Synthesis and biological evaluation of 4-hydroxy-4-(4-methoxyphenyl)- substituted proline and pyrrolidin-2-ylacetic acid derivatives as GABA uptake inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.11.015

A series of enantiomerically pure 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives have been synthesized starting from the respective N-protected 4-hydroxy derivatives via oxidation to the corresponding 4-oxo co

Full text of DOI:10.1016/j.bmc.2012.11.015

Bioorganic & Medicinal Chemistry 21 (2013) 470–484
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4-hydroxy-4-(4-methoxyphenyl)-
substituted proline and pyrrolidin-2-ylacetic acid derivatives as GABA
uptake inhibitors
⇑
Xueqing Zhao , Jörg Pabel, Georg C. Höfner, Klaus T. Wanner
Department für Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2012
Revised 7 November 2012
Accepted 8 November 2012
Available online 27 November 2012
A series of enantiomerically pure 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-
ylacetic acid derivatives have been synthesized starting from the respective N-protected 4-hydroxy
derivatives via oxidation to the corresponding 4-oxo compounds, subsequent addition of organometallic
reagents, final hydrolysis and deprotection. The major diastereoisomers obtained by the addition of the
Grignard reagents were found to have opposite stereoconfigurations depending on whether cerium
trichloride was present or absent as an additive. The final compounds were evaluated for their capability
to inhibit the GABA transport proteins GAT1 and GAT3. 4-Hydroxyproline derivatives substituted with
a tris(4-methoxyphenyl)methyloxyethyl residue at the nitrogen and a 4-methoxyphenyl group in
4-position showed, with the exception of the (2R,4R)-diastereomer, an improved inhibition at GAT3
compared to the derivatives missing the 4-methoxyphenyl group in 4-position. This may imply that an
appropriate lipophilic group at the C-4 position of the proline moiety is beneficial for potent inhibition
at GAT3.
Dedicated with warmest wishes to
Wolfgang Beck on the occasion of his 80th
birthday
Keywords:
GABA uptake inhibitors
GAT1
GAT3
Antiepileptic
Pyrrolidines
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
nervous system (CNS)22 and were found in close proximity to the
synapse or in the synapse itself. On the other hand, BGT-1 and
c
-Aminobutyric acid (GABA) is the dominant inhibitory neuro-
GAT2 are found mainly in peripheral organs, that is, in the liver
and in the kidneys, whereas in the brain significant concentrations
are restricted to the leptomeninges (GAT2 and BGT-1) and to cere-
bral blood vessels (GAT2), indicating that these transporters are
unlikely to play an important role for inactivation of the neuro-
transmitter GABA.23,24
Many potent uptake inhibitors selective for GAT1 like (R)-SK&F-
89976-A (1), SK&F-100591-A (2à),25 and Tiagabine (3) have been
discovered and investigated for their pharmacological properties
since 1985. As a GAT1 inhibitor, Tiagabine (GabitrilÒ) was firstly
marketed for add-on treatment of epilepsy in 1997, new indications
for the treatment of diabetic neuropathy and migraine are under
clinical trials.26 However, until now the number of inhibitors with
high affinity for the other GAT subtypes is still small, which is espe-
cially regrettable for GAT3, which, in addition, to GAT1 possesses
great importance for GABA inactivation in the brain.27–29 (S)-SNAP-
5114 (4)30,31 and NNC-05-2045 (5, Fig. 3)32,33 range among the most
potent GAT3 inhibitors known, but their potencies and subtype
selectivities are still relatively low. Therefore, there is still a great
need for more selective and more potent GAT3 inhibitors.
transmitter in the brain. GABAergic dysfunction has been found to
be involved in a number of CNS disorders, including epilepsy,1
Huntington’s chorea,2 migraine,3 Morbus Parkinson,4 and
depression.5–7 Enhancement of GABA function can be achieved
through several mechanisms, such as direct receptor agonism8 or
inhibition of enzymatic breakdown of GABA.9 But long-term
administration is often limited by rapid development of toler-
ance.10–12 Since the discovery of GABA transport proteins (GAT),
the inhibition of these GABA uptake proteins has become a new
and efficient approach to palliate GABA deficiency.13 Four distinct
GABA transporters have been characterized over the years. Follow-
ing a species independent nomenclature, also used by the Humane
Genome Organization (HUGO), these transporters are termed GAT1
(slc6a1), GAT2 (slc6a13), GAT3 (slc6a11), and BGT-1 (slc6a12),14–20
respectively (for a detailed discussion see Ref. 21). The four
subtypes differ in their distribution and their pharmacological role.
GAT1 and GAT3 are almost exclusively located in the central
⇑
Corresponding author. Tel.: +49 89 2180 77249; fax: +49 89 2180 77247.
E-mail address: Klaus.Wanner@cup.uni-muenchen.de (K.T. Wanner).
Present Address: Synthetic Group, Fujian Institute of Microbiology, Jinbu Road 25,
Fuzhou 350007, PR China.
à
Mixture of racemic diastereomers.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.015

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
471
O
O
O
OH
N
O
O
O
O
OH
OH
OH
OH
N
R
N
R
N
R
N
R
OH
OH
OH
N
N
R
(S)-6
(R)-6
(S)-7
(R)-7
GAT-1
GAT-3
2.56
309
2.97
231
0.396
64.8
3.05
189
b
S
S
0.645
>100
0.343
26.6
0.875
180
GAT-1
GAT-3
c
d
(R)-SK&F-89976-A (1)
GAT1: 0.11 µM25
SK&F-100591 (2)
Tiagabine (3)
123
57.7
143
18.5
35.4
28.7
67.8
3.10
GAT-1
GAT-3
GAT1: 0.26
µ
M25
GAT1: 0.159 µM34
GAT3: 483 µM34
For residues b-d see Fig. 3
H3CO
O
Figure 2. Structures of GABA uptake inhibitors with a pyrrolidine moiety. Struc-
tures of R are given in Figure 3 (IC50 unit: M).
OH
OH
l
H3CO
N
N
7c; best compounds at GAT3: (R)-6d, (R)-7d). Introduction of a hy-
droxy group into the 4-position of the pyrrolidine ring resulting in
compounds 8 and 9 (Fig. 4), however, was found to significantly
decrease the inhibitory potency even for the most potent stereoiso-
mers and at both, GAT1 and GAT3 ( Figs. 2 and 4; (2R,4R)-8b:
O
N
H3CO
OCH3
9.4
19.9
l
l
M
at GAT1; (2S,4R)-9c: 3.15
lM at GAT1; (2R,4S)-9d:
NNC-05-2045 (5)
(S)-SNAP-5114 (4)
GAT1: 83.3 µM34
GAT3: 1.08 µM34
M at GAT3).
GAT1: 27 µM32
Herein, we present the synthesis and biological evaluation of
new pyrrolidine derivatives with further structural modifications.
Motivated by the structural characteristics of NNC-05-2045 (5),
compound series 10a,b,d and 11a,c,d (Fig. 3) bearing a 4-methoxy-
phenyl group at the 4-position of the pyrrolidine ring were pre-
GAT3: 14 µM32
Figure 1. Representative known GABA uptake inhibitors (potencies are given as
IC50).
pared in
a stereoselective pure form. Since our previous
investigations had shown that 4-hydroxypyrrolidine derivatives
substituted with the 3-(carbazol-9-yl)propyl residue of NNC-05-
2045 (5, Fig. 1) exhibited only weak GAT inhibition,35 we decided
to employ only the lipophilic N-substituents b and c which are
typical for GAT1 inhibitors (Fig. 3). Residue d was chosen (Fig. 3)
as it is known to enhance GAT3 activity, as for example in the case
of (S)-SNAP-5114 (4).
Previously, we reported the synthesis and biological evaluation
of all stereoisomers of the pyrrolidine derivatives 6 to 9b–d (Figs. 2
and 4) as potential GABA uptake inhibitors.34,35 The compounds 6
and 7 which in contrast to 8 and 9 (Fig. 4) are devoid of a substitu-
ent in 4-position exhibited high selectivity for and potency at GAT1
and GAT3 depending on their stereo configuration and structure of
their N-substituent (Fig. 2; best compounds at GAT1: (S)-6b, (S)-
OCH3
OCH3
H3CO
H3CO
OH
OH
HO
HO
O
O
O
O
N
R
N
R
N
R
N
R
OH
OH
OH
OH
(2R,4S)-10a,b,d,e
(2R,4R)-10a,b,d,e
(2S,4S)-10a,b,d,e
(2S,4R)-10a,b,d,e
OCH3
OCH3
H3CO
H3CO
OH
O
OH
O
HO
O
HO
O
OH
OH
OH
OH
N
R
N
R
N
R
N
R
(2S,4S)-11a,c-e
(2S,4R)-11a,c-e
(2R,4S)-11a,c-e
(2R,4R)-11a,c-e
R:
a
b
c
d
e
H3CO
H
O
O
S
S
O
H3C
CH3
H3CO
OCH3
Figure 3. Structures of the target compounds.

472
X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
at the amino nitrogen, were obtained only in very low yields (10–
R'
HO
27%) by Swern oxidation of (2S,4R)-12e and (2S,4R)-15e, respectively.
Thus, compounds (S)-13e and (S)-16e were finally prepared by Jones
oxidation according to literature procedures.36,37
O
N
R
OH
GAT-1
GAT-3
R'=
R=
Similar to the aforementioned syntheses, the enantiomers
(R)-13b,d,e and (R)-16c–e were obtained from diastereomers
(2R,4R)-12b,d,e and (2R,4R)-15c–e, respectively (Schemes 2 and 3).
(2R,4R)-8b
(2R,4R)-8b
(2R,4S)-8b
(2R,4S)-8b
H
((22SS,4,4SS))--88bb
(2S,4R)-8b
(2S,4R)-8b
OCH3
(2R,4R)-10b
(2R,4R)-10b
2.2. Organometallic addition reactions
(2R,4S)-10b
(2R,4S)-10b
(2S,4S)-10b
(2S,4S)-10b
(2S,4R)-10b
(2S,4R)-10b
For the preparation of the 4-methoxyphenyl substituted deriv-
atives (2S,4R)-14b,d and (2S,4S)-14b,d as well as of (2S,4R)-17c,d
and (2S,4S)-17c,d, (4-MeOC6H4)MgBr was added to the 4-oxopro-
line derivatives (S)-13b,d and 4-oxopyrrolidin-2-ylacetic acid
derivatives (S)-16c,d yielding in each case the (2S,4S)-configured
diastereomers (2S,4S)-14b,d and (2S,4S)-17c,d as major products
( Schemes 2 and 3). With ratios of 89:11 [(2S,4S)-14b/(2S,4R)-
14b], 85:15 [(2S,4S)-14d/(2S,4R)-14d], 87:13 [(2S,4S)-17c/(2S,4R)-
17c] and 81:19 [(2S,4S)-17d/(2S,4R)-17d], respectively, the
diastereoselectivities were quite pleasing, but the yields of the
addition reaction were with 45–62% only moderate.39
Numerous examples exist, according to which the presence of
cerium trichloride may significantly alter the stereoselectivity of
Grignard addition reactions to ketones.40–43 Therefore,, a mixture
of 4-MeOC6H4MgBr and CeCl3 was added to (S)-13b,d and (S)-
16c,d in order to reverse the asymmetric induction (general proce-
dure 3A in Section 6.3). This time, indeed, the diastereomers
(2S,4R)-14b,d (Scheme 2) and (2S,4R)-17c,d, (Scheme 3) to which
the nucleophile had added from the side opposite to the ester func-
tion were formed as major products (ds 52:48–17:83).
Addition reactions to the N-Cbz-protected compounds (S)-13e
and (S)-16e showed compared to the N-alkyl substituted com-
pounds (S)-13b,d and (S)-16c,d a different outcome concerning
asymmetric induction. Here addition of (4-MeOC6H4)MgBr yielded
only the (2S,4R)-configured diastereomers (2S,4R)-14e and (2S,4R)-
17e (Scheme 2 and 3) as single diastereomers (ds >2:98) which
could be easily isolated in pure form (yield 38–56%). The sense of
asymmetric induction remained this time unchanged when CeCl3
was used as additive in the addition reaction.
For the preparation of the enantiomeric reaction products re-
quired for the biological testing an identical reaction sequence
was followed, in which, however, the starting materials had been
replaced by their enantiomeric counter parts, (R)-13b,d,e and (R)-
16c–e. The results obtained for these transformations were similar
to those of the original sequence.
(2R,4R)-8d
(2R,4R)-8d
(2R,4S)-8d
(2R,4S)-8d
H
H
CO
3
((22SS,,44SS))--88dd
(2S,4R)-8d
(2S,4R)-8d
O
OCH3
((22RR,,44RR))--1100dd
H
CO
3
(2R,4S)-10d
(2R,4S)-10d
OCH
3
(2S,4S)-10d
(2S,4S)-10d
((22SS,,44RR))--1100dd
R'
HO
O
OH
N
R
(2R,4R)-9c
(2R,4R)-9c
((22RR,,44SS))--99cc
H
(2S,4S)-9c
(2S,4S)-9c
(2S,4R)-9c
(2S,4R)-9c
S
OCH3
(2R,4R)-11c
(2R,4R)-11c
H3
C
((22RR,,44SS))--1111cc
CH3
S
(2S,4S)-11c
(2S,4S)-11c
(2S,4R)-11c
(2S,4R)-11c
((22RR,,44RR))--99dd
R
S 9d
(2R,4S)-9d
H
H3CO
(2S,4S)-9d
(2S,4S)-9d
(2S,4R)-9d
(2S,4R)-9d
O
OCH3
(2R,4R)-11d
(2R,4R)-11d
H3CO
(2R,4S)-11d
(2R,4S)-11d
OCH3
((22SS,,44SS))--1111dd
(2S,4R)-11d
(2S,4R)-11d
0
0
70
4I0C506[µM8]0
10 20 30
50
90
100 110 120
Figure 4. Diagram with IC50 values of the target compounds 10 and 11 compared to
the values of compounds 8 and 935 with no aryl substituent in 4-position. Values of
>100 lM are given as 100 lM.
2. Chemistry
The proline and pyrrolidine derivatives 12b,d,e and 15c–e
(Schemes 2 and 3), the syntheses of which had been described
previously,35–37 were chosen as starting materials to prepare the
target compounds.
The determination of the configuration of all of the newly gen-
erated compounds is described in Section 2.5.
2.3. Hydrolysis
The reaction routes to all of the target compounds are outlined
in Schemes 2 and 3. They comprise the following steps: (1)
oxidation of the 4-hydroxy group, (2) subsequent addition of an
organometallic reagent to the resulting keto group, (3) basic hydro-
lysis, and (4), in the case of Cbz-protected compounds, additionally
reductive removal of the Cbz group.
Each of the diastereomerically and enantiomerically pure prod-
ucts 14b,d and 17c,d was subjected to an alkaline hydrolysis in
methanol or ethanol, to give all stereoisomeres of the target
compounds 10b,d and 11c,d in 70–98% yield. Besides, also the
N-Cbz-protected carboxylic acids (2S,4R)-10e, (2S,4R)-11e and
2.1. Oxidation
OCH
3
OCH
OCH
O
3
3
Swern oxidation38 of the compounds (2S,4R)-12b,d and (2S,4R)-
15c,d provided the corresponding 4-oxoproline derivatives
(S)-13b,d as well as the 2-(4-oxopyrrolidin-2-yl)acetic acid deriva-
tives (S)-16c,d in 83–89% yields (Schemes 2 and 3). The C–O bond
present in the lipophilic side chain d was not affected under these
reaction conditions, even though it is highly sensitive to acids. The
Cbz-protected derivatives (S)-13e and (S)-16e, which were re-
quired to access the corresponding amino acids lacking a substituent
HO
i
ii
O
HO
HO
O
CH3
CH3
CH3
O
O
O
n
N
n
n
N
H
N
R
Cbz
n=1 (2S,4R)-17e
n=0 (2S,4R)-14e
(2S,4R)-18
(2S,4R)-19
(2S,4R)-17c
(2S,4R)-14b
Scheme 1. (i) H2/10%Pd-C, TEA, MeOH, rt; (ii) K2CO3, KI, RBr (R = c in the case of
(2S,4R)-18 and R = b in the case of (2S,4R)-19).

