Novel thienopyrimidine and thiazolopyrimidine kinase inhibitors with activity against Tie-2 in vitro and in vivo

Article abstract of DOI:10.1016/j.bmcl.2009.10.001

The SAR and improvement in potency against Tie2 of novel thienopyrimidine and thiazolopyrimidine kinase inhibitors are reported. The crystal structure of one of these compounds bound to the Tie-2 kinase domain is consistent with the SAR. These compounds h

Full text of DOI:10.1016/j.bmcl.2009.10.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6670–6674
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel thienopyrimidine and thiazolopyrimidine kinase inhibitors
with activity against Tie-2 in vitro and in vivo
a
a
a
a
a
Richard W. A. Luke a, , Peter Ballard , David Buttar , Leonie Campbell , Jon Curwen , Steve C. Emery ,
Alison M. Griffen a, Lorraine Hassall a, Barry R. Hayter a, Cliff D. Jones a, William McCoull a, Martine Mellor a,
Mike L. Swain a, Julie A. Tucker b
*
a Cancer and Infection Research Area, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG, UK
b CPSS, LG-DECS, AstraZeneca, 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:
The SAR and improvement in potency against Tie2 of novel thienopyrimidine and thiazolopyrimidine
kinase inhibitors are reported. The crystal structure of one of these compounds bound to the Tie-2 kinase
domain is consistent with the SAR. These compounds have moderate potency in cellular assays of Tie-2
inhibition, good physical properties, DMPK, and show evidence of in vivo inhibition of Tie-2.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 20 August 2009
Revised 30 September 2009
Accepted 1 October 2009
Available online 13 October 2009
Keywords:
Kinase
Tie-2
TEK
Inhibition
Angiogenesis
Small molecule
There is an ongoing need for novel approaches to treating hu-
man tumours and one of the most promising new approaches is
blocking angiogenesis.1 Tumours require a blood supply to grow
to a significant size and it is believed that blocking specific angio-
genic factors will prevent the development of this vascular net-
We recently described7 the SAR developed around an imidazole
vinyl pyrimidine HTS hit leading to 1 which had good potency
against Tie-2 in cells, lead-like properties and oral bioavailability.
However, these compounds have poor photostability and react
with glutathione so we sought more stable analogues. As both of
these stability issues appeared to be associated with the alkene lin-
ker we attempted to replace this with other groups but this led to
loss of potency or no improvement in glutathione reactivity.7 In the
work described here we explored conceptually fusing the alkene
with the pyrimidine ring to give bicyclic compounds (Fig. 1). The
directly fused compounds 2 were not easily accessible syntheti-
cally but the analogues 3, where one of the pyrimidine nitrogens
had been moved, were. This re-positioning of the pyrimidine nitro-
gen was well tolerated in the imidazole vinyl pyrimidine series.7
A range of different bicyclic pyrimidines was synthesized but
the thienopyrimidine and thiazolopyrimidine were most potent.
The thienopyrimidine 7 was synthesized by reaction of the imine
derived from thienopyrimidine 48 with phenyl-TosMIC reagent 8
and then oxidation of the SMe of 6 with MCPBA and displacement
with ammonia (Scheme 1). Full synthetic details for the com-
pounds reported in this paper have been described elsewhere.8
Thiazolopyrimidine 12 was prepared from phenyl-TosMIC reagent
8 by formation of alcohol 9 and then by oxidation and conversion
to the acid chloride 10, followed by amide coupling to give 11 and
work. The endothelial cell growth factor VEGF is
a potent
stimulus for angiogenesis and recent clinical trials have shown effi-
cacy of the antibody drug bevacizumab2 which binds to VEGF.
There is considerable work underway directed towards other angi-
ogenesis inhibitors and VEGFr inhibitors are the most advanced ki-
nase inhibitors in this field. Of the other kinases thought to be
involved in angiogenesis in human tumours, Tie-2, an endothelium
specific receptor tyrosine kinase, promotes tumour angiogenesis
through interaction with angiopoietin3 and plays an important role
in stabilizing the immature endothelial cell network, attracting
pericytes and maintaining vessel integrity.4 Although the details
of Tie receptor biology are still emerging5 it has attracted consider-
able interest, although to date only a small number of inhibitors
have been reported outside of patent literature.6
We wish to report here work leading to the identification of
bicyclic imidazoles as a novel class of kinase inhibitors which inhi-
bit Tie-2 in vitro, in cells and in vivo.
