Exploration of diverse hinge-binding scaffolds for selective Aurora kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.05.125

Four hinge-binding scaffolds have been explored for novel selective Aurora kinase inhibitors. The structure activity relationship, selectivity and pharmacokinetic profiles have been evaluated.

Full text of DOI:10.1016/j.bmcl.2012.05.125

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4528–4531
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of diverse hinge-binding scaffolds for selective Aurora
kinase inhibitors
⇑
Zhiqin Ji , Yujia Dai, Cele Abad-Zapatero, Daniel H. Albert, Jennifer J. Bouska, Keith B. Glaser,
Patrick A. Marcotte, Nirupama B. Soni, Terry J. Magoc, Kent D. Stewart, Ru-Qi Wei, Steve K. Davidsen,
Michael R. Michaelides
Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6100, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Four hinge-binding scaffolds have been explored for novel selective Aurora kinase inhibitors. The struc-
ture activity relationship, selectivity and pharmacokinetic profiles have been evaluated.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 9 May 2012
Revised 30 May 2012
Accepted 31 May 2012
Available online 7 June 2012
Keywords:
Hinge-binding scaffold
Aurora kinase inhibitor
Anticancer drug
Aurora kinases consisting of Aurora A, Aurora B and Aurora C
play critical roles during mitosis in chromosome segregation and
cell division.1–3 Aurora kinases are over-expressed in a variety of
human tumors and the increased expression correlates with ad-
vanced clinical progression in several tumor types.4–6 The inhibi-
tion of Aurora kinases is regarded as a promising approach for
the development of anticancer drugs.7–21 Several small molecule
Aurora selective inhibitors, such as VX-680(MK-0457),11
AZD-115212 and MLNM-823713 have demonstrated anti-tumor
efficacy in tumor models and advanced to clinical development.
In our previous work, we discovered that thienopyrimidine,
benzoisoxazole, benzoisothiazole, indazole and pyrrolotriazine
were hinge binding moieties in a program targeting KDR kinase
inhibitors.23,24 As a part of Aurora kinase program at Abbott Labora-
tories, amide-based thienopyrimidine (1) was employed as a favor-
able hinge-binding scaffold resulting in a series of potent Aurora A
and B inhibitors.22 As a continuation of this effort, we explored the
replacement of the thienopyrimidine core with other potential
hinge binding moieties. We anticipated that amide-based heterocy-
cles, namely benzoisoxazole (2), benzoisothiazole (3), indazole (4)
and pyrrolotriazine (5), were suitable scaffolds for Aurora inhibitors
(Fig. 1). The diverse hinge-binders provide us with more options to
optimize the potency, efficacy and physical properties.
lined in Scheme 1. The key intermediate iodides 6–8 were prepared
using literature reported procedures.23,24 Palladium-catalyzed car-
bonylation of 6–8 with carbon monoxide in a methanol solution
followed by saponification of generated esters 9–11 gave rise to
carboxylic acids 12–14. The anilines used for the last step were
either from commercial resources or prepared as previously
reported.22
Pyrrolotriazines (5) were synthesized via the route shown in
Scheme 2. Compound 15 was prepared using the literature proce-
dures25 and then converted to nitrile 16 through a two-step
reaction in one pot, via the corresponding oxime. N-amination of
16 followed by a cyclization reaction formed the intermediate
H
N
H
N
O
O
Ar
Ar
Ar
H2N
N
H2N
N
O
S
Ar
NH
Replacement of
NH2
O
S
Hinge binding Core
N
2
3
H
N
N
1
O
Ar
NH
O
H2N
N
NH2
N
The general synthesis of the amide-based inhibitors containing
benzoisoxazole (2), benzoisothiazole (3) and indazole (4) is out-
N
N
N
R
R= H, Me
4
5
⇑
Corresponding author.
E-mail address: zhiqin.ji@abbott.com (Z. Ji).
