Tyrosine kinase inhibitors. 6. Structure-activity relationships among N- and 3-substituted 2,2'-diselenobis(1H-indoles) for inhibition of protein tyrosine kinases and comparative in vitro and in vivo studies against selected sulfur congeners

Article abstract of DOI:10.1021/jm960689b

A small series of 2,2'-diselenobis(1H-indoles) was synthesized as redox- modified congeners of our earlier reported 2,2'-dithiobis(1H-indole) series. Utilizing chemistry similar to that developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indolecarboxylic acid precursors bearing various polar functionality at the C-3 position and small alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic source of diselenium, and a much improved process to a 2,2'-dithiobis(1H-indole) congener was developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine kinases, compounds in this series displayed broad inhibitory activity with IC₅₀ = 0.9 to > 100 μM vs EGFr, 3.4 to >50 μM vs PDGFr, and 0.4-6.7 μM vsv-src. In general, compounds derived from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-indolecarboxylic acids greater potency against PDGFr and v- src. Enzyme kinetics studies showed that both classes of compounds display primarily noncompetitive inhibition with respect to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium and disulfur series with the reversal of enzyme inhibition occurring less readily within the diselenium series. In whole cell studies, compounds of this class were growth inhibitory against Swiss 3T3 mouse fibroblasts with IC₅₀ values from 0.5 to 19.5 μM, and the observed SAR was different from that of the 2,2'-dithiobis(1H-indoles). A comparative study in the same cell line on the effects of the 2,2'-diselenobis(lH-indole) derived from (R)-tryptophan vs its disulfur congener on growth factor mediated tyrosine phosphorylation showed that this compound significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with complete suppression at 25 and 5 μM, respectively. Tyrosine phosphorylation of an 85 kDa protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at submicromolar concentrations. The disulfur congener exhibited a qualitatively similar pattern; however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses (1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was effective against the C6 glioma when administered intraperitoneally on a d 1-9 schedule. Studies were also carried out against the A431 epidermoid xenograft to evaluate the same pair of compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The maximum dose that could be administered daily was limited by compound solubility. Neither compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-day growth delay without appreciable treatment related weight loss. The in vitro and in vivo findings offer a mechanistic rationale as to why the 2,2'-diselenobis(1H-indoles) are more potent inhibitors than their disulfur congeners.

Full text of DOI:10.1021/jm960689b

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J . Med. Chem. 1997, 40, 413-426
413
Tyr osin e Kin a se In h ibitor s. 6. Str u ctu r e-Activity Rela tion sh ip s a m on g N- a n d
3-Su bstitu ted 2,2′-Diselen obis(1H-in d oles) for In h ibition of P r otein Tyr osin e
Kin a ses a n d Com p a r a tive in Vitr o a n d in Vivo Stu d ies a ga in st Selected Su lfu r
Con gen er s
H. D. Hollis Showalter,*,† Anthony D. Sercel,† Boguslawa M. Leja,† Craig D. Wolfangel,† Linda A. Ambroso,†
William L. Elliott,† David W. Fry,† Alan J . Kraker,† Curtis T. Howard,† Gina H. Lu,† Charles W. Moore,†
J ames M. Nelson,† Bill J . Roberts,† Patrick W. Vincent,† William A. Denny,‡ and Andrew M. Thompson‡
Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, 2800 Plymouth Road,
Ann Arbor, Michigan 48106-1047, and Cancer Research Laboratory, University of Auckland School of Medicine,
Private Bag 92019, Auckland, New Zealand
Received October 3, 1996X
A small series of 2,2′-diselenobis(1H-indoles) was synthesized as redox-modified congeners of
our earlier reported 2,2′-dithiobis(1H-indole) series. Utilizing chemistry similar to that
developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indole-
carboxylic acid precursors bearing various polar functionality at the C-3 position and small
alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived
from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic
source of diselenium, and a much improved process to a 2,2′-dithiobis(1H-indole) congener was
developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth
factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine
kinases, compounds in this series displayed broad inhibitory activity with IC50 ) 0.9 to >100
µM vs EGFr, 3.4 to >50 µM vs PDGFr, and 0.4-6.7 µM vs v-src. In general, compounds derived
from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-
indolecarboxylic acids greater potency against PDGFr and v-src. Enzyme kinetics studies
showed that both classes of compounds display primarily noncompetitive inhibition with respect
to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused
a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium
and disulfur series with the reversal of enzyme inhibition occurring less readily within the
diselenium series. In whole cell studies, compounds of this class were growth inhibitory against
Swiss 3T3 mouse fibroblasts with IC50 values from 0.5 to 19.5 µM, and the observed SAR was
different from that of the 2,2′-dithiobis(1H-indoles). A comparative study in the same cell line
on the effects of the 2,2′-diselenobis(1H-indole) derived from (R)-tryptophan vs its disulfur
congener on growth factor mediated tyrosine phosphorylation showed that this compound
significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with
complete suppression at 25 and 5 µM, respectively. Tyrosine phosphorylation of an 85 kDa
protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this
compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at
submicromolar concentrations.The disulfur congener exhibited a qualitatively similar pattern;
however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in
vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and
the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses
(1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was
effective against the C6 glioma when administered intraperitoneally on a d1-9 schedule. Studies
were also carried out against the A431 epidermoid xenograft to evaluate the same pair of
compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The
maximum dose that could be administered daily was limited by compound solubility. Neither
compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day
study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-
day growth delay without appreciable treatment related weight loss. The in vitro and in vivo
findings offer a mechanistic rationale as to why the 2,2′-diselenobis(1H-indoles) are more potent
inhibitors than their disulfur congeners.
In tr od u ction
domain of the receptor results in its activation and
ultimately leads to cell proliferation primarily via a
Protein phosphorylation plays a major role in the
response of cells to various regulatory signals.1
A
number of pathways by which such signaling is initiated
by external growth factors and transmitted to the
nucleus via protein kinases are now known.2-4 Binding
of the appropriate growth factor to the extracellular
* Author to whom correspondence should be addressed. Phone: 313-
996-7028. Fax: 313-996-7879. E-mail: showalh@aa.wl.com.
† Warner-Lambert Company.
‡ University of Auckland School of Medicine.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00689-9 CCC: $14.00 © 1997 American Chemical Society

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414 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Showalter et al.
cascade of receptor and nonreceptor protein tyrosine
kinases.5-8 Thus, these enzymes play a crucial role in
the normal functioning and development of cells. How-
ever, in a number of diseases including cancer, athero-
sclerosis, and psoriasis, the generation of mitogenic
signals from abnormally expressed protein tyrosine
kinases is believed to play a major role in the loss of
growth control.9,10 The concept that deregulated signal
transduction can drive cellular transformation is sup-
ported by many experimental findings,6-8 such as
studies on the role of the epidermal growth factor
receptor (EGFr) and p185c-erbB2 protein tyrosine kinases
in epithelial proliferation.11-14
A comparison of the primary amino acid sequence
within the kinase domain of members of the tyrosine
kinase gene family has revealed a high degree of
homology.15 Despite this and the lack until recently of
precise structural data concerning the active site con-
formation of the enzyme,16 numerous small molecule
inhibitors have emerged with many showing good
selectivity between different enzymes.17-20 Hence, this
has lent credence to a paradigm that the selective
interruption of signal transduction by specific tyrosine
kinase inhibitors could have therapeutic potential in the
control of certain proliferative diseases.21-24
Previous reports from our laboratories have detailed
the synthesis and inhibitory activity of a large series of
3-substituted 2,2′-dithiobis(1H-indoles), generalized by
1, both against the EGFr and the nonreceptor pp60v-src
tyrosine kinases.25-28 This class possesses low micro-
molar potency both against isolated enzyme and in cells
after relatively short times of treatment. Structure-
activity relationship (SAR) studies on this class of
compounds have shown that biological activity is af-
fected to some extent by the overall nature of the indole
substitution pattern, but more markedly by variation
of the 3-substituent. The mechanism of inhibition
toward the above kinases by this class has been shown
to be noncompetitive with respect to both ATP and
peptide substrate, and thiol reversal studies suggest
that one of the possible mechanisms of inhibition is via
thiol-disulfide exchange with a thiol-containing resi-
due(s) within the catalytic site (vide infra).
bond would have an effect on (a) the reduction potential
of the connector moiety (and thus interaction of 2 with
endogenous thiols), (b) the relative conformation of the
connecting indole rings, and (c) metal binding since
divalent magnesium and manganese are required for
catalysis. In this paper, we detail the synthesis and
SAR of this class of compounds against a range of
protein tyrosine kinases including the epidermal growth
factor receptor (EGFr), platelet-derived growth factor
receptor (PDGFr), and v-src, and for selected compounds
compare their inhibitory effects in cellular and receptor
thiol reversal studies against direct congeners of the
sulfur series, namely compounds 3 and 4. Lastly, we
present the first results of in vivo studies for selected
compounds from both of these series.
Ch em istr y
A common method of 2,2′-dithiobis(1H-indole) syn-
thesis is the direct thiation of suitably functionalized
oxindole precursors with P2S5 followed by oxidative
dimerization of the derived thione.25-28 However, this
was not applicable to compounds of the diselenium
series since the analogous selenium reagent (P2Se5) is
unknown. Instead, we utilized two other methods that
had also previously been applied to the disulfur series.
The first utilized chemistry in which a selenium nu-
cleophile attacked a 2-halogeno-3-indole precursor ac-
tivated by an electron-withdrawing carbonyl group at
the 3-position.28 The second proceeded from a C-3
alkylindole substrate in which installation of the di-
selenium connector at the 2-position was carried out by
electrophilic chemistry utilizing diselenium dichloride
(Se2Cl2).25,26
Most of the target 2,2′-diselenobis(1H-indoles) were
derived from a 2-halogeno-3-indolecarboxylic acid pre-
cursor and were synthesized as outlined in Scheme 1.
Thus, starting from either 1-methyloxindole (5)29 or
oxindole (6), the synthesis of intermediates 7-9 was
achieved under Vilsmeier conditions similar to those
previously described for 9 (method A).30 Modification
of this procedure utilizing the bromide salt of the
Vilsmeier reagent provided 7. The known aldehyde
930,31 was made in a much improved yield with a simple
modification of the workup. Alternatively, 8 could be
made from 9 in near quantitative yield via a modifica-
tion of the known alkylation conditions (method B).32
The carboxylic acids 10-12 were initially prepared by
KMnO4 oxidation of the 3-carboxaldehydes 7-9 by a
literature procedure.31 However, yields generally did
not exceed 50%. A better procedure was to employ
sodium chlorite oxidation conditions33 which provided
the acids 10-12 in 62-78% yield (method C). Esteri-
fication of 10 with tert-butyl alcohol was difficult to
achieve and was unsuccessful by a number of common
methodologies. However, utilization of bis(2-oxo-3-
oxazolindinyl)phosphinic chloride (BOP chloride) as the
condensing agent provided 13 in 63% yield (method D).
On the basis of Moody’s work in which a range of
standard nucleophiles had been introduced into the
2-position of a suitably functionalized 2-chloroindole-3-
carboxaldehyde,34 we extended this chemistry toward
the introduction of a selenium moiety. After evaluating
a number of different selenium nucleophiles, we eventu-
ally resorted to the chemistry of Tiecco35 who had
evaluated the displacement of a range of inactivated aryl
In an attempt to broaden our understanding of the
mechanism of action of the 2,2′-dithiobis(1H-indoles),
we decided to synthesize a smaller series of closely
related congeners, namely the 2,2′-diselenobis(1H-in-
doles), generalized by 2. We anticipated that replace-
ment of the bridging S-S bond with the longer Se-Se

