Inhibitors of the tyrosine kinase EphB4. Part 3: Identification of non-benzodioxole-based kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.08.100

Starting from the initial bis-anilinopyrimidine 1, good potency against EphB4 was retained when benzodioxole at C-4 was replaced by an indazole. The key interactions of the indazole with the protein were characterised by crystallographic studies. Further optimisation led to compound 20, a potent inhibitor of the EphB4 and Src kinases with good pharmacokinetics in various preclinical species and high fraction unbound in plasma. Compound 20 may be used as a tool for evaluating the potential of EphB4 kinase inhibitors in vivo.

Full text of DOI:10.1016/j.bmcl.2010.08.100

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6242–6245
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of the tyrosine kinase EphB4. Part 3: Identification
of non-benzodioxole-based kinase inhibitors
Catherine Bardelle b, Bernard Barlaam a, , Nigel Brooks b, Tanya Coleman b, Darren Cross b, Richard Ducray a,
⇑
Isabelle Green b, Christine Lambert-van der Brempt a, Annie Olivier a, 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:
Starting from the initial bis-anilinopyrimidine 1, good potency against EphB4 was retained when benzo-
dioxole at C-4 was replaced by an indazole. The key interactions of the indazole with the protein were
characterised by crystallographic studies. Further optimisation led to compound 20, a potent inhibitor
of the EphB4 and Src kinases with good pharmacokinetics in various preclinical species and high fraction
unbound in plasma. Compound 20 may be used as a tool for evaluating the potential of EphB4 kinase
inhibitors in vivo.
Received 27 May 2010
Revised 18 August 2010
Accepted 19 August 2010
Available online 16 September 2010
Keywords:
EphB4 kinase inhibitor
bis-Anilinopyrimidine
Ó 2010 Elsevier Ltd. All rights reserved.
Eph receptors constitute the largest family of tyrosine kinase
receptors, and together with their membrane-bound ephrin li-
gands, play a critical role in embryonic patterning, neuronal target-
ing,1 vascular development and adult neovascularisation.2 There is
growing evidence that Eph receptor signalling may contribute to
tumourigenesis in a wide variety of human cancers, either directly
in tumour cells or indirectly via tumour vascularisation: for
instance, Eph receptors and their ephrin ligands are frequently
overexpressed in various tumour types, including breast, prostate,
non-small cell lung and colon cancers.3
Method A
HetAr
HetAr
Cl
N
HN
HN
a
b
N
N
Ar
X
N
X
N
B
N
H
N
C
X= Cl or SMe
c
R
HetAr
N
R
HetAr
In particular, EphB4 has been one of the most studied receptor
of the Eph family: inhibition of EphB4 expression using interfering-
RNA or antisense oligonucleotides inhibited proliferation, survival
and invasion of PC3 prostate cancer cells in vitro and in vivo.4 Inhi-
N
N
b
N
Ar
N
N
D
X
N
H
Method B
Cl
Cl
HetAr
Cl
HN
R2
HN
d
O
e
N
N
N
O
Ar
N
Ar
MeSO2
N
N
N
N
N
C
H
R1
N
H
H
N
B'
1 R1= SO2Me, R2= H
Scheme 1. General synthesis of compounds 3–31. Reagents and conditions: (a) for
X = Cl: HetArNH2, DMF or alcohols, HCl (cat.) or NEtiPr2, heat; or Buchwald type
conditions (DBU, xantphos, Pd2dba3, dioxane, heat). For X = SMe: HetArNH2,
n-BuOH, HCl (cat.), 80 °C; (b) for X = Cl: ArNH2, alcohols, HCl (cat.), heat; or
Buchwald type conditions (DBU, xantphos, Pd2dba3, dioxane, heat); for X = SMe:
2 R1= R2= 4-morpholinyl
Figure 1. Compounds 1 and 2.
mCPBA (2 equiv), DMF, 0 °C; ArNH2, 2-pentanol, HCl (cat.), 150–170 °C, lw; (c) R–X
⇑
Corresponding author. Tel.: +33 3 26 61 68 97; fax: +33 3 26 61 68 42.