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
473
HO
O
N
R
(2R,4R)-12b,d,e
O
CH3
(2S,4R)-12b,d,e
i or ii
i or ii
O
O
(R)-13b,d,e
N
R
O
CH3
(S)-13b,d,e
iii or iv
iii or iv
OH
H3CO
OH
HO
(2R,4S)-14b,d,e
(2R,4R)-14b,d
+
O
O
+
N
R
N
R
O
O
CH3
CH3
v
v
(2S,4S)-14b,d
(2S,4R)-14b,d,e
v
v
OCH3
OCH3
H3CO
H3CO
OH
OH
HO
HO
O
O
O
O
N
R
N
R
N
R
N
R
OH
OH
R = e
OH
OH
R = e
(2S,4S)-10b,d
(2S,4R)-10b,d,e
(2S,4R)-10a
(2R,4S)-10a,b,d
(2R,4S)-10a
(2R,4R)-10b,d
vi
vi
Scheme 2. Scheme for the synthesis of the compound series 10. (i) Swern oxidation (81–92%); (ii) Jones oxidation (71–77%); (iii) 4-MeOC6H4MgBr/CeCl3, ꢀ60 to ꢀ78 °C, THF
(32–56%); (iv) 4-MeOC6H4MgBr, ꢀ78 °C, Et2O (37–65%); (v) 1.0 M NaOH (2–3 equiv), MeOH or EtOH, rt (73–98%); (vi) H2/10% Pd-C, TEA, MeOH, rt (91–100%).
HO
O
CH3
O
N
R
(2R,4R)-15c-e
(2S,4R)-15c-e
i or ii
i or ii
O
O
CH3
O
N
R
(R)-16c-e
(S)-16c-e
iii or iv
iii or iv
OH
H3CO
OH
HO
O
O
CH3
CH3
(2R,4S)-17c-e
(2R,4R)-17c,d
+
O
O
+
N
R
N
R
(2S,4S)-17c,d
(2S,4R)-17c-e
v
v
v
v
OCH3
OCH3
H3CO
H3CO
OH
OH
HO
HO
O
O
O
O
OH
OH
OH
OH
N
R
N
R
N
R
N
R
R = e
R = e
vi
(2S,4S)-11c,d
(2S,4R)-11c-e
(2S,4R)-11a
(2R,4S)-11c-e
(2R,4S)-11a
(2R,4R)-11c,d
vi
Scheme 3. Scheme for the synthesis of the compound series 11. For reaction conditions see Scheme 1.

474
X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
their enantiomers were obtained by alkaline hydrolysis from com-
pounds (2S,4R)-14e and (2R,4S)-14e, (2S,4R)-17e and (2R,4S)-17e,
with yields in the range of 80–91%.
cbsP2C) and finally porcine brain material (pfcP2B, pbsP2C)
was temporarily used for this test system instead of the
bovine brain material. The reliability of the newly developed
assay systems was evaluated using
a series of reference
2.4. Hydrogenolysis
compounds. Results were found to be in accordance with both,
the previously used assay based on bovine brain material as well
as the alternative assay systems published by other research
groups.
For the preparation of the N-unsubstitued amino acids (2S,4R)-
10a and (2R,4S)-10a, (2S,4R)-11a and (2R,4S)-11a the N-Cbz-pro-
tected amino acids (2S,4R)-10e and (2R,4S)-10e, (2S,4R)-11e, and
(2R,4S)-11e were subjected to catalytic hydrogenation over Pd-C
in the presence of TEA, affording the target compounds in high
yields (92–100%).
4. Results and discussion
The parent compounds (2S,4R)-10a and (2R,4S)-10a, (2S,4R)-
11a and (2R,4S)-11a with no lipophilic residue at the nitrogen were
found to be devoid of significant inhibitory potency at GAT1 as well
2.5. Stereochemical assignment for the compounds obtained by
organometallic additions
as GAT3, even when tested at a concentration of 100
Tables 1 and 2).
lM (see
To allow a better comparison of the biological data from Tables
1 and 2 with the already published35 affinities of compounds 8 and
9 bearing no aryl substituent in 4-position, these data are quoted in
this manuscript, too (Fig. 4). The bar diagrams (Fig. 4) display the
effects on the selectivities for and the potencies at GAT1 and
GAT3, when a 4-methoxyphenyl group is present in 4-position of
the 4-hydroxypyrrolidine and 4-hydroxyproline derivatives as
compared to compounds 8 and 9. For compounds which at a con-
NOE experiments performed on the sodium salt of (2S,4R)-11a
(Scheme 3)44 revealed the aromatic ring in 4-position being trans
to the carboxylic acid ester function. Given that the stereocenter
in 2-position must be S-configured, resulting from the stereo-
chemistry of the starting material (2S,4R)-15e used, the 4-position
must possess R-configuration. Taking into account that (2S,4R)-
11e and (2S,4R)-17e are synthetic precursors of (2S,4R)-11a and
(2R,4S)-11a, (2R,4S)-11e and (2R,4S)-17e are their enantiomeric
counter parts led to the stereochemistry of these compounds to
be either identical with the stereochemistry of (2S,4R)-11a or
opposite to it. In the next step, the N-Cbz protected product
(2S,4R)-17e with now known stereochemistry was transformed
into (2S,4R)-17c displaying a 4,4-bis(3-methylthiophen-2-yl)but-
3-en-1-yl moiety by N-deprotection and subsequent N-alkylation
(Scheme 1). The thus obtained compound (2S,4R)-17c was identi-
cal with the major isomer formed upon addition of 4-
MeOC6H4MgBr/CeCl3 to (S)-16c. By comparison of the 1H NMR
spectra of (2S,4R)-17c and (2S,4R)-17d the stereochemistry of
the latter compound could be identified to be identical to that
of the former compound. The stereochemistry of the remaining
stereoisomers of 17c, 17d, 11c, and 11d could, finally, be assigned
considering their stereochemical relation to (2S,4R)-17c and
(2S,4R)-17d, being either diastereomers or enantiomers of these
compound depending on their syntheses.
Following the same strategy, the stereochemistry could also
be assigned to the individual stereoisomers of 10 and 14
(Scheme 2). This time, however, the NOE experiments for the
determination of the relative configuration were performed on
the N-deprotected compound (2S,4R)-19. Taking into account
the results of these NOE experiments together with the fact that
(2S,4R)-19 had been delineated from (2S,4R)-12e exhibiting (2R)-
configuration the (2S,4R)-configuration of (2S,4R)-19 became
apparent. Serving as a precursor in the synthesis of (2S,4R)-19
the same absolute configuration had to be assigned to (2S,4R)-
14e. The stereochemical relation between the isomer (2S,4R)-
centration of 100 lM in preliminary GABA uptake experiments
remaining GABA uptake has been reduced to no less than 50% no
IC50 values have been determined (in Tables 1 and 2 listed as
IC50 >100
listed in Figure 4 with IC50s = 100
l
M). For the sake of simplicity, these compounds are
M.
l
For the 4,4-diphenylbut-3-en-1-yl substituted 4-hydroxypro-
lines 8b and the 4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl (c)
substituted 4-hydroxypyrrolidin-2-ylacetic acids 9c (Fig. 4) with
N-residues typical for GAT1 inhibitors, introduction of
a 4-
methoxyphenyl group resulting in 10b and 11c, respectively, led
either to no enhancement or even a significant decrease in the
inhibitory effect on GAT1 and GAT3 (IC50 >100 lM for all isomers
of 10b and 11c at GAT1 and GAT3; Tables 1 and 2, Fig. 4). Especially
the good potencies at and selectivities for GAT1 of (2S,4R)-9c of
series 9c (Fig. 4) was not reflected in any of the 4-aryl substituted
analogues of 11c (Table 2).
A somewhat different picture emerged for compounds 8d and
9d exhibiting a tris(4-methoxyphenyl)methoxyethyl moiety (d)
known from (S)-SNAP-5114 and analogous compounds to enhance
GAT3 potency. Upon introduction of a 4-(4-methoxyphenyl) resi-
due in the pyrrolidine ring the inhibitory potency at GAT1 proteins
increases, all isomers of 10d and 11d being more potent than their
analogues 8d and 9d devoid of the aforementioned 4-substituent
(Fig. 4).
Furthermore, all isomers of series 10d with exception of
(2R,4R)-10d showed also improved activity at GAT3 as compared
to compounds 8d (Fig. 4). Thereby, the cis-isomers (regarding the
4-hydroxy and 2-carboxy function) (2R,4S)-10d (IC50 at GAT3:
14e exhibiting
a N-Cbz protective group and the N-alkyl
38.0
inhibitors, at GAT3, than the corresponding trans-isomers
(2R,4R)-10d and (2S,4S)-10d (IC50 = 63.3 M and >100 M, respec-
tively). Of this series of compounds (2S,4R)-10d showed the best
uptake inhibitory potency, but not only at GAT3 (IC50 = 29.7 M),
but also at GAT1 (IC50 = 45.1 M).
lM) and (2S,4R)-10d (IC50 at GAT3: 29.7 lM) are stronger
derivatives 14b,d and thus also of the free amino acids 10b,d
was established via the 4-bromo-1,1-diphenylbutenyl substituted
compound (2S,4R)-14b (see Scheme 1).
l
l
l
3. Biological tests
l
Whereas for all isomers of 9d with no aryl group in 4-position the
inhibitory potency at GAT1 increases when switching to the 4-aryl-
substituted compounds 11d, the situation is less consistent for
GAT3. Thus, the potencies of the isomers (2R,4S)-11d and (2S,4S)-
11d are worse at GAT3 than those of the enantiomers 9d lacking a
4-aryl-substituent. In contrast, the potencies of (2S,4R)-11d and
(2R,4R)-11d are better at GAT3. Interestingly, albeit bearing a lipo-
philic residue typical for GAT3 selective inhibitors, all compounds
The final compounds 10a,b,d and 11a,c,d were tested for their
inhibitory potency at GAT1 and GAT3. For the evaluation of the
activity of the test compounds at the GABA transport proteins, an
assay developed in our group has been employed that is based
on subcellular membrane fractions from frontal cortex (bfcP2B)
and brain stem (bbsP2C) of bovine brain.45 Due to the emergence
of bovine spongiform encephalitis in Germany, calf brain (cfcP2B,

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
475
Table 1
Results of the biological testing of the compound series 10
OCH3
OCH3
H3CO
H3CO
OH
OH
HO
HO
O
O
O
O
N
N
R
N
R
N
R
OH
R
OH
OH
OH
R
IC50 SEM (lM)
H
GAT1
GAT3
—
>100c
>100d
>100c
–
>100d
(2S,4R)-10a
(2R,4S)-10a
>100a
>100b
(2S,4S)-10b
>100a
>100e
>100e
>100f
(2R,4R)-10b
GAT1
GAT3
237 160b
(2S,4R)-10b
>100f
(2R,4S)-10b
H CO
3
68.1 4.4a
63.3 5.8b
(2S,4S)-10d
45.1 2.7a
29.7 0.8b
(2S,4R)-10d
56 4.3c
71 1.4c
>100b
(2R,4R)-10d
O
GAT1
GAT3
38.0 5.8d
(2R,4S)-10d
H CO
3
OCH
3
Footnotes a–f specify the used biological materials: abfcP2B, bbbsP2C, cpfcP2B, dpbsP2C, ecfcP2B, fcbsP2C. All the results of the measurements were processed and evaluated in
triplicate unless otherwise stated. Each IC50 was given as mean SEM. For compounds where at a concentration of 100
l
M in preliminary GABA uptake experiments
remaining GABA uptake has been reduced to no less than 50% no IC50 values have been determined (IC50 >100 lM).
Table 2
Results of the biological testing of the compound series 10
OCH3
OCH3
H3CO
H3CO
OH
OH
O
O
HO
HO
O
O
OH
OH
OH
OH
N
R
N
R
N
R
N
R
R
IC50 SEM (
lM)
H
GAT1
GAT3
—
>100c
>100d
>100c
—
>100d
(2S,4R)-11a
(2R,4S)-11a
>100c
>100c
114 12.3c
>100d
(2R,4S)-11c
>100c
S
GAT1
GAT3
122 9d
124 15d
(2S,4R)-11c
>100d
(2S,4S)-11c
(2R,4R)-11c
H3C
CH3
S
H CO
3
75.2 8.8c
78.9 18.5d
(2S,4S)-11d
48.4 1.6c
66.7 11.2d
(2S,4R)-11d
59.2 4.5c
66.1 3.5d
(2R,4S)-11d
64.5 3.0c
65.4 5.1d
(2R,4R)-11d
O
GAT1
GAT3
H CO
3
OCH
3
Footnotes a–f specify the used biological materials: abfcP2B, bbbsP2C, cpfcP2B, dpbsP2C, ecfcP2B, fcbsP2C. All the results of the measurements were processed and evaluated in
triplicate unless otherwise stated. Each IC50 was given as mean SEM. For compounds where at a concentration of 100 M in preliminary GABA uptake experiments
remaining GABA uptake has been reduced to no less than 50% no IC50 values have been determined (IC50 >100 M)
l
l
of series 11d show only marginal to small differences with regard to
their potencies at both transporters (GAT1 and GAT3).
Besides, it should be noted that the differences of the proline
derivatives 10d and their homologues 11d with respect to
their inhibitory potencies at GAT1 and GAT3 are not very large
either.
5. Conclusion
Herein, we reported the synthesis and biological evaluation of
optically pure 4-(4-methoxyphenyl) substituted 4-hydoxyproline
and 4-hydroxypyrrolidin-2-ylacetic acid derivatives. The results
of the biological tests showed that none of the tested compounds