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Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 415
Sch em e 1
a
(a) POX3-DMF, CH2Cl2, 25-42 °C; (b) K2CO3, CH3I, acetone, 25 °C; (c) NaClO2, NaH2PO4, 2-methyl-2-butene, aqueous p-dioxane, 25
°C; (d) tert-butyl alcohol, BOPCl, NEt3, 1,2-DCE, reflux; (e) CH3SeLi, DMA, 0-25 °C; H2O2 or NaBO3, 0-25 °C; (f) SOCl2, 1,2-DCE,
reflux; aqueous NH2R2, 0 °C or NH2R2, CH2Cl2 or THF, 0°C; (g) Cl(CH2)2NEt2‚HCl, Cs2CO3, 3A molecular sieves, acetone, 25 °C; (h) TFA,
0 °C.
halides with alkane thiolate or selenolate anions. Thus,
treatment of ester 13 with in situ generated lithium
selenomethylate in DMA at 25 °C followed by workup
led to the bis-seleno ester 21 in 77% yield (method E).
A 2-fold excess of the selenium nucleophile was used so
that the nascent 2-methylseleno substituent would be
further demethylated to the arylselenide anion. In
contrast to the corresponding reaction with the meth-
anethiolate anion, as in the 2,2′-dithiobis(1H-indole)
series,28 it was not possible to isolate the resultant
arylselenol 14. Instead, upon acidification during work-
up, it spontaneously dimerized to the diselenide diester
21. Simple TFA hydrolysis of 21 gave diacid 22 in 78%
yield (method F). The carboxamide target compounds
(23-27) shown in Table 1 were derived from simple
Schotten-Bauman acylation of acids 10-12 to provide
monomeric amides 15-18 (method G). The addition of
an aqueous solubilizing amine side chain onto the indole
nitrogen of 18 to give 19 was carried out by alkylation
with 2-(N,N-diethylamino)ethyl chloride in acetone
utilizing Cs2CO3 as base (method H). Nucleophilic
displacement of the C-2 halogen of 15-19 with lithium
selenomethylate proceeded as described above to the
give target amide dimers 23-27 in 20-68% yields. The
physicochemical properties of compounds 21-27 are
listed in Table 1.
Compounds 30a , 32a , 32b, 33, and 34 of Table 1 were
synthesized starting from either (R)- or (S)-tryptophan
as outlined in Scheme 2 for the [R-(R*,R*)]-stereoisomer
33. Trifluoroacetylation of (R)-tryptophan utilizing the
literature procedure36 gave the known trifluoroacet-
amide 28a ,37 which was then coupled with benzylamine
via DCC/HBT condensation to provide 29a in 66% yield
(method I). Introduction of selenium into the 2-position
was accomplished with Se2Cl2, a reagent previously
used as an electrophilic source of selenium in reactions
with olefins38 and alkynes.39 We hoped that this
reagent would react with 29a analogous to the reaction
of 29a with S2Cl2.26 Thus, reaction of 29a with Se2Cl2
in THF at 0-5 °C provided a mixture of three products
by TLC: the diaryl selenide 30a , the presumed diaryl
triselenide 31a , and 32a as the major component
(method J ). This mixture was purified by treatment
with NaBH4 in cold methanol to leave a mixture of 30a
and a sodium aryl selenide monomer resulting from
reduction of the di- and triselenides 31a and 32a ,

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416 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Showalter et al.
Ta ble 1. Physicochemical Properties of 2,2′-Diselenobis(1H-indole-3-carboxamides) and Analogues
yield,a
%
no.
R1
Z
X
method
mp, °C
molecular formula
analysesb
21 CH3
22 CH3
23 CH3
24 CH3
CO2t-Bu
CO2H
CONHCH3
CONH(CH2)2NEt2
Se2
Se2
Se2
Se2
E
F
E
E
77
78
67
68
187-189
C28H32N2O4Se2‚0.2H2O C, H, N
C20H16N2O4Se2‚0.1H2O C, H, N
174c
225-230c C22H22N4O2Se2‚0.9H2O C, H, N
160-164d C32H44N6O2Se2‚2.0HCl‚ C, H, N, Cl
1.7H2O
25
26
H
H
CONHCH3
CONH(CH2)2NEt2
Se2
Se2
E
E
20
44
272-275c C20H18N4O2Se2‚0.9H2O C, H, N
257-259c,e C30H40N6O2Se2‚2.0 HCl‚ C, H, N
H2O
27 (CH2)2NEt2 CONHCH3
Se2
E
J
J
J
K
63
156-157
134-136
181-185
154-156
C32H44N6O2Se2‚0.5H2O C, H, N
30a
32a
32b
33
H
H
H
H
CH2CH(NHTFA)CONHCH2Ph[R-(R*,R*)]f,g Se
CH2CH(NHTFA)CONHCH2Ph[R-(R*,R*)]f,g Se2
CH2CH(NH-t-Boc)CONHCH2Ph[R-(R*,R*)]f,g Se2
C40H34N6O4F6Se‚0.3H2O C, H, N
45
6
57
C40H34N6O4F6Se2‚H2O
C46H54N6O6Se2‚H2O
C, H, N
C, H, N
CH2CH(NH2)CONHCH2Ph[R-(R*,R*)]g
Se2
172-174h C36H36N6O2Se2‚1.5H2O C, H, Nh
178-180i
173-175
34
H
CH2CH(NH2)CONHCH2Ph[S-(R*,R*)]j
Se2
K
50
C36H36N6O2Se2‚0.3H2O‚ C, H, N
k
0.2C6H14
a
b
d
Yields were not optimized. The analyses were within (0.4% of the theoretical values. c With decomposition. Free base, mp ) 128-
130 °C. e Free base, mp ) 225-226 °C. f TFA ) trifluoroacetyl. Derived from (R)-tryptophan. Derived from 32a . Derived from 32b.
g
h
i
Derived from (S)-tryptophan. n-Hexane detected by 1H NMR.
j
k
respectively. While carefully maintaining a nitrogen
environment, the mixture was diluted with water and
then extracted with diethyl ether to selectively remove
the neutral diaryl selenide 30a . Acidification of the
aqueous phase then resulted in the spontaneous dimer-
ization of the selenide to the dimer 32a in an overall
yield of 45%. To the best of our knowledge, this is the
first application of Se2Cl2 for the synthesis of diaryl
diselenides. The cleavage of the trifluoroacetate pro-
tecting group to give target compound 33 was then
carried out by NaBH4 in refluxing ethanol by a litera-
ture procedure40 (method K). While this reductive
procedure claims to be racemization free, we observed
10-15% of another diastereomer (due to epimerization
of one of the centers of chirality) by chiral HPLC.
Compound 33 is stable when stored as a solid in the
cold, but is sensitive to slow decomposition on standing
in the light at room temperature. The same sequence
of reactions was carried out starting from (S)-tryp-
tophan to provide the [S-(R*,R*)]-enantiomer 34 in a
comparable overall yield and chiral purity.
Since we were observing some apparent inversion of
one of the centers of chirality in the NaBH4 cleavage of
32a to 33, we decided to synthesize 33 by an alternate
procedure in which a more easily cleavable nitrogen
protecting group would be utilized. Thus, reaction of
N-t-Boc derivative 29b, made in a manner similar to
the literature procedure,41 with Se2Cl2 as described
above (method J ) followed by a similar extractive
purification provided the dimer 32b in only a 6% yield.
This reduced yield relative to 32a can likely be at-
tributed to the instability of the t-Boc protecting group
to the strongly acidic conditions of the Se2Cl2 reaction.
The cleavage of the N-t-Boc protecting group to give
target compound 33 was then carried out in 58% yield
under the mild conditions of trimethylsilyl triflate in
dichloromethane buffered with 2,6-lutidine at 0 °C42
(method K). Standard methods of N-t-Boc cleavage
(TFA in dichloromethane or HCl/methanol) resulted in
impure product along with the deposition of some
elemental selenium. Compound 33 obtained by this
procedure was shown to be 98.5% pure by chiral HPLC.
The reported synthesis26 of the 2,2′-dithiobis(1H-
indole) 4 was substantially improved to provide multi-
gram quantities necessary for the in vivo studies. Each
step in the original route was examined, achieving an
improvement in the overall yield of 4 from (R,S)-
tryptophan from 5% to 50%. The optimum route is
shown in Scheme 3 (method L). In the diethylphos-
phoryl cyanide (DEPC) coupling of the N-trifluoro-
acetamide of (R,S)-tryptophan (28a ) with benzylamine,
the yield of racemic 35 was increased from 50% to 83%
by adding DEPC directly to the protected tryptophan
acid, followed by benzylamine, with the base added last.
Presumably the amine initially acts as the base, forming
the amine salt and the carboxylate anion. The DMF
was then removed at 60 °C prior to aqueous workup,
which may have also helped by driving the reaction to
completion. The yield of dimer 35 from the amide 29a
via reaction with S2Cl2 was also markeldy improved
(from 44% to 81%), primarily by purification of the S2Cl2
by rapid distillation immediately prior to the reaction.
Maintaining strictly anhydrous conditions (dry ap-
paratus, solvent, and substrate and protection from
atmospheric moisture during the reagent addition and
the reaction) was also important. Finally, a major
improvement (from 22% to 75% yield) was achieved in
the reductive cleavage of the TFA group in 35 to give 4,
by employing a longer reaction time and higher tem-
perature for the reduction (3 h at 80 °C) and using H2O2
at pH 8 for the reoxidation of the acid soluble material,
instead of relying on aerial oxidation at pH 10. Com-
pound 4 obtained by this sequence of reactions was
shown to be a ca. 1:2:1 mixture of [S-(R*,R*)]:[R-
(R*,S*)]:[R-(R*,R*)] stereoisomers by chiral HPLC.
SAR vs Isola ted Tyr osin e Kin a ses
For the synthesized 2,2′-diselenobis(1H-indoles) listed
in Table 1, compounds 21-27 were derived from 2-ha-
logeno-3-indolecarboxylic acid precursors whereas 30,

