Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells

Article abstract of DOI:10.1016/j.bmc.2011.11.039

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2-3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC₅₀ of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood-brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cells, potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73) were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel therapeutic candidates for ALS.

Full text of DOI:10.1016/j.bmc.2011.11.039

Bioorganic & Medicinal Chemistry 20 (2012) 1029–1045
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent
protein aggregation and toxicity in PC12 cells
Wei Zhang a, , Radhia Benmohamed b, Anthony C. Arvanites b,à, Richard I. Morimoto c, Robert J. Ferrante d,e
,
Donald R. Kirsch b, Richard B. Silverman a,f,
⇑
a Department of Chemistry, Northwestern University, Evanston, IL 60208-3113, USA
b Cambria Pharmaceuticals, Cambridge, MA 02142, USA
c Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL 60208-3500, USA
d Neurological Surgery, Neurology, and Neurobiology Departments, University of Pittsburgh, Pittsburgh, PA 15213, USA
e The Geriatric Research Educational and Clinical Center (00-GR-H), V.A. Pittsburgh Healthcare System, 7180 Highland Drive, Pittsburgh, PA 15206, USA
f Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL
60208-3113, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive
loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that
drug only extends life, on average, by 2–3 months. Mutations in Cu/Zn superoxide dismutase (SOD1)
are found in familial forms of the disease and have played an important role in the study of ALS patho-
physiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several
bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A
concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural
modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC50 of 700 nM
having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic poten-
tial, and relatively good plasma stability. It was also found to efficiently penetrate the blood–brain bar-
rier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It
was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including
primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cells,
potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was
aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73)
were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar
potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel
therapeutic candidates for ALS.
Received 22 September 2011
Revised 15 November 2011
Accepted 19 November 2011
Available online 30 November 2011
Keywords:
Cyclohexane-1,3-dione
Amyotrophic lateral sclerosis
Mutant G93A SOD1
Blood–brain barrier penetration
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
familial, similar clinical and pathological features are exhibited in
familial and sporadic forms of the disease. Mutations in Cu/Zn
Amyotrophic lateral sclerosis (ALS), an orphan neurodegenera-
tive disease, is defined by the progressive loss of motor neurons.
There are an estimated 87,000 ALS patients worldwide, who sur-
vive, on average, only 2–5 years after diagnosis.1,2 Riluzole is the
only FDA approved drug for the treatment of ALS; however, it only
extends life by 2–3 months.1 Although only 5–10% of ALS cases are
superoxide dismutase (SOD1), which cause misfolding and aggre-
gation of the protein, are responsible for the disease in a subgroup
of patients with familial ALS.1,2 The cultured cell model, PC12-
G93A-YFP, developed by Matsumoto and co-workers,3 was utilized
to develop a high-throughput screen (HTS) as a cellular model of
ALS, which identified active compounds in the cytotoxicity protec-
tion assay.4 In this assay, G93A SOD1 aggregation is cytotoxic and
inhibition of SOD1 mutant protein aggregation is cytoprotective.
The percentage of cell survival is determined from the maximum
cell viability after exposure of test compounds and EC50 values
are determined from the decrease in the cytotoxicity of mutant
SOD1-G93A-YFP aggregation by the treatment with test com-
pounds. Three chemotypes were identified in this high-throughput
screen: arylsulfanyl pyrazolones,5 pyrimidine 2,4,6-triones,6 and
⇑
Corresponding author. Tel.: +1 847 491 5663; fax: +1 847 491 7713.
E-mail address: Agman@chem.northwestern.edu (R.B. Silverman).
Present address: State Key Laboratory of Organometallic Chemistry, Shanghai
Institute of Organic Chemistry, Chinese Academy of Sciences, No. 345, Lingling Road,
Shanghai 230032, China.
à
Present address: Harvard Stem Cell Institute, Harvard University, Cambridge, MA
92138, USA.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.039

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W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
Table 1
cyclohexane 1,3-diones (CHDs). They all showed 100% efficacy (cell
viability) compared with the positive control (radicicol, 85% effi-
cacy) in the cytotoxicity assay. EC50 values of the most potent CHDs
Selected examples from the preliminary HTS results4
a
CHD
EC50
(lM)
CHD
EC50
6.52
(lM)
ranged between 3 lM and 8 lM in the protection assay. Here, we
describe the SAR and pharmacokinetic studies of this class of com-
pounds as inhibitors of protein aggregation caused by mutant
SOD1.
4.57
4
6
2. Results and discussion
3
A concise and efficient synthetic route to CHD analogs is sum-
marized in Scheme 1.7 This two-step procedure made it possible
to apply parallel synthesis methods. Commercially available or
previously synthesized aldehydes (1) were used as starting materi-
als, which were conveniently converted to intermediates 2 in high
yields. CHDs were then obtained in one pot in satisfactory yields
and purity by treatment with diethyl malonate followed by hydro-
lysis and decarboxylation. More than 120 analogs were obtained
by this simple protocol.
8.30
3.33
5
a
An analog was considered to be inactive when EC50 >32 lM.
4 h, within the concentration plateau. At this time point the brain
concentration was determined to be 579 M and plasma concen-
tration 675 M, producing a very good brain-plasma ratio (B/P)
of 0.86. Compound 26 (10 M) was subjected to the LeadProfiling-
Screen of MDS Pharma Services, a panel of 68 radioligand receptor
assays, and none was positive (all exhibited <20% [mostly <10%]
inhibition). At 10 lM concentration, 26 inhibited the hERG channel
by only 7.8%, indicating a low probability of it leading to arrhyth-
mias in humans.
On the basis of their activity in the PC12-G93A-YFP high-
throughput screening assay,4 several active CHD analogs were
identified (Table 1). In this assay, G93A mutant SOD1 protein
aggregates are observed following treatment with the proteasome
inhibitor MG132. The formation of these SOD1 aggregates is cyto-
toxic in this model. Therefore, cell survival is measured to deter-
mine test compound efficacy. Active CHD analogs showed 100%
efficacy (cell viability) compared with the positive control radicicol
(85% efficacy). EC50 values of the most potent compounds ranged
l
l
l
Consequently, 26 was selected for testing in the ALS mouse
model.8 Compound 26 demonstrated no significant activity in the
mutant SOD1 G93A transgenic mice (Fig. 1). At the highest dose
(20 mg/kg) there was a non-significant 4.6% increase in survival.
We did not, however, use a dose that resulted in adverse events
or worsening survival, as compared to the untreated G93A mice
and, as such, the greatest optimal dose might not have been
achieved. Given the good potency, aqueous solubility, stability to
microsomes, high oral bioavailability, blood–brain barrier penetra-
tion, and volume of distribution (Vss), the in vivo inactivity was sur-
prising. One parameter that was different from that seen for other
compounds in our research program that were active in G93A
SOD1 ALS mice, was the smaller volume of distribution, which
was only ꢁ15–25% that of active compounds. We, therefore, be-
came concerned that the lack of efficacy might be the result of
the inability of 26 to penetrate into certain cells, particularly neu-
rons. To address this concern, a qualitative primary cortical neuron
assay was developed. As described in the experimental section, this
assay is not based on the ability of the compounds to reduce mu-
tant SOD1 aggregation, but rather on a related activity (protection
against MG132 toxicity), which made it possible to conveniently
test compounds on a wide variety of cell types. While the assay
measures activity and not cell penetration per se, it nevertheless
allowed us to determine whether compounds were active in the
target nerve cells and, if necessary, to guide analog synthesis for
identification of compounds with the desired activity. As can be
between 3 and 8 lM in the protection assay.
Various aromatic aldehydes were used to synthesize CHD ana-
logs, as all of the active CHD analogs contained a phenyl or substi-
tuted phenyl group. Size, electronic properties, and positional
effects were tested by modifying the substituents on the aromatic
ring. Several conclusions can be drawn regarding a structure–activ-
ity relationship (Table 2): (1) electronic properties of the substi-
tuted groups do not affect the activity of analogs; (2) the meta
position is much more important than the other positions; (3)
the size of the meta-substituents is very crucial; and (4) CF3 is a fa-
vored substituent to increase the potency. Finally, it was found that
the 3,5-CF3 substituted analog (26, Table 2) had superior potency
relative to the other analogs.
Pharmacokinetic studies with 26 showed that it had good solu-
bility (P500 lM), high human and mouse (both T1/2 P 180 min)
microsomal stability, and an oral bioavailability of 89% (Table 3a
and b). Caco-2 permeability (A?B, 38 cmꢀ6/s) indicated promising
ability for good absorption, and a relatively low flux in the B?A
direction (5.7 cmꢀ6/s), giving a low efflux ratio (B?A/A?B, 0.2).
A maximum tolerated dose for 26 of 80 mg/kg was determined
by intraperitoneal injection in mice starting at 5 mg/kg and dou-
bling the compound b.i.d. each day, once in the morning and once
mid afternoon. Plasma concentration and blood brain barrier pen-
etration of 26 were determined in G93A SOD1 transgenic mice.
Compound 26 produces stable plasma concentrations between 3
and 12 h and brain concentration was, therefore, determined at
Scheme 1. General synthetic route to cyclohexane 1,3-dione (CHD) analogs.

