Design, synthesis, and evaluation of indolinones as triple angiokinase inhibitors and the discovery of a highly specific 6-methoxycarbonyl-substituted indolinone (BIBF 1120)

Article abstract of DOI:10.1021/jm900431g

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a newtreatment modality in oncology. Preclinical findings suggest that blockade of additional pro-angiogenic kinases, such as fibroblast and platelet-derived growth factor receptors (FGFR and PDGFR), may improve the efficacy of pharmacological cancer treatment. Indolinones substituted in position 6 were identified as selective inhibitors of VEGF-, PDGF-, and FGF-receptor kinases. In particular, 6-methoxycarbonyl-substituted indolinones showed a highly favorable selectivity profile. Optimization identified potent inhibitors of VEGF-related endothelial cell proliferation with additional efficacy on pericyctes and smooth muscle cells. In contrast, no direct inhibition of tumor cell proliferation was observed. Compounds 2 (BIBF 1000) and 3 (BIBF 1120) are orally available and display encouraging efficacy in in vivo tumor models while being well tolerated. The triple angiokinase inhibitor 3 is currently in phase III clinical trials for the treatment of nonsmall cell lung cancer.

Full text of DOI:10.1021/jm900431g

4466 J. Med. Chem. 2009, 52, 4466–4480
DOI: 10.1021/jm900431g
Design, Synthesis, and Evaluation of Indolinones as Triple Angiokinase Inhibitors and the Discovery of a
Highly Specific 6-Methoxycarbonyl-Substituted Indolinone (BIBF 1120)†
,‡
‡
§
‡
‡
^
€
Gerald J. Roth,* Armin Heckel, Florian Colbatzky, Sandra Handschuh, Jorg Kley, Thorsten Lehmann-Lintz, Ralf Lotz,
Ulrike Tontsch-Grunt,# Rainer Walter,‡ and Frank Hilberg#
§
‡Department of Chemical Research and Department of Non-Clinical Drug Safety and Department of Lead Discovery and ^Department of Drug
Discovery Support, Boehringer Ingelheim Pharma GmbH & Co. KG, D-88397 Biberach, Germany, and #Boehringer Ingelheim RCV GmbH & Co.
KG, A-1121 Vienna, Austria
Received April 3, 2009
Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF)
signaling pathway is a new treatment modality in oncology. Preclinical findings suggest that blockade of
additional pro-angiogenic kinases, such as fibroblast and platelet-derived growth factor receptors
(FGFR and PDGFR), may improve the efficacy of pharmacological cancer treatment. Indolinones
substituted in position 6 were identified as selective inhibitors of VEGF-, PDGF-, and FGF-receptor
kinases. In particular, 6-methoxycarbonyl-substituted indolinones showed a highly favorable selectivity
profile. Optimization identified potent inhibitors of VEGF-related endothelial cell proliferation with
additional efficacy on pericyctes and smooth muscle cells. In contrast, no direct inhibition of tumor cell
proliferation was observed. Compounds 2 (BIBF 1000) and 3 (BIBF 1120) are orally available and
display encouraging efficacy in in vivo tumor models while being well tolerated. The triple angiokinase
inhibitor 3 is currently in phase III clinical trials for the treatment of nonsmall cell lung cancer.
Introduction
wide variability in response to treatment with antiangiogenic
drugs has been observed, both between patients and between
tumor types. Clearly, the development of drugs that simulta-
neously target several pro-angiogenic receptors (angioki-
nases) at the same time, while retaining a favorable overall
kinase selectivity profile, represents a promising avenue to-
ward a more effective and well tolerated cancer treatment.
In this paper, we report the identification of substituted
indolinones as inhibitors of VEGFR-2 kinase. Optimization
of an initial lead 1 led to 2 (BIBF 1000) and 3 (BIBF 1120)
(Chart 1), compounds targeting VEGFR, FGFR, and
PDGFR receptors with low cross-reactivity in the human
kinome. The triple angiokinase inhibitor 3 is currently in
phase III clinical trials in nonsmall cell lung cancer.8
The concept of tumor angiogenesis inhibition, interfering
with tumor blood vessel formation, as a new approach for
treating cancer was first proposed almost four decades ago.1
Several drugs targeting the tumor vasculature, notably the
monoclonal antibody to vascular endothelial growth factor
(VEGF) bevacizumab,2 as well as the small-molecule VEGF
receptor (VEGFRa) inhibitors sorafenib3 and sunitinib,4 have
been successfully introduced into clinical practice during the
past years. The pivotal role of VEGFR signaling in the
formation of blood vessels and its role in disease-associated
angiogenesis is well documented.5 Other signaling molecules,
including platelet-derived growth factor (PDGF) and its
receptors (PDGFR), are additional mediators of blood vessel
formation and stabilization via pathways that control the
proliferation and survival of perivascular cells such as peri-
cytes and smooth muscle cells.6 A potential escape mechan-
ism, whereby tumor cells switch from VEGF to FGF
(fibroblast growth factor) signaling upon sustained VEGFR
inhibition has been described recently.7 Although tumor
angiogenesis is an essential mechanism in all solid cancers, a
Chemistry
The compounds described in Tables 1 and 2 were typically
synthesized starting from the corresponding indolinones and
aromatic amines as building blocks. Because indolinones
substituted in position 6 are rarely described in literature,
different routes of synthesis had to be developed by adapting
known procedures, as exemplified for compounds 14-17 in
Scheme 1. The particular synthetic path was based on the
accessibility of the starting materials in each case. Aromatic
nitration of phenyl-acetic acids followed by reduction of the
nitro group in acidic media with subsequent ring closure
offered an easy approach, as shown for compound 14. Alter-
natively, the acetic acid moiety could be introduced by
vicarious nucleophilic substitution9 followed by reduction,
optional removal of protecting groups and subsequent ring
closure, as seen for compounds 15 and 16. Should the
respective 2-nitro halo benzenes be available, introduction
of malonic ester equivalents followed by decarboxylation was
*To whom correspondence should be addressed. Phone: +49-7351-540.
Fax: +49-7351-540. E-mail: gerald.roth@boehringer-ingelheim.com.
†The atomic coordinates for the X-ray structure of compound 3 have
been deposited in the Protein Data Bank: PDB code 3C7Q.
aAbbreviations: VEGFR, vascular endothelial growth factor
receptor; FGFR, fibroblast growth factor receptor; PDGFR, platelet-
derived growth factor recptor; CDK, cyclin-dependent kinase; HUVEC,
human umbilical vein endothelial cell; IGFR, insulin-like growth factor
receptor; InsR, insulin receptor; EGFR, epidermal growth factor
receptor; HER, human epidermal growth factor receptor; Plk, polo-like
kinase; HNSCC, head and neck squamous cell carcinoma; TBTU,
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate;
HOBT, hydroxy-benzotriazole.
r
pubs.acs.org/jmc
Published on Web 06/12/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4467
Table 1. VEGFR-2/HUVEC Inhibition of 6-Substituted Indolinones
Chart 1. VEGFR-2 Hit Compound 1 and Clinical Candidates 2
(BIBF 1000) and 3 (BIBF 1120)
VEGFR-2
IC50 (nM) a
HUVEC/VEGF
EC50 (nM) a
compd
R1
NO2
35
45
34
36
46
37
1
7 ( 9
36 ( 36
109 ( 3
60 ( 10
103 ( 11
47 ( 21
49 ( 54
414 ( 323
281 ( 98
342 ( 176
585 ( 156
1070 ( 565
542 ( 310
718 ( 165
NT b
COOMe
COOEt
Cl
129 ( 78
132 ( 34
248 ( 121
763 ( 198
791 ( 349
1230 ( 620
1312 ( 719
1447 ( 1751
1752 ( 433
2099 ( 1647
2112 ( 1215
41000
NH2
CN
CONH2
pyrrol-1-yl
CONHiPr
CONMe2
CONEtMe
COCH3
CONHCH3
H
48
41
42
43
38
44
39
40
47
1907 ( 806
NT b
COOH
NHCOCH3
NT b
43000
NT b
a Values are averages ( SD of at least three independent determina-
tions. Values “greater than” indicate that half-maximum inhibition was
not achieved at the highest concentration tested. b Not tested.
the method of choice,10 as exemplified for compound 17. The
anilines or cycloalkyl amines used as building blocks in
Scheme 3 were available by standard chemistry summarized
in Scheme 2 starting from commercially available precursors.
The majorityof the finalcompounds weresynthesizedusing
a two- or three-step sequence: N-acetylation of the corre-
sponding indolinones11 (22-25) activated the 3-position for
subsequent condensation with aryl ortho-esters.12 Both steps
couldbe combined in a practicalone-potsequence usingacetic
anhydride as a solvent (26-28). For large-scale synthesis,
however, a stepwise procedure is recommended, facilitating
workup due to the cleaner reaction progress (29-33). The
amino side chains were introduced in the final step by addition
and subsequent elimination of alcohol,13 followed by in situ
acetyl cleavage (2, 3, 34-39, 49-63). The double-bond geo-
metry in the final compounds is locked in a Z-conformation
due to an intramolecular hydrogen bond, as could clearly be
detected in the respective NMR spectra (upfield shift of the
proton in position 4 of the indolinone core due to magnetic
shielding by the central aryl group, see also ref 13). Because no
shielding was visible in the NMR spectra of intermediates 26-
33, the double bond seemed to adopt the E-conformation in
these compounds.
Several additional analogues were prepared with modifica-
tions at the C-6 position (Scheme 4). Amides and esters 1 and
41-45 were obtained by ester cleavage of 34 and subsequent
amide coupling or ester formation. Compounds 47 and 48
were accessiblebyacetylationor condensationreactionsof the
6-amino substituted indolinone 46, available by reduction of
35 with Raney nickel.
In general, most compounds with basic side chains dis-
played good aqueous solubility at pH 4.0 and moderate
solubility at higher pH values (e.g., for compound 2: 690
mg/mL at pH 4.0 and 22 mg/mL at pH 6.0, free base).
Results and Discussion
When evaluating compounds from our CDK4 kinase in-
hibitor project in a set of kinase selectivity assays, the 6-
amido-substituted indolinone 1 was identified as a nano-
molar inhibitor of VEGFR-2 (IC50=763 nM). Interestingly,
this compound was completely devoid of CDK4 inhibition, in
contrast to the related 5-amido substituted derivatives.14 In
addition, no other kinase included in the selectivity evaluation
was inhibited (IGF1R, InsR, CDK1, CDK2, CDK4, EGFR,
HER2, Plk1: IC50 4 10 μM).15 Compound 1 was therefore
chosen as an interesting starting point, complementing activ-
ities from a VEGFR-2 HTS campaign.
In a first attempt to evaluate the suitability of this structural
class for further investigation, a couple of derivatives with
modified basic groups were synthesized by introducing a
diverse set of anilines. Unfortunately, all initial derivatives
were less active than the hit compound (structures not shown),
showing that the 6-amido-substituted indolinone core might
not yet be optimal for improving the potency. Compound 1
was, however, more potent than the corresponding unsubsti-
tuted indolinone 39 and showed a favorable selectivity profile
when compared with 39, which inhibits CDK4, InsR, and
IGF1R in the same selectivity panel.15 We decided to use
computational modeling to explore the binding mode of 1 and
especially the role of the 6-amido group in more detail.16
Because indolinone-type kinase inhibitors had been reported
as ATP pocket binders before,10,13 1 was positioned in a similar
way in the homology model of the VEGFR-2 kinase
domain showing the typical canonical hydrogen bonds
between the lactam moiety and the hinge region (Figure 1A).

4468 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Roth et al.
