Structural studies on bioactive compounds. 32. Oxidation of tyrphostin protein tyrosine kinase inhibitors with hypervalent iodine reagents

Article abstract of DOI:10.1021/jm990947f

Hydroxylated styrenes (tyrphostins) undergo oxidation by hypervalent iodine oxidants such as [(diacetoxy)iodo]benzene (DAIB) to give a range of products depending on the structure of the phenolic substrate, the solvent, the oxidant stoichiometry, and the purification strategy. Conditions have been developed to modify the phenolic component of the tyrphostin without affecting the appended substituted-vinyl moiety. Novel products include: unstable 2-acyloxy-2-methoxy-4-(substituted-vinyl)cyclohexadienones and their rearrangement products 2-acyloxy-4-hydroxy-3-methoxy-1-(substituted- vinyl)benzenes; phenyliodoniophenolates and their rearrangement products iodophenoxytyrphostins; and 3,3'-dialkoxy-2,2'-dihydroxy-5,5'-di(substituted- vinyl)biphenyls. None of these oxidation products displayed enhanced activity in vitro in the NCI 60-cell line panel or in a panel of human breast cancer cell lines, compared to their tyrphostin precursors. The inhibitory activity of three representative tyrphostins (3e,n, 28) was not modulated by aerobic/anaerobic conditions in MCF-7 and MDA 468 cells and was independent of EGFR status in clones of ZR75B cells transfected with this receptor. Basal growth of MCF-7 cells was unaffected by co-administration of the growth factors EGF, TGF-α, IGF-I, and IGF-II, and the new agents did not inhibit EGFR and c-erbB2 autophosphorylation in cell lysates from MDA 468 or SkBr3 cells, respectively, suggesting that receptor tyrosine kinases are not targets for these compounds. Growth stimulation by the tyrphostin 3n in the ER⁺ breast cell lines MCF-7, T47D, and ZR75-1 was abolished by 1 μM tamoxifen, suggesting that this compound has estrogen agonist activity.

Full text of DOI:10.1021/jm990947f

1550
J . Med. Chem. 2000, 43, 1550-1562
Str u ctu r a l Stu d ies on Bioa ctive Com p ou n d s. 32.1 Oxid a tion of Tyr p h ostin
P r otein Tyr osin e Kin a se In h ibitor s w ith Hyp er va len t Iod in e Rea gen ts
Geoffrey Wells, Angela Seaton, and Malcolm F. G. Stevens*
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K.
Received November 5, 1999
Hydroxylated styrenes (tyrphostins) undergo oxidation by hypervalent iodine oxidants such
as [(diacetoxy)iodo]benzene (DAIB) to give a range of products depending on the structure of
the phenolic substrate, the solvent, the oxidant stoichiometry, and the purification strategy.
Conditions have been developed to modify the phenolic component of the tyrphostin without
affecting the appended substituted-vinyl moiety. Novel products include: unstable 2-acyloxy-
2-methoxy-4-(substituted-vinyl)cyclohexadienones and their rearrangement products 2-acyloxy-
4-hydroxy-3-methoxy-1-(substituted-vinyl)benzenes; phenyliodoniophenolates and their rear-
rangement products iodophenoxytyrphostins; and 3,3′-dialkoxy-2,2′-dihydroxy-5,5′-di(substituted-
vinyl)biphenyls. None of these oxidation products displayed enhanced activity in vitro in the
NCI 60-cell line panel or in a panel of human breast cancer cell lines, compared to their
tyrphostin precursors. The inhibitory activity of three representative tyrphostins (3e,n , 28)
was not modulated by aerobic/anaerobic conditions in MCF-7 and MDA 468 cells and was
independent of EGFR status in clones of ZR75B cells transfected with this receptor. Basal
growth of MCF-7 cells was unaffected by co-administration of the growth factors EGF, TGF-R,
IGF-I, and IGF-II, and the new agents did not inhibit EGFR and c-erbB2 autophosphorylation
in cell lysates from MDA 468 or SkBr3 cells, respectively, suggesting that receptor tyrosine
kinases are not targets for these compounds. Growth stimulation by the tyrphostin 3n in the
ER+ breast cell lines MCF-7, T47D, and ZR75-1 was abolished by 1 µM tamoxifen, suggesting
that this compound has estrogen agonist activity.
In tr od u ction
the functions of receptor PTKs: these include the
development and marketing of an antibody (herceptin)
raised against the extracellular domain of EGFR and
the clinical evaluation of a family of orally active
4-anilinoquinazolines which compete with ATP and
inhibit EGFR autophosphorylation with IC50 values in
the nanomolar range.8
Protein tyrosine kinases (PTKs) occupy critical loci
in cell signaling pathways. They catalyze the transfer
of the γ-phosphate group of ATP to the phenolic group
of a specific tyrosine residue in the substrate.2 Phos-
photyrosine-containing proteins then trigger a cascade
of signaling events downstream of the kinase. Although
less than 0.01% of intracellular phosphorylation in-
volves tyrosine residues,3 the involvement of PTKs and
their regulatory phosphatases in human cancer is
demonstrated by the fact that a large proportion of viral
oncogene products have been identified as constitutively
active PTKs, which often have human proto-oncogenic
counterparts.4
Certain peptidic growth factors, e.g. epidermal growth
factor (EGF), exert their stimulatory effects via tyrosine
kinase enzymes, with aberrant expression of the growth
factor or kinase being evident in many cancers. The
epidermal growth factor receptor (EGFR) is overex-
pressed in a range of tumor types, notably breast,
prostatic, hepatic, renal, bladder, esophageal, laryngeal,
stomach, lung, and ovarian cancers. For example, 57%
of primary breast tumor biopsies were shown to be
EGFR +ve; moreover, the receptor is overexpressed in
estrogen receptor (ER) -ve tumors5 which respond
poorly to hormonal therapy.6 Also, in ovarian cancer the
presence of EGFR mRNA correlates with poor prognosis
and reduced survival.7 Not surprisingly, there has been
great interest in therapeutic strategies to antagonize
After the identification of the flavone quercetin (1)
(Chart 1) as a PTK inhibitor,9 a diverse array of
structures ranging from complex natural product mac-
rocyclic quinones such as herbimycin A and geldana-
mycin to low molecular weight heterocycles to simple
phenols has been studied.3,10 In the phenolic class the
lead structure erbstatin (2) inhibits EGFR autophos-
phorylation in membrane fractions and intact cells by
partially competing with ATP and the substrate at the
enzyme active site.11 Structurally related hydroxylated
styrenes of general structure 3 (the ‘tyrphostins’ where
group R is often a carboxylic acid-derived moiety and
R1, R2, and R3 comprise at least one phenolic group)
developed by Gazit, Levitzky, and co-workers12 can
differentiate between the highly homologous EGFR and
c-erbB2 receptor in some cases.13
Our interest in phenolic PTK inhibitors has focused
on a characteristic property of the phenolic group - its
instability, especially under oxidizing conditions. Erb-
statin has a short half-life (<30 min) in fetal calf
serum,14 and the lack of correlation between the ac-
tivity of tyrphostins against isolated enzymes and their
effects in vitro and in vivo could be attributed to
oxidative decomposition or bioconversion of the inhibi-
* To whom correspondence should be addressed. Tel: 0115-9513404.
Fax: 0115-9513412. E-mail: malcolm.stevens@nottingham.ac.uk.
10.1021/jm990947f CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/31/2000

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1551
Ch a r t 1
Sch em e 1
ascertain whether the oxidative activation hypothesis
is sustainable.
Ch em istr y
At its simplest oxidation of a phenol (e.g. 4-meth-
ylphenol) by DAIB (4a ) in methanol generates a phe-
noxenium cation T π-carbocation reactive intermediate
5 which is quenched by methanol to yield the cyclo-
hexadienone 6 (Scheme 1).19 A wide range of nucleo-
philes can participate as trapping partners (including
intramolecular trapping leading to spirocyclic products),
and the reactive dienone products can subsequently
participate in a range of intermolecular processes, such
as dimerizations, rearrangements, and Diels-Alder
reactions. These reactions are exquisitely sensitive to
the nature of the phenolic substrate, solvent, participat-
ing nucleophile, reaction conditions, and isolation pro-
cedures but, notwithstanding these variables, have
great synthetic utility.20
Representative tyrphostins 3a -u required as starting
materials for this study were prepared by Knoevenagel
condensations between substituted benzaldehydes and
acetonitriles in ethanol-piperidine in near-quantitative
yields.
tors to other moieties more/or less bioactive. Perhaps
significantly, the di- and triphenolic tyrphostins 3p ,u
decompose in solution to chemical species which are
more active PTK inhibitors.15 The slow onset of action
of tyrphostin 3q16 may be explained by the agent
requiring prior oxidative activation: in contrast, tyr-
phostins devoid of hydroxy groups (e.g. 3r ) have a rapid
onset of activity in cellular systems.17 This raises the
intriguing possibility that the unstable phenolic sub-
strates might undergo redox interconversions between
the phenolic state and more bioactive quinone (or other)
oxidized states.
In this paper we describe our results using the
hypervalent iodine reagents [(diacetoxy)iodo]benzene
(DAIB, 4a) and [bis(trifluoroacetoxy)iodo]benzene (TAIB,
4b) to oxidize a range of tyrphostins and some simple
model phenols. These oxidants have been used to mimic
key steps in opioid biosynthesis as well as in the
construction of natural product skeletons and thus may
be regarded as ‘biomimetic’ in certain cases.18 The
biological properties of certain of the oxidized products
have been compared with the starting tyrphostins to
Oxid a tion of Mod el P h en ols a n d Tyr p h ostin s
w ith DAIB in Acetic Acid : F or m a tion a n d Rea r -
r a n gem en t of Cycloh exa d ien on es. Oxidations of
some model disubstituted phenols with DAIB gave clues
to the complexities of these reactions. Thus when a
dilute solution of vanillin (7a ) in an acetic acid-
nitromethane mixture was oxidized with 1.1 equiv of
DAIB at 25 °C the product was an unstable 2-acetoxy-
4-formyl-2-methoxycyclohexadienone (8a ) (72%). Ni-
tromethane is a solvent which facilitates oxidations of
phenols bearing EWGs,21 and high concentrations of
acetic acid ensure that the reactive intermediate is
efficiently quenched. In a consistent pattern, the reac-
tion can be extended to other phenols bearing EWGs
7b-d to yield cyclohexadienones 8b-d in >60% isolated
yields and also to the use of propionic acid as solvent,
but yields of propionyloxycyclohexadienones 8e-g were
lower (38-54%) (Scheme 2). The poorer yields are
probably due to a combination of steric effects and also
to the competitive intervention of a stoichiometric
amount of acetic acid liberated from the DAIB which

