Optimization of the indolyl quinolinone class of KDR (VEGFR-2) kinase inhibitors: Effects of 5-amido- and 5-sulphonamido-indolyl groups on pharmacokinetics and hERG binding

Article abstract of DOI:10.1016/j.bmcl.2003.11.007

Modifications to the basic side-chain of early lead structures of the indolyl quinolinone class of KDR kinase inhibitors resulted in improved pharmacokinetic and ancillary profiles. Specifically, compounds bearing 5-amido- and 5-sulphonamido-indolyl substituents exhibited lower plasma clearance and weaker binding affinity for the I_Kr potassium channel hERG. 2003 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/j.bmcl.2003.11.007

Bioorganic & Medicinal Chemistry Letters 14 (2004) 351–355
Optimization of the indolyl quinolinone class of KDR (VEGFR-2)
kinase inhibitors: effects of 5-amido- and 5-sulphonamido-indolyl
groups on pharmacokinetics and hERG binding
Mark E. Fraley,* Kenneth L. Arrington, Carolyn A. Buser, Patrice A. Ciecko,
Kathleen E. Coll, Christine Fernandes, George D. Hartman, William F. Hoffman,
Joseph J. Lynch, Rosemary C. McFall, Keith Rickert, Romi Singh, Sheri Smith,
Kenneth A. Thomas and Bradley K. Wong
Departments of Medicinal Chemistry, Cancer Research, Pharmacology, and Drug Metabolism, Merck Research Laboratories,
West Point, PA 19486, USA
Received 11 September 2003; accepted 4 November 2003
Abstract—Modifications to the basic side-chain of early lead structures of the indolyl quinolinone class of KDR kinase inhibitors
resulted in improved pharmacokinetic and ancillary profiles. Specifically, compounds bearing 5-amido- and 5-sulphonamido-indo-
lyl substituents exhibited lower plasma clearance and weaker binding affinity for the IKr potassiumchannel hERG. #2003 Elsevier
Science Ltd. All rights reserved.
# 2003 Elsevier Ltd. All rights reserved.
Angiogenesis, the formation of new capillaries from
established blood vessels, is an essential process for
normal growth and development that has also been
implicated in the pathogenesis of several diseases
including diabetic retinopathy,1 rheumatoid arthritis,2
osteoarthritis,3 psoriasis,4 and cancer.5 Specifically, the
growth and metastasis of solid tumors has been shown
to be dependent on angiogenesis at an early stage,6
while tumors that lack adequate vascularization become
necrotic or apoptotic and do not grow beyond a limited
size.7 Interest in inhibition of angiogenesis as a new
approach for the treatment of cancer has led to the elu-
cidation of key underlying molecular mechanisms that
control the angiogenic process. Angiogenesis is regu-
lated by the expression of a variety of growth factors
including vascular endothelial growth factor (VEGF), a
selective mitogen for endothelial cells whose mitogenic
signaling is mediated through the receptor tyrosine
kinase KDR (VEGFR-2).8 Several lines of evidence
indicate that expression and signaling of VEGF are
critical for tumor angiogenesis. Among these, anti-
bodies against VEGF9 and its receptor KDR,10 as well
as small molecule inhibitors of KDR kinase activity,11
have been shown to inhibit angiogenesis in tumor
xenograft models. Importantly, bevacizumab (Avastin;
Genentech, Inc.), a chimeric anti-VEGF monoclonal
antibody, prolonged the survival of patients with colo-
rectal cancer when administered in combination with
standard chemotherapy, providing valuable proof-of-
concept in a clinical setting.12 KDR kinase inhibitors
derived froma number of different structural classes,
including indolinones (SU5416 and SU11248), quinazo-
lines (ZD4190 and ZD6474), phthalazines (PTK-787),
and isothiazoles (CP-547,632), have progressed to the
clinical evaluation stage.13
We recently reported the discovery and evolution of the
indolyl quinolinone class of KDR kinase inhibitors
(Fig. 1).14 Briefly, the benzoannulenone core of screen-
ing lead 1 was replaced with the more chemically stable
2-quinolinone ring systemof 2 to provide an 8-fold
improvement in KDR kinase activity,15 a gain that we
attributed to the additional hydrogen bonding interac-
tion formed between the backbone and the quinolinone
NH. Potency was further optimized by substitution of
the benzimidazole moiety with an indolyl nucleus.
