Design and synthesis of tri-substituted chiral pyrrolidin-2-one derivatives as CCR4 antagonists

Article abstract of DOI:10.1002/cjoc.201300363

A series of tri-substituted chiral pyrrolidin-2-one derivatives have been designed and synthesized as CC chemokine receptor 4 (CCR4) antagonists. The structure of CCR4 was built by homology modeling. Asymmetric synthesis was applied to synthesize the R,R configuration chiral pyrrolidin-2-one scaffold. The stereoisomeric configurations of the compounds were identified by 2D ¹H-¹H COSY spectroscopy and 1D NOESY spectroscopy. This method was more economical and convenient than traditional X-ray single crystal diffraction. In addition, the interactions between these compounds and the N-terminal extracellular tail of CCR4 were studied using capillary zone electrophoresis. The CCR4 chemotaxis inhibition effect was tested in CCR4-transfected HEK293 cells. Several compounds showed potent activities as CCR4 antagonists. Among these compounds, 1c is the most active one. Its apparent binding constant of CZE experiment result is (1.569±0.11)×10 ⁵ L·mol^-1, and its percentage inhibition of the HEK293/CCR4 cells migration with the concentration of 1 μmol·L ^-1 in DMSO is 59%. And compound 1f has slightly higher affinity to N-terminal of CCR4 according to its apparent binding constant than 1b because of the introduced ester linkage. Further studies on the mechanism of these compounds are in progress. The structure of CCR4 based on bovine rhodopsin was built through homology modeling. And six tri-substituted chiral pyrrolidin-2-one derivatives have been designed and synthesized as CCR4 antagonists. Several of these compounds show high inhibitory effect in cell assays and higher affinity for the N-terminal of CCR4. Among the compounds, 1c exhibited excellent activity. Copyright

Full text of DOI:10.1002/cjoc.201300363

FULL PAPER
DOI: 10.1002/cjoc.201300363
Design and Synthesis of Tri-substituted Chiral Pyrrolidin-2-one
Derivatives as CCR4 Antagonists
Wei Sun,a Linjie Tian,c Hui Qi,c Dan Jiang,b Ying Wang,c
Song Li,b Junhai Xiao,*,b and Xiaohong Yang*,a
a School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin 130021, China
b Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology & Toxicology,
Beijing 100850, China
c Center for Human Disease Genomics, Peking University, Beijing 100083, China
A series of tri-substituted chiral pyrrolidin-2-one derivatives have been designed and synthesized as CC
chemokine receptor 4 (CCR4) antagonists. The structure of CCR4 was built by homology modeling. Asymmetric
synthesis was applied to synthesize the R,R configuration chiral pyrrolidin-2-one scaffold. The stereoisomeric con-
figurations of the compounds were identified by 2D 1H-1H COSY spectroscopy and 1D NOESY spectroscopy. This
method was more economical and convenient than traditional X-ray single crystal diffraction. In addition, the inter-
actions between these compounds and the N-terminal extracellular tail of CCR4 were studied using capillary zone
electrophoresis. The CCR4 chemotaxis inhibition effect was tested in CCR4-transfected HEK293 cells. Several
compounds showed potent activities as CCR4 antagonists. Among these compounds, 1c is the most active one. Its
apparent binding constant of CZE experiment result is (1.569±0.11)×105 L•mol−1, and its percentage inhibition of
the HEK293/CCR4 cells migration with the concentration of 1 μmol•L−1 in DMSO is 59%. And compound 1f has
slightly higher affinity to N-terminal of CCR4 according to its apparent binding constant than 1b because of the in-
troduced ester linkage. Further studies on the mechanism of these compounds are in progress.
Keywords CCR4 antagonists, tri-substituted chiral pyrrolidin-2-one derivatives, synthesis
Introduction
agents.
In the present study, the structure of CCR4 based on
Chemokines are a group of cytokines with similar
structure and function. They are the largest family of
cytokines. Chemokines are divided into four subfamilies
depending on the position of their first two N-terminal
cysteine residues, namely, CXC, CC, CX3C and C.
Chemokine receptors belong to G-protein-coupled re-
ceptors (GPCRs). They are also classified into CXCR1
－CXCR6, CCR1－CCR11, CX3CR1 and XCR1 ac-
cording to the chemokines they bind, among which CC
chemokine receptor 4 (CCR4) is mainly expressed on
Th2 cells and was found in 1995.[1] Thymus and activa-
tion regulated chemokines (TARC/CCL17), macro-
bovine rhodopsin (PDB ID: 1U19A) was built through
homology modeling. The modeled structure could pro-
vide theoretical basis for intensive study of CCR4 and
its antagonists. Two pockets (A and B) were found in
N-terminal extracellular tail of the modeled receptor
(Figure 1a). The interaction between CCR4 and its an-
tagonist, N-(3-chloro-2-methyl benzyl)-2-((3R,5R)-5-
(2,4-dichlorophenyl)-2-oxo-1-(2-oxo-2-(3-(piperidin-1-
yl)propylamino)ethyl)pyrrolidin-3-yl)acetamide,
a
known CCR4 antagonist that has high binding affinity,
excellent chemotaxis inhibitory activity and selectivity
toward CCR4[12] was analyzed by docking. The com-
pound can only bind pocket A (Figure 1b). Thus, a se-
ries of tri-substituted chiral pyrrolidin-2-one derivatives
was designed based on this compound. Ester linkage,
the bioisostere of the amidolinkage, was introduced to
some of these compounds to increase their flexibilities
and binding probabilities on both sites A and B. Using
capillary zone electrophoresis (CZE),[13] the trans-dia-
stereomer was verified to have more potent affinity with
phage-derived
chemokine
(MDC/CCL22),
and
chemokine-like factor 1 (CKLF1)[2-5] are the three spe-
cific natural ligands of CCR4. CCR4 and its ligands
have attracted significant attention because of their in-
volvement in mediating various autoimmune diseases,
allergic inflammations, and thrombotic diseases, among
others.[6-11] The development of selective small mole-
cule CCR4 antagonists facilitates the study of CCR4
biological function and development of new therapeutic
*
E-mail: yangxiaoh@jlu.edu.cn or xiaojunhai@139.com; Tel.: 0086-0431-85619660 or 0086-010-66930673-712
Received April 27, 2013; accepted June 15, 2013; published online July 5, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201300363 or from the author.
1144
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Design and Synthesis of Tri-substituted Chiral Pyrrolidin-2-one Derivatives as CCR4 Antagonists
NaOH solution and saturated salt solution. The resulting
CCR4 especially with R,R-isomer. Thus, asymmetric
synthesis was applied to synthesize the R,R configura-
tion chiral pyrrolidin-2-one scaffold. The stereoisomeric
configuration of the compound was identified by 2D
1H-1H COSY spectroscopy and 1D NOESY spectros-
copy, as previously reported.[14] This method was more
economical and convenient than traditional X-ray single
crystal diffraction. The interactions between these
compounds and the N-terminal extracellular tail of
CCR4 were studied by CZE. And the CCR4 chemotaxis
inhibition effect was tested in CCR4-transfected
HEK293 cells.
mixture was then dried with anhydrous sodium sulfate
and evaporated under reduced pressure. The residue was
purified through column chromatography on silica gel
to produce 36.93 g of compound 3 as a white solid with
72% yield. m.p. 50－52 ℃; 1H NMR (400 MHz,
CDCl3) δ: 7.69 (d, J＝8.10 Hz, 2H, ArH), 7.53 (d, J＝
8.10 Hz, 2H, ArH), 3.68 (s, 3H, CH3), 3.45 (t, J＝6.55
Hz, 2H, CH2), 2.58 (t, J＝6.55 Hz, 2H, CH2); FAB-MS
m/z: 272.1 [M＋H]＋.