* Corresponding author. Tel.: +44 1625 514495.
E-mail address: richard.luke@astrazeneca.com (R.W.A. Luke).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.001

R. W. A. Luke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6670–6674
6671
NH2
NH2
NH2
N
N
X
Y
N
X
Y
N
N
N
N
N
N
N
~5o
~30o
N
N
1
2
3
putative active
conformation
N shift tolerated
in alkene series
Figure 1. Design of bicyclic Tie-2 inhibitors.
SMe
SMe
Table 1
a, b
Thienopyrimidine and thiazolopyrimidine overall properties.
O
N
N
S
S
N
H
N
Property
Thienopyrimidine Thiazolopyrimidine
7
12
4
5
a
pTie-2 cell IC50
(
l
M)
1.8
34
10, 22
307, n.d.
84
2.7
20
84, 4.4
308, 2.7
160
a
Tie-2 enzyme IC50
Flt-1, KDR enzyme IC50
(l
M)
(
c, d
a
lM)
MW, log D
NH2
N
Ph
Solubility at pH7 (lM)
Plasma protein binding (rat%
SMe
e, f
Ph
N
N
95.5
94.8
N
N
N
S
bound)
N
S
PK (rat): F (%), t1/2 (h), Vdss (L/kg), >100, 1.8 c, 1.4, 39 >100, 1.6 c, 1.2, 30.1
N
7
Cl (mL/min/kg)b
6
n.d.: not determined.
Scheme 1. Thienopyrimidine synthesis. Reagents and conditions: (a) LDA, THF; (b)
DMF, 51%; (c) 4 Å mol. sieves, NH2Me, DCM, reflux, 100%; (d) PhTosMIC, piperazine,
THF, 90%; (e) mCPBA, DCM, 56%; (f) H3N, dioxane, 100%.
a
Values are geometric means of two or more experiments with a standard
deviation of < 0.3 log units.
b
Female rats dosed at 2 mg/kg iv and 5 mg/kg po.
Half life after oral dosing.
c
incorporation of sulphur and cyclization with phosphorus
pentasulphide to form 12 (Scheme 2).
with substitution on this ring to try to position favourable groups
in this pocket. Synthesis of the iodophenyl intermediate 13
(Scheme 3) was by the same route as used in Scheme 1. This inter-
mediate was elaborated to the thiotoluene 14 and aniline 15. Ani-
line 15 reacted with various isocyanates to give the aryl ureas 16–
18.
Generally the SAR was rather flat in this area with substitution
by relatively large groups such as the thiotoluene 14 and the phe-
nyl urea 16 being tolerated but not beneficial (Table 2). Phenyl ur-
eas with lipophilic meta or para substituents such as 17, however,
gave a significant improvement in potency. Unfortunately, the
The thienopyrimidine 7 and thiazolopyrimidine 12 showed no
reactivity with glutathione and were photostable (Hanau sun test).
These compounds had an attractive overall balance of properties
(Table 1). In a phospho Tie-2 cell assay9 they were reasonably po-
tent and selective. In contrast to their Tie-2 cell potency they were
less potent in Tie-2 enzyme assay which we believe is due to insen-
sitivity of this assay (assay conditions and further discussion on the
sensitivity of the enzyme assay is contained in our previous pa-
per7). They have good physical properties and good pharmacoki-
netics in the rat.
Our next aim was to improve the potency of these leads. We
anticipated that the phenyl ring would be close to, but not in, the
kinase selectivity pocket so we prepared analogues of 7 and 12
I
NH2
NH2
NH2
b, c
N
N
N
N
a
b, c
N
NC
SO2
N
S
N
N
Cl
N
S
N
N
N
O
13
HO
15
8
9
10
a
NH2
d
NH2
d
N
O
O
N
N
S
N Ar
NH2
NH2
N
NH2
N
N
N
N
N
NH2
N
N
e
N
N
N
N
N
S
N
N
N
S
N
S
O
O
N
16-18
14
12
11
Scheme 3. Synthesis of phenyl substituted thienopyrimidines. Reagents and
conditions: (a) NaSTol, CuI, 110 °C, DMF, 20%; (b) benzophenone imine, (Ph2P)fer-
rocene, (acac)2Pd, NaOtBu, 90 °C, dioxane; (c) 2 M HCl, THF, 50%; (d) Ar-NCO, THF,
67–90%.
