Design, synthesis and SAR of indazole and benzoisoxazole containing 4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.08.074

A novel series of 4-azetidinyl-1-aryl-cyclohexanes containing indazole or benzoisoxazole moiety have been identified as potent CCR2 antagonists with high selectivity versus hERG.

Full text of DOI:10.1016/j.bmcl.2011.08.074

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6042–6048
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and SAR of indazole and benzoisoxazole containing
4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists
⇑
Xuqing Zhang , Heather Hufnagel, Cuifen Hou, Evan Opas, Sandra McKenney, Carl Crysler,
John O’Neill, Dana Johnson, Zhihua Sui
Johnson & Johnson Pharmaceutical Research and Development, Welsh & McKean Roads, Spring House, PA 19477, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 4-azetidinyl-1-aryl-cyclohexanes containing indazole or benzoisoxazole moiety have
been identified as potent CCR2 antagonists with high selectivity versus hERG.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 19 June 2011
Revised 11 August 2011
Accepted 15 August 2011
Available online 22 August 2011
Keywords:
CCR2
Indazole
Benzoisoxazole
MCP-1 is a potent chemotactic factor for monocytes and mem-
ory T lymphocytes, and stimulates the movement of those cells
along a chemotactic gradient following binding to its cell-surface
receptor, CC chemokine receptor-2 (CCR2). MCP-1 interacts exclu-
sively with CCR2 and is therefore recognized as the prime CCR2 li-
gand.1–5 This ligand/receptor pair is overexpressed in numerous
inflammatory conditions wherein excessive monocyte recruitment
is observed. Indeed, CCR2- and MCP-1-deficient mice and CCR2 or
MCP-1 antibody-treated rodents show decreased recruitment of
monocytes and produce markedly attenuated inflammatory re-
sponses in animal models of multiple sclerosis, rheumatoid arthri-
tis, atherosclerosis, diabetes, asthma, allograft rejection, and
neuropathic pain.6–15 Clearly, these observations confirm the role
of CCR2 in the pathogenesis of several immune-based inflamma-
tory diseases and identify this chemokine receptor as a potentially
valuable therapeutic target. An antagonist of the binding of MCP-1
to its receptor CCR2 may be an effective treatment for any inflam-
matory disease in which monocytes, mast cells, or basophils play
major roles. As a result, there has been significant interest in the
design and synthesis of CCR2 antagonists.16–34
1 in our early series (Fig. 1). Installation of ortho-amino group at
the right side of the phenyl ring was well tolerated for CCR2
binding affinity and functional activities. Encouraged by this
finding, we investigated fusion strategy between amino group
and its neighboring amide group. Hence, compounds 2–5 bearing
5/6-fused ring systems have been synthesized and their CCR2
binding affinity is summarized in Table 1. Direct connection of
the ortho-amino and the amide nitrogen dramatically reduced
CCR2 binding affinity as shown in 2 with IC50 of 5.4 lM. To our
delight, moving the amide to the 3-position of the five-membered
heterocycle to form an aromatic system provided compounds
reasonable CCR2 binding affinity (compounds 3–5). Compound 5
bearing amino indazole moiety was particularly interesting
because it improved CCR2 binding affinity by threefold compared
to its diamide counterpart (1, IC50 46 nM).35 Detailed SAR and
O
We have recently described azetidinyl cyclohexane scaffold
bearing two consecutive amide bonds as potent CCR2 antagonists
with good selectivity versus hERG.35 However, this type of func-
tionality is prone to rapid metabolism in vivo, which might lead
to low exposure. We describe herein our exploration on amide
replacements.36,37 We were guided by the result from compound
N
NH HN
O
NH2
F3C
1
hCCR2 binding, IC50, 46 nM
hCCR2 function (CTX), IC50, 9 nM
⇑
Corresponding author.
E-mail address: xzhang5@prdus.jnj.com (X. Zhang).