Synthesis of 4-(4-bromophenyl)-4-oxobutanoic acid
(4)
Compound 3 (31.44 g, 0.12 mol) was dissolved in
600 mL of THF and 240 mL of H2O, then 390 mL of 1
mol•L−1 LiOH was added. The solution was stirred for 2
h at room temperature. To remove THF, it was then
evaporated under reduced pressure. Then, the water so-
lution was extracted with ethyl acetate. The water layer
was adjusted to pH 1 with 1 mol•L−1 HCl. The precipi-
tate was filtered to give 30.84 g of compound 4 as a
light yellow solid with 100% yield. m.p. 148－152 ℃;
1H NMR (400 MHz, CDCl3) δ: 9.03 (br, 1H, COOH),
7.67 (d, J＝8.10 Hz, 2H, ArH), 7.50 (d, J＝8.10 Hz, 2H,
ArH), 3.34 (t, J＝6.53 Hz, 2H, CH2), 2.69 (t, J＝6.53
Hz, 2H, CH2); FAB-MS m/z: 258.1 [M＋H]＋.
Figure 1 (a) Modeled receptor. (b) Docking picture between
the receptor and a known CCR4 antagonist.
Synthesis of (3S,7aS)-7a-(4-bromophenyl)-3-phenyl-
tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (5)
Experimental
A mixture of compound 4 (30.84 g, 0.12 mol) and
(S)-(＋)-2-phenylglycinol (18.12 g, 0.132 mol) in 600
mL of anhydrous toluene was heated to reflux and de-
hydrated using a water segregator for 16 h. Upon cool-
ing to room temperature, the solvent was removed under
reduced pressure. The residue was then purified through
column chromatography on silica gel to produce 42.12 g
of compound 5 as a white solid with 98% yield. m.p. 87
Materials and reagents
Melting points were determined on a YRT-3 melting
point detector and were uncorrected. The NMR spectra
were recorded on a JEOL JNM-ECA400 (400 MHz)
spectrometer. FAB mass spectra were determined using
a Micromass ZabSpec (1000) high-resolution mass
spectrometer.
1
－90 ℃; H NMR (400 MHz, CDCl3) δ: 7.46 (d, J＝
Synthesis of 3-methoxycarbonyl-propionyl chloride
(2)
8.40 Hz, 2H, ArH), 7.33 (d, J＝8.40 Hz, 2H, ArH), 7.21
－7.26 (m, 4H, ArH), 7.06－7.08 (m, 1H, ArH), 5.15 (t,
J＝8.27 Hz, 1H, 3-H), 4.66 (dd, J＝7.83, 1.12 Hz, 1H,
2-H), 3.58 (t, J＝8.83 Hz, 1H, 2-H), 2.91－2.94 (m, 1H,
7-H), 2.58－2.63 (m, 2H, 6-H), 2.28－2.31 (m, 1H,
7-H); FAB-MS m/z: 359.2 [M＋H]＋.
Mono-methyl succinate (26.4 g, 0.2 mol) and thionyl
chloride (14.5 mL, 0.2 mol) were placed in a round-
bottomed flask. The mixture was heated to 35 ℃ for 4
h. Excessive thionyl chloride was then removed by re-
ducing the pressure. Pressure was continuously reduced
and the distillate was collected at 92 to 93 ℃/2400 Pa
(18 mmHg). Up to 28.5 g of compound 2 was obtained
as a colorless liquid with 95% yield.
Synthesis of (R)-5-(4-bromophenyl)-1-((S)-2-hy-
droxy-1-phenylethyl)pyrrolidin-2-one (6)
Approximately 17 mL (152.1 mmol) of TiCl4 and 24
mL (152.1 mmol) of Ei3SiH were added into compound
5 solution (36.3 g, 101.4 mmol) in 500 mL anhydrous
CH2Cl2 at −78 ℃. The mixture was stirred for 16 h at
−78 ℃. Saturated ammonium chloride solution was
added to quench the reaction. The organic compounds
were extracted with CH2Cl2. The organic layer was
separated and dried with anhydrous sodium sulfate, after
which, the solvent was removed under reduced pressure.
The residue was then purified through column chroma-
tography on silica gel to produce 26.7 g of compound 6
Synthesis of methyl 4-(4-bromophenyl)-4-oxobutano-
ate (3)
Compound 2 (28.5 g, 0.19 mol) was dropped into the
mixture of anhydrous aluminum chloride (40.04 g, 0.3
mol) and bromobenzene (100 mL) at 0 ℃. The mixture
was then heated to 80 ℃ for 4 h. The resulting mixture
was poured into ice containing HCl and the organic
compounds were extracted with ethyl acetate. The or-
ganic layer was separated and then washed with 5%
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Sun et al.
FULL PAPER
1
as a white solid with 73% yield. H NMR (400 MHz,
CDCl3) δ: 7.49－7.50 (m, 2H, ArH), 7.29－7.32 (m, 3H,
ArH), 7.11－7.14 (m, 2H, ArH), 7.03－7.05 (m, 2H,
ArH), 4.35－4.37 (m, 1H, ArCH), 4.22－4.26 (m, 1H,
5-H), 4.02－4.06 (m, 1H, CH2OH), 3.90－3.93 (m, 1H,
CH2OH), 3.33 (br, 1H, OH), 2.59－2.62 (m, 2H, 3-H),
2.43－2.46 (m, 1H, 4-H), 1.90－1.93 (m, 1H, 4-H);
FAB-MS m/z: 361.2 [M＋H]＋.
1H, 1-H), 4.72－4.74 (m, 1H, 5-H), 2.56－2.58 (m, 1H,
4-H), 2.44－2.48 (m, 2H, 3-H), 1.90－19.4 (m, 1H,
4-H); FAB-MS m/z: 241.1 [M＋H]＋.
Synthesis of (R)-5-(4-bromophenyl)-1-(2-(tert-butyl-
dimethylsilyloxy)ethyl)pyrrolidin-2-one (10)
A 60% NaH solution (0.94 g, 23.5 mmol) was added
to compound 9 solution (4.72 g, 19.66 mmol) in 60 mL
of anhydrous DMF. Then (2-indoethoxy)-tert-butyl-
dimethyl silane (6.72 g, 23.5 mmol) in 15 mL of anhy-
drous DMF was dropped into the reactants. The mixture
was stirred overnight at room temperature and was
poured into ice. The organic compounds were then ex-
tracted with ethyl acetate, after which, the organic layer
was separated, washed with saturated salt solution, then
dried over anhydrous sodium sulfate and evaporated
under reduced pressure. The residue was then purified
through column chromatography on silica gel to give
3.66 g of compound 10 as slightly yellow oil with 47%
Synthesis of (R)-5-(4-bromophenyl)-1-((S)-2-chloro-
1-phenylethyl)pyrrolidin-2-one (7)
SOCl2 (5.5 mL) was added to compound 6 solution
(21.51 g, 59.67 mmol) in 400 mL THF at 0 ℃ and the
mixture was stirred for 2 h. The THF and superfluous
SOCl2 were then removed under reduced pressure.