* Corresponding author. Fax: +1-215-652-6345; e-mail: mark_fraley
@merck.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.007

352
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 14(2004) 351–355
Figure 1. Evolution of the indolyl quinolinone class.
Figure 2. Metabolic profile of 4 in human and dog liver microsomes.
Introduction of a basic side chain via an ether linkage at
the 5-position of the indolyl ring system, as in 4, pro-
vided compounds that exhibited improved physical
properties and excellent cellular activity.16
of KDR kinase, we turned our attention to the optimi-
zation of pharmacokinetics as well as the identification
of ancillary activity that might limit chronic oral dosing.
Compound 4 served as an assay and target validation
tool for our program. Oral dosing of 4 to HT1080
xenografted mice inhibited tumor growth in a dose-
proportional manner.17 Satisfyingly, tumor growth
inhibition correlated with systemic exposure, in vivo
inhibition of KDR phosphorylation in mouse lung, and
inhibition of tumor angiogenesis. Thus, with the identi-
fication of 4 as a potent and efficacious in vivo inhibitor
Compound 4 exhibited high plasma clearance in rat and
dog (Table 1). Human and dog liver microsome stability
studies indicated that although the relative stability
between the two species was similar, the metabolic pro-
file in each systemwas distinct ( Fig. 2). In dog liver
microsomes, N-oxidation predominated furnishing 5,
while in the human preparation N-demethylation was
the major metabolic pathway giving rise to 6. Encoura-
gingly, metabolism appeared primarily limited to the
side chain with only minor amounts of core oxidation
products detected. These results suggested that the core
was stable relative to the side chain and that modifi-
cations to the side chain might lead to reductions in
plasma clearance.
Table 1. Pharmacokinetics of 4 in rat and dog
Species
Cl
(mL/min/kg)
t1/2
(h)
Vdss
(L/kg)
Cmax
(nM)
%F
With regard to ancillary activity, 4 exhibited moderate
binding to the IKr potassiumchannel hERG (human
Ether-a-go-go Related Gene) with an inflection point (IP)
of 1.9 uM.18 Blockade of hERG has been implicated in
the drug-induced alteration of cardiac ventricular repo-
larization that is typically manifested in prolongation of
Rata
56
>50
1.4
<1.0
5.0
N.D.
470
N.D.
20
N.D.
Dogb
N.D., not determined
a Dosed 2 mpk iv (DMSO), 10 mpk po (1.0% methyl cellulose, pH 5).
b Dosed 0.25 mpk iv (DMSO) in a cocktail of six compounds.
Table 2. Properties of selected compounds of the ether series
Compd
R
KDR IC50 (nM)/
Cell Prolif IC50 (nM)
LogP
3.1
Dog Cl
(mL/min/kg)
t1/2 (h)
<1.0
hERG IP (nM)/
QTc EC10 (nM)
4a
7/8
4/4
>50
7.8
1950/1900
7b
8b
9a
3.2
0.9
3.7
2.2
3.2
1.3
520
8/41
4/17
7.7
3050/5200
980/640
>50
a Dosed 0.25 mpk iv (DMSO) in a cocktail of six compounds.
b Dosed 1.0 mpk iv (DMSO) as a single.

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 14(2004) 351–355
Table 3. Properties of selected compounds of the 5-amido series
353
Compd
R
KDR IC50 (nM)/
cell IC50 (nM)
LogP
2.6
Dog Cl
(mL/min/kg)
t1/2 (h)
hERG IP (nM)
10,800
10b
5/4
19
2.4
11a
5/4
5/4
1.7
27
26
38
4.2
1.1
1.7
>10,000
>10,000
13,700
12a (rac)
13a (rac)
N.D.