Table 2. Inhibitory Profile of 6-Methoxycarbonyl-Substituted Indolinones
IC50 (nM)
EC50 (nM)
compd
R2
VEGFR-2 a FGFR-1b PDGFRRb VEGFR-1 b VEGFR-3 b HUVEC/VEGF a
3
4-(NCH3)COCH2-(4-methyl-piperazin-1-yl)
4-(NCH3)COCH2-(imidazol-1-yl)
4-(NCOCH3)CH2CONMe2
4-(NCOCH3)(CH2)3NMe2
4-(NCOCH3)(CH2)2NMe2
4-(NSO2CH3)(CH2)2NMe2
4-CO(NCH3)(CH2)2NMe2
4-CH2(piperidin-1-yl)
5 ( 2
6 ( 4
8 ( 4
9 ( 6
38 ( 4
66 ( 1
54 ( 4
17 ( 4
18 ( 3
25 ( 5
88 ( 8
71 ( 20
50 ( 4
82 ( 6
219 ( 3
NT c
18 ( 0.1
8 ( 0.1
9 ( 1
104 ( 14
55 ( 10
61 ( 6
28 ( 1
35 ( 8
69 ( 11
325 ( 45
146 ( 13
82 ( 7
173 ( 73
278 ( 50
NT c
5 ( 1
7 ( 1
6 ( 1
3 ( 1
5 ( 0.3
10 ( 1
59 ( 4
32 ( 5
10 ( 0.4
73 ( 26
24 ( 5
NT c
10 ( 13
52 ( 19
37 ( 16
15 ( 5
15 ( 9
14 ( 9
25 ( 8
103 ( 11
22 ( 11
28 ( 11
83 ( 45
88 ( 64
NT c
49
50
51
52
53
54
45
2
7 ( 1
12 ( 4
8 ( 0.3
12 ( 1
11 ( 0.2
54 ( 9
20 ( 2
28 ( 3
164 ( 13
NT c
23 ( 10
24 ( 17
36 ( 36
61 ( 17
64 ( 44
83 ( 42
119 ( 82
150 ( 11
245 ( 43
248 ( 61
∼8000
4-(NCH3)COCH2NMe2
4-CH2NMe2
55
56
57
58
59
60
61
62
63
4-NHCOCH2-(4-methyl-piperazin-1-yl)
4-NHCOCH2NMe2
4-(NCH3)SO2Me
NT c
NT c
NT c
NT c
4-CH2(2-oxo-pyrrolidin-1-yl)
3-CO(NCH3)(CH2)2NMe2
H
NT c
NT c
NT c
NT c
NT c
NT c
NT c
NT c
NT c
172 ( 43
NT c
NT c
NT c
NT c
NT c
410000
410000
NT c
NT c
NT c
NT c
NT c
NT c
NT c
NT c
NT c
NT c
a Values are averages ( SD of at least three independent determinations. Values “greater than” indicate that half-maximum inhibition was not
achieved at the highest concentration tested. b Selectivity values are averages ( SD of at least two independent determinations. c Not tested.
Scheme 1. Synthesis of 6-Substituted Indolinonesa
a (a) NH4NO3, H2SO4, -5 ꢀC; (b) H2, Pd/C, AcOH, rt; (c) methyl chloroacetate, KOtBu, DMF, -10 ꢀC, then 6, -2 ꢀC; (d) tert-butyl chloroacetate,
KOtBu, DMF, then 8, -10 ꢀC; (e) H2, Ra-Ni, MeOH, rt; (f) 1 N HCl, MeOH, reflux; (g) dimethyl malonate, KOtBu, DMSO, rt, then 10, 100 ꢀC; (h)
LiCl, DMSO, H2O, 100 ꢀC; (i) H2, Lindlar catalyst, MeOH, rt.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4469
Scheme 2. Synthesis of Aniline or Cycloalkylamine Building Blocksa
a (a) N-Methylpiperazine or imidazole or dimethylamine HCl, K2CO3, acetone, then 64 or 68, rt; (b) H2, Pd/C, MeOH, rt; (c) Ac2O, 140 ꢀC;
3
(d) 2-chloro-N,N-dimethylethylamine HCl, K2CO3, NaI, acetone, 50 ꢀC; (e) MeI, KOtBu, DMSO, rt; (f) N,N,N0-trimethylpropane-1,3-diamine, NEt3,
3
CH2Cl2, 0 ꢀC, then 79 or 80, rt; (g) chloroacetyl chloride, NEt3, THF, rt; (h) dimethylamine HCl, K2CO3, acetone, rt; (i) trifluoroacetic acid, CH2Cl2, rt.
3
The 6-amido moiety points toward the VEGFR-2 specificity
pocket flanked by the gatekeeper Val916 and Lys868, which may
explain the favorable selectivity profile of 1. In addition, the
carbonyl oxygen of the amido group can form an additional
hydrogen bond with Lys868, probably accounting for the
higher potency of 1 compared with 39. The basic side chain
points toward the water phase of the enzyme. The specificity
pocket is defined by mainly hydrophobic amino acids
(Figure 1B), suggesting that more lipophilic substituents on
the indolinone core than an amido moiety might have the
potential for improved potency while retaining high selectivity.
Various 6-substituted derivatives of 1 were synthesized to
test this hypothesis (Table 1). For clarity of discussion, only a
limited set of representatives is discussed within this paper,
sufficient to explain structure-activity relationships. To
slightly increase lipophilicity while conserving the amide
hydrogen bond, substituted amides 41-44 were tested.
Disappointingly, all compounds were slightly less active,
probably due to steric hindrance within the specificity
pocket. In addition, 6-acetyl substituted 38 did not show
any improvement. Inversion of the amido moiety, as in 47,
led to complete loss of activity. The significantly more lipo-
philic6-ethoxycarbonyl- and 6-methoxycarbonyl-indolinones
34 and 45, however, were considerably more active than 1. By
far the most active compound in the whole series was the 6-
nitro substituted indolinone 35, showing single-digit nano-
molar activity. Surprisingly, several substituents without car-
bonyl moieties displayed high activities, too. The 6-chloro, 6-
amino, and 6-cyano indolinones 36, 46, and 37 were repre-
sentatives of this group of compounds. In contrast, attaching
a pyrrol-1-yl, as in 48, led to loss of activity. Taken together,
the structure-activity relationships in this series are complex.
A subtle interplay between steric requirements, polarity, and
hydrogen-bonding capability seemed to be decisive for good
potency. Not too bulky, preferably lipophilic substituents
with the ability to form an additional hydrogen bond,
such as in compounds 34, 35, and 45, represented the best
combination. The electronic influence of the substituent
on the indolinone core, modifying the ability of the lactam
to form hydrogen bonds to the hinge region, may also play

4470 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Scheme 3. Synthesis of Final Compoundsa
Roth et al.
a (a) Ac2O, 130 ꢀC; (b) (EtO)3CPh, Ac2O, 130 ꢀC; (c) (EtO)3CPh, Ac2O, 120 ꢀC; (d) R3NH2, DMF, 80-100 ꢀC, then piperidine, rt.
Scheme 4. Synthesis of Specific 6-Substituted Indolinonesa
a (a) 1 N NaOH, EtOH, 80 ꢀC; (b) R4NH2, TBTU, HOBT, NEt3, or EtN(iPr)2, DMF, rt; (c) CDI, DMF, 80 ꢀC, then MeOH, 50 ꢀC; (d) H2, Ra-Ni,
CH2Cl2/MeOH, rt; (e) AcCl, NEt3, CH2Cl2, rt; (f) 2,5-dimethoxy tetrahydrofuran, AcOH, 100 ꢀC.
a role. However, because electron-withdrawing as well as
electron-donating substituents as in 46 can display high
activities, this influence is probably minor.
The more potent compounds were also evaluated for their
ability to inhibit the VEGF-stimulated proliferation of human
umbilical vein endothelial cells (HUVEC). In general, the
trend for inhibition correlated with the biochemical activity
(Table 1). This demonstrated that the cellular inhibition is
specifically dependent on VEGFR-2 mediated signaling rather
than on a general cytostatic or cytotoxic effect. To additionally

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4471
Figure 1. Proposed binding mode of 1 in VEGFR-2. (A) The hydrogen bonds to the hinge region residues and the proposed additional
hydrogen bond of the amide moiety at the indolinone scaffold to Lys868 are shown. The gatekeeper Val916 and Phe1047 from the DFG motif are
additionally highlighted. (B) Zoom into the hydrophobic kinase selectivity pocket of the ATP pocket that binds to the back part of the
indolinone scaffold and the amide substituent. The coloring of the Connolly surface is shown as follows: hydrophobic (brown), hydrophilic
(blue), in between (white).
Table 3. Blood Levels After Single Oral Administration to Micea
verify this important issue, we tested the effects of compounds
34, 35, and 45 on the proliferation of the human epithelial
cancer cell line HeLa, which does not express detectable
amounts of VEGFR. None of the compounds inhibited pro-
liferation of HeLa cells when tested at similar concentrations
to those tested in HUVEC cells (EC50 4 1 μM).
compd
dose (mg/kg)
cplasma (nM) (2 h)
cplasma (nM) (24 h)
2
3
40
50
1252 ( 177
923 ( 313
16 ( 7
8 ( 5
a For pharmacokinetic analysis, blood was isolated at indicated
points in time from the retroorbital venous plexus of five- to six-week-
old athymic NMRI-nu/nu female mice and plasma was analyzed using
HPLC-MS/MS methodology. The compounds were administered in
0.5% (w/v) hydroxypropyl methylcellulose solution (free base). Char-
acteristic pharmacokinetic parameters have been evaluated for com-
pound 3 (CL 173 mL/min/kg, Vd = 32 l/kg, F 45%, cmax (po) 1091 nM,
Although we managed to improve the potency of the
indolinones by modifying the substituent in position 6 in
general, the most active compounds identified were disap-
pointingly not ideal candidates for further optimization at
first glance, with 35 containing a nitro group (mutagenic
potential) and 45 displaying an ester moiety usually prone to
metabolic degradation. On the other hand, selectivity testing
revealed that 45 did not inhibit any of 23 kinases tested on a
larger kinase panel, whereas 35 was not as selective.17 The
angiokinases FGFR-1 and PDGFRR were additionally tar-
geted by 45 (Table 2), contributing to what we considered an
overall very favorable selectivity profile. We were therefore
very pleased to learn that despite its ester moiety, 45 showed
significant plasma levels after single oral administration in
t1/2 (po) 5.0 h, AUC (po) 2746 nM h, dose: 50 mg/kg po, 10 mg/kg iv).
3
(compare 56 and 57 with 3 and 2). As an alternative explana-
tion, the additional methyl groups in 3 and 2 may increase the
potency by picking up lipophilic contacts to the enzyme. The
aromatic core of the aniline was essential for activity, as seen
when comparing 63 with 57. Although structure-activity
relationships were generally shallow in this region, additional
potency could be gained by special substituents. Compounds
3 and 49-51, for instance, were active in the single-digit
nanomolar region. As revealed by an X-ray structure,8 com-
pound 3 forms an additional hydrogen bond between the
methyl-piperazinyl moiety and Glu850, which can explain the
excellent IC50.
All derivatives tested additionally inhibited the angioki-
nases FGFR-1 and PDGFRR as well as the homologous
kinases VEGFR-1 and -3 in the same range as VEGFR-2
(Table 2), contributing to an overall attractive antiangiogenic
target profile. Bycontrast, selected compoundssuch as2 and 3
did not inhibit the 23 kinases17 mentioned before, neither did
they inhibit IGF1R, InsR, CDK1, CDK2, CDK4, EGFR,
HER2, or Plk1 (IC50 4 10 μM),8,15 confirming the low risk of
off-target effects for 6-methoxycarbonyl-substituted indoli-
nones in general. Again, correlation of VEGF-stimulated
HUVEC proliferation with biochemical data followed the
same trend as before (Table 2). As a consequence, our efforts
to modify the side chain furnished several highly potent
inhibitors of endothelial cell proliferation.
nude mice (cmax 952 ng/mL, t1/2 3.5 h, AUC 5246 ng h/mL,
dose 50 mg/kg po). Compound 45 was therefore chosen as a
lead for further optimization.
3
To further explore structure-activity relationships, the
basic side chain pointing toward the water phase of the
enzyme was chosen for modification (Table 2). A wide variety
of linkers between the aniline core and the basic moiety was
tolerated, as seen in 51-55. Various basic groups with differ-
ent pKa values were tolerated (3, 49, 45). In general, basicity
was not a prerequisite for high activity, as shown in 50 and 59.
Nevertheless, neutral compounds were inferior to charged
compounds in terms of low solubility and therefore not
followed up further. Anilines with smaller substituents (58)
or without any substitution (61) were clearly less active.
Shifting the substituent from the 4- into the 3-position was
also detrimental to activity (60 vs 54). Side chains positioned
in an orthogonal position to the aniline appeared to be more
active than side chains that adopt a coplanar geometry

4472 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Roth et al.
experiment were 31% (50 mg/kg qd), 13% (100 mg/kg qd),
and 11% (50 mg/kg bid).
Compound 2 was initially chosen as a preclinical candidate
and extensively profiled.18 It was later replaced by 3. The
efficacy of 3 was tested in a wide variety of tumor types,
summarized in Table 4.
A detailed pharmacological evaluation of 3 has been
described elsewhere.8 Compound 3 inhibited growth-depen-
dent proliferation in a wide range of endothelial cells, pericytes,
and smooth muscle cells. Inhibition usually resulted in apop-
tosis. The Src-family of kinases (IC50 Src 156 nM, Lck 16 nM,
Lyn 195 nM) and Flt-3 (IC50 26 nM) were identified as
additional targets of 3. Flt-3 inhibition might be an interesting
addition to the compound profile, opening up therapeutic
options in acute myelogenous leukemia (AML).19 Compound
3 yielded significant plasma levels after oral administration in
various species but is rapidly cleared from plasma by ester
cleavage at later points in time. Part of the in vivo efficacy may
be attributed to the sustained inhibition of VEGF receptor
phosphorylation by 3, lasting up to 32 h after compound
exposure.8 The mechanistic reason for this prolonged inhibi-
tion is currently under investigation. It is intriguing to spec-
ulate that prolonged receptor blockade in combination with
fast in vivo clearance after drug exposition20 may explain the
excellent efficacy of 3, being at the same time well tolerated in
different species.