Through ethyl acetate extraction, saturated salt solution
washing, anhydrous sodium sulfate drying, and evapo-
ration under reduced pressure, compound 7 (22.5 g) was
obtained as a yellow oil with 90% yield. 1H NMR (400
MHz, CDCl3) δ: 7.74 (d, J＝8.31 Hz, 2H, ArH), 7.47－
7.49 (m, 4H, ArH), 7.19－7.21 (m, 3H, ArH), 5.41－
5.44 (m, 1H, ArCH), 4.87－4.91 (m, 1H, 5-H), 3.91－
3.94 (m, 1H, CH2Cl),3.71－3.74 (m, 1H, CH2Cl), 2.43
－2.50 (m, 2H, 4-H), 2.44－2.46 (m, 1H, 3-H), 1.94－
1.98 (m, 1H, 3-H); FAB-MS m/z: 379.7 [M＋H]＋.
1
yield. H NMR (400 MHz, CDCl3) δ: 7.49－7.52 (m,
2H, ArH), 7.03－7.06 (m, 2H, ArH), 4.81－4.83 (m, 1H,
5-H), 3.74－3.76 (m, 2H, OCH2CH2), 3.59－3.60 (m,
1H, OCH2CH2), 2.69－2.70 (m, 1H, OCH2CH2), 2.44－
2.47 (m, 3H, 3-H, 4-H), 1.82－1.83 (m, 1H, 4-H), 0.87
[s, 9H, C(CH3)3], 0.02 [s, 6H, Si(CH3)2]; FAB-MS m/z:
399.4 [M＋H]＋.
Synthesis of (R)-5-(4-bromophenyl)-1-(1-phenyl-vi-
nyl)pyrrolidin-2-one (8)
Synthesis of (3S,5R)-3-allyl-5-(4-bromophenyl)-1-(2-
(tert-butyldimethylsilyloxy)ethyl)pyrrolidin-2-one (11)
Na (0.98 g, 41 mmol) was added to 100 mL of an-
hydrous ethanol. The solution was then added to a
compound 7 mixture (17.8 g, 35.8 mmol) in anhydrous
ethanol (150 mL). The reactant was then heated for 4 h
at 45 ℃. THF was removed under reduced pressure,
after which, the organic compounds were extracted with
ethyl acetate. The organic layer was separated, washed
with saturated salt solution, and dried with anhydrous
sodium sulfate. The solvent was then removed under
reduced pressure. Compound 8 (10.5 g) was obtained as
LDA (4 mL) and allyl bromide (1 mL, 15.0 mmol)
were added to compound 10 solution (3.6 g, 9.2 mmol)
in 50 mL of newly treated anhydrous THF at −78 ℃
under N2 atmosphere. The resulting solution was stirred
for 30 min. Then the temperature was allowed to rise to
room temperature and the mixture was stirred overnight.
Saturated ammonium chloride solution was then added
to quench the reaction. THF was removed under re-
duced pressure, after which, the organic compounds
were extracted with ethyl acetate. The organic layer was
separated, washed with saturated salt solution, and dried
with anhydrous sodium sulfate. The solvent was then
removed under reduced pressure. Compound 11 (3.54 g)
was obtained as a yellow oil with 88% yield. 1H NMR
(400 MHz, CDCl3) δ: 7.48－7.50 (m, 2H, ArH), 7.02－
7.06 (m, 2H, ArH), 5.73－5.75 (m, 1H, CH＝CH2),
5.07－5.09 (m, 2H, CH＝CH2), 4.80－4.81 (m, 1H,
5-H), 3.78－3.80 (m, 2H, OCH2CH2), 3.63－3.64 (m,
1H, OCH2CH2), 2.69－2.70 (m, 3H, 4-H, OCH2CH2,
3-H), 2.20－2.22 (m, 2H, CH2CH＝CH2), 1.97－1.97
(m, 1H, 4-H), 0.87 [s, 9H, C(CH3)3], 0.02 [s, 6H,
Si(CH3)2]; FAB-MS m/z: 439.5 [M＋H]＋.
1
a yellow oil with 86% yield. H NMR (400 MHz,
CDCl3) δ: 7.76 (d, J＝8.33 Hz, 2H, ArH), 7.66－7.69
(m, 2H, ArH), 7.33－7.40 (m, 3H, ArH), 7.16－7.18 (m,
2H, ArH), 5.37 (t, J＝5.39 Hz, 1H, 5-H ), 5.03 (s, 1H,
CH2＝), 4.88 (s, 1H, CH2＝), 2.53－2.63 (m, 2H, 3-H),
2.09－2.12 (m, 1H, 4-H), 1.61－1.63 (m, 1H, 4-H);
FAB-MS m/z: 243.2 [M＋H]＋.
Synthesis of (R)-5-(4-bromophenyl)pyrrolidin-2-one
(9)
A solution of 1 μmol•L−1 HCl (40 mL) was added to
compound 8 solution (10.0 g, 29.34 mmol) in 80 mL
THF and heated to reflux for 3 h. THF was then re-
moved under reduced pressure, after which, the organic
compounds were extracted with ethyl acetate. The or-
ganic layer was separated, washed with saturated salt
solution, and dried with anhydrous sodium sulfate. The
solvent was removed under reduced pressure. Com-
pound 9 (5.36 g) was obtained as white flakes with 76%
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-1-(2-
(tert-butyldimethylsilyloxy)ethyl)-2-oxopyrrolidin-3-
yl)acetaldehyde (12)
As an indicator, 2 mg of Sudan III was added to
compound 11 solution (3.50 g, 8 mmol) in 200 mL of
anhydrous CH2Cl2. Ozone was then added into the mix-
ture at −78 ℃ until the red color faded. After that, N2
1
yield. H NMR (400 MHz, CDCl3) δ: 7.50 (d, J＝8.30
Hz, 2H, ArH), 7.18 (d, J＝8.30 Hz, 2H, ArH), 6.00 (br,
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Chin. J. Chem. 2013, 31, 1144—1152

Design and Synthesis of Tri-substituted Chiral Pyrrolidin-2-one Derivatives as CCR4 Antagonists
was added into the reactant to deplete excess ozone.
Triphenylphosphine (3.52, 8.08 mmol) was added at
−78 ℃ and the mixture was stirred at this temperature
for 0.5 h. The temperature was then allowed to rise to
room temperature and the mixture was stirred overnight,
after which, the solvent was removed under reduced
pressure. The residue was purified through column
chromatography on silica gel to give 2.67 g of com-
1H, ArH), 6.92－6.96 (m, 2H, ArH), 4.83 (dd, J＝9.24,
3.32 Hz, 1H, 5-H), 3.65－3.67 (m, 2H, OCH2CH2), 3.15
－3.25 (m, 2H, OCH2CH2), 3.08－3.09 (m, 1H, CH2-
CONH), 2.45－2.49 (m, 1H, 3-H), 2.25－2.28 (m, 1H,
4-H), 2.19 (s, 3H, Ar-CH3), 2.01－2.03 (m, 1H, CH2-
CONH), 1.90 －1.92 (m, 1H, 4-H), 0.86 [s, 9H,
C(CH3)3], 0.03 [s, 6H, Si(CH3)2]; FAB-MS m/z: 581.0
[M＋H]＋.
