Exploration of (S)-3-aminopyrrolidine as a potentially interesting scaffold for discovery of novel Abl and PI3K dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.01.020

Based on the literature-reported compensatory effect of PI3K on Abl inhibition and the improved preclinical effect of drug combination of Abl and PI3K inhibitors, a series of compounds bearing novel scaffold of (S)-3-aminopyrrolidine was identified as Abl and PI3K dual inhibitors through support vector machine screening tool, which were subsequently synthesized and tested. Most compounds demonstrated promising cytoxicity against a CML leukemia cell-line K562 and moderate inhibition against Abl and PI3K kinases. These compounds induced no apoptosis in K562 cell-line, suggesting that their cytotoxic activities are unlikely duo to other known anti-CML mechanisms. Molecular docking study further showed that the compound 5k could bind with both Abl and PI3K, but the weaker binding with Abl compared to Imatinib is consistent with its low kinase inhibitory rates. These plus literature-reported evidences suggest that the promising cytotoxic effect of our novel compounds might be due to the collective effect of Abl and PI3K inhibition.

Full text of DOI:10.1016/j.ejmech.2011.01.020

European Journal of Medicinal Chemistry 46 (2011) 1404e1414
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Exploration of (S)-3-aminopyrrolidine as a potentially interesting scaffold for
discovery of novel Abl and PI3K dual inhibitors
,
Cunlong Zhang a b, Chunyan Tan b, Xuyu Zu b, Xin Zhai a, Feng Liu b, Bizhu Chu b, Xiaohua Ma c,
,b,
Yuzong Chen c, Ping Gong a, Yuyang Jiang d
*
a Key Laboratory of Original New Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang,
Liaoning 110016, China
b The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
c Bioinformatics and Drug Design Group, Department of Computational Science, National University of Singapore, Blk SOC1, Level 7, 3 Science Drive 2, Singapore 117543, Singapore
d School of Medicine, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the literature-reported compensatory effect of PI3K on Abl inhibition and the improved
preclinical effect of drug combination of Abl and PI3K inhibitors, a series of compounds bearing novel
scaffold of (S)-3-aminopyrrolidine was identified as Abl and PI3K dual inhibitors through support vector
machine screening tool, which were subsequently synthesized and tested. Most compounds demon-
strated promising cytoxicity against a CML leukemia cell-line K562 and moderate inhibition against Abl
and PI3K kinases. These compounds induced no apoptosis in K562 cell-line, suggesting that their
cytotoxic activities are unlikely duo to other known anti-CML mechanisms. Molecular docking study
further showed that the compound 5k could bind with both Abl and PI3K, but the weaker binding with
Abl compared to Imatinib is consistent with its low kinase inhibitory rates. These plus literature-reported
evidences suggest that the promising cytotoxic effect of our novel compounds might be due to the
collective effect of Abl and PI3K inhibition.
Received 27 September 2010
Received in revised form
29 December 2010
Accepted 12 January 2011
Available online 21 January 2011
Keywords:
(S)-3-Aminopyrrolidine
Abl
PI3K
K562
Dual kinase inhibitor
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
pathways of Abl kinase and related kinase families, it has been
shown that activation of phosphatidylinositol 3-kinase (PI3K)
Abelson (Abl) kinase is one of the important anti-tumor drug
targets for the treatment of chronic myeloid leukemia (CML),
because it plays key roles in cell growth, survival and proliferation.
Although Imatinib, a marketed drug by Novartis targeting Abl, is
effective in treating CML, patients with late-stage disease often
have a less durable response due to acquired resistance [1].
Extensive in vitro and in vivo studies have suggested that several
possible mechanisms might cause Imatinib’s resistance, such as
mutation of T315I [2,3] on Abl, gene amplification [4,5], redun-
dancies in signaling pathways and alternative signaling [6], etc.
Systems-oriented drug design, which considers network cross-
talk and regulation instead of an individual target, has thus become
a promising alternative approach in overcoming the problems of
targeting single preselected target [7]. By examining the signaling
signaling pathway in CML is crucial for survival and proliferation of
leukemia cells [8,9] and it compensates the effect of BcreAbl
inhibition. In addition, BcreAbl inhibition in CML progenitors
results in a growth factor-dependent compensatory increase in
Mitogen activated protein kinase (MAPK) activity and in only
partial inhibition of PI3K activity [10]. Preclinical studies also have
demonstrated the improved efficacy by combination of Abl and
PI3K inhibitors [11,12], and synergy has been revealed between Abl
inhibitor and inhibitor of PI3K/AKT signaling [13]. Based on the
literature work and preclinical study results, we proposed to
develop novel dual inhibitors targeting both Abl and PI3K.
It is challenging to rationally design selective Abl and PI3K dual
inhibitors with novel scaffolds, since these two kinases are from
different kinas families. It was reported that a series of tyrosine and
PI3K dual inhibitors based on pyrazole [3,4-d]pyrimidin-4-amine
scaffold were developed recently [14]. In the present study, we
combined two virtual screening methods, support vector machine
(SVM) [15e19] and molecular docking to screen Pubchem and in-
house chemical libraries to find Abl and PI3K dual inhibitors virtual
hits. A particular scaffold, (S)-3-aminopyrrolidine, was identified
* Corresponding author. The Key Laboratory of Chemical Biology, Guangdong
Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055,
China. Tel./fax: þ86 755 26036017.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.020

C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
1405
Table 1
from 240 virtual hits. 19 compounds containing such scaffold were
Inhibitory activity of compounds against K562 cell.
synthesized and their inhibitory activities against K562 cell-lines as
well as their kinase inhibitory activities against Abl and PI3K were
evaluated.
Compound
R
IC50 (m
M)a
5a
H
9.5
10.2
11.3
8.2
9.0
8.6
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
3-CF3
2,4,6-CH3
2,4-Cl
2,6-Cl
2-Cl-6-F
3-OCH3
4-OCH3
3,5-OCH3
3,4,5-OCH3
To further evaluate whether the activities against K562 cell-lines
are really due to Abl and PI3K inhibition, we conducted experi-
mental test to determine if these compounds can induce apoptosis
in K562 cell-line. Apart from the inhibition of the primary anti-CML
target Abl [20] and together their downstream effectors such as
PI3K [13,21], anti-CML activities can be induced or partially induced
by inhibition of histone deacetylase [22], dihydrofolate reductase
[23], aromatase [24], thymidylate synthase [25] and DNA top-
oisomerase [26], DNA binding [27], blocking of NF-kappaB signaling
[28], and modulation of bax:bcl-2 ratio [29]. All of these alternative
mechanisms are known to produce antiproliferative activities
primarily by inducing apoptosis. On the other hand, only highly
potent Abl inhibitors are capable of inducing apoptosis without
prolonged inhibitor exposure (an intermediately potent drug
Imatinib can only induce apoptosis after 16 h of exposure) [30].
Moreover, PI3K selective inhibitors induce apoptosis only at high
inhibition concentrations and with prolonged inhibitor exposure
[21]. Therefore, these alternative anti-CML mechanisms can be
excluded if our compounds can be shown to induce no apoptosis
under the condition of low inhibition concentrations and shorter
inhibitor exposure times.
9.2
12.6
13.2
14.3
3.6
3.3
4.3
28.4
65.8
60.8
50.9
33.8
93.0
5s
Imatinib
0.58
a
All values are means of three experiments.
3. Results and discussion
3.1. High-throughput virtual screening
Virtual screening against Pubchem and in-house libraries was
conducted using the similar procedure as recently published [15].
First, SVM models of Abl inhibitors [37] and PI3K inhibitors [38]
were used to initially screen the compounds, then the initially
selected SVM virtual hits were evaluated by Lipinsky’s rule of five,
those passed Lipinsky’s rule of five were subject to further and
more refined screening by using molecular docking.
2. Chemistry
The synthetic route to obtain the desired target compounds is
shown in Scheme 1. The reaction of benzylsulfonyl chloride and
(S)-tert-butyl pyrrolidin-3-ylcarbamate yielded the correspond-
ing (S)-tert-butyl (1-(benzylsulfonyl)pyrrolidin-3-yl)carbamate
(1) [31], which was then deprotected with the home-made HCl/
dioxane to afford (S)-1-(benzylsulfonyl)pyrrolidin-3-aminium
chloride (2) [32]. Compounds (4aes) were prepared according to
the reported chloroacetylation [33,34] of substituted benzylpi-
perazines (3aes), which were obtained by the reaction of benzyl
chloride and anhydrous piperazine under the reflux of distilled
THF [35]. The desired target compounds (5aes) were then
obtained by the reaction of compound (2) and compounds
(4aes) according to the modified procedure described in the
literature [36].
3.2. Cytotoxic activities and structureeactivity relationships
MTT assay was used to evaluate the anti-tumor activities of
compounds 5aes against K562 cells as method described [39].
After treating K562 cells with compounds (5aes) at the varying
concentrations for 48 h, the IC50 values of each compound were
determined based on the inhibition rates at each concentration. As
shown in Table 1, compounds 5aem exhibit better cytotoxic
Scheme 1. Synthesis of the desired compounds (5aes). Reagents and conditions: (a) (S)-tert-butyl pyrrolidin-3-ylcarbamate, EtOAc, K2CO3, THF, H2O; (b) HCl/dioxane, r.t.; (c)
Anhydrous piperazine, THF, reflux; (d) ClCH2COCl, CH2Cl2, 0 ꢀC to r.t.; (e) NEt3, KI, THF, r.t. to reflux.

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C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
activity with IC50 values at 3e15
m
M. Specifically, 5k, 5l and 5m
M, 3.3 M and
M, respectively. Compounds 5pes bearing methoxy groups
Similar to Imatinib, 5k also binds Abl kinase within this active
showed the best activity with the IC50 values at 3.6
4.3
m
m
pocket and the core of 5k, (S)-3-aminopyrrolidine, occupies the
same region as pyridine and pyrimidine rings of Imatinib binding.
Different from Imatinib, however, 5k forms only one hydrogen
bond through the benzylsulfonyl oxygen with Met318 of Abl
protein, and other hydrogen bonds which were proved to be
responsible for the activity of known inhibitors are not reproduced.