476
X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
exhibits very strong inhibitory activity at GAT1 or GAT3. The pres-
ence of a 4-methoxyphenyl unit in 4-position is clearly detrimental
for stereoisomers of series 10b and 11c as the inhibitory potency at
both GAT1 and GAT3 is far lower than that of the analogous com-
pounds 8b and 9c lacking this substituent. However, for the stereo-
isomers of the proline derivatives 10d (except stereoisomer
(2R,4R)-10d), the presence of this moiety in 4-position is beneficial
as it gives rise to improved inhibitory potencies at GAT3 and at
GAT1 as compared to compounds of series 8d. In most cases, this
is also true for series 11d compared to 9d. Especially for GAT3
for which potent uptake inhibitors are still missing, appropriately
4-substituted proline and pyrrolidin-2-ylacetic acid derivatives
might be a promising starting point for the search of new and
highly potent GAT3 inhibitors.
(3.7 mL, 3.2 mmol); purification by CC (n-heptane/acetone, 4:1).
Yield: 312 mg (87%) as a colorless oil. ½a D25
ꢃ
ꢀ32.4 (c 1.26, EtOH).
~
IR:
m
= 1761, 1737 cmꢀ1; MS (m/z): 350 [M+1]+. 1H NMR (CDCl3)
d: 2.26 (q, 2H, J = 7.4 Hz, NCH2CH2), 2.43 (dd, 1H, J = 18.0, 5.5 Hz,
NCHCH2), 2.54–2.62 (m, 2H, NCHCH2 and NCH2CH2), 2.77 (dt, 1H,
J = 12.1, 7.4 Hz, NCH2CH2), 2.91 (d, 1H, J = 17.2 Hz, NCH2CO), 3.29
(d, 1H, J = 17.2 Hz, NCH2CO), 3.65 (s, 3H, OCH3), 3.70 (dd, 1H,
J = 7.8, 5.5 Hz, NCH), 6.03 (t, 1H, J = 7.4 Hz, @CHCH2), 7.07–7.34
(m, 10H, Haromat). Calcd for C22H23NO3 (349.43): C, 75.62; H,
6.64; N, 4.01. Found: C, 75.79; H, 6.75; N, 3.73.
6.1.2. Methyl (2R)-1-(4,4-diphenylbut-3-en-1-yl)-4-
oxopyrrolidine-2-carboxylate [(2R)-13b]
From DMSO (62 mg, 0.79 mmol) and (COCl)2 (50 mg,
0.40 mmol) in CH2Cl2 (2 mL), (2R,4R)-12b (92 mg, 0.26 mmol) in
CH2Cl2 (2 mL); TEA (83 mg, 0.82 mmol), aq 1.0 M NaOH (0.80 mL,
0.80 mmol); purification by CC (n-heptane/EtOAc, 4:1). Yield:
6. Experimental part
78 mg (85%) as a colorless oil. ½a D20
ꢃ
+33.2 (c 0.60, EtOH). The spectra
In general, tetrahydrofuran, ether and diisopropylamine were
distilled from sodium under nitrogen. Triethylamine (TEA) was re-
fluxed with benzoyl chloride, followed by distillation under nitro-
gen. Other common solvents for recrystallization, column
chromatography, analytical HPLC, and preparative HPLC were dis-
tilled prior to use. Purchased chemical reagents were used without
further purification. TLC plates were made from silica gel 60 F254 on
aluminum sheets (Merck). Compounds were stained with a solu-
tion of 5% (NH4)6Mo7O24ꢁ4H2O, 0.2% Ce(SO4)2ꢁ4H2O, and 5% concd
H2SO4. If not stated otherwise, Merck silica gel (mesh 230–400)
was used as stationary phase for flash chromatography. Analytical
(1H NMR, IR, and MS) were identical with those of (2S)-13b. Calcd
for C22H23NO3 (349.43): C, 75.62; H, 6.64; N, 4.01. Found: C, 75.51;
H, 6.66; N, 3.87.
6.1.3. Methyl (2S)-4-oxo-1-{2-[tris(4-methoxyphenyl)methoxy]-
1-ethyl}pyrrolidine-2-carboxylate [(2S)-13d]
From DMSO (72 mg, 0.92 mmol) and (COCl)2 (61 mg,
0.46 mmol) in CH2Cl2 (2 mL), (2S,4R)-12d (160 mg, 0.307 mmol)
in of CH2Cl2 (1.5 mL), TEA (108 mg, 1.07 mmol), aq 0.85 M KOH
(1.2 mL, 1.0 mmol); purification by CC (Al2O3, pH 7.5 0.5; n-hep-
tane/EtOAc, 1:1). Yield: 132 mg (83%) as a colorless viscous oil.
~
HPLC: Column LiChrospher Si 60 (5
umn 4 ꢂ 4 mm). Prep. (preparative) HPLC: Column LiChrospher Si
60 (7
lm, 250 ꢂ 4 mm with precol-
½
a 2D9
ꢃ
ꢀ6.4 (c 1.83, EtOAc). IR:
m
= 1754, 1604 cmꢀ1. MS (m/z): 333
[Ar3C]+. 1H NMR (CDCl3) d: 2.48 (dd, 1H, J = 18.0, 5.5 Hz, NCHCH2),
2.65 (dd, 1H, J = 18.0, 7.8 Hz, NCHCH2), 2.83 (dt, 1H, J = 13.0, 5.7 Hz,
NCH2CH2), 2.94 (dt, 1H, J = 13.0, 5.7 Hz, NCH2CH2), 3.13 (d, 1H,
J = 17.4 Hz, NCH2CO), 3.24 (t, 2H, J = 5.7 Hz, NCH2CH2), 3.44 (d,
1H, J = 17.4 Hz, NCH2CO), 3.72 (s, 3H, COOCH3), 3.78 (s, 9H,
ArOCH3), 3.84 (dd, 1H, J = 7.8, 5.5 Hz, NCH), 6.79–6.83 (m, 6H,
l
m, 250 ꢂ 25 mm). Optical rotations: Polarimeter 241 MC
at k 589 cmꢀ1. Melting points (uncorrected) were determined on
a Büchi 510 Melting Point apparatus. Elementary analysis: Elemen-
taranalysator Rapid (Heraeus). IR spectroscopy: FT-IR Spectrome-
ter 1600 and Paragon 1000 (Perkin Elmer), oils as film, solid
samples as pellets for measurements. Mass spectroscopy: Mass
Spectrometer 5989 A with 59980 B particle beam LC/MS interface
(Hewlett Packard). NMR spectroscopy: NMR spectra were recorded
on JNMR-GX (Jeol, 400 and 500 MHz) with TMS as internal stan-
dard and integrated with the program of NMR-software Nuts (2D
Version 5.097, Acorn NMR, 1995).
Haromat), 7.29–7.32 (m, 6H, Haromat). Calcd for C30H33NO7 (519.59):
C, 69.35; H, 6.40; N, 2.70. Found: C, 69.53; H, 6.59; N, 2.33.
6.1.4. Methyl (2R)-4-oxo-1-{2-[tris(4-
methoxyphenyl)methoxy]-1-ethyl}pyrrolidine-2-carboxylate
[(2R)-13d]
From DMSO (93 mg, 1.2 mmol) and (COCl)2 (75 mg, 0.59 mmol)
in CH2Cl2 (5 mL), (2R,4R)-12d (206 mg, 0.395 mmol) in CH2Cl2
(5 mL); TEA (132 mg, 1.30 mmol), aq 1.0 M NaOH (1.2 mL,
1.2 mmol); purification (n-heptane/acetone = 3:1). Yield: 183 mg
6.1. General procedure 1 for the preparation of the 4-
oxopyrrolidine derivatives (2S)-13b,d, (2R)-13b,d, (2R)-16c,d and
(2R)-16c,d by Swern oxidation
20
(89%) as a colorless oil. ½alphaꢃD +4.3 (c 1.5, EtOAc). The spectra
(1H NMR, IR, and MS) were identical with those of (2S)-13d. Calcd
for C30H33NO7 (519.59): C, 69.35; H, 6.40; N, 2.70. Found: C, 69.42;
H, 6.75; N, 2.82.
To a solution of (COCl)2 (1.5 equiv) in CH2Cl2 at ꢀ78 °C, DMSO
(3 equiv) was added dropwise to generate an oxidizing reagent.
After 15 min, the respective 4-hydroxypyrrolidine derivative of
(2S,4R)-12b,d, (2R,4R)-12b,d, (2S,4R)-15c,d, and (2R,4R)-15c,d
(1 equiv) in CH2Cl2 was added to the oxidizing solution at
ꢀ78 °C. After 15 min (10 min for the derivatives with side chain
b) between ꢀ78 °C and –70 °C, TEA (3.2 equiv) was added. The stir-
ring was continued at rt for 15 min (10 min for the derivative with
side chain b). After adding water and aq KOH or NaOH (3.2 equiv),
the organic layer was isolated and the aqueous phase was ex-
tracted with CH2Cl2. The combined organic layers were washed
with water, dried (MgSO4), and concentrated in vacuum. The resid-
ual oil was purified by CC (column chromatography).
6.1.5. Methyl (2S)-4-oxo-1-{2-[tris(4-methoxyphenyl)methoxy]-
1-ethyl}pyrrolidin-2-acetate [(2S)-16d]
From (COCl)2 (65 mg, 0.51 mmol) and DMSO (80 mg, 1.0 mmol)
in CH2Cl2 (3 mL), (2S,4R)-15d (183 mg, 0.342 mmol) in CH2Cl2
(3 mL); TEA (114 mg, 1.14 mmol), aq 1.0 M NaOH (1.1 mL,
1.1 mmol); purification by CC (n-heptane/acetone = 3:2). Yield:
148 mg (81%) as a colorless oil. ½a D20
ꢃ
ꢀ45.2 (c 0.95, EtOAc). IR:
= 1756, 1738 cmꢀ1. MS (m/z): 333 [Ar3C]+. 1H NMR (CDCl3) d:
~
m
2.23 (dd, 1H, J = 18.4, 9.3 Hz, CH2COO), 2.41 (dd, 1H, J = 15.5,
8.8 Hz, CH2CHCH2COO), 2.49 (dt, 1H, J = 12.7, 5.7 Hz, NCH2CH2),
2.60 (dd, 1H, J = 18.4, 6.7 Hz, CH2COO), 2.79 (d, 1H, J = 17.7 Hz,
NCH2CO), 2.82 (dd, 1H, J = 15.5, 3.8 Hz, CH2CHCH2COO), 3.01–
3.07 (m, 1H, NCH2CH2), 3.12–3.28 (m, 3H, NCH and 2H of
NCH2CH2), 3.45 (d, 1H, J = 17.7 Hz, NCH2CO), 3.65 (s, 3H, COOCH3),
3.77 (s, 9H, ArOCH3), 6.78–6.84 (m, 6H, Haromat), 7.28–7.32 (m, 6H,
6.1.1. Methyl (2S)-1-(4,4-diphenylbut-3-en-1-yl)-4-
oxopyrrolidine-2-carboxylate [(2S)-13b]
From DMSO (227 mg, 2.91 mmol) and (COCl)2 (193 mg,
1.46 mmol) in CH2Cl2 (4.5 mL), (2S,4R)-12b (357 mg, 0.970 mmol)
in CH2Cl2 (1.5 mL); TEA (304 mg, 3.00 mmol), aq 0.85 M KOH

