Difluorophenylglycinols as New Modulators of Proteolytic Processing of Amyloid Precursor Proteins

Article abstract of DOI:10.1002/ardp.201300283

Synthesis and evaluation of difluorophenylglycinols as new modulators of proteolytic processing of the amyloid-β precursor proteins for Alzheimer's therapies were described. A range of N-substituted (R)- and (S)- difluorophenylglycinols, structured on the amino alcohol framework, were explored by incorporating the arylsulfonyl moieties and various N-substituents. Evans' chiral auxiliary strategy was employed for the asymmetric synthesis of these enantiomeric difluorophenylglycinols. Compounds with effects on the γ-secretase inhibition and ERK-mediated signaling pathways were evaluated on cell-based assays. Among them, N-cyclopropylmethyl derivatives R-12c and R-13c showed modest γ-secretase inhibition as well as ERK-dependent activation. A range of N-substituted (R)- and (S)-difluorophenylglycinols, structured on the amino alcohol framework, were explored by incorporating the arylsulfonyl moieties and various N-substituents. Compounds with effects on γ-secretase inhibition and ERK-mediated signaling pathways were evaluated on cell-based assays. The N-cyclopropylmethyl derivatives R-12c and R-13c showed modest γ-secretase inhibition and ERK-dependent activation.

Full text of DOI:10.1002/ardp.201300283

Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
161
Full Paper
Difluorophenylglycinols as New Modulators of Proteolytic
Processing of Amyloid Precursor Proteins
Chia-Yu Chen1, Yung-Feng Liao2*, Ming-Yun Chang2, and Ming-Kuan Hu1*
1
School of Pharmacy, National Defense Medical Center, Taipei, Taiwan
Laboratory of Molecular Neurobiology, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei,
2
Taiwan
Synthesis and evaluation of difluorophenylglycinols as new modulators of proteolytic processing of the
amyloid-b precursor proteins for Alzheimer’s therapies were described. A range of N-substituted (R)- and
(S)-difluorophenylglycinols, structured on the amino alcohol framework, were explored by
incorporating the arylsulfonyl moieties and various N-substituents. Evans’ chiral auxiliary strategy
was employed for the asymmetric synthesis of these enantiomeric difluorophenylglycinols.
Compounds with effects on the g-secretase inhibition and ERK-mediated signaling pathways were
evaluated on cell-based assays. Among them, N-cyclopropylmethyl derivatives R-12c and R-13c showed
modest g-secretase inhibition as well as ERK-dependent activation.
Keywords: Alzheimer’s disease / Asymmetric synthesis / ERK-mediated signaling / g-Secretase
Received: July 28, 2013; Revised: September 7, 2013; Accepted: September 17, 2013
DOI 10.1002/ardp.201300283
Introduction
neuronal protection [4]. One of the testimonies for the
beneficial mechanism was proven by certain N-propargyl-
containing compounds such as rasagiline and selegiline,
which improved the a-secretase-mediated cleavage of APP
through activating mitogen-activated protein kinases
(MAPKs)-dependent pathways under in vivo conditions [5, 6].
This process alternatively not only attenuates the b- and g-
secretase-mediated cleavage but also produces neuroprotec-
tive sAPPa. As far as how much Ab lowering was required to
restore cognitive deficits was still in debate, a recent study in
the Tg2576 mouse model disclosed that partial inhibition of
g-secretase activity would lower Ab adequately to reverse
cognitive impairment while avoiding Notch-related side
effects by allowing certain proteolytic processing of Notch [7].
This partial inhibition would be accessed by the g-secretase
inhibitors with Notch-sparing selectivity as well as decreasing
the b-/g-secretase-mediated proteolytic processing of APP
through augmenting ERK-mediated a-secretase activity [8, 9].
From the mechanism-of-action point of view, we planned to
develop new dual-acting modulators of proteolytic processing
of APP as potential agents for Alzheimer’s therapy.
The amyloid-b (Ab) peptide and its aggregated plaques in the
brain are considered the major hallmarks in the pathogenesis
of Alzheimer’s disease (AD). The Ab peptides (mainly Ab40 and
Ab42) originate from amyloid-b precursor protein (APP) by the
proteolytic cleavage steps involving enzymes b-secretase and
g-secretase [1, 2], whereas g-secretase drives the final step to
generate the more fibrillogenic Ab42, and it has thus been
regarded as a prime therapeutic target for AD therapy [3].
In addition to the known substrate APP, g-secretase also
processes Notch and the resulting Notch intracellular domain
(NICD) plays a vital role in the developmental signaling that
controls a cell’s fate during embryonic stage. Therefore, the
identity of g-secretase inhibitors with selectivity for APP over
Notch to minimize the impact on Notch signaling was thus
demanded in the drug discovery strategy. Contrary to the
sequential cleavage of APP by b- and g-secretases in the
generation of the fibrillogenic Ab, a-secretase is thought to
mediate a beneficial processing of APP and produces a
neuroprotective, soluble amyloid precursor protein a (sAPPa),
which precludes the Ab production and contributes to the
Aiming at developing dual-acting small molecules toward
AD therapies, we previously reported a type of N-propargyl
tetrahydroisoqunoline analogs that exhibited both g-secretase
Correspondence: Prof. Ming-Kuan Hu, School of Pharmacy, National
Defense Medical Center, Taipei 11490, Taiwan.
E-mail: hmk@ndmctsgh.edu.tw
Fax: þ886 2 87923169
ꢀThese two authors contributed equally to this work.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
Figure 1. Potent g-secretase inhibitors and the
designed structures.
inhibition and ERK-dependent sAPPa release [10]. These
molecules would act synergistically as they provide both
decreased Ab generation and neuronal protection. Mount of
investigations demonstrated that certain arylsulfonamide-
based small molecules are potent and selective g-secretase
inhibitors, such as GSI-953 (1a, begacestat, Fig. 1) and BMS-
708163 (1b, avagacestat) [11, 12]. Structurally, begacestat is a
simple amino alcohol and avagacestat is an amide derivative.
or 2b is chosen to be an alternative of the 5-chlorothiophene-
sulfonyl moiety of the synthesized amino alcohols to provide
the analogous compounds series-2. The synthesized difluoro-
phenylglycinols would directly exhibit g-secretase inhibition
as well as activation of ERK-signaling pathways, by which
they may decrease the generation of amyloidogenic Ab and
simultaneously increase the neuroprotective features.
They have been undergoing clinical investigations in AD Results and discussion
patients, while avagacestat was recently terminated after phase
II trials due to shortage of supported profiles for advanced
development [13]. However, they are valuable to be models for
further development of novel Notch-sparing g-secretase inhib-
itors with chemical modifications, based upon their structural
features and pharmaceutical properties. Other small molecules
such as BMS-433796 (2a), an alaninamide derivative, and a
cyclohexyl-based derivative, 1-(2,5-difluorobenzyl),1-(p-chloro-
benzenesulfonyl)-cyclohexane (2b, named PU-1 here) are also
potent g-secretase inhibitors (Ab40 IC50 ¼ 0.3 and 3 nM,
respectively) [14, 15]. Taken together, we discovered that the
difluorophenyl and chloro-substituted arylsulfonyl groups
appear to be important as the core structures to exhibit g-
secretase inhibition. In fact, the 3,5-difluorophenyl moiety of
BMS-433796 is also shared with another well-known inhibitor
DAPT [16]. Therefore, the 3,5-difluorophenyl moiety can be a
good starting point to explore new modulators of APP
processing. As a beginning, we choose the difluorophenyl
moiety of BMS-433796 to replace the side chain hexafluoro-
isopropyl group of the amino alcohol GSI-953 followed by
elaborating small N-propargyl-relative substituents to provide a
type of N-substituted amino alcohols as compounds series-1, and
secondly, the p-chlorobenzenesulfonyl group of avagacestat
Chemistry
The synthesis of these difluorophenylglycinols was accom-
plished by using modified methods of Evans’ chiral auxiliary
strategy for the asymmetric synthesis of unusual amino
acids [17, 18]. As described in Scheme 1, chiral oxazolidinone 4
was condensed with difluorophenylacetic acid 3 to give amide
5 via mixed anhydride intermediate. The next diastereose-
lective bromination of amide 5 was achieved by enolating
with dibutylboryl triflate in situ followed by treating the
established metal enolate with N-bromosuccinimide (NBS),
to give a highly diastereoselective b-bromocarboxamide 6,
which was readily separated by column chromatography
from minor a-isomer in 90% yield. By employing sodium azide
in the following azide substitution reaction, intriguingly
equal amounts of diastereomeric azidocarboxamides R-7
and S-7 were obtained in 60% total yields after silica gel
chromatography. The assignment of the absolute stereo-
chemistry for both azidocarboxamides was confirmed by the
spectroscopic characterization for one of the diastereomers
identical to the exclusive product a-azidocarboxamide R-7,
which was prepared from the potassium enolate of amide 5
and the hindered 2,4,6-triisopropylbenzenesulfonyl azide at
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Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
Enantiomeric Difluorophenylglycinols
163
Scheme 1. Synthesis of the diastereomeric
a-azidocarboxamides R-7 and S-7.
the kinetic control conditions (step c, Scheme 1) according the
method reported by Evans et al. [18].
arylsulfonyl chlorides to afford the corresponding arylsulfon-
amides R-10, R-11, and S-10, S-11 in satisfactory yields.
In order to gain an understanding of the requirement of the
simple, N-propargyl-relative substituents for the ERK-mediat-
ed activation for these difluorophenylglycinols, various N-
substituted derivatives R-12a–f and S-12a–f were synthesized
by either alkylation or carbonylation in situ followed by
Reductive aminolysis of each azidocarboxamide with
lithium borohydride followed by heterogeneous hydrogena-
tion (10% Pd-C, H2(g)) of the resulting azido alcohols R-8 and S-8
gave the key difluorophenylglycinols R-9 and S-9 (Scheme 2).