1552 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wells et al.
Sch em e 2a
a
Reagents and conditions: (a) DAIB, AcOH-MeNO2, 25 °C; (b) DAIB, EtCO2H-MeNO2, 25 °C; (c) BF3‚Et2O, Et2O; (d) AcOH, reflux;
(e) DAIB, EtCl2H-MeNO2, 25 °C, then reflux in AcOH.
Sch em e 3a
a
Reagents and conditions: (a) DAIB, AcOH, 50 °C, 2 h.
competes with propionic acid for the reactive intermedi-
ate and complicates chromatographic purification. Pos-
sibly use of TAIB as oxidant, which liberates a weaker
nucleophile trifluoroacetate, would widen the scope of
this reaction to participation of a range of acids.
(16%); from similar oxidation of 7d cyclohexadienone
8d (59%) and acetoxyphenol 9b (14%) were isolated. A
pure sample of 8a was rearranged to the phenol 9a by
boron tribromide etherate in ether, suggestive of an
intramolecular rearrangement. However, when 7d was
oxidized in propionic acid and then the product rapidly
isolated and washed with sodium bicarbonate to remove
residual acid, followed by refluxing in acetic acid, both
the acetoxyphenol 9b and the corresponding propionyl-
oxyphenol 9c were isolated in low yields, implying a
competitive intermolecular aromatization.
The cyclohexadienones are unstable yellow oils, which
solidify below 0 °C. They decompose readily on silica
gel, and isolated yields vary according to the speed with
which chromatography columns are eluted: faster elu-
tion gives higher yields. The 13C NMR spectra of 8a -g
show the dienone carbonyl at δ 189.2-191.0 and the
tetrahedral carbon C-2 at δ 91.8-93.1, characteristic of
analogous monoketal structures,22 as are the IR absorp-
tions at 1726-1744 and 1692-1701 cm-1 for the acyloxy
and dienone carbonyl groups, respectively.23 Further
evidence that the compounds are cyclohexadienones
comes from their acid-catalyzed rearrangement to 3-acyl-
oxy-2-methoxy-4-(substituted)phenols 9 (Scheme 2).
Thus, when vanillin (7a ) was oxidized with DAIB at 25
°C in acetic acid alone the expected cyclohexadienone
8a (49%) was accompanied by the acetoxyphenol 9a
Tyrphostins 3a -d bearing a 3-alkoxy-4-hydroxy sub-
stitution pattern do not oxidize to form isolable cyclo-
hexadienones, but the presence of these unstable inter-
mediates 10 can be inferred by the isolation of phenols
11-14 albeit in poor yields (27-50%) (Scheme 3). The
isomeric 4-alkoxy-3-hydroxytyrphostins 3f,g also gave
the 2-acetoxy-4-methoxy-5-hydroxytyrphostins 15 and
16 (30-40%). The poor isolated yields in these oxida-
tions reflect the fact that products 11-16 are still
phenolic and possible substrates for further oxidation,

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1553
Sch em e 4a
a
Reagents and conditions: (a) DAIB, AcOH, 25 °C, 3-9 days; (b) AcOH, reflux; (c) CF3CO2H.
as well as chromatographic difficulties in isolating the
main components of the reactions.
6.47-6.49 (Figure 1A). Protons in the ortho position of
the pendant phenyl ring absorb downfield at δ 8.03-
8.06 due to the effect of the positively charged iodine.
If the spectrum of 17 is run in TFA, the phenolate is
protonated (Scheme 4) which results in a downfield shift
of the H-6 proton from δ 6.49 to 7.14 for the trifluoro-
acetate salt 25 (Figure 1B). The ortho protons in the
phenoxy substituent of the iodophenoxytyrphostin 21
(Figure 1C) show a marked upfield shift to δ 7.14 in
DMSO-d6.
Oxid a tion of Tyr p h ostin s w ith DAIB in Acetic
Acid : F or m a tion a n d Rea r r a n gem en t of P h en yl-
iodon ioph en olates. When the monohydroxytyrphostins
3h -j were oxidized by DAIB in acetic acid over 3-9
days at 25 °C the reaction took a different path and the
products were yellow phenyliodoniophenolates 17-19
(61-80%) (Scheme 4). Similar products have been
isolated from phenols bearing conjugated EWGs24,25
where there is a balance between the nucleophilicity of
the phenol and the electron deficiency of the ring.
Although relatively stable when stored below 25 °C,
these zwitterions readily decompose when exposed to
light or heat or when stored in solution. Compound 17
rearranged to the iodophenoxytyrphostin 21 in hot
acetic acid. The mechanism may involve a 1,4-sigma-
tropic process24 or an intramolecular nucleophilic sub-
stitution via a spiro intermediate 20 (R ) CN). A ‘one-
pot’ process is a more efficient approach to form these
iodophenoxytyrphostins: thus phenols 3h -k could be
converted to iodoniophenolates with DAIB in acetic acid
at 25 °C; refluxing acetic acid then completed the
rearrangement to iodophenoxy compounds 21-24. Al-
though isolated yields were only moderate (33-70%)
these products could be separated readily from starting
phenols and iodoniophenolates by column chromatog-
raphy, the polar zwitterions remaining at the origin.
These novel stable tyrphostins could be chromato-
graphed to analytical purity and have further synthetic
potential for conversion to dibenzofuran derivatives by
photolytic, radical, or palladium(0)-mediated cycliza-
tions.
Although other monohydroxytyrphostins 3l-o did not
afford iodophenoxytyrphostins under similar DAIB/
acetic acid oxidations (no reaction with 3l,o; complex
mixture with 3m ,n ), an alternative route to such
compounds is feasible. For example, oxidation of 4-hy-
droxybenzaldehyde (26a ) in the ‘one-pot’ process gave
the iodophenoxybenzaldehyde 27a (32%); similarly meth-
yl 4-hydroxybenzoate (26b) afforded methyl 3-iodo-4-
phenoxybenzoate (27b) (76%) with DAIB/acetic acid.
Knoevenagel condensation of 27a with 2-pyridylaceto-
nitrile furnished the iodophenoxytyrphostin 28 which
could not be formed directly from 3n .
Oxidation of the dihydroxytyrphostin 3p with DAIB
resulted in black resinous material. The choice of
oxidant (DAIB, TAIB, DDQ, Fe3+, AcO2H), solvent
(MeOH, EtOH, EtOAc, AcMe, Et2O, DCM, THF, TFE,
1,1,1,3,3,3-hexafluoro-2-propanol), and reaction temper-
ature (0-50 °C) had no effect on the observed result.
The dihydroxy functionality is responsible for this
negative outcome, because tyrphostins 3r -t in which
both hydroxyl groups are protected are unreactive under
these conditions. Attempts to trap the presumed o-
quinone intermediates with nucleophiles26 or as Diels-
Alder adducts27 proved unsuccessful with these very
electron-deficient catechols.
The structure of the unstable phenyliodoniophenolate
17 was confirmed by the compound showing the correct
molecular ion (by HRMS) and by the 1H NMR spectrum
(in DMSO-d6) which showed an upfield resonance for
the H-6 proton adjacent to the phenolate oxygen at δ
Oxid a tion of Tyr p h ostin s w ith DAIB in Aceto-
n itr ile: F or m a tion of 2,2′-Dih yd r oxybip h en yls.
Oxidative phenolic coupling can be accomplished by a

1554 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wells et al.
Sch em e 5a
a
Reagents and conditions: (a) DAIB, AcOH, reflux; (b) 2-py-
ridylacetonitrile, piperidine, EtOH, reflux.
mol equiv) was added to a solution of vanillin (7a ) in
acetonitrile the color changed from green to yellow and
the biphenyl 29a precipitated (52%) (Scheme 6). Simi-
larly, phenols 7b,c afforded biphenyls 29b,c. The spar-
ingly soluble biphenyl 29a could be converted to a more
soluble monotosyl derivative 30 with tosyl chloride in
pyridine. Surprisingly, 30 underwent a double Knoev-
enagel condensation with methyl 2-cyanoacetate in
piperidine-EtOH to form the unusual bis(tyrphostin)
31 (52%) which, potentially, can exist as a pair of
separable enantiomers (atropisomers) noninterconvert-
ible by restricted rotation around the pivotal phenyl-
phenyl bond. Oxidation of the tyrphostins 3a -e simi-
larly gave bis(tyrphostins) 32-36 in yields ranging from
45% to 69%: in these cases the more insoluble products
precipitated in a pure state from the reaction medium.
This oxidative coupling is limited to 2-alkoxy-4-(substi-
tuted)phenols, but the reaction fails with 2-methoxy-4-
nitrophenol. Coupling, when it occurs, is exclusively
ortho-ortho to yield 2,2′-dihydroxybiphenyls: no para
coupling was observed when o-vanillin (2-hydroxy-3-
methoxybenzaldehyde) (37) was oxidized with DAIB in
acetonitrile. Instead, a small yield (10%) of 5-acetoxy-
2-hydroxy-3-methoxybenzaldehyde (38) was isolated
(Scheme 6), the source of the acetoxy group being the
oxidant DAIB.
Biologica l Resu lts a n d Discu ssion
In Vitr o Activity. Representative novel acetoxytyr-
phostins 11-16, iodoniophenolates 17-19, iodophe-
noxytyrphostins 21-24 and 28, and biphenyls 29a -c
and 32-36 were tested in the NCI panel of 60 human
cell lines.29 Mean GI50 values were in the range 20-80
µM with no notable pattern of selectivity in cells of a
particular tumor type. These values were comparable
to mean GI50 values for quercetin, erbstatin, and
representative 4-anilinoquinazolines extracted from the
NCI Developmental Therapeutics Program Internet
Site.30 The quinones herbimycin A and geldanamycin
gave mean GI50 values of 2.32 and 0.327 µM, respec-
tively. The poor activity of the tyrphostin type of PTK
inhibitors (GI50 generally > 100 µM)30 in this 48-h drug
F igu r e 1. A, 1H NMR spectrum of 17 in DMSO-d6; B, 1H
NMR spectrum of 25 in TFA; C, 1H NMR spectrum of 21 in
DMSO-d6.
range of oxidants, in aqueous or organic solvents or in
the solid state. Most recently 2-naphthols have been
converted to 1,1′-binaphthols (>90%) using atmospheric
oxygen catalyzed by solid Lewis acids.28 When DAIB (0.5

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1555
Sch em e 6a
a
Reagents and conditions: (a) DAIB, MeCN, 25 °C, 24 h; (b) tosyl chloride, pyridine, 25 °C; (c) methyl cyanoacetate, piperidine, EtOH,
reflux.
Ta ble 1. Activity of Tyrphostins Against Human Breast
Tumor Cell Lines in Vitro
exposure assay may reflect the fact that these com-
pounds require a prolonged drug exposure to exert
inhibitory effects on their target enzymes and thence
cell growth. Also, their profile of activity does not appear
to correlate with levels of EGFR and erbB-2 mRNA in
the cells lines.31 Evidently the new tyrphostins tested
in the present work, which are all variants of the
prototypic agents, albeit at a higher oxidation level, do
not have enhanced activity in this assay.
IC50 (µM)a
MDA MDA
compd MCF-7 MCF-7/adr 468
231 T47D SkBr3 ZR75-1
3e
3n
28
0.7
15.0
20.0
52.8
51.2
25.2
0.06 51.0
0.45 40.3
6.0
36.3
48.7
0.5
7.8
6.5
2.6
48.5
24.7
0.3
39.8
a
All IC50 values were the mean of at least three experiments.
Three tyrphostins (3e,n and 28) were evaluated by
MTT assay in a panel of human breast cell lines with
varying receptor characteristics32 in a 7-day exposure
assay (Table 1). All three compounds were less active
against MCF-7/adr than MCF-7 wt suggesting they can
act as substrates for P-glycoprotein-mediated efflux. The
most potent agent was tyrphostin 3e, and the most
sensitive cell line to the compounds was the ER-,EGFR+
MDA 468. Efficacy of 3e against MDA 468 was directly
related to drug exposure time: after a 24-h exposure to
drug the IC50 value was 100 µM; 48 h, 2 µM; 72 h, 0.7
µM; and 96 h, 0.5 µM. This corroborates our conclusion
that short-term assays (e.g. 48-h drug exposure) are
inappropriate as a primary screen for this class of
compound.
The activities of 3e,n and 28 were assessed in MTT
assays against MCF-7 and MDA 468 cells under aerobic
and anaerobic conditions where any observed differen-
tial cytotoxicity might indicate a requirement for oxida-
tive or reductive activation, respectively.33 However, no
differential activity was observed (data not shown).
E ffect s of Gr ow t h F a ct or s on t h e Act ivit y of
Tyr p h ostin s. As the activity of the tyrphostins against
the MDA 468 line may be related to its high EGFR
content, compounds 3e,n and 28 were evaluated against
clones of human breast ZR75B cells transfected with the

