Inhibitors of the tyrosine kinase EphB4. Part 4: Discovery and optimization of a benzylic alcohol series

Article abstract of DOI:10.1016/j.bmcl.2011.03.009

Optimization of our bis-anilino-pyrimidine series of EphB4 kinase inhibitors led to the discovery of compound 12 which incorporates a key m-hydroxymethylene group on the C4 aniline. 12 displays a good kinase selectivity profile, good physical properties a

Full text of DOI:10.1016/j.bmcl.2011.03.009

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2207–2211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of the tyrosine kinase EphB4. Part 4: Discovery and optimization
of a benzylic alcohol series
Bernard Barlaam a, Richard Ducray a, , Christine Lambert-van der Brempt a, Patrick Plé a,
⇑
Catherine Bardelle b, Nigel Brooks b, Tanya Coleman b, Darren Cross b, Jason G. Kettle b, Jon Read b
a AstraZeneca, Centre de Recherches, Z.I. la Pompelle, BP1050, 51689 Reims, Cedex 2, France
b AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of our bis-anilino-pyrimidine series of EphB4 kinase inhibitors led to the discovery of
compound 12 which incorporates a key m-hydroxymethylene group on the C4 aniline. 12 displays a good
kinase selectivity profile, good physical properties and pharmacokinetic parameters, suggesting it is a
suitable candidate to investigate the therapeutic potential of EphB4 kinase inhibitors.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 28 January 2011
Revised 1 March 2011
Accepted 2 March 2011
Keywords:
EphB4 kinase
Kinase inhibitor
Bis-anilinopyrimidine
The Eph receptors, which form the largest family of receptor
tyrosine kinases (RTK), are implicated in embryogenesis, neural
development, vascular development and adult neovascularisation.
In particular, EphB4 is increasingly recognized as a potential target
for treatment of solid cancers through angiogenesis inhibition.1
Several studies have shown that both EphB4 and its only cognate
ligand, EphrinB2, are essential to embryonic vasculature develop-
ment. Disruption of the EphB4–EphrinB2 interaction with soluble
extracellular domain of EphB4 has demonstrated anti-angiogenic
activity and tumor growth inhibition.2 More recently, similar
effects have been reported using EphB4 monoclonal antibodies.3
The EphB4 signaling could also interplay with the vascular endo-
thelial growth factor (VEGF) signaling, as EphrinB2 has been found
to control VEGF-induced angiogenesis and lymphangiogenesis.4
Unlike other RTKs, the EphB receptors sub-family can trigger a
bi-directional signaling when binding to the cell membrane bound
EphrinB1–3 ligands. Upon ligand-receptor interaction and subse-
quent dimerization, a ‘forward’ signaling is induced by auto-phos-
phorylation of the EphB receptor kinase domain while a ‘reverse’
signaling arises from the EphrinB ligand which contains a trans-
membrane domain. Because of the bi-directional nature of the
EphB4–EphrinB2 signaling axis, the effect of a small molecule
inhibitor of the EphB4 kinase on angiogenesis has remained un-
known until researchers at Novartis recently described for the first
time that such an agent inhibits VEGF driven angiogenesis in vivo,5
suggesting new therapeutic opportunities for cancer or angiogene-
sis related disorders.
In addition there have only been a limited number of reports on
small molecules specifically targeting EphB4 inhibition. We have
previously described the discovery of a series of bis-anilino-pyrim-
idines displaying excellent inhibitory activity against the EphB4
kinase, such as the benzodioxole 1 (Fig. 1)6 or the indazole 2.7
Other groups have reported structurally distinct inhibitors of the
EphB48,9 or EphB210 kinases and the marketed drug dasatinib is
also known to inhibit Eph kinases.11 However, some of these mol-
ecules have a multiple kinase inhibitory profile which would pos-
sibly make difficult the interpretation of any anti-angiogenic
activity.12
Compounds 1 and 2 have both shown a cytochrome P450 time
dependant inhibition7 (TDI) which can result in drug–drug interac-
tions in patients. In order to circumvent this potential risk and
expand the chemical space of our bis-anilino-pyrimidine series,
we explored the structure–activity relationship further. Since we
O
O
O
N
O
N
HN
N
N
NH
N
Cl
N
N
N
H
N
N
H
N
N
O
O
1
2
⇑
Corresponding author. Tel.: +33 3 26 61 69 03; fax: +33 3 26 61 68 42.
