 1.00, EtOH). ¹H NMR (CD₃OD): d = 1.29 (t, J = 7.4 Hz,

3H, CH₂CH₃), 1.79 (ddt, J = 14.1, 4.7, 1.9 Hz, 1H,

CH₂CHCH₂COO), 2.43 (ddd, J = 14.1, 9.8, 5.0 Hz, 1H,

CH₂CHCH₂COO), 2.92 (dd, J = 18.0, 5.2 Hz, 1H, CH₂COO),

2.98 (dd, J = 18.0, 9.4 Hz, 1H, CH₂COO), 3.22 (dd, J = 11.9,

3.6 Hz, 1H, NCH₂), 3.27 (dt, J = 11.9, 1.9 Hz, 1H, NCH₂),

4.00–4.08 (m, 1H, NCH), 4.21 (q, J = 7.4 Hz, 2H, CH₂CH₃),

6.1.33. (2R,4R)-4-Hydroxypyrrolidine-2-acetic acid

[(2R,4R)-9]

4.50–4.53 (m, 1H, CHOH). IR: ˜

v = 3303 cm^–1, 1721. MS;

m/z (%): 174 (100) [M – HCl]⁺. C₈H₁₆ClNO₃(209.67).

According to GP5 from 59 mg (0.210 mmol) of (2R,4R)-28

and 30 mg of 10% Pd-C (0.028 mmol) in 10 ml of MeOH,

reaction time: 1 h. Yield: 31 mg (100%); colorless crystals,

m.p. 210–212 °C (MeOH, decomp.). [␣]_D²⁰= –4.8 (c = 0.33,

6.1.37. Benzyl (2S,4R)-4-benzyloxy-2-diazoacetyl-

pyrrolidine-1-carboxylate [(2S,4R)-16]

1

H₂O). H NMR (D₂O): d = 1.77 (dddd, J = 14.2, 7.3, 3.6,

2.10 g (16.5 mmol) of (COCl)₂was added to a solution of

3.90 g (11.0 mmol) of (2S,4R)-15 [15] and 50 µl of DMF in

26 ml of CH₂Cl₂at 0 °C. The mixture was stirred for 1 h at

0 °C. Following evaporation under vacuo, the obtained yel-

low oil was dissolved in 4.5 ml of Et₂O. The mixture was

slowly added at 0 °C to a solution of 2.22 g (52.8 mmol) of

CH₂N₂in 150 ml of Et₂O. Following 1 h stirring, excess of

CH₂N₂was decomposed by addition of AcOH and the sol-

vent was evaporated. Puriﬁcation by CC (iso-hexane/acetone

1.4 Hz, 1H, CH₂CHCH₂COO), 2.55 (ddd, J = 14.2, 8.8,

6.0 Hz, 1H, CH₂CHCH₂COO), 2.72–2.74 (m, 2H, CH₂COO),

3.32 (ddd, J = 12.5, 2.4, 1.4 Hz, 1H, NCH₂), 3.38 (dd, J =12.5,

4.9 Hz, 1H, NCH₂), 3.93–3.99 (m, 1H, NCH), 4.62–4.65 (m,

1H, CHOH). IR: ˜

v = 3175 cm^–1, 1662. MS; m/z (%): 146

(100) [M + 1]⁺. C₆H₁₁NO₃(145.16).

6.1.34. (2S,4S)-4-Hydroxypyrrolidine-2-acetic acid

[(2S,4S)-9]

20

= 7:3). Yield: 3.33 g (80%); yellow oil. [␣]_D= –55.5

(c = 1.00, EtOAc). ¹H NMR (CDCl₃, –30 °C): Two rotamers

d = 2.01–2.07, 2.10–2.16 (m, 1H, NCHCH₂), 2.30–2.37, 2.41–

2.48 (m, 1H, NCHCH₂), 3.55–3.59, 3.61–3.65 (m, 1H,

NCH₂), 3.69–3.73, 3.88–3.92 (m, 1H, NCH₂), 4.14–4.23 (m,

1H, NCH₂CHO), 4.37–4.53 (m, 3H, NCH and PhCH₂OCH),

5.00–5.23 (m, 2H, PhCH₂OCO), 5.26, 5.54 (s, 1H, CHN₂),

According to GP5 from 80 mg (0.290 mmol) of (2S,4S)-28

and 40 mg of 10% Pd-C (0.038 mmol) in 8 ml of MeOH;

reaction time 1 h.Yield: 41 mg (99%); colorless crystals, m.p.

238–240 °C (MeOH, decomp.). [␣]_D²⁰= +5.3 (c = 0.32, H₂O).

The spectra (¹H NMR, IR, and MS) were identical with those

of (2R,4R)-9. C₆H₁₁NO₃(145.16).

7.28–7.38 (m, 10H, H_aromat). IR:˜

v= 2107 cm^–1, 1704, 1650.

MS; (m/z): 380 [M + 1]⁺. C₂₁H₂₁N₃O₄(379.42).

6.1.35. Methyl (2S,4R)-4-hydroxypyrrolidine-2-acetate

hydrochloride [(2S,4R)-12]

6.1.38. Methyl (2S,4R)-4-benzyloxy-1-benzyloxy-

carbonylpyrrolidine-2-acetate [(2S,4R)-17]

To a solution of 325 mg (0.848 mmol) of (2S,4R)-17 and

80 µl (0.960 mmol) of aq. 37% HCl in 10 ml of MeOH,

162 mg (0.153 mmol) of 10% Pd-C was added. The mixture

was subjected to hydrogen under ambient pressure at r.t. for

5 d and then ﬁltrated. The ﬁltrate was evaporated and its resi-

due was crystallized from iPr₂O. Yield: 149 mg (90%); col-

To a solution of 390 mg (1.03 mmol) of (2S,4R)-16 in 3 ml

of MeOH, a solution of 1.5 ml of MeOH containing 17 mg

(0.10 mmol) of AgOAc and 61 mg (0.61 mmol) of TEA was

added at r.t. The resulting mixture was warmed to 60 °C for

30 min. Following decolorization using charcoal, the solvent

was evaporated under vacuo. Puriﬁcation by CC (n-

heptane/acetone, 3:1). Yield: 317 mg (81%); colorless oil.

20

orless crystals, m.p. 116–117 °C (iPr₂O). [␣]_D= +42.8

(c = 0.43, MeOH). ¹H NMR (CD₃OD): d = 1.71 (ddd, J = 13.7,

11.4, 4.0 Hz, 1H, CH₂CHCH₂COO), 2.09 (ddt, J = 13.7, 6.4,

1.5 Hz, 1H, CH₂CHCH₂COO), 2.69 (dd, J = 18.0, 9.9 Hz,

1H, CH₂COO), 2.82 (dd, J = 18.0, 4.0 Hz, 1H, CH₂COO),

3.09 (dd, J = 12.2, 1.5 Hz, 1H, NCH₂), 3.25 (dd, J = 12.2,

3.7 Hz, 1H, NCH₂), 3.62 (s, 3H, CH₃), 3.99–4.07 (m, 1H,

20

1

[␣]_D= –33.8 (c = 0.95, EtOAc). H NMR (C₆D₅NO₂,

120 °C): d = 2.07–2.13 (m, 1H, CH₂CHCH₂COO), 2.41–

2.47 (m, 1H, CH₂CHCH₂COO), 2.62 (dd, J = 15.4, 8.4 Hz,

1H, CH₂COO), 3.07 (dd, J = 15.4, 3.7 Hz, 1H, CH₂COO),

3.62 (dd, J = 11.8, 4.8 Hz, 1H, NCH₂), 3.66 (s, 3H, CH₃),

3.91 (br. d, J = 11.8 Hz, 1H, NCH₂), 4.24–4.29 (m, 1H,

NCH₂CHO), 4.50–4.54 (m, 1H, NCH), 4.56 (s, 2H,

PhCH₂OCH), 5.26 (s, 2H, COOCH₂Ph), 7.25–7.45 (m, 10H,

NCH), 4.38–4.41 (m, 1H, CHOH). IR: ˜

v = 1735 cm^–1. MS;

m/z (%): 160 (100) [M – HCl]⁺. C₇H₁₄ClNO₃(195.46).

6.1.36. Ethyl (2R,4R)-4-hydroxypyrrolidine-2-acetate

hydrochloride [(2R,4R)-13]

H

_aromat). IR:˜

v= 1746 cm^–1, 1712. MS; m/z (%): 384 (22) [M

+ 1]⁺. C₂₂H₂₅NO₅(383.44).

To a solution of 187 mg (0.608 mmol) of (2R,4R)-21 in

10 ml of EtOH, 93 mg (0.087 mmol) of 10% Pd-C and 55 µl

(0.66 mmol) of aq. 37% HCl were added. The mixture was

subjected to hydrogen at r.t. under ambient pressure for 2 h,

and then ﬁltrated and ﬁnally concentrated. The residue was

recrystallized from EtOH/iPr₂O. Yield: 114 mg (89%); col-

orless crystals, m.p. 150–151 °C (EtOH/iPr₂O). [␣]_D²⁰= –34.0

6.1.39. Benzyl (2R,4R)-4-hydroxy-2-methoxypyrrolidine-1-

carboxylate [(2R,4R)-18] and benzyl (2S,4R)-4-hydroxy-

2-methoxypyrrolidine-1-carboxylate [(2S,4R)-18]

A solution of 145 mg (1.44 mmol) of TEA and 1.90 g

(7.17 mmol) of (2S,4R)-14 in 60 ml of MeOH was subjected

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

241

v = 3040 cm^–1, 1714. MS; m/z (%): 334 (1) [M-OCH₃]⁺.

to anodic oxidation at 10–15 °C (U = 13 V, platinum as

˜

cathode, a platinum wire as anode, for 7 h) followed by evapo-

ration of MeOH (T < 25 °C). Puriﬁcation and separation of

the diastereomers by CC (iPr₂O/EtOH, 92:8). Total yield:

1.74 g (97%). Analytical (n-heptane/EtOAc, 40:60;

1.5 ml min^–1, t_R= 10.5 min [Diastereomer I], 9.4 min [Dias-

tereomer II]): ds = 41:59 (Diastereomer II: Diastereomer I).

C₁₉H₃₁NO₄Si (365.55).

