A New Class of Potent Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitors: Structure-Activity Relationships for a Series of 9-Alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-c]carbazole-5-ones and the Identification of CEP-521

Article abstract of DOI:10.1021/jm0301641

A series of potent vascular endothelial growth factor R2 (VEGF-R2) tyrosine kinase inhibitors from a new indenopyrrolocarbazole template is reported. The structure-activity relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno [2,1-a] py

Full text of DOI:10.1021/jm0301641

J . Med. Chem. 2003, 46, 5375-5388
5375
A New Cla ss of P oten t Va scu la r En d oth elia l Gr ow th F a ctor Recep tor Tyr osin e
Kin a se In h ibitor s: Str u ctu r e-Activity Rela tion sh ip s for a Ser ies of
9-Alk oxym eth yl-12-(3-h yd r oxyp r op yl)in d en o[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on es
a n d th e Id en tifica tion of CEP -5214 a n d Its Dim eth ylglycin e Ester P r od r u g
Clin ica l Ca n d id a te CEP -7055
Diane E. Gingrich,† Dandu R. Reddy,† Mohamed A. Iqbal,† J asbir Singh,† Lisa D. Aimone,‡ Thelma S. Angeles,§
Mark Albom,§ Shi Yang,§ Mark A. Ator,§ Sheryl L. Meyer,# Candy Robinson,| Bruce A. Ruggeri,|
Craig A. Dionne,| J effry L. Vaught, J ohn P. Mallamo,† and Robert L. Hudkins*,†
Departments of Medicinal Chemistry, Pharmacology, Biochemistry, Protein Expression, Oncology, and Discovery Research,
Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380
Received April 10, 2003
A series of potent vascular endothelial growth factor R2 (VEGF-R2) tyrosine kinase inhibitors
from a new indenopyrrolocarbazole template is reported. The structure-activity relationships
for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-c]carbazole-5-ones
revealed an optimal R9 substitution with ethoxymethyl 19 (VEGF-R2 IC50 ) 4 nM) and
isopropoxymethyl 21 (VEGF-R2 IC50 ) 8 nM) being the most potent inhibitors in the series.
The VEGF-R2 activity was reduced appreciably by increasing the size of the R9 alkoxy group
or by R-methyl branching adjacent to the ring. The combined R9 alkoxymethyl and N12
hydroxypropyl substitutions were required for potent VEGF-R2 activity, and the corresponding
thioether analogues were weaker than their ether counterparts. Compound 21 (R9 isopropoxy-
methyl, CEP-5214) was identified as a potent, low-nanomolar pan inhibitor of human VEGF-R
tyrosine kinases, displaying IC50 values of 16, 8, and 4 nM for VEGF-R1/FLT-1, VEGF-R2/
KDR, and VEGF-R3/FLT-4, respectively, with cellular activity equivalent to the isolated enzyme
activity. Compound 21 exhibited good selectivity against numerous tyrosine and serine/
threonine kinases including PKC, Tie2, TrkA, CDK1, p38, J NK, and IRK. To increase water
solubility and oral bioavailability, the N,N-dimethylglycine ester 40 was prepared. In
pharmacokinetic studies in mice and rats, increased plasma levels of 21 were observed after
oral administration of 40. Compound 21 demonstrated significant in vivo antitumor activity
in numerous tumor models and was advanced into phase I clinical trials as the water-soluble
N,N-dimethylglycine ester prodrug 40 (CEP-7055).
In tr od u ction
factors, cytokines, and oncogenes increase its expres-
sion.7 VEGF drives the angiogenic cascade by promoting
vascular endothelial cell expression of invasive pro-
teases, activation of integrins, and the activation,
proliferation, and migration of endothelial cells.1-3,7 Two
high-affinity VEGF transmembrane receptor tyrosine
kinases, VEGF-R1 (Flt-1) and kinase insert domain-
containing receptor VEGF-R2 (Flk-1, KDR), are ex-
pressed on vascular endothelial cells.9 While VEGF-R1/
Flt-1 is also expressed on nonendothelial cells (monocytes,
dendritic cells, smooth muscle cells), VEGF-R2/KDR is
primarily restricted to vascular endothelial cells and is
the principal kinase receptor through which VEGFs
exert their mitogenic, chemotactic, and vascular per-
meabilizing effects on the host vasculature.9-11 A third
homologous receptor, VEGF-R3 (Flt-4), is constitutively
expressed on adult lymphatic endothelial cells and
supports lymphangiogenesis.12
Angiogenesis, the generation and growth of new blood
vessels from the endothelium of an existing vascular
network, is an essential event in a variety of physio-
logical and pathological processes.1-3 Physiological con-
ditions where angiogenesis occurs include embryogen-
esis, ovulation, and wound healing. Angiogenesis also
occurs in pathological processes such as inflammation,
retinopathies, rheumatoid arthritis, and psoriasis and
is associated with tumor growth and the formation of
metastases.4-6
Vascular endothelial growth factor (VEGF) is a key
mediator of angiogenesis,2,7 and evidence has accumu-
lated that tumor VEGF expression is clinically associ-
ated with disease progression in a wide range of solid
malignancies.8 Virtually all cell types can express
VEGF. Hypoxia and a variety of hormones, growth
The enhanced expression of VEGF and VEGF-R2 on
vascular endothelial cells during tumor angiogenesis on
a variety of human and rodent tumors correlates with
tumor growth rate, microvessel density and prolifera-
tion, and tumor metastatic potential.1-6,13 The specificity
of the receptor expression, the prerequisite of angio-
* To whom correspondence should be addressed. E-mail: rhudkins@
cephalon.com. Phone: 610-738-6283. Fax: 610-738-6558.
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
§ Department of Biochemistry.
#
Department of Protein Expression.
| Department of Oncology.
Department of Discovery Research.
10.1021/jm0301641 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/31/2003

5376 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Gingrich et al.
Ch a r t 1
F igu r e 1. Structures of 5-oxo (1) and 7-oxo (1a ) indenopyrrolo-
carbazoles.
Sch em e 1a
genesis for tumor growth, and the characteristic that
the endothelium of normal mature cells remains gener-
ally quiescent makes disruption of the VEGF-R signal-
ing pathway a highly attractive strategy for chronic oral
therapy.
Antiangiogenesis therapies directed against VEGF-R
kinases have been under active evaluation in clinical
trials.14,15 The preclinical profile has been reported for
several orally active small-molecule inhibitors of VEGF-R
kinases currently undergoing clinical evaluation, in-
cluding PTK78716 (phase III), ZD647417 (phase II), CP-
547,63218 (phase I/II), and SU-1124814c (phase I/II)
(Chart 1). The Pfizer compound CP-547,632, an 11 nM
inhibitor of VEGF-R2, is the first compound from a new
series of isothiazoles. PTK787 (VEGF-R2 IC50 ) 37 nM)
is an anilinophthalazine derivative from Novartis. SU-
11248 is the third indolinone to advance from Sugen/
Pharmacia, and the AstraZeneca compound ZD6474
(VEGF-R2 IC50 ) 40 nM) is an anilinoquinazoline.
Indenopyrrolocarbazole CEP-5214 (21; VEGF-R2 IC50
) 8 nM) demonstrates significant in vivo antitumor
activity in numerous tumor models, and the correspond-
ing water-soluble N,N-dimethylglycine ester prodrug
CEP-7055 (40) has advanced into phase I clinical
trials.19 We disclose here the synthesis and VEGF-R2
structure-activity relationships leading to the selection
and advancement of CEP-5214 (21).
a
Reagents and conditions: (a) NaH, MsO(CH2)nOBn, DMF,
90 °C; (b) 20% Pd(OH)2-C, H2, concentrated HCl, DMF; (c)
CH2dCHCO2Et, DBU, CH3CN, reflux; (d) LiBH4, MeOH/THF,
room temp.
tetrahydrocarbazole cyanoester regioisomer produced in
the Diels-Alder reaction.21 The N-substituted ethanol
(6) and propanol (5) derivatives (Scheme 1, method B)
were prepared from 1 by alkylation with the appropriate
benzyl protected mesylate reagents (NaH, DMF). The
benzyl protecting group was removed in high yield using
20% Pd(OH)2/H2 (Scheme 1, method A). A method to
obtain larger quantities of propanol 5 utilized a hetero-
geneous Michael reaction of 1 with ethyl acrylate and
DBU in acetonitrile to give ester 4, followed by lithium
borohydride reduction to alcohol 5 (Scheme 1, method
B). Schemes 2 and 3 outline the routes to prepare the
diol intermediates 8 and 16 required for the target ether
and thioether analogues. Two methods were used to
prepare the 9-hydroxymethyl intermediates (Scheme 2).
In method A, formylation of 4 with R,R-dichloromethyl
methyl ether22 and tin(IV) chloride produced aldehyde
7 in 79% yield, which was reduced to 8 using lithium
borohydride in a THF/methanol solution (96% yield).
Scheme 2, method B, outlines a route to 8 using a
palladium-catalyzed carbonylation sequence.23 Protec-
tion of the lactam N-H was required to circumvent the
major product being the reduced 9-H compound. The
base stable dimethoxybenzhydryl group was used be-
cause of the ease of synthesis and the improved solubil-
ity in organic solvents, which aided purification. The
Ch em istr y
The compounds described were prepared starting
from indenopyrrolocarbazole 1.20 The 5-oxo isomer 1
(6H,7H,12H,13H-indeno[2,1-a]pyrrolo[3,4-c]carbazole-
5(5H)one) (Figure 1) was prepared as described previ-
ously utilizing a regioselective Diels-Alder reaction
with 2-(2-indenyl)indole and ethyl cis-â-cyanoacrylate.21
In the Diels-Alder step, the desired tetrahydrocarbazole
diastereomer leading to 1 (Figure 1) was isolated,
aromatized to the cyanoester carbazole, and subjected
to a reductive cyclization to form the lactam (RaNi, H2,
DMF, MeOH). The 7-oxo regioisomer 1a (Figure 1) was
prepared in an analogous sequence after isolation of the

