Synthesis and structure-activity relationship of a novel series of heterocyclic sulfonamide γ-secretase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.04.052

γ-Secretase inhibitors have been shown to reduce the production of β-amyloid, a component of the plaques that are found in brains of patients with Alzheimer's disease. A novel series of heterocyclic sulfonamide γ-secretase inhibitors that reduce β-amyloid levels in cells is reported. Several examples of compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid precursor protein compared to Notch, an alternative γ-secretase substrate.

Full text of DOI:10.1016/j.bmc.2009.04.052

Bioorganic & Medicinal Chemistry 17 (2009) 4708–4717
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationship of a novel series of
heterocyclic sulfonamide c-secretase inhibitors
Jun Pu a, Anthony F. Kreft a, Suzan H. Aschmies b, Kevin P. Atchison b, Joshua Berkowitz a,
Thomas J. Caggiano a, Micheal Chlenov a, George Diamantidis a, Boyd L. Harrison a, Yun Hu b,
Donna Huryn a, J. Steven Jacobsen b, Mei Jin a, Kerri Lipinski b, Peimin Lu b, Robert L. Martone b,
Koi Morris a, June Sonnenberg-Reines b, Dave R. Riddell b, Joan Sabalski a, Shaiu-Ching Sun b,
Erik Wagner b, Yiqun Wang b, Zheng Xu b, Hua Zhou b, Lynn Resnick a,
*
a Chemical Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, USA
b Neuroscience Discovery Research, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
c
-Secretase inhibitors have been shown to reduce the production of b-amyloid, a component of the pla-
ques that are found in brains of patients with Alzheimer’s disease. A novel series of heterocyclic sulfon-
amide -secretase inhibitors that reduce b-amyloid levels in cells is reported. Several examples of
compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid pre-
cursor protein compared to Notch, an alternative -secretase substrate.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 March 2009
Revised 21 April 2009
Accepted 24 April 2009
Available online 3 May 2009
c
c
Keywords:
c
-Secretase inhibitors
b-Amyloid lowering agents
Alzheimer’s disease
1. Introduction
BMS-299897, a compound that prevents cleavage of APP 15-fold
more effectively than Notch in vitro, lowered Ab levels in mice
without any Notch related side effects.6
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder, which is the most common cause of dementia in the el-
derly population. To date, disease-modifying therapeutic agents
to treat this disease are not available.1 AD is characterized patho-
logically by abnormal deposits of extracellular plaques composed
of b-amyloid peptides (Ab) and intracellular neurofibrillary tangles
composed of hyperphosphorylated tau protein in the brain.2 Ab
peptides are released by sequential cleavage of the amyloid precur-
sor protein (APP) by two membrane associated aspartic proteases.
Firstly, b-secretase (BACE1) cleaves APP to form b-C-terminal frag-
Herein, we report a series of
c-secretase inhibitors that were
derived from a sulfonamide series represented by structure 1
(Fig. 1).7 This compound was shown to inhibit production of
Ab40 and Ab42 in CHO cells transfected with human APP with an
average EC50 value of 29 nM and increased b-CTF levels without
effecting APP levels, consistent with the mechanism of a c-secre-
tase inhibitor. Based on the sulfonamide series, we proposed a
new scaffold, whereby the alkyl alcohol moiety of structure 1 is re-
placed by a heterocycle. Initial lead 2, with a pyrazole in place of
the alkyl alcohol, had weak activity against Ab production
ment (bCTF) and then
c-secretase cleaves bCTF to form Ab pep-
tides, predominantly Ab40 and Ab42. Hindrance of Ab synthesis
(EC50 = 19
proved Ab lowering activity were identified and their mechanism
of action, as -secretase inhibitors, was demonstrated. Moreover,
lM). From this new starting point, analogs with im-
via inhibition of either of these secretases is an attractive strategy
for the treatment of AD.3,4 Several
c
-secretase inhibitors have been
c
tested in preclinical models and reductions in levels of Ab in plas-
compounds within this series demonstrated selectivity for APP
ma, brain and cerebrospinal fluid have been demonstrated. In addi-
processing over Notch processing.
tion to APP, c-secretase cleaves a range of transmembrane peptide
substrates including Notch. Inhibition of Notch processing is asso-
ciated with toxicity in the gastrointestinal tract, thymus and
spleen.5 Recently, it has been shown that the GSI clinical candidate
2. Chemistry
Several heterocyclic cores were chosen as alkyl alcohol replace-
ments of lead structure 2, including pyrazole, imidazole, triazole
and pyrrole. These derivatives were prepared with the general syn-
thetic sequence described in Scheme 1a. N-Substituted pyrazoles,
* Corresponding author. Tel.: +1 732 274 4780; fax: +1 732 274 4505.
E-mail address: resnicl@wyeth.com (L. Resnick).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.052

J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
4709
H
N
HO
N
N
Cl
O
S
O
Cl
N
O
S
O
S
O
S
S
S
HN
Cl
b
HN
HN
O
MeO
1
2
8
a
Figure 1. Structures of initial leads 1 and 2.
triazoles or imidazoles 4 (W = N, Y = CH; W = N, Y = N; W = CH,
Y = N) were prepared by treatment with base (NaH for pyrazoles
and imidazoles, DBU for triazoles) and appropriate alkyl halide or
sulfonyl chloride. Alternatively, phenyl pyrazoles 4 (R1 = phenyl),
were made by treatment of pyrazole with a phenyl boronic acid,
pyridine and cupric acetate.8 Intermediate 4 was treated with n-
BuLi9 or t-BuLi10 and the resulting lithiated heterocycle was re-
acted with an aldehyde to provide the corresponding alcohol 5. A
pyrrole analog was made from 1-(4-methoxybenzyl)-1H-pyrrole-
N
N
HN
O
S
N
O
S
S
HN
Cl
S
HN
Cl
O
O
HO
2
40
Scheme 2. Reagent and conditions: (a) TFA, anisole, CH2Cl2, reflux; (b) BBr3, CH2Cl2,
ꢀ78 °C to rt.
2-carbaldehyde (39) by treatment with
a Grignard reagent
(Scheme 1b). Conversion of pyrazole, pyrrole and imidazole alco-
hols 5 to azides 6 was performed with a Mitsunobu reaction.11
For triazole alcohols, 5, a 2-step sequence, which entailed forma-
tion of the mesylate, followed by treatment with sodium azide
was used. Standard catalytic hydrogenation of azide 6 provided
amines 7, which were converted to sulfonamides or amides by
treatment with base and a suitable sulfonyl chloride or carboxyl
chloride to give targets 8–38.
N
N
N
O
S
N
O
S
a
S
N
X
S
HN
X
O
O
OMe
OMe
Treatment of para-methoxybenzyl pyrazole 8 with TFA and ani-
sole removed the para-methoxybenzyl group to afford 2 (Scheme
2).9 Demethylation of pyrazole 8 was affected with BBr3 to afford
p-hydroxyl benzyl heterocycle 40. Analogously, p-hydroxyl phenyl
41 was made from methoxyl phenyl 28 (see Table 2 for structure).
N-Alkylation of sulfonamides 11 and 18 afforded 42 and 43
(Scheme 3).
11 (X = Cl)
18 (X = Br)
42 (X = Cl)
43 (X = Br)
Scheme 3. Reagent and conditions: (a) NaH, MeI, THF, 0 °C to rt.
chased or prepared from the corresponding amino acid,13 were
transformed to the corresponding Weinreb amides 45 by conven-
tional conditions. Treatment of 45 with ethynylmagnesium bro-
mide afforded ynones 46 which were subsequently converted to
enamino ketones 47 by reaction with diethylamine. Cycloconden-
sation with phenyl hydrazines gave pyrazoles 48 as single regioi-
somers. Deprotection by catalytic hydrogenation afforded free
amines, which were converted to sulfonamides 49–52. (See Table
4 for structures.) Hydroxyl phenyl 53 was prepared by demethyla-
tion of methoxyl phenyls 51 and 52, respectively. The correspond-
The synthesis of optically pure pyrazole targets was accom-
plished with methodology that was developed by Pirc et al.
(Scheme 4).12 N-CBz
L
-Amino acids 44, which were either pur-
Y
Y
Y
R2
OH
W
W
c
W
a
b
N
N
N
H
R1
R1
ing (R)-enantiomers were obtained from D-amino acids following
3
4
5
the same synthetic route (compounds 54, 55). The chiral purity
of these compounds were determined to be P95% as determined
by chiral HPLC.
The chiral approach was used for the synthesis of phenyl pyra-
zoles. For enantiomers 56–59 chiral HPLC was used to separate
enantiomers (Table 4).
Y
Y
Y
R2
R2
N3
R2
d
W
e
W
W
N
N
N
R1
R3
HN
NH2
Z
R1
R1
6
7
8-38
Scheme 1a. Reagents and conditions: (a) R1X, base (NaH or DBU), THF, rt or
ArB(OH)2, Cu(OAc)2, pyridine, rt; (b) (i) n-BuLi or t-BuLi, THF, ꢀ78 °C; (ii) R2CHO,
ꢀ78 °C to rt; (c) DPPA, DEAD, PPh3, THF, 0 °C to rt or (i) MsCl, pyr; (ii) NaN3, DMF;
(d) H2, Pd/C, MeOH, rt; (e) R3SO2Cl or R3COCl, Et3N, DMAP, CH2Cl2, rt.
3. Biology
Inhibition of Ab production was determined by two methods.
The first is a whole cell assay consisting of CHO cells transfected
with human APP. Compounds were assayed for inhibition of the
secretion of both Ab40 and Ab42 by a double sandwich ELISA assay,
whereby peptide was captured by monoclonal 6E10 (anchored by
goat anti-mouse IgG1) and detected by Ab40 and Ab42 carboxy
specific rabbit antibody. Secondary detection was by APL-donkey
anti-rabbit IgG.14
O
N
f
N
OH
Compounds with an EC50 of less than 10
recently developed electrochemiluminescent immuno assay
(MSD, Gaithersburg, MD). This method is an adaptation of the
sandwich ELISA assay, which is performed in liquid phase (see Sec-
lM were retested with
MeO
MeO
a
5 (pyrrole)
39
Scheme 1b. Reagents and conditions: (f) i-PrMgBr, THF, 0 °C to rt.