Figure 1. Rationale of inhibitor design.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.125

Zhiqin Ji et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4528–4531
4529
I
I
I
O
O
H2N
N
H2N
N
a,b
e,f
c,d
CHO
N
H
15
b
CN
I
a
N
H
16
X
X
6: X = O
9: X = O
7: X = S
NH2
I
10: X = S
8: X = NH, NMe
11: X = NH, NMe
N
g, h,
CN
O
N
N
NH2
17
H
O
OH
N
O
N
Ar
H2N
N
H2N
18
c
N
X
O
Ar
NH2
X
NH2
OH
NH
12: X = O
2: X = O
3: X = S
i
N
N
13: X = S
14: X = NH, NMe
N
N
N
N
4: X = NH, NMe
19
5
Scheme 1. Reagents and conditions: (a) CO, MeOH, PdCl2(dppf), Et3N; (b) LiOH,
MeOH–THF; (c) ArNH2, HATU, Et3N, DMF.
Scheme 2. Reagents and conditions: (a) NH2OH, pyridine; (b) Ac2O, 90 °C, 6 h; (c)
NaH, DMF RT; (d) Ph2PO(ONH2), RT; (e) HC(EtO)3, reflux 6 h ; (f) 7 M NH3 in MeOH;
(g) CO, MeOH, PdCl2(dppf), Et3N; (h) LiOH, MeOH, (i) Ar-NH2, HATU, Et3N, DMF.
pyrrolotriazine 18. Palladium-catalyzed carbonylation of 18 with
carbon monoxide in a methanol solution followed by saponifica-
tion of the generated ester gave rise to carboxylic acids 19, which
was then used in the last step for the synthesis of desired com-
pound 5.
The compounds with potent Aurora B inhibitory activity were
further evaluated for their ability to induce cellular nuclear poly-
ploidy,27 a phenotype associated with Aurora B kinase inhibition.28
These results are expressed as EC15 values and also included in Ta-
ble 1. In general, the cellular activity of these compounds reflected
well their enzymatic potency against Aurora B; many of these com-
pounds displayed single-digit or low double-digit nanomolar EC15
values in polyploidy induction.
After evaluating the benzoixozoles, we then looked briefly into
benzoisothioazoles and indazoles. Similar to the benzoisoxazole,
both benzoisothiazole and aminoindazole were as effective
hinge-binding scaffolds. Some representative examples are listed
in Table 3. For easy comparison, benzoisoxazoles 2j, 2k and 2l
are also included in Table 3. Benzoisothiazoles (3a–c) and inda-
zoles (4a–c) exhibited very similar potency against Aurora B as
their counterparts in the benzoisoxazole series (2j–l). The benzo-
isothiazole analogs are also very selective for Aurora B, having at
least 250-fold lower activity against Aurora A. Similar to the benz-
oisoxazoles 2j–l, benzoisothiazoles 3a–c also exhibited potent
activity in inducing cellular polyploidy. In general, the indazole
analogs while equipotent in the enzymatic assay are less active
in the cellular assay (4a and 4b vs 2j and 2l). Additionally, methyl-
ation of indazole NH led to significant deterioration of Aurora B po-
tency (4c and 4d).
We first synthesized a series of benzoisoxazole amide analogs
and evaluated their enzymatic activity against Aurora A and Aurora
B in the presence of 1 mM ATP using a homogenous time-resolved
fluroscence (HTRF) assay.26 As shown in Table 1, substitution of
benzoisoxazole for thienopyrimidine was generally well tolerated
in terms of Aurora B kinase inhibition and a series of benzoisoxaz-
ole amide-based ureas were identified as potent Aurora B inhibi-
tors. One carbon homologated meta ureas (2e–2l) are markedly
more potent than the para diphenyl ureas (2a–2d) against Aurora
B kinase inhibition. Among the four direct matched pairs included
in Table 1, compounds 2e, 2f, 2g, and 2h are 7-, 11-, 20- and 46-
fold more potent than 2a, 2b, 2c, and 2d, respectively. One notable
feature of this series of compounds is their selectivity for Aurora B
over Aurora A. All compounds listed in the Table 1 except for 2a
(Aurora A activity not available) are much more potent against
Aurora B than against Aurora A, with the selectivity ranging from
26-fold for 2b to over 1000-fold for 2k.
Consistent with the results observed in the thienopyrimidine
series, the urea link is important for potency.22 Replacement of
the urea link with either a sulfonamide (2n) or an amide (2o, 2p)
led to significant loss in Aurora B potency (Table 2).