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Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 417
Sch em e 2
a basic side chain nitrogen, showed the greatest potency
(IC50 ca. 1 µM). Against PDGFr, the pattern of inhibi-
tion differed from that of EGFr in that the greatest
potency was associated with compounds derived from
2-halogeno-3-indolecarboxylic acid precursors, especially
23 and 24 which have amide functionality at C-3 and
methyl substitution at N-1. Compounds derived from
(R)- or (S)-tryptophan were less potent, although 33
derived from (R)-tryptophan again displayed greater
potency than its enantiomer 34. The pattern of inhibi-
tion against the cytoplasmic tyrosine kinase v-src was
markedly different from the above two receptor kinases.
Within the 2,2′-diselenobis(1H-indoles), there was about
a 10-fold range in potencies (IC50 0.4-6.2 µM) with the
highest potency again associated with analogues derived
from 2-halogeno-3-indolecarboxylic acid precursors (e.g.,
21, 23, 24, 27).
A comparison of two pairs of diselenium-disulfur
congeners in Table 2 (3 vs 23; 4 vs 32 or 33) against
the three kinases demonstrates a 5-10-fold increase in
potency for diselenium compound 23 against PDGFr
and v-src and equivalent potency vs EGFr. Its indole
N-H congener 25 demonstrated a similar trend relative
to its sulfur congener28 (data not shown). For the
diselenium compound 33 (34) vs the disulfur congener
4, the opposite pattern was observed vs EGFr and v-src.
En zym e Kin etics
The inhibition kinetics of the 3/23 and 4/34 pairs of
disulfur/diselenium congeners are shown in Table 3. For
the 3 vs 23 pair, the potency of the diselenium com-
pound 23 was greater in the v-src assay (Ki ) 0.8 and
0.6 µM, respectively) than that of the disulfur compound
(Ki ) 8 and 1.4 µM, respectively) with respect to varying
ATP or the peptide polymer substrate, whereas in the
EGFr assay, little if any difference in the inhibition
kinetics was observed. In contrast, in the EGFr assay
for the 4/34 pair, the potency of the diselenium com-
pound 34 was markedly greater (Ki ) 2.7 and 5.6 µM,
respectively) than that of the disulfur congener 4 (Ki )
19.4 and 21.2 µM, respectively) with respect to varying
the same substrates. A curve-fitting analysis of the
inhibition (Grafit, Erithicus Software, Ltd.) showed
primarily noncompetitive inhibition by both classes of
compounds with respect to either ATP or peptide
substrate. Double reciprocal plots (not shown) confirm
this inhibition mechanism.
a
(a) HOBT, DCC, benzylamine, THF, 25 °C; (b) Se2Cl2, THF, 5
°C; NaBH4, MeOH, 5 °C; H3O+; (c) For 32a , NaBH4, EtOH, 80 °C;
for 32b, TMS triflate, 2,6-lutidine, CH2Cl2, 5 °C.
32a , 32b, 33, and 34 were derived from (R)- or (S)-
tryptophan. The C-3 side chain in compounds 21-27
has either acid, ester, or amide functionality, whereas
the C-3 side chain in compounds 30a , 32a , 32b, 33, and
34 possesses a terminal amine that is either free or
trifluoroacetylated and a benzyl amide moiety. In all
cases the substituent on the indole nitrogen is either
hydrogen or methyl except for 27, which has the 2-(N,N-
diethylamino)ethyl substituent. Two compounds (24,
26) possess this same tertiary amine functionality at
the 3-position whereas compounds 33 and 34 possess
the primary NH2 moiety in the side chain. All com-
pounds that were evaluated biologically possess the Se-
Se connector except for 30a , which was isolated as a
side product and has a single Se connector.
For activity against all kinases evaluated in Table 2,
there was a requirement for a diselenium connector
(compare 30a vs other congeners). Against the EGFr,
compounds 21-27 derived from 2-halogeno-3-indole-
carboxylic acid precursors showed generally equivalent
potency (IC50 3.5-6.1 µM) regardless of the C-3 or N-1
substitution pattern except for 21 (IC50 >100 µM) with
the bulky tert-butyl ester moiety and 25 (IC50 13 µM)
which is unsubstituted on N-1 and has a small amide
moiety at C-3. Compounds 33 and 34, derived from (R)-
and (S)-tryptophan, respectively, and each possessing
Th iol Rever sa l Stu d ies
The effect of the sulfhydryl reducing agent dithio-
threitol (DTT) on the inhibition of tyrosine kinases by
various compounds has been previously described.43-45
Figures 1 and 2 show the effect of increasing DTT on
the activity of diselenium compound 33 and disulfur
congener 4 in the EGFr kinase assay. For a given
inhibitor concentration, increasing concentrations of
DTT caused a general decrease in kinase inhibition.
This effect may be indirect (i.e., not via a direct disulfide
or seleno-sulfide exchange) since the stoichiometry of
DTT to inhibitor is much greater than 1:1 at 0%
inhibition. The reversal of enzyme inhibition by DTT
occurs somewhat less readily with the diselenium
compound 33 (Figure 1) than with the disulfur com-
pound 4 (Figure 2), both against the EGFr tyrosine
kinase and the v-src kinase (data not shown). This

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418 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Showalter et al.
Sch em e 3
a
(a) CF3CO2Et, NEt3, DMF, 20 °C, then DEPC, benzylamine, NEt3, DMF, 0-20 °C; (b) S2Cl2, THF, 20 °C; (c) NaBH4, EtOH, 80 °C,
then 35% H2O2.
Ta ble 2. Inhibition of Tyrosine Kinases by
2,2′-Selenobis(1H-indoles) in Vitro and the Ability to Inhibit the
Proliferation of Swiss 3T3 Mouse Fibroblasts (IC50, µM)a
no.
EGFr
6.9
7.4
>100
PDGFr
v-src
Swiss 3T3 cells
3
4
28.1
NDc
>31
17.4
4.7
6.7
1.5
1.5
2.4
0.4
1.8
2.8
3.6
1.7
1.8b
5.3d
ND
8.0
5.5
19.5
1.0
21
22
23
24
25
26
27
30a
32a
33
34
3.5
6.1
4.7
13
4.6
6.9
3.4
8.0
12.8
ca. 50
>50
>50
14.1
25.5
8.0
ND
ND
ND
0.5
>50
>50
7.0
0.9
1.3
6.2
2.0
3.8
0.6
a
IC50 values reported for kinase and cellular inhibition are
means of duplicates from at least two separate experiments with
typical variation less than 30% between values. The IC50 vs Swiss
3T3 cells represents the concentration of compound required to
F igu r e 1. Effect of DTT on the inhibition of EGFr tyrosine
kinase by compound 33. Data shown are duplicate determina-
tions in a representative experiment.
b
inhibit cell growth rate by 50%. Reference 26. c ND, not deter-
d
mined. Reference 28.
Ta ble 3. Inhibition Kinetics of Selected
2,2′-Diselenobis(1H-indoles) and Sulfur Congeners in EGFr and
v-Src Tyrosine Kinases Varying ATP and Peptide Polymer
Substratea
EGFr
Ki ATP
v-src
Ki ATP
EGFr
Ki peptide
v-src
Ki peptide
no.
3
23
4
2.3
3.1
19.4
2.7
8
3.9
3.9
21.2
5.6
1.4
0.8
NDb
NDb
0.6
NDb
NDb
34
a
Values given are micromolar and were determined by a
nonlinear regression analysis curve-fitting program (Grafit) of the
inhibition data using Cleland′s equations for enzyme inhibition.
The type of inhibition was determined to be substantially non-
competitive with respect to both ATP and peptide polymer
substrate based on comparisons of Ki values calculated from slope
and intercept determinations. Double-reciprocal plots of the
inhibitor data confirmed the curve-fitting analysis (data not
F igu r e 2. Effect of DTT on the inhibition of EGFr tyrosine
kinase by compound 4. Data shown are duplicate determina-
tions in a representative experiment.
b
shown). ND, not determined.
on the substitution pattern on the indole ring. It
appears that methyl substitution on the indole nitrogen
is detrimental to growth inhibition (compare 23 vs 25
and 24 vs 26). Surprisingly, the carboxylic acid 22 was
nearly as potent as methyl amide 23. This is a different
pattern than that found for the 2,2′-dithiobis(1H-in-
doles) where carboxylic acid substituted derivatives
were much less potent than alkyl amides or esters.26
The most potent compounds were 33 and 34. Since both
isomers were essentially equipotent, the [R-(R*,R*)]
isomer, 33, was selected for further testing. Figure 3
is an antiphosphotyrosine western blot showing the
effect of compound 33 on growth factor-mediated ty-
rosine phosphorylation in Swiss 3T3 murine fibroblasts.
The results indicate that this compound significantly
difference may be related to the relatively more stable
nature of the diselenide bond compared to the disulfide
moiety.46 The retention of inhibitory activity by 33 in
the presence of thiol suggested better cellular activity
for the 2,2′-diselenobis(1H-indoles) compared to their
sulfur congeners (vide infra).
Cellu la r Stu d ies
The 2,2′-diselenobis(1H-indoles) were also evaluated
in whole cell studies. Shown in Table 2 is the ability of
several compounds to inhibit the proliferation of Swiss
3T3 mouse fibroblasts. Compounds of this class were
growth inhibitory with IC50 values ranging from 0.5 µM
for 33 to 19.5 µM for 24, with potency highly dependent

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Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 419
F igu r e 3. Effect of compound 33 on growth factor mediated tyrosine phosphorylation in Swiss 3T3 murine fibroblasts. Cells
were treated for 2 h with varying concentrations of compound and then exposed to the indicated growth factor for 5 min. Extraction
and western blotting procedures are described in the Experimental Section.
Ta ble 4. Comparison of 2,2′-Diselenobis(1H-indole) 33 and
to growth factors in vitro. Compound 4 was ineffective
Sulfur Congener 4 in Selected Cellular Assaysa
against all three tumor models (Table 5). This was the
IC50 (µM)
4 33
case against B16 and C26 even though the administra-
tion sites for tumor implant and compound 4 were both
intraperitoneal. A q4h × 3 treatment schedule was
used in an attempt to maximize tumor cell exposure to
4 on each treatment day. This rigorous treatment
schedule did not improve the antitumor effectiveness
based upon either % T/C (B16 and C26) or tumor growth
delay (M5076). The maximum tolerated dose of 4 was
approximately 3.0 mg/kg/day for the daily treatment
schedule for 9 days and 9.0 mg/kg/day for the q4h × 3
daily for 9 days treatment schedule. It should be noted
that the maximum tolerated doses in the intraperitoneal
tumor implant studies were not necessarily the doses
producing the optimal (largest) % T/C values that are
shown in Table 5. The % T/C values against the B16
and C26 tumor models were in the range 85-95% when
4 was administered at a maximum tolerated dose.
cellular process
bFGF-mediated tyrosine phosphorylationb
PDGFr autophosphorylationb
EGFr autophosphorylationb
proliferationb
2.3
6.2
0.2
0.57
4.2
0.54
0.18
0.35
0.17
>100
5.3
DNA synthesisc
2.5
2.0
0.6
RNA synthesisc
protein synthesisc
a
IC50 values reported for kinase inhibition are means of
duplicates from at least two separate experiments with typical
b
variation less than 30% between values. See ref 27 for details of
experimental procedures. c See the Experimental Section for
details.
inhibits EGF receptor autophosphorylation at 5 µM with
complete suppression at 25 µM. Autophosphorylation
of the PDGF receptor in response to its ligand was even
more susceptible and was completely suppressed at 5
µM. Tyrosine phosphorylation of an 85 kDa protein
typically phosphorylated in response to bFGF was also
exquisitely sensitive to compound 33, and a significant
effect was seen as low as 0.2 µM.
Table 4 summarizes data from several cellular assays
for the diselenium compound 33 and compares this with
its disulfur congener 4. IC50 values generated from
quantitating the results of western blots similar to
Figure 3 indicate that compound 33 is indeed most
active against bFGF- and PDGF-mediated tyrosine
phosphorylation and produces a 50% inhibition at less
than 1 µM. The disulfur congener 4 exhibited a
qualitatively similar pattern; however, its potency was
10-fold less. Likewise, the inhibitory effects of 33 on
cellular proliferation and DNA, RNA, and protein
syntheses also occurred at submicromolar concentra-
tions. Compound 4 again produced similar effects but
was consistently 10-fold less potent.
The human tumor xenografts A431 epidermoid, C6
glioma, and MCF-7 used in these studies were selected
based upon the in vitro responsiveness of these tumors
to growth factors. Compound 4 was ineffective against
the MCF-7 breast xenograft on both of the administra-
tion schedules tested. At maximum tolerated doses,
neither 4 nor 33 was effective against the C6 glioma
when administered intraperitoneally on a daily treat-
ment schedule for 9 days. The maximum tolerated dose
for 4 administered on a daily treatment schedule was
>12.0 mg/kg/injection against the MCF-7 breast line,
but only 5.0 mg/kg/injection against the C6 glioma.
We then carried out studies to evaluate the effective-
ness of 4 and 33 administered by continuous subcutane-
ous infusion from Alzet miniosmotic pumps, based upon
studies of a quinazoline EGFr tyrosine kinase inhibitor
evaluated in our laboratories that showed the need for
continuous drug exposure.47 Compound solubility was
the limiting factor for the dose that could be delivered
to the animals. The doses in Table 5 for the infusion
studies represent the maximum dose that could be
administered daily without significant compound pre-
cipitation. The degree and amount of compound pre-
cipitation were reasonably consistent in the two studies
In Vivo Stu d ies
The murine tumors B16 melanoma, colon carcinoma
26, and M5076 sarcoma were selected for an initial
evaluation of disulfur compound 4 based upon their
growth properties and the response of similar histotypes

