Tyrphostins. 5. Potent inhibitors of platelet-derived growth factor receptor tyrosine kinase: Structure-activity relationships in quinoxalines, quinolines, and indole tyrphostins

Article abstract of DOI:10.1021/jm950727b

A series of 3-indoleacrylonitrile tyrphostins, 2-chloro-3- phenylquinolines, and 3-arylquinoxalines were prepared and tested for inhibition of platelet-derived growth factor receptor tyrosine kinase (PDGF- RTK) activity. The potency of the inhibitors was found to be quinoxalines > quinolines > indoles. Lipophilic groups (methyl, methoxy) in the 6 and 7 positions and phenyl at the 3 position of quinoxalines and quinolines were essential for potency, in contrast to the hydrophilic catechol group in tyrphostins active against EGFR kinase inhibition at different sites. The inhibitors showed selectivity for PDGF and were not active against EGF receptor and HER-2/c-ErbB-2 receptor.

Full text of DOI:10.1021/jm950727b

2170
J . Med. Chem. 1996, 39, 2170-2177
Tyr p h ostin s. 5. P oten t In h ibitor s of P la telet-Der ived Gr ow th F a ctor Recep tor
Tyr osin e Kin a se: Str u ctu r e-Activity Rela tion sh ip s in Qu in oxa lin es, Qu in olin es,
a n d In d ole Tyr p h ostin s
Aviv Gazit,† Harald App,‡ Gerald McMahon,‡ J efferey Chen,‡ Alexander Levitzki,*,† and Frank D. Bohmer§
Department of Biological Chemistry, The Alexander Silverman Institute of Life Sciences, The Hebrew University of J erusalem,
Givat Ram, J erusalem 91904, Israel, SUGEN, Inc., 515 Galveston Drive, Redwood City, California 94063-4720, and
Max-Planck Society, Research Group “Growth Factor Signal Transduction” Medical Faculty, Friedrich-Schiller University,
Drackendorfer Str. I D 07747, Germany
Received October 3, 1995X
A series of 3-indoleacrylonitrile tyrphostins, 2-chloro-3-phenylquinolines, and 3-arylquinoxalines
were prepared and tested for inhibition of platelet-derived growth factor receptor tyrosine kinase
(PDGF-RTK) activity. The potency of the inhibitors was found to be quinoxalines > quinolines
> indoles. Lipophilic groups (methyl, methoxy) in the 6 and 7 positions and phenyl at the 3
position of quinoxalines and quinolines were essential for potency, in contrast to the hydrophilic
catechol group in tyrphostins active against EGFR kinase inhibition at different sites. The
inhibitors showed selectivity for PDGF and were not active against EGF receptor and HER-
2/c-ErbB-2 receptor.
In tr od u ction
of quinoxaline compounds and on structure-activity
relationships (SAR) which are important for inhibition
of the PDGF receptor tyrosine kinase.
Platelet-derived growth factor (PDGF) is a potent
mitogen and mitogen for mesenchymal cells (for a recent
review, see ref 1). The PDGF AA, AB, and BB isoforms
interact differentially with PDGF R- and â-receptors.
Both PDGF receptor types are closely related trans-
membrane tyrosine kinases whose activation by ligand
binding is essential for cellular signaling. PDGF and
its receptors have been shown to be involved in regula-
tion of vital aspects of embryogenesis.2,3 In the adult,
most of the proposed functions of PDGF relate to
different responses to injury as wound healing and
inflammation.4,5
Ch em istr y
The tyrphostins of Table 1 were prepared by Knoev-
enagel condensation of the 3- or 5-formylindole with
substituted acetonitriles. The quinolines II in Table 2
were prepared by Vilsmeyer reaction of amides I:23
PDGF-induced cell proliferation is also likely to be
involved in a number of pathophysiological conditions
such as atherosclerosis, restenosis,6 and fibrosis as well
as in certain cancers such as gliomas. In atherosclerosis
and restenosis, it is believed that PDGF, which is
secreted by the injured wall of the blood vessel, induces
the proliferation and migration of smooth muscle cells
from the media to form the neointima.6
For various cancers it has been shown that the tumor
cells express functional PDGF and PDGF receptors,
suggesting that this autocrine loop may contribute to
tumor growth.7 Indeed, interference with PDGF signal-
ing led, in various tumor cell lines expressing PDGF and
PDGFR, to growth inhibition and reversion of the
transformed phenotype.8,9 Also, PDGF expression level
has been found to correlate with malignancy in a
number of human tumors.10-15
We and others have reported on the biological effects
of PDGF tyrosine kinase blockers from the benzenema-
lononitrile family,16 indole-containing blockers,17 quino-
line blockers,18,19 quinoxaline blockers,20,21 and ami-
nopyrimidine blockers.22 In this article we report on
the properties of indole, quinoline, and a large number
Quinolines 18 and 19 were obtained by condensation
of 3,4-dimethoxyaniline with the substituted â-chloro-
acrolein (ref 24). Quinoxalines were synthesized mostly
by condensation of the appropriate 1,2-diamino aromat-
ics with the 1,2-keto aldehyde or diketo compounds.
Several quinoxalines were prepared by exchange reac-
tion of the bis-thiosemicarbazones25 and condensation
with 1,2-phenylenediamine.
Resu lts a n d SAR
Our goal was to find inhibitors which would be both
potent and selective as blockers of the PDGF receptor
(PDGFR). Tyrphostins such as compound 1 and its
analogs containing a catechol ring were moderately
potent against PDGFR but not selective (Table 1).
* Address correspondence to this author. Tel: 972-2-585 404. Fax:
972-2-512 958. E-mail: LEVITZKI@vms.huji.ac.il.
† The Hebrew University of J erusalem.