Figure 2. Compound 2 inhibits human tumor xenograft growth
in a model of human head and neck SCC (FaDu). Effect of 2
(9 100 mg/kg qd, 2 2 ꢀ 50 mg/kg bid, and [ 50 mg/kg qd) or vehicle
(O) on growth of tumor xenografts. Xenografts were established sc
in athymic NMRI-nu/nu female mice and allowed to reach a volume
of ∼50 mm3 before treatment. Once or twice daily, oral adminis-
tration of compound 2 or vehicle then commenced and was con-
tinued for the duration of the experiment. Data points represent the
mean of five mice, bars represent the SD. The compound was
administered in 0.5% (w/v) hydroxypropyl methylcellulose solu-
tion.
Conclusions
6-Methoxycarbonyl-substituted indolinones are potent in-
hibitors of VEGFR-1/2/3, PDGFRR, and FGFR-1, with low
cross-reactivity against a panel of other kinases. This attrac-
tive antiangiogenic target profile translates well into cellular
efficacy, exemplified by VEGF-specific inhibition of HUVEC
proliferation, as well as inhibition of pericytes and smooth
muscle cells. At similar concentrations, no direct effects on the
proliferation of tumor cell lines can be observed. Typical
methoxycarbonyl-substituted compounds are orally available
but almost completely cleared from plasma 24 h after admin-
istration in mice. Oral application in tumor xenograft experi-
ments leads to complete inhibition of tumor growth after
continuous dosing. The triple angiokinase inhibitory pattern
in combination with good efficacy in vivo defines a unique
pharmacological target profile of this compound class, with
potential for improved efficacy in cancer treatment. Com-
pound 3 (BIBF 1120) is currently being evaluated in phase III
clinical trials in the treatment of nonsmall cell lung cancer21
and is in clinical development for other tumor types. Since it
was shown that 2 and 3 are also highly effective in an animal
model of lung fibrosis,22 clinical development of 3 in idio-
pathic pulmonary fibrosis was recently initiated.
Table 4. Efficacy of 3 in Various Tumor Models
model
FaDu
derivation
HNSCC
dose [mg/kg/d]
T/C value [%]a
100
50
11
14/27b
15
46
2 ꢀ 50
25
10
82
16
Caki-1
kidney
100
50
25
71
10
HT-29
colon
ovary
lung
100
50
16
19
SKOV-3
Calu-6
PAC-120
50
100
24
34
prostate
a Nude mice bearing established human xenografts (0.05-0.1 cm3
volume) were treated once or twice daily with compound 3 po or vehicle
control. For T/C values, median tumor volumes of each treatment group
were compared with the median of the control group at the end of the
experiment, depending on tumor growth kinetics. The compound was
administered in 0.5% (w/v) hydroxypropyl methylcellulose solution.
b Results of two separate experimental series.
Because of their cellular potency and attractive selectivity
profiles, compounds 2 and 3 were selected for in vivo testing.
Both compounds yielded good plasma levels 2 h after oral
administration to mice and were almost completely cleared
from plasma 24 h after administration (Table 3). As shown for
the lead structures, none of the compounds inhibited the
proliferation of VEGF-independent cell lines at similar con-
centrations to those tested in HUVEC cells (EC50 4 1 μM),
particularly HeLa, Calu-6, and FaDu tumor cell lines.
Inmice withestablished humanhead and neck FaDutumor
xenografts, once or twice daily po treatment with compound 2
resulted in significant inhibition of tumor growth after 21days
(Figure 2). Compound 2 was well tolerated, with no obvious
weight loss over the whole treatment period. Ratios of tumor
volumes of treated versus control animals (T/C) in this
Experimental Section
All starting materials and reagents were either commercially
available or their synthesis had been described in the literature
before. All purchased chemicals and solvents were used without
further purification. Reaction progresses were usually monitored
by TLC using Merck silica gel 60 F254 plates and UV light at
254 nm. All chromatographic purifications were conducted as
MPLC using DAVISIL LC60A silica gel (35-70 μm) unless
otherwise noted. Yields refer to purified products and were not
optimized. 1H NMR (400 MHz) spectra were recorded on a
Bruker DPX 400 spectrometer using DMSO-d6 as solvent and Si
(CH3)4 as an internal standard. Low resolution mass spectra
(MS) were run on a Micromass platform mass spectrometer.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4473
High resolution masses (HRMS) were determined on a Micro-
mass Q-Tof-2 mass spectrometer. Combustion analyses were
performed by InfraServ Knapsack on an Elementar Vario Macro
System and are within (0.4% of theoretical values. HPLC
retention times were recorded on a Waters 1515 apparatus using
a X-terra MS C18 column (2.5 μm, 4.6 mm ꢀ 30 mm) eluted with
a 3.1 min gradient from 5% to 98% B, wherein A=water/0.1%
formic acid and B=acetonitrile/0.1% formic acid.
4-Carboxymethyl-3-nitrobenzoic Acid Ethyl Ester (5). 4-Car-
boxymethylbenzoic acid ethyl ester23 4 (20.8 g, 100 mmol) was
dissolved in conc sulfuric acid (200 mL) and ammonium nitrate
(8.80 g, 110 mmol) was slowly added in a way that the reaction
temperature did not exceed -5 ꢀC. Stirring was continued for 2 h
at -5 ꢀC. After that time, the mixture was carefully poured into
ice water. The precipitate was filtered off, washed with water,
and dried at 80 ꢀC to give 22.9 g (91%) of 5. 1H NMR: δ 1.38
(t, 3H), 4.10 (s, 2H), 4.38 (q, 2H), 7.76 (d, 1H), 8.21 (d, 1H), 8.50
(s, 1H), 12.50 (br, 1H). MS: m/z 276 [M+Na]+.
4-Methoxycarbonylmethyl-3-nitrobenzoic Acid Methyl Ester
(7). Potassium tert-butylate (78.5 g, 700 mmol) was dissolved in
dimethylformamide (600 mL) and a solution of methyl chloro-
acetate (29.0 mL, 330 mmol) and 3-nitrobenzoic acid methyl
ester 6 (54.3 g, 300 mmol) in dimethylformamide (100 mL) was
slowly added at -10 ꢀC. Stirring was continued for 10 min at -
10 ꢀC. After that time, the mixture was poured into a mixture of
ice water (1.0 L) and conc hydrochloric acid (350 mL). The
precipitate was filtered off and washed with water. The residue
was recrystallized from methanol and dried at 40 ꢀC in vacuum
to give 47.8 g (63%) of 7. 1H NMR: δ 3.61 (s, 3H), 3.92 (s, 3H),
4.18 (s, 2H), 7.74 (d, 1H), 8.26 (d, 1H), 8.52 (s, 1H). MS: m/z 276
[M+Na]+.
2-(4-Cyano-2-nitrophenyl)acetic Acid Methyl Ester (13). 2-(4-
Cyano-2-nitrophenyl)malonic acid dimethyl ester 11 (20.0 g,
71.9 mmol) was dissolved in dimethyl sulfoxide (350 mL), and
lithium chloride (6.10 g, 143 mmol) and water (1.3 mL) were
added. The mixture was stirred for 3.5 h at 100 ꢀC. After being
cooled, the mixture was poured into ice water (1.8 L) and stirred
for 10 min. The precipitate was filtered off and washed with
water. The residue was triturated with water, filtered off, and
1
dried at 60 ꢀC to give 7.77 g (49%) of 13. H NMR: δ 3.55
(s, 3H), 4.21 (s, 2H), 7.80 (d, 1H), 8.22 (d, 1H), 8.64 (s, 1H). MS:
m/z 219 [M-H]-.
2-oxo-2,3-Dihydro-1H-indole-6-carboxylic Acid Ethyl Ester
(14). 4-Carboxymethyl-3-nitrobenzoic acid ethyl ester 5 (2.50 g,
10.0 mmol) was dissolved in acetic acid (50 mL) and hydro-
genated (50 psi) at room temperature for 1 h using 0.50 g
palladium on charcoal (10%) as catalyst. After that time, the
catalyst was filtered off and the solvent was removed by evapora-
tion. The residue was triturated with diethyl ether, filtered off,
and dried at 100 ꢀC under vacuum to give 1.70 g (83%) of 14.
1H NMR: δ 1.46 (t, 3H), 3.59 (s, 2H), 4.30 (q, 2H), 7.30 (s, 1H)
superimposed on 7.32 (d, 1H), 7.58 (d, 1H), 10.55 (s, 1H). MS:
m/z 205 [M]+.
2-oxo-2,3-Dihydro-1H-indole-6-carboxylic Acid Methyl Ester
(15). 4-Methoxycarbonylmethyl-3-nitrobenzoic acid methyl es-
ter 7 (38.7 g, 153 mmol) was dissolved in acetic acid (800 mL)
and hydrogenated (50 psi) at room temperature for 2.5 h
using 5.0 g palladium on charcoal (10%) as catalyst. After
that time, the catalyst was filtered off and the solvent was
removed by evaporation. The residue was triturated with to-
luene, filtered off, and dried at 100 ꢀC under vacuum to give 28.6
g (98%) of 15. 1H NMR: δ 3.56 (s, 2H), 3.84 (s, 3H), 7.33 (s, 1H)
superimposed on 7.34 (d, 1H), 7.57 (d, 1H), 10.53 (s, 1H). MS:
m/z 190 [M -H]-.
6-Acetylindolin-2-one (16). [2-Amino-4-(2-methyl-[1,3]dioxo-
lan-2-yl)phenyl]acetic acid tert-butyl ester 12 (780 mg, 2.66
mmol) was dissolved in a mixture of methanol (3 mL) and 1 N
hydrochloric acid (15 mL). The mixture was stirred at reflux for
1 h. After being cooled, methylene chloride was added. The
organic layer was separated and extracted with water. After
drying over sodium sulfate, the solvent was removed by eva-
poration to give 390 mg (84%) of 16. 1H NMR: δ 2.55 (s, 3H),
3.58 (s, 2H), 7.28 (s, 1H), 7.33 (d, 1H), 7.59 (d, 1H), 10.55 (s, 1H).
MS: m/z 176 [M+H]+.
6-Cyanoindolin-2-one (17). 2-(4-Cyano-2-nitrophenyl)acetic
acid methyl ester 13 (18.7 g, 85.0 mmol) was dissolved in methanol
(500 mL) and hydrogenated (44 psi) at room temperature for 4.0 h
using 7.0 g Lindlar catalyst. After that time, the catalyst was
filtered off and the solvent was removed by evaporation. The
residue was taken up in acetic acid (300 mL) and stirred for 0.5 h
at 100 ꢀC. After that time, the solvent was removed by evapora-
tion. The residue was washed with water and dried at 70 ꢀC to give
11.5 g (86%) of 17. 1H NMR: δ 3.60 (s, 2H), 7.12 (s, 1H), 7.39 (2xs,
2 ꢀ 1H), 10.30 (s, 1H). MS: m/z 157 [M-H]-.
[4-(2-Methyl-[1,3]dioxolan-2-yl)-2-nitrophenyl]acetic Acid tert-
Butyl Ester (9). Potassium tert-butylate (7.0 g, 62 mmol) was
dissolved in dimethylformamide (50 mL) and a solution of tert-
butyl chloroacetate (3.7 mL, 26 mmol) and 2-methyl-2-
(3-nitrophenyl)-[1,3]dioxolane24 (8) (5.0 g, 24 mmol) in di-
methylformamide (15 mL) was slowly added at -5 ꢀC. Stirring
was continued for 10 min at -2 ꢀC. After that time, the mixture
was poured into a mixture of ice water (1.6 L) and conc
hydrochloric acid (100 mL). Methylene chloride was added,
and the organic layer was separated and washed with water.
After drying over sodium sulfate, the solvent was evaporated
and the residue was purified by column chromatography (silica
gel), eluting with methylene chloride/cyclohexane (4/1) to give
1
2.4 g (31%) of 9. H NMR: δ 1.40 (s, 9H), 1.62 (s, 3H), 3.74
(t, 2H), 3.98 (s, 2H), 4.13 (t, 2H), 7.53 (d, 1H), 7.73 (d, 1H), 8.04
(s, 1H). MS: m/z 324 [M+H]+.
2-(4-Cyano-2-nitrophenyl)malonic Acid Dimethyl Ester (11).
Potassium tert-butylate (42.0 g, 112 mmol) was dissolved in
dimethyl sulfoxide (140 mL) at 20 ꢀC, and dimethyl malonate
(44.0 mL, 385 mmol) was slowly added. Stirring was continued
for 1 h at ambient temperature. After that time, 4-cyano-
2-nitrobenzonitrile 10 (20.0 g, 110 mmol) was slowly added
and the mixture was stirred at 100 ꢀC for 1 h. After being
cooled, the mixture was poured into water (1 L) and neu-
tralized with 1 N hydrochloric acid (24 mL). The precipitate
was filtered off and washed with water. The residue was taken
up in methylene chloride, extracted with water, and dried over
6-Nitroindolin-2-one (19). 3,3-Bis(methoxycarbonyl)-6-nitroin-
dolin-2-one25 18 (5.40 g, 18.4 mmol) was dissolved in a mixture of
formic acid (100 mL) and methanesulfonic acid (100 mL) and
refluxed at 110 ꢀC for 2.5 h. After that time, the solvents were
evaporated and water was added. The precipitate was filtered off,
dissolved in dichloromethane/methanol (1:1), and washed with
water. The aqueous phase was extracted with dichloromethane/
methanol (1:1) and ethyl acetate, and all combined organic layers
were dried over sodium sulfate. After filtration over celite, the
solvent was removed by evaporation to give 2.30 g (70%) of 19. 1H
NMR: δ 3.65 (s, 2H), 7.45 (d, 1H), 7.51 (s, 1H), 7.83 (d, 1H), 10.70
(s, 1H). MS: m/z 178 [M]+.