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420 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Showalter et al.
Ta ble 5. In Vivo Anticancer Activity of Compounds 4 and 33 against Murine Tumors and Human Tumor Xenograftsa
c
tumor
no.
dose (mg/kg)
schedule
weight change (g)
T/C or T - Cb
net cell kill (log10)
B16 melanoma
4
0.75d
3.0d
0.75d
1.5d
3.0e
10f
ip, q4h × 3; d1-9
+1.1
+1.4
+0.8
+1.8
-0.5
-1.2
-0.2
+
99
100
119
115
0.7
1.9
2.2
1.0
10.8
0
-1.3
-1.3
-0.2
-0.3
-0.8
-0.2
-0.2
-0.6
-0.4
ip, d1-9
colon 26
4
ip, q4h × 3; d1-9
ip, d1-9
M5076 sarcoma
A431 epidermoid
4
4
33
4
33
4
33
4
ip, q4h × 3; d1-9
sc Alzet d9-15
0.5f
9.0f
0.5f
5e
sc Alzet d1-27
+
C6 glioma
MCF-7
ip, D1-9
ip, D1-9
+
1.8e
3.0e
12.0g
-0.9
+
2.0
1.0
0
-0.2
-1.2
-
ip, q4h × 3; d5-11
4
ip, d5-11
-0.4
a
For B16 and colon 26 models 1.8 × 106 and 1.5 × 106 viable cells, respectively, were implanted ip on day 0. Tumor fragments of
b
A431, C6, MCF-7, or M5076 were implanted sc into the right axilla of mice on day 0. Endpoint for the B16 and C26 studies was % T/C,
while the endpoint in all other evaluations was tumor growth delay (T - C). c The net reduction in tumor burden (in logs) between the
first and last treatments. Optimal dose. The dose producing the largest % T/C from the dose response. e Maximum tolerated dose,
d
eLD10 (mg/kg/injection), from a complete dose response. f Dose represents the maximum amount of compound (mg/kg/day) that could be
g
delivered without significant precipitation after the test compound was extruded from the pump. Highest dose tested.
conducted against the A431 epidermoid xenograft.
Neither compound produced an antitumor effect in an
initial 7-day infusion study. A second study evaluated
the effect of a 27-day continuous infusion to determine
if a prolonged exposure time would produce a greater
effect. The miniosmotic pumps in this study were
removed and replaced with full pumps each week to
increase the infusion time to a total of 27 days. Treat-
ment with 4 over this time period resulted in no
antitumor effect. However, treatment with 33 on the
same schedule resulted in a 10.8-day growth delay. The
lack of any appreciable treatment-related weight loss
in the 27-day study indicated that more 33 could have
been delivered to the animals on a daily basis had
solubility not been a limiting factor.
10.8-day tumor growth delay against the A431 epider-
moid carcinoma compared with the inactivity of com-
pounds 4 and 33 against the other models evaluated
suggested that continuous exposure would be necessary
for in vivo antitumor effectiveness. However, the
reduced solubility of 33 prohibited the use of higher
infusion doses which might have increased antitumor
effectiveness. The weight loss data, as an indicator of
toxicity, showed that with the infusion doses used in
these studies there was little, if any, weight loss,
indicating that a larger dose might be tolerated and
contribute to greater antitumor effectiveness.
Our in vitro and in vivo findings offer a mechanistic
rationale as to why the 2,2′-diselenobis(1H-indoles) are
more potent inhibitors than their disulfur congeners.
In the presence of physiological intracellular glutathione
levels (ca. 1 mM), compounds of the diselenium series
might be less susceptible to thiol reduction, and thus
be more likely to be presented to a tyrosine kinase in
dimeric form where they could interact with a sulfhydryl
residue via an exchange mechanism. Our mechanistic
studies show that both the 2,2′-diselenobis(1H-indoles)
and 2,2′-dithiobis(1H-indoles) are noncompetitive in-
hibitors, with respect to both ATP and peptide sub-
strate. Over a 2-h period it appears that binding is
essentially irreversible, suggesting that these agents
might act either as selenolating or sulfenolating agents
on a sulfhydryl residue. Within the EGFr catalytic
domain, there are six sulfhydryl residues.15 Cysteine-
272 near the catalytic site is conserved over a broad
range of kinases and thus might be a site of selective
covalent modification by compounds of this general
class. Additional studies at the molecular level are
necessary to support this proposal.
Su m m a r y
We have synthesized a small series of 2,2′-diselenobis-
(1H-indoles) and have determined that their SAR pat-
tern is similar to that of the congeneric 2,2′-dithiobis-
(1H-indoles) against selected isolated tyrosine kinases.
However, against these kinases and in whole cells,
compounds of the diselenium series are generally more
potent (up to 10-fold). Additionally, the pattern of
inhibition of cellular phosphorylation by the diselenium
series is markedly different, and the reversal of inhibi-
tion by thiols such as DTT is markedly less for a given
concentration of inhibitor. Similar effects have been
observed in studies carried out in the presence of
glutathione (data not shown). This retention of inhibi-
tory activity by the diselenium series in the presence of
added thiol might relate in part to the better cellular
activity for this class compared to their disulfur con-
geners.
Two congeners from the disulfur and diselenium
series (compounds 4 and 33, respectively) have also been
evaluated for antitumor effectiveness against murine
and human tumor xenograft model systems. The modi-
fication from the S-S to Se-Se linkage appears to
increase the dose potency as reflected in a 3-fold lower
maximum tolerated dose for 33 compared to 4 in the
C-6 glioma tumor model (Table 5). However, 33 was
approximately 20-fold less soluble than 4, which limited
the amount of compound that could be continuously
delivered from a subcutaneously implanted Alzet mini-
osmotic pump. The observation that 33 produced a
Exp er im en ta l Section
Melting points are uncorrected. Proton nuclear magnetic
resonance (1H NMR) spectra were measured at 300 or 400
MHz. Chemical shifts are reported as δ values (parts per
million) downfield from internal Me4Si. The following ab-
breviations are used to describe peak patterns when appropri-
ate: br ) broad, s ) singlet, d ) doublet, t ) triplet, q )
quartet, m ) multiplet. Selenium nuclear magnetic resonance
(77Se NMR) spectra were recorded at a frequency of 76.3 MHz.
Chemical shifts are referenced to neat (CH3)2Se at 0 ppm with
(CD3)2SO in an external capillary. Mass spectra (MS) were
recorded in either the APCI or FAB mode in a matrix of MeOH/