Tyrphostins 2-4, in which a 5-indole ring was intro-
duced instead of a catechol ring, were either of low
potency (2), not selective (4), or even more potent
‡ SUGEN, Inc.
§ Friedrich-Schiller University.
X Abstract published in Advance ACS Abstracts, April 1, 1996.
S0022-2623(95)00727-8 CCC: $12.00 © 1996 American Chemical Society

Potent Inhibitors of PDGFR Tyrosine Kinase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2171
Ta ble 2. Quinoline-Containing Tyrosine Kinase Inhibitors
Ta ble 1. Indole-Containing Tyrosine Kinase Inhibitors
R
_
a
Determined as in Table 1. IC50 values from a representative
experiment. IC50 values are reproducible within <10% from
experiment to experiment.
None of compounds 1-12 were active in intact Swiss
3T3 cells as blockers of PDGFR. We therefore tried to
design more rigid tyrphostins. Recently two classes of
rigidified tyrphostins were shown to exhibit reasonable
inhibitory activity against p56lck (III)26 and EGFR16 in
vitro against the isolated kinases. III and its analogs
a
Inhibition of PDGFR and EGFR autophosphorylation in Swiss
3T3 membranes was conducted as described in ref 20. The IC50
values were determined in three independent experiments. IC50
values are reproducible within <7-8% between replicate experi-
ments.
against EGFR (3) than toward the PDGFR. The 3-in-
dole analog (5) exhibited both better selectivity and
potency. We therefore prepared a series of 3-indole
derivatives, with substituents that showed good efficacy
against PDGFR. The best were compounds 7 and 8 (R
) thioamide and amide, respectively), but both exhibited
reduced efficacy and selectivity compared to tyrphostin
5.
are obtained through incorporation of the cyanovinyl
group of tyrphostins into a heteroaromatic ring. In IV16
liphophilic dimethoxy (or dichloro) replaces dihydroxy
groups, and the second aryl is connected directly and

2172 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Gazit et al.
not through the amide group to the cyanovinyl phar-
macophore. The vinyl element also occurs in “stilbenic”
tyrosine kinase inhibitors. We, therefore, decided to
combine all these elements in our design to yield
compounds of general structure V.
dimethoxy groups, while substitution at the 5 or 8
position in compounds 15 and 16 reduced potency.
We first prepared a series of 2-chloro-3-phenylquino-
lines (V, X ) CH), presented in Table 2. Only one
compound in this series, 17, with 6,7-dimethoxy sub-
stitution was both active and selective against PDGFR.
Movement of the methoxy groups to different positions,
exchanging chlorine in the 2 position for a bulky phenyl
group (compound 18), or exchanging the 3-phenyl for
an ester or an amide group (compounds 19 and 20) all
resulted in loss of activity. The 4-quinolinylidenema-
lononitrile 21 inhibits both PDGFR and EGFR with
identical potency (IC50 ) 10 µM). The removal of the
nitrogen ring to yield naphthalene analogs 22 and 23
abolishes activity.
Another group18 described quinolines VII in which the
most potent inhibitors (IC50 values in the low nanomolar
range) had either 3-pyridyl, 3-thienyl, styryl, or 4-sub-
stituted phenyl at the 3-quinoline position and 6,7-
dimethoxy groups. Here, too, 5,7-dimethyl derivatives
were potent, while the 6,7-dihydroxy group abolished
activity.
The next logical step was to try the related quinoxa-
line system (V, X ) N), with the additional nitrogen
added at the 4 position. A large series of quinoxalines
was synthesized and is described in Table 3.
Discu ssion
In a previous study20 we have demonstrated that two
quinoxalines, compounds 25 (AG 1295) and 26 (AG
1296), were very potent and selective inhibitors of
PDGFR but inactive against EGF receptor kinase. The
selective potencies of AG 1295 and AG1296 were
demonstrated both in a cell free system and in intact
Swiss 3T3 cells. In the present study we report in more
detail on the potency of 46 quinoxalines as inhibitors of
the PDGFR kinase in intact cells. These compounds are
inactive against EGFR in biochemical assays and cel-
lular assays (ref 20 and data not shown). We show the
evolution of the rationale moving from first-generation
cis-arylidenemalononitrile (BMN) tyrphostins to closed
structures, the quinolines, moving on to quinoxalines.
Indeed, BMN kinase inhibitors exemplified by com-
pound 127 show no selectivity and block PDGFR and
EGFR equipotently in a biochemical assay, using the
Swiss 3T3 membrane preparation.20 Indole-containing
tyrphostins show some improvement in some cases
(compounds 5, 7, and 8, Table 1), but most compounds
are either inactive or possess low potency (Table 1).
Closing the pharmacophore of the tyrphostin into a
second-ring heterocycle, thus eliminating the cis-cyano
group in the arylidenemalononitrile, generates quino-
lines which yielded a lead structure (compound 17,
Table 2) with good efficacy against the PDGFR and
promising selectivity vis-a-vis the EGFR. The insertion
of another nitrogen into the quinoline ring yielding the
Inspection of the quinoxalines showed that the most
potent inhibitors of PDGFR are 25, 26, 54, and 66 with
IC50 ) 0.4, 0.6, 0.8, and 0.9 µM, respectively. These
compounds possess lipophilic dimethoxy, dimethyl, or
benzo groups at the 6 and 7 positions and phenyl at the
3 position. Interestingly, the 6,7-dichloro analog 52 was
inactive. Substitution of the 6 and 7 positions with
hydrophilic dihydroxy or hydroxymethoxy groups (43
and 39, with IC50 ) 100 and 100 µM) abolished activity.