The majority of contacts between 5k and protein are mediated by
van de Waals interactions. The reduced binding to the Abl protein
might explain why the inhibitory rate of synthesized compounds
m
show limited cytotoxic activities. By comparing their structures, we
obtained the following preliminary structureeactivity relation-
ships: 1) the strong electron donating groups, like methoxy group,
strongly hindered the activity no matter what position it
substituted on the benzene ring; 2) di-substitution at ortho posi-
tions of benzene ring by halogen could decrease the activity; 3)
there is no obvious position discrimination since introduction of
methyl or halogen groups to the different position on the benzene
ring (5aej) results in the similar cytotoxic activities. In addition,
introducing trifluoromethy group to the meta position on the
benzene ring (5k) can enhance 3 times activity in comparison with
methyl group (5c).
against Abl is much lower than Imatinib (IC50 ¼ 0.66
mM) [1].
As shown in Fig. 3, 5k can bind into the active pocket of PI3K
enzyme, but does not penetrate deeply as cocrystallized inhibitor.
Rightmost part of 5k projects upward to form hydrogen bonds with
Ser614 and Lys636 (not labeled) of protein, but not downward to
make H bonds with Asp644, Phe684 and Asp761 which were
proved to be important binding sites for known PI3K inhibitors. The
left part of 5k, trifluoromethyl benzene moiety, bends upward to
avoid clash with protein, but for cocrystallized inhibitor within this
sphere a hydrogen bond was formed between piperidyl oxygen and
Ile685.
3.3. Kinase inhibition
Compounds 5kem were further evaluated in Abl and PI3K
kinase inhibition assays [40] along with Staurosporine and PI103 as
controls for Abl and PI3K enzymes, respectively. As the kinase assay
results shown in Table 2, compounds 5kem displayed moderate
inhibitory activities against both Abl and PI3K kinases at 20 mM.
4. Concluding remarks
Specifically, three compounds showed relatively lower inhibitory
rates against Abl1 and Abl2 kinases in the range of 1.4e8.2%,
whereas better inhibition against PI3K (alpha) in the range of
12e16%.
In summary, we used SVM virtual screening and molecular
docking to discover a novel class of compounds bearing (S)-3-
aminopyrrolidine scaffold that displayed moderate anti-CML
cellular activities and moderate activities against Abl and PI3K
kinases. Apoptosis test further confirmed that the anti-CML activ-
ities of our compounds are unlikely due to other known anti-CML
mechanisms. The activities of the synthesized compounds against
the two kinases are weak, over 10 fold weaker than their cytotoxic
activities against K562 cell-lines. There are experimental evidences
that multi-target agents with moderate inhibition rates against
their targets are capable of producing w10 fold better cell-line
activities. For instance, the multi-target VEGFR2 and Raf inhibitor
3.4. Apoptosis test
To further evaluate whether these compounds could really
inhibit Abl kinase, we conducted flow cytometric analysis of
apoptosis [41]to determine if these compounds can induce
apoptosis in K562 cell-line (Fig. 1). Annexin-V conjugated with the
fluorochrome FITC serves as a marker for apoptotic cells because it
has a strong binding affinity to phosphatidylserine (PS), which re-
distributes from the inner to the outer layer of the plasma
membrane in apoptotic cells. Compared to blank and DMSO,
representative compounds 5kem could not induce apoptosis. The
results provide the further evidence that the anti-CML activities of
our compounds might be due to the collective effect of Abl and PI3K
inhibition.
CHIR-265/RAF-265 inhibits VEGFR2 and Raf at IC50 values of 1.3
mM
and 1.2 M respectively [43], while its IC50 values against SK-MEL-
m
28, Malme-3M and A375M cell-lines are in the range of
140e300 nM [44].
Additional evidences seem to support our conclusion that anti-
CML activities of our compounds are likely duo to collective inhi-
bition of Abl and PI3K instead of inhibition of one of the kinases, e.g.
PI3K. Apart from the fact that both kinases are moderately inhibited
by our compounds, literature-reported experimental studies of
PI3K specific inhibitors seem to indicate that under our experi-
mental conditions the anti-CML activities of our compounds are
unlikely due solely to PI3K inhibition even-though they were found
to inhibit PI3K at substantially higher inhibition rates than Abl. PI3K
specific inhibitors have been found to produce antiproliferative
effects against CML cell-lines only at high inhibition concentrations
(at 95% PI3K inhibition level) and with prolonged inhibitor expo-
sure [21]. While our compounds showed anti-CML activities at
12%e16% PI3K inhibition level and with shorter exposure 24 h.
The moderate anti-CML cellular activities of our compounds,
particularly of compounds 5k, 5l and 5m, suggest that the (S)-3-
aminopyrrolidine scaffold is potentially interesting in generating
novel dual inhibitors of Abl and PI3K. Further studies are in prog-
ress to optimize the present hits into more potent leads. Given the
difficulty to rationally design selective Abl and PI3K dual inhibitors,
particularly those of novel scaffolds, our scaffold together with
other discovered scaffolds [14] offer good starting points for
designing more effective therapeutic agents in the treatment
of CML.
3.5. Molecular docking
Docking studies of the representative compound 5k was per-
formed and compared to the positive control compound Imatinib.
As shown in Fig. 2, Imatinib penetrates through the central region
of the Abl kinase from one side to the other, forming hydrogen bond
with amino acids Glu286, Thr315, Met318, Asp381, His361 and
Ile360 of protein, respectively. The pyridine and pyrimidine rings of
Imatinib occlude the region where the adenine ring of ATP nor-
mally binds and the rest of the molecule stretches into the hydro-
phobic region of kinase to freeze the kinase conformation [42].
Table 2
Inhibitory activity of 5kem with two Abl isoforms and PI3K (alpha) (inhibitory
rate, %) at 20
mM.
Compound
Abl1
Abl2
PI3K (alpha)
5k
5l
5m
8.21
2.36
4.51
96.9
4.49
1.39
4.92
90.2
12.11
13.54
16.36
Staurosporine
PI103
98.1

C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
1407
Fig. 1. Flow cytometric analysis of phosphoatidylserine externalization (Annexin-V binding) and cell membrane integrity (PI staining). K562 cells were treated with compounds
5kem at 5 M for 24 h.
m
5. Experimental section
Chemical shifts are given in ppm (d) relative to SiMe3 as internal
standard. Coupling constants (J) are in hertz (Hz), and signals are
designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet;
br s, broad singlet, etc. The mass spectra were obtained on a Waters
Micromass Q-TOF Premier Mass Spectrometer. Melting points were
determined with an SGW X-4 digital apparatus and are
5.1. Chemistry
NMR spectra were recorded on a Bruker 500 (500 MHz) or
a Bruker 400 (400 MHz) spectrometer at room temperature.

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C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
Fig. 2. Docking of compound 5k and Imatinib on Abl. Molecules are colored by atom type and hydrogen bonds are represented by yellow dotted lines. 5k was represented as sticks
and Imatinib was represented as balls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
uncorrected. Thin layer chromatography was carried out using
plate silica gel F254. All chemical yields are unoptimized and
generally represent the result of a single experiment.
tetrahydrofuran (1:1), was slowly added dropwise at 0 ꢀC. After
being stirred at room temperature for 1 day, the reaction mixture
was concentrated till the 4/5 of original volume and additional
water (30 mL) and ethyl acetate (50 mL) was added. The resulting
mixture was separated and the organic phases was washed with
a saturated sodium bicarbonate aqueous solution (2 ꢁ 50 mL),
water (2 ꢁ 100 mL), brine (2 ꢁ 100 mL) and dried over anhydrous
sodium sulfate and concentrated in vacuo to give a colorless solid.
Yield 78.6%; m.p 131e132 ꢀC; HRMS (ESI): calcd for [C16H24N2O4S]þ
Naþ: 363.1354; found: 363.1340; 1H NMR (400 MHz, CDCl3)
5.1.1. General procedure for the synthesis of derivatives of (S)-tert-
butyl (1-(benzylsulfonyl) pyrrolidin-3-yl)carbamate (1)
Potassium carbonate (8.28 g, 60 mmol) was dissolved in water
(20 mL) and stirred for 5 min at room temperature. (S)-tert-Butyl
pyrrolidin-3-ylcarbamate (5.70 g, 30.6 mmol) in ethyl acetate
(70 mL) was added and stirred for additional 25 min. Benzylsulfonyl
chloride (6.42 g, 33 mmol), dissolved in 60 mL of ethyl acetate and
d
7.38e7.41(m, 5H), d 4.56 (br s, 1H), d 4.28 (s, 2H), d 4.13 (br s, 1H),
Fig. 3. Docking of compound 5k on PI3K. Molecules are colored by atom type and hydrogen bonds are represented by yellow dotted lines. 5k was represented as sticks and the
cocrystallized ligand was represented as balls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
1409
d
3.34 (dd, J ¼ 10.0, 6.4 Hz, 1H),
(br s,1H), 3.07 (br s,1H),
2.04 (dt, J ¼ 18.8, 7.6 Hz,1H),
(m, 1H), 1.44 (s, 9H);
13C NMR (100.6 MHz, CDCl3)
d
3.26 (dd, J ¼ 16.2, 7.6 Hz, 1H),
1.61e1.78
155.05,
d
3.15
1.4 Hz 1H),
d
7.15e7.25 (m, 2H),
d
3.60 (s, 2H),
d
2.89 (t, J ¼ 4.8 Hz,
d
d
d
4H), 2.48 (br s, 4H), d
d
1.90 (br s, 1H); 13C NMR (100.6 MHz, CDCl3)
d
d
d
d
135.62, 134.07, 130.46, 129.16, 127.80, 126.29, 59.59, 54.31, 45.88.
130.59, 129.27, 128.87, 79.96, 57.19, 53.65, 50.59, 46.13, 32.15, 28.36.