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5377
Sch em e 2a
a
Reagents and conditions: (a) Cl2CHOCH3, SnCl4, CH2Cl2, PhCH3; (b) LiBH4, MeOH/THF, room temp; (c) 4,4′-dimethoxybenzhydrol,
NMP, PhH, p-TsOH, reflux; (d) NBS, THF; (e) CO, PdCl2(PPh3)2, NaOAc, MeOCH2CH2OH, 155 °C; (f) thioanisole, TFA, CH2Cl2; (g)
DIBAL-H, CH2Cl2.
Sch em e 3a
a
Reagents and conditions: (a) CH3COCl, AlCl3, C2H4Cl2, 0 °C to reflux; (b) K2CO3, MeOH/THF, room temp; (c) LiBH4, THF, 0 °C to
room temp.
bromoalcohol intermediate 11 (Scheme 2, method B)
necessary for the carbonylation step was prepared by
protection of ester 4 with 4,4′-dimethoxybenzhydrol (9)
(NMP/benzene, p-TsOH), LiBH4 reduction to propanol
10, then regiospecific bromination using NBS in THF.20
The reaction of 11, carbon monoxide, and PdCl2(PPh3)2
catalyst in methoxyethanol at 155 °C in a sealed tube
produced the DMB protected methoxyethoxy ester 13
in 85% yield. Removal of the DMB group to 14 (TFA,
anisole, methylene chloride) followed by diisobutyl-
aluminum hydride reduction of the ester gave diol 8
(Scheme 2). Similarly, deprotection of DMB-11 provided
bromoalcohol 12. Ether derivatives containing an R-
methyl group adjacent to the ring were prepared from
16 (Scheme 3). Diol 16 was prepared by Friedel-Crafts
acylation of 5 (acetyl chloride, AlCl3) to give ketoester
15, followed by lithium borohydride reduction to diol 16,
or removal of the acetate group to give ketoalcohol 17
(Scheme 3). 9-Carboxaldehyde 39 (Scheme 4) was
prepared by first protecting 5 as the acetate (39a ),
formylation using R,R-dichloromethyl methyl ether and
tin(IV) chloride to produce aldehyde 39b, and then
deprotection of the acetate using sodium methoxide in
methanol. The 9-methyl-12-propanol derivative 41 was
prepared from 9-hydroxymethyl 8 using TFAA and
triethylsilane (Scheme 5).
The preferred approach to the target ethers and
thioethers (Scheme 6) as outlined using diols 8 and 16
was to treat the pertrifluoroacetate intermediates (8a ,
16a ) (trifluoroacetic anhydride, triethylamine) with an
appropriate alcohol or thiol reagent. In cases where the
N12 trifluoroacetate ester was isolated, treatment with
lithium hydroxide in THF readily produced the desired
target alcohol compound. Alternatively, the ether/thio-
ether formation could be effected by directly treating
the diol with camphor sulfonic acid in a solvent such as
methylene chloride.22 The dimethylglycine ester prodrug
40 was prepared by coupling 21 and N,N-dimethyl-
glycine with 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride in DMA.

5378 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Gingrich et al.
Sch em e 4a
a
Reagents and conditions: (a) Ac2O, triethylamine, NMP, DMAP, room temp; (b) Cl2CHOCH3, SnCl4, CH2Cl2, PhCH3; (c) NaOMe,
MeOH, NMP, room temp.
Sch em e 5a
18 displayed a VEGF-R2 IC50 of 17 nM, 3-fold more
potent than the lead 5 and 12-fold more potent than
hydroxymethyl 8. A number of analogues were prepared
to study the SAR of methyl ether 18. The ethyl ether
derivative 19 was the most potent VEGF-R2 inhibitor
in this series with an IC50 of 4 nM. Increasing the size
of the alkoxy group to propyl (20, 13 nM) or butyl (25,
18 nM), while tolerated, did not further improve the
potency. Branching was also explored on the methylene
(benzyl) position and in the alkoxy moiety. An SAR
observed with the size of the alkyloxy group indicated
an optimum steric requirement in the lipophilic pocket
at the R9 region. The isopropyl 21 (8 nM), sec-butyl 22
(12 nM), and tert-butyl 26 (11 nM) derivatives retained
potent VEGF-R2 activity, while further increasing the
alkyl length (for example, 27 and 28) resulted in a
decrease in activity. The stereochemistry in the alkyoxy
portion did not appear critical because both enantiomers
of 22 (23 and 24) displayed equivalent VEGF-R2
potency. However, R-methyl substitution adjacent to the
carbazole ring as in 32 produced a 20-fold loss in VEGF-
R2 activity compared to 19. The more potent isomer 33
displayed an IC50 of 65 nM, while the IC50 of 34 was
240 nM (ER ) 4). Compounds in the R-methyl series
containing branched or larger alkyloxy groups, such as
isopropoxy 36 and butyloxy 37, were weak inhibitors of
VEGF-R2.
a
Reagents and conditions: (a) trifluoroacetic anhydride, tri-
ethylsilane, CH2Cl2, 0 °C to room temp.
Resu lts a n d Discu ssion
In h ibition of VEGF -R2 Recep tor Tyr osin e Ki-
n a se Activity in Vitr o. The initial screening target
for optimization of the indenopyrrolocarbazoles was
inhibition of VEGF-R2 kinase activity. The VEGF-R2
assay utilized an enzyme linked immunosorbent assay
(ELISA) based format24 with time-resolved fluorescence
readout and recombinant human phospholipase C-γ/
glutathione S-transferase fusion protein as a substrate.
The IC50 values reported are the average of two to three
replications and are within 50% agreement. The VEGF-
R2 inhibitory data are shown in Table 1. Novel hetero-
cycles from our database were originally screened for
VEGF-R2 activity, identifying the 5-oxo-indenopyrrolo-
carbazole 1 (Figure 1) as a potent VEGF-R2 lead with
an IC50 of 110 nM. The 7-oxo regioisomer 1a (Figure 1)
was a weaker VEGF-R2 inhibitor (IC50 ) 414 nM). Lead
optimization of 1 was systematically conducted around
the B- and F-rings, C13 indene, and the N12 positions
(Figure 1). For brevity, the VEGF-R2 SAR of the
9-alkoxymethyl series and ensuing identification of the
9-isopropoxymethyl derivative (21 CEP-5214) will be
detailed. Substitutions at C13 and N6 were not favor-
able. A three-carbon alcohol at N12 was optimal because
5 displayed a VEGF-R2 IC50 of 48 nM. The ethanol
derivative 6 was equally potent compared to the parent
1. Exploring substitutions around the B-ring revealed
the 9-position as the most favorable position for syn-
thetic accessibility and enhancing VEGF-R2 potency.
Thorough lead optimization of the R9 substituent was
conducted on the N12-propanol lead 5. Relative to 5,
compounds with 9-aldehyde (39) (105 nM), 9-methyl (41)
(163 nM), 9-hydroxymethyl (8) (208 nM), 9-bromo (12)
(104 nM), and 9-methyl ketone (17) (187 nM) substitu-
tions tended to be weaker VEGF-R2 inhibitors. While
the hydroxymethyl 8 was a relatively weak inhibitor
with an IC50 of 208 nM, the methoxymethyl derivative
The 9-isopropoxymethyl-12H analogue 38 (VEGF-R2
IC50 ) 55 nM) demonstrated that both the R9 alkoxy-
methyl and N12 hydroxypropyl substitutions together
are required for potent VEGF-R2 activity. The corre-
sponding N-propanol thioether derivatives were pre-
pared for comparison with the oxygen series. The
ethylthiomethyl analogue 29 was about 5-fold weaker
than the corresponding ethoxymethyl derivative 19, and
sulfur oxidation further decreased activity (compare 30
to 31).
To assist in the design of indenopyrrolocarbazole
inhibitors and support the SAR, an inhibitor binding
model of VEGF-R2/KDR was built using the crystal
structure of the tyrosine kinase domain of fibroblast
growth factor receptor 1 as reported in complex with
Su4984 (PDB code ) 1AGW)25 and the crystal structure
of the human VEGF-R2/KDR kinase domain (PDB code
) 1VR2).26 The structures of a number of protein
kinases in complex with ATP, or ATP analogues, have
now been solved, which reveals the structural basis for
nucleotide binding. For example, with cAMP-dependent
protein kinase (PKA), the adenine moiety of ATP
anchors to the linker region connecting the small

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5379
Sch em e 6a
a
Reagents and conditions: (a) TFAA, triethylamine, CH2Cl2, 0 °C to room temp; (b) ROH or RSH, reflux.
Ta ble 1. VEGF-R2 Kinase Activity of 9-Substituted 5-Oxo-indenopyrrolocarbazoles
VEGF-R2a
IC50 (nM)
compd
R9
R12
1
5
6
12
41
39
17
16
8
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
40
H
H
H
Br
CH3
CHO
COCH3
CH(OH)CH3
CH2OH
CH2OCH3
CH2OCH2CH3
H
110
48
CH2CH2CH2OH
CH2CH2OH
107
104
163
105
187
111
208
17
4
13
8
12
19
12
18
11
35
276
21
57
309
83
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2OH
H
CH2OCH2CH2CH3
CH2OCH(CH3)2
(()-CH2OCH(CH3)CH2CH3
(S)-CH2OCH(CH3)CH2CH3
(R)-CH2OCH(CH3)CH2CH3
CH2OCH2CH2CH2CH3
CH2OC(CH3)3
CH2OCH(CH2CH3)2
CH2OCH2CH2CH(CH3)2
CH2SCH2CH3
CH2SCH(CH3)2
CH2SOCH(CH3)2
(()-CH(CH3)OCH2CH3
(chiral)-CH(CH3)OCH2CH3
(chiral)-CH(CH3)OCH2CH3
(()-CH(CH3)OCH3
(()-CH(CH3)OCH(CH3)2
(()-CH(CH3)OCH2CH2CH2CH3
CH2OCH(CH3)2
65
240
73
411
130
55
CH2OCH(CH3)2
CH2CH2CH2OCOCH2NMe2
18
a
The VEGF-R2/KDR assay was conducted as described in the Experimental Section.
N-terminal and large C-terminal lobes via hydrogen
bonds involving the N1 and N6 amino group with the
backbone carbonyl of Glu121 and the amide NH of
Val123.27 The hydrogen-bond donor-acceptor pair of
ATP binding to PKA is mimicked by the lactam NH and
carbonyl of pyrrolocarbazole staurosporine (1STC). The
VEGF-R2 cavity was identified using the coordinates
for staurosporine and Su4984, known ATP-competitive
inhibitors, and inhibitor 21 was docked using the FlexX
module in Sybyl6.8 and minimized in the model. A
binding mode including putative important interactions
of 21 with VEGF-R2/KDR is shown in Figure 2. The
proposed binding mode for 21 in this model is consistent
with the lactam moiety of the indenopyrrolocarbazole
mimicking the ATP donor-acceptor interactions, with
lactam N-H sharing a hydrogen with Glu917 carbonyl
and the lactam CdO hydrogen bonds with the backbone
amide of Cys919. The R9 alkoxymethyl occupies a
hydrophobic pocket, while the ether oxygen forms a
significant hydrogen bond with Asp1046 and Lys868.
The N-12 propyl alcohol forms key interactions with
Arg1032 and Asn923. Compound 21 makes significant
van der Waals contacts with the hydrophobic surface
of the cleft: Val848, Leu1035, Cys1045, and Leu840.
The decrease in activity of the R-methyl series can be
predicted by the model, where the R-R-methyl isomer