4710
J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
O
R2
O
N
R2
O
R2
b
a
c
NHCBz
MeO
N
NHCBz
HO
NHCBz
44
45
46
O
R2
NHCBz
R2
O
S
O
N
R2
d
e, f
N
N
R1
S
HN
Cl
R1
NHCBz
(Et)2N
49-52
47
48
Scheme 4. Reagents and conditions: (a) EDCI, NMM, MeNHOMe, CH2Cl2, rt; (b) ethynylmagnesium bromide, ꢀ78 °C to rt, THF; (c) NH(Et)2, CH2Cl2, 0 °C to rt; (d) R1NHNH2,
EtOH, HCl (37% aqueous), 80 °C; (e) H2, Pd/C, MeOH; (f) 5-chlorothiophene-2-sulfonyl chloride, TEA, CH2Cl2, rt. R1: substituted phenyl; R2: (CH3)2CH or (CH3CH2)2CH.
tion 6 for details). In general, EC50 values for Ab40 and Ab42 were
12, 13 and 11 versus 14–16. 5-Chlorothiophene or 5-bromothio-
similar, typically with less then twofold difference.
phene sulfonamides are more active than 4,5-dichlorothiophene
Notch receptor activity was assayed using a constitutively ac-
sulfonamide (11 and 18 vs 19). Replacement of the sulfonamide
tive Notch (mDE Notch) to drive expression of soluble alkaline
phosphatase (SEAP) reporter via transactivation of HES. SEAP levels
were measured in conditioned media by fluorescent assay.
linkage with an amide linkage leads to a significant decrease in
activity (12 vs 17). N-Alkylation of the sulfonamide decreases the
activity by approximately 40-fold (11 vs 42 and 18 vs 43). Similar
trends in the thiophene sulfonamide region of the molecule were
also observed in the original alkyl alcohol series represented by
lead 1.7
4. Discussion
The structure–activity relationships of the prepared compounds
will be discussed based on their Ab40-lowering effects. Measure-
ments of inhibition of Ab42 were found to be similar to that of
Ab40 (data not shown). Table 1 shows a set of pyrazole inhibitors.
From the start, it was apparent that N-substitution of the pyrazole
significantly improves activity compared to initial lead 2 and that a
variety of substituents are tolerated including, aliphatic, aromatic,
or sulfonyl (8–11). The preferred substitution on the aliphatic
nitrogen was shown to be thiophene sulfonyl. A change to phenyl
sulfonyl leads to a decrease of activity as exemplified by 8 versus
Additional targets were prepared to explore the effects of the R1
group in combination with variations of the R2 alkyl (Table 2). In
the benzyl pyrazole series, R2 can be methyl (20), straight chain al-
kyl (21), or branched chain alkyl (8, 22), with 3-pentyl derivative 8
possessing the highest activity (Ab40 EC50 = 0.13 lM). When
R1 = phenyl, the 3-pentyl alkyl chain did not provide an advantage
over the isopropyl group (11 vs 23) as it did with the benzyl deriv-
atives (8 vs 22). Replacement of the methoxyl of analog 8 with hy-
droxyl (compound 40) does not affect activity. Different R1 phenyl
substituents including 4-Me, 3-Me, 4-F, 4-CF3, 4-OMe and OH (24–
28, 41) were tolerated with the 4-Me analog 24 providing a 2.7-
fold improvement in activity compared to unsubstituted analog 23.
Replacement of the pyrazole core with other nitrogen-contain-
ing 5-membered heterocycles was investigated (Table 3). Imida-
zoles 29 and 30 and pyrrole 31 are weaker inhibitors (Ab40 EC50
Table 1
Effects of N-substituents on the pyrazole derivatives
>13 lM) than their corresponding pyrazole analogs. On the other
hand, compounds of the 1,2,4-triazole series (32–38) demonstrate
N
N
R1
R3
R4N
Z
Table 2
Structure–activity relationships of pyrazole derivatives: R1 and R2 variation
a
Compound R1
R3
R4
Z
Ab40 EC50
(lM)
R2
O
S
O
2
8
H
5-Cl-Thiophene
5-Cl-Thiophene
H
H
SO2 19.49b
N
N
R1
4-OMe-
Benzyl
Butyl
SO2
0.13
S
HN
Cl
9
5-Cl-Thiophene
5-Cl-Thiophene
5-Cl-Thiophene
4-Cl-Benzene
H
H
H
H
SO2
SO2
SO2
SO2
1.00
1.68
1.62
1.98
10
11
12
4-Me-PhSO2
Phenyl
4-OMe-
Benzyl
4-OMe-
Benzyl
Phenyl
Phenyl
Phenyl
4-OMe-
Benzyl
Phenyl
Phenyl
a
Compound
R1
R2
Ab40 EC50 (lM)
8
4-OMe-Benzyl
Phenyl
4-OMe-Benzyl
4-OMe-Benzyl
4-OMe-Benzyl
Phenyl
4-Me-Phenyl
3-Me-Phenyl
4-F-Phenyl
4-CF3-Phenyl
4-OMe-Phenyl
4-OH-Benzyl
4-OH-Phenyl
CH(CH2CH3)2
CH(CH2CH3)2
CH3
CH2CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
0.13
1.67
1.20
0.93
0.41
1.12
0.42
2.53
2.76
2.19
1.56
0.14
0.63
13
4-Br-Benzene
H
SO2
2.27
11
20
21
22
23
24
25
26
27
28
40
41
14
15
16
17
4-Cl-Benzene
H
H
H
H
SO2 22.97b
SO2 85.24b
SO2 25.51b
4-CN-Benzene
3,4-Di-Cl-benzene
4-Cl-Benzene
CO
42.36b
18
19
5-Br-Thiophene
4,5-Di-Cl-
thiophene
5-Cl-Thiophene
5-Br-Thiophene
H
H
SO2
1.53
SO2 13.36b
42
43
Phenyl
Phenyl
CH3 SO2 70.15b
CH3 SO2 65.12b
a
Unless otherwise noted, the MSD ELISA assay was used to measure Ab levels.
a
The MSD ELISA assay was used to measure Ab levels. Values are averages of
three experiments, with 10 data points in each experiment.
Values are averages of three experiments, with 10 data points in each experiment.
b
The double sandwich ELISA was used to measure Ab levels.

J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
4711
Table 3
Table 5
Heterocyclic variations
Notch selectivity
Notch sparing selectivityb
a
Y
Compound
Ab40 EC50
(lM)
Notch EC50 (lM)
R2
O
S
O
W
49
58
56
51
52
53
0.32
0.092
0.11
0.55
0.99
0.47
12.56
1.8
2.65
6.12
3.97
3.26
39
20
24
11
4
N
S
HN
Cl
R1
6.9
a
Compound
W
Y
R1
R2
Ab40 EC50 (lM)
a
Values are averages of 2 experiments with 10 data points in each experiment.
Notch sparing selectivity = notch EC50/Ab40 EC50
8
N
CH
N
N
CH
N
N
N
N
N
N
4-OMe-Benzyl
4-OMe-Benzyl
Benzyl
4-OMe-Benzyl
4-OMe-Benzyl
Benzyl
4-Me-Benzyl
4-OCF3-Benzyl
4-F-Benzyl
Benzyl
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH2CH3)2
CH(CH3)2
0.13
50.63b
19.4b
13.32b
0.94
1.01
0.88
0.99
0.48
b
.
29
30
31
32
33
34
35
36
37
38
CH
CH
CH
N
N
N
N
N
N
N
To determine the effect of stereochemistry on the activity, sev-
eral optically pure analogs were tested (Table 4). It was found that
the S enantiomers (compounds 49–53, 56, 58) are much more ac-
tive than the R enantiomers (e.g., 54, 55, 57, 59). This is in agree-
ment with the original alkyl alcohol series.7
Importantly, compounds within this series are selective for
inhibition of APP processing versus Notch processing, as calculated
by the Notch sparing selectivity, which is the ratio of Notch EC50 to
Ab40 EC50 (Table 5). Notably, compound 49 demonstrates 39-fold
selectivity and compounds 58 and 56 demonstrate ꢁ20-fold
selectivity.
3.36
2.89
N
4-Me-Benzyl
CH(CH3)2
a
Unless otherwise noted, the MSD ELISA assay was used to measure Ab levels.
Values are averages of three experiments, with 10 data points in each experiment.
b
The double sandwich ELISA was used to measure Ab levels.
activities that are similar to those of the pyrazole series. From
these results, it is evident that a nitrogen in the W position gives
improved activity compared to carbon–hydrogen in this position.
For the triazole series, as in the benzyl pyrazole series, compounds
with the CH(CH2CH3)2 side chain are more active than those with
the CH(CH3)2 side chain (34 vs 38 and 33 vs 37), although the fold
improvement is not as large (ꢁ7 vs 2–3).
To show that c-secretase is the mechanism by which this series
of compounds inhibits Ab production, experiments which measure
CTF and APP levels were run on representative compounds (data
not shown). Increased levels of CTF with no change in APP levels
were found, which is indicative of
c-secretase inhibition. Further
validation of mechanism was demonstrated with a
c-secretase li-
gand displacement assay. This assay employs membranes isolated
from human neuroblastoma SH-SY5Y cells and a proprietary triti-
Table 4
Stereochemistry
ated
c
-secretase inhibitor. Benchmark
c
-secretase inhibitors
-alanyl]-S-
including compound 1, N-[N-(3,5-diflurophenacetyl)-
L
phenylglycine-t-butylester (DAPT), LY41157515 and LY45013916
competitively displace this radiolabeled ligand at concentrations
comparable to their EC50 values for inhibition of Ab synthesis.
Compound 58 provides an IC50 value of 154 nM in this assay, which
is similar to its Ab lowering potency (EC50 = 92 nM). This result
R2
O
S
O
N
N
R1
S
HN
Cl
suggests that compound 58 and its analogs bind to c-secretase at
the same site as other benchmark inhibitors.
c
Compound Configuration Rotation R1
R2
Ab40 EC50
M)
(
l
49
50
51
52
53
54
55
56
57
58
59
Sa
Sa
Sa
Sa
Sa
Ra
Ra
Sb
Rb
Sb
Rb
+
4-Me-
Phenyl
4-F-
CH(CH3)2
CH(CH3)2
CH(CH3)2
0.32
1.01
0.55
5. Conclusions
+
Phenyl
4-OMe-
Phenyl
4-OMe-
Phenyl
4-OH-
Phenyl
4-Me-
Phenyl
4-F-
Phenyl
4-OMe-
Benzyl
4-OMe-
Benzyl
4-OH-
Benzyl
4-OH-
Benzyl
Starting from lead pyrazole 2, a series of small molecule hetero-
cyclic -secretase inhibitors has been discovered. Expansion of the
+
c
series, lead to compounds with up to 200-fold improvement in
activity compared to the lead. The elements to maintain good
activity in this series include N-substitution of the pyrazole, the
presence of 5-chlorothiophene sulfonamide, and (S)-configuration
at the chiral centre. Several potent analogs, including compounds
49, 56 and 58 show good selectivity for inhibition of APP versus
Notch processing. Unfortunately, characteristics such as poor solu-
bility and microsomal instability caused compounds within the
series to have poor in vivo efficacy. Further research on the series
is required to optimize drug-like characteristics.
+
CH(CH2CH3)2 0.99
+
CH(CH3)2
CH(CH3)2
CH(CH3)2
0.47
>15
ꢀ
ꢀ
+
>15d
CH(CH2CH3)2 0.11
CH(CH2CH3)2 >15
CH(CH2CH3)2 0.092
CH(CH2CH3)2 8.5
ꢀ
+
6. Experimental
ꢀ
6.1. APP stable cell line
a
Stereoisomers were obtained by chiral synthesis.
