Synthesis and biological evaluation of 4-fluoroproline and 4-fluoropyrrolidine-2-acetic acid derivatives as new GABA uptake inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.010

Preparation for the N-alkylated derivatives of enantiomerically pure (2S)-4-fluoroproline and (2S)-4-fluoropyrrolidine-2-acetic acid is described. The final compounds were evaluated as potential GAT-1 uptake inhibitors via cultured cell lines expressing m

Full text of DOI:10.1016/j.bmc.2009.08.010

Bioorganic & Medicinal Chemistry 17 (2009) 6540–6546
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4-fluoroproline and
4-fluoropyrrolidine-2-acetic acid derivatives as new GABA uptake inhibitors
b
b
a
a
Weiping Zhuang a, Xueqing Zhao a, , Gang Zhao , Lihe Guo , Yunyang Lian , Jingming Zhou ,
*
Dongsheng Fang a
a Fujian Institute of Microbiology, No. 25, Jinbu road, Fuzhou 350007, China
b Cell Star Bio-Technologies Co., Limited, No. 898 Halei Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Preparation for the N-alkylated derivatives of enantiomerically pure (2S)-4-fluoroproline and (2S)-4-flu-
oropyrrolidine-2-acetic acid is described. The final compounds were evaluated as potential GAT-1 uptake
inhibitors via cultured cell lines expressing mouse GAT-1. Compared with their corresponding 4-hydroxy
compounds, these derivatives exhibited slight improvement on their inhibitory potency, but still much
weaker than their corresponding compounds with no substituents at the C-4 of the pyrrolidine moiety,
with the most potent affinity being about 1/15 fold as that of Tiagabine. The drastic decrease of their
affinity may arise from sharp reduction of their basicity due to strong inductive effect of the 4-fluorine.
However the configuration of the C-4 linking fluorine did not have much influence on their affinity for
GAT-1.
Received 2 June 2009
Revised 3 August 2009
Accepted 4 August 2009
Available online 9 August 2009
Keywords:
GABA uptake inhibitor
GAT-1
4-Fluoropyroline
4-Fluoropyrrolidine-2-acetic acid
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The substitution of the nitrogen atoms in conformationally
strained GABA analogues with appropriate bulky lipophilic groups
c
-Aminobutyric acid (GABA) plays a significant role for the
led to the discovery of very potent GABA uptake inhibitors for GAT-
1, i.g. (R)-SK&F 89976-A (1), (RS)-cis-SK&F 100591-A (2)10 and
Tiagabine (3).11 Since then, many similar potent derivatives with
different bulky lipophilic groups from those of 1 and 3 have been
synthesized and intensively investigated for their pharmacology
(Fig. 1).12–15
inhibitory function in the mammalian central nervous system
(CNS), as approximate 40% of synapses in the CNS are GABA0er-
gic.1–3 A decrease of GABA’ergic neurotransmission appears to be
involved in the development and outbreak of several diseases, such
as epilepsy,4 Huntington’s chorea,5 and Parkinson’s disease.6 GABA
uptake protein (GAT) inhibitors have shown great promise for the
treatment of related diseases, as they have less influences on GABA
receptor system.7–9
Wanner’s group found that the novel compounds 4–7 with high
(2S)-homoproline in place of classic (R)-nipecotic acid, exhibited
selectively potent GAT-1 inhibition, the compound 7 being only
slightly weaker than Tiagabine (Fig. 2).16 While introduction of a
hydroxyl group into the C-4 position of the pyrrolidine moiety
led to a drastic decrease of its potency, for example, by comparison
of 4–7 with the compounds 8–10, respectively (Fig. 2).17
Due to the unique physicochemical properties of fluorine, such
as highest electronegativity, strong lipophilicity, extremely low
polarizability, and the ability to participate in a hydrogen bond,18
organofluorine compounds have exerted a range of applications
in medicines over the past several decades.19–21 Thus, following
my previous work, 4-hydroxy group at the C-4 position of the
pyrrolidine moiety was substituted by fluorine. Meantime, two
typically bulky lipophilic groups a–b were chosen as their N-sub-
stituents. Therefore, two different series of (2S)-pyrrolidine deriv-
atives (2S,4R)-11a–b and (2S,4S)-12a–b aimed at GAT-1, were
prepared in enantiomerically pure form and evaluated as potential
GAT-1 inhibitors (Fig. 3).
O
OH
COOH
COOH
OH
N
N
N
S
S
(R)-SK&F-89976-A (1)
(RS)-SK&F-100591 (2)
Tiagabine (3)
Figure 1. Representatives of potent GABA uptake inhibitors.
* Corresponding author. Tel.: +86 591 8347 5802; fax: +86 591 8316 6191.
E-mail address: wolfgangzhao@yahoo.com (X. Zhao).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.010

W. Zhuang et al. / Bioorg. Med. Chem. 17 (2009) 6540–6546
R2
6541
R2
R1
R2
R2
R1
R1
R1
COOH
COOH
COOH
COOH
N
N
N
N
S
S
S
S
R1 = R2 = H (7)
R1 = R2 = H (4)
R1 = R2 = H (5)
R1 = R2 = H (6)
IC50 GAT-1: 2.56; GAT-3: 309
GAT-1: 0.40; GAT-3: 64.8
R1 = OH, R2 = H [(2S,4R)-9]
GAT-1: 3.29; GAT-3: > 100
GAT-1: 0.65
GAT-1: 0.34; GAT-3: 26.6
R1 = OH, R2 = H [(2S,4R)-10]
GAT-1: 3.15; GAT-3: > 100
R1 = OH, R2 = H [(2S,4R)-8]
IC50 GAT-1: 79.6; GAT-3: >100
Figure 2. GABA uptake inhibitors with a pyrrolidine moiety (IC50 concentration: lM).
F
F
F
F
S
S
COOH
COOH
COOH
N
R
COOH
N
R
N
R
R: a
N
R
b
(2S,4R)-11a-b
(2S,4S)-11a-b
(2S,4R)-12a-b
(2S,4S)-12a-b
Figure 3. New target compounds.
can be achieved by Arndt–Eistert reaction. Thus, (2S,4R)-17a–b
was reacted with isobutyl chloroformate at ꢀ20 °C in the presence
of TEA, and further with CH2N2 to afforded (2S,4R)-18a–b. Then
wolff’s rearrangement of the diazo compounds in methanol cata-
lyzed by AgNO3–TEA led to the desired b-amino acid esters
(2S,4R)-19a–b. Finally (2S,4R)-20a–b were obtained by the same
procedure to synthesize (2S,4R)-16a–b.
Starting from (2S,4R)-14a–b (Scheme 2), the compounds
(2S,4S)-16a–b as well as (2S,4S)-20a–b could be obtained via the
same synthetic methods as described in Scheme 1.