6.1.41. Ethyl (2R,4R)-1-benzyloxycarbonyl-4-(tert-

butyl)dimethylsilyloxypyrrolidine-2-acetate [(2R,4R)-20]

and ethyl (2S,4R)-1-benzyloxycarbonyl-4-(tert-

butyl)dimethylsilyloxypyrrolidine-2-acetate [(2S,4R)-20]

To a solution of 3.00 (8.2 mmol) of a mixture of (2R,4R)-19

and (2S,4R)-19 and 8.60 g (purity: 61%, 32.8 mmol) of

1-ethoxy-1-(trimethylsilyloxy)ethene in 83 ml of CH₂Cl₂,

2.43 g (17.2 mmol) of BF₃·Et₂O was added at –78 °C. The

reaction was quenched after 4 h using 66.7 ml (48.3 mmol)

of aq. 5% K₂CO₃. The organic layer was separated, dried

(MgSO₄) and concentrated. Puriﬁcation by CC (n-heptane/

EtOAc, 88:12). Separation of the diastereomers by prep.

HPLC (n-heptane/EtOAc, 88:12; 12 ml min^–1; t_R= 32.7 min

[(2R,4R)-20], 27.0 min [(2S,4R)-20]). Analytical HLPC

(n-heptane/EtOAc, 88/12; 1.3 ml min^–1; ds [(2R,4R)-

20/(2S,4R)-20] = 87:13; t_R= 12.0 min [(2R,4R)-20], 9.5 min

20

Diastereomer I: Colorless oil. [␣]_D= +1.2 (c = 1.76,

EtOH). ¹H NMR (CDCl₃): Two rotamers d = 1.91–2.01 (m,

1H, NCHCH₂), 2.15–2.19 (m, 1H, NCHCH₂), 3.23, 3.37 (2s,

3H, OCH₃), 3.45–3.49, 3.67–3.71 (m, 2H, NCH₂), 4.60–4.64

(m, 1H, CHOH), 5.11–5.31 (m, 3H, NCH and OCH₂Ph),

7.29–7.36 (m, 5H, H_aromat). IR: ˜

v = 3429 cm^–1, 1704. MS;

m/z (%): 220 (100) [M-OCH₃]. C₁₃H₁₇NO₄(251.28) Calc. C

62.14, H 6.82, N 5.57. Found: C 62.00, H 7.00, N 5.53.

20

Diastereomer II: Colorless oil. [␣]_D= –31.7 (c = 1.20,

1

EtOH). H NMR (CDCl₃): two rotamers d = 1.91–1.97 (m,

1H, NCHCH₂), 2.12–2.18 (m, 1H, NCHCH₂), 3.10–3.24,

3.58–3.71 (m, 2H, NCH₂), 3.31–3.44 (m, 3H, OCH₃), 4.34–

4.37 (m, 1H, CHOH), 5.13–5.38 (m, 3H, NCH and OCH₂Ph),

[2S,4R)-20].

20

7.30–7.38 (m, 5H, H_aromat). IR: ˜

v = 3466 cm^–1, 1708. MS;

(2R,4R)-20: Yield: 2.72 g (79%); colorless oil. [␣]_D

=

1

+13.0 (c =1.02, EtOAc). H NMR (C₆D₅NO₂, 120 °C):

d = 0.13 (s, 3H, SiCH₃), 0.14 (s, 3H, SiCH₃), 0.94 (s, 9H,

C(CH₃)₃), 1.25 (t, J = 7.0 Hz, 3H, CH₂CH₃), 2.04 (dt, J = 13.5,

2.4 Hz, 1H, CH₂CHCH₂COO), 2.32 (ddd, J = 13.5, 8.1,

5.4 Hz, 1H, CH₂CHCH₂COO), 2.91 (dd, J = 15.1, 9.6 Hz,

1H, CH₂COO), 3.18 (dd, J = 15.1, 4.0 Hz, 1H, CH₂COO),

3.50 (ddd, J = 11.4, 2.9, 1.1 Hz, 1H, NCH₂), 3.82 (dd, J = 11.4,

5.4 Hz, 1H, NCH₂), 4.14–4.22 (m, 2H, CH₂CH₃), 4.43–4.53

(m, 2H, CHOSi and NCH), 5.26 (s, 2H, CH₂Ph), 7.24–7.45

m/z (%): 252 (92) [M-OCH₃]. C₁₃H₁₇NO₄(251.28).

6.1.40. Benzyl (2R,4R)-4-(tert-butyl)dimethylsilyloxy-2-

methoxypyrrolidine-1-carboxylate [(2R,4R)-19] and ben-

zyl (2S,4R)-4-(tert-butyl)dimethylsilyloxy-2-

methoxypyrrolidine-1-carboxylate [(2S,4R)-19]

A solution of 0.770 g (5.11 mmol) of TBDMSCl in 6 ml

of CH₂Cl₂was added to a solution of 0.986 g (3.93 mmol) of

a mixture of (2R,4R)-18 and (2S,4R)-18 and 0.401 g

(5.89 mmol) of imidazole in 14 ml of CH₂Cl₂at r.t. The mix-

ture was stirred for 16 h, washed with H₂O, dried (Na₂SO₄),

and evaporated to afford a colorless oil. Puriﬁcation by CC

(n-heptane/EtOAc, 7:1). Yield: 1.22 g (85%). Separation of

the diastereomers by prep. HPLC (n-heptane/EtOAc, 7:1;

12 ml min^–1; t_R= 21.6 min [Diastereomer I], 24.6 min [Dias-

tereomer II]).

(m, 5H, H_aromat). IR:˜

v= 1738 cm^–1, 1704. MS; m/z (%): 422

(36) [M + 1]⁺. C₂₂H₃₅NO₅(421.61).

20

(2S,4R)-20: Yield: 314 mg (9%); colorless oil. [␣]_D

–40.3 (c =1.00, EtOAc). H NMR (C₆D₅NO₂, 120 °C):

d = 0.06 (s, 6H, 2 SiCH₃), 0.90 (s, 9H, Si(CH₃)₃), 1.24 (t,

=

1

J

= 7.0 Hz, 3H, CH₂CH₃), 2.06–2.12 (m, 1H,

CH₂CHCH₂COO), 2.22–2.28 (m, 1H, CH₂CHCH₂COO),

2.62 (dd, J = 15.1, 8.5 Hz, 1H, CH₂COO), 3.08 (dd, J = 15.1,

2.2 Hz, 1H, CH₂COO), 3.62 (dt, J = 11.3, 2.2 Hz, 1H, NCH₂),

3.69 (br. d, J = 11.3 Hz, 1H, NCH₂), 4.17 (q, J = 7.0 Hz, 2H,

CH₂CH₃), 4.50–4.56 (m, 2H, NCH and CHOSi), 5.23 (d,

J = 12.9 Hz, 1H, CH₂Ph), 5.27 (d, J = 12.9 Hz, 1H, CH₂Ph),

20

Diastereomer I: Colorless oil. [␣]_D= –10.9 (c = 0.82,

1

EtOAc). H NMR (C₆D₅NO₂, 120 °C): d = 0.11 (s, 3H,

SiCH₃), 0.12 (s, 3H, SiCH₃), 0.93 [s, 9H, SiC(CH₃)₃], 2.00

(dt, J = 13.9, 1.5 Hz, 1H, NCHCH₂), 2.35 (dt, J = 13.9, 6.4 Hz,

1H, NCHCH₂), 3.41–3.45 (m, 4H, NCH₂and OCH₃), 3.99

(dd, J = 11.2, 6.6 Hz, 1H, NCH₂), 4.44–4.49 (m, 1H, CHOSi),

5.24 (d, J = 12.6 Hz, 1H, CH₂Ph), 5.28 (d, J = 12.6 Hz, 1H,

CH₂Ph), 5.36 (dd, J = 6.4, 1.5 Hz, 1H, NCH), 7.25–7.44 (m,

7.25–7.43 (m, 5H, H_aromat). IR: ˜

v = 1735 cm^–1, 1705. MS;

m/z (%): 422 (72) [M + 1]⁺. C₂₂H₃₅NO₅(421.61).

5H, H_aromat). IR:

˜

v= 3034 cm^–1, 1714. MS; m/z (%): 334 (1)

6.1.42. Ethyl (2R,4R)-1-benzyloxycarbonyl-4-hydroxy-

pyrrolidine-2-acetate [(2R,4R)-21]

[M-OCH₃]⁺. C₁₉H₃₁NO₄Si (365.55).

Diastereomer II: Colorless oil. [␣]_D²⁰: +14.4 (c = 1.07,

To a solution of 90 mg (0.21 mmol) of (2R,4R)-20 in 2 ml

of THF, 0.26 ml (1.0 M in THF, 0.26 mmol) of TBAF was

added. After 4.5 h. at r.t. 2.0 ml of 0.1% K₂CO₃was added.

The organic layer was separated, dried (Na₂SO₄), and evapo-

rated. Puriﬁcation by CC (iPr₂O/EtOH, 95:5). Yield: 58 mg

(88%); colorless oil. [␣]_D= +23.4 (c = 0.7, EtOAc). H

NMR (C₆D₅NO₂, 120 °C): d = 1.24 (t, J = 7.1 Hz, 3H,

CH₂CH₃), 2.09 (dtd, J = 13.8, 3.1, 1.3 Hz, 1H,

CH₂CHCH₂COO), 2.39 (ddd, J = 13.8, 8.4, 5.6 Hz, 1H,

1

EtOAc). H NMR (C₆D₅NO₂, 120 °C): d = 0.10 (s, 3H,

SiCH₃), 0.11 (s, 3H, SiCH₃), 0.91 (s, 9H, SiC(CH₃)₃), 2.00

(dd, J = 12.9, 7.2 Hz, 1H, NCHCH₂), 2.20 (ddd, J = 12.9, 5.8,

1.5 Hz, 1H, NCHCH₂), 3.38 (s, 3H, OCH₃), 3.50 (dd, J = 11.1,

5.2 Hz, 1H, NCH₂), 3.72 (dd, J = 11.1, 6.3 Hz, 1H, NCH₂),

4.66–4.73 (m, 1H, CHOSi), 5.25 (d, J = 12.7 Hz, 1H, CH₂Ph),

5.29 (d, J = 12.7 Hz, 1H, CH₂Ph), 5.38 (dd, J = 7.2, 1.5 Hz,

1H, NCH), 7.26–7.35, 7.42–7.44 (m, 5H, H_aromat). IR:

22

1

242

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

CH₂CHCH₂COO), 2.60 (s, 1H, OH), 2.93 (dd, J = 15.4,

9.2 Hz, 1H, CH₂COO), 3.17 (dd, J = 15.4, 4.1 Hz, 1H,

CH₂COO), 3.57 (ddd, J = 11.8, 2.9, 1.3 Hz, 1H, NCH₂), 3.83

(dd, J = 11.8, 5.4 Hz, 1H, NCH₂), 4.17 (q, J = 7.1 Hz, 2H,

CH₂CH₃), 4.41–4.48 (m, 1H, NCH), 4.54–4.58 (m, 1H,

CHOH), 5.25 (s, 2H, CH₂Ph), 7.25–7.45 (m, 5H, H_aromat).