1
pound 12 as yellow oil with 76% yield. H NMR (400
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-1-(2-
(tert-butyldimethylsilyloxy)ethyl)-2-oxopyrrolidin-3-
yl)-N-(4-(trifluoromethoxy)phenyl)acetamide 14b
MHz, CDCl3) δ: 9.83 (s, 1H, CHO), 7.49 (d, J＝8.10 Hz,
2H, ArH), 7.03 (d, J＝8.10 Hz, 2H, ArH), 4.88 (dd, J＝
8.57, 2.28 Hz, 1H, 5-H), 3.77－3.79 (m, 2H, OCH2CH2),
3.64－3.67 (m, 1H, 4-H), 3.09－3.13 (m, 2H, OCH2-
CH2), 2.77－2.78 (m, 1H, 4-H), 2.58－2.63 (m, 1H,
3-H), 2.20－2.22 (m, 1H, CH2CHO), 2.12－2.14 (m,
1H, CH2CHO), 0.87 [s, 9H, C(CH3)3], 0.04 [s, 6H,
Si(CH3)2]; FAB-MS m/z: 441.5 [M＋H]＋.
This was prepared from 13 (1.5 g, 3.3 mmol),
dicyclohexyl carbodiimide (750 mg, 3.6 mmol) and
4-(trifluoromethoxy)aniline (643 mg, 3.6 mmol) with
1
31% yield (629 mg) as a white solid. H NMR (400
MHz, CDCl3) δ: 9.11 (s, 1H, CONH), 7.75 (d, J＝8.77
Hz, 2H, ArH), 7.67 (d, J＝8.30 Hz, 2H, ArH), 7.28 (d,
J＝8.30 Hz, 2H, ArH), 7.20 (d, J＝8.77 Hz, 2H, ArH),
4.85 (dd, J＝9.21, 2.88 Hz, 1H, 5-H), 3.64－3.67 (m,
2H, OCH2CH2), 3.17－3.25 (m, 2H, OCH2CH2), 3.08－
3.13 (m, 1H, 3-H), 2.48－2.50 (m, 1H, CH2CON), 2.26
－2.29 (m, 1H, 4-H), 2.02－2.04 (m, 1H, CH2CON),
1.93－1.95 (m, 1H, 4-H), 0.89 [s, 9H, C(CH3)3], 0.04 [s,
6H, Si(CH3)2]; FAB-MS m/z: 616.6 [M＋H]＋.
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-1-(2-(tert-
butyldimethylsilyloxy)ethyl)-2-oxopyrrolidin-3-yl)-
acetic acid (13)
A 5% NaH2PO3 solution (30 mL) and 1 μmol•L−1
KMnO4 solution (15 mL) were added into compound 12
solution (2.67 g, 5.9 mmol) in 50 mL of t-butyl alcohol.
The mixture was then stirred for 5 h at room tempera-
ture. Then, calculated Na2SO3 was added to react with
the excessive KMnO4, after which, MnO2 was filtered
out and 1 μmol•L−1 HCl was added to the filtrate to ad-
just its pH to 4－5. The organic compounds were ex-
tracted with ethyl acetate. The organic layer was then
separated, washed with saturated salt solution, and dried
with anhydrous sodium sulfate. The solvent was re-
moved under reduced pressure. The residue was purified
through column chromatography on silica gel to pro-
duce 2.94 g of compound 13 as yellow oil with 86%
Synthesis of N-benzyl-2-((3R,5R)-5-(4-bromophenyl)-
1-(2-(tert-butyldimethylsilyloxy)ethyl)-2-oxopyrro-
lidin-3-yl) acetamide (14e)
EDCI•HCl (202 mg, 1.051 mmol), HoBt (142 mg,
1.051 mmol), and phenylmethanamine (103 mg, 0.964
mmol) were added to compound 13 solution (400 mg,
0.876 mmol) in 10 mL of anhydrous CH2Cl2 and the
mixture was stirred overnight at room temperature. The
solvent was then removed under reduced pressure. The
residue was purified through column chromatography
on silica gel to produce 172 mg of compound 14e as a
white solid with 36% yield. 1H NMR (400 MHz, CDCl3)
δ: 8.98 (s, 1H, CONH), 7.67 (d, J＝8.40 Hz, 2H, ArH),
7.36 (d, J＝8.40 Hz, 2H, ArH), 7.22－7.24 (m, 3H,
ArH), 7.10－7.11 (m, 2H, ArH), 4.81 (dd, J＝9.20, 2.96
Hz, 1H, 5-H), 4.13 (d, J＝4.67, 2H, Ar-CH2), 3.66 (t,
J＝5.80 Hz, 2H, OCH2CH2), 3.15－3.26 (m, 2H,
OCH2CH2), 3.10－3.15 (m, 1H, 3-H), 2.42－2.43 (m,
1H, CH2CON), 2.20－2.24 (m, 1H, 4-H), 2.02－2.04
(m, 1H, CH2CON), 1.93－1.95 (m, 1H, 4-H), 0.88 [s,
9H, C(CH3)3], 0.04 [s, 6H, Si(CH3)2]; FAB-MS m/z:
546.6 [M＋H]＋.
1
yield. H NMR (400 MHz, CDCl3) δ: 7.48－7.51 (m,
2H, ArH), 7.00－7.02 (m, 2H, ArH), 4.89－4.91 (m, 1H,
5-H), 3.78－3.80 (m, 2H, OCH2CH2), 3.65－3.68 (m,
1H, 4-H), 3.05－3.06 (m, 1H, 4-H), 2.85－2.87 (m, 2H,
OCH2CH2), 2.51－2.54 (m, 1H, 3-H), 2.21－2.22 (m,
2H, CH2COOH), 0.87 [s, 9H, C(CH3)3], 0.03 [s, 6H,
Si(CH3)2]; FAB-MS m/z: 457.4 [M＋H]＋.
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-1-(2-(tert-
butyldimethylsilyloxy)ethyl)-2-oxo-pyrrolidin-3-yl)-
N-(3-chloro-2-methylphenyl)acetamide (14a)
Dicyclohexyl carbodiimide (250 mg, 1.2 mmol) was
added to compound 13 solution (500 mg, 1.1 mmol) in
10 mL of anhydrous CH2Cl2 and the mixture was stirred
for 15 min at 0 ℃. 3-Chloro-2-methylaniline (171 mg,
1.2 mmol) was then added to the mixture at 0 ℃ and
stirred for 30 min. Then the mixture was allowed to set
to room temperature and was stirred overnight, after
which, the solvent was removed under reduced pressure.
The residue was purified through column chromatog-
raphy on silica gel to give 179 mg of compound 14a as
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-1-(2-
(tert-butyldimethylsilyloxy)ethyl)-2-oxopyrrolidin-3-
yl)acetate (14f)
EDCI•HCl (202 mg, 1.051 mmol), catalyst DMAP
and (2-methoxyphenyl)methanol (133 mg, 0.964 mmol)
were added to compound 13 solution (400 mg, 0.876
mmol) in 10 mL of anhydrous CH2Cl2 and the mixture
was stirred at room temperature overnight. The solvent
was then removed under reduced pressure. The residue
was purified through column chromatography on silica
1
a white solid with 28% yield. H NMR (400 MHz,
CDCl3) δ: 8.96 (s, 1H, CONH), 7.66 (d, J＝8.33 Hz, 2H,
ArH), 7.27 (d, J＝8.33 Hz, 2H, ArH), 7.05－7.06 (m,
Chin. J. Chem. 2013, 31, 1144—1152
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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1147

Sun et al.