It is know that Wolff’s rearrangement of diazoketones proceeds
occasionally with partial inversion of the chiral center next to the
carbonyl group, which depends on its applied catalysts, solvents or
structural characteristics.26–30 In our case no epimized products
were observed for the rearrangement of (2S,4S)-18a–b induced
by AgNO3 (0.2 equiv)–TEA (0.1 equiv) in methanol, whereas in
the same condition the rearrangement of (2S,4R)-18a as well as
(2S,4R)-18b gave (2S,4R)-19a (64%) and epimized (2R,4R)-19a
(12%), (2S,4R)-19b (54%) and epimized (2R,4R)-19b (9%), respec-
tively. We suspected that owing to the strong induction of the 4-
2. Chemistry
The acids (2S,4S)-13a–b were treated with CH2N222 in CH2Cl2 at
0 °C readily to give their corresponding esters in excellent yields
(Scheme 1). The conversion of the alcohol (2S,4S)-14a–b into their
corresponding fluorides with the stereo inversion at C-4 center was
accomplished in good yields by perfluoro-1-butanesulfonyl fluo-
ride and tetrabutylammonium triphenyldifluorosilicate (PBSF-
TBAT) in toluene at the presence of iPr2EtN according to the proce-
dure in our recent publication.23 The esters (2S,4R)-15a–b were
subject to sodium hydroxyl in ethanol, and salted by dried HCl
methanol solution in CH2Cl2 to give target compounds (2S,4R)-
16a–b.
(2S,4R)-16a–b were titrated with anhydrous 1 N NaOH ethanol
solution to offer the free acids (2S,4R)-17a–b. By employing
(COCl)2 (or SOCl2) and then CH2N2, however transformation of
the free acids (2S,4R)-17a–b into their diazoketones (2S,4R)-18a–
b failed. We speculated that the tertiary amine in the adjacent po-
sition of the acid group may affect the formation of the desired acid
chloride.24 Penke et al.25 reported that by applying a mixed carbox-
ylic-carbonic anhydride, the chain extension of the carboxylic acid
fluorine, the
a
-proton of the diazoketone became more active so
F
F
HO
HO
a
e
c, d
b
COOH
HCl
COOCH3
COOCH3
COOH
N
R
N
N
R
N
R
R
(2S,4R)-16a-b
(2S,4S)-14a-b
(2S,4S)-13a-b
(2S,4R)-15a-b
F
F
F
F
f, g
c, d
h
COOH
COOCH3
N
COCHN2
N
R
N
R
COOH
N
HCl
R
R
(2S,4R)-18a-b
(2S,4R)-19a-b
(2S,4R)-20a-b
(2S,4R)-17a-b
Scheme 1. Synthesis for (2S,4R)-16a–b and (2S,4R)-20a–b: (a) CH2N2, 0 °C; (b) PBSF-TBAT, iPr2EtN; (c) 4 N NaOH, C2H5OH, then 4 N HCl pH 5–6; (d) 37% HCl; (e) 1 N NaOH,
C2H5OH; (f) ibutyl chloroformate, TEA, ꢀ20 °C; (g) CH2N2, 0 °C; (h) AgNO3–TEA, CH3OH.

6542
W. Zhuang et al. / Bioorg. Med. Chem. 17 (2009) 6540–6546
F
HO
F
F
c, d
b
e
COOH
COOCH3
N
R
COOCH3
COOH
N
R
N
R
N
R
HCl
(2S,4S)-16a-b
(2S,4R)-14a-b
(2S,4S)-17a-b
(2S,4S)-15a-b
F
F
F
f, g
c, d
h
COOH
COOCH3
N
COCHN2
N
R
N
R
HCl
R
(2S,4S)-18a-b
(2S,4S)-19a-b
(2S,4S)-20a-b
Scheme 2. Synthesis for (2S,4S)-16a–b and (2S,4S)-20a–b: reagents and conditions for (a)–(h) were identical to those in Scheme 1.
F
F
F
F
F
TEA-H+
H
TEA
-H+
-
AgNO3
CH3OH
O
COOCH3
COCHN2
COCHN2
N
R
N
R
N
R
N
R
N
R
COCHN2
N2
(2R,4R)-19a-b
(2S,4R)-18a-b
21
(2R,4R)-18a-b
22
Scheme 3. Assumed epimized procedure during the Wolff’s rearrangement of (2S,4R)-18a–b.
that TEA as a base could deprive the proton to form carbonion 21,
whose tautomer 22 further afforded the epimer (2R,4R)-18a–b
(Scheme 3). To verify our assumption, a large amount of TEA
(2.0 equiv) was used in the rearrangement of (2S,4R)-19a–b, inter-
estingly, epimized (2R,4R)-19a (43%) and (2R,4R)-19b (45%)
respectively as major were obtained, but (2S,4R)-19a (35%) and
(2S,4R)-19b (29%), respectively as minor. From these results, we
may postulate that (2R,4R)-18a–b with the favorite cis conforma-
tion tended to become dominant at the presence of excessive
TEA (2.0 equiv), leading to the epimized products as major, in con-
trast (2S,4S)-19a–b with more stable cis conformation did not tend
to epimize to trans at the presence of a small amount of TEA
(0.1 equiv).
16b (IC50 = 18.0 lM), whatever its configuration of the C-4 is R or
S, exhibited sharp decrease in their inhibitory potency on GAT-1.
By comparison with the compound (2S,4R)-9 [IC50 of (2S,4R)-
9 = 3.29
presented approximately equivalent affinity [IC50 of (2S,4R)-
20a = 3.96 M; IC50 of (2S,4S)-20a = 5.81 M]. Furthermore, as re-
ferred to the compound (2S,4R)-10 [IC50 of (2S,4R)-10 = 3.15 M;
IC50 of (2S,4S)-10 = 5.14 M], this replacement of a hydroxyl group
with a fluorine at C-4 position only caused slightly improved affin-
ity [IC50 of (2S,4R)-20b = 2.97 M; IC50 of (2S,4S)-20b = 4.94 M].
Further contrary to the compounds 5 (IC50 = 0.40 M) and 7
(IC50 = 0.343 M), the tested 20a–b series of the 4-fluoro analogues
still displayed much weaker potency (IC50 ranged from 2.97 to
5.81 M). Moreover, it can be seen from Table 1 that the configu-
lM; IC50 of (2S,4S)-9 = 4.92 lM], their 4-fluoro derivatives
l
l
l
l
l
l
l
l
l
ration at C-4 linking fluorine appears to have no much influences
for their affinity.
3. Results and discussion
Because of the strong inductive effect, b-substituted fluorine of
an amino group tends to dramatically reduce its basicity.32,33 The
pKb data revealed that the basicity of b-trifluoroethylamine sharply
decreased nearly at five orders of magnitude in comparison with
that of ethylamine. From a series of statistical data,32,33 we may in-
fer that, in our case, this single fluorine b-substitution lead to a
drop of the basicity at not less than one order of magnitude. It is
known now that an amine functional group, preferably a secondary
amine, is an essentially pharmacophoric requirement for the affin-
ity of inhibitors to GABA transport proteins.34 Thus, we may as-
sume that the harsh reduction of their bascity accounts for the
big loss of their affinity to GAT-1.
The compounds (2S,4R)-16a–b, (2S,4S)-16a–b, (2S,4R)-20a–b
and (2S,4S)-20a–b were investigated as potential inhibitors of
GABA transport proteins on culture cell lines expressing mouse
GAT-1 in transport bioassay31 with (R)-Tiagabine (3) as a control.
The IC50 values of GABA uptake in vitro are presented in Table 1.