E-mail address: richard.ducray@astrazeneca.com (R. Ducray).
Figure 1. Structures of EphB4 kinase inhibitors 1 and 2.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.009

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B. Barlaam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2207–2211
originally attributed the TDI effect of compounds 1 and 2 to their
bicyclic ring,7,13 we decided to investigate mono-cyclic replace-
ments of the C4 aniline.
using Buchwald coupling conditions in the final stage. Further de-
tails on the synthesis and characterization of compounds 3–34 can
be found in the patent literature.14
The general synthetic routes to the compounds described here-
in are outlined in Scheme 1. Compounds 3–11 and 13–15 have
been obtained by addition of N-methyl anilines onto intermediate
35 under acidic catalysis. Compounds 12 and 18–32 have been pre-
pared by a three steps sequence: addition of (3-amino-4-methyl-
phenyl)-methanol onto 2,4-dichloropyrimidine followed by alkyl-
ation of the C4-nitrogen provided 2-chloropyrimidines which were
condensed with 3,5-disubstituted anilines under acidic conditions.
Compounds 33 and 34 have been prepared in a similar way but
The structure–activity relationship around the 4-anilino group
of the pyrimidine core has been assessed with compounds 3–17
and the results are summarized in Table 1. The unsubstituted phe-
nyl (compd 3) provided a good activity in both the enzymatic and
cellular assays, confirming the importance of the 3,5-bis-morpho-
lino-aniline and the C4-nitrogen methylation for EphB4 activity,
as already described in our previous publication.7 However, we
found that compound 3 also shows some activity against the
FGFR1 and VEGFR2 kinases (Table 1). These two RTKs are impli-
cated in the formation of new vasculature. In particular, VEGFR2
blockade by either antibodies or small molecule kinase inhibitors
has been validated in the clinic as an anti-angiogenic approach.
Recognizing the importance of avoiding such pharmacology, we
pursued our lead optimization efforts to obtain EphB4 inhibitors
with an improved selectivity profile.
The EphB4 inhibitory activity was maintained at a good level
with a range of anilines bearing small ortho or meta substituents
(compd 4–14) as well as 2- or 3-pyridyl groups (compd 15–
17).Introduction of a methyl on the 2- or 3- position of the aniline
(4, 5) slightly improved cellular activity over 3 with little impact on
FGFR1 or VEGFR2. Similarly a 3-chloro or 3-methoxy group (6, 7)
improved cellular activity but inhibition of FGFR1 and VEGFR2
was maintained. Combining substituents in a 2,5 pattern (8, 9)
was also well tolerated and the 2,5-dimethylphenyl 8 was particu-
larly interesting since the selectivity over FGFR1 and VEGFR2 was
increased. We found that a 3-hydroxymethyl group was also toler-
ated since compound 10 displayed the same enzymatic activity as
its methyl analogue 5. Although the cellular activity had decreased,
it was an interesting option to reduce the lipophilicity of the C4-
aniline. Again, combining two substituents on the phenyl ring
proved advantageous with compounds 11–13 being more potent
than 10 in the cellular assay. Consistent with our observation with
8, compound 12 had a significantly improved selectivity profile
over 10 with a 800-fold selectivity vs. FGFR1 and 200-fold versus
VEGFR2 when comparing enzymatic IC50. Expanding the size of
the benzyl alcohol was detrimental to EphB4 activity, as demon-
O
Cl
X4
N
N
a
N
N
S
O O
N
N
H
N
2
O
35: X= Cl-
b
3-11, 13-15: X= ArN(Me)-
R
OH
N
Cl
N
OH
d
H2N
e
N
12, 18-34
c
Cl
N
Cl
N
R= H
R= Alk
Scheme 1. Synthesis of compounds 3–15 and 18–34. Reagents and conditions: (a)
N-(3,5-dimorpholin-4-ylphenyl)formamide, NaH, THF, 0 °C to rt, 73%; (b)
ArNH(Me), HCl (cat.), pentanol (68% yield for compd 7); (c) TEA, ethanol, 39% (d)
Alk-X, Cs2CO3, DMF, 26–61%; (e) ArNH2, HCl (cat.), pentanol (62% yield for compd
12); for compd 33–34, (i) TBDMSCl, imidazole, DMF, (ii) HetNH2, K2CO3, Xantphos,
Pd2dba3, toluene, (iii) 2 N HCl, MeOH (35% overall yield).