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Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 421
CH3CN/DMSO or thioglycerol, respectively. Column chroma-
tography was carried out in the flash mode utilizing E. Merck
230-400 mesh silica gel. Analytical TLC was carried out on
E. Merck silica gel 60 F254 plates with detection by UV light.
HPLC was carried out on a Vydac 218 TP54 column with a
gradient mobile phase of 10-76% of 0.1% TFA in CH3CN:0.1%
TFA in H2O to assay for chemical purity, and on a 5 µM
Chiralpak AD column (serial no. 15-42-01225) with a mobile
phase of 5:30:65 MeOH:EtOH:hexanes containing 1.0 mL of
diethylamine/L at a flow rate of 0.7 mL/min to assay for chiral
purity. Combustion analyses were determined by Robertson
Microlit Laboratories, Inc., Madison, NJ . All reaction solvents
were reagent grade or distilled-in-glass and were stored over
activated 3A (for lower alcohols) or 4A molecular sieves. For
the scale-up synthesis of 4, DMF, triethylamine, and benzyl-
amine were additionally predried over molecular sieves. Fol-
lowing normal workup procedures, organic extracts were dried
over anhydrous Na2SO4 or Mg2SO4 prior to concentration.
Meth od A. 2-Br om o-1-m eth ylin d ole-3-ca r boxa ld eh yd e
(7). A three-neck round-bottomed flask was equipped with a
mechanical stirrer, pressure equalizing dropping funnel, and
N2 inlet. To a stirred 25 °C solution of 55 mL (744 mmol) of
DMF and 105 mL of CH2Cl2 was added dropwise a solution of
178 g (620 mmol) of POBr3 in 125 mL of CH2Cl2 at such a
rate to maintain a gentle reflux via the exothermic reaction
(ca. 1 h). The resulting thick tan mixture was stirred vigor-
ously for 10 min, and then 36.5 g (248 mmol) of N-methylox-
indole (5)29 was added portionwise over 20 min. The mixture
was stirred at 25 °C for 60 h at which time TLC (SiO2; 1:1
EtOAc:hexanes) showed clean conversion to product. The
solution was poured slowly onto 600 mL of crushed ice. The
resulting suspension was stirred for 30 min, and the phases
were then separated. The golden aqueous phase was extracted
with 200 mL of CH2Cl2 and then was allowed to stir at 25 °C
for 18 h. The flocculent tan precipitate that had formed was
collected by filtration and washed well with H2O. The aqueous
filtrate was stirred for 48 h during which additional precipitate
formed. The combined crops were dried at 70 °C over P2O5 to
leave 40.7 g (69%) of 7 as a light tan solid: mp 110-111.5 °C;
1H NMR (CDCl3) δ 10.04 (s, 1H, CHO), 8.33-8.31 (m, 1H, H-4),
7.34-7.29 (m, 3H, ArH), 3.86 (s, 3H, NCH3). Anal. (C10H8-
BrNO) C, H, N, Br.
2-Ch lor oin d ole-3-ca r boxa ld eh yd e (9). The following is
a modification of the procedure of Schulte.30 A 3 L three-neck
round-bottomed flask fitted with a pressure-equalizing addi-
tion funnel, an N2 inlet, and an internal thermometer was
charged with 1 L of CH2Cl2 and 233 mL of DMF. The solution
was cooled to 0 °C with an ice bath and treated dropwise over
25 min with 233 mL (2.5 mol) of POCl3. The solution was
brought to 25 °C and maintained there for 2 h. The clear
amber solution was cooled to 0 °C and charged over 35 min
with 137.3 g (1 mol) of oxindole (6) via a solid addition funnel.
The darkened mixture was stirred under N2 at 25 °C for 38 h
at which time TLC (1:1 hexanes:EtOAc) showed no starting
material. The mixture was cautiously poured into 1.7 L of wet
ice, and additional ice was kept available to control a subse-
quent exotherm. The mixture was transferred to a separatory
funnel and extracted with 200 mL of CH2Cl2 (during the
extraction the temperature of the aqueous layer rose and was
controlled with ice addition). The organic extract was back-
extracted with H2O (3 × 350 mL), and all of the aqueous layers
were combined and stirred at 25 °C overnight. The resultant
flocculent solids were collected by filtration, washed well with
H2O, air dried, and then dried over P2O5 to afford 131.8 g (73%)
of 9: mp 218-219 °C dec (lit. mp 232-235 °C30, 223-225 °C31).
The material was pure by TLC and used directly in the next
reaction.
residue that was stirred in 25 mL of H2O for 15 min. The
solids were collected by filtration, washed well with H2O, and
dried over P2O5 to afford 10.41 g (97%) of 8 as an off-white
solid: mp 97-98 °C (lit.32 mp 89-91 °C).
Meth od C. 2-Br om o-1-m eth ylin d ole-3-ca r boxylic Acid
(10). To a vigorously stirred solution of 39 g (164 mmol) of
2-bromo-1-methylindole-3-carboxaldehyde (7), 164 mL of 2-meth-
yl-2-butene, and 615 mL of p-dioxane at 25 °C was added
dropwise over ca. 30 min a solution made up of 81.9 g (902
mmol) of NaClO2 and 81.9 g (590 mmol) of NaH2PO4‚H2O in
410 mL of H2O. The mixture was treated with 20 g each of
solid NaClO2 and NaH2PO4‚H2O after 2.5 h and then 10 g each
of these reagents after 5 h. After a total reaction time of 6.5
h, the mixture was diluted with 1.1 L of EtOAc and stirred
for ca. 45 min. The layers were separated, and the aqueous
phase was extracted twice with EtOAc (2 × 300 mL). The
combined organic extracts were concentrated to 1 L and then
extracted with 1% aqueous NaOH (3 × 400 mL). The
combined aqueous extracts were cooled and acidified to ca. pH
2 with 6 N aqueous HCl. The resultant solids were collected,
washed well with H2O, and dried over P2O5 to leave 33 g (78%)
1
of 10 as a white solid: mp 198 °C dec; H NMR ((CD3)2SO) δ
12.45 (br s, 1H, CO2H), 8.04 (d, J ) 7.9 Hz, 1H, H-4), 7.60 (d,
J ) 7.9 Hz, 1H, H-7), 7.29-7.19 (m, 2H, ArH), 3.84 (s, 3H,
indole NCH3). Anal. (C10H8BrNO2) C, H, N, Br.
2-Ch lor oin d ole-3-ca r boxylic Acid (12). Application of
these reaction conditions to 15 g (83.5 mmol) of 2-chloroindole-
3-carboxaldehyde (9) afforded 10.1 g (62%) of 12 [mp 181.5 °C
(lit.31 mp 160 °C)] in two crops following crystallization from
7:3 acetone:hexanes.
Meth od D. 2-Br om o-1-m eth ylin dole-3-car boxylic Acid,
ter t-Bu tyl Ester (13). A suspension of 2.54 g (10 mmol) of
2-bromo-1-methylindole-3-carboxylic acid (10), 2.54 g (10
mmol) of bis(2-oxo-3-oxazolidinyl)phosphinic chloride, 1.13 mL
(12 mmol) of tert-butyl alcohol, 2.78 mL (20 mmol) of triethyl-
amine, and 25 mL of 1,2-dichloroethane was heated at reflux
for 1.5 h. The mixture was cooled, poured into 150 mL of 5%
aqueous NaHCO3, and stirred for 30 min. The mixture was
extracted with CH2Cl2 (3×), and the combined organic phase
was washed with H2O and brine, dried, and concentrated to
leave a red oil that was triturated in EtOAc:hexanes and
filtered. The filtrate was concentrated to a viscous oil that
was dissolved into CHCl3 and purified by column chromatog-
raphy, eluting with 95:5 hexanes:EtOAc. Product fractions
were pooled, concentrated, and triturated in isooctane to give
1
1.96 g (63%) of 13 as a white solid: mp 87-88 °C; H NMR
(CDCl3) δ 8.11 (dd, J ) 2.5, 6.5 Hz, 1 H, H-4), 7.40-7.19 (m,
3H, ArH), 3.84 (s, 3H, NCH3), 1.67 (s, 9H, C(CH3)3). Anal.
(C14H16BrNO2) C, H, N, Br.
Meth od E. 2,2′-Diselen obis[1-m eth yl-1H-in d ole-3-ca r -
boxylic a cid , ter t-bu tyl ester ] (21). An ice-cold suspension
of 119 mg (1.5 mmol) of elemental Se in 2 mL of THF under
N2 was treated dropwise with 1.1 mL of CH3Li‚LiBr complex
(1.5 M in ether). The flask was opened to the air, and with a
brisk stream of N2, the resultant white suspension was
warmed to ca. 85 °C to distill off the ether and most of the
THF. The residual semisolid was cooled in an ice bath and
diluted with 1.5 mL of DMA followed by 155 mg (0.5 mmol) of
2-bromo-1-methylindole-3-carboxylic acid, tert-butyl ester (13).
The resultant solution was stirred at room temperature for
24 h, cooled to 0 °C, and then treated with 2 mL of dilute
HOAc. The mixture was diluted with H2O and extracted with
CHCl3 (3 × 10 mL). The combined extracts were washed with
H2O (4×), dried, and concentrated to leave a golden solid. The
solid was suspended in 2.3 mL of 2:1 v/v HOAc:H2O, and the
suspension was treated with 154 mg of NaBO3‚4H2O and then
stirred at 25 °C for 30 min. The solids were collected by
filtration, washed with H2O, and dried to leave 119 mg of pure
21: 1H NMR (CDCl3) δ 8.13 (dd, J ) 0.7, 7.9 Hz, 2 × 1 H,
H-4), 7.31-7.19 (m, 2 × 3H, ArH), 3.63 (s, 2 × 3H, NCH3),
1.44 (s, 2 × 9H, C(CH3)3). Anal. (C28H32N2O4Se2‚0.2 H2O) C,
H, N.
Met h od B. 2-Ch lor o-1-m et h ylin d ole-3-ca r b oxa ld e-
h yd e (8). A solution of 10 g (55.7 mmol) of 2-chloroindole-3-
carboxaldehyde (9) in 100 mL of dry acetone was treated with
14.61 g (111 mmol) of finely powdered anhydrous K2CO3, and
the resulting suspension was stirred vigorously for 10 min
under an N2 atmosphere. Iodomethane (6.95 mL; 111 mmol)
was then added, and stirring was continued for 16 h. The
suspension was filtered and the filter cake washed with
acetone (3 × 30 mL). The filtrate was concentrated to a solid
2,2′-Dise le n ob is[N ,1-d im e t h yl-1H -in d ole -3-ca r b ox-
a m id e] (23). An ice-cold solution of 3 mmol of CH3SeLi in 2
mL of DMA, made up as described above, was treated with
267 mg (1.0 mmol) of 2-bromo-1-methylindole-3-(N-methyl-

