Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): Optimization for JNK potency and physicochemical properties

Article abstract of DOI:10.1016/j.bmcl.2013.04.029

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC₅₀ of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2013.04.029

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3565–3569
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of indole/indazole-aminopyrimidines as inhibitors
of c-Jun N-terminal kinase (JNK): Optimization for JNK potency
and physicochemical properties
a
a
a
a
a
Leyi Gong a, , Xiaochun Han , Tania Silva , Yun-Chou Tan , Bindu Goyal , Parch Tivitmahaisoon ,
Alejandra Trejo a, Wylie Palmer a, Heather Hogg a, Alam Jahagir a, Muzaffar Alam a, Paul Wagner a, Karin
Stein a, Lubov Filonova a, Brad Loe a, Ferenc Makra a, David Rotstein a, Lubica Rapatova a, James Dunn a,
Fengrong Zuo b, Joseph Dal Porto b, Brian Wong b, Sue Jin b, Alice Chang b, Patricia Tran b, Gary Hsieh b,
Linghao Niu b, Ada Shao c, Deborah Reuter a, Johaness Hermann a, Andreas Kuglstatter c, David Goldstein a
⇑
a Department of Medicinal Chemistry, Roche Palo Alto, Palo Alto, CA 94304, USA
b Inflammation Therapeutic Area, Roche Palo Alto, Palo Alto, CA 94304, USA
c Department of Discovery Technologies, Roche Palo Alto, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 February 2013
Revised 8 April 2013
Accepted 12 April 2013
Available online 21 April 2013
A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to
achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design
was used to optimize the series resulting in a significant potency improvement. The best compound
(17) has IC50 of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other
kinases with good physicochemical and pharmacokinetic properties.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
JNK
JNK inhibitors
Aminopyrimidine
c-Jun N-terminus kinase
Kinase inhibitors
The c-Jun N-terminal kinases (JNKs) are a family of serine/thre-
onine protein kinases of the mitogen-activated protein kinase
(MAPK) group along with p38 and ERK.1–3 JNKs can be expressed
as 10 different isoforms by mRNA alternative splicing of three
highly related genes, JNK1, JNK2 and JNK3.4 While JNK1 and
JNK2 are found to be ubiquitously expressed, JNK3 is principally
present in the brain, cardiac muscle, and testis.5 Based on the role
of JNK in regulating members of the activator protein-1 (AP-1)
transcription factors and other cellular factors implicated in gene
expression, cellular survival and proliferation in response to cyto-
kines and growth factors, inhibiting JNK may have many potential
therapeutic utilities.6,7 Consequently, many chemotypes of JNK
inhibitors have been reported in the literature.8–19
N
X
HN
N
N
R
OH
X=C, N
When X=N, R=OCH2CH2CH2SO2Me
JNK1/2 = 6.2/24 nM (1)
Figure 1. Aminopyrimidine JNK inhibitors.
interest for the treatment of rheumatoid arthritis and asthma.
Since both JNK1 and JNK2 are implicated, dual JNK1/2 inhibitors
were developed.
In our previous letter, we described our effort on optimizing ki-
nase selectivity with different heterocycles attached to the amino-
pyrimidine core shown in Figure 1 and demonstrated that indole
and indazole scaffolds afforded the favorable selectivity and phys-
icochemical properties we desired.20 Here, we wish to report our
JNK plays a critical role in T cell signaling and has been shown to
regulate the expression or function of a number of proinflamma-
tory cytokines (TNFa, IL-2, IL-6, etc.), that are central to many hu-
man inflammatory disorders.6 As such, JNK inhibitors have the
potential to be immuno-modulatory agents and are of therapeutic
⇑
Corresponding author. Tel.: +1 650 859 4306; fax: +1 650 859 3153.
E-mail address: leyi.gong@sri.com (L. Gong).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.029

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L. Gong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3565–3569
continued effort on further optimizing the potency and properties
of this series.