5380 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Gingrich et al.
F igu r e 2. Important interactions of 21 with VEGF-R2/KDR tyrosine kinase.
Ta ble 2. Inhibition of VEGF-Stimulated VEGF-R2
Autophosphorylation in HUVECs with Selected
Indenopyrrolocarbazolesa
concentration
compd
10 nM
3
50 nM
100 nM
4
200 nM
4
19
20
21
23
24
25
26
29
30
35
3
4
F igu r e 3. Inhibition of VEGF-stimulated VEGFR2 phos-
phorylation by compound 21 in HUVEC. Serum-starved
HUVEC cells were incubated in the presence of DMSO
(control) or different concentrations of 21 for 1 h at 37 °C prior
to treatment with 10 ng/mL VEGF for 5 min. Immunoprecipi-
tation was performed with anti-VEGFR peptide antibody from
1 mg of total protein. Separation was done on 6% Tris-glycine
SDS-PAGE and probed with antiphosphotyrosine antibody.
Detection was by enhanced chemiluminescence. Lane designa-
tions are as follows: (1) VEGF(-) control; (2) VEGF(+) control;
(3) 10 nM 21; (4) 100 nM 21; (5) 1 µM 21; (6) VEGF(+) control.
4
4
0
4
4
4
4
4
1
4
a
VEGF-R2/KDR autophosphorylation assay was conducted as
described in the Experimental Section. Data were scored on the
basis of a decrease in protein band density compared to that of
VEGF-stimulated control (no inhibitor) as follows: 0 ) no de-
crease; 1 ) 1-25%; 2 ) 26-50%; 3 ) 51-75%; 4 ) 76-100%.
endothelial cells in vitro. The cellular IC50 for compound
21 was estimated to be =10 nM in HUVECs (Figure 3),
indicating that the compound is highly cell-permeable
and closely comparable to an IC50 of 8 nM in the isolated
VEGF-R2 kinase assay. A time course study revealed
that the inhibitory activity of 21 on VEGF-R2/KDR
receptor phosphorylation in murine and human endo-
thelial cells was observed within 15 min of exposure to
cells. In a washout assay conducted to determine the
duration of inhibitory effects of 21 on VEGF-stimulated
VEGF-R2 phosphorylation, inhibition was sustained
maximally for 1 h, was apparent for 3 h following
washout, and then declined to basal levels by 6 h,
consistent with competitive and reversible inhibition of
VEGF-R2/KDR kinase activity in cells as was observed
in the human VEGF-R2 kinase assay.
would be the more active enantiomer. The decrease in
VEGF-R2 activity is most dramatic when comparing 21
(VEGF-R2 IC50 ) 8 nM) to its R-methyl counterpart (()-
36 (IC50 ) 411 nM). The R-methyl group forces the bulky
iPrO group to rotate above the 4-position to hydrogen-
bond with Asp1046, resulting in unfavorable steric
interactions with Val916, Leu889, and Phe1047.
In h ibition of VEGF -R2 Cell Activity in Vitr o. The
VEGF-R2 kinase inhibitory activity in cells was evalu-
ated for dose-related inhibition of VEGF-R2/KDR auto-
phosphorylation utilizing human umbilical vein endo-
thelial cells (HUVECs) monitoring inhibition of VEGF-
induced stimulation of VEGF-R2/KDR phosphoryl-
ation. Inhibitors from Table 2 screened in HUVECs at
concentrations of 100 nM or lower (compounds 19, 21,
23, 24, 26, 35) gave complete inhibition of VEGF-
stimulated VEGF-R2 phosphorylation in both the hu-
man (HUVECs) and murine (SVR data not shown)
P r od r u g for Or a l Ad m in istr a tion of Com p ou n d
21 (CEP -5214). The pharmacokinetics of compounds 19
and 21 were studied in vivo in rats and in vitro in rat

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5381
Ta ble 3. Kinase Inhibitory Profile of 21
kinase IC50 (nM)
(av ( SD)
VEGF-R1
VEGF-R2
VEGF-R3
Tie2
16 ( 1
8 ( 2
4 ( 2
>10000
1.6 ( 0.7
162 ( 37
406 ( 76
57 ( 5
Flt3
FGF-R1
PDGF-Râ
Kit
TrkA
âIrk
J NK1â1
CHK1
CDS1
Cdk1/cyclinB
rat brain PKC
(R, â, γ isozymes)
p38R
>3000
>10000
448 ( 102
>3000
>3000
>10000
>10000
F igu r e 4. Plasma levels of compound 21 (ng/mL) in rat,
administered as prodrug 40 (30 and 100 mg/kg) or parent 21
(30 mg/kg).
>10000
comparable to that observed in CD-1 mice, and was
∼20% in rats. In nude mice, plasma levels of 21 were
dose-proportional between 3.6 and 11.9 mg/kg of orally
administered 40 with Cmax plasma levels of about 100
ng/mL achieved.29 While cleavage in plasma cannot be
ruled out, it is believed that the prodrug ester is
converted primarily in the stomach. A manuscript is in
preparation describing the optimization, solubility, and
prodrug stability leading to the selection of ester 40
(CEP-7055) for advancement as a water-soluble form
of 21.
Sch em e 7a
a
Reagents and conditions: (a) N,N-dimethylglycine, EDCI,
Kin a se Selectivity P r ofile for CEP -5214 (21). The
inhibitory profile of 21 was subsequently determined in
a panel of in vitro kinase enzyme assays and is shown
in Table 3. As previously described, 21 is a potent, low
nanomolar inhibitor of the primary screening target,
human VEGF-R2 tyrosine kinase. It was also discovered
to be a potent, low nanomolar pan inhibitor of the
human VEGF-R tyrosine kinase family, displaying IC50
values of 16, 8, and 4 nM for VEGF-R1/Flt-1, VEGF-
R2/KDR, and VEGF-R3/Flt-4, respectively. The Hill
slopes of 21 for human VEGF-R2/KDR kinase ap-
proached unity. ATP competition studies confirmed
competitive binding. Compound 21 demonstrated weaker
inhibitions of the human c-Kit, PDGF-Râ, J NK1â1, and
FGF-R1 kinases, with IC50 values of 57, 406, 448, and
162 nM, respectively, and was a potent inhibitor of Flt3
kinase with an IC50 value of 1.6 nM. Compound 21 was
weak to inactive against the additional tyrosine and
serine/threonine kinases evaluated, including PKC,
Tie2, TrkA, CDK1, p38, and IRK (Table 3). Of the
VEGF-R2 inhibitors currently undergoing clinical evalu-
ation, compound 21 is the most potent pan inhibitor of
the human VEGF-R tyrosine kinase family members.
The Pfizer compound CP-547,632, SU-11248 (Sugen/
Pharmacia), and the AstraZeneca compound ZD6474 are
selective for VEGF-R2. The Novartis compound PTK787,
a 37 nM VEGF-R2 inhibitor, is about 2-fold selective
over VEGF-R1.
DMAP, DMA, 35-45 °C, 2 h.
liver S9 fraction for stability. An important objective was
to establish the metabolic stability of the benzylic ether
linkage in vitro. In the rat liver S9 fraction, on the basis
of mass spectral analysis after 2 h of incubation, 98%
of the branched isopropoxy compound 21 remained
intact in the absence of NADPH and 46% remained
intact in the presence of NADPH cofactor. The ethoxy
compound 19 was unstable in the S9 preparation in the
absence or presence of NADPH cofactor, showing 0%
and 2% remaining after a similar incubation period.
Oral dosing of 21 at 30 mg/kg to rats showed maximum
plasma levels of about 100 ng/mL (Figure 4). The low
aqueous solubility (10 µg/mL) was believed to be a
reason for the low oral bioavailability. Extensive evalu-
ations of different formulations for 21 in an attempt to
improve the oral bioavailability and increase the drug
plasma levels at high doses for toxicity studies were
unsuccessful. Therefore, to increase the aqueous solubil-
ity of 21, a series of prodrug esters28 were prepared and
screened for solubility and stability. The N,N-dimethyl-
glycine ester 40 (Scheme 7) as the hydrochloride showed
good solubility in water (40 mg/mL) and the free base
was readily soluble and stable as a 1% aqueous acetic
acid solution, allowing for parenteral or oral adminis-
tration. Pharmacokinetic studies in rats and nude mice
demonstrated that following oral administration of 40,
only 21 was detected in the plasma compartment; no
40 was detected within the shortest time frame (∼5 min
postdosing) evaluated. Moreover, oral administration of
40 produced better systemic exposure compared to 21
(Figure 4) at all doses. The oral bioavailability of 40 in
nude mice (measured as 21 in plasma) was ∼15%,
In Vitr o An tia n giogen esis Activity. On the basis
of its potent and selective inhibition of the VEGF-R
kinases and marked inhibition of VEGF-R2 phosphoryl-
ation in human and rodent endothelial cells, compound
21 was evaluated in in vitro and ex vivo bioassays of
angiogenesis. The rat aortic ring explant model in three-
dimensional collagen gel matrixes and the HUVEC

