Design and synthesis of 1-aryl-5-anilinoindazoles as c-Jun N-terminal kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.02.082

Starting from pyrazole HTS hit (1), a series of 1-aryl-1H-indazoles have been synthesized as JNK3 inhibitors with moderate selectivity against JNK1. SAR studies led to the synthesis of 5r as double digital nanomolar JNK3 inhibitor with good in vivo exposure.

Full text of DOI:10.1016/j.bmcl.2013.02.082

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2683–2687
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 1-aryl-5-anilinoindazoles as c-Jun N-terminal
kinase inhibitors
Rong Jiang, Bozena Frackowiak, Youseung Shin, Xinyi Song, Weimin Chen, Li Lin, Michael D. Cameron,
⇑
Derek R. Duckett, Theodore M. Kamenecka
Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #A2A, Jupiter, FL 33458, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from pyrazole HTS hit (1), a series of 1-aryl-1H-indazoles have been synthesized as JNK3
inhibitors with moderate selectivity against JNK1. SAR studies led to the synthesis of 5r as double digital
nanomolar JNK3 inhibitor with good in vivo exposure.
Received 14 December 2012
Revised 13 February 2013
Accepted 19 February 2013
Available online 27 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
The c-Jun N-terminal kinases (JNKs) are members of mitogen-
activated protein kinase (MAPK) family which are activated in re-
sponse to dual phosphorylation on threonine and tyrosine when
exposed to extracellular stimuli such as stress and cytokines.1 Acti-
vation of the JNK pathway leads to the phosphorylations of a num-
ber of transcription factors involved in cell proliferation and
apoptosis such as c-Jun,2 ATF-2,3 nuclear factor of activated T cells
(NFAT),4 and tumor suppressor p53.1a Ten different JNK isoforms
derived from three distinct genes (jnk1, jnk2, and jnk3) have been
identified. JNK1 and JNK2 are ubiquitously expressed in most tis-
sues, while JNK3 is primarily expressed in the brain and, to a lesser
extent, in the heart and testis.5,6 This variably localized expression,
together with the activation by different biochemical pathways,
indicates that different JNK isoforms have distinct biological func-
tions. For example, elevated JNK1 activity is shown to play a crucial
role in the biochemical pathway responsible for obesity-induced
insulin resistance in vivo,7 whereas JNK2 and JNK3 is activated in
the MPTP-induced Parkinson’s disease mouse model.8 Thus, devel-
oping JNK isoform-specific inhibitors as therapeutics has gained
considerable interest over the past few years despite of the chal-
lenge that the three JNK isoforms share more than 90% sequence
identity.9,10
NH
1
O
N
JNK3 1.1 µM
N
JNK1 > 20 µM
H
N
O
O
Given the floppy nature of the side chains, we sought to restrict
free rotation in an effort to introduce some conformational rigidity
into the molecule, and hopefully improve potency. There were two
potential cyclization manifolds that would lead to conformational-
ly restricted analogs of 1 as outlined in Figure 1. Route A removed
free rotation about the phenacetyl group and led to benzimidazole
2. Route B restricted rotation about the pyrazole linked amide to
give 5-substituted indazole 3.
Though only two compounds were synthesized, the results
were undeniable (Table 1). Benzimidazole 2 showed no inhibitory
activity against JNK3, while indazole 3 was a very potent JNK3
inhibitor with 10-fold selectivity against JNK1. Apparently, the 2-
aminobenzimidazole in 2 failed to mimic the m-tolyl acetamide
in 1, whereas 5-anilinoindazole 3 succeeded perhaps indicating a
conformational preference for the phenyl side chain only available
to 1 and 3.
As part of our effort to investigate potential therapeutical treat-
ments for Parkinson’s disease, a high throughput screening cam-
paign was conducted to indentify JNK3 specific inhibitors. Among
those, compound 1 was identified as a novel JNK3 selective inhib-
itor with IC50 values of 1.1
respectively.
lM and P20 lM against JNK3 and JNK1,
Encouraged by the promising indazole result, our initial SAR on
this scaffold was focused on modifying R1 and R2 as seen in com-
pound 5 maintaining the central part of molecule as a balanced
compromise between quick parallel synthesis and ability to confer
JNK3 activity (Scheme 1). Starting from commercially available
⇑
Corresponding author. Tel.: +1 561 228 2207.