Attaching dihydroxy groups on the other side of the
molecule, on the 3-phenyl, did not reduce potency
(compound 54 with IC50 ) 0.6 µM compared to com-
pound 25 with IC50 ) 0.4 µM). Polar groups at the 6
position lowered the IC50 (27, 6-COOH, has IC50 > 50
µM; 44, 6-NO2, IC50 ) 25 µM compared to 24, IC50 ) 10
µM), while the large 6-benzoyl derivative yields some-
what better inhibition (45, IC50 ) 6 µM).
Phenyl at the 3 position (compound 24) is much better
than 2-furan (32), 3-indole (33), or 2′-thiophene (31) and
its derivatives, while the 3-CH3 analog is inactive.
Adding to 3-phenylquinoxaline almost any substitu-
ent at the 2 position (methyl, Ph, furan, pyridyl, CHO,
ethyl ester, dimethylamino, or phosphate) reduced or
abolished activity; however, the 2-carboxy analog 56
retained potency (IC50 ) 1.0 µM).
The quinoxalines were found to be inactive against
EGFR and Flk-1/KDR when tested in cell free system
or intact Swiss 3T3 cells (data not shown, but see ref
20). Several compounds (59-69) related to quinoxalines
were also prepared, but none had improved IC50 values
compared to its appropriate quinoxaline analog. Also
three dimeric compounds (67-69), designed in the hope
they could interact with both sites on two PDGF
receptors, were not active. Compound 66 which is a
close analog of 25 and 26 was found to be active (IC50
) 0.9 M).
Recently, quinoline inhibitors were described by other
groups18,19 One group (ref 19) tested a series of 3-(4-
pyridinyl)quinolines VI and found that the 5,7-dimethoxy
derivative is the most potent (IC50 ) 0.08 µM) while 6,7-
dihydroxy yields IC50 ) 36 µM. These findings contrast
with our results where the most potent inhibitor in the
quinoline series (17, IC50 ) 1.5 µM) possesses 6,7-
quinoxaline moiety generated several compounds (25,
<
26, 54, 56, and 66) with excellent efficacy (IC50 1.0
)
µM) toward PDGFR in intact cells. These compounds
were all ineffective against the EGFR at 50-100 µM
(not shown). Elimination of the nitrogen atom from the
quinoline (compounds 22 and 23, Table 2) yielded
naphthalene compounds with no kinase inhibitory
activity.
Only a portion of the other compounds were tested
on Swiss 3T3 membranes, whereas all the quinoxalines
(Table 3) were tested for PDGFR inhibition in intact
cells. Thus one cannot exclude the possibility that some
of the compounds may exert their inhibitory effects via
indirect mechanisms. An interesting point is that the
most potent quinoxaline PDGFR inhibitors, 25, 26, and
66, are lipophilic compounds, in contrast to the hydro-
philic BMN tyrphostins, which contain the catecholic

Potent Inhibitors of PDGFR Tyrosine Kinase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2173
Ta ble 3. Inhibition of PDGFR Kinase in Intact Cells by Quinoxalines
a
IC50 values in repeated experiments vary <10% from experiment to experiment. The most potent compounds, 25 and 26, were run
in parallel to each set of compounds. The IC50 values for compounds 25 and 26 in repeated experiments vary <6-8% of the value quoted
in the table.

2174 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Gazit et al.
acetate. Evaporation yielded an oily solid which was triturated
with CH2Cl2 and filtered to give 160 mg, 72% yield, of an
orange solid: mp 228 °C; NMR (acetone-d6) δ 8.77 (1H, s), 8.54
(1H, s, vinyl), 7.90-7.0 (7H, m); MS m/e 304 (M+, 29), 137 (69),
130 (10), 117 (100), 109 (16).
Com p ou n d 7. 3-Formylindole (435 mg, 3 mM), 350 mg,
3.5 mM, of cyanothioacetamide, and 20 mg of â-alanine in 20
mL of ethanol were refluxed for 6 h. Cooling and filtering gave
0.415 g, 61% yield, of a yellow-orange solid: mp 238 °C; NMR
(acetone-d6) δ 8.74 (1H, s), 8.69 (1H, s), 7.92 (1H, m), 7.60 (1H,
m), 7.30 (2H, m); MS m/e 227(M+, 80), 226 (100), 194 (30), 193
(25), 140 (M - CSNH2 - HCN, 25).
The yields and melting points for the analogous compounds
were 6, 81%, 228 °C; 8, 77%, 293 °C; 9, 87%, 194 °C; 10, 47%,
242 °C;28 12, 80%, 302 °C.
Com p ou n d 11. 3-Formylindole (0.29 g, 2 mM), 0.29 g, 2
mM, of 3-amino-4-cyano-5-(cyanomethyl)-1,2-pyrazole (ref 29),
and 20 mg of â-alanine in 30 mL of ethanol were refluxed 4 h.
Cooling and filtering gave 0.34 g, 62% yield, of a yellow solid:
mp 281 °C; NMR (acetone-d6) δ 8.52 (1H, s, vinyl), 8.42 (1H,
s, H2), 7.79 (1H, m), 7.57 (1H, m), 7.27 (2H, m), 6.17 (1H, br,
s, NH); MS m/e 274 (M+, 100), 219 (14), 91 (35).