FULL PAPER
gel to produce 146 mg of compound 14f as a white solid
with 29% yield. 1H NMR (400 MHz, CDCl3) δ: 8.53 (s,
1H, CONH), 7.66 (d, J＝8.32 Hz, 2H, ArH), 7.28 (d,
J＝8.33 Hz, 2H, ArH), 7.16－7.26 (m, 5H, ArH), 4.83
(dd, J＝9.27, 3.12 Hz, 1H, 5-H), 4.13 (d, J＝4.67 Hz,
2H, Ar-CH2), 3.67－3.67 (m, 3H, Ar-OCH3), 3.65 (t,
J＝5.76 Hz, 2H, OCH2CH2), 3.15－3.25 (m, 2H,
OCH2CH2), 3.12－3.15 (m, 1H, 3-H), 2.35－2.36 (m,
1H, CH2CON), 2.20－2.23 (m, 1H, 4-H), 2.10－2.11
(m, 1H, CH2CON), 2.09－2.10 (m, 1H, 4-H), 0.78 [s,
9H, C(CH3)3], 0.29 [s, 6H, Si(CH3)2]; FAB-MS m/z:
577.6 [M＋H]＋.
1H, COOH), 8.03 (s, 1H, CONH), 7.62 (d, J＝8.29 Hz,
2H, ArH), 7.36 (d, J＝8.29 Hz, 2H, ArH), 7.16－7.26
(m, 5H, ArH), 4.88 (dd, J＝9.04, 3.13 Hz, 1H, 5-H),
4.13 (d, J＝9.22 Hz, 2H, Ar-CH2), 3.83－3.93 (d, J＝
14.33 Hz, 2H, CH2COOH), 3.12－3.14 (m, 1H, 3-H),
2.42－2.44 (m, 1H, CH2CONH), 2.23－2.24 (m, 1H,
CH2CONH), 1.80－1.82 (m, 1H, 4-H), 1.70－1.72 (m,
1H, 4-H); FAB-MS m/z: 446.3 [M＋H]＋.
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-3-(2-(2-
methoxybenzyloxy)-2-oxoethyl)-2-oxopyrrolidin-1-
yl)acetic acid (15f)
This was prepared from 14f (146 mg, 0.253 mmol)
and Jones reagent (0.2 mL) with 88% yield (106 mg) as
a white solid. 1H NMR (400 MHz, CDCl3) δ: 10.22 (br,
1H, COOH), 7.60 (d, J＝8.30 Hz, 2H, ArH), 7.43 (d,
J＝8.30 Hz, 2H, ArH), 6.72－7.22 (m, 4H, ArH),
5.12－5.16 (m, 2H, ArCH2), 4.81 (dd, J＝9.04, 3.13 Hz,
1H, 5-H), 3.80－3.90 (d, J＝14.33 Hz, 2H, CH2COOH),
3.68 (s, 3H, ArOCH3), 3.02－3.03 (m, 1H, 3-H), 2.25－
2.27 (m, 1H, CH2CONH), 2.05－2.06 (m, 1H, CH2CO-
NH), 2.00－2.01 (m, 1H, 4-H), 1.91－1.92 (m, 1H,
4-H); FAB-MS m/z: 477.3 [M＋H]＋.
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-3-(2-(3-
chloro-2-methylphenylamino)-2-oxoethyl)-2-oxopyr-
rolidin-1-yl)acetic acid (15a)
Jones reagent (0.2 mL) was added to compound 14a
solution (179 mg, 0.308 mmol) in 5 mL of acetone at 0
℃ and was mixed for 30 min. Then, the mixture was
allowed to set to room temperature and stirred for 3 h.
Trash ice was then added to the mixture. The organic
compounds were extracted with ethyl acetate. The or-
ganic layer was then separated, washed with saturated
salt solution, and dried with anhydrous sodium sulfate.
The solvent was then removed under reduced pressure.
The residue was purified through column chromatogra-
phy on silica gel to produce 117 mg of compound 15a
Synthesis of (3R,5R)-5-(4-bromophenyl)-1-(2-(2-
(morpholino-1-yl)ethyl)amino-2-oxo)ethyl-3-(2-(N-
(3-chloro-2-methylphenyl))amino-2-oxo)ethyl pyrro-
lidin-2-one (1a)
1
as a white solid with 79% yield. H NMR (400 MHz,
N,N′-Carbonyl diimidazo (48 mg, 0.3 mmol) was
added to compound 15a solution (117 mg, 0.279 mmol)
in 10 mL of anhydrous CH2Cl2 and the mixture was
stirred for 2 h at room temperature. Then, 2-morpholino
ethanamine (40 mg, 0.307 mmol) was added and the
mixture was continuously stirred overnight at room
temperature. Then, the resulting mixture was extracted
with saturated salt solution and the organic layer was
dried with anhydrous sodium sulfate. The solvent was
removed under reduced pressure. The residue was puri-
fied through column chromatography on silica gel to
produce 30 mg of compound 1a as a white solid with
CDCl3) δ: 9.93 (br, 1H, COOH), 7.82 (d, J＝8.10 Hz,
2H, ArH), 7.36 (d, J＝8.10 Hz, 2H, ArH), 7.23 (s, 1H,
CONH), 7.05－7.06 (m, 1H, ArH), 6.92－6.96 (m, 2H,
ArH), 4.88 (dd, J＝9.28, 3.08 Hz, 1H, 5-H), 3.83－3.93
(d, J＝14.88 Hz, CH2COOH), 3.11－3.15 (m, 1H, 3-H),
2.48－2.49 (m, 1H, CH2CONH), 2.28－2.30 (m, 1H,
CH2CONH), 2.20 (s, 3H, Ar-CH3), 2.12－2.14 (m, 1H,
4-H), 2.02－2.03 (m, 1H, 4-H); FAB-MS m/z: 480.8 [M
＋H]＋.
Synthesis of 2-((3R,5R)-5-(4-bromophenyl)-2-oxo-
3-(2-oxo-2-(4-(trifluoromethoxy)phenylamino)ethyl)-
pyrrolidin-1-yl)acetic acid 15b
1
18% yield. H NMR (400 MHz, CDCl3) δ: 7.91 (s, 1H,
This was prepared from 14b (629 mg, 1.022 mmol)
CONH), 7.49－7.51 (m, 3H, ArH), 7.22－7.26 (m, 2H,
ArH), 7.02－7.11 (m, 3H, ArH, CONH), 4.78 (dd, J＝
9.25, 3.08 Hz, 1H, 5-H), 4.46 (d, J＝16.80 Hz, 1H,
CH2-pyrrolidin-1), 3.63－3.66 (m, 4H, morpholine-3H,
5H), 3.40－3.32 (m, 1H, 4-H), 3.04－3.19 (m, 4H,
CH2-pyrrolidin-1,4-H, CH2CH2NHCO), 2.82－2.85 (m,
1H, 3-H), 2.31－2.54 (m, 11H, Ar-CH3, morpholine-2H,
6H, CH2CH2NHCO, pyrrolidin-3-CH2CONH); FAB-
MS m/z: 593.0 [M＋H]＋. Anal. calcd for C27H32BrCl-
N4O4 (591.92): C 50.79, H 5.45, N 9.47; found C 50.59,
H 5.42, N 9.39.
and Jones reagent (0.7 mL) with 85% yield (447 mg) as
1
a white solid. H NMR (400 MHz, CDCl3) δ: 11.0 (br,
1H, COOH), 7.63 (d, J＝8.75 Hz, 2H, ArH), 7.61 (d,
J＝8.10 Hz, 2H, ArH), 7.35 (d, J＝8.10 Hz, 2H, ArH),
7.18－7.20 (m, 3H, ArH, CONH), 4.86 (dd, J＝9.24,
3.33 Hz, 1H, 5-H), 3.83－3.93 (d, J＝14.26 Hz, 2H,
CH2COOH), 3.10－3.13 (m, 1H, 4-H), 2.47－2.49 (m,
1H, CH2CONH), 2.27－2.28 (m, 1H, CH2CONH),
2.11－2.12 (m, 1H, 4-H), 2.00－2.03 (m, 1H, 4-H);
FAB-MS m/z: 516.3 [M＋H]＋.