4.40–4.44 (m, 1H, CHOH), 6.83–6.79 (m, 6H, H_aromat), 7.29–

7.33 (m, 6H, H_aromat). IR: ˜

v = 3418 cm^–1, 1740. MS (ESI⁺);

m/z (%): 522 [M + 1]⁺(1). C₃₀H₃₅NO₇(521.61).

6.1.46. Methyl (2R,4R)-4-hydroxy-1-{2-[tris(4-methoxy-

phenyl)methoxy]-1-ethyl}pyrrolidine-2-carboxylate

[(2R,4R)-22c]

IR: ˜

v= 3442 cm^–1, 1731, 1682. MS; m/z (%): 308 (93) [M +

1]⁺. C₁₆H₂₁NO₅(307.35).

According to GP1 from 42 mg (0.25 mmol) of KI, 1.14 g

(2.50 mmol) of RBr (R = c) [11,22], 458 mg (2.50 mmol) of

(2R,4R)-11, 1.73 g (12.5 mmol) of K₂CO₃in 12 ml of acetone.

Yield: 429 mg (33%); colorless oil. [␣]_D²⁰= +30.8 (c = 1.45,

EtOH). ¹H NMR (CDCl₃): d = 1.88–1.93 (m, 1H, NCHCH₂),

2.32 (ddd, J = 14.2, 10.0, 5.9 Hz, 1H, NCHCH₂), 2.67 (dd,

J = 9.9, 4.1 Hz, 1H, NCH₂CHOH), 2.79 (dt, J = 12.4, 6.0 Hz,

1H, NCH₂CH₂), 2.94 (dt, J = 12.4, 6.0 Hz, 1H, NCH₂CH₂),

3.03 (s, 1H, OH), 3.06 (dt, J = 9.9, 1.4 Hz, 1H, NCH₂CHOH),

3.12–3.21 (m, 2H, NCH₂CH₂), 3.38 (dd, J = 10.0, 3.9 Hz,

1H, NCH), 3.66 (s, 3H, COOCH₃), 3.77 (s, 9H, ArOCH₃),

4.20–4.24 (m, 1H, CHOH), 6.78–6.82 (m, 6H, H_aromat), 7.28–

6.1.43. Methyl (2S,4R)-1-(4,4-diphenylbut-3-en-1-yl)-4-

hydroxypyrrolidine-2-carboxylate [(2S,4R)-22a]

According to GP1 from 50 mg (0.30 mmol) of KI, 454 mg

(1.50 mmol) of RBr (R = a) [11,21], 274 mg (1.0 mmol) of

(2S,4R)-11 and 691 mg (5.0 mmol) of K₂CO₃in 8 ml of

acetone. Yield: 273 mg (52%); colorless oil. [␣]_D²⁰= –53.3

(c = 0.6, EtOAc). ¹H NMR (CDCl₃): d = 2.03 (ddd, J = 13.7,

7.6, 3.1 Hz, 1H, NCHCH₂), 2.19 (dd, J = 13.7, 7.6 Hz, 1H,

NCHCH₂), 2.30 (q, J = 7.5 Hz, 2H, NCH₂CH₂), 2.41 (dd,

J = 10.1, 3.6 Hz, 1H, NCH₂CHOH), 2.62 (dt, J = 12.2, 7.5 Hz,

1H, NCH₂CH₂), 2.82 (dt, J = 12.2, 7.5 Hz, 1H, NCH₂CH₂),

3.33 (dd, J = 10.1, 5.5 Hz, 1H, NCH₂CHOH), 3.53 (t,

J = 7.6 Hz, 1H, NCH), 3.67 (s, 3H, OCH₃), 4.40–4.45 (m,

1H, CHOH), 6.07 (t, J = 7.5 Hz, 1H, =CHCH₂), 7.15–7.38

7.32 (m, 6H, H_aromat). IR: ˜

v = 3444 cm^–1, 1732. MS (ESI⁺);

m/z (%): 522 (1). C₃₀H₃₅NO₇(521.61).

6.1.47. (1S,4S)-5-(4,4-Diphenylbut-3-en-1-yl)-2-oxa-5-

azabicyclo[2.2.1]heptane-3-one [(1S,4S)-23a]

(m, 10H, H_aromat). IR: ˜

v = 3418 cm^–1, 1738. MS; m/z (%):

352 (67) [M + 1]⁺. C₂₂H₂₅NO₃(351.45).

According to GP4 from 195 mg (0.578 mmol) of

(2S,4R)-7a and 304 mg (1.160 mmol) of Ph₃P, 151 mg

(0.867 mmol) of DEAD in 10 ml of THF; reaction time: 13 h.

Yield: 157 mg (85%); colorless crystals, m.p. 85–86 °C

(n-heptane/acetone = 4:1). [␣]_D²¹= –60.7 (c = 0.84, EtOH).

¹H NMR (CDCl₃): d = 1.85 (dd, J = 10.5, 1.6 Hz, 1H,

NCHCH₂), 1.92–2.08 (m, 1H, NCHCH₂), 2.15 (dd, J = 10.6,

1.2 Hz, 1H, NCH₂CHO), 2.17–2.31 (m, 2H, NCH₂CH₂), 2.43

(dt, J = 11.8, 6.6 Hz, 1H, NCH₂CH₂), 2.68 (dt, J = 11.8,

7.1 Hz, 1H, NCH₂CH₂), 3.22 (dd, J = 10.6, 0.9 Hz, 1H,

NCH₂CHO), 3.48 (br. s, 1H, NCH), 4.78 (br. s, 1H,

NCH₂CHO), 6.02 (t, J = 7.3 Hz, 1H, =CHCH₂), 7.09–7.30

6.1.44. Methyl (2R,4R)-1-(4,4-diphenylbut-3-en-1-yl)-4-

hydroxypyrrolidine-2-carboxylate [(2R,4R)-22a]

According to GP1 from 50 mg (0.30 mmol) of KI, 936 mg

(3.09 mmol) of RBr (R = a) [11,21], 549 mg (3.00 mmol) of

(2R,4R)-11, 2.07 g (15.0 mmol) of K₂CO₃in 12 ml of acetone.

Yield: 432 mg (41%); colorless oil. [␣]_D²⁰= +45.4 (c = 1.00,

EtOAc). ¹H NMR (CDCl₃): d = 1.92 (ddt, J = 14.2, 3.6, 1.5 Hz,

1H, NCHCH₂), 2.30 (q, J = 7.5 Hz, 2H, NCH₂CH₂), 2.29–

2.37 (m, 1H, NCHCH₂), 2.58 (dd, 1H, J = 9.9, 4.0 Hz,

NCH₂CHOH), 2.63 (dt, J = 12.0, 7.5 Hz, 1H, NCH₂CH₂),

2.81 (dt, J = 12.0, 7.5 Hz, 1H, NCH₂CH₂), 3.00 (dt, J = 9.9,

1.5 Hz, 1H, NCH₂CHOH), 3.26 (dd, J = 9.9, 3.6 Hz, 1H,

NCH), 3.70 (s, 3 H, OCH₃), 4.23–4.26 (m, 1H, CHOH), 6.08

(t, J = 7.5 Hz, 1H, =CHCH₂), 7.16–7.40 (m, 10H, H_aromat).

(m, 10H, H_aromat). IR: ˜

v = 1766 cm^–1. MS; m/z (%): 320

(100) [M + 1]⁺. C₂₁H₂₁NO₂(319.40).

6.1.48. Methyl (2R,4S)-4-acetoxy-1-(4,4-diphenylbut-3-en-

1-yl)pyrrolidine-2-carboxylate [(2R,4S)-24a]

IR: ˜

v = 3424 cm^–1, 1732. MS; m/z (%): 352 (100) [M + 1]⁺.

C₂₂H₂₅NO₃(351.45).

According to GP4 from 120 mg (0.352 mmol) of (2R,4R)-

22a, 134 mg (0.513 mmol) of Ph₃P, 243 µl (38–40% in PhCH₃,

0.513 mmol) of DEAD, 29 mg (0.510 mmol) of AcOH in

5 ml of THF; reaction time: for 6 h; puriﬁcation by CC

(n-heptane/EtOAc, 3:1) and prep. HPLC (n-heptane/EtOAc,

75:25; 12 ml min^–1; t_R= 37.8 min).Yield: 92 mg (69%); col-

orless oil. [␣]_D= +43.1 (c = 1.02, EtOAc). H NMR

(CDCl₃): d = 2.02 (s, 3H, CH₃COO), 2.12 (ddd, J = 13.9, 7.5,

2.9 Hz, 1H, NCHCH₂), 2.25–2.33 (m, 3H, NCHCH₂and

NCH₂CH₂), 2.50 (dd, J = 10.8, 3.3 Hz, 1H, NCH₂CHO), 2.59

(dt, J = 12.0, 7.6 Hz, 1H, NCH₂CH₂), 2.79 (dt, J = 12.0,

8.0 Hz, 1H, NCH₂CH₂), 3.43 (dd, J = 10.8, 6.2 Hz, 1H,

NCH₂CHO), 3.49 (t, J = 7.5 Hz, 1H, NCH), 3.68 (s, 3H,

COOCH₃), 5.17–5.22 (m, 1H, NCH₂CHO), 6.06 (t,

J = 7.4 Hz, 1H, =CHCH₂), 7.14–7.38 (m, 10H, H_aromat). IR:

6.1.45. Methyl (2S,4R)-4-hydroxy-1-{2-[tris(4-methoxy-

phenyl)methoxy]-1-ethyl}pyrrolidine-2-carboxylate

[(2S,4R)-22c]

According to GP1 from 50 mg (0.30 mmol) of KI, 682 mg

(1.49 mmol) of RBr (R = c) [11,22], 273 mg (1.49 mmol) of

(2S,4R)-11, 680 mg (6.85 mmol) of K₂CO₃in 6 ml of acetone.