The enantiomers R-9 and S-9 were each treated with
Scheme 2. Synthesis of enantiomeric N-substituted N-arylsulfonyldifluorophenylglycinols R-12a–f, S-12a–f, R-13a–f, and S-13a–f.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
amidation on the sulfonamide nitrogen under basic
conditions.
analogs, only N-ethyl derivative S-12a showed about 40%
activity of PU-1, while others were inferior. These results
suggested that the replacement of the 3,5-difluorophenyl
group for the hexafluoroisopropyl moiety of GSI-953 was
not an effective approach to improve g-secretase inhibition,
postulating that a bulky amino alcoholic side-chain similar to
hexafluoroisopropyl moiety might be necessary for fitting to
the binding pocket of the g-secretase to exert the inhibitory
activity, and the plane structure of 3,5-difluorophenyl
group of compounds series-1 did not fully match the
requisite binding space. The results also indicated that the
difluorophenylglycinol derivatives did not show preferred
Biological evaluation
The results of initial investigation on the g-secretase
inhibition of these synthesized difluorophenylglycinols
through luciferase reporter gene assays are listed in Table 1.
Surprisingly, most of the (R)-difluorophenylglycinols lacked
significant g-secretase inhibition with <30% activity of PU-1 at
10 mM. The N-cyclopropylmethyl derivative R-12c had about
28% of potency, whereas the N-propargyl compound R-12b had
only 17% activity, compared to PU-1. For the (S)-enantiomeric
Table 1. Inhibition of g-secretase-mediated C99 cleavage by N-substituted (R)- and (S)-N-(5-chlorothiophen-2-yl)sulfonyl phenyl-
glycinols using luciferase reporter assay.
Reduction of C99
cleavage (%)
Reduction of C99
cleavage (%)
Compound
X
Compound
X
R-12a
0.0
S-12a
38.9
16.7
R-12b
R-12c
16.7
S-12b
S-12c
27.8
5.6
0.0
R-12d
S-12d
11.1
R-12e
R-12f
PU-1
11.1
11.1
S-12e
S-12f
22.2
11.1
–
100.0
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Enantiomeric Difluorophenylglycinols
165
stereochemistry at the b-center of the amino alcohols, with
chirality of this center exhibiting little preference for both
enantiomer pairs on the g-secretase inhibition. The loss of
stereoselectivity of these difluorophenylglycinol analogs for
the g-secretase inhibition may be attributed to the worse
binding ability to the active site of g-secretase.
Next, our attention turned to the replacement of the p-
chlorobenzenesulfonyl group for the 5-chlorothiophenesul-
fonyl moiety of these (R)- and (S)-3,5-difluorophenylglycinols.
Thus, these chlorobenzenesulfonamides R-13a–f and S-13a–f
were synthesized (Scheme 2) and the results of g-secretase
inhibition are listed in Table 2. Among them, (R)-N-
cyclopropylmethyl derivative R-13c exhibited the substantial
potency with 78% activities of PU-1 and about threefold
improvement compared to R-12c, while S-13c is about fourfold
less active than R-13c but showed a small increase in potency
compared to the corresponding S-12c (Table 2). (S)-N-Alkylur-
eido derivatives S-13d and S-13f still showed 33.3% reduction
of C99 cleavage, while the corresponding (R)-analogs had
virtually no potency. Similar to the above thiophenesulfon-
amide analogs, the chirality of the b-center of these amino
alcohols is also not a mandatory requisite for g-secretase
inhibition as R-13c, S-13d, and S-13f all showed modest
potency.
From the structure–activity relationship point of view, the
p-chlorobenzenesulfonyl group of the compounds series-2 was
Table 2. Inhibition of g-secretase-mediated C99 cleavage by N-substituted (R)- and (S)-N-(p-chlorobenzenesulfonyl)difluorophenyl-
glycinols using luciferase reporter assay.
Compound
X
Reduction of C99 cleavage (%) Compound
X
Reduction of C99 cleavage (%)
R-13a
11.1
16.7
S-13a
S-13b
ꢁ5.6
R-13b
R-13c
5.6
77.8
0.0
S-13c
S-13d
22.2
R-13d
33.3
R-13e
R-13f
PU-1
11.1
5.6
S-13e
S-13f
5.6
33.3
–
100.0
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Table 3. IC50 values for selected difluorophenylglycinols on g-secretase-mediated inhibition.
IC50 (mM)
Compound
Ar
X
S3 cleavage
C99 cleavage
R-12c
S-12a
R-13c
S-13c
5-Chlorothiophen-2-yl
5-Chlorothiophen-2-yl
p-Chlorophenyl
c-Propylmethyl
Ethyl
c-Propylmethyl
c-Propylmethyl
16.5
47.9
8.3
11.7
43.6
8.7
p-Chlorophenyl
30.2
29.3
a little more favorable than the chlorothiophenesulfonyl
moiety for the g-secretase inhibition, and led to modest
potency. When the X group is N-cyclopropyomethyl, both (R)-
difluorophenylglycinols maintained 28–78% inhibitory activ-
ity; propargyl analogs were a little worse in potency. Other (R)-
difluorophenylglycinols with X ¼ N-alkylureido such as 12d–f
and 13d–f were detrimental to the inhibitory activity.
However, regarding the (S)-difluorophenylglycinols, N-alkylur-
eido analogs such as 13d and 13f were favorable with 33%
inhibitory activity of PU-1, while N-alkyl analogs were inferior
except the ethyl analog 12a with 39% activity.
We further examined whether these arylsulfonylphenyl-
glycinols with modest g-secretase inhibition exhibited selec-
tive Notch-sparing effects. Thus, their capability to process g-
secretase-mediated S3 cleavage of Notch was examined by
using a stable cell line (N7) constitutively expressing NDE as
previously reported [19]. As shown in Table 3, the IC50 values
of the selected compounds R-12c, S-12a, R-13c, and S-13c were
quite close to those values against g-secretase-mediated C99
cleavage of APP. The results indicated that these arylsulfonyl
analogs did not exhibit substantial selectivity for the g-
secretase-mediated proteolytic processing of APP over Notch.
In the following step, we investigate whether these
compounds can significantly modulate the MEK–ERK signal
pathway because the a-secretase-mediated processing of APP
would be enhanced directly or indirectly through the
activation of MEK pathway by certain propargylamine-related
compounds. Thus, the moderate g-secretase inhibitors R-12c,
S-12a, R-13c, and S-13c, which possessed propargyl-related
substitution, were chosen for further investigation of the
relative levels of ERK activation (p-ERK/ERK, %) in g-30 cells
as previously described [10, 20]. The results are described in
Table 4 and showed that N-cyclopropylmethyl derivative R-13c
was 1.1-fold more potent than rasagiline on ERK-mediated
activation at 10 mM, while others exhibited similar potency.
The results revealed that the introduction of an N-cyclo-
propylmethyl substituent to the difluorophenylglycinols 10
and 11 led to the activation effects on the ERK-mediated
signaling. Structurally, the molecular size and chemical
reactivity of the N-cyclopropylmethyl group in the com-
pounds R-12c and R-13c are similar to the prevailing
propargylamine moiety, which is responsible for stimulating
the processing of amyloid precursor protein to sAPPa through
MAP kinase-dependent activation of a-secretase as previously
described [5, 20, 21]. Thus, the activation of the ERK-mediated
signaling of R-12c and R-13c was considerably attributed to
the N-cyclopropylmethyl moiety and would evoke additional
Table 4. The relative levels of ERK activation of certain N-
arylsulfonyl-difluorophenylglycinols in treated cells compared with
nontreated cells.
Compound
Activation of ERK1/2 (p-ERK/ERK, %)a)
D þ Tb)
Rasagiline
R-12c
S-12a
R-13c
100
119 ꢂ 22
117 ꢂ 23
124 ꢂ 11
132 ꢂ 23
117 ꢂ 13
S-13c
a)
Each value represents the mean ꢂ SD of three experiments.
b)
D, DMSO; T, tetracycline.
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Enantiomeric Difluorophenylglycinols
167
a-secretase stimulation. These effects would subsequently
enhance sAPPa release and eventually provide neuronal
protection accompanied by decreased Ab production.
mixture was extracted with CH2Cl2, washed with NaOH and
KHSO4, and dried over MgSO4. The residue was purified by
silica gel column chromatography to give 5 (4.67 g, 50%) as
1
yellow oil; TLC, Rf 0.3 (EtOAc/n-hexane ¼ 1:5); H NMR (300 MHz,
CDCl3) d 2.78 (1H, dd, J ¼ 9.6, 11.8 Hz, CHHAr), 3.24 (1H, dd, J ¼ 3.3,
12.4 Hz, CHHAr), 4.16–4.29 (4H, m, CH2O and CH2), 4.64–4.71
(1H, m, CHCH2O), 6.69–6.76 (1H, m, Ar–H), 6.80–6.91 (2H, m,
Ar–H), 7.11–7.32 (5H, m, Ar–H); FABMS: m/z [MþH]þ 332.
Conclusion
In this study, we developed a type of difluorophenylglycinol
derivatives and investigated their effects on the modulation of
the proteolytic processing of APP, including g-secretase-
mediated C99 cleavage and ERK-mediated activation. We
presented the identification of the N-cyclopropylmethyl
derivatives R-12c and R-13c that exhibited dual-acting
activities with modest g-secretase inhibition as well as
substantial ERK activation, while they were compromised
by poor selectivity of APP processing over Notch. The dual
actions of the N-cyclopropylmethyl derivative would eventu-
ally avoid the generation of Ab and thus provide the beneficial
effects from neuronal protection. From the chemical feature
for the biological results point of view, development of a type
of optimized bulky alkyl amino alcohols as new modulators
of APP processing will be the next course to explore selective
g-secretase inhibition with dual-acting activities for AD
therapies.
