1,4-Dihydroindeno[1,2-c]pyrazoles with acetylenic side chains as novel and potent multitargeted receptor tyrosine kinase inhibitors with low affinity for the hERG ion channel

Article abstract of DOI:10.1021/jm061223o

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED₅₀ = 3 mg/kg) superior to Sutent (ED₅₀ = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg·day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC₅₀ of 11.6 μM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

Full text of DOI:10.1021/jm061223o

J. Med. Chem. 2007, 50, 2011-2029
2011
Articles
1,4-Dihydroindeno[1,2-c]pyrazoles with Acetylenic Side Chains as Novel and Potent
Multitargeted Receptor Tyrosine Kinase Inhibitors with Low Affinity for the hERG Ion
Channel
Ju¨rgen Dinges,*,† Daniel H. Albert,† Lee D. Arnold,‡,§ Kimba L. Ashworth,† Irini Akritopoulou-Zanze,† Peter F. Bousquet,‡
Jennifer J. Bouska,† George A. Cunha,‡ Steven K. Davidsen,† Gilbert J. Diaz,† Stevan W. Djuric,† Alan F. Gasiecki,†
Gary A. Gintant,† Vijaya J. Gracias,† Christopher M. Harris,‡ Kathryn A. Houseman,† Charles W. Hutchins,† Eric F. Johnson,†
Hu Li,† Patrick A. Marcotte,† Ruth L. Martin,† Michael R. Michaelides,† Michelle Nyein,† Thomas J. Sowin,† Zhi Su,†
Paul H. Tapang,† Zhiren Xia,† and Henry Q. Zhang†
Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois 60064-6217, and
Abbott Bioresearch Center, 100 Research DriVe, Worcester, Massachusetts 01605-5314
ReceiVed October 18, 2006
The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is
described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral
activity in an estradiol-induced murine uterine edema model (ED50 ) 3 mg/kg) superior to Sutent (ED50
)
9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth
model (tumor growth inhibition ) 90% at 25 mg/kg‚day po). The compound was docked into a homology
model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve
its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652,
and Ser624 yielded 90, which only showed an IC50 of 11.6 µM in the hERG patch clamp assay. The selectivity
profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with
low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.
Introduction
growth signals, insensitivity to antigrowth signals, evasion of
apoptosis, unlimited replicative potential, sustained angiogenesis,
and tissue invasion and metastasis.5 Members of the PDGF
(PDGFR-R, PDGFR-â, FLT3, CSF1R, and cKit) and VEGF
(KDR, FLT1, and FLT4) RTK subfamilies appear to play
essential roles in all stages of tumor angiogenesis, are able to
form autocrine loops which mediate cancer cell growth and
survival, and drive hematologic malignancies.6 Initial strategies
to target these subfamilies for the development of anticancer
therapies have focused on the selective inhibition of signaling
by individual RTKs and have resulted in the approval of Avastin
for the treatment of metastatic colorectal cancer7 and Gleevec
for cKit-driven gastrointestinal tumors.8 However, more recent
preclinical studies have demonstrated that simultaneous inhibi-
tion of multiple kinases by single-agent kinase inhibitors has
the potential to greatly enhance antitumor activities, while still
maintaining acceptable toxicity profiles. Proof-of-concept for
this approach can be derived from Sunitinib maleate (Sutent,
SU11248), a multitargeted (class III/V) RTK inhibitor, which
was efficacious in clinical trials for the treatment of gastrointes-
tinal stromal tumors and advanced renal-cell carcinoma.9
Protein kinases are a large (about 1.7% of all human genes)
and diverse multigene family of enzymes, which mediate most
of the signal transduction events in eukaryotic cells. They are
characterized by their ability to transfer the γ-phosphate group
of ATP to hydroxyl groups on their target proteins. All of the
518 protein kinases encoded in the human genome have been
classified based on the phylogeny of their catalytic domains
and distinct structural characteristics.1 Approximately 90 protein
kinases selectively catalyze the phosphorylation of tyrosine
hydroxyl groups, and 58 of those protein tyrosine kinases
were categorized as receptor tyrosine kinases (RTKs) due to
the presence of a transmembrane spanning domain.2 Further
differentiation of the RTKs was achieved through comparison
of their extracellular domains. For example, the members of
the PDGF and VEGF receptor tyrosine kinase subfamilies
contain five (class III RTKs) or seven (class V RTKs)
immunoglobulin-like domains, respectively.3,4 RTKs allosteri-
cally transmit extracellular signals through the plasma membrane
and activate downstream signaling cascades which mediate
growth, differentiation, and developmental signals in cells.
Disregulation of those tightly controlled processes contributes
significantly to the six hallmarks of cancer: self-sufficiency in
Recently, we disclosed 1,4-dihydroindeno[1,2-c]pyrazoles as
a novel class of KDR kinase inhibitors.10 Attachment of a urea-
based side chain to access the hydrophobic specificity pocket
in KDR kinase then led to a series of potent multitargeted (KDR,
FLT1, cKit, Tie2) receptor tyrosine kinase inhibitors.11 Disap-
pointingly, when dosed intraperitoneally (ip) in a mouse uterine
edema (UE) model, these compounds displayed only mediocre
in ViVo efficacies (Table 1). In addition, a detailed analysis of
* Corresponding author. Target and Lead Discovery, Dept. R43G, Bldg.
AP52, 100 Abbott Park Rd., Abbott Park, IL 60064-6217. Phone: (847)
935-1448. Fax: (847) 938-2756, e-mail: jurgen.dinges@abbott.com.
† Abbott Laboratories.
‡ Abbott Bioresearch Center.
§ Present location: OSI Pharmaceuticals, Inc.
10.1021/jm061223o CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/11/2007

2012 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
Scheme 1a
Table 1. In Vitro Potencies and in ViVo Efficacies of a Selected Set of
1,4-Dihydroindeno[1,2-c]pyrazoles with Urea-type Side Chains
a Reagents and conditions: (a) (i) NaH, benzene, reflux, (ii) H2NNH2‚H2O,
HOAc, EtOH, reflux, 83%; (b) phenyl propargyl ether, PdCl2(PPh3)2, CuI,
PPh3, Et3N, DMF, microwave 120 °C, 62%.
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate, variability around the mean
value was <50%. b Intraperitoneal dosing, data reported as an average of
five animals with associated p-values vs vehicle in parentheses. c Determined
by ELISA. d Determined by Western blot analysis.
Scheme 2a
the ADMET (Absorption, Distribution, Metabolism, Excretion,
Toxicity) profile of a selected set of ureas and their precursors
showed that the urea-based side chain gave rise to unfavorable
pharmacokinetic properties (short half-lives, low oral bioavail-
abilities). Attempts to substitute this side chain with urea
mimetics and bioisosteres, or to replace it with substituents based
on other functional groups, fortuitously led to the discovery of
a new series of alkynes. In this report we now describe the
structure-activity relationships (SARs) that were developed for
1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side
chains and our efforts to improve the cardiac safety profile of
this class of compounds by minimizing their affinity for the
hERG potassium channel.
a Reagents and conditions: (a) phenyl propargyl ether, PdCl2(PPh3)2,
CuI, PPh3, Et3N, DMF, microwave 120 °C, 86%; (b) 5, NaH, benzene,
reflux, (ii) H2NNH2‚H2O, HOAc, EtOH, reflux, 54%.
5-fluoroindanone (16) with N-methylpiperazine (Method B)
produced indanone 17 without a tether to the N-methylpiperazine
group. The next higher homologue of 5 could be obtained via
lithium-halogen exchange of the protected bromoindanone 11,10
followed by treatment with ethylene oxide14 to furnish alcohol
18 (Method C). Hydrolysis of the dioxolane (19), mesylation
of the OH-group (20), and subsequent nucleophilic substitution
with N-methylpiperazine afforded 21 in 11% overall yield. For
the preparation of an indanone with a C-linked basic side chain,
the hydroxymethyl indanone 13 was mesylated and then
converted to the phosphonate 22 in 78% yield (Method D).
Successive Horner-Wadsworth-Emmons reaction with 1-methyl-
4-piperidone followed by hydrogenation of the generated C-C-
double bond and hydrolysis of the dioxolane yielded the
piperidine-type indanone 23. Palladium-mediated carboxami-
nation of 10 gave rise to indanone 24 (Method E) with a
carbonyl functionality as a tether to the N-methylpiperazine
group. O-Alkylation of hydroxyindanone 25 with alkyl halides
in the presence of potassium carbonate led to indanone ethers
as, for example, 26.
The highest yields in the coupling reaction to form the 1,4-
dihydroindeno[1,2-c]pyrazole ring system were achieved when
the aryl carboxylates were used in form of their phenol esters.
Preparation of these phenol esters followed a standard protocol
(DCC, DMAP, CH2Cl2/ether, 0 °C to r.t.). The precursor for 6,
5-bromothiophene-3-carboxylic acid, was synthesized following
Campaigne et al.’s15 conditions for the bromination of thiophene-
3-carboxylic acid.
The applied routes for the syntheses of modified acetylenes
are illustrated in Scheme 4. Alcohol 28 was accessed from
propargyl bromide (27) as described by Kitching et al. (Method
1).16 Alkylation with 27 was the method of choice for the
Chemistry
The synthesis of 1,4-dihydroindeno[1,2-c]pyrazoles with
acetylene-type side chains is exemplified with the synthesis of
8 (Scheme 1). Cyclization of indanone 5 with the bromo-
thiophene ester 6 provided 1,4-dihydroindeno[1,2-c]pyrazole 7
as a key intermediate for the preparation of various target
compounds with modified acetylenic side chains. For instance,
Sonogashira coupling of 7 with phenyl propargyl ether under
microwave conditions12 afforded alkyne 8. Alternatively, it was
also possible to reverse the two synthesis steps (Scheme 2). As
such, Sonogashira coupling of the bromothiophene ester 6 with
phenyl propargyl ether furnished alkyne 9, which served as a
key building block for target compounds with modifications of
the basic side chain. Cyclization of 9 with indanone 5, for
example, also gave access to the 1,4-dihydroindeno[1,2-c]-
pyrazole 8. The yields for the latter route were commonly
slightly lower than for the previous approach. Analogues of 8,
with the polar side chain in the 7-position, were generated in
analogy to Schemes 1 and 2.
The syntheses of indanones with modified side chains are
illustrated in Scheme 3. The majority of indanones were
generated according to Method A, using commercially available
aliphatic or aromatic heterocycles, as described previously.10
One heterocycle, 1-methylpiperazin-2-one, was prepared ac-
cording to Cusic et al.13 Nucleophilic aromatic substitution of

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2013
Scheme 4. Synthesis of Acetylenesa
Scheme 3. Synthesis of Indanone Precursorsa
a Reagents and conditions: (a) (i) Al-powder, HgCl2, THF, 45 °C; (ii)
acetone, THF, 0 to 45 °C, 70%; (b) 3-hydroxypyridine, KOH, DMF, 5 °C
to r.t., 10%; (c) aniline, EtOH, r.t., 91%; (d) 2-chlorophenol, K2CO3, acetone,
56 °C, 50%; (e) BzCl, Et3N, CH2Cl2, 0 °C to r.t., 34%; (f) morpholine,
MP-borohydride, MeOH, r.t. 98%; (g) propargyl alcohol (38), THF, PS-
PPh3, DEAD, r.t. 93%; (h) (i) NaH, THF, 5 °C, (ii) 2-bromoethyl methyl
ether, 5 °C to r.t. 56%.
a Reagents and conditions: (a) ethylene glycol, p-TsOH, benzene, reflux
(Dean Stark trap), 82%; (b) (i) n-BuLi, THF, -78 °C; (ii) DMF, THF,
-78 °C to r.t., 78%; (c) NaBH4, MeOH/THF (10:1), 0 °C to r.t., 85%; (d)
p-TsOH, acetone/H2O (4:1), reflux, 69%; (e) MsCl, Et3N, THF, 0 °C, 96%;
(f) N-methylpiperazine, K2CO3, EtOH, 0 °C to r.t., 68%; (g) N-methylpip-
erazine, 100 °C, 60%; (h) (i) n-BuLi, THF, -78 °C; (ii) ethylene oxide,
ether, -78 °C to 0 °C, 42%; (j) HPO(OEt)2, NaH, THF, reflux, 78%; (k)
(i) NaH, DME, 0 °C; (ii) 1-methyl-4-piperidone, reflux, 54%; (l) H2 (60
psi), 10% Pd/C, MeOH, r.t., 89%; (m) CO (130 psi), N-methylpiperazine,
PdCl2(dppf)‚CH2Cl2, Et3N, 110 °C, 40%; (n) 2-chloroethyl methyl ether,
K2CO3, CH3CN, reflux, 84%.
propargyl alcohol (38) afforded acetylene 37 (Method 6).
Aliphatic ethers of propargyl alcohol, for example 39, were
prepared by alkylation of 38 with alkyl halides (Method 7).
In several cases, acetylenic side chains were further modi-
fied after coupling to the 1,4-dihydroindeno[1,2-c]pyrazole
precursor 7 (Scheme 5). For instance, Sonogashira coupling of
7 with trimethylsilylacetylene and subsequent desilylation of
40 with tetra-n-butylammonium fluoride (TBAF) gave rise to
compound 41 with a terminal alkyne group (Method I). The
amine intermediate 43 was synthesized by coupling of 7 with
N-propargylphthalimide (89% yield), protection of the pyra-
zole-NH group in 42 by employing 4,4′-dimethoxybenz-
hydryl chloride and deprotection of the primary amine func-
tionality with anhydrous hydrazine. Starting from 43, the
sulfonamide 44 (Method II), the carbamate 45 (Method III) and
the urea 46 (Method IV) could be obtained via application of
production of propargyl ethers of aromatic heterocycles like 2917
(Method 2) and secondary propargyl amines such as 30 (Method
3). In contrast, optimum yields for the synthesis of aryl propargyl
ethers like 32 were obtained when the propargyl toluenesulfonate
31a was used as alkylating agent (Method 4). Acylation of amine
33 with benzoyl chloride generated amide 34 (Method 5).18
Reductive amination of 35 with morpholine through treatment
with macroporous triethylammonium methylpolystyrene boro-
hydride, followed by Mitsunobu reaction of the phenol 36 with

2014 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Scheme 5. Modifications of Acetylene Side Chainsa
Dinges et al.
a Reagents and conditions: (a) PdCl2(PPh3)2, CuI, PPh3, Et3N, DMF, microwave 120 °C, 75%; (b) TBAF‚H2O, THF, r.t., 72%; (c) 4,4′-dimethoxybenzhydryl
chloride, Et3N, THF, 50 °C, 60%; (d) H2NNH2, EtOH/THF (1:1), r.t., 46%; (e) (i) PhSO2Cl, pyridine, r.t.; (ii) 4M HCl in dioxane, r.t., 47%; (f) (i) PhOCOCl,
Et3N, CH2Cl2, r.t.; (ii) 37% HCl in EtOH/EtOAc, r.t., 11%; (g) (i) PhNCO, THF, r.t.; (ii) 4M HCl in dioxane, r.t., 30%; (h) 2M NH3 in MeOH, r.t., 68%.
well-established reaction conditions. Conversion of the ester
47 to the amide 48 was achieved by stirring of the starting
material for 3 days in a 2 M solution of ammonia in methanol
(Method V).
Ahx-AEEEYFFLFA-amide) by KDR kinase in a homogeneous
time-resolved fluorescence (HTRF) assay at physiological
relevant concentrations (1.0 mM) of adenosine-5′-triphosphate
(ATP). Potent compounds (IC50 < 100 nM) were then evaluated
for their effectiveness to inhibit VEGF-induced KDR phospho-
rylation in a 3T3 murine fibroblast cell line, stably transfected
with full length human KDR.
Previously established SAR demonstrated the importance of
reoptimizing the substitution pattern on the thiophene spacer
and the position of the basic side chain. The results are listed
in Table 2. In contrast to the urea-type 1,4-dihydroindeno[1,2-
c]pyrazoles, a 3′,5′-disubstitution pattern on the thienyl spacer
(8) now appears to provide a better trajectory to accommodate
the more rigid acetylenic side chain in the hydrophobic
specificity pocket of KDR kinase. Compound 52, with a 2′,5′-
disubstituted thienyl, which was the preferred constellation for
the urea-type analogues, is about 4-fold less active than 8, and
53, with a 2′,4′-substitution pattern on the thienyl group, is about
8-fold less potent. Attempts to replace the thienyl moiety with
a phenyl group (54 and 55) turned out to be detrimental. A
comparison between compounds 8 and 56 illustrates that the
Two compounds with modifications of the general chemotype
were synthesized as shown in Scheme 6. The indeno[1,2-c]-
pyrazol-4(1H)-one 50 was obtained through air-oxidation of the
4-methylene group19 in 7 and Sonogashira coupling of the
resulting intermediate 49 with alkyne 39. For the preparation
of the 1,4-dihydroindeno[1,2-c]pyrazol-3-amine 51, the sodium
enolate of 5 was treated with phenyl isothiocyanate, and the
resulting thioamide intermediate was methylated in situ. The
crude material was then refluxed in ethanol in the presence of
hydrazine to give the target compound.20
Results and Discussion
Structure-Activity Studies. Initially, the 1,4-dihydroindeno-
[1,2-c]pyrazoles were optimized for inhibition of the unactivated
form of human KDR kinase. The activities of the target
compounds were assessed based on their ability to inhibit the
phosphorylation of a biotinylated polypeptide substrate (biotin-

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2015
Scheme 6. Synthesis of Related Chemotypes 50 and 51a
Figure 1. Effects of 8 on the growth of MX-1 human xenografts.
Dosing (BID) started on day seven. Tumor volumes are expressed as
means ( SEM; n ) 7-10 per group. The percent inhibition on Day
28 for each dosage compared to control is shown in parentheses in the
legend. Significant differences (p < 0.01 for all groups compared to
vehicle controls (Mann-Whitney)) in mean tumor volume were
observed for all treatment groups by day 18.
The model is based on the fact that a bolus injection of estradiol
into a mouse uterus results in upregulation of VEGF. As a
consequence, vascular permeability is increased, leading to the
development of edema. The efficacy of KDR kinase inhibitors
can be determined based on their capacity to reduce the acute
increase in uterine weight. With an ED50 of 3 (0.1-5.6)
mg/kg,21 8 turned out to be more potent than Sutent (ED50 ) 9
mg/kg) in the same assay. This outstanding result and an
acceptable pharmacokinetic profile (F ) 75% after a 10 mg/kg
oral (po) dose, and T1/2 ) 5.5 h after a 3 mg/kg intravenous
(iv) dose into CD-1 mice)22 qualified the compound to be
evaluated for its antitumor efficacy against tumors derived from
MX-1 human breast carcinoma implanted subcutaneously into
nude mice. The tumor growth inhibition was determined to be
90% at 25 mg/kg‚day po (Figure 1) (PTK787: ED75 > 100
mg/kg‚day po).
a Reagents and conditions: (a) air, Cs2CO3, DMF, 90 °C, 61%; (b) 39,
PdCl2(PPh3)2, CuI, PPh3, Et3N, DMF, microwave 120 °C, 55%; (c) (i)
phenyl isothiocyanate, NaH, DMF, 0 °C to r.t.; (ii) MeI, r.t.; (iii) H2NNH2,
EtOH, reflux, 30%.
Table 2. Optimization of the Spacer in 3-Position and Identification of
the Preferred Regiochemistry for the Basic Side Chain
Maintaining the optimized substitution pattern described for
8, we then focused on the optimization of the acetylenic
substituent of our inhibitors (Table 3). A comparison of the
terminal alkyne 41 and its n-propyl analogue 57 showed that a
minimum size of the substituent was required to maintain potent
KDR activity. However, steric bulk from branched chains or
aliphatic ring systems was not well tolerated. The site was also
sensitive to the introduction of polar functional groups (no
examples shown). One of the few functionalities that could be
utilized was the alcohol 58. The compound was extremely potent
in both the enzyme and the whole cell assays but then failed to
demonstrate sufficient in ViVo efficacy in the uterine edema
model (54 (0.002) % inhibition at 100 mg/kg ip).21 Assuming
that rapid metabolism of the primary hydroxy group could be
responsible for this result, the geminal dimethyl analogue 59
was synthesized. However, this modification led to a dramatic
deterioration of KDR activity. The methyl ether 60 was more
successful with an ED50 of 12.6 (10-120) mg/kg21 after oral
dosing in the UE model. Homologation of 8 to 61 only resulted
in diminished KDR inhibitory activity. Small hydrophobic
substituents on the phenyl ether portion of 8 (62 to 64) were
best tolerated in the meta-position, while the meta- and para-
positions were preferred for disubstituted phenyl ethers (65).
Unfortunately, evaluation of a set of compounds in the UE
model demonstrated that these substitutions on the terminal
phenyl ring generally resulted in reduced in ViVo efficacies (UE
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate, variability around the mean
c
value was <50%. bDetermined by ELISA. Determined by Western blot
analysis.
basic side chain is now preferred in the 6-position, as compared
to the 7-position in the urea series. The in Vitro (IC50 ) 6 nM)
and whole cell (IC50 ) 40 nM) potencies of 8 were comparable
to more advanced competitor compounds like Sutent (in Vitro
IC50 ) 18 nM, whole cell IC50 ) 22 nM), and therefore the
compound was further evaluated in the uterine edema model.