E-mail address: kameneck@scripps.edu (T.M. Kamenecka).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.082

2684
R. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2683–2687
H
N
H
N
NH
NH
N
Cyclization Route A
Cyclization Route B
N
O
N
N
A
N
N
N
H
N
B
R
H
N
H
N
R
O
R
O
3
2
1
O
Figure 1. Potential cyclization routes from lead pyrazole 1.
exhibited good JNK3 inhibition. The morpholinopropyl analog
(5k) was equipotent to lead indazole 3 and 3-fold more potent
against JNK3 than its one carbon shorter homolog (5g). Actually,
no side chain was needed at all as the analog containing a simple
primary amide (5p) was quite potent against JNK3 as well. Tertiary
amides and esters, on the other hand, led to 1000-fold loss of JNK3
inhibition (data not shown). This may imply the importance of the
free NH group in the amide to act as a hydrogen bond donor. As
seen in Table 2, this series of compounds was also somewhat selec-
tive (5- to 25-fold) for JNK3 versus JNK1 much like the pyrazole-
based lead structure 1.
Table 1
Indazole and benzimidazole JNK inhibitors
JNK311 IC50
(
lM)
JNK111 IC50
(l
M)
a
Compd
R
OMe
OMe
2
>20
NTb
OMe
OMe
OMe
3
0.024
0.203
The next round of modifications involved 1-pyridyl-1H-inda-
zole and 1H-pyrazolo[3,4]pyridine-1-yl substitutions (Table 3).
Compounds 6a–f were synthesized following chemistry detailed
in Scheme 1 using the appropriate precursors. The pyrazolopyri-
dines were made as described in the literature or purchased.14
The 20- and 40-pyridyl replacements (6a, 6b) resulted in a 10-
fold drop in JNK3 activity as compared to 5g and 5h. In these exam-
ples, an intramolecular hydrogen bond between the pyridine ring
nitrogen atom and the amide side chain NH is possible to form a
5-membered ring chelate. This might disrupt hydrogen bonding
between the inhibitor and the enzyme, hence, the loss in potency.
Perhaps it is no coincidence that this same disruption in binding in
both molecules (6a, 6b) causes the same fold drop in JNK3 inhibi-
tion. With the 50- and 60-pyridyl analogs (6c, 6d), there was only a
slight loss in JNK3 inhibition (twofold) compared to phenyl deriv-
ative 5g. Incorporation of nitrogen into the indazole ring itself led
to a 2- to 3-fold drop in JNK3 inhibition (6e–f). All analogs tested
still exhibited the 10- to 20-fold selectivity for JNK3 versus JNK1.
Substitution on the N-phenyl pyrazole ring was conducted (20–60
positions, data not shown), but this did not result in any improve-
ment in potency.10e
OMe
The IC50 values are averages of two or more experiments. All standard errors
a
612%.
b
NT = not tested.
5-nitroindazole, Ullman coupling with ethyl 3-bromobenzoate
followed by hydrogenation provided ethyl 3-(5-amino-1H-inda-
zol-1-yl)benzoate (4) which was subjected to Buchwald amination
with aryl halides (R1). The resulting ethyl esters were hydrolyzed
and coupled with various amines to furnish 5.
Introduction of a small ortho-substituent like fluorine (5a) and
chlorine (5b) enhanced the inhibitory activity against JNK3 com-
pared to parent analog 3 whereas meta-substitution (5d) resulted
in a small drop in potency (Table 2).12 While the trimethoxyphenyl
group in 3 was very potency enhancing, solubility and metabolic
stability precluded its use in future analogs.13 Fortunately, this part
of the molecule was quite tolerant to modifications and a range of
side chains were acceptable without sacrificing too much potency.