Enantiomers were isolated from racemates by chiral HPLC; stereochemical
b
The characteristics of a CHO-cell line stably expressing an APP
reporter construct containing the Swedish KM/NL mutation (APP-
Rep 751NL) were reported previously.17 Cells were plated in 96-
well plates and allowed to adhere overnight in Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10% certified fetal
assignment deduced from optical rotation compared with similar analogs of known
stereochemical assignment.
c
Unless otherwise noted, the MSD ELISA assay was used to measure Ab levels.
Values are averages of three experiments, with 10 data points in each experiment.
d
The double sandwich ELISA was used to measure Ab levels.

4712
J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
bovine serum. For compound testing, compounds were diluted
from stock solutions in dimethylsulfoxide (DMSO) to yield a final
concentration of 0.1% DMSO in media. Cells were treated for 24 h
at 37 °C with compounds.
0–2
92 pM GS. Following 1 h incubation at room temperature with shak-
ing, 180 L reactions were transferred to the filter plate. The filter
plate was then vacuum filtrated, followed by washing 4 times with
200 L wash buffer (5 mM Tris–HCl, pH 7.4) and vacuum dried for 5
addition min. The filter plate bottom was removed and the plate was
lM or 0–10 lM test compound, 1% DMSO and estimated
l
l
6.2. Cellular Ab assay
blotted dry with paper towel. Microscint-20 (30 lL) was added and
Ab in conditioned media was measured by sandwich Ab40 and
Ab42 end-specific ELISA using monoclonal antibody 6E10 (Signet/
Covance Labs) for capture, and rabbit C-terminal specific antibod-
ies (Biosource) coupled with an alkaline phosphatase-conjugated
anti-Rabbit detection antibody and attophos (alkaline phosphatase
substrate) for detection. ELISA plates were read using a Cytofluor
fluorescence plate reader to determine the concentration of the
fluorescent product of the alkaline phosphatase activity. Standard
curves were made using synthetic Ab40 and Ab42 peptides (Ana-
Spec). Reductions in Ab levels were measured relative to control
cells treated with 0.1% DMSO and expressed as a percentage inhi-
bition. Data from 5 doses in triplicate were fitted to a four-param-
eter logistical model using LSW software in order to determine
EC50 values. Cells from the same assay were washed 2ꢂ in PBS
and a CellTiter 96 Aqueous Non-Radioactive Cell Proliferation As-
say (MTS: Promega, Madison, WI) solution was plated onto the
cells to assess cell viability. After 1 h, plates were read on a plate
reader at 560 nm.
3H radioactivity was counted on a Microbeta (Wallac Model# 1450).
Data were analyzed in GaphPad Prism 4.0 using the following equa-
tion: Counts ¼ a þ ðb ꢀ aÞ ꢃ ICn50=ðConcn þ IC5n0Þ, in which n is the
Hill number and IC50 is the compound concentration that yields
50% competition.
6.5. Notch functional assay
A constitutively active mouse Notch construct with a deletion of
the extracellular domain containing the M1726V mutation as pre-
viously described18 and a reporter construct employing soluble
alkaline phosphatase driven by the Hes1 promoter (pSEAP-Basic
vector, BD Biosciences) were generated. CHO K1 cells were tran-
siently transfected with both constructs using Polyfect (Qiagen)
transfection reagent in Opti-MEM media. Cells were plated in 96
well plates and treated with compounds for 48 h. SEAP levels in
the conditioned media were assessed using the Great EscAPe SEAP
Chemiluminescence detection kit (Clontech) according to manu-
facturer’s instruction. Briefly, 15 lL of conditioned media were
6.3. MDS cellular Ab assay
mixed with a dilution buffer and incubated at 65 °C for 45 min.
After cooling, assay buffer was added, and the samples were incu-
bated with CSPD substrate. Luminescence was measured in a Wal-
lac 1450 Victor luminescence counter.
Human Ab40 and Ab42 in conditioned medium were measured
by a double-sandwich Meso Scale Discovery (MSD) based homog-
enous immunoassay. Briefly, 96-well streptavidin coated MSD
plates (MSD, Gaithersburg, MD) were washed three times in Tris
buffered saline containing 0.1% Tween 20 (TBST). Twenty microli-
tres samples or standards were loaded into wells together with
6.6. Chemistry—general details
NMR spectra were determined on a 400 MHz Varian Unity Plus.
Mass spectra were recorded on a Micromass LCT mass spectrome-
ter. HRMS were recorded in FAB mode on an Agilent mass spec-
trometer. Elemental analysis was taken by Robertson Microlit
Inc. using Perkin–Elmer elemental 2400 CHN analyzer. Optical
rotation was recorded with a Jasco P-1020 polarimeter with a
30 mm cell. IR was obtained on an Avatar 360 FT-IR. Melting points
were taken on a Mel-Temp 3.0 capillary melting point apparatus
and were uncorrected. All reagents were purchased from commer-
cial sources and used without further purification.
20 lL antibody mastermix [Ab40: 1/1000 biotinylated 6E10 (Sig-
net), 1/5000 anti-human Ab40 rabbit polyclonal antibody (Bio-
source) and 1/2000 anti-rabbit MSD-Tag (MSD) in MSD blocker
A; or Ab42: 1/500 biotinylated 6E10 (Signet), 1/625 anti-human
Ab42 rabbit polyclonal antibody (Biosource) and 1/1250 anti-rab-
bit MSD-SulfoTag (MSD) in MSD blocker A]. Plates were incubated
overnight at 4 °C while shaking. The plates were then washed three
times in TBST and 150
lL of MSD read buffer-T was added to each
well. Plates were read in a Sector Imager 6000.
6.4. Radiolabeled c-secretase binding and displacement assay
6.6.1. General synthesis for pyrazoles 8–28 and imidazoles 29,
30
A tritiated analog of compound 1 ([3H] 1-analog) was prepared
in-house as an 11.00 mCi/mL ethanol stock solution with the spe-
cific activity of 26.6 Ci/mol and the radiochemical purity of >99%.
Step A. To the corresponding N-substituted pyrazole8,9 or imid-
azole (4) (12.9 mmol) in THF (100 mL) at ꢀ78 °C was added a solu-
tion of n-BuLi (2 M in cyclohexane, 7.1 mL, 14.2 mmol) dropwise.
After 75 min, the corresponding aldehyde (10.6 mmol) was added.
After 45 min the reaction mixture was quenched with saturated
aqueous ammonium chloride (50 mL), extracted with EtOAc
(3 ꢂ 100 mL), dried (Na2SO4) and concentrated. Column chroma-
tography provided alcohol 5.
c-Secretase enzyme was prepared in-house as CHAPSO-solubilized
microsomes from human neuroblastoma SH-SY5Y cells. Filter
plates were purchased from Millipore. Microscint-20 was pur-
chased from Perkin–Elmer. Polyethyleneimine solution (PEI, 50%
solution in water) was purchased from Sigma. Multiscreen 96-well
FB filter plate was prepared by incubating with 200
v) PEI solution at 4 °C overnight. The filter plate was vacuum fil-
trated and washed twice with 200 L of water and twice with
200 L of assay buffer (25 mM MES, pH 6.5, 1 mM EDTA, 0.01% b-
lL of 0.6% (w/
Step B. To the appropriate alcohol 5 (4.20 mmol) in THF (40 mL)
at 0 °C was added triphenylphosphine (1.65 g, 6.29 mmol), diethyl
azodicarboxylate (1.0 mL, 6.3 mmol) and diphenylphosphoryl
azide (1.4 mL, 6.3 mmol). The reaction mixture was warmed to
room temperature and stirred for 15.5 h. The solvent was removed
in vacuo and the resulting residue was purified by column chroma-
tography to give azide 6.
Step C. Azide 6 (2.39 mmol) in MeOH (24 mL) with 5% Pd/C
(75 mg) was hydrogenated under atm pressure for 24 h. The reac-
tion mixture was filtered through a plug of Celite and the filtrate
was concentrated to give amine 7 as an oil.
l
l
mercaptoethanol, 0.01% BSA). Reactions were set up in Costar 96-
well clear polypropylene plates (Corning) in the following addition
sequence: 160
in the assay buffer, 20
DMSO and 90% assay buffer) and 20
l
L
c
-secretase containing microsomes diluted 1:10
L test compound serial dilution in 10%
L 250 nM [3H] GSI-Analog
l
l
in the assay buffer. Each plate contained 3 test compounds and
cold-compound 1-analog (as positive control) in duplicate. The
final concentrations in the reactions were 25 nM [3H] 1-analog,

J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
4713
StepD. To amine 7 (1.74 mmol) in CH2Cl2 (15 mL) was added tri-
ethylamine (0.29 mL, 2.1 mmol) and the appropriate sulfonyl chlo-
ride or carbonyl chloride (2.09 mmol). After 1 h to 2 days the
reaction mixture was diluted with EtOAc (30 mL), washed with
1 N HCl (30 mL), dried (Na2SO4) and concentrated. Column chro-
matography provided target compounds.
6.6.1.7.
4-Chloro-N-[2-ethyl-1-(2-phenyl-2H-pyrazol-3-yl)-
butyl]-benzenesulfonamide (14). 1H NMR (400 MHz, DMSO-d6):
d 8.34 (d, 1H, J = 6.86 Hz), 7.63–7.61 (m, 2H), 7.56–7.51 (m, 4H),
7.49–7.44 (d, 1H, J = 7.5 Hz), 7.38 (s, 1H), 7.24 (d, 2H, J = 7.47 Hz),
6.27 (s, 1H), 4.48 (m, 1H), 1.33 (m, 1H), 1.19 (m, 2H), 1.09–1.02 (m,
2H), 0.56 (t, 3H, J = 6.9 Hz), 0.46 (t, 3H, J = 7.01 Hz). MS (+ESI):
418.1 (M+H)+. Anal. Calcd for C21H24ClN3O2S: C, 60.35; H, 5.79; N,
10.05. Found: C, 60.11; H, 6.00; N, 10.11.
6.6.1.1. 5-Chloro-N-{2-ethyl-1-[1-(4-methoxybenzyl)-1H-pyra-
zol-5-yl]butyl}thiophene-2-sulfonamide (8). 1H NMR (400 MHz,
DMSO-d6): d 0.62 (t, 3H, J = 7.4 Hz), 0.70 (t, 3H, J = 7.3 Hz), 0.93–
1.04 (m, 2H), 1.16–1.40 (m, 3H), 3.71 (s, 3H), 4.46 (m, 1H), 5.20
(q, 2H, J = 15.8 Hz), 6.12 (d, 1H, J = 1.8 Hz), 6.84 (d, 1H, J = 4.1 Hz),
6.88 (d, 2H, J = 8.7 Hz), 6.95 (d, 1H, J = 4.0 Hz), 7.05 (d, 2H,
J = 8.7 Hz), 7.24 (d, 1H, J = 1.8 Hz), 8.46 (d, 1H, J = 9.5 Hz); MS
(ꢀESI): 466 (MꢀH)ꢀ. Anal. Calcd for C21H26ClN3O3S2: C, 53.89; H,
5.60; N, 8.98. Found: C, 53.96; H, 5.72; N, 8.66.