Table 1
EphB4, FGFR1 and VEGFR2 kinase inhibition and EphB4 cellular activity for compounds 3–17
O
N
R
N
N
N
N
N
O
a
a
a
a,b
Compound
R
EphB4 enz. IC50
(
lM)
FGFR1 enz. IC50
(l
M)
VEGFR2 enz. IC50
(lM)
p-EphB4 IC50 (lM)
3
4
5
6
7
Phenyl
o-Tolyl
m-Tolyl
3-Chlorophenyl
3-Methoxyphenyl
2,5-Dimethylphenyl
2-Methyl-5-methoxyphenyl
3-(Hydroxymethyl)-phenyl
3-(Hydroxymethyl)-5-methoxyphenyl
2-Methyl-5-(hydroxymethyl)-phenyl
3-(Hydroxymethyl)-4-chlorophenyl
2-Methyl-5-(1-hydroxyethyl)-phenyl
4-Methoxy-2-pyridyl
6-Methoxy-2-pyridyl
5-Methoxy-3-pyridyl
0.017
0.010
0.008
0.006
0.002
0.019
0.010
0.010
0.002
0.004
0.002
0.035
0.013
0.006
0.002
0.313
0.662
0.501
0.112
0.183
1.58
0.350
0.738
0.447
3.34
0.056
1.55
1.38
0.998
0.140
0.094
0.092
0.115
0.027
0.100
0.965
0.231
0.141
0.190
0.818
0.022
0.589
0.084
0.028
0.028
0.012
0.010
0.038
0.021
0.114
0.007
0.012
0.013
0.180
0.041
0.028
0.030
8
9
10
11
12
13
14
15
16
17
0.989
0.430
a
n P 2, standard error less than 0.3 log unit.
Inhibition of ephB4 autophosphorylation (engineered CHO-K1 cell line stably expressing EphB4).
b

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2207–2211
2209
strated with the racemic compound 14 which is approximately 10
times less potent than 12 in enzymatic and cellular assays. Finally,
the methoxy-pyridyl derivatives 15–17 had an interesting activity
and selectivity profile although they were slightly inferior to com-
pound 12 in the cellular assay.
We pursed our optimization work around 12 by modifying one
of the morpholino groups on the C2-aniline. Replacement of mor-
pholine with other cyclic amines (Table 2, compd 18–22) main-
tained a good level of cellular activity but no improvement over
12 has been achieved. Only piperidine 19 was as potent as 12
but the increased lipophilicity was not desirable. Homologation
of the morpholine into a benzylic amine (23) induced a small drop
in activity with no impact on log D. In contrast, a carbonyl linker
(24) had a significant effect on log D but this modification led to
a large decrease in EphB4 cellular activity. Introduction of a phenyl
ether instead of a morpholine provided reasonably active com-
pounds (25–27) but more lipophilic than 12. Interestingly, the
methylsulfone 28 was as active as 12 with no increase in log D.