2016 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
Table 3. SAR for 1,4-Dihydroindeno[1,2-c]pyrazoles with a Selected Set of Acetylenic Substituents
a Each IC50 determination was performed with seven concentrations, and each assay point was determined in duplicate, variability around the mean value
was <50%. bDetermined by ELISA. cDetermined by Western blot analysis. dValues are means of two experiments, standard deviation is given in parentheses.
(65): 32 (0.025) % inhibition at 10 mg/kg po).21 Poor efficacies
were also observed for heterocyclic ethers. For example the
3-pyridyl ether 66 only produced 24 (0.040) % inhibition at 10
mg/kg po21 in the UE model. Isosteric replacement of the ether
oxygen in the parent compound 8 with a methylene group led
to the phenethyl analogue 67, which exhibited low whole cell
activity. Substitution with sulfur (not shown) resulted in
markedly reduced potencies as well, but replacement with
nitrogen appeared to be beneficial as long as the basicity of the
resulting amines could be held to a minimum (68, 69, and
44-46). The benzamide 69 and the urea 46 were exceptionally
potent but then could not reach relevant plasma concentrations
after oral dosing (10 mg/kg) into CD-1 mice (F ) 17%, AUC
(po) ) 3.5 µg‚h/mL and F ) 0%, respectively).22
Cardiac Safety Studies. Drug-induced blockade of the hERG
(human ether-a`-go-go)-encoded potassium channel has received
increased attention over the past few years since it can lead to
delayed repolarization and prolongation of the QT interval on
the electrocardiogram. QT prolongation, in turn, is associated
with a predisposition for the polymorphous ventricular arrhyth-
mia Torsades de Pointes, which may degenerate into ventricular
fibrillation and sudden death.23 The [3H]dofetilide membrane
binding assay provides a useful high throughput platform to
assess the potential of early lead compounds to block the hERG
K+ channel.24 In this radioligand binding assay, the test com-
pounds compete with [3H]dofetilide for binding to homogenized
membrane aliqoutes, derived from hERG-transfected HEK 293
cells. Table 3 shows that our KDR inhibitors displayed sig-

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2017
ionic current was measured using hERG-transfected HEK 293
cells.27 At a concentration of 2 µM, 77 did not affect a block of
the tail current in a statistically significant manner (3.7% block
compared to 64% block at 0.5 µM for 8) and only showed an
IC50 of 53 µM.28 Compared to 76, 77 had a slightly better
pharmacokinetic profile (F ) 50% at 10 mg/kg po in CD-1
mice),22 and the results in the UE model were comparable for
both compounds (ED50 ) 5.5 (3-7.7) mg/kg for 76 and 5.7
(3.5-10) mg/kg po for 77).21 Therefore, 77 was further
evaluated for its antitumor activity in an HT1080 fibrosarcoma
mouse flank xenograft model. However, when orally dosed
twice a day with a total daily dose of 10 mg/kg, 77 inhibited
tumor growth only 29% (p-value vs vehicle <0.001) compared
to the vehicle control (Sutent: ED75 ) 65 mg/kg‚day po).
Compounds 79 to 81 are examples where the piperazine moiety
could be replaced with other aliphatic rings and chains to obtain
analogues with acceptable KDR inhibitory potency but no
affinity for the hERG K+ channel. Unfortunately, none of those
compounds showed sufficient efficacy in the UE model (46
(0.007) %, 8 (0.633) %, and 9 (0.510) % inhibition at 10
mg/kg po).21 Replacements with aromatic heterocycles are
exemplified with 82 to 85. The imidazole (82) and 1,2,3-triazole
(84) analogues only displayed low KDR activity, the pyrazole
83 failed to demonstrate sufficient efficacy in the UE model
(14 (0.664) % inhibiton at 10 mg/kg po),21 but the 1,2,4-triazole
85 was potent in Vitro and in ViVo (UE: 96 (<0.001) %
inhibition at 10 mg/kg po),21 with reduced binding to the hERG
channel (patch clamp IC50 ) 1.7 µM).28 Nonetheless, further
evaluations were put on hold because of the compound’s
marginal pharmacokinetic profile (Cl ) 2.5 L/kg‚h, T1/2 ) 0.8
h at 3 mg/kg iv in CD-1 mice).22
Figure 2. Model of 8 (gray) docked into the inner cavity of the hERG
K+ channel (closed state, homology model based on KcsA, PDB entry
1BL8). One of the four subunits was removed for better visibility. Key
binding sensitive residues (Phe656, Tyr652, Ser624) are shown for one
monomer (cyan) as space filling models (orange). Surface (green) is
shown for two other monomers (light green and purple).
nificant hERG affinities. To address this problem, we con-
structed a homology model of the homo-tetrameric pore do-
main of hERG in its closed state (drug trapping mode), based
on the crystal structure of the bacterial KcsA potassium channel
(Figure 2).25,26 The 1,4-dihydroindeno[1,2-c]pyrazole 8 was then
docked into the inner ion conduction cavity. The best fit was
obtained by orienting 8 parallel to the pore axis, such that the
more hydrophobic acetylenic ether moiety was located at the
intracellular mouth. This allowed the π-stacking interaction
between the 1,4-dihydroindeno[1,2-c]pyrazole core and Phe656
in the S6 helix. The N-methylpiperazine-containing side chain,
which is protonated under physiological pH, aligned with its
internal nitrogen atom such that π-cation interactions with
Tyr652 were possible, while the external nitrogen was able to
interact with Ser624 in the pore helix of the selectivity filter.
Based on the binding mode of 8, three strategies were developed
to interrupt the binding to the hERG K+ channel.
First, the basic side chain was modified (Table 4). Compound
70 demonstrated that the hERG affinity was not intrinsic to the
chemotype but also that a polar side chain was required for
potent KDR activity. Extending the tether to the N-methylpip-
erazine group (72) reduced hERG activity more than completely
removing it (71) but at the same time led to a loss of whole
cell activity. Selective isosteric replacement of either basic
nitrogen atom with a CH group (73 and 74) revealed that both
nitrogens contribute to the hERG binding; the internal nitrogen
(73), however, appeared to play a more important role. This
result is in agreement with our computer model predictions,
since both nitrogens interact with the key binding-sensitive
residues Tyr652 and Ser624, but interaction with Tyr652 is
considered to provide the more significant contribution to the
binding energy. The basicity of either nitrogen atom was then
modulated through the introduction of neighboring carbonyl
groups (75 to 78). In the case of 75 and 78, with the carbonyl
group external to the piperazine ring, the priority of the nitrogen
atoms for binding to hERG was reversed compared to 73 and
74. This might be due to conformational changes since 78
contained an sp2-configured tether. Gratifyingly, both 76 and
77 had excellent activities against KDR but did not display any
affinity for hERG. This result was confirmed for 77 in a whole
cell patch clamp assay, where the inhibition of the actual hERG
The second strategy to reduce the affinity of our KDR kinase
inhibitors to bind to the hERG K+ channel was based on
increasing the polarity of the acetylenic side chain (Table 5).
Attachment, for example, of a morpholine group in the para-
postion of the phenyl ether (86) had no beneficial effect on
reducing hERG affinity. Using 60, one of the least hERG active
compounds from the initial optimization of the acetylenic side
chain (Table 3), as a parent compound, the methyl ether was
extended with additional polar functionalities. This strategy
generally led to a reduction of hERG channel affinity, but in
most cases those compounds also lost their inhibitory potency
against KDR (48). The only exception was 87; the glycol ether
moiety of this compound clearly reduced hERG binding and at
the same time maintained acceptable KDR activity in Vitro. In
ViVo, 87 exhibited 71 (0.001) % inhibition at 25 mg/kg po21 in
the uterine edema model and 76% tumor growth inhibition at
70 mg/kg‚day po (p-value vs vehicle <0.001) in the MX-1
tumor xenograft model. Reoptimization of the regiochemistry
of the polar side chain (87 vs 88) and combination with the
earlier identified 1,2,4-triazole resulted in 89 and 90 (patch
clamp IC50 > 150 µM and 11.6 µM, respectively).28 The
pharmacokinetic profile for 89 was characterized by a relatively
high clearance rate, a short half-life, and low oral bioavailability
(Cl ) 3.4 L/h‚kg and T1/2 ) 0.3 h at 3 mg/kg iv, and F ) 22%
at 10 mg/kg po in CD-1 mice).22 The profile was slightly better
for 90 (Cl ) 2.2 L/h‚kg and T1/2 ) 0.3 h at 3 mg/kg iv, and F
) 64% at 10 mg/kg po in CD-1 mice)22 and the compound
achieved 57 (<0.001) % inhibition at 10 mg/kg po21 in the
uterine edema model.
Finally, we investigated whether two common variations of
the 1,4-dihydroindeno[1,2-c]pyrazole core have the potential to
disrupt the proposed π-stacking interactions with Phe656 either
sterically or electronically (Figure 3). Introduction of a carbonyl

2018 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
Table 4. Selected Modifications of the Polar Side Chain in 8 and their Effect on KDR Inhibitory Potency and Binding to the hERG K+ Channel
a Each IC50 determination was performed with seven concentrations, and each assay point was determined in duplicate, variability around the mean
c
value was <50%. bDetermined by ELISA. Determined by Western blot analysis. dValues are means of two experiments, standard deviation is given in
parentheses.
functionality into the methylene-bridge of the pharmacophore,
exemplified by 50, was tolerated but did not result in any
considerable improvement (UE: 54 (0.010) % inhibition at 10
mg/kg po).21 Insertion of an NH group between the pharma-
cophore and the thiophene spacer (51 vs 9110) rendered the
compound inactive.
The selectivity profile of the two key compounds 8 and 90
are listed in Table 6. Both compounds can clearly be character-
ized as potent multitargeted (class III/V) receptor tyrosine kinase
inhibitors with comparable potencies against both, the kinases
of the VEGFR and PDGFR subfamilies. On the other hand,
the compounds are much less active against kinases that are
structurally less homologous such as Tie2, LCK, FYN, SRC,
and HCK.
inhibition ) 90% at 25 mg/kg‚day po). However, the compound
also showed a strong affinity for the hERG potassium channel.
Docking of 8 into a homology model of the homo-tetrameric
pore domain of hERG allowed the development of strategies
to systematically interrupt key binding interactions. Replacement
of the 4-methylpiperazine moiety in 8 with 1,2,4-triazole
minimized interactions with Tyr652 and Ser624, and changing
the phenyl propargyl ether to its methoxyethyl analogue
disrupted the binding in the intracellular mouth of the hERG
channel. Alternatively, attempts to reduce the π-stacking
interactions between the 1,4-dihydroindeno[1,2-c]pyrazole core
and Phe656 turned out to be less successful. Combination of
the improved features ultimately led to the discovery of 90,
which, compared to 8, displayed a strongly reduced hERG
affinity (IC50 ) 11.6 µM in the hERG patch clamp assay) but
also a lower in ViVo efficacy. The selectivity profile for the key
compounds 8 and 90 revealed that both are multitargeted
receptor kinase inhibitors with low nanomolar potencies against
other structurally related members of the VEGFR- and PDGFR
kinase subfamilies. Studies to improve the in ViVo efficacy
profile of 90 will be published in due time.
Conclusion
A series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-
type side chains was discovered as novel KDR kinase inhibitors.
Extensive SAR studies established a 3′,5′-disubstitution pattern
for the thienyl spacer between the pharmacophore and the
alkyne. A second substituent, containing a 4-methylpiperazine
moiety, was preferred in the 6-position of the 1,4-dihydroindeno-
[1,2-c]pyrazole core. Optimization of the acetylene-type side
chain identified the phenylpropargyl ether 8, which displayed
an oral activity in the estradiol-induced murine uterine edema
model (ED50 ) 3 mg/kg) superior to Sutent (ED50 ) 9 mg/kg)
and potent antitumor efficacy against the MX-1 human breast
carcinoma xenograft tumor growth model (tumor growth
Experimental Section
Chemistry. All reactions were carried out under inert atmosphere
(N2). Solvents and reagents were obtained commercially and were
used without further purification. Normal-phase flash chromatog-
raphy was carried out using Merck Kieselgel 60 (230-400 mesh)
or was performed on an Analogix IntelliFlash system with pre-
packed columns. Reverse-phase chromatography was carried out

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2019
Table 5. Selected Modifications of the Propargyl Ether and the Polar
Chain in 8 and Their Effect on KDR Inhibitory Potency and Binding to
the hERG K+ Channel
MHz), or Varian UNITY plus or Mercury (300 MHz) spectrometer.
The chemical shifts are reported as δ values (ppm) relative to
tetramethylsilane as an internal standard. Mass spectra were
obtained on a Finnigan MAT SSQ700 instrument. High resultion
mass spectra were generated on an Agilent MSDTOF instrument.
The above spectral data were obtained through the Department of
Structural Chemistry at Abbott Laboratories. Elemental analysis
was performed by Robertson Microlit Laboratories, Inc. Madison,
NJ, or Quantitative Technologies, Inc. Whitehouse, NJ, and the
results indicated by elemental symbols are within (0.4% of
theoretical values. Analytical HPLC was performed on a Waters
2690 HPLC system, peaks were acquired at λ ) 254 nm (given)
and 220 nm (see Supporting Information). The methods used were
(A) a YMC ODS-A C18 column (4.6 × 150 mm, 5.5 µm particle
size) using a gradient of 10-100% acetonitrile and 0.1% trifluo-
roacetic acid in water at a flow rate of 1 mL/min over 20 min and
(B) a Xterra RP18 column (4.6 × 150 mm, 5 µm particle size)
using a gradient of 20-100% methanol and water at a flow rate of
1 mL/min over 20 min.
3-(5-Bromothiophen-3-yl)-6-((4-methylpiperazin-1-yl)methyl)-
1,4-dihydroindeno[1,2-c]pyrazole (7). A mixture of 510 (360 mg,
1.47 mmol), 610 (416 mg, 1.47 mmol), and 60% NaH in mineral
oil (147 mg, 3.68 mmol) in benzene (5.5 mL) was heated to reflux
for 2.5 h. The reaction was cooled and quenched by dropwise
addition of 50% aq HOAc. The solvents were evaporated, and the
residue was dried in high vacuum for 1 h. The crude intermediate
was taken up in ethanol (5.5 mL), hydrazine monohydrate (107
µL, 2.21 mmol), and HOAc (252 µL, 4.41 mmol) were added, and
the mixture was heated to reflux for 4 h. The mixture was cooled
and concentrated, and the crude product was purified by flash
chromatography on silica gel eluting with 10:1 dichloromethane/
methanol + 1% ammonium hydroxide to provide the title compound
(524 mg, 83%). 1H NMR (500 MHz, CDCl3) δ 2.32 (s, 3H), 2.4-
2.65 (m, 8H), 3.58 (s, 2H), 3.70 (s, 2H), 7.29 (d, J ) 7.8 Hz, 1H),
7.37 (d, J ) 1.3 Hz, 1H), 7.40 (d, J ) 1.6 Hz, 1H), 7.49 (s, 1H),
7.61 (d, J ) 7.8 Hz, 1H). MS (ESI) m/z 429, 431 (M + H)+.
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-(3-phenoxyprop-1-
ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(8). A mixture of 7 (200 mg, 0.47 mmol), phenyl propargyl ether
(68 mg, 0.51 mmol), triphenylphosphine (24 mg, 0.09 mmol),
dichlorobis(triphenylphosphine)palladium(II) (16 mg, 0.023 mmol),
copper iodide (4 mg, 0.023 mmol), and triethylamine (0.97 mL,
6.99 mmol) in DMF (2 mL) was stirred under nitrogen in a heavy
walled process vial in a microwave synthesizer at 120 °C for 25
min. The reaction mixture was concentrated, and the crude product
was purified by preparative reverse phase HPLC to give the title
compound (239 mg, 62%). 1H NMR (500 MHz, DMSO-d6) δ 2.81
(s, 3H), 3.00-3.50 (m, 8H), 3.83 (s, 2H), 3.89 (bs, 2H), 5.11 (s,
2H), 6.99-7.02 (m, 1H), 7.05-7.06 (m, 2H), 7.33-7.37 (m, 2H),
7.40 (d, J ) 7.8 Hz, 1H), 7.59 (s, 1H), 7.67 (d, J ) 7.8 Hz, 1H),
7.74 (d, J ) 1.3 Hz, 1H), 7.90 (d, J ) 1.3 Hz, 1H). MS (ESI) m/z
481 (M + H)+. Anal. (C29H28N4OS‚3.5CF3CO2H) C, H, N.
Phenyl 5-(3-Phenoxyprop-1-ynyl)thiophene-3-carboxylate (9).
Compound 9 was prepared using the same procedure as described
for the synthesis of 8 by substituting 6 for 7. The title compound
was obtained after purification by flash chromatography on silica
gel using 5:1 hexane/ethyl acetate as the mobile phase (3.0 g, 86%).
1H NMR (500 MHz, DMSO-d6) δ 5.10 (s, 2H), 6.98-7.01 (m,
1H), 7.04-7.05 (m, 2H), 7.25-7.27 (m, 2H), 7.29-7.36 (m, 3H),
7.45-7.48 (m, 2H), 7.76 (d, J ) 1.3 Hz, 1H), 8.62 (d, J ) 1.3 Hz,
1H). MS (DCI) m/z 352 (M + NH4)+.
a Each IC50 determination was performed with seven concentrations, and
each assay point was determined in duplicate, variability around the mean
c
value was <50%. bDetermined by ELISA. Determined by Western blot
analysis. dValues are means of two experiments, standard deviation is given
in parentheses.
Figure 3. Modifications of the 1,4-dihydroindeno[1,2-c]pyrazole core.
Each KDR IC50 determination was performed with seven concentrations,
and each assay point was determined in duplicate. The IC50 values from
the [3H]dofetilide binding assay and the hERG patch clamp assay are
the means of two experiments.
5-(4-Methylpiperazin-1-yl)indan-1-one (17). A solution of
5-fluoro-1-indanone (3.5 g, 23.3 mmol) in 1-methylpiperazine (20
mL) was heated to 100 °C for 20 h. The reaction mixture was
concentrated, and the crude product was purified by flash chro-
matography on silica gel eluting with ethyl acetate/methanol
on a Waters HPLC system equipped with a Gilson FC204 fraction
collector. Samples were purified on a Waters Symmetry C8 column
(25 mm x 100 mm, 7 µm particle size) using a gradient of 10% to
100% acetonitrile and 0.1% aqueous TFA over 8 min (10 min run
time) at a flow rate of 40 mL/min. 1H NMR spectra were recorded
on a Varian UNITY or Inova (500 MHz), Varian UNITY (400
1
(97:3) to provide the title compound (3.2 g, 60%). H NMR (300
MHz, CD3OD) δ 2.35 (s, 3H), 2.57-2.63 (m, 6H), 3.03-3.07
(m, 2H), 3.43-3.46 (m, 4H), 6.94 (d, J ) 2.4 Hz, 1H), 6.98 (dd,
J ) 8.6, 2.4 Hz, 1H), 7.56 (d, J ) 8.6 Hz, 1H). MS (ESI) m/z 231
(M + H)+.