Synthesis of 2-((3R,5R)-3-(2-(benzylamino)-2-oxo-
ethyl)-5-(4-bromophenyl)-2-oxopyrrolidin-1-yl)acetic
acid (15e)
Synthesis of (3R,5R)-5-(4-bromophenyl)-1-(2-oxo-2-
(4-(piperidin-1-yl)butylamino))ethyl-3-(2-(N-(4-(tri-
fluoromethoxy)phenylamino)-2-oxo)ethyl pyrro-
lidin-2-one (1b)
This was prepared from 14e (172 mg, 0.316 mmol)
and Jones reagent (0.2 mL) with 80% yield (112 mg) as
a white solid. H NMR (400 MHz, CDCl3) δ: 9.93 (br,
This was prepared from 15b (149 mg, 0.289 mmol),
N,N′-carbonyldiimidazo (50 mg, 0.313 mmol), and
1
1148
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1144—1152

Design and Synthesis of Tri-substituted Chiral Pyrrolidin-2-one Derivatives as CCR4 Antagonists
4-(piperidin-1-yl)butan-1-amine (50 mg, 0.318 mmol)
with 12% yield (23 mg) as a slight yellow solid. H
EDCI•HCl (58 mg, 0.302 mmol), catalyst DMAP, and
1
(2-methoxyphenyl)methanol (38 mg, 0.277 mmol) with
16% yield (23 mg) as a white solid. 1H NMR (400 MHz,
CDCl3) δ: 7.47 (d, J＝8.4 Hz, 2H, ArH), 7.22－7.30 (m,
7H, ArH), 7.02 (d, J＝8.4 Hz, 2H, ArH), 6.87－6.88 (m,
2H, ArH), 6.71 (br, 1H, CONH), 5.14－5.18 (m, 2H,
ArCH2O), 4.72 (dd, J＝8.40, 4.00 Hz, 1H, 5-H), 4.51 (d,
1H, J＝17.60 Hz, CH2-pyrrolidin-1), 4.43－4.49 (m,
2H, CONHCH2Ar), 3.81 (s, 3H, CONHCH2Ar), 3.31 (d,
J＝17.60 Hz, 1H, CH2-pyrrolidin-1), 3.10－3.12 (m,
1H, 4-H), 2.70－2.76 (m, 1H, 3-H), 2.20－2.37 (m, 3H,
4-H, pyrrolidin-3-CH2CONH); FAB-MS m/z: 567.0 [M
＋H]＋. Anal. calcd for C29H29BrN2O5 (632.47): C 61.60,
H 5.17, N 4.95; found C 61.05, H 5.11, N 5.00.
NMR (400 MHz, CDCl3) δ: 9.17 (s, 1H, CONH),
7.76－7.77 (m, 1H, CONH), 7.49－7.53 (m, 4H, ArH),
7.04－7.05 (m, 4H, ArH), 4.75 (dd, J＝9.24, 3.64 Hz,
1H, 5-H), 4.45 (d, J＝16.53 Hz, 1H, CH2-pyrrolidin-1),
2.98 － 3.18 (m, 5H, CH2-pyrrolidin-1, piperidine
CH2CH2CH2CH2, 4-H), 2.83 －2.85 (m, 1H, 3-H),
2.26－2.47 (m, 8H, piperidine-2H,6H, piperidine-CH2,
pyrrolidin-3-CH2CONH), 1.25－1.53 (m, 10H, piperi-
dine-3H,4H,5H, piperidine CH2CH2CH2CH2); FAB-MS
m/z: 653.0 [M＋H]＋. Anal. calcd for C30H36BrF3N4O4
(653.53): C 55.13, H 5.55, N 8.57; found C 55.13, H
5.58, N 8.50.
Synthesis of (3R,5R)-5-(4-bromophenyl)-1-(2-oxo-2-
(4-(pyrrolidin-1-yl)butylamino))ethyl-3-(N-(4-(tri-
fluoromethoxy)phenylamino)-2-oxo)ethylpyrrolidin-
2-one (1c)
Synthesis of (3R,5R)-5-(4-bromophenyl)-1-(2-N-(2-
morpholinoethylamino)-2-oxo)ethyl-3-(2-(methoxy)-
benzyloxo-2-oxo)ethylpyrrolidin-2-one (1f)
This was prepared from 15f (146 mg, 0.307 mmol),
N,N′-carbonyldiimidazo (53 mg, 0.332 mmol), and
2-morpholino-ethanamine (44 mg, 0.338 mmol) with
This was prepared from 15b (149 mg, 0.289 mmol),
N,N′-carbonyldiimidazo (50 mg, 0.313 mmol) and
4-(pyrrolidin-1-yl)butan-1-amine (45 mg, 0.318 mmol)
1
15% yield (27 mg) as a slight yellow solid. H NMR
1
with 19% yield (35 mg) as a slight yellow solid. H
(400 MHz, CDCl3) δ: 7.48－7.51 (m, 2H, ArH), 7.26－
7.28 (m, 2H, ArH), 7.10 (br, 1H, CONH), 7.01－7.03
(m, 2H, Ar-H), 6.90－6.93 (m, 2H, Ar-H), 5.13－5.17
(m, 2H, COOCH2), 4.76 (dd, J＝9.24, 2.52 Hz, 1H,
5-H), 4.47 (d, J＝16.53 Hz, 1H, CH2-pyrrolidin-1), 3.84
(s, 3H, ArOCH3), 3.70 (s, 4H, morpholine-3H,5H),
3.33－3.52 (m, 2H, morpholine CH2CH2), 3.17 (d, J＝
16.53 Hz, 1H, CH2-pyrrolidin-1), 2.98－3.07 (m, 2H,
pyrrolidin-3-CH2CONH), 2.80－2.83 (m, 1H, 4-H),
2.45 －4.48 (m, 7H, 3-H, morpholine-2H,6H, mor-
pholine-CH2CH2), 2.15－4.18 (m, 1H, 4-H); FAB-MS
m/z: 588.2 [M＋H]＋. Anal. calcd for C28H34BrN3O6
(588.49): C 57.15, H 5.82, N 7.14; found C 56.59, H
5.88, N 7.11.
NMR (400 MHz, CDCl3) δ: 9.03 (s, 1H, CONH),
7.86－7.88 (m, 1H, CONH), 7.49－7.54 (m, 4H, ArH),
7.03－7.15 (m, 4H, ArH), 4.77 (dd, J＝9.20, 3.64 Hz,
1H, 5-H), 4.42 (d, J＝16.42 Hz, 1H, CH2-pyrrolidin-1),
2.84－3.28 (m, 6H, 3-H, CH2-pyrrolidin-1, tetrahydro-
pyrrole-CH2CH2CH2CH2,
pyrrolidin-3-CH2CONH),
2.43－2.49 (m, 7H, tetrahydropyrrole-2H,5H, tetrahy-
dropyrrole-CH2, 4-H), 2.17－2.19 (m, 1H, 4-H), 1.56－
1.73 (m, 8H, tetrahydropyrrole-3H,4H, tetrahydropyr-
role-CH2CH2CH2CH2); FAB-MS m/z: 639.1 [M＋H]＋.