E-mail address: bernard.barlaam2@astrazeneca.com (B. Barlaam).
(X = Br or I), K2CO3 or Cs2CO3, DMF or MeCN, rt; (d) ArNHCHO, NaH, THF, 0 °C to rt;
i
(e) HetArNH2, PrOH, HCl (cat.), 90 °C.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.100

C. Bardelle et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6242–6245
6243
bition of EphB4 signalling using soluble extracellular-domains of
EphB4 have also been shown to inhibit tumour growth in xenograft
studies.5 Transgenic studies have shown that EphB46 and its asso-
ciated ligand ephrin B27 cause embryonic lethality associated with
vascular modelling defects. Finally, interference with endothelial
EphB4 signalling altered tumour blood vessel morphology in tu-
mour models.8 These observations suggest that EphB4 in particular
may play a critical role in tumour angiogenesis and tumour
growth.
drug–drug interactions or toxicological events.12a This potential
risk was confirmed by the following evidence: 1 and 2 showed
some CYP inhibition (resp. IC50 2.6 and 0.9 lM against 3A4, fluoro-
metric assay) with evidence of time dependant inhibition.12b X-ray
crystal structure of 1 bound to the EphB4 kinase domain11b has
established that the benzodioxole group is buried in the selectivity
pocket, the hydrophobic region beyond the Thr693 gatekeeper res-
idue. As a consequence, variation of this group may highly impact
EphB4 potency and selectivity versus other kinases.
However, the functional significance of EphB4 is far from being
fully understood: for instance, tumour-suppressor as well as tu-
mour-promoting activities have been proposed for EphB4 in breast
cancer.9 In addition, by contrast with most receptor tyrosine ki-
nases, signalling can originate from the ephrin ligands as well as
from the Eph receptors; bi-directional signalling has emerged as
an important mechanism by which Eph receptors and ephrin li-
gands control the output signal in processes of cell–cell communi-
cation. The relative contribution of these ‘forward’ and ‘reverse’
signals in tumour vascularisation and tumour growth has not been
fully clarified.10,3b
In our search for EphB4 kinase inhibitors as potential oncology
drugs, we had recently identified 4-(benzodioxolyl)amino pyrimi-
dines such as compounds 1 and 2 as potent EphB4 kinase inhibitors
(Fig. 1).11 Previous reports have linked the benzodioxole scaffold
with mechanism based P450 inhibition, which might lead to
In this publication, we describe the replacement of the benzodi-
oxole by other bicyclic heteroaryl groups as EphB4 kinase
inhibitors.
In general terms, compounds 3–31 have been made by one of
the following methods,13 as described in Scheme 1. From readily
available 2,4-dichloropyrimidine (or the 4-chloro-2-methylthio-
pyrimidine), displacement of the more reactive 4-chloro by the
heteroarylamino group under acidic, basic or Buchwald type condi-
tions gave B; optional alkylation of B followed by displacement of
the 2-chloro (or the 2-sulfonyl after oxidation of the 2-methylthio
group with m-CPBA) under acidic or Buchwald type conditions
gave the desired compound C or D (method A). Alternatively, the
sequence of addition can be reversed: treatment of the 4-chloro-
2-methylsulfonylpyrimidine14 with the anion of the desired for-