Scheme 2. Thiazolopyrimidine synthesis. Reagents and conditions: (a) MeNH2,
(CHO–CHO)2, 80%; (b) KMnO4, K2CO3, Me2CO, H2O; (c) (COCl)2, DCM, DMF, 60%; (d)
NMP, 24%; (e) P2S5, pyridine, 110 °C, 19%.

6672
R. W. A. Luke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6670–6674
Table 2
Table 3
Phenyl ring substitution
N1-imidazole substitution
R
N
NH2
N
N
S
NH2
N
N
N
N
N
N
R
S
Compd
R
pTie-2 cells
Solubility Protein binding
a
Compd
R
pTie-2 cells IC50 (lM)
a
IC50
(lM)
(
lM)
rat (% bound)
7
H
1.8
1.4
12
26
27
28
Methyl
Ethyl
–CH2–cyclohexyl
–Isobutyl
2.7
3.0
0.54
0.49
160
171
10
94.6
n.d.
98.4
94.1
14
S
*
*
70
16
1.4
NHCONH
NHCONH
H
O
CF3
29
0.37
0.44
682
0.9
80
C
*
H2
17
0.07
*
*
H2
C
F
*
O
N
30
94.2
18
0.34
NHCONH
N
O
a
Values are geometric means of two or more experiments with a standard
n.d.: not determined.
deviation of < 0.3 log units.
a
Values are geometric means of two or more experiments with a standard
deviation of < 0.3 log units.
physical properties of 17 were poor: aqueous solubility 0.5 lM and
rat plasma protein binding 99.97%. By reducing the lipophilicity of
the phenyl ring or its substituents the physical properties
could be improved, but this also led to reduced potency. One of
the best compromises between potency and properties was 18,
With improvements in Tie-2 cell potency in hand we investi-
gated the DMPK properties of these new leads. The isoxazole urea
18 gave very low levels on dosing orally in the rat (Cmax 0.009 lM
from a 1 mg/kg dose). Compound 30 had no oral bioavailability in
the rat but was evaluated using iv dosing in a haemodynamic mod-
el12 for Tie-2 activity. This assay, in the anaesthetized rat, tests the
ability of compounds to reverse kinase signalling-induced hypo-
tension following stimulation by vasoactive ligands such as VEGF
and Ang-1. The methodology for this model has previously been
described for a similar VEGF response13 and has been shown to
predict the haemodynamic outcome in conscious animals to dos-
ing by oral gavage.14 Dosing 30 at 20 mg/kg iv produced complete
reversal of Ang-1 induced hypotension but, as expected, did not re-
which had solubility of 7.2
99.85%.
lM and rat plasma protein binding of
We next explored substitution on the N1 of the imidazole. To
provide an efficient route we first prepared the aldehyde 21 from
the amino-dithiopyrimidine 19 by cyclization to form the thiazole
ring, followed by methylation and oxidation (Scheme 4). The imi-
nes formed from aldehyde 21 and amines reacted with phenyl-Tos-
MIC reagent to give the imidazoles 22–25. Oxidation of the
thiomethyl group and then displacement with ammonia led to
compounds 26–29.
In the N1 position, small substituents such as ethyl 26 had little
effect on potency (Table 3). Larger lipophilic groups were beneficial
such as cyclohexylmethyl 27, isobutyl 28, methyl-THF 29, and,
benzyl morpholine 30. The methyl-THF compound 29 was particu-
larly interesting as it retained potency and had good solubility and
free drug levels in rat plasma. The cyclohexylmethyl compound 27
was crystallized with Tie-2 kinase domain10 (Fig. 2). This confirmed
the expected binding mode with the pyrimidine forming two H-
bonds to the hinge region of the kinase and the cyclohexyl group
in the ribose binding pocket. As expected the phenyl ring is just
outside the selectivity pocket.