N.D.
6/53
a Dosed 0.25 mpk iv (DMSO) in a cocktail of six compounds.
b Dosed 1.0 mpk iv (DMSO) as a single.
Table 4. Properties of selected compounds of the 5-sulphonamido series
Compd
R
KDR IC50 (nM)/
cell IC50 (nM)
LogP
Dog Cl
(mL/min/kg)
t1/2 (h)
hERG IP (nM)
14b
10/31
11/30
16/27
14/97
3.0
0.5
3.5
11
6.4
12.7
3.4
3440
2450
2650
2835
15b
2.3
16a (rac)
17a (rac)
N.D.
N.D.
0.4
9.5
a Dosed 0.25 mpk iv (DMSO) in a cocktail of six compounds.
b Dosed 1.0 mpk iv (DMSO) as a single.
the heart rate-corrected QT interval (QTc).19 The ability
of 4 to bind to hERG was corroborated by an electro-
physiologic, rising-dose infusion study in anesthetized
dogs which demonstrated that 4 caused 10% QTc inter-
val prolongation at a plasma concentration of 1.9 uM.
Because prolongation of the QTc interval may potenti-
ate cardiac arrhythmia in patients and lead to torsades
de points or sudden death, we closely monitored hERG
binding affinity during our optimization efforts.
clearance and hERG activity compared to analogue 9,
perhaps as a result of its greater polarity.20 We hypo-
thesized at this juncture that further optimization of
pharmacokinetics and reduction in hERG binding affi-
nity would require a design strategy focused on increas-
ing the polarity of this inhibitor class. Initially, we chose
to investigate a 5-amido group as a linkage between the
indolyl core and requisite basic amine. Gratifyingly, this
modification resulted in more polar, equipotent com-
pounds that exhibited reduced binding affinity for
hERG (Table 3).
Attempts to optimize both plasma clearance and IKr
profile simultaneously in the ether series yielded only
limited success (Table 2). For example, although com-
pound 7 exhibited improved plasma clearance, the level
of hERG binding remained a potential issue for this
compound. Interestingly, compound 8 showed lower
While compounds 10–13 showed moderate to high
plasma clearance in dogs, the corresponding analogues
in the 5-sulphonamido series exhibited low clearance
and long half-lives (Table 4). However, the improve-

354
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 14(2004) 351–355
Acknowledgements
Table 5. Pharmacokinetics of 10 and 14 in rat, dog, and monkey
Compd Species Cl (mL/min/kg) t1/2 (h) Vdss (L/kg) Cmax (nM) %F
We thank Matt Zrada and Ken Anderson for logP
determinations and Elaine Walker for assistance in the
preparation of this manuscript.
10
14
10
14
10
14
rata
rata
3.8
29
19
0.5
3.4
12
2.3
1.1
2.4
6.4
2.8
3.4
0.7
0.9
2.9
0.3
0.9
2.1
5790
4800
160
5200
1250
630
31
54
15
63
46
40
dogb
dogb
monkeyb
monkeyb
References and notes
a Mesylate salt dosed 2 mg/kg iv (DMSO), 10 mg/kg po (0.05 M citric
acid).
b Mesylate salt dosed 1 mg/kg iv (DMSO), 1 mg/kg po (0.05 M citric
acid).
1. Adamis, A. P.; Shima, D. T.; Yeo, K. T.; Yeo, T. K.;
Brown, L. F.; Berse, B.; D’Amore, P. A.; Folkman, J.
Biochem. Biophys. Res. Commun. 1993, 193, 631.
2. Giatromanolaki, A.; Sivridis, E.; Athanassou, N.; Zois,
E.; Thorpe, P. E.; Brekken, R. A.; Gatter, K. C.; Harris,
A. L.; Koukourakis, I. M.; Koukourakis, M. I. J. Path.
2001, 194, 101.