1556 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wells et al.
Ta ble 2. Comparisons of Basal Cell Growth of MCF-7 Human
Breast Tumor Cells with Growth Factor Stimulation in the
Presence of Tyrphostins
IC50 (µM)a
basal
compd
growth
+EGF
+TGF-R
+IGF-I
+IGF-II
3e
3n
28
2.7
73.8
41.8
2.1
71.7
45.8
2.3
67.7
41.0
2.9
72.8
39.1
3.2
77.1
46.5
a
All IC50 values are the mean of at least three experiments.
F igu r e 2. Effect of tamoxifen on the growth stimulation
caused by 3n in MCF-7 wt cells.
human EGF receptor and the pSV2-neo selectable
marker.34 The ZR75B parental line had 2 × 104 receptor
sites/cell; clone 2, 4.3 × 104 sites (marker transfect only);
clone 13, 3.1 × 105 sites; and clone 11, >1 × 106 sites.
The overexpressing clones have been reported to be
more resistant to doxorubicin, vinblastine, and 5-FU34
but had not previously been used to evaluate potential
EGFR inhibitors to which they should be more sensitive.
However, clones 13 and 11 do not show hypersensitivity
to any of the compounds or reference tyrphostin 3u
(data not shown).
F igu r e 3. (A) Inhibitory effect of tyrphostins on EGFR
autophosphorylation. Results are expressed as % band inten-
sity relative to the control and are the mean of at least three
experiments (EGFR band ca. 170 kDa). (B) Inhibitory effect
of tyrphostins on c-erbB2 autophosphorylation. Results are
expressed as % band intensity relative to the control and are
the mean of at least three experiments (c-erbB2 band ca. 185
kDa).
Because the growth of MCF-7 cells can be sustained
in media deprived of growth factors (phenol red-free
medium supplemented with charcoal-stripped FCS) the
effects of individual exogenously applied RTK ligands
(e.g. EGF, TGF-R, IGF-I, IGF-II) on tyrphostin inhibi-
tion of basal cell growth can be evaluated. Stimulated
MCF-7 cell growth was compared with basal growth.
No modulation of the activity of compounds 3e,n and
28 was observed (Table 2), suggesting that the com-
pounds exhibit no selectivity toward any of these
phosphorylation pathways.
Of the new compounds 3n elicited growth stimulation
in three of the panel of breast cells lines when cultured
in phenol red-free medium supplemented with charcoal-
stripped FCS. Significantly, the mitogenic effect was
restricted to the ER+ cell lines MCF-7, T47D, and ZR75-
1. For example, growth stimulation of MCF-7 cells was
completely abolished in the presence of 1 µM tamoxifen,
suggesting that this monophenolic compound has es-
trogen agonist activity (Figure 2).
shown to be selective tyrosine kinase inhibitors able to
distinguish between the highly homologous EGFR and
c-erbB2.12,13 Novel compounds 3e,n and 28 were as-
sessed (Western blotting followed by densitometry) for
their ability to inhibit EGFR and c-erbB2 autophospho-
rylation in cell lysates from MDA 468 (overexpress
EGFR) or SkBr3 cells (overexpress c-erbB2). Whereas
the known EGFR inhibitor 3u (IC50 3 µM)12 in this study
partially inhibited EGFR autophosphorylation (70% of
control at 100 µM) none of the test compounds were
inhibitory at this concentration over a 24-h drug expo-
sure time (Figure 3A).
The tyrphostin AG825 (3v) is known to be a selective
inhibitor of c-erbB2, exhibiting an IC50 value of 0.35
µM (cf. 19 µM against EGFR).13 In the present work 3v
only partially inhibited c-erbB2 autophosphorylation
(69% of control at 100 µM) (Figure 3B). In contrast,
compounds 3e,n and 28 had no effects at the same
concentration. These results suggests that EGF-stim-
ulated EGFR and c-erbB2 are not targets for these
agents.
Tyr osin e Kin a se In h ibitor y Effects of Novel
Oxid ized Tyr p h ostin s. Certain tyrphostins have been

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1557
namide, 3i; methyl 2-cyano-3-(4-hydroxyphenyl)propenoate, 3j;
2-cyano-3-(4-hydroxyphenyl)propenethioamide, 3l; 2-amino-
1,1,3-tricyano-4-(4-hydroxyphenyl)-1,3-butadiene, 3m ; 3-(4-
hydroxyphenyl)-2-(2-pyridyl)propenenitrile, 3n ; 4-hydroxy-3-
nitrobenzylidenemalonitrile, 3o; 3,4-dihydroxybenzylidenema-
lonitrile, 3p ; 4-acetoxy-3-methoxybenzylidenemalonitrile, 3q;
3,4-diacetoxybenzylidenemalonitrile, 3r .
Con clu sion s
The DAIB oxidation chemistry serves to demonstrate
both the diversity of products that can be synthesized
from tyrphostin type bioactive phenols and the marked
dependency of the reactions on phenol substitution,
solvent, and oxidant stoichiometry. Phenyliodoniophe-
nolates can be formed from 4-hydroxytyrphostins and
2-acyloxy-2-alkoxycyclohexadienones or 3,3′-dialkoxy-
2,2′-dihydroxybiphenyls from 3-alkoxy-4-hydroxytyr-
phostins. The initial products of oxidation may under-
go subsequent rearrangement. Thus phenyliodoniophe-
nolates rearrange to iodophenoxytyrphostins when
heated, and 2-acyloxy-2-alkoxycyclohexadienones aro-
matize to 2-acyloxy-4-hydroxy-3-methoxy-1-(substituted)
benzenes in acid. It is therefore apparent that, depend-
ing on the choice of phenol and conditions, a range of
oxidation products can be synthesized.
3-(4-Hyd r oxy-3-m eth oxyp h en yl)-2-(2-p yr id yl)p r op en i-
tr ile, 3e. A mixture of vanillin (1.0 g, 6.6 mmol) and 2-py-
ridylacetonitrile (0.82 g, 6.9 mmol) in ethanol (15 mL) was
reacted for 18 h according to general method A to give a white
solid (1.53 g, 92%): mp 121 °C; IR 3520, 2211 (CN), 1586, 1518,
1439, 1298, 1169, 779 cm-1; δH (DMSO-d6) 10.08 (brs, 1H, OH),
8.62 (m, 1H, pyridine H-6), 8.34 (s, 1H, vinylic H), 7.90 (m,
1H, pyridine H-4), 7.74 (m, 2H, pyridine H-3 or 5, H-2), 7.53
(dd, 1H, J ) 1.9, 8.3, H-6), 7.37 (m, 1H, pyridine H-3 or 5),
6.94 (d, 1H, J ) 8.3, H-5), 3.84 (s, 3H, OCH3); δC (DMSO-d6)
152.4 (C), 151.2 (C), 150.4 (CH), 148.5 (C), 146.0 (CH), 138.5
(CH), 125.8 (CH), 125.4 (C), 124.1 (CH), 120.9 (CH), 119.2 (C),
116.7 (CH), 113.9 (CH), 106.7 (C), 56.4 (CH3); m/z 253 (M+
+
1), 221 (M+ - OCH3). Anal. (C15H12N2O2‚0.5H2O) C, H, N.
The oxidation products generally proved to be poor