Figure 1. Design of 5,6-fused ring system.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.074

X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6042–6048
6043
R1 group was well tolerated for CCR2 binding affinity,38 as
evidenced by compounds bearing electron neutral (13a), electron
rich (13b and 13e), polar phenyl ring (13c), or heterocyclic ring
(13d). Unfortunately, these compounds exhibited strong hERG
binding affinity which could be related to the chemo-type contain-
ing a central basic amine flanked by two aromatic rings. While dis-
playing weaker CCR2 binding and functional activity,39 compound
13f was quite interesting since it also presented weaker hERG
Table 1
N
NH
O
R
A
ID
R
CCR2 bindinga IC50 (nM)
binding affinity (IC50 of 34 l
M).40 Structurally, compound 13f con-
O
CF3
tained a partially unsaturated pyridone substitution at the R1 posi-
tion, which indicated that nonaromatic R1 might be tolerated for
CCR2 activity and may increase selectivity versus hERG. To explore
this hypothesis, we synthesized a sub-set of derivatives (14a–14n)
bearing nonaromatic R1 group and evaluated both CCR2 and hERG
affinity. In contrast to their diamide analogs which were usually
2
5,400c
290c
510c
12b
N
N
H
S
N
CF3
CF3
CF3
3
4
N
H
inactive in CCR2 (binding IC50 >25 lM, data not shown), nonaro-
matic R1 substituted indazoles 14 displayed moderate to good
CCR2 binding and functional activity. As illustrated by 14a and
14b, installation of i-Pr or CN group at the R1 position exhibited
O
N
CCR2 binding affinity to sub-lM. Incorporation of more lipophilic
N
H
cyclohexanyl group (14c) slightly increased CCR2 binding affinity
as compared to 14a. To our delight, ethyl carboxylate substituted
indazole 14d displayed comparable CCR2 antagonistic activity as
its aromatic substituted analogs (binding IC50 of 13 nM, CTX IC50
of 40 nM). Unfortunately, 14d showed no improvement on attenu-
ating hERG affinity. While significantly decreasing hERG affinity,
substitution at the R1 group with polar groups such as carboxylic
acid (14e), carbamide (14f) or hydroxyl (14j) failed to maintain
good CCR2 binding affinity. Alcohol 14g was promising, as it dis-
played potent CCR2 binding affinity (IC50 of 44 nM) with promising
(ꢀ80-fold) selectivity over hERG. To circumvent the phase II
metabolism liability of 14g due to the primary hydroxyl group,
ethyl (14h) and allyl ethers (14i) were evaluated. These modifica-
tions maintained good CCR2 binding affinity but increased hERG
inhibition as compared to 14g. Similar operation by masking alco-
hol 14j with ethyl (14k) or allyl group (14l) only yielded moderate
enhancement on CCR2 binding affinity. Substitution containing
amino group either dramatically reduced or completely abolished
CCR2 binding affinity as shown by 14m and 14n.
NH
5
N
N
H
a
MCP-1 receptor binding assay in THP-1 cells, see Ref. 38.
IC50 value is reported as the average of two separate determinations with
b
variation of 15%.
c
IC50 values are reported from single determination.
optimization of this novel series containing 5/6-fused ring system
were warranted. Since this scaffold is derived from the diamide
series, we faced similar challenges in hERG liability. Hence, our
goal is to identify lead compounds with potent CCR2 activity and
high selectivity with respect to hERG activity.