Com p ou n d 17 (2-Ch lor o-3-p h en yl-6,7-d im eth oxyqu in -
olin e). a . To 3 g, 19.6 mM, of 3,4-dimethoxyaniline and 3.5
g, 22.6 mM, of phenylacetyl chloride in 60 mL of CH2Cl2 was
added 5 mL of pyridine. After 2 h stirring at room tempera-
ture, water was added and the organic phase was evaporated
to give a violet solid. Trituration with ethanol gave 3.0 g, 57%
yield, of a white solid: mp 147 °C; NMR (CDCl3) δ 7.40 (5H,
m), 7.0 (1H, m), 6.75 (2H, m), 3.86, 3.84 (6H, 2 s, OCH3), 3.73
(2H, s, CH2Ph).
b. To 3 mL of DMF and 16 mL of POCl3 was added 2.7 g,
10 mM, of amide from part a. The reaction was heated at 90
°C for 4 h, decanted on ice, filtered, and washed with water to
give 2.9 g, 96% yield, of a white solid: mp 234 °C; NMR
(CDCl3) δ 8.26 (1H, s, H4), 8.0 (1H, s, H8), 7.51 (5H, s, Ph),
7.15 (1H, s, H5), 4.13, 4.05 (6H, 2s, OCH3); MS m/e 301, 299
(M+, 33, 100), 286, 284 (M - CH3, 2, 6), 258, 256 (6, 18), 220
(9), 215, 213 (4, 13).
ring. Exchanging the dimethoxy groups at 6 and 7
positions of 26 to dihydroxy groups yields compound 43
with greatly reduced potency. Quinoxalines 54 and 56
possess the 5,6-dimethyl lipophilic region, which seems
to be the critical pharmacophore for potency and selec-
tivity of the PDGFR. These inhibitors possess also
additional polar groups on the other side of the molecule.
The quinoxaline pharmacophore seems, so far, to give
the most potent and most selective PDGFR inhibitors.
In this regard the quinoxaline compounds are superior
to AG 17 (RG 50872) and AG 370 (compound 2) we
reported previously. AG 17 is highly potent in its
antimitogenic activity against PDGF-stimulated growth
of rabbit vascular smooth muscle cells as well as a
PDGFR kinase blockers in intact cells. Interestingly,
the most potent and selective inhibitors of the EGFR
kinase are quinazoline derivatives, in which the 1,4-
diaza structure of the quinaxoline is changed to 1,3-
diazanaphthalene. These derivatives block the EGFR
kinase in intact cells with IC50 ∼ 1-400 nM and are
devoid of activity against the PDGFR kinase (Gazit et
al., unpublished; Ben Bassat et al., unpublished). This
difference in selectivity between quinoxalines and
quinazolines probably reflects the structural difference
between the kinase domains of PDGFR and EGFR.
It remains to be seen whether relatively nonselective
PTK inhibitors such as AG 17 (RG 50872) or selective
PDGFR blockers will be more effective as agents to
combat various disorders in which PDGF is involved.
Exp er im en ta l Section
Meth od s a n d Ma ter ia ls. General directions have ap-
peared previously.27 Microanalyses were performed by The
Hebrew University Analytical Services and are given in the
Supporting Information.
Syn th etic Meth od s. For each class of compounds, a
general procedure is given followed by yield and melting points
(mp) for the compounds in that class. NMR and MS data for
these compounds is given in the Supporting Information.
Starting materials for which no procedure or ref is given were
obtained from Aldrich.
The yields and melting points for the analogous quinolines
were 13, 20%, 120 °C; 14, 84%, 167 °C; 15, 85%, 103 °C; 16,
10%, 120 °C.
Com p ou n d 18 (2,3-Dip h en yl-6,7-d im eth oxyqu in olin e).
3,4-Dimethoxyaniline (0.76 g, 5 mM) and 1 g, 4 mM, of
R-phenyl-â-chlorocinnamaldehyde (1:1 Z:E mixture) (ref 24)
in 20 mL of ethanol were stirred overnight at room tempera-
ture. Filtering and washing with ethanol gave 6.8 g, 59%
yield, of a bright yellow solid: mp 210 °C; NMR (CDCl3) δ 8.58
(1H, br, s), 8.35 (1H, s), 7.50-7.19 (11H, m), 4.16, 4.08 (6H,
2s, OCH3).
Com p ou n d 19 (2-(p-Nitr op h en yl)-3-ca r bom eth oxy-6,7-
d im eth oxyqu in olin e). 3,4-Dimethoxyaniline (0.35 g, 2.3
mM) and 0.54 g, 2 mM, of (Z)-p-nitro-1-carbomethoxy-2-
chlorocinnamaldehyde (ref 24) in 30 mL of ethanol was stirred
overnight at room temperature. Filtering gave 0.47 g, 66%
yield, of a light-brown solid: mp 237 °C; NMR (acetone-d6) δ
8.75 (1H, s, H4), 8.36, 7.89 (4H, ABq, J AB ) 8.8 Hz), 7.55 (1H,
s, H5), 7.53 (1H, s, H8), 4.07, 4.05 (6H, 2s, OCH3), 3.97 (3H, s,
COOCH3).
Com p ou n d 20. To 0.86 g, 6 mM, of 3-aminoquinoline and
1.2 g, 7.7 mM, of phenylacetyl chloride in 40 mL of CH2Cl2
was added 1 mL of pyridine. After 2 h at room temperature,
water was added and the reaction mixture extracted with CH2-
Cl2. Evaporation and trituration with ethanol gave 0.7 g, 50%
yield, of a white solid: mp 147 °C; NMR (CDCl3) δ 8.71 (1H,
d, J ) 2.5 Hz), 8.57 (1H, d, J ) 2.5 Hz), 8.0 (1H, m), 7.80-
7.40 (8H, m), 3.83 (2H, s, CH2).
Com p ou n d 21 (4-Qu in ylid en em a lon on itr ile). 4-For-
mylquinoline (0.54 g, 2.9 mM) and 0.3 g, 4.5 mM, of malono-
nitrile in 20 mL of ethanol, under N2, were refluxed for 3 h.
