The combi-targeting concept: Chemical dissection of the dual targeting properties of a series of "combi-triazenes"

Article abstract of DOI:10.1021/jm030142e

The combi-targeting concept postulates that a molecule termed a "combi-molecule" designed to interact with an oncoreceptor on its own and allowed to further degrade to another more stable inhibitor of the latter receptor + a DNA-damaging species should be more potent than the individual combination of the same inhibitor with a DNA-damaging agent in cells expressing the targeted receptor. Recently, using the epidermal growth factor receptor (EGFR) as a target, we demonstrated the feasibility of combi-molecules with dual EGFR/DNA-targeting properties and with the ability to degrade to another potent inhibitor of EGFR. However, despite a clear demonstration of their superior potency when compared with classical combinations in EGFR-expressing cells, the true contribution of each fragment of the combi-molecules to their overall antiproliferative activity remained elusive. Here, we report a structure-function approach whereby a series of quinazoline-based "combi-triazenes" were altered to either abrogate the affinity of the EGFR-targeting quinazoline head or to suppress the DNA-damaging property of the triazene tail. The results showed that (a) inactivation of the quinazoline head by appending an N-methylaniline group to its 4-position reduced EGFR tyrosine kinase (TK) inhibitory activity by ca. 200-fold and decreased the ability of the combi-molecule to block serum-induced growth stimulation in c-erbB2 transfected NIH3T3 cells by ca. 10-fold, (b) abrogation of the alkylating activity or the DNA-damaging potential of the triazene tail by forming 3,3-dimethyltriazenes did not suppress EGFR TK inhibitory affinity but decreased the antiproliferative activity in basal growth assays, and (c) the antiproliferative activities of the monoalkyltriazenes that possessed binary EGFR TK inhibitory and alkylating activities were superior to those of their monotargeted counterparts. The results in toto suggest that each component of the dual targeting property of combi-triazenes plays a critical role in their overall antiproliferative activity.

Full text of DOI:10.1021/jm030142e

J . Med. Chem. 2003, 46, 4313-4321
4313
Th e Com bi-Ta r getin g Con cep t: Ch em ica l Dissection of th e Du a l Ta r getin g
P r op er ties of a Ser ies of “Com bi-Tr ia zen es”
Zakaria Rachid, Fouad Brahimi, Athanasia Katsoulas, Nicole Teoh, and Bertrand J . J ean-Claude*
Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/
Royal Victoria Hospital, Montreal, H3A1A1, Quebec, Canada
Received March 28, 2003
The combi-targeting concept postulates that a molecule termed a “combi-molecule” designed
to interact with an oncoreceptor on its own and allowed to further degrade to another more
stable inhibitor of the latter receptor + a DNA-damaging species should be more potent than
the individual combination of the same inhibitor with a DNA-damaging agent in cells expressing
the targeted receptor. Recently, using the epidermal growth factor receptor (EGFR) as a target,
we demonstrated the feasibility of combi-molecules with dual EGFR/DNA-targeting properties
and with the ability to degrade to another potent inhibitor of EGFR. However, despite a clear
demonstration of their superior potency when compared with classical combinations in EGFR-
expressing cells, the true contribution of each fragment of the combi-molecules to their overall
antiproliferative activity remained elusive. Here, we report a structure-function approach
whereby a series of quinazoline-based “combi-triazenes” were altered to either abrogate the
affinity of the EGFR-targeting quinazoline head or to suppress the DNA-damaging property
of the triazene tail. The results showed that (a) inactivation of the quinazoline head by
appending an N-methylaniline group to its 4-position reduced EGFR tyrosine kinase (TK)
inhibitory activity by ca. 200-fold and decreased the ability of the combi-molecule to block serum-
induced growth stimulation in c-erbB2 transfected NIH3T3 cells by ca. 10-fold, (b) abrogation
of the alkylating activity or the DNA-damaging potential of the triazene tail by forming 3,3-
dimethyltriazenes did not suppress EGFR TK inhibitory affinity but decreased the antipro-
liferative activity in basal growth assays, and (c) the antiproliferative activities of the
monoalkyltriazenes that possessed binary EGFR TK inhibitory and alkylating activities were
superior to those of their monotargeted counterparts. The results in toto suggest that each
component of the dual targeting property of combi-triazenes plays a critical role in their overall
antiproliferative activity.
In tr od u ction
The fundamental premises outlining the combi-
targeting concept are extensively discussed elsewhere.6
Briefly, the combi-targeting approach is based on equi-
libria outlined in Scheme 1. In EGFR-overexpressing
cells, the combi-molecule (TZ-I) enters the cells by
passive diffusion and in the cytosol (TZ-I) can either
directly bind to the EGFR ATP-binding site (path 1) or
degrade to a cytotoxic TZ + an EGFR TK inhibitor I
(path 2). While the TZ will exert cytotoxic activity by
damaging DNA, the generated inhibitor I will further
induce EGF-dependent (autocrine or paracrine) growth
inhibition via binding to EGFR. Recent studies demon-
strated the irreversible EGFR TK inhibitory effects of
a TZ-I (BJ 2000) of the triazene class,6 suggesting that
the TZ may also damage the receptor as in path 3.
These effects may lead to enhanced antiproliferative
activity of the TZ-I conjugate in high EGFR-expressing
cells. In contrast, in non-EGF-dependent cells, because
of the absence of an inhibitory target for TZ-I and I,
these cells will be less sensitive to the action of TZ-I
conjugates. Thus, the combi-targeting concept is a
tandem approach to chemosensitivity and chemoselec-
tivity.
Overexpression of EGFR and its closest homologue
p185neu, the HER2 (erbB2) gene product, is known to
induce aggressive tumor progression and reduced sen-
sitivity to antitumor drugs.1-4 We recently reported a
novel tumor-targeting strategy termed the “combi-
targeting concept” that sought to combine inhibitors of
EGFR tyrosine kinase (TK) with DNA-damaging agents
in order to irreversibly block EGF-dependent tumor
progression. The combi-targeting approach consists of
synthesizing agents termed “combi-molecules” that are
not only capable of inhibiting EGFR-mediated signaling
on their own but also to be hydrolyzed under physi-
ological conditions to release another EGFR TK inhibi-
tor + a cytotoxic DNA-alkylating species.5-8 This mul-
tiple “knock out” of the EGFR TK activity added to the
DNA damage induced by the alkylating species is
expected to culminate in a sustained antiproliferative
activity in EGFR-overexpressing cells. It should be
remembered that while a great number of highly specific
and potent inhibitors of EGFR TK have now been
synthesized, the emphasis is now placed on their
combination with other cytotoxic drugs in order to
enhance their efficacy in vivo.9
The feasibility of the combi-targeting concept has now
been demonstrated with two molecular probes of the
triazene class SMA415 (9) and BJ 20006 (10) that showed
a dual ability to block signaling mediated by the
* Tel: (514) 842-1231, ext 35841. Fax: (514) 843-1475. E-mail:
J acques.jeanclaude@mcgill.ca.
10.1021/jm030142e CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/03/2003

4314 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Rachid et al.
Sch em e 1
Sch em e 2
epidermal growth factor receptor (EGFR) and to sig-
nificantly damage DNA in high EGFR-expressing hu-
man carcinoma of the vulva A431 cells. As depicted in
Scheme 2, in these models the TZ-I represents the
triazenoquinazoline, TZ the released methyldiazonium,
and the I the released aminoquinazolines. The primary
models focused on 3-monoalkyltriazenes, since 3,3-
dialkyltriazenes, despite their superior stability and
clinical activity, require metabolic activation to ulti-
mately generate the cytotoxic alkyldiazonium.10-12 This
type of compound (12-14) will be used in this study as
examples of non-DNA-damaging combi-molecules.