2020 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
Table 6. Kinase Inhibition Profiles of 1,4-Dihydroindeno[1,2-c]pyrazoles 8 and 90
IC50, nMa
VEGFR family
FLT1
PDGFR family
FLT3
other families
FYN
Cmpds
KDR
FLT4
PDGFRb
CSF1R
cKit
Tie2
LCK
SRC
HCK
8
90
6
5
2
<3
3
4
58
84
9
<3
8
9
26
12
4600
8100
>50000
11700
27300
>50000
30400
>50000
>50000
>50000
a Each IC50 determination was performed with seven concentrations, and each assay point was determined in duplicate; variability around the mean value
was <50%. b Values of cellular phosphorylation by ELISA assay.
2-(Spiro[1,3dioxolane-2,1′-Indane]-5′-yl)ethanol (18). To a solu-
tion of 1110 (470 mg, 1.84 mmol) in THF (5.5 mL) was added a
1.6 M solution of n-butyllithium in hexanes (1.44 mL, 2.3 mmol)
dropwise at -78 °C. To this mixture was then added a solution of
ethylene oxide (1.21 g, 27.5 mmol) in diethyl ether (2.1 mL) at
-78 °C. The mixture was allowed to warm to 0 °C over 2 h, and
stirring at 0 °C was continued for an additional 2 h. The reaction
was quenched by addition of H2O (10 mL), and the mixture was
extracted four times with ethyl acetate. The combined organic
extracts were dried (MgSO4), filtered, and evaporated, and the crude
product was purified by flash column chromatography on silica
gel using hexane/ethyl acetate (1:1) as the mobile phase to give
the title compound (170 mg, 42%). 1H NMR (300 MHz, CDCl3) δ
2.28-2.32 (m, 2H), 2.91-2.98 (m, 4H), 3.92 (t, J ) 6.4 Hz, 2H),
4.05-4.21 (m, 4H), 7.25 (d, J ) 7.8 Hz, 1H), 7.36 (s, 1H), 7.71
(d, J ) 7.8 Hz, 1H). MS (DCI) m/z 221 (M + H)+.
5-(2-Hydroxyethyl)indan-1-one (19). A mixture of 18 (170 mg,
0.77 mmol) and p-TsOH (147 mg, 0.77 mmol) in 4:1 acetone/H2O
(2.5 mL) was refluxed for 1 h. The reaction mixture was
concentrated, diluted with H2O, and neutralized by addition of
K2CO3. The aqueous layer was extracted five times with ethyl
acetate, and the combined organic extracts were washed with brine,
dried (MgSO4), and concentrated to provide the title compound
(121 mg, 89%). 1H NMR (300 MHz, CDCl3) δ 2.67-2.71 (m, 2H),
2.96 (t, J ) 6.4 Hz, 2H), 3.10-3.14 (m, 2H), 3.92 (t, J ) 6.4 Hz,
2H), 7.24 (d, J ) 7.8 Hz, 1H), 7.35 (s, 1H), 7.71 (d, J ) 7.8 Hz,
1H). MS (DCI) m/z 177 (M + H)+.
suspension of NaH in mineral oil (252 mg, 6.3 mmol) in THF (5
mL) at room temperature. The reaction mixture was refluxed for 1
h, cooled, and quenched with H2O. The layers were separated, and
the aqueous phase was extracted three times with ethyl acetate.
The combined organic extracts were dried (MgSO4) and evaporated,
and the crude product was purified by flash column chromatography
on silica gel using ethyl acetate as the mobile phase to provide the
1
title compound (954 mg, 78%). H NMR (300 MHz, CDCl3) δ
1.25 (t, J ) 7.1 Hz, 6H), 2.27-2.32 (m, 2H), 2.91-2.95 (m, 2H),
3.14 (d, J ) 31.7 Hz, 2H), 3.95-4.05 (m, 4H), 4.06-4.21 (m,
4H), 7.16-7.19 (m, 2H), 7.29 (d, J ) 8.1 Hz, 1H). MS (DCI) m/z
327 (M + H)+.
4-((Spiro[1,3dioxolane-2,1′-indane]-5′-yl)methylene)-1-meth-
ylpiperidine (92). To a 60% suspension of NaH in mineral oil (90
mg, 2.24 mmol) in 1,2-dimethoxyethane (3.5 mL) was added
dropwise a solution of 22 (665 mg, 2.04 mmol) in 1,2-dimethoxy-
ethane (2.0 mL) at 0 °C. The mixture was stirred for 15 min before
a solution of 1-methyl-4-piperidone (231 mg, 2.04 mmol) in 1,2-
dimethoxyethane (2.0 mL) was added. The mixture was heated to
reflux overnight, cooled, quenched with H2O, and concentrated.
The residue was partitioned between ethyl acetate and H2O. The
organic layer was separated, dried (MgSO4), and concentrated. The
crude product was purified by flash column chromatography on
silica gel using 10:1 dichloromethane/methanol + 1% ammonium
hydroxide as the mobile phase to provide the title compound (313
mg, 54%). 1H NMR (300 MHz, CDCl3) δ 2.28-2.33 (m, 2H), 2.38
(s, 3H), 2.43-2.67 (m, 8H), 2.91-2.96 (m, 2H), 4.05-4.22 (m,
4H), 6.33 (s, 1H), 7.05-7.08 (m, 2H), 7.31 (d, J ) 7.5 Hz, 1H).
MS (DCI) m/z 286 (M + H)+.
2-(1-Oxo-indan-5-yl)ethyl Methanesulfonate (20). To a solution
of 19 (110 mg, 0.62 mmol) in THF (5 mL) was added triethylamine
(130 µL, 0.94 mmol), followed by methanesulfonyl chloride (58
µL, 0.75 mmol) at 0 °C. The mixture was stirred for 1 h, while
being allowed to warm to ambient temperature, poured into H2O,
and extracted five times with ethyl acetate. The combined organic
extracts were dried (MgSO4) and concentrated to provide the title
4-((Spiro[1,3dioxolane-2,1′-indane]-5′-yl)methyl)-1-methylpi-
peridine (93). To a solution of 92 (350 mg, 1.35 mmol) in methanol
(70 mL) was added 10% palladium on charcoal (350 mg), and the
mixture was hydrogenated at 60 psi for 2 h. The mixture was filtered
through Celite, and the filtrate was concentrated to provide the title
1
1
compound (150 mg, 95%). H NMR (300 MHz, CDCl3) δ 2.68-
compound (344 mg, 89%). H NMR (300 MHz, CDCl3) δ 1.49-
2.72 (m, 2H), 2.93 (s, 3H), 3.12-3.17 (m, 4H), 4.46 (t, J ) 6.4
Hz, 2H), 7.25 (d, J ) 7.8 Hz, 1H), 7.37 (s, 1H), 7.73 (d, J ) 7.8
Hz, 1H). MS (DCI) m/z 255 (M + H)+.
1.57 (m, 2H), 1.68-1.72 (m, 2H), 2.02-2.16 (m, 1H), 2.27-2.31
(m, 4H), 2.41 (s, 3H), 2.55-2.57 (m, 2H), 2.89-2.94 (m, 2H),
3.01-3.05 (m, 2H), 4.05-4.12 (m, 2H), 4.13-4.21 (m, 2H),
7.00-7.03 (m, 2H), 7.27 (d, J ) 7.5 Hz, 1H). MS (DCI) m/z 288
(M + H)+.
5-(2-(4-Methylpiperazin-1-yl)ethyl)indan-1-one (21). To a
suspension of 20 (150 mg, 0.59 mmol) and potassium carbonate
(163 mg, 1.18 mmol) in 1:1 ethanol/THF (8 mL) was added
1-methylpiperazine (131 µL, 1.18 mmol). The mixture was refluxed
overnight and concentrated, and the crude product was purified by
flash column chromatography on silica gel eluting with 10:1
dichloromethane/methanol + 1% ammonium hydroxide to provide
5-((1-Methylpiperidin-4-yl)methyl)indan-1-one (23). A solu-
tion of 93 (340 mg, 1.18 mmol) and p-toluenesulfonic acid
monohydrate (225 mg, 1.18 mmol) in a mixture of 5:1 acetone/
H2O (6 mL) was refluxed for 1 h. The mixture was cooled,
concentrated, and partitioned between ethyl acetate and a saturated
aqueous solution of potassium carbonate. The layers were separated,
and the aqueous phase was extracted five times with ethyl acetate.
The combined organic extracts were dried (MgSO4) and evaporated,
and the crude product was purified by flash column chromatography
on silica gel using 10:1 dichloromethane/methanol + 1% am-
monium hydroxide as the mobile phase to provide the title
1
the title compound (20 mg, 13%). H NMR (500 MHz, CDCl3) δ
2.33 (s, 3H), 2.45-2.67 (m, 8H), 2.63-2.67 (m, 2H), 2.67-2.69
(m, 2H), 2.87-2.90 (m, 2H), 3.09-3.12 (m, 2H), 7.21 (d, J ) 7.8
Hz, 1H), 7.30 (s, 1H), 7.67 (d, J ) 7.8 Hz, 1H). MS (DCI) m/z
259 (M + H)+.
Diethyl (Spiro[1,3dioxolane-2,1′-indane]-5′-yl)methylphospho-
nate (22). A solution of methanesulfonyl chloride (0.38 mL, 4.9
mmol) in dichloromethane (5 mL) was added dropwise to a solution
of 1310 (1.0 g, 4.85 mmol) and triethylamine (0.73 mL, 5.25 mmol)
in dichloromethane (10 mL) at 0 °C. The mixture was stirred for
30 min, while being allowed to warm to ambient temperature and
was then washed once with a saturated aqueous solution of sodium
bicarbonate. The organic layer was dried (Na2SO4) and evaporated.
The residue was taken up in THF (5 mL) and was slowly added to
a mixture of diethyl phosphite (0.82 mL, 6.4 mmol) and a 60%
1
compound (166 mg, 58%). H NMR (300 MHz, CDCl3) δ 1.43-
1.68 (m, 5H), 1.95-2.03 (m, 2H), 2.33 (s, 3H), 2.63 (d, J ) 6.8
Hz, 2H), 2.66-2.70 (m, 2H), 2.91-2.95 (m, 2H), 3.09-3.13 (m,
2H), 7.16 (d, J ) 7.8 Hz, 1H), 7.24 (s, 1H), 7.68 (d, J ) 7.8 Hz,
1H). MS (DCI) m/z 244 (M + H)+.
5-(4-Methylpiperazine-1-carbonyl)indan-1-one (24). To 10
(497 mg, 2.35 mmol) were added triethylamine (10 mL), 1-meth-
ylpiperazine (0.5 mL, 4.5 mmol), and [1,1′bis(diphenylphosphino)-
ferrocene]dichloropalladium(II) complex with dichloromethane

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2021
(1:1) (41.3 mg, 0.05 mmol), and the mixture was carbonylated at
130 psi and about 110 °C for 20 h. The mixture was filtered through
Celite and concentrated, and the crude product was purified by flash
column chromatography on silica gel using dichloromethane/
methanol (20:1) as the mobile phase to provide the title compound
and the crude product was purified by flash chromatography on
silica gel using diethyl ether/n-pentane (1:3) as eluent to provide
1
the title compound (3.8 g, 56%). H NMR (300 MHz, CD3OD) δ
2.74 (t, J ) 2.4 Hz, 1H), 3.37 (s, 3H), 3.47-3.56 (m, 2H), 3.64-
3.74 (m, 2H), 4.15 (d, J ) 2.4 Hz, 2H). MS (DCI) m/z 115 (M +
H)+.
1
(250 mg, 40%). H NMR (400 MHz, DMSO-d6) δ 2.31 (s, 3H),
2.40-2.58 (m, 4H), 2.66-2.69 (m, 2H), 3.12-3.15 (m, 2H), 3.25-
3.35 (m, 4H), 7.40 (d, J ) 7.7 Hz, 1H), 7.58 (s, 1H), 7.69 (d, J )
7.7 Hz, 1H). MS (DCI) m/z 259 (M + H)+.
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-((trimethylsilyl)ethy-
nyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole (40). Com-
pound 40 was prepared using the same procedure as described for
the synthesis of 8 by substituting trimethylsilylacetylene for phenyl
propargyl ether. The title compound was obtained after purifica-
tion by flash chromatography on silica gel using 10:1 dichlo-
romethane/methanol + 1% ammonium hydroxide as the mobile
5-(2-Methoxyethoxy)indan-1-one (26). To a solution of 25 (870
mg, 5.9 mmol) in DMF (20 mL) were added 1-bromo-2-methoxy-
ethane (1.8 g, 12.9 mmol) and potassium carbonate (2.0 g, 14.5
mmol), and the mixture was heated to reflux for 2 h. The solvent
was evaporated, and the crude product was purified by flash column
chromatography on silica gel using hexane/ethyl acetate (9:1) as
1
phase (150 mg, 75%). H NMR (500 MHz, DMSO-d6) δ 0.00 (s,
9H), 2.54 (s, 3H), 2.75-3.2 (m, 8H), 3.58 (s, 2H), 3.68 (bs, 2H),
7.13 (d, J ) 7.8 Hz, 1H), 7.32 (s, 1H), 7.41 (d, J ) 7.8 Hz, 1H),
7.48 (d, J ) 1.3 Hz, 1H), 7.62 (d, J ) 1.3 Hz, 1H). MS (ESI) m/z
447 (M + H)+.
1
the mobile phase to provide the title compound (1.0 g, 84%). H
NMR (300 MHz, CDCl3) δ 2.65-2.69 (m, 2H), 3.06-3.10 (m,
2H), 3.46 (s, 3H), 3.77-3.80 (m, 2H), 4.18-4.21 (m, 2H),
6.92-6.95 (m, 2H), 7.68 (d, J ) 9.5 Hz, 1 H). MS (ESI) m/z 207
(M + H)+.
3-(5-Ethynylthiophen-3-yl)-6-((4-methylpiperazin-1-yl)methyl)-
1,4-dihydroindeno[1,2-c]pyrazole (41). A solution of 40 (130 mg,
0.29 mmol) and tetrabutylammonium fluoride hydrate (250 mg,
0.96 mmol) in THF (3.0 mL) was stirred at room temperature for
30 min. The solvent was evaporated, and the crude product was
purified by flash column chromatography on silica gel using ethyl
acetate/methanol (95:5) as the mobile phase to provide the title
compound (78 mg, 72%). 1H NMR (500 MHz, DMSO-d6) δ 2.14
(s, 3H), 2.22-2.45 (m, 8H), 3.49 (s, 2H), 3.78 (s, 2H), 4.63 (s,
1H), 7.27 (d, J ) 7.8 Hz, 1H), 7.46 (s, 1H), 7.57 (d, J ) 7.8 Hz,
1H), 7.73 (d, J ) 1.3 Hz, 1H), 7.83 (d, J ) 1.3 Hz, 1H). MS (ESI)
m/z 375 (M + H)+. HRMS (FAB) m/z 375.16506; calcd,
C22H23N4S: 375.16379 (M + H)+. HPLC 99% purity (A), 99%
purity (B).
2-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyl)isoindoline-1,3-di-
one (42). Compound 42 was prepared using the same procedure
as described for the synthesis of 8 by substituting N-propar-
gylphthalimide for phenyl propargyl ether. The title compound was
obtained after purification by flash chromatography on silica gel
using 10:1 dichloromethane/methanol + 1% ammonium hydroxide
as the mobile phase (2.2 g, 89%). 1H NMR (300 MHz, DMSO-d6)
δ 2.15 (s, 3H), 2.25-2.45 (m, 8H), 3.50 (s, 2H), 3.78 (s, 2H),
4.70 (s, 2H), 7.27 (d, J ) 7.8 Hz, 1H), 7.47 (s, 1H), 7.58 (d, J )
7.8 Hz, 1H), 7.62 (d, J ) 1.7 Hz, 1H), 7.81 (d, J ) 1.7 Hz, 1H),
7.87-7.90 (m, 2H), 7.93-7.96 (m, 2H). MS (ESI) m/z 534 (M +
H)+.
N-(Prop-2-ynyl)aniline (30). A solution of 27 (0.11 mL, 1
mmol) and aniline (0.46 mL, 5 mmol) in ethanol (2 mL) was stirred
at room temperature for 5 d. The solvent was evaporated, and the
crude product was purified by flash chromatography on silica gel
using hexane/ethyl acetate (20:1) as eluent to provide the title
1
compound (120 mg, 91%). H NMR (500 MHz, CD3OD) δ 2.47
(t, J ) 2.5 Hz, 1H), 3.87 (d, J ) 2.5 Hz, 2H), 6.66-6.71 (m, 3H),
7.11-7.14 (m, 2H). MS (ESI) m/z 132 (M + H)+.
1-Chloro-2-(prop-2-ynyloxy)benzene (32). A mixture of 31a
(1.0 g, 4.76 mmol), 2-chlorophenol (0.6 g, 4.76 mmol), and
potassium carbonate (1.0 g, 7.14 mmol) in acetone (25 mL) was
stirred at 56 °C for 2 d. The mixture was cooled, filtered, and the
solvent was carefully distilled off at atmospheric pressure. The crude
product was purified by flash chromatography on silica gel using
diethyl ether/n-pentane (1:9) as eluent to provide the title compound
1
(400 mg, 50%). H NMR (400 MHz, CDCl3) δ 2.53 (t, J ) 2.5
Hz, 1H), 4.76 (d, J ) 2.5 Hz, 2H), 6.94 (dt, J ) 1.5, 7.8, 7.8 Hz,
1H), 7.07 (dd, J ) 8.3, 1.5 Hz, 1H), 7.21 (ddd, J ) 8.3, 7.8, 1.5
Hz, 1H), 7.36 (dd, J ) 7.8, 1.5 Hz, 1H). MS (DCI) m/z 166, 168
(M + H)+.
4-(Morpholinomethyl)phenol (36). To a solution of 35 (500
mg, 4.09 mmol) and morpholine (356 mg, 4.09 mmol) in methanol
(10 mL) was added macroporous triethylammonium methylpoly-
styrene borohydride (2.0 g, 6.0 mmol), and the mixture was stirred
at ambient temperature for 2 d. The mixture was filtered and
evaporated in vacuum to provide the title compound (780 mg, 98%).
1H NMR (300 MHz, CDCl3) δ 2.44-2.48 (m, 4H), 3.44 (s, 2H),
3.70-3.73 (m, 4H), 6.70-6.73 (m, 2H), 7.14-7.17 (m, 2H). MS
(ESI) m/z 194 (M + H)+.
4-(4-(Prop-2-ynyloxy)benzyl)morpholine (37). To a solution
of 36 (780 mg, 4.04 mmol) in THF (20 mL) were added propargyl
alcohol (229 mg, 4.09 mmol), diphenylphosphino-polystyrene (5.1
g, 8.18 mmol), and diethyl azodicarboxylate (1.06 g, 6.13 mmol).
After being stirred overnight at room temperature, the mixture was
filtered and the filtrate was concentrated. The crude product was
purified by flash chromatography on silica gel using diethyl ether/
n-pentane (1:3) as eluent to provide the title compound (869 mg,
93%). 1H NMR (300 MHz, CDCl3) δ 2.42-2.43 (m, 4H), 2.52 (t,
J ) 2.4 Hz, 1H), 3.45 (s, 2H), 3.69-3.72 (m, 4H), 4.68 (d, J )
2.4 Hz, 2H), 6.92-6.94 (m, 2H), 7.24-7.27 (m, 2H). MS (DCI-
NH3) m/z 232 (M + H)+.
3-(2-Methoxyethoxy)prop-1-yne (39). A solution of 38 (5.0 g,
89.2 mmol) in THF (20 mL) was added dropwise to a 60%
suspension of NaH in mineral oil (3.55 g, 89.2 mmol) in THF (100
mL) at 5 °C, and the mixture was stirred for 30 min. Then,
2-bromoethyl methyl ether (5.6 mL, 59.5 mmol) was added, and
the mixture was stirred overnight at room temperature. The reaction
was quenched by addition of H2O, and the layers were separated.
The aqueous layer was extracted with diethyl ether, and the
combined organic extracts were dried (MgSO4) and filtered. The
organic solvents were carefully distilled off at atmospheric pressure,
2-(3-(4-(1-(Bis(4-methoxyphenyl)methyl)-6-((4-methylpiper-
azin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)thiophen-
2-yl)prop-2-ynyl)isoindoline-1,3-dione (94). To 4,4′-dimethoxy-
benzhydrol (1.8 g, 7.3 mmol) was carefully added thionyl chloride
(6.3 mL, 86.9 mmol), and the mixture was heated to reflux for 1 h.
The solution was cooled, evaporated to dryness, and the residue
was taken up in THF (15 mL). This solution was then added to a
solution of 42 (2.2 g, 4.1 mmol) and triethylamine (1.7 mL, 12.4
mmol) in THF (25 mL). The reaction mixture was heated to 50 °C
for 2 h, cooled, diluted with H2O, and extracted six times with
ethyl acetate. The combined organic extracts were dried (Na2SO4)
and evaporated, and the crude product was purified by flash
chromatography on silica gel using dichloromethane/methanol
1
(10:1) as eluent to provide the title compound (1.9 g, 60%). H
NMR (500 MHz, DMSO-d6) δ 2.14 (s, 3H), 2.20-2.45 (m, 8H),
3.47 (s, 2H), 3.72 (s, 6H), 3.77 (s, 2H), 4.69 (s, 2H), 6.90-6.93
(m, 4H), 7.10 (s, 1H), 7.17 (d, J ) 7.9 Hz, 1H), 7.25-7.27 (m,
4H), 7.37 (d, J ) 7.9 Hz, 1H), 7.47 (s, 1H), 7.50 (d, J ) 1.5 Hz,
1H), 7.72 (d, J ) 1.5 Hz, 1H), 7.87-7.89 (m, 2H), 7.93-7.95 (m,
2H). MS (ESI) m/z 760 (M + H)+.
3-(4-(1-(Bis(4-methoxyphenyl)methyl)-6-((4-methylpiperazin-
1-yl)methyl)-1 ,4-dihydroindeno[1,2-c]pyrazol-3-yl)thiophen-2-
yl)prop-2-yn-1-amine (43). A solution of 94 (2.9 g, 3.8 mmol)
and anhydrous hydrazine (0.53 mL, 16.9 mmol) in 1:1 ethanol/
THF (60 mL) was stirred at ambient temperature overnight. THF
(30 mL) was added, and the mixture was filtered. The filtrate was