5382 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Gingrich et al.
F igu r e 6. Effect of 21 and prodrug 40 on the growth of SVR
angiosarcoma xenografts in nude mice. Murine endothelial
SVR cells (1 × 106) were injected into the right flank of female
BALB/c nude mice. At day 6 postimplantation when palpable
tumors were confirmed, mice were randomized into vehicle and
treatment groups (n ) 8 or 9) and the compounds were
administered at the specified concentration, orally, twice a day.
Statistical analyses were done using the Mann-Whitney sum
test: (/) p < 0.05; (//) p < 0.01.
F igu r e 5. Dose-related effects of 21 on VEGF-stimulated
microvessel growth in rat aortic ring collagen gel explant
cultures. Shown is the effect of increasing concentrations of
VEGFR kinase inhibitor 21 on angiogenesis in serum-free
collagen gel cultures of rat aortic ring explants maintained in
serum-free MCDB 131 medium. Aortic rings were stimulated
with 4 ng/mL recombinant murine VEGF for 24 h prior to the
addition of compound. Values are the mean ( SE of micro-
vessel outgrowths, n ) 3 per time point. (/) p < 0.05; (//) p <
0.01; (///) p < 0.01. The p values are relative to untreated
controls by the Student-Newman-Keuls method.
significant inhibition of 21 in this murine SVR angio-
sarcoma xenograft model was 1-3 mg/kg b.i.d. admin-
istered over a 10-day dosing regimen. The maximum
efficacy achieved in this model was ∼70% inhibition of
tumor growth at a dose of 23.8 mg/kg b.i.d. (equivalent
to 20 mg/kg 21) of 40. No deaths occurred at the high
dose. Results of antitumor efficacy after longer dosing
in multiple tumor models have been reported.31
capillary tube formation assay on a Matrigel synthetic
basement membrane matrix are two widely used ex vivo
and in vitro angiogenesis systems.30 Both assays are
sensitive to the angiogenic effects of VEGF and to
VEGF-R2 inhibitors. Compound 21 displayed statisti-
cally significant dose-related inhibition of complete
HUVEC capillary tube formation in the absence of
apparent endothelial cell cytotoxicity. Compound 21
inhibited VEGF-induced capillary tube formation by
21% (40 nM), 75% (100 nM), and 89% (400 nM),
respectively. The antiangiogenic response to 21 was
further evaluated ex vivo in rat aortic ring explant
cultures over a 19-day time course in the presence of
exogenous VEGF stimulation, where dose-related in-
hibitory effects on microvessel growth were observed
from 4 to 400 nM (Figure 5). At days 8 and 11, when
microvessel growth was at its peak in control cultures,
statistically significant effects were observed at 40 nM
of 21. Concentrations of 100 and 400 nM significantly
inhibited vessel outgrowth relative to near controls in
the absence of apparent cytotoxicity in the primary
aortic ring explants.
In Vivo An t it u m or Act ivit y. Detailed oral anti-
tumor efficacy of 21 or the prodrug 40 in a variety of
established murine and human tumor models in nude
mice along with the pharmacokinetics of 40 in nude
mice is reported elsewhere.29,31 The antitumor activity
of the indenopyrrolocarbazole compounds reported here
were routinely evaluated using the SVR murine angio-
sarcoma xenograft model.32 Compound 21 administered
orally (Figure 6) at a dose of 10 mg/kg b.i.d. resulted in
significant inhibition of SVR tumor growth relative to
vehicle-treated controls as early as day 4 of dosing and
extending to day 10, with a maximum inhibition of 57%
( 5%. A comparable effect was observed with prodrug
40 at the equivalent dose of 21. In multiple, independent
experiments, no toxicity or morbidity was observed. The
minimum effective dose (MED) for consistent and
Con clu sion s
Potent VEGF-R2 tyrosine kinase inhibitors from a
new indenopyrrolocarbazole template are reported. The
structure-activity relationships revealed a size require-
ment in the R9 pocket with the ethoxymethyl (19) and
isopropoxymethyl (21) being the most potent inhibitors
from the series. Increasing the alkyl size at R9 or
R-methyl branching adjacent to the ring significantly
reduced VEGF-R2 activity in this series. The ether
series was more potent for VEGF-R2 than the corre-
sponding sulfur analogues. In addition, both the R9
alkoxymethyl and N12 hydroxypropyl substitutions
together were required for potent VEGF-R2 activity. An
important characteristic of this series was that several
compounds, including 19, 21, 23, 24, and 26, displayed
activity in cells approximately equivalent to their
isolated enzyme IC50
values. Compound 21 (CEP-5214)
was also a potent, pan inhibitor of the human VEGF-R
tyrosine kinase family, displaying comparable IC50
values for VEGF-R1, VEGF-R2, and VEGF-R3. Com-
pound 21 demonstrated selectivity against a number of
tyrosine and serine/threonine kinases including PKC,
Tie2, TrkA, CDK1, p38, and IRK. To increase water
solubility, the N,N-dimethylglycine ester 40 was pre-
pared. In pharmacokinetic studies in mice and rats,
increased plasma levels of 21 were observed after oral
administration of 40. Compound 21 demonstrated sig-
nificant in vivo antitumor activity at relevant oral doses
and has advanced into phase I clinical trials as the
water-soluble N,N-dimethylglycine ester prodrug 40.

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5383
(d, 1H, J ) 6 Hz), 7.5 (d, 1H, J ) 6 Hz), 8.0 (d, 1H, J ) 6 Hz),
8.57 (s, 1H), 9.5 (d, 1H, J ) 7 Hz). MS (m/z): 355 (M + 1).
Exp er im en ta l Section
Ch em istr y. All reagents and solvents were obtained from
commercial sources and used as received. The 1H NMR spectra
were recorded at 300 or 400 MHz in the solvent indicated with
tetramethylsilane as an internal standard. Coupling constants
(J ) are given in hertz (Hz). Analytical HPLC purity was carried
out on a Zorbax RX8, 5 mm × 150 mm, with product eluted
with a mixture of acetonitrile and water containing 0.1%
trifluroacetic acid with a gradient of 10-100% and a flow rate
of 1 mL/min over 20 min. Column chromatography was
performed on silica gel 60 (230-400 mesh). M-Scan Inc., West
Chester, PA, performed high-resolution mass spectra (FAB).
12-(3-Ben zyloxyp r op yl)-6H,7H,13H-in d en o[2,1-a ]p yr -
r olo[3,4-c]ca r ba zole-5-on e (2). Meth od A. To a solution of
1 (1.0 g, 3.2 mmol) in dry DMF (60 mL) was added solid NaH
(60%, 130 mg, 3.2 mmol) at room temperature. After the
mixture was stirred for 30 min, 2-benzyloxypropyl methane-
sulfonate was added (828 mg, 3.4 mmol), followed by heating
at 90 °C for 4 h. The solution was cooled to room temperature
and the DMF was removed at reduced pressure to leave a
semisolid. Ethyl acetate (100 mL) was added and the resulting
suspension was stirred and collected to give 1.2 g (82%) of 2
as a yellowish solid. 1H NMR (DMSO-d6): δ 2.09 (m, 2H), 3.54
(m, 2H), 4.45 (s, 2H), 4.51 (s, 2H), 4.82 (m, 2H), 4.93 (s, 2H),
7.30-7.53 (m, 10H), 7.73 (d, 1H, J ) 8 Hz), 8.01 (d, 1H, J )
8 Hz), 8.59 (s, 1H), 9.50 (d, 1H, J ) 7.5 Hz). MS (m/z): 459 (M
+ 1).
9-H yd r oxym et h yl-12-(3-h yd r oxyp r op yl)-6H ,7H ,13H -