Most of compounds in this study were synthesized by appropri-
ate substitution of 4-chloro-2-thiomethyl-diaminopyrimidine
according to the Schemes 1–4.
Analogs (7a–f) were synthesized according to Scheme 1. Dis-
placement of 4-Cl-2-thiomethyl-pyrimidine (2) with 4-bromoin-
dole and sodium hydride in DMF afforded the intermediate (4).
Amination of 4 with secondary amines such as piperazine and
piperidine using traditional conditions such as Pd(PPh3)4 and
NaOMe, yielded mostly the des-bromo indole by-product and gave
low yields of the desired products (5a–f). This is presumably due to
a
slow reductive elimination step in the Pd-catalytic cycle
presented by the steric bulkiness of the secondary amine (e.g.,
piperidine methyl-sulfone). To facilitate the rate of the elimination
step, steric hindered phosphine ligand (t-Bu3P) was used.21 Using
the pre-formed bis(tri-t-butylphosphine)-palladium(0) in combi-
nation with sodium t-butoxide was the most effective condition
for this transformation and improved the yields. The methyl-sul-
fide 5a–f was then oxidized using 3-chloro-perbenzoic acid (m-
CPBA) and the resulting sulfoxide or sulfone was displaced with
1-methyl sulfonylamino-4-aminocyclohexane without further iso-
lation to give the final products (7a–f).
Scheme 1. Reagents and conditions: (i) NaH in DMF (0–25 °C), 50–60%; (ii) amine
(R1NH), NaOtBu, bis(tri-tBu-phosphine)Pd (0) in dioxane, 40–50%; (iii) 4-Cl-PhCO3H
in CH2Cl2, 0 °C, 2 h, 95–100% without purification; (iv) 4–5 equiv of NEt2(iPr), 100 °C
in NMP, 60–70%.
Scheme 2. Reagents and conditions: (i) NaH in DMF, 0–25 °C, 55%; (ii) BBr3 in CH2Cl2, 0–25 °C, 2 h, 85–95%; (iii) K2CO3, 100 °C in NMP, 45–55%; (iv) 4-Cl-PhCO3H in CH2Cl2,
0 °C, 2 h, 95–100% without purification; (v) 4–5 equiv of NEt2(iPr), 100 °C in NMP, 60–70%.
N
N
N
N
ii
i
N
S
N
N
S
N
2
Cl
S
N
N
12
NHNH2
O
F
Br
Br
13
N
N
N
iii
iv
SO2Me
S
N
N
S
N
O
O
MeO2S
NH
N
SO2Me
N
15
14
N
N
v
HN
N
N
H2N
OH
N
SO2Me
OH
16
Scheme 3. Reagents and conditions: (i) 4–5 equiv of NEt2(iPr), 100 °C in NMP, 60–70%; (ii) 5 equiv NaOH in EtOH, 80 °C, 95%; (iii) NaOtBu, bis(tri-tBu-phosphine)Pd (0) in
dioxane, 50%; (iv) 4-Cl-PhCO3H in CH2Cl2, 0 °C, 2 h, 95–100% without purification; (v) 4–5 equiv of NEt2(iPr), 100 °C in NMP, 60–70%.

L. Gong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3565–3569
3567
N
N
O
HN
N
N
S
N
N
i
O
N
SO2Me
N
SO2Me
O
H2N
OEt
O
OEt
6a
21
N
HN
N
N
ii-iii
N
SO2Me
H
N
O
N
OH
23a
OH
Scheme 4. Reagents and conditions: (i) 4–5 equiv of NEt2(iPr), 100 °C in NMP, 60–70%; (ii) 5 equiv NaOH in EtOH, 80 °C, 95%; (iii) EDCI in CH2Cl2, rt, overnight, 80–85%.