1-Acetyl-6-chloroindolin-2-one (22). 6-Chloroindolin-2-one 20
(100 g, 168 mmol) was suspended in acetic anhydride (200 mL)
and refluxed at 130 ꢀC for 7 h. After that time, the mixture was
allowed to cool and the precipitate was filtered off and washed
with petroleum ether to give 109 g (87%) of 22. 1H NMR: δ 2.50
1
sodium sulfate to give 27.0 g (88%) of 11. H NMR: δ 3.72
(s, 6H), 5.65 (s, 1H), 7.80 (d, 1H), 8.27 (d, 1H), 8.66 (s, 1H). MS:
m/z 301 [M+Na]+.
[2-Amino-4-(2-methyl-[1,3]dioxolan-2-yl)phenyl]acetic Acid tert-
Butyl ester (12). [4-(2-Methyl-[1,3]dioxolan-2-yl)-2-nitrophe-
nyl]acetic acid tert-butyl ester 9 (3.1 g, 9.6 mmol) was dissolved
in methanol (50 mL) and hydrogenated (50 psi) at room
temperature for 4.5 h using 1.6 g Raney nickel as catalyst. After
that time, the catalyst was filtered off and the solvent was
1
removed by evaporation to give 2.6 g (92%) of 12. H NMR:
δ 1.40 (s, 9H), 1.51 (s, 3H), 3.37 (s, 2H), 3.67 (t, 2H), 3.94 (t, 2H),
6.56 (d, 1H), 6.73 (s, 1H), 6.89 (d, 1H). MS: m/z 294 [M+H]+.

4474 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Roth et al.
(s, 3H), 3.80 (s, 2H), 7.25 (d, 1H), 7.35 (d, 1H), 8.05 (s, 1H). MS:
m/z 210 [M+H]+.
(E)-1-Acetyl-3-(methoxy-phenyl-methylene)-2-oxo-2,3-dihydro-1H-
indole-6-carboxylic Acid Methyl Ester (33). Compound 33
was prepared using the same procedure as described for the
synthesis of 29 by substituting 25 for 22 and ortho-benzoic
acid triethyl ester for ortho-benzoic acid trimethyl ester. 1H
NMR: δ 2.44 (s, 3H), 3.75 (s, 3H), 3.87 (s, 3H), 7.45-7.60
(m, 5H), 7.86 (d, 1H), 8.06 (d, 1H), 8.73 (s, 1H). MS: m/z 374
[M+Na]+.
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Ethyl Ester (34). (E)-
1-Acetyl-3-(ethoxy-phenyl-methylene)-2-oxo-2,3-dihydro-1H-
indole-6-carboxylic acid ethyl ester (26) (1.50 g, 3.95 mmol) and
4-piperidin-1-ylmethyl-phenylamine26 (1.13 g, 5.93 mmol) were
dissolved in dimethylformamide (15 mL). The mixture was
stirred at 100 ꢀC for 0.5 h. After that time, heating was
suspended and piperidine (5 mL) was added at room tem-
perature and stirring was continued for 3.0 h. After that time,
the solvent was removed by evaporation. The residue
was purified by column chromatography (aluminum oxide,
activity 2-3) eluting with methylene chloride/ethanol (100/3)
to give 1.10 g (58%) of 34. 1H NMR: δ 1.28 (t, 3H), 1.30-1.50
(m, 6H), 2.23 (m, 4H), 3.30 (s, 2H), 4.22 (q, 2H), 5.80 (d, 1H),
6.80 (d, 2H), 7.07 (d, 2H), 7.18 (d, 1H), 7.40-7.65 (m, 6H), 10.92
(s, 1H), 12.22 (s, 1H). MS: m/z 481 [M]+. Anal. (C30H31N3O3)
C, H, N.
1,6-Diacetylindolin-2-one (23). Compound 23 was prepared
using the same procedure as described for the synthesis of 22 by
1
substituting 16 for 6-chloroindolin-2-one 20. H NMR: δ 2.50
(s, 3H), 2.60 (s, 3H), 3.91 (s, 2H), 7.50 (d, 1H), 7.86 (d, 1H), 8.61
(s, 1H). MS: m/z 240 [M+Na]+.
1-Acetylindolin-2-one (24). Compound 24 was prepared using
the same procedure as described for the synthesis of 22 by
substituting indolin-2-one 21 for 6-chloroindolin-2-one 20. H
1
NMR: δ 2.50 (s, 3H), 3.81 (s, 2H), 7.20 (t, 1H), 7.31 (1xd/1xt, 2 ꢀ
1H), 8.07 (d, 1H). MS: m/z 176 [M+H]+.
6-Acetyl-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid Methyl
Ester (25). 2-oxo-2,3-Dihydro-1H-indole-6-carboxylic acid
methyl ester 15 (11.5 g, 60.2 mmol) was suspended in acetic
anhydride (100 mL) and refluxed at 130 ꢀC for 8 h. After
that time, the mixture was allowed to cool and the
precipitate was filtered off and washed with petroleum ether
to give 10.2 g (73%) of 25. 1H NMR: δ 2.56 (s, 3H), 3.87 (s, 3H),
3.88 (s, 2H), 7.47 (d, 1H), 7.80 (d, 1H), 8.62 (s, 1H). MS: m/z 256
[M+Na]+.
(E)-1-Acetyl-3-(ethoxy-phenyl-methylene)-2-oxo-2,3-dihydro-
1H-indole-6-carboxylic Acid Ethyl Ester (26). 2-oxo-2,3-Dihy-
dro-1H-indole-6-carboxylic acid ethyl ester 14 (12.9 g, 62.9
mmol) was dissolved in acetic anhydride (130 mL) and ortho-
benzoic acid triethyl ester (42.7 mL, 189 mmol) was added. The
mixture was stirred at 110 ꢀC for 3.5 h. After that time, the
solvent was removed by evaporation. The residue was triturated
with petroleum ether, filtered off, and dried at 50 ꢀC under
vacuum to give 18.2 g (76%) of 26. 1H NMR: δ 1.32 (m, 2 ꢀ 3H),
2.48 (s, 3H), 4.02 (q, 2H), 4.32 (q, 2H), 7.40-7.60 (m, 5H), 7.88
(d, 1H), 8.08 (d, 1H), 8.76 (s, 1H). MS: m/z 380 [M+H]+.
(E)-1-Acetyl-3-(ethoxy-phenyl-methylene)-6-nitroindolin-2-one
(27). Compound 27 was prepared using the same procedure as
(Z)-6-Nitro-3-[phenyl-(4-piperidin-1-ylmethyl-phenylamino)-
methylene]-indolin-2-one (35). Compound 35 was prepared using
the same procedure as described for the synthesis of 34 by
1
substituting 27 for 26. H NMR: δ 1.30-1.50 (m, 6H), 2.20
(m, 4H), 3.30 (s, 2H), 5.80 (d, 1H), 6.84 (d, 2H), 7.08 (d, 2H),
7.45-7.70 (m, 7H), 11.18 (s, 1H), 12.39 (s, 1H). MS: m/z 455
[M+H]+. Anal. (C27H26N4O3 0.25H2O) C, H, N.
3
(Z)-6-Chloro-3-[phenyl-(4-piperidin-1-ylmethyl-phenylamino)-
methylene]-indolin-2-one (36). Compound 36 was prepared using
the same procedure as described for the synthesis of 34 by
1
described for the synthesis of 26 by substituting 19 for 14. H
1
NMR: δ 1.43 (t, 3H), 2.55 (s, 3H), 4.11 (q, 2H), 7.50-7.70 (m,
5H), 8.20 (m, 2 ꢀ 1H), 8.93 (s, 1H). MS: m/z 353 [M+H]+.
(E)-1-Acetyl-6-cyano-3-(ethoxy-phenyl-methylene)-indolin-2-
one (28). Compound 28 was prepared using the same procedure
as described for the synthesis of 26 by substituting 17 for 14.
1H NMR: δ 1.35 (t, 3H), 2.43 (s, 3H), 4.04 (q, 2H), 7.45-7.60
(m, 5H), 7.73 (d, 1H), 8.12 (d, 1H), 8.42 (s, 1H). MS: m/z 355
[M+Na]+.
(E)-1-Acetyl-6-chloro-3-(ethoxy-phenyl-methylene)-indolin-2-
one (29). 1-Acetyl-6-chloroindolin-2-one 22 (41.9 g, 200 mmol)
was dissolved in acetic anhydride (150 mL), and ortho-benzoic
acid triethyl ester (136 mL, 600 mmol) was added. The mixture
was stirred at 120 ꢀC for 6 h. After that time, the solvent was
removed by evaporation. The residue was triturated with pet-
roleum ether (100 mL), filtered off, and dried at 80 ꢀC under
vacuum to give 38.0 g (56%) of 29. 1H NMR: δ 1.31 (t, 3H), 2.42
(s, 3H), 3.96 (q, 2H), 7.29 (d, 1H), 7.45-7.60 (m, 5H), 7.93
(d, 1H), 8.14 (s, 1H). MS: m/z 342 [M+H]+.
substituting 29 for 26. H NMR: δ 1.30-1.50 (m, 6H), 2.20
(m, 4H), 3.18 (s, 2H), 5.70 (d, 1H), 6.57 (d, 1H), 6.77 (d, 2H), 6.85
(s, 1H), 7.05 (d, 2H), 7.40-7.60 (m, 5H), 10.82 (s, 1H), 11.98
(s, 1H). MS: m/z 444 [M + H]+. HPLC: 495% (tR=2.3 min).
HRMS (ES+) calcd for C27H26ClN3O [M + H]+ m/e 444.1843,
found m/e 444.1839.
(Z)-6-Cyano-3-[phenyl-(4-piperidin-1-ylmethyl-phenylamino)-
methylene]-indolin-2-one (37). Compound 37 was prepared using
the same procedure as described for the synthesis of 34 by
1
substituting 28 for 26. H NMR: δ 1.30-1.50 (m, 6H), 2.20
(m, 4H), 3.28 (s, 2H), 5.77 (d, 1H), 6.79 (d, 2H), 6.98 (d, 1H), 7.08
(d, 2H), 7.17 (s, 1H), 7.45-7.65 (m, 5H), 11.03 (s, 1H), 12.28
(s, 1H). MS: m/z 435 [M + H]+. HPLC: 495% (tR=2.2 min).
HRMS (ES+) calcd for C28H26N4O [M + H]+ m/e 435.2185,
found m/e 435.2178.
(Z)-6-Acetyl-3-[phenyl-(4-piperidin-1-ylmethyl-phenylamino)-
methylene]-indolin-2-one (38). Compound 38 was prepared using
the same procedure as described for the synthesis of 34 by
1
(E)-1,6-Diacetyl-3-(ethoxy-phenyl-methylene)-indolin-2-one
(30). Compound 30 was prepared using the same procedure as
described for the synthesis of 29 by substituting 23 for 22. H
substituting 30 for 26. H NMR: δ 1.30-1.80 (m, 6H), 2.43
(s, 3H), 2.75 (m, 2H), 3.18 (m, 2H), 4.12 (s, 2H), 5.82 (d, 1H),
1
6.88 (d, 2H), 7.23 (d, 1H), 7.40-7.70 (m, 8H), 11.02 (s, 1H),
NMR: δ 1.36 (t, 3H), 2.45 (s, 3H), 2.60 (s, 3H), 4.02 (q, 2H),
7.45-7.55 (m, 5H), 7.95 (d, 1H), 8.10 (d, 1H), 8.70 (s, 1H). MS:
m/z 350 [M+H]+.
12.26 (s, 1H). MS: m/z 451 [M]+. Anal. (C29H29N3O2 0.25H2O)
C, H, N.
3
(Z)-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methylene]-
indolin-2-one (39). Compound 39 was prepared using the same
procedure as described for the synthesis of 34 by substituting 31
(E)-3-(Ethoxy-phenyl-methylene)-indolin-2-one (31). Comp-
ound 31 was prepared using the same procedure as described
for the synthesis of 29 by substituting 24 for 22. 1H NMR: δ 1.33
(t, 3H), 2.43 (s, 3H), 3.94 (q, 2H), 7.25 (m, 2H), 7.45-7.55
(m, 5H), 7.98 (d, 1H), 8.17 (d, 1H). MS: m/z 330 [M+Na]+.