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1031
M)
Table 2
Selected examples of synthetic CHD analogs in the PC12-G93A-YFP cell-based assay
a
CHD
EC50
(lM)
CHD
EC50
>32
(lM)
CHD
EC50
9.93
(l
>32
7
8
9
7.01
>32
>32
>32
8.20
>32
12
10
11
14
13
16
19
15
18
>32
>32
>32
17
20
28.7
24.3
2.40
21
2.35
3.20
6.21
0.70
2.71
2.22
22
23
24
25
26
27
30
2.22
3.16
1.23
2.70
4.81
2.01
3.39
2.39
1.58
29
32
28
31
33
34
36
35
3.51
2.37
2.88
37
38
39
(continued on next page)

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W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
Table 2 (continued)
a
CHD
EC50
3.60
(
lM)
CHD
EC50
1.16
(lM)
CHD
EC50
2.18
(lM)
41
42
45
40
6.66
3.07
4.09
1.56
1.30
3.99
43
44
48
46
47
1.49
8.29
5.20
51
49
50
3.09
2.53
2.56
0.73
2.71
1.56
53
52
54
55
56
59
57
1.10
2.04
1.24
2.15
58
60
61
a
An analog was considered to be inactive when EC50 >32 lM.
seen in Table 4, although 26 is active in PC12 cells, which was the
cell line used in the high-throughput screen of compounds, it is
inactive in a variety of other cell types, including neuronal cells.
Whether this is due to cell penetration or other factors remains
an open question. However, lack of activity on the target cell type,
neurons, provides a plausible explanation for lack of activity in the
ALS mouse model. Further modification of CHD analogs was aimed
at improving their neuronal cell activity as well as maintaining
their potency. To that end, different linkages between the cyclo-
hexane 1,3-dione core and the substituted phenyl group were syn-
thesized and tested. This would change the geometry of the
molecule as well as make it more flexible, which might have an ef-
fect on cell permeability (Fig. 2).
The synthetic route to the CHD analogs shown in Scheme 1, also
was applicable to the synthesis of the modified scaffolds in Figure
1. The R groups selected were 3-Cl, 3-CF3, 3,5-Cl, or 3,5-CF3, be-
cause previous analogs with these groups were among the most
potent analogs. On the basis of PC12-G93A-YFP high-throughput
screening assay,4 activities of some of the new CHD analogs were
measured (Table 5). All of these analogs were also screened in
the primary cortical neuron assay. Only two CHD scaffolds (71
and 73) showed consistently positive results in all cell lines,

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1033
Table 3
(A) Intravenous pharmacokinetics of 26 (1 mg/kg) in Sprague-Dawley rat; (B) oral pharmacokinetics of 26 (1 mg/kg) in Sprague-Dawley rat
(A)
AUCa last (ng h/mL)
AUC/dose (ng h/mL/dose)
1996
Cl (mL/min/kg)
8.4
T1/2 (h)
Vss (L/kg)
1.5
1982
3.7
(B)
b
AUC last (ng h/mL)
AUC/dose (ng h/mL/dose)
1774
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
F (%)
1764
468
0.5
3.2
89
a
AUC (area under the curve), Cl (clearance), T1/2 (half life), Vss (volume of distribution at steady state).
Cmax (maximum concentration), Tmax (time to achieve maximum concentration following dosing), F (bioavailability).
b
O
O
O
O
O
CF3
F3C
F3C
CF3
( )-73
( )-trans-71
3. Summary
More than 120 CHD analogs have been synthesized, and the
structural modification of this scaffold led to the discovery of a
more potent analogue (26) with an EC50 of 700 nM. Although the
CHD scaffold showed promise for the potential treatment of ALS
on the basis of its in vitro efficacy and pharmacokinetic properties,
26 showed very poor activity on a variety of cells, including pri-
mary cortical neurons, which may account for its inability to ex-
tend the life in the ALS mouse model. Modification of the core
structure, however, led to two structures (71 and 73) with good
potency and the ability to cross into neurons. These compounds
are being investigated further.
Figure 1. Kaplan–Meier curve of the effect of 26 on the G93A SOD1 transgenic
mouse model for ALS. Untreated: 126.4 4.2; Group 1 (1 mg/kg): 128.9 6.1;
Group 2 (10 mg/kg): 130.5 5.8; Group 3 (20 mg/kg):132.3 4.7.
including cortical neurons. The cortical neuron assay does not pro-
duce quantitative results and as a result we can only determine
whether a scaffold is active or inactive. The four most promising
compounds were 67, 69, 71, and 73, which have potencies in the
cytotoxicity protection assay comparable to that of 26; however,
69 was inactive in the cortical neuron assay and 67 is not chiral,
and we thought that one enantiomer of 71 or 73 would have great-
er potency and would be more promising to pursue than 67. Com-
pound 73 also has acceptable mouse (t1/2 45 min) and human (t1/2
71 min) microsomal stability, good plasma stability (t1/2 >60 min),
and an excellent efflux ratio (0.1; A?B, 24.8 cmꢀ6/s; B?A, 3.1 cm
ꢀ6/s)).
4. Experimental section
4.1. General methods
All reagents were purchased from Aldrich, Alfa Aesar, Oakwood
Inc., or Maybridge Ltd and were used without further purification,
unless stated otherwise. Tetrahydrofuran was distilled under nitro-
Table 4
Activity of 26 on different cell types
Compound
ALS mouse activity
PC12
+
SY5Y
+
HeLa
+
HEK293
+
Primary cortical neurons
+
O
Cl
O
NH
a
N
H
+
Cl
O
O
b
N
N
+
+
+
+
+
+
+
O
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
26
The compounds in the first two rows were found earlier to be active in the G93A ALS mouse model.
a
Refs. 5 and 12.
Ref. 6 and unpublished observations.
b

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W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
Figure 2. Modified CHD scaffolds.
Table 5
New synthetic CHD analogs in the PC12-G93A-YFP cell-based assay
a
CHD
EC50
(lM)
CHD
EC50
0.90
(lM)
CHD
EC50
5.85
1.82
( )-64
62
63
3.43
6.28
>32
0.70
2.65
65
66
67
0.70
68
70
69
0.70
1.41
0.70
( )-72
( )-73
71
a
An analog was considered to be inactive when EC50 >32 lM.
gen from sodium/benzophenone. Thin-layer chromatography was
carried out on E. Merck precoated Silica Gel 60 F254 plates. Com-
pounds were visualized with ferric chloride reagent or a UV lamp.
Column chromatography was performed with E. Merck Silica Gel
60 (230–400 mesh). Proton nuclear magnetic resonances (1H
NMR) were recorded in deuterated solvents on a Varian Inova 400
(400 MHz), a Varian Inova 500 (500 MHz), or a Bruker Ag500
(500 MHz) spectrometer. Chemical shifts are reported in parts per
million (ppm, d) using various solvents as internal standards (CDCl3,
d 7.26 ppm; DMSO-d6, d 2.50 ppm). 1H NMR splitting patterns are
designated as singlet (s), doublet (d), triplet (t), or quartet (q). Split-
ting patterns that could not be interpreted or easily visualized were
recorded as multiplet (m) or broad (br). Coupling constants are re-
ported in Hertz (Hz). Proton-decoupled carbon (13C NMR) spectra
were recorded on a Varian Inova 500, a Varian Inova 400, or a Bruker
Ag500 (125.7, 100.6, and 125.7 MHz, respectively) spectrometer
and are reported in ppm using various solvents as internal standards
(CDCl3, d 77.23 ppm; DMSO-d6, d 39.52 ppm). High-resolution mass
spectra were recorded using a VG70-250SE mass spectrometer.
4.2. General procedure to prepare cyclohexane 1,3-dione (CHD)
analogs7
All aldehydes were previously prepared or purchased. Aldehyde
1 (5.0 mmol) was dissolved in THF (20 mL), and 1-(triphenylphos-