The synthesis of the target compounds in this series is shown in
Scheme 1. 2-Fluoro-benzonitrile 6 was condensed with commer-
cially available hydrazine (R2NHNH2) in refluxing IPA to afford
the corresponding indazoles 7 (77–95% yield). A second route to
indazoles 7 was developed to synthesize analogs inaccessible from
direct coupling of 6 and substituted hydrazines. Thus, 6 was re-
acted with NH2NH2 to form un-substituted indazole intermediate
(92% yield), which was then protected as phthalimide 6a by treat-
ing with phthalic anhydride (70% yield), followed by sulfonylation
(for compound 14t, MsCl, TEA, 81% yield), acyclation (for com-
pounds 13h, 13i, 14s, 16l, t-Bu-NCO, 85% yield) or alkylation (for
compounds 14q, 14r, 14u, and 14v, NaH, R2Br, 65–75% yield). After
de-protection of phthalimide (50–72% yield), the corresponding
Next we conducted SAR studies at the R2 substitution on inda-
zole ring. 3,4-Methlenedioxy-phenyl and ethyl carboxylate groups
were selected as two representative R1 substitutions for R2 SAR
studies. As illustrated in Table 3, benzoisoxazoles 15a and 15b of-
fered no improvement on dialing out hERG compared to their cor-
responding indazoles 13b and 14o. Substitution on 1-position of
the indazole ring indicated there is room for modifications. Small
lipophilic alkyl groups (14d, 14p, and 14q) as well as hydroxyethyl
group (13g) were well tolerated for CCR2 activity. Surprisingly, tri-
fluoroethyl substituted indazole 14r displayed more than 10-fold
potency drop compared with its hydrocarbon analog 14p. In an at-
tempt to improve CCR2/hERG selectivity, polar substitutions were
introduced onto the indazole ring. Urea substituted indazole 13i
and 14s displayed good CCR2 activity and trend to decrease hERG
indazoles
7 were obtained. For the benzoisoxazole analogs,
compound 6 was reacted with acetohydroxamic acid by treatment
with t-BuOK to afford intermediate 8 (71% yield). The resulting
5/6-fused amino indazoles 7 or benzoisoxazoles 8 were converted
to acids 9 by reductive amination with glyoxylic acid using
NaBCNH3 (75–80% yield). Azetidine intermediates 11 were
obtained by coupling of 9 and amino azetidine 10 under EDCI
(65–88% yield). Target compounds were obtained by reductive
amination with NaBH(OAc)3 of azetidines 11 and ketones 12 in
55–85% overall yield. The cis isomer (13–16) and trans isomers
(17) were separated in 2.5:1 to 1:1 ratio and evaluated individually
in the biological assays. Ketones 12 were synthesized according to
our early Letter.35
binding affinity (IC50 of 12 and 20 lM). Methyl sulfonylation of
indazole ring substantially reduced CCR2 binding affinity as shown
in 14t (IC50 of 920 nM). Steric bulky substitutions such as benzyl or
cyclopentyl ring also decreased CCR2 activity (14u and 14v). It was
apparent that polar substitutions especially with H-bonding do-
nors could significantly suppress hERG affinity. However, com-
pounds containing this feature possess potential poor physical
characteristics such as low aqueous solubility, poor intestinal per-
meability (uera), and conjugate formation (primary alcohol).41
Inspired by our early findings on azetidinyl diamide series that
incorporation of 1-hydroxy group at the cyclohexanyl ring effi-
ciently suppressed hERG affinity, we prepared a sub-set of com-
pounds bearing 1-hydroxy substitution on the cyclohexanyl ring
We began our SAR exploration on the left side R1 group of inda-
zoles B shown in Table 2. Consistent with the SAR of the diamide
series, modification on the left side aromatic or hetero-aromatic

6044
X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6042–6048
(Table 4). While in diamide series the hydroxy analogs generally
displayed weaker CCR2 activity as compared to the corresponding
des-oxy derivatives, introduction of 1-hydroxyl group into
5,6-fused indazoles and benzoisoxazoles was well tolerated for
good CCR2 activity. For examples, indazoles 16a and 16b exhibited
slight gain in CCR2 binding affinity over their corresponding dia-
mide analogs (IC50 of 19 and 5 nM vs 36 and 15 nM).35 For electron
deficient R1 group such as pyridinyl substitution, hydroxyl inda-
zole 16c gained sixfold in affinity for CCR2 as compared to its dia-
mide analog (IC50 of 190 nM) without increasing hERG affinity (IC50
these compounds exhibited an increase in hERG potency compared
to 16j as indicated by both hERG binding affinity and hERG patch
express test. Substitutions at the R1 position with more polar
five-membered heterocycles such as oxazole (16q), imidazole
(16r), or pyrazole (16s) significantly reduced CCR2 binding affinity.