Unfortunately, metabolic stability was still quite poor.13 Com-
pounds containing both aromatic and alkyl secondary amides
H
N
H2N
O2N
a,b
c,d,e
R1
N
N
N
N
N
H
N
NH R2
OEt
4
5
O
O
Scheme 1. Reagents and conditions: (a) ethyl 3-bromobenzoate, CuI, (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine, Cs2CO3, Dioxane; (b) Pd/C (10%), H2, MeOH and
EtOAc; (c) R1Br or R1I, Pd2(dba)3, Xantphos, Cs2CO3, DME; (d) LiOH (1N), THF and (e) R2NH2, HATU, triethylamine, DMF.

R. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2683–2687
2685
Table 2
Inhibition of JNK3/1 by 1H-indazole benzamides
a
Compd
R1
R2
JNK311 IC50
(lM)
JNK111 IC50
(lM)
OMe
OMe
3
Phenyl
0.024
0.009
0.20
OMe
OMe
OMe
5a
2-Fluorophenyl
0.073
OMe
OMe
OMe
5b
5c
2-Chlorophenyl
2-Fluorophenyl
0.012
0.046
0.093
0.43
OMe
OMe
OEt
OMe
OMe
5d
5e
2-Methylphenyl
2-Chlorophenyl
0.059
0.032
NTb
OMe
0.16
O
O
N
5f
2-Fluorophenyl
2-Chlorophenyl
0.038
0.054
0.17
0.75
N
5g
O
O
N
5h
5i
2,6-Dichlorophenyl
2-Methoxyphenyl
2-Fluorophenyl
0.077
0.043
0.022
0.015
1.38
NTb
N
O
O
O
N
N
N
5j
NTb
5k
2-Chlorophenyl
0.41
5l
2-Fluorophenyl
0.12
0.47
5m
5n
2-Methylphenyl
2-Chlorophenyl
0.25
NTb
OH
N
H
0.035
0.80
5o
5p
2-Pyridinyl
1.4
7.1
O
N
2-Fluorophenyl
H
0.063
0.73
5q
5r
2,6-Dichlorophenyl
2-Cl,3-Me-phenyl
0.050
0.04
1.2
N
N
N
N
0.97
a
The IC50 values are averages of two or more experiments. All standard errors 615%.
NT = not tested.
b

2686
R. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2683–2687
Table 5
H
N
JNK3, JNK1 and p38 inhibition of selected compounds
R1
JNK311 IC50
(lM)
JNK111 IC50
(l
M)
p3811 IC50
(lM)
N
a
Compd
6
N
7
5a
5g
5f
6b
6d
6f
7
0.009
0.054
0.038
0.64
0.12
0.17
0.073
0.75
0.17
NTb
2.3
1.2
0.03
0.04
0.018
0.48
1.3
2'
H
N
6'
R2
6
5'
4'
O
0.14
0.17
0.033
0.92
Table 3
a
1-Pyridyl-1H-indazole JNK inhibitors
The IC50 values are averages of two or more experiments. All standard errors
625%.
Compd Atom R1
R2
JNK311
JNK111
M) IC50 M)
b
NT = not tested.
a
IC50
(l
(l
5g
6a
6b
6c
6d
6e
6f
—
2-Chlorophenyl
0.054
0.64
0.64
0.11
0.12
0.08
0.17
0.75
7.01
O
N
N
N
N
N
N
N
Table 6
In vivo profile of selected JNK3 inhibitors16
20-N 2-Chlorophenyl
40-N 2,6-Dichlorophenyl
50-N 2,6-Dichlorophenyl
60-N 2-Chlorophenyl
6-N 2-Chlorophenyl
7-N Phenyl
O
O
N
O
O
O
Compd
Clpa
t
(min)
AUC
M h)
F (%)
Plasma
M)b
Brain
(lM)
½
(mL/min/kg)
(l
(l
NTb
NTb
5a
5g
5k
5l
5q
5r
6d
7
57
NT
123
79
1.5
NT
0.5
0.3
2
0.05
NT
0.01
5
NT
6
0.79
0.56
0.28
0.35
2.12
1.2
0.05
0.55
0.57
0.03
0.81
3.9
0.5
7
34
0.38
0.72
0.01
20
27
5
25
54
3.3
0.5
2.3
0.53
0.24
1.4
0.69
NT
NT
NT
NT
1.3
1.2
a
1 mg/kg IV, 2 mg/kg PO.