A significant body of work has accumulated to suggest
that, on one hand, combi-molecules block EGFR-medi-
ated signaling like other known EGFR inhibitors and,
on the other hand, significantly damage DNA according
to a mechanism similar to that of classical triazenes.5-7
However, the precise contribution of the triazene tail
and the quinazoline head to the mixed potency of the
combi-molecules remained elusive. Here, we report a
structure-function approach designed to define the role
of each of the two major targeting components (i.e. DNA-
damaging triazene and the EGFR inhibitory quinazo-
line) in the overall antiproliferative activity of a series
of combi-triazenes. To clearly dissect the effects of their
reactive alkylating DNA-damaging component from that
of their EGFR inhibitory one, we synthesized three
categories of agents: (a) molecules on which the DNA-
damaging function is abrogated by appending a 3,3-
dimethyltriazene moiety to the 6-position of the quinazo-
lines, (b) molecules in which the quinazoline moiety is
tethered to an N-methylaniline or benzylamine at the
4-position in order to attenuate the EGFR-inhibitory
properties, and (c) dual targeting monoalkyltriazenes
with various substituents on both the anilinoquinazoline
backbone and the 3-position of the monoalkyltriazene
side chain. The latter class of combi-molecules is de-
signed to degrade as outlined in Scheme 2.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of these compounds pro-
ceeded as described in Scheme 3. Briefly, compound 2
was obtained as described by Roth et al.13 by treating
the 5-nitroanthranilonitrile 1 with a sulfuric acid/formic
acid mixture. The resulting quinazoline 2 was heated
with phosphorus pentachloride to provide 3, which was
treated with benzylamine or substituted anilines. The
nitro groups of the resulting compounds were reduced
by catalytic hydrogenation to give 6-aminoquinazolines
4-8. Amine 4 bearing X ) Br was obtained by reduction
with Fe in ethanol as previously described.14 Diazoti-
zation of amines 4-8 using NOBF4 in acetonitrile
followed by addition of a series of primary or secondary
amines and neutralization with triethylamine gave
9-24 as pure solids after purification by column chro-
matography on basic alumina. Their structures were
confirmed by 1H, 13C NMR and high-resolution mass
spectrometry (HRMS).
It is important to note that monoalkyltriazenes are
unstable molecules that rapidly decompose under acidic
conditions. The 6-alkyltriazene-substituted quinazolines
reported herein degraded on silica gel and even neu-
tralization of the eluent with triethylamine did not
improve the yield of purification. It was found that only
a rapid elution on basic alumina could provide pure
compounds in a reasonable yield. On the other hand,
diazotization of the aminoquinazoline in acetonitrile
with NOBF4 has proven more effective than aqueous
reaction with NaNO2/H+, perhaps due to a greater
stability of the diazonium tetrafluoroborate complex in
an organic solvent.
Biology. Effects of th e Qu in a zolin e Hea d . One of
the premises of the combi-targeting concept is to design
the combi-molecule to be small enough to interact with
the receptor on its own prior to degradation. Thus, we
tested the ability of each agent of the series to block

The Combi-Targeting Concept
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4315
Sch em e 3a
a
Reagents: (i) H2SO4/formic acid/heat; (ii) PCl5/heat; (iii) ArNH2/iPrOH/rt; (iv) BnNH2/iPrOH/rt; (v) H2/Pd-C or Fe/ethanol/AcOH/
heat; (vi) CH3CN/NOBF4/-5 °C/ether/Et3N/alkylamine.
isolated EGFR tyrosine kinase in a short 8 min exposure
assay at room temperature in order to prevent partial
degradation of the combi-molecules prior to binding to
the ATP site of EGFR. It is important to note that the
significant number of structure-activity relationship
(SAR) studies on anilinoquinazolines14-16 and pyrido-
[d]pyrimidines15,16 as EGFR TK inhibitors are consistent
with their binding to the ATP site of EGFR. Molecular
modeling suggests that the N-1 atom accepts an H-bond
from Met-769 and N-3 from the side chain of Thr-766.
The anilino moiety binds in an adjacent hydrophobic
pocket and the 6- and 7-positions are positioned at the
entrance of the binding cleft. The SAR in the current
series of 6-triazene-substituted quinazolines paralleled
those of previously reported quinazolines14-16 (Table 1).
Compounds with X ) Me were less active than their
bromo or chloro analogues. As an example, in the
monomethyltriazene series 9-11, a 0.039 µM IC50 was
observed for the bromo analogue 11, whereas the 3′-
Me substituted combi-molecule 9 was 5 times less active
(IC50 ) 0.2 µM). Although electron-donating substitu-
ents at the 6-position are known to significantly enhance
binding affinity,14-16 the 3-mono- and 3,3-dialkyltri-
azenoquinazolines of these series showed surprisingly
higher EGFR TK inhibitory potency than their parental
6-aminoquinazolines (4-8). The amino group being a
stronger electron-donating group than the 3-alkyl-1,2,3-
triazene moiety, one would expect the former to confer
a greater EGFR affinity than the latter. We believe that
the conformation of the triazene tail is such that it may
stabilize the binding of the structure to the ATP site.
While compounds substituted with a benzylamino group
at the 4-position (7, 23, 24) retain EGFR TK inhibitory
activity (IC50 ) 0.3-0.5 µM), those substituted with an
N-methylanilino group (18, 21, 22) were virtually in-
active (IC50 ) 8-13 µM). A similar effect has already
been reported for 4-(N-methylanilino)quinazolines in
earlier structure optimization studies.14 Thus, these
series of molecules have provided a sufficiently broad
spectrum of EGFR TK activities to test the role of the
EGFR-targeting component in the antiproliferative
activity of combi-triazenes.
The modulation of the EGFR targeting component of
the series was further tested in serum-stimulated
growth assays using NIH3T3 and NIH3T3neu cells
transfected with the HER2 (erbB2) gene, a close homo-
logue of the EGFR gene that confers a significant growth
advantage to its host transfectant. We have already
demonstrated that inhibition of serum-stimulation in
this pair of isogenic cell lines was a good cell-based assay
to correlate EGFR TK inhibitory activities with inhibi-
tion of growth signaling.7 Interestingly, a statistically
significant correlation (Pearson r ) 0.8, p < 0.001) was
obtained between IC50 values of EGFR TK inhibition
and those for inhibition of serum-stimulated growth in
the NIH3T3neu (Figure 1 and Table 2). More impor-
tantly, when IC50 values for growth inhibition of NIH3T3
wild type were compared with those of its transfected
counterpart NIH3T3neu (a test for selective targeting),
the combi-molecules possessing affinity for the ATP site

4316 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Rachid et al.
Ta ble 1. EGFR Tyrosine Kinase (TK) Inhibition and Antiproliferative Data for Combi-Molecules of the Triazene Class
IC50 (µM)
EGFR enzyme
assaya
inhibition of A431
basal growthb
no.
R1
R2
R3
X
4
5
6
7
8
H
H
H
Br
Me
Cl
0.044
1
0.2
0.508
8.4
0.2
45
65
47
25
115.9
23.22
16.67
16
93.97
100
100
40
20
11.40
25.14
12.40
33
89.46
29.76
20
Me
H
H
H
H
H
H
H
H
H
9 (SMA41)
10 (BJ 2000)
H
H
H
Me
Me
Me
H
H
H
H
H
H
H
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Cl
Br
Me
Cl
Br
Br
Br
Br
H
Br
Br
H
H
H
0.1
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0.039
0.024
0.014
0.011
0.071
0.025
0.238
9.3
0.038
0.064
8
13.35
0.335
0.482
methoxyethyl
benzyl
(2-pyridyl)methyl
(2-pyridyl)methyl
N,N-dimethylaminoethyl
N-morpholinoethyl
Me
Me
Me
Me
H
Me
H
H
Me
Me
H
H
H
50.29
a
IC50 (mM) to inhibit the phosphorylation of poly(L-glutamic acid-L-tyrosine) by EGFR isolated form A431 carcinoma cells. See the
b
Experimental Section for details. IC50 (mM) of inhibition of A431 carcinoma cells basal growth. Cell growth was measured using SRB
assay. See the Experimental Section for details. Values are the average of two independent experiments run in triplicate. Variation was
generally (1%.
6-aminoquinazoline.5,6 In contrast, dialkyltriazenes (e.g.