Compared with the compound (2S,4R)-8 (IC50 = 79.6
(2S,4R)-16a with fluorine in place of hydroxyl group exhibited a
slight improvement on its potency (IC50 = 67.4 M, IC50 of Tiga-
bine = 0.191 M as a control). But in contrast with the compound
6 (IC50 = 0.645 M as a control),
M;16 IC50 of Tiagabine = 0.159
M) and (2S,4S)-
lM),
l
l
l
l
both new derivatives (2S,4R)-16b (IC50 = 12.8
l
Table 1
4. Conclusion
GABA uptake IC50 of compounds 16a–b and 20a–b
No.
Compound no.
IC50 (lM)
In conclusion, the introduction of fluorine into the C-4 position
of the (2S)-proline derivatives and the (2S)-pyrrolidine-2-acetic
acid derivatives drastically decreased their inhibitory potency at
GAT-1, but exhibited slight improvement on their potency com-
pared with those corresponding 4-hydroxy derivatives. We as-
sumed that strong induction of the fluorine at b-position of the
amine functional group may cause drastic drop of their basicity
and decrease their affinity to GAT-1. Our results manifested that
weak basicity of the amino functional group is detrimental to their
1
2
3
4
5
6
7
8
9
3 (Tiagabine)
(2S,4R)-16a
(2S,4S)-16a
(2S,4R)-16b
(2S,4S)-16b
(2S,4R)-20a
(2S,4S)-20a
(2S,4R)-20b
(2S,4S)-20b
0.191
67.4
>100
12.8
18.0
3.96
5.81
2.97
4.94

W. Zhuang et al. / Bioorg. Med. Chem. 17 (2009) 6540–6546
6543
potent inhibition but the configuration of the carbon-4 linking
fluorine did not have large impact.
of alcohol) were added in turns at rt. Then PBSF (2.2 equiv) was
introduced into the resulting mixture. The stirring was continued
until TLC revealed complete conversion. The reaction mixture
was concentrated under reduced pressure. The residual was mixed
with a little bit of silica gel, dried in ventilation, and finally purified
by flash chromatography to give the corresponding fluoride.
5. Experimental part
5.1. General mark
(2S,4R)-15a: slightly yellow oil; yield: 82%;
½
a 1D8
ꢁ
¼ ꢀ43
~
(c = 0.88, ethyl acetate); IR:
m
3054, 2952, 1734, 1443, 1200, 761,
Reagents were purchased from commercial sources and used
without further purification unless otherwise indicated. Toluene
and diisopropylethylamine (iPr2NEt) were distilled from sodium un-
der nitrogen; Dichloromethane was distilled from CaH2 under nitro-
gen, while triethylamine (TEA) was refluxed with benzoyl chloride
and distilled under nitrogen before use. TBAT was prepared from tri-
phenylsilanol according the literature method.35 Melting points
were determined with a Büchi 540 Melting Point apparatus and
are uncorrected. If nothing else is stated, all packing for flash chro-
matography was silica gel (mesh 300–400). Optical rotation was
measured at k 589 cmꢀ1 of Na light. IR spectra were run on FI-IR
Spectrometer (Perkin Elmer) with oily samples as film and with solid
samples as pellets. 1H NMR spectra were recorded on Unity-600 if
nothing else is specified, and MS spectra on Agilent 1100 Series.
701 cmꢀ1; MS (m/z) ESI+: 354 [M+1]+; 1H NMR (CDCl3): d 2.11–
2.23 (m, 1H, NCHCH2), 2.28–2.38 (m, 3H, 2H of NCH2CH2 and 1H
of NCHCH2), 2.63 (dt, 1H, J = 11.9, 7.5 Hz, NCH2CH2), 2.71 (dd, 1H,
J = 30.8, 12.4 Hz, NCH2CHF), 2.84 (dt, 1H, J = 11.9, 7.5 Hz, NCH2CH2),
3.41 (ddd, 1H, J = 30.2, 11.5, 5.0 Hz, NCH2CHF), 3.57 (t, 1H,
J = 7.3 Hz, NCHCO), 3.69 (s, 3H, OCH3), 5.18 (d, 1H, J = 55.4 Hz,
CHF), 6.08 (t, 1H, J = 7.3 Hz, @CHCH2), 7.15–7.39 (m, 10H, Haromat.);
Calcd C22H24FNO2 (353.24): C, 74.76; H, 6.85; N, 3.96. Found: C,
74.48; H, 7.22; N, 3.81.
(2S,4S)-15a: slightly yellow oil; yield: 85%; ½a D21
ꢁ
¼ ꢀ29 (c = 0.92,
~
m
ethyl acetate); IR:
3104, 3058, 2930, 2854, 1739, 1436, 1158,
711 cmꢀ1; MS (m/z) ESI+: 354 [M+1]+; 1H NMR (CDCl3): d 2.20–
2.36 (m, 3H, 2H of NCH2CH2 and 1H of NCHCH2), 2.43–2.56 (m,
3H, NCHCH2, NCH2CH2 and NCH2CHF), 2.92 (dt, 1H, J = 12.4,
7.3 Hz, NCH2CH2), 3.19 (t, 1H, J = 7.4 Hz, NCHCO), 3.35 (dd, 1H,
J = 21.0, 11.5 Hz, NCH2CHF), 3.71 (s, 3H, OCH3), 5.10 (dt, 1H,
J = 54.5, 5.0 Hz, CHF), 6.07 (t, 1H, J = 7.3 Hz, @CHCH2), 7.15–7.38
(m, 10H, Haromat.); Calcd for C22H24FNO2 (353.44): C, 74.76; H,
6.85; N, 3.96. Found: C, 74.69; H, 7.28; N, 3.73.
5.2. General procedure 1 for the preparation of (2S,4S)-methyl
1-(4,4-diphenyl-3-butenyl)-4-hydroxylpyrrolidine -2-
carboxylate [(2S,4S)-14a] and (2S,4S)-methyl 1-[4,4-bis(3-
methyl-2-thienyl)-3-butenyl]-4-hydroxylpyrrolidine -2-
carboxylate [(2S,4S)-14b]
(2S,4R)-15b: yellow oil; yield: 78%; ½a D18
ꢁ
¼ ꢀ47 (c = 1.2, ethyl
~
acetate); IR:
m
3105, 3060, 2952, 1738, 1435, 1199, 713 cmꢀ1; MS
To a solution of the respective 13a–b (1 equiv) in CH2Cl2 (5 ml
per mmol), freshly prepared CH2N2 (3 equiv) in CH2Cl2 (4 ml per
mmol) was added at 0 °C. The excessive CH2N2 was decomposed
by slow addition of acetic acid. The reaction mixture was evapo-
rated to leave an oil, which was purified by flash chromatography.