Table 1
Enzymatic and cellular activities of benzoisoxazole urea analogs
H
N
H
N
H
N
R
H
O
O
N
H2N
N
O
H2N
N
H
N
H
N
R
O
O
O
Structure A
Structure B
Compd
Structure
R
IC50, nM
AurA/AurB
Polyploidy (EC15, nM)
AurB
AurA
2a
2b
2c
2d
2e
2f
2g
2h
2i
A
A
A
A
B
B
B
B
B
B
B
B
H
p-F
p-Me
p-Cl
H
127
480
82
140
18
41
4
Not tested
>12,500
>12,500
>12,500
3790
12,200
586
166
>12,500
3180
12,500
4780
Not tested
Not tested
Not tested
Not tested
47
30
1
12
124
3
>26
>152
>89
210
298
146
55
227
54
>1000
696
p-F
p-Me
p-Cl
o-F
m-F
p-CF3
3
55
59
12
7
2j
2k
2l
13
15
3.4-di-F

4530
Zhiqin Ji et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4528–4531
Table 2
Table 4
Enzymatic activity of benzoisoxazole non-urea analogs
Enzymatic and cellular activities of pyrrolotriazines
H
HN
O
R
HN
O
R
H
N
O
N
HN
H2N
N
O
O
R
NH2
NH
NH2
O
NH
N
N
Compd
R
IC50, nM
N
N
N
N
AurB
525
AurA
2m
2n
NH2
>12,500
Structure A
Structure B
H
N
Compd Structure
R
IC50, nM
AurB AurA
AurA/AurB
Polyploidy
(EC15, nM)
>12,500
>12,500
S
O
O
5a
5b
5c
5d
5e
5f
A
A
A
A
A
B
B
B
p-Cl-Ph
p-CF3-Ph
p-Me-Ph
m-F-Ph
m-Cl-Ph
m-Me-Ph
m-F-Ph
p-F-Ph
0.9
24
1
4
10
38
9
46
786
46
51
33
46
16
19
4.5
1
6
1
2
2
9
3
16
O
2o
2p
450
>12,500
>12,500
N
H
F
67
O
186
171
NA
88
>12,500
N
H
F
5g
5h
36
2.4
1.1
5i
5j
B
B
33
17
37
8
15
6
Table 3
S
0.5
Enzymatic and cellular activities of compounds with different scaffolds
H
N
O
H
N
H
N
H2N
N
R
O
X
Compd
X
R
IC50, nM
AurA/AurB Polyploidy (EC15, nM)
AurB AurA
2j
2k
2l
3a
3b
3c
4a
4b
4c
4d
O
O
O
S
S
S
m-F
p-CF3
3,4-di-F
m-F
p-CF3
3,4-di-F
m-F
3,4-di-F
59
12
7
3180
12,500 1041
4870
12,500
12,500 1042
12,500
1279
1623
12,500
12,500
54
3
13
15
18
18
21
111
143
696
250
50
12
36
18
28
938
172
347
71
58
13
73
NH
NH
NMe m-F
Not tested
Not tested
NMe p-CF3
Based on the similarity between pyrrolotriazine and thienopyr-
imidine, the amide ureas of pyrrolotriazine were also expected to
be active against Aurora kinases. Similar to thienopyrimidine
amide ureas but unlike the bezoisoxazoles and benzoisothiazoles,
these pyrrolotriazine analogs are potent inhibitors of both Aurora
A and B kinases (Table 4). Consistent with their Aurora B enzymatic
activity, many of these pyrrolotriazine amide ureas effectively in-
duce cellular polyploidy at nanomolar concentrations.
Compounds 2j and 5g were selected for modeling analysis, and
the proposed binding orientations are shown in Figure 2.29 Both
bind to the ‘inactive’ confirmation of Aurora B kinase, with the ex-
pected canonical hinge hydrogen bonds to Glu 155 and Ala 157. An
intramolecular hydrogen bond between the amino group and the
amide carbonyl that is present in both hinge heterocycles (benzois-
oxazole and pyrrolotriazine, respectively) likely positions the di-
phenyl urea portion of the molecule into the back pocket. The
conserved Lys residue, Lys 106, can donate a hydrogen bond to
the inhibitor urea carbonyl. A hydrophobic pocket surrounds the
terminal meta-fluoro-phenyl group of each inhibitor. Overlay of
Aurora A and B structures indicates that one amino acid sequence
difference is found in this hydrophobic pocket: Ile 113 in Aurora
B/Leu 169 in Aurora A. The model indicates that differential occu-
pation of this pocket and interaction with this varying residue
Figure 2. (a). Model of compound 2j bound to Aurora B kinase (top picture).