+
+
422 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Showalter et al.
carboxamide) (15). The resultant solution was stirred at room
temperature for 3.5 h, cooled to 0 °C and then treated with
5% aqueous HCl. The mixture was extracted with CH2Cl2 (2
× 10 mL), and the combined extracts were washed with H2O
(2×) and then concentrated in vacuo to leave an oil that was
dissolved in MeOH. The solution was ice-cooled and treated
with 113 µL of 30% aqueous H2O2. After being stirred for 10
min, the resultant suspension was filtered, and the solids were
washed with 2-propanol and dried to leave 183 mg of pure 23
as a yellow solid: 1H NMR (CDCl3 + (CD3)2SO) δ 7.97 (d, J )
7.9 Hz, 2 × 1H, H-4), 7.39-7.18 (m, 2 × 3H, ArH), 6.84 (s, 2
× 1H, NHCH3), 3.85 (s, 2 × 3H, indole NCH3), 2.12 (d, J ) 4.5
Hz, 2 × 3H, NHCH3); FABMS m/ z (relative intensity) 535
(MH+, 10). Anal. (C22H22N4O2Se2‚0.9H2O) C, H, N.
2,2′-Diselen ob is[N-[2-(d iet h yla m in o)et h yl]-1-m et h yl-
1H-in d ole-3-ca r boxa m id e] (24): 1H NMR ((CD3)2SO) δ 10.13
(s, 2 × 1H, +NH(CH2CH3)2), 8.14-8.11 (m, 2 × 1H, CONH),
7.89 (d, J ) 8.2 Hz, 2 × 1H, H-4), 7.57 (d, J ) 8.4 Hz, 2 × 1H,
H-7), 7.34-7.17 (m, 2 × 2H, ArH), 3.63 (s, 2 × 3H, NCH3),
3.17-3.14 (m, 2 × 2H, CONHCH2), 3.06-3.00 (m, 2 × 4H,
N(CH2CH3)2), 2.86 (t, J ) 6.5 Hz, 2 × 2H, CONHCH2CH2),
1.16 (t, J ) 7.2 Hz, 2 × 6H, N(CH2CH3)2). Anal. (C32H44N6O2-
Se2‚2.0HCl‚1.7H2O) C, H, N, Cl-.
2,2′-Diselen ob is[N-m et h yl-1H -in d ole-3-ca r b oxa m id e]
(25): 1H NMR ((CD3)2SO) δ 12.36 (s, 2 × 1H, indole NH), 7.83
(d, J ) 7.7 Hz, 2 × 1H, H-4), 7.79 (d, J ) 4.1, 2 × 1H, NHCH3),
7.48 (d, J ) 7.7 Hz, 2 × 1H, H-7), 7.16-7.07 (m, 2 × 2H, ArH),
2.90 (d, J ) 4.1 Hz, 2 × 3H, NHCH3). Anal. (C20H18N4O2-
Se2‚0.9H2O) C, H, N.
2,2′-Diselen obis[N-[2-(d ieth yla m in o)eth yl]-1H-in d ole-
3-ca r boxa m id e] (26): 1H NMR ((CD3)2SO) δ 12.75 (s, 2 ×
1H, indole NH), 10.08 (s, 2 × 1H, +NH(CH2CH3)2), 8.09 (t, J
) 5.7 Hz, 2 × 1H, CONH), 7.93 (d, J ) 8.9 Hz, 2 × 1H, H-4),
7.51 (d, J ) 6.8 Hz, 2 × 1H, H-7), 7.19-7.12 (m, 2 × 2H, ArH),
3.78-3.73 (m, 2 × 2H, CONHCH2), 3.32 (t, J ) 6.5 Hz, 2 ×
2H, CONHCH2CH2), 3.29-3.20 (m, 2 × 4H, N(CH2CH3)2), 1.26
(t, J ) 7.2 Hz, 2 × 6H, N(CH2CH3)2). Anal. (C30H40N6O2-
Se2‚2.0HCl‚1.0H2O) C, H, N, Cl-.
2,2′-Diselen ob is[1-[2-(d iet h yla m in o)et h yl]-N-m et h yl-
1H-in d ole-3-ca r boxa m id e] (27): 1H NMR (CDCl3) δ 8.17 (d,
J ) 8.2 Hz, 2 × 1H, H-4), 7.35-7.20 (m, 2 × 3H, ArH), 6.61
(d, J ) 4.6 Hz, 2 × 1H, CONHCH3), 4.28 (t, J ) 7.0 Hz, 2 ×
2H, 1-NCH2), 2.62 (t, J ) 7.0 Hz, 2 × 2H, 1-NHCH2CH2), 2.45
(q, J ) 7.0 Hz, 2 × 4H, N(CH2CH3)2), 2.39 (d, J ) 4.6 Hz, 2 ×
3H, CONHCH3), 0.90 (t, J ) 7.1 Hz, 2 × 6H, N(CH2CH3)2).
Anal. (C32H44N6O2Se2‚0.5H2O) C, H, N.
Meth od F . 2,2′-Diselen obis[1-m eth yl-1H-in d ole-3-ca r -
boxylic a cid ] (22). To an ice-cold solution of 4 mL of TFA
under N2 was added 420 mg (0.68 mmol) of 2,2′-diselenobis-
[1-methyl-1H-indole-3-carboxylic acid, tert-butyl ester] (21).
The suspension was maintained at 0 °C for 3 h and then
poured into ice H2O. The solids were collected by filtration,
washed well with H2O, and then suspended into 80 mL of 10%
aqueous NH4OH. The suspension was filtered, and filtrate
was adjusted to pH 3 with 6 N aqueous HCl. The precipitated
solids were collected by filtration, washed with H2O, and dried
to leave 268 mg of pure 22 as an orange solid: 1H NMR ((CD3)2-
SO) δ 12.35 (s, 2 × 1H, CO2H), 8.04 (d, J ) 7.9 Hz, 2 × 1H,
H-4), 7.56 (d, J ) 8.4 Hz, 2 × 1H, H-7), 7.31-7.20 (m, 2 × 2H,
ArH), 3.63 (s, 2 × 3H, NCH3). Anal. (C20H16N2O4Se2‚0.1H2O)
C, H, N.
3H, indole NCH3), 3.05 (s, 3H, NHCH3). Anal. (C11H11N2OBr)
C, H, N, Br.
2-Ch lor o-1-m eth ylin d ole-3-[N-(2-(d ieth yla m in o)eth yl)-
ca r boxa m id e] (16). Similar reaction of 2-chloro-1-methyl-
indole-3-carboxylic acid (11)31 with SOCl2 and further reaction
with 3 equiv of N,N-diethylethylenediamine in CH2Cl2 at 0
°C gave 16 as an oil in 68% yield, further characterized as the
hydrochloride salt: mp 170-171 °C; 1H NMR ((CD3)2SO) δ
10.33 (br s, exchanges with D2O, 1H, HN+(CH2CH3)2), 8.07 (t,
J ) 5.7 Hz, exchanges with D2O, 1H, CONH), 7.95 (d, J ) 7.9
Hz, 1H, H-4), 7.57 (d, J ) 8.2 Hz, 1H, H-7), 7.28 (t, J ) 7 Hz,
1H, ArH), 7.21 (t, J ) 7 Hz, 1H, ArH), 3.80 (s, 3H, indole
NCH3), 3.68 (q, J ) 6.3 Hz, 2H, CONHCH2), 3.27-3.15 (m,
6H, CH2N(CH2CH3)2), 1.25 (t, J ) 7.2 Hz, 6H, N(CH2CH3)2.
Anal. (C16H22ClN3O‚HCl) C, H, N.
2-C h lo r o in d o le -3-[N -(2-(d ie t h y la m in o )e t h y l)c a r -
boxa m id e] (17). Similar reaction of 2-chloroindole-3-car-
boxylic acid (12) with SOCl2 and further reaction with 3 equiv
of N,N-diethylethylenediamine in ether gave 17 in 38% yield:
1
mp 99-108 °C; H NMR (CDCl3) δ 11.50 (s, 1H, indole NH),
8.19 (d, J ) 6.5 Hz, 1H, H-4), 7.33 (d, J ) 8.4 Hz, 1H, H-7),
7.21-7.15 (m, 3H, ArH and CONH), 3.54 (q, J ) 5.3 Hz, 2H,
CONHCH2), 2.69 (t, J ) 6.0 Hz, 2H, CONHCH2CH2), 2.59 (q,
J
) 7.2 Hz, 4H, N(CH2CH3)2, 1.05 (t, J ) 7.2 Hz, 6H,
N(CH2CH3)2). The product was further characterized as the
HCl salt: mp 164-165 °C. Anal. (C15H20ClN3O‚HCl) C, H,
N.
2-Ch lor oin d ole-3-(N-m eth ylca r boxa m id e) (18). Similar
reaction of 2-chloroindole-3-carboxylic acid (12) with SOCl2 and
further reaction with
a saturated solution of anhydrous
methylamine in THF at 0 °C gave 18 in 51% yield: mp 234-
1
236 °C; H NMR (CD3)2SO) δ 7.85 (d, J ) 7.5 Hz, 1H, H-4),
7.59 (br d, J ) 4.3 Hz, exchanges with D2O, 1H, CONH), 7.34
(d, J ) 8.7 Hz, 1H, H-7), 7.22-7.04 (m, 2H, ArH), 2.80 (d, J )
4.6 Hz, collapses to s with D2O wash, 1H, CONHCH3). Anal.
(C10H9N2OCl) C, H, N.
Meth od H. 1-[2-(Dieth yla m in o)eth yl]-N-m eth yl-1H-
in d ole-3-ca r boxa m id e (19). A mixture of 2.09 g (10 mmol)
of 2-chloroindole-3-(N-methylcarboxamide) (18), 1.72 g (10
mmol) of 2-(N,N-diethylamino)ethyl chloride hydrochloride, 7.5
g (23 mmol) of anhydrous Cs2CO3, 3 g of activated 3A
molecular sieves, and 20 mL of acetone was stirred under N2
at 25 °C for 16 h. The mixture was filtered through Celite,
and the filtrate was concentrated to a solid that was parti-
tioned between CHCl3 and H2O. The organic phase was dried
and concentrated to a residue that was crystallized from
EtOAc:hexanes (5:8). The solid was collected and dried to
leave 1.43 g (46%) of 19: mp 103-104 °C; 1H NMR (CDCl3) δ
8.24 (d, J ) 8.0 Hz, 1H, H-4), 7.33-7.21 (m, 3H, ArH), 6.35 (s,
1H, CONHCH3), 4.27 (t, J ) 7.6 Hz, 2H, 1-NCH2), 3.06 (d, J
) 4.8 Hz, 3H, CONHCH3), 2.73 (t, J ) 7.5 Hz, 2H,
1-NHCH2CH2), 2.62-2.55 (m, 4H, N(CH2CH3)2), 1.02 (t, J )
7.0 Hz, 6H, N(CH2CH3)2). Anal. (C16H22N3OCl) C, H, N.
Meth od I. (R)-N-(P h en ylm eth yl)-r-[(tr iflu or oa cetyl)-
a m in o]-1H-in d ole-3-p r op a n a m id e (29a ). An ice-cold solu-
tion of 15 g (50 mmol) of the N-trifluoroacetamide of (R)-
tryptophan (28a ),37 synthesized by the general literature
procedure,36 in 50 mL of THF under N2 was treated sequen-
tially with 7.1 g (52.5 mmol) of 1-hydroxybenzotriazole and
then 10.83 g (52.5 mmol) of 1,3-dicyclohexylcarbodiimide.
After 15 min, the solution was treated with 5.74 mL (52.6
mmol) of benzylamine. The solution was maintained at 0-5
°C for 1 h and then stirred at room temperature overnight.
The mixture was filtered, and the collected solid was washed
with EtOAc. The filtrate was concentrated to an oil that was
dissolved in 250 mL of EtOAc. The solution was washed
sequentially with 250 mL portions of 10% aqueous HOAc, H2O,
5% aqueous NaHCO3, H2O, and brine, then dried, and con-
centrated to a solid. Crystallization from 170 mL of 65:35
2-propanol:petroleum ether afforded 12.81 g (66%) of 29a as
Meth od G. 2-Br om o-1-m eth ylin d ole-3-(N-m eth ylca r -
boxa m id e) (15). A 25 °C suspension of 2.79 g (11 mmol) of
2-bromo-1-methylindole-3-carboxylic acid (10) in 13 mL of 1,2-
dichloroethane was treated dropwise with 2.41 mL (33 mmol)
of SOCl2. The mixture was heated at 75 °C for 2 h. The
solution was concentrated to a solid that was coevaporated
once with CH2Cl2. The solid was ice cooled and treated rapidly
with 26 mL of 40% aqueous methylamine. The bath was
removed and the suspension was stirred at 25 °C for 2 h. The
solids were collected by filtration, washed well with H2O, and
dried over P2O5 to leave 2.2 g (75%) of product, mp 154-157
°C. Recrystallization from MeOH provided 1.91 g of 15 as a
an off-white solid: mp 186-188 °C; [R]23 -5.6° (c ) 1.0,
D
MeOH). The 1H NMR was essentially identical to that
reported previously for the racemate.26 Anal. (C20H18F3N3O2)
C, H, N.
1
beige solid in three crops: mp 159-160 °C; H NMR (CDCl3)
(R)-[1-(1H-In d ol-3-ylm eth yl)-2-oxo-2-[(p h en ylm eth yl)-
a m in o]et h yl]ca r b a m ic Acid , 1,1-Dim et h ylet h yl E st er
δ 8.25-8.14 (m, 1H, H-4), 7.31-7.18 (m, 3H, ArH), 3.78 (s,