2022 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
concentrated, and the crude product was purified by flash chro-
matography on silica gel using 10:1 dichloromethan/methanol +
1% ammonium hydroxide as eluent to provide the title compound
(1.1 g, 46%). 1H NMR (500 MHz, DMSO-d6) δ 2.14 (s, 3H), 2.20-
2.45 (m, 8H), 3.47 (s, 2H), 3.54 (s, 2H), 3.72 (s, 6H), 3.78 (s,
2H), 6.91-6.93 (m, 4H), 7.09 (s, 1H), 7.17 (d, J ) 7.8 Hz, 1H),
7.25-7.27 (m, 4H), 7.37 (d, J ) 7.8 Hz, 1H), 7.47 (s, 1H), 7.50
(d, J ) 1.3 Hz, 1H), 7.68 (d, J ) 1.3 Hz, 1H). MS (ESI) m/z 630
(M + H)+.
N-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyl)benzenesulfona-
mide TFA Salt (44). To benzenesulfonyl chloride (11 µL, 0.09
mmol) was added a solution of 43 (50 mg, 0.08 mmol) in pyridine
(1 mL), and the mixture was agitated at room temperature for 2.5
h. The mixture was evaporated in vacuum. To the residue was added
a 4 M solution of hydrogen chloride in 1,4-dioxane (1 mL), and
the mixture was agitated at room temperature for 6 h. The mixture
was concentrated, and the crude product was purified by preparative
reverse phase HPLC to give the title compound (18 mg, 47%).
1H NMR (500 MHz, CD3OD) δ 2.28 (s, 3H), 2.40-2.65 (m, 8H),
3.61 (s, 2H), 3.78 (s, 2H), 4.09 (s, 2H), 7.34 (d, J ) 7.8 Hz, 1H),
7.38 (s, 1H), 7.55-7.66 (m, 6H), 7.92-7.94 (m, 2H). MS (ESI)
m/z 544 (M + H)+. HRMS (FAB) m/z 544.18469; calcd,
C29H30N5O2S2: 544.18354 (M + H)+. HPLC 97% purity (A), 96%
purity (B).
Ethyl 2-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihy-
droindeno[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyloxy)ac-
etate (47). Compound 47 was prepared using the same procedure
as described for the synthesis of 8 by substituting 95 for phenyl
propargyl ether. The title compound was obtained after purification
by flash chromatography on silica gel using 10:1 dichloromethane/
methanol + 1% ammonium hydroxide as the mobile phase (270
mg, 59%). 1H NMR (500 MHz, DMSO-d6) δ 1.22 (t, J ) 7.2 Hz,
3H), 2.81 (s, 3H), 3.00-3.50 (m, 8H), 3.84 (s, 2H), 4.02 (bs, 2H),
4.14 (s, 2H), 4.16 (q, J ) 7.2 Hz, 2H), 4.53 (s, 2H), 7.41 (d, J )
7.8 Hz, 1H), 7.60 (s, 1H), 7.68 (d, J ) 7.8 Hz, 1H), 7.75 (s, 1H),
7.90 (s, 1H). MS (ESI) m/z 491 (M + H)+.
2-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyloxy)acetamide TFA
Salt (48). To 47 (120 mg, 0.24 mmol) was added a 2 M solution
of ammonia in methanol (2 mL), and the mixture was stirred at
room temperature for 3 d. The mixture was concentrated, and the
crude product was purified by preparative reverse phase HPLC to
give the title compound (130 mg, 68%). 1H NMR (500 MHz,
DMSO-d6) δ 2.81 (s, 3H), 3.00-3.50 (m, 8H), 3.84 (s, 2H), 3.95
(s, 2H), 4.00 (bs, 2H), 4.53 (s, 2H), 7.26 (m, 2H), 7.40 (d, J ) 7.8
Hz, 1H), 7.60 (s, 1H), 7.68 (d, J ) 7.8 Hz, 1H), 7.75 (s, 1H), 7.90
(s, 1H). MS (ESI) m/z 462 (M + H)+. Anal. (C25H27N5O2S‚3.2CF3-
CO2H) C, H, N.
3-(5-Bromothiophen-3-yl)-6-((4-methylpiperazin-1-yl)methyl)-
indeno[1,2-c]pyrazol-4(1H)-one (49). To a solution of 7 (800 mg,
1.87 mmol) in DMF (180 mL) was added cesium carbonate (2 g,
6.1 mmol), and the mixture was heated to 90 °C. Air was bubbled
through the vigorously stirred mixture, and heating was continued
overnight. The reaction mixture was cooled and filtered, and the
filtrate was evaporated. The crude product was purified by flash
chromatography on silica gel using 9:1 ethyl acetate/methanol +
1% ammonium hydroxide as eluent to provide the title compound
Phenyl 3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihy-
droindeno[1,2-c]pyrazol-3-yl )thiophen-2-yl)prop-2-ynylcarbam-
ate TFA Salt (45). To phenyl chloroformate (11 µL, 0.09 mmol)
was added a solution of 43 (50 mg, 0.08 mmol) and triethylamine
(22 µL) in dichloromethane (0.7 mL), and the mixture was shaken
at room temperature overnight. The mixture was concentrated, and
the residue was taken up in ethyl acetate (1 mL). A 37% solution
of hydrochloric acid in ethanol (1 mL) was added and the mixture
was shaken at room temperature overnight. The mixture was
concentrated, and the crude product was purified by preparative
1
(503 mg, 61%). H NMR (300 MHz, DMSO-d6) δ 2.15 (s, 3H),
2.22-2.47 (m, 8H), 3.50 (s, 2H), 7.43-7.50 (m, 2H), 7.51 (s, 1H),
7.87 (d, J ) 1.7 Hz, 1H), 8.35 (d, J ) 1.7 Hz, 1H). MS (ESI) m/z
443, 445 (M + H)+.
1
reverse phase HPLC to give the title compound (4 mg, 11%). H
NMR (500 MHz, CD3OD) δ 2.28 (s, 3H), 2.42-2.63 (m, 8H), 3.61
(s, 2H), 3.80 (s, 2H), 4.25 (s, 2H), 7.13-7.16 (m, 2H), 7.22-7.23
(m, 1H), 7.34 (d, J ) 8.1 Hz, 1H), 7.36-7.39 (m, 2H), 7.55 (s,
1H), 7.63 (s, 1H), 7.64 (d, J ) 8.1 Hz, 1H), 7.69 (s, 1H). MS (ESI)
m/z 524 (M + H)+. Anal. (C30H29N5O2S‚3.5CF3CO2H) C, H, N.
1-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyl)-3-phenylurea TFA
Salt (46). To phenyl isocyanate (10 µL, 0.09 mmol) was added a
solution of 43 (50 mg, 0.08 mmol) in THF (1 mL), and the mixture
was agitated at room temperature overnight. The mixture was
concentrated; the residue was suspended in a 4 M solution of
hydrogen chloride in 1,4-dioxane (1 mL) and was agitated at room
temperature overnight. The mixture was concentrated, and the crude
product was purified by preparative reverse phase HPLC to give
the title compound (32 mg, 30%). 1H NMR (500 MHz, DMSO-d6)
δ 2.80 (s, 3H), 3.00-3.50 (m, 8H), 3.83 (s, 2H), 3.96 (bs, 2H),
4.20 (d, J ) 5.5 Hz, 2H), 6.67 (t, J ) 5.5 Hz, 1H), 6.91-6.94 (m,
1H), 7.23-7.26 (m, 2H), 7.38 (d, J ) 7.6 Hz, 1H), 7.41-7.43 (m,
2H), 7.58 (s, 1H), 7.66-7.67 (m, 2H), 7.84 (s, 1H), 8.70 (s, 1H).
MS (ESI m/z 523 (M + H)+. Anal. (C30H30N6OS‚4.5CF3CO2H) C,
H, N.
Ethyl 2-(Prop-2-ynyloxy)acetate (95). To a solution of ethyl
glycolate (3.12 g, 30.0 mmol) in THF (80 mL) was added a 60%
suspension of NaH in mineral oil (1.28 g, 32.0 mmol), and the
mixture was stirred for 1 h at ambient temperature. Propargyl
bromide (4.08 g, 35.0 mmol) was added dropwise, and stirring was
continued for 3 d. The mixture was concentrated; the residue was
taken up in diethyl ether (150 mL) and was washed twice with
H2O. The organic layer was dried (MgSO4) and evaporated, and
the crude product was purified by flash chromatography on silica
gel using pentane/diethyl ether (5:1) as eluent to provide the title
compound (2.7 g, 63%). 1H NMR (300 MHz, CDCl3) δ 1.30
(t, J ) 7.1 Hz, 3H), 2.48 (t, J ) 2.4 Hz, 1H), 4.20 (s, 2H), 4.24 (q,
J ) 7.1 Hz, 2H), 4.32 (d, J ) 2.4 Hz, 2H). MS (ESI) m/z 143 (M
+ H)+.
3-(5-(3-(2-Methoxyethoxy)prop-1-ynyl)thiophen-3-yl)-6-((4-
methylpiperazin-1-yl)methyl)indeno[1,2-c]pyrazol-4(1H)-one TFA
Salt (50). Compound 50 was prepared using the same procedure
as described for the synthesis of 8 by substituting 49 for 7 and 39
1
for phenyl propargyl ether (59 mg, 55%). H NMR (500 MHz,
DMSO-d6) δ 2.79 (s, 3H), 2.95-3.15 (m, 8H), 3.27 (s, 3H), 3.50-
3.51 (m, 2H), 3.63-3.64 (m, 2H), 3.74 (s, 2H), 4.47 (s, 2H), 7.51-
7.56 (m, 2H), 7.61 (s, 1H), 7.97 (s, 1H), 8.37 (s, 1H), 13.79 (bs,
1H). MS (ESI) m/z 477 (M + H)+. HRMS (FAB) m/z 477.19585;
calcd, C26H29N4O3S: 477.19549 (M + H)+. HPLC 96% purity (A),
95% purity (B).
6-((4-Methylpiperazin-1-yl)methyl)-N-phenyl-1,4-dihydroin-
deno[1,2-c]pyrazol-3-amine TFA Salt (51). To a solution of 510
(200 mg, 0.82 mmol) and phenyl isothiocyanate (98 µL, 0.82 mmol)
in DMF (0.6 mL) was added a 60% suspension of NaH in mineral
oil (36 mg, 0.9 mmol) at 0 °C. The mixture was then stirred for
1.5 h at room temperature, methyl iodide (56 µL, 0.9 mmol) was
added, and stirring was continued for 1 h. The mixture was
concentrated; the residue was taken up in diethyl ether (10 mL)
and was washed with H2O and brine. The organic layer was dried
(MgSO4) and concentrated. The crude intermediate was dissolved
in ethanol (1.4 mL), hydrazine monohydrate (60 µL, 1.23 mmol)
was added, and the mixture was refluxed overnight. The solvent
was evaporated, and the crude product was purified by preparative
reverse phase HPLC to provide the title compound (200 mg, 30%).
1H NMR (400 MHz, DMSO-d6) δ 2.80 (s, 3H), 2.90-3.40 (m,
8H), 3.49 (s, 2H), 3.94 (bs, 2H), 6.74-6.78 (m, 1H), 7.16-7.23
(m, 4H), 7.37 (d, J ) 7.7 Hz, 1H), 7.55-7.58 (m, 2H), 8.45 (bs,
1H). MS (ESI) m/z 360 (M + H)+. Anal. (C22H25N5‚3.4CF3CO2H)
C, H, N.
3-(5-Bromothiophen-2-yl)-6-((4-methylpiperazin-1-yl)methyl)-
1,4-dihydroindeno[1,2-c]pyrazole (97). Compound 97 was pre-
pared using the same procedure as described for the synthesis of 7
by substituting phenyl 5-bromothiophene-2-carboxylate10 for 6 (2.4