The methyl on the 4-amino group of the pyrimidine could be ex-
tended to other small alkyl chains (29–31) without loss of activity.
This was especially interesting for compound 31 as it is slightly less
lipophilic than 12. However, 31 was less selective than 12 with an
Figure 2. Crystal structure of compound 32 bound to EphB4 kinase domain. The
compound is colored with green carbon atoms. Hydrogen bonds are shown as blue
dotted lines. The protein carbon atoms are shown in white and the backbone is
depicted as
contoured at 1
a
cartoon. Electron density (2f0fc) is shown as
. The pyrimidine group is engaged in the canonical hydrogen bond
a wire mesh and
r
IC50 against FGFR1 and VEGFR2 of 0.72 and 0.39 lM, respectively.
donor acceptor motif with the hinge region of the protein at the bottom of the view.
The hydroxyl group of 32 at the top of the view makes hydrogen bonds with the
carboxylate side chain of E664 and the backbone NH of D758 from the DFG motif.
Picture produced using PYMOL.20
Compound 32 confirmed the interesting replacement of the mor-
pholine with a methylsulfone since it is as potent and less lipo-
philic compared to its analogue 30.
We have been able to obtain a crystal structure15 of 32 bound to
the kinase domain of ephB4 and the binding mode shown on Fig-
ure 2 is consistent with our previously reported crystal struc-
tures6a,b,7 of related bis-anilinopyrimidines. It is noteworthy that
the benzylic hydroxyl group makes two hydrogen bonds with the
carboxylate side chain of E664 and the backbone NH of D758. This
may help to explain why the compounds retain affinity for the ki-
nase despite the presence of a polar group in the relatively lipo-
philic environment of the back pocket of the ATP site.
Direct analogues of 12 in which the C2-aniline is replaced by
the corresponding 4-aminopyridine (33) or the 4-aminopyrimidine
(34) have also been prepared. Compound 33 was remarkably po-
tent with an IC50 of 8 nM whereas the pyrimidine 34 was signifi-
cantly less potent in the cellular assay. Surprisingly, the
measured log D of these compounds indicate that lipophilicity in-
creases from phenyl (3.1) to pyridine (3.4) to pyrimidine (3.9),
which is opposite to what is typically observed with such modifi-
Table 2
EphB4 kinase inhibition, cellular activity and log D for compounds 18–34
O
R2
R1
N
N
N
N
N
OH
OH
N
X
Y
N
N
N
N
N
O
O
12, 18-32
33: X=N, Y=CH
34: X=N, Y=N
a
a,b
Compound
R1
R2
EphB4 enz. IC50
(lM)
p-EphB4 IC50
(lM)
Log D
12
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Morpholin-4-yl
Pyrrolidin-1-yl
Piperidin-1-yl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
0.004
0.016
0.010
0.009
0.004
0.007
0.006
0.017
0.008
0.008
0.005
0.003
0.004
0.005
0.012
0.002
0.014
0.015
0.012
0.027
0.011
0.028
0.032
0.045
0.025
0.459
0.023
0.035
0.032
0.011
0.014
0.013
0.012
0.007
0.008
0.106
3.1
3.8
4.2
3.3
2.6
2.8
3.0
2.2
3.7
3.3
3.9
2.9
3.8
3.9
2.8
3.4
3.4
3.9
1,4-Oxazepan-4-yl
4-Methyl-piperazin-1-yl
4-Hydroxy-piperidin-1-yl
(Morpholin-4-yl)-methyl
(Morpholin-4-yl)-carbonyl
(Tetrahydropyran-4-yl)-oxy
2-Methoxyethoxy
Methoxy
Methylsulfonyl
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
Methylsulfonyl
—
i-Pr
2-methoxyethyl
i-Pr
-
-
—
a
n P 2, standard error less than 0.3 log unit.