in d en o[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on e (8). Meth od
A. To a stirred suspension of ester 4 (10 g, 24.3 mmol) and
1,1-dichloromethyl methyl ether (55.6 g, 488 mmol) in meth-
ylene chloride (250 mL) and toluene (40 mL) was added tin(IV)
chloride (95.1 g, 365 mmol) (1 M solution in methylene
chloride). After 4.5 h at room temperature, the reaction was
quenched with aqueous 2 N HCl (150 mL) and the methylene
chloride/toluene layer was removed under vacuum. To the
crude residue, 2 N HCl (350 mL) and tert-butyl methyl ether
(200 mL) were added. The suspension was stirred at room
temperature for 14 h, collected, washed with tert-butyl methyl
ether, and then resuspended in ethyl acetate/tetrahydrofuran
(1:1; 250 mL) and stirred for 14 h at room temperature. The
solid was collected, washed with cold ethyl acetate, and dried
1
to give 8.4 g (79% yield, 99% purity) of compound 7. H NMR
(DMSO-d6): δ 0.98 (t, 3H), 2.92 (t, 2H), 3.88 (q, 2H), 4.65 (s,
2H), 4.92-5.08 (m, 4H), 7.25-7.45 (m, 2H), 7.75 (d, 1H), 7.85
(d,1H), 8.05 (d, 1H), 8.52 (s, 1H), 8.69 (s, 1H), 8.95 (s, 1H),
10.18 (s, 1H). MS (m/z): 439 (M + 1). To a suspension of
intermediate 7 (2.4 g, 5 mmol) in THF (50 mL) at 0 °C under
nitrogen was added lithium borohydride (25 mL of 2 M solution
in THF). The reaction mixture was stirred for 3.5 h at room
temperature and cooled to 0 °C, and methanol was added
slowly until no evolution of gas was observed. The mixture
was concentrated at reduced pressure to give a yellow solid,
which was triturated with water and ether, collected by
filtration, and dried to yield 2.0 g (96%, purity 96.5%) of diol
12-(3-Hydr oxypr opyl)-6H,7H,13H-in den o[2,1-a ]pyr r olo-
[3,4-c]ca r ba zole-5-on e (5). A solution of 2 (1.0 g, 2.2 mmol),
20% Pd(OH)2 (200 mg), and concentrated HCl (3 drops) in DMF
(100 mL) was hydrogenated on a Parr apparatus for 14 h. The
catalyst was removed by filtration through Celite and the
solvent was concentrated at reduced pressure to leave a solid
that was triturated with methanol, collected, and dried to give
700 mg (86%, 96% purity) of 5 as a white solid, mp 280-284
1
8. H NMR (DMSO-d6): δ 1.92 (m, 2H), 3.46 (m, 2H), 4.50 (s,
2H), 4.65 (s, 2H), 4.71 (m, 2H), 4.88 (s, 2H), 7.32-7.39 (m, 2H),
7.47 (d, 1H, J ) 8.3 Hz), 7.65 (m, 2H), 7.89 (s, 1H), 8.53 (s,
1H), 9.46 (d, 1H, J ) 7.4 Hz). MS (m/z): 399 (M + 1). HRMS
(FAB) calcd for C25H22N2O3: 399.1708; found 399.1709.
Diol 8. Meth od B. To a suspension of ester 4 (5.62 g, 13.7
mmol) in benzene (300 mL) and N-methylpyrrolidine (60 mL)
at room temperature under nitrogen was added p-toluene-
sulfonic acid monohydrate (2.48 g, 13.0 mmol) and 4,4′-
dimethoxybenzhydrol (3.19 g, 13.0 mmol). The mixture was
heated to reflux for 8 h, cooled to room temperature, and
diluted with ethyl acetate (300 mL). The ethyl acetate layer
was washed with a saturated sodium bicarbonate solution,
water, and brine and then dried over magnesium sulfate. The
drying agent was removed by filtration and the solvent was
concentrated to give 8.31 g (95%) of intermediate 9 as an
orange solid. 1H NMR (CDCl3): δ 1.18 (t, 3H, J ) 7.1 Hz),
2.84 (m, 2H), 3.80 (6H, s), 4.12 (q, 2H, J ) 7.1 Hz), 4.38 (s,
2H), 4.72 (2H, s), 4.94 (m, 2H), 6.90 (d, 4H, J ) 8.5 Hz), 6.96
(s, 1H), 7.26 (d, 4H, J ) 8.5 Hz), 7.34-7.49 (m, 5H), 7.61 (d,
1H, J ) 7.4 Hz), 7.69 (d, 1H, J ) 7.7 Hz), 9.65 (d, 1H, J ) 7.8
Hz).
To a stirred solution of intermediate 9 (7.8 g, 12.2 mmol) in
THF (480 mL) and methanol (93 mL) was added lithium
borohydride (18.9 mL of a 2.0 M solution in THF) dropwise.
The reaction was monitored by HPLC until completion, the
mixture was cooled on an ice bath, and the reaction was then
quenched with 2 N HCl (60 mL). Water (750 mL) was added
to the mixture and the precipitate was collected by filtration
and dried to give 7.2 g (99%) of 10 as white solid. 1H NMR
(DMSO-d6): δ 1.93 (m, 2H), 3.66 (m, 2H), 3.71 (s, 6H), 4.55 (s,
2H), 4.73 (m, 2H), 4.79 (s, 2H), 6.70 (s, 1H), 6.93 (d, 4H, J )
8.44 Hz), 7.22 (d, 4H, J ) 8.4 Hz), 7.26 (m, 1H), 7.34-7.46 (m,
2H), 7.49 (m, 1H), 7.65 (d,1H, J ) 7.0 Hz), 7.70 (d, 1H, J )
8.3 Hz), 7.86 (d, 1H, J ) 7.8 Hz), 9.49 (d, 1H, J ) 7.5 Hz).
1
°C (dec). H NMR (DMSO-d6): δ 1.96 (m, 2H), 3.52 (m, 2H),
4.57 (s, 2H), 4.78 (m, 2H), 4.95 (s, 2H), 7.30-7.46 (m, 3H), 7.55
(m, 2H), 7.67 (d, 1H, J ) 8 Hz), 7.75 (d, 1H, J ) 8 Hz), 8.0 (d,
1H, J ) 8 Hz), 8.60 (s, 1H), 9.51 (d, 1H, J ) 7.5 Hz). MS (m/z):
369 (M + 1).
Meth od B. To a suspension of 1 (8.0 g, 25.8 mmol) in
acetonitrile (300 mL) was added ethyl acrylate (4.19 mL, 38.7
mmol) followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
(1.93 mL, 0.013 mmol) under a nitrogen atmosphere. The
heterogeneous reaction mixture was heated to reflux for 18 h,
then cooled to room temperature. The solid was collected by
filtration, washed with acetonitrile, and dried to yield 5.4 g
1
(78%) of 4. H NMR (DMSO-d6): δ 9.72 (t, 3H, J ) 6. 8 Hz),
2.87 (m, 2H), 3.89 (q, 2H, J ) 6.8 Hz), 4.49 (s, 2H), 4.88 (s,
2H), 4.92 (m, 2H), 7.29-7.48 (m, 3H), 7.50-7.73 (m, 3H), 7.96
(d, 1H, J ) 7.3 Hz), 8.56 (s, 1H), 9.47 (d, 1H, J ) 7.3 Hz). MS
(m/z): 411 (M + 1). To a stirred solution 4 (1.0 g, 2.4 mmol) in
THF/methanol (150 mL, 4:1) was added lithium borohydride
(7.2 mL of 2 M solution in THF). The mixture was stirred
overnight. Then the reaction was quenched by addition of 2 N
HCL (15 mL), the mixture was diluted with an equal volume
of water, and the product 5 was collected and dried to give
760 mg (86%). Compound 5 prepared by method B showed
physical and spectral characteristics identical to those pre-
pared by method A.
12-(2-Ben zyloxyeth yl)-6H,7H,13H-in den o[2,1-a ]pyr r olo-
[3,4-c]ca r ba zole-5-on e (6). This compound was prepared by
the same general procedure as for 5 (method A). Intermediate
3 was prepared by alkylation of 1 with 2-benzyloxyethyl
1
methanesulfonate. MS (m/z): 445 (M + 1). H NMR (DMSO-
To a suspension of intermediate 10 (2.0 g, 3.4 mmol) in THF
(131 mL) at room temperature under nitrogen was added solid
N-bromosuccinimide (630 mg, 3.6 mmol) in one portion. The
reaction mixture was stirred at room temperature overnight,
at which time the solvent was removed in vacuo leaving a pale-
yellow solid. This solid was triturated with cold methanol,
collected by filtration, and dried to give 1.98 g (87%) of bromo
intermediate 11. 1H NMR (DMSO-d6): δ 1.91 (m, 2H), 3.44
(m, 2H), 3.72 (s, 6H), 4.53 (s, 2H), 474 (m, 2H), 4.87 (s, 2H),
d6): δ 3.58 (m, 2H), 4.48 (s, 2H), 4.51 (s, 2H), 4.80 (m, 2H),
4.95 (s, 2H), 7.30-7.58 (m, 10H), 7.75 (d, 1H, J ) 8 Hz), 8.00
(d, 1H, J ) 8 Hz), 8.6 (s, 1H), 9.50 (d, 1H, J ) 7.5 Hz).
Intermediate 3 was deprotected using 20% Pd(OH)2 (100 mg)
and concentrated HCl (3 drops) in DMF (60 mL). Compound
1
6 (85% yield, 97% purity): mp >300 °C. H NMR (DMSO-d6):
δ 3.8-3.9 (b, 2H), 4.55 (s, 2H), 4.77 (t, 2H), 4.9 (s, 2H), 5.0 (b,
1H, D2O exchange), 7.3-7.45 (m, 3H), 7.5-7.57 (t, 1H), 7.67