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2023
1
g, 55%). H NMR (300 MHz, DMSO-d6) δ 2.15 (s, 3H), 2.23-
3-(5-Bromothiophen-3-yl)-7-((4-methylpiperazin-1-yl)methyl)-
1,4-dihydroindeno[1,2-c]pyrazole (101). Compound 101 was
prepared using the same procedure as described for the synthesis
of 7 by substituting 6-((4-methylpiperazin-1-yl)methyl)indan-1-one10
2.48 (m, 8H), 3.50 (s, 2H), 3.73 (s, 2H), 7.18-7.35 (m, 3H), 7.49
(s, 1H), 7.52-7.62 (m, 1H). MS (ESI) m/z 429, 431 (M + H)+.
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-(3-phenoxyprop-1-
ynyl)thiophen-2 -yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(52). Compound 52 was prepared using the same procedure as
described for the synthesis of 8 by substituting 97 for 7 (141 mg,
1
for 5 (3.25 g, 86%). H NMR (300 MHz, DMSO-d6) δ 2.15 (s,
3H), 2.25-2.45 (m, 8H), 3.52 (s, 2H), 3.78 (s, 2H), 7.20 (d, J )
7.8 Hz, 1H), 7.49 (d, J ) 7.8 Hz, 1H), 7.58 (s, 1H), 7.64 (s, 1H),
7.84 (s, 1H). MS (ESI) m/z 429, 431 (M + H)+.
1
74%). H NMR (300 MHz, DMSO-d6) δ 2.79 (s, 3H), 3.00-3.50
(m, 8H), 3.78 (s, 2H), 3.84 (bs, 2H), 5.10 (s, 2H), 6.94-7.06 (m,
3H), 7.11 (s, 1H), 7.28 (s, 1H), 7.32-7.42 (m, 3H), 7.58 (s, 1H),
7.63 (d, J ) 7.8 Hz, 1H). MS (ESI) m/z 481 (M + H)+. Anal.
(C29H28N4OS‚2.9CF3CO2H) C, H, N.
3-(4-Bromothiophen-2-yl)-6-((4-methylpiperazin-1-yl)methyl)-
1,4-dihydroindeno[1,2-c]pyrazole (98). Compound 98 was pre-
pared using the same procedure as described for the synthesis of 7
by substituting phenyl 4-bromothiophene-2-carboxylate for 6 (850
mg, 87%). 1H NMR (300 MHz, DMSO-d6) δ 2.15 (s, 3H), 2.25-
2.45 (m, 8H), 3.51 (s, 2H), 3.77 (s, 2H), 7.30 (d, J ) 7.8 Hz, 1H),
7.40 (s, 1H), 7.49 (s, 1H), 7.56 (d, J ) 7.8 Hz, 1H), 7.68 (s, 1H).
MS (ESI) m/z 429, 431 (M + H)+.
6-((4-Methylpiperazin-1-yl)methyl)-3-(4-(3-phenoxyprop-1-
ynyl)thiophen-2 -yl)-1,4-dihydroindeno[1,2-c]pyrazole (53). Com-
pound 53 was prepared using the same procedure as described for
the synthesis of 8 by substituting 98 for 7. The title compound
was obtained after purification by flash chromatography on silica
gel using 10:1 dichloromethane/methanol + 1% ammonium
hydroxide as the mobile phase (35 mg, 31%). 1H NMR (400 MHz,
DMSO-d6) δ 2.23 (s, 3H), 2.35-2.55 (m, 8H), 3.76 (s, 2H), 4.01
(s, 2H), 5.05 (s, 2H), 6.98-7.01 (m, 1H), 7.04-7.06 (m, 2H), 7.29
(d, J ) 7.7 Hz, 1H), 7.32-7.36 (m, 3H), 7.49 (s, 1H), 7.52-7.61
(m, 1H), 7.76 (bs, 1H), 13.22 (s, 1H). MS (ESI) m/z 481 (M +
H)+. Anal. (C29H28N4OS‚0.4CH2Cl2) C, H, N.
7-((4-Methylpiperazin-1-yl)methyl)-3-(5-(3-phenoxyprop-1-
ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(56). Compound 56 was prepared using the same procedure as
described for the synthesis of 8 by substituting 101 for 7 (57 mg,
1
34%). H NMR (500 MHz, DMSO-d6) δ 2.81 (s, 3H), 3.00-3.50
(m, 8H), 3.84 (s, 2H), 4.07 (bs, 2H), 5.11 (s, 2H), 6.99-7.02 (m,
1H), 7.05-7.06 (m, 2H), 7.33-7.37 (m, 3H), 7.59 (d, J ) 7.7 Hz,
1H), 7.74 (m, 2H), 7.89 (d, J ) 1.3 Hz, 1H). MS (ESI) m/z 481
(M + H)+. Anal. (C29H28N4OS‚3.7CF3CO2H) C, H, N.
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-(pent-1-ynyl)thiophen-
3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt (57). Compound
57 was prepared using the same procedure as described for the
synthesis of 8 by substituting 1-pentyne for phenyl propargyl ether
1
(49 mg, 32%). H NMR (500 MHz, DMSO-d6) δ 0.99-1.02 (m,
3H), 1.55-1.62 (m, 2H), 2.15 (s, 3H), 2.25-2.45 (m, 8H), 2.46-
2.48 (m, 2H), 3.50 (s, 2H), 3.79 (m, 2H), 7.27 (d, J ) 7.8 Hz, 1H),
7.47 (s, 1H), 7.56-7.63 (m, 2H), 7.75 (s, 1H). MS (ESI) m/z 417
(M + H)+. HRMS (FAB) m/z 417.21150; calcd, C25H29N4S:
417.21074 (M + H)+. HPLC 95% purity (A), 95% purity (B).
4-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)but-3-yn-1-ol TFA Salt (58).
Compound 58 was prepared using the same procedure as described
for the synthesis of 8 by substituting 3-butyn-1-ol for phenyl
1
propargyl ether (25 mg, 28%). H NMR (400 MHz, DMSO-d6) δ
2.62 (t, J ) 6.5 Hz, 2H), 2.79 (s, 3H), 3.00-3.50 (m, 8H), 3.60 (t,
J ) 6.5 Hz, 2H), 3.82 (s, 2H), 3.91 (bs, 2H), 7.37 (d, J ) 7.8 Hz,
1H), 7.56 (s, 1H), 7.61 (d, J ) 1.2 Hz, 1H), 7.66 (d, J ) 7.8 Hz,
1H), 7.78 (d, J ) 1.2 Hz, 1H). MS (ESI) m/z 419 (M + H)+. Anal.
(C24H26N4OS‚2.7CF3CO2H) C, H, N.
Phenyl 3-(3-Phenoxyprop-1-ynyl)benzoate (99). Compound 99
was prepared using the same procedure as described for the
synthesis of 9 by substituting phenyl 3-bromobenzoate for 6 (160
mg, 83%). 1H NMR (300 MHz, CDCl3) δ 4.94 (s, 2H), 6.98-7.05
(m, 3H), 7.19-7.22 (m, 2H), 7.26-7.35 (m, 3H), 7.41-7.49 (m,
3H), 7.69 (dt, J ) 7.8, 1.7, 1.7 Hz, 1H), 8.15 (dt, J ) 7.8, 1.7, 1.7
Hz, 1H), 8.28 (t, J ) 1.7 Hz, 1H). MS (ESI) m/z 329 (M + H)+.
6-((4-Methylpiperazin-1-yl)methyl)-3-(3-(3-phenoxyprop-1-
ynyl)phenyl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt (54).
Compound 54 was prepared using the same procedure as described
for the synthesis of 7 by substituting 99 for 6. The crude product
was purified by preparative reverse phase HPLC to provide the
title compound (125 mg, 42%). 1H NMR (500 MHz, DMSO-d6) δ
2.79 (s, 3H), 3.00-3.50 (m, 8H), 3.86 (bs, 2H), 3.89 (s, 2H), 5.09
(s, 2H), 6.99-7.02 (m, 1H), 7.07-7.09 (m, 2H), 7.34-7.39 (m,
3H), 7.43 (d, J ) 7.8 Hz, 1H), 7.53 (t, J ) 7.8 Hz, 1H), 7.58 (s,
1H), 7.66 (d, J ) 7.8 Hz, 1H), 7.84 (d, J ) 7.8 Hz, 1H), 7.89 (s,
1H). MS (ESI) m/z 475 (M + H)+. HRMS (FAB) m/z 475.25057;
calcd, C31H31N4O: 475.24924 (M + H)+. HPLC 97% purity (A),
96% purity (B).
2-Methyl-5-(4-(6-((4-methylpiperazin-1-yl)methyl)-1,4-dihy-
droindeno[1,2-c]pyrazol-3-yl)thiophen-2-yl)pent-4-yn-2-ol TFA
Salt (59). Compound 59 was prepared using the same procedure
as described for the synthesis of 8 by substituting 2-methylpent-
1
4-yn-2-ol16 for phenyl propargyl ether (190 mg, 60%). H NMR
(500 MHz, CD3OD) δ 1.36 (s, 6H), 2.63 (s, 2H), 2.90 (s, 3H),
3.02-3.14 (m, 4H), 3.34-3.42 (s, 4H), 3.82 (s, 2H), 4.00 (s, 2H),
7.43 (d, J ) 7.8 Hz, 1H), 7.54 (s, 1H), 7.63 (s, 2H), 7.73 (d, J )
7.8 Hz, 1H). MS (ESI) m/z 447 (M + H)+. HRMS (FAB) m/z
447.22172; calcd, C26H31N4OS: 447.22131 (M + H)+. HPLC 96%
purity (A), 96% purity (B).
3-(5-(3-Methoxyprop-1-ynyl)thiophen-3-yl)-6-((4-methylpip-
erazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(60). Compound 60 was prepared using the same procedure as
described for the synthesis of 8 by substituting methyl propargyl
ether for phenyl propargyl ether (88 mg, 54%). 1H NMR (500 MHz,
DMSO-d6) δ 2.81 (s, 3H), 3.20-3.45 (m, 8H), 3.35 (s, 3H), 3.84
(s, 2H), 3.99 (bs, 2H), 4.39 (s, 2H), 7.40 (d, J ) 7.8 Hz, 1H), 7.60
(s, 1H), 7.68 (d, J ) 7.8 Hz, 1H), 7.74 (d, J ) 1.3 Hz, 1H), 7.89
(d, J ) 1.3 Hz, 1H). MS (ESI) m/z 419 (M + H)+. HRMS (FAB)
m/z 419.19022; calcd, C24H27N4OS: 419.19001 (M + H)+. HPLC
96% purity (A), 97% purity (B).
Phenyl 4-(3-Phenoxyprop-1-ynyl)benzoate (100). Compound
100 was prepared using the same procedure as described for the
synthesis of 9 by substituting phenyl 4-bromobenzoate for 6 (2.2
1
g, 71%). H NMR (500 MHz, CDCl3) δ 4.95 (s, 2H), 7.00-7.05
(m, 3H), 7.20-7.21 (m, 2H), 7.25-7.29 (m, 1H), 7.32-7.35 (m,
2H), 7.41-7.44 (m, 2H), 7.55-7.57 (m, 2H), 8.13-8.15 (m, 2H).
MS (ESI) m/z 329 (M + H)+.
(But-3-ynyloxy)benzene (102). Compound 102 was prepared
using the same procedure as described for the synthesis of 32 by
substituting 3-butynyl p-toluenesulfonate for propargyl p-toluene-
sulfonate and phenol for 2-chlorophenol (170 mg, 52%). 1H NMR
(300 MHz, CDCl3) δ 2.04 (t, J ) 2.7 Hz, 1H), 2.68 (dt, J ) 7.1,
2.7 Hz, 2H), 4.08 (t, J ) 7.1 Hz, 2H), 6.90-6.99 (m, 3H), 7.26-
7.31 (m, 2H). MS (ESI) m/z 147 (M + H)+.
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-(4-phenoxybut-1-
ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(61). Compound 61 was prepared using the same procedure as
described for the synthesis of 8 by substituting 102 for phenyl
propargyl ether (106 mg, 55%). 1H NMR (500 MHz, DMSO-d6) δ
6-((4-Methylpiperazin-1-yl)methyl)-3-(4-(3-phenoxyprop-1-
ynyl)phenyl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt (55).
Compound 55 was prepared using the same procedure as described
for the synthesis of 7 by substituting 100 for 6. The crude product
was purified by preparative reverse phase HPLC to provide the
title compound (110 mg, 70%). 1H NMR (500 MHz, DMSO-d6) δ
2.78 (s, 3H), 3.00-3.50 (m, 8H), 3.88 (s, 4H), 5.06 (s, 2H), 6.98-
7.01 (m, 1H), 7.05-7.06 (m, 2H), 7.32-7.35 (m, 2H), 7.37 (d, J
) 7.5 Hz, 1H), 7.56-7.57 (m, 3H), 7.66 (d, J ) 7.5 Hz, 1H), 7.81-
7.83 (m, 2H). MS (ESI) m/z 475 (M + H)+. HRMS (FAB) m/z
475.24940; calcd, C31H31N4O: 475.24924 (M + H)+. HPLC 99%
purity (A), 99% purity (B).

2024 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
2.78 (s, 3H), 2.98 (t, J ) 6.6 Hz, 2H), 3.00-3.50 (m, 8H), 3.82 (s,
4H), 4.18 (t, J ) 6.6 Hz, 2H), 6.95-6.98 (m, 1H), 6.99-7.00 (m,
2H), 7.30-7.33 (m, 2H), 7.36 (d, J ) 7.5 Hz, 1H), 7.55 (s, 1H),
7.64-7.66 (m, 2H), 7.80 (s, 1H). MS (ESI) m/z 495 (M + H)+.
Anal. (C30H30N4OS‚2.6CF3CO2H) C, H, N.
1H), 7.74 (d, J ) 1.3 Hz, 1H), 7.90 (d, J ) 1.3 Hz, 1H). MS (ESI)
m/z 509 (M + H)+. HRMS (FAB) m/z 509.23907; calcd, C31H33N4-
OS: 509.23696 (M + H)+. HPLC 96% purity (A), 95% purity
(B).
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-(3-(pyridin-3-yloxy)-
prop-1-ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA
Salt (66). Compound 66 was prepared using the same procedure
as described for the synthesis of 8 by substituting 3-(prop-2-
ynyloxy)pyridine for phenyl propargyl ether (85 mg, 52%). 1H NMR
(500 MHz, DMSO-d6) δ 2.78 (s, 3H), 3.00-3.50 (m, 8H), 3.81 (s,
2H), 3.90 (bs, 2H), 5.24 (s, 2H), 7.37 (d, J ) 7.2 Hz, 1H), 7.50
(dd, J ) 8.4, 4.7 Hz, 1H), 7.56 (s, 1H), 7.63-7.66 (m, 2H), 7.74
(d, J ) 1.3 Hz, 1H), 7.90 (d, J ) 1.3 Hz, 1H), 8.30 (d, J ) 4.7 Hz,
1H), 8.46 (d, J ) 2.8 Hz, 1H). MS (ESI) m/z 482 (M + H)+. Anal.
(C28H27N5OS‚4.5CF3CO2H) C, H, N.
3-(5-(3-(2-Chlorophenoxy)prop-1-ynyl)thiophen-3-yl)-6-((4-
methylpiperazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyra-
zole TFA Salt (62). Compound 62 was prepared using the same
procedure as described for the synthesis of 8 by substituting 32 for
phenyl propargyl ether (106 mg, 55%). 1H NMR (500 MHz,
DMSO-d6) δ 2.76 (s, 3H), 3.00-3.50 (m, 8H), 3.73 (s, 2H), 3.81
(s, 2H), 5.24 (s, 2H), 7.03 (dt, J ) 1.5, 7.8, 7.8 Hz, 1H), 7.31 (dd,
J ) 7.8, 1.5 Hz, 1H), 7.33 (d, J ) 7.8 Hz, 1H), 7.38 (ddd, J ) 8.1,
7.8, 1.5 Hz, 1H), 7.47 (dd, J ) 8.1, 1.5 Hz, 1H), 7.53 (s, 1H), 7.63
(d, J ) 7.8 Hz, 1H), 7.75 (d, J ) 1.2 Hz, 1H), 7.90 (d, J ) 1.2 Hz,
1H). MS (ESI) m/z 515, 517 (M + H)+. HRMS (FAB) m/z
515.16690; calcd, C29H28ClN4OS: 515.16669 (M + H)+. HPLC
95% purity (A), 95% purity (B).
6-((4-Methylpiperazin-1-yl)methyl)-3-(5-(4-phenylbut-1-ynyl)-
thiophen-3-yl) -1,4-dihydroindeno[1,2-c]pyrazole TFA Salt (67).
Compound 67 was prepared using the same procedure as described
for the synthesis of 8 by substituting 4-phenyl-1-butyne for phenyl
1-Chloro-3-(prop-2-ynyloxy)benzene (103). Compound 103
was prepared using the same procedure as described for the
synthesis of 32 by substituting 3-chlorophenol for 2-chlorophenol
1
propargyl ether (38 mg, 40%). H NMR (500 MHz, DMSO-d6) δ
2.77-2.80 (m, 5H), 2.88 (t, J ) 7.5 Hz, 2H), 3.00-3.50 (m, 8H),
3.81 (s, 2H), 3.89 (bs, 2H), 7.22-7.25 (m, 1H), 7.31-7.33 (m,
4H), 7.38 (d, J ) 7.7 Hz, 1H), 7.57 (m, 2H), 7.67 (d, J ) 7.7 Hz,
1H), 7.77 (s, 1H). MS (ESI) m/z 479 (M + H)+. Anal.
(C30H30N4S‚2.4CF3CO2H) C, H, N.
1
(280 mg, 63%). H NMR (300 MHz, CDCl3) δ 2.55 (t, J ) 2.4
Hz, 1H), 4.73 (d, J ) 2.4 Hz, 2H), 7.15 (dd, J ) 8.1, 2.4 Hz, 1H),
7.21-7.27 (m, 2H), 7.42 (t, J ) 8.1 Hz, 1H). MS (DCI) m/z 166,
168 (M + H)+.
3-(5-(3-(3-Chlorophenoxy)prop-1-ynyl)thiophen-3-yl)-6-((4-
methylpiperazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyra-
zole TFA Salt (63). Compound 63 was prepared using the same
procedure as described for the synthesis of 8 by substituting 103
N-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyl)aniline TFA Salt
(68). Compound 68 was prepared using the same procedure as
described for the synthesis of 8 by substituting 30 for phenyl
1
for phenyl propargyl ether (78 mg, 45%). H NMR (500 MHz,
1
propargyl ether (38 mg, 40%). H NMR (500 MHz, CD3OD) δ
DMSO-d6) δ 2.81 (s, 3H), 3.00-3.50 (m, 8H), 3.83 (s, 2H), 3.98
(bs, 2H), 5.17 (s, 2H), 7.04 (dd, J ) 8.4, 0.6 Hz, 1H), 7.07 (dd, J
) 7.8, 0.6 Hz, 1H), 7.16 (t, J ) 2.2 Hz, 1H), 7.37 (t, J ) 8.1 Hz,
1H), 7.40 (d, J ) 7.5 Hz, 1H), 7.59 (s, 1H), 7.67 (d, J ) 7.5 Hz,
1H), 7.75 (d, J ) 1.3 Hz, 1H), 7.91 (d, J ) 1.3 Hz, 1H). MS (ESI)
m/z 515, 517 (M + H)+. HRMS (FAB) m/z 515.16773; calcd,
C29H28ClN4OS: 515.16669 (M + H)+. HPLC 97% purity (A), 97%
purity (B).
2.33 (s, 3H), 2.45-2.65 (m, 8H), 3.61 (s, 2H), 3.77 (s, 2H), 4.17
(s, 2H), 6.70-6.73 (m, 1H), 6.77-6.78 (m, 2H), 7.16-7.19 (m,
2H), 7.33 (d, J ) 7.5 Hz, 1H), 7.53 (s, 1H), 7.54 (s, 1H), 7.63 (m,
2H). MS (ESI) m/z 480 (M + H)+. HRMS (FAB) m/z 480.22241;
calcd, C29H30N5S: 480.22164 (M + H)+. HPLC 95% purity (A),
96% purity (B).
N-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroindeno-
[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyl)benzamide TFA
Salt (69). To benzoyl chloride (10 µL, 0.09 mmol) was added a
solution of 43 (50 mg, 0.08 mmol) in pyridine (1 mL), and the
mixture was agitated for 2.5 h. The mixture was evaporated, a 4
M solution of hydrogen chloride in 1,4-dioxane (1 mL) was added,
and the mixture was agitated at room temperature for 6 h. The
mixture was concentrated, and the crude product was purified by
preparative reverse phase HPLC to provide the title compound (14
1-Chloro-4-(prop-2-ynyloxy)benzene (104). Compound 104
was prepared using the same procedure as described for the
synthesis of 32 by substituting 4-chlorophenol for 2-chlorophenol
1
(297 mg, 67%). H NMR (300 MHz, CDCl3) δ 2.52 (t, J ) 2.4
Hz, 1H), 4.66 (d, J ) 2.4 Hz, 2H), 6.88-6.93 (m, 2H), 7.22-7.28
(m, 2H). MS (DCI) m/z 166, 168 (M + H)+.
3-(5-(3-(4-Chlorophenoxy)prop-1-ynyl)thiophen-3-yl)-6-((4-
methylpiperazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyra-
zole TFA Salt (64). Compound 64 was prepared using the same
procedure as described for the synthesis of 8 by substituting 104
1
mg, 39%). H NMR (500 MHz, CD3OD) δ 2.28 (s, 3H), 2.40-
2.65 (m, 8H), 3.60 (s, 2H), 3.79 (s, 2H), 4.44 (s, 2H), 7.33 (d, J )
7.8 Hz, 1H), 7.47-7.50 (m, 2H), 7.54-7.57 (m, 2H), 7.62 (s, 1H),
7.64 (d, J ) 7.8 Hz, 1H), 7.68 (s, 1H), 7.86-7.88 (m, 2H). MS
(ESI) m/z 508 (M + H)+. HRMS (FAB) m/z 508.21644; calcd,
C30H30N5OS: 508.21656 (M + H)+. HPLC 97% purity (A), 97%
purity (B).
1
for phenyl propargyl ether (54 mg, 31%). H NMR (500 MHz,
DMSO-d6) δ 2.80 (s, 3H), 3.00-3.50 (m, 8H), 3.83 (s, 2H), 3.97
(bs, 2H), 5.13 (s, 2H), 7.07-7.11 (m, 2H), 7.39-7.41 (m, 3H),
7.59 (s, 1H), 7.67 (d, J ) 7.8 Hz, 1H), 7.74 (s, 1H), 7.91 (s, 1H).
MS (ESI) m/z 515, 517 (M + H)+. HRMS (FAB) m/z 515.16774;
calcd, C29H28ClN4OS: 515.16669 (M + H)+. HPLC 97% purity
(A), 98% purity (B).
3-(5-(3-Phenoxyprop-1-ynyl)thiophen-3-yl)-1,4-dihydroindeno-
[1,2-c]pyrazole (70). Compound 70 was prepared using the same
procedure as described for the synthesis of 7 by substituting
1,2-Dimethyl-4-(prop-2-ynyloxy)benzene (105). Compound 105
was prepared using the same procedure as described for the
synthesis of 32 by substituting 3,4-dimethylphenol for 2-chlorophe-
1
1-indanone for 5 and 9 for 6 (46 mg, 36%). H NMR (500 MHz,
DMSO-d6) δ 3.82 (s, 2H), 5.11 (s, 2H), 6.99-7.02 (m, 1H), 7.05-
7.06 (m, 2H), 7.28-7.30 (m, 1H), 7.33-7.38 (m, 3H), 7.56 (d, J
) 7.8 Hz, 1H), 7.66 (m, 1H), 7.74 (bs, 1H), 7.89 (bs, 1H). MS
(ESI) m/z 369 (M + H)+. Anal. (C23H16N2OS‚0.1H2O) C, H, N.
1
nol (400 mg, 49%). H NMR (300 MHz, CDCl3) δ 2.19 (s, 3H),
2.24 (s, 3H), 2.49 (t, J ) 2.4 Hz, 1H), 4.65 (d, J ) 2.4 Hz, 2H),
6.72 (dd, J ) 8.1, 2.7 Hz, 1H), 6.77 (d, J ) 2.7 Hz, 1H), 7.05 (d,
J ) 8.1 Hz, 1H). MS (DCI) m/z 161 (M + H)+.
6-(4-Methylpiperazin-1-yl)-3-(5-(3-phenoxyprop-1-ynyl)-
thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt (71).
Compound 71 was prepared using the same procedure as described
for the synthesis of 7 by substituting 17 for 5 and 9 for 6. The
crude product was purified by preparative reverse phase HPLC to
3-(5-(3-(3,4-Dimethylphenoxy)prop-1-ynyl)thiophen-3-yl)-6-
((4-methylpiperazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyra-
zole TFA Salt (65). Compound 65 was prepared using the same
procedure as described for the synthesis of 8 by substituting 105
1
1
for phenyl propargyl ether (42 mg, 49%). H NMR (500 MHz,
provide the title compound (49 mg, 30%). H NMR (500 MHz,
DMSO-d6) δ 2.16 (s, 3H), 2.21 (s, 3H), 2.82 (s, 3H), 3.00-3.50
(m, 8H), 3.83 (s, 2H), 4.06 (bs, 2H), 5.04 (s, 2H), 6.77 (dd, J )
8.1, 2.4 Hz, 1H), 6.85 (d, J ) 2.4 Hz, 1H), 7.08 (d, J ) 8.1 Hz,
1H), 7.42 (d, J ) 7.8 Hz, 1H), 7.62 (s, 1H), 7.68 (d, J ) 7.8 Hz,
DMSO-d6) δ 2.87 (s, 3H), 2.98-3.02 (m, 2H), 3.15-3.21 (m, 2H),
3.52-3.55 (m, 2H), 3.74 (s, 2H), 3.87-3.90 (m, 2H), 5.09 (s, 2H),
6.97-7.01 (m, 2H), 7.03-7.05 (m, 2H), 7.22 (s, 1H), 7.32-7.35
(m, 2H), 7.51 (d, J ) 7.8 Hz, 1H), 7.70 (d, J ) 1.3 Hz, 1H), 7.84