(m/z) ESI+: 394 [M+1]+; 1H NMR (CDCl3): d 2.01 (s, 3H, thienyl–
CH3), 2.04 (s, 3H, thienyl–CH3), 2.11–2.24 (m, 1H, NCHCH2), 2.29–
2.39 (m, 3H, 2H of NCH2CH2 and 1H of NCHCH2), 2.60–2.66 (m,
1H, NCH2CH2), 2.73 (dd, 1H, J = 30.9, 11.4 Hz, NCH2CHF), 2.82–
2.89 (m, 1H, NCH2CH2), 3.43 (ddd, 1H, J = 30.6, 11.6, 4.5, NCH2CHF),
3.58 (t, 1H, J = 7.2 Hz, NCHCO), 3.71 (s, 3H, OCH3), 5.20 (br d, 1H,
J = 55.3 Hz, CHF), 6.05 (t, 1H, J = 7.1 Hz, @CHCH2), 6.75 (d, 1H,
J = 4.9 Hz, SC@CH), 6.84 (d, 1H, J = 4.9 Hz, SC@CH), 7.05 (d, 1H,
J = 4.9 Hz, SCH), 7.21 (d, 1H, J = 4.9 Hz, SCH); Calcd for
C20H24FNO2S2 (393.53): C, 61.04; H, 6.15; N, 3.56; S, 16.30. Found:
C, 60.84; H, 6.34; N, 3.37; S, 15.93.
(2S,4S)-14a: yellow oil; yield: 93%; ½a D20
ꢁ
¼ ꢀ45 (c = 1.1, ethyl
~
acetate) IR:
m
3307, 2982, 1728, 1233, 1110, 764 cmꢀ1; MS (m/z)
ESI+: 352 [M+1]+; 1H NMR (CDCl3): d 1.93 (dd, 1H, J = 14.1,
2.4 Hz, NCHCH2), 2.28–2.39 (m, 3H, 1H of NCHCH2 and 2H of
NCH2CH2), 2.57 (dd, 1H, J = 10.0, 4.2 Hz, NCH2CHOH), 2.60–2.66
(m, 1H, NCH2CH2), 2.82 (dt, 1H, J = 12.1, 7.9 Hz, NCH2CH2), 3.01
(d, 1H, J = 9.9 Hz, NCH2CHOH), 3.26 (dd, 1H, J = 9.3, 3.7 Hz, NCH),
3.71 (s, 3H, OCH3), 4.26 (t, 1H, J = 4.4 Hz, CHOH), 6.09 (t, 1H,
J = 7.2 Hz, @CHCH2), 7.16–7.41 (m, 10H, Haromat.); Calcd for
C22H25NO3 (351.44): C, 75.19; H, 7.17; N, 3.99. Found: C, 74.97;
H, 7.29; N, 3.75.
(2S,4S)-15b: yellow oil; yield: 80%; 1H NMR and IR spectra were
identical with those in the literature.23
5.4. General procedure 3 for the preparation of 2-diazoacetyl-1-
(4,4-diphenyl-3-butenyl)-4-fluoropyrrolidine (18a) and 1-[4,4-
bis(3-methyl-2-thienyl)-3-butenyl]-2-diazoacetyl-4-
fluoropyrrolidine (18b)
(2S,4S)-14b: yellow oil; yield: 95%; ½a D20
¼ ꢀ51 (c = 0.95, ethyl
ꢁ
acetate); IR:
3258, 3035, 2980, 1739, 1520, 1259, 1110 cmꢀ1
~
m ;
MS (m/z) ESI+: 392 [M+1]+; 1H NMR (CDCl3): d 2.00 (s, 3H, thie-
nyl–CH3), 2.04 (s, 3H, thienyl–CH3), 2.29–2.39 (m, 3H, 1H of
NCHCH2 and 2H of NCH2CH2), 2.56 (dd, 1H, J = 10.0, 4.6 Hz,
NCH2CHOH), 2.61 (dt, 1H, J = 12.4, 7.3 Hz, NCH2CH2), 2.82 (dt, 1H,
J = 12.4, 8.2 Hz, NCH2CH2), 3.03 (d, 1H, J = 10.0 Hz, NCH2CHOH),
3.26 (dd, 1H, J = 10.0, 3.9 Hz, NCH), 3.72 (s, 3H, OCH3), 4.26 (t,
1H, J = 4.7 Hz, CHOH), 6.05 (t, 1H, J = 7.3 Hz, @CHCH2), 6.76 (d,
1H, J = 5.5 Hz, SC@CH), 6.85 (d, 1H, J = 5.5 Hz, SC@CH), 7.05 (d,
1H, J = 5.5 Hz, SCH), 7.22 (d, 1H, J = 5.5 Hz, SCH); Calcd for
C20H25NO3S2 (391.54): C, 61.35; H, 6.44; N, 3.58; S, 16.38. Found:
C, 61.04; H, 6.18; N, 3.25; S, 16.59.
The respective salt of 16a–b (1 equiv) was titrated with 1 N
NaOH (1 equiv) in anhydrous ethanol, and the free acid 17a–b
was obtained after removal of ethanol. The acid was then dissolved
in CH2Cl2 solution (5 ml per mmol), TEA (1.2 equiv) was added and
then isobutanyl chloroformate (1.1 equiv) was introduced at
ꢀ20 °C. After stirring was continued for 1.5 h, freshly prepared
CH2N2 (5 equiv) in CH2Cl2 was added at 0 °C. Two hours later, the
reaction mixture was evaporated under reduced pressure at rt in
a hood with good ventilation. Purification by flash chromatography
gave each diazo compound.
(2S,4R)-18a: yellow oil; yield: 81%; ½a D18
ꢁ
¼ ꢀ53 (c = 0.90, ethyl
5.3. General procedure 2 for the preparation of methyl 1-(4,4-
diphenyl-3-butenyl)-4-fluoropyrrolidine-2-carboxylate (15a)
and methyl 1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-
fluoropyrrolidine-2-carboxylate (15b)
acetate); MS (m/z) ESI+ 364 [M+1]+, 386 [M+Na]+; IR:
3101,
~
m
3023, 2964, 2104, 1639, 1343, 762, 701 cmꢀ1 1H NMR (CDCl3): d
;
1.80–1.93 (m, 1H, NCHCH2), 2.29 (q, 2H, J = 7.2 Hz, NCH2CH2),
2.38 (ddd, 1H, J = 20.6, 14.7, 6.9 Hz, NCHCH2), 2.56–2.66 (m, 2H,
NCH2CHF and NCH2CH2), 2.78 (dt, 1H, J = 11.6, 7.3 Hz, NCH2CH2),
3.34 (ddd, 1H, J = 32.0, 12.4, 4.6 Hz, NCH2CHF), 3.43 (dd, 1H,
To a mixture of the respective alcohols 14a–b (1 equiv) and
TBAT (0.8 equiv), iPr2EtN (2.5 equiv) and toluene (8 ml per mmol

6544
W. Zhuang et al. / Bioorg. Med. Chem. 17 (2009) 6540–6546
J = 9.8, 7.3 Hz, NCHCO), 5.08 (d, 1H, J = 55.4 Hz, CHF), 5.82 (s, 1H,
CHN2), 6.11 (t, 1H, J = 7.3 Hz, @CHCH2), 7.16–7.41 (m, 10H,
Haromat.); Calcd for C22H22FN3O (363.44): C, 72.71; H, 6.10; N,
11.56. Found: C, 72.56; H, 6.26; N, 11.34.
3H, OCH3), 5.08 (dt, 1H, J = 55.0, 5.0 Hz, CHF), 6.08 (t, 1H,
J = 7.3 Hz, @CHCH2), 7.15–7.40 (m, 10H, Haromat.); Calcd for
C23H26FNO2 (367.46): C, 75.18; H, 7.13; N, 3.81. Found: C, 74.85;
H, 7.42; N, 3.66.