3. Enomoto, H.; Inoki, I.; Komiya, K.; Shiomi, T.; Ikeda,
E.; Obata, K.; Matsumoto, H.; Toyama, Y.; Okada, Y.
Am. J. Pathol. 2003, 162, 171.
4. Detmar, M. Dermatol. Sci. 2000, 24, S78.
5. For reviews, see: (a) Carmeliet, P.; Jain, R. K. Nat. 2000,
407, 249. (b) Folkman, J. Nat. Med. 1995, 1, 27.
6. Zetter, B. R. Annu. Rev. Med. 1998, 49, 407.
7. (a) Hanahan, D.; Folkman, J. Cell 1996, 86, 353. (b)
Holmgen, L.; O’Reilly, M. S.; Folkman, J. Nat. Med.
1995, 1, 149.
8. (a) Veikkola, T.; Karkkainen, M.; Claesson-Welsh, L.;
Alitalo, K. Cancer Res. 2000, 60, 203. (b) Thomas, K. A.
J. Biol. Chem. 1996, 271, 603.
9. Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.;
Phillips, H. S.; Ferrara, N. Nature 1993, 362, 841.
10. Witte, L.; Hicklin, D.; Zhu, Z.; Pytowski, B.; Kotanides,
H.; Rockwell, P.; Bohlen, P. Cancer Metastasis Rev. 1998,
17, 155.
Figure 3. Synthesis of 14.
ment in pharmacokinetics with the 5-sulphoamido lin-
ker came at the expense of decreased KDR kinase
activity and greater binding affinity for hERG. The dif-
ference in hERG activity between the two series may be
related, in part, to their relative lipophilicity; the logP
values of sulphonamides 14 and 15 measured approxi-
mately one-half log unit higher than their direct amide
counterparts 10 and 11.
11. (a) Mendel, D. B.; Laird, A. D.; Xin, X.; Louie, S. G.;
Christensen, J. G.; Li, G.; Schreck, R. E.; Abrams, T. J.;
Ngai, T. J.; Lee, L. B.; Murray, L. J.; Carver, J.; Chan, E.;
¨
Moss, K. G.; Haznedar, J. O.; Sukbuntherng, J.; Blake,
R. A.; Sun, L.; Tang, C.; Miller, T.; Shirazian, S.;
McMahon, G.; Cherrington, J. M. Clin. Cancer Res.
2003, 327. (b) 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.; Gra-
ham, 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,
62, 4645. (c) Drevs, J.; Hofmann, I.; Hugenschmidt, H.;
The oral pharmacokinetic profiles for compounds 10
and 14 in rat, dog, and monkey are found in Table 5.
Sulphonamide 14 showed greater bioavailability in rat
and dog than did amide 10. That amide 10 exhibited
moderate plasma clearance relative to hepatic blood
flow in the dog suggested that the low bioavailability in
this species was due to a combination of incomplete
absorption and first-pass hepatic elimination.
Wittig, C.; Madjar, H.; Muller, M.; Wood, J.; Martiny-
¨
´
Baron, G.; Unger, C.; Marme, D. Cancer Res. 2000, 60,
4819.
12. Hurwitz, H.; Fehrenbacher, L.; Cartwright, T.; Hains-
worth, J.; Heim, W.; Berlin, J.; Griffing, S.; Novotny, W.;
Holmgren, E.; Kabbinavar, F. Proc. Am. Soc. Clin.
Oncol. 2003, 22; Abstract 3646.
13. For recent reviews, see: (a) Connell, R. D. Expert Opin.
Ther. Pat. 2002, 12, 1763. (b) Boyer, S. J. Curr. Top. Med.
Chem. 2002, 2, 973.