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2025
(d, J ) 1.3 Hz, 1H). MS (ESI) m/z 467 (M + H)+. Anal. (C28H26N4-
OS‚2.7CF3CO2H) C, H, N.
(s, 1H), 7.99 (s, 1H), 13.10 (bs, 1H). MS (ESI) m/z 495 (M + H)+.
HRMS (FAB) m/z 495.18527; calcd, C29H27N4O2S: 495.18492 (M
+ H)+. HPLC 99% purity (A), 99% purity (B).
1-Methyl-4-((1-oxo-indan-5-yl)methyl)piperazin-2-one (108).
Compound 108 was prepared using the same procedure as described
for the synthesis of 106 by substituting 1-methylpiperazin-2-one13
for 4-methylpiperidine (380 mg, 30%). 1H NMR (300 MHz, CDCl3)
δ 2.69-2.73 (m, 4H), 2.88 (s, 3H), 3.11-3.17 (m, 4H), 3.31-
3.39 (m, 2H), 3.64 (s, 2H), 7.35 (d, J ) 7.8 Hz, 1H), 7.46 (s, 1H),
7.72 (d, J ) 7.8 Hz, 1H). MS (ESI) m/z 259 (M + H)+.
1-Methyl-4-((3-(5-(3-phenoxyprop-1-ynyl)thiophen-3-yl)-1,4-
dihydroindeno[1,2-c]pyrazol-6-yl)methyl)piperazin-2-one TFA
Salt (76). Compound 76 was prepared using the same procedure
as described for the synthesis of 7 by substituting 108 for 5 and 9
for 6. The crude product was purified by preparative reverse phase
HPLC to provide the title compound (50 mg, 27%). 1H NMR (300
MHz, DMSO-d6) δ 2.87 (s, 3H), 3.44-3.51 (m, 4H), 3.76-3.80
(m, 2H), 3.87 (s, 2H), 4.32 (bs, 2H), 5.11 (s, 2H), 6.98-7.03 (m,
1H), 7.04-7.07 (m, 2H), 7.32-7.38 (m, 2H), 7.47 (d, J ) 7.5 Hz,
1H), 7.67 (s, 1H), 7.72-7.75 (m, 2H), 7.91 (d, J ) 1.3 Hz, 1H).
MS (ESI) m/z 495 (M + H)+. Anal. (C29H26N4O2S‚1.7CF3CO2H)
C, H, N.
4-Methyl-1-((1-oxo-indan-5-yl)methyl)piperazin-2-one (109).
Compound 109 was prepared using the same procedure as described
for the synthesis of 106 by substituting 4-methylpiperazin-2-one
for 4-methylpiperidine (380 mg, 55%). 1H NMR (300 MHz, CDCl3)
δ 2.38 (s, 3H), 2.66-2.72 (m, 4H), 3.11-3.14 (m, 2H), 3.25 (s,
2H), 3.29-3.33 (m, 2H), 4.68 (s, 2H), 7.25 (d, J ) 7.8 Hz, 1H),
7.38 (s, 1H), 7.72 (d, J ) 7.8 Hz, 1H). MS (DCI) m/z 259 (M +
H)+.
4-Methyl-1-((3-(5-(3-phenoxyprop-1-ynyl)thiophen-3-yl)-1,4-
dihydroindeno[1,2-c]pyrazol-6-yl)methyl)piperazin-2-one TFA
Salt (77). Compound 77 was prepared using the same procedure
as described for the synthesis of 7 by substituting 109 for 5 and 9
for 6. The crude product was purified by preparative reverse phase
HPLC to provide the title compound (17 mg, 23%). 1H NMR (300
MHz, DMSO-d6) δ 2.86 (s, 3H), 3.48-3.53 (m, 4H), 3.81 (s, 2H),
3.92-4.08 (m, 2H), 4.62-4.73 (m, 2H), 5.11 (s, 2H), 6.98-7.03
(m, 1H), 7.04-7.07 (m, 2H), 7.29 (d, J ) 7.8 Hz, 1H), 7.32-7.38
(m, 2H), 7.49 (s, 1H), 7.64 (d, J ) 7.8 Hz, 1H), 7.73 (d, J ) 1.4
Hz, 1H), 7.88 (d, J ) 1.4 Hz, 1H). MS (ESI) m/z 495 (M + H)+.
Anal. (C29H26N4O2S‚1.8CF3CO2H) C, H, N.
(4-Methylpiperazin-1-yl)(3-(5-(3-phenoxyprop-1-ynyl)thiophen-
3-yl)-1,4-dihydroindeno[1,2-c]pyrazol-6-yl)methanone TFA Salt
(78). Compound 78 was prepared using the same procedure as
described for the synthesis of 7 by substituting 24 for 5 and 9 for
6. The crude product was purified by preparative reverse phase
HPLC to provide the title compound (130 mg, 53%). 1H NMR (400
MHz, DMSO-d6) δ 2.20 (s, 3H), 2.28-2.38 (m, 4H), 3.30-3.65
(m, 4H), 3.86 (s, 2H), 5.11 (s, 2H), 6.98-7.02 (m, 1H), 7.05-
7.07 (m, 2H), 7.31-7.39 (m, 3H), 7.57 (s, 1H), 7.69 (d, J ) 7.7
Hz, 1H), 7.74 (s, 1H), 7.90 (s, 1H). MS (ESI) m/z 495 (M + H)+.
Anal. (C29H26N4O2S‚0.5CF3CO2H) C, H, N.
6-(2-(4-Methylpiperazin-1-yl)ethyl)-3-(5-(3-phenoxyprop-1-
ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole (72). Com-
pound 72 was prepared using the same procedure as described for
the synthesis of 7 by substituting 21 for 5 and 9 for 6 (32 mg,
1
28%). H NMR (500 MHz, DMSO-d6) δ 2.14 (s, 3H), 2.25-2.48
(m, 8H), 2.50-2.54 (m, 2H), 2.75-2.78 (m, 2H), 3.75 (s, 2H),
5.09 (s, 2H), 6.98-7.01 (m, 1H), 7.04-7.05 (m, 2H), 7.20 (d, J )
7.8 Hz, 1H), 7.32-7.35 (m, 2H), 7.39 (s, 1H), 7.53 (d, J ) 7.8 Hz,
1H), 7.71 (s, 1H), 7.86 (s, 1H), 13.05 (s, 1H). MS (ESI) m/z 495
(M + H)+. HRMS (FAB) m/z 495.22215; calcd, C30H31N4OS:
495.22131 (M + H)+. HPLC 98% purity (A), 98% purity (B).
5-((4-Methylpiperidin-1-yl)methyl)indan-1-one (106). To a
suspension of 1510 (200 mg, 0.83 mmol) and potassium carbonate
(276 mg, 2.0 mmol) in ethanol (6 mL) was added 4-methylpiperi-
dine (0.24 mL, 2.0 mmol) dropwise at 0 °C. The mixture was stirred
at room temperature for 3 h, concentrated, diluted with H2O, and
extracted with ethyl acetate. The combined organic extracts were
dried (MgSO4), filtered, and evaporated. The crude product was
purified by flash column chromatography on silica gel using
dichloromethane/methanol (5:1) as the mobile phase to provide the
1
title compound (195 mg, 87%). H NMR (500 MHz, CDCl3) δ
0.92 (d, J ) 6.6 Hz, 3H), 1.22-1.30 (m, 2H), 1.34-1.39 (m, 1H),
1.59 (t, J ) 13.7 Hz, 2H), 1.98 (t, J ) 10.6 Hz, 2H), 2.67-2.70
(m, 2H), 2.82-2.84 (m, 2H), 3.12 (t, J ) 5.6 Hz, 2H), 3.54 (s,
2H), 7.33 (d, J ) 7.8 Hz, 1H), 7.45 (s, 1H), 7.69 (d, J ) 7.8 Hz,
1H). MS (ESI) m/z 244 (M + H)+.
6-((4-Methylpiperidin-1-yl)methyl)-3-(5-(3-phenoxyprop-1-
ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(73). Compound 73 was prepared using the same procedure as
described for the synthesis of 7 by substituting 106 for 5 and 9 for
6. The crude product was purified by preparative reverse phase
HPLC to provide the title compound (48 mg, 54%). 1H NMR (500
MHz, DMSO-d6) δ 0.91 (d, J ) 6.6 Hz, 3H), 1.28-1.36 (m, 2H),
1.55-1.66 (m, 2H), 1.80-1.82 (m, 3H), 2.91-2.98 (m, 2H), 3.87
(s, 2H), 4.33 (s, 2H), 5.11 (s, 2H), 6.99-7.02 (m, 1H), 7.05-7.06
(m, 2H), 7.33-7.37 (m, 2H), 7.48 (d, J ) 7.8 Hz, 1H), 7.68 (s,
1H), 7.73-7.75 (m, 2H), 7.91 (s, 1H). MS (ESI) m/z 480 (M +
H)+. HRMS (FAB) m/z 480.21062; calcd, C30H30N3OS: 480.21041
(M + H)+. HPLC 97% purity (A), 98% purity (B).
6-((1-Methylpiperidin-4-yl)methyl)-3-(5-(3-phenoxyprop-1-
ynyl)thiophen-3 -yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt
(74). Compound 74 was prepared using the same procedure as
described for the synthesis of 7 by substituting 23 for 5 and 9 for
6. The crude product was purified by preparative reverse phase
HPLC to provide the title compound (47 mg, 22%). 1H NMR (300
MHz, DMSO-d6) δ 1.31-1.44 (m, 2H), 1.78-1.82 (m, 3H), 2.60
(d, J ) 6.1 Hz, 2H), 7.73 (s, 3H), 2.81-2.93 (m, 2H), 3.37-3.41
(m, 2H), 3.78 (s, 2H), 5.11 (s, 2H), 6.98-7.03 (m, 1H), 7.04-
7.07 (m, 2H), 7.17 (d, J ) 7.4 Hz, 1H), 7.31-7.38 (m, 3H), 7.57
(d, J ) 7.4 Hz, 1H), 7.73 (d, J ) 1.4 Hz, 1H), 7.88 (d, J ) 1.4 Hz,
1H). MS (ESI) m/z 480 (M + H)+. HRMS (FAB) m/z 480.21245;
calcd, C30H30N3OS: 480.21041 (M + H)+. HPLC 97% purity (A),
96% purity (B).
5-(Thiazolidin-3-ylmethyl)indan-1-one (110). Compound 110
was prepared using the same procedure as described for the
synthesis of 106 by substituting thiazolidine for 4-methylpiperidine
(660 mg, 69%). 1H NMR (300 MHz, CDCl3) δ 2.69-2.73 (m, 2H),
2.97-3.01 (m, 2H), 3.12-3.16 (m, 4H), 3.67 (s, 2H), 4.06 (s, 2H),
7.40 (d, J ) 7.8 Hz, 1H), 7.53 (s, 1H), 7.73 (d, J ) 7.8 Hz, 1H).
MS (DCI) m/z 234 (M + H)+.
5-((4-Formylpiperazin-1-yl)methyl)indan-1-one (107). Com-
pound 107 was prepared using the same procedure as described
for the synthesis of 106 by substituting 1-formylpiperazine for
1
4-methylpiperidine (200 mg, 77%). H NMR (500 MHz, DMSO-
d6) δ 2.63-2.65 (m, 2H), 2.75-2.76 (m, 4H), 3.09-3.11 (m, 2H),
3.42-3.44 (m, 4H), 3.73 (s, 2H), 7.37 (d, J ) 7.8 Hz, 1H), 7.52
(s, 1H), 7.62 (d, J ) 7.8 Hz, 1H), 8.03 (s, 1H). MS (ESI) m/z 259
(M + H)+.
4-((3-(5-(3-Phenoxyprop-1-ynyl)thiophen-3-yl)-1,4-dihydroin-
deno[1,2-c]pyrazol-6-yl)methyl)piperazine-1-carbaldehyde (75).
Compound 75 was prepared using the same procedure as described
for the synthesis of 7 by substituting 107 for 5 and 9 for 6 (50 mg,
24%). 1H NMR (300 MHz, DMSO-d6) δ 2.33-2.42 (m, 4H), 3.35-
3.41 (m, 4H), 3.56 (s, 2H), 3.81 (s, 2H), 5.11 (s, 2H), 6.98-7.03
(m, 1H), 7.04-7.07 (m, 2H), 7.30 (d, J ) 7.5 Hz, 1H), 7.32-7.38
(m, 2H), 7.49 (s, 1H), 7.61 (d, J ) 7.5 Hz, 1H), 7.74 (s, 1H), 7.89
3-((3-(5-(3-Phenoxyprop-1-ynyl)thiophen-3-yl)-1,4-dihydroin-
deno[1,2-c]pyrazol-6-yl)methyl)thiazolidine TFA Salt (79). Com-
pound 79 was prepared using the same procedure as described for
the synthesis of 7 by substituting 110 for 5 and 9 for 6. The crude
product was purified by preparative reverse phase HPLC to provide
1
the title compound (112 mg, 28%). H NMR (300 MHz, DMSO-
d6) δ 3.00-3.08 (m, 2H), 3.23-3.34 (m, 2H), 3.84 (s, 2H), 4.02
(bs, 2H), 4.18 (s, 2H), 5.11 (s, 2H), 6.98-7.03 (m, 1H), 7.04-
7.07 (m, 2H), 7.32-7.38 (m, 2H), 7.41 (d, J ) 7.8 Hz, 1H), 7.62
(s, 1H), 7.67 (d, J ) 7.8 Hz, 1H), 7.74 (d, J ) 1.4 Hz, 1H), 7.89