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1035
phoranylidene)-2-propanone (1.60 g, 5.0 mmol) was added. The
mixture was allowed to stir at room temperature for 12 h. Solvent
was removed under vacuum, and the residue was purified by flash
column chromatography to provide 2, which was used directly in
the next step, in almost quantitative yield.
Compound 9 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.17 (br s, 1H), 7.33–7.31 (m, 2H), 7.25–7.23 (m, 2H), 5.28 (s,
1H), 3.26–3.22 (m, 1H), 2.57–2.52 (m, 2H), 2.41–2.37 (m, 2H),
1.27 (s, 9H); 13C NMR (125 MHz, DMSO-d6, d): 148.79, 140.47,
126.52, 125.16, 103.22, 38.22, 34.08, 30.97.
Compound 2 (2.0 mmol) and diethyl malonate (2.0 mmol) were
dissolved in EtOH (5 mL), and a NaOEt solution (21 wt % in ethanol;
1.50 mL, 4.0 mmol) was added. After being stirred for 12 h, 3 N aq
NaOH (20 mL) was added, and stirring was continued for another
12 h. Aqueous 3 N HCl was added until the solution turned cloudy.
Ether was used to extract the cloudy mixture until the aqueous
layer was clear. The combined organic portions were combined
and allowed to stir for 4 h until decarboxylation was complete.
Heating might be required in some cases when decarboxylation
was not complete at room temperature. The solvent was evapo-
rated, and the crude product was then purified by recrystallization
or flash chromatography. The CHD product was typically a white
solid, obtained in about an 80% yield, and had an Rf = 0.6 (dichloro-
methane/methanol = 8:1).
10
Compound 10 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.31 (br s, 1H), 7.73 (s, 2H), 5.31 (s, 1H), 4.19–4.15 (m, 1H),
3.24–3.20 (m, 2H), 2.24–2.20 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 134.99, 132.71, 130.17, 128.68, 103.40, 35.07.
4.3. Selected analog characterization
11
7
Compound 11 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.22 (br s, 1H), 7.29 (d, J = 8 Hz, 2H), 7.22 (d, J = 8 Hz, 2H), 5.28
(s, 1H), 3.30–3.24 (m, 1H), 2.59–2.53 (m, 2H), 2.45 (s, 3H), 2.39–
2.36 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 140.35, 136.07,
127.62, 126.26, 103.56, 38.24, 14.91.
Compound 7 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.30 (br s, 1H), 7.51–7.46 (m, 1H), 7.25–7.20 (m, 1H), 7.11–7.07
(m, 1H), 5.31 (s, 1H), 3.57–3.52 (m, 1H), 2.65–2.60 (m, 2H), 2.42–
2.38 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 161.99, 161.89,
161.02, 160.93, 160.04, 159.94, 159.05, 158.96, 129.40, 129.35,
129.32, 129.28, 126.23, 126.20, 126.11, 126.08, 111.57, 111.54,
111.40, 111.38, 104.07, 103.87, 103.65, 103.41, 31.67.
Compound 12 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.30 (br s, 1H), 7.14 (d, J = 6.5 Hz, 2H), 7.10–7.06 (m, 1H), 5.28
(s, 1H), 3.38–3.34 (m, 1H), 2.63–2.58 (m, 2H), 2.40–2.73 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 163.40, 163.29, 161.45, 161.34,
148.27, 148.20, 148.13, 110.44, 110.40, 110.29, 110.25, 103.42,
102.23, 102.02, 101.82, 38.39.
8
Compound 8 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.21 (br s, 1H), 6.82 (s, 2H), 5.29 (s, 1H), 3.73–3.65 (m, 1H),
2.88–2.83 (m, 2H), 2.32 (s, 3H), 2.17 (s, 6H), 2.16–2.12 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 135.93, 135.21, 135.04, 103.47,
34.09, 21.38, 20.21.
13
Compound 13 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.34 (br s, 1H), 7.36–7.32 (m, 1H), 7.10–7.07 (m, 2H), 5.30 (s,
1H), 3.65–3.61 (m, 1H), 2.79–2.75 (m, 2H), 2.33–2.30 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 161.78, 161.72, 159.83, 159.76,
9

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W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
129.36, 129.28, 129.19, 117.67, 117.53, 117.39, 112.25, 112.22,
112.08, 112.05, 103.61, 103.37, 103.10, 28.94.
DMSO-d6, d): 140.11, 132.60, 129.62, 128.47, 128.00, 127.71,
103.44, 35.50.
14
18
Compound 14 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.29 (br s, 1H), 7.43–7.41 (m, 1H), 7.32–7.28 (m, 1H), 7.20–7.16
(m, 2H), 5.30 (s, 1H), 3.59–3.53 (m, 1H), 2.65–2.59 (m, 2H), 2.42–
2.38 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 161.13, 159.19,
129.91, 129.80, 128.65, 128.58, 128.31, 128.27, 124.70, 124.68,
115.56, 115.38, 103.50, 32.16.
Compound 18 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.35 (br s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 7.5 Hz, 1H),
7.41–7.38 (m, 1H), 7.22–7.18 (m, 1H), 5.31 (s, 1H), 3.66–3.60 (m,
1H), 2.65–2.59 (m, 2H), 2.42–2.39 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 141.66, 132.94, 128.84, 128.30, 128.10, 123.66,
103.44, 38.24.
19
15
Compound 19 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.26 (br s, 1H), 7.57 (s, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.37 (d,
J = 7.5 Hz, 1H), 7.30–7.27 (m, 1H), 7.22–7.18 (m, 1H), 5.28 (s, 1H),
3.36–3.30 (m, 1H), 2.63–2.57 (m, 2H), 2.40–2.36 (m, 2H); 13C
NMR (125 MHz, DMSO-d6, d): 146.47, 130.71, 129.93, 129.58,
126.21, 121.89, 103.56, 38.41.
Compound 15 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.27 (br s, 1H), 7.44–7.43 (m, 2H), 7.40–7.37 (m, 2H), 7.33–7.32
(m, 1H), 7.25 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 5.30 (s,
1H), 5.07 (s, 2H), 3.26–3.20 (m, 1H), 2.54–2.50 (m, 2H), 2.37–2.34
(m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 157.05, 137.25,
135.86, 128.49, 127.98, 127.84, 127.69, 114.74, 103.57, 69.17,
38.04.
20
Compound 20 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.49 (br s, 1H), 7.49 (m, 2H), 7.34–7.30 (m, 1H), 5.32 (s, 1H),
4.22–4.18 (m, 1H), 3.26–3.20 (m, 2H), 2.23–2.20 (m, 2H); 13C
NMR (125 MHz, DMSO-d6, d): 135.78, 135.02, 134.37, 130.85,
129.67, 129.22, 103.52, 35.41.
16
Compound 16 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.22 (br s, 1H), 7.58 (s, 1H), 7.50 (s, 1H), 6.52 (s, 1H), 5.25 (s,
1H), 3.38–3.33 (m, 1H), 3.21–3.16 (m, 2H), 2.50–2.45 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 143.25, 138.47, 127.51, 109.47,
103.45, 29.47.
21
Compound 21 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.19 (br s, 1H), 6.92 (s, 2H), 6.84 (s, 1H), 5.28 (s, 1H), 3.21–3.17
(m, 1H), 2.54–2.52 (m, 2H), 2.36–2.33 (m, 2H), 2.23 (s, 6H); 13C
NMR (125 MHz, DMSO-d6, d): 143.38, 137.32, 127.97, 124.62,
103.46, 38.66, 30.96. Elemental analysis: Comb. Anal. Calcd for
17
Compound 17 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.37 (br s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H),
7.37–7.34 (m, 1H), 7.30–7.27 (m, 1H), 5.31 (s, 1H), 3.71–3.64 (m,
1H), 2.65–2.60 (m, 2H), 2.42–2.39 (m, 2H); 13C NMR (125 MHz,
C14H16O2: C, 77.75; H, 7.46; O, 14.80. Found: C, 77.55; H, 7.35. Pur-
ity: >95%.