Anal. calcd for C29H34BrF3N4O4 (639.50): C 54.47, H
5.36, N 8.76; found C 54.15, H 5.18, N 8.78.
Synthesis of (3R,5R)-5-(4-bromophenyl)-1-(2-oxo-2-
(3-phenylpropyl)amino)ethyl)-3-(N-(4-(trifluorometh-
oxy)phenylamino)-2-oxo)ethyl pyrrolidin-2-one (1d)
Biological Methods
Materials and reagents
This was prepared from 15b (149 mg, 0.289 mmol),
N,N′-carbonyldiimidazo (50 mg, 0.313 mmol) and
3-phenylpropan-1-amine (43 mg, 0.318 mmol) with
14% yield (26 mg) as a white solid. 1H NMR (400 MHz,
CDCl3) δ: 8.60 (s, 1H, CONH), 7.48－7.50 (m, 2H,
ArH), 7.43 (m, 2H, ArH), 7.11－7.31 (br, 6H, CONH,
ArH), 7.00－7.02 (m, 4H, ArH), 4.71 (dd, J＝9.24, 2.80
Hz, 1H, 5-H), 4.47 (d, J ＝16.81 Hz, 1H, CH2-
pyrrolidin-1), 3.36－3.38 (m, 1H, 3-H), 3.14－3.18 (m,
3H, CH2-pyrrolidin-1, ArCH2CH2CH2), 3.10－3.11 (m,
1H, 4-H), 2.59－2.75 (m, 4H, pyrrolidin-3-CH2CONH,
ArCH2CH2CH2), 2.14－2.15 (m, 1H, 4-H), 1.86－1.88
(m, 2H, ArCH2CH2CH2); FAB-MS m/z: 634.1 [M＋H]＋.
Anal. calcd for C30H29BrF3N3O4 (632.47): C 56.97, H
4.62, N 6.64; found C 57.00, H 4.66, N 6.60.
CZE experiments were performed using a Beckman
P/ACETM MDQ system (Beckman Coulter, Fullerton,
CA, USA) equipped with a photodiode array detector
and 32 Karat™ software version 5.0 (Beckman). Detec-
tion wavelength was 214 nm. The equivalent peptide
derived from ML40 was synthesized by the Chinese
Peptide Company in Hangzhou, China and was subse-
quently purified (purity ≥95%) and characterized us-
ing RP HPLC and MS. The synthesized amino acid se-
quence of the human CCR4 amino terminus was
MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIK-
AFGEL.
RPMI 1640 medium and fetal bovine serum (FBS)
were purchased from Life Technologies, Inc. The
48-well chemotaxis chambers and membranes were
purchased from Neuroprobe (Gaithersburg, MD).
Synthesis of (3R,5R)-5-(4-bromophenyl)-1-(2-(meth-
oxy)benzyloxo-2-oxo)ethyl-3-(N-(2-(benzylamino)-
2-oxo)ethyl) pyrrolidin-2-one (1e)
CZE experiments
This was prepared from 15e (112 mg, 0.251 mmol),
To study the interaction of ML40 and the synthetic
Chin. J. Chem. 2013, 31, 1144—1152
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1149

Sun et al.
FULL PAPER
compounds, the cartridge and sample room were kept at
25 and 4 ℃, respectively. Before each measurement,
the capillary was rinsed with running buffer (30 mmol/L
Tris-HAc, pH＝7.3). Samples containing the mixtures
of ML40 and the synthetic compounds were injected
using pressure injection mode at 0.5 psi for 10 s (1 psi＝
6894.76 Pa). The applied voltages on the ML40 and the
synthetic compounds were at −10 and 30 kV, respec-
tively. Between runs, the capillary was washed with the
running buffer for 5 min at 20 psi. Each concentration
was run in duplicate. The binding constant of the inter-
action between the compounds and ML40 was calcu-
lated from a Scatchard plot by regression.
tagonist binds only with pocket A through hydrogen
bonding. The 2,4-dichlorophenyl group in this com-
pound combined with the receptor (TYR159) by hy-
drophobic interaction. The electrostatic potential inter-
action between the compound and the receptor was not
obvious. The aim of this study was to search for com-
pounds with more flexibility to combine with the re-
ceptor. Therefore, an ester linkage was introduced, as
well as a bioisostere of amidolinkage to some com-
pounds and the side chain of R2 was lengthened.
The final compounds 1a－1f have two chiral car-
bons. A general asymmetric synthetic procedure is il-
lustrated in Scheme 1. The commercially available
bromobenzene was coupled with 3-methoxycarbonyl-
propionyl chloride 2 through Friedel-Crafts acylation
reaction to afford the intermediate 3. 3 was hydrolyzed
to obtain 4, which was then converted to 5 through de-
hydration with (S)-(＋)-2-phenylglycinol. The cis-con-
figuration of the phenyl and 4-bromophenyl, which was
substituted at tetrahydropyrrolo[2,1-b]-oxazol-5(6H)-
Chemotaxis assay
The chemotaxis assay was performed using a
48-well microchemotaxis chamber. Chemotatic factor
MDC was then diluted in HEPES-buffered RPMI 1640
medium supplemented with 0.1% BSA to 10 ng/mL.
The solution was added to the lower wells (27.5
μL/well), which were separated from the upper wells by
a 10 μm pore size filter membrane. The HEK293 cells
were then transfected with pcDI-CCR4, incubated for
36 h, and digested. The cells were then resuspended in
HEPES-buffered RPMI 1640 medium supplemented
with 10% FBS and incubated with rotation for 6.5 h at
37 ℃. The cells were then washed with HEPES-buff-
ered RPMI 1640 twice and resuspended in HEPES-
buffered RPMI 1640 medium supplemented with 0.1%
BSA to 1×106 cells/mL. HEK293 cells transfected with
pcDI-CCR4 and the compound (1 μmol•L−1 in DMSO)
were incubated for 0.5 h at room temperature. The re-
sulting solution was then added to the upper wells (55
μL/well). The chamber was incubated for 4 h at 37 ℃
in 5% CO2 and 95% air. The filters were then removed
from the chamber, washed, fixed, and stained using the
Three-Step Stain Set. The migrated cells were counted
in five randomly selected high-power fields (400×) per
well. All samples were assayed at least twice. The ex-
periments were divided into three groups, i.e., MDC/
HEK293 cells transfected with pcDI-CCR4 group (posi-
tive control), HEK293 cells transfected with pcDI-
CCR4/HEPES-buffered RPMI 1640 medium/0.1% BSA
group (negative control), and compound/MDC/HEK293
cells transfected with pcDI-CCR4 group. The number of
migrating HEK293/CCR4 cells in the positive control
was set as 100%. The percentage inhibition of HEK293/
CCR4 cell migration was defined as the ratio of the
number of detected migrating cells subtracted from the
number of migrating cells in the negative control to that
in the positive control.