manilide, followed by saponification afforded the corresponding
2-anilinopyrimidine B0 with excellent regioselectivity15; B0 was
Table 1
Inhibition data in an EphB4 kinase enzyme assay (using acoustic dispensing) and in a cellular assay of phospho-EphB4 inhibition for compounds 1–31
R3
R2
N
N
N
R1
N
H
a
a,b
Compound R1
R2
R3
Synthesis
method
EphB4 enz. IC50
M)
p-EphB4 IC50
M)
(
l
(l
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
3-MeSO2-Ph
3,5-Di-(morpholin-4-yl)-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
3-MeSO2-Ph
5-Cl-1,3-benzodioxol-4-yl
5-Cl-1,3-benzodioxol-4-yl
1,3-Benzodioxol-4-yl
6-Cl-benzofuran-7-yl
Benzofuran-7-yl
2,3-Dihydrobenzofuran-7-yl
Benzoxazol-7-yl
Benzoxazol-4-yl
Indazol-4-yl
H
H
H
H
H
H
H
H
0.025c
0.004c
0.034
0.300
0.205
1.16
0.187
0.014
0.186
0.897
0.460
1.72
1.35
1.76
2.53
A
B
B
B
B
B
B
B
A
A
A
A
B
B
B
B
A
A
A
A
A
A
A
A
B
B
A
A
A
0.219
0.549
0.061
0.913
0.036
0.095
0.246
0.121
0.011
0.089
0.117
0.115
0.036
0.0013
0.204
0.021
0.246
0.029
0.084
0.013
0.067
0.055
0.0009
0.0026
0.432
H
H
3-Cl-indol-7-yl
5-Cl-1,3-benzodioxol-4-yl
5-Cl-1,3-benzodioxol-4-yl
5-Cl-1,3-benzodioxol-4-yl
5-Cl-1,3-benzodioxol-4-yl
Indazol-4-yl
Indazol-7-yl
Indol-4-yl
Benzotriazol-4-yl
Indazol-4-yl
Indazol-4-yl
Indazol-4-yl
Indazol-4-yl
Indazol-4-yl
Indazol-4-yl
Indazol-4-yl
Indazol-4-yl
3-Me-indazol-4-yl
3-Cl-indazol-4-yl
Indazol-4-yl
Me
CH2CN
CH2CH2OMe
CH2CH2OH
H
H
H
H
Me
Me
H
Me
H
Me
H
Me
H
0.042
0.190
0.378
0.187
0.302
3.5
0.856
8.6
0.051
0.009
4.4
3-MeSO2-Ph
3,5-Di-(morpholin-4-yl)-Ph
3,5-Di-(morpholin-4-yl)-Ph
3,5-Di-(morpholin-4-yl)-Ph
3,5-Di-(morpholin-4-yl)-Ph
3-MeSO2-Ph
3,5-Di-(morpholin-4-yl)-Ph
3-(CH2OH)-Ph
3-(CH2OH)-Ph
3-(CONMe2)-Ph
3-(CONMe2)-Ph
3-(NHSO2Me)-Ph
3-(NHSO2Me)-Ph
3,5-Di-(morpholin-4-yl)-Ph
3,5-Di-(morpholin-4-yl)-Ph
2,6-Di-(morpholin-4-yl)-pyridin-4-yl
2,6-Di-(morpholin-4-yl)-pyrimidyl-4-yl
4,6-Di-(morpholin-4-yl)-pyrimidyl-2-yl
0.232
10.4
0.453
8.42
0.513
0.227
0.005
0.024
H
Me
Me
Me
Indazol-4-yl
Indazol-4-yl
12
a
b
c
Standard error is typically 0.3 log unit.
EphB4 cellular autophosphorylation assay (engineered CHO-K1 cell line stably expressing EphB4).
Previously reported EphB4 enzyme IC50 (using serial aqueous dilution)13 for 1 and 2 was resp. 90 nM and 2 nM.11b

6244
C. Bardelle et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6242–6245
then reacted with the desired heteroarylamino derivative to give C
(method B).
Compounds 1–31 in Table 1 were evaluated in an EphB4 kinase
enzyme assay (measuring the inhibition of phosphorylation of a
synthetic peptide substrate at Km ATP concentration using acous-
tic dispensing)16 and in a cellular assay with a phospho-EphB4
similar to the one observed for compound 1. However, the indazole
adopts a different and flipped orientation compared to the benzo-
dioxole 1. Here the indazole N01 NH and N02 nitrogen make hydro-
gen donor and acceptor bonds to the side chain carbonyl of Glu664
and the backbone NH of Asp758, respectively. The 3-position of the
indazole explores the same area as the 5-chloro of the benzodiox-
ole (see Fig. 2). As observed for 1, the C-2 aniline of 9 adopts a dual
conformation within the protein structure, with the methyl sulfo-
nyl pointing towards the hinge region or towards the Gly-rich loop.