(E)-1-Acetyl-3-(ethoxy-phenyl-methylene)-2-oxo-2,3-dihydro-
1H-indole-6-carboxylic Acid Methyl Ester (32). Compound 32
was prepared using the same procedure as described for the
synthesis of 29 by substituting 25 for 22. 1H NMR: δ 1.46 (t, 3H),
2.48 (s, 3H), 3.88 (s, 3H), 4.03 (q, 2H), 7.45-7.60 (m, 5H), 7.88
(d, 1H), 8.09 (d, 1H), 8.74 (s, 1H). MS: m/z 366 [M+H]+.
for 26. 1H NMR:
δ 1.30-1.50 (m, 6H), 2.10-2.30
(m, 4H), 3.38 (m, 2H), 5.76 (d, 1H), 6.51 (t, 1H), 6.72 (d, 2H),
6.86 (m, 2H), 7.04 (d, 2H), 7.40-7.60 (m, 5H), 10.69 (s, 1H),
12.03 (s, 1H). MS: m/z 409 [M]+. Anal. (C27H27N3O) C, H, N.
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid (40). (Z)-2-oxo-3-
[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methylene]-2,3-
dihydro-1H-indole-6-carboxylic acid ethyl ester (34) (800 mg,
1.66 mmol) was dissolved in ethanol (30 mL) and 1 N NaOH

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4475
(8.3 mL) was added. The mixture was stirred at 80 ꢀC for 1 h.
After that time, heating was suspended and the mixture
was neutralized using 1 N HCl. The precipitate was filtered
off, washed with water, ethanol, and diethyl ether, and dried at
100 ꢀC under vacuum to give 670 mg (89%) of 40. 1H NMR: δ
1.25-1.50 (m, 6H), 2.15-2.30 (m, 4H), 3.30 (s, 2H), 5.78 (d, 1H),
6.77 (d, 2H), 7.05 (d, 2H), 7.14 (d, 1H), 7.40-7.60 (m, 6H), 10.88
(s, 1H), 12.22 (s, 1H). MS: m/z 453 [M]+. HPLC: 495%
(tR=2.0 min). HRMS (ES+) calcd for C28H27N3O3 [M+H]+
m/e 454.2131, found m/e 454.2123.
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Methylamide Hydro-
chloride (44). Compound 44 was prepared using the same
procedure as described for the synthesis of 41 by sub-
stituting methylamine hydrochloride for isopropylamine. 1H
NMR: δ 1.30-1.80 (m, 6H), 2.75 (d, 3H) superimposed
on 2.60-2.90 (m, 2H), 3.10-3.30 (m, 2H), 4.09 (m, 2H), 5.74
(d, 1H), 6.83 (d, 1H), 6.82 (d, 2H), 7.15 (d, 1H), 7.30-7.70
(m, 7H), 8.20 (q, 1H), 10.35 (br, 1H), 10.98 (s, 1H), 12.14 (s, 1H).
MS: m/z 467 [M+H]+. Anal. (C29H30N4O2 1.0HCL 0.75H2O)
3
3
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Amide (1). (Z)-2-oxo-
3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methylene]-2,3-
dihydro-1H-indole-6-carboxylic acid (40) (907 mg, 2.00 mmol),
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluor-
oborate (TBTU, 771 mg, 2.40 mmol), and hydroxy-benzotria-
zole (HOBT, 367 mg, 2.40 mmol) were suspended in
dimethylformamide (25 mL) and triethylamine (1.0 mL) was
added. The mixture was stirred at room temperature for
0.25 h. After that time, the mixture was cooled to 0 ꢀC and
ammonia was bubbled through the solution for 15 min in a
way that the reaction temperature did not exceed 15 ꢀC.
Stirring was continued for 1.5 h at room temperature. The
precipitate was filtered off, washed with water, ethanol, and
diethyl ether and dried at 100 ꢀC under vacuum to give 578 mg
(64%) of 1. 1H NMR: δ 1.30-1.50 (m, 6H), 2.15-2.30 (m, 4H),
3.30 (s, 2H), 5.72 (d, 1H), 6.76 (d, 2H), 7.05 (d, 2H) super-
imposed on 7.08 (d, 1H), 7.38 (s, 1H), 7.45-7.75 (m, 5H), 10.88
C, H, N.
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Methyl Ester (45).
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic acid (40) (900 mg,
1.98 mmol) was suspended in dimethylformamide (35 mL),
and N,N0-carbonyldiimidazole (385 mg, 2.38 mmol) was added.
The mixture was stirred at 80 ꢀC for 14 h. After that time,
methanol (20 mL) was added and stirring was continued for
3 h at 50 ꢀC. After that time, the solvent was removed by
evaporation. The residue was purified by column chromato-
graphy (aluminum oxide, activitiy 2-3) eluting with methylene
chloride/methanol (3/1) to give to give 450 mg (49%) of 45. 1H
NMR: δ 1.30-1.50 (m, 6H), 2.20 (m, 4H), 3.23 (m, 2H), 3.78
(s, 3H), 5.79 (d, 1H), 6.79 (d, 2H), 7.03 (d, 2H), 7.19 (d, 1H), 7.39
(s, 1H) superimposed on 7.40-7.65 (m, 5H), 10.94 (s, 1H), 12.24
(s, 1H). MS: m/z 468 [M + H]+. Anal. (C29H29N3O3 0.25H2O)
3
C, H, N.
(Z)-6-Amino-3-[phenyl-(4-piperidin-1-ylmethyl-phenylamino)-
methylene]-indolin-2-one (46). (Z)-6-Nitro-3-[phenyl-(4-piperi-
(s, 1H), 12.13 (s, 1H). MS: m/z 452 [M]+. Anal. (C28H28N4O2
3
0.25H2O) C, H, N.
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Isopropylamide (41).
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic acid (40) (454 mg, 1.00
mmol), O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tet-
rafluoroborate (TBTU, 385 mg, 1.20 mmol), and hydroxy-benzo-
triazole (HOBT, 184 mg, 1.20 mmol) were suspended in
dimethylformamide (10 mL) and N-ethyl diisopropylamine (2.8
mL) was added. Isopropylamine (128 mL, 1.50 mmol) was
added, and the mixture was stirred at room temperature for 1 h.
After that time, water was added, the precipitate was filtered
off, washed with water, acetone, and diethyl ether, and dried
at 100 ꢀC under vacuum to give 368 mg (74%) of 41. 1H NMR:
δ 1.10 (d, 6H), 1.30-1.50 (m, 6H), 2.15-2.25 (m, 4H), 3.23 (s, 2H),
4.03 (sept, 1H), 5.70 (d, 1H), 6.78 (d, 2H), 7.04 (s, 1H) super-
imposed on 7.06 (d, 2H), 7.32 (s, 1H), 7.40-7.60 (m, 5H), 10.88
din-1-ylmethyl-phenylamino)-methylene]-indolin-2-one
(35)
(850 mg, 1.87 mmol) was dissolved in methylene chloride (50
mL) and methanol (50 mL) and hydrogenated at room tem-
perature and atmospheric pressure for 24 h using 0.05 g Raney
nickel as catalyst. After that time, the catalyst was filtered off
and the solvent was removed by evaporation to give 675 mg
(85%) of 46. 1H NMR: δ 1.25-1.65 (m, 6H), 2.40-2.70 (m, 4H),
3.30 (s, 2H), 5.55 (d, 1H), 5.78 (dd, 1H), 6.14 (d, 1H), 6.63 (d,
2H), 7.12 (m, 2H), 7.40-7.60 (m, 5H), 10.43 (s, 1H), 11.55 (s,
1H). MS: m/z 425 [M + H]+. HPLC: 495% (tR =1.6 min).
HRMS (ES+) calcd for C27H28N4O [M+H]+ m/e 425.2341,
found m/e 425.2335.
(Z)-N-{2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-
methylene]-2,3-dihydro-1H-indol-6-yl}-acetamide Hydrochloride
(47). (Z)-6-Amino-3-[phenyl-(4-piperidin-1-ylmethyl-phenyl-
amino)-methylene]-indolin-2-one (46) (500 mg, 1.18 mmol)
was dissolved in methylene chloride (20 mL), and triethylamine
(0.2 mL) was added. Acetylchloride (0.1 mL, 1.2 mmol) was
added, and the mixture was stirred for 5 h at ambient tempera-
ture. After that time, the solvent was removed by evaporation
and the residue was purified by column chromatography (silica
gel) eluting with methylene chloride/methanol (4/1) to give 360
mg (66%) of 47. 1H NMR: δ 1.40-1.80 (m, 6H), 1.98 (s, 3H),
2.60-2.90 (m, 2H), 3.30-3.40 (m, 2H), 3.97 (m, 2H), 5.67 (d,
1H), 6.62 (d, 1H), 6.74 (d, 2H), 7.30 (d, 2H), 7.45-7.65 (m, 6H),
9.90 (s, 1H), 10.50 (br, 1H), 10.73 (s, 1H), 11.86 (s, 1H). MS: m/z
467 [M + H]+. HPLC: 495% (tR = 2.1 min). HRMS (ES+)
calcd for C29H30N4O2 [M + H]+ m/e 467.2447, found m/e
467.2441.
(s, 1H), 12.10 (s, 1H). MS: m/z 494 [M]+. Anal. (C31H34N4O2 0.5-
3
H2O) C, H, N.
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Dimethylamide Hy-
drochloride (42). Compound 42 was prepared using the same
procedure as described for the synthesis of 41 by sub-
1
stituting dimethylamine hydrochloride for isopropylamine. H
NMR: δ 1.30-1.80 (m, 6H), 2.90 (s, 6H) superimposed on 2.60-
2.90 (m, 2H), 3.10-3.30 (m, 2H), 4.12 (s, 2H), 5.78 (d, 1H), 6.62
(d, 1H), 6.82 (d, 2H) superimposed on 6.86 (s, 1H), 7.25 (d, 2H),
7.45-7.65 (m, 5H), 9.10 (br, 1H), 10.88 (s, 1H), 12.15 (s, 1H).
MS: m/z 480 [M]+. HPLC: 495% (tR=2.0 min). HRMS (ES+)
calcd for C30H32N4O2 [M + H]+ m/e 481.2604, found m/e
481.2595.
(Z)-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methylene]-6-
pyrrol-1-yl-indolin-2-one (48). (Z)-6-Amino-3-[phenyl-(4-piperi-
din-1-ylmethyl-phenylamino)-methylene]-indolin-2-one
(Z)-2-oxo-3-[Phenyl-(4-piperidin-1-ylmethyl-phenylamino)-methy-
lene]-2,3-dihydro-1H-indole-6-carboxylic Acid Ethylmethylamide
(43). Compound 43 was prepared using the same procedure
as described for the synthesis of 41 by substituting N-methyl-
ethylamine for isopropylamine. 1H NMR: δ 1.04 (t, 3H), 1.35-
1.45 (m, 6H), 2.22 (m, 4H), 2.85 (s, 3H), 3.28 (q, 3H) super-
imposed on 3.29 (m, 2H), 5.77 (d, 1H), 6.57 (d, 1H), 6.75
(d, 2H), 6.85 (s, 1H), 7.04 (d, 2H), 7.45-7.55 (m, 5H), 10.78
(46)
(500 mg, 1.18 mmol) were dissolved in acetic acid (20 mL) and
2,5-dimethoxy tetrahydrofuran (160 mL, 1.18 mmol) were
added. The mixture was refluxed for 1 h. After that time, the
solvent was removed by evaporation. The residue was taken up
in water (20 mL) and stirred for 2.0 h at ambient temperature.
After that time, the mixture was neutralized with 1 N sodium
hydroxide solution. Methylene chloride was added, and the
organic layer was separated and dried over sodium sulfate.
(s, 1H), 12.10 (s, 1H). MS: m/z 494 [M]+. Anal. (C31H34N4O2
0.25H2O) C, H, N.
3

4476 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Roth et al.
The solvent was removed by evaporation, and the residue was
purified by column chromatography (silica gel) eluting with
methylene chloride/methanol (4/1) to give 200 mg (36%) of 48.
1H NMR: δ 1.25-1.55 (m, 6H), 2.20-2.30 (m, 4H), 3.30 (m, 2H),
5.78 (d, 1H), 6.18 (s, 2H), 6.72 (s, 1H), 6.76 (s, 2H), 6.95 (s, 1H), 7.04
(d, 2H), 7.16(s, 2H), 7.40-7.65(m, 5H), 10.87(s, 1H), 11.94(s,1H).
(Z)-3-({4-[(3-Dimethylaminopropyl)-methyl-carbamoyl]-phenyl-
amino}-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carbo-
xylic Acid Methyl Ester (54). Compound 54 was prepared using
the same procedure as described for the synthesis of 3 by
1
substituting 93 for 84. H NMR (2 rotamers): δ 1.52/1.68 (m,
2H), 1.95/2.10 (s, 6H) superimposed on 1.90-2.20 (t, 2H,
incompletely resolved), 2.80/2.86 (s, 3H), 3.10/3.35 (t, 2H),
3.79 (s, 3H), 5.87 (d, 1H), 6.68 (d, 2H), 7.19 (d, 2H) super-
imposed on 7.21 (d, 1H), 7.42 (s, 1H), 7.45-7.65 (m, 5H), 11.00
(s, 1H), 12.25 (s, 1H). MS: m/z 513 [M+H]+. HPLC: 495%
(tR=2.2 min). HRMS (ES+) calcd for C30H32N4O4 [M+H]+
m/e 513.2502, found m/e 513.2490.