Introduction of a nonaromatic acetylene substitution (16t) abol-
ished CCR2 affinity as shown in 16t. As for benzoisoxazoles 15c,
15d, and 15e, they maintained good CCR2 activity as compared
to their indazole analogs. Once again, stronger hERG binding affin-
ity and hERG functional signals in patch express study were ob-
served. Overall, through the SAR study, 5-thiazole as the R1
substitution in hydroxyl indazoles afforded the resulting com-
pounds with high selectivity over the hERG ion channel.
Thiazole 16j had overall the best profile and was selected for
pharmacokinetic studies.43 As shown in Table 5, compound 16j
has high clearance and low bioavailability in rats but low clearance
(CL, 9.80 mL/min/kg) and good oral bioavailability (F, 75.7%) in dogs.
In summary, we have demonstrated that 5/6-fused rings such as
indazole and benzoisoxazole are viable diamide replacements in
4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists. Optimiza-
tion of indazole series according to divergent SARs on both CCR2
and hERG produced compound 16j with potent CCR2 activity and
good selectivity over hERG. Furthermore, the PK profile of 16j
of 36 l
M).35 Encouraged by this finding, we then turned our effort
to refine SAR of various heterocyclic substitutions at the R1 posi-
tion. Installation of electron donating group such as methoxy
(16g) or methyl (16h) on pyridinyl ring exhibited an increase in
CCR2 affinity as compared to its un-substituted analog 16f. Both
2-thiazoyl (16i) and 5-thiazoyl (16j) substituted indazoles dis-
played good CCR2 binding affinity with satisfactory selectivity ver-
sus hERG as indicated by hERG binding and patch express
studies.42 As expected, methyl (16k), hydroxyethyl (16m), or urea
(16l) substitution on the indazole was tolerated for CCR2 affinity.
Substitutions of thiazole ring with lipophilic alkyl groups gave
compounds 16n, 16o, and 16p that were equipotent to the un-
substituted thiazole analog 16j versus CCR2. Unfortunately again,
O
N
O
H2N
N
N
O
NC
F
CF3
O
NC
F
CF3
CF3
CF3
iv)
iii)
CF3
i)
i)
N
N
ii)
N
N
H
N
R2
R2
6
6
6
7
6a
6b
O
H2N
N
HO
NC
F
CF3
CF3
v)
NH
H2N
CF3
+
BocN
NH2
CF3
O
vi)
N
N
A
A
8
10
7, A = NR2
8, A = O
9
H
N
O
R1
X
ix)
vii) and viii)
NH
HN
O
CF3
+
N
A
12
11
R1
X
N
N
R1
X
A
A
N
NH HN
O
N
NH HN
O
+
F3C
F3C
13, R1 = Ar, HetAr, A = NR2, X = H
14, R1 = non-Ar, A = NR2, X = H
15, A = O
17
16, A = NR2, X = OH
Scheme 1. Reagents and conditions: (i) NH2NHR2, IPA, reflux (77–95%); (ii) phthalic anhydride (70%); (iii) for 14t, MsCl, TEA(81%); for13h, 13i, 14s, 16l, t-BuNCO, and then TFA
(ꢀ85%); for 14q, 14r, 14u, 14v, NaH, BrCH2CH@CH2 or ICH2CF3 or BnBr or cyclopentyl-OTs (65–75%); (iv) NH2NH2 (50–72%); (v) MeCONHOH, t-BuOK (71%); (vi) glyoxylic acid
monohydrate, NaBCNH3, MeOH (75–80%); (vii) EDCI, HOBt, TEA; (vii) TFA (65–88%); (ix) NaBH(OAc)3, AcOH, silica gel column separation 5% 7 N NH3/MeOH in DCM (55–85%).