(R)-4-Benzyl-3-((R)-2-bromo-2-(3,5-difluorophenyl)acetyl)-
oxazolidin-2-one (6)
To a solution of 5 (3.96 g, 12 mmol) in CH2Cl2 (50 mL), DIEA
(2.36 mL, 14.3 mmol) was added followed by dropwise addition of
n-Bu2BOTf (12.6 mL, 12.6 mmol) at ꢁ78°C under Ar(g). The pale
yellow solution was stirred at ꢁ78°C for 15 min and then at 0°C
for 1 h. The resulting boron enolate solution was then precooled
to ꢁ78°C and added by cannula to a slurry solution of NBS (2.3 g,
13 mmol) in CH2Cl2 at ꢁ78°C under Ar(g). The mixture was stirred
at ꢁ78°C for 2 h and quenched by aqueous KHSO4–brine solution.
The resulting mixture was extracted with EtOAc, washed with
Na2S2O3 and brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography to give 6 (1.97 g, 40%) as brown oil. TLC, Rf 0.4
(EtOAc/n-hexane ¼ 1:5); 1H NMR (300 MHz, CDCl3) d 2.70–2.91
(1H, m, CHHAr), 3.21–3.34 (1H, m, CHHAr), 4.27–4.30 (2H, m,
CH2O), 4.67–4.79 (1H, m, CHCH2O), 6.76–6.86 (3H, m, Ar–H), 7.04–
7.08 (1H, m, CHBr), 7.17–7.39 (5H, m, Ar–H); FABMS: m/z [MþH]þ
411.
Experimental
(R)-3-((S)-2-Azido-2-(3,5-difluorophenyl)acetyl)-4-
benzyloxazolidin-2-one (R-7)
Chemistry
To a solution of 5 (3.96 g, 14.4 mmol) in THF (50 mL), potassium
hexamethyldisilamide (KHMDS, 31.6 mL, 15.8 mmol) was added
at ꢁ78°C under Ar(g) for 30 min. The resulting potassium enolate
solution was added via cannulation to a precooled solution of
2,4,6-triisopropylbenzenesulfonyl azide (5.7 g, 18.7 mmol) in THF
(50 mL) at ꢁ78°C under Ar(g). After 10 min, the reaction was
quenched with glacial acetic acid (3.7 mL), and stirred at room
temperature for 12 h. The solution was partitioned with CH2Cl2.
The organic phases was washed with NaHCO3, dried over MgSO4,
and evaporated under reduced pressure. The residue was purified
by silica gel column chromatography to give R-7 (2.68 g, 50%)
as white solid. TLC, Rf ¼ 0.6 (CH2Cl2/n-hexane ¼ 2:1); 1H NMR
(300 MHz, CDCl3) d 2.87 (1H, dd, J ¼ 9.6, 13.5 Hz, CHHAr), 3.38 (1H,
dd, J ¼ 3, 13.5 Hz, CHHAr), 4.14–4.24 (2H, m, CH2O), 4.64–4.72
(1H, m, CHCH2O), 6.13 (1H, s, CHN3), 6.81–6.88 (1H, m, Ar–H),
6.97–7.04 (2H, m, Ar–H), 7.23–7.39 (5H, m, Ar–H); FABMS:
m/z [MþH]þ 373.
All reagents were commercial materials and were used directly
unless otherwise noted. DMF was dehydrated over 4 Å molecular
sieves. NMR spectra were recorded on a Varian Gemini at 300 MHz
for 1H and at 75 MHz for 13C. Chemical shifts are reported in parts
per million (d) downfield from tetramethylsilane. The deuterated
NMR solvent contained 99.8–99.9% deuterium in the indicated
position and was obtained from CIL, Inc. (Massachusetts, USA).
1H NMR coupling constants (J values) are listed in Hertz (Hz)
and spin multiplicities are reported as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broad (br). Elemental
analyses were determined using a Perkin-Elmer 240 EA analyzer.
Fast atom bombardment mass spectra (FABMS) were acquired on
a Finnigan Mat 95S mass spectrometer. Specific rotations were
measured with an optical activity AA-5 polarimeter. Chromatog-
raphy refers to flash chromatography on silica gel (silica gel 60,
230–400 mesh ASTM, E. Merck, Darmstadt, Germany). Melting
points were recorded on a Thomas Hoover capillary melting point
apparatus in open capillary tubes and are uncorrected.
(R)-3-((S)-2-Azido-2-(3,5-difluorophenyl)acetyl)-4-
benzyloxazolidin-2-one (R-7); (S)-3-((S)-2-azido-2-(3,5-
(R)-4-Benzyl-3-(2-(3,5-difluorophenyl)acetyl)oxazolidin-2-
difluorophenyl)acetyl)-4-benzyloxazolidin-2-one (S-7)
To a solution of 6 (0.62 g, 1.5 mmol) in acetonitrile (20 mL), NaN3
(0.39 mg, 6 mmol) was added in an ice bath. The reaction mixture
was stirred for 1 h at 0°C, and then at room temperature for 6 h
and quenched by saturated aqueous NaHCO3. The resulting
mixture was extracted with CH2Cl2, dried over MgSO4, and
purified by silica gel column chromatography to give R-7 (168 mg,
30%) that is identical to the above product as white solid; S-7
(168 mg, 30%) as yellow oil. TLC, Rf ¼ 0.4 (CH2Cl2/n-hexane ¼ 2:1);
one (5)
To a solution of 3 (5.1 g, 29.6 mmol) and triethylamine (3.6 g,
35.6 mmol) in 250 mL of THF, trimethylacetyl chloride (3.76 g,
31.2 mmol) was added, and stirred at ꢁ78°C under Ar(g). A
solution of metallated oxazolidinone prepared by the addition of
n-butyllithium (17.5 mL, 28.2 mmol) to a solution of 4-(R)-4-
phenylmethyl-2-oxazolidinone (4, 5.0 g, 28.2 mmol) in THF at
ꢁ78°C was added via cannula, and stirred for 30 min at 0°C then
quenched by saturated aqueous ammonium chloride. The
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1H NMR (300 MHz, CDCl3) d 2.68 (1H, dd, J ¼ 9.3, 12.9 Hz, CHHAr),
3.21 (1H, dd, J ¼ 3.6, 12.9 Hz, CHHAr), 4.16–4.31 (2H, m, CH2O),
4.76–4.82 (1H, m, CHCH2O), 6.12 (1H, s, CHN3), 6.85–6.93 (1H, m,
Ar–H), 7.03–7.09 (4H, m, Ar–H), 7.23–7.26 (3H, m, Ar–H); FABMS:
m/z [MþH]þ 373.
3.85 (1H, dd, J ¼ 3.9, 11.4 Hz, CHHOH), 4.46–4.51 (1H, m, CHN),
5.74 (1H, d, J ¼ 6 Hz, NH), 6.68–6.78 (3H, m, Ar–H), 6.81 (1H, d,
J ¼ 4.2 Hz, thiophene-H), 7.25 (1H, d, J ¼ 4.2 Hz, thiophene-H);
13C NMR (75 MHz, CDCl3) d 61.52, 62.36, 103.83, 111.01, 126.45,
131.64, 137.34, 139.43, 140.28, 163.06; [a]D ꢁ37° (c 0.006, MeOH);
FABMS: m/z [MþH]þ 354; HRFABMS: calcd. for C12H11ClF2NO3S2
[MþH]þ 353.9838, found 353.9839.
(R)-2-Azido-2-(3,5-difluorophenyl)ethanol (R-8)
To a solution of R-7 (1.7 g, 4.5 mmol) and water (0.08 mL,
(S)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-
thiophene-2-sulfonamide (S-10)
4.5 mmol) in THF (20 mL) at 0°C,
a solution of lithium
borohydride (2 M in THF, 9 mmol) was added. The reaction
mixture stood to warm for 30 min and was stirred for 1 day at
room temperature. The mixture was quenched with methanol
and sodium hydroxide was added. The resulting mixture was
extracted with CH2Cl2, dried over MgSO4, and then purified by
silica gel column chromatography to give R-8 (0.63 g, 70%) as
yellow oil. TLC, Rf 0.4 (CH2Cl2/n-hexane ¼ 3:1); 1H NMR (300 MHz,
CDCl3) d 2.98 (1H, br, OH), 3.68 (1H, dd, J ¼ 7.8, 11.7 Hz, CHHOH),
3.76 (1H, dd, J ¼ 4.2, 11.7 Hz, CHHOH), 4.62 (1H, dd, J ¼ 4.5, 7.9 Hz,
CHN3), 6.75–6.90 (3H, m, Ar–H); FABMS: m/z [MþH]þ 200.
According to the above method, S-9 (0.59 g, 3.4 mmol) was
converted to S-10 (0.54 g, 45%) as white powder. TLC, Rf 0.5 (EtOAc/
n-hexane ¼ 1:1); m.p. 118–120°C; 1H NMR (300 MHz, CDCl3) d 2.05
(1H, br, OH), 3.72 (1H, dd, J ¼ 6, 11.1 Hz, CHHOH), 3.85 (1H, dd,
J ¼ 3.9, 12 Hz, CHHOH), 4.46–4.51 (1H, m, CHN), 5.75 (1H, d,
J ¼ 6.3 Hz, NH), 6.69–6.78 (3H, m, Ar–H), 6.81 (1H, d, J ¼ 4.2 Hz,
thiophene-H), 7.25 (1H, d, J ¼ 4.2 Hz, thiophene-H); 13C NMR
(75 MHz, CDCl3) d 61.52, 62.36, 103.85, 111.03, 126.42, 131.65,
137.33, 139.42, 140.35, 163.07; [a]D þ37°C (c 0.006, MeOH);
FABMS: m/z [MþH]þ 354; HRFABMS: calcd. for C12H11ClF2NO3S2
[MþH]þ 353.9838, found 353.9836.