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1037
132.94, 128.76, 126.83, 126.60, 126.37, 126.14, 125.75, 125.71,
125.67, 125.62, 125.58, 123.40, 103.20, 35.14.
22
Compound 22 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.28 (br s, 1H), 7.46 (s, 3H), 5.28 (s, 1H), 3.39–3.33 (m, 1H),
2.66–2.64 (m, 2H), 2.39–2.36 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 147.85, 134.01, 126.30, 126.10, 103.45, 38.21.
Elemental analysis: Anal. Calcd for C12H10Cl2O2: C, 56.06; H,
3.92; Cl, 27.58; O, 12.45. Found: C, 56.13; H, 3.77; Cl, 27.47. Purity:
>95%.
26
Compound 26 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. Mp: 209–211 °C; 1H NMR (500 MHz,
DMSO-d6, d): 11.30 (br s, 1H), 8.12 (s, 2H), 7.95 (s, 1H), 5.31 (s, 1H),
3.62–3.57 (m, 1H), 2.77–2.71 (m, 2H), 2.46–2.43 (m, 2H); 13C NMR
(125 MHz, DMSO-d6, d): 146.97, 130.69, 130.43, 130.17, 129.91,
128.23, 126.61, 124.44, 122.27, 120.41, 120.39, 103.46, 38.28. Ele-
mental analysis: Anal. Calcd for C14H10F6O2: C, 51.86; H, 3.11; F,
35.16; O, 9.87. Found: C, 51.79; H, 3.11; F, 35.31. Purity: >95%.
23
Compound 23 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.30 (br s, 1H), 7.87–7.83 (m, 3H), 7.79 (s, 1H), 7.57–7.53 (m,
1H), 7.50–7.44 (m, 2H), 5.34 (s, 1H), 3.49–3.44 (m, 1H), 2.73–
2.67 (m, 2H), 2.49–2.47 (m, 2H); 13C NMR (125 MHz, DMSO-d6,
d): 141.16, 133.11, 132.01, 128.05, 127.64, 127.51, 126.19,
125.82, 125.68, 125.01, 103.64, 38.92.
27
Compound 27 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.23 (br s, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8 Hz, 2H),
5.31 (s, 1H), 3.28–3.23 (m, 1H), 2.59–2.53 (m, 4H), 2.40–2.36 (m,
2H), 1.16 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6, d):
142.07, 140.86, 127.92, 126.89, 103.62, 38.52, 27.87, 15.75.
24
28
Compound 24 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.20 (br s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8 Hz, 2H),
5.30 (s, 1H), 3.30–3.26 (m, 1H), 2.60–2.54 (m, 4H), 2.40–2.36 (m,
2H), 1.56–1.51 (m, 2H), 1.30–1.25 (m, 2H), 0.90 (t, J = 6.0 Hz, 3H);
13C NMR (125 MHz, DMSO-d6, d): 140.72, 140.51, 128.31, 126.72,
103.48, 38.36, 34.40, 30.66, 21.90, 13.88.
Compound 28 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.23 (br s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8 Hz, 2H),
5.30 (s, 1H), 3.29–3.24 (m, 1H), 2.60–2.51 (m, 4H), 2.41–2.37 (m,
2H), 1.59–1.54 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6, d): 140.85, 140.43, 128.45, 126.79, 103.58,
38.46, 36.96, 24.22, 13.79.
25
29
Compound 25 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.40 (br s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.70–7.68 (m, 2H), 7.48–
7.50 (m, 1H), 5.30 (s, 1H), 3.54–3.50 (m, 1H), 2.80–2.76 (m, 2H),
2.30–2.26 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 141.98,
Compound 29 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.33 (br s, 1H), 7.03–6.98 (m, 3H), 5.30 (s, 1H), 3.73–3.69 (m,
1H), 2.92–2.86 (m, 2H), 2.35 (s, 6H), 2.22–2.18 (m, 2H); 13C NMR

1038
W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
(125 MHz, DMSO-d6, d): 138.31, 136.21, 126.39, 103.56, 34.44,
21.51.
34
30
Compound 34 was purified by recrystallization (ether/hex-
anes) and obtained as a white solid. 1H NMR (500 MHz, DMSO-
d6, d): 11.23 (br s, 1H), 7.70–7.67 (m, 2H), 7.50–7.32 (m, 7H),
5.30 (s, 1H), 3.35–3.33 (m, 1H), 2.70–2.64 (m, 2H), 2.48–2.43
(m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 144.26, 140.36,
140.18, 129.05, 128.86, 127.41, 126.79, 126.03, 125.49, 124.98,
103.49, 38.82.
Compound 30 was purified by recrystallization (ether/
hexanes) and obtained as a white solid. 1H NMR (500 MHz,
DMSO-d6, d): 11.21 (br s, 1H), 7.36–7.34 (m, 1H), 7.19–7.10
(m, 3H), 5.31 (s, 1H), 3.52–3.46 (m, 1H), 2.60–2.54 (m, 2H),
2.33–2.29 (m, 2H), 2.29 (s, 3H); 13C NMR (125 MHz, DMSO-d6,
d): 141.48, 135.24, 130.40, 126.34, 126.26, 125.69, 103.43,
34.66, 18.95.
31
35
Compound 31 was purified by recrystallization (ether/hex-
anes) and obtained as a white solid. 1H NMR (500 MHz, DMSO-
d6, d): 11.22 (br s, 1H), 7.41–7.40 (m, 1H), 7.19–7.15 (m, 3H),
5.31 (s, 1H), 3.53–3.47 (m, 1H), 2.65–2.61 (m, 4H), 2.29–2.26
(m, 2H), 1.13 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6,
d): 141.19, 140.93, 128.85, 126.58, 126.23, 126.19, 103.42,
34.16, 25.13, 16.10.
Compound 35 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.20 (br s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8 Hz, 2H),
5.30 (s, 1H), 3.29–3.23 (m, 1H), 2.60–2.51 (m, 4H), 2.41–2.36 (m,
2H), 1.57–1.52 (m, 2H), 1.33–1.24 (m, 4H), 0.87 (t, J = 6.5 Hz, 3H);
13C NMR (125 MHz, DMSO-d6, d): 140.79, 140.61, 128.37, 126.78,
103.56, 38.44, 34.78, 31.00, 30.76, 22.02, 13.95.
32
36
Compound 32 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.20 (br s, 1H), 7.21–7.18 (m, 1H), 7.14–7.11 (m, 2H), 7.04–7.03
(m, 1H), 5.28 (s, 1H), 3.27–3.22 (m, 1H), 2.60–2.54 (m, 2H), 2.38–
2.35 (m, 2H), 2.28 (s, 3H); 13C NMR (125 MHz, DMSO-d6, d):
143.53, 137.56, 128.42, 127.66, 127.27, 123.97, 103.55, 38.76,
21.14.
Compound 36 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.20 (br s, 1H), 7.23–7.21 (m, 2H), 7.14–7.09 (m, 2H), 5.28 (s,
1H), 3.29–3.25 (m, 1H), 2.89–2.81 (m, 1H), 2.61–2.56 (m, 2H),
2.39–2.36 (m, 2H), 1.19 (dd, J = 7.0 Hz, 1.0 Hz, 6H); 13C NMR
(125 MHz, DMSO-d6, d): 148.64, 143.55, 128.45, 125.20, 124.50,
124.27, 103.54, 38.89, 33.53, 23.97.
33
Compound 33 was purified by recrystallization (ether/hex-
anes) and obtained as a white solid. 1H NMR (500 MHz, DMSO-
d6, d): 11.19 (br s, 1H), 7.17–7.16 (m, 1H), 7.07–7.02 (m, 2H),
5.29 (s, 1H), 3.56 (m, 1H), 2.56–2.50 (m, 2H), 2.33–2.29 (m,
2H), 2.23 (s, 3H), 2.17 (s, 3H); 13C NMR (125 MHz, DMSO-d6, d):
141.18, 136.54, 133.81, 127.99, 125.54, 123.40, 103.42, 34.87,
20.72, 14.34.
37
Compound 37 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.25 (br s, 1H), 7.73–7.71 (m, 2H), 7.66 (s, 1H), 7.54–7.51 (m,
3H), 7.43–7.40 (m, 1H), 7.37–7.36 (m, 1H), 5.30 (s, 1H), 3.41
(m, 1H), 2.69 (m, 2H), 2.45–2.43 (m, 2H); 13C NMR (125 MHz,