Modification of the C-2 aniline had identified the 3,5-(dimorph-
olin-4-yl)aniline as a more active aniline compared to the m-meth-
endpoint (engineered CHO-K1 cell line stably expressing
a
EphB4-Myc-His construct).13
Replacement of the benzodioxole of 1 with other various het-
erocycles significantly reduced EphB4 activity (compounds 3–8,
10). But the indazole 9 retained good potency in the enzyme assay.
In an attempt to understand the particular role of the indazole,
the crystal structure of 9 in complex with EphB4 was deter-
mined.17 This structure, shown in Figure 2, confirms the predicted
binding mode of 9 in the ATP binding site, with the pyrimidine N-1
and C-2 anilino N–H making hydrogen acceptor and donor bonds
to the hinge region at Met696 and the indazole heterocycle buried
in the selectivity pocket, resulting in a ‘S-shaped’ conformation
ylsulfonylaniline (2 vs 1) with the chlorobenzodioxole at C-4.11b
A
similar result was observed with the 4-indazole at C-4 (15 vs 9).
Variation of the C-4 aniline in the presence of the 3,5-(dimorpho-
lin-4-yl)aniline at C-2 was explored. Reduced activity was ob-
served with the 7-indazole 16, the indole 17 and the
benzotriazole 18 compared to the 4-indazole 15, as anticipated
from the key hydrogen bonds of the 4-indazole moiety with the
protein. Substitution at the 3-position of the indazole with methyl
or chlorine (27 and 28) also reduced potency slightly. Alkylation of
the N-4 nitrogen on the pyrimidine was tolerated with methyl
being optimal (11–13 vs 1) in the benzodioxole subseries. In the
indazole subseries, methylation of the N-4 nitrogen showed a
marked improvement of activity (19, 20, 22, 24, 26 vs 9, 15, 21,
23, 25).
Additive SAR was observed when combining the three key ele-
ments: N-4 methylation and 4-indazole at C-4 and 3,5-(dimorpho-
lin-4-yl)aniline at C-2 of the pyrimidine culminated in the
identification of 20 as a potent inhibitor of the EphB4 kinase.
Replacement of the phenyl of 3,5-(dimorpholin-4-yl) aniline by a
4-pyridine (29) or a 4-pyrimidine (30) was tolerated, but not by
a 2-pyrimidine (31). In the case of 31, we hypothesised that repul-
sion between the lone pairs of the N-3 nitrogen of the central
pyrimidine and the N-2 nitrogen of the dimorpholino-pyrimidine
forces the dimorpholino-pyrimidine out of the plane of the central
pyrimidine into a conformation that cannot be accommodated in
the kinase.
Selectivity of 20 was evaluated against a panel of 68 kinases
(Dundee panel) at 10 lM: only 12 out 68 kinases were inhibited
by more than 80%, including Src-family kinases (99% and 92% of
inhibition Src and Lck, respectively). At the cellular level, 20
showed good inhibition of proliferation of c-Src transfected 3T3
cells18 (IC50 2 nM) as well as autophosphorylation of EphB4 in
transfected CHO-K1 cells (IC50 9 nM). Some selectivity at the cellu-
lar level was observed versus other kinases involved in vascular
modulation (inhibition of p-KDR in HUVEC IC50 240 nM; inhibition
of p-PDGFR-b in MG63 cell line IC50 58 nM). It is interesting to note
that scientists at GlaxoSmithKline had also identified related inda-
zoles as inhibitors of Lck, where the indazole works as a phenol iso-
stere.19 The binding mode they propose based on docking studies
in Lck is very similar to the binding mode we observed for 9 in
EphB4.