Yield: 414 mg (53%); colorless oil. [␣]_D²²= –24.5 (c = 0.55,

EtOH). ¹H NMR (CDCl₃): d = 2.00–2.07 (m, 1H, NCHCH₂),

2.16 (ddt, J = 13.6, 7.2, 3.3 Hz, 1H, NCHCH₂), 2.57 (dd,

J = 10.2, 3.4 Hz, 1H, NCH₂CHOH), 2.83 (m, 1H, NCH₂CH₂),

2.95 (m, 1H, NCH₂CH₂), 3.20 (t, J = 6.1 Hz, 2H, NCH₂CH₂),

3.38 (dd, J = 10.2, 5.4 Hz, 1H, NCH₂CHOH), 3.64–3.66 (m,

1H, NCH), 3.65 (s, 3H, COOCH₃), 3.78 (s, 9H, ArOCH₃),

20

1

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

243

v = 1738 cm^–1, 1718. MS; m/z (%): 394 (64) [M + 1]⁺.

NCH), 3.33 (dd, J = 10.2, 6.1 Hz, 1H, NCH₂CHOH), 3.65 (s,

3H, OCH₃), 4.32–4.37 (m, 1H, CHOH), 6.04 (t, J = 7.3 Hz,

1H, =CHCH₂), 6.75 (d, J = 5.1 Hz, 1H, SC=CH), 6.83 (d,

J = 5.1 Hz, 1H, SC=CH), 7.04 (d, J = 5.1 Hz, 1H, SCH=),

˜

C₂₄H₂₇NO₄(393.48).

6.1.49. Methyl (2R,4S)-4-acetoxy-1-{2-[tris(4-methoxy-

phenyl)methoxy]-1-ethyl}pyrrolidine-2-carboxy-late

[(2R,4S)-24c]

7.20 (d, J = 5.1 Hz, 1H, SCH=). IR: ˜

v = 3410 cm^–1, 1738.

MS; m/z (%): 406 (46) [M + 1]⁺. C₂₁H₂₇NO₃S₂(405.57).

According to GP4 from 100 mg (0.192 mmol) of (2R,4R)-

22c and 79 mg (0.290 mmol) of Ph₃P, 139 µl (38–40% in

toluene, 0.288 mmol) of DEAD, 13 mg (0.220 mmol) of

AcOH in 3.5 ml of THF; reaction time: 4 h; puriﬁcation by

CC (n-heptane/acetone, 3:1) and prep. HPLC (n-heptane/

EtOAc, 60:40; 12 ml min^–1; t_R= 32.1 min). Yield: 60 mg

6.1.52. Methyl (2S,4R)-4-hydroxy-1-{2-[tris(4-methoxy-

phenyl)methoxy]-1-ethyl}pyrrolidine-2-acetate [(2S,4R)-

25c]

According to GP1 from 195 mg (1.00 mmol) of (2S,4R)-

12, 457 mg (1.00 mmol) of RBr (R = c) [11,22], 690 mg

(5.00 mmol) of K₂CO₃and 16 mg of KI in 3 ml of acetone.

Yield: 210 mg (39%); colorless oil. [␣]_D²⁰= –28.2 (c = 1.00,

20

1

(56%); colorless oil. [␣]_D= +21.3 (c = 1.10, EtOAc). H

NMR (CDCl₃): d = 2.02 (s, 3H, CH₃COO), 2.12 (ddd,

J = 13.9, 7.5, 3.0 Hz, 1H, NCHCH₂), 2.27 (dt, J = 13.9, 7.5 Hz,

1H, NCHCH₂), 2.64 (dd, J = 11.0, 3.3 Hz, 1H, NCH₂CHO),

2.79 (dt, J = 12.6, 6.2 Hz, 1H, NCH₂CH₂), 2.89–2.94 (m, 1H,

NCH₂CH₂), 3.19 (t, J = 6.2 Hz, 2H, NCH₂CH₂), 3.48 (dd,

J = 11.0, 6.2 Hz, 1H, NCH₂CHO), 3.59 (t, J = 7.5 Hz, 1H,

NCH), 3.67 (s, 3H, COOCH₃), 3.78 (s, 9H, ArOCH₃), 5.17–

5.21 (m, 1H, NCH₂CHO), 6.78–6.81 (m, 6H, H_aromat), 7.29–

1

EtOAc). H NMR (CDCl₃): d = 1.81 (ddd, J = 13.5, 8.4,

7.0 Hz, 1H, CH₂CHCH₂COO), 1.95 (ddd, J = 13.5, 7.0,

3.1 Hz, 1H, CH₂CHCH₂COO), 2.23 (dd, J = 15.0, 9.1 Hz,

1H, CH₂COO), 2.32 (dd, J = 10.4, 4.4 Hz, 1H, NCH₂CHOH),

2.56–2.66 (m, 2H, CH₂COO and NCH₂CH₂), 2.93 (dt,

J = 12.6, 6.3 Hz, 1H, NCH₂CH₂), 3.10–3.20 (m, 3H, NCH

and NCH₂CH₂), 3.36 (dd, J = 10.4, 5.8 Hz, 1H, NCH₂CHOH),

3.63 (s, 3H, COOCH₃), 3.77 (s, 9H,ArOCH₃), 4.33–4.38 (m,

1H, CHOH), 6.78–6.82 (m, 6H, H_aromat), 7.30–7.34 (m, 6H,

7.32 (m, 6H, H_aromat). IR:

(563.65).

˜

v= 1738 cm^–1, 1732. C₃₂H₃₇NO₈

H

_aromat). IR: ˜

v= 3444 cm^–1, 1735. C₃₁H₃₇NO₇(535.64).

6.1.50. Methyl (2S,4R)-1-(4,4-diphenylbut-3-en-1-yl)-4-

hydroxypyrrolidine-2-acetate [(2S,4R)-25a]

6.1.53. Methyl (2S,4R)-1-[3-(carbazol-9-yl)-1-propyl]-4-

hydroxypyrrolidine-2-acetate [(2S,4R)-25d]

According to GP1 from 81 mg (0.410 mmol) of (2S,4R)-

12, 129 mg (0.426 mmol) of RBr (R = a) [11,21], 228 mg

(1.65 mmol) of K₂CO₃and 10 mg of KI; reaction time: 3 d.

Yield: 89 mg (59%); colorless oil. [␣]_D²⁰= –72.8 (c = 0.87,

According to GP1 from 166 mg (0.847 mmol) of (2S,4R)-

12, 406 mg (purity: 70%, 0.85 mmol) of RBr (R = d) [24],

574 mg (1.240 mmol) of K₂CO₃in 3.5 ml of acetone. Yield:

1

20

CHCl₃). H NMR (CDCl₃): d = 1.83 (ddd, J = 13.6, 8.4,

170 mg (57%); yellowish oil. [␣]_D= –51.0 (c = 1.05,

7.0 Hz, 1H, CH₂CHCH₂COO), 1.93 (ddd, J = 13.6, 7.0,

3.1 Hz, 1H, CH₂CHCH₂COO), 2.16 (dd, J = 10.1, 4.4 Hz,

1H, NCH₂CHOH), 2.19–2.33 (m, 3H, CH₂COO and

NCH₂CH₂), 2.39 (ddd, J = 12.0, 8.8, 4.9 Hz, 1H, NCH₂CH₂),

2.58 (dd, J = 15.0, 4.2 Hz, 1H, CH₂COO), 2.81 (dt, J = 12.0,

7.3 Hz, 1H, NCH₂CH₂), 3.02–3.09 (m, 1H, NCH), 3.29 (dd,

J = 10.1, 6.0 Hz, 1H, NCH₂CHOH), 3.62 (s, 3H, OCH₃),

4.29–4.35 (m, 1H, CHOH), 6.06 (t, J = 7.2 Hz, 1H, =CHCH₂),

EtOAc). ¹H NMR (CDCl₃, 500 MHz): d = 1.89 (ddd, J = 13.6,

8.2, 7.0 Hz, 1H, CH₂CHCH₂COO), 1.94–2.11 (m, 3H,

CH₂CHCH₂COO and 2H of CHNCH₂CH₂), 2.17–2.24 (m,

2H, CH₂COO and NCH₂CHOH), 2.38 (ddd, J = 12.0, 7.1,

4.7 Hz, 1H, CH₂NCH), 2.50 (dd, J = 15.0, 4.9 Hz, 1H,

CH₂COO), 2.78 (dt, J = 12.0, 7.9 Hz, 1H, CH₂NCH), 3.05–

3.11 (m, 1H, NCH), 3.40 (dd, J = 10.2, 6.0 Hz, 1H,

NCH₂CHOH), 3.61 (s, 3H, OCH₃), 4.30 (dt, J = 14.9, 7.4 Hz,

1H, CH₂-carbazole), 4.37–4.43 (m, 2H, CHOH and CH₂-

carbazole), 7.23 (t, J = 7.4 Hz, 2H, H_aromat), 7.41 (br. d,

J = 7.9 Hz, 2H, H_aromat), 7.44–7.47 (m, 2H, H_aromat), 8.10 (br.

7.14–7.37 (m, 10H, H_aromat). IR: ˜

v = 1738 cm^–1. MS; m/z

(%): 366 (57) [M + 1]⁺. C₂₃H₂₇NO₃(365.47).

6.1.51. Methyl (2S,4R)-1-[4,4-di(3-methylthien-2-yl)but-3-

en-1-yl]-4-hydroxypyrrolidine-2-acetate [(2S,4R)-25b]

According to GP1 from 94 mg (0.480 mmol) of (2S,4R)-

12, 156 mg (0.480 mmol) of RBr (R = b) [10,23], 330 mg

(2.40 mmol) of K₂CO₃and 11 mg of KI in 3 ml of acetone;

reaction time: 3 d; puriﬁcation by CC (n-heptane/acetone,

d, J = 7.9 Hz, 2H, H_aromat). IR:˜

v= 3417 cm^–1, 1732. MS; m/z

(%): 367 (100) [M + 1]⁺. C₂₂H₂₆N₂O₃(366.46).