Table 1
Cyclic side chains
N
N
N
HN
N
N
HN
N
N
R1
R1
NH
OH
S
O
O
(7a-f, 11)
1
R1
JNK1/JNK2 IC50 (nM)a
c-Jun IC50
1.0
(l
M)a
O
O
O
S
1
6.2/24
O
O
O
7a
3/24
0.71
na
S
N
O
S
7b
7c
29/180
5/36
Figure 2. X-ray structure of compound 1 bound to JNK1b.20,22
N
N
N
O
N
N
0.20
the subsequent replacement with (trans)-N-methylsulfonyl diami-
nocyclohexane described in Scheme 2.
CN
O
O
The indazole analogs were synthesized via a slightly different
route shown in Scheme 3. Regioisomers were obtained if indazole
was reacted with 2 directly under a basic condition such as NaH.
Therefore, 4-chloro-2-thiomethyl pyrimidine was mixed with
hydrazine to give 12, which upon reacting with 2-bromo-6-fluro-
benzaldehyde was converted to 13 in a region-specific and high
yielding fashion. Similar palladium mediated aromatic amination
shown in Scheme 1, procedure (ii) was used for the transformation
of 13 and 14. Subsequent oxidation afforded 15 and nucleophilic
replacement with 4-hydroxy-cyclohexyl amine provided 16.
Analogs with modification at the distal end of cyclohexyl ring in
Table 2 were synthesized according to Scheme 4.
Nucleophilic substitution of 6a by various cyclohexyl derived
amines gave compounds 17–22c. For example, the use of trans-4-
hydroxy-cyclohexylamine afforded 17 whereas the use of 4-acetyl-
amino-cyclohexylamine and 4-OH-4-Me-cyclohexylamine pro-
vided 18 and 19, respectively. On the other hand, trans-4-
ethylcarboxyl-cyclohexylamine gave 20 and 21. Compound 21
then underwent saponification and EDCI mediated amide coupling
to produce compound 22a–c as described in Scheme 4.
7d
7e
7f
10/68
0.82
na
N
N
94/650
44/280
O
O
O
S
na
N
S
11
6.8/35
0.104
O
O
Cellular activity was measured based on the description in Ref. 24; na = no data
obtained.
a
Compounds were characterized by mass spectral, 1H NMR, elemental analysis
and mp. IC50 values are an average of multiple determinations (n P 2). Assay
conditions are described in Ref. 23.
Similarly, 4-methoxyindole with NaH in DMF readily gave the
intermediate (8), which was reacted with boron tribromide to give
the de-methylated phenol derivative (9). Alkylation of 9 with
chloromethyl tetrahydro-thiophene dioxide at 100 °C in dioxane
in the presence of potassium carbonate yielded compound 10,
which was converted to the final product (11) by oxidation and
X-ray crystal structure of compound 1 bound to the JNK1b
shown in Figure 2 provided the basis for the design of analogs with
improved potency and physicochemical properties.

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L. Gong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3565–3569
Figure 3. Overlay of the liner side chain in 1 (in pink color) with the cyclic side chain in 7b in gray color.
Table 3
Physicochemical properties
Table 2
Activities of compounds with varying front groups
b
Compds
PSA
Solubilitya
CACO2 (AB)
ERc
N
16
17
22a
22c
98
84
101
113
3.5
5.2
43
3.8
10.5
0.4
5
2
53
94
X
HN
Y
N
N
N
SO2Me
21
0.1
a
Solubility was measured in a standard PO4 buffer system with pH of 6.4, unit:
ug/mL.
b
Unit: Â10À6 cm/min.
Compds
X
Y
JNK1/2 IC50 (nM)a
c-Jun IC50, (l
M)a
c
ER = AB/BA.