6.6.1.8.
4-Cyano-N-[2-ethyl-1-(2-phenyl-2H-pyrazol-3-yl)-
butyl]-benzenesulfonamide (15). 1H NMR (400 MHz, DMSO-
d6): d 8.51 (d, 1H, J = 9.61 Hz), 7.97 (d, 2H, J = 8.39 Hz), 7.75 (d,
2H, J = 8.08 Hz), 7.57 (d, 2H, J = 7.4 Hz), 7.55 (s, 1H), 7.35 (s,
1H), 7.25 (d, 2H, J = 6.2 Hz), 6.21 (s, 1H), 4.52 (m, 1H), 1.36 (m,
1H), 1.11 (m, 2H), 1.05–1.02 (m, 2H), 0.59 (t, 3H, J = 6.7 Hz)
0.48 (t, 3H, J = 7.05 Hz). MS (+ESI): 409.1 (M+H)+. Anal. Calcd for
C22H24N4O2S: C, 64.68; H, 5.92; N, 13.71. Found: C, 64.36; H,
5.86; N, 13.70.
6.6.1.2. N-[1-(1-Butyl-1H-pyrazol-5-yl)-2-ethylbutyl]-5-chloro-
thiophene-2-sulfonamide (9). 1H NMR (400 MHz, DMSO-d6): d
0.76 (t, 3H, J = 7.2 Hz), 0.82 (t, 3H, J = 7.4 Hz), 0.88 (t, 3H,
J = 7.4 Hz), 1.07–1.19 (m, 1H), 1.20–1.33 (m, 4H), 1.34–1.48 (m,
2H), 1.59–1.70 (m, 2H), 3.81–3.94 (m, 2H), 4.44 (m, 1H), 6.09 (d,
1H, J = 1.7 Hz), 7.02 (d, 1H, J = 4.1 Hz), 7.10 (d, 1H, J = 4.0 Hz),
7.19 (d, 1H, J = 1.7 Hz), 8.53 (d, 1H, J = 9.17 Hz); MS (ꢀESI): 402
(MꢀH)ꢀ. Anal. Calcd for C19H22ClN3O3S2: C, 50.54; H, 6.49; N,
10.40. Found: C, 50.58; H, 6.65; N, 10.31.
6.6.1.9. 3,4-Dichloro-N-[2-ethyl-1-(2-phenyl-2H-pyrazol-3-yl)-
butyl]-benzenesulfonamide (16). 1H NMR (400 MHz, DMSO-d6):
d 8.44 (d, 1H, J = 8.23 Hz), 7.75 (d, 1H, J = 8.4 Hz), 7.72 (1H, d,
J = 2.2 Hz), 7.58–7.48 (m, 4H), 7.38 (s, 1H), 7.25 (d, 2H, J = 7.6 Hz),
6.27 (s, 1H), 4.48 (m, 1H), 1.39–1.31 (m, 1H), 1.28–1.15 (m, 2H),
1.13–1.07 (m, 2H), 0.61 (t, 3H, J = 6.89 Hz), 0.51 (t, 3H,
J = 7.07 Hz). MS (+ESI): 452.1 (M+H)+. Anal. Calcd for C21H23N3O2S:
C, 55.75; H, 5.12; N, 9.29. Found: C, 55.72; H, 5.15; N, 9.08.
6.6.1.3. 5-Chloro-N-(2-ethyl-1-{1-[(4-methylphenyl)sulfonyl]-
1H-pyrazol-5-yl}butyl)thiophene-2-sulfonamide (10). 1H NMR
6.6.1.10. 4-Chloro-N-{2-ethyl-1-[2-(4-methoxy-benzyl)-2H-pyr-
azol-3-yl]-butyl}-benzamide (17). 1H NMR (400 MHz, DMSO-
d6): d 8.69 (1H, d, J = 9.01 Hz), 7.79 (2H, d, J = 8.56 Hz), 7.50
(2H, d, J = 8.55 Hz), 7.41 (1H, s), 7.08 (2H, d, J = 8.56 Hz), 6.83
(2H, d, J = 8.55 Hz), 6.29 (1H, s), 5.40 (2H, q, J = 15.5 Hz), 5.20
(1H, t, J = 9.4 Hz), 3.68 93H, s), 1.87 (1H, m), 1.55 (1H, m), 1.35
(1H, m), 0.99 (1H, m), 0.89 (1H, m), 0.75 (3H, t, J = 7.3 Hz),
0.58 (3H, t, J = 7.2 Hz). MS (+ESI): 426.3 (M+H)+. Calcd for
C24H28ClN3O2.
(400 MHz, DMSO-d6):
d 0.68 (t, 3H, J = 7.4 Hz), 0.94 (t, 3H,
J = 7.4 Hz), 1.10–1.30 (m, 4H), 1.42–1.57 (m, 1H), 2.41 (s, 3H),
5.28 (dd, 1H, J = 4.2, 10.0 Hz), 6.37 (s, 1H), 6.91 (d, 1H, J = 4.0 Hz),
6.95 (d, 1H, J = 4.0 Hz), 7.51 (d, 2H, J = 8.2 Hz), 7.61 (s, 1H), 7.81
(d, 2H, J = 8.2 Hz), 8.51 (d, 1H, J = 10.2 Hz). MS (ꢀESI): 500 (MꢀH)ꢀ.
Anal. Calcd for C20H24ClN3O4S3: C, 47.85; H, 4.82; N, 8.37. Found: C,
48.00; H, 4.75; N, 8.38.
6.6.1.4.
5-Chloro-N-[2-ethyl-1-(1-phenyl-1H-pyrazol-5-yl)-
(11). 1H
NMR (400 MHz,
butyl]thiophene-2-sulfonamide
6.6.1.11.
butyl]thiophene-2-sulfonamide
5-Bromo-N-[2-ethyl-1-(1-phenyl-1H-pyrazol-5-yl)-
(18). 1H
NMR (400 MHz,
DMSO-d6): d 8.60 (d, 1H, J = 9.3 Hz), 7.57 (t, 2H, J = 7.6 Hz), 7.51
(m, 1H), 7.47 (m, 1H), 7.34 (m, 2H), 7.22 (d, 1H, J = 4.0 Hz), 7.12
(d, 1H, J = 4.0 Hz), 6.36 (m, 1H), 4.57 (m, 1H), 1.31 (m, 1H), 1.16
(m, 2H), 1.04 (m, 2H), 0.58 (t, 3H, J = 7.3 Hz), 0.48 (t, 3H,
J = 7.3 Hz); MS (+ESI): 424 (MꢀH)ꢀ. Anal. Calcd for C19H22N3O2ClS2:
C, 53.82; H, 5.23; N, 9.91. Found: C, 54.07; H, 5.31; N, 9.87.
DMSO-d6): d 8.58 (d, 1H, J = 8.7 Hz), 7.59–7.52 (m, 2H), 7.50–7.49
(d, 1H, J = 7.2 Hz), 7.46 (s, 1H), 7.34 (d, 2H, J = 8.2 Hz), 7.21 (d,
1H, J = 5.02 Hz), 7.17 (d, 1H, J = 5.01 Hz), 6.35 (s, 1H), 4.56 (m,
1H), 1.34–1.32 (m, 1H), 1.19–1.11 (m, 2H), 1.10–1.01 (m, 2H),
0.55 (t, 3H, J = 7.05 Hz), 0.49 (t, 3H, J = 6.99 Hz). MS (+ESI): 468.1
(M+H)+. Anal. Calcd for C19H22BrN3O2S2: C, 48.72; H, 4.73; N,
8.97. Found: C, 48.72; H, 4.31; N, 8.84.
6.6.1.5. 4-Chloro-N-{2-ethyl-1-[2-(4-methoxy-benzyl)-2H-pyra-
zol-3-yl]-butyl}-benzenesulfonamide (12). 1H NMR (400 MHz,
DMSO-d6): d 0.61 (t, 3H, J = 7.2 Hz), 0.65 (t, 3H, J = 7.5 Hz), 0.79–
0.99 (m, 2H), 1.17–1.36 (m, 3H), 3.72 (s, 3H), 4.39 (t, 1H,
J = 7.6 Hz), 5.02 (q, 2H, J = 15.8 Hz), 6.01 (d, 1H, J = 1.4 Hz), 6.87
(d, 2H, J = 8.8 Hz), 6.99 (d, 2H, J = 8.8 Hz), 7.14 (d, 1H, J = 1.9 Hz),
7.39 (s, 4H), 8.19 (d, 1H, J = 8.6 Hz); MS (+ESI): 462.1 (M+H)+. Anal.
Calcd for C23H28ClN3O3S2: C, 59.79; H, 6.11; N, 9.10. Found: C,
54.69; H, 5.89; N, 8.95.
6.6.1.12. 4,5-Dichloro-N-[2-ethyl-1-(1-phenyl-1H-pyrazol-5-yl)-
butyl]thiophene-2-sulfonamide (19). 1H NMR (400 MHz, DMSO-
d6): d 8.76 (d, 1H, J = 9.4 Hz), 7.60–7.53 (m, 2H), 7.51–7.48 (m, 2H),
7.36 (d, 2H, J = 8.4 Hz), 7.29 (s, 1H), 6.35 (s, 1H), 4.55 (m, 1H), 1.37–
1.31 (m, 1H), 1.29–1.24 (m, 2H), 1.19–1.15 (m, 1H), 1.11–1.05 (m,
1H), 0.62 (t, 3H, J = 7.01 Hz), 0.52 (t, 3H, J = 6.93 Hz). MS (+ESI):
458.1 (M+H)+. Anal. Calcd for C19H21Cl2N3O2S2: C, 49.78; H, 4.62;
N, 9.17. Found: C, 51.52; H, 4.97; N, 8.56. 1H NMR is included in
the Supplementary data as an indication of purity.
6.6.1.6. 4-Bromo-N-{2-ethyl-1-[1-(4-methoxybenzyl)-1H-pyra-
zol-5-yl]butyl}-benzene sulfonamide (13). 1H NMR (400 MHz,
DMSO-d6): d 0.61 (t, 3H, J = 7.3 Hz), 0.65 (t, 3H, J = 7.3 Hz), 0.84–
0.99 (m, 2H), 1.17–1.36 (m, 3H), 3.72 (s, 3H), 4.40 (t, 1H,
J = 7.5 Hz), 5.05 (q, 2H, J = 15.7 Hz), 6.01 (d, 1H, J = 1.5 Hz), 6.87
(d, 2H, J = 8.6 Hz), 6.99 (d, 2H, J = 8.6 Hz), 7.14 (d, 1H, J = 1.7 Hz),
7.32 (d, 2H, J = 8.4 Hz), 7.53 (d, 2H, J = 8.4 Hz), 8.21 (d, 1H,
8.9 Hz); MS (+ESI): 506 (M+H)+. Anal. Calcd for C23H28BrN3O3S2C:
54.55; H, 5.57; N, 8.30. Found: C, 54.73; H, 5.56; N, 8.21.
6.6.1.13.