6.1.54. (1S,5S)-6-(4,4-Diphenylbut-3-en-1-yl)-2-oxa-6-

azabicyclo[3.2.1]octane-3-one [(1S,5S)-26a]

According to GP4 from 97 mg (0.270 mmol) of (2S,4R)-8a

and 112 mg (0.411 mmol) of Ph₃P in 5 ml of THF, 198 µl

(38–40% in toluene, 0.410 mmol) of DEAD; reaction time:

4 h; puriﬁcation by CC (n-heptane/acetone, 3:1) and prep.

HPLC (n-heptane/EtOAc, 1:1; 12 ml min^–1; t_R= 35.8 min).

Yield: 65 mg (76%); colorless crystals, m.p. 72–74 °C.

[␣]_D²⁰= +4.6 (c = 1.00, EtOAc). ¹H NMR (CDCl₃, 500 MHz):

d = 1.98 (br. s, 2H, CH₂CHCH₂COO), 2.26 (q, J = 7.3 Hz,

2H, NCH₂CH₂), 2.43 (dd, J = 18.6, 3.7 Hz, 1H, CH₂COO),

20

3:1). Yield: 80 mg (41%); slight yellow oil. [␣]_D= –84.6

(c = 1.00, EtOAc). ¹H NMR (CDCl₃): d = 1.83 (ddd, J = 13.5,

8.3, 7.1 Hz, 1H, CH₂CHCH₂COO), 1.95 (ddd, J = 13.5, 7.1,

3.2 Hz, 1H, CH₂CHCH₂COO), 2.00 (s, 3H, thienyl-CH₃),

2.03 (s, 3H, thienyl-CH₃), 2.16–2.32 (m, 4H, NCH₂CHOH,

CH₂COO and NCH₂CH₂), 2.41 (ddd, J = 11.9, 8.2, 5.0 Hz,

1H, NCH₂CH₂), 2.62 (dd, J = 15.0, 4.0 Hz, 1H, CH₂COO),

2.83 (dt, J = 11.9, 8.2 Hz, 1H, NCH₂CH₂), 3.05–3.12 (m, 1H,

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X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

2.67–2.78 (m, 3H, CH₂COO and NCH₂CH₂), 2.95 (d,

J = 11.9 Hz, 1H, NCH₂CHO), 3.06 (dd, J = 11.9, 3.7 Hz, 1H,

NCH₂CHO), 3.37–3.41 (m, 1H, NCH), 4.84–4.86 (m, 1H,

NCH₂CHO), 6.09 (t, J = 7.3 Hz, 1H, =CHCH₂), 7.15–7.39

(m, 4H, CH₂CHCH₂COO and CHNCH₂CH₂), 2.41–2.51 (m,

2H, CH₂COO and CH₂NCH), 2.58–2.64 (m, 1H, CH₂NCH),

2.73–2.79 (m, 1H, CH₂COO), 3.00 (br. d, J = 11.8 Hz, 1H,

NCH₂CHO), 3.06 (dd, J = 11.8, 3.5 Hz, 1H, NCH₂CHO),

3.28–3.31 (m, 1H, NCH), 4.36–4.49 (m, 2H, CH₂-carbazole),

4.8–4.91 (m, 1H, NCH₂CHO), 7.21–7.26 (m, 2H, H_aromat),

7.41–7.48 (m, 4H, H_aromat), 8.09–8.12 (m, 2H, H_aromat). IR:

(m, 10H, H_aromat). IR: ˜

v = 1732 cm^–1. MS; m/z (%): 334

(100) [M + 1]⁺. C₂₂H₂₃NO₂(333.43).

˜

v = 1738 cm^–1. MS; m/z (%): 335 (100) [M + 1]⁺.

6.1.55. (1S,5S)-6-[4,4-Di(3-methylthien-2-yl)but-3-en-1-

yl]-2-oxa-6-azabicyclo[3.2.1]octane-3-one [(1S,5S)-26b]

C₂₁H₂₂N₂O₂(334.42).

According to GP4 from 111 mg (0.284 mmol) of

(2S,4R)-8b and 154 mg (0.568 mmol) of Ph₃P in 6 ml of THF,

269 µl (38–40% in toluene, 0.570 mmol) of DEAD; reaction

time: 4 h; puriﬁcation by CC (n-heptane/acetone, 3:1) and

prep. HPLC (n-heptane/EtOAc, 35:65; 12 ml min^–1; t_R

= 42.7 min).Yield: 74 mg (70%); colorless oil. [␣]_D²²= +6.9

(c = 1.00, EtOAc). ¹H NMR (CDCl₃): d = 1.97–2.00 (m, 2H,

CH₂CHCH₂COO), 2.00 (s, 3H, thienyl-CH₃), 2.03 (s, 3H,

thienyl-CH₃), 2.28 (q, J = 7.3 Hz, 2H, NCH₂CH₂), 2.45 (dd,

J = 18.7, 3.7 Hz, 1H, CH₂COO), 2.68–2.81 (m, 3H, CH₂COO

and NCH₂CH₂), 2.97 (br. d, J = 11.9 Hz, 1H, NCH₂CHO),

3.06 (dd, J = 11.9, 3.7 Hz, 1H, NCH₂CHO), 3.38–3.41 (m,

1H, NCH), 4.85–4.87 (m, 1H, NCH₂CHO), 6.06 (t, J = 7.3 Hz,

1H, =CHCH₂), 6.76 (d, J = 5.2 Hz, 1H, SC=CH), 6.84 (d,

J = 5.2 Hz, 1H, SC=CH), 7.05 (d, J = 5.2 Hz, 1H, SCH=),

6.1.58. (2S,4R)-1-Benzyloxycarbonyl-4-hydroxypyrroli-

dine-2-acetic acid [(2S,4R)-28]

According to GP3 from 312 mg (1.06 mmol) of (2S,4R)-27

20

[20]. Yield: 290 mg (98%); colorless oil. [␣]_D= –61.2

(c = 1.22, MeOH). Analytical data were identical with those

given in the literature {Ref. [␣]_D²⁰= –62.4 (c = 3, MeOH)}

[19a].

6.1.59. (2R,4R)-1-Benzyloxycarbonyl-4-hydroxypyrroli-

dine-2-acetic acid [(2R,4R)-28]

According to GP3 from 86 mg (0.28 mmol) of (2R,4R)-

21, 0.57 ml (0.57 mmol) of aq. 1.0 M NaOH in 3 ml of MeOH;

reaction time: 23 h. Yield: 75 mg (91%); colorless oil.

20

1

[␣]_D= +17.2 (c = 1.41, MeOH). H NMR (C₆D₅NO₂,

120 °C): d = 2.11–2.17 (m, 1H, CH₂CHCH₂COO), 2.37–

2.46 (m, 1H, CH₂CHCH₂COO), 3.04 (dd, J = 15.8, 8.9 Hz,

1H, CH₂COO), 3.28 (dd, J = 15.8, 3.1 Hz, 1H, CH₂COO),

3.59–3.67 (m, 1H, NCH₂), 3.79–3.85 (m, 1H, NCH₂), 4.44–

4.51 (m, 1H, NCH), 4.57–4.62 (m, 1H, CHOH), 5.27 (s, 2H,

7.21 (d, J = 5.2 Hz, 1H, SCH=). IR: ˜

v= 1738 cm^–1. MS; m/z

(%): 374 (32) [M + 1]⁺. C₂₀H₂₃NO₂S₂(373.53).

6.1.56. (1S,5S)-6-{2-[Tris(4-methoxyphenyl)methoxy]-1-

ethyl}-2-oxa-6-azabicyclo[3.2.1]octane-3-one [(1S,5S)-

26c]

CH₂Ph), 7.25–7.46 (m, 5H, H_aromat). IR: ˜

v = 3296 cm^–1,

1716. MS (EI, 70 eV); m/z (%): 279 (5) M⁺. C₁₄H₁₇NO₅

According to GP4 from 96 mg (0.18 mmol) of (2S,4R)-8c

and 97 mg (0.37 mmol) of Ph₃P in 4 ml of THF, 174 µl (38–

40% in toluene, 0.37 mmol) of DEAD; reaction time: 4 h;

puriﬁcation by CC (n-heptane/acetone, 3:1) and prep. HPLC

(n-heptane/EtOAc, 8:2; 12 ml min^–1; t_R= 32.4 min). Yield:

43 mg (46%); colorless oil. [␣]_D²⁰= +2.4 (c = 0.55, EtOAc).

¹H NMR (CDCl₃): d = 1.99 (br. s, 2H, CH₂CHCH₂COO),

2.47 (dd, J = 18.6, 3.5 Hz, 1H, CH₂COO), 2.82–2.88 (m, 3H,

CH₂COO, NCH₂CH₂and NCH₂CHO), 3.07 (d, J = 12.0 Hz,

1H, NCH₂CHO), 3.14–3.18 (m, 3H, NCH₂CH₂and

NCH₂CH₂), 3.48–3.51 (m, 1H, NCH), 3.78 (s, 9H,ArOCH₃),

4.85–4.87 (m, 1H, NCH₂CHO), 6.79–6.83 (m, 6H, H_aromat),

(279.30).

6.1.60. (2S,4S)-1-Benzyloxycarbonyl-4-hydroxypyrroli-

dine-2-acetic acid [(2S,4S)-28]

According to GP3 from 98 mg (0.38 mmol) of (1S,5S)-29,

0.77 ml (0.77 mmol) of aq. 1.0 M NaOH in 8.3 ml of MeOH;

reaction time: 4 d. Yield: 98 mg (94%); colorless oil.

[␣]_D²⁰= –17.6 (c = 1.03, MeOH). The spectra (¹H NMR, IR,

and MS) were identical with those of (2R,4R)-27. C₁₄H₁₇NO₅

(279.30).

6.1.61. (2R,4S)-1-Benzyloxycarbonyl-4-hydroxypyrroli-

dine-2-acetic acid [(2R,4S)-28]

7.28–7.32 (m, 6H, H_aromat). IR:

(503.59).