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1039
DMSO-d6, d): 144.45, 139.04, 139.02, 132.36, 129.23, 128.86,
128.62, 126.49, 125.51, 124.99, 103.55, 38.88.
Compound 41 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.33 (br s, 1H), 7.50–7.48 (m, 1H), 7.28–7.21 (m, 3H), 7.19–7.16
(m, 1H), 7.02–7.00 (m, 2H), 6.84–6.83 (m, 1H), 5.26 (s, 1H), 3.59–
3.56 (m, 1H), 2.69–2.63 (m, 2H), 2.39–2.36 (m, 2H); 13C NMR
(125 MHz, DMSO-d6, d): 158.95, 157.05, 154.14, 153.25, 153.23,
133.90, 128.27, 128.08, 124.18, 119.75, 119.68, 118.75, 116.75,
116.56, 103.37, 32.74.
38
Compound 38 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.26 (br s, 1H), 7.35 (d, J = 9.0 Hz, 2H), 7.23–7.21 (m, 2H), 7.06–
7.03 (m, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.30 (s, 1H), 3.33–3.26 (m,
1H), 2.60–2.54 (m, 2H), 2.41–2.37 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 159.18, 157.27, 155.75, 152.74, 152.71, 138.62,
128.58, 120.67, 120.61, 118.15, 116.71, 116.52, 103.59, 38.10.
42
Compound 42 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.27 (br s, 1H), 7.43–7.41 (m, 2H), 7.36–7.33 (m, 1H), 7.16–7.15
(m, 1H), 7.08 (s, 1H), 7.03–7.01 (m, 2H), 6.87–6.85 (m, 1H), 5.27
(s, 1H), 3.35–3.29 (m, 1H), 2.62–2.56 (m, 2H), 2.40–2.37 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 156.30, 155.67, 146.16, 130.22,
129.93, 127.12, 122.57, 120.18, 117.73, 116.88, 103.54, 38.57.
43
39
Compound 43 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.27 (br s, 1H), 7.41–7.36 (m, 2H), 7.24–7.22 (m, 1H), 7.16 (s,
1H), 7.03 (s, 2H), 6.97–6.95 (m, 1H), 5.27 (s, 1H), 3.35–3.33 (m,
1H), 2.63–2.57 (m, 2H), 2.42–2.38 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 158.69, 155.10, 146.45, 134.96, 130.44, 123.61,
122.88, 118.54, 117.72, 116.95, 103.50, 38.51.
Compound 39 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.23 (br s, 1H), 7.42–7.36 (m, 4H), 7.02–6.98 (m, 4H), 5.30 (s,
1H), 3.35–3.29 (m, 1H), 2.61–2.55 (m, 2H), 2.42–2.38 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 155.86, 154.77, 139.21, 129.90,
128.68, 127.06, 120.06, 118.96, 103.58, 38.10.
44
40
Compound 44 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.19 (br s, 1H), 7.25 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H),
5.28 (s, 1H), 3.29–3.22 (m, 1H), 2.87–2.82 (m, 1H), 2.59–2.54 (m,
2H), 2.39–2.36 (m, 2H), 1.19 (t, J = 7.0 Hz, 6H); 13C NMR
(125 MHz, DMSO-d6, d): 146.63, 140.96, 126.84, 126.37, 103.54,
38.40, 33.08, 23.95.
Compound 40 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.25 (br s, 1H), 7.39–7.38 (m, 3H), 7.18 (d, J = 8.0 Hz, 2H), 7.02–
6.00 (m, 3H), 6.95–6.92 (m, 3H), 5.31 (s, 1H), 3.36–3.29 (m, 1H),
2.62–2.56 (m, 2H), 2.43–2.40 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 158.10, 154.30, 139.57, 134.01, 131.47, 128.75,
123.17, 119.32, 118.10, 116.82, 103.59, 38.12.
41
45

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W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
Compound 45 was purified by recrystallization (ether/hexanes)
Compound 49 was purified by recrystallization (ether/hex-
anes) and obtained as
white solid. 1H NMR (500 MHz,
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.28 (br s, 1H), 7.30–7.27 (m, 2H), 7.26–7.22 (m, 4H), 7.20–7.17
(m, 1H), 6.88 (d, J = 8.5 Hz, 2H), 5.30 (s, 1H), 3.94 (t, J = 6.5 Hz,
2H), 3.27–3.21 (m, 1H), 2.74 (t, J = 7.5 Hz, 2H), 2.58–2.51 (m, 2H),
2.39–2.35 (m, 2H), 2.03–1.97 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 157.33, 141.45, 135.57, 128.40, 127.95, 125.89,
114.41, 103.57, 66.62, 38.03, 31.53, 30.47.
a
DMSO-d6, d): 11.17 (br s, 1H), 7.11 (d, J = 8.5 Hz, 1H), 6.51 (s,
1H), 6.46 (d, J = 8.5 Hz, 1H), 5.27 (s, 1H), 4.04–3.97 (m, 4H),
3.51–3.45 (m, 1H), 2.58–2.51 (m, 2H), 2.36–2.33 (m, 2H), 1.33–
1.29 (m, 6H); 13C NMR (125 MHz, DMSO-d6, d): 158.42, 156.86,
127.43, 123.13, 104.99, 103.41, 99.69, 63.30, 63.02, 32.45,
14.72, 14.70.
46
Compound 46 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.30 (br s, 1H), 7.86–7.83 (m, 4H), 7.74 (s, 1H), 7.64 (s, 2H),
7.34–7.30 (m, 4H), 5.32 (s, 1H), 3.37 (m, 1H), 2.82–2.76 (m, 2H),
2.51–2.48 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 162.96,
161.01, 145.10, 140.04, 136.59, 136.57, 129.11, 129.04, 124.63,
123.46, 115.71, 115.54, 103.45, 38.95.
50
Compound 50 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.29 (br s, 1H), 7.19 (d, J = 8.5 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H),
5.26 (s, 1H), 3.44–3.39 (m, 4H), 3.20 (m, 1H), 3.05–3.03 (m, 4H),
2.55–2.50 (m, 2H), 2.36–2.33 (m, 2H), 1.41 (s, 9H); 13C NMR
(125 MHz, DMSO-d6, d): 153.83, 149.65, 134.53, 127.40, 116.15,
103.49, 78.96, 48.80, 37.94, 28.04.
47
Compound 47 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.25 (br s, 1H), 7.32–7.25 (m, 4H), 6.99–6.92 (m, 5H), 5.27 (s, 1H),
4.29 (s, 4H), 3.28–3.23 (m, 1H), 2.58–2.52 (m, 2H), 2.37–2.34 (m,
2H); 13C NMR (125 MHz, DMSO-d6, d): 158.31, 157.01, 135.91,
129.59, 128.01, 120.77, 114.47, 114.46, 103.51, 66.31, 66.18, 38.01.
51
Compound 51 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.20 (br s, 1H), 6.51 (s, 2H), 6.36 (s, 1H), 5.28 (s, 1H), 3.71 (s,
6H), 3.25–3.20 (m, 1H), 2.61–2.57 (m, 2H), 2.38–2.33 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 160.45, 145.93, 104.82, 103.41,
98.42, 55.05, 39.03.
48
Compound 48 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.24 (br s, 1H), 7.00–6.97 (m, 1H), 6.91–6.88 (m, 2H), 5.30 (s,
1H), 4.03–3.94 (m, 4H), 3.66–3.61 (m, 1H), 2.58–2.53 (m, 2H),
2.31–2.29 (m, 2H), 1.35 (t, J = 7.0 Hz, 3H), 1.27 (t, J = 7.0 Hz, 3H);
13C NMR (125 MHz, DMSO-d6, d): 151.61, 145.41, 136.72, 123.84,
118.56, 112.01, 103.46, 68.28, 63.63, 32.49, 15.69, 14.81.
52
Compound 52 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.29 (br s, 1H), 8.13–8.11 (m, 2H), 7.58–7.52 (m, 2H), 7.45–7.42
(m, 2H), 7.19–7.16 (m, 1H), 5.34 (s, 1H), 4.43 (q, J = 7.0 Hz, 2H),
3.49–3.42 (m, 1H), 2.76–2.70 (m, 2H), 2.50–2.47 (m, 2H), 1.30 (t,
J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6, d): 139.84, 138.51,
134.12, 125.69, 124.97, 122.22, 122.13, 120.33, 118.62, 118.43,
109.10, 109.08, 103.60, 39.08, 36.97, 13.76.
49