5-Chloro-N-{1-[1-(4-methoxybenzyl)-1H-pyrazol-5-
yl]ethyl}thiophene-2-sulfonamide (20). 1H NMR (400 MHz,
DMSO-d6): d 1.16 (d, 3H, J = 8.4 Hz) 3.71 (s, 3H), 4.59 (m, 1H),
5.23 (m, 2H), 6.15 (d, 1H, J = 1.7 Hz), 6.86 (d, 1H, J = 8.7) 7.05 (d,
2H, J = 8.7 Hz), 7.11 (d, 1H, J = 4.0 Hz), 7.21 (d, 1H, J = 4.1 Hz),
7.29 (d, 1H, J = 1.7 Hz), 8.64 (br s, 1H); MS (ꢀESI): 410 (MꢀH)ꢀ.
Anal. Calcd for C17H18ClN3O3S2: C, 49.47; H, 4.40; N, 10.20. Found:
C, 49.48; H, 4.38; N, 9.87.

4714
J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
6.6.1.14. 5-Chloro-N-{1-[1-(4-methoxybenzyl)-1H-pyrazol-5-
yl]butyl}thiophene-2-sulfonamide (21). 1H NMR (400 MHz,
DMSO-d6): d 0.62 (t, 3H, J = 7.4 Hz), 1.00–1.11 (m, 2H), 1.30–1.37
(m, 1H), 1.42–1.49 (m, 1H), 3.71 (s, 3H), 4.40 (br s, 1H), 5.17–
5.23 (m, 2H), 6.11 (d, 1H, J = 1.7 Hz), 6.88 (d, 2H, J = 8.7 Hz), 7.04
(d, 2H, J = 8.6 Hz), 7.05 (d, 1H, J = 4.4 Hz), 7.09 (d, 1H, J = 4.1 Hz),
7.27 (d, 1H, J = 1.5 Hz), 8.61 (br s, 1H); MS (+ESI): 440 (M+H)+. Anal.
Calcd for C19H22ClN3O3S2: C, 51.87; H, 5.04; N, 9.55. Found: C,
52.00; H, 5.10; N, 9.48.
4.36; N, 8.28. 1H NMR is included in the Supplementary data as
an indication of purity.
6.6.1.21. 5-Chloro-N-{2-ethyl-1-[1-(4-methoxyphenyl)-1H-pyr-
azol-5-yl]butyl}thiophene-2-sulfonamide
(28). 1H
NMR
(400 MHz, DMSO-d6): d 8.57 (d, 1H, J = 9.5 Hz), 7.41 (m, 1H), 7.24
(m, 3H), 7.10 (m, 3H), 6.31 (m, 1H), 4.50 (m, 1H), 3.83 (s, 3H),
1.31 (m, 1H), 1.19 (m, 2H), 1.06 (m, 2H), 0.59 (t, 3H, J = 7.3 Hz),
0.50 (t, 3H, J = 7.3 Hz); MS (ESI+): 454 (M+H)+. Anal. Calcd for
C20H24N3O3S2Cl: C, 52.91; H, 5.33; N, 9.26. Found: C, 53.08; H,
5.33; N, 9.16.
6.6.1.15.
5-Chloro-N-{1-[1-(4-methoxybenzyl)-1H-pyrazol-5-
yl]-2-methylpropyl}thiophene-2-sulfonamide (22). 1H NMR
(400 MHz, DMSO-d6): d 0.57 (d, 3H, J = 6.7 Hz), 0.78 (d, 3H,
J = 6.7 Hz), 1.72–1.78 (m, 1H), 3.71 (s, 3H), 4.27 (m, 1H), 5.12–
5.18 (m, 2H), 6.11 (d, 1H, J = 1.7 Hz), 6.87 (d, 2H, J = 8.6 Hz), 6.93
(d, 1H, J = 4.0 Hz), 6.97 (d, 1H, J = 4.0 Hz), 7.11 (d, 2H, J = 8.6 Hz),
7.25 (d, 1H, J = 1.8 Hz), 8.52 (d, 1H, J = 7.9 Hz); MS (+ESI): 440
(M+H)+. Anal. Calcd for C19H22ClN3O3S2: C, 51.87; H, 5.04; N,
9.55. Found: C, 51.80; H, 5.05; N, 9.32.
6.6.1.22. 5-Chloro-N-{2-ethyl-1-[1-(4-methoxybenzyl)-1H-imi-
dazol-2-yl]butyl}thiophene-2-sulfonamide
(29). 1H
NMR
(400 MHz, DMSO-d6): d 8.57 (br m, 1H), 7.08 (d, 2H, J = 8.5 Hz),
7.03 (m, 2H), 6.99 (m, 1H), 6.91 (d, 2H, J = 8.9 Hz), 6.77 (m, 1H),
5.02 (m, 2H), 4.40 (d, 1H, J = 8.2 Hz), 3.73 (s, 3H), 1.59 (m, 1H),
1.47 (m, 1H), 1.31 (m, 1H), 0.79 (m, 2H), 0.63 (t, 3H, J = 7.4 Hz),
0.57 (t, 3H, J = 7.4 Hz); MS (ES+): 468 (M+H)+. Anal. Calcd for
C21H26ClN3O3S2: C, 53.89; H, 5.60; N, 8.98. Found: C, 53.76; H,
5.41; N, 8.87.
6.6.1.16.
5-Chloro-N-[2-methyl-1-(1-phenyl-1H-pyrazol-5-
yl)propyl]thiophene-2-sulfonamide (23). 1H NMR (400 MHz,
DMSO-d6): d 8.71 (m, 1H), 7.57 (m, 2H), 7.51 (m, 2H), 7.33 (d,
2H, J = 7.1 Hz), 7.14 (d, 1H, J = 4.0 Hz), 7.11 (d, 1H, J = 4.0 Hz),
6.36 (m, 1H), 4.23 (m, 1H), 1.80 (m, 1H), 0.76 (d, 3H, J = 6.6 Hz),
0.63 (d, 3H, J = 6.7 Hz); MS (+ESI): 396 (M+H)+. Anal. Calcd for
C17H18N3O2ClS2: C, 51.57; H, 4.58; N, 10.61. Found: C, 51.53; H,
4.61; N, 10.55.
6.6.1.23.
N-[1-(1-Benzyl-1H-imidazol-2-yl)-2-ethylbutyl]-5-
chlorothiophene-2-sulfonamide (30). 1H NMR (400 MHz,
DMSO-d6): d 8.51 (br s, 1H), 7.29 (m, 2H), 7.26 (m, 1H), 7.08 (m,
2H), 6.99 (m, 3H), 6.75 (s, 1H), 5.05 (m, 2H), 4.33 (d, 1H,
J = 8.05 Hz), 1.51 (m, 1H), 1.41 (m, 1H), 1.26 (m, 1H), 0.69 (m,
2H), 0.55 (t, 3H, J = 7.44 Hz), 0.48 (t, 3H, J = 7.38 Hz). MS (ES+):
438 (M+H)+. Anal. Calcd for C20H24ClN3O2S2: C, 54.84; H, 5.52; N,
9.59. Found: C, 54.64; H, 5.58; N, 9.51.
6.6.1.17. 5-Chloro-N-{2-methyl-1-[1-(4-methylphenyl)-1H-pyr-
azol-5-yl]propyl}thiophene-2-sulfonamide
(24). 1H
NMR
(400 MHz, DMSO-d6): d 8.69 (d, 1H, J = 8.1 Hz), 7.48 (m, 1H),
7.36 (d, 2H, J = 7.9 Hz), 7.18 (d, 2H, J = 8.2 Hz), 7.13 (d, 1H,
J = 4.1 Hz), 7.11 (d, 1H, J = 4.1 Hz), 6.34 (m, 1H), 4.21 (m, 1H),
2.40 (s, 3H), 1.80 (m, 1H), 0.76 (d, 3H, J = 6.7 Hz), 0.63 (d, 3H,
J = 6.7 Hz); MS (ꢀESI): 408 (MꢀH)ꢀ. Anal. Calcd for
C18H20N3O2ClS2: C, 52.74; H, 4.92; N, 10.25. Found: C, 51.55; H,
5.0; N, 9.19.
6.6.1.24. 5-Chloro-thiophene-2-sulfonic acid [2-ethyl-1-(2H-
pyrazol-3-yl)-butyl]-amide (2). To
a solution of 8 (0.09 g,
0.192 mmol) in anhydrous CH2Cl2 (2 mL) was added anisole
(0.21 mL, 1.92 mmol) and trifluoroacetic acid (0.074 mL,
0.96 mmol). The mixture was heated to reflux for 18 h. The solvent
was removed under reduced pressure. Column chromatography
(EtOAc/hexane, 1:1) afforded 2 (0.04 g, 60%) as a white solid. 1H
NMR (400 MHz, DMSO-d6): d 12.41 (br s, 1H), 8.29 (br s, 1H), 7.45
(s, 1H), 7.07 (s, 1H), 6.98 (s, 1H), 5.99 (s, 1H), 4.36 (m, 1H), 1.48–
1.19 (m, 3H), 1.02–0.91 (m, 2H), 0.70 (t, 3H, J = 7.3 Hz), 0.62 (t, 3H,
J = 7.4 Hz); MS (+ESI) 348.0 (M+H)+. Anal. Calcd for C13H18ClN3O2S2:
C, 44.88; H, 5.22; N, 12.08. Found: C, 45.13; H, 5.10; N, 12.06.
6.6.1.18. 5-Chloro-N-{2-methyl-1-[1-(3-methylphenyl)-1H-pyr-
azol-5-yl]propyl}thiophene-2-sulfonamide
(25). 1H
NMR
(400 MHz, DMSO-d6): d 8.73 (br s, 1H), 7.51 (m, 1H), 7.44 (t, 1H,
J = 7.8 Hz), 7.31 (d, 1H, J = 7.6 Hz), 7.12 (m, 3H), 7.08 (s, 1H), 6.38
(m, 1H), 4.23 (m, 1H), 2.39 (s, 3H), 1.81 (m, 1H), 0.77 (d, 3H,
J = 6.7 Hz), 0.64 (d, 3H, J = 6.8 Hz). MS (ꢀESI): 408 (MꢀH)ꢀ. Anal.
Calcd for C18H20N3O2ClS2: C, 52.74; H, 4.92; N, 10.25. Found: C,
52.71; H, 4.94; N, 9.95.
6.6.1.25. 5-Chloro-N-{1-[1-(4-methoxybenzyl)-1H-pyrrol-2-yl]-
2-methylpropyl}thiophene-2-sulfonamide
(31). 1H
NMR
(400 MHz, DMSO-d6): d 0.53 (d, 3H, J = 6.6 Hz), 0.80 (d, 3H,
J = 6.6 Hz), 1.73–1.77 (m, 1H), 3.73 (s, 3H), 4.11 (t, 1H, J = 7.8 Hz),
5.87 (s, 2H), 5.86–5.89 (m, 1H), 5.91–5.94 (m, 1H), 6.57 (s, 1H),
6.87 (d, 2H, J = 8.7 Hz), 6.92 (d, 1H, J = 4.0 Hz), 7.01 (d, 2H,
J = 8.7 Hz), 8.34 (d, 1H, J = 9.0 Hz). MS (+ESI): 439 (M+H)+. Anal.