Compounds dosed 10 mpk IP; Plasma and brain drug levels measured at t = 2 h;
b
NT = not tested.
a
The IC50 values are averages of two or more experiments. All standard
errors 6 15%.
b
NT = not tested.
showed 10-fold JNK3/p38 selectivity whereas most compounds
were actually more potent against p38 than JNK3. Previously,
researchers at AstraZeneca reported a related series of 6-anilinoin-
dazole JNK3 inhibitors that also inhibit p38.10e Attempts to under-
stand and improve the JNK3/p38 selectivity in this series are still
ongoing.
Treating CNS disorders requires blood brain barrier (BBB) pene-
tration.15 Early assessment in this series with one of the more po-
tent analogs (5a) was disappointing (Table 6). The other N-aryl
amide, 5l, also had poor CNS penetration. Fortunately, compounds
containing aliphatic amide side chains (5g, 5k, 5q, 5r, 6d, 7) all
exhibited very good brain exposure typically with more drug in
brain than in plasma at t = 2 h.16 In terms of rat pharmacokinetics,
most analogs examined had poor exposure, characterized by high
clearance rates, short half-lives, and poor oral absorption. How-
ever, the two piperazine containing analogs (5q–r) showed mark-
edly improved profiles. Clearance rates seem high, but this is due
to higher than average volume of distributions (5–6L).17
While most analogs carry the amide side chain at the meta-po-
sition of the N1-linked phenyl ring, SAR indicates that substitution
at the para-position is also viable (Table 4, 7). While this minor
structural modification may not have dramatic effects on JNK
activity, it may play a role in brain penetration and or DMPK
properties.
Cl
H
N
N
N
3
NH
R
4
O
Select compounds were tested in vitro for their ability to inhibit
Table 4
the phosphorylation of JNK3 in cells.18 Compounds showed a con-
Indazole ortho-, meta- and para-amide isomers
siderable drop in potency (5g IC50 = 4.1
lM; 6d IC50 = 5.3 lM; 5n, 7
a
Compd
R
JNK311 IC50
(
lM)
JNK111 IC50
0.41
(lM)
IC50’s P 20 M). It’s not clear why there is such a large difference
l
in biochemical versus cell-based potency as these compounds
should surely penetrate the cell, however, drops in potency of this
type are not uncommon in the literature.19 This loss of potency has
not been observed in all series studied, and efforts to understand
this observation are on-going.
In summary, a novel series of 1-aryl-5-anilinoindazole JNK3
inhibitors were developed from the high throughput screening
lead 1. Lead compound optimization produced potent JNK3 inhib-
itors that showed up to 25-fold selectivity for JNK1, although 10-
fold selectivity was more common. Incorporation of aliphatic
amide side chains instead of aromatic amides resulted in com-
pounds with improved PK and CNS penetration. Further optimiza-
tion is required, however, to increase JNK3-p38 selectivity.
O
O
5k
7
0.015
N
N
0.033
0.92
a
The IC50 values are averages of two or more experiments. All standard errors
615%.
In testing compounds for JNK3 and JNK1 inhibition, we rou-
tinely counter-screened against the closely related MAP kinase
p38. Significant inhibition against p38 was observed in most com-
pounds in this series (Table 5). Interestingly, 6-pyridyl analog 6d

R. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2683–2687
2687
S.; Bahmanyar, S.; Chamberlain, P.; Muir, J.; Cathers, B. E.; Giegel, D.; Xu, L.;
Celeridad, M.; Moghaddam, M.; Khatsenko, O.; Omholt, P.; Katz, J.; Pai, S.; Fan,
R.; Tang, Y.; Shirley, M. A.; Benish, B.; Blease, K.; Raymon, H.; Bhagwat, S.;
Henderson, I.; Cole, A. G.; Bennett, B.; Satoh, Y. Bioorg. Med. Chem. Lett. 2012, 22,
1427.