12-14) are stable molecules that require oxidative
demethylation in order to generate the corresponding
monoalkyltriazene and the ultimate DNA-damaging
alkyldiazonium.10-12 Thus, the dimethyltriazenes were
tested as isosteric and perhaps isolectronic non-DNA-
damaging analogues of the combi-triazenes. Their in-
ability to damage DNA under the conditions of the study
was confirmed by comparing the DNA-damaging poten-
tial of 14 with that of 11. A comet assay demonstrated
that in contrast to 11, which showed a dose-dependent
increase in DNA strand breaks following a 30 min drug
exposure, the dimethyltriazene 14 did not show any
DNA damage in the A431 carcinoma cells (Figure 3).
We have already demonstrated in a similar fashion that
6-aminoquinazolines do not damage DNA.5-8 Thus 4-8
were studied as the nonisoelectronic, non-DNA-damag-
ing EGFR inhibitors deriving from aqueous degradation
of combi-triazenes. The release of aminoquinazolines
from SMA41 and BJ 2000 has already been confirmed
by HPLC on earlier studies.5,6 In this study the conver-
sion of the combi-triazenes to their corresponding ami-
noquinazolines was determined by spectrofluorometer
as the latter amines were fluorescent (absorption 290
nm, emission 450 nm) and the rates of amine released
were in the range of 0.6-2.5 × 10-2 min-1 (see 11, 16,
19, Figure 4). These results confirmed that regardless
of the 3-alkyl substituents, the combi-triazenes are
indeed prodrugs of the aminoquinazolines 4-8, and
even after their degradations, another stable inhibitor
F igu r e 1. Correlation between the IC50 values for EGFR TK
inhibition and blockade of serum-stimulated growth of
NIH3T3neu (NIH3T3 transfected with erbB2 gene). Pearson
correlation coefficient r ) 0.834, p < 0.001. See the Experi-
mental Section for details.
of EGFR were selectively more potent against the
transfectant and selectivity (expressed as IC50 NIH3T3/
IC50 NIH3T3neu) linearly increased with EGFR affinity
of the drugs. A statistically significant correlation
(Pearson r ) 0.8, p < 0.001) was obtained (Figure 2 and
Table 2). The significant substituent effects on EGFR
TK IC50 values, selectivity, and subsequent inhibition
of growth stimulation by the combi-molecules is strong
evidence of the targeting role of the quinazoline head
in their ultimate antiproliferative activities.
Effects of th e Tr ia zen e Ta il. As per Scheme 2, the
monoalkyltriazenes (9-11, 15-21, 23) are designed to
be hydrolyzed to a cytotoxic alkyldiazonium and a

The Combi-Targeting Concept
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4317
Ta ble 2. Selective Inhibition of Serum-Stimulated Growth by Combi-Molecules of the Triazene Class in NIH3T3 Isogenic Cells
inhibition of serum-stimula ted
growth, IC50 (µM)a
IC50 NIH3T3/
no.
R1
R2
R3
X
NIH3T3neu
NIH3T3
IC50 NIH3T3neu
4
5
6
7
8
H
H
H
Br
Me
Cl
4.10
7.20
4.93
33.79
99.37
9.69
4.16
4.32
6.94
3.09
3.74
2.98
11.23
4.06
64.09
3.40
52.89
90
12.92
12.51
7.94
2.86
0.72
7.05
7.71
7.64
7.31
19.53
14.02
14.41
5.36
7.53
0.62
4.91
7.61
0.38
1.49
1.44
1.98
39.19
96.74
71.35
68.29
32.09
33.03
50.75
60.38
52.45
42.94
60.18
30.55
39.42
16.71
22.24
30.46
47
Me
H
H
H
H
H
H
H
H
H
9 (SMA41)
10 (BJ 2000)
H
H
H
Me
Me
Me
H
H
H
H
H
H
H
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Cl
Br
Me
Cl
Br
Br
Br
Br
Br
Br
Br
H
11
12
13
14
15
16
17
18
19
20
21
22
23
24
methoxyethyl
benzyl
(2-pyridyl)methyl
(2-pyridyl)methyl
N,N-dimethylaminoethyl
N-morpholinoethyl
Me
Me
Me
Me
H
Me
H
H
2.92
Me
Me
H
81.05
31.64
34.91
33.90
H
H
H
50.27
67.29
H
a
Cell growth was measured using the SRB assay. See the Experimental Section for details. Values are the average of two independent
b
experiments run in triplicate. Variation was generally (1%. IC50 ratio of inhibition of serum-stimulated growth in NIH3T3/NIH3T3neu.
F igu r e 4. Formation of fluorescent aminoquinazolines from
combi-triazenes (11, 16, 19). Experiments were carried out by
spectrofluorometry as described in the Experimental Section.
The rates of amine released from 11, 16, and 19 were 1.4 ×
10-2, 0.71 × 10-2, and 0.69 × 10-2 min-1, respectively.
F igu r e 2. Correlation between the ratio IC50 NIH3T3 (wild
type)/IC50 NIH3T3neu (erbB2 transfectant) for inhibition of
serum-stimulated growth (selective targeting) and IC50 values
for EGFR TK inhibition. Pearson correlation coefficient r )
0.827, p < 0.001. See the Experimental Section for details.
(12-14, 22, and 24), whose activities were solely de-
pendent on their ability to block EGFR-mediated cell
signaling, were in the 50-100 µM range in a basal
growth assay. In contrast, all monoalkyltriazenes were
more potent than their blocked counterpart (Table 1).
As depicted in Figure 5, where only compounds with
methylating properties are compared, agents such as
Temozolomide (TEM),17-19 methylnitrosourea (MNU),
or 21, which only possess methylating properties, had
little antiproliferative activity against A431 cells that
express O6-methylguanine transferase, a DNA-repair
enzyme capable of repairing the cytotoxic O6-meth-
ylguanine adduct. In contrast, the methylating combi-
triazenes 9-11 and 23, which possess mixed targeting
properties, were significantly more potent in this refrac-
tory tumor cell line. Thus, combi-triazenes that combine
multiple properties appear to be more potent than their
monotargeted counterparts.
F igu r e 3. Quantitation of DNA damage using the alkaline
comet assay. Tail moment was used as a parameter for the
detection of DNA damage in A431 cells exposed to 11 and 14
for 30 min. See the Experimental Section for details.
(the released aminoquinazoline) may remain in the cell
culture medium.
Using the A431 carcinoma of the vulva cell line that
coexpresses EGFR and its ligand TGFR and aggres-
sively proliferates by autocrine induction, we found that
the aminoquinazolines (4-8) and dimethyltriazenes
Th e Com bi-Molecu les. The current study demon-
strated that the combination of an alkyltriazene with
an aminoquinazoline in order to simultaneously target
DNA and EGFR is an at least additive process in which

4318 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Rachid et al.
3-bromoaniline (1 mL, 9.5 mmol) and the resulting mixture
stirred at room temperature overnight under argon. The
precipitate that formed was filtered and washed with water
and ether, after which it was dried under vacuum to give
6-nitro-4-(3-bromophenylamino)quinazoline (4 g, 82%). This
compound was found to be sufficiently pure to be used for the
1
next step: mp 267-270 °C; H NMR (400 MHz, DMSO-d6) δ
11.95 (br s, 1H, NH), 9.87 (d, 1H, J ) 2.6 Hz, H-5), 9.0 (s, 1H,
H-2), 8.73 (dd, 1H, J ) 12.2, 2.6 Hz, H-7), 8.15 (d, 1H, J )
12.2 Hz, H-8), 8.04 (s, 1H, H-2′), 7.79 (d, 1H, J ) 10.4, H-6′),
7.16-7.14 (m, 2H, H-4′,5′); 13C NMR (100 MHz, DMSO-d6) δ
159.2, 158.0, 153.2, 145.2, 140.7, 131.1, 130.0, 127.6, 127.5,
125.5, 122.0, 121.9, 121.5, 115.0.