inhibitors of cell growth in the NCI screen. This result
is not entirely unsurprising in view of the relatively poor
performance of other known PTK inhibitors and tyr-
phostin type compounds in the panel and may reflect
the relatively slow onset of PTK inhibition with these
types of compound. Compounds 3e,n and 28 showed
some selective toxicity in a panel of breast cancer cell
lines, being especially active in MDA468 cells (EGFR+).
However the compounds did not affect EGFR or erbB-2
receptor autophosphorylation or modulate MCF-7 cell
growth induced by EGF, TGF-R, IGF-I, or IGF-II,
suggesting that they are not receptor tyrosine kinase
inhibitors. The growth inducing effects of 3n in MCF-7
cells grown in phenol red-free medium could be reversed
by the antiestrogen tamoxifen, suggesting 3n can
behave as an estrogenic mitogen.
2-Cya n o-3-(4-h yd r oxyp h en yl)-N-(3-p h en ylp r op yl)p r o-
p en a m id e, 3k . A mixture of 4-hydroxybenzaldehyde (0.40 g,
3.3 mmol) and N-(3-phenylpropyl)-2-cyanoethanamide (0.66 g,
3.3 mmol) in ethanol (40 mL) was reacted for 18 h according
to the general method A to give a yellow solid (0.61 g, 60%):
mp 158-60 °C; IR 3235, 2214 (CN), 1655, 1545, 1510, 1289,
1173, 837 cm-1; δH (DMSO-d6) 10.51 (brs, 1H, OH), 8.32 (brs,
1H, NH), 8.02 (s, 1H, vinylic-H), 7.87 (d, 2H, J ) 10.0, H-2,6),
7.23 (m, 5H, Ph-H), 6.92 (d, 2H, J ) 10.0, H-3,5), 3.23 (q, 2H,
J ) 7.5, CH2), 2.60 (t, 2H, J ) 7.5, CH2), 1.80 (q, 2H, J ) 7.5
Hz, CH2); δC (DMSO-d6) 162.6 (C), 162.4 (C), 151.0 (CH), 142.5
(C), 133.7 (CH), 129.1 (CH), 126.6 (CH), 123.8 (C), 118.1 (C),
117.1 (CH), 102.2 (C), 40.2 (CH2), 33.4 (CH2), 31.5 (CH2); m/z
307 (M+ + 1). Anal. (C19H18N2O2) C, H, N.
Gen er a l Meth od B for Syn th esis of 2-Acyloxy-2-m eth -
oxy-4-(su bstitu ted )cycloh exa -3,5-d ien on es. To a solution
of 2-methoxy-4-(substituted)phenol (0.50 g) in nitromethane
(15 mL) and a carboxylic acid (5 mL) was added solid DAIB
(1.1 mol equiv). After 10 min the solvent was removed by
vacuum evaporation and the resultant yellow oil was purified
by rapid flash column chromatography (EtOAc-hexane, 1:1)
to give the product.
Exp er im en ta l Section
Gen er a l. Melting points were obtained using a Gallenkamp
melting point apparatus and are uncorrected. IR spectra were
measured using a Mattson 2020 Galaxy Series FT-IR spec-
trometer as KBr disks. 1H and 13C NMR spectra were acquired
using a Bruker ARX250 spectrometer at 250.13 and 62.9 MHz,
respectively. Coupling constants are in hertz (Hz). Mass
spectra were recorded using a Micromass platform spectrom-
eter or an AEI MS-902 spectrometer using electron impact
(EI), electrospray (ES), chemical ionization (CI), or atmospheric
pressure CI (AP) techniques. Nominal mass spectra were
obtained using an AP+ ionization technique and accurate mass
spectra using an EI+ technique unless otherwise stated. Flash
column chromatography refers to medium-pressure silica gel
(C60 (40-60 µm)) preparative column chromatography, unless
otherwise stated. Petrol ether refers to the fraction which boils
between 60 and 80 °C.
Gen er a l Meth od A for Syn th esis of Tyr p h ostin s. To the
substituted benzaldehyde (2.0 g) dissolved in ethanol (20 mL)
were added the 1-substituted acetonitrile (1.1 mol equiv) and
piperidine (5-10 drops). The mixture was refluxed for 0.5-
18 h, then allowed to cool. Addition of water precipitated the
desired tyrphostin which was collected on a filter, washed with
water and dried in vacuo.
2-Ac e t o x y -4-fo r m y l-2-m e t h o x y -3,5-c y c lo h e x a d i -
en on e, 8a . A mixture of vanillin (7a ) (0.50 g, 3.3 mmol), DAIB
(1.17 g, 3.6 mmol) and acetic acid (5 mL) was reacted according
to general method B to give a yellow oil (yellow needles below
0 °C) (0.50 g, 72%): IR 3379, 2861, 1720, 1691, 1251, 1103,
950, 824 cm-1; δH (CDCl3) 9.63 (s, 1H, CHO), 7.41 (dd, 1H, J
) 2.1, 10.1, H-5), 6.92 (d, 1H, J ) 2.1, H-3), 6.28 (d, 1H, J )
10.1, H-6), 3.51 (s, 3H, OCH3), 2.16 (s, 3H, s, CH3CO2); δC
(CDCl3) 190.9 (C), 189.4 (C), 170.3 (C), 147.9 (CH), 136.5 (C),
134.0 (CH), 127.8 (CH), 93.1 (C), 52.2 (CH3), 20.8 (CH3); m/z
(EI+) 168 (M+ - C2H2O), 151 (-OH), 136 (-CH3), 108 (-CO),
79 (-CHO).
2-Acetoxy-4-acetyl-2-m eth oxy-3,5-cycloh exadien on e, 8b.
A mixture of acetovanillinone (7b) (0.50 g, 3.3 mmol), DAIB
(1.07 g, 3.6 mmol) and acetic acid (5 mL) was reacted according
to general method B to give a yellow oil (0.45 g, 66%): IR 3065,
1730, 1696, 1682, 1360, 1253, 1113, 960 cm-1; δH (CDCl3) 7.56
(dd, 1H, J ) 2.2, 10.2, H-5), 6.95 (d, 1H, J ) 2.2, H-3), 6.24 (d,
1H, J ) 10.2, H-6), 3.51(s, 3H, OCH3), 2.46 (s, 3H, s, CH3CO),
2.18 (s, 3H, CH3CO2); δC (CDCl3) 195.5 (C), 190.9 (C), 170.2
(C), 141.1 (CH), 136.6 (C), 135.8 (CH), 126.7 (CH), 93.0 (C),
52.0 (CH3), 25.8 (CH3), 20.9 (CH3); m/z (EI+) 182 (M+ - C2H2O),
165 (-OH), 151 (-CH3), 122 (-CO), 107 (-CH3), 79 (-CO).
The following known tyrphostins were synthesized and had
melting points and spectroscopic properties consistent with
literature values: 4-hydroxy-3-methoxybenzylidenemalononi-
trile, 3a ; 3-ethoxy-4-hydroxybenzylidenemalononitrile, 3b; 2-
cyano-3-(4-hydroxy-3-methoxyphenyl)propenamide, 3c; methyl
2-cyano-3-(4-hydroxy-3-methoxyphenyl)propenoate, 3d ; 3-hy-
droxy-4-methoxybenzylidenemalononitrile, 3f; 2-cyano-3-(3-
hydroxy-4-methoxyphenyl)propenamide, 3g; 4-hydroxybenz-
yl-denemalononitrile, 3h ; 2-cyano-3-(4-hydroxyphenyl)prope-
2-Acetoxy-2-m eth oxy-4-m eth oxycar bon yl-3,5-cycloh exa-
d ien on e, 8c. A mixture of methyl vanillate (7c) (0.50 g, 2.7
mmol), DAIB (0.97 g, 3.0 mmol) and acetic acid (5 mL) was
reacted according to general method B to give a yellow oil (0.40
g, 61%): IR 3435, 1730, 1692, 1439, 1240, 1072, 1017, 766
cm-1; δH (CDCl3) 7.45 (dd, 1H, J ) 2.1, 10.2, H-5), 7.17 (d, 1H,
J ) 2.1, H-3), 6.23 (d, 1H, J ) 10.2, H-6), 3.86 (s, 3H, OCH3),
3.51 (s, 3H, OCH3), 2.15 (s, 3H, CH3CO2); δC (CDCl3) 191.0