5384 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Gingrich et al.
6.71 (s, 1H), 6.93 (d, 4H, J ) 8.1 Hz), 7.25 (d, 4H, J ) 8.1 Hz),
7.37 (m, 2H), 7.59-7.69 (m, 3H), 8.08 (s, 1H), 9.50 (d, 1H, J )
7.0 Hz).
2H), 4.91 (s, 2H), 7.36 (m, 3H), 7.48 (d, 1H), 7.64 (m, 2H), 7.90
(s, 1H), 8.55 (s, 1H), 9.47 (d, 1H). MS (m/z): 427 (M + 1).
HRMS (FAB) calcd for C27H26N2O3: 427.2022; found 427.2034.
Com p ou n d 20. 1H NMR (DMSO-d6): δ 0.88 (t, 3H), 1.55
(m, 2H), 1.933 (m, 2H), 3.36-3.58 (m, 4H), 4.53 (s, 2H), 4.61
(s, 2H), 4.73 (m, 3H), 4.90 (s, 2H), 7.33-7.39 (m 2H), 7.47
(d, 1H), 7.62-7.70 (m, 2H), 8.54 (s, 1H), 9.47 (d, 1H). MS
(m/z): 441 (M + 1), 462 (M + Na). HRMS (FAB) calcd for
A Schlenk tube was charged with 11 (790 mg, 1.7 mmol),
methoxyethanol (25 mL), sodium acetate (570 mg, 7.0 mmol),
and dichlorobis(triphenylphosphine)palladium(II) (82 mg, 0.12
mmol). The tube was successively evacuated and filled with
carbon monoxide, then heated at 155 °C in an oil bath for 3 h.
The reaction mixture was cooled to room temperature, ad-
ditional CO and PdCl2(PPh3)2 were added, and the mixture
continued heating for 4 h. The reaction mixture was cooled to
room temperature, diluted with methylene chloride, and
filtered through Celite. The filtrate was concentrated in vacuo,
and the residue was dissolved in ethyl acetate and washed
with water. The organic layer was dried over magnesium
sulfate, filtered, and concentrated to a solid. The solid was
triturated with ethyl ether, collected by filtration, and dried
to give 700 mg (85%) of 13 as a light-orange solid. 1H NMR
(CDCl3): δ 2.14 (m, 2H), 3.44 (s, 3H), 3.67-3.78 (m, 4H), 3.81
(s, 6H), 4.44 (s, 2H), 4.51 (m, 2H), 4.81 (m, 4H), 6.91 (d, 4H, J
) 8.5 Hz), 6.98 (s, 1H), 7.28 (d, 4H, 8.6), 7.34-7.7.61 (m, 4H),
8.21 (d, 1H, J ) 8.3 Hz), 8.42 (s, 1H), 9.67 (d, 1H, J ) 7.6 Hz).
To a solution of intermediate 13 (960 mg, 1.38 mmol) in
CH2Cl2 (30 mL) at 0 °C under nitrogen was added thioanisole
(3.2 mL, 110 mmol) followed by trifluoroacetic acid (8.5 mL,
27.6 mmol). The mixture was stirred at 0 °C for 1 h, then
allowed to warm to room temperature overnight. The solvent
was removed under vacuum, leaving a dark-red oil. Ethyl ether
was added and a tan solid was collected to give 600 mg (92%)
C
28H28N2O3: 441.2178; found 441.2171.
Com p ou n d 21. Mp 224-228 °C. 1H NMR (DMSO-d6): δ
1.15 (d, 6H, 1.92 (m, 2H), 3.45 (m, 2H), 3.67 (m, 1H), 4.52 (s,
2H), 4.61 (s, 2H), 4.73 (m, 2H), 4.89 (s, 2H), 7.3-7.39 (m, 2H0,
7.47 (d, 1H), 7.62-7.69 (m, 2H), 7.89 (s, 1H), 8.54 (s, 1H), 9.47
(d, 1H). MS (m/z): 441 (M + 1). HRMS (FAB) calcd for
C
28H28N2O3: 441.2178; found 441.2183.
Com p ou n d (()-22. H NMR (CDCl3): δ 0.98 (t, 3H), 1.26
1
(d, 3H), 1.65 (m, 2H), 2.03 (m, 2H), 3.56 (m, 2H), 4.095 (m,
1H), 4.24 (s, 2H), 4.57 (m 2H), 4.70 (m, 2H), 4.71 (s, 2H), 6.12
(s, 1H), 7.33 (t, 1H), 7.42-7.58 (m, 4H), 7.75 (s, 1H), 9.48
(d, 1H). MS (m/z): 455 (M + 1). HRMS (FAB) calcd for
C
29H30N2O3: 455.2335; found 455.2342.
Com p ou n d (S)-23. H NMR (CDCl3): δ 0.98 (t, 3H), 1.26
1
(d, 3H), 1.65 (m, 2H), 2.03 (m, 2H), 3.56 (m, 2H), 4.095 (m,
1H), 4.24 (s, 2H), 4.57 (m 2H), 4.70 (m, 3H), 4.71 (s, 2H), 6.12
(s, 1H), 7.33 (t, 1H), 7.42-7.58 (m, 4H), 7.75 (s, 1H), 9.48
(d, 1H). MS (m/z): 455 (M + 1). HRMS (FAB) calcd for
C
29H30N2O3: 455.2335; found 455.2322.
Com p ou n d (R)-24. 1H NMR (CDCl3): δ 0.98(t, 3H), 1.26
(d, 3H), 1.65 (m, 2H), 2.03 (m, 2H), 3.56 (m, 2H), 4.095 (m,
1H), 4.24 (s, 2H), 4.57 (m 2H), 4.70 (m, 2H), 4.71 (s, 2H), 6.12
(s, 1H), 7.33 (t, 1H), 7.42-7.58 (m, 4H), 7.75 (s, 1H), 9.48
(d, 1H). MS (m/z): 455 (M + 1). HRMS (FAB) calcd for
1
of 14. H NMR (DMSO-d6): δ 2.29 (m, 2H), 3.3 (m, 2H), 3.73
(m, 2H), 4.45 (m, 2H), 4.54 (m, 3H), 4.82 (m, 2H), 4.99 (s, 2H),
7.40 (m, 2H), 7.58 (d, 1H), 7.85 (d, 1H), 8.13 (d, 1H), 8.52 (s,
1H), 8.6 (s, 1H), 9.49 (d, 1H).
C
29H30N2O3: 455.2335; found 455.2339.
Com p ou n d 25. H NMR (DMSO-d6): δ 0.854 (t, 3H), 1.34
Diol 8 (Meth od B). To a stirred suspension of 14 (4.4 g,
9.35 mmol) in CHCl2 (220 mL) at 0 °C under nitrogen was
added DIBAL-H (10.3 mL of 1 M solution) dropwise. The
reaction mixture gradually became homogeneous, and the
mixture was stirred at 0 °C for 1 h and then warmed to room
temperature for 6 h. The mixture was cooled to 0 °C on an ice
bath, and water (50 mL) was slowly added. An aqueous
solution of NaOH (1 M, 300 mL) was added, and the reaction
mixture was stirred at room temperature for 1 h. The
precipitate was collected by filtration to yield 3.6 g (96%) of 8
as a tan solid. This compound was identical in its physical and
spectral properties as the material prepared by method A.
P r ep a r a tion of Tr itr iflu or oa ceta te In ter m ed ia te 8a .
To a suspension of 8 (450 mg, 1.1 mmol) in methylene chloride
(30 mL) at 0 °C under nitrogen was added trifluoroacetic
anhydride (714 mg, 3.4 mmol) followed by triethylamine (325
mg, 3.4 mmol). The reaction mixture gradually became homo-
geneous, was stirred at 0 °C for 1 h, and then warmed to room
temperature overnight. The mixture was diluted with meth-
ylene chloride and washed with water and brine. The organic
phase was dried over magnesium sulfate, filtered, and con-
centrated in vacuo to a solid. This solid was used in the next
sequence without further purification.
1
(m, 2H), 1.52 (m, 2H), 1.93 (m, 2H), 3.48 (m, 2H), 4.52 (s, 2H),
4.60 (s, 2H), 4.73 (m, 3H), 4.89 (s, 2H), 7.30-7.42 (m, 2H), 7.47
(d, 1H), 7.62-7.70 (m, 2H), 7.89 (s, 1H), 8.54 (s, 1H), 9.47
(d, 1H). MS (m/z): 455 (M + 1). HRMS (FAB) calcd for
C
29H30N2O3: 455.2335; found 455.2337.
Com p ou n d 26. H NMR (DMSO-d6): δ 1.28 (s, 9H), 1.97
1
(m, 2H), 3.62 (m, 2H), 4.56 (s, 2H), 4.52 (s, 2H), 4.77 (m, 3H),
4.94 (s, 2H), 7.35-7.72 (3m, 3H), 7.72 (m, 2H), 7.90 (s, 1H),
8.8.57 (s, 1H), 9.50 (d, 1H). MS (m/z): 455 (M + 1), 477 (M +
Na). HRMS (FAB) calcd for C29H30N2O3: 455.2335; found
455.2350.
Com p ou n d 27. 1H NMR (DMSO-d6): δ 0.87 (t, 6H), 1.50
(m, 4H), 1.93 (m, 2H), 3.46 (m, 2H), 4.52 (s, 2H), 4.62 (s, 2H),
4.73 (m, 3H), 4.89 (s, 2H), 7.37 (m, 2H), 7.48 (m, 1H), 7.66 (m,
2H), 7.91 (s, 1H), 8.54 (s, 1H), 9.47 (d, 1H). MS (m/z): 469 (M
+ 1). HRMS (FAB) calcd for C30H32N2O3: 469.2491; found
469.2496.
1
Com p ou n d 28. H NMR (DMSO-d6): δ 0.89 (d, 6H), 1.47
(m, 2H), 1.70 (m, 1H), 1.96 (m, 2H), 3.52 (m, 4H), 4.56 (s, 2H),
4.64 (s, 2H), 4.77 (m, 2H), 4.93 (s, 2H), 7.40 (m, 2H), 7.51 (d,
1H), 7.69 (m, 2H), 7.94 (s, 1H), 8.58 (s, 1H), 9.48 (d, 1H). MS
(m/z): 469 (M + Na). HRMS (FAB) calcd for C30H32N2O3:
469.2491; found 469.2491.
Gen er a l P r oced u r e for Eth er s a n d Th ioeth er s 18-31.
8a was dissolved in the appropriate alcohol (0.025 M) or thiol
and heated to 80 °C in an oil bath. The reaction mixture was
monitored for disappearance of starting material by HPLC.
The mixture was cooled to room temperature and the solvent
was removed in vacuo, leaving a solid. The resulting solid was
triturated with ether and collected by filtration. The products
were further purified using silica gel column chromatography
with CH2Cl2/MeOH (95:5) as solvent. The HPLC purity of the
final products was >96%.
Com p ou n d 29. 1H NMR (DMSO-d6): δ 1.17 (t, 3H), 1.93
(m, 2H), 2.42 (q, 2H), 3.48 (m, 2H), 3.93 (s, 2H), 4.52 (s, 2H),
4.72 (m, 3H), 4.89 (s, 2H), 7.33-7.49 (m, 3H), 7.65 (m, 2H),
7.88 (s, 1H), 8.56 (s, 1H), 9.46 (d, 1H). MS (m/z): 443 (M + 1).
HRMS (FAB) calcd for C27H26N2O2S: 443.1793; found 443.2174.
1
Com p ou n d 30. H NMR (CDCl3): δ 1.31 (d, 6H), 2.34 (m,
2H), 2.86 (m, 1H), 3.98 (s, 2H), 4.29 (s, 2H), 4.45 (m, 1H), 4.74
(m, 2H), 4.92 (s, 2H), 6.07 (s, 1H), 7.39 (m, 2H), 7.51 (m, 2H),
7.57 (m, 1H), 7.80 (s, 1H), 9.53 (d, 1H). MS (m/z): 457 (M +
1), 479 (M + Na). HRMS (FAB) calcd for C28H28N2O2S:
457.1950; found 457.1945.
1
Com p ou n d 18. H NMR (DMSO-d6): δ 1.99 (m, 2H), 3.36
(s, 3H), 3.54 (m, 2H), 4.58 (s, 2H), 4.66 (s, 2H), 4.79 (m, 2H),
4.96 (s, 2H), 7.40-7.49 (m, 2H), 7.52 (d, 1H), 7.65-7.84 (m,
2H), 7.98 (s, 1H), 8.60 (s, 1H), 9.51 (d, 1H). MS (m/z): 413 (M
+ 1), 435 (M + Na). HRMS (FAB) calcd for C26H24N2O3:
413.1865; found 413.1852.
Com p ou n d 31. This compound was isolated by chroma-
tography from the reaction of 30. 1H NMR (DMSO-d6): δ 1.21
(dd, 6H), 1.93 (m, 2H), 2.82 (m, 1H), 3.49 (m, 2H), 4.12 (d, 1H),
4.23 (d, 1H), 2.52 (s, 2H), 4.75 (m, 3H), 4.88 (s, 2H), 7.33-
7.45 (m, 2H), 7.55 (d, 1H), 7.65 (d, 1H), 7.71 (d, 1H), 7.94 (s,
1
Com p ou n d 19. H NMR (DMSO-d6): δ 1.148 (t, 3H), 1.94
(m, 2H), 3.46-3.52 (m, 4H), 4.53 (s, 2H), 4.60 (s, 2H), 4.73 (m,

