The combi-targeting concept: Synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR)

Article abstract of DOI:10.1021/jm0600390

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3′-Cl and Br series) with small angles (0.5-3°) were generally stronger EGFR TK inhibitors than those with large angles (18-21°). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC₅₀ values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t_1/2), EGFR TK inhibitory potency (IC₅₀), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

Full text of DOI:10.1021/jm0600390

3544
J. Med. Chem. 2006, 49, 3544-3552
The Combi-Targeting Concept: Synthesis of Stable Nitrosoureas Designed to Inhibit the
Epidermal Growth Factor Receptor (EGFR)
Juozas Domarkas, Fabienne Dudouit, Christopher Williams, Qiu Qiyu, Ranjita Banerjee, Fouad Brahimi, and
Bertrand Jacques Jean-Claude*
Cancer Drug Research Laboratory, Department of Medicine, DiVision of Medical Oncology, McGill UniVersity Health Center/
Royal Victoria Hospital, Montreal, H3A1A1, Quebec, Canada
ReceiVed January 12, 2006
According to the “combi-targeting” concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds
termed “combi-molecules” is critical for selective growth inhibition of tumor cells with disordered expression
of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory
potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and
ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their
EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the
ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3′-Cl and Br series) with small
angles (0.5-3°) were generally stronger EGFR TK inhibitors than those with large angles (18-21°). This
was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant
binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative
studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the
erbB2 oncogene, showed that IC50 values for inhibition of EGFR TK could be good predictors of their
selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r ) 0.8).
On the basis of stability (t1/2), EGFR TK inhibitory potency (IC50), and selective erbB2 targeting, compound
23, a stable nitrosourea, was considered to have the structural requirements for further development.
1. Introduction
The combi-targeting concept postulates that molecules termed
“combi-molecules”, designed to block growth factor induced
signaling on their own and to further degrade to a DNA-
damaging species, should induce significant cell-killing in
tumors resistant to classical alkylating agents of the same class.
SMA41 and BJ2000 (Figure 1), the first molecular probes
designed to verify the combi-targeting postulates, were shown
to strongly block the epidermal growth factor receptor tyrosine
kinase (EGFR TK) activity on their own in a short exposure
Figure 1. Structures of the first molecular probes SMA41 and BJ2000.
enzyme assay.1,2 In addition, these molecules degraded in cell
culture medium to generate a free aminoquinazoline, also
capable of blocking EGFR TK on its own, and a DNA-alkylating
methyldiazonium species. The formation of the latter has been
proven by radiotracing.3 Thus, as outlined in Scheme 1, the
combi-molecules TZ-I block EGFR TK on their own and
degrade to another inhibitor I plus TZ, a DNA-damaging species.
Both combi-molecules, SMA41 and BJ2000, were shown to
possess significantly stronger potency than the clinical drug
Temozolomide in cells coexpressing the DNA repair enzyme
O6-alkylguanine transferase (AGT) and EGFR.4 SMA41 and
BJ2000 being 1,2,3-triazenes, their half-lives were less than 1.5
h. Thus, to study the combi-targeting principles with more stable
probes and to enhance their potency by releasing a bifunctional
chloroethylating agent, we synthesized FD137 (22), a nitrosourea
designed to release, like the triazenes, a free aminoquinazoline
and a DNA-alkylating species.5,6 While FD137 was extremely
Scheme 1. Hydrolytic Pathway and Interactions Involved in the
“Combi-Targeting” Strategy
synthesizing a series of compounds with variable substituents
at R on the ureido moiety and X on the aniline portion.
Molecular modeling was used to rationalize the results and to
develop a predictive equation for EGFR TK inhibitory potency
of these agents based upon protein/ligand interaction energy
(Eint), ligand bond stretch (Estr) and total strain energy (dEstrain).
We also studied the correlation between the IC50 values for
EGFR TK inhibition and the erb selectivity of these compounds
in a pair of NIH3T3 wild-type and erbB2-transfected (NIH3T3/
neu) cells. It should be remembered herein that the erbB2
oncogene is overexpressed in a variety of solid tumors including
breast, ovary, and prostate cancer cells and that this is associated
with aggressive tumor progression and poor prognosis in the
clinic.7-10
stable (t1/2 ) 41 h), its EGFR TK inhibitory activity (IC50
)
1000 nM) was more than 5-fold weaker than those of the
triazenes. Here, we attempted to optimize the EGFR TK potency
of this new class of molecules, termed combi-nitrosoureas, by
* Corresponding author. Phone: (514) 934-1934 ext. 35841. Fax: (514)
843-1475. E-mail: bertrandj.jean-claude@mcgill.ca.
10.1021/jm0600390 CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/20/2006