(S)-2-Azido-2-(3,5-difluorophenyl)ethanol (S-8)
According to the above method, azide S-7 (1.7 g, 4.5 mmol) was
converted to S-8 (0.62 g, 70%) as yellow oil. TLC, Rf 0.4 (CH2Cl2/
n-hexane ¼ 3:1); 1H NMR (300 MHz, CDCl3) d 2.36 (1H, t, J ¼ 5.7 Hz,
OH), 3.69–3.77 (2H, m, CH2OH), 4.64 (1H, dd, J ¼ 4.2, 7.8 Hz, CHN3),
6.76–6.92 (3H, m, Ar–H); FABMS: m/z [MþH]þ 200.
(R)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-
benzenesulfonamide (R-11)
According to the above method, R-9 (86.6 mg, 0.5 mmol) was
converted to R-11 (75 mg, 43%) as white powder. TLC, Rf 0.5 (EtOAc/
n-hexane ¼ 1:1); m.p. 129–131°C; 1H NMR (300 MHz, CDCl3) d 1.95
(1H, t, J ¼ 5.1 Hz, OH), 3.67–3.81 (2H, m, CH2OH), 4.44 (1H, dd,
J ¼ 4.2, 6.3 Hz, CHN), 5.60 (1H, d, J ¼ 6.3 Hz, NH), 6.65–6.70 (3H, m,
Ar–H), 7.39 (2H, d, J ¼ 9 Hz, Ar–H), 7.65 (2H, d, J ¼ 9.3 Hz, Ar–H);
13C NMR (75 MHz, CDCl3) d 61.36, 62.24, 103.70, 110.92, 128.63,
129.36, 139.22, 139.33, 140.55, 163.04; [a]D ꢁ56.0°C (c 0.005,
MeOH); FABMS: m/z [MþH]þ 348; HRFABMS: calcd. for
(R)-2-Amino-2-(3,5-difluorophenyl)ethanol (R-9)
A solution of R-8 (0.19 g, 1.1 mmol) in 15 mL of methanol was
flushed with inert gas and treated with 10% Pd on carbon (53 mg).
The mixture was placed under an atmosphere of H2 (g), stirred for
4 h, then filtered through a pad of celite, and concentrated under
reduced pressure to give R-9 (143 mg, 75%) as pale yellow powder.
C
14H13ClF2NO3S [MþH]þ 348.0274, found 348.0275.
1
TLC, Rf 0.4 (EtOAc); H NMR (300 MHz, CDCl3) d 2.13 (3H, br, OH
and NH2), 3.51 (1H, dd, J ¼ 7.8, 10.8 Hz, CHHOH), 3.73 (1H, dd,
J ¼ 3.9, 10.5 Hz, CHHOH), 4.04 (1H, dd, J ¼ 4.2, 7.5 Hz, CHNH2),
6.67–6.73 (2H, m, Ar–H), 6.86–6.90 (1H, m, Ar–H); FABMS: m/z
[MþH]þ 173.
(S)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-
benzenesulfonamide (S-11)
According to the above method, amine S-9 (86.6 mg, 0.5 mmol)
was converted to give S-11 (86 mg, 50%) as white powder. TLC, Rf
0.5 (EtOAc/n-hexane ¼ 1:1); m.p. 128–130°C; 1H NMR (300 MHz,
CDCl3) d 2.27 (1H, t, J ¼ 5.1 Hz, OH), 3.67–3.83 (2H, m, CH2OH), 4.45
(1H, dd, J ¼ 6.3, 10.8 Hz, CHN), 5.82 (1H, d, J ¼ 6.6 Hz, NH), 6.63–
6.70 (3H, m, Ar–H), 7.37 (2H, d, J ¼ 8.7 Hz, Ar–H), 7.66 (2H, d,
J ¼ 9 Hz, Ar–H); 13C NMR (75 MHz, CDCl3) d 61.33, 62.04, 103.69,
110.92, 128.64, 129.37, 139.27, 139.32, 140.58, 163.04; [a]D
þ57.5°C (c 0.0065, MeOH); FABMS: m/z [MþH]þ 348; HRFABMS:
calcd. for C14H13ClF2NO3S [MþH]þ 348.0274, found 348.0272.
(S)-2-Amino-2-(3,5-difluorophenyl)ethanol (S-9)
According to the above method, azide S-8 (0.19 g, 1.1 mmol) was
converted to amine S-9 (143 mg, 75%) as pale powder. TLC, Rf 0.4
(EtOAc); 1H NMR (300 MHz, CDCl3) d 2.11 (3H, br, OH and NH2),
3.50 (1H, dd, J ¼ 7.8, 10.8 Hz, CHHOH), 3.72 (1H, dd, J ¼ 3.9,
10.5 Hz, CHHOH), 4.04 (1H, dd, J ¼ 4.2, 7.5 Hz, CHNH2), 6.66–6.73
(2H, m, Ar–H), 6.85–6.90 (1H, m, Ar–H); FABMS: (m/z) C8H10F2NO
[MþH]þ 173.16.
(R)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-
(R)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
thiophene-2-sulfonamide (R-10)
ethylthiophene-2-sulfonamide (R-12a)
To a solution of R-9 (0.36 g, 2.1 mmol) in CH2Cl2 (6 mL) 0.5 mL of
pyridine was added. The mixture was stirred for 15 min at room
temperature and then added 5-chlorothiophene-2-sulfonyl chlo-
ride (0.2 mL, 1.7 mmol). The reaction mixture was stirred for
1 day, the resulting mixture was extracted with CH2Cl2, and dried
over MgSO4. The residue was purified by silica gel column
chromatography to give R-10 (0.29 g, 40%) as white powder. TLC, Rf
0.5 (EtOAc/n-hexane ¼ 1:1); m.p. 118–120°C; 1H NMR (300 MHz,
CDCl3) d 2.02 (1H, br, OH), 3.72 (1H, dd, J ¼ 6, 11.4 Hz, CHHOH),
To a mixture of R-10 (0.08 g, 0.23 mmol) in DMF (6 mL), potassium
carbonate (0.188 g, 1.36 mmol) and iodoethane (0.018 mL,
0.23 mmol) were added at room temperature. The mixture was
stirred for 12 h, poured into brine, then extracted with EtOAc. The
organic layer was separated, dried over MgSO4, and concentrated.
The residue was purified by silica gel column chromatography to
give R-12a (40 mg, 46%) as pale yellow powder. TLC, Rf 0.4 (EtOAc/n-
hexane ¼ 1:2); m.p. 58.0–60.0°C; 1H NMR (300 MHz, CDCl3) d 1.13
(3H, t, J ¼ 7.2 Hz, CH3), 2.01 (1H, br, OH), 3.17 (1H, sextet,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
Enantiomeric Difluorophenylglycinols
169
J ¼ 7.2 Hz, CH2CH3), 3.32 (1H, sextet, J ¼ 7.2 Hz, CH2CH3), 4.04–
4.10 (2H, m, CH2OH), 5.00 (1H, t, J ¼ 6.6 Hz, CHN), 6.72–6.79
(3H, m, Ar–H), 6.92 (1H, d, J ¼ 4.2 Hz, thiophene-H), 7.38 (1H, d,
J ¼ 3.9 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 16.13, 40.83,
61.66, 62.07, 103.82, 111.03, 126.58, 131.63, 137.30, 139.40,
140.29, 163.01; UV lmax (MeOH) nm: 287.6 (0.626); [a]D ꢁ38°C
(c 0.005, MeOH); c log p ¼ 3.65; FABMS: m/z 382 [MþH]þ; HRFABMS:
calcd. for C14H15ClF2NO3S2 [MþH]þ 382.0151, found 382.0152.
(EtOAc/n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 0.15–0.20
(2H, m, cyclopropane-H), 0.48–0.53 (2H, m, cyclopropane-H), 0.87–
0.89 (1H, m, cyclopropane-H), 2.10 (1H, br, OH), 2.93 (1H, dd,
J ¼ 6.9, 15 Hz, NCH2), 3.20 (1H, dd, J ¼ 6.6, 15.3 Hz, NCH2), 4.10–
4.12 (2H, m, CH2OH), 5.03 (1H, t, J ¼ 6.9 Hz, CHN), 6.72–6.77
(3H, m, Ar–H), 6.91 (1H, d, J ¼ 3.9 Hz, thiophene-H), 7.38 (1H, d,
J ¼ 3.9 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 4.94, 5.25,
11.42, 29.67, 50.60, 61.58, 62.22, 103.73, 111.07, 126.50, 131.70,
137.33, 139.58, 140.30, 163.03; UV lmax (MeOH) nm: 297 (1.58);
[a]D ꢁ22.2°C (c 0.0054, MeOH); c log p ¼ 4.04; FABMS: m/z 408
[MþH]þ; HRFABMS: calcd. for C16H17ClF2NO3S2 [MþH]þ 408.0308,
found 408.0307.
(S)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
ethylthiophene-2-sulfonamide (S-12a)
According to the above method, S-10 (0.08 g, 0.23 mmol) was
converted to S-12a (42%) as pale yellow powder. TLC, Rf 0.4 (EtOAc/
n-hexane ¼ 1:2); m.p. 55.0–57.0°C; 1H NMR (300 MHz, CDCl3) d
1.12 (3H, t, J ¼ 7.2 Hz, CH3), 2.10 (1H, br, OH), 3.19 (1H, sextet,
J ¼ 7.2 Hz, CH2CH3), 3.31 (1H, sextet, J ¼ 7.2 Hz, CH2CH3), 4.04–
4.09 (2H, m, CH2OH), 5.00 (1H, t, J ¼ 6.9 Hz, CHN), 6.72–6.78
(3H, m, Ar–H), 6.92 (1H, d, J ¼ 4.2 Hz, thiophene-H), 7.37 (1H, d,
J ¼ 3.9 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 16.13, 40.83,
61.66, 62.07, 103.82, 111.03, 126.58, 131.63, 137.30, 139.41,
140.29, 163.02; UV lmax (MeOH) nm: 287 (0.459); [a]D þ38°C
(c 0.005, MeOH); c log p ¼ 3.65; FABMS: m/z 382 [MþH]þ; HRFABMS:
calcd. for C14H15ClF2NO3S2 [MþH]þ 382.0151, found 382.0153.