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
477
Haromat). Calcd for C31H35NO7 (533.62): C, 69.78; H, 6.61; N, 2.62.
Found: C, 69.29; H, 6.75; N, 2.54.
(2R,4R)-15e (1 equiv) in acetone (15 mL), chromic acid in aq
H2SO4 (2.67 M, 8.50 equiv) was added at rt for 5 min. The brown
mixture was stirred at rt for 30 min followed by the addition of
MeOH (9 equiv) to destroy excessive oxidizing agent. The superna-
tant was decanted, diluted by CH2Cl2 (70 mL), washed with sat. aq
NH4Cl, dried (MgSO4) and concentrated in vacuo. The residual oil
was purified by CC (n-heptane/acetone, 3:1).
6.1.6. Methyl (2S)-1-[4,4-bis(3-methylthiophen-2-yl)but-3-en-1-
yl]-4-oxopyrrolidin-2-acetate [(2S)-16c]
From (COCl)2 (56 mg, 0.44 mmol) and DMSO (69 mg,
0.87 mmol) in CH2Cl2 (2.5 mL), (2S,4R)-15c (118 mg, 0.291 mmol)
in CH2Cl2 (2.5 mL); TEA (98 mg, 0.96 mmol), aq 1.0 M NaOH
(0.92 mL, 0.92 mmol); purification by CC (n-heptane/acetone,
6.2.1. Methyl (2S)-1-benzyloxycarbonyl-4-oxopyrrolidine-2-
carboxylate [(2S)-13e]
3:1). Yield: 108 mg (92%) as a slightly yellow oil. ½a D20
ꢃ
+68.3 (c
~
0.75, EtOAc). IR:
m
= 1760, 1732 cmꢀ1. MS (m/z): 404 [M+1]+. 1H
From (2S,4R)-12e (500 mg, 1.79 mmol) in acetone (28 mL),
2.67 M chromic acid in aq H2SO4 (1.90 mL, 15.2 mmol). Yield:
NMR (CDCl3) d: 2.00 (s, 3H, thienyl-CH3), 2.02 (s, 3H, thienyl-
CH3), 2.23–2.43 (m, 5H, CH2COO, NCH2CH2, CH2CHCH2COO and
2H of NCH2CH2), 2.59–2.66 (m, 2H, NCH2CO and CH2COO), 2.80
(dd, 1H, J = 15.4, 3.9 Hz, CH2CHCH2COO), 2.99 (dt, 1H, J = 11.6,
7.9 Hz, NCH2CH2), 3.17–3.23 (m, 1H, NCH), 3.35 (d, 1H,
J = 17.5 Hz, NCH2CO), 3.67 (s, 3H, OCH3), 6.05 (t, 1H, J = 7.3 Hz,
@CHCH2), 6.76 (d, 1H, J = 5.2 Hz, SCH), 6.84 (d, 1H, J = 5.2 Hz,
SCH), 7.05 (d, 1H, J = 5.2 Hz, SCH@CH), 7.21 (d, 1H, J = 5.2 Hz,
SCH@CH). Calcd for C21H25NO3S2 (403.560): C, 62.50; H, 6.24; N,
3.47. Found: C, 62.25; H, 5.97; N 3.40.
354 mg (71%); colorless oil. ½a D20
ꢃ
+17.8 (c 1.10, CHCl3) {Lit.30b
:
½
a 2D0
+18.5 (c 1.0, CHCl3)}.
ꢃ
6.2.2. Methyl (2R)-1-benzyloxycarbonyl-4-oxopyrrolidine-2-
carboxylate [(2R)-13e]
From (2R,4R)-12e (800 mg, 2.87 mmol), 2.67 M chromic acid in
aq H2SO4 (3.04 mL, 24.3 mmol). Yield: 623 mg (78%); colorless oil.
½
a 2D0
ꢃ
ꢀ18.8 (c 1.31, CHCl3) {lit.30b: ½a D20
ꢀ19.4 (c 1.0, CHCl3)}.
ꢃ
6.1.7. Ethyl (2R)-4-oxo-1-{2-[tris(4-methoxyphenyl)methoxy]-
1-ethyl}pyrrolidin-2-acetate [(2R)-16d]
6.2.3. Methyl (2S)-1-benzyloxycarbonyl-4-oxopyrrolidin-2-
acetate [(2S)-16e]
From (COCl)2 (69 mg, 0.54 mmol) and DMSO (85 mg, 1.1 mmol)
in CH2Cl2 (2 mL), (2R,4R)-15d (149 mg, 0.271 mmol) in CH2Cl2
(2.5 mL); TEA (121 mg, 1.19 mmol), aq 1.0 M NaOH (1.1 mL,
1.1 mmol); purification by CC (n-heptane/acetone, 3:2). Yield:
From (2S,4R)-15e (300 mg, 1.02 mmol) in acetone (16 mL),
2.67 M chromic acid in aq H2SO4 (1.08 mL, 8.7 mmol). Yield:
225 mg (76%) as colorless crystals. mp 53–54 °C. ½a D20
ꢃ
: +10.7 (c
~
1.06, CHCl3). IR:
m
= 1765, 1732 cmꢀ1. MS (m/z): 292 [M+1]+. 1H
130 mg (87%) as a pale yellow oil. ½a 20
ꢃ
578: +56.9 (c 1.53, EtOAc).
NMR (C6D5NO2, 120 °C) d: 2.59 (d, 1H, J = 18.4 Hz, CH2COO),
2.83–2.99 (m, 3H, CH2COO and 2H of CH2CHCH2COO), 3.63 (s,
3H, OCH3), 3.88 (d, 1H, J = 18.1 Hz, NCH2), 4.02 (d, 1H, J = 18.1 Hz,
NCH2), 4.75–4.82 (m, 1H, NCH), 5.27 (s, 2H, CH2Ph), 7.27–7.44
(m, 5H, Haromat). Calcd for C15H17NO5 (291.31): C, 61.85; H, 5.88;
N, 4.81. Found: C, 61.77; H, 6.07; N 4.74.
IR:
= 1760, 1732, 1608 cmꢀ1. MS (m/z): 333 [Ar3C]+. 1H NMR
~
m
(CDCl3) d: 1.23 (t, 3H, J = 7.2 Hz, CH2CH3), 2.23 (dd, 1H, J = 18.4,
9.3 Hz, CH2COO), 2.40 (dd, 1H, J = 15.5, 8.8 Hz, CH2CHCH2COO),
2.48 (dt, 1H, J = 12.7, 5.7 Hz, NCH2CH2), 2.60 (dd, 1H, J = 18.4,
6.7 Hz, CH2COO), 2.79 (d, 1H, J = 17.7 Hz, NCH2CO), 2.81 (dd, 1H, J
=15.5, 3.8 Hz, CH2CHCH2COO), 3.02–3.08 (m, 1H, NCH2CH2),
3.13–3.29 (m, 3H, NCH and 2H of NCH2CH2), 3.45 (d, 1H,
J = 17.7 Hz, NCH2CO), 3.77 (s, 9H, ArOCH3), 4.06–4.17 (m, 2H,
CH2CH3), 6.79–6.82 (m, 6H, Haromat), 7.29–7.23 (m, 6H, Haromat).
Calcd for C32H37NO7 (547.65): C, 70.18; H, 6.81; N, 2.56. Found:
C, 69.98; H, 6.79; N 2.43.
6.2.4. Ethyl (2R)-1-benzyloxycarbonyl-4-oxopyrrolidin-2-
acetate [(2R)-16e]
From (2R,4R)-15e (192 mg, 0.625 mmol), 2.67 M chromic acid
in aq H2SO4 (0.69 mL, 5.5 mmol). Yield: 147 mg (77%) as a colorless
= 1766, 1732 cmꢀ1. MS (m/z):
~
m
oil. ½a 2D0
ꢃ
ꢀ11.3 (c 1.38, CHCl3). IR:
306 [M+1]+. 1H NMR (CDCl3) d: 1.66–1.70 (m, 3H, CH2CH3), 3.06–
3.12 (m, 1H, CH2COO), 3.31–3.37 (m, 1H, CH2COO), 3.39–3.47 (m,
2H, CH2CHCH2COO), 4.36 (d, 1H, J = 18.6 Hz, NCH2), 4.50 (d, 1H,
J = 18.6 Hz, NCH2), 4.58–4.64 (m, 2H, CH2CH3), 5.23–5.29 (m, 1H,
NCH), 5.74 (s, 2H, CH2Ph), 7.72–7.84 (m, 5H, Haromat). Calcd for
6.1.8. Ethyl (2R)-1-[4,4-bis(3-methylthiophen-2-yl)but-3-en-1-
yl]-4-oxopyrrolidin-2-acetate [(2R)-16c]
From (COCl)2 (140 mg, 1.10 mmol) and DMSO (173 mg,
2.20 mmol) in CH2Cl2 (3 mL), (2R,4R)-15c (230 mg, 0.548 mmol)
in CH2Cl2 (3 mL); TEA (246 mg, 2.42 mmol), aq 1.0 M NaOH
(2.3 mL, 2.3 mmol); purification by CC (n-heptane/acetone, 3:1).
C16H19NO5 (305.34): C, 62.94; H, 6.27; N, 4.59. Found: C, 62.76;
H, 6.28; N, 4.55.
Yield: 201 mg (88%) as a yellow oil. ½a D20
ꢃ
ꢀ59.4 (c 2.00, EtOAc).
~
IR:
m
= 1759, 1732 cmꢀ1. MS (m/z): 418 [M+1]+. 1H NMR (CDCl3,
6.3. General procedure 3 (GP3) for the preparation of the
optically pure 4-hydroxy-4-(4-methoxyphenyl)pyrrolidine
derivatives 14b,d and 17c,d by organometallic addition
500 MHz) d: 1.25 (t, 3H, J = 7.2 Hz, CH2CH3), 2.00 (s, 3H, thienyl-
CH3), 2.02 (s, 3H, thienyl-CH3), 2.22–2.42 (m, 5H, CH2COO,
NCH2CH2, CH2CHCH2COO and 2H of NCH2CH2), 2.59–2.66 (m, 2H,
NCH2CO and CH2COO), 2.80 (dd, 1H, J = 15.4, 3.9 Hz,
CH2CHCH2COO), 2.99 (dt, 1H, J = 11.6, 7.9 Hz, NCH2CH2), 3.17–
3.23 (m, 1H, NCH), 3.35 (d, 1H, J = 17.5 Hz, NCH2CO), 4.09–4.18
(m, 2H, CH2CH3), 6.05 (t, 1H, J = 7.3 Hz, @CHCH2), 6.76 (d, 1H,
J = 5.2 Hz, SCH), 6.84 (d, 1H, J = 5.2 Hz, SCH), 7.05 (d, 1H,
J = 5.2 Hz, SCH@CH), 7.21 (d, 1H, J = 5.2 Hz, SCH@CH). Calcd for
Method A: To a mixture of Mg (266 mg, 11.1 mmol) and THF
(22 mL), 4-MeOC6H5Br (1.43 mL, 2.08 g, 11.1 mmol) was added
for 40 min to yield 4-MeOC6H4MgBr (0.48 M in THF). The resulting
46
anhydrous CeCl3 (0.3–2.0 equiv) was dried in vacuo at elevated
temperature for 15 min before use and then cooled to 0 °C. THF
(10–20 mL mmolꢀ1) and 4-MeOC6H4MgBr (1.0–2.1 equiv, 0.48 M
in THF) were added. The resulting white suspension was stirred
for 1 h at 0 °C and then cooled to the temperature given. A solution
of the respective 4-oxopyrrolidine (1 equiv) in THF (10–
20 mL mmolꢀ1) was added. Stirring was continued for the time gi-
ven. The reaction was quenched with aq sat. NH4Cl. The organic
layer was separated and the aqueous layer was extracted with
Et2O. The combined organic layers were dried (MgSO4) and con-
centrated in vacuo. The residual oil was subjected to analytical
C22H 27NO3S2 (417.59): C, 63.28; H, 6.52; N, 3.35; S 15.35. Found:
C, 63.20; H, 6.53; N, 3.20; S 14.92.
6.2. General procedure 2 for the preparation of the Cbz-
protected 4-oxopyrrolidine derivatives (2S)-13e, (2R)-13e, (2S)-
16e and (2R)-16e by Jones oxidation
To a solution of the respective N-protected 4-hydroxypyrrol-
idine derivative of (2S,4R)-12e, (2R,4R)-12e, (2S,4R)-15e, and

478
X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
HPLC, purified by CC and by prep. (preparative) HPLC. Unconsumed
4-oxopyrrolidine derivative was recovered.
(2R,4S)-14b: 116 mg (28%) as colorless crystals. mp 105–107 °C
(iPr2O). ½a 2D0
ꢃ
+47.9 (c 0.90, CHCl3). The spectra (1H NMR, IR, and
Method B: 4-MeOC6H4Br (1.43 mL, 2.08 g, 11.1 mmol) was
added for 40 min to a mixture of Mg (266 mg, 11.1 mmol) in
Et2O (25 mL). The resulting solution of 4-MeOC6H4MgBr (0.44 M
in Et2O, 1.80 equiv) was added dropwise to a solution of the
respective 4-oxopyrrolidine (1 equiv) in Et2O (35 mL mmolꢀ1) at
the temperature given and stirring was continued for the time gi-
ven. Workup and purification were similar to GP3A.
MS) were identical with those of (2S,4R)-14b. Calcd for C29H31NO4
(457.57): C, 76.12; H, 6.83; N, 3.06. Found: C, 76.03; H, 6.86; N, 3.02.
(2R,4R)-14b: 82 mg (20%) as colorless crystals. mp 103–104 °C
(iPr2O). ½a 2D0
ꢃ
+1.6 (c 0.80, CHCl3). The spectra (1H NMR, IR, and
MS) were identical with those of (2S,4S)-14b. Calcd for
C
29H31NO4 (457.57): C, 76.12; H, 6.83; N, 3.06. Found: C, 76.07;
H, 6.83; N, 3.09.
6.3.1. Methyl (2S,4S)-1-(4,4-diphenylbut-3-en-1-yl)-4-hydroxy-
4-(4-methoxyphenyl)pyrrolidine-2-carboxylate [(2S,4S)-14b]
and methyl (2S,4R)-1-(4,4-diphenylbut-3-en-1-yl)-4-hydroxy-4-
(4-methoxyphenyl)pyrrolidine-2-carboxylate [(2S,4R)-14b]
According to GP3A: from CeCl3 (148 mg, 0.601 mmol) and 4-
MeOC6H4MgBr (1.25 mL, 0.48 M in THF, 0.60 mmol) in THF
(8 mL); (2S)-13b (150 mg, 0.429 mmol) in THF (7 mL); reaction:
at ꢀ78 °C for 20 h; analytical HPLC (n-heptane/EtOAc/iso-propanol,
88:12:0.77; 1.3 mL minꢀ1) ds: (2S,4R)-14b /(2S,4S)-14b = 70:30
[(2S,4R)-14b tR = 22.8 min, (2S,4S)-14b tR = 28.4 min]; prep. HPLC
(n-heptane/EtOAc/isopropanol, 90:10:1.0; 12 mL minꢀ1) yielded
(2S,4R)-14b (tR = 39.7 min) and (2S,4S)-14b (tR = 49.5 min).
(2S,4R)-14b: 54 mg (28%) as colorless crystals. mp 105–106 °C
~
(m/z): 458 [M+1]+. 1H NMR (CDCl3) d: 2.15 (ddd, 1H, J = 13.9, 3.2,
2.0 Hz, NCHCH2), 2.25 (q, 2H, J = 7.5 Hz, NCH2CH2), 2.49 (dd, 1H,
J = 13.9, 10.7 Hz, NCHCH2), 2.63–2.70 (m, 2H, NCH2CH2 and
NCH2COH), 2.80 (dt, 1H, J = 12.1, 7.5 Hz, NCH2CH2), 3.09 (dd, 1H,
J = 9.2, 2.0 Hz, NCH2COH), 3.38 (dd, 1H, J = 10.7, 3.2 Hz, NCH),
3.66 (s, 3H, COOCH3), 3.72 (s, 3H, ArOCH3), 3.92 (s, 1H, OH), 6.05
(t, 1H, J = 7.5 Hz, @CHCH2), 6.78–6.81 (m, 2H, Haromat.), 7.09–7.33
(m, 12H, Haromat.). Calcd for C29H31NO4 (457.57): C, 76.12; H,
6.83; N, 3.06. Found: C, 76.03; H, 6.89; N, 3.09.
6.3.3. Methyl (2S,4S)-4-hydroxy-4-(4-methoxyphenyl)-1-{2-[tris
(4-methoxyphenyl)methoxy]ethyl}pyrrolidine-2-carboxylate
[(2S,4S)-14d] and methyl (2S,4R)-4-hydroxy-4-(4-methoxyphen
yl)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}pyrrolidine-2-
carboxylate [(2S,4R)-14d]
According to GP3A from CeCl3 (130 mg, 0.527 mmol) and 4-
MeOC6H4MgBr (0.80 mL, 0.67 M in THF, 0.53 mmol) in THF
(5 mL); (2S)-13d (196 mg, 0.377 mmol) in THF (6 mL); reaction:
at ꢀ60 °C for 20 h; analytical HPLC (n-heptane/EtOAc, 50:50), ds:
(2S,4S)-14d/(2S,4R)-14d
=
48:52 [(2S,4S)-14d tR = 7.6 min,
(2S,4R)-14d tR = 11.9 min]. Purification by CC (n-heptane/EtOAc,
3:2) and separation by prep. HPLC (n-heptane/EtOAc, 55:45) affor-
ded (2S,4S)-14d (tR = 19.7 min) and (2S,4R)-14d (tR = 24.6 min).
(iPr2O). ½a 2D0
ꢃ
ꢀ49.4 (c 0.815, CHCl3). IR:
m
= 3471, 1720 cmꢀ1. MS
(2S,4S)-14d: 57 mg (24%) as a colorless viscous oil. ½a D20
ꢃ
ꢀ7.1 (c
~
1.10, acetone). IR:
m
= 3478, 1738 cmꢀ1. MS (m/z): 333 [Ar3C]+. 1H
NMR (CDCl3) d: 2.34–2.44 (m, 2H, NCHCH2), 2.54 (s, 1H, OH),
2.94–3.00 (m, 2H, NCH2COH and NCH2CH2), 3.04–3.10 (m, 1H,
NCH2CH2), 3.17–3.26 (m, 2H, NCH2CH2), 3.49 (d, 1H, J = 10.7 Hz,
NCH2COH), 3.69 (s, 3H, COOCH3), 3.78 (s, 9H, ArOCH3), 3.79 (s,
3H, Ar‘OCH3), 3.94 (t, 1H, J = 7.6 Hz, NCH), 6.79–6.83 (m, 6H,
Haromat), 6.85–6.88 (m, 2H, Haromat), 7.31–7.34 (m, 6H, Haromat),
7.38–7.42 (m, 2H, Haromat). Calcd for C37H41NO8 (627.73): C,
70.80; H, 6.58; N, 2.23. Found: C, 70.33; H, 7.07; N, 2.22.
(2S,4S)-14b: 24 mg (12%) as colorless crystals. mp 103–104 °C
(2S,4R)-14d: 62 mg (26%) as a colorless viscous oil. ½a D20
ꢃ
ꢀ22.7 (c
(iPr2O). ½a 2D0
ꢃ
ꢀ1.8 (c 1.00, CHCl3). IR:
m
= 3424, 1742 cmꢀ1. MS
= 3476, 1733 cmꢀ1. MS (m/z): 333 [Ar3C]+. 1H
~
1.66, acetone). IR: m
~
(m/z): 458 [M+1]+. 1H NMR (CDCl3): d 2.27 (q, 2H, J = 7.5 Hz,
NCH2CH2), 2.34 (d, 2H, J = 7.7 Hz, NCHCH2), 2.67–2.75 (m, 2H,
NCH2CH2 and NCH2COH), 2.85 (dt, 1H, J = 12.0, 7.5 Hz, NCH2CH2),
3.35 (d, 1H, J = 10.3 Hz, NCH2COH), 3.63 (s, 3H, COOCH3), 3.71 (s,
3H, ArOCH3), 3.78 (t, 1H, J = 7.7 Hz, NCH), 6.05 (t, 1H, J = 7.5 Hz,
@CHCH2), 6.76–6.81 (m, 2H, Haromat.), 7.09–7.37 (m, 12H, Haromat.).
Calcd for C29H31NO4 (457.57): C, 76.12; H, 6.83; N, 3.06. Found: C,
76.23; H, 6.86; N 2.92.
NMR (CDCl3) d: 2.23 (dt, 1H, J = 13.9, 3.0 Hz, NCHCH2), 2.56 (dd,
1H, J = 13.9, 10.7 Hz, NCHCH2), 2.87–2.95 (m, 2H, NCH2COH and
NCH2CH2), 2.98–3.05 (m, 1H, NCH2CH2), 3.19–3.24 (m, 3H,
NCH2COH and 2H of NCH2CH2), 3.63 (dd, 1H, J = 10.7, 3.0 Hz,
NCH), 3.71 (s, 3H, COOCH3), 3.79 (s, 9H, ArOCH3), 3.81 (s, 3H, Ar‘-
OCH3), 6.81–6.84 (m, 6H, Haromat), 6.86–6.90 (m, 2H, Haromat),
7.31–7.35 (m, 6H, Haromat), 7.37–7.40 (m, 2H, Haromat). Calcd for
C37H41NO8 (627.73): C, 70.80; H, 6.58; N, 2.23. Found: C, 70.51;
H, 6.62; N, 2.15.
According to GP3B: from (2S)-13b (150 mg, 0.429 mmol) in
Et2O (15 mL), 4-MeOC6H4MgBr (1.76 mL, 0.44 M in Et2O,
0.77 mmol); reaction: at ꢀ78 °C for 20 h; analytical HPLC (n-hep-
tane/EtOAc/iso-propanol, 88:12:0.77; 1.3 mL minꢀ1), ds: (2S,4R)-
6.3.4. Methyl (2R,4S)-4-hydroxy-4-(4-methoxyphenyl)-1-{2-
[tris(4-methoxyphenyl)methoxy]ethyl}pyrrolidine-2-
carboxylate [(2R,4S)-14d] and methyl (2R,4R)-4-hydroxy-4-(4-
methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidine-2-carboxylate
[(2R,4R)-14d]
14b
/(2S,4S)-14b = 11:89
[(2S,4R)
tR = 23.1 min,
(2S,4S)
tR = 28.4 min];
prep. HPLC
(n-heptane/EtOAc/isopropanol,
90:10:1.0; 12 mL minꢀ1) yielded (2S,4R)-13b (12 mg, yield: 6%;
tR = 39.0 min) and (2S,4S)-13b (109 mg, yield: 56%; tR = 53.6 min).
According to GP3A from CeCl3 (165 mg, 0.669 mmol) and 4-
MeOC6H4MgBr (1.40 mL, 0.48 M in THF, 0.67 mmol) in THF
(10 mL); (2R)-13d (248 mg, 0.478 mmol) in THF (10 mL); reaction:
at ꢀ60 °C for 20 h. Analytical HPLC (n-heptane/EtOAc/iso-propanol,
70:30:1.0; 1.3 mL minꢀ1), ds: (2R,4R)-14d/(2R,4S)-14d = 45:55
[(2R,4R)-14d tR = 17.5 min, (2R,4S)-14d tR = 22.9 min]. Purification
by CC (n-heptane/acetone, 2:1) and separation by prep. HPLC (n-
6.3.2. Methyl (2R,4S)-1-(4,4-diphenylbut-3-en-1-yl)-4-hydroxy-
4-(4-methoxyphenyl)pyrrolidine-2-carboxylate [(2R,4S)-14b]
and methyl (2R,4R)-1-(4,4-diphenylbut-3-en-1-yl)-4-hydroxy-4-
(4-methoxyphenyl)pyrrolidine-2-carboxylate [(2R,4R)-14b]
According to GP3A from CeCl3 (315 mg, 1.28 mmol) and 4-
MeOC6H4MgBr (2.67 ml, 0.48 M in THF, 1.3 mmol) in THF
(14 mL); (2R)-13b (320 mg, 0.915 mmol) in THF (10 mL); reaction:
at ꢀ60 °C for 20 h; analytical HPLC (n-heptane/EtOAc/iso-propanol,
88:12:0.77), ds: (2R,4S)-14b /(2R,4R)-14b = 58:42 [(2R,4S)-14b
tR = 21.8 min, (2R,4R)-14b tR = 27.2 min]. Purification by CC (n-hep-
tane/EtOAc, 3:1) and separation by prep. HPLC (n-heptane/EtOAc/
iso-propanol, 86:14:0.9) yielded (2R,4S)-14b (tR = 26.0 min) and
(2R,4R)-14b (tR = 37.1 min); (2R)-13b (25 mg, 7.8%) was recovered.
heptane/EtOAc, 60:40; 12 mL minꢀ1
)
yielded (2R,4R)-14d
(tR = 29.3 min) and (2R,4S)-14d (tR = 43.7 min); (2R)-13d (19 mg,
7.7%) was recovered.
(2R,4R)-14d: 57 mg (19%) as a colorless viscous oil. ½a D23
ꢃ
+7.6 (c
0.93, acetone). The spectra (1H NMR, IR, and MS) were identical
with those of (2S,4S)-14d. Calcd for
C37H41NO8 (627.73): C,
70.80; H, 6.58; N, 2.23. Found: C, 70.47; H, 6.62; N, 2.13.