1558 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wells et al.
(C), 170.2 (C), 164.6 (C), 141.8 (CH), 137.5 (CH), 129.7 (C),
126.7 (CH), 92.7 (C), 53.1 (CH3), 52.2 (CH3), 20.8 (CH3); m/z
(EI+) 198 (M+ - C2H2O), 181 (-CH3CO2), 166 (-CH3), 138
(-CO), 123 (-CH3).
5 min with water (50 mL). The mixture was extracted with
EtOAc (2 × 50 mL) and the organic layer was washed with
water (2 × 50 mL), then dried (MgSO4). Removal of the solvent
followed by flash column chromatography (eluted with EtOAc-
hexane, 1:4) gave 9a as a white crystalline solid (0.31 g, 45%),
the product being contaminated with a small amount of a
compound tentatively identified as 2,4-dihydroxy-3-methoxy-
benzaldehyde.
(iii) To compound 8a (0.10 g, 0.5 mmol) dissolved in dry
ether (2 mL) was added boron trifluoride etherate (0.4 mL)
under nitrogen. The reaction was stirred at 25 °C for 3 h,
diluted with ether (20 mL) and washed with water (2 × 20
mL). The organic layer was dried (MgSO4) and the solvent
removed to give a white solid identified as primarily 9a (1H
NMR).
2-Acetoxy-4-cya n o-2-m eth oxyp h en ol, 9b. DAIB (1.19 g,
3.7 mmol) in acetic acid (10 mL) was added to 4-hydroxy-3-
methoxybenzaldehyde (0.50 g, 3.4 mmol) in acetic acid (20 mL)
at 25 °C. The reaction was left to stand for 3 days, the solvent
was removed and the products were purified by flash column
chromatography (eluted with EtOAc-hexane, 1:4) to give 8d
as a yellow oil (0.41 g, 59%) and 9b as a white solid (97 mg,
14%): mp 67 °C; IR 3239, 2249 (CN), 1777, 1601, 1317, 1168,
1059, 808 cm-1; δH (DMSO-d6) 11.06 (brs, 1H, OH), 7.45 (d,
1H, J ) 8.6, H-5), 6.91 (d, 1H, J ) 8.6, H-6), 3.73 (s, 3H, OCH3),
2.39 (s, 3H, CH3CO2); δC (DMSO-d6) 169.0 (C), 157.4 (C), 147.1
(C), 141.1 (C), 129.2 (CH), 116.5 (C), 116.2 (CH), 97.6 (C), 61.1
(CH3), 21.0 (CH3); m/z 208 (M+ + 1), 166 ([M+ + 1] - C2H4O).
Anal. (C10H9NO4) C, H, N.
2-Cya n o-5-h yd r oxy-6-m eth oxyp h en yl P r op ion a te, 9c.
To TAIB (0.88 g, 2.0 mmol) in acetonitrile (10 mL) was added
propionic acid (1 mL) with stirring at 25 °C. A solution of
4-hydroxy-3-methoxybenzonitrile (0.298 g, 2.0 mmol) dissolved
in acetonitrile (10 mL) was then added dropwise over 10 min.
After a further 10 min the reaction was diluted with ether (50
mL) and extracted with saturated sodium hydrogen carbonate
solution (50 mL), then washed with water (2 × 50 mL) and
dried (MgSO4). Removal of the solvent gave a yellow oil which
was mixed with acetic acid (50 mL) and heated at reflux for 2
h. Purification of the products by flash column chromatography
(eluted with EtOAc-hexane, 3:7) gave two products, 9c as a
white crystalline solid (42 mg, 9%): mp 121 °C; IR 3252, 2241
(CN), 1771, 1599, 1462, 1319, 1117, 810 cm-1; δH (DMSO-d6)
11.09 (brs, 1H, OH), 7.46 (d, 1H, J ) 8.6, H-5), 6.92 (d, 1H, J
) 8.9, H-6), 3.74 (s, 3H, OCH3), 2.71 (q, 2H, J ) 7.5, CH2-
CH3), 1.21 (t, 3H, J ) 7.5, CH2CH3); δC (DMSO-d6) 171.8 (C),
154.8 (C), 146.0 (C), 140.4 (C), 129.1 (CH), 115.7 (C), 114.5
(CH), 100.2 (C), 61.9 (CH3), 27.9 (CH2), 9.4 (CH3); m/z 222 (M+
+ 1), 196 ([M+ + 1] - CN). Anal. (C11H11NO4) C, H, N.
Also isolated was 9b (53 mg, 13%), identical (1H NMR) to
the sample characterized (above).
2-Acetoxy-4-cyan o-2-m eth oxy-3,5-cycloh exadien on e, 8d.
A mixture of 4-hydroxy-3-methoxybenzonitrile (7d ) (0.50 g, 3.4
mmol), DAIB (1.19 g, 3.7 mmol) and acetic acid (5 mL) was
reacted according to general method B to give a yellow oil
(yellow needles below 0 °C) (0.50 g, 73%): IR 2953, 2232 (CN),
1744, 1690, 1242, 1181, 1080, 960, 831 cm-1; δH (DMSO-d6)
7.65 (d, 1H, J ) 2.0, H-3), 7.24 (dd, 1H, J ) 2.0, 10.1, H-5),
6.36 (d, 1H, J ) 10.1, H-6), 3.43 (s, 3H, OCH3), 2.11 (s, 3H, s,
CH3CO2); δC (CDCl3) 190.2 (C), 170.8 (C), 149.5 (CH), 137.5
(CH), 127.7 (CH), 117.1 (C), 112.9 (C), 92.5 (C), 53.1 (CH3),
20.9 (CH3); m/z (EI+) 165 (M+ - C2H2O), 148 (-OH), 133
(-CH3), 106 (-HCN).
4-F or m yl-2-m eth oxy-2-p r op ion yloxy-3,5-cycloh exa d i-
en on e, 8e. A mixture of vanillin (7a ) (0.50 g, 3.3 mmol), DAIB
(1.17 g, 3.6 mmol) and propionic acid (5 mL) was reacted
according to general method B to give a yellow oil (0.40 g,
54%): IR 2947, 1730, 1697, 1460, 1181, 1074, 1003, 824 cm-1
;
δH (CDCl3) 9.65 (s, 1H, CHO), 7.44 (dd, 1H, J ) 2.0, 10.1, H-5),
6.93 (d, 1H, J ) 2.0, H-3), 6.31 (d, 1H, J ) 10.1, H-6), 3.53 (s,
3H, OCH3), 2.41 (qd, 2H, J ) 7.5, CH2CH3), 1.16 (t, 3H, J )
7.5, CH2CH3); δC (CDCl3) 191.0 (C), 189.5 (CH), 173.9 (C), 148.0
(CH), 136.4 (C), 134.0 (CH), 127.8 (CH), 92.9 (C), 52.2 (CH3),
27.4 (CH2), 9.0 (CH3). EI (M+) C11H12O5 Requires: 224.0685.
Found: 224.0678.
2-Meth oxy-4-m eth oxyca r bon yl-2-p r op ion yloxy-3,5-cy-
cloh exa d ien on e, 8f. A mixture of methyl vanillate (7c) (0.50
g, 2.7 mmol), DAIB (0.97 g, 3.0 mmol) and propionic acid (5
mL) was reacted according to general method B to give a
yellow oil (0.39 g, 38%): IR 2953, 1720, 1692, 1439, 1248, 1071,
1005, 974 cm-1; δH (CDCl3) 7.47 (dd, 1H, J ) 2.1, 10.2, H-5),
7.18 (d, 1H, J ) 2.1, H-3), 6.25 (d, 1H, J ) 10.2, H-6), 3.88 (s,
3H, OCH3), 3.52 (s, 3H, OCH3), 2.46 (qd, 2H, J ) 7.6, CH2-
CH3), 1.16 (t, 3H, J ) 7.6, CH2CH3); δC (CDCl3) 190.9 (C), 180.5
(C), 170.8 (C), 164.2 (C), 142.0 (CH), 129.9 (C), 126.7 (CH),
93.1 (C), 53.1 (CH3), 27.6 (CH2), 9.0 (CH3); m/z (EI+) 198 ([M+
+ 1] - EtCO).
4-Cya n o-2-m et h oxy-2-p r op ion yloxy-3,5-cycloh exa d i-
en on e, 8g. A mixture of 4-hydroxy-3-methoxybenzonitrile (7d )
(0.50 g, 3.4 mmol), DAIB (1.19 g, 3.7 mmol) and propionic acid
(5 mL) was reacted according to general method B to give a
yellow oil (0.33 g, 44%): IR 2949, 2232 (CN), 1730, 1701, 1460,
1269, 1177, 1074, 824 cm-1; δH (CDCl3) 6.89 (dd, 1H, J ) 2.1,
10.6, H-5), 6.88 (d, 1H, J ) 2.3, H-3), 6.31 (d, 1H, J ) 10.6,
H-6), 3.51 (s, 3H, OCH3), 2.46 (qd, 2H, J ) 7.5, CH2CH3), 1.21
(t, 3H, J ) 7.5, CH2CH3); δC (CDCl3) 189.2 (C), 180.6 (C), 173.9
(C), 146.9 (CH), 135.7 (C), 128.4 (CH), 116.2 (C), 113.5 (C),
91.8 (C), 52.1 (CH3), 27.3 (CH2), 9.0 (CH3). EI (M+) C11H11NO4
Requires: 221.0688. Found: 221.0685.
Syn th esis of 3-Acyloxy-2-m eth oxy-4-(su bstitu ted )p h e-
n ols: 2-Acetoxy-4-h yd r oxy-3-m eth oxyben za ld eh yd e, 9a .
(i) To vanillin (0.50 g, 2.3 mmol) in acetic acid (30 mL) was
added DAIB (1.17 g, 2.6 mmol) in acetic acid (20 mL). After
mixing the reaction was maintained at 25 °C for 3 days, solvent
was removed by vacuum evaporation and the products were
purified by flash column chromatography (eluted with ethyl
acetate-hexane, 3:7) to give 8a as a yellow oil (0.34 g, 49%)
and 9a as white needles (0.11 g, 16%): mp 115 °C; IR 3079,
1775, 1667, 1570, 1319, 1180, 1086, 822 cm-1; δH (DMSO-d6)
10.96 (brs, 1H, OH), 9.83 (s, 1H, CHO), 7.50 (d, 1H, J ) 8.6,
H-6), 6.94 (d, 1H, J ) 8.6, H-5), 3.72 (s, 3H, OCH3), 2.36 (s,
3H, CH3CO2); δC (DMSO-d6) 189.2 (CH), 169.6 (C), 158.3 (C),
146.5 (C), 140.7 (C), 127.9 (CH), 121.9 (C), 115.2 (CH), 61.1
(CH3), 21.2 (CH3); m/z 211 (M+ + 1), 169 ([M+ + 1] - C2H4O).
Anal. (C10H10O5) C, H, N.
Gen er al Meth od C for Syn th esis of 2-Acetoxy-3-alkoxy-
4-h yd r oxytyr p h ostin s. To the phenol in acetic acid was
added DAIB (1.1 mol equiv) and the mixture was stirred at
50 °C for 2 h. After removal of solvent the product was purified
by flash column chromatography (EtOAc-hexane) to give the
acetoxytyrphostin as a white or yellow solid.
2-Acet oxy-4-h yd r oxy-3-m et h oxyb en zylid en em a lon i-
tr ile, 11. Tyrphostin 3a (1.00 g, 5.0 mmol) and DAIB (1.62 g,
5.0 mmol) in acetic acid (100 mL) were oxidized according to
general method C to give a yellow solid (0.35 g, 27%): mp 138-
139 °C; IR 3351, 2221 (CN), 1757, 1572, 1501, 1202, 895, 826
cm-1; δH (CDCl3) 8.12 (d, 1H, J ) 9, H-6), 7.69 (s, 1H, vinylic-
H), 7.01 (d, 1H, J ) 9.0, H-5), 3.88 (s, 3H, OCH3), 2.46 (s, 3H,
CH3CO2); δC (CDCl3) 168.0 (C), 156.3 (C), 152.2 (CH), 145.0
(C), 140.0 (C), 125.5 (CH), 118.3 (C), 115.2 (CH), 114.5 (C),
112.9 (C), 81.3 (C), 61.9 (CH3), 21.0 (CH3); m/z (AP-) 257 (M-
- 1). Anal. (C13H10N2O4) C, H, N.
(ii) Compound 9a can also be prepared in situ after the
oxidation of vanillin in the presence of acetic acid. To vanillin
(0.50 g, 3.3 mmol) dissolved in nitromethane (15 mL) and
acetic acid (5 mL) was added DAIB (1.17 g, 3.6 mmol) with
stirring at 25 °C. After 15 min the reaction was cooled to 0 °C
and boron trifluoride etherate (1 mL) was added, followed after
2-Cyan o-3-(2-acetoxy-4-h ydr oxy-3-m eth oxyph en yl)pr o-
p en a m id e, 12. Compound 3b (0.50 g, 2.3 mmol) and DAIB
(0.81 g, 2.5 mmol) in acetic acid (100 mL) were reacted
according to general method C to give a white solid (0.25 g,
40%): mp 175-176 °C; IR 3374, 3163, 2212 (CN), 1777, 1691,
1497, 1171, 822 cm-1; δH (DMSO-d6) 10.88 (brs, 1H, OH), 8.07