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5385
1H), 8.58 (s, 1H), 9.47 (d, 1H). MS (m/z): 494 (M + Na). HRMS
(FAB) calcd for C28H28N2O3S: 473.1899; found 473.1886.
9-(1-Oxoeth yl)-12-(3-a ceoxyp r op yl)-6H,7H,13H-in d en o-
[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on e (15). To a suspension
of aluminum chloride (335 mg, 2.2 mmol) in 1,2-dichloro-
ethane/methylene chloride (1:1, 8 mL) was added acetyl
chloride (172 mg, 2.2 mmol) under nitrogen. The reaction
mixture was cooled to 0 °C in an ice bath, and a suspension of
5 (310 mg, 0.84 mmol) in methylene chloride (3 mL) was added
dropwise. The ice bath was removed, and the reaction mixture
was warmed to room temperature and heated to reflux for 2
h. The mixture was cooled to room temperature and poured
over ice/water, and concentrated HCl (5 mL) was added. The
precipitate was collected by filtration and dried to give 340
(d, 1H), 7.88 (s, 1H), 8.58 (s, 1H), 9.49 (d, 1H). MS (m/z): 427
(M + 1). HRMS (FAB) calcd for C27H26N2O3: 427.2022; found
427.2035.
Com p ou n d (()-36. 1H NMR (DMSO-d6): δ 1.01 (d, 3H),
1.10 (d, 3H), 1.38 (d, 3H), 1.95 (m, 2H), 3.47 (m, 2H), 3.98 (q,
1H), 4.26 (m, 1H), 4.52 (s, 2H), 4.74 (m, 3H), 4.90 (m, 2H),
7.33-7.39 (m, 2H), 7.48 (d, 1H), 7.62-7.69 (m, 2H), 7.87 (s,
1H), 8.54 (s, 1H), 9.47 (d, 1H). MS (m/z): 455 (M + 1). HRMS
(FAB) calcd for C29H30N2O3: 455.2335; found 455.2321.
Com p ou n d (()-37. 1H NMR (DMSO-d6): δ 0.81 (t, 3H),
1.27-1.43 (m, 7H), 1.93 (m, 2H), 3.48 (m, 2H), 4.53 (s, 2H),
4.58 (m, 1H), 4.73 (m, 4H), 4.92 (m, 2H), 7.33-7.39 (m, 2H),
7.46 (d, 1H), 7.63 (d, 1H), 7.69 (d, 1H), 7.86 (s, 1H), 8.55 (s,
1H), 9.47 (d, 1H). MS (m/z): 469 (M + 1). HRMS (FAB) calcd
for C30H32N2O3: 469.2491; found 469.2503.
1
mg (89%) of 15. H NMR (DMSO-d6): δ 2.02 (s, 3H), 2.18 (m,
2H), 2.74 (s, 3H), 4.12 (m, 2H), 4.56 (s, 2H), 4.83 (m, 2H), 5.05
(s, 2H), 7.43 (m, 2H), 7.68 (d, 1H), 7.86 (d, 1H), 8.17 (d, 1H),
8.56 (s, 1H), 8.72 (1H), 9.53 (d, 1H). MS (m/z): 453 (M + 1).
9-(1-Hyd r oxyeth yl)-12-(3-h yd r oxyp r op yl)-6H,7H,13H-
in d en o[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on e (16). To a sus-
pension of 15 (82 mg, 0.18 mmol) in THF (6 mL) under
nitrogen was added lithium borohydride (0.9 mL of 2 M
solution in THF) at 0 °C. The reaction mixture was stirred at
0 °C for 1 h, then warmed to room temperature for 4 h. The
mixture was cooled to 0 °C, methanol was added slowly
dropwise, and then the solution was stirred at room temper-
ature overnight. The reaction mixture was diluted with ethyl
acetate, washed with water and brine, dried (magnesium
sulfate), and concentrated in vacuo to 69 mg (90%) of 16 as a
white solid. 1H NMR (DMSO-d6): δ 1.41 (d, 3H), 1.92 (m, 2H),
3.46 (m, 2H), 4.52 (s, 2H), 4.71 (m, 3H), 4.89 (s, 3H), 5.18 (s,
1H), 7.32-7.39 (m, 2H), 7.50 (d, 1H), 7.64 (m, 2H), 7.89 (s,
1H), 8.55 (s, 1H), 9.46 (d, 1H). MS (m/z): 413 (M + 1), 435 (M
+ Na).
9-(1-Oxoeth yl)-12-(3-h ydr oxypr opyl)-6H,7H,13H-in den o-
[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on e (17). To a stirred solu-
tion of 15 (55 mg, 0.122 mmol) in methanol/THF (8 mL, 3:1)
at room temperature was added potassium carbonate (16.8 mg,
0.122 mmol), and the mixture was heated to reflux for 30 min.
The reaction mixture was cooled to room temperature over-
night and the solvent was removed in vacuo, giving a white
solid. The solid was triturated with water and collected by
filtration. The solid was purified by flash chromatography on
silica gel using ethyl acetate (100%), then methanol/ethyl
acetate (10%) to give 38 mg (76%, 96% purity by HPLC) of 17.
1H NMR (DMSO-d6): δ 1.95 (m, 2H), 2.69 (s, 3H), 3.47 (m,
2H), 4.54 (s, 2H), 4.77 (m, 2H), 5.00 (s, 2H), 7.37 (m, 2H), 7.64
(m, 1H), 7.82 (d, 1H), 8.11 (d, 1H), 8.51 (s, 1H), 8.66 (s, 1H),
9.48 (d, 1H). MS (m/z): 411 (M + 1).
9-(Isop r op oxym et h yl)-6H ,7H ,12H ,13H -in d en o[2,1-a ]-
p yr r olo[3,4-c]ca r ba zole-5-on e (38). This compound was
prepared from 9-(hydroxymethyl)indeno[2,1-a]pyrrolo[3,4-c]-
carbazole-5-one according to the general procedure for ether
formation for tritrifluoroacetate intermediate 8a . Precipitation
from DMF/ether gave 38 as a white solid (85%, 97% purity).
1H NMR (DMSO-d6): δ 1.15 (d, 6H), 3.68 (m, 1H), 4.13 (s, 2H),
4.59 (s, 2H), 4.89 (s, 2H), 7.28-7.42 (m, 3H), 7.54 (d, 1H), 7.64
(d, 1H), 7.88 (s, 1H), 8.49 (s, 1H), 9.35 (d, 1H), 11.87 (s, 1H).
MS (m/z): 383 (M + 1). HRMS (FAB) calcd for C25H22N2O2:
383.1759; found 383.1754.
9-(Ca r boxa ld eh yd e)-12-(3-h yd r oxyp r op yl)-6H,7H,13H-
in d en o[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on e (39). Step 1.
To a well-stirred suspension of 5 (4.4 g, 11.9 mmol) in a
mixture of 1-methyl-2-pyrrolidinone (51 mL) and methylene
chloride (51 mL) were added sequentially triethylamine (2.99
g, 29.6 mmol), a catalytic amount of 4-(dimethylamino)pyridine
(0.2 g), and acetic anhydride (3.04 g, 29.8 mmol) at room
temperature. After 16 h at room temperature, the reaction was
quenched with water (150 mL) and methylene chloride was
removed under vacuum. The solid was filtered, washed with
water, and dried to give 4.5 g (90%) of the O-acetate interme-
1
diate 39a . H NMR (DMSO-d6): δ 2.01 (s, 3H), 2.06-2.18 (m,
2H), 4.06-4.16 (m, 2H), 4.51 (s, 2H), 4.75-4.87 (m, 2H), 4.92
(s, 2H), 7.27-7.78 (m, 6H), 8.00 (d, 1H), 8.54 (s, 1H), 9.51 (d,
2H).
Step 2. To a well-stirred suspension of 39a (0.5 g, 1.21
mmol) and 1,1-dichloromethyl methyl ether (1.39 g, 12.19
mmol) in a mixture of methylene chloride (42 mL) and toluene
(5 mL) was added tin(IV) chloride (2.37 g, 9.11 mmol) (1 M
solution in methylene chloride) at room temperature. The
reaction mixture was further stirred at room temperature for
4 h and at 50 °C for 45 min, then quenched with aqueous 2 N
HCl (25 mL) at room temperature. Methylene chloride was
removed under vacuum, and the aqueous layer was triturated
with tert-butylmethyl methyl ether (30 mL) at room temper-
ature for 14 h. The solid was filtered, washed with water and
tert-butylmethyl methyl ether, and then dried to obtain a crude
product, which was triturated with tert-butylmethyl methyl
ether (20 mL), to give 430 mg (81%) of aldehyde 39b. 1H NMR
(DMSO-d6): δ 2.00 (s, 3H), 2.15-2.27 (m, 2H), 4.10-4.16 (m,
2H), 4.48 (s, 2H), 4.76-4.90 (m, 2H), 4.95 (s, 2H), 7.33-7.45
(m, 2H), 7.64 (d, 1H), 7.89 (d, 1H), 8.04 (d, 1H), 8.51 (s, 1H),
8.64 (s, 1H), 9.51 (d, 1H), 10.11 (s, 1H). MS m/z 439 (M + 1).
Step 3. To a well-stirred solution of 39b (0.021 g, 0.048
mmol) in 1-methyl-2-pyrrolidinone (2 mL) was added sodium
methoxide in methanol (2 mL, 2 M solution) at room temper-
ature, and the resulting mixture was stirred at room temper-
ature for 45 min, then quenched with water (50 mL). The solid
was filtered, washed with water, and dried to give 17 mg (93%)
of 39 as an orange solid. 1H NMR (DMSO-d6): δ 1.98-2.00
(m, 2H), 3.50-3.54 (m, 2H), 4.54 (s, 2H), 4.77-4.81 (m, 2H),
4.98 (s, 2H), 7.36-7.45 (m, 2H), 7.665 (d, 1H), 7.90 (d, 1H),
8.04 (d, 1H), 8.53 (s, 1H), 8.64 (s, 1H), 9.51 (d, 1H), 10.11 (s,
1H). MS (m/z): 397 (M + 1).
Compounds 32-37 were prepared by the method described
for 18-31 using 16 according to the general procedure for
tritrifluoroacetate intermediate 8a . The products were further
purified using silica gel column chromatography with CH2Cl2/
MeOH (95:5) as solvent. The HPLC purity of the final products
was >96%.
Com p ou n d (()-32. 1H NMR (DMSO-d6): δ 1.08 (t, 3H),
1.41 (d, 3H), 1.93 (m, 2H), 3.47 (m, 2H), 4.52 (s, 2H), 4.60 (m,
1H), 4.73 (m, 2H), 4.90 (m, 2H), 7.33-7.39 (m, 2H), 7.47 (d,
1H), 7.63 (d, 1H), 7.69 (d, 1H), 7.86 (s, 1H), 8.55 (s, 1H), 9.47
(d, 1H). MS (m/z): 441 (M + 1), 395 (M - OCH2CH3). HRMS
(FAB) calcd for C28H28N2O3: 441.2178; found 441.2159.
Ch ir a l HP LC Sep a r a tion of (()-32. Racemic 32 (15 mg)
was separated on a Chiralcel OD column (1 cm × 25 cm) using
hexanes/ethanol (1:4, flow rate 0.5 mL/min) solvent system to
give enantiomer 33 (3.6 mg) and 34 (2.8 mg). Compound 33
(chiral): tR ) 27.9 min. MS (m/z): 441 (M + 1), 395 (M -
OCH2CH3). Compound 34 (chiral): tR ) 50.9 min. MS (m/z)
441 (M + 1), 395 (M - OCH2CH3). Because of the low recovery
under the conditions to achieve separation of the enantiomers,
only milligram quantities could be obtained for kinase screen-
ing.
Dim eth ylglycin e Ester 40. An oven-dried, 3 L three-
necked, round-bottomed flask equipped with a mechanical
stirrer, a three-way stopcock connected to an argon balloon,
and an immersion thermometer was charged with 21 (65.4 g,
Com p ou n d (()-35. 1H NMR (DMSO-d6): δ 1.45 (d, 3H),
1.98 (m, 2H), 3.14 (s, 3H), 3.50 (m, 2H), 4.58 (m, 3H), 7.75 (m,
2H), 4.93 (s, 2H), 7.33 (m, 2H), 7.48 (d, 1H), 7.67 (d, 1H), 7.72