Inhibition of ephB4 autophosphorylation (engineered CHO-K1 cell line stably expressing EphB4).
b

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B. Barlaam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2207–2211
Table 3
DMPK properties of compounds 12, 28 and 33
a
b
Compound
Human plasma
Fu (% free)
Rat pharmacokinetics
Dog pharmacokinetics
Cl (%lbf)
Vdss (L/kg)
F%
Cl (%lbf)
Vdss (L/kg)
F%
12
28
33
11
8.5
5.2
11
16
9
0.5
0.4
0.7
78
17
64
51
—
57
2.1
—
1.6
58
—
22
a
Male Han Wistar rats dosed at 1 or 2.5
Mean values for male and female beagle dogs dosed at 2 lmol/kg iv and 5 lmol/kg p.o.
l
mol/kg iv and 5 or 10 lmol/kg p.o.
b
cations. We believe this is a consequence of the higher sp2 charac-
ter of the morpholine nitrogen atom when its lone pair is conju-
gated with a heterocycle, thus reducing its solvation state in
water. The increased lipophilicity of 33 versus 12 is also apparent
from their relative protein binding in human plasma (see Table 3).
Compounds 12, 28 and 33 have been further evaluated in vivo
and Table 3 summarizes their pharmacokinetic properties. Com-
pound 28 demonstrated a modest oral bioavailability in the rat de-
spite a low plasma clearance whereas 12 and 33 displayed good
oral exposure. In the dog, both 12 and 33 had a moderate plasma
clearance but 12 proved to be superior to 33 with a better oral bio-
availability. When dosed orally in nude mouse, compound 12 dis-
Acknowledgements
We would like to thank the following people for the prepara-
tion, purification and analysis of the compounds described herein:
Dominique Boucherot, Christian Delvare, Delphine Dorison-Duval,
Patrice Koza, Antoine Le Griffon, Françoise Magnien, Mickaël Mau-
det, Rémy Morgentin, Annie Olivier, Marie-Jeanne Pasquet, Jacques
Pelleter and Fabrice Renaud. We also acknowledge Sara Davenport
and Jon Wingfield for kinase activity determination, Eileen McCall
for insect cell supply, Anna Valentine, Hannah Pollard, Caroline
Trueman and Heather Haye for protein supply and Claire Brassing-
ton for protein crystallization.
played a very good exposure at 100 and 200
lmol/kg, with a
greater than proportional increase in AUC from a 10
lmol/kg dose
References and notes
(Table 4).
1. For recent reviews see: (a) Pasquale, E. B. Nat. Rev. Cancer 2010, 10, 165; (b)
Noren, N. K.; Pasquale, E. B. Cancer Res. 2007, 67, 3994.
2. Kertesz, N.; Krasnoperov, V.; Reddy, R.; Leshanski, L.; Kumar, S. R.; Zozulya, S.;
Gill, P. S. Blood 2006, 107, 2330.
3. Krasnoperov, V.; Kumar, S. R.; Ley, E.; Li, X.; Scehnet, J.; Liu, R.; Zozulya, S.; Gill,
P. S. Am. J. Pathol. 2010, 176, 2029.
The broad kinase selectivity profile of 12 was assessed by mea-
suring its inhibitory activity in a panel of 70 protein kinases at a
concentration of 10 lM. Only three kinases were inhibited by more
than 80%, namely Src, LCK and CSK. These activities have been con-
firmed with a dose–response and 12 was found to be more potent
4. Wang, Y.; Nakayama, M.; Pitulescu, M. E.; Schmidt, T. S.; Bochenek, M. L.;
Sakakibara, A.; Adams, S.; Davy, A.; Deutsch, U.; Luethi, U.; Barberis, A.;
Benjamin, L. E.; Maekinen, T.; Nobes, C. D.; Adams, R. H. Nature 2010, 465, 483.