˜

v = 1737 cm^–1. C₃₀H₃₃NO₆

According to GP3 from 73 mg (0.22 mmol) of (2R,4S)-

32, 0.89 ml (0.89 mmol) of aq. 1.0 M NaOH in 9.1 ml of

MeOH; reaction time: 3 d.Yield: 60 mg (98%); colorless oil.

6.1.57. (1S,5S)-6-[3-(Carbazol-9-yl)-1-propyl]-2-oxa-6-

azabicyclo[3.2.1]octane-3-one [(1S,5S)-26d]

20

1

[␣]_D= +59.6 (c = 1.05, MeOH). H NMR (C₆D₅NO₂,

120 °C): d = 2.10–2.20 (m, 1H, CH₂CHCH₂COO), 2.36–

2.44 (m, 1H, CH₂CHCH₂COO), 2.72 (dd, J = 15.6, 8.2 Hz,

1H, CH₂COO), 3.20 (dd, J = 15.6, 3.8 Hz, 1H, CH₂COO),

3.70 (dd, J = 11.5, 4.4 Hz, 1H, NCH₂), 3.79 (d, J = 11.5 Hz,

1H, NCH₂), 4.53–4.62 (m, 2H, CHOH and NCH), 5.25 (s,

According to GP4 from 142 mg (0.403 mmol) of

(2S,4R)-8d and 212 mg (0.806 mmol) of Ph₃P in 8 ml of THF,

383 µl (38–40% in toluene, 0.810 mmol) of DEAD; reaction

time: 3 h; puriﬁcation by CC (n-heptane/acetone, 3:2) and

prep. HPLC (n-heptane/EtOAc, 2:8; 16.5 ml min^–1;

t_R= 36.0 min). Yield: 55 mg (41%); colorless oil. [␣]_D

2H, CH₂Ph), 7.24–7.45 (m, 5H, H_aromat). IR:

1704. MS; m/z (%): 280 (44) [M + 1]⁺. C₁₄H₁₇NO₅(279.30).

˜

v= 3423 cm^–1,

20

= +6.2 (c = 0.97, EtOAc). ¹H NMR (CDCl₃): d = 1.95–2.07

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

245

6.1.62. Benzyl (1S,5S)-3-oxo-2-oxa-6-azabicyclo[3.2.1]-

octane-6-carboxylate [(1S,5S)-29]

NCH₂CHOH), 4.05–4.18 (m, 3H, CHOH and 2H of

CH₂CH₃), 6.03–6.07 (m, 1H, =CHCH₂), 6.75 (d, J = 5.2 Hz,

1H, SC=CH), 6.84 (d, J = 5.2 Hz, 1H, SC=CH), 7.04 (d,

J = 5.2 Hz, 1H, SCH=), 7.21 (d, J = 5.2 Hz, 1 H, SCH=). IR:

According to GP4 from 290 mg (1.04 mmol) of (2S,4R)-

28, 491 mg (1.87 mmol) of Ph₃P in 25 ml of THF, 886 µl

(38–40% in PhCH₃, 1.9 mmol) of DEAD; reaction time: 7 h;

puriﬁcation by CC (n-heptane/acetone, 3:1) and separation

by prep. HPLC (n-heptane/EtOAc/iso-propanol, 55:45:1.0;

12 ml min^–1; t_R= 53.8 min). Yield: 191 mg (70%); colorless

oil. [␣]_D²⁰= –63.4 (c = 1.22, CHCl₃). ¹H NMR (C₆D₅NO₂,

120°C): d = 2.19 (br. s, 2H, CH₂CHCH₂COO), 2.70–2.77

(m, 1H, CH₂COO), 3.04 (br. d, J = 18.5 Hz, 1H, CH₂COO),

3.67–3.71 (m, 1H, NCH₂), 3.88 (d, J = 12.2 Hz, 1H, NCH₂),

4.52 (br. s, 1H, NCH), 5.06 (br. s, 1H, NCH₂CHO), 5.24 (br.

s, 2H, CH₂Ph), 7.25–7.44 (m, 5H, H_aromat). IR:

˜

v = 3433 cm^–1, 1732. MS; m/z (%): 420 (29) [M + 1]⁺.

C₂₂H₂₉NO₃S₂(419.60).

6.1.65. Ethyl (2R,4R)-4-hydroxy-1-{2-[tris(4-methoxy-

phenyl)methoxy]-1-ethyl}pyrrolidine-2-acetate [(2R,4R)-

30c]

According to GP1 from 136 mg (0.608 mmol) of (2R,4R)-

13, 278 mg (0.608 mmol) of RBr (R = c) [11,22], 420 mg

(3.040 mmol) of K₂CO₃and 15 mg of KI in 4 ml of acetone.

Yield: 175 mg (52%); colorless oil. [␣]_D²⁰= +22.5 (c = 1.65,

1

˜

v = 1747 cm^–1, 1709. MS; m/z (%): 262 (100) [M + 1]⁺.

EtOAc). H NMR (CDCl₃): d = 1.20 (t, J = 7.1 Hz, 3H,

C₁₄H₁₅NO₄(261.28).

CH₂CH₃), 1.69 (ddt, J = 14.2, 6.2, 1.7 Hz, 1H,

CH₂CHCH₂COO), 2.29 (dd, J = 10.1, 4.2 Hz, 1H,

NCH₂CHOH), 2.38 (ddd, J = 14.2, 9.5, 6.6 Hz, 1H,

CH₂CHCH₂COO), 2.41–2.48 (m, 2H, NCH₂CH₂and

CH₂COO), 2.64 (dd, J = 15.8, 3.4 Hz, 1H, CH₂COO), 2.70–

2.77 (m, 1H, NCH), 2.96 (dt, J = 12.6, 6.3 Hz, 1H,

NCH₂CH₂), 3.05 (br. d, J = 10.1 Hz, 1H, NCH₂CHOH), 3.12

(dt, J = 9.5, 5.8 Hz, 1H, NCH₂CH₂), 3.19 (dt, J = 9.5, 6.1 Hz,

1H, NCH₂CH₂), 3.77 (s, 9H, ArOCH₃), 4.01–4.14 (m, 2H,

CH₂CH₃), 4.13–4.18 (m, 1H, CHOH), 6.79–6.82 (m, 6H,

6.1.63. Ethyl (2R,4R)-1-(4,4-diphenylbut-3-en-1-yl)-4-

hydroxypyrrolidine-2-acetate [(2R,4R)-30a]

According to GP1 from 92 mg (0.440 mmol) of (2R,4R)-

13, 132 mg (0.439 mmol) of RBr (R = a) [11,21], 298 mg

(2.160 mmol) of K₂CO₃and 10 mg of KI in 2.7 ml of acetone.

Yield: 115 mg (69%); colorless oil. [␣]_D²⁰= +58.9 (c = 0.85,

EtOAc). ¹H NMR (CDCl₃, 500 MHz): d = 1.20 (t, J = 7.1 Hz,

3H, CH₂CH₃), 1.69 (ddt, J = 14.3, 6.1, 1.7 Hz, 1H,

CH₂CHCH₂COO), 2.16 (dd, J = 10.0, 4.2 Hz, 1H,

NCH₂CHOH), 2.23–2.33 (m, 3H, NCH₂CH₂and NCH₂CH₂),

2.37 (ddd, J = 14.3, 9.2, 6.5 Hz, 1H, CH₂CHCH₂COO), 2.43

(dd, J = 15.3, 7.8 Hz, 1H, CH₂COO), 2.62 (dd, J = 15.3,

3.3 Hz, 1H, CH₂COO), 2.63–2.70 (m, 2H, NCH and OH),

2.80–2.90 (m, 1H, NCH₂CH₂), 2.99 (d, J = 10.0 Hz, 1H,

NCH₂CHOH), 4.05–4.15 (m, 3H, CHOH and CH₂CH₃),

6.05–6.09 (m, 1H, =CHCH₂), 7.14–7.37 (m, 10H, H_aromat).

¹³C NMR (CDCl₃, 125.8 MHz): d = 14.2 (CH₃), 28.8

(NCH₂CH₂), 38.8 (CH₂COO), 40.9 (1 C, CH₂CHCH₂COO),

53.3 (NCH₂CH₂), 59.5 (NCH), 60.4 (OCH₂CH₃), 62.1

(NCH₂CHO), 70.1 (CHOH), 127.0, 127.1 (2 C, C_aromat), 127.3

(=CHCH₂), 128.2, 128.3 (4 C, C_aromat), 129.9 (4 C, C_aromat),

140.0 (2 C, C=CHCH₂), 142.5 142.7 (2 C, C_aromat), 172.3

H_aromat), 7.30–7.33 (m, 6 H, H_aromat). IR:

˜

v = 3440 cm^–1,

1731. C₃₂H₃₉NO₇(549.66).

6.1.66. Ethyl (2R,4R)-1-[3-(carbazol-9-yl)-1-propyl]-4-

hydroxypyrrolidine-2-acetate [(2R,4R)-30d]

According to GP1 from 122 mg (0.528 mmol) of (2R,4R)-

13, 279 mg (purity: 70%, 0.53 mmol) of RI (R = d) [25] and

400 mg (2.910 mmol) of K₂CO₃in 3 ml of acetone. Yield:

8.2 g; yellow oil [purity: 70% (w/w); I/Cl = 1.6:1 (mol/mol),

determined by ¹H NMR].Yield: 193 mg (87%); colorless oil.

20

[␣]_D= +56.8 (c = 0.96, EtOAc). ¹H NMR (CDCl₃,

500 MHz): d = 1.23 (t, J = 7.2 Hz, 3H, CH₂CH₃), 1.74 (ddt,

J = 14.3, 6.2, 1.7 Hz, 1H, CH₂CHCH₂COO), 1.99–2.14 (m,

2H, CHNCH₂CH₂), 2.21 (dd, J = 10.0, 4.1 Hz, 1H,

NCH₂CHOH), 2.23–2.29 (m, 1H, CH₂NCH), 2.38–2.46 (m,

2H, CH₂CHCH₂COO and CH₂COO), 2.56 (dd, J = 15.6,

3.6 Hz, 1H, CH₂COO), 2.64–2.71 (m, 1H, NCH), 2.84 (ddd,

J = 15.9, 8.7, 7.3 Hz, 1H, CH₂NCH), 3.11 (br. d, J = 10.0 Hz,

1H, NCH₂CHOH), 4.07–4.17 (m, 2H, CH₂CH₃), 4.19–4.23

(m, 1H, CHOH), 4.28–4.35 (m, 1H, CH₂-carbazole), 4.42–

4.49 (m, 1H, CH₂-carbazole), 7.25 (ddd, J = 8.0, 6.9, 1.1 Hz,

2H, H_aromat), 7.44 (br. d, J = 8.0 Hz, 2H, H_aromat), 7.49 (ddd,

J = 8.0, 6.9, 1.1 Hz, 2H, H_aromat), 8.12 (br. d, J = 8.0 Hz, 2H,

(COO). IR: ˜

v= 3415 cm^–1, 1731. MS; m/z (%): 380 (82) [M

+ 1]⁺. C₂₄H₂₉NO₃(379.50).