(2S,4S)-18a: yellow oil; yield: 56%; ½a D21
ꢁ
¼ ꢀ56 (c = 1.1, ethyl
(2R,4R)-19a by Method A from (2S,4R)-18a: yellow oil, Rf large;
~
acetate); MS (m/z) ESI+: 364 [M+1]+; IR:
m
3116, 3055, 2969,
1H NMR (CDCl3): d 2.08
yield: 12%; ½a 2D2
ꢁ
¼ þ47 (c = 0.88, ethyl acetate); IR:
m 3058, 3023,
~
2808, 2103, 1635, 1348, 763, 702 cmꢀ1
;
2951, 2847, 1738, 1443, 1163, 763, 701 cmꢀ1; MS (m/z) ESI+: 368
[M+1]+; 1H NMR (CDCl3): d 1.85 (ddd, 1H, J = 31.6, 15.0, 6.9 Hz,
NCHCH2), 2.22–2.49 (m, 6H, NCHCH2, NCH2CH2, CH2COO, NCH2CHF
and 2H of NCH2CH2), 2.70 (dd, 1H, J = 15.0, 3.9 Hz, CH2COO), 2.78–
2.91 (m, 2H, NCH2CH2 and NCHCH2), 3.26 (dd, 1H, J = 20.2, 11.5 Hz,
NCH2CHF), 3.66 (s, 3H, OCH3), 5.06 (dt, 1H, J = 55.5, 4.8 Hz, CHF),
6.08 (t, 1H, J = 7.3 Hz, @CHCH2), 7.16–7.39 (m, 10H, Haromat.).
(2S,4R)-19a by Method B from (2S,4R)-18a; yield: 35%.
(2R,4R)-19a by Method B from (2S,4R)-18a: yield: 43%.
(2S,4S)-19a by Method A from (2S,4S)-18a: yellow oil; yield:
~
(ddd, 1H, J = 27.0, 15.6, 5.0 Hz, NCHCH2), 2.28–2.39 (m, 3H, 2H of
NCH2CH2 and 1H of NCHCH2), 2.43–2.59 (m, 2H, NCH2CH2 and
NCH2CHF), 2.79 (dt, 1H, J = 12.0, 8.0 Hz, NCH2CH2), 3.11 (dd, 1H,
J = 11.0, 5.0 Hz, NCHCO), 3.32 (dd, 1H, J = 17.5, 12.0 Hz, NCH2CHF),
5.08 (dt, 1H, J = 53.1, 3.8 Hz, CHF), 6.05 (s, 1H, CHN2), 6.11 (t, 1H,
J = 7.2 Hz, =CHCH2), 7.14–7.44 (m, 10H, Haromat.); Calcd for
C22H22FN3O (363.44): C, 72.71; H, 6.10; N, 11.56. Found: C,
72.38; H, 6.36; N, 11.29.
(2S,4R)-18b: yellow oil; yield: 67%; ½a D24
ꢁ
¼ ꢀ62 (c = 0.89, ethyl
acetate); MS (m/z) ESI+: 402 [Mꢀ1]+; IR:
3106, 2823, 2828,
82%; ½a 2D0
ꢁ
¼ ꢀ45 (c = 1.3, ethyl acetate); IR:
m 3023, 2951, 2801,
~
m
2105, 1635, 1346, 714 cmꢀ1
;
1H NMR (CDCl3): d 1.81–1.95 (m,
1739, 1436, 762, 702 cmꢀ1; MS (m/z) ESI+: 368 [M+1]+; 1H NMR
(CDCl3, 400 Mz): d 1.84–1.96 (m, 1H, NCHCH2), 2.27–2.62 (m, 6H,
NCHCH2, NCH2CH2, CH2COO, NCH2CHF and 2H of NCH2CH2), 2.75
(dd, 1H, J = 15.0, 3.9 Hz, CH2COO), 2.80–2.99 (m, 2H, NCH2CH2
and NCHCH2), 3.28 (dd, 1H, J = 17.5, 11.6 Hz, NCH2CHF), 3.69 (s,
3H, OCH3), 5.08 (br d, 1H, J = 54.8 Hz, CHF), 6.09–6.14 (m, 1H,
@CHCH2), 7.16–7.43 (m, 10H, Haromat.); Calcd for C23H26FNO2
(367.46): C, 75.18; H, 7.13; N, 3.81. Found: C, 75.43; H, 6.92; N,
3.59.
4H, 1H of NCHCH2 and 3H of thienyl–CH3), 2.05 (s, 3H, thienyl–
CH3), 2.28–2.44 (m, 3H, 2H of NCH2CH2 and 1H of NCHCH2),
2.55–2.66 (m, 2H, NCH2CH2 and NCH2CHF), 2.78–2.84 (m, 1H,
NCH2CH2), 3.36 (ddd, 1H, J = 32.1, 12.5, 4.4 Hz, NCH2CHF), 3.44
(dd, 1H, J = 9.2, 7.8 Hz, NCHCO), 5.10 (d, 1H, J = 54.5 Hz, CHF),
5.93 (s, 1H, CHN2), 6.10 (t, 1H, J = 7.3 Hz, @CHCH2), 6.76 (d, 1H,
J = 5.0 Hz, SCH), 6.86 (d, 1H, J = 5.0 Hz, SCH), 7.06 (d, 1H,
J = 5.2 Hz, SC@CH), 7.24 (d, 1H, J = 5.2 Hz, SC@CH); Calcd for
C20H22FN3OS2 (403.53): C, 59.53; H, 5.50; N, 10.41; S, 15.89. Found:
C, 59.41; H, 5.64; N, 10.18; S, 15.60.
(2S,4R)-19b by Method A from (2S,4R)-18b: yellow oil, Rf small;
~
m 3101, 2924,
yield: 54%; ½a 3D2
ꢁ
¼ ꢀ40 (c = 0.58, ethyl acetate); IR:
(2S,4S)-18b: yellow oil: yield: 62%; ½a D18
ꢁ
¼ ꢀ75 (c = 0.80, ethyl
2855, 1743, 1456, 710 cmꢀ1; MS (m/z) ESI+: 408 [M+1]+; 1H NMR
(CDCl3): d 1.70–1.82 (m, 1H, NCHCH2), 2.00 (s, 3H, thienyl–CH3),
2.03 (s, 3H, thienyl–CH3), 2.23–2.34 (m, 4H, 2H of NCH2CH2, 1H
of NCHCH2 and 1H of CH2COO), 2.42–2.56 (m, 2H, NCH2CH2 and
NCH2CHF), 2.65 (dd, 1H, J = 14.8, 3.9 Hz, CH2COO), 2.86 (dt, 1H,
J = 11.9, 8.2 Hz, NCH2CH2), 3.08–3.14 (m, 1H, NCHCH2), 3.41 (ddd,
1H, J = 28.0, 11.6, 5.2 Hz, NCH2CHF), 3.65 (s, 3H, OCH3), 5.09 (br
d, 1H, J = 55.4 Hz, CHF), 6.04 (d, 1H, J = 7.3 Hz, @CHCH2), 6.76 (d,
1H, J = 5.0 Hz, SCH), 6.84(d, 1H, J = 5.0 Hz, SCH), 7.05 (d, 1H,
J = 5.1 Hz, SC@CH), 7.21 (d, 1H, J = 5.1 Hz, SC@CH); Calcd for
C21H26FNO2S2 (407.56): C, 61.89; H, 6.43; N, 3.45; S, 15.73. Found:
61.81; H, 6.21; N, 3.28; S, 15.46.