5.1.3.6. 1-(3-Chlorobenzyl)piperazine (3f). Yield 74%; 1H NMR
5.1.2. General procedure for the synthesis of derivatives of (S)-1-
(benzylsulfonyl) pyrrolidin-3-aminium chloride (2)
(400 MHz, CDCl3) 7.34 (br s,1H), 7.18e7.44 (m, 3H), 3.46 (s, 2H),
2.41 (br s, 4H),
1.74 (s, 1H); 13C NMR
140.46, 134.14, 129.43, 129.06, 127.19, 63.03,
d
d
d
d
2.90 (t, J ¼ 4.8 Hz, 4H),
d
d
To
a
round-bottomed flask charged with (S)-tert-butyl
(100.6 MHz, CDCl3)
54.46, 46.07.
d
(1-(benzylsulfonyl) pyrrolidin-3-yl)carbamate (4.9 g, 14.4 mmol)
was poured 70 mL of HCl/dioxane solution, which was prepared in
Lab, and sealed immediately with a rubber stopper. The reaction
mixture was stirred at room temperature till the milky solution
appeared. The milky mixture was then carefully concentrated
under reduced pressure and recrystallized from ethyl acetate to
give pure light brown solid. Yield 93.2%; m.p 185e186 ꢀC; HRMS
(ESI): calcd for [C11H17ClN2O2SꢂClꢂ]þ: 241.1005; found: 241.1003;
5.1.3.7. 1-(4-Chlorobenzyl)piperazine (3g). Yield 70%; 1H NMR
(400 MHz, CDCl3) 7.24e7.29 (m, 4H), 3.44 (s, 2H), 2.88 (t,
J ¼ 4.8 Hz, 4H), 2.39 (br s, 4H),
1.72 (s, 1H); 13C NMR (100.6 MHz,
CDCl3) 136.74, 132.70, 130.43, 128.33, 62.87, 54.44, 46.08.
d
d
d
d
d
d
5.1.3.8. 1-(2-Fluorobenzyl)piperazine (3h). Yield 41%; 1H NMR
(500 MHz, CDCl3) 7.20e7.24 (m, 1H),
7.37 (dd, J ¼ 7.4, 6.3 Hz, 1H),
7.10 (t, J ¼ 7.5 Hz, 1H), 7.00e7.04 (m, 1H), 3.57 (s, 2H), 2.89 (t,
J ¼ 4.8 Hz, 4H), 2.46 (br s, 4H),
1.76 (br s, 1H); 13C NMR
(100.6 MHz, CDCl3) 164.66, 131.32, 128.42, 124.3, 123.51,
114.92, 55.53, 54.89, 53.87, 52.46, 45.75.
1H NMR (400 MHz, DMSO-d6)
7.35e7.41 (m, 3H), 4.51 (s, 2H),
J ¼ 10.8, 6.8 Hz, 1H), 3.41 (dd, J ¼ 16.6, 7.2 Hz, 1H),
1H), 3.23e3.29 (m, 1H),
2.17 (dt, J ¼ 20.0, 6.8 Hz, 1H),
J ¼ 20.0, 6.0 Hz, 1H);
13C NMR (100.6 MHz, DMSO-d6)
130.07, 128.88, 128.64, 54.34, 51.11, 49.76, 46.26, 29.97.
d
8.57 (br s, 3H),
3.75e3.81 (m, 1H),
3.39e3.46 (m,
1.99 (dt,
131.38,
d
7.43e7.45 (m, 2H),
d
d
d
d
d
d
3.52 (dd,
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
5.1.3.9. 1-(3-Fluorobenzyl)piperazine (3i). Yield 88%; 1H NMR
5.1.3. General procedure for the synthesis of various substituted
benzylpiperazines (3aes)
(500 MHz, CDCl3)
d
7.23e7.25 (m, 1H),
d
7.06e7.09 (m, 2H),
3.48 (s, 2H), 2.88 (t,
1.73 (br s, 1H); 13C NMR
d
6.91e6.94 (td, J ¼ 8.4, 2.0 Hz, 1H),
d
d
Anhydrous piperazine (6.89 g, 80 mmol) was dissolved in 40 mL
of freshly distilled THF. Once the piperazine was fully dissolved,
2.303 mL (20 mmol) of benzyl chloride was added dropwise. The
reaction mixture was then refluxed for about 4 h until benzyl
chloride disappeared, as assessed by TLC. The stirring mixture was
allowed to cool, and then filtered. The filtrate was concentrated in
a rotary evaporator and then diluted with EtOAc (100 mL) and
water (50 mL), which was then made basic (pH>12) with a satu-
rated 1 N NaOH aqueous solution and separated. The organic phase
was washed with water (4 ꢁ 100 mL), brine (2 ꢁ 100 mL), dried
over Na2SO4 and concentrated to yield an oil. Column chromatog-
raphy (PE/EtOAc ¼ 1:1 to EtOAc/MeOH ¼ 5:1) afforded a pale
yellow liquid.
J ¼ 4.8 Hz, 4H),
d
2.41 (br s, 4H),
d 163.92, 161.48, 140.76, 129.33, 124.34, 115.53,
d
(100.6 MHz, CDCl3)
113.63, 62.81, 54.21, 45.80.
5.1.3.10. 1-(4-Fluorobenzyl)piperazine (3j). Yield 74%; 1H NMR
(400 MHz, CDCl3)
d
7.26e7.29 (m, 2H),
2.39 (br s, 4H),
161.95, 133.85, 130.60, 114.95, 62.86,
d
7.01 (t, J ¼ 8.4 Hz, 2H),
d
3.44 (s, 2H),
d
2.85 (t, J ¼ 4.8 Hz, 4H),
d
d
1.65 (s,1H);
13C NMR (100.6 MHz, CDCl3)
54.45, 46.11.
d
5.1.3.11. 1-(3-(Trifluoromethyl)benzyl)piperazine (3k). Yield 96%; 1H
NMR (500 MHz, CDCl3) 7.59 (s, 1H), 7.49e7.53 (m, 2H), 7.42 (t,
J ¼ 7.7 Hz, 1H), 3.54 (s, 2H), 2.89 (t, J ¼ 4.9 Hz, 4H), 2.41 (br s,
4H), 139.05, 132.16,
1.69 (br s, 1H); 13C NMR (100.6 MHz, CDCl3)
d
d
d
d
d
d
d
d
5.1.3.1. 1-Benzylpiperazine (3a). Yield 85.8%; 1H NMR (500 MHz,
130.33, 128.40, 125.41, 123.68, 123.65, 122.65, 62.81, 54.14, 45.72.
CDCl3)
d
7.29e7.32 (m, 4H),
d
7.23e7.25 (m, 1H),
2.41 (br s, 4H),
1.77 (br s, 1H); 13C NMR
138.04, 129.10, 128.07, 126.89, 63.61, 54.44,
d 3.49 (s, 2H), d 2.87
(t, J ¼ 4.9 Hz, 4H),
(125.8 MHz, CDCl3)
46.02.
d
d
5.1.3.12. 1-(2,4,6-Trimethylbenzyl)piperazine (3l). Yield 94.3%; 1H
d
NMR (400 MHz, CDCl3)
J ¼ 4.4 Hz, 4H), 2.40 (br s, 4H),
1H); 13C NMR (100.6 MHz, CDCl3)
d
6.82(s, 2H),
2.34 (s, 6H),
138.13, 138.10, 136.27, 131.80,
d
3.41 (s, 2H),
d
2.80 (t,
d 1.84 (s,
d
d
d
2.25 (s, 3H),
d
5.1.3.2. 1-(2-Methylbenzyl)piperazine (3b). Yield 80.6%; 1H NMR
128.82, 128.78, 56.42, 55.83, 54.10, 53.11, 46.37, 20.89, 20.07, 20.01.
(400 MHz, CDCl3)
(s, 2H),
2.86 (d, J ¼ 4.8 Hz, 4H),
(s, 1H); 13C NMR (100.6 MHz, CDCl3)
d
7.26 (t, J ¼ 3.6 Hz, 1H),
d
7.13e7.15 (m 3H),
2.35 (s, 3H),
d
3.43
1.75
d
d
2.41 (br s, 4H),
d
d
5.1.3.13. 1-(2,4-Dichlorobenzyl)piperazine (3m). Yield 97%; 1H NMR
d
137.32, 136.18, 129.99, 129.60,
(400 MHz, CDCl3)
d
d
7.43 (d, J ¼ 8.4 Hz, 1H),
7.21 (dd, J ¼ 8.4, 2.0 Hz, 1H), 3.56 (s, 2H),
2.50 (br s, 4H),
d
7.36 (d, J ¼ 2.0 Hz, 1H),
126.73, 125.16, 61.24, 54.39, 53.11, 45.98, 19.01.
d
d
2.89 (t, J ¼ 4.8 Hz, 4H),
d
d
1.63 (s,1H); 13C NMR (100.6 MHz, CDCl3)
d 134.83,
5.1.3.3. 1-(3-Methylbenzyl)piperazine (3c). Yield 68%; 1H NMR
134.67, 132.99, 131.43, 129.14, 126.86, 59.26, 58.58, 54.55, 53.12,
46.15.
(500 MHz, CDCl3)
J ¼ 7.5 Hz, 1H), 3.46 (s, 2H),
2.34 (s, 3H),
1.43 (br s, 1H); 13C NMR (100.6 MHz, CDCl3)
d
7.19 (t, J ¼ 7.5 Hz,1H),
d
7.11e7.13 (m, 2H),
2.40 (br s, 4H),
137.65,
d 7.05 (d,
d
d
2.88 (t, J ¼ 4.8 Hz, 4H),
d
d
d
d
5.1.3.14. 1-(2,6-Dichlorobenzyl)piperazine (3n). Yield 95.7%; 1H
137.55, 129.76, 127.82, 127.54, 126.13, 63.51, 54.29, 45.80, 21.15.
NMR (400 MHz, CDCl3)
3.72 (s, 2H),
2.83 (t, J ¼ 4.8 Hz, 4H),
(br s, 1H); 13C NMR (100.6 MHz, CDCl3)
d
7.27e7.30 (m, 2H),
2.53 (t, J ¼ 4.4 Hz, 4H),
137.01, 134.39, 128.71,
d
7.09e7.14 (m, 1H),
d
d
d
d
1.79
5.1.3.4. 1-(4-Methylbenzyl)piperazine (3d). Yield 62%; 1H NMR
d
(400 MHz, CDCl3)
2H),
2.87 (t, J ¼ 4.8 Hz, 4H),
1H); 13C NMR (100.6 MHz, CDCl3)
d
7.17e7.21 (m, 2H),
d
7.09e7.13 (m, 2H),
2.32 (s, 3H),
d
3.47 (s,
1.65 (s,
128.66, 128.30, 57.05, 56.41, 54.39, 53.08, 46.15.
d
d
2.39 (br s, 4H), d
d
d
136.57, 134.97, 129.21, 128.85,
5.1.3.15. 1-(2-Chloro-6-fluorobenzyl)piperazine (3o). Yield 87%; 1H
63.44, 62.82, 54.52, 53.06, 46.13, 21.09.