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1559
(brs, 1H, NH), 7.97 (s, 1H, vinylic-H), 7.89 (d, 1H, J ) 8.9,
H-6), 7.77 (brs, 1H, NH), 6.98 (d, 1H, J ) 8.9, H-5), 3.79 (s,
3H, s, OCH3), 2.43 (s, 3H, s, CH3CO2); δC (DMSO-d6) 169.3
(C), 163.7 (C), 156.4 (C), 145.7 (C), 144.0 (CH), 140.7 (C), 124.2
(CH), 117.6 (C), 117.5 (C), 115.7 (CH), 105.9 (C), 60.8 (CH3),
21.2 (CH3); m/z (AP-) 275 (M- - 1). Anal. (C13H12N2O5) C, H,
N.
121.2 (C), 118.0 (C), 117.1 (C), 117.0 (C), 113.7 (C), 64.1 (C).
EI (M+) C16H9IN2O Requires: 371.9760. Found: 371.9769.
4-(2-Ca r ba m oyl-2-cya n ovin yl)-2-p h en yliod on iop h en o-
la te, 18. Compound 3i (1.0 g, 5.3 mmol) and DAIB (1.71 g,
5.3 mmol) in acetic acid (10 mL) were reacted for 3 days
according to general method D to give a mustard yellow solid
(1.37 g, 65%): mp 105-106 °C; IR 3391, 2211 (CN), 1688, 1576,
1470, 1388, 1254, 1200 cm-1; δH (DMSO-d6) 8.06 (m, 2H, Ph-
H), 8.00 (d, 1H, J ) 2.3, H-3), 7.86 (dd, 1H, J ) 2.3, H-5), 7.83
(s, 1H, s, vinylic-H), 7.66 (m, 1H, Ph-H), 7.51 (m, 2H, Ph-H),
7.33 (brs, 2H, NH2), 6.47 (d, 1H, J ) 9.0, H-6); δC (DMSO-d6)
156.1 (CH), 136.3(CH), 132.4 (CH), 120.1 (CH), 119.6 (C), 118.8
(C), 117.3 (C), 113.1 (C), 93.6 (C). EI (M+) C16H11IN2O2
Requires: 390.9944. Found: 390.9950.
4-(C y a n o -2-m e t h o x y c a r b o n y lv in y l)-2-p h e n y lio d o -
n iop h en ola te, 19. Compound 3j (0.50 g, 2.5 mmol) and DAIB
(0.83 g, 2.6 mmol) in acetic acid (100 mL) were reacted for 3
days according to general method D to give a yellow solid (0.61
g, 61%): mp 105-106 °C; IR 3440, 2214 (CN), 1719, (CO2Me),
1562, 1491, 1280, 1190, 829 cm-1; δH (DMSO-d6) 8.31 (d, 1H,
J ) 1.75, H-3), 8.04 (m, 3H, Ph-H, H-5), 7.91 (s, 1H, s, vinylic-
H), 7.64 (m, 1H, Ph-H), 7.50 (m, 2H, Ph-H), 6.49 (d, 1H, J )
9.0, H-6); δC (DMSO-d6) 165.5 (C), 153.3 (CH), 136.0(CH), 132.4
(CH), 120.9 (C), 119.1 (C), 116.8 (C), 116.2 (C), 113.8 (C), 87.6
(C), 53.2 (CH3). EI (M+) C17H12INO3 Requires: 405.9940.
Found: 405.9928.
Meth yl 2-Cya n o-3-(2-a cetoxy-4-h yd r oxy-3-m eth oxy-
p h en yl)p r op en oa te, 13. Compound 3c (0.50 g, 2.1 mmol) and
DAIB (0.76 g, 2.4 mmol) in acetic acid (100 mL) were reacted
according to general method C to give a white solid (0.31 g,
50%): mp 131 °C; IR 3269, 2228 (CN), 1782, 1698, 1586, 1238,
1061, 806 cm-1; δH (DMSO-d6) 11.10 (brs, 1H, OH), 8.11 (s,
1H, vinylic-H), 7.98 (d, 1H, J ) 8.9, H-6), 7.01 (d, 1H, J ) 8.9,
H-5), 3.83 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 2.39 (s, 3H, CH3-
CO2); δC (DMSO-d6) 169.3 (C), 163.5 (C), 157.9 (C), 147.8 (CH),
146.5 (C), 140.8 (C), 124.8 (CH), 116.9 (C), 116.7 (C), 116.1
(CH), 100.6 (C), 60.9 (CH3), 54.1 (CH3), 21.0 (CH3); m/z 292
(M+ + 1), 250 ([M+ + 1] - C2H4O). Anal. (C14H13NO6) C, H, N.
2-Ace t oxy-3-e t h oxy-4-h yd r oxyb e n zylid e n e m a lon i-
tr ile, 14. Tyrphostin 3d (0.50 g, 2.3 mmol) and DAIB (0.79 g,
2.5 mmol) in acetic acid (100 mL) were reacted according to
general method C to give a yellow solid (0.32 g, 50%): mp 120-
121 °C; IR 3283, 2239 (CN), 1740, 1572, 1321, 1190, 1061, 824
cm-1; δH (DMSO-d6) 11.33 (brs, 1H, OH), 8.34 (s, 1H, vinylic-
H), 7.93 (d, 1H, J ) 9.0, H-6), 7.02 (d, 1H, J ) 9.0, H-5), 3.99
(q, 2H, J ) 7.0, CH2), 2.39 (s, 3H, CH3CO2), 1.25 (t, 3H, J )
7.0, CH3); δC (DMSO-d6) 169.3 (C), 158.9 (C), 154.7 (CH), 146.8
(C), 139.8 (C), 124.6 (CH), 117.4 (C), 116.1 (CH), 115.6 (C),
114.8 (C), 79.0 (C), 68.9 (CH2), 21.5 (CH3), 16.3 (CH3); m/z 231
([M+ + 1] - C2H4O), 185 (-EtOH). Anal. (C14H12N2O4) C, H,
N.
Gen er a l Meth od E for Syn th esis of 3-Iod o-4-p h en oxy-
1-(vin yl-su bstitu ted )ben zen es. To the 4-(substituted)phenol
dissolved in acetic acid was added DAIB (1.1 mol equiv) in
acetic acid. The mixture was stirred at 25 °C in the dark for
24 h, then refluxed (24 h). Removal of solvent in vacuo was
followed by isolation of the product by column chromatography
(elution with DCM-hexane).
Syn th esis of 2-Acetoxy-5-h yd r oxy-4-m eth oxytyr p h os-
tin s: 2-Acetoxy-5-h yd r oxy-4-m eth oxyben zylid en em a lon i-
tr ile, 15. Oxidation of tyrphostin 3f (0.50 g, 2.5 mmol) and
DAIB (0.89 g, 2.8 mmol) in acetic acid (100 mL) according to
general method C gave a yellow solid (0.24 g, 38%): mp 161
°C; IR 3441, 2226 (CN), 1750, 1588, 1512, 1312, 1229, 918
cm-1; δH (DMSO-d6) 9.96 (brs, 1H, OH), 8.26 (s, 1H, vinylic-
H), 7.67 (s, 1H, H-6), 7.00 (s, 1H, H-3), 3.85 (s, 3H, OCH3),
2.34 (s, 3H, OCH3); δC (DMSO-d6) 170.3 (C), 155.4 (C), 154.3
(CH), 146.4 (C), 145.6 (C), 117.1 (C), 115.6 (C), 114.6 (C), 112.6
(CH), 108.4 (CH), 79.2 (C), 57.3 (CH3), 21.9 (CH3); m/z 259 (M+
+ 1), 217 ([M+ + 1] - C2H4O). Anal. (C13H10N2O4) C, H, N.
2-Cyan o-3-(2-acetoxy-5-h ydr oxy-4-m eth oxyph en yl)pr o-
p en a m id e, 16. Compound 3g (0.50 g, 2.3 mmol) and DAIB
(0.78 g, 2.4 mmol) in acetic acid (10 mL) were reacted according
to general method C to give 16 as pale yellow needles (0.19
g, 30%): mp 171 °C; IR 3435, 2214 (CN), 1757, 1670, 1512,
1219, 1181, 922 cm-1; δH (DMSO-d6) 9.70 (brs, 1H, OH), 8.00
(brs, 1H, NH), 7.94 (s, 1H, vinylic-H), 7.72 (brs, 1H, NH),
7.68 (s, 1H, H-6), 6.96 (s, 1H, H-3), 3.84 (s, 3H, OCH3), 2.33
(s, 3H, CH3CO2); δC (DMSO-d6) 170.2 (C), 163.7 (C), 153.1 (C),
145.4 (C), 145.2 (C), 143.9 (CH), 117.4 (C), 117.3 (C), 113.4
(CH), 108.1 (CH), 105.7 (C), 57.0 (CH3), 21.6 (CH3); m/z 277
(M+ + 1), 235 ([M+ + 1] - C2H4O). Anal. (C13H12N2O5‚0.5H2O)
C, H, N.
3-Iod o-4-p h en oxyben zylid en em a lon itr ile, 21. (i) Com-
pound 3h (1.00 g, 5.9 mmol) and DAIB (1.89 g, 5.9 mmol) in
acetic acid (200 mL) were reacted according to general method
E to give a white solid (1.53 g, 70%): mp 110-112 °C; IR 2226
(CN), 1574, 1478, 1402, 1265, 1196, 745, 691 cm-1; δH (d6-
DMSO) 8.49 (d, 1H, J ) 2.2, H-2), 8.41 (s, 1H, vinylic-H), 7.96
(dd, 1H, J ) 2.2, 8.7, H-5), 7.49 (m, 2H, Ph-H), 7.30 (m, 1H,
Ph-H), 7.14 (m, 2H, Ph-H), 6.93 (d, 1H, J ) 8.7, H-6); δH
(CDCl3) 8.32 (d, 1H, J ) 2.0, H-2), 7.92 (dd, 1H, J ) 2.0, 10.0,
H-5), 7.62 (s, 1H, vinylic-H), 7.45 (m, 2H, Ph-H), 7.31 (m, 1H,
Ph-H), 7.10 (m, 2H, Ph-H), 6.77 (d, 1H, J ) 10.0, H-6); δC
(CDCl3) 162.9 (C), 157.4 (CH), 154.8 (C), 143.6 (CH), 132.2
(CH), 130.8 (CH), 127.4 (C), 126.3 (C), 120.9 (CH), 116.6 (CH),
114.1 (C), 113.0 (C), 87.9 (C), 81.8 (C); m/z 372 (M+ ). Anal.
(C16H9IN2O) C, H, N.
Compound 21 was also prepared (50%) by rearrangement
of the phenyliodoniophenolate 17 (0.1 g, 0.4 mmol) in refluxing
acetic acid (50 mL) for 18 h.
2-Cya n o-3-(3-iod o-4-p h en oxyp h en yl)p r op en a m id e, 22.
Compound 3i (1.00 g, 5.4 mmol) and DAIB (1.71 g, 5.4 mmol)
in acetic acid (200 mL) were reacted according to general
method E to give a white solid (0.71 g, 33%): mp 145-147 °C;
IR 3412, 2211 (CN), 1690, 1591, 1576, 1371m 1246, 752 cm-1
;
δH (CDCl3) 8.40 (d, 1H, J ) 2.0, H-2), 8.20 (s, 1H, vinylic-H),
7.92 (dd, 1H, J ) 2.0, 10.0, H-5), 7.43 (m, 2H, Ph-H), 7.28 (m,
1H, Ph-H), 7.08 (m, 2H, Ph-H), 6.80 (d, 1H, J ) 7.5, H-6), 6.33
(brs, 1H, NH), 5.92 (brs, 1H, NH); δC (CDCl3) 162.1 (C), 161.5
(C), 155.4 (C), 151.8 (CH), 143.3 (CH), 132.3 (CH), 130.7 (CH),
128.3 (C), 125.8 (CH), 120.6 (CH), 117.4 (C), 117.1 (CH), 102.5
(C), 87.9 (C), 88.1 (C); m/z 391 (M+ + 1). Anal. (C16H11IN2O2)
C, H, N.
Meth yl 2-Cyan o-3-(3-iodo-4-ph en oxyph en yl)pr open oate,
23. Prepared from 3j (0.5 g) and DAIB (0.83 g) g) in acetic
acid (50 mL) according to general method E, the propenoate
23 was isolated as a white solid (0.60 g, 60%): mp 121-123
°C; IR 2218 (CN), 1721 (CO2Me), 1576, 1481, 1258, 1208, 976,
764 cm-1; δH (CDCl3) 8.38 (d, 1H, J ) 2.2, H-2), 8.11 (s, 1H,
vinylic-H), 7.99 (dd, 1H, J ) 2.2, 8.7, H-5), 7.41 (m, 2H, Ph-
H), 7.25 (m, 1H, Ph-H), 7.07 (m, 2H, Ph-H), 6.80 (d, 1H, J )
7.5, H-6), 3.92 (s, 3H, OCH3); δC (CDCl3) 163.4 (C), 161.8 (C),
155.3 (C), 153.0 (CH), 143.8 (CH), 132.5 (CH), 130.7 (CH),
Gen er a l Meth od D for Syn th esis of 4-(Su bstitu ted )-
p h en yliod on iop h en ola tes. To the substituted phenol dis-
solved in acetic acid was added DAIB (1 mol equiv) in acetic
acid for 3-9 days in darkness at 25 °C. Removal of solvent
gave a yellow oil which solidified on addition of EtOAc or petrol
ether. The solid was collected, washed with petrol ether and
dried in vacuo over KOH.
4-(2,2-Dicya n ovin yl)-2-p h en yliod on iop h en ola t e, 17.
Compound 3h (0.50 g, 2.9 mmol) and DAIB (0.95 g, 2.9 mmol)
in acetic acid (65 mL) were reacted for 9 days according to
general method D to give a mustard yellow solid (0.88 g,
80%): mp 104-105 °C; IR 2226 (CN), 1574, 1470, 1402, 1263,
1210, 1190, 745 cm-1; δH (DMSO-d6) 8.16 (d, 1H, J ) 2, H-3),
8.03 (m, 2H, Ph-H), 7.82 (m, 1H, H-5), 7.76 (s, 1H, s, vinylic-
H), 7.64 (m, 1H, Ph-H), 7.50 (m, 2H, Ph-H), 6.49 (d, 1H, J )
10, H-6); δC (DMSO-d6) 157.0 (CH), 136.1(CH), 132.4 (CH),