(Z)-3-({4-[(2-Dimethylamino-acetyl)-methyl-amino]-phenyl-
amino}-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carbo-
xylic Acid Methyl Ester (2). Compound 2 was prepared using the
same procedure as described for the synthesis of 3 by substitut-
ing 86 for 84. 1H NMR: δ 2.03 (s, 6H), 2.68 (s, 2H), 3.33 (s, 3H),
3.78 (s, 3H), 5.82 (d, 1H), 6.89 (d, 2H), 7.12 (d, 2H), 7.19 (d, 1H),
7.41 (s, 1H), 7.45-7.65 (m, 5H), 10.98 (s, 1H), 12.22 (s, 1H). MS:
m/z 485 [M+H]+. Anal. (C28H28N4O4) C, H, N.
(Z)-3-[(4-Dimethylaminomethyl-phenylamino)-phenyl-methy-
lene]-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid Methyl Es-
ter (55). Compound 55 was prepared using the same procedure
as described for the synthesis of 3 by substituting 95 for 84. 1H
NMR: δ 2.05 (s, 6H), 3.22 (s, 2H), 3.77 (s, 3H), 5.82 (d, 1H), 6.90
(d, 2H), 7.06 (d, 2H), 7.19 (d, 1H), 7.41 (s, 1H), 7.45-7.65
(m, 5H), 10.95 (s, 1H), 12.25 (s, 1H). MS: m/z 428 [M+H]+.
Anal. (C26H25N3O3) C, H, N.
MS: m/z 475 [M+H]+. Anal. (C31H30N4O 0.25H2O) C, H, N.
3
(Z)-3-[(4-{Methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino}-
phenylamino)-methylene]-2-oxo-2,3-dihydro-1H-indole-6-carboxylic
Acid Methyl Ester (3). (E)-1-Acetyl-3-(methoxy-phenyl-methy-
lene)-2-oxo-2,3-dihydro-1H-indole-6-carboxylic acid methyl es-
ter (33) (10.5 g, 30.0 mmol) and N-(4-aminophenyl)-N-methyl-
2-(4-methylpiperazin-1-yl)acetamide (84, 8.60 g, 33.0 mmol)
were dissolved in dimethylformamide (80 mL). The mixture
was stirred at 80 ꢀC for 1 h. After that time, heating was
suspended and piperidine (6.5 mL) was added at room tempera-
ture and stirring was continued for 2.0 h. After that time,
water was added and the precipitate was filtered off and
washed with water. The residue was triturated with methanol,
filtered off, washed with cold methanol and diethyl ether, and
1
dried at 110 ꢀC under vacuum to give 12.4 g (77%) of 3. H
NMR: δ 2.00-2.35 (m, 11H), 2.70 (s, 2H), 3.06 (s, 3H), 3.76
(s, 3H), 5.82 (d, 1H), 6.87 (d, 2H), 7.11 (d, 2H), 7.17 (d, 1H),
7.40-7.60 (m, 6H), 10.94 (s, 1H), 12.22 (s, 1H). MS: m/z 540
[M+H]+. Anal. (C31H33N5O4 0.25H2O) C, H, N.
3
(Z)-3-({4-[(2-Imidazol-1-yl-acetyl)methylamino]-phenylamino}-
phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid
Methyl Ester (49). Compound 49 was prepared using the same
procedure as described for the synthesis of 3 by substituting 85
for 84. 1H NMR: δ 3.10 (s, 3H), 3.79 (s, 3H), 4.50 (s, 2H), 5.84 (d,
1H), 6.80 (s, 1H), 6.92 (m, 3H), 7.18 (d, 1H) superimposed on
7.24 (m, 2H), 7.41 (m, 2H), 7.50-7.70 (m, 5H), 11.00 (s, 1H),
12.29 (s, 1H). MS: m/z 508 [M+H]+. HPLC: 495% (tR=2.4
min). HRMS (ES+) calcd for C29H25N5O4 [M + H]+ m/e
508.1985, found m/e 508.1974.
(Z)-3-({4-[2-(4-Methylpiperazin-1-yl)acetylamino]-phenylamino}-
phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid
Methyl Ester (56). Compound 56 was prepared using the
same procedure as described for the synthesis of 3 by sub-
stituting 87 for 84. 1H NMR: δ 2.13 (s, 3H), 2.30-2.55
(m, 8H), 3.13 (s, 2H), 3.78 (s, 3H), 5.82 (d, 1H), 6.84 (d, 2H),
7.19 (d, 1H), 7.40-7.65 (m, 8H), 9.60 (s, 1H), 10.92 (s, 1H), 12.18
(Z)-3-{[4-(N-Acetyl-dimethylcarbamoylmethyl-amino)-pheny-
lamino]-phenyl-methylene}-2-oxo-2,3-dihydro-1H-indole-6-carbo-
xylic Acid Methyl Ester (50). Compound 50 was prepared using
the same procedure as described for the synthesis of 3 by
(s, 1H). MS: m/z 526 [M+H]+. Anal. (C30H31N5O4 1.0H2O) C,
3
H, N.
(Z)-3-{[4-(2-Dimethylaminoacetylamino)-phenylamino]-phenyl-
methylene}-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid
Methyl Ester (57). Compound 57 was prepared using the
same procedure as described for the synthesis of 3 by substitut-
ing N-(4-aminophenyl)-2-dimethylamino-acetamide27 for 84.
1H NMR: δ 2.20 (s, 6H), 3.00 (s, 2H), 3.78 (s, 3H), 5.81 (d,
1H), 6.83 (d, 2H), 7.19 (d, 1H), 7.40-7.65 (m, 8H), 9.70 (s, 1H),
10.96 (s, 1H), 12.18 (s, 1H). MS: m/z 470 [M]+. Anal.
1
substituting 88 for 84. H NMR: δ 1.71 (s, 3H), 2.78 (s, 3H),
2.92 (s, 3H), 3.78 (s, 3H), 4.33 (s, 2H), 5.82 (d, 1H), 6.84 (d, 2H),
7.19 (d, 1H), 7.41 (s, 1H), 7.50-7.70 (m, 5H), 10.98 (s, 1H), 12.27
(s, 1H). MS: m/z 535 [M+Na]+. Anal. (C29H28N4O5) C, H, N.
(Z)-3-({4-[N-Acetyl-(3-dimethylaminopropyl)-amino]-phenyl-
amino}-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carbo-
xylic Acid Methyl Ester (51). Compound 51 was prepared using
the same procedure as described for the synthesis of 3 by
(C27H26N4O4 0.25H2O) C, H, N.
3
(Z)-3-{[4-(N-Methanesulfonyl-methylamino)-phenylamino]-phenyl-
methylene}-2-oxo-2,3-dihydro-1H-indole-6-carboxylicAcidMethyl
Ester (58). Compound 58 was prepared using the same proce-
dure as described for the synthesis of 3 by substituting 92
for 84. 1H NMR: δ 2.85 (s, 3H), 3.13 (s, 3H), 3.80 (s, 3H), 5.82
(d, 1H), 6.86 (d, 2H), 7.20 (d, 2H) superimposed on 7.22
(d, 1H), 7.42 (s, 1H), 7.50-7.70 (m, 5H), 10.96 (s, 1H), 12.28
(s, 1H). MS: m/z 478 [M+H]+. Anal. (C25H23N3O5S) C, H, N.
(Z)-2-oxo-3-{[4-(2-oxo-Pyrrolidin-1-ylmethyl)-phenylamino]-phenyl-
methylene}-2,3-dihydro-1H-indole-6-carboxylic Acid Methyl Ester (59).
Compound 59 was prepared using the same procedure as
described for the synthesis of 3 by substituting 1-(4-aminoben-
zyl)-pyrrolidin-2-one28 for 84. 1H NMR: δ 1.88 (m, 2H), 2.26 (t,
2H), 3.17 (t, 2H), 3.78 (s, 3H), 4.23 (s, 3H), 5.81 (d, 1H), 6.82 (d,
2H), 7.03 (d, 2H), 7.19 (d, 1H), 7.42 (s, 1H), 7.50-7.65 (m, 5H),
10.98 (s, 1H), 12.24 (s, 1H). MS: m/z 468 [M + H]+. Anal.
1
substituting 89 for 84. H NMR: δ 1.42 (m, 2H), 1.63 (s, 3H),
1.99 (s, 6H), 2.09 (t, 2H), 3.52 (t, 2H), 3.78 (s, 3H), 5.84 (d, 1H),
6.90 (d, 2H), 7.10 (d, 2H), 7.20 (d, 1H), 7.41 (s, 1H), 7.50-7.65
(m, 5H), 10.98 (s, 1H), 12.25 (s, 1H). MS: m/z 513 [M + H]+.
Anal. (C30H32N4O4 0.25H2O) C, H, N.
3
(Z)-3-({4-[N-Acetyl-(2-dimethylaminoethyl)-amino]-phenyl-
amino}-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carbo-
xylic Acid Methyl Ester (52). Compound 52 was prepared using
the same procedure as described for the synthesis of 3 by
1
substituting 90 for 84. H NMR: δ 1.62 (s, 3H), 2.02 (s, 6H),
2.13 (t, 2H), 3.58 (t, 2H), 3.78 (s, 3H), 5.86 (d, 1H), 6.88 (d, 2H),
7.10 (d, 2H), 7.18 (d, 1H), 7.41 (s, 1H), 7.50-7.70 (m, 5H), 10.93
(s, 1H), 12.22 (s, 1H). MS: m/z 499 [M+H]+. Anal. (C29H30N4O4
0.25H2O) C, H, N.
3
(Z)-3-({4-[(2-Dimethylaminoethyl)-methanesulfonyl-amino]-
phenylamino}-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-
6-carboxylic Acid Methyl Ester (53). Compound 53 was pre-
pared using the same procedure as described for the synthesis of
3 by substituting 91 for 84. 1H NMR: δ 2.04 (s, 6H), 2.13 (t, 2H),
2.92 (s, 3H), 3.58 (t, 2H), 3.78 (s, 3H), 5.81 (d, 1H), 6.87 (d, 2H),
7.16 (d, 2H) superimposed on 7.19 (d, 1H), 7.41 (s, 1H), 7.50-
7.65 (m, 5H), 11.00 (s, 1H), 12.15 (s, 1H). MS: m/z 535 [M +
(C29H26N2O4 0.5H2O) C, H, N.
3
(Z)-3-({3-[(3-Dimethylaminopropyl)-methyl-carbamoyl]-phenyl
amino}-phenyl-methylene)-2-oxo-2,3-dihydro-1H-indole-6-carbo-
xylic Acid Methyl Ester (60). Compound 60 was prepared using
the same procedure as described for the synthesis of 3 by
1
substituting 94 for 84 and 32 for 33. H NMR (2 rotamers): δ
1.45/1.65 (m, 2H), 1.98/2.17 (s, 6H) superimposed on 2.00-2.30
(t, 2H, incompletely resolved), 2.58/2.88 (s, 3H) superimposed
H]+. Anal. (C28H30N4O5S 1.0H2O) C, H, N.
3

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4477
on 2.90/3.20 (t, 2H, incompletely resolved), 3.78 (s, 3H), 5.83
(d, 1H), 6.40-7.30 (m, 5H), 7.42 (s, 1H), 7.40-7.65 (m, 5H),
11.00 (s, 1H), 12.14 (s, 1H). MS: m/z 513 [M + H]+. Anal.
(s, 3H), 4.10 (d, 2H), 6.74 (d, 2H), 7.20 (t, 1H), 8.00 (d, 2H). MS:
m/z 224 [M+H]+.
2-[Acetyl-(4-nitrophenyl)amino]-N,N-dimethylacetamide (73).
N,N-Dimethyl-2-(4-nitrophenylamino)acetamide (70, 450 mg,
2.00 mmol) was dissolved in acetic anhydride (5 mL). The
mixture was stirred at 140 ꢀC for 1.5 h. After that time, the
solvent was removed by evaporation. The residue was triturated
with diethyl ether, filtered off, and dried at 60 ꢀC under vacuum
to give 464 mg (88%) of 73. 1H NMR: δ 1.96 (s, 3H), 2.83 (s, 3H),
2.98 (s, 3H), 4.58 (s, 2H), 7.61 (d, 2H), 8.28 (d, 2H). MS: m/z 288
[M+Na]+.
(C30H32N4O4 1.5H2O) C, H, N.
3
(Z)-2-oxo-3-(Phenyl-phenylamino-methylene)-2,3-dihydro-1H-
indole-6-carboxylic Acid Methyl Ester (61). Compound 61 was
prepared using the same procedure as described for the synthesis
of 3 by substituting aniline for 84. 1H NMR: δ 3.78 (s, 3H), 5.82
(d, 1H), 6.86 (d, 2H), 7.14 (m, 1H), 7.19 (m, 3H), 7.42 (s, 1H),
7.45-7.65 (m, 5H), 10.94 (s, 1H), 12.24 (s, 1H). MS: m/z 371
[M+H]+. Anal. (C23H18N2O3 0.25H2O) C, H, N.