NMR (400 MHz, CDCl3)
3.69 (s, 2H),
2.85 (d, J ¼ 9.2 Hz, 4H),
1H); 13C NMR (100.6 MHz, CDCl3)
162.13, 136.72, 129.03, 125.39,
123.90, 113.90, 54.15, 53.01, 52.79, 52.31, 46.14.
d
7.17e7.18 (m, 2H),
d
6.95e7.00 (m, 1H),
d
d
d
2.54 (br s, 4H),
d
1.63 (br s,
5.1.3.5. 1-(2-Chlorobenzyl)piperazine (3e). Yield 81%; 1H NMR
d
(400 MHz, CDCl3)
d
7.47 (dd, J ¼ 7.5, 1.7 Hz, 1H),
d
7.34 (dd, J ¼ 7.8,

1410
C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
5.1.3.16. 1-(3-Methoxybenzyl)piperazine (3p). Yield 74%; 1H NMR
(500 MHz, CDCl3) 6.90e6.91 (m, 2H),
3.46 (s, 2H), 2.88 (t,
1.82 (s, 1H); 13C NMR (125.8 MHz,
J ¼ 4.8 Hz, 2H),
d
3.50e3.52 (m, 4H),
d
2.46 (dt, J ¼ 14, 4.8 Hz, 4H),
165.01, 137.02, 134.36,
d
7.22 (t, J ¼ 8.0 Hz, 1H),
3.80 (s, 3H),
d
d
2.34 (s, 3H); 13C NMR (100.6 MHz, CDCl3)
d
d
6.79 (dd, J ¼ 8.1, 2.1 Hz, 1H),
d
d
d
129.10, 129.06, 62.53, 52.85, 52.43, 46.35, 42.22, 40.90, 21.11.
J ¼ 4.9 Hz, 4H),
d 2.41 (br s, 4H), d
CDCl3)
d
159.58, 139.76, 129.03, 121.41, 114.56, 112.36, 63.49, 55.10,
5.1.4.5. 2-Chloro-1-(4-(2-chlorobenzyl)piperazin-1-yl)ethanone
54.31, 45.96.
(4e). Yield 87%; HRMS (ESI): calcd for [C13H16Cl2N2O þ H]þ:
287.0718; found: 287.0712; 1H NMR (400 MHz, CDCl3)
d
7.44e7.46
(m, 1H), 4.07 (s, 2H),
3.64 (s, 4H), 2.55 (dt, J ¼ 16.0, 4.8 Hz, 4H);
13C NMR (100.6 MHz, CDCl3)
d 164.79, 134.99, 134.22, 130.50,
5.1.3.17. 1-(4-Methoxybenzyl)piperazine (3q). Yield 71.1%; 1H NMR
d
7.35e7.38 (m, 1H),
d 7.21e7.27 (m, 2H), d
(400 MHz, CDCl3)
d
7.22 (d, J ¼ 8.8 Hz, 2H),
3.42 (s, 2H),
2.87 (t, J ¼ 4.8 Hz, 4H),
1.66 (s, 1H); 13C NMR (100.6 MHz, CDCl3)
158.70, 130.38, 130.10,
d
6.84 (d, J ¼ 8.8 Hz, 2H),
d
d
3.53 (t, J ¼ 4.8 Hz 2H),
d
d
3.79 (s, 2H),
d
d
d
2.39 (br s, 4H),
d
d
129.38, 128.25, 126.45, 58.44, 52.66, 52.25, 46.14, 42.01, 40.66.
113.55, 63.08, 55.24, 54.44, 46.12.
5.1.4.6. 2-Chloro-1-(4-(3-chlorobenzyl)piperazin-1-yl)ethanone
5.1.3.18. 1-(3,5-Dimethoxybenzyl)piperazine (3r). Yield 83.9%; 1H
(4f). Yield 88.2%; HRMS (ESI): calcd for [C13H16Cl2N2O þ H]þ:
NMR (400 MHz, CDCl3)
d
6.50 (t, J ¼ 2.4 Hz, 2H),
3.44 (s, 2H),
2.89 (t, J ¼ 4.8 Hz, 4H),
1.70 (s, 1H); 13C NMR (100.6 MHz, CDCl3)
160.70, 140.79,
d
6.35 (t, J ¼ 2.4 Hz,
287.0718; found: 287.0721; 1H NMR (400 MHz, CDCl3)
d
7.34 (s, 1H),
3.63 (t,
2.46 (dt, J ¼ 12.8, 5.2 Hz, 4H);
d 165.00, 139.83, 134.32, 129.63,
1H),
4H),
d
3.78 (s, 6H),
d
d
d
2.41 (br s,
d
7.24e7.27 (m, 2H),
J ¼ 4.8 Hz, 2H), 3.50e3.54 (m, 4H),
13C NMR (100.6 MHz, CDCl3)
d 7.19e7.22 (m, 1H), d 4.06 (s, 2H), d
d
d
d
d
106.93, 98.95, 63.76, 63.14, 55.31, 54.56, 53.16, 46.14.
128.94, 127.52, 127.07, 62.10, 52.85, 52.49, 46.28, 41.15, 40.88.
5.1.3.19. 1-(3,4,5-Trimethoxybenzyl)piperazine (3s). Yield 82.8%; 1H
NMR (400 MHz, CDCl3)
d
6.57 (s, 2H),
2.42 (br s, 4H),
153.07, 136.92, 134.00, 105.70, 63.89, 60.83,
d
3.86 (s, 6H),
d
3.84 (s, 3H),
5.1.4.7. 2-Chloro-1-(4-(4-chlorobenzyl)piperazin-1-yl)ethanone
d
2.90 (t, J ¼ 4.8 Hz, 4H),
d
d
1.72 (s, 1H); 13C NMR
(4g). Yield 92%; HRMS (ESI): calcd for [C13H16Cl2N2O þ H]þ:
(100.6 MHz, CDCl3)
d
287.0718; found: 287.0716; 1H NMR (400 MHz, CDCl3)
d
7.30 (d,
56.13, 54.50, 53.15, 46.10.
J ¼ 8.4 Hz, 2H),
J ¼ 4.8 Hz, 2H),
13C NMR (100.6 MHz, CDCl3)
d
7.25 (d, J ¼ 8.4 Hz, 2H),
3.49e3.53 (m, 4H),
165.02, 136.12, 133.08, 130.31, 128.54,
d 4.06 (s, 2H), d 3.63 (t,
2.46 (dt, J ¼ 13.2, 5.2 Hz, 4H);
d
d
5.1.4. General procedure for the 2-chloroacetylation of various
substituted benzylpiperazines (4aes)
d
61.97, 52.84, 46.30, 42.16, 40.87.
To a solution of chloroacetic chloride (1.67 mL, 20.64 mmol) in
CH2Cl2 (10 mL) at 0 ꢀC was added 1-benzylpiperazine (17.2 mmol)
which was dissolved in CH2Cl2 (10 mL). The reaction mixture was
stirred at room temperature for about 30 min until no 1-benzyl-
piperazine remained, as monitored by TLC. The mixture was poured
into cold H2O (50 mL) and rendered alkaline with a 10% NaHCO3
aqueous solution and separated. The organic layer, dried over
Na2SO4, was evaporated under reduced pressure to give pure
compound as a yellow oil.
5.1.4.8. 2-Chloro-1-(4-(2-fluorobenzyl)piperazin-1-yl)ethanone
(4h). Yield 90%; HRMS (ESI): calcd for [C13H16ClFN2O þ H]þ:
271.1013; found: 271.1009; 1H NMR (400 MHz, CDCl3)
d
7.35 (dd,
7.04
3.62 (d,
2.51 (dt, J ¼ 15.6, 4.8 Hz,
d 161.16, 131.24, 128.88, 123.88,
J ¼ 7.4,1.4 Hz,1H),
d
7.23e7.29 (m,1H),
d
7.12 (t, J ¼ 7.2 Hz,1H),
d
(t, J ¼ 9.2 Hz, 1H),
d
4.05 (s, 2H),
d
3.64 (d, J ¼ 5.2 Hz, 2H),
d
J ¼ 4.4 Hz, 2H), 3.52 (t, J ¼ 5.0 Hz, 2H),
d
4H); 13C NMR (100.6 MHz, CDCl3)
123.75, 115.27, 115.05, 54.87, 52.41, 52.00, 46.06, 41.93, 40.67.
5.1.4.1. 1-(4-Benzylpiperazin-1-yl)-2-chloroethanone
(4a). Yield
5.1.4.9. 2-Chloro-1-(4-(3-fluorobenzyl)piperazin-1-yl)ethanone
(4i). Yield 91%; HRMS (ESI): calcd for [C13H16ClFN2O þ H]þ:
77%; HRMS (ESI): calcd for [C13H17ClN2O þ H]þ: 253.1108; found:
253.1116; 1H NMR (400 MHz, CDCl3)
d
7.30e7.35 (m, 4H),
271.1013; found: 271.1009; 1H NMR (400 MHz, CDCl3)
d
(m, 1H),
4.07
3.53 (d, J ¼ 6.5 Hz, 4H),
2.44e2.50 (m, 4H); 13C NMR (100.6 MHz, CDCl3)
164.80, 163.95,
d
7.26e7.29 (m,1H),
d
4.05 (s, 2H),
d
3.63 (t, J ¼ 4.8 Hz, 2H),
d
3.51 (m,
d
7.26e7.31 (m, 1H),
d 7.06e7.09 (m, 2H), d 6.94e6.98 (m, 1H), d
4H),
d
d
2.47(dt, J ¼ 12.8, 5.2 Hz, 4H); 13C NMR (100.6 MHz, CDCl3)
(s, 2H),
d
d
3.63 (t, J ¼ 4.9 Hz, 2H),
d
165.00, 137.50, 129.09, 128.37, 127.35, 62.78, 52.88, 52.48, 46.32,
d
42.20, 40.89.
161.51, 140.17, 129.59, 124.24, 115.40, 114.00, 61.88, 52.42, 46.05,
41.92, 40.50.