SH
N
SMe
SH
N
a, b
c, d
NH2
SH
O
H
N
OH
N
S
N
N
N
S
N
19
20
21
e, f
SMe
NH2
N
Ph
Ph
g, h
N
N
S
N
N
N
N
N
R
S
N
R
N
22-25
26-29
Scheme 4. Synthesis of N1-imidazole substituted thiazolopyrimidines. Reagents
and conditions: (a) acetoxyacetyl chloride, pyridine; (b) 2 N HCl, reflux, 97%; (c)
Me–I, 2 N NaOH, 53%; (d) MnO2, 100 °C, dioxane, 91%; (e) RNH2, THF; (f) PhTosMIC,
morpholine, ꢀ58%; (g) mCPBA, DCM; (h) NH3, 25–64%,
Figure 2. Crystal structure10 of human Tie2 kinase domain complexed with 27
showing final 2Fo À Fc electron density (yellow, 1.0
r level). Selected nearby protein
residues are shown. Hydrogen bonding interactions with the protein are indicated
as dashed yellow lines. The figure was prepared using PyMol.11

R. W. A. Luke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6670–6674
6673
(a)
160
140
120
100
BP
80
60
40
20
2 mins
mmHg
Time
(b)
(c)
160
140
120
100
80
BP
60
40
20
00
2 mins
mmHg
Time
Compound Vehicle
I.V.
Ang-1 I.V.
Ang-1 I.V.
30 (20mg/kg I.V.)
(d)
(e)
160
140
120
BP 100
80
60
40
2 mins
mmHg20
00
Time
VEGF I.V. compound vehicle
I.V.
VEGF I.V. 30 (20mg/kg I.V.)
Figure 3. Hypotension assay12 results for compound 30. (a) Ang-1 (40–60
lg) induced a fall in blood pressure (BP) (upper line is systolic pressure, lower line is diastolic
pressure); (b) compound vehicle was administered, showing an injection spike, but did not reverse the Ang-1 induced fall in blood pressure; (c) compound 30 reversed the
Ang-1 induced fall in blood pressure; (d) VEGF (10 g) induced a fall in blood pressure; (e) compound 30 did not reverse the VEGF induced fall in blood pressure.
l
verse VEGF induced hypotension (Fig. 3). These initial results are
consistent with 30 selectively inhibiting Tie-2 activity and demon-
strate that a methodology is available for assessing the in vivo effi-
cacy and selectivity of other targeted compounds. However as the
most potent compounds such as 30 were not orally bioavailable,
and so were not considered drug candidates, we did not test them
in further in vivo angiogenesis or tumour xenograft models but
developed a new series of orally bioavailable compounds which
will be reported in a further paper (in preparation).
In conclusion we have shown that the attractive, moderately
potent, but unstable alkene imidazoles can be converted to bicy-
clic thieno- and thiazolopyrimidines with retention of potency
and overall properties but with improved stability. The potency
of these bicyclic compounds was increased by substituting on
the N1 of the imidazole or the phenyl ring although this led to
loss of oral bioavailability. The benzyl morpholine 30 was shown
to reverse Tie-2 mediated hypotension in vivo following iv
administration.
Acknowledgements
We would like to thank Rob Bradbury for reviewing the draft
manuscript, Colin Rodgers and the in vivo pharmacokinetic
support team, and, the following people for assistance with the
crystallography: Cheryl Forder and Martina Fitzek (molecular
biology); Eileen McCall (insect cell cultures); Dougie Paterson
(protein purification); Jason Breed (crystal growth); Helen McMi-
ken and Claire Brassington (preparation of crystals for data collec-
tion); Richard Norman and Siân Rowsell (diffraction data
collection).
References and notes
1. Folkman, J. Annu. Rev. Med. 2006, 57, 1.
2. Morgensztern, D.; Govindan, R. Exp. Rev. Anticancer Ther. 2006, 6, 545.
3. Kim, I.; Kim, H. G.; Moon, S.-O.; Chac, S. W.; So, J.-N.; Koh, K. N.; Ahn, B. C.; Koh,
G. Y. Circ. Res. 2000, 86, 952.
4. Thurston, G. Cell Tissue Res. 2003, 314, 61.

6674
R. W. A. Luke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6670–6674
5. (a) Kim, K. L.; Shin, I.-S.; Kim, J.-M.; Choi, J.-H.; Byun, J.; Jeon, E.-S.; Suh, W.; Kim,
D.-K. Cardiovascular Res. 2006, 72, 394; (b) Miyazaki, Y.; Matsunaga, S.; Tang, J.;
Maeda, Y.; Nakano, M.; Philippe, R. J.; Shibahara, M.; Liu, W.; Sato, H.; Wang, L.;
Nolte, R. T. Bioorg. Med. Chem. Lett. 2005, 15, 2203.