To a solution of the isolated 6-nitro-4-(3-bromophenylami-
no)quinazoline (5 g, 0.015 mol) in aqueous ethanol (1:2, 300
mL) and acetic acid (15 mL) was added Fe (10 g. 0.18 mol),
and the resulting cloudy mixture was kept under reflux for 1
h. The reaction was cooled to room temperature, alkalinized
with concentrated ammonia, and filtered through Celite. The
filtrate was evaporated and the resulting solid washed with
water and further dried to give 4 (5.7 g, 80%) as a yellow solid:
mp 204-206 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H,
NH), 8.37 (s, 1H, H-2), 8.20 (s, 1H, H-2′), 7.85 (d, 1H, J ) 8.0
Hz, H-6′), 7.53 (d, 1H, J ) 8.8 Hz, H-8), 7.32-7.20 (m, 4H,
H-5,7,4′,5′), 5.66 (s, 2H, NH2); 13C NMR (100 MHz, DMSO-d6)
δ 166.4, 156.2, 148.9, 143.7, 143.3, 131.5, 128.7, 124.9, 123.9,
121.8, 118.4, 114.9, 114.1, 99.5.
6-Am in o-4-[(3-m eth ylp h en yl)a m in o]qu in a zolin e (5). A
suspension of 6-nitro-4-[(3-methylphenyl)amino]quinazoline
obtained as previously described14 (5.49 g, 0.02 mol) and
palladium (5% charcoal, 1 g, 9.4 mmol) in MeOH (100 mL)
was hydrogenated at 2 bar for 1 h at room temperature.
Filtration, evaporation, and recrystallization from neat ethyl
acetate gave 5 (5 g, 90%): mp 245-248 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.18 (br s, 1H, NH), 8.30 (s, 1H, H-2), 7.65-7.63
(m, 2H, H-5,7), 7.5 (d, 1H, J ) 8.8 Hz, H-8), 7.37 (s, 1H, H-2′),
7.24-7.21 (m, 2H, H-4′,5′), 6.86 (d, 1H, J ) 7.6 Hz, H-6′), 5.49
(br s, 2H, NH2); 2.31 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-
d6) δ 156.7, 150.5, 147.8, 143.1, 140.5, 138.1, 129.3, 128.8,
124.3, 124.2, 122.9, 119.7, 117.4, 101.9, 22.1.
F igu r e 5. Comparison of the antiproliferative activities of
monotargeted methylating agents (EGFR-targeting or DNA-
targeting) with mixed DNA/EGFR-targeting methylating combi-
molecules (combi-targeting) in the A431 human carcinoma of
the vulva cell line. See the Experimental section for details.
each individual component TZ and I plays a distinct
antiproliferative role. This study also demonstrated
that, as postulated, molecules designed to enhance both
chemoselectivity and chemosensitivity in growth factor-
dependent tumors can be achieved. Although the impact
of each of these components may vary with the cell
phenotype, the combi-molecular approach possesses
significant pharmacological advantages over mono-
targeted agents, since where DNA repair enzymes
deplete the cytotoxic activity associated with DNA
alkylation, the signaling inhibitory component will exert
significant antiproliferative effects. Where DNA repair
status is deficient and EGFR/erbB2 confers a growth
advantage, a tandem block can be inflicted to both
growth factor-stimulated proliferation and uncontrolled
DNA replication in refractory tumor cells. More impor-
tantly, this combination of two major antiproliferative
properties are imprinted into single and small struc-
tures: the “combi-molecules”.
6-Am in o-4-[(3-ch lor op h en yl)a m in o]q u in a zolin e (6).
6-Aminoquinazoline 6 was obtained as described for 5 (4.5 g,
1
80%): mp 235-237 °C; H NMR (400 MHz, DMSO-d6) δ 10.5
Exp er im en ta l Section
(br s, 1H, NH), 8.56 (s, 1H, H-2), 8.00 (br s, 1H, H-2′), 7.74
(dd, 1H, J ) 8, 0.8 Hz, H-4′), 7.68 (d, 1H, J ) 8.8 Hz, H-8),
7.56 (d, 1H, J ) 1.6 Hz, H-5), 7.42-7.35 (m, 2H, H-7,5′), 7.21
(dt, 1H, J ) 8.0, 1.0 Hz, H-6′), 5.50 (br s, 2H, NH2); 13C NMR
(100 MHz, DMSO-d6) δ 157.8, 149.5, 147.7, 140.4, 135.3, 133.3,
130.8, 125.7, 125.0, 124.3, 123.4, 122.4, 116.6, 102.3.
Ch em istr y. 1H NMR spectra and 13C NMR spectra were
recorded on a Varian 400.14 or 300.06 spectrometer. Chemicals
shifts are expressed in parts per million (ppm) relative to the
internal standard tetramethylsilane. Mass spectrometry was
performed by the McGill University Mass spectroscopy Center.
Electrospray ionization (ESI) and atmospheric pressure chemi-
cal ionization (APCI) spectra were performed on Finnigan LC
QDUO spectrometers. High-resolution mass spectra (HMRS)
were obtained from a ZAB-E4F analytical mass spectrometer.
Data are reported as m/z (intensity relative to base peak )
100). The compounds were purified by flash chromatography
using Caledon 50-200 µm alumina (basic). Melting points
were determined in open capillary tubes on a Meltemp melting
point apparatus and were uncorrected. Intermediates 2-8
were synthesized as described previously.14-16
4-Hyd r oxy-6-n itr oqu in a zolin e (2). 5-Nitroanthraniloni-
trile 1 (25 g, 0.15 mol) was added by portions over 1 h to a
mildly refluxing mixture of 88% formic acid (300 mL) and
sulfuric acid (18 mL). After an additional 30 min, the mixture
was cooled to 0 °C and poured into ice-water. The resulting
precipitate was collected by filtration, washed with water, and
dried under vacuum to give 2 (34 g, 86%) as a yellow solid:
mp 283-285 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.72 (br s,
1H, OH), 8.76 (d, 1H, J ) 3.0 Hz, H-5), 8.50 (m, 1H, H-7),
8.28 (s, 1H, H-2), 7.83 (d, 1H, J ) 8.7 Hz, H-8); 13C NMR (100
MHz, DMSO-d6) δ 159.7, 152.5, 148.6, 144.6, 128.7, 128.0,
122.4, 121.6.
6-Am in o-4-[(p h en ylm et h yl)a m in o]qu in a zolin e (7). 6-
Aminoquinazoline 7 was obtained as described for 5 (3 g, 85%):
1
mp 170 °C; H NMR (400 MHz, DMSO-d6) δ 9.65 (br s, 1H,
NH), 8.43 (s, 1H, H-2), 7.56-7.23 (m, 8H, ArH), 5.90 (br s,
2H, NH2), 4.81 (d, 2H, J ) 4.0 Hz, CH2); 13C NMR (100 MHz,
DMSO-d6) δ 159.2, 149.0, 148.6, 139.3, 129.2, 129.0 (2C
overlap), 128.0 (2C overlap), 127.6, 124.8, 124.2, 116.0, 102.4,
44.8.
6-Am in o-4-[(N-m eth yl-N-ph en yl)am in o]qu in azolin e (8).
6-Aminoquinazoline 8 was obtained as described for 5 (3.5 g,
1
75%): mp 220-222 °C; H NMR (300 MHz, DMSO-d6) δ 8.66
(s, 1H, H-2), 7.59 (d, 1H, J ) 9.0 Hz, H-7), 7.45-7.12 (m, 7H,
ArH), 5.93 (s, 2H, NH2), 3.58 (s, 3H, CH3); 13C NMR (75 MHz,
DMSO-d6) δ 160.7, 148.3, 147.8, 147.6, 139.6, 130.4 (2C
overlap), 126.8, 126.3, 125.3 (2C overlap), 124.8, 118.2, 105.1,
43.4.
1-{4-[(3-Br om op h en yl)a m in o]-6-qu in a zolin yl}-3-m eth -
yltr ia zen e (11). 6-Aminoquinazoline 4 (100 mg, 0.28 mmol)
was stirred in dry acetonitrile (10 mL) under argon. There-
after, it was cooled to -5 °C and nitrosonium tetrafluoroborate
(67 mg, 0.58 mmol) in acetonitrile was added. The resulting
solution was stirred for 1 h at -5 °C and added dropwise to a
mixture of cold ether (10 mL), water (2 mL), triethylamine
(0.5 mL), and methylamine (2.0 M solution in THF, 1.1 mL,
2.24 mmol). The mixture was kept at 0 °C for 2 h more and
6-Am in o-4-[(3-b r om op h en yl)a m in o]q u in a zolin e (4).