5.1.4.2. 2-Chloro-1-(4-(2-methylbenzyl)piperazin-1-yl)ethanone
(4b). Yield 91.3%; HRMS (ESI): calcd for [C14H19ClN2O þ H]þ:
5.1.4.10. 2-Chloro-1-(4-(4-fluorobenzyl)piperazin-1-yl)ethanone
(4j). Yield 92.5%; HRMS (ESI): calcd for [C13H16ClFN2O þ H]þ:
267.1264; found: 267.1259; 1H NMR (400 MHz, CDCl3)
d 7.21e7.26
(m, 1H),
3.49 (s, 4H),
(100.6 MHz, CDCl3)
d
7.12e7.19 (m, 3H),
2.45e2.50 (m, 4H),
164.99, 137.58, 135.58, 130.42, 129.91, 127.39,
d
4.06 (s, 2H),
d
3.61 (t, J ¼ 4.4 Hz, 2H),
271.1013; found: 271.1013; 1H NMR (400 MHz, CDCl3)
d 7.27e7.30
d
d
d
2.37 (s, 3H); 13C NMR
(m, 2H),
2H), 3.49e3.53 (m, 4H),
(100.6 MHz, CDCl3) d 165.01, 162.14, 133.24, 130.59, 130.51, 115.31,
d
7.01 (t, J ¼ 8.4 Hz, 1H),
d
4.06 (s, 2H),
d
3.63 (t, J ¼ 4.8 Hz,
d
d
d
2.46 (dt, J ¼ 14.0, 5.2 Hz, 4H); 13C NMR
125.59, 60.66, 52.89, 52.54, 46.39, 42.28, 40.89, 19.21.
115.09, 61.93, 52.80, 52.40, 46.20, 42.16, 40.88.
5.1.4.3. 2-Chloro-1-(4-(3-methylbenzyl)piperazin-1-yl)ethanone
(4c). Yield 89.2%; HRMS (ESI): calcd for [C14H19ClN2O þ H]þ:
5.1.4.11. 2-Chloro-1-(3-(trifluoromethyl)benzyl)piperazine
267.1264; found: 267.1265; 1H NMR (400 MHz, CDCl3)
d
7.21 (t,
3.64 (t, J ¼ 5.2 Hz,
2.35 (s, 3H); 13C
164.99, 137.99, 129.84, 128.24, 128.09,
(4k). Yield 98％; HRMS (ESI): calcd for [C14H16ClF3N2O þ H]þ:
J ¼ 7.2 Hz,1H),
2H), 3.49e3.53 (m, 4H),
NMR (100.6 MHz, CDCl3)
d
7.07e7.13 (m, 3H),
d
4.06 (s, 2H),
d
321.0982; found: 321.0975; 1H NMR (500 MHz, CDCl3)
d
7.59 (s, 1H),
3.64 (t,
2.45e2.50
d 164.97, 138.78, 132.17,
d
d
2.44e2.49 (m, 4H),
d
d
7.50e7.53 (m, 2H),
J ¼ 5.0 Hz, 2H), 3.57 (s, 2H),
(m, 4H). 13C NMR (125.8 MHz, CDCl3)
d
7.44 (t, J ¼ 7.8 Hz, 1H),
d 4.05 (s, 2H), d
d
d
d
3.53 (t, J ¼ 5.0 Hz, 2H)
d
126.20, 62.81, 52.94, 52.52, 46.31, 42.19, 40.90, 21.39.
128.78, 125.49, 125.46, 124.16, 124.13, 64.09, 52.82, 52.46, 46.21,
42.08, 40.80.
5.1.4.4. 2-Chloro-1-(4-(4-methylbenzyl)piperazin-1-yl)ethanone
(4d). Yield 90%; HRMS (ESI): calcd for [C14H19ClN2O þ H]þ:
267.1264; found: 267.1263; 1H NMR (400 MHz, CDCl3)
d
7.20 (d,
3.64 (t,
5.1.4.12. 2-Chloro-1-(4-(2,4,6-trimethylbenzyl)piperazin-1-yl)etha-
J ¼ 8.0 Hz, 2H),
d
7.13 (d, J ¼ 8.0 Hz, 2H),
d
4.05 (s, 2H),
d
none (4l). Yield 96.1%; HRMS (ESI): calcd for [C16H23ClN2O þ H]þ:

C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
1411
295.1577; found: 295.1571; 1H NMR (400 MHz, CDCl3)
4.05 (s, 2H), 3.43e3.48 (m, 4H),
3.56 (t, J ¼ 4.8 Hz, 2H),
(dt, J ¼ 14.0, 4.8 Hz, 4H), 2.34 (s, 6H),
2.26 (s, 3H); 13C NMR
(100.6 MHz, CDCl3) 164.99, 138.07, 136.73, 131.01, 129.21, 129.01,
d
6.85 (s, 2H),
2.46
5.1.5. General procedure for the synthesis of target compounds
(5aes)
d
d
d
d
d
d
To a solution of (S)-1-(benzylsulfonyl)-3-pyrrolidinamine,
d
hydrochloride (0.331 g, 1.2 mmol) in tetrahydrofuran (30 mL) was
added triethylamine (0.56 mL, 4.8 mmol) and stirred at room
temperature. When the solution became clear, potassium iodide
55.62, 52.50, 52.08, 46.62, 42.50, 40.90, 20.90, 20.05, 19.15.
5.1.4.13. 2-Chloro-1-(4-(2,4-dichlorobenzyl)piperazin-1-yl)ethanone
(0.498 g, 3 mmol) and 1-(4-benzylpiperazin-1-yl)-2-chloro-
(4m). Yield 79%; HRMS (ESI): calcd for [C13H15Cl3N2O þ H]þ:
ethanone (0.267 g, 1 mmol) were added. After refluxing for about
20 h, the reaction mixture was evaporated to remove the solvent
and then diluted with EtOAc (50 mL) and water (30 mL). The
organic layer was washed with additional water (3 ꢁ 50 mL), brine
(2 ꢁ 50 mL), and dried over Na2SO4, concentrated to give a brown
oil which was then purified by TLC (EtOAc/MeOH ¼ 20:1) to afford
the target compound as yellow oil.
321.0328; found: 321.0323; 1H NMR (400 MHz, CDCl3)
d
7.38e7.41
3.64 (t,
2.53 (dt,
d 156.03, 135.01,
(m, 2H),
J ¼ 4.8 Hz, 2H),
d
7.23 (dd, J ¼ 8.4, 1.6 Hz, 1H),
d 4.07 (s, 2H), d
3.53 (t, J ¼ 4.8 Hz, 2H),
d
3.60 (s, 2H),
d
d
J ¼ 16.4, 4.8 Hz, 4H); 13C NMR (100.6 MHz, CDCl3)
133.95, 133.50, 131.46, 129.37, 127.03, 58.49, 52.87, 52.46, 46.33,
42.20, 40.85.
5.1.4.14. 2-Chloro-1-(4-(2,6-dichlorobenzyl)piperazin-1-yl)ethanone
5.1.5.1. (S)-1-(4-Benzylpiperazin-1-yl)-2-((1-(benzylsulfonyl)pyrroli-
(4n). Yield 92.5%; HRMS (ESI): calcd for [C13H15Cl3N2O þ H]þ:
din-3-yl)amino)ethanone (5a). Yield 50.2%; HRMS (ESI): calcd for
321.0328; found: 321.0332; 1H NMR (400 MHz, CDCl3)
d
7.32 (d,
3.78(s, 2H),
2.60 (dt, J ¼ 14.0,
164.97, 136.97, 133.71,
[C24H32N4O3S
(400 MHz, CDCl3)
þ
H]þ: 457.2273; found: 457.2286; 1H NMR
J ¼ 8.0 Hz, 2H),
3.60 (t, J ¼ 4.8 Hz, 2H),
4.8 Hz, 4H); 13C NMR (100.6 MHz, CDCl3)
d
7.17 (t, J ¼ 8.0 Hz, 1H),
d
4.06 (s, 2H),
d
d
7.41e7.43 (m, 2H),
d
7.35e7.39 (m, 3H),
4.25 (s, 2H), 3.64 (t,
3.34e3.37 (m,
3.25e3.29 (m, 3H), 2.96
d
d
3.48 (t, J ¼ 4.8 Hz, 2H),
d
d
7.29e7.35 (m, 4H),
d
7.26e7.28 (m, 1H),
d
d
d
J ¼ 4.8 Hz 2H),
d
3.53 (s, 2H),
d 3.34e3.38 (m, 1H), d
129.11, 128.44, 56.19, 52.73, 52.36, 46.39, 42.28, 40.89.
3H),
d
3.32 (br s, 1H),
d
3.31 (br s, 1H),
d
d
(dd, J ¼ 9.0, 4.8 Hz, 4H),
6.0 Hz, 1H), 1.84 (br s, 1H),
(100.6 MHz, CDCl3)
128.57,128.39,127.40, 62.84, 57.82, 56.55, 53.58, 53.43, 52.71, 48.60,
46.58, 44.41, 41.99, 32.41.
d
2.44 (t, J ¼ 4.4 Hz, 4H), 2.00 (dt, J ¼ 18.0,
d
5.1.4.15. 2-Chloro-1-(4-(2-chloro-6-fluorobenzyl)piperazin-1-yl)ethanone
(4o). Yield 99.3%; HRMS (ESI): calcd for [C13H15Cl2FN2O þ H]þ:
d
d
1.74 (dt, J ¼ 18.8, 6.4 Hz, 1H); 13C NMR
d
168.81, 137.36, 130.78, 129.39, 129.14, 128.72,
305.0624; found: 305.0622; 1H NMR (400 MHz, CDCl3)
d
7.17e7.25
3.73 (s, 2H), 3.61
2.58 (dt, J ¼ 14.0,
164.96, 162.05, 136.64,
(m, 2H),
(t, J ¼ 4.8 Hz, 2H),
5.2 Hz, 4H); 13C NMR (100.6 MHz, CDCl3)
d
6.94e7.04 (m, 1H),
d
4.05 (s, 2H),
d
d
d
3.49 (t, J ¼ 4.8 Hz, 2H),
d
d
5.1.5.2. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(2-
methylbenzyl)piperazin-1-yl)ethanone (5b). Yield 52.8%; HRMS
(ESI): calcd for [C25H34N4O3S þ H]þ: 471.2430; found: 471.2423; 1H
129.44, 125.53, 123.33, 114.04, 52.58, 52.22, 52.18, 46.33, 42.22,
40.87.