Hingehydrogen bonds to Glu 155 backbone C@O and Ala 157 N–H are shown with
black dashed lines. An intramolecular H-bond between the amine and amide carbonyl
exocyclic on the benzoisoxazole is shown. Lys 106 (the ‘conserved’ Lys) donates an H-
bond to the urea carbonyl of the ligand. The fluorophenyl group projects into a
hydrophobic pocket comprised of Leu 108, Ile 113, and Leu 122 in Aurora B kinase,
corresponding to Leu 164, Leu 169, and Leu 178 in Aurora A kinase. Ile 113 is
highlighted as it shows a point of sequence variation between the two kinases. (b)
Model of compound 5g with features highlighted as in (a) (bottom picture).

Zhiqin Ji et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4528–4531
4531
Table 5
5. Zeng, W. F.; Navaratne, K.; Prayson, R. A.; Weil, R. J. J. Clin. Pathol. 2007, 60, 218.
6. Lee, E. C. Y.; Frolov, A.; Li, R.; Ayala, G.; Greenberg, N. M. Cancer Res. 2006, 66,
4996.
7. Gritsko, T. M.; Coppola, D.; Paciga, J. E.; Yang, L.; Sun, M.; Shelley, S. A.; Fiorica, J.
V.; Nicosia, S. V.; Cheng, J. Q. Clin. Cancer Res. 2003, 9, 1420.
8. Carvajal, R. D.; Tse, A.; Schwartz, G. K. Clin. Cancer Res. 2006, 12, 6869.
9. Keen, N.; Taylor, S. Nat. Rev. Cancer 2004, 4, 927.
10. Fancelli, D.; Moll, J. Expert Opin. Ther. Patents 2005, 15, 1169.
11. Bebbington, D.; Binch, H.; Charrier, J.-D.; Everitt, S.; Fraysse, D.; Golec, J.; Kay,
D.; Knegtel, R.; Mak, C.; Mazzei, F.; Miller, A.; Mortimore, M.; O’Donnell, M.;
Patel, S.; Pierard, F.; Pinder, J.; Pollard, J.; Ramaya, S.; Robinson, D.; Rutherford,
A.; Studley, J.; Westcott, J. Bioorg. Med. Chem. Lett. 2009, 19, 3586.
12. Mortlock, A. A.; Foote, K. M.; Heron, N. M.; Jung, F. H.; Pasquet, G.; Lohmann, J.-
J. M.; Warin, N.; Renaud, F.; Savi, C. D.; Roberts, N. J.; Johnson, T.; Dousson, C. B.;
Hill, G. B.; Perkins, D.; Hatter, G.; Wilkinson, R. W.; Wedge, S. R.; Heaton, S. P.;
Odedra, R.; Keen, N. J.; Crafter, C.; Brown, E.; Thompson, K.; Brightwell, S.;
Khatri, L.; Brady, M. C.; Kearney, S.; McKillop, D.; Rhead, S.; Parry, T.; Green, S. J.
Med. Chem. 2007, 50, 2213.
Kinome selectivity profile of compound 5ga
Kinase
IC50 (nM)
Kinase
IC50 (nM)
Aur A
Aur B
Alk1
Camk4
Chk1
EGFR
Erk2
<3
<3
Jak2
Jak3
KDR
lck
Jnk1
c-Met
Plk4
P38
>10,000
8140
1180
>10,000
>10,000
>10,000
2090
296
1100
5340
>10,000
340
566
>10,000
>10,000
<3
>10,000
>10,000
>10,000
1870
Flt3
Gsk3a
IGF1R
Irak4
Rock2
Src
TrkA
a
Assay was done in the presence of 10 lM ATP.
13. Qi, W.; Cooke, L. S.; Liu, X.; Rimsza, L.; Roe, D. J.; Manziolli, A.; Persky, D. O.;
Miller, T. P.; Mahadevan, D. Biochem. Pharmacol. 2011, 81, 881.