1560 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wells et al.
128.3 (C), 125.8 (CH), 120.7 (CH), 117.0 (C), 115.8 (C), 101.0
(C), 87.9 (C), 53.9 CH3); m/z 405 (M+ ). Anal. (C17H12INO3) C,
H, N.
(s, 6H, CH3), 3.79 (s, 6H, CH3); δC (DMSO-d6) 167.0 (C), 149.7
(C), 148.3 (C), 126.2 (CH), 125.2 (C), 120.3 (C), 111.7 (CH),
56.9 (CH3), 52.7 (CH3); m/z 363 (M+ + 1). Anal. (C18H18O8) C,
H, N.
2-Cya n o-3-(3-iod o-4-p h en oxyp h en yl)-N-(3-p h en ylp r o-
p yl)p r op en a m id e, 24. Compound 3k (0.20 g, 0.7 mmol) and
DAIB (0.21 g, 0.7 mmol) in acetic acid (100 mL) were reacted
according to general method E to give a white solid (0.11 g,
34%): mp 116-118 °C; IR 3376, 2214 (CN), 1680, 1528, 1470,
1252, 1198, 692 cm-1; δH (CDCl3) 8.36 (d, 1H, J ) 2.3, H-2),
8.17 (s, 1H, vinylic-H), 7.88 (dd, 1H, J ) 2.3, 9.0, H-6), 7.40
(m, 2H, Ph-H), 7.23 (m, 6H, Ph-H), 7.07 (m, 2H, Ph-H), 6.78
(d, 1H, J ) 8.8, H-5), 6.32 (brt, 1H, NH), 3.46 (q, 2H, J ) 6.0,
NHCH2), 2.70 (t, 2H, J ) 7.3, CH2CH2CH3), 1.96 (q, 2H, J )
7.8, CH2CH2CH3); δC (CDCl3) 161.2 (C), 160.4 (C), 155.5 (C),
150.6 (CH), 143.0 (CH), 141.4 (C), 132.1 (CH), 130.6 (CH),
129.0 (CH), 128.8 (CH), 128.6 (C), 126.6 (CH), 125.6 (CH),
120.6 (CH), 117.3 (C), 117.2 (CH), 103.5 (C), 88.1 (C), 77.6 (C),
40.6 (CH2), 33.5 (CH2), 31.3 (CH3); m/z 509 (M+ + 1).
3-Iod o-4-p h en oxyben za ld eh yd e, 27a . A mixture of 4-hy-
droxybenzaldehyde (26a ) (2.00 g, 16.4 mmol) and DAIB (5.54
g, 17.2 mmol) in acetic acid (100 mL) was reacted according
to general method E to give a white solid (1.68 g, 32%): mp
58-59 °C; IR 2851, 1670, 1580, 1479, 1252, 1192, 891, 750
cm-1; δH (CDCl3) 9.86 (s, 1H, CHO), 8.39 (d, 1H, J ) 2.0, H-2),
7.75 (dd, 1H, J ) 2.0, 7.5, H-5), 7.44 (m, 1H, Ph-H), 7.28 (m,
2H, Ph-H), 7.09 (m, 2H, Ph-H), 6.82 (d, 1H, J ) 7.5, H-6); δC
(CDCl3) 189.8 (CH), 162.6 (C), 155.5 (C), 142.1 (C), 133.2 (C),
131.6 (C), 130.7 (CH), 125.7 (CH), 120.6 (CH), 116.9 (CH), 88.0
(C); m/z 325 (M+ + 1). Anal. (C13H9IO2) C, H, N.
Meth yl 3-Iod o-4-p h en oxyben zoa te, 27b. Similarly pre-
pared, from methyl 4-hydroxybenzoate (26b) (2.00 g, 13.2
mmol) and DAIB (4.66 g, 14.5 mmol) in acetic acid (100 mL)
according to general method E, the white crystalline benzoate
27b (3.57 g, 76%) had: mp 57 °C (lit. mp 63 °C);35 IR 2945,
1709, 1584, 1479, 1260, 1192, 1119, 756 cm-1; δH (CDCl3) 8.40
(d, 1H, J ) 2.0, H-2), 7.91 (dd, 1H, J ) 2.1, 8.6, H-6), 7.47 (m,
2H, Ph-H), 7.29 (m, 1H, Ph-H), 7.09 (m, 2H, Ph-H), 6.86 (d,
1H, J ) 8.6, H-5), 3.85 (s, 3H, OCH3); δC (CDCl3) 165.3 (C),
161.2 (C), 156.0 (C), 141.4 (CH), 132.1 (CH), 131.3 (CH), 130.8
(CH), 126.9 (C), 125.7 (CH), 120.2 (CH), 118.1 (CH), 116.1
(CH), 89.1 (C), 53.2 (CH3); m/z 355 (M+ + 1).
3-(3-Iod o-4-p h en oxyp h en yl)-2-(p yr id in -2-yl)p r op en e-
n itr ile, 28. Compound 27a (0.32 g, 1.0 mmol) and 2-pyridyl-
acetonitrile (0.12 g, 1.0 mmol) in ethanol (10 mL) were reacted
at room temperature for 3 days according to general method
A to give a white solid (0.17 g, 40%): mp 139-140 °C; IR 2209
(CN), 1578, 1470, 1256, 1200, 1034, 887, 777 cm-1; δH (CDCl3)
8.70 (m, 1H, pyridine H-6), 8.62 (d, 1H, J ) 2.2, H-2), 8.43 (s,
1H, vinylic-H), 8.06 (dd, 1H, J ) 2.2, 8.8, H-6), 7.97 (dd, 1H,
J ) 1.8, 7.5, pyridine H-4), 7.87 (m, 1H, pyridine H-3 or H-5),
7.48 (m, 3H, pyridine H-5 or H-3, Ph-H), 7.25 (m, 1H, Ph-H),
7.12 (m, 2H, Ph-H), 7.01 (d, 1H, J ) 8.6, H-5); δC (CDCl3) 159.1
(C), 156.4 (C), 151.9 (C), 150.5 (CH), 143.4 (CH), 141.8 (CH),
138.7 (CH), 132.4 (CH), 131.2 (CH), 125.3 (CH), 124.9 (CH),
121.4 (CH), 119.8 (CH), 119.2 (CH), 118.2 (C), 111.2 (C), 90.2
(C); m/z 425 (M+ + 1), 297 (M+ - HI). Anal. (C20H13IN2O) C,
H, N.
5,5′-Difor m yl-2-h yd r oxy-3,3′-d im eth oxy-2′-tosyloxybi-
p h en yl, 30. To compound 29a (0.25 g, 0.8 mmol) suspended
in pyridine (20 mL) was added tosyl chloride (0.17 g, 0.8 mmol).
After 10 min the resulting clear solution was concentrated in
vacuo and triturated with water and the white precipitate was
collected, washed with water and dried to give 30 as a white
solid (0.32 g, 83%): mp 151-152 °C; IR (KBr disk) 3412, 1688,
1593, 1341, 1132, 1090, 855, 721 cm-1; δH (DMSO-d6) 10.00
(brs, 1H, OH), 9.98 (s, 1H, CHO), 9.71 (s, 1H, CHO), 7.65 (d,
1H, J ) 1.8, H-6 or 6′), 7.51 (d, 1H, J ) 1.8, H-6 or 6′), 7.34 (d,
2H, J ) 8.3, tosyl-H-2,6), 7.27 (d, 1H, J ) 1.8, H-4 or 4′), 7.20
(d, 1H, J ) 1.7, H-4 or 4′), 7.15 (d, 2H, J ) 8.5, tosyl-H-3,5),
3.88 (s, 3H, CH3), 3.86 (s, 3H, CH3), 2.33 (s, 3H, tosyl CH3); δC
(DMSO-d6) 192.9 (CH), 191.8 (CH), 154.2 (C), 150.7 (C), 148.7
(C), 145.5 (C), 141.6 (C), 135.8 (C), 134.4 (C), 134.1 (C), 130.4
(CH), 128.8 (C), 128.7 (CH), 127.8 (CH), 126.5 (CH), 123.6 (C),
112.3 (CH), 110.1 (CH), 57.3 (CH3), 56.8 (CH3), 21.9 (CH3); m/z
457 (M+ + 1), 303 (M+ - C7H7SO2). Anal. (C23H20O8S) C, H,
N.
5,5′-Di(2-cya n o-2-m et h oxyca r b on ylvin yl)-2-h yd r oxy-
3,3′-d im eth oxy-2′-tosyloxybip h en yl, 31. Compound 30 (0.10
g, 0.2 mmol) and methyl cyanoacetate (28 mg, 0.3 mmol) in
EtOH (40 mL) were reacted for 4 h according to general
method A to give a yellow solid (46 mg, 52%): mp 224-225
°C; IR (KBr disk) 3434, 2218 (CN), 1736, 1584, 1418, 1260,
1094, 856 cm-1; δH (DMSO-d6) 10.31 (brs, 1H, OH), 8.45 (s,
1H, vinylic-H), 8.17 (s, 1H, vinylic-H), 7.94 (d, 1H, J ) 1.8,
H-6 or 6′, or H-4,4′), 7.66 (d, 2H, H-6 or 6′, or H-4,4′), 7.35 (d,
2H, J ) 8.6, tosyl-H-2,6), 7.16 (d, 2H, J ) 8.4, tosyl-H-3,5),
3.88 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.86 (s, 3H, CH3), 3.85 (s,
3H, CH3), 2.3 (s, 3H, tosyl-CH3); δC (DMSO-d6) 164.0 (C), 163.0
(C), 162.5 (C), 155.6 (CH), 154.7 (CH), 153.8 (C), 150.5 (C),
148.2 (C), 145.4 (C), 140.5 (C), 134.5 (C), 133.7 (C), 131.3 (C),
130.3 (CH), 129.9 (CH), 127.7 (CH), 126.7 (C), 124.1 (C), 123.1
(C), 117.4 (C), 116.3 (C), 115.6 (CH), 113.4 (CH), 104.4 (C),
104.1 (C), 98.0 (C), 57.3 (CH3), 56.7 (CH3), 54.3 (CH3), 54.0
(CH3), 22.0 (CH3); m/z 619 (M+ + 1), 447 (M+ - C7H7SO2). Anal.
(C31H26N2O10S) C, H, N.
5,5′-Di(2,2-d icya n ovin yl)-2,2′-d ih yd r oxy-3,3′-d im et h -
oxybip h en yl, 32. Compound 3a (0.50 g, 2.5 mmol) and DAIB
(0.40 g, 1.3 mmol) in acetonitrile (20 mL) were reacted
according to general method F to give a yellow solid (0.30 g,
59%): mp >300 °C; IR 3389, 2226 (CN), 1595, 1566, 1411,
1298, 1188, 629 cm-1; δH (DMSO-d6) 8.32 (s, 2H, vinylic-H),
7.72 (d, 2H, J ) 2.0, H-6,6′), 7.46 (d, 2H, J ) 2.0, H-4,4′), 3.90
(s, 6H, CH3); δC (DMSO-d6) 161.3 (CH), 152.3 (C), 148.7 (C),
130.1 (CH), 125.7 (C), 123.1 (C), 115.9 (C), 115.2 (C), 112.6
(CH), 76.3 (C), 56.8 (CH3); m/z 398 (M+), 381 (M+ - OH). Anal.
(C22H14N4O4‚0.5H2O) C, H, N.
5,5′-Di(2-ca r ba m oyl-2-cya n ovin yl)-2,2′-d ih yd r oxy-3,3′-
d im eth oxybip h en yl, 33. Compound 3b (0.25 g, 1.1 mmol)
and DAIB (0.19 g, 0.6 mmol) in acetonitrile (10 mL) were
reacted according to general method F to give a yellow solid
(0.17 g, 69%); mp >300 °C; IR (KBr disk) 3368, 2216 (CN),
1690, 1574, 1279, 1180, 1042, 631 cm-1; δH (DMSO-d6) 9.67
(brs, 2H, OH), 8.07 (s, 2H, vinylic-H), 7.71 (d, 2H, J ) 2.0,
H-6,6′), 7.55 (brs, 4H, NH2), 7.45 (d, 2H, J ) 2.0, H-4,4′), 3.91
(s, 6H, CH3); δC (DMSO-d6) 164.1 (C), 151.6 (CH), 150.0 (C),
148.6 (C), 128.5 (CH), 126.0 (C), 123.2 (C), 118.3 (C), 112.8
(CH), 102.5 (C), 56.8 (CH3); m/z (AP-) 433 (M- - 1). Anal.
(C22H18N4O6‚0.5H2O) C, H, N.
Gen er a l Meth od F for Syn th esis of 2,2′-Dih yd r oxybi-
p h en yls. To the 2-alkoxy-4-(substituted)phenol in acetonitrile
was added DAIB (0.505 mol equiv) in acetonitrile. The mixture
was stirred at 25 °C in the dark for 24 h. The precipitated
biphenyl was collected, washed with more acetonitrile and
dried in vacuo.
The following known biphenyls were prepared: (from 7a )
5,5′-diformyl-2,2′-dihydroxy- 3,3′-dimethoxybiphenyl (29a ), mp
296 °C (56%); and (from 7b) 5,5′-diacetyl-2,2′-dihydroxy-3,3′-
dimethoxybiphenyl (29b), mp >300 °C (43%).
Dim eth yl 2,2′-Dih yd r oxy-3,3′-d im eth oxybip h en yl-5,5′-
d ica r boxyla te, 29c. A mixture of methyl vanillate (7c) (0.50
g, 2.7 mmol) and DAIB (0.45 g, 1.4 mmol) in acetonitrile (40
mL) was reacted according to general method F to give a white
solid (0.20 g, 40%): mp 230-231 °C; IR 3420, 2959, 1711, 1591,
1425, 1231, 1047, 762 cm-1; δH (DMSO-d6) 9.54 (brs, 2H, OH),
7.46 (d, 2H, J ) 2.0, H-6,6′), 7.44 (d, 2H, J ) 2.0, H-4,4′), 3.90
2,2′-Dih yd r oxy-3,3′-d im et h oxy-5,5′-(2-cya n o-2-m et h -
oxyca r bon ylvin yl)bip h en yl, 34. Compound 3c (0.50 g, 2.1
mmol) and DAIB (0.35 g, 1.1 mmol) in acetonitrile (30 mL)
were reacted according to general method F to give a yellow
solid (0.34 g, 69%): mp 244-245 °C; IR 3391, 2220 (CN), 1729,
1582, 1415, 1252, 1098, 1047 cm-1; δH (DMSO-d6) 10.20 (brs,
2H, OH), 8.29 (s, 2H, vinylic-H), 7.83 (d, 2H, J ) 2.0, H-6,6′),
7.64 (d, 2H, J ) 2.0, H-4,4′), 3.91 (s, 6H, CH3), 3.83 (s, 6H,
CH3); δC (DMSO-d6) 164.0 (C), 155.9 (CH), 151.2 (C), 148.6 (C),