6.1.64. Ethyl (2R,4R)-1-[4,4-di(3-methylthien-2-yl)but-3-

en-1-yl]-4-hydroxypyrrolidine-2-acetate [(2R,4R)-30b]

According to GP1 from 92 mg (0.440 mmol) of (2R,4R)-

13, 143 mg (0.439 mmol) of RBr (R = b) [10,23], 298 mg

(2.160 mmol) of K₂CO₃and 10 mg of KI in 2.7 ml of acetone.

20

Yield: 95 mg (52%); yellow oil. [␣]_D= +69.8 (c = 0.80,

EtOAc). H NMR (CDCl₃): d = 1.23 (t, J = 7.3 Hz, 3H,

H_aromat). IR:˜

v= 3427 cm^–1, 1732. MS; m/z (%): 381 (27) [M

1

CH₂CH₃), 1.69 (ddt, J = 14.3, 6.1, 1.7 Hz, 1H,

CH₂CHCH₂COO), 1.99 (s, 3H, thienyl-CH₃), 2.04 (s, 3H,

thienyl-CH₃), 2.20 (dd, J = 10.0, 4.1 Hz, 1H, NCH₂CHOH),

2.25–2.40 (m, 4H, NCH₂CH₂, CH₂CHCH₂COO and

NCH₂CH₂), 2.43 (dd, J = 15.4, 7.8 Hz, 1H, CH₂COO), 2.52

(br. s, 1H, OH), 2.62–2.72 (m, 2H, NCH and CH₂COO), 2.81–

2.92 (m, 1H, NCH₂CH₂), 3.01 (br. d, J = 10.0 Hz, 1H,

+ 1]⁺. C₂₃H₂₈N₂O₃(380.49).

6.1.67. Ethyl (2R,4S)-4-acetoxy-1-(4,4-diphenylbut-3-en-1-

yl)pyrrolidine-2-acetate [(2R,4S)-31a]

According to GP4 from 50 mg (0.13 mmol) of (2R,4R)-

30a and 52 mg (0.20 mmol) of Ph₃P in 2 ml of THF, 94 µl

(38–40% in toluene, 0.20 mmol) of DEAD and 11 mg

246

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

(0.18 mmol) of AcOH; reaction time: 23 h; puriﬁcation

(n-heptane/EtOAc, 3:1) and prep. HPLC (n-heptane/EtOAc,

75:25; 12 ml min^–1; t_R= 37.8 min).Yield: 40 mg (72%); col-

NCH₂CH₂), 3.46 (dd, J = 10.9, 6.3 Hz, 1H, NCH₂CHO), 3.78

(s, 3H, ArOCH₃), 4.06–4.14 (m, 2H, CH₂CH₃), 5.06–5.12

(m, 1H, NCH₂CHO), 6.78–6.82 (m, 6H, H_aromat), 7.29–7.33

20

1

(m, 6H, H_aromat). IR: ˜

v= 1733 cm^–1. C₃₄H₄₁NO₈(591.70).

orless oil. [␣]_D= +57.9 (c = 0.98, EtOAc). H NMR

(CDCl₃): d = 1.24 (t, J = 7.0 Hz, 3H, CH₂CH₃), 1.89 (ddd,

J = 13.9, 9.0, 7.6 Hz, 1H, CH₂CHCH₂COO), 2.01 (s, 3H,

CH₃COO), 2.06 (ddd, J = 13.9, 6.6, 2.6 Hz, 1H,

CH₂CHCH₂COO), 2.17–2.40 (m, 5H, NCH₂CHO,

NCH₂CH₂, CH₂COO and NCH₂CH₂), 2.61 (dd, J = 15.0,

4.2 Hz, 1H, CH₂COO), 2.80–2.87 (m, 1H, NCH₂CH₂), 2.97–

3.04 (m, 1H, NCH), 3.42 (dd, J = 10.8, 6.4 Hz, 1H,

NCH₂CHO), 4.05–4.17 (m, 2H, CH₂CH₃), 5.06–5.11 (m, 1H,

NCH₂CHO), 6.06 (t, J = 7.2 Hz, 1H, =CHCH₂), 7.14–7.38

6.1.70. Ethyl (2R,4S)-4-acetoxy-1-[3-(carbazol-9-yl)-1-

propyl]pyrrolidine-2-acetate [(2R,4S)-31d]

According to GP4 from 110 mg (0.289 mmol) of (2R,4R)-

30d and 152 mg (0.578 mmol) of Ph₃P in 4 ml of THF, 269 µl

(38–40% in toluene, 0.580 mmol) of DEAD and 26 mg

(0.430 mmol) of AcOH; reaction time: 3 h; puriﬁcation by

CC (n-heptane/acetone, 4:1) and prep. HPLC (n-

heptane/EtOAc, 60:40; 12 ml min^–1; t_R= 34.2 min). Yield:

104 mg (85%); colorless oil. [␣]_D²⁰= +43.6 (c = 1.04, EtOAc).

¹H NMR (CDCl₃, 500 MHz): d = 1.22 (t, J = 7.1 Hz, 3H,

CH₂CH₃), 1.93–2.12 (m, 7H, CH₂CHCH₂COO,

CHNCH₂CH₂and CH₃COO), 2.19 (dd, J = 15.0, 8.6 Hz, 1H,

CH₂COO), 2.29 (dd, J = 10.8, 4.3 Hz, 1H, NCH₂CHO), 2.32–

2.35 (m, 1H, CH₂NCH), 2.48 (dd, J = 15.0, 4.4 Hz, 1H,

CH₂COO), 2.77 (dt, J = 12.1, 7.8 Hz, 1H, CH₂NCH), 3.00–

3.05 (m, 1H, NCH), 3.53 (dd, J = 10.8, 6.3 Hz, 1H,

NCH₂CHO), 4.08 (q, J = 7.1 Hz, 2H, CH₂CH₃), 4.29–4.35

(m, 1H, CH₂-carbazole), 4.37–4.43 (m, 1H, CH₂-carbazole),

5.14–5.18 (m, 1H, NCH₂CHO), 7.21–7.25 (m, 2H, H_aromat),

7.41–7.42 (m, 2H, H_aromat), 7.45–7.48 (m, 2H, H_aromat), 8.09–

(m, 10H, H_aromat). IR: ˜

v = 1740 cm^–1, 1732. MS; m/z (%):

422 (46), [M + 1]⁺. C₂₆H₃₁NO₄(421.54).

6.1.68. Ethyl (2R,4S)-4-acetoxy-1-[4,4-di(3-methylthien-2-

yl)but-3-en-1-yl]pyrrolidine-2-acetate [(2R,4S)-31b]

According to GP4 from 117 mg (0.279 mmol) of (2R,4R)-

30b and 108 mg (0.419 mmol) of Ph₃P in 4 ml of THF, 199 µl

(38–40% in toluene, 0.420 mmol) of DEAD and 24 mg

(0.400 mmol) of AcOH; reaction time: 3 h; puriﬁcation by

CC (n-heptane/EtOAc, 4:1) and prep. HPLC (n-

heptane/EtOAc, 75:25; 12 ml min^–1; t_R= 27.3 min). Yield:

81 mg (63%); colorless oil. [␣]_D²⁰= +86.8 (c = 0.94, EtOAc).

¹H NMR (CDCl₃): d = 1.24 (t, J = 7.1 Hz, 3H, CH₂CH₃),

1.87 (ddd, J = 13.9, 9.0, 7.6 Hz, 1H, CH₂CHCH₂COO), 2.00–

2.09 (m, 10H, CH₂CHCH₂COO, CH₃COO and 2 × thienyl-

CH₃), 2.18–2.42 (m, 5H, NCH₂CHO, NCH₂CH₂, CH₂COO

and NCH₂CH₂), 2.63 (dd, J = 15.1, 4.2 Hz, 1H, CH₂COO),

2.83 (dt, J = 11.5, 8.1 Hz, 1H, NCH₂CH₂), 2.97–3.05 (m, 1H,

NCH), 3.44 (dd, J = 10.8, 6.4 Hz, 1H, NCH₂CHO), 4.12 (q,

J = 7.1 Hz, 2H, CH₂CH₃), 5.06–5.11 (m, 1H, NCH₂CHO),

6.02 (t, J = 7.2 Hz, 1H, =CHCH₂), 6.75 (d, J = 5.1 Hz, 1H,

SC=CH), 6.83 (d, J = 5.1 Hz, 1H, SC=CH), 7.05 (d,

J = 5.1 Hz, 1H, SCH=), 7.20 (d, J = 5.1 Hz, 1H, SCH=). IR:

8.11 (m, 2H, H_aromat). IR:˜

v= 1737 cm^–1, 1732. MS; m/z (%):

423 (100) [M + 1]⁺. C₂₅H₃₀N₂O₄(422.53).