Synthesis and characterization of these compounds is in progress
and will be reported in due course.
References and notes
11. Biochemical IC50’s for JNK3, JNK1 and p38 were determined using HTRF. Briefly,
final assay concentrations of JNK3, JNK1, biotinylated-ATF2 and ATP were 0.3,
1. (a) Kyriakis, J. M.; Avruch, J. J. Biol. Chem. 1990, 265, 17355; (b) Hibi, M.; Lin, A.;
Smeal, T.; Minden, A.; Karin, M. Genes Dev. 1993, 7, 2135; (c) Adler, V.;
Polotskaya, A.; Wagner, F.; Kraft, A. S. J. Biol. Chem. 1992, 267, 17001; (d)
Derijard, B.; Hibi, M.; Wu, I. H.; Barrett, T.; Su, B.; Deng, T.; Karin, M.; Davis, R. J.
Cell 1994, 76, 1025; (e) Kyriakis, J. M. et al Nature 1994, 369, 156; (f) Davis, R. J.
Cell 2000, 103, 239.
2. (a) Yang, D. D.; Juan, C. Y.; Whimarsh, A. J.; Rincon, M.; Zheng, T. S.; Davis, R. J.;
Rakic, P.; Flavell, R. A. Nature 1997, 389, 865; (b) Wilhem, D.; van Dam, H.; Herr,
I.; Baumann, B.; Herrlich, P.; Angel, P. Immunobiology 1995, 193, 143.
3. (a) Hu, M. C.; Qiu, W. R.; Wang, Y. P. Oncogene 1997, 15, 2277; (b) Livingstone,
C.; Patel, G.; Jones, N. EMBO J. 1995, 14, 1785.
0.1, 0.4 and 1
lM, respectively. Final assay concentrations of p38, biotinylated-
ATF2 and ATP were 1, 0.4 and 11.5
lM, respectively. In both assays, the
phosphor-Thr71-ATF-2 product was detected by a Europium-cryptate labeled
anti-phospho-Thr71-ATF-2 antibody. Streptavidin-allophycocyanin-XL was
used as the acceptor. A 10-point dose-response curve for each compound
was generated in duplicate and data was fit to a four parameter logistic.
12. In a structurally related series (not yet disclosed), all 4-substituted analogs
showed a drop in JNK3 inhibition. Consequently, no para-substituted analogs
were made in this indazole series.
13. (a) The t1/2 of 3 in rat and human liver microsomes was 5.8 and 12.8 min,
respectively; (b) The t1/2 of 5k and 5l in rat and human liver microsomes was:
5.6, 6.0, 8.6 min and 8.9 min, respectively.
4. Chow, C. W.; Rincon, M.; Cavanogh, J.; Dickens, M.; Davis, R. J. Science 1997, 278,
1638.
14. Wenglowsky, S.; Ren, L.; Ahrendt, K. A.; Laird, E. R.; Aliagas, I.; Alicke, B.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Feng, B.; Gloor, S. L.; Gould, S. E.; Gross,
S.; Gunzner-Toste, J.; Hansen, J. D.; Hatzivassiliou, G.; Liu, B.; Malesky, K.;
Mathieu, S.; Newhouse, B.; Raddatz, N. J.; Ran, Y.; Rana, S.; Randolph, N.; Risom,
T.; Rudolph, J.; Savage, S.; Selby, L. T.; Shrag, M.; Song, K.; Sturgis, H. L.; Voegtli,
W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.-I.; Young, W. B.; Grina, J.
ACS Med. Chem. Lett. 2011, 2, 342.