Gen er a l Exa m p le of th e Cou p lin g P r oced u r e. To a
solution of 314 (2 g, 9.5 mmol) in 2-propanol was added

The Combi-Targeting Concept
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4319
further was extracted with ethyl acetate. The organic layer
was dried over potassium carbonate and evaporated to give
11 as a brown residue, which was purified by flash chroma-
tography on basic alumina (1:4 cyclohexane-AcOEt). Triazene
11 was obtained as an oil that solidified upon addition of 1:1
ether/petroleum ether (100 mg, 88%): mp 167-169 °C; FABMS
m/z 356.9 (MH+ with 79Br), 358.9 (MH+ with 81Br), 329 (M -
N2, 10.7), 315 (M - methyldiazonium, 19.8), 299 (M -
methyltriazene, 11.9) m/z 357.046 331 (MH+), C15H13N6Br
requires m/z 357.046 200; 1H NMR (400 MHz, DMSO-d6) δ 9.88
(br s, 1H, -NdN-NH), 9.28 (s 1H, NH), 8.57 (s, 1H, H-2),
8.45 (d, 1H, J ) 1.6 Hz, H-2′), 8.27 (s, 1H, H-5), 8.00-7.94 (m,
2H, H-7,6′), 7.74 (d, 1H, J ) 9.2 Hz, H-8), 7.32-7.26 (m, 2H,
H-5′,4′), 3.08 (d, 3H, J ) 3.6 Hz, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 158.0, 153.7, 149.6, 148.7, 141.8, 130.9, 129.5,
126.4, 125.5, 124.6, 121.8, 121.1, 116.3, 115.2, 31.5.
1-{4-[(3-Met h ylp h en yl)a m in o]-6-q u in a zolin yl}-3,3-d i-
m eth yltr ia zen e (12). 6-Aminoquinazoline 5 (100 mg, 0.4
mmol) was stirred in dry acetonitrile (10 mL) under argon.
Thereafter, it was cooled to -5 °C, and nitrosonium tetrafluo-
roborate (94 mg, 0.8 mmol) in acetonitrile was added. The
resulting solution was stirred for 1 h at -5 °C and added
dropwise to a mixture cold of ether (10 mL), water (2 mL),
triethylamine (0.5 mL), and aqueous dimethylamine (40%) (0.3
mL). The mixture was further kept at 0 °C, and 2 h later, the
precipitate was filtered and washed with ether and AcOEt to
give 12 as a brown powder (80 mg, 65%): mp 165-167 °C;
FABMS 307 (MH+), 235 (M - 3,3-dimethyltriazene, 36.2) m/z
307.167120 (MH+), C17H18N6 requires m/z 307.167 000; 1H
NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H, NH), 8.69 (s, 1H,
H-2), 8.00 (d, 1H, J ) 11.8 Hz, H-7), 7.86 (d, 1H, J ) 11.8 Hz,
H-8), 7.80 (s, 1H, H-5), 7.56-7.53 (m, 2H, H-4′,2′), 7.29 (t, 1H,
J ) 9.9 Hz, H-5′), 6.98 (d, 1H, J ) 9.9 Hz, H-6′), 3.56 (br, 3H,
N(Me)2), 3.29 (br, 3H, N(Me)2), 2.41 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6) δ 158.2, 153.9, 149.1, 148.5, 139.9, 138.1,
129.3, 128.8, 125.1, 124.8, 123.4, 120.1, 116.4, 115.2, 43.8, 36.9,
22.1.
1-{4-[(3-Ch lor op h en yl)a m in o]-6-q u in a zolin yl}-3,3-d i-
m eth yltr ia zen e (13). As described for 12, triazene 13 was
obtained as a pale brown powder (84 mg, 70%), using 6 (100
mg, 0.4 mmol) and aqueous dimethylamine (40%) (0.97 mL,
2.8 mmol) in ether: mp 192-194 °C; FABMS 327 (MH+), 255
(M - 3,3-dimethyltriazene, 39.9) m/z 327.112497 (MH+),
C16H15N6Cl requires m/z 327.112 520; 1H NMR (400 MHz,
DMSO-d6) δ 9.87(s, 1H, NH), 8.56 (s, 1H, H-2), 8.41 (d, 1H, J
) 2.8 Hz, H-5), 8.14 (t, 1H, J ) 1.4 Hz, H-2′), 7.96 (dd, 1H, J
) 2.8, 11.9 Hz, H-7), 7.88 (dd, 1H, J ) 1.4, 10.8 Hz, H-4′),
7.74 (d, 1H, J ) 11.9 Hz, H-8), 7.38 (t, 1H, J ) 10.9 Hz, H-5′),
7.13 (dd, 1H, J ) 1.4, 10.9 Hz, H-6′), 3.56 (br, 3H, N(Me)2),
3.23 (br, 3H, N(Me)2); 13C NMR (100 MHz, DMSO-d6) δ 157.9,
153.6, 149.2, 148.5, 141.6, 133.3, 130.7, 129.4, 125.3, 123.5,
121.8, 120.8, 116.4, 115.0, 43.9, 37.1.
1-{4-[(3-Br om op h en yl)a m in o]-6-q u in a zolin yl}-3,3-d i-
m eth yltr ia zen e (14). As described for 12, triazene 14 was
obtained as a brown powder (85 mg, 72%), using 4 (100 mg,
0.32 mmol) and dimethylamine (40%) (0.3 mL) in ether: mp
179-180 °C; FABMS m/z 371.1 (MH+ with 79Br), 373.0 (MH+
with 81Br), 299 (M - dimethyltriazene, 39.6) m/z 371.061981
(MH+), C16H15N6Br requires m/z 371.061 980; 1H NMR (400
MHz, DMSO-d6) δ 8.72 (s, 1H, NH), 8.09 (s, 1H, H-2), 8.00-
7.51 (m, 5H, H-8,7,5,6′,2′), 7.26 (m, 2H, H-4′,5′), 3.58 (s, 3H,
N(Me)2), 3.29 (s, 3H, N(Me)2); 13C NMR (100 MHz, DMSO-d6)
δ 157.9, 153.6, 149.2, 148.5, 141.7, 130.9, 129.4, 126.3, 125.3,
124.6, 121.8, 121.1, 116.3, 115.0, 43.8, 37.0.
7.41 (m, 4H, H-5,7,8,6′), 7.26-7.24 (m, 2H, H-4′,5′), 3.91 (m,
2H, CH2CH2OCH3), 3.72 (t, 2H, J ) 6.8 Hz, CH2CH2OCH3),
3.43 (s, 3H, CH2CH2OCH3); 13C NMR (100 MHz, CDCl3) δ
157.9, 153.7, 149.5, 148.7, 141.8, 130.9, 129.4, 126.3, 125.4,
124.6, 121.8, 121.1, 116.3, 115.2, 68.7, 58.8, 44.0.
1-{4-[(3-Br om op h en yl)a m in o]-6-q u in a zolin yl}-3-b en -
zyltr ia zen e (16). As described for 11, triazene 16 was
obtained as a pale brown powder (90 mg, 65%), using 4 (100
mg, 0.32 mmol) and benzylamine (0.24 mL, 2.24 mmol) in
ether: mp 132-133 °C; FABMS m/z 433 (MH+ with 79Br), 435
(MH+ with 81Br), 405 (M - N2, 5.6), 315 (M - benzyldiazonium,
16.0), 299 (M - benzyltriazene, 13.9) m/z 433.077631 (MH+),
C
21H17N6Br requires m/z 433.077 730; 1H NMR (400 MHz,
DMSO-d6) δ (-NdN-NH signal not observed) 9.80 (s, 1H,
NH), 8.73 (s, 1H, H-2), 8.07-7.21 (m, 13H, ArH), 4.86 (s, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) δ 158.0, 153.8, 149.3,
148.8, 141.8, 137.1, 130.9 (2C overlap), 129.5, 129.1 (2C
overlap), 128.7, 127.9, 126.4, 125.6, 124.7, 121.8, 121.2, 116.3,
115.3, 48.1.