acetate); MS (m/z) ESI+: 404 [M+1]+; IR:
3111, 2922, 2811,
~
m
2103, 1688, 1635, 1348, 712 cmꢀ1 1H NMR (CDCl3): d 1.95 (s,
;
3H, thienyl–CH3), 2.04 (s, 3H, thienyl–CH3), 2.05–2.15 (m, 1H,
NCHCH2), 2.32–2.42 (m, 3H, 2H of NCH2CH2 and 1H of NCHCH2),
2.44–2.55 (m, 2H, NCH2CH2 and NCH2CHF), 2.82 (dt, 1H, J = 12.0,
8.0 Hz, NCH2CH2), 3.11 (dd, 1H, J = 10.4, 5.5 Hz, NCHCO), 3.34
(ddd, 1H, J = 18.3, 11.4, 1.5 Hz, NCH2CHF), 5.09 (dt, 1H, J = 53.5,
4.0 Hz, CHF), 6.08–6.14 (m, 2H, @CHCH2 and CHN2), 6.76 (d, 1H,
J = 5.2 Hz, SCH), 6.86 (d, 1H, J = 5.2 Hz, SCH), 7.06 (d, 1H,
J = 5.2 Hz, SC@CH), 7.23 (d, 1H, J = 5.2 Hz, SC@CH); Calcd for
C20H22FN3OS2 (403.53): C, 59.53; H, 5.50; N, 10.41; S, 15.89. Found:
C, 59.33; H, 5.83; N, 10.25; S, 15.52.
(2R,4R)-19b by Method A from (2S,4R)-18b: yellow oil, Rf large;
~
m 3101, 2925,
yield: 9%; ½a 3D2
ꢁ
¼ þ75 (c = 0.96, ethyl acetate); IR:
2855, 1733, 1436, 712 cmꢀ1; MS (m/z) ESI+: 408 [M+1]+; 1H NMR
(CDCl3): d 1.84 (ddd, 1H, J = 31.2, 15.1, 6.6 Hz, NCHCH2), 2.03 (s,
3H, thienyl–CH3), 2.04 (s, 3H, thienyl–CH3), 2.26–2.50 (m, 6H,
NCHCH2, NCH2CH2, CH2COO, NCH2CHF and 2H of NCH2CH2), 2.72
(dd, 1H, J = 15.2, 4.1 Hz, CH2COO), 2.78–2.92 (m, 2H, NCH2CH2
and NCHCH2), 3.29 (dd, 1H, J = 20.3, 11.5 Hz, NCH2CHF), 3.67 (s,
OCH3), 5.08 (dt, 1H, J = 54.5, 4.6 Hz, CHF), 6.05 (t, 1H, J = 7.3 Hz,
@CHCH2), 6.76 (d, 1H, J = 5.5 Hz, SCH), 6.84 (d, 1H, J = 5.0 Hz,
SCH), 7.05 (d, 1H, J = 5.5 Hz, SC@CH), (d, 1H, J = 5.0 Hz, SC@CH);
Calcd for C21H26FNO2S2 (407.56): C, 61.89; H, 6.43; N, 3.45; S,
15.73. Found: C, 61.95; H, 6.24; N, 3.17; S, 15.42.
5.5. General procedure 4 for the preparation of methyl 1-(4,4-
diphenyl-3-butenyl)-4-fluoropyrrolidine-2-acetate (19a) and
methyl 1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-
fluoropyrrolidine-2-acetate (19b)
Method A: To a solution of the respective 18a–b (1 equiv) in
MeOH (7 ml), a solution of AgNO3 (0.2 equiv) and TEA (0.1 equiv)
in MeOH (4 ml) was added at rt. The mixture turned to be brown
within a few minutes, and then warmed to 60 °C for 30 min. The
reaction mixture was decolorized with charcoal and concentrated.
The resulting oil was purified by flash chromatography to give the
desired ester.
(2S,4R)-19b by Method B from (2S,4R)-18b: yield: 29%.
(2R,4R)-19b by Method B from (2S,4R)-18b: yield: 45%.
(2S,4S)-19b by Method A from (2S,4S)-18b: yellow oil; yield:
~
Method B is the same as Method A but TEA (2.0 equiv) was
employed.
75%; ½a 2D0
ꢁ
¼ ꢀ72 (c = 1.1, ethyl acetate); IR:
m 3087, 3063, 2952,
2836, 1727, 739, 700 cmꢀ1; MS (m/z) ESI+: 408 [M+1]+; 1H
NMR (CDCl3, 400Mz): d 1.80–1.96 (m, 1H, NCHCH2), 1.98 (s,
3H, thienyl–CH3), 2.06 (s, 3H, thienyl–CH3), 2.27–2.65 (m, 6H,
NCHCH2, NCH2CH2, CH2COO, NCH2CHF and 2H of NCH2CH2),
2.67–2.98 (m, 3H, CH2COO, NCH2CH2 and NCHCH2), 3.30 (dd,
1H, J = 17.5, 11.0 Hz, NCH2CHF), 3.70 (s, 3H, OCH3), 5.03–5.18
(m, 1H, CHF), 6.04–6.09 (m, 1H, @CHCH2), 6.78 (d, 1H,
J = 5.1 Hz, SCH), 6.86 (d, 1H, J = 5.1 Hz, SCH), 7.07(d, 1H,
(2S,4R)-19a by Method A from (2S,4R)-18a: yellow oil, Rf small;
~
yield: 64%; ½a 2D0
ꢁ
¼ ꢀ37 (c = 0.92, ethyl acetate); IR:
m 3058, 3021,
2949, 2854, 1735, 1443, 1158, 761, 704 cmꢀ1; MS (m/z) ESI+: 368
[M+1]+; 1H NMR (CDCl3): d 1.72–1.84 (m, 1H, NCHCH2), 2.21–
2.32 (m, 4H, 2H of NCH2CH2, 1H of NCHCH2 and 1H of CH2COO),
2.41–2.53 (m, 2H, NCH2CH2 and NCH2CHF), 2.63 (dd, 1H, J = 13.5,
3.9 Hz, CH2COO), 2.83–2.89 (m, 1H, NCH2CH2), 3.07–3.13 (m, 1H,
NCHCH2), 3.39 (ddd, 1H, J = 28.0, 11.9, 5.0 Hz, NCH2CHF), 3.65 (s,

W. Zhuang et al. / Bioorg. Med. Chem. 17 (2009) 6540–6546
6545
J = 5.1 Hz, SC@CH), 7.23 (d, 1H, J = 5.1 Hz, SC@CH); Calcd for
C21H26FNO2S2 (407.56): C, 61.89; H, 6.43; N, 3.45; S, 15.73.
Found: 61.72; H, 6.34; N, 3.68; S, 15.57.