(S)-5-Chloro-N-(cyclopropylmethyl)-N-(1-(3,5-
difluorophenyl)-2-hydroxyethyl)thiophene-2-sulfonamide
(S-12c)
According to the above method, S-10 (0.1 g, 0.3 mmol) was
converted to S-12c (71 mg, 58%) as yellow oil. TLC, Rf 0.5 (EtOAc/
n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 0.16–0.21 (2H, m,
cyclopropane-H), 0.49–0.54 (2H, m, cyclopropane-H), 0.87–0.90
(1H, m, cyclopropane-H), 1.99 (1H, t, J ¼ 5.7 Hz, OH), 2.93 (1H, dd,
J ¼ 6.9, 15.3 Hz, NCH2), 3.21 (1H, dd, J ¼ 6.6, 15.3 Hz, NCH2), 4.10–
4.16 (2H, m, CH2OH), 5.03 (1H, t, J ¼ 7.2 Hz, CHN), 6.72–6.78
(3H, m, Ar–H), 6.92 (1H, d, J ¼ 3.9 Hz, thiophene-H), 7.39 (1H, d,
J ¼ 4.2 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 4.94, 5.25,
11.42, 29.67, 50.60, 61.58, 62.22, 103.73, 111.07, 126.50, 131.70,
137.33, 139.58, 140.30, 163.03; UV lmax (MeOH) nm: 297.0 (1.58);
[a]D þ16°C (c 0.005, MeOH); c log p ¼ 4.04; FABMS: m/z 408 [MþH]þ;
HRFABMS: calcd. for C16H17ClF2NO3S2 [MþH]þ 408.0308, found
408.0310.
(R)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(prop-2-ynyl)thiophene-2-sulfonamide (R-12b)
According to the above method, R-10 (0.2 g, 0.56 mmol) was
converted to R-12b (50%) as dark yellow oil. TLC, Rf 0.4 (EtOAc/
n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 2.07 (1H, t, J ¼ 5.4 Hz,
OH), 2.31 (1H, t, J ¼ 2.7 Hz, CCH), 3.78 (1H, dd, J ¼ 2.4, 18.9 Hz,
CHHOH), 4.11–4.19 (2H, m, NCH2CCH), 4.36 (1H, dd, J ¼ 2.7,
18.9 Hz, CHHOH), 5.14 (1H, t, J ¼ 5.4 Hz, CHN), 6.78–6.89 (3H, m,
Ar–H), 6.94 (1H, d, J ¼ 4.2 Hz, thiophene-H), 7.49 (1H, d, J ¼ 4.2 Hz,
thiophene-H); 13C NMR (75 MHz, CDCl3) d 33.78, 61.87, 73.69,
78.53, 104.02, 111.03, 126.70, 132.57, 137.92, 138.56, 139.46;
163.16; UV lmax (MeOH) nm: 287.4 (0.673); [a]D ꢁ38.8°C (c 0.0067,
MeOH); c log p ¼ 3.6; FABMS: m/z 392 [MþH]þ; HRFABMS: calcd. for
C15H13ClF2NO3S2 [MþH]þ 391.9994, found 391.9995.
(R)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(isobutylcarbamoyl)thiophene-2-sulfonamide (R-12d)
To a stirred solution of triphosgene (78.3 mg, 0.26 mmol) in
CH2Cl2 a solution of isobutylamine (0.033 mL, 0.33 mmol) and
DIEA (0.05 mL) in CH2Cl2 was slowly added over a period of 20 min
using a syringe pump. After a further 30 min of stirring, a
solution of R-10 (118 mg, 0.33 mmol) and DIEA (0.11 mL,
0.66 mmol) in CH2Cl2 (10 mL) was added in one portion. The
mixture was stirred for 12 h, poured into water, then extracted
with CH2Cl2. The organic layer was separated, dried over MgSO4,
and concentrated. The residue was purified by silica gel column
chromatography to give R-12d (15 mg, 10%) as white powder. TLC,
Rf 0.5 (EtOAc/n-hexane ¼ 1:2); m.p. 102.0–103.5°C; 1H NMR
(300 MHz, CDCl3) d 0.91 (6H, d, J ¼ 6.6 Hz, CH(CH3)2), 1.75 (1H,
septet, J ¼ 6.3 Hz, CH(CH3)2), 2.99 (2H, t, J ¼ 6.6 Hz, CH2), 4.10 (1H,
dd, J ¼ 3.6, 12 Hz, CHHOH), 4.23 (1H, dd, J ¼ 8.1, 12 Hz, CHHOH),
4.61 (1H, br, CHN), 4.80 (1H, br, OH), 6.23 (1H, br, NH), 6.70–6.80
(3H, m, Ar–H), 6.82 (1H, d, J ¼ 3.9 Hz, thiophene-H), 7.24 (1H, d,
J ¼ 3.9 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 19.86, 28.63,
29.69, 48.66, 57.71, 66.46, 103.71, 110.07, 126.45, 131.87, 132.19,
139.13, 141.03, 156.54, 163.03; UV lmax (MeOH) nm (A): 286.4
(0.413); [a]D ꢁ15°C (c 0.004, MeOH); c log p ¼ 3.86; FABMS: m/z 453
[MþH]þ; HRFABMS: calcd. for C17H20ClF2N2O4S2 [MþH]þ
453.0523, found 453.0523.
(S)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(prop-2-ynyl)thiophene-2-sulfonamide (S-12b)
According to the above method, S-10 (0.2 g, 0.56 mmol) was
converted to S-12b (62%) as yellow oil. TLC, Rf 0.4 (EtOAc/
1
n-hexane ¼ 1:2); H NMR (300 MHz, CDCl3) d 2.08 (1H, t, J ¼ 6 Hz,
OH), 2.31 (1H, t, J ¼ 2.4 Hz, CCH), 3.79 (1H, dd, J ¼ 2.4, 18.6 Hz,
CHHOH), 4.11–4.19 (2H, m, NCH2CCH), 4.36 (1H, dd, J ¼ 2.7,
18.9 Hz, CHHOH), 5.14 (1H, t, J ¼ 5.7 Hz, CHN), 6.75–6.88 (3H, m,
Ar–H), 6.94 (1H, d, J ¼ 4.2 Hz, thiophene-H), 7.49 (1H, d, J ¼ 3.9 Hz,
thiophene-H); 13C NMR (75 MHz, CDCl3) d 33.78, 61.63, 61.87,
73.69, 78.53, 104.02, 111.03, 126.70, 132.57, 137.92, 138.56,
139.48; 163.17; UV lmax (MeOH) nm: 287.2 (0.516); [a]D þ26°C
(c 0.005, MeOH); c log p ¼ 3.6; FABMS: m/z 392 [MþH]þ; HRFABMS:
calcd. for C15H13ClF2NO3S2 [MþH]þ 391.9994, found 391.9990.
(R)-5-Chloro-N-(cyclopropylmethyl)-N-(1-(3,5-
difluorophenyl)-2-hydroxyethyl)thiophene-2-sulfonamide
(S)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(R-12c)
(isobutylcarbamoyl)thiophene-2-sulfonamide (S-12d)
According to the above method, S-10 (140 mg, 0.39 mmol) was
converted to S-12d (23 mg, 13%) as pale yellow powder. TLC, Rf 0.5
According to the above method, R-10 (0.1 g, 0.3 mmol) was
converted to R-12c (50 mg, 41%) as pale yellow oil. TLC, Rf 0.5
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170
C-Y. Chen et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
(EtOAc/n-hexane ¼ 1:2); m.p. 104.0–106.0°C; 1H NMR (300 MHz,
CDCl3) d 0.90 (6H, d, J ¼ 6.6 Hz, CH(CH3)2), 1.74 (1H, septet,
J ¼ 6.9 Hz, CH(CH3)2), 2.98 (2H, t, J ¼ 6.6 Hz, CH2), 4.12 (1H, dd,
J ¼ 3.9, 12 Hz, CHHOH), 4.21–4.30 (1H, m, CHHOH), 4.64 (1H, br,
OH), 4.90 (1H, br, CHN), 6.46 (1H, br, NH), 6.69–7.23 (4H, m, Ar–H),
7.25 (1H, d, J ¼ 3.9 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d
19.86, 28.63, 29.69, 48.66, 57.71, 66.46, 103.71, 110.06, 126.60,
131.87, 132.19, 139.13, 141.03, 156.54, 163.04; UV lmax (MeOH)
nm: 285.6 (0.143); [a]D þ11.7°C (c 0.006, MeOH); c log p ¼ 3.86;
HRFABMS: calcd. for C17H20ClF2N2O4S2 [MþH]þ 453.0523, found
453.0523.
(S)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(prop-2-ynylcarbamoyl)-thiophene-2-sulfonamide (S-12f)
According to the above method, S-10 (108 mg, 0.3 mmol) was
converted to S-12f (50 mg, 38%) as yellow oil. TLC, Rf 0.3 (EtOAc/
n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 2.27 (1H, br, CCH),
3.92–3.95 (2H, m, NCH2CCH), 4.15–4.25 (2H, m, CH2OH), 4.65 (1H,
br, OH), 5.20 (1H, br, CHN), 6.44 (1H, d, J ¼ 6 Hz, NH), 6.68–6.75
(3H, m, Ar–H), 6.80 (1H, d, J ¼ 3.9 Hz, thiophene-H), 7.24 (1H, d,
J ¼ 4.2 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 29.58, 57.00,
66.63, 71.95, 79.11, 103.60, 110.00, 126.57, 131.90, 137.53, 138.90,
140.80, 155.68, 162.90; UV lmax (MeOH) nm: 288.0 (0.775); [a]D
þ20°C (c 0.009, MeOH); c log p ¼ 2.85; FABMS: m/z 435 [MþH]þ.