5. Martiny-Baron, G.; Holzer, P.; Billy, E.; Schnell, C.; Brueggen, J.; Ferretti, M.;
Schmiedeberg, N.; Wood, J. M.; Furet, P.; Imbach, P. Angiogenesis 2010, 13, 259.
6. (a) Bardelle, C.; Cross, D.; Davenport, S.; Kettle, J. G.; Ko, E. J.; Leach, A. G.;
Mortlock, A.; Read, J.; Roberts, N. J.; Robins, P.; Williams, E. J. Bioorg. Med. Chem.
Lett. 2008, 18, 2776; (b) Bardelle, C.; Coleman, T.; Cross, D.; Davenport, S.;
Kettle, J. G.; Ko, E. J.; Leach, A. G.; Mortlock, A.; Read, J.; Roberts, N. J.; Robins, P.;
Williams, E. J. Bioorg. Med. Chem. Lett. 2008, 18, 5717.
7. Bardelle, C.; Barlaam, B.; Brooks, N.; Coleman, T.; Cross, D.; Ducray, R.; Green, I.;
Lambert-van der Brempt, C.; Olivier, A.; Read, J. Bioorg. Med. Chem. Lett. 2010,
20, 6242.
8. Mitchell, S. A.; Danca, M. D.; Blomgren, P. A.; Darrow, J. W.; Currie, K. S.; Kropf, J.
E.; Lee, S. H.; Gallion, S. L.; Xiong, J.-M.; Pippin, D. A.; DeSimone, R. W.; Brittelli,
D. R.; Eustice, D. C.; Bourret, A.; Hill-Drzewi, M.; Maciejewski, P. M.; Elkin, L. L.
Bioorg. Med. Chem. Lett. 2009, 19, 6991.
on Src and LCK (IC50 <0.001
0.08 M). The activity of 12 against Src was further evaluated in
cell by measuring inhibition of proliferation of c-Src transfected
3T3 cells16 (IC50 0.010
M). Therefore compound 12 can be consid-
lM for both kinases) than on CSK (IC50
l
l
ered as a dual ephB4-Src kinase inhibitor with an excellent overall
selectivity profile.17
Finally, compound 12 was assessed in our CYP450 TDI assay,
with the expectation that removal of the bicyclic aniline present
in 1 and 2 would alleviate the tendency for such compounds to in-
duce TDI. To our surprise, 12 displayed a similar effect on 3A4 com-
pared to our previous leads,18 invalidating our first hypothesis. The
3,5-bis-morpholinoaniline being a common feature to all three
compounds, we suspect it could actually be the cause of the TDI ef-
fect. Effectively, it has been reported in the literature that oxidative
metabolism on a morpholine ring, producing a reactive species,
was responsible for a TDI effect of CYP 3A4.19 Although we did
not produce experimental evidence to support this hypothesis, this
suggests that a similar process might occur with our compounds.
Compound 12 also showed a modest reversible inhibition of
9. Lafleur, K.; Huang, D.; Zhou, T.; Caflisch, A.; Nevado, C. J. Med. Chem. 2009, 52,
6433.
10. Choi, Y.; Syeda, F.; Walker, J. R.; Finerty, P. J.; Cuerrier, D.; Wojciechowski, A.;
Liu, Q.; Dhe-Paganon, S.; Gray, N. S. Bioorg. Med. Chem. Lett. 2009, 19, 4467.
11. See Supplementary data in: Karaman, M. W.; Herrgard, S.; Treiber, D. K.;
Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.;
Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.;
Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar,
P. P. Nat. Biotechnol. 2008, 26, 127.
12. The compounds described in reference 8 are also inhibitors of the VEGFR2 and
Tie2 receptor tyrosine kinases, both of which are linked to angiogenic
mechanisms.
13. The benzodioxole ring is known to produce mechanism based inhibition of
P450 enzymes, see: Fontana, E.; Dansette, P. M.; Poli, S. M. Curr. Drug Met. 2005,
6, 413.