14. Pollard, J. R.; Mortimore, M. J. Med. Chem. 2009, 52, 2629.
Table 6
Mouse pharmacokinetic profiles of selected compounds
15. Carpinelli, P.; Ceruti, R.; Giorgini, M. L.; Capprlla, P.; Gianellini, L.; Croci, V.;
Degrassi, A.; Texido, G.; Rocchetti, M.; Vianello, P.; Rusconi, L.; Storici, P.;
Zugnoni, P.; Arrigoni, C.; Soncini, C.; Alli, C.; Patton, V.; Marsiglio, A.; Ballinari,
D.; Pesenti, E.; Fancelli, D.; Moll, J. Mol. Cancer Ther. 2007, 6, 3158.
16. Oslob, J. D.; Heumann, S. A.; Yu, C. H.; Allen, D. A.; Baskaran, S.; Bui, M.;
Delarosa, E.; Fung, A. D.; Hashash, A.; Hau, J.; Ivy, S.; Jacobs, J. W.; Lew, W.;
Maung, J.; McDowell, R. S.; Ritchie, S.; Romanowski, M. J.; Silverman, J. A.; Yang,
W.; Zhong, M.; Fuchs-Knotts, T. Bioorg. Med. Chem. Lett. 2009, 19, 1409.
17. Prime, M. E.; Courtney, S. M.; Brookfield, F. A.; Marston, R. W.; Walker, V.;
Warne, J.; Boyd, A. E.; Kairies, N. A.; Von der Saal, W.; Limberg, A.; Georges, G.;
Engh, R. A.; Goller, B.; Rueger, P.; Rueth, M. J. Med. Chem. 2011, 54, 312.
18. Kerekes, A. D.; Esposite, S. J.; Doll, R. J.; Tagat, J. R.; Yu, T.; Xiao, Y.; Zhang, Y.;
Prelusky, D. B.; Tevar, S.; Gray, K.; Terracina, G. A.; Lee, S.; Jones, J.; Liu, M.;
Basso, A. D.; Smith, E. B. J. Med. Chem. 2011, 54, 201.
Compd
iv (3 mg/kg)
Cl (L/h kg)
po (10 mg/kg)
AUC (lM h/mL)
F (%)
Vd (L/kg)
td (h)
2j
2k
2l
0.54
0.23
0.96
0.53
0.13
0.39
0.7
1.3
1.7
5
61
8
28
77
31
might provide the basis for the selective binding observed for these
compounds. A more detailed analysis at the atomic level will re-
quire crystallographic studies.
19. VanderPorten, E. C.; Taverna, P.; Hogan, J. N.; Ballinger, M. D.; Flanagan, W. M.;
Fucini, R. V. Mol. Cancer. Ther. 2009, 8, 930.
In general these compounds are highly selective for Aurora
kinases. The selectivity is exemplified by the inhibition profile of
compound 5g against a selected panel of kinases as shown in Table 5.
A number of selected benzoisoxazole amide ureas were evalu-
ated for their mouse pharmacokinetic profiles (Table 6). In general,
these benzoisoxaole amide ureas possess a low plasma clearance
after iv administration and good oral bioavailability, ranging from
28% for 2j to 77% for 2k. Pharmacokinetic evaluation of pyrrolotri-
azines was done employing an oral cassette dosing protocol. Com-
pound 5a–c and 5e were dosed in one cassette at 10 mpk, resulting
20. Rawson, T. E.; Rüth, M.; Blackwood, E.; Burdick, D.; Corson, L.; Dotson, J.;
Drummond, J.; Fields, C.; Georges, G. J.; Goller, B.; Halladay, J.; Hunsaker, T.;
Kleinheinz, T.; Krell, H.-W.; Li, J.; Liang, J.; Limberg, A.; McNutt, A.; Moffat, J.;
Phillips, G.; Ran, Y.; Safina, B.; Ultsch, M.; Walker, L.; Wiesmann, C.; Zhang, B.;
Zhou, A.; Zhu, B.-Y.; Rüger, P.; Cochran, A. G. J. Med. Chem. 2008, 51, 4465.
21. Belanger, D. B.; Curran, P. J.; Hruza, A.; Voigt, J.; Meng, Z.; Mandal, A. K.;
Siddiqui, M. A.; Basso, A. D.; Gray, K. Bioorg. Med. Chem. Lett. 2010, 20, 5170.
22. McClellan, W. J.; Dai, Y.; Abad-Zapatero, C.; Albert, D. H.; Bouska, J. J.; Glaser, K.
B.; Magoc, T. J.; Marcotte, P. A.; Osterling, D. J.; Stewart, K. D.; Davidsen, S. K.;
Michaelides, M. R. Bioorg. Med. Chem. Lett. 2011, 21, 5620.