7a
16
17
18
19
20
21
C
N
C
C
C
N
C
NHSO2Me
OH
OH
NHCOMe
Me(OH)
CO2Et
3/24
6/17
3/20
7/49
12/66
na/27
7/40
0.7
0.29
0.24
0.7
1.3
1.7
Table 4
Pharmacokinetic data of 17
Cla
T1/2
% Fc
b
Species
Rat
Route
CO2Et
1.3
IVd
POe
IVd
POe
47
–
10.6
–
0.7
2
4.5
3.4
–
100
–
O
N
22a
22b
C
C
8/33
0.65
0.42
Dog
O
15
O
a
b
c
Unit: ml/min-kg.
Unit: h.
Oral bioavailability.
Dose: 0.5 mpk.
Dose: 2 mpk.
12/86
N
O
N
d
e
N
22c
C
na/12
0.22
N
N
O
a
Values are means of three experiments (na = not measured).
lines in black color) in a classical donor-acceptor pair hinge inter-
action. A closer examination of the bound conformation of 1 re-
vealed that the side chain was tucked under the glycine-rich-
loop via hydrophobic interaction. Furthermore, the side chain
(OCH2CH2CH2SO2Me) adopted the gauche conformation. It was
rationalized that cyclic ring systems, particularly six member ring
systems in chair conformation could mimic the linear guache con-
formation. In addition, cyclic side chains would provide different
kinase selectivity profile and different physicochemical properties
than the linear ones. As such, a number of cyclic ring systems were
evaluated using ROCS, the modeling tool from OpenEye Scientific
Software. Shown in Figure 3 is the overlay view of a linear side
chain with a cyclic one using ROCS.
As Figure 2 indicated, the indazole-aminopyrimidine of 1 inter-
acted with the backbone amino acids (Met111, Leu110 and
Glu109) via two hydrogen bonds (as highlighted by the dashed
Table 5
Kinase selectivity profile of 17c
Kinase
Folda
Kinase
Folda
Kinase
Folda
JNK1
JNK2
IKK-b
1b
6
46
71
71
107
p38-d
IRAK1
DAPK3
MAPK15
DAPK2
MAP2K4
150
193
264
364
371
393
SgK085
MAPK7
DCAMKL3
RIOK2
CDK7
RPS6KA1
700
786
786
857
929
1786
p38-c
STK17A
DAPK1
a
b
c
Fold = Kd of a kinase/Kd of JNK1.
Kd of JNK1 = 0.14 nM.
The rest of kinases in the commercial kinase panel (unlisted in this table) have
Figure 4. X-ray structure of compound 17 bound to JNK1b.25
fold over JNK1 of >2000; all Kds were determined in ambit.

L. Gong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3565–3569
3569
10. Liu, G.; Zhao, H.; Liu, B. Bioorg. Med. Chem. Lett. 2007, 17, 495.
Compounds (7a–f, 11) in Table 1 were synthesized based on the
near perfect shape match in Figure 3.
11. Szczepankiewicz, B. G.; Kosogof, C.; Nelson, L. T.; 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.
12. Zhao, H.; Serby, M. D.; Xin, Z.; Szczepankiewicz, B. G.; Liu, M.; Kosogof, C.; Liu,
B.; Nelson, L. T. J.; Johnson, E. F.; Wang, S.; Pederson, T.; Gum, R. J.; Clampit, J.
E.; Haasch, D. L.; Abad-Zapatero, C.; Fry, E. H.; Rondinone, C.; Trevillyan, J. M.;
Sham, H. J.; Liu, G. J. Med. Chem. 2006, 49, 4455.
13. Liu, G.; Zhao, H.; Liu, B.; Xin, Z.; Liu, M.; Serby, M. D.; Lubbers, N. L.; Widomski,
D. L.; Polakowski, J. S.; Beno, D. W. A.; Trevillyan, J. M.; Sham, H. L. Bioorg. Med.
Chem. Lett. 2006, 16, 5723.
14. Christopher, J. A.; Atkinson, F. L.; Bax, B. D.; Brown, M. J.; Champigny, A. C.;
Chuang, T. T.; Jones, E. J.; Mosley, J. E.; Musgrave, J. R. Bioorg. Med. Chem. Lett.