NMR (400 MHz, CDCl3)
7.30 (d, J ¼ 7.2 Hz, 2H),
s, 2H), 3.48 (s, 2H), 3.34e3.38 (m,1H),
(d, J ¼ 6.0 Hz, 2H), 3.27 (br s, 1H), 2.96e3.00 (m, 1H),
J ¼ 4.8 Hz, 4H), 2.37 (s, 3H), 1.96e2.04 (m, 2H), 1.73 (dt, J ¼ 18.0,
6.4 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
168.75, 137.60, 135.58,
d
7.41e7.43 (m, 2H),
7.14e7.21 (m, 3H),
3.31e3.33 (m, 4H),
2.44 (t,
d
7.35e7.39 (m, 3H),
4.25 (s, 2H), 3.61 (br
3.28
5.1.4.16. 2-Chloro-1-(4-(3-methoxybenzyl)piperazin-1-yl)ethanone
d
d
d
d
(4p). Yield 93%; HRMS (ESI): calcd for [C14H19ClN2O2þH]þ:
d
d
d
d
283.1213; found: 283.1212; 1H NMR (500 MHz, CDCl3)
d
7.23 (t,
d
d
d
J ¼ 8.0 Hz, 1H),
1H), 4.05 (s, 2H),
J ¼ 4.9 Hz, 4H),
2.44e2.49 (m, 4H); 13C NMR (125.8 MHz, CDCl3)
164.89, 159.66, 139.17, 129.21, 121.24, 114.53, 112.55, 62.58, 55.11,
d
6.89 (d, J ¼ 6.5 Hz, 2H),
d
6.81 (dd, J ¼ 8.2, 1.8 Hz,
d
d
d
d
d
3.80 (s, 3H),
d
3.63 (t, J ¼ 4.8 Hz, 2H),
d
3.51 (t,
d
d
130.77, 130.43, 129.91, 129.39, 128.71, 128.57, 127.41, 125.60, 60.74,
57.81, 56.53, 53.56, 52.92, 52.71, 48.57, 46.58, 44.50, 42.11, 32.38,
19.23.
d
52.79, 52.41, 46.24, 42.13, 40.81.
5.1.4.17. 2-Chloro-1-(4-(4-methoxybenzyl)piperazin-1-yl)ethanone
5.1.5.3. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(3-
methylbenzyl)piperazin-1-yl)ethanone (5c). Yield 50.2%; HRMS
(ESI): calcd for [C24H32N4O3S þ H]þ: 471.2430; found: 471.2429; 1H
(4q). Yield 89.5%; HRMS (ESI): calcd for [C14H19ClN2O2þH]þ:
283.1213; found: 283.1210; 1H NMR (400 MHz, CDCl3)
d
7.22 (d,
3.80 (s, 3H),
3.47 (s, 2H), 2.45
164.99,
J ¼ 8.4 Hz, 2H),
d
6.86 (d, J ¼ 8.4 Hz, 2H),
d
4.05 (s, 2H),
d
NMR (400 MHz, CDCl3)
2H),
7.22 (t, J ¼ 7.6 Hz, 2H),
3.49 (s, 2H),
3.26e3.31 (m, 3H),
2.35 (s, 3H),
d
7.41e7.43 (m, 2H),
d
7.19 (dt, J ¼ 8.0, 2.0 Hz,
4.25 (s, 2H),
d
3.62 (t, J ¼ 4.8 Hz 2H),
d
3.50 (t, J ¼ 4.8 Hz, 2H),
d
d
d
d
7.08e7.12 (m, 3H), d
(dt, J ¼ 13.2, 4.8 Hz, 4H); 13C NMR (100.6 MHz, CDCl3)
d
d
3.64 (t, J ¼ 4.8 Hz, 2H),
d
d
3.34e3.38 (m, 3H),
d
3.32 (d,
2.44 (t,
158.94, 130.28, 129.50, 113.74, 62.17, 55.27, 52.78, 52.37, 46.34,
42.22, 40.90.
J ¼ 6.0 Hz, 2H),
J ¼ 4.8 Hz, 4H),
d
d 2.95e2.99 (m, 1H),
2.04 (s, 1H),
d
d
d
d
1.96e2.03 (m, 1H),
d
1.70e1.77 (m, 1H); 13C NMR (100.6 MHz, CDCl3)
d 168.77, 138.02,
5.1.4.18. 2-Chloro-1-(4-(3,5-dimethoxybenzyl)piperazin-1-yl)etha-
137.19, 130.76, 129.89, 12938, 128.71, 128.56, 128.25, 128.13, 126.24,
62.86, 57.79, 56.50, 53.56, 52.86, 52.62, 48.56, 46.57, 44.37, 41.96,
32.38, 21.39.
none (4r). Yield 99.5%; HRMS (ESI): calcd for [C15H21ClN2O3þH]þ:
313.1319; found: 313.1318; 1H NMR (400 MHz, CDCl3)
d
6.49 (d,
3.63 (t,
1.70 (s, 1H);
J ¼ 2.0 Hz, 2H),
d
6.37 (s, 1H),
3.51 (t, J ¼ 4.8 Hz, 2H),
d
4.06 (s, 2H),
d
3.79 (s, 6H), d
J ¼ 4.8 Hz, 2H),
d
d
3.46 (s, 4H),
d
5.1.5.4. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(4-
methylbenzyl)piperazin-1-yl)ethanone (5d). Yield 53.2%; HRMS
(ESI): calcd for [C24H32N4O3S þ H]þ: 471.2430; found: 471.2431; 1H
13C NMR (100.6 MHz, CDCl3)
d
165.06, 160.85, 140.15, 160.88, 99.10,
62.84, 55.33, 52.90, 52.54, 46.37, 42.24, 40.91.
NMR (400 MHz, CDCl3)
d
7.35e7.43 (m, 5 H),
4.25 (s, 2H), 3.63 (br s, 2H),
3.30 (s, 1H), 3.25e3.29
d
7.19 (d, J ¼ 8.0 Hz,
5.1.4.19. 2-Chloro-1-(4-(3,4,5-trimethoxybenzyl)piperazin-1-yl)etha-
2H),
2H),
d
7.14 (d, J ¼ 8.0 Hz, 2H),
d
d
d
3.49 (s,
none (4s). Yield 83.4%; HRMS (ESI): calcd for [C16H23ClN2O4þH]þ:
d
3.34e3.37 (m, 3H),
2.42 (br s, 4H),
1.85 (br s, 1H),
(100.6 MHz, CDCl3)
d
d
3.31 (s, 1H),
2.34(s, 3H),
d
d
343.1425; found: 343.1423; 1H NMR (400 MHz, CDCl3)
d
7.56 (s, 2H),
(m, 3H),
1H),
d
d
2.00 (dt, J ¼ 18.8, 7.2 Hz,
d
4.07 (s, 2H),
3.53 (t, J ¼ 4.8 Hz, 2H),
165.05, 153.23, 137.20, 133.39, 105.81, 63.04,
60.86, 56.16, 52.90, 52.57, 46.37, 42.23, 40.90.
d
3.84 (s, 6H),
d
3.83 (s, 3H),
d
3.65 (t, J ¼ 4.8 Hz, 2H),
d
d
1.72 (dt, J ¼ 19.2, 6.4 Hz, 1H); 13C NMR
d
d
3.46 (s, 2H),
d
2.45e2.50 (m, 4H); 13C NMR
d
164.79, 137.08, 130.78, 130.70, 129.39, 129.14,
(100.6 MHz, CDCl3)
d
129.07,128.71,128.57, 62.56, 57.82, 56.54, 53.59, 52.80, 52.54, 48.60,
46.58, 44.39, 41.97, 32.41, 21.11.

1412
C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
5.1.5.5. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(2-
chlorobenzyl)piperazin-1-yl)ethanone (5e). Yield 45%; HRMS (ESI):
calcd for [C24H31ClN4O3S þ H]þ: 491.1884; found: 491.1881; 1H
calcd for [C24H31FN4O3S þ H]þ: 475.2179; found: 475.2175; 1H NMR
(400 MHz, CDCl3)
d
d
7.41e7.43 (m, 2H),
7.00 (t, J ¼ 8.4 Hz 2H),
d
d
7.35e7.40 (m, 3H),
d
7.26e7.30 (m, 2H),
4.25 (s, 2H),
3.33e3.37
2.97 (dd, J ¼ 9.6,
1.98e2.05 (m, 1H), 1.70e1.77 (m,
2H); 13C NMR (100.6 MHz, CDCl3)
168.71, 162.16, 133.13, 130.77,
d 3.64 (t,
NMR (400 MHz, CDCl3)
7.19e7.25 (m, 2H), 4.25 (s, 2H),
3.33 (s, 1H),
3.00 (dd, J ¼ 9.8, 4.8 Hz, 1H),
1.93 (br s, 1H),
1.75(dt, J ¼ 19.2, 6.4 Hz, 1H);
13C NMR (100.6 MHz, CDCl3)
168.77, 135.12, 134.47, 130.78, 130.70,
d
7.39e7.46 (m, 3H),
3.65 (s, 4H),
3.32 (br s, 1H),
2.50 (t, J ¼ 4.4 Hz, 4H),
d
7.36e7.38 (m, 4H),
3.37 (t, J ¼ 4.8 Hz,
3.26e3.29 (m,
J ¼ 4.4 Hz, 2H),
d 3.58 (br s, 1H), d 3.46e3.49 (m, 3H), d
d
d
d
d
(m, 3H),
d
3.31 (br s, 1H),
d
3.26e3.29 (m, 2H),
d
d
2H),
3H),
d
3.34 (br s, 2H),
d
d
d
4.8 Hz, 1H),
d
2.42 (br s, 4H),
d
d
d
d
d
1.97e2.04 (m, 1H),
d
d
130.61, 130.53, 129.38, 128.72, 128.58, 115.32, 115.11, 62.01, 57.81,
56.58, 53.56, 52.94, 52.80, 52.54, 48.60, 46.57, 44.40, 41.97, 32.41.
d
129.63, 129.38, 128.78, 128.72, 128.58, 128.52, 126.70, 59.11, 57.81,
56.55, 53.55, 52.87, 52.63, 48.56, 46.58, 44.45, 42.04, 32.36.