Calcd for C20H23ClN2O3S2: C, 54.72; H, 5.28; N, 6.38. Found: C,
55.28; H, 5.41; N, 6.24.
6.6.1.19. 5-Chloro-N-{1-[1-(4-fluorophenyl)-1H-pyrazol-5-yl]-
2-methylpropyl}thiophene-2-sulfonamide
(26). 1H
NMR
(400 MHz, DMSO-d6): d 8.72 (m, 1H), 7.51 (m, 1H), 7.42 (m, 2H),
7.37 (m, 2H), 7.16 (d, 1H, J = 4.1 Hz), 7.12 (d, 1H, J = 4.1 Hz), 6.35
(m, 1H), 4.16 (m, 1H), 1.80 (m, 1H), 0.77 (d, 3H, J = 6.7 Hz), 0.63
(d, 3H, J = 6.7 Hz); MS (ꢀESI): 412 (MꢀH)ꢀ. Anal. Calcd for
C17H17N3O2ClFS2: C, 49.33; H, 4.14; N, 10.15. Found: C, 49.41; H,
3.99; N, 9.97.
6.6.2. General procedure for triazoles 32–38
Step A. To
a stirred solution of 1,2,4-triazole (0.803 g,
11.6 mmol) in THF (100 mL) at 0 °C was added the corresponding
benzyl bromide (10.6 mmol), then 1,8-diazabicyclo[5.4.0]undec-
7-ene (1.9 mL, 12.7 mmol) and the mixture was slowly warmed
to room temperature. The solution was concentrated in vacuo, then
purified by column chromatography (SiO2, MeOH/EtOAc, 5:95).
StepB. To a stirred solution of benzyl triazole 4 (2.82 mmol) in THF
(30 mL) at ꢀ78 °C was added a solution of n-butyllithium (2.5 M in
hexanes, 1.1 mL, 2.9 mmol) dropwise. The mixture was stirred at
ꢀ78 °C for 1.5 h, then the corresponding aldehyde (3.4 mmol) was
added. The solution was slowly warmed to room temperature and
6.6.1.20. 5-Chloro-N-(2-ethyl-1-{1-[4-(trifluoromethyl)phenyl]-
1H-pyrazol-5-yl}butyl)thiophene-2-sulfonamide (27). 1H NMR
(400 MHz, DMSO-d6): d 8.62 (d, 1H, J = 8.0 Hz), 7.93 (d, 2H,
J = 8.4 Hz), 7.56 (d, 2H, J = 8.2 Hz), 7.51 (m, 1H), 7.19 (d, 1H,
J = 4.0 Hz), 7.06 (d, 1H, J = 4.1 Hz), 6.37 (m, 1H), 4.50 (m, 1H),
1.28 (m, 1H), 1.19 (m, 2H), 1.01 (m, 2H), 0.54 (t, 3H, J = 7.3 Hz),
0.48 (t, 3H, J = 7.3 Hz); MS (ꢀESI): 490 (MꢀH)ꢀ. Anal. Calcd for
C20H21N3O2ClF3S2: C, 48.83; H, 4.30; N, 8.54. Found: C, 49.26; H,

J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
4715
stirred for 18 h. It was then quenched with saturated aqueous NH4Cl
and extracted twice with EtOAc. The organic extracts were dried
(MgSO4), filtered, concentrated in vacuo and purified by column
chromatography (SiO2, EtOAc/hexane, 1:1) to give alcohol 5.
Step C. To a stirred solution of alcohol 5 (1.32 mmol) in pyridine
(15 mL) at 0 °C was added methanesulfonyl chloride (0.61 mL,
7.9 mmol) and the solution was slowly warmed to room tempera-
ture. The mixture was diluted with H2O, extracted twice with
EtOAc and dried (MgSO4), filtered and concentrated in vacuo to
give methane sulfonate which was used in the next step without
further purification.
C
20H22N4O3ClF3S2: C, 45.93; H, 4.24; N, 10.71. Found: C, 46.35; H,
4.11; N, 10.63.
6.6.2.5.
5-Chloro-N-{2-ethyl-1-[1-(4-fluorobenzyl)-1H-1,2,4-
triazol-5-yl]butyl}thiophene-2-sulfonamide
(36). 1H
NMR
(400 MHz, DMSO-d6): d 8.83 (br m, 1H), 7.79 (s, 1H), 7.22 (m,
2H), 7.11 (m, 3H), 7.01 (d, 1H, J = 4.0 Hz), 5.30 (s, 2H), 4.47 (d,
1H, J = 8.4 Hz), 1.56 (m, 1H), 1.39 (m, 1H), 1.29 (m, 1H), 0.79 (m,
1H), 0.61 (m, 1H), 0.59 (t, 3H, J = 7.4 Hz), 0.52 (t, 3H, J = 7.3 Hz);
MS (+ESI): 457 (M+H)+. Anal. Calcd for C19H22N4O2S2ClF: C,
49.94; H, 4.85; N, 12.26. Found: C, 50.24; H, 4.84; N, 12.23.
Step D. To a stirred solution of the methane sulfonate pre-
pared above (1.42 mmol) in DMF (10 mL) was added sodium
azide (0.277 g, 4.26 mmol) and the mixture was heated to reflux
for 3 h. After cooling, the reaction mixture was diluted with
H2O and extracted with EtOAc twice. The organic extracts were
dried (MgSO4), filtered, concentrated in vacuo and purified by
column chromatography (SiO2, EtOAc/hexane, 1:9) to give azide
6.
Step E. Azide 6 (2.39 mmol) in MeOH (24 mL) with 5% Pd/C
(75 mg) was hydrogenated under atm pressure for 24 h. The reac-
tion mixture was filtered through a plug of Celite and the filtrate
was concentrated to give amine 7. To amine 7 (1.74 mmol) in
CH2Cl2 (15 mL) was added triethylamine (0.29 mL, 2.1 mmol) and
5-chloro-2-sulfonyl chloride (2.09 mmol). After 1–24 h the reac-
tion mixture was diluted with EtOAc (30 mL), washed with 1 N
HCl (30 mL), dried (Na2SO4) and concentrated. Column chromatog-
raphy provided target compounds.
6.6.2.6. N-[1-(1-Benzyl-1H-1,2,4-triazol-5-yl)-2-methylpropyl]-
5-chlorothiophene-2-sulfonamide (37). 1H NMR (400 MHz,
DMSO-d6): d 0.46 (d, 3H, J = 6.7 Hz), 0.85 (d, 3H, J = 6.6 Hz), 1.85–
2.02 (m, 1H), 4.22–4.26 (m, 1H), 5.38 (d, 2H, J = 7.5 Hz), 7.08 (d,
1H, J = 4.0 Hz), 7.18 (s, 1H), 7.25 (d, 2H, J = 6.9 Hz), 7.31–7.36 (m,
3H), 7.85 (s, 1H), 8.84 (s, 1H); MS (ꢀESI): 409 (MꢀH)ꢀ. Anal. Calcd
for C17H19ClN4O2S2: C, 49.69; H, 4.66; N, 13.63. Found: C, 49.85; H,
4.71; N, 13.55.
6.6.2.7. 5-Chloro-N-{2-methyl-1-[1-(4-methylbenzyl)-1H-1,2,4-
triazol-5-yl]propyl}thiophene-2-sulfonamide (38). General pro-
cedure I from 5w to form azide, then general procedure C to form
the amine, followed by general procedure D. 1H NMR (400 MHz,
DMSO-d6): d 0.85 (d, 6H, J = 7.8 Hz), 1.97–2.02 (m, 1H), 2.28 (s,
3H), 4.38 (t, 1H, J = 7.8 Hz), 5.31 (d, 2H, J = 5.7 Hz), 7.08 (d, 1H,
J = 4.0 Hz), 7.15–7.17 (m, 5H), 7.83 (s, 1H), 8.88 (d, 1H,
J = 7.0 Hz); MS (+ESI): 425 (M+H)+. Anal. Calcd for C18H21ClN4O2S2:
C, 50.87; H, 4.98; N, 13.18. Found: C, 51.01; H, 4.91; N, 13.15.
6.6.2.1. 5-Chloro-N-{2-ethyl-1-[1-(4-methoxybenzyl)-1H-1,2,4-
triazol-5-yl]butyl}thiophene-2-sulfonamide
(32). 1H
NMR
(400 MHz, DMSO-d6): d 0.58 (t, 3H, J = 7.4), 0.64 (t, 3H, J = 7.5 Hz),
0.71–0.86 (m, 2H), 1.24–1.37 (m, 1H), 1.40–1.47 (m, 1H), 1.62 (br
s, 1H), 3.73 (s, 3H), 4.52 (d, 1H, J = 8.1 Hz), 5.26 (s, 2H), 6.91 (d,
2H, J = 8.7 Hz), 7.05 (d, 1H, J = 4.0 Hz), 7.11 (d, 1H, J = 4.1 Hz),
7.18 (d, 2H, J = 8.7 Hz), 7.81 (s, 1H), 8.87 (br s, 1H); MS (+ESI):
469 (M+H)+. Anal. Calcd for C20H25ClN4O3S2: C, 51.22; H, 5.37; N,
11.95. Found: C, 51.35; H, 5.38; N, 11.87.
6.6.3. General procedure for phenols 40, 41, 53
To the corresponding methoxyphenyl analog (121
CH2Cl2 (3 mL) at ꢀ78 °C was added a solution of boron tribromide
(1 M in CH2Cl2, 400 L, 400 mol) dropwise. The reaction mixture
lmol) in
l
l
was warmed to room temperature gradually over several hours
and allowed to stir overnight. Water (10 mL) was added slowly
to the mixture. It was then extracted with EtOAc (3 ꢂ 20 mL), dried
(Na2SO4) and concentrated. Column chromatography (EtOAc/hex)
provided the target compounds.
6.6.2.2.
N-[1-(1-Benzyl-1H-1,2,4-triazol-5-yl)-2-ethylbutyl]-5-
chlorothiophene-2-sulfonamide (33). 1H NMR (400 MHz,
DMSO-d6): d 0.55 (t, 3H, J = 7.4 Hz), 0.61 (t, 3H, J = 7.4 Hz), 0.70–
0.82 (m, 2H), 1.29–1.37 (m, 1H), 1.40–1.46 (m, 1H), 1.60 (br s,
1H), 4.51 (t, 1H, J = 8.1 Hz), 5.33 (d, 1H, J = 15.9 Hz), 5.37 (d, 1H,
J = 15.9 Hz), 7.05 (d, 1H, J = 4.0 Hz), 7.13 (d, 1H, J = 4.1 Hz), 7.22
(d, 2H, J = 8.1 Hz), 7.30–7.38 (m, 3H), 7.84 (s, 1H), 8.87 (d, 1H,
J = 7.9 Hz); MS (+ESI): 439 (M+H)+. Anal. Calcd for C19H23ClN4O2S2:
C, 51.98; H, 5.28; N, 12.76. Found: C, 51.86; H, 5.43; N, 12.68.