We then looked at the pharmacokinetic properties of 20, with
the aim of using this dual Src/EphB4 kinase inhibitor as an in vivo
tool. Oral administration of 20 in nude mice at 100 mg/kg gave
excellent plasma levels (Table 2). Finally, 20 exhibited good
pharmacokinetic parameters in rat and dog (Table 3) and rela-
tively high fraction unbound in plasma (fu nude mouse: 7%, fu hu-
man: 6%)
Figure 2. (a) Crystal structure (pdb:2xf9) of compound
9 bound to EphB4.
As expected, 20 showed only modest inhibition of CYP P450
Compound is shown in green. The protein backbone cartoon is represented in
white. The hinge region at the bottom, E664 and D758 backbone above are
represented as sticks. Refined electron density (2fofc) contoured at 1.0 sigma
represented as wire mesh. Both conformations of the C-2 aniline are shown. (b)
(IC50
5 lM against 2C9 and 3A4, >10 lM against 1A4, 2D6 and
2C19; fluorometric assay). However, we were surprised still to
see evidence of time dependent inhibition with 20.12b
Crystal structures of compounds
1 (white, pdb:2vwu) and 9 (wheat). Protein
In conclusion, starting from the bis-anilinopyrimidine core, we
have identified a potent inhibitor 20 of the EphB4 and Src kinases
in a non-benzodioxole related series. This compound 20 may be
structures overlaid using backbone main-chain atoms. The second, minor confor-
mation of the C-2 aniline for both compound structures is not shown for clarity.
Pictures produced using PYMOL.20

C. Bardelle et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6242–6245
6245
Table 2
10. Pasquale, E. B. Cell 2008, 133, 38.
AUC0–24 and plasma concentrations after p.o. administrationa of 20 (100 mg/kg) in
11. (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.
12. (a) Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.; Callegari, E.; Henne,
K. R.; Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.; Nakai, Y.; O’Donnell, J. P.; Boer, J.;
Harriman, S. P. Current Drug Metab. 2005, 6, 161; (b) Murray, M. Current Drug
Metab. 2000, 1, 67. CYP Time dependant inhibition (TDI) was evaluated by
nude mice
AUC (
l
M h)
Plasma concentration (lM) at
1 h
11
4 h
1.9
24 h
1.8
48.7
a
Administered as an HPMC Tween suspension.
preincubating the compound (10 lM) with HLM (human liver microsomes) for
30 min in the presence versus absence of NADPH, followed by incubation with
a specific CYP substrate with NADPH. Compound 1 gave 26% TDI against
CYP3A4 and 2 gave 42% and 40% TDI resp. against CYP3A4 and 2C9. Compound
20 showed 62% TDI against CYP3A4..
Table 3
Pharmacokinetic parameters for compound 20
13. For detailed conditions for the synthesis of compounds 3–31 and description of
biological assays, see: Kettle, J. G.; Read, J.; Leach, A.; Barlaam, B. C.; Ducray, R.;
Lambert-Van Der Brempt, C. M. P. PCT Int. Appl. WO2007085833. Protecting
groups for the indazolyl endocyclic nitrogen were used for compounds 19, 20,
a
Rat/dog Cla (%hbf)
19/80
Rat/dog Vdss (L/kg)
Rat/dog Fa (%)
65/63
0.8/2.6
22, 24, 26 (benzyl) or 29–31 (4-methoxybenzyl). For
7 and 8, the
a
Female Han Wistar rats dosed at 1.2 mg/kg iv and 5 mg/kg p.o.; mean values for
aminobenzoxazole was introduced as the 2-Boc-NH 6-aminophenol and the
2-Boc-NH 3-aminophenol. Removal of the Boc protecting group with TFA and
formation of the benzoxazole ring by treatment with trimethyl orthoformate
with p-toluenesulfonic acid gave the expected benzoxazoles.
male and female beagle dogs dosed at 1 mg/kg iv and 2 mg/kg p.o. Cl expressed in %
of hepatic blood flow.