2009, 19, 2230.
Compounds 7a containing the 4-SO2Me-piperidine and 11
incorporating an oxymethyl tetrahydrothiophene dioxide cyclic
side chains are equipotent with 1 containing a liner side chain.
Furthermore, it was evident that a H-bond acceptor at the end
of a side chain was important for the potency as one compares
compound 7a with 7e. The difference in potency between com-
pound 7a and 7b or 7a and 7f can be explained on the basis of
the orientation of the acceptor. In addition, acetyl piperazine side
chains in 7c and 7d were slightly less potent than sulfonyl piperi-
dine (7a).
To further explore the SAR around this series, modification of
the group at the distal end of cyclohexyl amine, labeled as Y were
pursued (Table 2). In general, a diverse set of Y groups were well
tolerated (16–22c). With Figures 2 and 4, one could rationalize this
based on the fact that Y groups were situated in a more solvent ex-
posed region. The impact of a Y group was more reflected on polar-
ity and molecular weight of a compound, thus the physicochemical
properties, and less on the potency. Moreover, indole and indazole
analogs were equipotent (16 vs 17 and 20 vs 21).
15. 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.
16. Stocks, M. J.; Barber, S.; Ford, R.; Leroux, F.; St-Gallay, S.; Teague, S.; Xue, Y.
Bioorg. Med. Chem. Lett. 2005, 15, 3459.
17. De, S. K.; Chen, V.; Stebbins, J. L.; Chen, L.; Cellitti, J. F.; Machleidt, T.; Barile, E.;
Riel-Mehan, M.; Dahl, R.; Yang, L.; Emdadi, A.; Murphy, R. Bioorg. Med. Chem.
2010, 18, 590.
18. Kamenecka, T.; Jiang, R.; Song, X.; Duckett, D.; Chen, W.; Ling, Y. Y.; Habel, J.;
Laughlin, J. D.; Chambers, J.; Figuera-Losada, M.; Cameron, M. D.; Lin, L.; Ruiz, C.
H.; LoGrasso, P. V. J. Med. Chem. 2010, 53, 419.
Figure 4 confirms that the bound conformation of cyclic piper-
idine side chain in 17 in X-ray structure of JNK1b protein perfectly
mimics the conformation of the linear side chain in 1 (Fig. 2).
Table 3 exhibits some of the physicochemical properties of rep-
resentative compounds including solubility, permeability and the
efflux ratio (ER).
It is apparent that the intrinsic permeability of these com-
pounds decreased when their PSA exceeded 100 (Table 3). It ap-
pears that as this series of compounds got more polar, it was
more likely to be a substrate of PGP transporters based on the high
efflux ratio. Compound 17 displayed overall balanced profile and
was chosen for further evaluation. Table 4 highlights some of the
PK parameters obtained for 17 in both rats and dogs. The rat clear-
ance rate for 17 was high, but dog clearance rate was medium. The
oral bioavailability in both species was good enough to enable fur-
ther in-vivo characterization of 17 in these species.
Kinase selectivity of 17 was then profiled against 317 kinases in
a commercial kinase panel.26,27 17 exhibited 40-fold selectivity for
the JNK subfamily over other kinases shown in Table 5.
Based on its good pharmacokinetic property demonstrated in
rat and in dogs as well as its excellent kinase selectivity profile,
compound (17) serves as a superb tool molecule for studying JNK
pharmacology. The efficacy of 17 in various disease models will
be presented in due course.
In summary, a series of potent and selective indole-aminopy-
rimidine inhibitors was discovered and developed using structure
based drug design. Compound 17, a potent and selective JNK inhib-
itor from the series, exhibited excellent physicochemical and phar-
macokinetic properties.