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
479
(2R,4S)-14d: 63 mg (21%) as a colorless oil. ½a D20
ꢃ
24: +22.9 (c
and NCH), 3.64 (s, 3 H, COOCH3), 3.78 (s, 3H, ArOCH3), 6.09 (t,
1H, J = 7.2 Hz, @CHCH2), 6.76 (d, 1H, J = 5.2 Hz, SCH), 6.82–6.86
(m, 3H, SCH and 2 H of Haromat), 7.05 (d, 1H, J = 5.2 Hz, SCH@CH),
7.19 (d, 1H, J = 5.2 Hz, SCH@CH), 7.37–7.41 (m, 2H, Haromat). Calcd
for C28H33NO4S2 (511.70): C, 65.72; H, 6.50; N, 2.74; S, 12.53.
Found: C, 65.97; H, 6.84; N, 2.56; S, 12.35.
D
1.12, acetone). The spectra (1H NMR, IR, and MS) were identical
with those of (2S,4R)-14d. Calcd for C37H41NO8 (627.73): C,
70.80; H, 6.58; N, 2.23. Found: C, 70.61; H, 6.58; N, 2.15.
6.3.5. Methyl (2S,4R)-4-hydroxy-4-(4-methoxyphenyl)-1-{2-[tris
(4-methoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetate [(2S,4R)
-17d] and methyl (2S,4S)-4-hydroxy-4-(4-methoxyphenyl)-1-{2-
[tris(4-methoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetate
[(2S,4S)-17d]
(2S,4R)-17c: 45 mg (24%) as colorless crystals. mp 99–100 °C
= 3523, 1731 cmꢀ1. MS
~
+73.1 (c 1.00, CHCl3). IR: m
(iPr2O). ½a 2D0
ꢃ
(m/z): 512 [M+1]+. 1H NMR (CDCl3) d: 2.00 (s, 3H, thienyl-CH3),
2.03 (s, 3H, thienyl-CH3), 2.09 (ddd, 1H, J = 14.2, 5.2, 2.0 Hz,
CH2CHCH2COO), 2.29–2.37 (m, 2H, NCH2CH2), 2.39–2.46 (m, 2H,
NCH2CH2 and NCH2COH), 2.50–2.60 (m, 2H, CH2CHCH2COO and
CH2COO), 2.73 (dd, 1H, J = 15.7/3.5 Hz, CH2COO), 2.90–3.02 (m,
2H, NCH2CH2 and NCH), 3.13 (dd, 1H, J = 9.4, 2.0 Hz, NCH2COH),
3.42 (s, 1H, OH), 3.66 (s, 3H, COOCH3), 3.79 (s, 3H, ArOCH3), 6.08
(t, 1H, J = 7.2 Hz, @CHCH2), 6.76 (d, 1H, J = 5.2 Hz, SCH), 6.83 (d,
1H, J = 5.2 Hz, SCH), 6.84–6.88 (m, 2H, Haromat), 7.05 (d, 1H,
J = 5.2 Hz, SCH@CH), 7.20 (d, 1H, J = 5.2 Hz, SCH@CH), 7.36–7.40
(m, 2H, Haromat). Calcd for C28H33NO4S2 (511.70): C, 65.72; H,
6.50; N, 2.74; S, 12.53. Found: C, 66.07; H, 6.70; N, 2.70; S, 12.48.
According to GP3A from CeCl3 (110 mg, 0.45 mmol) and 4-
MeOC6H4MgBr (0.93 mL, 0.48 M in THF, 0.45 mmol) in THF
(8 mL); (2S)-16d (170 mg, 0.320 mmol) in THF (4 mL); reaction:
at ꢀ78 °C for 20 h; analytical HPLC (n-heptane/EtOAc/iso-propanol,
54:45:1.2; 1.3 mL minꢀ1
)
ds: (2S,4S)-17d/(2S,4R)-17d = 17:83
[(2S,4S)-17d tR = 10.0 min, (2S,4R)-17d tR = 14.1 min]; purification
by CC (n-heptane/acetone, 3:1) and separation by prep. HPLC (n-
heptane/EtOAc/isopropanol, 54:45:1.2; 12 mL minꢀ1
)
yielded
(2S,4S)-17d (tR = 20.4 min) and (2S,4R)-17d (tR = 28.0 min). (2S)-
16b (35 mg, 21%) was recovered.
(2S,4S)-17d: 11 mg (5%) as a colorless oil. ½a D20
ꢃ
ꢀ14.4 (c 0.95,
~
acetone). IR:
m
= 3462, 1732, 1608 cmꢀ1. MS (m/z): 333 [Ar3C]+.
6.3.7. Ethyl (2R,4R)-4-hydroxy-4-(4-methoxyphenyl)-1-{2-[tris
(4-methoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetate [(2R,
4R)-17d] and ethyl (2R,4S)-4-hydroxy-4-(4-methoxyphenyl)-1-
{2-[tris(4-methoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetate
[(2R,4S)-17d]
1H NMR (CDCl3) d: 2.05 (dd, 1H, J = 13.0, 9.6 Hz, CH2CHCH2COO),
2.28–2.37 (m, 2H, CH2CHCH2COO and CH2COO), 2.69–2.77 (m,
2H, NCH2CH2 and CH2COO), 2.82 (d, 1H, J = 10.9 Hz, NCH2COH),
2.99–3.06 (m, 1H, NCH2CH2), 3.10–3.20 (m, 2H, NCH2CH2), 3.43
(d, 1H, J = 10.9 Hz, NCH2COH), 3.44–3.51 (m, 1H, NCH), 3.63 (s,
3H, COOCH3), 3.77 (s, 9H, Ar’OCH3), 3.79 (s, 3H, ArOCH3), 6.78–
6.82 (m, 6H, Haromat), 6.83–6.86 (m, 2H, Haromat), 7.31–7.35 (m,
6H, Haromat), 7.36–7.40 (m, 2H, Haromat). Calcd for C38H43NO8
(641.77): C, 71.12; H, 6.75; N, 2.18. Found: C, 71.10; H, 6.86; N,
2.11.
According to GP3A from CeCl3 (76 mg, 0.31 mmol) and 4-
MeOC6H4MgBr (0.63 mL, 0.48 M in THF, 0.30 mmol) in THF
(5 mL), (2R)-16d (119 mg, 0.217 mmol) in THF (4 mL); reaction:
at ꢀ78 °C for 20 h. Purification by CC (iso-hexane/acetone, 3:1)
and separation by prep. HPLC (n-heptane/EtOAc/iso-propanol,
60:40:0.88; 12 mL minꢀ1.) yielded (2R,4R)-17d (tR = 23.0 min) and
(2R,4S)-17d (tR = 30.4 min). (2R)-16d (20 mg, 17%) was recovered.
(2S,4R)-17d: 88 mg (43%) as a colorless oil. ½a D20
ꢃ
ꢀ36.8 (c 0.88,
acetone). IR:
= 3481, 1732, 1607 cmꢀ1. MS; (m/z): 333 [Ar3C]+.
(2R,4R)-17d: 2 mg (1%) as a colorless oil. ½a D20
ꢃ
+14.6 (c 2.18,
~
m
1H NMR (CDCl3) d: 2.08 (ddd, 1H, J = 14.1, 5.2, 1.8 Hz,
= 3492, 1729, 1608 cmꢀ1. MS (m/z): 333 [Ar3C]+. 1H
~
EtOAc). IR: m
CH2CHCH2COO), 2.51–2.61 (m,
4
H, CH2CHCH2COO, CH2COO,
NMR (CDCl3) d: 1.25 (t, 3H, J = 7.1 Hz, CH2CH3), 2.10 (br. s, 1H,
OH), 2.05 (dd, 1H, J = 13.1, 9.7 Hz, CH2CHCH2COO), 2.28–2.35 (m,
2H, CH2CHCH2COO and CH2COO), 2.67–2.77 (m, 2H, NCH2CH2
and CH2COO), 2.82 (d, 1H, J = 10.8 Hz, NCH2COH), 3.00–3.07 (m,
1H, NCH2CH2), 3.10–3.20 (m, 2H, NCH2CH2), 3.42 (d, 1H,
J = 10.8 Hz, NCH2COH), 3.44–3.51 (m, 1H, NCH), 3.77 (s, 9H, Ar’-
OCH3), 3.79 (s, 3H, ArOCH3), 4.05–4.13 (m, 2H, CH2CH3), 6.77–
6.81 (m, 6H, Haromat), 6.82–6.86 (m, 2H, Haromat), 7.31–7.35 (m,
6H, Haromat), 7.36–7.40 (m, 2H, Haromat). Calcd for C39H45NO8
(655.80): C 71.43, H 6.92, N 2.14. Found: C 71.43, H 7.17, N 2.05.
NCH2CH2 and NCH2COH), 2.72 (dd, 1H, J = 16.0, 3.5 Hz, CH2COO),
2.99–3.08 (m, 2H, NCH2CH2 and NCH), 3.12–3.25 (m, 3H, NCH2COH
and 2H of NCH2CH2), 3.55 (s, 1H, OH), 3.64 (s, 3H, COOCH3), 3.77 (s,
9H, Ar’OCH3), 3.79 (s, 3H, ArOCH3), 6.80–6.88 (m, 8H, Haromat),
7.30–7.34 (m, 6H, Haromat), 7.35–7.39 (m, 2H, Haromat). Calcd for
C38H43NO8 (641.77): C, 71.12; H, 6.75; N, 2.18. Found: C, 71.06;
H, 7.08; N, 2.14.
6.3.6. Methyl (2S,4R)-1-[4,4-bis(3-methylthiophen-2-yl)but-3-
en-1-yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-acetate
[(2S,4R)-17c] and methyl (2S,4S)-1-[4,4-bis(3-methylthiophen-
2-yl)but-3-en-1-yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-
2-acetate [(2S,4S)-17c]
(2R,4S)-17d: 63 mg (44%) as a colorless oil. ½a D20
ꢃ
+36.5 (c 0.85,
~
EtOAc). IR:
m
= 3462, 1728, 1608 cmꢀ1. MS (m/z): 333 [Ar3C]+. 1H
NMR (CDCl3) d: 1.22 (t, 3H, J = 7.2 Hz, CH2CH3), 2.08 (dd, 1H,
J = 14.1, 5.2, 1.8 Hz, CH2CHCH2COO), 2.51–2.60 (m, 4H,
CH2CHCH2COO, CH2COO, NCH2CH2 and NCH2COH), 2.72 (dd, 1H,
J = 15.9, 3.3 Hz, CH2COO), 2.99–3.08 (m, 2H, NCH2CH2 and NCH),
3.12–3.25 (m, 3H, NCH2COH and 2H of NCH2CH2), 3.54 (s, 1H,
OH), 3.77 (s, 9H, Ar’OCH3), 3.79 (s, 3H, ArOCH3), 4.05–4.16 (m,
2H, CH2CH3), 6.80–6.88 (m, 8H, Haromat), 7.30–7.34 (m, 6H, Haromat),
7.35–7.39 (m, 2H, Haromat). Calcd for C39H45NO8 (655.80): C, 71.43;
H, 6.92; N, 2.14. Found: C, 71.43; H, 7.17; N, 2.05.
According to GP3A from CeCl3 (127 mg, 0.521 mmol) and
4-MeOC6H4MgBr (1.1 mL, 0.48 M in THF, 0.52 mmol) in THF
(8 mL), (2S)-16c (150 mg, 0.372 mml) in THF (7 mL); reaction: at
ꢀ78 °C for 20 h; analytical HPLC (n-heptane/EtOAc/iso-propanol,
75:25:0.5; 1.3 mL minꢀ1
) ds: (2S,4S)-17c/(2S,4R)-17c = 21:79
[(2S,4S)-17c tR = 16.7 min, (2S,4R)-17c tR 19.6 min;]. Purification
by CC (n-heptane/acetone, 3:1) and separation by prep. HPLC (n-
heptane/EtOAc/iso-propanol, 75:25:0.5; 12 mL minꢀ1
)
yielded
(2S,4S)-17c (tR = 39.1 min) and (2S,4R)-17c (tR = 46.2 min). (2S)-
16c (52 mg, 35%) was recovered.
6.3.8. Ethyl (2R,4S)-1-[4,4-bis(3-methylthiophen-2-yl)but-3-en-
1-yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-acetate
[(2R,4S)-17c] and ethyl (2R,4R)-1-[4,4-bis(3-methylthiophen-2-
yl)but-3-en-1-yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-
acetate [(2R,4R)-17c]
According to GP3A from CeCl3 (146 mg, 0.601 mmol) and 4-
MeOC6H4MgBr (1.30 mL, 0.48 M in THF, 0.60 mmol) in THF
(12 mL), (2R)-16c (176 mg, 0.421 mmol) in THF (6 mL); reaction: at
ꢀ78 °C for 20 h; analytical HPLC ds: (2R,4R)-17c/(2R,4S)-17c = 19:81
(2S,4S)-17c: 16 mg (8%) as a colorless oil. ½a D20
ꢃ
ꢀ50.4 (c 1.09,
~
CHCl3). IR:
m
= 3493, 1732, 1613 cmꢀ1. MS (m/z): 512 [M+1]+. 1H
NMR (CDCl3) d: 2.00 (s, 3H, thienyl-CH3), 2.02 (s, 3H, thienyl-
CH3), 2.04 (dd, 1H, J = 13.2, 9.5 Hz, CH2CHCH2COO), 2.27–2.35 (m,
4H, CH2CHCH2COO, CH2COO and 2H of NCH2CH2), 2.54–2.61 (m,
1H, NCH2CH2), 2.67–2.72 (m, 2H, CH2COO and NCH2COH), 2.90
(dt, 1H, J = 12.0, 8.0 Hz, NCH2CH2), 3.35–3.42 (m, 2H, NCH2COH