2026 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
(d, J ) 1.4 Hz, 1H). MS (ESI) m/z 470 (M + H)+. Anal. (C27H23N3-
OS2‚CF3CO2H) C, H, N.
m/z 449 (M + H)+. HRMS (FAB) m/z 449.14307; calcd, C27H21N4-
OS: 449.14306 (M + H)+. HPLC 98% purity (A), 99% purity
(B).
6-(2-Methoxyethoxy)-3-(5-(3-phenoxyprop-1-ynyl)thiophen-
3-yl)-1,4-dihydroindeno[1,2-c]pyrazole (80). Compound 80 was
prepared using the same procedure as described for the synthesis
5-((1H-1,2,3-Triazol-1-yl)methyl)indan-1-one (114). Com-
pound 114 was prepared using the same procedure as described
for the synthesis of 106 by substituting 1,2,3-triazole for 4-meth-
1
of 7 by substituting 26 for 5 and 9 for 6 (37 mg, 33%). H NMR
1
ylpiperidine (120 mg, 45%). H NMR (400 MHz, DMSO-d6) δ
(500 MHz, DMSO-d6) δ 3.31 (s, 3H), 3.66-3.68 (m, 2H), 3.75 (s,
2H), 4.12-4.14 (m, 2H), 5.09 (s, 2H), 6.92 (d, J ) 8.1 Hz, 1H),
6.98-7.01 (m, 1H), 7.03-7.05 (m, 2H), 7.15 (s, 1H), 7.32-7.35
(m, 2H), 7.51 (d, J ) 8.1 Hz, 1H), 7.69 (s, 1H), 7.83 (s, 1H). MS
(ESI) m/z 443 (M + H)+. HRMS (FAB) m/z 443.14265; calcd,
C26H23N2O3S: 443.14239 (M + H)+. HPLC 97% purity (A), 98%
purity (B).
N,N-Dimethyl-2-(1-oxo-indan-5-yloxy)acetamide (111). Com-
pound 111 was prepared using the same procedure as described
for the synthesis of 26 by substituting 2-chloro-N,N-dimethylac-
etamide for 1-bromo-2-methoxy-ethane (1.4 g, 60%). 1H NMR (500
MHz, DMSO-d6) δ 2.57-2.59 (m, 2H), 2.85 (s, 3H), 3.00 (s, 3H),
3.02-3.05 (m, 2H), 4.95 (s, 2H), 6.94 (dd, J ) 8.6, 2.1 Hz, 1H),
7.04 (d, J ) 2.1 Hz, 1H), 7.54 (d, J ) 8.6 Hz, 1H). MS (ESI) m/z
234 (M + H)+.
N,N-Dimethyl-2-(3-(5-(3-phenoxyprop-1-ynyl)thiophen-3-yl)-
1,4-dihydroindeno[1,2-c]pyrazol-6-yloxy)acetamide TFA Salt
(81). Compound 81 was prepared using the same procedure as
described for the synthesis of 7 by substituting 111 for 5 and 9 for
6. The crude product was purified by preparative reverse phase
HPLC to provide the title compound (15 mg, 21%). 1H NMR (500
MHz, DMSO-d6) δ 2.86 (s, 3H), 3.02 (s, 3H), 3.77 (s, 2H), 4.84
(s, 2H), 5.11 (s, 2H), 6.92 (dd, J ) 8.2, 2.1 Hz, 1H), 6.99-7.02
(m, 1H), 7.05-7.06 (m, 2H), 7.14 (d, J ) 2.1 Hz, 1H), 7.33-7.36
(m, 2H), 7.52 (d, J ) 8.2 Hz, 1H), 7.71 (d, J ) 1.3 Hz, 1H), 7.85
(d, J ) 1.3 Hz, 1H). MS (ESI) m/z 470 (M + H)+. HRMS (FAB)
m/z 470.15328; calcd, C27H24N3O3S: 470.15329 (M + H)+. HPLC
98% purity (A), 97% purity (B).
5-((1H-Imidazol-1-yl)methyl)indan-1-one (112). Compound
112 was prepared using the same procedure as described for the
synthesis of 106 by substituting imidazole for 4-methylpiperidine
(650 mg, 75%). 1H NMR (500 MHz, DMSO-d6) δ 2.61-2.63 (m,
2H), 3.06-3.08 (m, 2H), 5.32 (s, 2H), 6.94 (s, 1H), 7.21 (s, 1H),
7.27 (d, J ) 7.8 Hz, 1H), 7.38 (s, 1H), 7.62 (d, J ) 7.8 Hz, 1H),
7.79 (s, 1H). MS (ESI) m/z 213 (M + H)+.
6-((1H-Imidazol-1-yl)methyl)-3-(5-(3-phenoxyprop-1-ynyl)thio-
phen-3-yl)-1, 4-dihydroindeno[1,2-c]pyrazole (82). Compound 82
was prepared using the same procedure as described for the
synthesis of 7 by substituting 112 for 5 and 9 for 6 (50 mg, 37%).
1H NMR (500 MHz, DMSO-d6) δ 3.83 (s, 2H), 5.11 (s, 2H), 5.48
(s, 2H), 6.99-7.02 (m, 1H), 7.04-7.06 (m, 2H), 7.33-7.35 (m,
2H), 7.42 (d, J ) 8.4 Hz, 1H), 7.63 (s, 1H), 7.68-7.70 (m, 2H),
7.73 (s, 1H), 7.81 (s, 1H), 7.89 (s, 1H), 9.23 (s, 1H). MS (ESI) m/z
449 (M + H)+. HRMS (FAB) m/z 449.14303; calcd, C27H21N4OS:
449.14306 (M + H)+. HPLC 99% purity (A), 99% purity (B).
5-((1H-Pyrazol-1-yl)methyl)indan-1-one (113). Compound 110
was prepared using the same procedure as described for the
synthesis of 106 by substituting pyrazole for 4-methylpiperidine
(582 mg, 45%). 1H NMR (300 MHz, CDCl3) δ 2.66-2.70 (m, 2H),
3.08-3.12 (m, 2H), 5.41 (s, 2H), 6.36 (t, J ) 2.0 Hz, 1H), 7.19 (d,
J ) 7.8 Hz, 1H), 7.22 (s, 1H), 7.45 (d, J ) 2.4 Hz, 1H), 7.58 (d,
J ) 1.7 Hz, 1H), 7.71 (d, J ) 7.8 Hz, 1H). MS (DCI) m/z 213 (M
+ H)+.
2.61-2.64 (m, 2H), 3.06-3.09 (m, 2H), 5.75 (s, 2H), 7.30 (d, J )
7.8 Hz, 1H), 7.43 (s, 1H), 7.63 (d, J ) 7.8 Hz, 1H), 7.78 (d, J )
0.9 Hz, 1H), 8.23 (d, J ) 0.9 Hz, 1H). MS (DCI) m/z 214 (M +
H)+.
6-((1H-1,2,3-Triazol-1-yl)methyl)-3-(5-(3-phenoxyprop-1-ynyl)-
thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole (84). Compound
84 was prepared using the same procedure as described for the
synthesis of 7 by substituting 114 for 5 and 9 for 6 (92 mg, 36%).
1H NMR (500 MHz, DMSO-d6) δ 3.81 (s, 2H), 5.10 (s, 2H), 5.68
(s, 2H), 6.99-7.02 (m, 1H), 7.04-7.06 (m, 2H), 7.31 (d, J ) 7.8
Hz, 1H), 7.33-7.36 (m, 2H), 7.50 (s, 1H), 7.64 (d, J ) 7.8 Hz,
1H), 7.72 (s, 1H), 7.75 (d, J ) 0.9 Hz, 1H), 7.87 (s, 1H), 8.21 (d,
J ) 0.9 Hz, 1H), 13.16 (bs, 1H). MS (ESI) m/z 450 (M + H)+.
Anal. (C26H19N5OS) C, H, N.
5-((1H-1,2,4-Triazol-1-yl)methyl)inden-1-one (115). Compound
115 was prepared using the same procedure as described for the
synthesis of 106 by substituting 1,2,4-triazole for 4-methylpiperidine
1
(8.6 g, 68%). H NMR (300 MHz, CDCl3) δ 2.69-2,73 (m, 2H),
3.11-3.15 (m, 2H), 5.44 (s, 2H), 7.26 (d, J ) 7.8 Hz, 1H), 7.32
(s, 1H), 7.76 (d, J ) 7.8 Hz, 1H), 8.02 (s, 1H), 8.21 (s, 1H). MS
(ESI) m/z 214 (M + H)+.
6-((1H-1,2,4-Triazol-1-yl)methyl)-3-(5-(3-phenoxyprop-1-ynyl)-
thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole (85). Compound
85 was prepared using the same procedure as described for the
synthesis of 7 by substituting 115 for 5 and 9 for 6 (20 mg, 13%).
1H NMR (500 MHz, DMSO-d6) δ 3.80 (s, 2H), 5.11 (s, 2H), 5.48
(s, 2H), 6.99-7.02 (m, 1H), 7.04-7.06 (m, 2H), 7.29 (d, J ) 7.8
Hz, 1H), 7.33-7.36 (m, 2H), 7.48 (s, 1H), 7.62 (d, J ) 7.8 Hz,
1H), 7.72 (s, 1H), 7.87 (s, 1H), 8.00 (s, 1H), 8.70 (s, 1H). MS
(ESI) m/z 450 (M + H)+. Anal. (C26H19N5OS) C, H, N.
4-(4-(3-(4-(6-((4-Methylpiperazin-1-yl)methyl)-1,4-dihydroin-
deno[1,2-c]pyrazol-3-yl)thiophen-2-yl)prop-2-ynyloxy)benzyl)-
morpholine TFA Salt (86). Compound 86 was prepared using the
same procedure as described for the synthesis of 8 by substituting
37 for phenyl propargyl ether (80 mg, 75%). 1H NMR (400 MHz,
DMSO-d6) δ 2.84 (s, 3H), 3.00-3.50 (m, 12H), 3.60-3.70 (m,
2H), 3.84 (s, 2H), 3.90-4.00 (m, 2H), 4.15 (bs, 2H), 4.31 (s, 2H),
5.16 (s, 2H), 7.14-7.16 (m, 2H), 7.44 (d, J ) 7.9 Hz, 1H), 7.48-
7.50 (m, 2H), 7.64 (s, 1H), 7.70 (d, J ) 7.9 Hz, 1H), 7.76 (d, J )
1.2 Hz, 1H), 7.92 (d, J ) 1.2 Hz, 1H). MS (ESI) m/z 580 (M +
H)+. HRMS (FAB) m/z 580.27470; calcd, C34H38N5O2S: 580.27407
(M + H)+. HPLC 97% purity (A), 97% purity (B).
3-(5-(3-(2-Methoxyethoxy)prop-1-ynyl)thiophen-3-yl)-6-((4-
methylpiperazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyra-
zole TFA Salt (87). Compound 86 was prepared using the same
procedure as described for the synthesis of 8 by substituting 39 for
phenyl propargyl ether (60 mg, 75%). 1H NMR (400 MHz, DMSO-
d6) δ 2.83 (s, 3H), 3.26 (s, 3H), 3.00-3.50 (m, 8H), 3.48-3.50
(m, 2H), 3.62-3.64 (m, 2H), 3.83 (s, 2H), 4.19 (s, 2H), 4.44 (s,
2H), 7.44 (d, J ) 7.9 Hz, 1H), 7.64 (s, 1H), 7.69 (d, J ) 7.9 Hz,
1H), 7.73 (d, J ) 1.2 Hz, 1H), 7.88 (d, J ) 1.2 Hz, 1H). MS (ESI)
m/z 463 (M + H)+. HRMS (FAB) m/z 463.21700; calcd,
C26H31N4O2S: 463.21622 (M + H)+. HPLC 98% purity (A), 97%
purity (B).
3-(5-(3-(2-Methoxyethoxy)prop-1-ynyl)thiophen-3-yl)-7-((4-
methylpiperazin-1-yl)methyl)-1,4-dihydroindeno[1,2-c]pyra-
zole TFA Salt (88). Compound 88 was prepared using the same
procedure as described for the synthesis of 8 by substituting 101
for 7 and 39 for phenyl propargyl ether. The crude product was
purified by preparative reverse phase HPLC to provide the title
compound (24 mg, 23%). 1H NMR (400 MHz, DMSO-d6) δ 2.84
(s, 3H), 3.00-3.50 (m, 8H), 3.27 (s, 3H), 3.50-3.52 (m, 2H), 3.63-
3.66 (m, 2H), 3.86 (s, 2H), 4.21 (s, 2H), 4.46 (s, 2H), 7.37 (d, J )
7.9 Hz, 1H), 7.62 (d, J ) 7.9 Hz, 1H), 7.74 (s, 1H), 7.78 (s, 1H),
6-((1H-Pyrazol-1-yl)methyl)-3-(5-(3-phenoxyprop-1-ynyl)thio-
phen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA Salt (83).
Compound 83 was prepared using the same procedure as described
for the synthesis of 7 by substituting 113 for 5 and 9 for 6. The
crude product was purified by preparative reverse phase HPLC to
1
provide the title compound (42 mg, 39%). H NMR (300 MHz,
DMSO-d6) δ 3.79 (s, 2H), 5.11 (s, 2H), 5.39 (s, 2H), 6.28 (t, J )
2.0 Hz, 1H), 6.97-7.02 (m, 1H), 7.04-7.06 (m, 2H), 7.22 (d, J )
7.8 Hz, 1H), 7.32-7.37 (m, 2H), 7.40 (s, 1H), 7.47 (dd, J ) 2.0,
0.7 Hz, 1H), 7.60 (d, J ) 7.8 Hz, 1H), 7.72 (d, J ) 1.4 Hz, 1H),
7.85 (dd, J ) 2.4, 0.7 Hz, 1H), 7.87 (d, J ) 1.4 Hz, 1H). MS (ESI)

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2027
7.89 (s, 1H). MS (ESI) m/z 463 (M + H)+. HRMS (FAB) m/z
463.21634; calcd, C26H31N4O2S: 463.21622 (M + H)+. HPLC 95%
purity (A), 96% purity (B).
µL/well of 0.5 M ethylenediaminetetraacetic acid (EDTA), and then
75 µL of buffer containing streptavidin-allophycocyanin (Prozyme)
(1.1 µg/mL) and PT66 antibody europium cryptate (Cis-Bio) (0.1
µg/mL) was added to each well. The plates were read from 1 to 4
h after addition of the detection reagents, and the time-resolved
fluorescence (665:615 ratio) was measured using a Packard
Discovery instrument. The amount of each tyrosine kinase added
to the wells was calibrated to give a control (no inhibitor) to
background (prequenched with EDTA) ratio of 10-15, and it was
shown to be in the low nanomolar concentration range for each
kinase. The inhibition of each well was calculated using the control
and background readings for that plate. Each IC50 determination
was performed with seven concentrations, and each assay point
was determined in duplicate.
Cellular KDR Phosphorylation Assay. KDR Phosphorylation
Determined by ELISA.29 NIH3T3 cells stably transfected with
full length human KDR (VEGFR2) were maintained in a Dulbec-
co’s modified Eagle’s medium (DMEM) with 10% fetal bovine
serum and 500 µg/mL geneticin. KDR cells were plated at 20000
cells per well into duplicate 96-well tissue culture plates and
6-((1H-1,2,4-Triazol-1-yl)methyl)-3-(5-bromothiophen-3-yl)-
1,4-dihydroindeno[1,2-c]pyrazole (116). Compound 116 was
prepared using the same procedure as described for the synthesis
1
of 7 by substituting 115 for 5 (1.3 g, 70%). H NMR (300 MHz,
DMSO-d6) δ 3.80 (s, 2H), 5.48 (s, 2H), 7.29 (d, J ) 7.8 Hz, 1H),
7.48 (s, 1H), 7.62-7.64 (m, 2H), 7.83 (s, 1H), 7.99 (s, 1H), 8.69
(s, 1H), 13.24 (bs, 1H). MS (ESI) m/z 398, 400 (M + H)+.
6-((1H-1,2,4-Triazol-1-yl)methyl)-3-(5-(3-(2-methoxyethoxy)-
prop-1-ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole (89).
Compound 89 was prepared using the same procedure as described
for the synthesis of 8 by substituting 116 for 7 and 39 for phenyl
1
propargyl ether (90 mg, 49%). H NMR (500 MHz, DMSO-d6) δ
3.26 (s, 3H), 3.48-3.50 (m, 2H), 3.62-3.64 (m, 2H), 3.80 (s, 2H),
4.44 (s, 2H), 5.47 (s, 2H), 7.28 (d, J ) 7.8 Hz, 1H), 7.47 (s, 1H),
7.63 (d, J ) 7.8 Hz, 1H), 7.70 (s, 1H), 7.85 (s, 1H), 7.98 (s, 1H),
8.67 (s, 1H), 13.16 (bs, 1H). MS (ESI) m/z 432 (M + H)+. Anal.
(C23H21N5O2S) C, H, N.
6-((1H-1,2,4-Triazol-1-yl)methyl)indan-1-one (117). Com-
pound 117 was prepared using the same procedure as described
for the synthesis of 106 by substituting (3-oxo-indan-5-yl)methyl
methanesulfonate for 15 and 1,2,4-triazole for 4-methylpiperidine
cultured overnight in an incubator at 37 °C with 5% CO2 and 80%
humidity. The growth medium was replaced with a serum-free
growth medium for 2 h prior to compound addition. Compounds
in DMSO were diluted in the serum-free growth medium (final
DMSO concentration 1%) and added to cells for 20 min prior to
stimulation for 10 min with VEGF (50 ng/mL). Cells were lysed
by addition of RIPA buffer (50 mM Tris-HCl (pH 7.4), 1%
IGEPAL, 150 mM NaCl, 1 mM EDTA, and 0.25% sodium
deoxycholate) containing protease inhibitors (Sigma cocktail), NaF
(1 mM), and Na3VO4 (1 mM) and placed in a microtiter plate shaker
for 10 min. The lysates from duplicate wells were combined, and
170 µL of the combined lysate was added to the KDR ELISA plate.
The KDR ELISA plate was prepared by adding an anti-VEGFR2
antibody (1 µg/well, R&D Systems) to an unblocked plate and
incubated overnight at 4 °C. The plate was then blocked for at least
1 h with 200 µL/well of 5% dry milk in PBS. The plate was washed
two times with PBS containing 0.1% Tween 20 (PBST) before
addition of the cell lysates. Cell lysates were incubated in the KDR
ELISA plate with constant shaking on a microtiter plate shaker for
2 h at room temperature. The cell lysate was then removed, and
the plate was washed five times with PBST. Detection of phospho-
KDR was performed using a 1:2000 dilution of biotinylated 4G10
anti-phosphotyrosine (UBI, Lake Placid, NY). The wells were
incubated with constant shaking for 1.5 h at room temperature and
washed five times with PBST, and for detection, a 1:2000 dilution
of streptavidin-HRP (UBI, Lake Placid, NY) was added and
incubated with constant shaking for 1 h at room temperature. The
wells were then washed five times with PBST, and K-Blue HRP
ELISA substrate (Neogen) was added to each well. The develop-
ment time was monitored at 650 nm in a SpectraMax Plus plate
reader until 0.4-0.5 absorbance units were obtained (approximately
10 min) in the VEGF only wells. Phosphoric acid (1 M) was added
to stop the reaction, and the plate was read at 450 nm. The percent
inhibition was calculated using the VEGF only wells as 100%
controls and wells containing 5 µM pan-kinase inhibitor as 0%
controls (no VEGF wells were used to monitor the endogenous
phosphorylation state of the cells). IC50 values were calculated by
nonlinear regression analysis of the concentration response curve.
Each IC50 determination was performed with five concentrations,
and each assay point was determined in duplicate.
1
(3.7 g, 60%). H NMR (300 MHz, CDCl3) δ 2.70-2.74 (m, 2H),
3.13-3.17 (m, 2H), 5.40 (s, 2H), 7.50-7.51 (m, 2H), 7.67 (s, 1H),
7.98 (s, 1H), 8.10 (s, 1H). MS (DCI) m/z 214 (M + H)+.
7-((1H-1,2,4-Triazol-1-yl)methyl)-3-(5-bromothiophen-3-yl)-
1,4-dihydroindeno[1,2-c]pyrazole (118). Compound 118 was
prepared using the same procedure as described for the synthesis
1
of 7 by substituting 117 for 5 (3.0 g, 75%). H NMR (300 MHz,
DMSO-d6) δ 3.79 (s, 2H), 5.50 (s, 2H), 7.24 (d, J ) 7.8 Hz, 1H),
7.53-7.55 (m, 2H), 7.63 (s, 1H), 7.83 (s, 1H), 8.00 (s, 1H), 8.71
(s, 1H), 13.21 (bs, 1H). MS (ESI) m/z 398, 400 (M + H)+.
7-((1H-1,2,4-Triazol-1-yl)methyl)-3-(5-(3-(2-methoxyethoxy)-
prop-1-ynyl)thiophen-3-yl)-1,4-dihydroindeno[1,2-c]pyrazole TFA
Salt (90). Compound 90 was prepared using the same procedure
as described for the synthesis of 8 by substituting 118 for 7 and 39
for phenyl propargyl ether. The crude product was purified by
preparative reverse phase HPLC to provide the title compound (46
mg, 22%). 1H NMR (400 MHz, DMSO-d6) δ 3.27 (s, 3H), 3.49-
3.51 (m, 2H), 3.63-3.65 (m, 2H), 3.81 (s, 2H), 4.45 (s, 2H), 5.50
(s, 2H), 7.24 (d, J ) 7.8 Hz, 1H), 7.53-7.57 (m, 2H), 7.72 (s,
1H), 7.87 (s, 1H), 7.99 (s, 1H), 8.70 (s, 1H), 13.18 (s, 1H). MS
(ESI) m/z 432 (M + H)+. HRMS (FAB) m/z 432.14888; calcd,
C23H22N5O2S: 432.14887 (M + H)+. HPLC 98% purity (A), 97%
purity (B).
Computational Analysis. The hERG pore homology model in
its closed state was constructed (Insight II software, HOMOLOGY
module, Accelrys, San Diego, CA) using the homologous pore
region of the KcsA potassium channel (PDB entry 1BL8) following
methodologies described earlier.25,26 The structure of 8 was built
in Insight II and was energy minimized. Its piperazine moiety, which
is protonated at physiological pH, was maintained in a protonated
form. Compound 8 was docked manually into the hERG pore
homology model, following previously established orientations for
compounds with comparable shape and charge.25,26 Energy mini-
mization using the CFF force field (25 iterations with the static
pore model, followed by 50 iterations of full ligand-protein
minimization) provided the final model shown if Figure 2. No
solvation was included.
KDR Phosphorylation Determined by Western Blot Analy-
sis.29 NIH3T3 cells stably transfected with full length human KDR
(VEGFR2) were cultured in T75 or T150 flasks; compounds were
added in the serum-free growth medium for 20 min prior to
stimulation for 3 min with VEGF (50 ng/mL). The cells were
washed one time with PBS and lysed with 1 mL/flask of RIPA
buffer containing protease inhibitors, NaF, and Na3VO4 for 5 min
on ice. Cells were scraped, and the lysate was incubated for an
additional 20 min in ice with occasional mixing in a 2 mL
microcentrifuge tube. The lysate was centrifuged at 20000g for 20
min at 4 °C, and the supernatant was used to immunoprecipitate
Homogeneous Time-Resolved Fluorescence (HTRF) Assays
of Receptor Tyrosine Kinases.29 Assays were performed in a total
of 40 µL in 96-well Costar black half-volume plates using HTRF
technology. A peptide substrate (biotin-Ahx-AEEEYFFLFA-amide)
at 4 µM, 1 mM ATP, enzyme, and inhibitors were incubated for 1
h at ambient temperature in 50 mM HEPES/NaOH (pH 7.5), 10
mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.1 mM orthovanadate,
and 0.01% bovine serum albumin (BSA). Inhibitors were added to
the wells at a final concentration of 3.2 nM to 50 µM with 5%
DMSO added as a cosolvent. The reactions were stopped with 10