HRFABMS: calcd. for C16H14ClF2N2O4S2 [MþH]þ 435.0053, found
435.0051.
(S)-Methyl-2-(3-(5-chlorothiophen-2-ylsulfonyl)-3-((R)-1-
(3,5-difluorophenyl)-2-hydroxy-ethyl)ureido)propanoate
(R-12e)
(R)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
ethylbenzenesulfonamide (R-13a)
According to the above method, R-10 (140 mg, 0.39 mmol) was
converted to R-12e (92 mg, 49%) as yellow oil. TLC, Rf 0.3 (EtOAc/
n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 1.24–1.43 (3H, m,
CH3), 2.94 (1H, br, OH), 3.76 (3H, s, OCH3), 4.13–4.33 (2H, CH2OH),
4.60–4.66 (1H, m, CHCH3), 5.62 (1H, d, J ¼ 7.8 Hz, CHN), 6.33 (1H, d,
J ¼ 6.9 Hz, NH), 6.65–6.80 (4H, m, Ar–H), 7.23 (1H, d, J ¼ 4.2 Hz,
thiophene-H); 13C NMR (75 MHz, CDCl3) d 23.25, 36.78, 51.25,
57.40, 66.44, 103.71, 110.07, 126.45, 131.87, 132.19, 139.13,
141.03, 156.54, 163.06, 173.25; UV lmax (MeOH) nm: 287.0 (0.666);
[a]D ꢁ7.9°C (c 0.0076, MeOH); c log p ¼ 2.65; FABMS: m/z 483
[MþH]þ; HRFABMS: calcd. for C17H18ClF2N2O6S2 [MþH]þ
483.0265, found 408.0263.
According to the method for the preparation of R-12a, R-11 (82 mg,
0.24 mmol) was converted to R-13a (43 mg, 47%) as white powder.
TLC, Rf 0.5 (EtOAc/n-hexane ¼ 1:2); m.p. 66.0–67.5°C; 1H NMR
(300 MHz, CDCl3) d 1.07 (3H, t, J ¼ 7.2 Hz, CH3), 2.03 (1H, br, OH),
3.15 (1H, sextet, J ¼ 7.2 Hz, CH2CH3), 3.32 (1H, sextet, J ¼ 7.2 Hz,
CH2CH3), 4.01–4.08 (2H, m, CH2OH), 5.02 (1H, t, J ¼ 6.9 Hz, CHN),
6.65–6.76 (3H, m, Ar–H), 7.48 (2H, d, J ¼ 9 Hz, Ar–H), 7.79 (2H, d,
J ¼ 8.7 Hz, Ar–H); TLC, Rf 0.5 (EtOAc/n-hexane ¼ 1:2); 13C NMR
(75 MHz, CDCl3) d 16.20, 40.44, 61.33, 62.04, 103.69, 110.90,
128.64, 129.37, 139.26, 139.32, 140.58, 163.04; UV lmax (MeOH)
nm: 286.6 (0.114); [a]D ꢁ37.8°C (c 0.0045, MeOH); c log p ¼ 3.72;
FABMS: m/z 376 [MþH]þ; HRFABMS: calcd. for C16H17ClF2NO3S
[MþH]þ 376.0587, found 376.0588.
(S)-Methyl 2-(3-(5-chlorothiophen-2-ylsulfonyl)-3-((S)-1-
(3,5-difluorophenyl)-2-hydroxyethyl)ureido)propanoate
(S-12e)
(S)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
According to the above method, S-10 (140 mg, 0.39 mmol) was
converted to S-12e (109 mg, 58%) as yellow oil. TLC, Rf 0.3 (EtOAc/
n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 1.42 (3H, d, J ¼ 7.2 Hz,
CH3), 2.86 (1H, br, OH), 3.77 (3H, s, OCH3), 4.17–4.33 (2H, CH2OH),
4.63–4.66 (1H, m, CHCH3), 5.55 (1H, d, J ¼ 7.8 Hz, CHN), 6.28 (1H, d,
J ¼ 6.3 Hz, NH), 6.66–6.80 (4H, m, Ar–H), 7.23–7.27 (1H, m,
thiophene-H); 13C NMR (75 MHz, CDCl3) d 23.25, 36.78, 51.25,
57.40, 66.44, 103.71, 110.03, 126.60, 131.87, 132.19, 139.13,
141.03, 156.54, 163.05, 173.24; UV lmax (MeOH) nm: 287.0 (0.166);
[a]D þ21.1°C (c 0.009, MeOH); c log p ¼ 2.65; FABMS: m/z 483
[MþH]þ; HRFABMS: calcd. for C17H18ClF2N2O6S2 [MþH]þ
483.0265, found 483.0261.
ethylbenzenesulfonamide (S-13a)
According to the above method, S-11 (82 mg, 0.24 mmol) was
converted to S-13a (56 mg, 62%) as yellow oil. TLC, Rf 0.5 (EtOAc/
n-hexane ¼ 1:2); m.p. 63.0–65.0°C; 1H NMR (300 MHz, CDCl3) d
1.08 (3H, t, J ¼ 7.2 Hz, CH3), 1.93 (1H, br, OH), 3.16 (1H, sextet,
J ¼ 7.2 Hz, CH2CH3), 3.31 (1H, sextet, J ¼ 7.2 Hz, CH2CH3), 4.01–
4.11 (2H, m, CH2OH), 5.02 (1H, t, J ¼ 6.9 Hz, CHN), 6.67–6.76
(3H, m, Ar–H), 7.49 (2H, d, J ¼ 8.7 Hz, Ar–H), 7.78 (2H, d, J ¼ 8.7 Hz,
Ar–H); 13C NMR (75 MHz, CDCl3) d 16.20, 40.44, 61.33, 62.04,
103.69, 110.92, 128.64, 129.37, 139.27, 139.31, 140.58, 163.03; UV
lmax (MeOH) nm: 288.4 (0.150); [a]D þ36°C (c 0.005, MeOH);
c log p ¼ 3.72; FABMS: m/z 376 [MþH]þ; HRFABMS: calcd. for
C16H17ClF2NO3S [MþH]þ 376.0587, found 376.0589.
(R)-5-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-
N-(prop-2-ynylcarbamoyl)-thiophene-2-sulfonamide
(R)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(R-12f)
(prop-2-ynyl)benzenesulfonamide (R-13b)
According to the above method, R-10 (210 mg, 0.59 mmol) was
converted to R-12f (110 mg, 43%) as yellow oil. TLC, Rf 0.3 (EtOAc/
n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 2.28 (1H, br, CCH),
3.93–3.96 (2H, m, NCH2CCH), 4.19–4.26 (2H, m, CH2OH), 4.67
(1H, br, OH), 5.27 (1H, t, J ¼ 5.4 Hz, CHN), 6.55 (1H, br, NH), 6.69–
6.75 (3H, m, Ar–H), 6.81 (1H, d, J ¼ 3.9 Hz, thiophene-H), 7.25
(1H, d, J ¼ 3.9 Hz, thiophene-H); 13C NMR (75 MHz, CDCl3) d 29.58,
57.00, 66.63, 71.95, 79.11, 103.60, 110.01, 137.53, 138.90, 140.80,
155.68, 162.91; UV lmax (MeOH) nm: 288 (0.85); [a]D ꢁ16°C
(c 0.0075, MeOH); c log p ¼ 2.85; FABMS: m/z 435 [MþH]þ;
HRFABMS: calcd. for C16H14ClF2N2O4S2 [MþH]þ 435.0053, found
435.0056.
According to the above method, R-11 (0.130 g, 0.375 mmol) was
converted to R-13b (82 mg, 57%) as pale yellow powder. TLC, Rf 0.5
(EtOAc/n-hexane ¼ 1:2); m.p.: 82.5–84.0°C; 1H NMR (300 MHz,
CDCl3) d 1.93 (1H, t, J ¼ 6 Hz, OH), 2.22 (1H, t, J ¼ 2.4 Hz, CCH), 3.75
(1H, dd, J ¼ 2.4, 16.5 Hz, CHHOH), 4.05–4.13 (2H, m, NCH2CCH),
4.40 (1H, dd, J ¼ 2.4, 18 Hz, CHHOH), 5.15 (1H, t, J ¼ 5.7 Hz, CHN),
6.73–6.88 (3H, m, Ar–H), 7.49 (2H, d, J ¼ 9 Hz, Ar–H), 7.88 (2H, d,
J ¼ 8.7 Hz, Ar–H); 13C NMR (75 MHz, CDCl3) d 39.37, 61.48, 61.63,
73.26, 78.85, 103.94, 110.92, 129.15, 138.70, 139.63, 163.16; UV
lmax (MeOH) nm: 300.4 (0.253); [a]D ꢁ47.3°C (c 0.0055, MeOH);
c log p ¼ 3.9; FABMS: m/z 386 [MþH]þ; HRFABMS: calcd. for
C17H15ClF2NO3S [MþH]þ 386.0430, found 386.0426.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
Enantiomeric Difluorophenylglycinols
171
(S)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
J ¼ 8.7 Hz, Ar–H), 7.65 (2H, d, J ¼ 8.7 Hz, Ar–H); 13C NMR (75 MHz,
CDCl3) d 19.81, 28.60, 29.66, 48.60, 57.38, 66.42, 103.55, 110.04,
128.41, 129.07, 138.84, 139.57, 141.02, 156.41, 162.96; UV lmax
(MeOH) nm: 290.6 (0.155); [a]D ꢁ28.3°C (c 0.006, MeOH);
c log p ¼ 3.93; FABMS: m/z 447 [MþH]þ; HRFABMS: calcd. for
C19H22ClF2N2O4S [MþH]þ 447.0959, found 447.0956.