Hampton Research Additive Screen). Crystals were cryoprotected with 2,3-
butanediol (17% v/v) in mother liquor and flash cooled in a cryostream at
100 K. Diffraction data were collected on beamline PX14.2 at the SRS,
Daresbury, at 100 K. Data processing, data reduction and structure solution
by molecular replacement were carried out using programs from the CCP4
suite.15 Compound 27 was modelled into the electron density using QUANTA.16
The protein-compound complex model was refined using CNX,17 Refmac18 and
Buster19 with intermediate rounds of model building in QUANTA16 and Coot.20
The final structure21 has been deposited in the Protein Data Bank (2wqb)
together with structure factors and detailed experimental conditions.
11. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San
Carlos, CA, USA, 2002. http://www.pymol.org.
6. (a) Ji, Z.; Ahmed, A. A.; Albert, D. H.; Bouska, J. J.; Bousquet, P. F.; Cunha, G. A.;
Glaser, K. B.; Guo, J.; Li, J.; Marcotte, P. A.; Moskey, M. D.; Pease, L. J.; Stewart, K.
D.; Yates, M.; Davidsen, S. L.; Michaelides, M. R. Bioorg. Med. Chem. Lett. 2006,
16, 4326; (b) Hodous, B. L.; Geuns-Meyer, S. D.; Hughes, P. E.; Albrecht, B. K.;
Bellon, S.; Bready, J.; Caenepeel, S.; Cee, V. J.; Chaffee, S. C.; Coxon, A.; Emery,
M.; Fretland, J.; Gallant, P.; Gu, Y.; Hoffman, D.; Johnson, R. E.; Kendall, R.; Kim,
J. L.; Long, A. M.; Morrison, M.; Olivieri, P. R.; Patel, V. F.; Polverino, A.; Rose, P.;
Tempest, P.; Wang, L.; Whittington, D. A.; Zhao, H. J. Med. Chem. 2007, 50, 611;
(c) Cee, V. J.; Albrecht, B. K.; Geuns-Meyer, S.; Hughes, P.; Bellon, S.; Bready, J.;
Caenepeel, S.; Chaffee, S. C.; Coxon, A.; Emery, M.; Fretland, J.; Gallant, P.; Gu,
Y.; Hodous, B. L.; Hoffman, D.; Johnson, R. E.; Kendall, R.; Kim, J. L.; Long, A. M.;
McGowan, D.; Morrison, M.; Olivieri, P. R.; Patel, V. F.; Polverino, A.; Powers, D.;
Rose, P.; Wang, L.; Zhao, H. J. Med. Chem. 2007, 50, 627; (d) Hodous, B. L.;
Geuns-Meyer, S. D.; Hughes, P. D.; Albrecht, B. K.; Bellon, S.; Caenepeel, S.; Cee,
V. J.; Chaffee, S. C.; Emery, M.; Fretland, J.; Gallant, P.; Gu, Y.; Johnson, R. E.; Kim,
J. L.; Long, A. M.; Morrison, M.; Olivieri, P. R.; Patel, V. F.; Polverino, A.; Rose, P.;
Wang, L.; Zhao, H. Bioorg. Med. Chem. Lett. 2007, 17, 2886.
12. Hypotension assay: Angiopoietin-1 (Ang-1) activates the tyrosine kinase
receptor, Tie2 and this has been shown to influence the tone of blood
vessels, inducing dilation and leading to
a hypotensive response. Full
experimental details for the procedure using VEGF are given in a previous
publication13 and the same methodology was used for the Ang-1 response.
Ang-1 was expressed in insect cells using a baculovirus system in the same
way13 as for VEGF. Briefly, anesthesia was induced in male Alderley Park rats
using a-chloralose (iv) and then maintained with thiopentone (ip). The carotid
artery was cannulated to enable blood pressure recording using a pressure
transducer. The jugular vein was cannulated to allow VEGF or Ang-1
administration as a bolus injection in 0.85% sodium chloride. To give the
most comparable data the dose of Ang-1 used was adjusted to induce a
consistent blood pressure fall of between 10 and 20 mmHg to control for slight
7. Buttar, D.; Edge, M.; Emery, S. C.; Fitzek, M.; Forder, C.; Griffen, A.; Hayter, B.;
Hayward, C. F.; Hopcroft, P. J.; Luke, R. W. A.; Page, K.; Stawpert, J.; Wright, A.
Bioorg. Med. Chem. Lett. 2008, 18, 4723.