1-{4-[(3-Br om op h en yl)a m in o]-6-qu in a zolin yl}-3-(2′-p y-
r id ylm eth yl)tr ia zen e (17). As described for 11, triazene 17
was obtained as a brown powder (80 mg, 58%), using 4 (100
mg, 0.32 mmol) and 2-(aminomethyl)pyridine (0.23 mL, 2.24
mmol) in ether: mp 145-147 °C; FABMS m/z 433.92 (MH+
with 79Br), 435.95 (MH+ with 81Br), 406 (M - N2, 3.7), 315 (M
- pyridylmethyldiazonium, 21.5), 297 (M - pyridylmethyl-
triazene, 3.8) m/z 434.072880 (MH+), C20H16N7Br requires m/z
434.072 820; 1H NMR (400 MHz, DMSO-d6) δ (-NdN-NH
signal not observed) 9.89 (s, 1H, NH), 8.55 (d, 1H, J ) 9.2 Hz,
Py-H), 8.53 (s, 1H, H-2), 8.39 (s, 1H, H-2′), 8.23 (s, 1H, H-5),
7.94-7.90 (m, 2H, H-7, 6′), 7.76-7.74 (m, 2H, H-8, Py-H),
7.36-7.24 (m, 4H, H-5′, H4′, Py-H), 4.89 (s, 2H, CH2); 13C NMR
(100 MHz, DMSO-d6) δ 158.0, 156.7, 153.8, 149.7, 149.1, 148.8,
141.7, 137.4, 130.9, 129.5, 126.4, 125.6, 124.7, 123.0, 122.7,
121.8, 121.2, 116.3, 115.5, 49.6.
1-{4-[(N-Meth yl-N-p h en yl)a m in o]-6-qu in a zolin yl}-3-(2′-
p yr id ylm eth yl)tr ia zen e (18). As described for 11, triazene
18 was obtained as a brown powder (80 mg, 58%), using 8 (100
mg, 0.32 mmol) and 2-(aminomethyl)pyridine (0.23 mL, 2.24
mmol) in ether: mp 145-147 °C; FABMS 370 (MH+), 342 (M
- N2, 49.4), 251 (M - pyridylmethyldiazonium, 100.0), 235
(M - pyridylmethyltriazene, 47.6) m/z 370.178019 (MH+),
C
21H19N7 requires m/z 370.178 150; 1H NMR (400 MHz,
DMSO-d6) δ (-NdN-NH signal not observed) 8.77 (s, 1H,
H-2), 8.5-7.18 (m, 12H, ArH), 4.81 (s, 2H, CH2), 3.66 (s, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) δ 158.1, 153.8, 149.4,
149.2, 141.7, 137.1, 133.3, 130.7, 129.5, 129.2 (2C overlap),
129.1 (2C overlap), 127.9, 125.7, 123.6, 121.9, 120.9, 116.3,
115.3, 48.2.
1-{4-[(3-Br om oph en yl)am in o]-6-qu in azolin yl}-3-(2-N,N-
d im eth yla m in oeth yl)tr ia zen e (19). As described for 11,
triazene 19 was obtained as a pale brown powder (100 mg,
76%) using 4 (100 mg, 0.32 mmol) and N,N-dimethylethylene-
diamine (200 mg, 2.24 mmol) in ether: mp 130-132 °C;
FABMS m/z 414 (MH+ with 79Br), 416 (MH+ with 81Br), 386
(M - N2, 1.5), 315 (M - N,N-dimethylaminoethyldiazonium,
16.7), 299 (M - N,N-dimethylaminoethyltriazene, 7.6) m/z
1
414.104180 (MH+), C18H20N7Br requires m/z 414.103 990; H
NMR (400 MHz, DMSO-d6) δ 9.88 (br s, 1H, NdN-NH), 8.57
(s, 1H, NH), 8.41 (s, 1H, H-2), 8.26 (br s, 1H, H-2′), 7.95-7.73
(m, 3H, H-8, 7,6′), 7.32-7.24 (m, 3H, H-5,5′,4′), 3.65 (br, 2H,
CH2CH2N(CH3)2), 2.56 (t, 2H, J ) 6.8 Hz, CH2CH2N(CH3)2),
2.19 (s, 6H, N(CH3)2); 13C NMR (100 MHz, DMSO-d6) δ 157.1,
152.8, 148.8, 147.8, 141.0, 130.1, 128.6, 125.5, 124.6, 123.8,
121.0, 120.3, 115.5, 114.3, 55.3, 54.2 (2C overlap), 41.5.
1-{4-[(3-Br om op h en yl)a m in o]-6-qu in a zolin yl}-3-[2-(4-
m or p h olin o)eth yl]tr ia zen e (20). As described for 11, tri-
azene 20 was obtained as a brown powder (95 mg, 66%), using
4 (100 mg, 0.32 mmol) and 4-(2-aminoethyl)morpholine (229
mg, 2.24 mmol) in ether: mp 150-153 °C; FABMS m/z 456
(MH+ with 79Br), 458 (MH+ with 81Br), 429 (M - N2, 1.5), 328
(M - morpholinoethyltriazene, 2.0), 317 (M - morpholino-
ethyldiazonium, 6.4) m/z 456.114745 (MH+), C20H22N7OBr
requires m/z 456.114 620; 1H NMR (400 MHz, DMSO-d6) δ
1-{4-[(3-Br om oph en yl)am in o]-6-qu in azolin yl}-3-(2-m eth -
oxyeth yl)tr ia zen e (15). As described for 11, triazene 15 was
obtained as a brown powder (100 mg, 79%), using 4 (100 mg,
0.32 mmol) and 2-methoxyethylamine (0.21 mL, 2.24 mmol)
in ether: mp 141-143 °C; FABMS m/z 401 (MH+ with 79Br),
403 (MH+ with 81Br), 373 (M - N2, 9.0), 315 (M - methoxy-
ethyldiazonium, 42.1), 299 (M - methoxyethyltriazene, 38.3)
m/z 401.072546 (MH+), C17H17N6OBr requires m/ z, 401.072 420;
1H NMR (400 MHz, CDCl3) δ (-NdN-NH signal not observed)
8.73 (s, 1H, NH), 8.10 (s, 1H, H-2), 7.90 (s, 1H, H-2′), 7.82-

4320 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Rachid et al.
(-NdN-NH signal not observed) 8.74 (s, 1H, NH), 8.60 (s,
1H, H-2), 8.10-7.53 (m, 5H, H-8, 7, 5, 6′, 2′), 7.27-7.25 (m,
2H, H-5′, 4′), 3.77-3.74 (m, 6H, 3CH2), 2.74 (t, 2H, J ) 6.0
Hz, CH2), 2.53 (br, 4H, 2CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.0, 153.8, 149.6, 148.7, 141.8, 131.0, 129.5, 126.4, 125.5,
124.6, 121.8, 121.2, 116.3, 115.1, 67.0 (2C overlap), 55.3, 54.1
(2C overlap), 41.5.
1-{4-[(N-Met h yl-N-p h en yl)a m in o]-6-q u in a zolin yl}-3-
m eth yltr ia zen e (21). As described for 11, triazene 21 was
obtained as a brown powder (90 mg, 77%) using 8 (100 mg,
0.4 mmol) and methylamine (2.0 M solution in THF, 1.4 mL,
2.8 mmol) in ether: mp 152-154 °C; FABMS m/z 293.1 (MH+),
250 (M - methyldiazonium, 42.3), 235 (M - methyltriazene,
26.8) m/z 293.151470 (MH+), C16H16N6 requires m/z 293.151 380;
1H NMR (400 MHz, DMSO-d6) δ 10.41 (br s, 1H, NdN-NH),
8.75 (s, 1H, H-2), 7.70-6.85 (m, 8H, ArH), 3.54 (s, 3H, CH3),
2.85 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 161.0,
151.2, 149.0, 147.0, 143.5, 129.3 (2C overlap), 129.1, 126.0,
116.9, 115.3, 112.3 (2C overlap), 112.0, 43.9, 32.5.