(prop-2-ynyl)benzenesulfonamide (S-13b)
According to the above method, S-11 (0.130 g, 0.375 mmol) was
converted to give S-13b (92 mg, 64%) as pale yellow powder. TLC, Rf
1
0.5 (EtOAc/n-hexane ¼ 1:2); m.p. 80.5–82.0°C; H NMR (300 MHz,
CDCl3) d 1.92 (1H, t, J ¼ 7.5 Hz, OH), 2.22 (1H, t, J ¼ 2.7 Hz, CCH),
3.75 (1H, dd, J ¼ 2.4, 16.5 Hz, CHHOH), 4.05–4.13 (2H, m,
NCH2CCH), 4.40 (1H, dd, J ¼ 2.4, 17.1 Hz, CHHOH), 5.15 (1H, t,
J ¼ 5.7 Hz, CHN), 6.73–6.87 (3H, m, Ar–H), 7.48 (2H, d, J ¼ 8.7 Hz,
Ar–H), 7.88 (2H, d, J ¼ 9 Hz, Ar–H); 13C NMR (75 MHz, CDCl3) d
39.37, 61.48, 61.63, 73.26, 78.85, 103.94, 110.93, 129.15, 138.70,
139.63, 163.15; UV lmax (MeOH) nm: 278.6 (0.253); [a]D þ40°C
(c 0.005, MeOH); c log p ¼ 3.9; FABMS: m/z 402 [MþH]þ; HRFABMS:
calcd. for C18H19ClF2NO3S [MþH]þ 402.0744, found 402.0744.
(S)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(isobutylcarbamoyl)benzenesulfonamide (S-13d)
According to the above method, S-11 (95 mg, 0.27 mmol) was
transferred to S-13d (15 mg, 12%) as white powder. TLC, Rf 0.5
(EtOAc/n-hexane ¼ 1:2); m.p. 110–111.5°C; 1H NMR (300 MHz,
CDCl3) d 0.89 (6H, d, J ¼ 6.6 Hz, CH(CH3)2), 1.71 (1H, m, CH(CH3)2),
2.95 (2H, t, J ¼ 6.6 Hz, CH2), 4.06 (1H, dd, J ¼ 3.9, 12 Hz, CHHOH),
4.19 (1H, dd, J ¼ 8.1, 11.7 Hz, CHHOH), 4.59 (1H, br, CHN), 4.78 (1H,
br, OH), 6.24 (1H, br, NH), 6.64–6.73 (3H, m, Ar–H), 7.37 (2H, d,
J ¼ 8.7 Hz, Ar–H), 7.65 (2H, d, J ¼ 8.7 Hz, Ar–H); 13C NMR (75 MHz,
CDCl3) d 19.81, 28.60, 29.66, 48.60, 57.38, 66.42, 103.55, 110.04,
128.41, 129.07, 138.84, 139.16, 140.92, 156.41, 162.96; UV lmax
(MeOH) nm: 290.6 (0.173); [a]D þ26.7°C (c 0.006, MeOH);
c log p ¼ 3.93; FABMS: m/z 447 [MþH]þ; HRFABMS: calcd. for
C19H22ClF2N2O4S [MþH]þ 447.0959, found 447.0959.
(R)-4-Chloro-N-(cyclopropylmethyl)-N-(1-(3,5-
difluorophenyl)-2-hydroxyethyl)benzenesulfonamide
(R-13c)
According to the above method, R-11 (0.11 g, 0.32 mmol) was
converted to R-13c (37 mg, 29%) as pale yellow oil. TLC, Rf 0.5
(EtOAc/n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 0.11–0.15
(2H, m, cyclopropane-H), 0.42–0.48 (2H, m, cyclopropane-H), 0.79–
0.84 (1H, m, cyclopropane-H), 2.20 (1H, br, OH), 2.90 (1H, dd,
J ¼ 6.9, 15.3 Hz, NCH2), 3.19 (1H, dd, J ¼ 6.6, 15.3 Hz, NCH2), 4.07–
4.14 (2H, m, CH2OH), 5.05 (1H, t, J ¼ 6.9 Hz, CHN), 6.69–6.75
(3H, m, Ar–H), 7.46 (2H, d, J ¼ 8.7 Hz, Ar–H), 7.80 (2H, d, J ¼ 8.7 Hz,
Ar–H); 13C NMR (75 MHz, CDCl3) d 4.88, 5.23, 11.49, 50.31, 61.26,
62.22, 103.87, 110.97, 126.11, 128.68, 131.25, 139.44, 162.94; UV
lmax (MeOH) nm: 296 (1.326); [a]D ꢁ34.6°C (c 0.0052, MeOH);
c log p ¼ 4.11; FABMS: m/z 402 [MþH]þ; HRFABMS: calcd. for
C18H19ClF2NO3S [MþH]þ 402.0744, found 402.0744.
(S)-Methyl-2-(3-(4-chlorophenylsulfonyl)-3-((R)-1-(3,5-
difluorophenyl)-2-hydroxyethyl)-ureido)propanoate
(R-13e)
According to the above method, R-11 (107 mg, 0.31 mmol) was
converted to R-13e (66 mg, 45%) as white powder. TLC, Rf 0.3
(EtOAc/n-hexane ¼ 1:2); mp 122.5–124.0°C; 1H NMR (300 MHz,
CDCl3) d 1.41 (3H, d, J ¼ 7.2 Hz, CH3), 2.24 (1H, br, OH), 3.78
(3H, s, OCH3), 4.11–4.31 (2H, m, CH2OH), 4.47–4.62 (1H,
m, CHCH3), 5.41 (1H, d, J ¼ 7.5 Hz, CHN), 6.13 (1H, d, J ¼ 6.9 Hz,
NH), 6.64–6.72 (3H, m, Ar–H), 7.37 (2H, d, J ¼ 9 Hz, Ar–H), 7.66 (2H,
d, J ¼ 9 Hz, Ar–H); 13C NMR (75 MHz, CDCl3) d 23.25, 36.78, 51.25,
57.40, 66.44, 103.55, 110.08, 128.43, 129.07, 139.16, 141.00,
156.41, 163.05, 173.22; UV lmax (MeOH) nm (A): 289.8 (0.178); [a]D
ꢁ29.6°C (c 0.004, MeOH); c log p ¼ 2.73; HRFABMS: calcd. for
C19H20ClF2N2O6S [MþH]þ 477.0701, found 477.0697.
(S)-4-Chloro-N-(cyclopropylmethyl)-N-(1-(3,5-
difluorophenyl)-2-hydroxyethyl)benzenesulfonamide
(S-13c)
According to the above method, S-11 (0.11 g, 0.32 mmol) was
converted to S-13c (60 mg, 47%) as pale yellow oil. TLC, Rf 0.5
(EtOAc/n-hexane ¼ 1:2); 1H NMR (300 MHz, CDCl3) d 0.08–0.17
(2H, m, cyclopropane-H), 0.43–0.48 (2H, m, cyclopropane-H), 0.80–
0.85 (1H, m, cyclopropane-H), 2.11 (1H, br, OH), 2.90 (1H, dd,
J ¼ 7.2, 15.1 Hz, NCH2), 3.20 (1H, dd, J ¼ 6.6, 15.1 Hz, NCH2), 4.07–
4.15 (2H, m, CH2OH), 5.05 (1H, t, J ¼ 7.2 Hz, CHN), 6.69–6.75
(3H, m, Ar–H), 7.47 (2H, d, J ¼ 8.7 Hz, Ar–H), 7.80 (2H, d, J ¼ 8.7 Hz,
Ar–H); 13C NMR (75 MHz, CDCl3) d 4.88, 5.23, 11.49, 50.31, 61.26,
62.22, 103.87, 110.97, 126.11, 128.68, 131.25, 139.25, 162.96; UV
lmax (MeOH) nm: 296.2 (1.322); [a]D þ24.3°C (c 0.0074, MeOH);
c log p ¼ 4.11; FABMS: m/z 402 [MþH]þ; HRFABMS: calcd. for
C18H19ClF2NO3S [MþH]þ 402.0744, found 402.0740.
(S)-Methyl-2-(3-(4-chlorophenylsulfonyl)-3-((S)-1-(3,5-
difluorophenyl)-2-hydroxyethyl)ureido)propanoate (S-13e)
According to the above method, S-11 (105 mg, 0.3 mmol) was
transferred to S-13e (57 mg, 40%) as white powder. TLC, Rf 0.3
(EtOAc/n-hexane ¼ 1:2); m.p. 122.0–123.5°C; 1H NMR (300 MHz,
CDCl3) d 1.40 (3H, d, J ¼ 7.2 Hz, CH3), 2.62 (1H, br, OH), 3.77 (3H, s,
OCH3), 4.11–4.28 (2H, m, CH2OH), 4.43–4.47 (2H, m, CHCH3 and
CHN), 6.05 (1H, d, J ¼ 6.9 Hz, NH), 6.61–6.72 (3H, m, Ar–H), 7.34–
7.39 (2H, m, Ar–H), 7.63–7.68 (2H, m, Ar–H); 13C NMR (75 MHz,
CDCl3) d 23.25, 36.78, 51.25, 57.40, 66.44, 103.55, 110.09, 128.43,
129.07, 138.85, 140.93, 156.41, 163.03, 173.22; UV lmax (MeOH)
nm: 289.8 (0.170); [a]D þ33.3°C (c 0.0057, MeOH); c log p ¼ 2.73;
FABMS: m/z 477 [MþH]þ; HRFABMS: calcd. for C19H20ClF2N2O6S
[MþH]þ 477.0701, found 477.0698.