5386 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Gingrich et al.
148.6 mmol) followed by anhydrous N,N-dimethylacetamide
(654 mL). 4-(Dimethylamino)pyridine (9 g, 73.7 mmol), N,N-
dimethylglycine (38.2 g, 370.8 mmol), and 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (70.3 g, 368
mmol) were added sequentially at 35 °C. The suspension was
heated to 42-45 °C for 2 h, and additional quantities of
4-(dimethylamino)pyridine (1.5 g, 12.2 mmol), N,N-dimethyl-
glycine (8 g, 77.6 mmol), and 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (15 g, 78.5 mmol) were added
sequentially at 42 °C. After 1.5 h, the reaction mixture was
cooled to 0-5 °C and the reaction was quenched with water
(1310 mL, added dropwise for 35 min). The cooling bath was
removed, and the resulting pale-yellow suspension was stirred
at room temperature for 1 h. The suspension was filtered,
washed with water (2600 mL) to pH 8, and dried overnight.
The solid was dissolved in CH2Cl2 (800 mL) and washed with
water and brine (2 × 200 mL), dried (MgSO4), filtered, and
concentrated. The crude material was dissolved in CH2Cl2 (217
mL) and transferred into a 3 L three-necked round-bottomed
flask, which was equipped with a mechanical stirrer. Ethyl
acetate (1000 mL) was added dropwise at room temperature
to the clear-red solution for 1 h and 10 min, and then the
mixture was stirred for 2.5 h at room temperature and filtered.
The solid was washed sequentially with ethyl acetate (80 mL),
ethyl acetate (75 mL)/methyl tert-butyl ether (50 mL), and
methyl tert-butyl ether (2 × 60 mL) and dried to obtain 62 g
(97.8% purity, 78% yield) of 40 as an off-white solid, mp 193-
94 °C. 1H NMR (DMSO-d6): δ 1.195 (d, 6H), 2.13-2.18 (m,
2H), 2.21 (s, 6H), 3.10 (s, 2H), 3.70-3.76 (m, 2H), 4.10-4.13
(m, 2H), 4.46 (s, 2H), 4.64 (s, 2H), 4.71-4.75 (m, 2H), 4.87 (s,
2H), 7.35-7.69 ((m, 4H), 7.89 (s, 1H), 8.53 (s, 1H), 9.50 (d,
1H). MS (m/z): 527 (M + 1).
the procedure of Rotin et al.33 Histone H-1 and MBP were
purchased from Fluka Chemical Corp. (Milwaukee, WI) and
Upstate Biotechnology, Inc. (Lake Placid, NY), respectively.
Time-resolved fluorescence experiments were performed using
Eu-N1 antiphosphotyrosine antibody, Eu-N1 antiphospho-
threonine antibody, Eu-N1 antirabbit IgG, and DELFIA
enhancement solution purchased from Perkin-Elmer Life
Sciences (Gaithersburg, MD).
Recep tor -Lin k ed Tyr osin e Kin a se Assa ys. Enzyme
inhibition studies were performed as described for VEGFR-2,
using a modification of the ELISA described for trkA kinase
at the Km level of ATP.24 Inhibition curves were generated by
plotting percent control activity versus log of the concentration
of compound. IC50 values were calculated by nonlinear regres-
sion using the sigmoidal dose-response (variable slope) equa-
tion in GraphPad Prism. IC50 values were reported as the
average of at least three separate determinations.
Hu m a n En d oth elia l Cell-Ba sed Recep tor P h osp h or yl-
a tion Assa y. Subconfluent HUVECs were serum-starved by
replacing media with EBM-2 (endothelial cell basal medium,
serum-free; Clonetics) containing 0.05% BSA for 1 h at 37 °C,
during which time a range of concentrations of drug or DMSO
(control) were added to the cells. Human VEGF (Clonetics) was
then added to HUVECs at a concentration of 10 ng/mL for 5
min. Cells were lysed in RIPA buffer containing 1 mM
activated sodium vanadate and protease inhibitors (Protease
Inhibitor Cocktail Set III, Calbiochem), sheared with a 27-
gauge syringe, then centrifuged at 12000g for 15 min. Clarified
cell lysates were normalized to protein using the BCA method
(Pierce), immunoprecipitated with anti-VEGF-R2 antibody
(CEP-133) for 1 h, followed by incubation with protein A
sepharose for another hour at 4 °C. Immunoprecipitates were
washed three times with RIPA buffer, 4X sample buffer was
added, and the mixture was heated for 5 min at 85 °C. Samples
were separated on a 6% Tris-glycine gel (NOVEX) or NuPAGE
7% Tris-acetate gel (NOVEX) and transferred using a wet blot
system onto a Millipore PVDF membrane. The membrane was
blocked with SuperBlock buffer (Pierce) containing 0.05%
Tween-20, immunoblotted with 4G10 antiphosphotyrosine
antibody (UBI; diluted 1:1000 in TBS-T containing 3% BSA)
for 1-2 h, followed by incubation with horseradish peroxidase
coupled goat antimouse IgG (BioRad; diluted 1:20000 in TBS-T
containing 3% BSA) for 1 h. Phosphorylated proteins were
visualized using enhanced chemiluminescence (Amersham).
The percent inhibition was calculated by analyzing scanned
autoradiographs on a densitometer. Scores were based on a
decrease in protein band density compared to that of VEGF-
stimulated control (no inhibitor) as follows: 0 ) no decrease;
1 ) 1-25%; 2 ) 26-50%; 3 ) 51-75%; 4 ) 76-100%.
9-(Met h yl)-12-(3-h yd r oxyp r op yl)-6H ,7H ,13H -in d en o-
[2,1-a ]p yr r olo[3,4-c]ca r ba zole-5-on e (41). To a stirred sus-
pension of 8 (60 mg, 0.151 mmol) in methylene chloride (6 mL)
at 0 °C under nitrogen was added trifluoroacetic anhydride
(11.4 µL, 0.181 mmol) followed by triethylsilane (28 µL, 0.181
mmol). The homogeneous mixture was stirred at 0 °C for 1 h,
then warmed to room temperature overnight. The reaction
solvent was removed in vacuo, leaving a tan solid, which was
triturated with ether and collected by filtration. The solid was
purified by flash chromatography on silica gel using methanol/
1
chloroform (5%) to give 47 mg (82%) of 41. H NMR (DMSO-
d6): δ 1.91 (m, 2H), 2.46 (s, 3H), 3.45 (m, 2H), 4.51 (s, 2H),
4.70 (m, 2H), 4.89 (s, 2H), 7.35 (m, 3H), 7.61 (m, 2H), 7.77 (s,
1H), 8.53 (s, 1H), 9.46 (d, 1H). MS (m/z): 383 (M + 1).
VEGF -R2 Recep tor -Lin k ed Tyr osin e Kin a se Assa y.
Enzyme inhibition studies were performed using a modifica-
tion of the ELISA described for trkA kinase.24 Briefly, the 96-
well microtiter plate (FluoroNUNC or Costar High Binding)
was coated with 10 µg/mL recombinant human PLC-γ/GST.
Kinase assays were performed in 100 µL reaction mixtures
containing 50 mM HEPES (pH 7.4), Km level of ATP, 10 mM
MnCl2, 0.1% BSA, 2% DMSO, and various concentrations of
drug. The reaction was initiated by adding baculoviral recom-
binant human enzyme VEGF-R2 and allowed to proceed for
15 min at 37 °C. The detection antibody, Eu-N1 antiphospho-
tyrosine (PT66) antibody, was added. After 1 h of incubation
at 37 °C, 100 µL of enhancement solution was added and the
plate was gently agitated. After 5 min, the fluorescence of the
resulting solution was measured using the Victor2 multilabel
counter.
Recom bin a n t P r otein s a n d Bioch em ica l Kin a se As-
sa ys. En zym es a n d Su bstr a tes. The cytoplasmic domains
of recombinant human tyrosine kinases (FGF-R1, Flt-3, PDGF-
Râ, Tie2, TrkA, VEGF-R1, VEGF-R2, VEGF-R3) and serine/
threonine kinases (CHK1, CDK1/cyclinB, CDS1) were ex-
pressed in a baculovirus insect cell system. Recombinant â-
insulin receptor kinase (âIrk) was purchased from Stratagene
(La J olla, CA). Purified rat brain protein kinase C (mixture of
Ca2+-dependent isozymes R, â, and γ) and activated recombi-
nant human GST-p38R were obtained from Upstate Biotech-
nology, Inc. (Lake Placid, NY). Phospholipase C-γ was gener-
ated as a fusion protein with glutathione S-transferase following
In Vit r o Ca p illa r y Tu be F or m a t ion Assa y w it h
HUVECs on Ma tr igel a n d ex Vivo Ra t Aor tic Rin g
Exp la n t Assa y in Colla gen Gel Ma tr ixes. The ability of
21 to inhibit angiogenesis in vitro in a capillary tube formation
assay utilizing HUVECs cultured on a synthetic basement
membrane matrix and ex vivo in rat aortic ring explant
cultures was conducted using published methods.30
P h a r m a cok in etics in Ra t. Single-dose oral administration
of test compounds in fasted adult male Sprague-Dawley rat
(n ) 3) was delivered via oral gavage in a dose volume of 2
mL/kg. Compound 21 was delivered in a 3:1 geluceire/PG
vehicle. Compound 40 was delivered in distilled water or 1%
HOAc. A dose of 1.19 mg/kg 40 is equivalent to a 1.0 mg/kg
dose of 21. The blood samples were placed on wet ice, and
plasma was collected after centrifugation. Plasma samples
were frozen on dry ice and stored frozen at -20 °C prior to
analysis. Plasma was prepared for high-performance liquid
chromatography (HPLC)/mass spectrometric analysis by pro-
tein precipitation with two volumes of acetonitrile (200 µL)
per 100 µL sample of plasma. The plasma samples were then
analyzed for both CEP-7055 and CEP-5214 via HPLC coupled
with tandem mass spectrometry. The data were entered into
an Excel spreadsheet, and pharmacokinetic parameters were
determined by WinNonLin.

Tyrosine Kinase Inhibitors
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Tumor measurements were expressed as absolute volumes, as
well as normalized to individual tumor volumes at day 1, the
initiation of dosing (relative tumor volumes) to assess changes
in the rate of tumor growth relative to treatment. Statistical
analyses of tumor data were done using the Mann-Whitney
rank sum test or, when appropriate for the data set, by one-
way ANOVA and the Dunnet’s multiple comparison test, with
p < 0.05 being deemed significant. Animal body weights were
determined and analyzed over a similar time course.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the scientific support and consultation of Stuart
Dodson, Mary Ann Harris, J oe Herman, Beverly
Holskin, J ean Husten, Nelson Landmesser, Chung Ho
Park, Alyssa Reiboldt, Damaris Rolon-Steele, and
Santhe Spais.
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