5.1.5.11. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(3-
(trifluoromethyl)benzyl)piperazin-1-yl)ethanone (5k). Yield 53.6%;
HRMS (ESI): calcd for [C24H31FN4O3S þ H]þ: 524.2069; found:
5.1.5.6. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(3-
chlorobenzyl)piperazin-1-yl)ethanone (5f). Yield 44.8%; HRMS (ESI):
calcd for [C24H31ClN4O3S þ H]þ: 491.1884; found: 491.1879; 1H
525.2146; 1H NMR (400 MHz, CDCl3)
d
7.60 (br s, 1H),
d
d
7.30 (t,
4.26 (s,
J ¼ 8.0 Hz, 2H),
2H), 3.65 (br s, 2H),
3.35 (s, 1H),
(dd, J ¼ 9.6, 4.4 Hz, 1H),
J ¼ 18.8, 7.2 Hz, 1H),
(100.6 MHz, CDCl3)
d
7.41e7.47 (m, 3H),
d
7.36e7.38 (m, 3H),
NMR (400 MHz, CDCl3)
7.34 (br s, 1H),
(s, 2H), 3.64 (br s, 2H),
s, 1H), 3.23e3.29 (m, 3H),
J ¼ 4.4 Hz, 4H),
d
7.41e7.43 (m, 2H),
7.25 (d, J ¼ 5.6 Hz, 2H), 7.19e7.20 (m, 1H),
3.50 (s, 2H), 3.34e3.38 (m, 4H), 3.31 (br
2.43 (t,
1.73 (dt, J ¼ 19.2, 6.4 Hz, 1H);
d
7.35e7.39 (m, 3H),
d
d
3.47e3.57 (m, 3H),
d
3.36e3.38 (m, 2H),
2.99
2.00 (dt,
d
d
d
d
4.25
d
d
3.33 (s, 1H),
d
3.29 (s, 1H),
d
3.27e3.28 (m, 1H),
2.15 (br s, 2H),
d
d
d
d
d
d
2.43 (br s, 4H),
d
d
d
d
2.97 (dd, J ¼ 9.8, 4.4 Hz, 1H),
d
d
1.74 (dt, J ¼ 19.0, 6.0 Hz, 1H); 13C NMR
d
1.96e2.04 (m, 2H),
d
d
168.79, 138.72, 132.23, 130.99, 130.76, 130.70,
13C NMR (100.6 MHz, CDCl3)
d
168.81, 139.74, 134.36, 130.77, 129.65,
129.39, 128.75, 125.54, 124.24, 122.79, 62.23, 57.80, 56.60, 53.51,
52.86, 52.65, 48.56, 46.56, 44.39, 41.95, 32.34.
129.39, 128.98, 128.72, 128.58, 127.56, 127.10, 62.19, 57.80, 56.57,
53.56, 53.43, 52.86, 52.63, 48.58, 46.58, 44.40, 41.97, 32.39.
5.1.5.12. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-
(2,4,6-trimethylbenzyl)piperazin-1-yl)ethanone (5l). Yield 65.6%;
HRMS (ESI): calcd for [C27H38FN4O3S þ H]þ: 499.2743; found:
5.1.5.7. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(4-
chlorobenzyl)piperazin-1-yl)ethanone (5g). Yield 52.1%; HRMS
(ESI): calcd for [C24H31ClN4O3S þ H]þ: 491.1884; found: 491.1880;
499.2744; 1H NMR (400 MHz, CDCl3)
d
7.36e7.43 (m, 5H),
3.47 (s, 2H),
3.36 (dd, J ¼ 9.6,
3.24e3.29 (m, 5H), 2.97 (dd,
2.33 (s, 6H), 2.27 (s, 3H),
1.89 (s, 1H),
1.73 (dt, J ¼ 18.8,
6.4 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
168.73, 138.06, 136.73,
d 6.84 (s,
1H NMR (400 MHz, CDCl3)
d
d
7.39e7.42 (m, 2H),
d
7.35e7.38 (m, 3H),
4.27 (s, 2H),
2H),
6.0 Hz, 2H),
J ¼ 9.6, 4.8 Hz, 1H),
2.00 (dt, J ¼ 19.2, 6.8 Hz, 1H),
d
4.25 (s, 2H),
3.31 (d, J ¼ 7.2 Hz, 2H),
2.42 (br s, 4H),
d
3.56 (br s, 2H),
d
d
d
d
d
d
7.35 (d, J ¼ 8.4 Hz, 2H),
3.63 (br s, 2H),
3.22e3.29 (m, 3H),
1.98 (dt, J ¼ 19.6, 6.0 Hz, 1H),
7.25 (d, J ¼ 8.4 Hz, 2H),
d
d
d
d
d
3.48 (s, 2H),
d
3.33e3.37 (m, 4H),
d
3.31 (br s, 1H),
2.40 (br s, 4H),
d
d
d
d
2.97 (dd, J ¼ 9.6, 4.8 Hz, 1H),
d
d
d
d
d
1.93 (br s, 1H), 1.73 (dt, J ¼ 19.2,
d
6.0 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
d
168.84, 136.08, 133.08,
131.01, 130.78, 129.40, 129.00, 128.71, 128.56, 57.82, 56.52, 55.69,
53.59, 52.50, 52.28, 48.56, 46.58, 44.70, 42.32, 32.40, 20.90, 20.04.
130.76, 130.69, 130.32, 129.38, 128.71, 128.66, 128.56, 128.54, 62.03,
57.79, 56.65, 56.51, 53.57, 52.83, 52.58, 48.59, 46.57, 44.39, 44.97,
32.40.
5.1.5.13. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(2,4-
dichlorobenzyl)piperazin-1-yl)ethanone (5m). Yield 49.9%; HRMS
(ESI): calcd for [C24H30Cl2N4O3S þ H]þ: 525.1494; found: 525.1495;
5.1.5.8. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(2-
fluorobenzyl)piperazin-1-yl)ethanone (5h). Yield 60%; HRMS (ESI):
calcd for [C24H31FN4O3S þ H]þ: 475.2179; found: 475.2185; 1H NMR
1H NMR (400 MHz, CDCl3)
d
7.41e7.45 (m, 2H),
7.23 (dd, J ¼ 8.2, 1.6 Hz, 1H),
3.63 (s, 2H), 3.36e3.60 (m, 3H),
3.26e3.30 (m, 1H),
d 7.39 (br s, 2H),
d
d
d
d
d
d
7.35e7.37 (m, 3H),
3.64 (br s, 2H),
3.32 (s, 1H),
d
d
4.25 (s, 2H),
(400 MHz, CDCl3)
d
7.40e7.43 (m, 2H),
d
7.34e7.39 (m, 4H),
7.04 (t, J ¼ 8.8 Hz, 1H),
3.31 (d,
2.96 (dd, J ¼ 9.8, 4.4 Hz, 1H),
1.95e2.04 (m, 2H),
1.72 (dt, J ¼ 18.0,
6.4 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
168.81, 161.41, 131.51,
d
d
d 3.34 (s, 1H),
d
7.26e7.29 (m, 1H),
4.25 (s, 2H),
d
7.13 (t, J ¼ 7.6 Hz 1H),
d
d
d
2.98 (dd, J ¼ 9.8, 4.8 Hz, 1H),
d
d
3.60e3.65 (m, 4H),
d
3.34e3.37 (m, 3H),
d
2.48 (t, J ¼ 4.8 Hz, 4H),
d
2.01 (dt, J ¼ 18.8, 6.8 Hz,1H),
d 1.90 (s, 1H),
J ¼ 6.4 Hz, 2H),
d
3.23e3.29 (m, 3H),
d
1.74 (dt, J ¼ 19.2, 6.4 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
d
2.47 (t, J ¼ 4.4 Hz, 4H),
d
d
168.85, 135.01, 133.91, 133.53, 131.49, 130.77, 130.70, 129.39,
d
128.78, 128.72, 128.57, 127.05, 58.55, 57.80, 56.57, 53.57, 52.87,
52.62, 48.60, 46.58, 44.43, 42.01, 32.41.
130.73, 129.40, 129.20, 129.11, 128.71, 128.56, 124.00, 123.87, 115.52,
115.30, 57.60, 56.56, 55.17, 53.6, 52.61, 52.36, 48.57, 46.58, 44.40,
41.98, 32.40.
5.1.5.14. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(2,6-
dichlorobenzyl)piperazin-1-yl)ethanone (5n). Yield 42.1%; HRMS
(ESI): calcd for [C24H30Cl2N4O3S þ H]þ: 525.1494; found: 525.1497;
5.1.5.9. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(3-
fluorobenzyl)piperazin-1-yl)ethanone (5i). Yield 36.6%; HRMS (ESI):
calcd for [C24H31FN4O3S þ H]þ: 475.2179; found: 475.2190; 1H NMR
1H NMR (400 MHz, CDCl3)
d
7.39e7.42 (m, 2H),
7.17 (t, J ¼ 8.0 Hz, 1H),
3.60 (br s, 2H), 3.36e3.39 (m, 1H),
3.26e3.27 (m, 3H),
2.97 (dd, J ¼ 9.6, 4.8 Hz, 1H),
d
7.36 (d, J ¼ 5.2 Hz,
4.25 (s, 2H),
3.30e3.35
3H), 7.32 (d, J ¼ 8.0 Hz, 2H),
3.78 (br s, 2H),
(m, 4H),
d
d
(400 MHz, CDCl3)
7.26e7.31 (m, 1H),
1H), 4.25 (s, 2H),
3.36 (br s, 1H),
d
d
7.40e7.43 (m, 2H),
d
7.35e7.39 (m, 3H),
6.95 (dt, J ¼ 7.6, 1.6 Hz,
3.37 (t, J ¼ 4.8 Hz, 2H),
3.25e3.30 (m, 3H), 2.98 (dd,
1.96e2.05 (m, 2H), 1.74 (dt,
168.78, 163.00,
d
d
d
d
d
7.06e7.09 (m, 2H),
d
d
d
d
d
3.64 (t, J ¼ 4.4 Hz, 2H),
d
d
d
d
2.54e2.58 (m, 4H),
d
2.00 (dt, J ¼ 18.8, 6.8 Hz, 1H),
d 1.91 (s, 1H),
d
d
3.34 (br s, 1H),
d
d
1.74 (dt, J ¼ 18.8, 6.4 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
J ¼ 10.4, 4.8 Hz, 1H),
d
2.44 (br s, 4H),
d
d
168.73, 136.96, 133.71, 130.78, 129.40, 129.13, 128.71, 128.57,
J ¼ 19.6, 6.0 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
d
128.46, 57.82, 56.52, 56.27, 53.60, 53.43, 52.74, 52.55, 48.56, 46.58,
44.50, 42.11, 32.40.
140.22, 130.77, 129.60, 129.38, 128.64, 124.48, 115.68, 114.39, 114.18,
62.19, 57.81, 56.56, 53.54, 52.86, 52.61, 48.59, 46.58, 44.40, 41.97,
32.37.