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1041
Compound 56 was purified by recrystallization (ether/hex-
anes) and obtained as a white solid. 1H NMR (500 MHz, DMSO-
d6, d): 11.29 (br s, 1H), 7.99 (s, 1H), 7.83 (s, 1H), 7.78–7.76 (m,
2H), 7.73 (s, 1H), 7.52–7.49 (m, 2H), 7.44–7.41 (m, 1H), 5.31 (s,
1H), 3.56–3.50 (m, 1H), 2.79–2.74 (m, 2H), 2.50–2.45 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 145.92, 141.53, 138.66,
130.14, 129.88, 129.74, 129.64, 129.10, 128.30, 127.17, 125.35,
123.19, 122.86, 122.83, 122.81, 122.78, 121.58, 121.55, 121.51,
103.53, 38.67.
53
Compound 53 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.44 (br s, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H),
7.98 (s, 1H), 5.33 (s, 1H), 3.62–3.57 (m, 1H), 2.86–2.81 (m, 2H),
2.31–2.27 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 146.82,
130.45, 129.80, 129.78, 128.31, 128.05, 127.92, 127.78, 127.68,
127.52, 127.45, 127.21, 126.83, 126.57, 124.65, 124.40, 122.72,
122.47, 122.24, 120.28, 120.07, 103.23, 35.30.
57
Compound 57 was purified by recrystallization (ether/
hexanes) and obtained as a white solid. 1H NMR (500 MHz,
DMSO-d6, d): 11.32 (br s, 1H), 8.06 (s, 1H), 7.91–7.90 (m, 2H),
7.77–7.76 (m, 2H), 7.54–7.47 (m, 2H), 5.32 (s, 1H), 3.55–3.50
(m, 1H), 2.82–2.77 (m, 2H), 2.48–2.44 (m, 2H); 13C NMR
(125 MHz, DMSO-d6, d): 146.03, 140.73, 139.91, 133.94, 130.87,
130.47, 130.22, 129.97, 129.93, 129.72, 128.13, 127.44, 126.98,
125.90, 125.28, 123.50, 123.47, 123.11, 121.86, 121.83, 120.94,
103.52, 38.69.
54
Compound 54 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.29 (br s, 1H), 7.72 (s, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.44–7.40
(m, 1H), 7.31 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.31 (d,
J = 3.5 Hz, 1H), 5.27 (s, 1H), 3.53–3.49 (m, 1H), 2.67–2.58 (m,
4H); 13C NMR (125 MHz, DMSO-d6, d): 157.18, 150.16, 133.79,
132.32, 130.81, 126.88, 122.67, 121.69, 108.07, 107.38, 103.69,
32.25.
58
Compound 58 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.31 (br s, 1H), 7.72–7.70 (m, 1H), 7.64 (s, 1H), 7.58 (s, 1H),
7.49–7.44 (m, 3H), 7.41–7.38 (m, 2H), 5.31 (s, 1H), 3.44–3.37 (m,
1H), 2.74–2.68 (m, 2H), 2.50–2.43 (m, 2H); 13C NMR (125 MHz,
DMSO-d6, d): 146.70, 142.44, 138.72, 133.81, 129.04, 128.16,
127.02, 125.98, 124.75, 124.47, 103.56, 38.66.
55
Compound 55 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.30 (br s, 1H), 7.80–7.78 (m, 4H), 7.65 (s, 2H), 7.46–7.44 (m,
4H), 7.41–7.39 (m, 3H), 5.31 (s, 1H), 3.50–3.45 (m, 1H), 2.80–2.75
(m, 2H), 2.51–2.48 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d):
145.00, 141.08, 140.19, 128.65, 127.55, 127.04, 124.70, 123.53,
103.47, 38.95.
59
Compound 59 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.27 (br s, 1H), 7.75 (s, 1H), 7.61–7.60 (m, 1H), 7.49–7.47 (m,
2H), 7.44–7.39 (m, 2H), 7.23–7.19 (m, 1H), 5.32 (s, 1H), 3.43–
3.35 (m, 1H), 2.74–2.68 (m, 2H), 2.46–2.43 (m, 2H); 13C NMR
(125 MHz, DMSO-d6, d): 163.94, 163.84, 161.99, 161.89, 144.57,
143.93, 143.85, 143.77, 137.81, 137.79, 137.77, 129.28, 127.44,
125.77, 125.22, 110.02, 109.98, 109.87, 109.82, 103.56, 102.94,
102.74, 102.53, 38.94.
56

1042
W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
64
60
Compound 64 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.03 (br s, 1H), 7.32–7.29 (m, 2H), 7.22–7.19 (m, 3H), 5.16 (s,
1H), 2.64–2.58 (m, 1H), 2.41–2.38 (m, 1H), 2.16–2.08 (m, 2H),
1.96–1.91 (m, 1H), 1.82–1.79 (m, 1H), 1.22 (d, J = 7.0 Hz, 3H); 13C
NMR (125 MHz, DMSO-d6, d): 145.33, 128.42, 127.41, 126.24,
103.38, 43.80, 39.56, 18.79.
Compound 60 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.24 (br s, 1H), 8.38 (s, 2H), 8.09 (s, 1H), 7.91 (s, 1H), 7.74–7.73
(m, 1H), 7.50–7.45 (m, 2H), 5.32 (s, 1H), 3.46–3.40 (m, 1H), 2.80–
2.64 (m, 2H), 2.46–2.36 (m, 2H); 13C NMR (125 MHz, DMSO-d6,
d): 144.78, 142.75, 137.15, 131.29, 131.03, 130.77, 130.51,
129.41, 127.73, 127.59, 126.69, 126.28, 125.72, 124.52, 123.14,
122.35, 120.95, 120.91, 120.88, 120.18, 105.85, 103.50.
65
61
Compound 65 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.06 (br s, 1H), 7.36–7.35 (m, 4H), 7.29–7.26 (m, 4H), 7.18–7.14
(m, 2H), 5.13 (s, 1H), 4.11 (t, J = 8.0 Hz, 1H), 2.28 (m, 2H), 2.12–
2.09 (m, 4H), 1.77–1.74 (m, 1H); 13C NMR (125 MHz, DMSO-d6,
d): 144.80, 128.51, 127.61, 126.17, 103.46, 99.57, 47.32, 40.19,
31.36.
Compound 61 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.34 (br s, 1H), 7.65–7.63 (m, 2H), 7.42–7.35 (m, 3H), 5.31 (s,
1H), 3.60–3.54 (m, 1H), 2.69–2.64 (m, 2H), 2.33–2.30 (m, 2H);
13C NMR (125 MHz, DMSO-d6, d): 146.02, 135.55, 128.62, 128.57,
127.93, 123.23, 121.19, 120.88, 119.15, 117.11, 103.42, 32.18.
62
66
Compound 66 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.19 (br s, 1H), 7.30–7.26 (m, 2H), 6.95–6.91 (m, 3H), 5.24 (s,
1H), 3.93 (d, J = 6.0 Hz, 2H), 2.52–2.46 (m, 1H), 2.41–2.38 (m,
2H), 2.28–2.23 (m, 2H); 13C NMR (125 MHz, DMSO-d6, d): 158.45,
129.56, 120.70, 114.50, 103.66, 70.12, 33.25.
Compound 62 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.14 (br s, 1H), 7.31–7.29 (m, 2H), 7.22–7.17 (m, 3H), 5.18 (s,
1H), 2.63 (d, J = 7.0 Hz, 2H), 2.25–2.23 (m, 1H), 2.18–2.10 (m,
4H); 13C NMR (125 MHz, DMSO-d6, d): 139.54, 129.03, 128.35,
126.13, 103.59, 40.80, 35.22.
63
67
Compound 63 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.02 (br s, 1H), 6.83 (s, 1H), 6.78 (s, 2H), 5.18 (s, 1H), 2.54 (d,
J = 7.0 Hz, 2H), 2.23 (s, 6H), 2.21–2.08 (m, 5H); 13C NMR
(125 MHz, DMSO-d6, d): 139.33, 137.19, 127.55, 126.78, 103.59,
40.73, 35.20, 20.96.
Compound 67 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.25 (br s, 1H), 7.62–7.60 (m, 3H), 5.25 (s, 1H), 4.14 (d,
J = 6.5 Hz, 2H), 2.55–2.51 (m, 1H), 2.42–2.38 (m, 2H); 13C NMR
(125 MHz, DMSO-d6, d): 159.53, 159.46, 132.01, 131.75, 131.49,