3
(Z)-3-[(1-Methylpiperidin-4-ylamino)-phenyl-methylene]-2-oxo-2,3-
dihydro-1H-indole-6-carboxylic Acid Methyl Ester (62). Compound
62 was prepared using the same procedure as described for
the synthesis of 3 by substituting 1-methylpiperidin-4-ylamine
for 84 and 32 for 33. 1H NMR: δ 1.55 (m, 2H), 1.75-1.85
(m, 4H), 2.08 (s, 3H), 2.57 (m, 2H), 3.11 (m, 1H), 3.78 (s, 3H),
5.46 (d, 1H), 7.16 (d, 1H), 7.38 (s, 1H), 7.50-7.70
(m, 5H), 10.62 (d, 1H) superimposed on 10.66 (s, 1H). MS: m/
z 392 [M+H]+. Anal. (C23H25N3O3) C, H, N.
N-(3-Dimethylaminopropyl)-N-(4-nitrophenyl)acetamide (74).
Compound 74 was prepared using the same procedure as
described for the synthesis of 73 by substituting N,N-dimethyl-
N0-(4-nitrophenyl)-propane-1,3-diamine31 71 for 70. 1H NMR:
δ 1.54 (m, 2H), 1.90 (s, 3H), 2.04 (s, 6H), 2.16 (t, 2H), 3.73
(t, 2H), 7.63 (d, 2H), 8.27 (d, 2H). MS: m/z 266 [M + H]+.
N-(2-Dimethylaminoethyl)-N-(4-nitrophenyl)acetamide (75).
Compound 75 was prepared using the same procedure as
described for the synthesis of 73 by substituting N,N-dimethyl-
N0-(4-nitrophenyl)-ethane-1,2-diamine31 (72) for 70. 1H NMR:
δ 1.88 (s, 3H), 2.09 (s, 6H), 2.28 (t, 2H), 3.79 (t, 2H), 7.62 (d, 2H),
8.28 (d, 2H). MS: m/z 252 [M+H]+.
(Z)-3-{[4-(2-Dimethylaminoacetylamino)-cyclohexylamino]-phenyl-
methylene}-2-oxo-2,3-dihydro-1H-indole-6-carboxylic Acid Methyl
Ester (63). Compound 63 was prepared using the same proce-
dure as described for the synthesis of 3 by substituting 99 for 84.
1H NMR: δ 1.10 (m, 2H), 1.48 (m, 2H), 1.70-1.90 (m, 4H), 2.13
(s, 6H), 2.76 (s, 2H), 3.03 (m, 1H), 3.57 (m, 1H), 3.76 (s, 3H), 5.45
(d, 1H), 7.09 (d, 1H), 7.34 (s, 1H) superimposed on 7.35 (d, 1H),
7.45-7.70 (m, 5H), 10.50 (d, 1H), 10.69 (s, 1H). MS: m/z 477
N-(2-Dimethylaminoethyl)-N-(4-nitrophenyl)methanesulfona-
mide (77). N-(4-Nitrophenyl)methanesulfonamide32 (76) (21.6
g, 100 mmol) was dissolved in acetone (1.0 L), and 2-chloro-
N,N-dimethylethylamine hydrochloride (21.6 g, 150 mmol),
potassium carbonate (35.9 g, 260 mmol), and sodium iodide
(3.0 g, 20 mmol) were added. The mixture was stirred at 50 ꢀC
for 3 days. After that time, the precipitates were filtered off
and the solvent was evaporated from the filtrate. The residue
was taken up in ethyl acetate (300 mL) and extracted with water
(3 ꢀ 200 mL). The organic layer was separated and dried over
sodium sulfate. The solvent was removed by evaporation, and
the residue was recrystallized from 2-propanol to give 15.2 g
(53%) of 77. 1H NMR: δ 2.08 (s, 6H), 2.29 (t, 2H), 3.12 (s, 3H),
3.84 (t, 2H), 7.66 (d, 2H), 8.27 (d, 2H). MS: m/z 288 [M + H]+.
N-Methyl-N-(4-nitrophenyl)methanesulfonamide (78). N-(4-
Nitrophenyl)methanesulfonamide32 76 (4.32 g, 20.0 mmol)
was dissolved in dimethylsulfoxide (40 mL), and potassium
tert-butyrate (2.50 g, 22.0 mmol) was added. The mixture was
stirred at ambient temperature for 1 h. After that time, a solution
of methyl iodide (4.20 g, 18.0 mmol) in dimethylsulfoxide
10 mL) was slowly added. Stirring was continued for 8 h at
ambient temperature. After that time, the mixture was poured
onto ice water (120 mL) and ethyl acetate was added. The
organic layer was separated and extracted with water (3ꢀ).
Finally, the organic layer was dried over sodium sulfate. The
solvent was removed by evaporation, and the residue was
triturated with diethyl ether and filtered off to give 4.20 g
[M+H]+. Anal. (C27H32N4O4 0.25H2O) C, H, N.
3
N-Methyl-2-(4-methylpiperazin-1-yl)-N-(4-nitrophenyl)-acet-
amide (65). 1-Methylpiperazine (14.8 mL, 133 mmol) and
potassium carbonate (37.6 g, 205 mmol) were dissolved in
acetone (500 mL), and 2-bromo-N-methyl-N-(4-nitrophenyl)
acetamide29 64 (28.0 g, 103 mmol) was gradually added. The
mixture was stirred for 3 h at ambient temperature. After that
time, the precipitates were filtered off and the solvent was
evaporated from the filtrate. The residue was taken up in ethyl
acetate and extracted with water. After drying over sodium
sulfate, the solvent was removed by evaporation to give 29.5 g
(98%) of 65. 1H NMR: δ 2.00-2.40 (m, 11H), 3.12 (s, 2H), 3.31
(s, 3H), 7.64 (d, 2H), 8.26 (d, 2H). MS: m/z 293 [M+H]+.
2-Imidazol-1-yl-N-methyl-N-(4-nitrophenyl)acetamide (66).
Compound 66 was prepared using the same procedure as
described for the synthesis of 65 by substituting imidazole for
1-methylpiperazine. 1H NMR: δ 3.33 (s, 3H), 4.92 (s, 2H), 6.85
(s, 1H), 7.05 (s, 1H), 7.51 (s, 1H), 7.72 (d, 2H), 8.31 (d, 2H). MS:
m/z 261 [M+H]+.
2-Dimethylamino-N-methyl-N-(4-nitrophenyl)acetamide (67).
Compound 67 was prepared using the same procedure as
described for the synthesis of 65 by substituting dimethylamine
hydrochloride for 1-methylpiperazine. 1H NMR: δ 2.18 (s, 6H),
3.09 (s, 2H), 3.32 (s, 3H), 7.63 (d, 2H), 8.25 (d, 2H). MS: m/z 238
[M+H]+.
2-(4-Methylpiperazin-1-yl)-N-(4-nitrophenyl)acetamide (69).
Compound 69 was prepared using the same procedure as
described for the synthesis of 65 by substituting 2-bromo-N-
(4-nitrophenyl)acetamide30 (68) for 2-bromo-N-methyl-N-
(4-nitrophenyl)acetamide (64). 1H NMR: δ 2.13 (s, 3H), 2.30-
2.55 (m, 8H), 3.18 (s, 2H), 7.90 (d, 2H), 8.22 (d, 2H), 10.32
(s, 1H).
N,N-Dimethyl-2-(4-nitrophenylamino)acetamide (70). 2-(4-
Nitrophenylamino)-acetic acid (5.30 g, 27.0 mmol), O-(benzo-
triazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate
(TBTU, 10.4 g, 32.4 mmol), hydroxy-benzotriazole (HOBT,
4.97 g, 32.4 mmol), and dimethylamine hydrochloride (13.2 g,
162 mmol) were suspended in dimethylformamide (50 mL), and
triethylamine (37.5 mL) was added at 0 ꢀC. The mixture was
stirred at room temperature for 8 h. After that time, the
precipitate was filtered off, washed with water, and dried at
100 ꢀC to give 4.70 g (78%) of 70. 1H NMR: δ 2.88 (s, 3H), 3.03
1
(91%) of 78. H NMR: δ 3.18 (s, 3H), 3.35 (s, 3H), 7.64 (d,
2H), 8.28 (d, 2H). MS: m/z 229 [M - H]-.
N-(3-Dimethylaminopropyl)-N-methyl-4-nitrobenzamide (81).
N,N,N0-Trimethylpropane-1,3-diamine (4.80 g, 41.3 mmol) was
dissolved in methylene chloride (150 mL), and triethylamine
(11.5 mL, 82.6 mmol) was added. 4-Nitrobenzoyl chloride 79
(7.67 g, 41.3 mmol) was added at 0 ꢀC. The mixture was stirred
for 5 min at 0 ꢀC and for 20 min at room temperature. After
that time, the precipitates were filtered off and the filtrate
was extracted three times with water. The organic layer was
separated and dried over sodium sulfate. The solvent was
removed by evaporation to give 10.33 g (94%) of 81. 1H
NMR (2 rotamers): δ 1.61/1.73 (m, 2H), 1.98/2.06 (s, 6H),
2.04/2.28 (t, 2H), 2.86/3.00 (s, 3H), 3.16/3.48 (t, 2H), 7.78 (d,
2H), 8.29 (d, 2H). MS: m/z 266 [M + H]+.
N-(3-Dimethylaminopropyl)-N-methyl-3-nitrobenzamide (82).
Compound 82 was prepared using the same procedure as
described for the synthesis of 81 by substituting 3-nitrobenzoyl
1
chloride 80 for 4-nitrobenzoyl chloride 79. H NMR (2 rota-
mers): δ 1.62/1.78 (m, 2H), 2.00/2.23 (s, 6H), 2.10/2.43 (t, 2H),

4478 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Roth et al.
2.88/2.98 (s, 3H), 3.19/3.49 (t, 2H), 7.60-7.90 (m, 2H, incom-
pletely resolved), 8.15-8.35 (m, 2H, incompletely resolved).
MS: m/z 266 [M+H]+.
4-Amino-N-(3-dimethylaminopropyl)-N-methylbenzamide (93).
Compound 93 was prepared using the same procedure as
described for the synthesis of 84 by substituting 81 for 65. H
1
Dimethyl-(4-nitrobenzyl)amine (83). A solution of dimethyl-
amine in water (40%, 114 mL, 900 mmol) was dissolved in
methyl-iso-butylketone (600 mL) and a solution of 4-nitroben-
zyl chloride (52.0 g, 300 mmol) in methyl-iso-butylketone
(300 mL) was slowly added over 30 min. The mixture was stirred
at ambient temperature for 2.0 h and at 35 ꢀC for 0.5 h. After
that time, ethyl acetate (100 mL) was added and the organic
layer was separated. The organic layer was extracted with brine
(4ꢀ), separated, and dried over sodium sulfate. The solvent was
removed by evaporation, The residue was triturated with diethyl
ether, filtered, and the solvent was removed by evaporation to
give 55.1 g (99%) of 83. 1H NMR: δ 2.17 (s, 6H), 3.52 (s, 2H),
7.57 (d, 2H), 8.18 (d, 2H). MS: m/z 181 [M+H]+.
N-(4-Aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acet-
amide (84). N-Methyl-2-(4-methylpiperazin-1-yl)-N-(4-nitro-
phenyl)acetamide (65, 29.5 g, 101 mmol) was dissolved in
methanol (350 mL) and hydrogenated (50 psi) at room tem-
perature for 1.5 h using 3.0 g palladium on charcoal (10%) as
catalyst. After that time, the catalyst was filtered off and the
solvent was removed by evaporation. The residue was triturated
with diethyl ether, filtered off, and dried at 80 ꢀC under vacuum
to give 22.1 g (83%) of 84. 1H NMR: δ 2.00-2.40 (m, 11H), 2.79
(s, 2H), 3.04 (s, 3H), 5.20 (s, 2H), 6.55 (d, 2H), 6.90 (d, 2H). MS:
m/z 263 [M+H]+.
NMR: δ1.66 (m, 2H), 2.08 (s, 6H), 2.17 (t, 2H), 2.91 (s, 3H), 3.30 (t,
2H), 5.40 (s, 2H), 6.53 (d, 2H), 7.10 (d, 2H). MS: m/z 236 [M+H]+.
3-Amino-N-(3-dimethylaminopropyl)-N-methylbenzamide (94).
Compound 94 was prepared using the same procedure as
1
described for the synthesis of 84 by substituting 82 for 65. H
NMR (2 rotamers): δ 1.55/1.65 (m, 2H), 2.00/2.12 (s, 6H)
superimposed on 2.00-2.30 (t, 2H, incompletely resolved),
2.88/3.00 (s, 3H), 3.20-3.40 (t, 2H, incompletely resolved),
5.35 (br, 2H), 6.40-6.60 (m, 3H, incompletely resolved), 7.0-
7.10 (m, 1H, incompletely resolved). MS: m/z 236 [M+H]+.