14. (a) Fraley, M. E.; Antanavage, J.; Arrington, K. L.;
Ciecko, P. A.; Coll, K. E.; Gibbs, J. B.; Hambaugh, S. R.;
Hartman, G. D.; Heimbrook, D. C.; Hoffman, W. F.;
Hungate, R. W.; Kohl, N. E.; Mao, X.; McFall, R. C.;
Rands, E.; Rickert, K.; Sepp-Lorenzino, L.; Shipman,
J. M.; Tebben, A. J.; Thomas, K. A. Presented at the
224th National Meeting of the American Chemical
Society, Boston, MA, August 2002; Abstract MEDI-221.
(b) Fraley, M. E.; Hoffman, W. F.; Arrington, K. L.;
Hungate, R. W.; Hartman, G. D.; McFall, R. C.; Coll,
The synthesis of 14 is depicted in Figure 3. Key steps
included the sulfonylation reaction of 1-(tri-
fluoroacetyl)indoline21 and the Suzuki cross-coupling
reaction between the intermediate 2-indolyl boronic
acid and 3-iodo-2-quinolinone22 to furnish the indolyl
quinolinone ring system.
In summary, exchange of the ether linkage in our first
generation KDR kinase inhibitors of the indolyl quino-
linone class for 5-amido and 5-sulfonamido tethers
provided potent compounds that exhibited more favor-
able pharmacokinetic properties and potentially safer
ancillary profiles with lower binding affinity for the IKr
potassiumchannel hERG.

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 14(2004) 351–355
355
K. E.; Rickert, K.; Thomas, K. A.; McGaughey, G. B.
Curr. Med. Chem., in press.
Mao, X.; Connolly, B.; Shipman, J.; Antanavage, J.; Hill,
S.; Davis, L.; Beck, S.; Rickert, K.; Coll, K.; Ciecko, P.;
Fraley, M.; Hoffman, W.; Hartman, G.; Heimbrook, D.;
Gibbs, J.; Kohl, K.; Thomas, K. Cancer Res., in press.
18. The hERG IP values were determined through binding
competition experiments using membrane preparations
from human embryonic kidney cells that constitutively
express hERG. For an overview of currently available
drug cardiotoxicity assays, see Netzer, R.; Ebneth, A.;
Bischoff, U.; Pongs, O. Drug Discov. Today 2001, 6, 78.
19. For a recent review, see: De Ponti, F.; Poluzzi, E.; Cavalli,
A.; Recanatini, M.; Montanaro, N. Drug Safety 2002, 25,
263.
15. The KDR IC50 value represents biochemical inhibition of
phosphorylation of poly-Glu/Tyr (4:1) peptide substrate
by isolated KDR kinase (cloned and expressed as a GST-
fusion protein): see, Kendall, R. L.; Rutledge, R. Z.; Mao,
X.; Tebben, A. J.; Hungate, R. W.; Thomas, K. A. J. Biol.
Chem. 1999, 274, 6453. Values are reported as the average
of at least two determinations. Standard deviations are
typically within 25% of the IC50 value.
16. The EC proliferation IC50 value represents the inhibition
of VEGF-stimulated mitogenesis as determined in human
umbilical vein endothelial cells. Refer to U.S. Patent
6,306,874 for assay details. Values are reported as the
average of at least two determinations.
20. Partition coefficients were determined by HPLC analysis
using the octanol/aqueous shake flask method.
17. (a) Sepp-Lorenzino, L.; Mao, X.; Rands, E.; Connolly,
B.; Antanavage, J.; Shipman, J.; Hoffman, W.; Fraley,
M.; Ciecko, P.; Gibbs, J.; Hartman, G.; Heimbrook, D.;
Kohl, N.; Thomas, K. Proc. Am. Assoc. Cancer Res. 2002,
43; Abstract 5346. (b) Sepp-Lorenzino, L.; Rands, E.;
21. Borror, A. L.; Chinoporos, E.; Filosa, M. P.; Herchen,
S. R.; Petersen, C. P.; Stern, C. A.; Onan, K. D. J. Org.
Chem. 1988, 53, 2047.
22. Marsais, F.; Godard, A.; Queguiner, G. J. Heterocycl.
Chem. 1989, 26, 1589.