+
+
Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 423
(29b). To a -8 °C mechanically stirred solution of 60 g (197
mmol) of the N-BOC derivative of (R)-tryptophan (28b) in 200
mL of THF under N2 was added sequentially 28 g (207 mmol)
of HOBT and 42.7 g (207 mmol) of DCC. After 0.5 h, the
solution was treated with 22.6 mL (207 mmol) of benzylamine,
and then the mixture was allowed to warm to 25 °C. After 3
d of stirring, the mixture was filtered and the collected solid
was washed with EtOAc (3 × 50 mL). The filtrate was
concentrated to an oil that was dissolved in 250 mL of EtOAc.
The organic phase was washed sequentially with 300 mL
portions of 5% aqueous HOAc (2×), H2O, 5% aqueous NaHCO3,
H2O, and brine, dried, and concentrated to a solid. Crystal-
lization from 350 mL of 6:1 EtOAc:hexanes afforded 69.2 g
(89%) of 29b as a white solid: mp 136-139 °C (lit.41 mp 142.5-
143.5 °C); [R]23 +0.02° (c ) 1.0, MeOH) [lit.41 [R]23 +8° (c )
[R -(R *,R *)]-[D i s e le n o b i s [1H -i n d o l-2,3-y li d e n e [1-
[[(p h en ylm et h yl)a m in o]ca r b on yl]-2,1-et h a n ed iyl]]]b is-
ca r ba m ic Acid , Bis(1,1-d im eth yleth yl) Ester (32b). To a
-5 °C solution of 33 g (84 mmol) of (R)-[1-(1H-indol-3-
ylmethyl)-2-oxo-2-[(phenylmethyl)amino]ethyl]carbamic acid,
1,1-dimethylethyl ester (29b) in 170 mL of THF under N2 was
added rapidly 3.52 mL (42 mmol) of Se2Cl2. The resultant deep
red suspension was stirred at -5 °C for 2 h, and then quenched
with 160 mL of 1 N aqueous NaOH prechilled to -5 °C. The
mixture was filtered through a pad of SiO2, and the filter pad
was washed with 1.4 L of EtOAc. The filtrate was diluted with
700 mL of H2O, the phases were separated, and the aqueous
phase was extracted again with EtOAc. The combined organic
phases were washed with brine, dried, and concentrated to
leave 32 g of an orange oil. The crude material was dissolved
in 120 mL of MeOH, and the solution under N2 was cooled in
an ice bath. NaBH4 (ca. 1.2 g) was added portionwise until
there was no more color discharge. The mixture was poured
immediately into a N2-purged separatory funnel containing
250 mL of ether. A small charge of additional NaBH4 was
added, and the solution was then diluted with 250 mL of H2O.
The funnel was shaken, the phases were separated, and the
aqueous phase was extracted again with a small portion of
ether. The aqueous phase was ice-cooled and carefully acidi-
fied to pH 3.7 with 6 N aqueous HCl. The resultant yellow
suspension was stirred at 0-5 °C for 2 h, and then the solid
was collected, washed well with H2O, and dried to leave a
yellow solid that was slightly impure by TLC (6:1 CH2Cl2:
EtOAc). The solid was dissolved in a minimum volume of hot
CHCl3, and the solution was purified by column chromatog-
raphy, eluting with ca. 500 mL of CH2Cl2 and then with 4:1
CH2Cl2:EtOAc until all 32b had been stripped off. The
combined product fractions were concentrated to leave 2.8 g
of a viscous orange oil. A small drop of this was scratched on
a watch glass with some tert-butylmethyl ether. Upon stand-
ing, crystallization commenced. The bulk oil was seeded and
then diluted with MeOH. The precipitated solid was collected,
washed with MeOH, and dried to leave 2.28 g of pure 32b as
a yellow-orange powder: 1H NMR (CDCl3) δ 9.39 (br s, 2 ×
1H, indole NH), 7.61 (d, J ) 6.3 Hz, 2 × 1H, ArH), 7.40-6.90
(m, 2 × 8H, ArH), 6.05 (br s, 2 × 1H, CHNH), 5.49 (d, J ) 8.4
Hz, 2 × 1H, NHCH2), 4.75 (br m, 2 × 1H, 3-CH2CH), 4.41 (br
m, 2 × 2H, NHCH2), 3.48 (d, J ) 12.3 Hz, 2 × 1H, 3-CH), 3.22
(d, J ) 8.7 Hz, 2 × 1H, 3-CH), 1.23 (s, 2 × 9H, C(CH3)3);
D
D
0.25, 1:1 DMF:MeOH)]; 1H NMR (CDCl3) δ 8.00 (br s, 1H, NH),
7.67 (d, J ) 8 Hz, 1H, ArH), 7.36 (d, J ) 8.2 Hz, 1H, ArH),
7.23-7.19 (m, 4H, ArH), 7.15 (t, J ) 7.0 Hz, 1H, ArH), 7.00-
6.95 (m, 3H, ArH), 5.96 (br s, 1H, CHNH), 5.16 (br s, 1H,
NHCH2), 4.46-4.40 (m, 1H, 3-CH2CH), 4.38-4.22 (m, 2H,
NHCH2), 3.35 (dd, J ) 14.6, 5.4 Hz,1H, 3-CH), 3.17 (dd, J )
14.4, 7.7 Hz, 1H, 3-CH), 1.41 (s, 9H, C(CH3)3). The mother
liquor was processed to leave 5.2 g (7%) of a second crop after
two crystallizations: mp 132-136 °C.
Met h od J . [R-(R*,R*)]-2,2′-d iselen ob is[N-(p h en yl-
m e t h yl)-r-[(t r iflu or oa ce t yl)a m in o]-1H -in d ole -3-p r o-
p a n a m id e] (32a ) a n d [R-(R*,R*)]-2,2′-selen obis[N-(p h en -
ylm et h yl)-r-[(t r iflu or oa cet yl)a m in o]-1H -in d ole-3-p r o-
p a n a m id e] (30a ). To an ice-cold solution of 10 g (25.7 mmol)
of (R)-N-(phenylmethyl)-R-[(trifluoroacetyl)amino]-1H-indole-
3-propanamide (29a ) in 70 mL of THF under N2 was added
dropwise 1.1 mL (13.1 mmol) of Se2Cl2. The resultant deep
red suspension was stirred at 0-5 °C for 4 h, and then
quenched with 300 mL of H2O. The solid was collected by
filtration, washed well with H2O, and air dried to leave 12 g
of impure product as an orange solid. A portion of this
material (10.7 g) was dissolved in 100 mL of MeOH, and the
solution under N2 was cooled in an ice bath. NaBH4 (ca. 1 g)
was added portionwise until there was no more color discharge.
The mixture was poured immediately into a N2-purged sepa-
ratory funnel containing 200 mL of ether. The mixture was
diluted with 200 mL of H2O, the mixture was shaken, and the
phases were separated. The aqueous layer was treated with
a small portion of additional NaBH4, extracted again with
ether, ice cooled, and then acidified to pH 1 with concentrated
HCl. The aqueous phase was extracted with EtOAc (2×), and
then the combined extracts were dried and filtered through a
pad of SiO2. The filtrate was concentrated to leave 5.91 g of
a foam that was dissolved in ca. 40 mL of absolute EtOH. The
solution was kept at 25 °C for several hours to initiate
crystallization and then stored at 5 °C. The solid was collected
by filtration, washed with 2-propanol, and dried to leave 4.23
g of pure 32a as a powdery yellow solid: 1H NMR (CDCl3) δ
8.68 (s, 2 × 1H, indole NH), 7.49 (d, J ) 7.9 Hz, 2 × 1H,
CHNH), 7.38 (d, J ) 7.9 Hz, 2 × 1H, ArH), 7.24-7.18 (m, 2 ×
5H, ArH), 7.11 (t, J ) 7.9 Hz, 2 × 1H, ArH), 6.79-6.76 (m, 2
× 2H, ArH), 5.25 (t, J ) 5.2 Hz, 2 × 1H, NHCH2), 4.33 (m, 2
× 1H, 3-CH2CH), 4.17 (dd, J ) 14.8, 5.8 Hz, 2 × 1H, NHCH2),
4.05 (dd, J ) 14.5, 4.9 Hz, 2 × 1H, NHCH2), 2.99 (dd, J )
14.0, 8.4 Hz, 2 × 1H, 3-CH), 2.88 (dd, J ) 14.0, 5.9 Hz, 2 ×
1H, 3-CH); FABMS m/ z (relative intensity) 937 (MH+, 52);
[R]23D -132° (c ) 0.94, MeOH). Anal. (C40H34N6O4F6Se2‚H2O)
C, H, N. Further processing of the filtrate by column chro-
matography eluting first with 100:0 and then 93:7 CH2Cl2:
EtOAc provided an additional 671 mg of 32a following
crystallization: mp 180-183 °C.
APCIMS m/ z (relative intensity) 944 (M+, 10); [R]23 -1.0° (c
D
) 1.05, MeOH). Anal. (C46H52N6O6Se2‚H2O) C, H, N. Proc-
essing of the mother liquor gave 97 mg of a second crop: mp
145-153 °C.
Met h od K. [R-(R*,R*)]-2,2′-Diselen ob is[r-a m in o-N-
(ph en ylm eth yl)-1H-in dole-3-pr opan am ide] (33). (a) Fr om
32a . A suspension of 233.5 mg (0.25 mmol) of [R-(R*,R*)]-
2,2′-diselenobis[N-(phenylmethyl)-R-[(trifluoroacetyl)amino]-
1H-indole-3-propanamide] (32a ) in 4.5 mL of dry absolute
EtOH was treated with 95 mg (2.5 mmol) of NaBH4. The
mixture was heated to reflux and then treated with 95 mg of
additional portions of NaBH4 after 15 min and 1.25 h. After
being heated for a total of 2 h, the mixture was cooled to 25
°C, diluted with MeOH, and poured into an ice-cold stirring
mixture of 5% aqueous HCl and EtOAc. The resultant mixture
was stirred vigorously for 15 min and filtered, the phases were
separated, and the aqueous layer was extracted once more with
EtOAc. The combined EtOAc phases were then back-extracted
with H2O (2×). The aqueous phases were combined and
diluted with an equal volume of EtOAc. While the pH was
carefully monitored, the stirred solution was treated carefully
with 10% aqueous NaOH to pH 9.5. The resultant yellow
precipitate was collected by filtration, washed well with H2O,
and dried to leave 90 mg of pure 33 as a yellow powder: 1H
NMR ((CD3)2SO) δ 11.62 (s, 2 × 1H, NH), 8.23 (t, J ) 5.1 Hz,
2 × 1H, NHCH2), 7.61 (d, J ) 8.0 Hz, 2 × 1H, ArH), 7.38 (d,
J ) 8.2 Hz, 2 × 1H, ArH), 7.35-6.95 (m, 2 × 7H, ArH), 4.20,
4.17 (2 × dd, J ) 15.2, 5.8 Hz, 4 × 1H, NHCH2), 3.46-3.40
(br m, 2 × 1H, Ar-CH2CH), 3.04-2.98 (br m, 2 × 1H, Ar-CH),
2.75-2.68 (br m, 2 × 1H, Ar-CH), 1.70 (br s, 2 × 2H, NH2);
77Se NMR (CD3OD) δ 383.8 (s); FABMS m/ z (relative inten-
The ether layer from above was concentrated to leave a solid
residue that was crystallized from EtOAc:petroleum ether to
give pure 30a : 1H NMR [(CD3)2SO; 60 °C] δ 10.84 (s, 2 × 1H,
NH), 9.48 (d, J ) 7.7 Hz, 2 × 1H,CHNHCOF3), 8.42 (t, J )
5.3 Hz, 2 × 1H, NHCH2), 7.61 (d, J ) 7.9 Hz, 2 × 1H, ArH),
7.24-7.15(m, 2 × 4H, ArH), 7.06-7.01 (m, 2 × 3H, ArH), 6.95
(t, J ) 7.2 Hz, 2 × 1H, ArH), 4.79-4.73 (m, 2 × 1H, 3-CH2CH),
4.28-4.14 (m, 2 × 1H, NHCH2Ph), 3.55 (dd, J ) 14.1, 7.5 Hz,
2 × 1H, dd, 3-CHCH2), 3.29 (dd, J ) 14.2, 7.5 Hz, 2 × 1H,
3-CHCH2). Anal. (C40H34N6O4F6Se‚0.3H2O) C, H, N.
sity) 743 [(M - 1)+, 6]; [R]23 +0.42° (c ) 1.0, DMF); HPLC
D