1-{4-[(N-Meth yl-N-p h en yl)a m in o]-6-qu in a zolin yl}-3,3-
d im eth yltr ia zen e (22). As described for 12, triazene 22 was
obtained as a pale brown powder (55 mg, 45%) using 8 (100
mg, 0.4 mmol) and dimethylamine (40%) (0.4 mL, 2.8 mmol)
in ether: mp 140-142 °C; FABMS m/z 307 (MH+), 235 (M -
dimethyltriazene, 92.8), m/ z 307.167000 (MH+), C16H16N6
requires m/z 307.167 120; 1H NMR (400 MHz, DMSO-d6) δ 8.66
(s, 1H, H-2), 7.53-7.17 (m, 8H, ArH), 3.54 (s, 3H, CH3), 3.35
(br, 3H, N(Me)2), 3.10 (br, 3H, N(Me)2); 13C NMR (100 MHz,
DMSO-d6) δ 161.6, 153.5, 149.6, 148.5, 147.6, 130.7 (2C
overlap), 129.3, 127.0, 126.9, 126.3 (2C overlap), 117.3, 115.8,
43.2, 43.0, 37.4
1-{4-[(P h en ylm eth yl)a m in o]-6-qu in a zolin yl}-3-m eth yl-
tr ia zen e (23). As described for 11, triazene 23 was obtained
as a pale brown powder (97 mg, 83%), using 7 (100 mg, 0.4
mmol) and methylamine (2.0 M solution in THF, 1.4 mL, 2.8
mmol) in ether: mp 144-146 °C; FABMS m/z 293 (MH+), 265
(M-N2, 2.9), 250 (M - methyldiazonium, 30.2), 235 (M -
methyltriazene, 13.2) m/z 293.151470 (MH+), C16H16N6 re-
quires m/z 293.151 380; 1H NMR (400 MHz, DMSO-d6) δ 10.58
(br, s, 1H, NdN-NH), 8.86 (br, s, 1H, NH), 8.34 (s, 1H, H-2),
8.20 (s, 1H, H-5), 7.84 (d, 1H, J ) 11.4 Hz, H-7), 7.62 (d, 1H,
J ) 11.4 Hz, H-8), 7.33-7.20 (m, 5H, ArH), 4.75 (d, 2H, J )
6.8 Hz, CH2), 3.04 (d, 3H, J ) 3.2 Hz, CH3); 13C NMR (100
MHz, DMSO-d6) δ 160.0, 154.7, 149.0, 148.0, 140.2, 129.1,
128.9 (2C overlap), 127.9 (2C overlap), 127.3, 125.1, 116.0,
114.9, 44.3, 31.4.
1-{4-[(P h e n ylm e t h yl)a m in o]-6-q u in a zolin yl}-3,3-d i-
m eth yltr ia zen e (24). As described for 12, triazene 24 was
obtained as a brown powder (97 mg, 83%), using 7 (90 mg,
0.36 mmol) and dimethylamine (40%) (0.3 mL) in ether: mp
144 °C; FABMS m/z 307 (MH+), 235 (M - dimethyltriazene,
30.9), m/z 307.167000 (MH+), C16H16N6 requires m/z
307.167 120; 1H NMR (400 MHz, DMSO-d6) δ 8.83 (t, 1H, J )
6.0 Hz, NH), 8.34 (s, 1H, H-2), 8.17 (d, 1H, J ) 2.0 Hz, H-5),
7.84 (dd, 1H, J ) 2.0, 8.9 Hz, H-7), 7.62 (d, 1H, J ) 8.9 Hz,
H-8), 7.35-7.20 (m, 5H, ArH), 4.75 (d, 2H, J ) 6.0 Hz, CH2),
3.52 (br, 3H, N(Me)2), 3.18 (br, 3H, N(Me)2); 13C NMR (100
MHz, DMSO-d6) δ 159.9, 154.6, 148.6, 147.9, 140.2, 129.0,
128.9 (2C overlap), 127.9 (2C overlap), 127.3, 125.0, 116.0,
114.6, 44.3, 43.9, 37.5.
Cell Cu ltu r e. The human epidermoid carcinoma of the
vulva A431 was obtained from the American Type Culture
Collection (Manassas, VA). The mouse fibroblasts NIH3T3 and
NIH3T3neu (NIH3T3 cells stably transfected with ErbB2
gene) were generous gifts from Dr. Moulay Aloui-J amali of the
Montreal J ewish General Hospital. The A431 cell line was
maintained in RPMI-1640 supplemented with 10% fetal bovine
serum (FBS) and antibiotics as described previously.6 NIH3T3
and NIH3T3neu cells were maintained in DMEM supple-
mented with 10% FBS and antibiotics. All cells were main-
tained in an atmosphere of 5% CO2.
3.9-mm column (reverse phase). The operating mode was
isocratic and two different systems of solvents were used: the
first one was 57% acetonitrile and 43% water, and the second
system was 70% methanol and 30% water (pH8) with a 0.5
mL/min flow rate at 250 nm.
Degr a d a tion . The study of the conversion of the combi-
triazenes to their corresponding aminoquinazolines was per-
formed by spectrofluorometer as the latter amines were
fluorescent (absorption 290 nm, emission 450 nm). Briefly, 125
µM of the combi-triazenes was added to RPMI-1640 with 10%
FBS and incubated for 4 h at 37 °C in a microplate spectro-
fluorometer (SpectraMax Gemini fluorescence reader, Molec-
ular Device, CA). The data were analyzed by the SoftMaxPro
(Molecular Device, CA) and GraphPad software packages.
Tyr osin e Kin a se Assa ys. This assay is similar to the one
described previously.6 Nunc Maxisorp 96-well plates were
incubated overnight at 37 °C with 100 µL/well of 0.25 mg/mL
PGT in PBS. The kinase reaction was performed by using 4.5
ng/well EGFR affinity-purified from A431 cells. The compound
was added and phosphorylation initiated by the addition of
ATP (50 µM). After 8 min at room temperature with constant
shaking, the reaction was terminated by aspiration mixture
and by rinsing the plate four times with wash buffer. Phos-
phorylated PGT was detected following a 25 min incubation
with 50 µL/well of HRP-conjugated PY20 anti-phosphotyrosine
antibody (Santa Cruz Biotechnology) diluted to 0.2 µg/mL in
blocking buffer (3% bovine serum albumin, 0.05% Tween 20
in PBS). Antibody was removed by aspiration, and the plate
washed four times with wash buffer. The signals were devel-
oped by the addition of 50 µL/well of 3,3′,5,5′-tetramethylben-
zidine peroxidase substrate (Kirkegaard and Perry Laborato-
ries, Gaithersberg, MD), and following blue color development,
50 µL of H2SO4 (0.09 M) was added per well, and plates were
read at 450 nm using a Bio-Rad ELISA reader (model 2550).
In Vitr o Gr ow th In h ibition Assa y. (A) To study the effect
of our compounds on serum stimulated-proliferation, cells
(NIH3T3 and NIH3T3neu) were grown to 70% of confluence
in 96-well plates and washed twice with PBS after which they
were exposed to serum-free medium for 18 h. Cells were
exposed to each drug and serum for 72 h and cell growth
measured using the sulforhodamine B (SRB) assay.20
(B) To study the antiproliferative effects of our compounds
in basal growth condition and under continuous exposure,
A431 cells were grown to 70% of confluence in 96-well plates
for 24 h. Thereafter, cells were exposed to different concentra-
tions of each drug for 96 h. Growth inhibition was evaluated
using the SRB assay.
Alk a lin e Com et Assa y for Qu a n tita tion of DNA Da m -
a ge. The alkaline comet assay was performed as previously
described.6 The cells were exposed to drugs for 30 min,
harvested with trypsin-EDTA, and resuspended in PBS. Cell
suspensions were diluted to approximately 106 cells and mixed
with agarose (1%) in PBS at 37 °C in a 1:10 dilution. The gels
were cast on Gelbond strips (Mandel Scientific, Guelph,
Canada) using gel casting chambers and then immediately
placed into a lysis buffer [2.5 M NaCl, 0.1 M tetrasodium-
EDTA, 10 mM Tris-base, and 1% (v/v) Triton X-100, pH 10.0].