4-(Dimethylaminomethyl)phenylamine (95). Compound 95
was prepared using the same procedure as described for the
synthesis of 84 by substituting 83 for 65 and Raney nickel for
1
palladium on charcoal. H NMR: δ 2.07 (s, 6H), 3.16 (s, 2H),
4.88 (s, 2H), 6.49 (d, 2H), 6.89 (d, 2H). MS: m/z 151 [M+H]+.
trans-[4-(2-Chloroacetylamino)cyclohexyl]carbamic Acid tert-
Butyl Ester (97). trans-(4-Aminocyclohexyl)carbamic acid tert-
butyl ester (96) (2.50 g, 11.7 mmol) was suspended in tetrahy-
drofuran (500 mL) and triethylamine (2.43 mL, 17.5 mmol) was
added. After stirring for 2 h at ambient temperature, chloro
acetyl chloride (0.93 mL, 11.7 mmol) was added. The mixture
was stirred for 8 h at ambient temperature. After that time, the
solvent was removed by evaporation and the residue was taken
up in methylene chloride. The organic layer was extracted with
2 N hydrochloric acid and a saturated solution of sodium
hydrogen carbonate. The organic layer was separated and dried
over sodium sulfate. Finally, the solvent was removed by
N-(4-Aminophenyl)-2-imidazol-1-yl-N-methylacetamide (85).
Compound 85 was prepared using the same procedure as
described for the synthesis of 84 by substituting 66 for 65.
1H NMR: δ 3.12 (s, 3H), 4.54 (s, 2H), 5.33 (s, 2H), 6.62
(d, 2H), 6.82 (s, 1H), 7.01 (s, 1H), 7.07 (d, 2H), 7.46 (s, 1H).
MS: m/z 231 [M+H]+.
1
evaporation to give 3.40 g (99%) of 97. H NMR: δ 1.21 (m,
4H), 1.37 (s, 9H), 1.76 (m, 4H), 3.20 (m, 1H), 3.45 (m, 1H), 3.98
(s, 2H), 6.71 (d, 1H), 8.05 (d, 1H). MS: m/z 289 [M - H]-.
trans-{4-[2-(Dimethylamino)acetylamino]-cyclohexyl}-carba-
mic Acid tert-Butyl Ester (98). trans-[4-(2-Chloroacetylamino)
cyclohexyl]carbamic acid tert-butyl ester 97 (2.62 g, 9.00 mmol)
was suspended in acetone (50 mL) and potassium carbonate
(3.73 mL, 27.0 mmol), and dimethylamine hydrochloride (1.10 g,
13.5 mmol) were added. The mixture was stirred for 8 h at
ambient temperature. After that time, the precipitates were
filtered off and the solvent was removed by evaporation to give
1.65 g (61%) of 98. 1H NMR: δ 1.20 (m, 4H), 1.36 (s, 9H), 1.74
(m, 4H), 2.17 (s, 6H), 2.73 (s, 2H), 3.18 (m, 1H), 3.50 (m, 1H),
6.67 (d, 1H), 7.44 (d, 1H). MS: m/z 300 [M+H]+.
N-(4-Aminophenyl)-2-dimethylamino-N-methylacetamide (86).
Compound 86 was prepared using the same procedure as
described for the synthesis of 84 by substituting 67 for 65. H
1
NMR: δ 2.21 (s, 6H), 2.78 (s, 2H), 3.14 (s, 3H), 5.25 (s, 2H), 6.54
(d, 2H), 6.91 (d, 2H). MS: m/z 208 [M+H]+.
N-(4-Aminophenyl)-2-(4-methylpiperazin-1-yl)acetamide (87).
Compound 87 was prepared using the same procedure as
described for the synthesis of 84 by substituting 69 for 65. H
1
NMR: δ 2.16 (s, 3H), 2.30-2.55 (m, 8H), 3.04 (s, 2H), 4.80
(br, 2H), 6.51 (d, 2H), 7.24 (d, 2H), 9.19 (s, 1H). MS: m/z 249
[M+H]+.
2-[Acetyl-(4-aminophenyl)-amino]-N,N-dimethylacetamide (88).
Compound 88 was prepared using the same procedure as
described for the synthesis of 84 by substituting 73 for 65. H
trans-N-(4-Aminocyclohexyl)-2-(dimethylamino)acetamide (99).
trans-{4-[2-(Dimethylamino)acetylamino]cyclohexyl}carbamic
acid tert-butyl ester 98 (1.80 g, 6.01 mmol) was dissolved in
methylene chloride (50 mL), and trifluoroacetic acid (20.0 mL)
was added. The mixture was stirred for 0.5 h at ambient
temperature. After that time, the solvent was removed by
evaporation and the residue was taken up in methylene chlor-
ide/methanol (1:1). The solution was neutralized using a basic
MP resin. The solvent was removed by evaporation to give
1.20 g (100%) of 99. 1H NMR: δ 1.34 (m, 4H), 1.70-1.90 (m,
4H), 2.28 (s, 6H), 2.95 (m, 1H) superimposed on 2.98 (s, 2H),
3.17 (br, 2H), 3.53 (m, 1H), 7.62 (d, 1H). MS: m/z 200 [M+H]+.
Computational Methods. All binding mode models have been
built using the MOE (MOE revision 2008.10, Chemical Com-
puting Group Inc., Montreal, Quebec, 2008) software package.
The X-ray structure of 3 in complex with VEGFR-28 served as a
template for all initial binding conformations (PDB code
3C7Q). The final binding mode has been calculated using the
MMFF94x force field and a distance dependent dielectric
1
NMR: δ 1.73 (s, 3H), 2.80 (s, 3H), 2.91 (s, 3H), 4.30 (s, 2H), 5.20
(s, 2H), 6.53 (d, 2H), 7.02 (d, 2H). MS: m/z 258 [M+Na]+.
N-(4-Aminophenyl)-N-(3-dimethylaminopropyl)acetamide (89).
Compound 89 was prepared using the same procedure as
1
described for the synthesis of 84 by substituting 74 for 65. H
NMR: δ 1.50 (m, 2H), 1.68 (s, 3H), 1.94 (s, 6H), 2.15 (t, 2H), 3.49
(t, 2H), 5.20 (br, 2H), 6.57 (d, 2H), 6.86 (d, 2H). MS: m/z 236
[M+H]+.
N-(4-Aminophenyl)-N-(2-dimethylaminoethyl)acetamide (90).
Compound 90 was prepared using the same procedure as
described for the synthesis of 84 by substituting 75 for 65. H
1
NMR: δ 1.68 (s, 3H), 2.10 (s, 6H), 2.23 (t, 2H), 3.58 (t, 2H), 5.20
(br, 2H), 6.56 (d, 2H), 6.88 (d, 2H). MS: m/z 222 [M + H]+.
N-(4-Aminophenyl)-N-(2-dimethylaminoethyl)methanesulfonamide
(91). Compound 91 was prepared using the same procedure
as described for the synthesis of 84 by substituting 77 for 65.
1H NMR: δ 2.08 (s, 6H), 2.24 (t, 2H), 2.91 (s, 3H), 3.56 (t, 2H),
5.25 (br, 2H), 6.53 (d, 2H), 6.99 (d, 2H). MS: m/z 258 [M+H]+.
N-(4-Aminophenyl)-N-methylmethanesulfonamide (92). Com-
pound 92 was prepared using the same procedure as described
for the synthesis of 84 by substituting 78 for 65. 1H NMR: δ 2.85
(s, 3H), 3.10 (s, 3H), 5.22 (s, 2H), 6.54 (d, 2H), 7.02 (d, 2H). MS:
m/z 200 [M]+.
˚
constant. The ligand and a shell of 4 A around the ligand has
˚
been free to move, a second shell of 4 A has been tethered by a
force constant of 100, and the rest of the VEGFR-2 protein has
been fixed. All pictures have been rendered using PyMol 1.1r1
(DeLano Scientific LLC).
In Vitro VEGFR-2 Kinase Assay. The cytoplasmic kinase
domain of VEGFR-2 (residues 797 to 1335 according to sequence

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4479
deposited in databank SWISS-PROT P35968) was cloned into
pFastBac fused to Glutathion-S-transferase (GST). The GST-
fusion protein was expressed in SF-9 insect cells and extracted
with HEPEX (20 mM HEPES pH 7.4, 100 mM NaCl, 10 mM
ss-glycerophosphate, 10 mM para-nitro-phenylphosphate, 30 mM
NaF, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 1 mM
Na3VO4, 0.1% SDS, 0.5 μg/mL pepstatin A, 2.5 μg/mL 3,4-
dichloroisocoumarin, 2.5 μg/mL trans-epoxysuccinyl-L-leucyl-
L-amido butane, aprotinin 20 KIU/mL, leupeptin 2 μg/mL, ben-
zamidine 1 mM and 0.002% PMSF). Enzyme activity was assayed
in the presence or absence of serial dilutions of the inhibitor
performed in 25% DMSO. Each microtiter plate contained inter-
nal controls such as blank, maximum reaction, and historical
reference compound. All incubations were conducted at room
temperature on a rotation shaker. Ten μL of each inhibitor dilution
was added to 10 μL of diluted kinase (0.8 μg/mL VEGFR-2,
10 mM Tris pH 7.5, 2 mM EDTA, 2 mg/mL BSA) and preincu-
bated for 1 h. The reaction was started by addition of 30 μL
of substrate mix containing 62.4 mM Tris pH 7.5, 2.7 mM DTT,
5.3 mM MnCl2, 13.3 mM Mg-acetate, 0.42 mM ATP, 0.83 mg/mL
Poly-Glu-Tyr(4:1), and 1.7 μg/mL Poly-Glu-Tyr(4:1)-biotin
and incubated for 1 h. The reaction was stopped by addition of
50 μL of 250 mM EDTA, 20 mM HEPES, pH 7.4. Then 90 μL of
stopped solution was transferred to a streptavidin plate and
incubated for 1-2 h. After three washes with PBS the EU-labeled
antibody, PY20 was added (recommended dilution 1:2000 of
0.5 mg/mL labeled antibody in DELFIA assay buffer). Exces-
sive detection antibody was removed by three washes of
DELFIA washing buffer. Then 10 minutes before measurement
on the multilabel reader VICTOR, each well was incubated with
100 μL of DELFIA enhancement solution. IC50 values were
calculated by using a sigmoidal curve analysis program (Graph
Pad Prism) using the nonlinear regression analysis with variable
slope.
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Edelstein, M.; Frye, S. V.; Gampe, R. T.; Harris, P. A.; Hassell,
A.; Holmes, W. D.; Hunter, R. N.; Lackey, K. E.; Lovejoy, B.;
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Shewchuk, L.; Veal, J. M.; Walker, D. H.; Kuyper, L. F. Oxi-
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In Vitro VEGFR-1, VEGFR-3, FGFR-1, PDGFRr Kinase
Assay. IC50 in these assays were determined by Invitrogen Ltd.,
United Kingdom. For details please refer to www.invitrogen.
com.
Cellular Assays. All cellular assays (HUVEC, HeLa, Calu-6,
FaDu) were conducted as previously described in ref 8.
In Vivo Tumor Models. All in vivo experiments were con-
ducted as previously described in ref 8 and complied with the
Declaration of Helsinki and European Policy Legislations
(FELASA and GV-SOLAS) on the Care and Use of Laboratory
Animals.
(15) Assay conditions as described in ref 8.
(16) Computational modeling was originally performed using a homol-
ogy model based on the X-ray structure of FGFR-1. For better
accuracy, the representations shown in this paper have been retro-
spectively modeled using the X-ray structure of VEGFR-2 recently
reported in ref (8).
(17) Twenty-three kinases were analyzed at 10 μmol/L with 100 μmol/L
ATP as described in ref 8: GSK3β, ROCKII, DYRK1A, PKCR,
MAPK2ERK2, MSK1, PDK1, CHK1, MAPKAPK2, SAP-
K2AP38, S6K1, SGK, CK1, CK2, PKA, SAPK2BP38β2,
SAPK3P38γ, JNK1A1, SAPK4P38δ, PHK, PKBR, CSK, PRAK.
In contrast to 45, 35 inhibited CHK1, DYRK1A, MSK1, and PHK
in the same panel significantly (<10% CTRL at 10 μmol/L).
(18) (a) Bisping, G.; Kropff, M.; Wenning, D.; Dreyer, B.; Bessonov, S.;
Hilberg, F.; Roth, G. J.; Munzert, G.; Stefanic, M.; Stelljes, M.;
Acknowledgment. We thank Elke Honold, Simone Linke,
Evelin Rehm, and Josef Zeiler for their excellent technical
expertise in the synthesis of most of the final compounds, as
well as Nicole Reiners, Saskia Stenkamp, and Thomas Veser
for the synthesis of selected compounds. We additionally
thank Helmut Wittneben for performing initial computer
modeling studies.
€
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