+
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424 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Showalter et al.
100% chemical purity and 90% chiral purity. Anal.
(C36H36N6O2Se2‚1.5H2O) C, H, N. Processing of the EtOAc
layer from the base treatment provided 15 mg of additional
33, mp 165-171 °C.
and extracted with EtOAc (3 × 500 mL), back-washing the
combined extracts with 500 mL of 0.1 M aqueous HCl. The
combined aqueous acid portions were neutralized with NaH-
CO3 and treated with 0.90 mL (9.3 mmol) of 35% H2O2,
followed by stirring at 20 °C for 10 min. The resulting solution
was adjusted to pH 10 with Na2CO3 and then extracted with
EtOAc (3 × 500 mL), back-washing the combined extracts with
H2O. Evaporation under reduced pressure gave a yellow oil
that was crystallized from CH2Cl2/petroleum ether to give 5.52
g (75%) of 4: mp 141 °C dec (lit.26 mp 147-150 °C); chiral
HPLC shows 4 to be a ca. 1:2:1 mixture of [S-(R*,R*)]:[R-
(R*,S*)]:[R-(R*,R*)] stereoisomers.
En zym e Assa ys. Epidermal growth factor receptor from
human A431 carcinoma cell-shed membrane vesicles and
protein from v-src baculovirus-infected insect cells were pre-
pared as previously described.27 A full-length cDNA for the
mouse PDGF-â receptor tyrosine kinase was obtained from J .
Escobedo (UCSF) and was used to overexpress the protein in
baculovirus infected insect cells as previously described.49,50
The assay was performed in 96-well plates using cell lysates.
The kinase activity of the enzyme lysate was assayed by
measuring the incorporation of 32P-labeled phosphate from
ATP into trichloroacetic acid precipitable random copolymer
of glutamate/tyrosine(4:1 ratio) in vitro.
Cell Cu ltu r e a n d Gr ow th In h ibition Assa ys. Studies
in Swiss 3T3 mouse fibroblasts and A431 human epidermoid
carcinoma were carried out as previously described.27
In cor p or a tion of Ra d ioa ctive P r ecu r sor s in to Ma cr o-
m olecu les. The incorporation of radiolabeled precursors into
whole-cell DNA, RNA, and protein was monitored by exposing
drug-treated (2 h) or control logarithmically growing cells to
[methyl-3H]thymidine, [5-3H]uridine or (S)-[4,5-3H]leucine (all
from ICN Biomedicals, Costa Mesa, CA) at a concentration of
1 µM and a specific activity of 1 µCi/nmol. After 2 h the cells
were trypsinized and injected into 2 mL of ice cold 15%
trichloroacetic acid (TCA). The resulting precipitate was
collected on glass fiber filters, washed with 2 mL aliquots of
ice-cold 15% TCA (5×), dried, and placed in scintillation vials
along with 10 mL of Ready Gel (Beckman, Irvine, CA).
Radioactivity was determined in a Beckman LS 6800 scintil-
lation counter.
F r om 32b. To a 0-5 °C suspension of 3.63 g (3.85 mmol)
of [R-(R*,R*)]-[diselenobis[1H-indol-2,3-ylidene[1-[[(phenyl-
methyl)amino]carbonyl]-2,1-ethanediyl]]]biscarbamic acid, bis-
(1,1-dimethylethyl) ester (32b) and 1.35 mL (11.6 mmol) of 2,6-
lutidine in 31 mL of CH2Cl2 was added dropwise 2.25 mL (11.6
mmol) of trimethylsilyl triflate. The solution was stirred under
N2 at 0 °C for 15 h and concentrated at 25 °C. The residue
was diluted with MeOH and the solution poured into ice-cold
5% aqueous HCl. The aqueous phase was washed with
CH2Cl2, then with cooling and rapid stirring layered with
EtOAc and carefully adjusted to pH 9.5 with aqueous NaOH.
The precipitate was collected, washed with 2-propanol, and
dried to give 2.24 g of a deep orange solid, mp 262-264 °C.
The solid was further purified by suspension in 50 mL of ice-
cold MeOH and then treatment with portionwise addition of
NaBH4 until the color had discharged. The resultant solution
was poured slowly into a rapidly stirring solution of 300 mL
of ice-cold 1:1 5% aqueous HCl:EtOAc. The precipitate was
collected, washed well with H2O, and dried to leave 1.74 g of
pure 33 as a deep yellow solid in two crops: HPLC 100%
chemical purity and 98.5% chiral purity.
Meth od L. (R,S)-N-(P h en ylm eth yl)-r-[(tr iflu or oa cet-
yl)a m in o]-1H-in d ole-3-p r op a n a m id e (35). To a stirred
solution of 50.0 g (0.245 mol) of (R,S)-tryptophan (previously
dried at 60 °C/1 d) and 45 mL (0.32 mol) of dry triethylamine
in 80 mL of dry DMF was added 37 mL (0.31 mol) of ethyl
trifluoroacetate (dried over P2O5 and freshly distilled). The
flask was sealed and purged with N2, and the mixture was
stirred at 20 °C for 2 d. Excess reagents were removed under
reduced pressure, and then the derived racemic trifluoro-
acetamide (28a ) was diluted with 100 mL of DMF and cooled
to 0 °C (ice bath) under N2. Diethylphosphoryl cyanide (DEPC)
(46 mL of 93%, 0.28 mol) was added to the stirred solution,
followed by 56 mL (0.51 mol) of benzylamine and then 84 mL
(0.60 mol) of triethylamine. The resulting mixture was allowed
to warm to 20 °C over several hours and then stirred at 20 °C
for 3 d. The solution was concentrated under reduced pressure
at 60 °C (oil pump), then diluted with 300 mL of H2O, and
extracted with EtOAc (3 × 100 mL), back-washing the
combined extracts with dilute aqueous NaHCO3 and H2O. The
combined extracts were dried and evaporated to leave 116 g
of a solid that was recrystallized first from EtOAc/CH2Cl2 and
then CH2Cl2/petroleum ether to give 76.5 g (80%) of pure 35:
mp 181-183 °C (lit.26 mp 181-183 °C). Chromatography of
the liquors, eluting with CH2Cl2/petroleum ether (1:2) and then
CH2Cl2, followed by crystallization from CH2Cl2/petroleum
ether, gave 2.9 g (3%) of additional 35.
Im m u n op r ecip ita tion a n d Wester n Blottin g. These
studies were conducted as previously described.26
In Vivo Ch em oth er a p y. Immune competent mice were
housed in wire cages on automatic flush racks. Immune
deficient mice were housed in microisolator cages within a
barrier facility. All animals were maintained on a 12 h light/
dark cycle and received food and H2O ad libitum. Animal
housing was in accord with AAALAC guidelines. All experi-
mental protocols involving animals were approved by the
institutional animal care and use committee.
2,2′-Dit h iob is[N-(p h en ylm et h yl)-r-[(t r iflu or oa cet yl)-
a m in o]-1H-in d ole-3-p r op a n a m id e] (36). A solution of 2.06
mL (25.8 mmol) of freshly purified48 S2Cl2 in 30 mL of dry THF
was added dropwise over 10 min to a stirred, ice-cold solution
of 20.0 g (51.4 mmol) of (R,S)-N-(phenylmethyl)-R-[(trifluoro-
acetyl)amino]-1H-indole-3-propanamide (35) in 150 mL of dry
THF. After 1 d at 20 °C, the mixture was diluted with 200
mL of H2O, neutralized with NaHCO3, and extracted with
CH2Cl2 (3 × 200 mL). Evaporation under reduced pressure
gave a yellow oil that was crystallized directly from EtOH and
EtOH/H2O to leave 17.4 g (76%) of pure 36 as a single pair of
diastereoisomers solvated with 1 equiv of EtOH: mp 176.5-
178.5 °C (lit.26 mp 160-164 °C). Chromatography of the
liquors, eluting with CH2Cl2 and then 5% EtOAc/CH2Cl2,
followed by crystallization (EtOH/H2O) gave 1.04 g (5%) of
additional 36.
B16 melanoma, colon carcinoma 26, and M5076 sarcoma
were maintained by serial transplantation of tumor fragments
(∼30 mg) every 2 weeks into naive mice. Passage mice for
B16 melanoma and M5076 sarcoma were C57BL/6, while
BALB/c mice were the host for colon carcinoma 26. F1 hybrid
mice were used for in vivo anticancer agent evaluations;
B6C3F1 for B16 and M5076; and CD2F1 for colon 26.
The A431 epidermoid carcinoma, C6 glioma, and MCF-7
breast carcinoma human tumor xenografts were maintained
by serial passage as described above in nude mice (NCr nu/
nu). Nude mice were also used as tumor hosts for anticancer
agent evaluations against these human tumor xenografts.
Estrogen-containing pellets (Innovative Research of America,
60 day release) were implanted into the left axilla region (side
opposite the tumor) of animals bearing the MCF-7 breast
xenograft to support its growth.
2,2′-Dith iobis[r-a m in o-N-(p h en ylm eth yl)-1H-in d ole-3-
p r op a n a m id e] (4). A suspension of 10.0 g (11.3 mmol) of 2,2′-
dithiobis[N-(phenylmethyl)-R-[(trifluoroacetyl)amino]-1H-indole-
3-propanamide] (36) in 200 mL of absolute EtOH was treated
cautiously with 4.5 g (0.12 mol) of NaBH4, and then the
mixture was stirred at 80 °C for 15 min. Further NaBH4 (4.5
g, 0.12 mol) was added, and the mixture was stirred at 80 °C
for another 3 h. The reaction was quenched with 1.0 L of H2O,
and the mixture was acidified with concentrated HCl to pH 1
In each experiment for anticancer evaluation, test mice
weighing 18-22 g were randomized and implanted intra-
peritoneally (ip) with counted numbers of tumor cells or
subcutaneously (sc) in the region of the right axilla with tumor
fragments on day 0. Animals were treated on the basis of
average cage weight on the days indicated in Table 5. The
vehicles for compounds 4 and 33 were distilled H2O and 10%
DMA in 50 mM lactic acid buffer, pH 4.0, respectively.
Compounds were administered as ip bolus injections or

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Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 425
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continuously by an sc implanted Alzet miniosmotic pump
(model 2001). General anesthesia, ketamine-xylazine, was
used for the implant of miniosmotic pumps. Local anesthesia,
lidocaine-epinephrine, was used for pump removal and re-
placement when a longer duration of therapy was desired. Host
body weight change data are reported as the difference
between mean group weights on the last and first days of
treatment for the B16 and colon 26 life span assays, or as the
maximum treatment related weight loss in the M5076, A431,
C6, and MCF-7 growth delay assays. Calculation of the
median day of death, % T/C (B15 and C26), T - C (M5076,
A431, MCF-7, C6), and net logs of tumor cell kill was
performed as described previously.51-54 A positive net cell kill
indicates that the tumor burden at the end of therapy was
less than at the beginning of therapy. A negative net log cell
kill indicates that the tumor grew during treatment.
Ack n ow led gm en t. We thank Dr. J . H. Hartenstein
for stimulating discussions and administrative support,
Prof. H. Rapoport for helpful suggestions, and numerous
colleagues for their technical assistance in various facets
of this research.
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