6.6.3.1. 5-Chloro-N-{2-ethyl-1-[1-(4-hydroxybenzyl)-1H-pyra-
zol-5-yl]butyl}thiophene-2-sulfonamide
(40). Mp = 90–92 °C;
1H NMR (400 MHz, DMSO-d6): d 0.59 (t, 3H, J = 7.4 Hz), 0.67 (t,
3H, J = 7.3 Hz), 0.87–0.99 (m, 2H), 1.11–1.32 (m, 3H), 4.43 (br s,
1H), 5.04 (q, 2H, J = 15.8 Hz), 6.06 (d, 1H, J = 1.8 Hz), 6.66 (d, 2H,
J = 8.5 Hz), 6.80 (d, 1H, J = 4.0 Hz), 6.89 (d, 1H, J = 4.0 Hz), 6.90 (d,
2H, J = 8.5 Hz), 7.18 (d, 1H, J = 1.8 Hz), 8.38 (br s, 1H), 9.34 (s, 1H);
MS (ꢀESI): 452 (MꢀH)ꢀ. Anal. Calcd for C20H24ClN3O3S2: C, 52.91;
H, 5.33; N, 9.26. Found: C, 52.67; H, 5.41; N, 8.86.
6.6.2.3.
triazol-5-yl]butyl}thiophene-2-sulfonamide
(400 MHz, DMSO-d6): 0.57 (t, 3H, J = 7.3 Hz), 0.63 (t, 3H,
5-Chloro-N-{2-ethyl-1-[1-(4-methylbenzyl)-1H-1,2,4-
(34). 1H
NMR
d
6.6.3.2. 5-Chloro-N-{2-ethyl-1-[1-(4-hydroxyphenyl)-1H-pyra-
J = 7.5 Hz), 0.73–0.87 (m, 2H), 1.29–1.35 (m, 1H), 1.41–1.47 (m,
1H), 1.60 (br s, 1H), 2.28 (s, 3H), 4.52 (t, 1H, J = 8.2 Hz), 5.29 (s,
2H), 7.05 (d, 1H, J = 4.1 Hz), 7.10–7.17 (m, 5H), 7.82 (s, 1H), 8.86
(d, 1H, J = 8.2 Hz); mp 103–104 °C; MS (+ESI): 453 (M+H)+. Anal.
Calcd for C20H25ClN4O2S2: C, 53.03; H, 5.56; N, 12.37. Found: C,
52.63; H, 5.63; N, 12.21.
zol-5-yl]butyl}thiophene-2-sulfonamide
(41). 1H
NMR
(400 MHz, DMSO-d6): d 9.87 (s, 1H), 8.54 (d, 1H, J = 7.9 Hz), 7.38
(d, 1H, J = 1.7 Hz), 7.21 (d, 1H, J = 4.1 Hz), 7.10 (m, 3H), 6.89 (d,
2H, J = 8.9 Hz), 6.29 (m, 1H), 4.49 (m, 1H), 1.31 (m, 1H), 1.16 (m,
1H), 1.11 (m, 1H), 1.08 (m, 2H), 0.59 (t, 3H, J = 7.2 Hz), 0.50 (t,
3H, J = 7.3 Hz); MS (+ESI): 440 (M+H)+. Anal. Calcd for
C19H22N3O3S2Cl: C, 51.87; H, 5.04; N, 9.55. Found: C, 51.77; H,
5.04; N, 9.40.
6.6.2.4.
oxy)benzyl]-1H-1,2,4-triazol-5-yl}butyl)thiophene-2-sulfon-
amide (35). 1H NMR (400 MHz, DMSO-d6):
8.90 (d, 1H,
5-Chloro-N-(2-ethyl-1-{1-[4-(trifluorometh-
d
6.6.3.3. 5-Chloro-N-{(1S)-1-[1-(4-hydroxyphenyl)-1H-pyrazol-
5-yl]-2-methylpropyl}thiophene-2-sulfonamide (53). 1H NMR
(400 MHz, DMSO-d6): d 9.80 (br s, 1H), 8.59 (d, 1H, J = 9.2 Hz),
7.37 (d, 1H, J = 1.9 Hz), 7.09 (d, 1H, J = 4.1 Hz), 7.06 (d, 1H,
J = 8.4 Hz), 7.87 (s, 1H), 7.37 (m, 4H), 7.17 (m, 1H), 7.07 (m, 1H),
5.42 (m, 2H), 4.50 (t, 1H, J = 8.4 Hz), 1.60 (m, 1H), 1.42 (m, 1H),
1.32 (m, 1H), 0.77 (m, 1H), 0.71 (m, 1H), 0.62 (t, 3H, J = 7.4 Hz),
0.54 (t, 3H, J = 7.4 Hz); MS (ꢀESI): 521 (MꢀH)ꢀ. Anal. Calcd for

4716
J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
J = 4.0 Hz), 7.04–7.00 (m, 2H), 6.84 (d, 2H, J = 8.8 Hz), 6.25 (d, 1H,
J = 1.9 Hz), 4.10 (m, 1H), 1.79–1.69 (m, 1H), 0.71 (d, 3H,
J = 6.75 Hz), 0.58 (d, 3H, J = 6.82 Hz). MS (ꢀESI): 410.1 (MꢀH)ꢀ.
Anal. Calcd for C17H18ClN3O3S2: C, 49.57; H, 4.40; N, 10.20. Found:
ane, 1:1) to afford 45b (1.58 g, 54% yield), which solidified on stand-
ing. 1H NMR (400 MHz, DMSO-d6): d 7.39–7.24 (m, 5H), 6.60 (br s,
1H), 4.99 (m, 2H), 4.58 (m, 1H), 3.70 (s, 3H), 3.07 (s, 3H), 1.59–1.51
(m, 1H), 1.41–1.21 (m, 3H), 1.19–1.06 (m, 1H), 0.80 (t, 3H,
J = 7.45 Hz), 0.74 (t, 3H, J = 7.43 Hz). MS (+ESI): 323.2 (M+H)+.
C, 49.35; H, 4.03; N, 9.86. ½a D25
¼ þ66:0 (c 1.0, CHCl3).
ꢄ
6.6.3.4.
5-Chloro-N-[2-ethyl-1-(1-phenyl-1H-pyrazol-5-yl)bu-
6.6.4. General procedure for the chiral synthesis of 49–52, 54,
55
tyl]-N-methylthiophene-2-sulfonamide (42). To a solution of
11 (0.238 g, 0.51 mmol) in anhydrous THF (10 mL) at 0 °C under
N2 was added NaH (0.289 g, 2.03 mmol, 60% in mineral oil). The
mixture was stirred for 30 min at 0 °C. Iodomethane (0.049 g,
2.03 mmol) was added to this mixture. After 12 h the mixture
was diluted with H2O (10 mL), then extracted with EtOAc
(2 ꢂ 30 mL) and washed with brine (30 mL). The organic extract
was dried (Na2SO4) and concentrated under reduced pressure.
Chromatography purification (15% EtOAc/hexane) afforded the ti-
tle compound (0.128 g, 52%) as a white solid. 1H NMR (400 MHz,
DMSO-d6): d 7.61s, 1H), 7.53–7.45 (m, 2H), 7.44–7.42 (m, 1H),
7.29 (d, 2H, J = 5.01 Hz), 7.14 (d, 1H, J = 4.01 Hz), 6.97 (d, 1H,
J = 4.2 Hz), 6.56 (s, 1H), 5.04 (d, 1H, J = 11.3 Hz), 2.75 (s, 3H), 2.00
(m, 1H), 1.42–1.38 (m, 1H), 1.31–1.22 (m, 1H), 1.02–0.98 (m,
2H), 0.72 (t, 3H, J = 7.01 Hz), 0.62 (t, 3H, J = 6.99 Hz). MS (+ESI):
438.1 (M+H)+. Anal. Calcd for C20H24N3O2S2: C, 54.84; H, 5.52; N,
9.59. Found: C, 55.16; H, 5.25; N, 9.34.
StepA. To a solution of 45a19 or 45b (3.4 mmol) in anhydrous THF
(20 mL) at ꢀ78 °C was added a solution of ethynylmagnesium bro-
mide (0.5 M in THF, 27 mL, 14 mmol) dropwise over a period of
10 min. The reaction mixture was warmed to room temperature
and stirred for 16 h. The solution was cooled to 0 °C and quenched
with saturated aqueous NaHSO4 (10 mL). The solution was stirred
for 1 h and then extracted with EtOAc (3 ꢂ 60 mL). The combined or-
ganic layers were washed with saturated aqueous NaHCO3 (50 mL)
and brine (50 mL), dried (Na2SO4) and concentrated. Column chro-
matography (EtOAc/hexane, 3:7) afforded title compound 46.
Step B. Diethylamine (1.1 mL, 11 mmol) was added dropwise
over 5 min to a solution of 46 (10 mmol) in anhydrous CH2Cl2
(40 mL) at 0 °C under N2 atmosphere. The mixture was allowed
to warm to 25 °C overnight (16 h). The solution was diluted with
H2O (50 mL). The aqueous and organic layers were separated and
the aqueous layer was extracted with CH2Cl2 (2 ꢂ 100 mL). The
combined organic layers were dried over anhydrous Na2SO4, fil-
tered and concentrated under reduced pressure to obtain a yel-
low-oil. The crude product was purified by flash chromatography
(EtOAc/hexane, 1:1) to afford title compound 47.
6.6.3.5.
5-Bromo-N-[2-ethyl-1-(1-phenyl-1H-pyrazol-5-yl)bu-
tyl]-N-methylthiophene-2-sulfonamide (43). This compound
was prepared with a similar procedure to that described for 42,
but used 18 as the starting material. 1H NMR (400 MHz, DMSO-
d6): d 7.65 (s, 1H), 7.56–7.53 (m, 1H), 7.50–7.47 (m, 1H), 7.35 (d,
1H, J = 7.2 Hz), 7.25 (d, 1H, J = 4.2 Hz), 6.96 (d, 1H, J = 4.3 Hz),
6.61 (s, 1H), 5.06 (d, 1H, J = 11.1 Hz), 2.79 (s, 3H), 2.19–2.02 (m,
1H), 1.52–1.42 (m, 1H), 1.32–1.25 (m, 1H), 1.09–1.01 (m, 2H),
0.75 (t, 3H, J = 7.05 Hz), 0.68 (t, 3H, J = 6.97 Hz). MS (+ESI): 482.1
(M+H)+. Anal. Calcd for C20H24BrN3O2S2: C, 49.79; H, 5.01; N,
8.71. Found: C, 50.48; H, 4.89; N, 8.46.
Step C. To a solution of 47 (6.0 mmol) in EtOH (30 mL) was
added the corresponding phenyl hydrazine hydrochloride
(6.32 mmol) and aqueous HCl (37%, 0.5 mL, 6.3 mmol). The solu-
tion was heated to reflux for 6 h. The reaction mixture was cooled
to room temperature and the solvent was removed under reduced
pressure. The mixture was then diluted with H2O (50 mL), and ex-
tracted with EtOAc (3 ꢂ 60 mL). The combined organic layers were
washed with saturated aqueous NaHCO3 (50 mL) and brine
(50 mL), dried over Na2SO4, filtered and concentrated under re-
duced pressure. The crude product was purified by flash chroma-
tography (EtOAc/hexane, 1:1) to give 48.