15. Hitchcock, S. A.; Pennington, L. D. J. Med. Chem. 2006, 49, 7559.
16. Rat pharmacokinetics and mouse brain penetration: pharmacokinetics of test
compounds was assessed in Sprague–Dawley rats (n = 3). Compounds were
dosed intravenously at 1 mg/kg and orally by gavage at 2 mg/kg. Blood was
taken at eight time points (5, 15, 30 min, 1, 2, 4, 6, and 8 h) and collected into
EDTA containing tubes and plasma was generated using standard
centrifugation techniques. Plasma proteins were precipitated with
acetonitrile and drug concentrations were determined by LC–MS/MS. Data
5. Gupta, S.; Barrett, T.; Whitmarsh, A. J.; Cavanagh, J.; Sluss, H. K.; Derijard, B.;
Davis, R. J. EMBO J. 1996, 15, 2760.
6. (a) Mohit, A. A.; Martin, J. H.; Miller, C. A. Neuron 1995, 14, 67; (b) Martin, J. H.;
Mohit, A. A.; Miller, C. A. Brain Res. Mol. Brain Res. 1996, 35, 47.
7. (a) Aguirre, V.; Uchida, T.; Yenush, L.; Davis, R.; White, M. F. J. Biol. Chem. 2000,
275, 9047; (b) Hirosumi, J.; Tuncman, G.; Chang, L.; Gorgun, C. Z.; Uysal, K. T.;
Maeda, K.; Karin, M.; Hotamisligil, G. S. Nature 2002, 402, 333; (c) Bennet, B. L.;
Satoh, Y.; Lewis, A. J. Curr. Opin. Pharmacol. 2003, 3, 420.
8. (a) Hunot, S.; Vila, M.; Teismann, P.; Davis, R. J.; Hirsch, E. C.; Przedborski, D.;
Rakic, P.; Flavell, R. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 665; (b) Xia, X. G.;
Harding, T.; Weller, M.; Bieneman, A.; Uney, J. B.; Schulz, J. B. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 10433.
9. Manning, A. M.; Davis, R. J. Nat. Rev. 2003, 2, 554.
10. (a) Graczyk, P. P.; Khan, A.; Bhatia, G. S.; Palmer, V.; Medland, D.; Numata, H.;
Oinuma, H.; Catchich, J.; Dunne, A.; Ellis, M.; Smales, C.; Whitfield, J.; Neame, S.
J.; Shah, B.; Wilton, D.; Morgan, L.; Patel, T.; Chung, R.; Desmond, H.; Staddon, J.
M.; Sato, N.; Inoue, A. Bioorg. Med. Chem. Lett. 2005, 15, 4666; (b)
Szczepankiewicz, B. G.; Kosogof, C.; Nelson, L. T. J.; Liu, G.; Liu, B.; Zhao, H.;
Serby, M. D.; Xin, Z.; Liu, M.; Gum, R. J.; Haasch, D. L.; Wang, S.; Clampit, J. E.;
Johnson, E. F.; Lubben, T. H.; Stashko, M. A.; Olejniczak, E. T.; Sun, C.; Dorwin, S.
A.; Haskins, K.; Abad-Zapatero, C.; Fry, E. H.; Hutchins, C. W.; Sham, H. L.;
Rondinone, C. M.; Trevillyan, J. M. J. Med. Chem. 2006, 49, 3563; (c) Jiang, R.;
Duckett, D.; Chen, W.; Habel, J.; Ling, Y. Y.; Lograsso, P.; Kamenecka, T. M.
Bioorg. Med. Chem. Lett. 2007, 17, 6378; (d) Asano, Y.; Kitamura, S.; Ohra, T.;
Aso, K.; Igata, H.; Tamura, T.; Kawamoto, T.; Tanaka, T.; Sogabe, S.; Matsumoto,
S.; Yamaguchi, M.; Kimura, H.; Itoh, F. Bioorg. Med. Chem. Lett. 2008, 16, 4715;
(e) Kamenecka, T.; Habel, J.; Duckett, D.; Chen, W.; Ling, Y. Y.; Frackowiak, B.;
Jiang, R.; Shin, Y.; Song, X.; Lograsso, P. J. Biol. Chem. 2009, 284, 12853; (f) Hom,
R. K.; Bowers, S.; Sealy, J. M.; Truong, A. P.; Probst, G. D.; Neitzel, M. L.; Neitz, R.
J.; Fang, L.; Brogley, L.; Wu, J.; Konradi, A. W.; Sham, H. L.; Toth, G.; Pan, H.; Yao,
N.; Artis, D. R.; Quinn, K.; Sauer, J.-M.; Powell, K.; Ren, Z.; Bard, F.; Yednock, T.