14. Davey, D. D.; Adler, M.; Arnaiz, D.; Eagen, K.; Erickson, S.; Guilford, W.; Kenrick,
M.; Morrissey, M. M.; Ohlmeyer, M.; Pan, G.; Paradkar, V. M.; Parkinson, J.;
Polokoff, M.; Saionz, K.; Santos, C.; Subramanyam, B.; Vergona, R.; Wei, R. G.;
Whitlow, M.; Ye, B.; Zhao, Z.; Devlin, J. J.; Phillips, G. J. Med. Chem. 2007, 50,
1146.
used as a ‘relatively selective’ tool for evaluating the potential of
EphB4 kinase inhibitors in vivo.
15. For a previous report of analogous regioselective reactions, see: Ito, S.; Sumi, K.;
Masuda, K.; Kojima, Y.; Sawai, N. Jpn. Kokai Tokkyo Koho JP62106084.
16. For description of the EphB4 enzyme assay (using acoustic dispensing), see:
Barlaam, B. C.; Ducray, R. PCT Int. Appl. WO2008132505.
17. Human EphB4 (598–892; Y774E) was crystallized as described in Ref. 11b.
Detailed protein preparation, crystallization and freezing protocols are
included in the Supplementary data of Ref. 11b. Diffraction data for complex
of EphB4 with 9 were collected on a Rigaku FRe X-ray generator equipped with
Acknowledgements
We warmly thank Andrew Mortlock and Jason Kettle for fruitful
discussions and the following for technical assistance: Dominique
Boucherot, Sara Davenport, Christian Delvare, Delphine Dorison-
Duval, Patrice Koza, Antoine Le Griffon, Françoise Magnien, Mickaël
Maudet, Marie-Jeanne Pasquet, Jacques Pelleter, Fabrice Renaud,
and Jon Wingfield. We acknowledge Eileen McCall for insect cell
supply, Anna Valentine, Hannah Pollard, Caroline Trueman and
Heather Haye for protein supply and Claire Brassington for protein
crystallization.
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 MOSFLM
and SCALA and reduced using CCP4 software.20 The structures were solved by
molecular replacement using coordinates of the EphB4 kinase domain11b as a
trial model using CCP4 software. Protein and inhibitor were modelled into the
electron density using, COOT21 and AFITT.22 The model was refined using
Refmac.23 Atomic coordinates24 and structure factors for the EphB4 complexes
with compounds 9 have been deposited in the Protein Data Bank (2xf9)
together with structure factors and detailed experimental conditions.
18. 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.
19. Bamborough, P.; Angell, R. M.; Bhamra, I.; Brown, D.; Bull, J.; Christopher, J. A.;
Cooper, A. W. J.; Fazal, L. H.; Giordano, I.; Hind, L.; Patel, V. K.; Ranshaw, L. E.;
Sims, M. J.; Skone, P. A.; Smith, K. J.; Vickerstaff, E.; Washington, M. Bioorg. Med.
Chem. Lett. 2007, 17, 4363.
20. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, USA, 2002. http://www.pymol.org; CCP4 Acta Crystallogr., Sect. D
1994, D50, 760.
21. Coot; Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D 2004, 60, 2126.
22. AFITT, Openeye.
23. Refmac version 5.1.17: Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta
Crystallogr., Sect. D 1997, 53, 240.
24. Crystallographic statistics for the EphB4/compound 9 complex are as follows:
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3994.
Space group P21, unit cell 47.1, 52.5, 61.4 Å,
b 112.1, Resolution 56.89–
1.75(1.89–1.75) Å, 26301(2619) unique reflections with an overall redundancy
of 3.3(2.2) give 93.5(64.8)% completeness with Rmerge of 6.3(35.4)% and mean I/
r
(I) of 12.9(2.0). The final model containing 2108 protein, 263 solvent, and 38
compound atoms has an R-factor of 15.7% (Rfree 19.4%) using 5% of the data.
Mean temperature factors for the protein and the ligand are 17 and 18 Å2,
respectively.