480
X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
[(2R,4R)-17c tR = 13.9 min, (2R,4S)-17c tR = 17.7 min; n-heptane/
EtOAc/iso-propanol, 75:25:0.5; 1.3 mL minꢀ1]. Purification by
CC (iso-hexane/acetone, 3:1) and separation by prep. HPLC (n-hep-
tane/EtOAc/iso-propanol, 80:20:0.5, 12 mL minꢀ1) yielded (2R,4R)-
17c (tR = 40.5 min) and (2R,4S)-17c (tR = 45.6 min). (2R)-16c
(46 mg, 26%) was recovered.
reaction: at ꢀ78 °C for 4 h; purification by CC (n-heptane/acetone,
80:20) and separation by prep. HPLC (n-heptane/EtOAc/iso-propa-
nol, 78:22:0.5; tR = 64.2 min; 12 mL minꢀ1). (2S)-16e (91 mg, 54%)
was recovered. Yield: 92 mg (38%) as a colorless oil. ½a D20
ꢃ
ꢀ29.1 (c
~
0.95, CHCl3). IR:
m
= 3440, 1734, 1700 cmꢀ1. MS (m/z): 382 [M+1]+.
1H NMR (C6D5NO2, 120 °C) d: 2.40 (d, 1H, J = 13.7 Hz,
CH2CHCH2COO), 2.68 (dd, 1H, J = 13.7, 9.1 Hz, CH2CHCH2COO),
2.78 (s, 1H, OH), 3.17 (dd, 1H, J = 15.4, 3.3 Hz, CH2COO), 3.32 (dd,
1H, J = 15.4, 3.3 Hz, CH2COO), 3.70 (s, 3H, ArOCH3), 3.76 (s, 3H,
COOCH3), 3.90–4.00 (m, 1H, NCH), 4.56–4.64 (m, 2H, NCH2), 5.29,
5.34 (2s, 2H, CH2Ph), 6.86–6.91 (m, 2H, Haromat), 7.25–7.48 (m,
7H, Haromat). Calcd for C22H25NO6 (399.45): C, 66.15; H, 3.51; N,
6.31. Found: C, 66.45; H, 3.44; N, 6.67.
(2R,4R)-17c: 16 mg (7%) as a colorless oil. ½a D20
ꢃ
+51.0 (c 1.07,
~
CHCl3). IR:
m
= 3482, 1732 cmꢀ1. MS (m/z): 526 [M+1]+. 1H NMR
(CDCl3) d: 1.23 (t, 3H, J = 7.1 Hz, CH2CH3), 2.00 (s, 3H, thienyl-
CH3), 2.02 (s, 3H, thienyl-CH3), 2.00–2.07 (m, 1H, CH2CHCH2COO),
2.26–2.34 (m, 4H, CH2COO, CH2CHCH2COO and 2H of NCH2CH2),
2.53–2.61 (m, 1H, NCH2CH2), 2.65–2.72 (m, 2H, CH2COO and
NCH2COH), 2.91 (dt, 1H, J = 12.0, 8.0 Hz, NCH2CH2), 3.34–3.42 (m,
2H, NCH2COH and NCH), 3.78 (s, 3H, ArOCH3), 4.11 (q, 2H,
J = 7.1 Hz, CH2CH3), 6.09 (t, 1H, J = 7.2 Hz, @CHCH2), 6.76 (d, 1H,
J = 5.2 Hz, SCH), 6.82–6.87 (m, 3H, SCH and Haromat), 7.05 (d, 1H,
J = 5.2 Hz, SCH@CH), 7.19 (d, 1H, J = 5.2 Hz, SCH@CH), 7.37–7.41
(m, 2 H, Haromat). Calcd for C29H35NO4S2 (525.73): C, 66.25; H,
6.71; N, 2.66; S, 12.20. Found: C, 66.50; H, 6.79; N, 2.51; S, 11.89.
6.3.12. Ethyl (2R,4S)-1-benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidin-2-acetate [(2R,4S)-17e]
According to GP3B from (2R)-16e (120 mg, 0.393 mmol) and 4-
MeOC6H4MgBr (0.94 mL, 0.44 M in Et2O, 0.41 mmol) in Et2O
(20 mL); reaction: at ꢀ78 °C for 20 h; purification by CC (n-hep-
tane/acetone, 3:1) and separation by prep. HPLC (n-heptane/
EtOAc/iso-propanol, 78:22:0.5; tR = 47.4 min; 12 mL minꢀ1). (2R)-
16e (54 mg, 45%) was recovered. Yield: 60 mg (37%) as a colorless
~
(2R,4S)-17c: 55 mg (25%) as a colorless oil. ½a D20
ꢃ
ꢀ77.9 (c 0.67,
~
CHCl3). IR:
m
= 3468, 1713 cmꢀ1. MS (m/z): 527.1 [M+1]+. 1H NMR
(CDCl3) d: 1.24 (t, 3H, J = 7.3 Hz, CH2CH3), 1.99 (s, 3H, thienyl-
CH3), 2.03 (s, 3H, thienyl-CH3), 2.09 (ddd, 1H, J = 14.2, 5.2, 2.0 Hz,
CH2CHCH2COO), 2.30–2.37 (m, 2H, NCH2CH2), 2.39–2.46 (m, 2H,
NCH2CH2 and NCH2COH), 2.50–2.60 (m, 2H, CH2CHCH2COO and
CH2COO), 2.73 (dd, 1H, J = 15.7, 3.5 Hz, CH2COO), 2.90–3.02 (m,
2H, NCH2CH2 and NCH), 3.12 (dd, 1H, J = 9.4, 2.0 Hz, NCH2COH),
3.43 (s, 1H, OH), 3.79 (s, 3H, ArOCH3), 4.09–4.17 (m, 2H, CH2CH3),
6.07 (t, 1H, J = 7.2 Hz, @CHCH2), 6.76 (d, 1H, J = 5.0 Hz, SCH), 6.82–
6.88 (m, 3H, SCH and 2 H of Haromat), 7.05 (d, 1H, J = 5.0 Hz,
SCH@CH), 7.20 (d, 1H, J = 5.0 Hz, SCH@CH), 7.36–7.40 (m, 2H,
oil. ½a 2D0
ꢃ
+26.1 (c 1.05, CHCl3). IR: m
= 3440, 1730, 1700 cmꢀ1. MS
(m/z): 396 [M+1]+. 1H NMR (C6D5NO2, 120 °C) d: 1.22–1.29 (m,
3H, CH2CH3), 2.43 (d, 1H, J = 13.7 Hz, CH2CHCH2COO), 2.65–2.72
(m, 1H, CH2CHCH2COO), 2.80 (s, 1H, OH), 3.18 (dd, 1H, J = 15.4,
3.3 Hz, CH2COO), 3.27–3.34 (m, 1H, CH2COO), 3.76, 3.78 (2s, 3H,
ArOCH3), 3.91–4.00 (m, 1H, NCH), 4.17–4.24 (m, 2H, CH2CH3),
4.57–4.65 (m, 2H, NCH2), 5.30, 5.34 (2s, 2H, CH2Ph), 6.86–6.90
(m, 2H, Haromat), 7.26–7.48 (m, 7H, Haromat). Calcd for C23H27NO6
(413.48): C, 66.81; H, 6.58; N, 3.39. Found: C, 66.95; H, 6.77; N,
3.20.
Haromat). Calcd for C29H35NO4S2 (525.73): C, 66.25; H, 6.71; N,
2.66; S, 12.20. Found: C, 66.28; H, 6.86; N, 2.56; S, 11.99.
6.4. General procedure 4 for the preparation of the N-alkylated
4-hydroxy-4-(4-methoxyphenyl)-substituted proline and
pyrrolidin-2-acetic acid derivatives (10b,d and 11c,d)
6.3.9. Methyl (2S,4R)-1-benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylate [(2S,4R)-14e]
According to GP3A from CeCl3 (330 mg, 1.34 mmol) and 4-
MeOC6H4MgBr (2.80 mL, 0.48 M in THF, 1.34 mmol) in THF
(20 mL), (2S)-14e (354 mg, 1.28 mmol) in THF (5 mL); reaction:
at ꢀ60 °C for 4.5 h; purification by CC (n-heptane/acetone, 3:1).
(2S)-14e (61 mg, 17%) was recovered. Yield: 276 mg (56%) as a col-
The respective ester of 14b,d and 17c,d (1 equiv) was hydro-
lyzed in EtOH or MeOH (10–50 mL mmolꢀ1) at rt with aq 0.85 M
KOH or aq 1.0 M NaOH (3 equiv) and for the time given. The mix-
ture was neutralized with aq 1.0 M HCl to pH 7 and followed by
addition of buffer (pH 5.5–6.6). The mixture was concentrated in
vacuo and the residue was purified by CC and recrystallization.
orless oil. ½a 2D0
ꢃ
ꢀ23.5 (c 1.36, EtOAc). IR:
m
= 3438, 1756, 1706 cm
.
ꢀ1
~
MS (m/z): 385 [M+1]+. 1H NMR (C6D5NO2, 120 °C) d: 2.51 (dd, 1H,
J = 13.6, 1.8 Hz, NCHCH2), 2.83 (ddd, 1H, J = 13.6, 9.7, 1.5 Hz,
NCHCH2), 3.78–3.81 (m, 6H, COOCH3 and ArOCH3), 3.93 (dd, 1H,
J = 11.4, 1.5 Hz, NCH2), 4.07 (dd, 1H, J = 11.4, 1.3 Hz, NCH2), 4.77
(dd, 1H, J = 9.7, 1.3 Hz, NCH), 5.23 (d, 1H, J = 12.6 Hz, PhCH2), 5.32
(d, 1H, J = 12.6 Hz, PhCH2), 6.89–6.94 (m, 2H, Haromat), 7.27–7.50
(m, 7H, Haromat). Calcd for C21H23NO6 (385.42): C, 65.44; H, 6.02;
N, 3.63. Found: C, 65.11; H, 6.19; N, 3.70.
6.4.1. (2S,4S)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylic acid [(2S,4S)-10b]
From (2S,4S)-14b (50 mg, 0.11 mmol), aq 0.85 M KOH (0.39 mL,
0.33 mmol) in EtOH (1.7 mL); reaction time: 1 h; purification by CC
(gradient elution, iPr2O?EtOH). Yield: 36 mg (74%) as colorless
crystals. mp 173–175 °C (MeOH). ½a D20
ꢃ
ꢀ28.0 (c 0.64, MeOH). IR:
= 3254, 1622 cmꢀ1. MS (m/z): 444 [M+1]+. 1H NMR (CD3OD) d:
~
m
6.3.10. Methyl (2R,4S)-1-benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylate [(2R,4S)-14e]
2.42 (dd, 1H, J = 13.3, 12.0 Hz, NCHCH2), 2.57 (q, 2H, J = 7.6 Hz,
NCH2CH2), 2.65 (ddd, 1H, J = 13.3, 6.7, 2.0 Hz, NCHCH2), 3.26–3.33
(m, 1H, NCH2COH), 3.36–3.40 (m, 1H, NCH2CH2), 3.55 (dt, 1H,
J = 12.2, 7.6 Hz, NCH2CH2), 3.68 (d, 1H, J = 12.3 Hz, NCH2COH),
3.78 (s, 3H, ArOCH3), 4.25 (dd, 1H, J = 12.0, 6.7 Hz, NCH), 6.12 (t,
1H, J = 7.6 Hz, @CHCH2), 6.90–6.93 (m, 2H, Haromat), 7.17–7.44 (m,
12H, Haromat). Calcd for C28H29NO4 (443.54): C, 75.82; H, 6.59; N,
3.16. Found: C, 76.04; H, 6.84; N, 3.06.
According to GP3A from CeCl3 (399 mg, 1.62 mmol) and 4-
MeOC6H4MgBr (3.38 mL, 0.48 M in THF, 1.62 mmol) in THF
(25 mL), (2R)-14e (427 mg, 1.54 mmol); reaction: at ꢀ60 °C for
5.5 h; purification by CC (n-heptane/acetone, 3:1). (2R)-14e
(84 mg, 20%) was recovered. Yield: 265 mg (45%) as a colorless oil.
½
a 2D0 +24.8 (c 1.08, EtOAc). The spectra (1H NMR, IR, and MS) were
identical with those of (2S,4R)-14e. Calcd for C21H23NO6 (385.42):
ꢃ
C, 65.44; H, 6.02; N, 3.63. Found: C, 65.48; H, 6.16; N, 3.57.
6.4.2. (2R,4R)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylic acid [(2R,4R)-10b]
From (2R,4R)-14b (62 mg, 0.14 mmol), aq 1 M NaOH (0.42 mL,
0.42 mmol) in MeOH (3.6 mL); reaction time: 12 h; purification
by recrystallization from MeOH. Yield: 56 mg (94%) as colorless
6.3.11. Methyl (2S,4R)-1-benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidin-2-acetate [(2S,4R)-17e]
According to GP3B from (2S)-16e (168 mg, 0.611 mmol) in Et2O
(30 ml), 4-MeOC6H4MgBr (1.53 mL, 0.44 M in Et2O, 0.67 mmol);
crystals. mp 179–181 °C (MeOH). ½a D20
ꢃ
+28.0 (c 0.50, MeOH). The