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1043
131.23, 126.46, 124.29, 122.12, 119.94, 115.66, 115.63, 113.83,
113.81, 113.77, 103.68, 71.45, 33.09.
(500 MHz, DMSO-d6, d): 11.09 (br s, 1H), 7.24–7.21 (m, 2H),
7.12–7.09 (m, 1H), 7.06–7.05 (m, 2H), 5.19 (s, 1H), 3.77 (s, 2H),
2.39–2.26 (m, 4H), 2.15–2.11 (m, 1H), 1.60–1.54 (m, 1H), 1.26–
1.21 (m, 1H), 1.18–1.14 (m, 1H), 1.10–1.05 (m, 1H); 13C NMR
(125 MHz, DMSO-d6, d): 147.37, 130.61, 130.35, 130.09, 129.83,
126.73, 126.19, 126.16, 124.55, 122.39, 120.22, 118.75, 118.72,
118.69, 118.65, 103.69, 38.13, 29.66, 20.91, 15.29.
68
Compound 68 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.15 (br s, 1H), 7.29–7.26 (m, 2H), 6.94–6.92 (m, 3H), 5.23 (s,
1H), 3.77 (s, 2H), 2.50–2.45 (m, 2H), 2.17–2.14 (m, 2H), 1.09 (s,
3H); 13C NMR (125 MHz, DMSO-d6, d): 158.70, 129.52, 120.73,
114.55, 102.61, 74.13, 36.10, 22.71.
72
Compound 72 was purified by flash column chromatography
(ethyl acetate) and obtained as a white solid. 1H NMR (500 MHz,
DMSO-d6, d): 11.17 (br s, 1H), 7.13 (s, 1H), 7.07 (s, 1H), 5.22 (s,
1H), 4.54–4.52 (m, 1H), 2.40–2.37 (m, 1H), 2.28–2.24 (m, 4H),
1.22 (d, J = 6.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6, d): 159.15,
134.66, 120.29, 114.76, 103.49, 76.17, 38.46, 16.26.
69
Compound 69 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.20 (br s, 1H), 7.64 (s, 2H), 7.62 (s, 1H), 5.24 (s, 1H), 3.98 (s,
2H), 2.50 (m, 2H), 2.19–2.15 (m, 2H), 1.10 (s, 3H); 13C NMR
(125 MHz, DMSO-d6, d) 159.68, 159.65, 131.97, 131.71, 131.45,
131.19, 126.45, 124.28, 122.10, 119.94, 115.73, 115.63, 113.86,
113.83, 113.79, 102.67, 75.30, 36.06, 22.51.
73
Compound ( )-73 was purified by flash column chromatogra-
phy (ethyl acetate) and obtained as a white solid. Mp: 137–
138 °C; 1H NMR (500 MHz, DMSO-d6, d): 11.17 (br s, 1H), 7.62 (s,
2H), 7.60 (s, 1H), 5.22 (s, 1H), 4.75–4.73 (m, 1H), 2.43–2.41 (m,
1H), 2.34–2.28 (m, 4H), 1.25 (d, J = 6.0 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6, d): 158.65, 132.04, 131.78, 131.52, 131.26,
126.36, 124.19, 122.02, 119.85, 116.38, 113.62, 103.50, 76.31,
38.53, 16.08. Elemental analysis: Anal. Calcd for C16H14F6O3: C,
52.18; H, 3.83; F, 30.95. Found: C, 52.28; H, 3.70; F, 30.69. Purity:
>95%.
70
Compound 70 was purified by recrystallization (ether/hexanes)
and obtained as a white solid. 1H NMR (500 MHz, DMSO-d6, d):
11.09 (br s, 1H), 7.79 (s, 1H), 7.76 (s, 2H), 5.20 (s, 1H), 3.77 (s,
2H), 2.38–2.34 (m, 2H), 2.26–2.21 (m, 2H), 1.81–1.77 (m, 1H),
1.55–1.49 (m, 1H), 1.03–0.98 (m, 1H), 0.90–0.87 (m, 2H); 13C
NMR (125 MHz, DMSO-d6, d): 143.22, 128.25, 125.55, 125.27,
103.65, 38.25, 28.21, 21.31, 14.23.
4.4. In vitro pharmacokinetic studies
Studies were performed at Apredica, Inc. (Watertown, MA).
4.5. Chromatography and mass spectrometry
Samples were analyzed by LC-MS/MS using an Agilent 6410
mass spectrometer coupled with an Agilent 1200 HPLC and a CTC
PAL chilled auto sampler, all controlled by MassHunter software
(Agilent). After separation on a C18 reverse phase HPLC column
(Agilent, Waters, or equivalent) using a 4 min acetonitrile–water
gradient system, peaks were analyzed by mass spectrometry
(MS) using ESI ionization in the MRM mode. The mass spectrome-
ter gas flows and voltages were individually tuned to provide opti-
mal signal for each compound. Trial spectra were obtained to
determine the best conditions for data collection. The transition(s)
that gave the best signal/noise ratio were used for data analysis.
See Supplementary data for representative MS spectra and
chromatography data.
71
Compound ( )-71 was purified by recrystallization (ether/hex-
anes) and obtained as a white solid. Mp: 154–156 °C; 1H NMR