(415.97) 0.8CH3OH: C, 53.85; H, 5.98; N, 3.17; S, 14.52. Found: C,
53.60; H, 6.24; N, 3.01; S, 14.20.
(2S,4R)-20a: yellow foam; yield: 85%; ½a D22
ꢁ
¼ ꢀ12 (c = 0.85, eth-
anol); IR:
m
3022, 2927, 2855, 1732, 1444, 1149, 703 cmꢀ1; MS (m/
~
5.6. General procedure 5 for the preparation of 1-(4,4-diphenyl-
3-butenyl)-4-fluoropyrrolidine-2-carboxylic acid hydrochloride
(16a) and 1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-
fluoropyrrolidine-2-carboxylic acid hydrochloride (16b); 1-(4,4-
diphenyl-3-butenyl)-4-fluoropyrrolidine-2-acetic acid
z) ESI+: 354 [MꢀCl]+; 1H NMR (CD3OD): d 1.88–2.07 (m, 1H,
NCHCH2), 2.44–2.62 (m, 3H, 2H of NCH2CH2 and 1H of NCHCH2),
2.75 (dd, 1H, J = 17.9, 6.0 Hz, CH2COO), 2.85 (dd, 1H, J = 17.9,
6.0 Hz, CH2COO), 3.18–3.25 (m, 1H, NCH2CH2), 3.47 (dd, 1H,
J = 20.8, 14.4 Hz, NCH2CHF), 3.54–3.61 (m, 1H, NCH2CH2), 3.68
(dd, 1H, J = 34.4, 14.4, 3.9 Hz, NCH2CHF), 4.00–4.02 (m, 1H,
NCHCH2), 5.23 (dt, 1H, J = 52.2, 3.7 Hz, CHF), 5.99 (t, 1H,
J = 7.5 Hz, @CHCH2), 7.10–7.37 (m, 10H, Haromat.); Calcd for
C22H25ClFNO2 (388.89) 0.4CH3OH: C, 66.93; H, 6.66; N, 3.48.
Found: C, 66.98; H, 6.37; N, 3.16.
hydrochloride (20a) and 1-[4,4-bis(3-methyl -2-thienyl)-3-
butenyl]-4-fluoropyrrolidine-2-acetic acid hydrochloride (20b)
The respective ester of 15a–b or 19a–b (1 eq mmol) in ethanol
(10 ml/mmol) was subject to 4.0 M NaOH (3 equiv) at rt for 24 h.
Then the solution was acidified to pH 5–6 with aq 1.0 M HCl. After
ethanol had been removed in vacuo, the residue was extracted
with CH2Cl2, dried over Na2SO4 and then filtrated. After 1.0 M dried
HCl CH3OH solution (1 equiv) was added into the filter, solvents
were removed under reduced pressure to afford the desired prod-
uct, which further dried in vacuo.
(2S,4S)-20a: yellow foam; yield: 83%; ½a D24
ꢁ
¼ ꢀ15 (c = 0.76, eth-
~
anol); IR:
m
3419, 2924, 1729, 1408, 1196, 716 cmꢀ1; MS (m/z) ESI+:
354 [MꢀCl]+; 1H NMR (CD3OD): d 2.01 (ddd, 1H, J = 30.0, 15.1,
7.3 Hz, NCHCH2), 2.49–2.78 (m, 5H, 1H of NCHCH2, 2H of NCH2CH2
and 2H of CH2COO), 2.89–2.94 (m, 1H, NCH2CH2), 3.01 (ddd, 1H,
J = 34.8, 12.8, 4.1 Hz, NCH2CHF), 3.49–3.60 (m, 3H, NCHCH2,
NCH2CH2 and NCH2CHF), 5.25 (dt, 1H, J = 53.2, 4.6 Hz, CHF), 6.08
(t, 1H, J = 7.3 Hz, =CHCH2), 7.19–7.45 (m, 10H, Haromat.); Calcd for
C22H25ClFNO2 (388.89) 0.7CH3OH: 67.34, H, 6.92; N, 3.46. Found:
C, 67.46; H, 7.16; N, 3.08.
(2S,4R)-16a: gray crystals; yield: 76%; mp 125–129 °C;
~
m 3027, 2939, 1727, 1446, 1360,
½
a 2D1
ꢁ
¼ ꢀ15 (c = 1.1, ethanol); IR:
1213, 768, 703 cmꢀ1; MS (m/z) ESI+: 340 [MꢀCl]+; 1H NMR
(CD3OD):
d 2.19–2.32 (m, 1H, NCHCH2), 2.44–2.53 (m, 2H,
NCH2CH2), 2.68–2.77 (m, 1H, NCHCH2), 3.25–3.34 (m, 1H,
NCH2CH2), 3.44–3.55 (m, 2H, NCH2CH2 and NCH2CHF), 3.83 (ddd,
1H, J = 34.2, 14.1, 4.4, Hz, NCH2CHF), 4.55 (dd, 1H, J = 12.2, 7.1 Hz,
NCHCO), 5.36 (d, 1H, J = 53.0 Hz, CHF), 6.00 (t, 1H, J = 7.3 Hz,
@CHCH2), 7.08–7.20 (m, 6H, Haromat.), 7.26–7.38 (m, 4H, Haromat);
C21H23ClFNO2 (375.89) 1.1CH3OH: C, 64.21; H, 6.71; N, 3.40.
Found: C, 63.90; H, 6.95; N, 3.12.
(2S,4R)-20b: gray foam; yield: 93%; ½a D22
ꢁ
¼ ꢀ20 (c = 0.95, etha-
~
nol), IR:
m
3101, 2923, 2855, 1737, 1425, 1029, 722 cmꢀ1; MS (m/
z) ESI+: 394 [MꢀCl]+; 1H NMR (CD3OD): d 1.89 (s, 3H, thienyl–
CH3), 1.95 (s, 3H, thienyl–CH3), 1.92–2.05 (m, 1H, NCHCH2), 2.45–
2.56 (m, 3H, 1H of NCHCH2 and 2H of NCH2CH2), 2.69–2.78 (m,
2H, CH2COO), 3.12 (dt, 1H, J = 12.5, 7.3 Hz, NCH2CH2), 3.38 (dd,
1H, J = 22.5, 13.7 Hz, NCH2CHF), 3.47 (dt, 1H, J = 12.5, 8.0 Hz,
NCH2CH2), 3.76 (ddd, 1H, J = 33.7, 13.7, 3.8 Hz, NCH2CHF), 3.86–
3.92 (m, 1H, NCHCH2), 5,25 (br d, 1H, J = 52.7 Hz, CHF), 5.95 (t,
1H, J = 7.3 Hz, @CHCH2), 6.69 (d, 1H, J = 5.5 Hz, SCH), 6.82 (d, 1H,
J = 5.0 Hz, SCH), 7.07 (d, 1H, J = 5.5 Hz, SC@CH), 7.28 (d, 1H,
J = 5.0 Hz, SC@CH); Calcd for C20H25ClFNO2S2 (430.01) 0.9CH3OH:
C, 54.70; H, 6.28; N, 3.05; S, 13.98. Found: C, 54.42; H, 6.05; N,
2.88; S, 13.81.