14. PCT international applications WO/2008-104754 (for compd 3–14, 18-32),
WO/2008-132505 (for compd 33–34), and WO/2009-010794 (for compd 15–
17).
CYP3A4 when assessed in a fluorometric assay (7
3A4, >10 M against 1A2, 2C9, 2C19 and 2D6).
lM against
l
In conclusion, we have identified compound 12 as a potent and
selective inhibitor of the EphB4 and Src kinase families with good
pharmacokinetic properties in mouse, rat and dog. While 12 still
carries a risk of drug-drug interaction, this compound is potentially
useful to investigate the in vivo pharmacology of inhibitors of the
Eph receptor tyrosine kinases.
15. Human EphB4 (598–892; Y774E) was crystallized as described in Ref. 6b.
Detailed protein preparation, crystallization and freezing protocols are
included in the Supplementary data of Ref. 6b. Diffraction data for complex
of EphB4 with 32 were collected on a Rigaku FRe X-ray generator equipped
Table 4
Oral exposure of compound 12 in nude mouse.
with a Saturn 944 CCD detector, using a CuK
a wavelength of 1.54178 Å,
focused using Osmic Varimax HF mirrors at 100 K. Data were processed using
d⁄trek (Pflugrath, J.W. Acta Crystallogr., Sect. D 1999, 55, 1718) and reduced
using CCP4 software (Acta Crystallogr., Sect. D 1994, 50, 760). The structures
were solved by molecular replacement using coordinates of the EphB4 kinase
domain6b as a trial model using CCP4 software. Protein and inhibitor were
modeled into the electron density using, COOT (Emsley, P.; Cowtan, K. Acta
Dose (
l
mol/kg)
AUC (
lM h)
Cmax (lM)
10
100
200
1.6
63
155
1.9
15
17

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2207–2211
2211
Crystallogr., Sect. D 2004, 60, 2126) and AFITT (OpenEye). The model was
refined using Refmac (Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta
Crystallogr., Sect. D 1997, 53, 240). Atomic coordinates21 and structure factors
for the EphB4 complex with compound 32 has been deposited in the Protein
Data Bank (2xvd) together with structure factors and detailed experimental
conditions.
incubation with a specific CYP substrate with NADPH. Compound 12 gave 50%
TDI on CYP3A4 with no significant effect on the 4 other isoforms. In the same
assay, 1 gave, respectively, 40% and 42% TDI against CYP2C9 and 3A4 and 2
gave 62% TDI against CYP3A4 (see Ref. 7).
19. Zhang, X. et al J. Med. Chem. 2008, 51, 7099.
20. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, USA, 2002.
21. Crystallographic statistics for the EphB4/compound 32 complex are as follows:
Space group P21, unit cell 46.7, 53.3, 61.3 Å, b 111.7°, Resolution 43–1.7 (1.76–
1.7) Å, 28,154 unique reflections with an overall redundancy of 3.3(2.1) give
16. For a description of this assay see: Ple, P. A.; Green, T. P.; Hennequin, L. F.;
Curwen, J.; Fennell, M.; Allen, J.; Lambert-van der Brempt, C.; Costello, G. J. Med.
Chem. 2004, 47, 871.
17. In addition to ephB4, compound 12 is likely to inhibit other members of the
Eph receptors family, given the high structural homology of their kinase
domains.
90.9(46.9)% completeness with Rmerge of 4.6(36.3)% and mean I/r(I) of
14.3(2.0). The final model containing 2078 protein, 217 solvent, and 36
compound atoms has an R-factor of 17.5% (Rfree 20.9%) using 5% of the data.
Mean temperature factors for the protein and the ligand are 34 and 47 Å2,
respectively.
18. Time dependant inhibition (TDI) of CYP450 (isoforms 1A2, 2C9, 2C19, 2D6 and
3A4) was evaluated by pre-incubating the compound (10
lM) with human
liver microsomes for 30 min in the presence vs. absence of NADPH, followed by