A.; Griswold-Prenner, I. Bioorg. Med. Chem. Lett. 2010, 20, 7303; (g) Bowers, S.;
Truong, A. P.; Neitz, R. J.; Hom, R. K.; Sealy, J. M.; Probst, G. D.; Quincy, D.;
Peterson, B.; Chan, W.; Galemmo, R. A., Jr.; Konradi, A. W.; Sham, H. L.; Tóth, G.;
Pan, H.; Lin, M.; Yao, N.; Artis, D. R.; Zhang, H.; Chen, L.; Dryer, M.; Samant, B.;
Zmolek, W.; Wong, K.; Lorentzen, C.; Goldbach, E.; Tonn, G.; Quinn, K. P.;
Sauerc, J. M.; Wright, S.; Powell, K.; Ruslim, L.; Ren, Z.; Bard, F.; Yednock, T. A.;
Griswold-Prenner, I. Bioorg. Med. Chem. Lett. 2011, 21, 5521; (h) Krenitsky, V. P.;
Delgado, M.; Nadolny, L.; Sahasrabudhe, K.; Ayala, L.; Clareen, S. S.; Hilgraf, R.;
Albers, R.; Kois, A.; Hughes, K.; Wright, J.; Nowakowski, J.; Sudbeck, E.; Ghosh,
was fit by WinNonLin using
a noncompartmental model and basic
pharmacokinetic parameters including oral bioavailability (F%), exposure
(AUC), half-life (t1/2), clearance (Clp), and volume of distribution (Vd) were
calculated. CNS exposure was evaluated in C57Bl6 mice (n = 3). Compounds
were dosed at 10 mg/kg intraperitoneally and after 2 h blood and brain were
collected. Plasma was generated and the samples were frozen at ꢀ80 °C. The
plasma and brain were mixed with acetonitrile (1:5 v/v or 1:5 w/v,
respectively). The brain sample was sonicated with a probe tip sonicator to
break up the tissue, and samples were analyzed for drug levels by LCMS/MS.
Plasma drug levels were determined against standards made in plasma and
brain levels against standards made in blank brain matrix. All procedures were
approved by the Scripps Florida IACUC.
17. Ritschel, W. A.; Kearns, G. L. In Handbook of Basic Pharmacokinetics; Graubert, J.
I., Ed.; American Pharmaceutical Association, 1999. Chapter 16.
18. For cell-based assay conditions see: Shin, Y.; Chen, W.; Habel, J.; Duckett, D.;
Ling, Y. Y.; Koenig, M.; He, Y.; Vojkovsky, T.; Lograsso, P.; Kamenecka, T. M.
Bioorg. Med. Chem. Lett. 2009, 19, 3344 (reference 24).
19. Alam, M.; Beevers, R. E.; Ceska, T.; Davenport, R. J.; Dickson, K. M.; Fortunato,
M.; Gowers, L.; Haughan, A. F.; James, L. A.; Jones, M. W.; Kinsella, N.; Lowe, C.;
Meissner, J. W. G.; Nicolas, A.; Perry, B. G.; Phillips, D. J.; Pitt, W. R.; Platt, A.;
Ratcliffe, A. J.; Sharpe, A.; Tait, L. J. Bioorg. Med. Chem. Lett. 2007, 17, 3463; Liu,
M.; Wang, S.; Clampit, J. E.; Gum, R. J.; Haasch, D. L.; Rondinone, C. M.;
Trevillyan, J. M.; Abad-Zapatero, C.; Fry, E. H.; Sham, H. L.; Liu, G. Bioorg. Med.
Chem. Lett. 2007, 17, 668.