(2S,4S)-16a: yellow crystals; yield: 85%; mp: 160–165 °C;
~
m 3050, 2979, 2604, 2497,
½
a 1D8
ꢁ
¼ ꢀ11 (c = 0.94, ethanol); IR:
1705, 1442, 1208, 699 cmꢀ1; MS (m/z) ESI: 340 [MꢀCl]+; 1H NMR
(CD3OD): d 2.43–2.58 (m, 3H, 2H of NCH2CH2 and 1H of NCHCH2),
2.78–2.91 (m, 1H, NCHCH2), 3.29–3.49 (m, 3H, 2H of NCH2CH2 and
1H of NCH2CHF), 3.86 (t, 1H, J = 15.2 Hz, NCH2CHF), 4.41 (dd, 1H,
J = 11.0, 3.7 Hz, NCHCO), 5.32 (d, 1H, J = 52.0 Hz, CHF), 6.01 (t, 1H,
J = 7.3 Hz, @CHCH2), 7.10–7.39 (m, 10H, Haromat.); C21H23ClFNO2
(375.89) 1.2C2H5OH: C, 65.17; H, 7.06; N, 3.25. Found: C, 65.01;
H, 7.33; N, 2.98.
(2S,4S)-20b: gray foam; yield: 85%; ½a D24
¼ ꢀ24 (c = 0.83, etha-
ꢁ
nol); IR:
3440, 3100, 2945, 2560, 1726, 1457, 1171, 718 cmꢀ1
~
m ;
MS (m/z) ESI+: 394 [MꢀCl]+; 1H NMR (CD3OD): d 1.91–2.02 (m,
1H, NCHCH2), 1.99 (s, 3H, thienyl–CH3), 2.04 (s, 3H, thienyl–CH3),
2.50–2.75 (m, 5H, 2H of NCH2CH2, 2H of CH2COO and 1H of
NCHCH2), 2.80–2.86 (m, 1H, NCH2CH2), 2.97 (ddd, 1H, J = 34.8,
12.8, 4.1 Hz, NCH2CHF), 3.38–3.46 (m, 2H, NCH2CH2 and NCHCH2),
3.65 (dd, 1H, J = 18.6, 12.8 Hz, NCH2CHF), 5.24 (dt, 1H, J = 53.2,
5.0 Hz, CHF), 6.05 (t, 1H, J = 7.3 Hz,@CHCH2), 6.78 (d, 1H,
J = 5.0 Hz, SCH), 6.90 (d, 1H, J = 5.0 Hz, SCH), 7.15 (d, 1H,
J = 5.0 Hz, SC@CH), 7.35 (d, 1H, J = 5.0 Hz, SC@CH), Calcd for
C20H25ClFNO2S2 (430.01) 0.5CH3OH: C, 55.20; H, 6.10; N, 3.14; S,
14.91. Found: C, 55.47; H, 5.95; N, 2.83; S, 14.67.
(2S,4R)-16b: Gray crystals; yield: 92%; mp: 106–110 °C;
~
m 3421, 4104, 2922, 1740, 1710,
½
a 2D1
ꢁ
¼ ꢀ22 (c = 1.2, ethanol); IR:
1407, 1368, 1202, 741, 722 cmꢀ1; MS (m/z) ESI+: 380 [MꢀCl]+; 1H
NMR (CDCl3): d 2.00 (s, 3H, thienyl–CH3), 2.05 (s, 3H, thienyl–
CH3), 2.30–2.44 (m, 1H, NCHCH2), 2.58–2.66 (m, 2H, NCH2CH2),
2.78–2.86 (m, 1H, NCHCH2), 3.39 (ddd, 1H, J = 12.4, 9.6, 6.6 Hz,
NCH2CH2), 3.56 (ddd, J = 12.4, 9.6, 6.6 Hz, NCH2CH2), 3.65 (dd, 1H,
J = 22.5, 13.9 Hz, NCH2CHF), 3.99 (ddd, 1H, J = 34.0, 13.9, 3.6 Hz,
NCH2CHF), 4.68 (dd, 1H, J = 12.0, 7.0 Hz, NCHCO), 5.43 (dt, 1H,
J = 52.2, 3.6 Hz, CHF), 6.04 (t, 1H, J = 7.3 Hz, @CHCH2), 6.79 (d, 1H,
J = 5.0 Hz, SCH), 6.93 (d, 1H, J = 5.0 Hz, SCH), 7.18 (d, 1H,
J = 5.0 Hz, SC@CH), 7.39 (d, 1H, J = 5.0 Hz, SC@CH); C19H23ClFNO2S2
(415.97) 0.8CH3OH: C 53.85, H 5.98, N 3.17, S 14.52; found. C53.60,
H 6.24, N 3.01, S 14.20.2
5.7. Bioassay
D8 cells, grown in RMP I1640 medium (Gibco-BRL Life Technol-
ogies) containing 10% FBS (Gibco-BRL Life Technologies) to near
confluence in 48-well tissue culture plates (Costar) (approximately
60,000 cells per well in 48-well plates), were rinsed once with
(2S,4S)-16b: Gray crystals; yield: 87%; mp: 123–127 °C;
~
m 3410, 3096, 2969, 2492,
½
a 1D8
ꢁ
¼ ꢀ16 (c = 0.94, ethanol); IR:
1746, 1636, 1372, 1201, 719 cmꢀ1; MS (m/z) ESI+: 380 [MꢀCl]+,
402 [MꢀCl+Na]+; 1H NMR (CD3OD): d 1.90 (s, 3H, thienyl–CH3),
1.97 (s, 3H, thienyl–CH3), 2.44–2.61 (m, 3H, 2H of NCH2CH2 and
1H of NCHCH2), 2.78–2.91 (m, 1H, NCHCH2), 3.26–3.47 (m, 3H,
2H of NCH2CH2 and 1H of NCH2CHF), 3.89 (t, 1H, J = 14.5 Hz,
NCH2CHF), 4.45 (dd, 1H, J = 11.0, 4.6 Hz, NCHCO), 5.33 (d, 1H,
J = 50.9 Hz, CHF), 5.93 (t, 1H, J = 7.3 Hz, @CHCH2), 6.71 (d, 1H,
J = 5.0 Hz, SCH), 6.84 (d, 1H, J = 5.0 Hz, SCH), 7.11 (d, 1H,
J = 5.0 Hz, SC@CH),7.30 (d, 1H, J = 5.0 Hz, SC@CH); C19H23ClFNO2S2
phosphate buffered saline (PBS) and pre-incubated in 100 lL
Hanks, balanced salt solution (HBSS) for 10 min at room tempera-
ture. [3H] GABA (Amersham Pharmacia Biotech) was added to final
concentrations of 151 nM. The cells were incubated for 20 min at
room temperature. The reaction was terminated by aspiration of
the HBSS and the cells were washed three times rapidly (10 s/
wash) with cold PBS. The cells were then solubilized in 2 N NaOH
and an aliquot was measured by liquid scintillation counting

6546
W. Zhuang et al. / Bioorg. Med. Chem. 17 (2009) 6540–6546
(Beckman LS 5000 TA) to quantify the uptake of [3H] GABA. Inhibi-
tion studies were performed by adding inhibitor at the beginning
of the transport assay and IC50 values were determined by nonlin-
ear regression analysis.
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Acknowledgment
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