X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6042–6048
6045
Table 2
Table 3
F3C
N
F3C
R1
H
R1
H
N
NH
O
N
NH
O
HN
A
HN
N
R2
N
C
ID
R1
A
CCR2
CTX
IC50
(nM)
hERG
binding
B
bindinga
a
IC50 (nM)
IC50
(lM)
ID
R1
R2
CCR2 bindinga
IC50 (nM)
CTXa,b
IC50 (nM)
hERG
bindingc
O
O
O
O
O
IC50 (lM)
13b
15a
13g
13h
13i
NH
O
6
36
21
55
82
3
1.1
O
O
O
O
O
13a
13b
H
H
12
6
9
3
1.20
O
20
20
19
34
1.9
7.6
4.7
12
1.1
9.5
O
O
NCH2CH2OH
NCONH-t-Bu
NCONH2
13c
H
12
35
NH2
MeO
13d
13e
H
12
13
2
8
2.5
N
O
O
Me
0.84
14o –CO2Et
15b –CO2Et
14d –CO2Et
14p –CO2Et
14q –CO2Et
14r –CO2Et
14s –CO2Et
14t –CO2Et
14u –CO2Et
NH
O
NMe
NEt
NCH2CH@CH2
NCH2CF3
NCONH2
NSO2Me
Bn
27
29
13
15
53
230
56
920
170
15
34
40
7
28
nt
14
nt
67
9.1
5.6
8.0
7.6
3.7
2.5
20
N
O
13f
H
63
170
34
14a i-Pr
14b CN
H
Me
370
330
ntd
nt
6.2
24
14c
Me
120
nt
3.1
25
1.4
14d CO2Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
13
2,600
510
44
27
32
530
140
200
40
nt
nt
47
86
66
nt
nt
nt
nt
nt
8.0
25
32
14e COOH
14f CONH2
14g CH2OH
14h CH2OEt
14i CH2OCH2CH@CH2
14j OH
N
14v –CO2Et
650
nt
4.1
32
a
5.6
5.0
30
4.5
7.4
>50
23
IC50 values are reported as the average of at least two separate determinations if
IC50 <100 nM with a typical variation of less than 25%, for IC50 >100 nM, only one
test was conducted for IC50 determination.
14k OEt
14l OCH2CH@CH2
14m NH2
14n NHSO2Me
Me >25,000
Me 2080
a
IC50 values are reported as the average of at least two separate determinations if
IC50 <100 nM with a typical variation of less than 25%, for IC50 >100 nM, only one
test was conducted for IC50 determination.
b
MCP-1 induced chemotaxis in THP-1 cells (Ref. 39).
hERG 3H-astemizole binding activity on HEK-293 cell (Ref. 40).
nt, not tested
c
d
Table 4
F3C
R1
HO
N
NH
O
HN
A
N
D
ID
16a
R1
A
CCR2 binding IC50 (nM)a
19
CTX IC50 (nM)a
30
hERG binding IC50
(l
M)
hERG patchb % @ 3
76 (17)
lM (sov. con.)
NMe
10
12
16b
NMe
5
4
61 (11)
Me2N
(continued on next page)

6046
X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6042–6048
Table 4 (continued)
ID
R1
A
CCR2 binding IC50 (nM)a
30
CTX IC50 (nM)a
31
hERG binding IC50
36
(l
M)
hERG patchb % @ 3
31 (15)
lM (sov. con.)