19. Noël, R.; Shin, Y.; Song, X.; Koenig, M.; Chen, W.; Ling, Y. Y.; Lin, L.; Ruiz, C. H.;
LoGrasso, P.; Cameron, M. D.; Duckett, D. R.; Kamenecka, T. M. Bioorg. Med.
Chem. Lett. 2011, 21, 2732.
20. Palmer, W. S.; Alam, M.; Arzeno, H. B.; Chang, K. C.; Dunn, J. P.; Goldstein, D. M.;
Gong, L.; Goyal, B.; Hermann, J. C.; Hogg, J. H.; Hsieh, G.; Jahangir, A.; Janson, C.;
Jin, S.; Ursula, K. R.; Kuglstatter, A.; Lukacs, C.; Michoud, C.; Niu, L.; Reuter, D.
C.; Shao, A.; Silva, T.; Trejo-Martin, T. A.; Stein, K.; Tan, Y.; Tivitmahaisoon, P.;
Tran, P.; Wagner, P.; Weller, P.; Wu, S. Bioorg. Med. Chem. Lett. 2013, 23(5),
1486.
21. Tolman, C. A. Chem. Rev. 1977, 77, 313–348.
22. PDB accession number: 4HYU, 2.15 Å resolution.
23. JNK activity was measured by phosphorylation of GST-ATF2 (19–96) with
[c-
33P] ATP. The enzyme reaction was conducted at Km concentrations of ATP
and the substrate final volume of 40
7.5, 2 mM dithiothreitol, 150 mM NaCl, 20 mM MgCl2, 0.001% TweenÒ 20, 0.1%
BSA and 10% DMSO. Human JNK2 2 assay contains 1 nM enzyme, 1 M ATF2,
33P] ATP. Human JNK1
1 assay contains 2 nM enzyme,
M ATP with 1 Ci [
33P] ATP. The enzyme assay was carried
ll in buffer containing 25 mM HEPES, pH
a
l
8
1
l
l
M ATP with 1
M ATF2, 6
lCi [
c
-
a
l
l
c-
out in the presence or absence of several compound concentrations. JNK and
compound were pre-incubated for 10 min, followed by initiation of the
enzymatic reaction by adding ATP and the substrate. The reaction mixture
was incubated at 30 °C for 30 min. At the end of incubation, the reaction was
terminated by transferring 25 ll of the reaction mixture to 150 ll of 10%
glutathione SepharoseÒ slurry (Amersham #27-4574-01) containing 135 mM
EDTA. The reaction product was captured on the affinity resin, and washed on a
filtration plate (Millipore, MABVNOB50) with phosphate buffered saline for six
times to remove free radionucleotide. The incorporation of 33P into ATF2 was
quantified on
a microplate scintillation counter (Packard Topcount).
Compound inhibition potency on JNK was measured by IC50 value generated
from 10 concentration inhibition curves fitted into the 3-parameter model: %
inhibition = maximum/(1 + (IC50/[inhibitor])slope). Data were analyzed on
Microsoft Excel for parameter estimation. For details, please see: Gong, L.
WO Patent 138,340, 2009.
24. c-Jun activation assay in TNFa-induced human chondrosarcoma SW1353 cells:
SW1353 cells are grown in DMEM (invitrogen) with 10% fetal bovine serum
(invitrogen). Ascorbic acid (15 mg/ml), penicillin (100 U/ml) and streptomycin
(100 mg/ml) (invitrogen). Cells are plated at a density of 8000 cell per well in
100 ml of growth media for 24 h before the compound treatment. Immediately
before the treatment, media is replaced with 90 ml of fresh media, then add
10 ml of 10Â concentrated compound solution and allowed to pre-incubate
with cells for 30 min. The vehicle (DMSO) is maintained at a final concentration
of 0.5% in all samples. After 30 min, the cells are activated with 10 ng/ml of
References and notes
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25. PDB accession number: 4IZY, 2.3 Å resolution.
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