Workup and extraction with CH2Cl2 gave 0.22 g, 37% yield, of
a yellow solid: mp 136 °C; NMR (CDCl3) δ 9.09 (1H, d, J )
4.6 Hz), 8.63 (1H, s,vinyl), 8.25 (1H, d, J ) 8.2 Hz), 7.93-7.68
(4H, m); MS m/e 205 (M+, 100), 204 (60), 179 (M - CN, 30),
178 (M - CN, 68), 152 (M - CN - HCN, 10), 151 (22).
Compounds 1 and 2 were described by us previously (ref
27). Compound 10 was prepared according to literature (ref
28).
Com p ou n d 3. 5-Formylindole (0.3 g, 2 mM) and 0.4 g, 2
mM, of R-methylbenzyl cyanoacetamide (compound 43 in ref
27) in 15 mL of ethanol and 2 drops of piperidine were refluxed
for 3 h. Water and HCl were added, and the reaction mixture
was extracted with EtAc to yield a viscous oil. Chromatog-
raphy on silica gel gave 0.42 g, 66% yield, of a pale-yellow
solid: mp 76 °C; NMR (CDCl3) δ 8.44 (1H, s), 8.25 (1H, s,
vinyl), 7.88 (1H, dd, J ) 8.6, 2.0 Hz), 7.68 (1H, d, J ) 2.0 Hz),
7.4-7.2 (5H, m), 6.66-6.50 (2H, m), 5.26 (1H, quin, J ) 7.1
Hz, CHCH3), 1.60 (3H, d, J ) 7.1 Hz, CH3); MS m/e 315 (M+,
24), 196 (M - NCH(CH3)C6H5, 22), 195 (25), 188 (21), 173 (24),
168 (13), 149 (57), 145 (100), 134 (92), 116 (53).
Com p ou n d 4. 5-Formylindole (130 mg, 1 mM), 180 mg, 1
mM, of 2-cyano-3′,4′-dihydroxyacetophenone (ref 27), and 30
mg of â-alanine were refluxed for 3 h; water was added and
the reaction mixture extracted with EtAc to give an oily solid.
Chromatography gave an orange solid: 86 mg, 28% yield; mp
185 °C; NMR (acetone-d6) δ 8.40 (1H, d, J ) 1.6 Hz, H4), 8.18
(1H, s, vinyl), 8.03 (1H, dd, J ) 8.6, 1.6 Hz, H6), 7.63 (1H, d,
J ) 8.6 Hz, H7), 7.54-7.40 (3H, m, H3, H2′5′), 7.0 (1H, d, J )
8.5 Hz, H6), 6.69 (1H, d, J ) 3.2 Hz, H2); MS m/e 304 (M+, 8),
177 (29), 137(C6H3(OH)2CO+, 100), 117 (12), 116 (indole+, 15),
109 (93).
Com p ou n d 5. 3-Formylindole (105 mg, 0.7 mM), 130 mg,
0.7 mM, of 2-cyano-3′,4′-dihydroxyacetophenone, and 20 mg
of â-alanine in 30 mL of ethanol were refluxed for 5 h. Water
was added and the reaction mixture extracted with ethyl

Potent Inhibitors of PDGFR Tyrosine Kinase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2175
The yields and melting points for compounds 22 and 23 were
22, 94%, 175 °C; 23, 83%, 194 °C.
(DMSO-d6) δ 7.85 (1H, s), 7.80 (1H, m), 7.53 (5H, m), 3.46 (2H,
d, J ) 22.0 Hz), 2.48 (6H, s).
Com p ou n d 25 (3-P h en yl-6,7-d im et h ylq u in oxa lin e).
Phenylglyoxal (2.4 g, 16 mM) and 2.2 g, 16 mM, of 4,5-
dimethyl-1,2-phenylenediamine in 20 mL of ethanol were
refluxed 1.5 h. Cooling and filtering gave 3.25 g, 88% yield,
of a white solid: mp 124 °C; NMR (CDCl3) δ 9.23 (1H, s, H2),
8.19 (1H, d, J ) 1.7 Hz), 8.15 (1H, d, J ) 1.7 Hz), 7.90 (2H, d,
J ) 9.0 Hz), 7.57 (3H, m), 2.52 (6H, s, CH3); MS m/e 234 (M+,
100), 219 (M - CH3, 11), 207 (M - HCN, 12), 165 (M - 2HCN
- CH3,2), 131 (M - Ph-CN, 3).
Com p ou n d 54 (3-(3′,4′-Dih yd r oxyp h en yl)-6,7-d im eth -
ylqu in oxa lin e). 4,5-Dimethyl-1,2-phenylenediamine (1.9 g,
14 mM) and 1.9 g, 10.2 mM, of R-chloro-3,4-dihydroxyac-
etophenone in 15 mL DMSO and 25 mL of ethanol were
refluxed for 2 h. Cooling and filtering gave 0.76 g, 18% yield,
of a deep-yellow solid: mp 278 °C; HCl salt; NMR (DMSO-d6)
δ 9.47 (1H, s, H2), 9.31 (1H, s, OH), 9.29 (1H, s, OH), 7.79 (2H,
s, H5,8), 7.75 (1H, d, J ) 2.2 Hz, H2′), 7.62 (1H, dd, J ) 8.7, 2.2
Hz, H6′), 6.90 (1H, d, J ) 8.7 Hz, H5′), 2.44 (6H, s, CH3).
Treatment of 0.5 g with Na2CO3 and extraction with ethyl
acetate gave 0.4 g of a yellow-green solid: mp 276 °C; NMR
(DMSO-d6) δ 9.30 (1H, s, H2), 7.81 (2H, s, H5,8), 7.75 (1H, d, J
) 2,2 Hz, H2′), 7.62 (1H, dd, J ) 8.3, 2.2 Hz, H6′), 6.90 (1H, d,
J ) 8.3 Hz, H5′), 2.44 (6H, s, CH3).