16c
NH
N
MeO
16d
16e
16f
16g
16h
16i
NH
16
6
10
11
11
15
7
32
27
24
12
18
>50
44
30 (15)
23 (15)
52 (16)
68 (16)
56 (11)
17 (17)
27 (19)
N
N
Me
NH
N
NMe
NMe
NMe
NH
56
9
N
MeO
Me
N
7
N
16
28
11
14
S
N
16j
NMe
S
N
16k
16l
NMe
13
29
29
17
7
27
44 (17)
38 (17)
21 (19)
S
N
NCONH2
CH2CH2OH
31
S
N
16m
22
>50
S
N
N
16n
16o
NMe
NMe
19
18
33
7
25
23
48 (17)
62 (19)
Me
S
N
Et
S
16p
16q
NMe
NMe
5
12
nt
17
nt
55 (11)
nt
S
N
480
O
N
16r
NMe
1,400
nt
nt
nt
N
Me
16s
16t
15c
NMe
NMe
O
200
10,000
23
140
nt
>50
>50
8.7
nt
N
N
Me
nt
MeO
N
20
72 (11)
N
15d
15e
O
O
31
31
37
27
16
11
55 (11)
72 (18)
S
N
S
a
IC50 values are reported as the average of at least two separate determinations if IC50 <100 nM with a typical variation of less than 25%, for IC50 >100 nM, only one test
was conducted for IC50 determination.
b
The membrane K+ current IKr in HERG-transfected HEK293 cells (solvent control), see Ref. 42.

X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6042–6048
6047
Table 5
Rat and dog PK data for compound 16j
Species
POa (mpk)
t1/2 (h)
Cmax (ng/
l
L)
AUClast (hÃng/
l
L)
IVb (mpk)
Vss (L/kg)
CL (mL/min/kg)
F (%)
Rat
Dog
10
10
—
37 14
2973 320
56 29
12913 2450
2
2
7.61 2.62
1.81 0.45
56.0 16.4
9.80 3.25
2.35 0.4
75.7 12.3
5.2 2.1
a
PO (10 mg/kg) in 0.5% Methocel (n = 4)
V (2 mg/kg) in 20% HPBCD (n = 4)
b
26. Pinkerton, A. B.; Huang, D.; Cube, R. V.; Hutchinson, J. H.; Struthers, M.; Ayala, J.
M.; Vicario, P. P.; Patel, S. R.; Wisniewski, T.; DeMartino, J. A.; Vernier, J.-M.
Bioorg. Med. Chem. Lett. 2007, 17, 807.
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proved to be amenable with moderate clearance and volume distri-
bution and high oral bioavailability in dogs. The lead compound
from this series therefore deserves to be further explored in
in vivo efficacy models to exploit the therapeutic potential for
inflammation and metabolic diseases.
Acknowledgments
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32. Xia, M.; Hou, C.; DeMong, D. E.; Pollack, S. R.; Pan, M.; Brackley, J. A.; Jain, N.;
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Storace, L.; Shao, L.; Li, M.; Brodmerkel, C. M.; Covington, M.; Scherle, P.;
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We thank Lead Generation Biology, Center of Excellence for
Cardiovascular Safety Research and ADME/PK teams at J&J PRD
for their technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.074.
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38. MCP-1 receptor binding assay in THP-1 cells:
THP-1 cells were obtained from American Type Culture Collection (Manassas,
VA, USA). The THP-1 cells were grown in RPMI-1640 supplemented with 10%
fetal bovine serum in a humidified 5% CO2 atmosphere at 37 °C. The cell
density was maintained at 0.5Á106 cells/mL. THP-1 cells were incubated with
0.5 nM 125I labeled MCP-1 (Perkin–Elmer Life Sciences Inc., Boston, MA) in the
presence of varying concentrations of either unlabeled MCP-1 (R&D Systems,
Minneapolis, MN) or test compound for 2 h at 30 °C in a 96 well plates. Cells
were then harvested onto a filter plate, dried, and 20 lL of Microscint 20 was
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added to each well. Plates were counted in a TopCount NXT, Microplate
Scintillation & Luminescence Counter (Perkin–Elmer Life Sciences Inc., Boston,
MA). Blank values (buffer only) were subtracted from all values and drug
treated values were compared to vehicle treated values. 1
used for nonspecific binding.
lM cold MCP-1 was
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39. MCP-1 induced chemotaxis in THP-1 cells:
MCP-1 induced chemotaxis was run in a 24-well chemotaxis chamber. MCP-1
(0.01 g/mL) was added to the lower chamber and 100 L of THP-1 cells
l
l
(1 Â 10 7 cell/mL) was added to the top chamber. Varying concentrations of
test compound were added to the top and bottom chambers. Cells were
allowed to chemotax for 3 h at 37 °C and 5% CO2. An aliquot of the cells which
had migrated to the bottom chamber was taken and counted then compared to
vehicle.