23. Ji, Z.; Ahmed, A. A.; Albert, D. H.; Bouska, J. J.; Bousquet, P. F.; Cunha, G. A.; Diaz,
G.; Glaser, K. B.; Guo, J.; Harris, C. M.; Li, J.; Marcotte, P. A.; Moskey, M. D.; Oie,
T.; Pease, L.; Soni, N. B.; Stewart, K. D.; Davidsen, S. K.; Michaelides, R. M. J. Med.
Chem. 2008, 51, 1231.
24. Dai, Y.; Hartandi, K.; Ji, Z.; Ahmed, A. A.; Albert, D. H.; Bauch, J. L.; Bouska, J. J.;
Bousquet, P. F.; Cunha, G. A.; Glaser, K. B.; Harris, C. M.; Hickman, D.; Guo, J.; Li,
J.; Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Martin, R. L.; Olson, A. M.;
Osterling, D. J.; Pease, L. J.; Soni, N. B.; Stewart, K. D.; Stoll, V. S.; Tapang, P.;
Reuter, D. R.; Davidsen, S. K.; Michaelides, M. R. J. Med. Chem. 2007, 50, 1584.
25. Ghosez, L.; Franc, C.; Denonne, F.; Cuisinier, C.; Touillaux, R. Can. J. Chem. 2001,
79, 1827.
in a good oral exposure AUC of 112, 185, 9.3 and 20.6
respectively.
lM h/mL,
Additionally, compound 2l was evaluated for its in vivo target
modulation through inhibition of Histone 3 phosphorylation in
leukemia engrafted mouse model. 87% inhibition of H3 phosphor-
ylation was observed in 4 h after 50 mpk IP dosing.
Selected compounds were evaluated in vivo in tumor growth
inhibition models. However, the overall safety and efficacy profile
did not compare favorably with other series of inhibitors devel-
oped in our laboratories.30
26. To determine the activity of Aurora A and B kinases, a homogenous time-
resolved fluorescence (HTRF) in vitro kinase assay was used: (a) Mathis, G.
HTRF(R) Technol. J. Biomol. Screen. 1999, 4, 309; (b) Kolb, A. J.; Kaplita, P. V.;
Hayes, D. J.; Park, Y-W; Pernell, C.; Major, J. S.; Mathis, G. Drug Discovery Today
1998, 3, 333.
In summary, we have prepared a series of potent Aurora inhib-
itors based on a heterocycle amide linked diaryl urea motif by suc-
cessfully replacing the previously disclosed thienopyrimidine core
with alternative hinge-binding moieties. The benzoisoxazole, ben-
zoisothiazole and indazole series show potent Aurora B inhibition,
while the pyrrotriazines potently inhibit both Aurora A and B.
Additionally all series, with the exception of the indazoles are po-
tent Aurora B cell inhibitors as measured by their ability to effec-
27. To measure the induction of polyploidy: NCI-H1299 were seeded (4 K/well)
into 96-well culture plates (tissue culture grade, black, flat-clear bottom) and
incubated overnight to produce cell-to-plate adherence. Test drugs at varying
concentrations were added into duplicate wells containing cells and culture
media (RPMI 1640, 10% fetal calf serum) and incubated at 37 °C for 48 h. The
plates were then washed with PBS and the adherent cells fixed by incubating
with 3% formalin for 1 h. After washing four times with PBS, the cells were then
stained with Hoechst and subjected to fluorescent (360i/460e) microscopic
high content analysis to determine the effect of test drug on nuclear size.
Polyploid cells (P4 N) were defined as those having nuclear area >750 l2. Test
drug potency was expressed as the concentration of drug necessary to induce
polyploidy in 15% of cells (EC15) and was calculated from least squares
analysis of the log dose-response.
tively induce polyploidy. Selected benzoisoxazoles display
a
favorable pharmacokinetic profile with good oral biobioavailability
in mice.
28. Hauf, S.; Cole, R. W.; LaTerra, S.; Zimmer, C.; Schnapp, G.; Walter, R.; Heckel. A.,
van Meel. J.; Rieder, C. L.; Peters, J.-M. J. Cell Biol. 2003, 161, 281.
29. Energy minimized structures of compounds 2j and 5g were dockedinto a
homology model of human Aurora B. The model was built using PDB entry
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