Com pou n d 55 (2-Car beth oxy-3-ph en yl-6,7-dim eth ylqu i-
n oxa lin e). a . To 10 g of benzoylethyl acetate in 20 mL of
acetic acid cooled with ice was added 3.7 g of NaNO2. After
10 min 5 mL of water was added. After 3 h 100 mL of water
was added and the solid filtered to give 7.7 g, 84% yield, of a
white solid: mp 110 °C; NMR (CDCl3) δ 7.90 (2H, m), 7.6-7.5
(3H, m), 4.30 (2H, q, J ) 7.4 Hz), 1.24 (3H, t, J ) 7.4 Hz).
b. Oxime (5 g, 22.6 mM) from part a and 3.1 g, 22.8 mM, of
4,5-dimethyl-1,2-phenylenediamine in 20 mL of ethanol and
5 mL of HCl were refluxed for 6 h. Workup (H2O, NaHCO3,
CH2Cl2), chromatography, and trituration with hexane gave
2 g, 29% yield, of a white solid: mp 100 °C; NMR (CDCl3) δ
7.96 (1H, s), 7.93 (1H, s), 7.7 (2H, m), 7.5 (3H, m), 4.30 (2H, q,
J ) 7.0 Hz), 2.53 (6H, s), 1.17 (3H, t, J ) 7.0 Hz).
Com p ou n d 56 (2-Ca r boxy-3-p h en yl-6,7-d im eth ylqu i-
n oxa lin e). Compound 55 (2.4 g) and 5 g of KOH in 20 mL of
ethanol and 20 mL of H2O were stirred for 20 h at room
temperature. Acidification with HCl, filtering, and washing
with water gave 2.1 g, 96% yield, of a light-yellow solid: mp
153 °C; NMR (acetone-d6) δ 7.92 (1H, s), 7.90 (1H, s), 7.85 (2H,
m), 7.50 (3H, m) 2.56 (6H, s).
Com p ou n d 57 (2-For m yl-3-p h en yl-6,7-dim eth ylqu in ox-
a lin e). Compound 46 (0.8 g) in 10 mL of DMSO was heated
for 40 min at 90 °C. Workup and chromatography gave from
first fractions 40 mg, 4% yield, of a white solid: mp 148 °C;
2-dibromomethyl derivative; NMR (CDCl3) δ 8.03 (1H, s), 7.90
(1H, s), 7.60 (5H, m), 6.96 (1H, s, CHBr2), 2.52 (6H, s).
Following fractions gave the 2-formylquinoxaline: white
solid; mp 150 °C; 0.3 g, 47% yield; NMR (CDCl3) δ 11.30 (1H,
s, CHO), 8.05 (1H, s), 7.96 (1H, s), 7.67 (2H, m), 7.55 (3H, m),
2.55 (6H, s).
Com p ou n d 58 (2-((Dim eth yla m in o)m eth yl)-3-p h en yl-
6,7-d im eth ylqu in oxa lin e). Compound 46 (0.6 g, 1.8 mM)
and 5 mL of 25% dimethylamine (in water) in 20 mL of
acetonitrile were stirred for 14 h at room temperature.
Workup (H2O, CH2Cl2) and recrystallization with hexane gave
80 mg, 15% yield, of a white solid: mp 67 °C; NMR (CDCl3) δ
7.86 (4H, m), 7.50 (3H, m), 3.70 (2H, s, CH2N), 2.53 (6H, s,
CH3), 2.30 (6H, s, N(CH3)2).
Com pou n d 60 (1,2-Dih ydr o-2-keto-3-ph en yl-6,7-dim eth -
oxyqu in oxa lin e). Compound 26 (0.3 g, 1.13 mM) and 1.5
mL, 5 mM, of BBr3 in 30 mL of CH2Cl2 under argon were
stirred for 2 h at room temperature. Workup and chromatog-
raphy gave 30 mg of a yellow solid, 11% yield: mp >300 °C;
NMR (CDCl3) δ 9.37 (1H, s), 8.51 (1H, s), 7.93 (2H, m), 7.54
(3H, m), 4.24 (3H, s, OCH3), 4.12 (3H, s, OCH3); MS m/e 282
(M+, 11), 280 (36), 266 (M - O, 10), 167 (46), 149 (100), 133
(6), 125 (13), 119 (20).
Com p ou n d 61 (1,2-Dih yd r o-2-k et o-3-p h en yl-6,7-d i-
m eth ylqu in oxalin e). 4,5-Dimethyl-1,2-diaminobenzene (0.56
g, 4 mM) and 0.6 g, 4 mM, of benzoylformic acid in 15 mL of
ethanol were refluxed for 5 h. Cooling and filtering gave 0.8
g, 80% yield, of a yellow solid: mp 275 °C; NMR (CDCl3) δ
8.38 (2H, m), 7.70 (1H, s), 7.51 (3H, m), 7.06 (1H, s), 2.40 (3H,
s), 2.37 (3H, s); irradiation at 8.38 ppm gave a singlet at 7.51
ppm.
Com p ou n d 63 (1,2-Dih yd r o-2-k et o-3-p h en yl-6,7-d i-
m eth ylqu in oxa lin e 4-N-Oxid e). a . To 3.6 g, 22 mM, of
2-nitro-4,5-dimethylaniline and 3.8 g, 25 mM, of phenylacetyl
chloride in 50 mL of CH2Cl2 was added 5 mL of pyridine. After
Com p ou n d 26 (3-P h en yl-6,7-d im eth oxyqu in oxa lin e).