8. Luke, R. W. A.; Jones, C. D.; McCoull, W.; Hayter, B. R. WO Patent 2004013141,
2004.
differences in the level of functional Ang-1 across batches (40–60 lg of Ang-1
9. The phospo Tie-2 cell assay used in this work has been described previously.7
In the development of this assay we used an anti-Tie-2 antibody to show that,
unlike phospho-Tie-2, total Tie-2 levels were not reduced by a diverse set of
Tie-2 inhibitors.
depending on the batch). Compound 30 or vehicle alone [25% (w/v)
hydroxypropyl-b-cyclodextrin in Sorensons phosphate buffer (pH 5.5)] was
administered iv and blood pressure recorded. Consistent effects on blood
pressure were seen in repeat experiments.
10. Protein and crystals were obtained as follows: A baculovirus directing expression
of 6His-TEV-Tie2(802-1124)D964 N was generated using the Bac-to-Bac
method from Invitrogen. Protein was expressed in Sf9 insect cells infected
with high titre baculovirus at an MOI of 2, and cultured in SF900II media
(Invitrogen) for 48 h before harvesting by centrifugation and storage of the cell
pellets at À80 °C. Protein was purified by immobilized metal affinity and size
exclusion chromatography: frozen cell pellets were lysed by sonication in
13. Wedge, S. R.; Ogilvie, D. J.; Dukes, M.; Kendrew, J.; Chester, R.; Jackson, J. A.;
Boffey, S. J.; Valentine, P. J.; Curwen, J. O.; Musgrove, H. L.; Graham, G. A.;
Hughes, G. D.; Thomas, A. P.; Stokes, E. S. E.; Curry, B.; Richmond, G. H. P.;
Wadsworth, P. F.; Bigley, A. L.; Hennequin, L. F. Cancer Res. 2002, 4645.
14. Curwen, J. O.; Musgrove, H. L.; Kendrew, J.; Richmond, G. H. P.; Ogilvie, D.;
Wedge, S. R. Clin. Can. Res. 2008, 14, 3124.
15. CCP4 Acta Crystallogr., Sect. D 1994, D50, 760–763.
buffer
A
(50 mM Tris pH8, 300 mM NaCl, 5 mM DTT, containing Roche
16. QUANTA2000; Accelrys.
Complete EDTA-free protease inhibitor tablets), the lysate clarified by
centrifugation for 30 min at 48,000g, and the lysate supernatant stirred
overnight at 4 °C with Ni-NTA beads equilibrated with buffer A. The beads were
loaded into a column, washed with buffer A, then buffer A containing 50 mM
17. CNX Version 2000.1; Accelrys.
18. Refmac version 5.1.17: Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta
Crystallogr., Sect. D 1997, 53, 240.
19. Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.; Paciorek, W.;
Roversi, P.; Smart, O.; Vonrhein, C.; Womack, T. Buster Version 1.3.1; Global
Phasing Limited, 2008.
20. Coot; Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D 2004, 60, 2126.
21. Crystallographic statistics for the Tie2 K-compound 27 complex are as follows:
Space group C2221, unit cell 80.1, 108.8, 101.6 Å, resolution 2.95 Å, 9451
reflections from 49,960 observations give 96.0% completeness with Rmerge of
imidazole, and bound proteins eluted with buffer
A containing 500 mM
imidazole. The N-terminal His-tag was cleaved with rTEV protease, and the tag
removed by subtractive NiNTA chromatography. Pooled fractions containing
6His-TEV-Tie2(802-1124)D964 N were concentrated using a YM30 membrane
before loading on a Supderdex75 sizing column pre-equilibrated with buffer B
(20 mM HEPES pH 7.5, 300 mM NaCl, 5 mM DTT). Co-crystals with compound
27 were grown by incubating purified protein at 5 mg/ml with 1 mM
compound (1% DMSO) on ice for 30 min, and then setting up a hanging-drop
vapour diffusion experiment at 293 K using a reservoir of 5% (w/v) PEG6000, 5%
10% and mean I/r(I) of 3.4. The final model containing 2286 protein, 20 water,
and 28 compound atoms has an R-factor of 19.5% (Rfree using 5% of the data
25.8%). Mean temperature factors for the protein and the ligand are 42 and
46 Å2, respectively.
(v/v) MPD and 100 mM MOPS pH 7.5. The drop was composed of
5
lL
protein + compound, reservoir and additive (1 M glycine from
5
lL
1 lL