1
one, was confirmed by 2D H-1H COSY spectroscopy
and 1D NOESY spectroscopy. Therefore, we concluded
that another chiral carbon in the S configuration is lo-
cated at the 7a position of 5. After that, 5 was converted
into the optically pure intermediate (R)-5-(4-bromophe-
nyl)-pyrrolidin-2-one (9) through a ring opening reac-
tion, halogenation, elimination, and de-alkylation. Then,
9 was treated with NaH and (2-indoethoxy)-tert-butyldi-
methyl silane in DMF to yield 10. Then 10 was allylated
with allyl bromide at −78 ℃ in anhydrous THF to
yield 11. Compound 11 was subjected to two-step oxi-
dation, substituted with pyrrolidone carboxylic acid to
obtain 13. In the presence of condensation reagents, 13
was coupled with amines (R1NH2) or alcohols (R1OH)
to yield 14. Compound 14 was then deprotected and
oxidized in one step with Jones reagent to yield 15.
Through the same synthetic conditions of 14, the final
compound 1 was obtained.[12] By the same method, the
4-bromophenyl and amide side chain or ester side chain
contained R1, which was substituted at pyrrolidin-2-one,
were confirmed to have a trans-configuration. Hence,
the R,R configuration of the final product 1 was ob-
tained.
The interaction of N-terminal extracellular tail of
CCR4 and the synthetic compound was studied using
CZE experiments. Compounds 1b, 1c, and 1f showed
positive results. Their binding constants were (2.970±
0.86)×104, (1.569±0.11)×105, and (4.94±0.34)×104
L•mol−1, respectively. Compounds 1a and 1d showed
negative result. In addition, compound 1e was not solu-
ble in alcohol, the detection solvent. The apparent bind-
ing constants of CZE experiment are shown in Table 1.
The chemotaxis inhibition effects of these com-
pounds were determined in the CCR4-transfected
HEK293 cells. The percentage of migrating cells was
given in the result (Table 1). As shown in Table 1,
compounds 1a－1d, and 1f displayed suppression ac-
tivities under this condition. The percentage of migrat-
Results and Discussion
There are two pockets in N-terminal extracellular do-
main of the modeled receptor. A known CCR4 antago-
nist was docked to the receptor and the interaction be-
tween CCR4 and its antagonist was analyzed. The an-
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1144—1152

Design and Synthesis of Tri-substituted Chiral Pyrrolidin-2-one Derivatives as CCR4 Antagonists
ing cells in these compounds were 32%, 45%, 59%,
41%, and 39%, respectively. Among the five, 1c was
the most active compound, which is concordant with the
CZE experiment result.
only difference between compounds 1b and 1c is the
heterocyclic at the end of carbon chain, implying that
five-member ring is more favorable. Compounds 1b, 1c,
and 1d are more active than the others, implying that the
electrophilic group, 4-(trifluoromethoxy)phenyl in R2
probably enhances the activity.
The results illustrated that lengthening the R2 carbon
chain enhanced the activities of the compounds. The
Scheme 1 Synthetic route for tri-substituted chiral pyrrolidin-2-one derivatives
Br
Br
N
Br
Br
O
Br
O
Cl
b
c
a
O
d
e
N
+
O
O
HO
O
HO
O
O
O
O
O
3
4
2
5
6
Br
Br
Br
Br
N
Br
j
i
g
f
h
N
N
N
Cl
Si
O
Si
O
O
O
O
O
HN
O
11
10
7
8
9
Br
Br
Br
O
C X R1
m
CHO
l
COOH
k
N
N
N
Si
O
Si
O
Si
O
O
O
O
12
14a, 14b, 14e, 14f
13
Br
N
Br
O
O
C X R1
C X R1
n
O
N
R2
HOOC
Y
O
O
1a－1f
15a, 15b, 15e, 15f
H3C
Cl
, R2 =
a: R1 =
3 , R2 =
OCF
O
N
b: R1 =
, X = NH, Y = NH;
, X = NH, Y = NH;
OCF
O
N
, X = NH, Y = HN;
3 , R2 =
OCF
3 , R2 =
1
c:
, X = NH, Y = NH; d: R1 =
OCH3
, X = NH, Y = O; f: R1 =
N
R =
OCH3
, R2 =
, R2 =
e: R1 =
, X = O, Y = NH
N
1
Reagents and conditions: (a) aluminum chloride, 0 ℃, 0.5 h, then 80 ℃, 3 h; (b) 1 mol•L− LiOH (aq.), THF, H2O, r.t., 2 h; (c)
(S)-(＋)-2-phenylglycinol, toluene, refluxed overnight using a water segregator; (d) TiCl4, dry CH2Cl2, Et3SiH, −78 ℃ to r.t., 16 h; (e) THF,
SOCl2, ice bath to r.t., 2 h; (f) Na, EtOH, 45 ℃, 4 h; (g) 1 mol•L−1 HCl (aq.), THF, reflux, 3 h; (h) NaH, DMF, (2-indoethoxy)-tert-butyldimethyl
silane, r.t., overnight; (i) LDA, dry THF, −78 ℃, 15 min, then allyl bromide −78 ℃to r.t., overnight; (j) O3, CH2Cl2, Sudan III −78 ℃, until the red
color fades, then PhP3, −78 ℃ to r.t., overnight; (k) KMnO4, 5% NaH2PO4 (aq.), t-BuOH, r.t., 5 h; (l) X＝NH, dry CH2Cl2, DCC, R1NH2, r.t.,
overnight; X＝O, dry CH2Cl2, EDCI•HCl, DMAP, R1OH, r.t., overnight; (m) Jones reagent, acetone, ice-bath to r.t., 3 h; (n) Y＝NH, dry CH2Cl2,
EDCI•HCl, HoBt, R2NH2, r.t., overnight; Y＝O, dry CH2Cl2, EDCI•HCl, DMAP, R2OH, r.t., overnight.
Chin. J. Chem. 2013, 31, 1144—1152
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1151

Sun et al.
FULL PAPER
Table 1 CZE experiment result and chemotaxis assay result of compounds 1a－1f
CZE experiment result (apparent binding constant)
Percentage inhibition of the HEK293/CCR4 cells
migrationa/%
Compd.
K/(L•mol−1)
(1.06±0.11)×105
－
Positive compd.b
63
32
45
59
41
－
39
1a
1b
1c
1d
1e
(2.970±0.86)×104
(1.569±0.11)×105
－
N.D.
(4.94±0.34)×104
1f
a
b
The concentration of test compounds is 1 μmol•L−1 in DMSO;
Positive compound is N-(3-chloro-2-methyl benzyl)-2-
((3R,5R)-5-(2,4-dichlorophenyl)-2-oxo-1-(2-oxo-2-(3-(piperidin-1-yl)propylamino)ethyl)pyrrolidin-3-yl)acetamide.
Conclusions
Technology of Jilin Province (Grant no. 201201020).
In summary, several tri-substituted chiral pyrrolidin-
2-one derivatives have been designed and synthesized
as CCR4 antagonists. Several of these compounds show
high inhibitory effect in cell assays and high affinity for
the N-terminal of CCR4. Among the compounds, 1c
exhibited excellent activity. Results of this study are of
great significance for the design of new compounds
with reasonable activity and drug similarity. Further
studies on the mechanism of these compounds are in
progress.
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We are grateful to the PKU Human Disease Genom-
ics Center for their unconditional support in the work
during the biology assays. This work was financially
supported by the National Natural Science Foundation
of China (Nos. 30472093, 30872292 and 90813025), the
National Major Science & Technology Specific Project
(No. 2009ZX09103-024), and Young Scientific Re-
search Fund Project for Department of Science and
(Zhao, C.)
1152
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1144—1152