6.1.71. Ethyl (2R,4S)-1-benzyloxycarbonyl-4-formyloxy-

pyrrolidine-2-acetate [(2R,4S)-32]

According to GP4 from 106 mg (0.345 mmol) of

(2R,4R)-21 and 135 mg (0.517 mmol) of Ph₃P in 5 ml of

THF, 245 µl (38–40% in toluene, 0.720 mmol) of DEAD;

reaction time: 16 h; puriﬁcation by CC (n-heptane/acetone,

3:1) and prep. HPLC (n-heptane/EtOAc/iso-propanol,

78:22:0.8; 12 ml min^–1; t_R= 22.7 min). Yield: 92 mg (80%);

20

1

v = 1738 cm^–1, 1732. MS; m/z (%): 462 (12) [M + 1]⁺.

colorless oil. [␣]_D= +36.4 (c = 1.64, CHCl₃). H NMR

(C₆D₅NO₂, 120 °C): d = 1.21–1.26 (m, 3H, CH₂CH₃), 2.25–

2.33 (m, 1H, CH₂CHCH₂COO), 2.41–2.49 (m, 1H,

CH₂CHCH₂COO), 2.64–2.72 (m, 1H, CH₂COO), 3.04–3.10

(m, 1H, CH₂COO), 3.75–3.80 (m, 1H, NCH₂), 3.89 (d,

J = 12.1 Hz, 1H, NCH₂), 4.13–4.20 (m, 2H, CH₂CH₃), 4.45–

4.52 (m, 1H, NCH), 5.26 (s, 2H, CH₂Ph), 5.45–5.49 (m, 1H,

NCH₂CHO), 7.27–7.36, 7.40–7.44 (m, 5H, H_aromat), 8.03 (s,

˜

C₂₄H₃₁NO₄S₂(461.64).

6.1.69. Ethyl (2R,4S)-4-acetoxy-1-{2-[tris(4-methoxy-

phenyl)methoxy]-1-ethyl}pyrrolidine-2-acetate [(2R,4S)-

31c]

According to GP4 from 147 mg (0.267 mmol) of (2R,4R)-

30c and 105 mg (0.401 mmol) of Ph₃P in 5 ml of THF, 190 µl

(38–40% in toluene, 0.400 mmol) of DEAD and 19 mg

(0.320 mmol) of AcOH; reaction time: 2 h; puriﬁcation by

CC (n-heptane/acetone, 3:1) and prep. HPLC (n-

heptane/EtOAc, 60:40; 12 ml min^–1; t_R= 32.1 min). Yield:

50 mg (32%); colorless oil. [␣]_D²⁰= +23.4 (c = 1.51, EtOAc).

¹H NMR (CDCl₃): d = 1.23 (t, J = 7.2 Hz, 3H, CH₂CH₃),

1.86 (ddd, J = 13.9, 9.1, 7.4 Hz, 1H, CH₂CHCH₂COO), 2.01

(s, 3H, CH₃COO), 2.06 (ddd, J = 13.9, 6.6, 2.6 Hz, 1H,

CH₂CHCH₂COO), 2.21 (dd, J = 15.1, 9.1 Hz, 1H, CH₂COO),

2.34 (dd, J = 10.9, 4.4 Hz, 1H, NCH₂CHO), 2.48–2.55 (m,

1H, NCH₂CH₂), 2.65 (dd, J = 15.1, 4.1 Hz, 1H, CH₂COO),

2.90–2.97 (m, 1H, NCH₂CH₂), 3.03–3.18 (m, 3H, NCH and

1H, HCOO). IR: ˜

v = 1728 cm^–1, 1705. MS; m/z (%): 308

(79). C₁₇H₂₁NO₆(335.36).

6.2. Biological test

6.2.1. Preparation of subcellular membrane suspensions

Two subcellular membrane pellets, termed bfcP2B (from

bovine frontal cortex)^aand bbsP2C (from bovine brain stem)^b,

respectively, were prepared according to literature [20]. Their

suspensions were prepared and measured as described by

Bradford [26]. cfcP2B (from calf frontal cortex)^cand cbsP2C

(from calf brain stem)^d, pfcP2B (from porcine frontal cor-

X. Zhao et al. / European Journal of Medicinal Chemistry 40 (2005) 231–247

247

tex)^eand pbsP2C (from porcine brain stem)^fwere applied

alternatively instead of bfcP2B and bbsP2C, respectively.

[6] L.A. Borden, K.E. Smith, P.R. Hartig, T.A. Branchek, R.L. Weins-

hank, J. Biol. Chem. 267 (1992) 21098.

[7] A. Yamauchi, S. Uchida, H.M. Kwon, A.S. Preston, R.B. Robey,

A. Garcia-Perez, M.B. Burg, J.S. Handler, J. Biol. Chem. 267 (1992)

649.

6.2.2. Inhibition of GAT-1 mediated GABA-uptake

[8] (a) L.A. Borden, Neurochem. Int. 29 (1996) 335.

(b) J. Guastella, N. Nelson, H. Nelson, L. Czyzyk, S. Keinan, M.C.

Miedel, N. Davidson, H.A. Lester, B.I. Kanner, J. Biol. Chem. 25

(1986) 15437.

(c) L.A. Borden, K.E. Smith, P.J.-J. Vaysse, E.L. Gustafson, E.L.

Weinshank, T.A. Branchek, Recept. Channels 3 (1995) 129.

[9] F.E. Ali, W.E. Bondinell, P.A. Dandridge, J.S. Frazee, W. Garvey,

G.R. Girard, C. Kaiser, T.W. Ku, J.J. Lafferty, G.I. Moonsammy,

H.-J. Oh, J.A. Rush, P.E. Setler, O.D. Stringer, J.W. Venslavsky,

B.W. Volpe, L.M. Yunger, C.L. Zirkle, J. Med. Chem. 28 (1985) 653.

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E.B. Nielsen, U. Sonnewald, P.O. Sørensen, P.D. Suzdak, L.J.S. Knut-

sen, J. Med. Chem. 36 (1993) 1716.

[11] T. Dhar, L. Borden, K. Smith, T. Branchek, R. Weinshank, C. Glu-

chowski, J. Med. Chem. 37 (1994) 2334.

[12] (a) G. Hoefner, G. Fuelep, K. Wanner, WO0014060 (2000).

(b) G.H. Fülep, Ph.D. Thesis, LMU München, 1998.

[13] (a) S.C. Mayer, J. Ramanjulu, M.D. Vera, A.J. Pﬁzenmayer, M. Joul-

lie, J. Org. Chem. 59 (1994) 5192.

Aliquots of about 50–100 µg protein bfcP2B (alterna-

tively cfcP2B or pfcP2B) were preincubated with 10 µM ami-

nooxyacetic acid and a test compound in 200 µl of buffer

(119 mM NaCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM

KH₂PO₄, 4.7 mM KCl, 11 mM glucose and 25 mM Tris–HCl

pH 7.2) for 10 min at 37 °C. 25 µl of 12.5 nM [³H] GABA

and 25 µl of 250 nM GABA were added and the sample was

incubated at 37 °C for 4 min. The incubation was terminated

by ﬁltration in a Brandel M-24R Harvester through What-

man GF/C ﬁlters, which had been immersed in 0.9% NaCl

for 1 h. The ﬁlters were washed with 0.9% NaCl (4 × 2 ml)

and then measured in 3 ml of Rotiszint Eco Plus by the use of

a Packard TriCarb 1600 Counter. Speciﬁc uptake was deﬁned

as difference between entire uptake and non-speciﬁc uptake,

which was determined with identical samples lacking NaCl.

(b) M. Kaname, S. Yoshifuji, Tetrahedron Lett. 33 (1992) 8103.

(c) A.G.M. Barrett, D. Pilipauskas, J. Org. Chem. 55 (1990) 5194.

[14] (a) D.S. Robinson, J.P. Greenstein, J. Biol. Chem. 195 (1952) 383.

(b) G. Lowe, T. Vilaivan, J. Chem. Soc. Perkin Trans. 1 (1997) 539.

[15] (a) R. Beeli, M. Steger,A. Linden, J.A. Robinson, Helv. Chim.Acta 79

(1996) 2235.

6.2.3. Inhibition of GAT-3 mediated GABA-uptake

Aliquots of about 50–100 µg protein bbsP2C (alterna-

tively cbsP2C or pbsP2C) were preincubated with 10 µM ami-

nooxyacetic acid, 10 µM NNC-711 and a test compound in

200 µl of buffer (119 mM NaCl, 2.5 mM CaCl₂, 1.2 mM

MgSO₄, 1.2 mM KH₂PO₄, 4.7 mM KCl, 11 mM glucose and

25 mM Tris–HCl pH 7.2) for 10 min at 37 °C. Next, 25 µl of

50 nM [³H] GABA and 25 µl of 1 µM GABA were added and

the sample was incubated for 4 min at 37 °C. The subsequent

procedure was the same as described above in Section 6.2.2.

(b) E.M. Smith, G.F. Swiss, B.R. Neustadt, E.H. Gold, J.A. Sommer,

A.D. Brown, P.J.S. Chiu, R. Moran, E.J. Sybertz, T. Baum, J. Med.

Chem. 31 (1988) 875.

[16] (a) P. Renaud, D. Seebach, Synth. Commun. 16 (1986) 424.

(b) L.-G. Wistrand, M. Skrinjar, Tetrahedron 47 (1991) 573.

(c) H. Horikawa, T. Iwasaki, K. Matsumoto, M. Miyoshi, J. Org.

Chem. 43 (1978) 335.

(d) T. Shono, Tetrahedron 40 (1984) 811.

[17] P. Renaud, D. Seebach, Helv. Chim. Acta 69 (1986) 1704.

[18] (a) M. Thaning, L.-G. Wistrand, Helv. Chim. Acta 69 (1986) 1711.

(b) T. Rosen, D.T.W. Chu, I.M. Lico, P.B. Fernandes, K. Marsh, L.

Shen, V.G. Cepa, A.G. Pernet, J. Med. Chem. 31 (1988) 1598.

(c) H. Rueeger, M. Benn, Heterocycles 19 (1982) 23.

[19] (a) O. Mitsunobu, Synthesis 1 (1981) 1.

(b) M.M. Bowers-Nemia, M.M. Joullie, Heterocycles 20 (1983) 817.

(c) D. Papaioannou, G. Stavropoulos, K. Karagiannis, G.W. Francis,

T. Brekke, W. Dagﬁnn, Acta Chem. Scand. 44 (1990) 243.

[20] P.R. Dodd, J.A. Hardy, A.E. Oakley, J.A. Edwardson, E.K. Perry,

J.-P. Delaunoy, Brain Res. 226 (1981) 107.

Acknowledgements

Financial support of this work by the Deutsche Forschungs-

gemeinschaft, the Fonds der Chemischen Industrie and the

BMBF is gratefully acknowledged.

References

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Article Doi

Synthesis and biological evaluation of new GABA-uptake inhibitors derived from proline and from pyrrolidine-2-acetic acid

DOI: 10.1016/j.ejmech.2004.11.004

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