4,5-Dimethoxy-1,2-dinitrobenzene (2.3 g, 10.1 mM) (ref 32) was
hydrogenated over 0.2 g of Pd/C for 3 h, in 30 mL of acetic
acid and 30 mL of ethanol. To the filtered light-red solution
was added 1.6 g, 10.5 mM, of phenylglyoxal hydrate, and the
reaction mixture was refluxed for 2 h. Ammonia was added
to the cooled solution. Extraction with CH2Cl2 gave 2.1 g of a
light-brown solid. Recrystallization of 0.4 g from ethanol gave
0.16 g of a white solid. Chromatography of 1.7 g of the brown
solid gave 0.55 g of the white solid: yield 27%, mp 134 °C;
NMR (CDCl3) δ 9.13 (1H, s, H2), 8.16 (1H, d, J ) 1.6 Hz), 8.12
(1H, d, J ) 1.6 Hz), 7.60-7.40 (5H, m), 4.09 (6H, s, OCH3);
MS m/e 266 (M+, 100), 251 (M - CH3, 12), 223 (M - CH3
CO, 13), 196 (M - CH3 - CO - HCN, 5).
-
The yields and melting points for the quinoxalines were 24,
46%, 65 °C; 27, 47%, 278 °C; 28, 63%, 102 °C; 29, 62%, 121 °C
(29 is available form Aldrich); 30, 42%, 128 °C; 38, 51%, oil;
39, 40%, oil; 40, 31%, 114 °C; 41, 91%, 179 °C; 42, 78%, 192
°C; 44, 90%, 203 °C; 45, 69%, 133 °C; 46, 51%, 144 °C; 47,
62%, 98 °C; 52, 86%, 155 °C; 53, 70%, 192 °C; 59, 49%, 282
°C; 62, 88%, 278 °C, (62 is available from Aldrich); 64, 77%,
135 °C; 65, 67%, >300 °C (from 5-bromoisatin).
Com p ou n d 34 (3-(3-Th ien yl)qu in oxa lin e). 3-Thiophene
bis(thiosemicarbazone)25 (1.7 g, 6 mM) and 0.8 g, 7.4 mM, of
1,2-phenylenediamine in 15 mL of acetic acid were refluxed
for 6 h. Workup and chromatography gave 0.2 g of a light-
yellow solid: mp 80 °C; 15% yield; NMR (CDCl3) δ 9.25 (1H,
s, H2), 8.20-8.06 (3H, m), 7.92 (1H, dd, J ) 5.2, 1.2 Hz), 7.75
(2H, m), 7.52 (1H, m); MS m/e 212 (M+, 100), 185 (M - HCN,
71), 159 (9), 140 (19), 109 (38).
The yields and melting points for the quinoxalines obtained
from the ketoaldehydes bis-thiosemicarbazones were 31, 23%,
104 °C; 32, 40%, 94 °C; 33, 97%, 202 °C; 35, 32%, 70 °C; 36,
21%, 114 °C; 37, 3%, 146 °C.
Com p ou n d 43 (3-P h en yl-6,7-d ih yd r oxyq u in oxa lin e).
Compound 26 (0.15 g) in 5 mL of 48% HBr was refluxed for
23 h. Cooling and filtering gave a green-yellow solid, 95 mg,
53% yield, HBr salt by elemental analysis: mp 280 °C; NMR
(DMSO-d6) δ 9.25 (1H, s, H2), 8.24 (1H, d, J ) 1.9 Hz), 8.20
(1H, d, J ) 1.9 Hz), 7.50 (3H, m), 7.35 (2H, m).
Mother liquid was neutralized with NaHCO3. Extraction
with EtAc gave 20 mg, 15% yield, of an orange solid: mp 305
°C; free base; NMR (acetone-d6) δ 9.19 (1H, s, H2), 8.29 (1H,
d, J ) 1.5 Hz), 8.25 (1H, d, J ) 1.5 Hz), 7.6 (3H, m), 7.40 (2H,
m).
Com p ou n d 48 (2-Ch lor o-3-p h en yl-6,7-d im eth ylqu in ox-
a lin e). Compound 61 (0.3 g) and 3 mL POCl3 in 10 mL of
trichlorethylene were refluxed for 3 h. Workup and chroma-
tography gave 0.13 g, 40% yield, of a white solid: mp 128 °C;
NMR (CDCl3) δ 7.85 (1H, s), 7.79 (1H, s), 7.83 (2H, m), 7.50
(3H, m), 2.51 (3H, s), 2.49 (3H, s); MS m/e 268, 270 (M+, 76,
25), 233 (M - Cl, 100), 183 (30), 77 (50).
Com p ou n d 49 (2-((Dim eth oxyp h osp h in yl)m eth yl)-3-
p h en yl-6,7-d im eth ylqu in oxa lin e). Compound 46 (0.7 g, 2.1
mM) and 0.5 g, 5 mM, of trimethyl phosphite in 15 mL of
toluene were refluxed for 5 h. Evaporation and chromatog-
raphy gave a viscous oil: 0.36 g, 45% yield; NMR (CDCl3) δ
7.8-7.5 (7H, m), 3.72 (6H, d, J ) 11 Hz), 3.70 (2H, d, J ) 22.0
Hz), 2.51 (3H, s), 2.49 (3H, s).
Com p ou n d 50 (2-(P h osp h on om et h yl)-3-p h en yl-6,7-
d im eth ylqu in oxa lin e). Compound 49 (0.4 g, 1.1 mM) and
0.5 g of trimethyl bromosilane in 30 mL of CH2Cl2 were
refluxed for 4 h. Evaporation gave a viscous oil which
solidified on standing. Trituration with CH2Cl2 gave 0.33 g,
90% yield, of a greenish-yellow solid: mp 168 °C; NMR

2176 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11
Gazit et al.