(R)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(isobutylcarbamoyl)benzenesulfon amide (R-13d)
According to the method for the preparation of R-12d, R-11
(95 mg, 0.27 mmol) was converted to R-13d (29 mg, 24%) as white
powder. TLC, Rf 0.5 (EtOAc/n-hexane ¼ 1:2); m.p. 111–113°C; 1H
NMR (300 MHz, CDCl3) d 0.88 (6H, d, J ¼ 6.6 Hz, CH(CH3)2), 1.71
(1H, septet, J ¼ 6.6 Hz, CH(CH3)2), 2.94 (2H, t, J ¼ 6 Hz, CH2), 4.07
(1H, dd, J ¼ 4.2, 12 Hz, CHHOH), 4.20 (1H, dd, J ¼ 7.8, 11.7 Hz,
CHHOH), 4.57–4.63 (1H, m, CHN), 4.84 (1H, t, J ¼ 5.4 Hz, OH), 6.37
(1H, d, J ¼ 6.3 Hz, NH), 6.63–6.73 (3H, m, Ar–H), 7.36 (2H, d,
(R)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(prop-2-ynylcarbamoyl)benzenesulfonamide (R-13f)
According to the above method, R-11 (127 mg, 0.36 mmol) was
converted to R-13f (53 mg, 69%) as pale yellow powder. TLC, Rf 0.35
(EtOAc/n-hexane ¼ 1:2); m.p. 85.0–86.5°C; 1H NMR (300 MHz,
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172
C-Y. Chen et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
CDCl3) d 2.28 (1H, t, J ¼ 2.1 Hz, CCH), 3.91–3.93 (2H, m, NCH2CCH),
4.10–4.22 (2H, m, CH2OH), 4.59–4.61 (1H, m, CHN), 5.17 (1H,
t, J ¼ 5.4 Hz, OH), 6.37 (1H, d, J ¼ 6.3 Hz, NH), 6.64–6.73 (3H, m,
Ar–H), 7.37 (2H, d, J ¼ 8.7 Hz, Ar–H), 7.65 (2H, d, J ¼ 8.7 Hz, Ar–H);
13C NMR (75 MHz, CDCl3) d 30.80, 56.87, 66.69, 71.97, 79.11,
103.55, 110.09, 128.46, 129.13, 138.60, 139.23, 140.76, 155.67,
162.86; UV lmax (MeOH) nm: 288.0 (0.310); [a]D ꢁ24°C (c 0.005,
MeOH); c log p¼ 2.92; FABMS: m/z 429 [MþH]þ; HRFABMS:
calcd. for C18H16ClF2N2O4S [MþH]þ 429.0489, found 429.0485.
gene (e.g., under control of a CMV promoter) was performed as a
control panel.
To examine compound inhibition of g-secretase-dependent S3
cleavage of Notch, we generated a HEK293-derived stable line (N7)
that was constitutively expressing NDE as previously de-
scribed [22]. N7 cells were plated onto 12-well microplates in
1 mL/well DMEM supplemented with 10% FBS at 5 ꢃ 105 cells/well.
After incubation at 37°C overnight, cells were treated with
compounds at 10 mM as described above and incubated at 37°C
for 24 h. Treated cells were harvested using PBS containing 20 mM
EDTA and dissolved in 50 mL of 1ꢃ PLB, followed by centrifugation
at 13200g for 5 min to remove cell debris. The protein
concentrations of clarified supernatants were determined using
a BCA protein assay reagent kit, and cell extracts containing
equivalent amounts of proteins were resolved by SDS–polyacryl-
amide electrophoresis gels (SDS–PAGE) and analyzed by Western
blotting using an anti-Notch (Val1744) polyclonal antibody.
(S)-4-Chloro-N-(1-(3,5-difluorophenyl)-2-hydroxyethyl)-N-
(prop-2-ynylcarbamoyl)benzenesulfonamide (S-13f)
According to the above method, S-11 (127 mg, 0.36 mmol)
was transferred to S-13f (83 mg, 54%) as yellow powder. TLC, Rf
0.35 (EtOAc/n-hexane ¼ 1:2); m.p. 85.0–87.0°C; 1H NMR (300 MHz,
CDCl3) d 2.28 (1H, t, J ¼ 2.1 Hz, CCH), 3.90–3.93 (2H, m, NCH2CCH),
4.12 (1H, dd, J ¼ 3.9, 11.7 Hz, CHHOH), 4.22 (1H, dd, J ¼ 7.8,
11.7 Hz, CHHOH), 4.59–4.61 (1H, m, CHN), 5.17 (1H, t, J ¼ 5.4 Hz,
OH), 6.36 (1H, d, J ¼ 6.9 Hz, NH), 6.64–6.73 (3H, m, Ar–H), 7.37
(2H, d, J ¼ 8.7 Hz, Ar–H), 7.65 (2H, d, J ¼ 8.7 Hz, Ar–H); 13C NMR
(75 MHz, CDCl3) d 30.80, 56.87, 66.69, 71.97, 79.11, 103.55,
110.06, 128.40, 129.08, 138.54, 139.24, 140.76, 155.67, 162.83;
UV lmax (MeOH) nm: 288.2 (0.301); [a]D þ22.2°C (c 0.0054, MeOH);
c log p ¼ 2.92; FABMS: m/z 429 [MþH]þ; HRFABMS: calcd. for
C18H16ClF2N2O4S [MþH]þ 429.0489, found 429.0485.
Cell culture and cell lines
Human embryonic kidney cells (HEK293) were incubated in
DMEM supplemented with 10% FBS and 0.1 mg/mL penicillin and
streptomycin. T-REx293 cells were purchased from Invitrogen
and cultured in DMEM supplemented with 10% FBS and 5 mg/mL
blasticidin. The generation of stably transfected cell lines, T20 and
g-30, has been described previously [23]. Cells were incubated in a
humidified incubator at 37°C in 5% CO2.
Pharmacological evaluation
Reagents
Assay for extracellular signal-regulated kinase (ERK)
activity
Anti-MEK antibody and anti-ERK1/2 MAP kinase antibody were
purchased from Cell Signaling Technology, Inc. Horseradish
peroxidase-conjugated anti-rabbit IgG was obtained from Santa
Cruz Biotechnology, Inc. Horseradish peroxidase-conjugated
anti-mouse IgG and ECL Western blotting detection reagents
were obtained from Amersham Biosciences. Dulbecco’s modi-
fied Eagle’s medium (DMEM) and fetal bovine serum (FBS) were
obtained from Invitrogen and Biological Industries Ltd.
(Kibbutz Beit Haemek, Israel), respectively. All other reagents
were reagent grade and obtained from standard suppliers
abroad.
The stimulation of the ERKs was measured by using anti-phospho-
ERK1/2 and anti-ERK1/2 antibodies (Cell Signaling Technology,
Danvers, MA, USA). Briefly, g-30 cells were grown in six-well plates
at 5 ꢃ 105 cells/well and incubated with culture medium at 37°C
for 18 h, followed by an additional incubation with culture
medium containing 1 mg/mL tetracycline at 37°C for 18 h.
Before the experiments, we replaced the medium with DMEM
containing 0.5% FBS and treated it with the tested compounds
(10 mM each). After treatment, reactions were stopped by placing
cells on ice and aspirating the medium. Cells were harvested and
lysed in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA,
1 mM sodium orthovanadate, 1% Triton X-100, and protease-
and phosphatase-inhibitor cocktails. Protein concentration was
determined by the BCA assay (Pierce, Rockford, IL, USA). Each
cell lysate, which contained 50 mg proteins, was separated
on 12% SDS–PAGE, immunoblotted, and identified using anti-
phospho-ERK1/2 or anti-ERK1/2 antibody.
Cell-based g-secretase assays
The production of T20 cells has been reported previously [22].
T20 cells were maintained in DMEM supplemented with 10%
FBS, 200 mg/mL hygromycin, 5 mg/mL blasticidin, and 250 mg/
mL zeocin (DMEM-HZB). The stably transfected T20 cells were
detached from culture dishes by trypsinization, washed with
PBS, and resuspended in DMEM-HZB, followed by plating onto
96-well microplates (2 ꢃ 104 cells/50 mL/well) and incubating at
37°C for 24 h. Tested compounds diluted in DMEM-HZB were
added to a final concentration of 10 mM in the presence of
tetracycline (1 mg/mL). Treatments were terminated after
incubation at 37°C for 24 h by directly adding an equivalent
volume of the Steady-Glo luciferase assay reagent (Promega,
Madison, WI, USA), and luciferase signals from each well were
performed immediately with the luminescence plate reader
available in our Institute. Triplicates of each compound
treatment were assayed. Luciferase signals from the stable line
without tetracycline induction and tested compound treatment
were referred to as onefold of activation. Parallel testing with a
cell line constitutively expressing only the luciferase reporter
Data analysis
The data presented are means ꢂ SD and triplicates of each
compound treatment were measured in the experiments. The
effects of the tested compounds on the biological outcome were
statistically examined using a one-way analysis of variance.
Dunnets’s test was applied to compare individual compounds.
In all cases, p < 0.05 was accepted to denote significance.
This work was supported by the National Science Council, Taiwan,
of the Republic of China (NSC 98-2320-B-016-004-MY3 to M.-K. Hu
and NSC 98-2320-B-001-014-MY2 to Y.-F. Liao). High-resolution mass
spectra analyses performed by the National Science Council Regional
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 161–173
Enantiomeric Difluorophenylglycinols
173
Instruments Center at National Chiao Tung University are also
gratefully acknowledged.
J. H. Toyn, S. B. Roberts, K. A. Lentz, J. G. Houston, R. Zaczek,
C. F. Albright, C. P. Decicco, J. E. Macor, R. E. Olson, ACS Med.
Chem. Lett. 2010, 1, 120–124.
[13] Bristol-Myers Squibb Company. Available online: http://www.
bms.com/news/features/2012/Pages/AvagacestatDevelopment-
Status.aspx.
The authors have declared no conflict of interest.
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ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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