1044
W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
4.6. Microsomal stability
Caco-2 results are reported as apparent permeability, Papp, ex-
dQ=dt
pressed in units of 10ꢀ6 cm/s: Papp
¼
where dQ/dt is the rate
c0
A
Test compounds were incubated in duplicate at 5
lM concen-
of permeation (mol test compound transported per unit time), c0
is the initial concentration of test compound, and A is the area of
the cell monolayer. See Supplementary data for Caco-2 permeabil-
ity of CMB-086874.
tration with human or mouse microsomes at 37 °C. The reaction
mixture contained 0.275 mg/mL microsomal protein in 100 mM
potassium phosphate, 2 mM NADPH, 3 mM MgCl2, pH 7.4. A con-
trol was run for each compound omitting NADPH to detect
NADPH-independent degradation. After 15 s, 10 min, 20 min,
40 min, and 60 min incubation, aliquots were removed from each
experimental and control reaction and mixed with an equal vol-
ume of ice-cold Stop Solution (0.3% acetic acid in acetonitrile con-
4.8. Solubility screen
The solubility of the test compounds in PBS was estimated by
absorbance. Threefold serial dilutions were prepared in DMSO at
100 times the final concentration. This DMSO stock was diluted
to the final concentration in PBS, the plate was incubated at room
temperature for 1 h, then it was examined visually for the forma-
tion of precipitate, and the absorbance at 450 nm was measured.
For non-colored compounds the absorbance value was used to de-
tect solubility, while for highly colored compounds, visual inspec-
tion was used. The highest concentration that was not visibly
cloudy and whose absorbance was not different from background
was judged to be the solubility limit (see Supplementary data).
taining 0.3 lM haloperidol as an internal standard). Stopped
reactions were incubated at least 10 min at ꢀ20 °C, and an addi-
tional volume of water was added. The samples were centrifuged
to remove precipitated protein, and the supernatants were
analyzed by LC–MS/MS to quantify the remaining parent molecule.
Excel was used for the calculations. The Response Ratio (RR) was
calculated using Mass Hunter software by dividing the peak area
of the analyte by the peak area of the internal standard. The rela-
tive ratio was calculated by dividing the RR with NADPH by the
RR from the –NADPH control at the same time point.
4.9. Stability in plasma and PBS
ðRelative Ratio ¼ PAþNADPH=PAꢀNADPHÞ
The % compound remaining (RE) was calculated by dividing the
The test compounds, with warfarin and diltiazem as controls,
relative ratio of the sample in question by A0,
were incubated in mouse plasma and PBS in duplicate at 5 lM con-
centration. The time-zero samples were taken immediately (15 s)
after adding the compounds. The plate containing the plasma
and PBS samples was incubated at 37 °C, and then time points
were taken at 10, 20, 40, and 60 min in the same way. The plasma
samples and PBS samples were precipitated with acetonitrile con-
taining haloperidol as an internal standard and either centrifuged
or filtered through a Varian Captiva plate (following the manufac-
turer’s directions) to remove precipitated protein. The samples
were then analyzed by LC–MS/MS.
The response ratio (RR = area of analyte peak divided by the
area of the internal standard peak), as calculated by the LC–MS
software, was used for determining the plasma stability. The rela-
tive ratio was calculated by dividing the RR from plasma by the RR
from the PBS control at the same time point.
RE ¼ RR=A0
where A0 is a correction factor for small differences in the starting
concentration and extraction efficiency. Curve fitting was used to
determine A0, k (first-order rate constant) using an exponential fit
with 1/Y weighing. The half-life was calculated as: T1/2 = ln(2)/k.
The intrinsic clearance was calculated as follows: CL0int ¼ ð0:693=
T1=2Þ ꢂ (mL incubation/mg microsomal protein) ꢂ (mg microsomal
protein/g liver) ꢂ (g liver/kg bodyweight). The scale-up factor for
microsomes protein to g of liver is 45 mg/g of liver. Liver weights used
for human and mice were 20 g/kg body weight and 34 g/kg body
weight, respectively. The units on the CLint are mL/min/kg. See Sup-
plementary data for human/mouse microsomal stability results with
CMB-086874.
ðRelative Ratio ¼ RRplasma=RRPBS
Þ
4.7. Caco-2 monolayer permeability
The % compound remaining (RE) was calculated as described above
Caco-2 permeability is measured by detecting the amount of
compound that permeates through a confluent monolayer of
Caco-2 cells. Caco-2 cells grown in tissue culture flasks were tryp-
sinized, suspended in medium, and the suspensions were applied
to wells of a collagen-coated BioCoat Cell Environment in a 24-well
format (BD Biosciences). The cells were allowed to grow and differ-
entiate for three weeks, feeding at 2-day intervals.
for microsomes. To determine PBS half-life, the RR was fit to a first-
PBSt
order exponential curve to determine APBS and kPBS: RR ¼ APBSeꢀk
.
The half-life was calculated as above: T1/2 = In(2)/kPBS. See Supple-
mentary data for mouse plasma stability of CMB-086874.
4.10. Primary cortical neuron assay
Stock solutions of 10 mM test compound or control prepared in
DMSO were used to prepare solutions for the Caco-2 permeability
studies. For apical to basolateral (A?B), the apical (A-side) buffer
Primary rat cortical tissue was purchased from Neuromics Inc.,
Edina, MN and used to initiate primary cortical neuron cultures.
The tissue was isolated from micro-surgically dissected E18
embryonic Sprague/Dawley or Fischer 344 rat brain and shipped
in a nutrient rich medium under refrigeration.
To isolate neurons, the tissue was incubated with papain at a
concentration of 2 mg/mL in Hibernate without calcium for
30 min at 37 °C. The enzymatic solution was then removed, and
1 mL of culture media (Neurobasal, B27, 0.5 mM glutamine) was
added. A sterile Pasteur pipette was used to gently disperse the
cells, which were then washed, re-suspended and counted. The
contained 50 lM test compound or control and 100 lM Lucifer yel-
low dye in the transport buffer (1.98 g/L glucose in 10 mM HEPES,
1 ꢂ Hank’s Balanced Slat Solution) at pH 6.5; the basolateral (B-
side) buffer was the transport buffer at pH 7.4. For B?A studies,
the A-side buffer contained 100
buffer, pH 6.5, and the B-side buffer contained 50
l
M Lucifer yellow in the transport
M test com-
l
pound or control in the transport buffer, pH 7.4. The Caco-2 cells
were incubated with these buffers for 2 h, and the cell buffers
and dosing solutions were removed for analysis.
cells were plated on poly-D-lysine coated 96-well plates at a
To verify that the Caco-2 cell monolayer was properly formed,
an aliquot of the cell buffer from each well was analyzed by
fluorescence to determine the transport of the impermeable Luci-
fer Yellow dye. In all cases, acceptable transport of Lucifer Yellow
was observed (<1% transport in 2 h).
density of 20,000 cells/well and incubated at 37 °C in a 5%
CO2-humidified atmosphere for 5 days prior to use in compound
testing. By microscopic inspection, the resulting cultures consisted
of ꢁ90% neurons.

W. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 1029–1045
1045
Test compounds were assayed in six-point dose response exper-
iments. The highest compound concentration tested was 100 M,
4.14. Plasma concentration and blood brain barrier penetration
of 26
l
which was then diluted by approximately one-third with each sub-
sequent dose. After 24 h incubation with the compounds, MG132
was added at a final concentration of 100 nM, a dose that produces
an approximately 70% loss of viability. Cell viability was measured
48 h later using the fluorescent viability probe, Calcein-AM (Molec-
ular Probes, Invitrogen, Carlesbad, CA). Briefly, cells were washed
twice with PBS, Calcein-AM was added at a final concentration of
Compound 26 was administered in mice as a single bolus intra-
peritoneal injection of 50 mg/kg. A group of 18 mice were used and
blood and brain tissue samples were immediately quenched in li-
quid nitrogen and stored at ꢀ80 °C for analysis. Samples were col-
lected at 0 h (no drug), 1 h, 3 h, 6 h, 12 h, and 24 h.
1 lM for 20 min at room temperature, and fluorescence intensity
4.15. Effect of 26 in ALS mouse model
was then read in a POLARstar fluorescence plate reader (BMG).
Compounds that restored viability at any dose to a level equal or
higher than 5 standard deviations from MG132 controls were con-
sidered active.
A dose–response curve was implemented with 5 cohorts of
mice (littermate wild type untreated mice, untreated mutant
SOD 1 G93A mice, and 3 treated cohorts of mutant SOD 1 G93A
mice at 1 mg/kg, 10 mg/kg, and 20 mg/kg). Mice were weighed
and administered drug daily via intraperitoneal injection at the
same time of day. The mice were monitored twice daily for any ad-
verse events and were euthanized at end stage disease using the
righting mechanism as a surrogate measure of death.
4.11. Radioligand binding assays
These studies were carried out by MDS Pharma Services.
4.12. Effect of compound 26 on hERG potassium channels
Acknowledgments
FASTPatch hERG screen assay was carried out by ChanTest Cor-
poration (Cleveland, OH). Cloned hERG potassium channels were
expressed in human embryonic kidney cells (HEK293).
We thank the National Institutes of Health [1R43NS057849],
the ALS Association (TREAT program), the Department of Defense
[AL093052], and the Veterans Administration Pittsburgh Health-
care System, 7180 Highland Drive, Pittsburgh, PA for their gener-
ous support of the research project. The authors are grateful to
Biogen Idec, Inc. for carrying out the in vivo rat PK profiling tests,
in vitro P450 reversible inhibition study, the plasma binding
affinity assay, the hERG inhibition assay, and for funding to MDS
Pharma Service (now Ricerca Biosciences, LLC), who carried out
the LeadProfilingScreen of 26.
4.13. General procedures for in vivo experiments
Male transgenic ALS mice of the G93A H1 high-expressor strain
(Jackson Laboratories, Bar Harbor, ME, USA) were bred with fe-
males having a similar background (B6/SJLF1). Offspring were gen-
otyped using a PCR assay on tail DNA. To ensure homogeneity of
the cohorts tested, we developed a standardized method by which
to select mice. Mice were randomized from 24 litters all within
4 days of age from the same ‘f’ generation removed from the found-
ing mice in our colony. Bodyweights were taken at 20 days, and
mice were equally distributed according to weight within each
experimental cohort. Mice under 8 g at 20 days were excluded
from the experiments. Only male mice were used in the treatment
studies because we have observed gender differences in survival in
the G93A transgenic ALS mouse model.9 These experiments were
carried out in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals, and were
approved by both the Veterans Administration and the University
References and notes
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Animal Care Committees. Based upon previous studies,10,11
a
dose–response study was performed. Groups (n = 10) of wild-type
(WT), untreated G93A, and G93A mice received 1, 10, and 20 mg/kg
of 26 injected intraperitoneally once a day in the morning. Mice
were observed twice daily, morning and afternoon, for any adverse
events. Compound 26 was dissolved in phosphate-buffered saline
(PBS) with 0.1% DMSO, and fresh solutions were prepared daily.
Treatment was started at 42 days, as our studies demonstrate that
clinical disease onset begins on or about this age. The ALS mice
were euthanized when they were unable to right themselves after
being placed on their back for 30 s. Some deaths occurred over-
night and were recorded the following morning. Two independent
observers who were blinded to the treatment assignment agreed
when animals should be euthanized. Survival data were analyzed
by means of Kaplan–Meier survival curves.
11. Ryu, H.; Ferrante, R. J. MRMC 2007, 7, 141.
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Kirsch, D. R.; Ferrante, R. J.; Silverman, R. B. J. Med. Chem. 2012, in press.