After being kept on ice for 30 min, the gels were gently rinsed
with distilled water and immersed in a second lysis buffer (2.5
M NaCl, 0.1 M tetrasodium-EDTA, 10 mM Tris-base) contain-
ing 1 mg/mL proteinase K for 60 min at 37 °C. Thereafter,
the gels were rinsed with distilled water, incubated in alkaline
electrophoresis buffer for 30 min at 37 °C, and electrophoresed
at 19 V for 20 min. The gels were subsequently rinsed with
distilled water and placed in 1 M ammonium acetate for 30
min. Thereafter, they were soaked in 100% ethanol for 2 h,
dried overnight, and stained with SYBR Gold (1/10000 dilution
of stock supplied from Molecular Probes, Eugene, OR) for 20
min. Comets were visualized at 330× magnification, and DNA
damage was quantitated using the Tail Moment parameter
(i.e., the distance between the barycenter of the head and the
tail of the comet multiplied by the percentage of DNA within
the tail of the comet). A minimum of 50 cell comets were
P u r ity of Com p ou n d s. Purity analyses were performed
by high performance liquid chromatography (HPLC) using a
Spectrasystem (Thermoquest) and a Waters C4 15-µm 300 ×

The Combi-Targeting Concept
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4321
sensitive tyrosine kinase inhibitor. Clin. Cancer Res. 2001, 6,
2053-2063.
analyzed for each sample, using ALKOMET version 3.1 image
analysis software.
(10) Hemens, C. M.; Manning, H. W.; Vaughan, K.; LaFrance, R. J .;
Tang, Y. Open-chain nitrogen compounds. Part V. Hydroxy-
methyltriazenes: synthesis of some new alkyl homologues of the
antitumour 3-methyl-3-hydroxymethyltriazenes and preparation
of the derived acetoxymethyl-, benzoyloxymethyl- and meth-
oxymethyltriazenes. Can. J . Chem. 1984, 62, 741-748.
(11) Lassiani, L.; Nisi, C.; Sigon, F.; Sava, G.; Giraldi, T. Synthesis
of 1-aryl-3-formyl-3-methyltriazenes. Potential metabolites of
1-aryl-3,3-dimethyltriazenes. J . Pharm. Sci. 1980, 69, 1098-
1099.
(12) Kolar, G. F.; Schlesiger, J . Urinary metabolites of 3,3-dimethyl-
1-phenyltriazene. Chem. Biol. Int. 1976, 14, 301-311.
(13) Roth, G. A.; Tai, J . J . A new synthesis of aryl substituted
quinazolin-4(1H)-ones. J . Heterocycl. Chem. 1996, 33, 2051-
2053.
(14) Rewcastle, G. W.; Denny, W. A.; Bridges, A. J .; Hairong, Z.; Cody,
D. R.; McMichael, A.; Fry, D. W. Tyrosine kinase inhibitor.
Synthesis and structure-activity relationships for 4-[(phenyl-
methyl)amino]- and 4-(phenylamino)quinazolines as potent ad-
enosine 5′-triphosphate binding site inhibitors of the tyrosine
kinase domain of the epidermal growth factor receptor. J . Med.
Chem. 1995, 38, 3482-3487.
(15) Rewcastle, G. W.; Murray, D. K.; Elliott, W. L.; Fry, D. W.;
Howard, C. T.; Nelson, J . M.; Roberts, B. J .; Vincent, P. W.;
Showalter, H. D.; Winters, T. R., Denny, W. A. Tyrosine kinase
inhibitors. 14. Structure-activity relationships for methyl-
amino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-
(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780) a potent and
specific inhibitor of the tyrosine kinase activity of receptors for
the EGF family of growth factors. J . Med. Chem. 1998, 41, 742-
751.
(16) Rewcastle, G. W.; Bridges, A. J .; Fry, D. W.; Rubin, J . R.; Denny,
W. A. Tyrosine kinase inhibitors. Synthesis and structure-
activity relationships for 6-substituted 4-(phenylamino)pyrimidino-
[5,4d]pyrimidines designed as inhibitors of the epidermal growth
factor receptor. J . Med. Chem. 1997, 40, 1820-1826.
(17) Bull, V. L.; Tisdale, M. J . Antitumor imidazotetrazines XVI:
Macromolecular alkylation by 3-substituted imidazotetrazinones.
Biochem. Pharmacol. 1987, 36, 3215-3220.
(18) Baig, G. U.; Stevens, M. F. G. Antitumor imidazotetrazines. Part
12. Reactions of mitozolomide and its 3-alkyl congeners with
oxygen, nitrogen, halogen, and carbon nucleophiles. J . Chem.
Soc., Perkin. Trans 1 1987, 665-667.
(19) Catapano, C. V.; Broggini, M.; Erba, E.; Ponti, M.; Mariani, L.;
Citti, L.; D’incalci, M. In vitro and in vivo methazolastone-
induced DNA damage and repair in L-1210 leukemia sensitive
and resistant to chloroethylnitrosoureas. Cancer. Res. 1987, 47,
4884-4889.
(20) Skehan, P.; Storeng, R.; Scudiero D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anti-cancer drug screen-
ing.J . Natl. Cancer Inst. 1990, 82, 1107-1112.
Ack n ow led gm en t. We thank the Canadian Insti-
tute of Health Research (CIHR) for financial support.
We also acknowledge the National Cancer Institute of
Canada (NCIC) for an equipment grant that supported
the purchase of our scanning microplate reader. We are
grateful to the Cancer Research Society Inc for its
continuous support of the development of the “combi-
targeting” concept.
Su ppor tin g In for m ation Available: NMR spectra, HRMS
data, and HPLC chromatograms. This material is available
free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Meden, H.; Kuhn, W. Overexpression of the oncogene c-erbB-2
(HER2, neu) in ovarian cancer: A new prognostic factor. Eur.
J . Obstetr. Gynecol. Reprod. Biol. 1997, 71, 173-179.
(2) Modjtahedi, H.; Dean, C. The receptor for EGF and its ligands:
expression, prognostic value and target for tumour therapy. Int.
J . Oncol. 1998, 4, 277-296.
(3) Xinmei, C.; Yeung, T. K.; Wang, Z. Enhanced drug resistance
in cells coexpressing erb2 with EGF receptor or erb3. Biochem.
Bioph. Res. Commun. 2000, 277, 757-763.
(4) Hsieh, S. S.; Malerczyk, C.; Aigner, A.; Czubayko, F. ErbB-2
expression is rate-limiting for epidermal growth factor-mediated
stimulation of ovarian cancer cell proliferation. J . Cancer 2000,
86, 644-651.
(5) Matheson, S.; McNamee, J . P.; J ean-Claude, B. J . Design of a
chimeric 3-methyl-1,2,3-triazene with mixed receptor tyrosine
kinase and DNA damaging properties: A novel tumour targeting
strategy. J . Pharm. Exp. Ther. 2001, 296, 832-840.
(6) Brahimi, F.; Matheson, S.; McNamee, J . P.; Tari, A.; J ean-
Claude, B. J . Inhibition of epidermal growth factor receptor-
mediated signaling by “combi-triazene” BJ 2000, a new probe for
the Combi-Targeting postulates. J . Pharm. Exp. Ther. 2002, 303,
238-246.
(7) Matheson, S. L.; McNamee, J . P.; J ean-Claude, B. J . Differential
responses of EGFR-/-AGT-expressing cells to the “combi-tri-
azene” SMA41. Cancer Chemother. Pharmacol. 2003, 51, 11-
20.
(8) Qiu, Q.; Dudouit, F.; Matheson, S. L.; Brahimi, F.; Banerjee, R.;
McNamee, J . P.; J ean-Claude, B. J . The Combi-Targeting
Concept:
a novel 3,3-disubstituted nitrosourea with EGFR
tyrosine kinase inhibitory properties. Cancer Chemother. Phar-
macol. 2003, 51, 1-10.
(9) Ciardiello, F.; Caputo, R.; Bianco, R.; Damianco, V.; Pomatico,
G.; De Placido, S.; Bianco, A. R.; Tortora, G. Antitumour effect
and potentiation of cytotoxic drugs activity in human cancer cells
by ZD-1839 (Iressa), an epidermal growth factor receptor-
J M030142E