2028 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Dinges et al.
KDR. The lysate, 500 µg, was precleared with Protein-G agarose
beads (Calbiochem) for 30 min at 4 °C while rotating. The lysate
was centrifuged (20000g for 1 min), and the supernatant was
immunoprecipitated with 4 µg of an anti-VEGFR2 antibody (R&D
Systems) overnight at 4 °C while rotating. After incubation, 50 µL
of Protein-G agarose was added and incubated for 1 h at 4 °C while
rotating. Beads were washed five times with PBS containing
Na3VO4 (1 mM) and protease inhibitors (Sigma). The washed beads
were resuspended in 50 µL of sample buffer, electrophoresed on a
8-16% Tris-glycine gel (Invitrogen), transferred to a nitrocellulose
membrane, blocked with 5% milk in PBST, and probed with an
anti-KDR antibody (sc-315, Santa Cruz). KDR was visualized with
enhanced chemoluminescence detection, and IC50 values were
determined by comparison of the treated samples to the vehicle
(DMSO) treated samples.
Estradiol-Induced Murine Uterine Edema Assay.29 Twelve
week old balb/c female mice (Taconic, Germantown, NY) were
pretreated with 10 units of pregnant mare’s serum gonadotropin
(PMSG) (Calbiochem) intraperitoneally (ip) administered 72 and
24 h prior to estradiol. Mice were randomized the day of the experi-
ment. Test compounds were formulated in a variety of vehicles
and administered po 30 min prior to stimulation with an intraperi-
toneal injection of water-soluble 17â-estradiol (20-25 µg/mouse).
Animals were sacrificed and uteri removed 2.5 h following estra-
diol stimulation by cutting just proximal to the cervix and at the
fallopian tubes. After the removal of fat and connective tissue, uteri
were weighed, squeezed between filter paper to remove fluid, and
weighed again. The difference between wet and blotted weights
represented the fluid content of the uterus. Compound treated groups
were compared to vehicle treated groups of animals after subtracting
the background water content of unstimulated uteri. The experi-
mental group size was five or six.
HT1080 Tumor Growth Inhibition Model.29 The 1080 human
fibrosarcoma cells were obtained from the American Type Tissue
Culture Collection and maintained in DMEM, supplemented with
10% fetal bovine serum and antibiotics. For tumor xenograft studies,
cells were suspended in PBS, mixed with an equal volume of
matrigel (phenol red free) to a final concentration of 2 million cells/
mL, and inoculated (0.25 mL) into the flank of SCID-beige mice.
One week after inoculation, tumor-bearing animals were divided
into groups (n ) 10), and administration of vehicle (2% EtOH,
5% Tween 80, 20% PEG 400, and 73% saline) or inhibitor at the
indicated dose was initiated. Tumor growth was assessed every 2-3
days by measuring tumor size and calculating tumor volume using
the formula [length × width2]/2.
protein concentration of 30 µg per well). The plates were incubated
at ambient temperature for 45 min, aspirated onto GF/B filter plates
(Perkin-Elmer), and washed with 2 mL of cold wash buffer. After
allowing the plates to dry, 50 µL of scintillant (Perkin-Elmer
MicroScint 20) was added to each well, and the radioactivity was
counted in a Perkin-Elmer Topcount NXT scintillation counter. IC50
determinations were calculated from competition curves using six
drug concentrations, half-log apart, starting at a high concentration
of 100 µM (final assay DMSO concentration ) 1%) using a four-
parameter logistic equation.
hERG Patch Clamp Assay. hERG channels stably expressed
in human embryonic kidney cells (HEK-293 cells)30 were main-
tained in culture. For electrophysiology recordings, cells were
briefly trypsinized to release the cells from the plates, pelleted by
centrifugation (1000g), resuspended in culture media, and studied
within 8 h. HERG current was recorded using standard patch clamp
techniques as described previously.31 Currents were recorded using
either an Axopatch 200A or Axopatch Multiclamp 700A amplifier
along with pClamp data acquisition software (version 8, Molecular
Devices). Drug effects were assessed using a voltage clamp protocol
that sequentially stepped to -25, 0, 25 or 50 mV for 3 s, followed
by a step to -50 mV for 4 s from a holding potential of -80 mV;
clamp pulses were applied once every 15 s. IC50 values for block
of hERG current were calculated from tail currents measured at
-50 mV following conditioning pulses to 0 mV. Patch pipettes
were constructed with borosilicate glass capillary tubes (resistance
1.8-3.8 M-ohm)). The pipet solution contained (in mM): K+
aspartate 125, KCl 20, EGTA 10, MgCl2 1, HEPES 5, MgATP 5
(pH ) 7.3). The bath solution contained the following (in mM):
NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 5, HEPES 20, pH )
7.4. Experiments were performed at 36.5-37 °C.
Supporting Information Available: Combustion analysis and
high resolution MS and HPLC data. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. The Protein Kinase Complement of the Human Genome. Science
2002, 298, 1912-1934.
(2) Robinson, D. R.; Wu, Y.-M.; Lin, S.-F. The Protein Tyrosine Kinase
Family of the Human Genome. Oncogene 2000, 19, 5548-5557.
(3) Ullrich, A.; Schlessinger, J. Signal Transduction by Receptors with
Tyrosine Kinase Activity. Cell 1990, 61, 203-212.
(4) Rousset, D.; Agnes, F.; Lachaume, P.; Andre, C.; Galibert, F.
Molecular Evolution of the Genes Encoding Receptor Tyrosine
Kinase with Immunoglobulinlike Domains. J. Mol. EVol. 1995, 41,
421-429.
(5) Hanahan, D.; Weinberg, R. The Hallmarks of Cancer. Cell 2000,
100, 57-70.
(6) Potapova, O.; Laird, A. D.; Nannini, M. A.; Barone, A.; Li, G.; Moss,
K. G.; Cherrington, J. M.; Mendel, D. B. Contribution of Individual
Targets to the Antitumor Efficacy of the Multitargeted Receptor
Tyrosine Kinase Inhibitor SU11248. Mol. Cancer Ther. 2006, 5,
1280-1289.
(7) Rueegg, C.; Hasmim, M.; Lejeune, F. J.; Alghisi, G. C. Antiangio-
genic Peptides and Proteins: From Experimental Tools to Clinical
Drugs. Biochim. Biophys. Acta 2006, 1765, 155-177.
(8) Dagher, R.; Cohen, M.; Williams, G.; Rothmann, M.; Gobburu, J.;
Robbie, G.; Rahman, A.; Chen, G.; Staten, A.; Griebel, D.; Pazdur,
R. Approval Summary: Imatinib Mesylate in the Treatment of
Metastatic and/or unresectable Malignant Gastrointestinal Stromal
Tumors. Clin. Cancer Res. 2002, 8, 3034-3038.
(9) Atkins, M.; Jones, C.; A.; Kirkpatrick, P. Sunitinib Maleate. Nature
ReV., Drug DiscoVery 2006, 5, 279-280.
MX-1 Tumor Growth Inhibition Model. MX-1 human mam-
mary carcinoma was obtained from the tumor repository of the
National Cancer Institute (Bethesda, MD). Solid tumors were
maintained in ViVo by serial passage in specific pathogen free female
athymic Ncr-nude mice (Taconic). Donor tumors were harvested
when they reached 1-2 grams and 50 mg pieces of non-necrotic
tissue were implanted sc into anesthetized recipient mice through
a small skin incision in the flank which was subsequently closed
with a wound clip. Animals were randomized into groups of 5-10
when tumors reached a size of approximately 100 mg (5-7 days)
and treatment with vehicle or inhibitor was initiated. Tumor growth
was monitored 2-3 times per week by caliper measurements and
converted to volume using the formula: V ) width2 × length/2.
All animal procedures were reviewed and approved by the Animal
Care and Use Committee.
[3H]Dofetilide/HEK-293 Membrane Competition Binding
Assay.24 The affinity of test drugs for the hERG cardiac K+ channel
was determined by their ability to displace tritiated dofetilide (a
class III antiarrhythmic drug and potent hERG blocker) in
membrane homogenates from HEK-293 cells heterogenously
expressing the hERG channel. Drug dilutions were prepared from
10 mM DMSO stocks and the following were added to a 96-well
polystyrene plate (Perkin-Elmer Optiplate): 20 µL of assay binding
buffer (for total bounds), or 1 µM astemizole (for nonspecific
bounds), or test drug, 50 µL of [3H]dofetilide (20 nM, 85 Ci/mmol
specific activity), and 130 µL of membrane homogenate (final
(10) Dinges, J.; Akritopoulou-Zanze, I.; Arnold, L. D.; Barlozzari, T.;
Bousquet, P. F.; Cunha, G. A.; Ericsson, A. M.; Iwasaki, N.;
Michaelides, M. R.; Ogawa, N.; Phelan, K. M.; Rafferty, P.; Sowin,
T. J.; Stewart, K. D.; Tokuyama, R.; Xia, Z.; Zhang, H. Q. Hit-to-
Lead Optimization of 1,4-Dihydroindeno[1,2-c]pyrazoles as a Novel
Class of KDR Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2006,
16, 4371-4375.
(11) Dinges, J.; Ashworth, K. L.; Akritopoulou-Zanze, I.; Arnold, L. D.;
Baumeister, S. A.; Bousquet, P. F.; Cunha, G. A.; Davidsen, S. K.;
Djuric, S. W.; Gracias, V. J.; Michaelides, M. R.; Rafferty, P.; Sowin,
T. J.; Stewart, K. D.; Xia, Z.; Zhang, H. Q. 1,4-Dihydroindeno[1,2-
c]pyrazoles as Novel Multitargeted Receptor Tyrosine Kinase Inhibi-
tors. Bioorg. Med. Chem. Lett. 2006, 16, 4266-4271.

Multitargeted Receptor Tyrosine Kinase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2029
(12) Erde´lyi, M.; Gogoll, A. Rapid Homogeneous-Phase Sonogashira
Coupling Reactions Using Controlled Microwave Heating. J. Org.
Chem. 2001, 66, 4165.
(13) Cusic, J. W.; Lowrie, H. S.; Sause, H. W. U.S. Patent 2,787,617,
1957.
(14) Hosoya, T.; Takashiro, E.; Matsumoto, T.; Suzuki, K. Total Synthesis
of the Gilvocarcins. J. Am. Chem. Soc. 1994, 116, 1004-1015.
(15) Campaigne, E.; Bourgeois, R. C. 3-Substituted Thiophenes. VI.
Substitution Reactions of 3-Thenoic Acid. J. Am. Chem. Soc. 1954,
76, 2445-2447.
(16) Tu, Y. Q.; Hu¨bener, A., Zhang, H., Moore, C. J.; Fletcher, M. T.;
Hayes, P.; Dettner, K.; Francke, W.; McErlean, C. S. P.; Kitching,
W. Synthesis and Stereochemistry of Insect Derived Spiroacetals with
Branched Carbon Skeletons. Synthesis 2000, 13, 1956-1978.
(17) Rawat, D. S.; Zaleski, J. M. A Convenient Method for the Synthesis
of 1,8-Bis(pyridin-3-oxy)oct-4-ene-2,6-diyne. Synth. Commun. 2002,
32, 1489-1494.
(18) Wipf, P.; Aoyama, Y.; Benedum. T. E. A Practical Method for
Oxazole Synthesis by Cycloisomerization of Propargyl Amides. Org.
Lett. 2004, 6, 3593-3595.
(19) Tao, Z.-F.; Sowin, T. J.; Lin, N.-H. A facile Synthesis of Antitumoral
Indeno[1,2-c]pyrazol-4-one by mild Oxidation with molecular Oxy-
gen. Tetrahedron Lett. 2005, 46, 7615-7618.
(20) Reich, S. H.; Wallace, M. B. PCT Int. Appl. WO 079198, 2001.
(21) Data reported as an average of five animals; p-values vs vehicle are
given in parentheses for percent inhibition data, and 95% confidence
levels are given in parenthesis for ED50 values.
(22) Pharmacokinetic data are reported as an average of three animals.
Variability around the mean value was <40 %. Vehicle: iv, 2.5 %
DMSO, 2.5% Tween-80, 25% PEG400, 70% PBS; po, 5% Tween-
80, 2.5% ethanol, 25% PEG400, 67.5% PBS.
(24) Diaz, G. J.; Daniell, K.; Leitza, S. T.; Martin, R. L.; Su, Z.;
McDermott, J. S.; Cox, B. F.; Gintant, G. A. The [3H]Dofetilide
Binding is a predictive Screening Tool for hERG Blockade and
Proarrythmia: Comparison of intact Cell and Membrane Preparations
and Effect of altering [K+]o. J. Pharmacol. Toxicol. Meth. 2004, 50,
187-199.
(25) Mitcheson, J. S.; Chen, J.; Lin, M.; Culberson, C.; Sanguinetti, M.
C. A Structural Basis for Drug-Induced long QT Syndrome. Proc.
Natl. Acad. Sci. U.S.A. 2000, 24, 12329-12333.
(26) Witchel, H. J.; Dempsey, C. E.; Sessions, R. B.; Perry, M.; Milnes,
J. T.; Hancox, J. C.; Mitcheson, J. S. The Low-Potency, Voltage-
Dependent hERG Blocker Propafenone - Molecular Determinants
and Drug Trapping. Mol. Pharmacol. 2004, 66, 1201-1212.
(27) Lacerda, A. E.; Kramer, J.; Shen, K.-Z.; Thomas, D.; Brown, A. M.
Comparison of block among cloned cardiac potassium channels by
non-antiarrhytmic drugs. Eur. Heart J. Suppl. 2001, 3, K23-K30.
(28) The hERG patch clamp IC50 values are means of two experiments;
variability around the mean value was <50%.
(29) Dai, Y.; Guo, Y.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Ahmed, A. A.;
Albert, D. H.; Arnold, L.; Arries, S. S.; Barlozzari, T.; Bauch, J. L.;
Bouska, J. J.; Bousquet, P. F.; Cunha, G. A.; Glaser, K. B.; Guo, J.;
Li, J.; Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Pease, L. J.;
Stewart, K. D.; Stoll, V. S.; Tapang, P.; Wishart, N.; Davidsen, S.
K.; Michaelides, M. R. Thienopyrimidine Ureas as Novel and Potent
Multitargeted Receptor Tyrosine Kinase Inhibitors. J. Med. Chem.
2005, 48, 6066-6083.
(30) Zhou, Z.; Gong, Q.; Ye, B.; Fan, Z.; Makielski, J. C.; Robertson, G.
A.; January, C. T. Properties of hERG Channels Stably Expressed
in HEK 293 Cells Studied at Physiological Temperature. Biophys.
J. 1998, 74, 230-241.
(31) Martin, R. L.; McDermott, J. S.; Salmen, H. J.; Pallmatier, J.; Cox,
B. F.; Gintant, G. A. The Utility of hERG and Repolarization Assays
in Evaluating Delayed Cardiac Repolarization: Influence of Multi-
Channel Block. J. CardioVasc. Pharmacol. 2004, 43, 369-379.
(23) Recanatini, M.; Poluzzi, E.; Masetti, M.; Cavalli, A.; De Ponti, F.
QT Prolongation Through hERG K+ Channel Blockade: Current
Knowledge and Strategies for the Early Prediction During Drug
Development. Med. Res. ReV. 2005, 25, 133-166.
JM061223O