40. hERG [3H]-astemizole binding experiment: This assay is a 384well in-plate
vacuum filtration binding assay. Assay reagents are added into a prepared/
blocked 384 well assay plate in the following order: (1) hERG Membrane diluted
in assay buffer; (2) test compound; and (3) 3H astemizole diluted in assay buffer.
Assay reagents are incubated in the filter plate for 1 h and then washed 6Â with
ice-cold wash buffer. Plates are allowed to dry overnight at room temperature.
The following morning, plates are sealed and scintillant is added to each well.
Following a 2-h incubation with scintillant, plates are placed on the TopCount
and counted 1 min per well. Data is calculated using raw CPM. Where applicable,
IC50 values are calculated using raw CPM values. Curves are fitted individually
from singlet 11 point dosing curves + 1% DMSO control.
41. Compound 13i: kinetic solubility at pH 2, 3.5
l
M, Caco-2 (Papp, 10–6 cm/s),
M, Caco-2
0.15/2.44, ratio, 16.2; compound 14s: kinetic solubility at pH 2, 8.9
l

6048
X. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6042–6048
(Papp, 10–6 cm/s): 0.32/3.50, ratio: 10.9; compound 13g, microsomal stability
in rat liver microsome, 25% remain @ 10 min.
42. Patch express experiment: Experiments were performed using HEK293 cells
stably expressing the HERG potassium channel. Cells were grown at 37 °C and
5% CO2 in culture flasks in MEM medium supplemented with 10% heat-
and digitized by a Multiclamp amplifier, stored and analyzed by using the
PatchXpress, DataXpress software and Igor 5.0 (Wavemetrics). The holding
potential was À80 mV. The HERG current (K+-selective outward current) was
determined as the maximal tail current at À40 mV after a 2 s depolarization to
+60 mV. Pulse cycling rate was 15 s. Before each test pulse a short pulse (0.5 s)
from the holding potential to À60 mV was given to determine (linear) leak
current. After establishing whole-cell configuration and a stability period, the
vehicle was applied for 5 min followed by the test substance by increasing
concentrations of 3 Â 10À6 M, 10À5 M and 3 Â 10À5 M. Each concentration of
the test substance was applied twice. The effect of each concentration was
determined after 5 min as an average current of three sequential voltage
pulses. To determine the extent of block the residual current was compared
with vehicle pre-treatment. Data are presented as mean values standard
error of the mean (SEM).
inactivated fetal bovine serum, 1%
L-glutamine–penicillin–streptomycin-
solution, 1% nonessential amino acids (100Â), 1% sodium pyruvate (100 mM)
and 0.8% geneticin (50 mg/ml). Before use the cells were subcultured in MEM
medium in the absence of 5 ml L-glutamine–penicillin–streptomycin. For use
in the automated patch-clamp system PatchXpress 7000A (Axon Instruments)
cells were harvested to obtain cell suspension of single cells. Extracellular
solution contained (mM): 150 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 5
glucose (pH 7.4 with NaOH). Pipette solution contained (mM): 120 KCl, 10
HEPES, 5 EGTA, 4 ATP-Mg2, 2 MgCl2, 0.5 CaCl2 (pH 7.2 with KOH). Patch-clamp
experiments were performed in the voltage-clamp mode and whole-cell
currents were recorded with an automated patch-clamp assay utilizing the
PatchXpress 7000A system (Axon Instruments). Current signals were amplified
43. Compound 16j, cryptochrome p450 Inhibition IC50 (lM): 6.8 (3A4), >10 (2C9,
2C19, 2D6, and 1A2).