6.6.3.6. (S)-2-(Benzyloxycarbonylamino)-3-ethylpentanoic acid
(44b). (S)-2-Amino-3-ethyl-pentanoic
acid13
(1.32 g,
9.1
mmol) was dissolved in a mixed solvent of 1,4-dioxane/H2O (1:1,
20/20 mL). A 1 N sodium hydroxide solution (18.2 mL, 18.2 mmol)
was added slowly to the stirred solution and benzyl chloroformate
(1.54 mL, 11.0 mmol) was added dropwise. The mixture was stir-
red at room temperature for 36 h, during which time a white pre-
cipitate formed. A 2 N hydrochloric acid solution (approximately
50 mL) was added to adjust pH to 1–2. The mixture was extracted
with ethyl acetate (3 ꢂ 80 mL). The combined organic layers were
washed with brine (50 mL), dried over Na2SO4, filtered and concen-
trated under reduced pressure to give 2.54 g of an oil, which was
used in the next step without further purification. 1H NMR
(400 MHz, DMSO-d6): d 12.22 (1H, br s), 7.36–7.25 (m, 5H), 5.00
(m, 2H), 4.05 (m, 1H), 1.61–1.58 (m, 1H), 1.31–1.18 (m, 4H),
0.82–0.69 (m, 6H). MS (+ESI): 280.2 (M+H)+.
Step D. Pyrazole 48 (2 mmol) in MeOH (10 mL) with Pd/C (10%,
0.1 g) was hydrogenated under atm pressure for 16 h. The reaction
mixture was filtered through a plug of Celite and the filtrate was
concentrated to give amine intermediate which was used in the
next step without further purification.
Step E. To a solution of amine (2.0 mmol) in CH2Cl2 (20 mL) was
added triethylamine (0.83 mL, 6.0 mmol) and 5-chloro-thiophene-
2-sulfonyl chloride (0.54 mL, 4.0 mmol). The mixture was stirred at
room temperature for 16 h, then diluted with CH2Cl2 (50 mL) and
washed with aqueous HCl (1 N, 20 mL). The aqueous layer was ex-
tracted with CH2Cl2 (3 ꢂ 50 mL). The combined organic layers were
washed with saturated aqueous NaHCO3 (30 mL), and brine
(30 mL), then dried over Na2SO4, filtered and concentrated under
reduced pressure. The crude product was purified by flash chroma-
tography (EtOAc/hexane, 1:1) to give target compound.
6.6.3.7. (S)-Benzyl 3-ethyl-1-(methoxy(methyl)amino)-1-oxop-
entan-2-ylcarbamate (45b). Toa solutionof 44b(2.55 g, 9.1 mmol)
in anhydrous CH2Cl2 (40 mL) was added N-methylmorpholine
(2.1 mL, 18.3 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide) hydrochloride (2.63 g, 13.7 mmol) and N,O-dimethyl-
hydroxylamine hydrochloride (1.34 g, 13.7 mmol). The mixture
was stirred at room temperature for 16 h. Saturated aqueous NaH-
CO3 (50 mL)wasaddedcarefullyandtheorganiclayerwasseparated
from aqueous layer and the aqueous layer was extracted with ethyl
acetate (3 ꢂ 80 mL). The combined organic layer was dried over
Na2SO4, filtered and concentrated under reduced pressure. The
crude product was purified by flash chromatography (EtOAc/hex-
6.6.4.1. 5-Chloro-N-{(1S)-2-methyl-1-[1-(4-methylphenyl)-1H-
pyrazol-5-yl]propyl}thiophene-2-sulfonamide (49). 1H NMR
(same as 24). MS (+ESI): 410.2 (M+H)+. Anal. Calcd for
C18H20ClN3O2S2: C, 52.74; H, 4.92; N, 10.25. Found: C, 52.87; H,
4.82; N, 10.08. ½a D25
¼ þ67 (c 1.0, CHCl3). Chiral purity = 99.2% (Chi-
ꢄ
ralcel AD column, 0.45 ꢂ 25 cm, 30% EtOH in hexane, racemate
used as reference).
6.6.4.2. 5-Chloro-N-{(1S)-1-[1-(4-fluorophenyl)-1H-pyrazol-5-
yl]-2-methylpropyl}thiophene-2-sulfonamide (50). 1H NMR
(same as 26). MS (+ESI): 414.2 (M+H)+. Anal. Calcd for

J. Pu et al. / Bioorg. Med. Chem. 17 (2009) 4708–4717
4717
C17H17ClFN3O2S2: C, 49.33; H, 4.14; N, 10.15. Found: C, 49.42; H,
½
a 2D5
ꢄ
¼ þ79:0 (c 1.0, CHCl3). Purity determined by LC–MS at 254
4.14; N, 9.99. ½a D25
ꢄ
¼ þ21 (c 1.0, CHCl3). Chiral purity = 100% (Chi-
and 215 nm: >99%.
ralcel AD column, 0.45 ꢂ 25 cm, 30% EtOH in hexane, racemate
used as reference).
6.6.4.10. 5-Chloro-N-{(1R)-2-ethyl-1-[1-(4-hydroxybenzyl)-1H-
pyrazol-5-yl]butyl}thiophene- 2-sulfonamide (59). Racemate
40 was separated into enantiomers by chiral HPLC (Chiralcel AD-
H, 2 ꢂ 25 cm, 10% MeOH/EtOH—8/2 in hexane/DEA; retention
time: 7.59 min); 1H NMR (same as 40); MS (ꢀESI): 452 (MꢀH)ꢀ.
6.6.4.3. (5-Chloro-N-{(1R)-1-[1-(4-methoxyphenyl)-1H-pyrazol-
5-yl]-2-methylpropyl}thiophene-2-sulfonamide (51). 1H NMR
(400 MHz, DMSO-d6): d 8.64 (d, 1H, J = 8.95 Hz), 7.42 (dHz, 1H,
J = 1.7), 7.17 (d, 2H, J = 8.93 Hz), 7.02–7.04 (m, 4H), 6.18 (d, 1H,
J = 1.9 Hz), 4.14–4.12 (m, 1H), 3.80 (s, 3H), 1.79–1.70 (1H, m),
0.72 (d, 3H, J = 6.73 Hz), 0.60 (d, 3H, J = 6.84 Hz). MS (ꢀESI):
424.0 (MꢀH)ꢀ. Anal. Calcd for C18H20ClN3O3S2: C, 50.76; H, 4.73;
½
a 2D5
ꢄ
¼ ꢀ80:0 (c 1.0, CHCl3). Purity determined by LC–MS at 254
and 215 nm: >99%.
Acknowledgements
N, 9.86. Found: C, 50.89; H, 4.58; N, 9.86. ½a D25
¼ þ66 (c 1.0, CHCl3).
ꢄ
The authors thank colleagues in Discovery Analytical Chemistry
at Wyeth Research for compound characterization data. Special
thanks also to the Wyeth Postdoctoral Program.
6.6.4.4. 5-Chloro-N-{(1S)-2-ethyl-1-[1-(4-methoxyphenyl)-1H-
pyrazol-5-yl]butyl}thiophene-2-sulfonamide
(52). 1H NMR
(same as 28); MS (+ESI): 454.1 (M+H)+. Anal. Calcd for
C20H24ClN3O3S2: C, 52.91; H, 5.33; N, 9.26. Found: C, 53.24; H,
5.33; N, 8.85. ½a D25
¼ þ63:0 (c 1.0, CHCl3).
ꢄ
Supplementary data
Synthetic details and characterization of intermediates 4–7 and
1H NMR and HPLC data for compounds 56–59 for proof of purity
and 1H NMR of compounds 19 and 27 are included. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.bmc.2009.04.052.
6.6.4.5. 5-Chloro-N-{(1R)-2-methyl-1-[1-(4-methylphenyl)-1H-
pyrazol-5- yl]propyl}thiophene-2-sulfonamide (54). 1H NMR
(same as 24). MS (+ESI): 410.2 (M+H)+. Anal. Calcd for
C18H20ClN3O2S2: C, 52.74; H, 4.92; N, 10.25. Found: C, 52.97; H,
4.81; N, 10.16. ½a D25
¼ ꢀ66 (c 1.0, CHCl3). Chiral purity = 99.2% (Chi-
ꢄ
ralcel AD column, 0.45 ꢂ 25 cm, 30% EtOH in hexane, racemate
References and notes
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(same as 26). MS (+ESI): 414.2 (M+H)+. Anal. Calcd for
C17H17ClFN3O2S2: C, 49.33; H, 4.14; N, 10.15. Found: C, 49.65; H,
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4.24; N, 10.13. ½a D25
¼ ꢀ17 (c 1.0, CHCl3). Chiral purity = 100% (Chi-
ꢄ
ralcel AD column, 0.45 ꢂ 25 cm, 30% EtOH in hexane, racemate
used as reference).
The following compounds were isolated by chiral chromatogra-
phy (56–59).
6.6.4.7. 5-Chloro-N-{(1S)-2-ethyl-1-[1-(4-methoxybenzyl)-1H-
pyrazol-5-yl]butyl}thiophene- 2-sulfonamide (56). Racemate 8
was separated into enantiomers by chiral HPLC (Chiralcel AD-H,
2 ꢂ 25 cm, 10% MeOH/EtOH—8/2 in hexane/DEA; retention
time = 5.03 min); 1H NMR (same as 8); MS (ꢀESI): 466 (MꢀH)ꢀ.
½
a 2D5
ꢄ
¼ þ30:0 (c 1.0, CHCl3). Purity determined by LC–MS at 254
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6.6.4.8. 5-Chloro-N-{(1R)-2-ethyl-1-[1-(4-methoxybenzyl)-1H-
pyrazol-5-yl]butyl}thiophene- 2-sulfonamide (57). Racemate 8
was separated into enantiomers by chiral HPLC (Chiralcel AD-H,
2 ꢂ 25 cm, 10% MeOH/EtOH—8/2 in hexane/DEA; retention
time = 6.18 min); 1H NMR (same as 8); MS (ꢀESI): 466 (MꢀH)ꢀ.
½
a 2D5
ꢄ
¼ ꢀ25:0 (c 1.0, CHCl3). Purity determined by LC–MS at 254
and 215 nm: >99%.
6.6.4.9.
5-Chloro-N-{(1S)-2-ethyl-1-[1-(4-hydroxybenzyl)-1H-
pyrazol-5-yl]butyl}thiophene- 2-sulfonamide (58). Racemate
40 was separated into enantiomers by chiral HPLC (Chiralcel AD-
H, 2 ꢂ 25 cm, 10% MeOH/EtOH—8/2 in hexane/DEA; retention
time = 6.51 min); 1H NMR (same as 40); MS (ꢀESI): 452 (MꢀH)ꢀ.
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