5.1.5.15. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(2-
chloro-6-fluorobenzyl)piperazin-1-yl)ethanone (5o). Yield 66.5%;
HRMS (ESI): calcd for [C24H30ClFN4O3S þ H]þ: 509.1789; found:
5.1.5.10. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(4-
fluorobenzyl)piperazin-1-yl)ethanone (5j). Yield 20.2%; HRMS (ESI):
509.1786; 1H NMR (400 MHz, CDCl3)
d 7.40e7.42 (m, 2H),

C. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 1404e1414
1413
d
7.35e7.39 (m, 3H), 7.22e7.23 (m, 2H),
(s, 2H), 3.73 (br s, 2H), 3.61 (br s, 2H),
3.31e3.34 (m, 4H), 3.24e3.29 (m, 3H),
2.96 (dd, J ¼ 9.8, 4.8 Hz,
d
6.98e7.02 (m, 1H),
d
4.25
models of compounds on Abl and PI3K and to better understand
how molecules can inhibit both enzymes, and may shed light into
inhibition activity. The following crystal structures from the Broo-
khaven Protein Data bank were used as raw models for the docking
studies: Abl (PDB entry 1IEP)[42] and PI3K (PDB entry 3LS8) (to be
published by Tresaugues L. et al.). Hydrogen atoms were added and
water molecules cocrystallized with the protein were removed
from the original structure. The cocrystallized ligands served as
templates for initial conformation and for the manual docking of
representative compounds. All docking calculations were carried
out with Surflex-Dock. The standard docking protocol was followed
as (1) ligand and receptor preparation, (2) protomol generation, (3)
docking and scoring. (4) analysis of results.
d
d
d
3.36e3.38 (m, 1H),
d
d
d
1H),
d
d
2.54 (br s, 4H),
d
2.00 (dt, J ¼ 18.8, 6.8 Hz, 1H),
d 1.89 (s, 1H),
1.72 (dt, J ¼ 19.2, 6.0 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
d
168.68, 162.07, 136.64, 130.78, 129.45, 128.71, 128.57, 125.55,
123.35, 123.17, 114.08, 57.81, 56.53, 53.58, 52.55, 52.33, 52.27, 48.53,
46.58, 42.03, 32.38.
5.1.5.16. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(3-
methoxybenzyl)piperazin-1-yl)ethanone (5p). Yield 59.6%; HRMS
(ESI): calcd for [C25H34N4O4S þ H]þ: 487.2379; found: 487.2377; 1H
NMR (400 MHz, CDCl3)
7.24 (t, J ¼ 8.0 Hz, 1H),
2.4 Hz, 1H), 4.25 (s, 2H),
2H), 3.41e3.38 (m, 3H),
(m, 3H),
1.84 (br s,1H),
CDCl3)
d
7.39e7.41 (m, 2H),
d
7.35e7.38 (m, 3H),
6.82 (dd, J ¼ 8.0,
3.64 (br s, 2H), 3.51 (s,
3.31 (s, 1H), 3.25e3.29
d
6.90 (d, J ¼ 5.6 Hz, 2H),
d
d
d
3.81(s, 3H),
3.33 (s, 1H),
d
d
d
5.3. Biological activity
d
d
d
d
2.43 (d, J ¼ 4.8 Hz, 4H),
d
2.01 (dt, J ¼ 18.8, 6.8 Hz, 4H),
5.3.1. Cell-growth inhibition assay
The MTT assays were performed to assess the viability of tumor
cells treated with the synthesized compounds. Cell line was
d
d
1.73 (dt, J ¼ 18.8, 6.4 Hz,1H); 13C NMR (100.6 MHz,
d
168.82, 159.75, 130.78, 129.40, 129.35, 128.71, 128.57,
121.39, 114.70, 112.64, 62.76, 57.82, 56.55, 55.24, 53.60, 52.87, 52.64,
48.60, 46.58, 44.42, 42.01, 32.42.
cultured in DMEM, with 10% fetal bovine serum (FBS), 100
mg/mL
penicillin, and 100
m
g/mL streptomycin in humidified air at 37 ꢀC
with 5% CO2. The cell was then seeded in 96-well tissue culture
plate and treated with the synthesized compounds at different
5.1.5.17. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(4-
methoxybenzyl)piperazin-1-yl)ethanone (5q). Yield 57.5%; HRMS
(ESI): calcd for [C25H34N4O4S þ H]þ: 487.2379; found: 487.2379; 1H
concentrations. After 48 h treatment, 15
mL) was added to each well and incubated for 4 h at 37 ꢀC, 5% CO2.
The formazan precipitate was dissolved in 100 L DMSO and the
mL of MTT solution (5 mg/
NMR (400 MHz, CDCl3)
d
7.40e7.43 (m, 2H),
6.86 (d, J ¼ 8.4 Hz, 2H),
3.63 (br s, 2H), 3.47 (s, 2H), 3.34e3.38 (m, 3H),
3.29 (s, 1H), 3.25e3.30 (m, 3H),
2.96 (dd, J ¼ 9.8,
d
7.35e7.39 (m, 3H),
m
d
d
d
7.22 (d, J ¼ 8.4 Hz, 2H),
d
d
4.25 (s, 2H),
absorbance at 495 nm of each well was measured by Multimode
Detector DTX880 (Beckman Coulter) [39].
3.81 (s, 3H),
3.32 (s, 1H),
d
d
d
d
d
d
4.4 Hz, 1H),
d
d
2.41 (d, J ¼ 4.0 Hz, 4H), 2.00 (dt, J ¼ 18.8, 6.8 Hz, 1H),
d
5.3.2. In vitro kinase assays on Abl and PI3K (alpha)
1.86 (br s, 1H), 1.72 (dt, J ¼ 18.8, 6.4 Hz, 1H); 13C NMR (100.6 MHz,
In vitro kinase assays on Abl and PI3K (alpha) inhibition were
tested by HD Biosciences Co., Ltd. in Shanghai, China. General
procedures are as the following: Kinases were incubated with
substrates, compounds and ATP in a final buffer of 25 mM HEPES
CDCl3)
d 168.80, 158.95, 130.77, 130.32, 129.38, 128.71, 128.56,
113.74, 62.23, 57.80, 56.511, 55.28, 53.59, 52.75, 52.49, 48.58, 46.58,
44.41, 41.99, 32.41.
(pH 7.4), 10 mM MgCl2, 0.01% Triton X-100, 100
DTT in 384-well plate with the total volume of 10
m
g/mL BSA, 2.5 mM
5.1.5.18. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-(3,5-
dimethoxybenzyl)piperazin-1-yl)ethanone (5r). Yield76.6%; HRMS
(ESI): calcd for [C26H36N4O5S þ H]þ: 517.2485; found: 517.2494; 1H
mL. The assay
plate was incubated at 30 ꢀC for 1 h and stopped with the addition
of equal volume of kinase glo plus reagent. The luminescence was
read at envision. The signal was correlated with the amount of ATP
present in the reaction and was inversely correlated with the kinase
activity [40].
NMR (400 MHz, CDCl3)
6.49 (s, 2H), 6.38 (s, 2H),
2H), 3.47 (s, 2H), 3.34e3.37 (m, 3H),
3.26e3.29 (m, 3H),
2.97 (dd, J ¼ 9.8, 4.4 Hz, 1H),
d
7.38e7.42 (m, 2H),
4.25 (s, 2H), 3.79 (s, 6H),
3.32 (s, 1H),
2.43 (d, J ¼ 4.4,
1.80 (br s, 1H), 1.73 (dt,
168.82, 160.85,
d
7.36e7.37 (m, 3H),
3.64 (br s,
3.31 (s, 1H),
d
d
d
d
d
d
d
d
d
d
d
4H),
d
2.00 (dt, J ¼ 18.8, 6.8 Hz, 1H),
d
d
5.3.3. Flow cytometric analysis of apoptosis
J ¼ 19.2, 6.4 Hz, 1H); 13C NMR (100.6 MHz, CDCl3)
d
Untreated and drug-treated cells were collected by centrifuga-
tion and washed twice with ice-cold PBS. Surface exposure of
phosphatidylserine in apoptotic cells was measured by Annexin V-
FITC/PI apoptosis detection kit（Beyotime Company）according to
the protocol described using MoFlo/XDP cell sorter flow cytometry
(Beckman) [41].
139.96, 130.78, 129.39, 128.72, 128.58, 106.93, 62.89, 57.82, 56.54,
55.35, 53.60, 53.43, 52.86, 52.66, 48.61, 46.58, 44.42, 42.00, 32.42.
5.1.5.19. (S)-2-((1-(Benzylsulfonyl)pyrrolidin-3-yl)amino)-1-(4-
(3,4,5-trimethoxybenzyl)piperazin-1-yl)ethanone (5s). Yield 48.2%;
HRMS (ESI): calcd for [C27H38N4O6S þ H]þ: 546.2512; found:
547.2598; 1H NMR (400 MHz, CDCl3)
d
7.41e7.42 (m, 2H),
3.86 (s, 6H), 3.84
3.35e3.38 (m, 3H),
3.27 (br s, 1H), 3.23e3.26
2.00
Acknowledgement
d
7.36e7.40 (m, 3H), 6.56 (s, 2H),
(s, 3H), 3.65 (br s, 2H), 3.46 (s, 2H),
3.31e3.34 (m, 2H), 3.29 (br s, 2H),
d
4.25 (s, 2H),
d
d
d
d
d
The authors thank Tripos associate Inc., St. Louis, MO, USA for
providing Sybyl-X 1.1 software. The authors would like to thank the
financial supports from the Ministry of Science and Technology of
China (2007AA02Z160, 2009ZX09501-004), the Chinese National
Natural Science Foundation (20872077, 90813013).
d
d
d
d
(m, 1H),
d
2.98 (dd, J ¼ 9.8, 4.4 Hz, 1H),
d
2.40 (d, J ¼ 4.4, 4H),
d
(dt, J ¼ 18.8, 6.8 Hz, 1H),
d
1.73 (dt, J ¼ 19.6, 6.0 Hz, 2H); 13C NMR
(100.6 MHz, CDCl3)
d
168.85, 153.22, 137.18, 133.24, 130.77, 130.69,
129.37, 128.72, 128.67, 128.58, 63.09, 60.87, 57.80, 56.61, 56.16,
53.56, 52.78, 51.47, 48.61, 46.53, 44.42, 41.99, 34.82, 32.42.
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