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
481
spectra (1H NMR, IR, and MS) were identical with those of (2S,4S)-
10b. Calcd for C28H29NO4 (443.54)ꢁ1.0H2O: C, 72.86; H, 6.77; N,
3.03. Found: C, 72.77; H, 6.57; N, 2.95.
3.16–3.22 (m, 2H, NCH2COH), 3.25–3.32 (m, 2H, NCH2CH2), 3.49–
3.62 (m, 2H, NCH2CH2), 3.67 (s, 9H, Ar’OCH3), 3.70 (s, 3H, ArOCH3),
4.06 (dd, 1H, J = 11.5, 2.1 Hz, NCH), 6.77–6.84 (m, 8H, Haromat),
7.22–7.25 (m, 2H, Haromat), 7.28–7.32 (m, 6H, Haromat). Calcd for
6.4.3. (2S,4R)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylic acid [(2S,4R)-10b]
From (2S,4R)-14b (20 mg, 0.044 mmol), aq 0.85 M KOH
(0.15 mL, 0.13 mmol) in EtOH (1 mL); reaction time: 1 h; purifica-
tion by CC (gradient elution iPr2O?EtOH). Yield: 17 mg (88%) as
C
36H39NO8 (613.71)ꢁ1.0H2O: C, 68.45; H, 6.54; N, 2.22. Found: C,
68.62; H, 6.40; N, 2.18.
6.4.8. (2R,4S)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidine-2-carboxylic acid
[(2R,4S)-10d]
colorless crystals. mp 168–173 °C. ½a D20
ꢃ
ꢀ39.3 (c 0.81, MeOH). IR:
= 3431, 1612 cmꢀ1. MS (m/z): 444 [M+1]+. 1H NMR (CD3OD) d:
~
m
From (2R,4S)-14d (60 mg, 0.096 mmol), aq 1.0 M NaOH
(0.29 mL, 0.29 mmol) in MeOH (3 mL); reaction time: 3 h; buffer
(0.5 mL, pH 6.6). Yield: 52 mg (89%) as colorless crystals. mp
2.54–2.71 (m, 3H, NCHCH2 and 2H of NCH2CH2), 2.92 (dd, 1H,
J = 13.5, 11.4 Hz, NCHCH2), 3.33–3.53 (m, 3H, NCH2COH and 2H
of NCH2CH2), 3.62 (dd, 1H, J = 11.2, 1.8 Hz, NCH2COH), 3.78 (s,
3H, ArOCH3), 4.39 (dd, 1H, J = 11.4, 1.8 Hz, NCH), 6.09 (t, 1H,
J = 7.5 Hz, @CHCH2), 6.90–6.95 (m, 2H, Haromat), 7.16–7.45 (m,
12H, Haromat). Calcd for C28H29NO4 (443.54): C, 75.82; H, 6.59; N,
3.16. Found: C, 75.71; H, 6.82; N, 2.96.
158–160 °C (MeOH/H2O). ½a D20
ꢀ1.6 (c 0.75, MeOH). The spectra
ꢃ
(1H NMR, IR, and MS) were identical with those of (2S,4R)-10d.
Calcd for C36H39NO8 (613.71)ꢁ0.5H2O: C, 69.44; H, 6.68; N, 2.25.
Found: C, 69.31; H, 6.51; N, 2.17.
6.4.9. (2S,4S)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-met
hoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetic acid [(2S,4S)-
11d]
6.4.4. (2R,4S)-1-(4,4-Diphenylbut-3-en-1-yl)-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylic acid [(2R,4S)-10b]
From (2R,4S)-14b (88 mg, 0.19 mmol), aq 1 M NaOH (0.57 mL,
0.57 mmol) in MeOH (6 mL); reaction time: 12 h; purification by
recrystallization from MeOH/iPr2O. Yield: 72 mg (84%) as colorless
From (2S,4S)-17d (70 mg, 0.11 mmol), aq 1.0 M NaOH (0.33 mL,
0.33 mmol) in MeOH (2.7 mL); reaction time: 2 days; purification
by CC (MeOH-H2O, 4:1) and recrystallization from C6H6/iPr2O.
Yield: 50 mg (73%) as a colorless powder. mp 117–121 °C (C6H6/
~
crystals. mp 165–170 °C (MeOH/iPr2O). ½a D20
ꢃ
+40.4 (c 1.55, MeOH).
The spectra (1H NMR, IR, and MS) were identical with those of
(2S,4R)-10b. Calcd for C28H29NO4 (443.54)ꢁ0.4H2O: C, 74.56; H,
6.62; N, 2.92. Found: C, 74.61; H, 6.66; N, 3.11.
iPr2O). ½a 2D0
ꢃ
ꢀ9.4 (c 0.50, MeOH). IR:
m
= 3422, 1608 cmꢀ1. MS
(ESI+) (m/z): 628 [M+1]+. 1H NMR (CD3OD) d: 2.28–2.42 (m, 2H,
CH2CHCH2COO), 2.46 (dd, 1H, J = 16.9, 1.5 Hz, CH2COO), 2.85 (dd,
1H, J = 16.9, 5.1 Hz, CH2COO), 3.23 (dd, 1H, J = 12.4, 1.5 Hz,
NCH2COH), 3.30–3.34 (m, 1H, NCH2CH2), 3.38 (d, 1H, J = 12.4 Hz,
NCH2COH), 3.43–3.68 (m, 3H, NCH2CH2 and 2H of NCH2CH2),
3.74 (s, 9H, Ar’OCH3), 3.79 (s, 3H, ArOCH3), 4.05–4.12 (m, 1H,
NCH), 6.84–6.88 (m, 6H, Haromat), 6.90–6.94 (m, 2H, Haromat),
7.31–7.37 (m, 8H, Haromat). Calcd for C37H41NO8 (627.74)ꢁ0.8 H2O:
C, 69.21; H, 6.69; N, 2.18. Found: C, 69.22; H, 6.78; N, 2.02.
6.4.5. (2S,4S)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidine-2-carboxylic acid
[(2S,4S)-10d]
From (2S,4S)-14d (30 mg, 0.048 mmol), aq 0.85 M KOH
(0.39 mL, 0.33 mmol) in EtOH (1 mL); reaction time: 1.75 h; buffer
(1 mL, pH 6.6). Yield: 26 mg (89%) as colorless crystals. mp 138–
~
m = 3425,
139 °C (EtOH/H2O).
½
a 2D0
ꢃ
ꢀ3.9 (c 0.85, MeOH). IR:
1630 cmꢀ1. MS (ESI+) (m/z): 614 [M+1]+. 1H NMR (CD3OD) d: 2.32
(dd, 1H, J = 13.3, 11.6 Hz, NCHCH2), 2.57 (ddd, 1H, J = 13.3, 7.1,
1.5 Hz, NCHCH2), 3.14 (dd, 1H, J = 12.5, 2.1 Hz, NCH2COH), 3.30–
3.51 (m, 5H, NCH2COH and 2H of NCH2CH2 and 2H of NCH2CH2),
3.65 (s, 9H, ArOCH3), 3.69 (s, 3H, ArOCH3), 4.29 (dd, 1H, J = 11.6,
7.1 Hz, NCH), 6.73–6.76 (m, 6H, Haromat), 6.78–6.81 (m, 2H, Haromat),
7.23–7.27 (m, 8H, Haromat). Calcd for C36H39NO8 (613.71): C, 70.46;
H, 6.41; N, 2.28. Found: C, 70.18; H, 6.62; N, 2.35.
6.4.10. (2R,4R)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetic acid [(2R,4R
)-11d]
From (2R,4R)-17d (42 mg, 0.065 mmol), aq 1.0 M NaOH
(0.20 mL, 0.20 mmol) in MeOH (3 mL); reaction time: 2 days; puri-
fication by recrystallization from C6H6/iPr2O. Yield: 38 mg (93%) as
a colorless powder. mp 110–117 °C (C6H6/iPr2O). ½a D20
ꢃ
+9.5 (c 0.62,
MeOH). The spectra [1H NMR (500 MHz), IR, and MS] were identi-
cal with those of (2S,4S)-11d. Calcd for C37H41NO8 (627.74)ꢁ0.9
H2O: C, 69.01; H, 6.67; N, 2.18. Found: C, 68.85; H, 6.56; N, 2.21.
6.4.6. (2R,4R)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidine-2-carboxylic acid
[(2R,4R)-10d]
6.4.11. (2S,4R)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetic acid [(2S,4R
)-11d]
From (2R,4R)-14d (56 mg, 0.089 mmol), aq 1.0 M NaOH
(0.27 mL, 0.27 mmol) in MeOH (3 mL); reaction time: 5 h; buffer
(0.5 mL, pH 6.6). Yield: 48 mg (88%) as colorless crystals. mp
158–160 °C (MeOH/H2O). ½a D20
ꢃ
+3.6 (c 0.50, MeOH). The spectra
From (2S,4R)-17d (115 mg, 0.179 mmol), aq 1.0 M NaOH
(0.54 mL, 0.54 mmol) in MeOH (4.5 mL); reaction time: 2 days;
purification by recrystallization from C6H6/iPr2O. Yield: 97 mg
(1H NMR, IR, and MS) were identical with those of (2S,4S)-10d.
Calcd for C36H39NO8 (613.71)ꢁ0.3H2O: C, 69.84; H, 6.45; N, 2.26.
Found: C, 69.76; H, 6.67; N, 2.15.
(86%) as a colorless powder. mp 106–111 °C (C6H6/iPr2O). ½a D20
ꢃ
ꢀ20.5 (c 0.81, MeOH). IR:
= 3421, 1608 cmꢀ1. MS (ESI+, m/z):
~
m
628 (M+1)+. 1H NMR (CD3OD, 500 MHz) d: 2.19 (ddd, 1H, J = 14.1,
6.0, 1.4 Hz, CH2CHCH2COO), 2.70 (dd, 1H, J = 16.5, 4.1 Hz, CH2COO),
2.74–2.82 (m, 2H, CH2CHCH2COO and CH2COO), 3.19–3.25 (m, 1H,
NCH2CH2), 3.33 (d, 1H, J = 11.1 Hz, NCH2COH), 3.44–3.49 (m, 3H,
NCH2COH and 2 H of NCH2CH2), 3.62–3.67 (m, 1H, NCH2CH2),
3.74 (s, 9H, Ar’OCH3), 3.78 (s, 3H, ArOCH3), 3.87–3.93 (m, 1H,
NCH), 6.83–6.86 (m, 6H, Haromat), 6.89–6.92 (m, 2H, Haromat),
7.31–7.38 (m, 8H, Haromat). Calcd for C37H41NO8 (627.74)ꢁ0.7 H2O:
C, 69.40; H, 6.67; N, 2.19. Found: C, 69.45; H, 6.94; N, 2.19.
6.4.7. (2S,4R)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-
methoxyphenyl)methoxy]ethyl}pyrrolidine-2-carboxylic acid
[(2S,4R)-10d]
From (2S,4R)-14d (62 mg, 0.099 mmol), aq 0.85 M KOH
(0.47 mL, 0.40 mmol) in EtOH (2 mL); reaction time: 3.5 h; buffer
(2.0 mL, pH 6.6). Yield: 54 mg (89%) as colorless crystals. mp
105–108 °C. ½a 2D0
ꢃ
+1.5 (c 1.15, MeOH). IR:
= 3421, 1637 cm
.
ꢀ1
~
m
MS (ESI+) (m/z): 614 [M+1]+. 1H NMR (CD3OD) d: 2.48 (dd, 1H,
J = 13.5, 2.1 Hz, NCHCH2), 2.75 (dd, 1H, J = 13.5, 11.5 Hz, NCHCH2),