2.5 h at room temperature, water was added. Evaporation of
the organic phase, trituration with ethanol, and filtering gave
4.7 g, 76% yield, of a yellow solid: mp 166 °C; NMR (CDCl3)
δ 9.34 (1H, br s, NH), 8.57 (1H, s, H3), 7.92 (1H, s, H6), 7.40
(5H, m), 3.80 (2H, s), 2.32 (3H, s), 2.25 (3H, s).
b. Material (1 g) from part a and 0.5 g of KOH in 40 mL of
ethanol were refluxed for 1 h. Workup (HCl, H2O, CH2Cl2)
gave an orange solid: 0.525 g, 56% yield; mp 103 °C; NMR
(CDCl3) δ 7.86 (1H, s, H5), 7.30 (5H, br s, Ph), 6.58 (1H, s, H8),
3.65 (1H, br s, NH), 2.22 (3H, s), 2.17 (3H, s).
Com p ou n d 66 (3-P h en yl-1,4-d ia za a n th r a cen e). 2,3-
Diaminonaphthalene (0.47 g, 3 mM) and 0.47 g of phenylgloxal
hydrate in 20 mL of ethanol were refluxed for 1.5 h. Cooling
and filtering gave 0.5 g, 65%, of a light-green solid: mp 163
°C; NMR (CDCl3) δ 9.38 (1H, s, H2), 8.71, 8.67 (2H, 2d, H5,10),
8.25, 8.10 (4H, AA′ BB′ m, H6-9), 7.58 (5H, m, Ph); MS m/e
256 (M+, 100), 229 (M - CN, 12), 126 (C10H6+, 71).
Com p ou n d 67. Compound 46 (0.33 g, 1 mM), 0.08 g, 0.74
mM, of 1,3-propanedithiol, and 0.1 g of KOH, in 25 mL of
ethanol were stirred for 24 h at room temperature. Workup
(H2O, CH2Cl2) and trituration with hexane gave 0.18 g, 60%
yield, of a white solid: mp 165 °C; NMR (CDCl3) δ 7.85 (2H,
s), 7.82 (2H, s), 7.70 (4H, m), 7.48 (6H, m), 3.96 (4H, s), 2.61
(4H, t, J ) 7.6 Hz); MS m/e 328 (M - 272, 8), 248 (38), 247
(100), 232 (7), 189 (15).
Su p p or tin g In for m a tion Ava ila ble: NMR and MS data
for tyrphostins (3 pages). Ordering information can be found
on any current masthead page.
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Dimers 68 and 69 were prepared similarly: 68, 41%, 216
°C; 69, 55%, 153.
Bioch em ica l Meth od s. Methods used are identical with
those described in our previous publication.20
Mem br a n e Au top h osp h or yla tion Assa ys. Membranes
were prepared from confluent cultures of Swiss 3T3 cells as
described.30 For measuring receptor autophosphorylation, 10
µg of membrane protein/assay was incubated for 20 min on
ice in the presence of 1.2 µg/mL EGF or 2 µg/mL PDGF, or
both, 50 mM Hepes (pH 7.5), and 3 mM MnCl2 (final concen-
trations) in a volume of 45 µL. As was previously demon-
strated (ref 20 Figure 1), no PDGFR autophosphorylation
occurs without PDGF. In order to test the effects of tyr-
phostins, these were added in a volume of 0.5 mL (in DMSO,
final concentration 0.5%) 15 min before addition of the growth
factors. Phosphorylation was initiated by addition of [γ-32P]-
ATP (5 mL, 3-5 µCi, final concentration 2 µM) and terminated
after 2 min by addition of 10 mL of a solution containing 6%
SDS, 30% â-mercaptoethanol, 40% glycerol, and 0.5 mg/mL
bromophenol blue. The samples were heated for 5 min at 95
°C and subjected to SDS-PAGE according to the method of
Laemmli31 using 10% acrylamide gels. The gels were stained,
dried, and subjected to autoradiographic analysis. Only a
portion of the tyrphostins examined were tested on Swiss 3T3
membranes, and the results are published in ref 20.
For quantification of radioactivity in electrophoresis gels, a
PhosphorImager (Molecular Dynamics, Fuji or BioRad) was
used according to the instructions of the manufacturers. To
obtain autoradiograms, objects were exposed to X-ray film (Fuji
RX or Kodak X-OMAT) with intensifying screens at -70 °C.
Assa y of R ecep t or Au t op h osp h or yla t ion in In t a ct
Cells. Confluent Swiss 3T3 cells in 24-well plates (Nunc) were
incubated for 20-24 h in serum free DMEM. Subsequently,
tyrphostins were added at concentrations ranging from 0 to
100 µM (final DMSO concentration 0.5%), and the incubation
was continued for 6-8 h. The cells were then stimulated with
100 ng/mL PDGF-BB for 5 min at room temperature. The
growth factor treatment was terminated by washing twice with
ice-cold PBS, and the cells were scraped off the wells in 60
mL of lysis buffer containing 20 mM Hepes, pH 7.4, 150 mM
NaCl, 1% Triton X-100, 10 mM sodium pyrophosphate, 50 mM
NaF, 2 mM sodium orthovanadate, 20 mM zinc acetate, 10
mM EDTA, 2 mM EGTA, 1 mM PMSF, and 5 µg/mL leupeptin.
The cell lysates were clarified by centrifugation (cooled mi-
crofuge, 17 000 rpm, 15 min) and analyzed by SDS-PAGE
(6.5% gels) and immunoblotting with anti-phosphotyrosine
antibodies (either PY 20 or ICN and subsequently a peroxi-
dase-coupled secondary antibody or RC20-peroxidase conju-
gate; Affiniti, Nottingham, U.K.). The blots were developed
with a chemiluminescence detection system (Western light,
Tropix or ECL, Amersham).

Potent Inhibitors of PDGFR Tyrosine Kinase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 11 2177
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previous work and prepared from thiosemicarbazone and the
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J M950727B