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1561
129.6 (CH), 125.9 (C), 122.9 (C), 117.5 (C), 113.8 (CH), 97.8
(C), 56.8 (CH3), 53.9 (CH3); m/z 465 (M+ + 1). Anal. (C24H20N2O8‚
H2O) C, H, N.
removed and replaced with 250 µL of medium containing the
test drug. Plates were placed in gastight Perspex boxes and
gassed with either air or nitrogen with 5% CO2 for 3 h at 37
°C. The drug-containing medium was removed by aspiration
and 500 µL of fresh medium was added to each well. The plates
were returned to the incubator for a further 3 days, after which
time an MTT assay was used (see above) to assess viable cell
numbers.
5,5′-Di(2,2-d icya n ovin yl)-3,3′-d ieth oxy-2,2′-d ih yd r oxy-
bip h en yl, 35. Compound 3d (0.50 g, 2.3 mmol) and DAIB
(0.38 g, 1.2 mmol) in acetonitrile (25 mL) were reacted
according to general method E to give a yellow solid (0.23 g,
45%): mp 294-295 °C; IR 3293, 2231 (CN), 1570, 1431, 1290,
1186, 1074, 625 cm-1; δH (DMSO-d6) 10.28 (brs, 2H, OH),
8.32 (s, 2H, vinylic-H), 7.73 (d, 2H, J ) 1.8, H-6,6′), 7.45 (d,
2H, J ) 1.9, H-4,4′), 4.18 (q, 4H, J ) 7.0, CH2), 1.44 (t, 6H, J
) 7.0, CH3); δC (DMSO-d6) 161.3 (CH), 152.4 (C), 147.8 (C),
130.0 (CH), 125.8 (C), 123.2 (C), 115.9 (C), 115.1 (C), 113.3
(CH), 76.3 (C), 65.3 (CH2), 15.3 (CH3); m/z 426 (M+). Anal.
(C24H18N4O4) C, H, N.
Effects of Gr ow th F a ctor s on th e Activity of Tyr -
p h ostin s. MCF-7 cells (5 × 103) were seeded into sterile flat-
bottomed 96-well plates (volume/well 160 µL) in phenol red-
free RPMI 1640 medium supplemented with 5% charcoal-
stripped FCS, 1% penicillin/streptomycin solution and 300 µg/
mL L-glutamine. Cells were allowed to attach overnight, then
20 µL of the required growth factor solution (in RPMI 1640
medium) and 20 µL of the appropriate drug solution were
added. Control wells received medium and all wells contained
a total volume of 200 µL. Plates were returned to the incubator
5,5′-Di[2-cya n o-2-(p yr id in -2-yl)vin yl]-2,2′-d ih yd r oxy-
3,3′-d im eth oxybip h en yl, 36. Compound 3e (0.50 g, 2.0
mmol) and DAIB (0.32 g, 1.0 mmol) in acetonitrile (50 mL)
were reacted according to general method F to give a yellow
solid (0.27 g, 55%): mp 267-268 °C; IR 3516, 2209 (CN), 1581,
1408, 1181, 1148, 1045, 777 cm-1; δH (DMSO-d6) 9.67 (brs, 2H,
OH), 8.63 (m, 2H), 8.36 (s, 2H, vinylic-H), 7.92 (m, 2H), 7.81
(m, 4H), 7.58 (d, 2H), 7.39 (m, 2H), 3.94 (s, 6H, CH3); δC
(DMSO-d6) 152.5 (C), 150.4 (CH), 148.7 (C), 148.5 (C), 145.9
(CH), 138.6 (CH), 127.7 (CH), 126.2 (C), 124.5 (C), 124.1 (CH),
120.9 (CH), 119.2 (C), 112.6 (CH), 106.9 (C), 56.8 (CH3); m/z
(AP-) 501 (M- - 1). Anal. (C30H22N4O4‚0.5H2O) C, H, N.
Oxid a tion of 2-Hyd r oxy-3-m eth oxyben za ld eh yd e, 37.
o-Vanillin (0.50 g, 3.3 mmol) in acetonitrile (20 mL) was added
to DAIB (0.54 g, 1.7 mmol) in acetonitrile (20 mL). The reaction
was left to stand overnight, after which the solvent was
removed in vacuo and the product purified by flash column
chromatography (EtOAc-hexane, 1:4), to give (probably) 5-ac-
etoxy-2-hydroxy-3-methoxybenzaldehyde (38) as a yellow oil
(69 mg, 10%): IR 2851, 1748, 1669, 1470, 1221, 1021, 910, 743
cm-1; δH (CDCl3) 11.02 (brs, 1H, OH), 9.89 (s, 1H, CHO), 6.98
(d, 1H, J ) 2.6, H-6), 6.89 (d, 1H, J ) 2.6, H-4), 3.94 (s, 3H, s,
OCH3), 2.34 (s, 3H, CH3CO2); δC (CDCl3) 196.2 (C), 170.0 (C),
150.0 (C), 149.4 (C), 143.3 (C), 120.0 (C), 115.9 (CH), 112.8
(CH), 56.9 (CH3), 21.4 (CH3); m/z (EI+) 211 (M+ + 1).
NCI in Vitr o Cytotoxicity Assa ys. The cytotoxicity of test
agents was assessed in a panel of 60 cell lines using a
sulforhodamine B assay.29 Briefly, cell lines were inoculated
into a series of 96-well microtiter plates, with varied seeding
densities depending on the growth characteristics of the
particular cell lines. Following a 24-h drug-free incubation, test
agents were added routinely at five 10-fold dilutions with a
maximum concentration of 10-4 M. After 2 days of drug
exposure, the change in protein stain optical density allowed
the inhibition of cell growth to be analyzed.
MTT Color im etr ic Assa ys. Cells were seeded in 96-well
microtiter plates at densities of 250-300 cells/well for 7-day
assays in 10% FCS/1% penicillin/streptomycin solution supple-
mented RPMI 1640 medium (volume/well 180 µL). Cells were
allowed to adhere during a 24-h drug-free incubation period
at 37 °C/5% CO2. Drug dilutions (20 µL) were added to the
wells to give a concentration range of 1 nM-100 µM. After
drug exposures of 7 days, MTT was added to each well at a
final concentration of 400 µg/mL. After a 4-h incubation,
allowing metabolism of MTT by mitochondrial dehydrogenase
to an insoluble formazan product, medium was aspirated and
formazan solubilized by the addition of 125 µL of DMSO-
glycine buffer (4:1). Cell viability was determined as absor-
bance at 550 nm, read on an Anthos Labtec systems plate
reader.
for
a further 7 days and numbers of viable cells were
determined by MTT assays (see above).
Wester n Blot Assa ys To Deter m in e Tyr osin e Kin a se
In h ibitor y Activity of Tyr p h ostin s. Lysates from MDA 468
cells were used as a source of EGF receptors36 and SkBr3 cells
as a source of c-erbB2 tyrosine kinase.37 In both cases cells
were stimulated with EGF.37 Cells were grown to +70%
confluency in 75-cm2 flasks in RPMI 1640 medium supple-
mented with 10% FCS and 1% penicillin/streptomycin solution.
Medium was replaced with fresh RPMI 1640 medium supple-
mented with 0.1% FCS and 1% penicillin/streptomycin solution
and cells were incubated for 24 h before addition of test drug
(100 µM). Following drug addition, cells were incubated
overnight before stimulation with EGF (20 ng mL-1) for 10
min at 37 °C. Cells were washed twice with ice-cold PBS, lysed
at 4 °C for 20 min, transferred to eppendorfs, sonicated to
ensure complete lysis, then finally centrifuged at 13000 rpm
for 10 min. The cell lysates were mixed with sample buffer
(0.125 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.2 M
dithiothreitol, 0.25 mg/mL bromophenol blue) and boiled for
5 min to denature the proteins.
Protein content of lysates was measured using the Bio-Rad
protein assay;38 1:200 protein dilutions were prepared in
cuvettes containing 0.8 mL of distilled water and 0.2 mL of
Bradford reagent. Absorbance at 595 nm was read on a
Pharmacia Biotech Ultrospec 2000 UV/vis spectrophotometer.
Protein content was calculated from a BSA protein standard
curve (1-25 µg/mL).
For the detection of proteins containing phosphorylated
tyrosine residues, 50 µg of cellular proteins was separated on
denaturing polyacrylamide gels (10% for EGFR, 7.5% for
c-erbB2). Phosphorylated A431 protein preparation was in-
cluded as a positive control. Electrophoresis was performed
at 0.06 A for 1 h. Separated proteins were transferred to
polyvinylidene fluoride (PVDF) membranes (100 V, 1 h) then
probed for phosphotyrosine-containing proteins according to
manufacturer’s protocol (Upstate biotechnology catalog). Pro-
tein bands were subjected to densitometry using a Sharp
J X330P scanner and Phoretix ID Advanced V.400 software.
Ack n ow led gm en t. This work has been supported
by The Cancer Research Campaign, U.K. We thank
Prof. I. J . Stratford (University of Manchester) and Dr.
S. A. Watson (University of Nottingham) for helpful ex-
perimental assistance and Dr. Ven Narayanan and his
colleagues (NCI) for access to the NCI in vitro screening
facility.
Test agents were stored as 10 mM stock solutions in DMSO
at 4 °C protected from light. Serial dilutions were prepared in
media prior to each assay, with final DMSO concentration <
0.25%.
Differ en tia l Hyp oxic Cytotoxicity. The method of Strat-
ford and Stephens33 was used. Cells were seeded into glass
wells at an initial seeding density of 5 × 103 cells (MCF-7) or
6 × 103 cells (MDA 468) in 500 µL of medium and incubated
at 37 °C/5% CO2 for 3 h to allow them to adhere. Medium was
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