482
X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
6.4.12. (2R,4S)-4-Hydroxy-4-(4-methoxyphenyl)-1-{2-[tris(4-me
thoxyphenyl)methoxy]ethyl}pyrrolidin-2-acetic acid
[(2R,4S)-11d]
6.4.16. (2R,4R)-1-[4,4-Bis(3-methylthiophen-2-yl)but-3-en-1-
yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-acetic acid
[(2R,4R)-11c]
From (2R,4S)-17d (125 mg, 0.191 mmol), aq 1.0 M NaOH
(0.58 mL, 0.58 mmol) in MeOH (6.4 mL); reaction time: 2 days;
purification by CC (MeOH-H2O, 4:1) and recrystallization from
C6H6/iPr2O. Yield: 107 mg (89%) as colorless crystals. mp
From (2R,4R)-17c (61 mg, 0.12 mmol), aq 1.0 M NaOH (0.35 mL,
0.35 mmol) in MeOH (4 mL); reaction time: 2 days; purification by
recrystallization from C6H6/iPr2O. Yield: 55 mg (95%) as a slight yel-
low powder. mp 70–74 °C (C6H6/iPr2O). ½a D20
ꢃ
+40.1 (c 0.7, MeOH).
116–122 °C (C6H6/iPr2O). ½a D20
ꢃ
+20.3 (c 0.69, MeOH). The spectra
The spectra [1H NMR (500 MHz), IR, and MS] were identical with
those of (2S,4S)-11c. Calcd for C27H31NO4S2 (497.67)ꢁ0.9H2O: C,
65.11; H, 6.43; N, 2.73. Found: C, 65.09; H, 6.24; N, 2.64.
(1H NMR, IR, and MS) were identical with those of (2S,4R)-11d.
Calcd for C37H41NO8 (627.74)ꢁ0.6 H2O: C, 69.60; H, 6.66; N, 2.19.
Found: C, 69.50; H, 6.75; N, 2.08.
6.5. General procedure 5 for the preparation of the N-Cbz-
protected 4-hydroxy-4-(4-methoxyphenyl)-substituted prolines
[10e] and pyrrolidin-2-acetic acids [11e]
6.4.13. (2S,4R)-1-[4,4-Bis(3-methylthiophen-2-yl)but-3-en-1-
yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-acetic acid
[(2S,4R)-11c]
The respective ester of 14e and 17e (1 equiv) was hydrolyzed
with 1.0 M NaOH (2.0–3.0 equiv) in MeOH (10–50 mL mmolꢀ1) at
rt 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
CH2Cl2 and the combined extracts were dried (Na2SO4) and con-
centrated in vacuo to yield the carboxylic acid.
From (2S,4R)-17c (83 mg, 0.16 mmol), aq 1.0 M NaOH (0.49 mL,
0.49 mmol) in MeOH (4.5 mL); reaction time: 3 days; purification
by CC (MeOH) and recrystallization from C6H6/iPr2O. Yield: 56
mg (70%) as a colorless powder. mp 73–76 °C (C6H6/iPr2O). ½a D20
ꢃ
ꢀ37.9 (c 0.61, MeOH). IR:
= 3385, 1609 cmꢀ1. MS (m/z): 498
~
m
[M+1]+. 1H NMR (CD3OD, 500 MHz) d: 1.96 (s, 3H, thienyl-CH3),
2.02 (s, 3H, thienyl-CH3), 2.13–2.18 (m, 1H, CH2CHCH2COO), 2.60
(q, 2H, J = 7.4 Hz, NCH2CH2), 2.69–2.81 (m, 3H, CH2CHCH2COO
and 2H of CH2COO), 3.08–3.12 (m, 1H, NCH2CH2), 3.18–3.22 (m,
1H, NCH2COH), 3.54–3.62 (m, 2H, NCH2CH2 and NCH2COH), 3.77
(s, 3H, ArOCH3), 3.86–3.90 (m, 1H, NCH), 6.04 (t, 1H, J = 7.4 Hz,
@CHCH2), 6.78 (d, 1H, J = 5.2 Hz, SCH), 6.88–6.93 (m, 3H, SCH and
2 H of Haromat), 7.16 (d, 1H, J = 5.2 Hz, SCH@CH), 7.34 (d, 1H,
6.5.1. (2S,4R)-1-Benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylic acid [(2S,4R)-10e]
From (2S,4R)-14e (131 mg, 0.340 mmol), aq 1.0 M NaOH
(1.02 mL, 1.02 mmol) in MeOH (4 mL); reaction time: 3 h. Yield:
111 mg (88%) as a colorless foam. mp 62–65 °C. ½a D20
ꢃ
ꢀ25.0 (c
~
1.00, MeOH). IR:
m
= 3420, 1705, 1611 cmꢀ1. MS (m/z): 370 [M-
J = 5.2 Hz, SCH@CH), 7.38–7.40 (m, 2H,
27H31NO4S2 (497.67)ꢁ0.5 H20: C, 64.00; H, 6.37; N, 2.76. Found:
C, 63.84; H, 6.67; N, 2.54.
Haromat). Calcd for
1]+. 1H NMR (C6D5NO2, 120 °C) d: 2.74–2.89 (m, 2H, NCHCH2),
3.77 (s, 3H, ArOCH3), 3.91 (d, 1H, J = 11.0 Hz, NCH2), 4.09 (d, 1H,
J = 11.0 Hz, NCH2), 4.88 (d, 1H, J = 9.4 Hz, NCH), 5.29–5.36 (m, 2H,
CH2Ph), 6.89–6.93 (m, 2H, Haromt), 7.26–7.52 (m, 7H, Haromat). Calcd
for C20H21NO6 (371.40): C, 64.68; H, 5.70; N, 3.77. Found: C, 64.85;
H, 5.42; N, 3.53.
C
6.4.14. (2R,4S)-1-[4,4-Bis(3-methylthiophen-2-yl)but-3-en-1-
yl]-4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-acetic acid
[(2R,4S)-11c]
From (2R,4S)-17c (87 mg, 0.17 mmol), aq 1.0 M NaOH (0.50 mL,
0.50 mmol) in MeOH (4.5 mL); reaction time: 2 days; purification
by recrystallization from C6H6/iPr2O. Yield: 68 mg (82%) as a
6.5.2. (2R,4S)-1-Benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidine-2-carboxylic acid [(2R,4S)-10e]
From (2R,4S)-14e (107 mg, 0.278 mmol), aq 1.0 M NaOH
(0.83 mL, 0.83 mmol) in MeOH (4.2 mL); reaction time: 4 h. Yield:
slightly yellow powder. mp 69–73 °C (C6H6/iPr2O). ½a D20
ꢃ
+34.0 (c
0.85, MeOH). The spectra [1H NMR (500 MHz), IR, and MS] were
83 mg (80%); colorless foam. mp 58–62 °C. ½a D20
ꢃ
+25.8 (c 0.64,
identical with those of (2S,4R)-11c. Calcd for
C27H31NO4S2
MeOH). The spectra (1H NMR, IR, and MS) were identical with
those of (2S,4R)-10e. Calcd for C20H21NO6 (371.40): C, 64.68; H,
5.70; N, 3.77. Found: C, 64.74; H, 5.93; N, 3.56.
(497.67)ꢁ0.6 H2O: C, 63.78; H, 6.38; N, 2.75; S, 12.61. Found: C,
63.60; H, 6.38; N, 2.67; S, 12.43.
6.4.15. (2S,4S)-1-[4,4-Bis(3-methylthiophen-2-yl)but-3-en-1-yl]-
4-hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-acetic acid [(2S,4S
)-11c]
6.5.3. (2S,4R)-1-Benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidin-2-acetic acid [(2S,4R)-11e]
From (2S,4R)-17e (93 mg, 0.23 mmol) and aq 1.0 M NaOH
(0.48 mL, 0.48 mmol) in MeOH (4.5 mL); reaction time: 4 days.
From (2S,4S)-17c (60 mg, 0.12 mmol), aq 1.0 M NaOH (0.35 mL,
0.35 mmol) in MeOH (3.6 mL); reaction time: 3 days; purification
by recrystallization from C6H6/iPr2O. Yield: 57 mg (98%) as a col-
Yield: 73 mg (81%) as a colorless foam. ½a D20
ꢀ30.0 (c 0.80, MeOH).
ꢃ
1H NMR (C6D5NO2, 120 °C) d: 2.40–2.51 (m, 1H, CH2CHCH2COO),
2.68–2.74 (m, 1H, CH2CHCH2COO), 3.27 (dd, 1H, J = 16.0, 8.9 Hz,
CH2COO), 3.41 (dd, 1H, J = 16.0, 3.8 Hz, CH2COO), 3.77 (s, 3H, Ar-
OCH3), 3.93 (d, 1H, J = 11.5 Hz, NCH2), 4.00 (d, 1H, J = 11.5 Hz,
NCH2), 4.59–4.68 (m, 1H, NCH), 5.26–5.33 (m, 2H, CH2Ph), 6.85–
~
m = 3424,
orless powder. mp 73–76 °C (C6H6/iPr2O). ½a D20
ꢃ
ꢀ38.6 (c 0.70,
~
MeOH). IR:
m
= 3420, 1610 cmꢀ1. MS (m/z): 498 [M+1]+. 1H NMR
(CD3OD, 500 MHz) d: 1.98 (s, 3H, thienyl-CH3), 2.03 (s, 3H, thie-
nyl-CH3), 2.24–2.31 (m, 1H, CH2CHCH2COO), 2.39 (ddd, 1H,
J = 13.3, 6.0, 1.9 Hz, CH2CHCH2COO), 2.51 (dd, 1H, J = 16.7, 2.3 Hz,
CH2COO), 2.61 (q, 2H, J = 7.4 Hz, NCH2CH2), 2.79 (dd, 1H, J = 16.7,
5.8 Hz, CH2COO), 3.25–3.31 (m, 2H, NCH2CH2 and NCH2COH),
3.52–3.61 (m, 1H, NCH2CH2), 3.64 (d, 1H, J = 12.4 Hz, NCH2COH),
3.77 (s, 3H, ArOCH3), 4.05–4.12 (m, 1H, NCH), 6.07 (t, 1H,
J = 7.4 Hz, @CHCH2), 6.77 (d, 1H, J = 5.2 Hz, SCH), 6.89–6.92 (m,
3H, SCH and 2H of Haromat), 7.15 (d, 1H, J = 5.2 Hz, SCH@CH),
7.34–7.39 (m, 3H, SCH@CH and 2H of Haromat). Calcd for
6.91 (m, 2H, Haromat), 7.25–7.51 (m, 7H, Haromat). IR:
1702, 1677 cmꢀ1. MS (m/z): 368 [M-OH]+. Calcd for C21H23NO6
(385.42): C, 65.44; H, 6.02; N, 3.63. Found: C, 65.22; H, 6.14; N,
3.40.
6.5.4. (2R,4S)-1-Benzyloxycarbonyl-4-hydroxy-4-(4-
methoxyphenyl)pyrrolidin-2-acetic acid [(2R,4S)-11e]
From (2R,4S)-17e (40 mg, 0.097 mmol) and aq 1.0 M NaOH
(0.20 mL, 0.20 mmol) in MeOH (2.8 mL); reaction: 4 days. Yield:
C
27H31NO4S2 (497.67)ꢁ0.5 H20: C, 64.00; H, 6.37; N, 2.76. Found:
34 mg (91%) as a colorless foam. ½a D20
ꢃ
+29.2 (c 0.49, MeOH). The
C, 64.27; H, 6.59; N, 2.47.

X. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 470–484
483
spectra (1H NMR, IR, and MS) were identical with those of (2S,4R)-
6.6.5. Methyl (2S,4R)-4-hydroxy-4-(4-
11e. Calcd for C21H23NO6 (385.42): C, 65.44; H, 6.02; N, 3.63.
methoxyphenyl)pyrrolidine-2-carboxylate [(2S,4R)-19]
Found: C, 65.56; H, 6.32; N, 3.37.
From (2S,4R)-14e (144 mg, 0.374 mmol) and TEA (11 mg,
0.11 mg) in EtOAc (15 mL), 10% Pd-C (74 mg, 0.070 mmol); reac-
tion time: 6 h. Yield: 94 mg (100%) as colorless crystals. mp 96–
a 2D0
+27.3 (c 1.00, CHCl3). IR:m = 3303, 1740,
~
6.6. General procedure 6 for the preparation of the 4-hydroxy-4-
(4-methoxyphenyl)-substituted proline [10a] and pyrrolidin-2-
acetic acid [11a]
97 °C (EtOAc).
½
ꢃ
1612 cmꢀ1. MS (m/z): 252 [M+1]+. 1H NMR (CDCl3, NOE for the
determination of the C-4 configuration) d: 2.35 (dt, 1H, J = 13.7,
2.5 Hz, NCHCH2), 2.51 (dd, 1H, J = 13.7, 10.0 Hz, NCHCH2), 3.07
(br. s, 1H, OH), 3.10 (d, 1H, J = 11.8 Hz, NCH2), 3.27 (dd, 1H,
J = 11.8, 2.1 Hz, NCH2), 3.77 (s, 3H, COOCH3), 3.79 (s, 3H, ArOCH3),
3.99 (dd, 1H, J = 10.0, 2.5 Hz, NCH), 6.85–6.88 (m, 2H, Haromat),
7.33–7.37 (m, 2H, Haromat). Calcd for C13H17NO4 (251.29): C,
62.14; H, 6.82; N, 5.57. Found: C, 62.11; H, 6.97; N, 5.53.
To a solution of the respective N-protected pyrrolidine of 10e
and 11e (1 equiv) and TEA (0.33–20 equiv) in MeOH or EtOAc
(40–50 mL mmolꢀ1)), 10% Pd-C was added. This mixture was sub-
jected to hydrogen at rt under ambient pressure for the time given.
The reaction mixture was filtrated and concentrated in vacuo to
give the respective N-deprotected amine.
6.6.1. (2S,4R)-4-Hydroxy-4-(4-methoxyphenyl)pyrrolidine-2-
carboxylic acid [(2S,4R)-10a]
6.7. Biological test
From (2S,4R)-10e (140 mg, 0.377 mmol) and TEA (12 mg,
0.12 mmol) in MeOH (17 mL), 10% Pd-C (70 mg, 0.066 mmol);
reaction time: 1.5 h. Yield: 82 mg (92%) as colorless crystals. mp
6.7.1. Preparation of subcellular membrane suspensions
Two subcellular membrane pellets, termed bfcP2B (a) (from bo-
vine frontal cortex) and bbsP2C (b) (from bovine brain stem),
respectively, were prepared according to literature.37 Their suspen-
sions were prepared and measured as described by Bradford47
pfcP2B (c) (from porcine frontal cortex) and pbsP2C (d) (from por-
cine brain stem), cfcP2B (e) (from calf frontal cortex) and cbsP2C (f)
(from calf brain stem), were applied alternatively instead of bfcP2B
and bbsP2C, respectively.
244–247 °C (MeOH, decomp.).
m
½
a 2D0
+0.7 (c 0.45, H2O). IR:
ꢃ
= 3383, 1614 cmꢀ1. MS (m/z): 237 M+. 1H NMR (D2O) d: 2.47
~
(dt, 1H, J = 14.0, 2.2 Hz, NCHCH2), 2.63 (dd, 1H, J = 14.0, 11.0 Hz,
NCHCH2), 3.40 (d, 1H, J = 12.2, NCH2), 3.55 (dd, 1H, J = 12.2,
2.2 Hz, NCH2), 3.67 (s, 3H, ArOCH3), 4.23 (dd, 1H, J = 11.0, 2.2 Hz,
NCH), 6.85–6.89 (m, 2H, Haromat), 7.26–7.30 (m, 2H, Haromat). Calcd
for C12H15NO4 (237.25)ꢁ0.2MeOH: C, 60.13; H, 6.53; N, 5.74. Found:
C, 60.00; H, 6.47; N, 5.76.
6.7.2. Inhibition of GAT1 mediated GABA-uptake
Aliquots of about bfcP2B protein (50–100
cfcP2B or pfcP2B) were preincubated with 10 M aminooxyacetic
acid and a test compound in the buffer (200 L, a solution of
lg, alternatively
6.6.2. (2R,4S)-4-Hydroxy-4-(4-methoxyphenyl)pyrrolidine-2-
carboxylic acid [(2R,4S)-10a]
l
l
From (2R,4S)-11e (66 mg, 0.17 mmol) and TEA (5.8 mg,
0.056 mmol) in EtOAc (8 mL), 10% Pd-C (33 mg, 0.031 mmol); reac-
tion time: 4 h. Yield: 38 mg (96%) as colorless crystals. mp 244–
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. Following the addition of 12.5 nM [3H] GABA
246 °C (MeOH, decomp.). ½a D20
ꢃ
ꢀ0.9 (c 0.76, H2O). The spectra (1H
(25 lL) and 250 nM GABA (25 lL), the sample was incubated at
NMR (500 MHz), IR, and MS) were identical with those of
(2S,4R)-10a. Calcd for C12H15NO4 (237.25)ꢁ0.4MeOH: C, 59.56; H,
6.69; N, 5.60. Found: C, 59.32; H, 6.54; N, 5.68.
37 °C for 4 min. The incubation was terminated by filtration in a
Brandel M-24R Harvester through Whatman GF/C filters, which
had been immersed in 0.9% NaCl for 1 h. The filters were washed
with 0.9% NaCl (4 ꢂ 2 mL) and then measured in Rotiszint Eco Plus
(3 mL) 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.
6.6.3. (2S,4R)-4-Hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-
acetic acid [(2S,4R)-11a]
From (2S,4R)-11e (59 mg, 0.15 mmol), TEA (0.20 mL,
1.4 mmol) in MeOH (5 mL), 10% Pd-C (40 mg, 0.038 mmol);
reaction time: 3 h. Yield: 38 mg (100%) as colorless crystals.
6.7.3. Inhibition of GAT3 mediated GABA-uptake
mp 261–262 °C (MeOH, decomp.). ½a D20
ꢃ
+5.6 (c 0.50, 0.05 M
Aliquots of about bbsP2C protein (50–100
cbsP2C or pbsP2C) were preincubated with 10 M aminooxyacetic
acid, 10 M NNC-711 and a test compound in the buffer (200 L, a
lg, alternatively
= 3424, 1648, 1610 cmꢀ1. MS (m/z): 252
l
~
m
NaOH in MeOH). IR:
[M+1]+. 1H NMR (its sodium salt in CD3OD, 500 MHz; NOE) d:
1.93 (ddd, 1H, J = 13.9, 6.4, 1.8 Hz, CH2CHCH2COO), 2.43 (dd, 1H,
J = 13.9, 9.3 Hz, CH2CHCH2COO), 2.51 (dd, 1H, J = 15.4, 6.8 Hz,
CH2COO), 2.60 (dd, 1H, J = 15.4, 5.4 Hz, CH2COO), 2.90 (d, 1H,
J = 12.0 Hz, NCH2), 3.04 (dd, 1H, J = 12.0, 1.8 Hz, NCH2), 3.54–3.61
(m, 1H, NCH), 3.77 (s, 3H, ArOCH3), 6.85–6.89 (m, 2H, Haromat),
7.38–7.41 (m, 2H, Haromat). Calcd for C13H17NO4 251.4191 HRMS
l
l
solution of 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. The additions of 50 nM [3H] GABA (25
lL)
and 1
lM GABA (25 lL) were followed.
Acknowledgment
+
(DEI+): M 251.4235.
Financial support of this work by the Deutsche Forschungs-
gemeinschaft, the Fonds der Chemischen Industrie and the BMBF
are gratefully acknowledged.
6.6.4. (2R,4S)-4-Hydroxy-4-(4-methoxyphenyl)pyrrolidin-2-
acetic acid [(2R,4S)-11a]
From (2R,4S)-11e (73 mg, 0.19 mmol) and TEA (0.50 mL,
3.5 mmol) in MeOH (10 mL), 10% Pd-C (38 mg, 0.036 mmol);
reaction time: 1 h. Yield: 45 mg (95%) as colorless crystals. mp
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251–253 °C (MeOH, decomp.). ½a D20
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