Combi-Targeting Synthesis of Nitrosoureas
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3545
Scheme 2. Synthesis of Amines 7-12a
a (i) H2SO4/formic acid/reflux; (ii) PCl5/160 °C; (iii) 3-methylaniline/2-propanol/60 °C; (iv) DMF-dimethyl acetal/reflux; (v) ArNH2/AcOH/reflux; (vi)
Pd(5%)/H2(2 bar)/room temperature (X ) CH3) or Fe/AcOH/EtOH/H2O/reflux (X ) Cl, Br); (vii) EtOCOCl/pyridine/0 °C; (viii) LiAlH4/THF/reflux.
Scheme 3. Synthesis of Ureas 13-18 and Nitrosoureas 19-24a
a (i) 2-Chloroethylisocyanate/THF/0 °C; (ii) NOBF4/AcOH/CH3CN/0 °C.
2. Results and Discussion
giving amidine 3, which was cyclized in refluxing acetic acid
in the presence of corresponding aniline to provide 6-nitro-
quinazolines 5 and 6. Reduction of these nitro compounds with
Fe gave the targeted amines 8 and 9 in good yield. 6-Amino-
quinazolines 7-9 were converted to their N-methyl analogues
10-12 following a slightly modified procedure published by
Bridges et al.15 First, amines 7-9 were treated with ethyl
chloroformate, and then the resulting carbamates were reduced
with LiAlH4. The aminoquinazolines 7-12 thus generated were
treated with 2-chloroethylisocyanate to provide ureas 13-18,
which were subsequently nitrosated with NOBF4/AcOH to give
nitrosoureas 19-24. The latter compounds were purified by
2.1. Chemistry. The synthesis of the series proceeded as
depicted in Schemes 2 and 3. 6-Aminoquinazolines 7-9 were
prepared in three steps from nitroanthranilonitrile 1 as previously
described.11 Briefly, anthranilonitrile 1 was heated at reflux in
formic acid containing concentrated H2SO4 to give 4-oxo-
quinazoline 2,11 which was chlorinated with PCl5 in fusion.12
The resulting 4-chloroquinazoline was treated with 3-methyl-
aniline to yield 6-nitroquinazoline 4 which was catalytically
reduced to amine 7.13 Aminoquinazolines 8 and 9 were
synthesized as per the procedure described by Tsou et al.14
Briefly, anthranilonitrile 1 was condensed with DMF-acetal

3546 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Domarkas et al.
Figure 2. Three-dimensional views of energy-minimized structures of 19 (N3-H) and 22 (N3-methylated) nitrosoureas. Minimization was done
and views generated by CS Chemdraw 3D Ultra 7.0 software, using the empirical MM2 method. See the Experimental Section for details.
Table 1. Half-lives (t1/2) of the Combi-Nitrosoureas
half-life
Table 2. Biological Activities of Aminoquinazolines,
Ureidoquinazolines and Their Corresponding Combi-Nitrosoureas
compd
(t1/2)
19
20
21
22
23
24
30 min
15 min
26 min
41 ha
21 h 27 min
26 h
a See ref 5.
IC50, µMb
column chromatography on silica gel with a mixture of ethyl
acetate/petroleum ether as eluent. The structures of various
compounds were confirmed by 1H, 13C NMR, and high-
resolution mass spectrometry (HRMS).
2.2. Biology. 2.2.1. Stability of Combi-Nitrosoureas. As
expected, the NH-unsubstituted nitrosoureas 19-21 were sig-
nificantly less stable (t1/2 ) 15-30 min) than their N3-Me
analogues 22-24, the most stable compound remaining the 3′-
methylanilinonitrosourea 22 (t1/2 ) 41 h) (Table 1). The half-
lives of the 3′-halogenated compounds 23 and 24 were shorter
(t1/2 ) 21-26 h), perhaps due to a more electron-withdrawing
effect on the quinazoline ring that may translate into a relatively
decreased electron density at the ureido N3 nitrogen.
2.2.2. EGFR Inhibitory Activity. Structure-activity rela-
tionships in the quinazolines already demonstrated that com-
pounds containing a 3′-X ) Br or Cl are consistently more
potent than those with a methyl group.13 This trend was observed
in all the series of compounds used herein whether they were
aminoquinazolines, ureas or nitrosoureas. Many compounds with
3′-X ) Me showed IC50 in the 1 µM range, indicating little
tolerance of bulkiness in the hydrophobic pocket of the ATP
site of EGFR. In the amine series, the N-methylaminoquinazo-
lines 10-12 were consistently more active than the primary
amino ones 7-9. This is in agreement with previous studies
that showed significantly lower IC50 values for N-monoalkylated
aminoquinazolines when compared with those of the corre-
sponding primary amines.15 Carbamoylation of the primary
6-aminoquinazolines resulted in compounds with increased
EGFR TK inhibitory potency. In contrast, carbamoylation of
N-methylaminoquinazolines consistently decreased EGFR TK
inhibitory activities with a more pronounced effect in the
3′-methyl analogues. Finally, the nitrosation of N1 in the ureas,
which slightly increases the bulkiness of the ureido moiety, did
not show a consistent trend in the series.
angle IC50 nM
no. R1
R2
X
dega EGFR TK NIH3T3 NIH3T3/neu
7
8
9
H
H
H
H
H
H
H
H
H
Me
Cl
Br
Me
Cl
Br
1000
200
44
11
4
44.2
97.0
26.7
42.8
49.8
17.6
31.0
157.1
35.9
21.8
150.0
60.5
78.5
157.0
36.4
16.5
11.7
10.8
6.39
3.29
0.67
2.07
0.89
0.17
2.10
1.35
1.77
11.80
23.03
11.08
12.18
5.40
1.15
6.88
0.68
3.06
10 Me
11 Me
12 Me
5
13
14
15
H
H
H
ClEtNHCO
ClEtNHCO
ClEtNHCO
Me 0.69
676
22
18
Cl
Br
2.92
0.53
16 Me ClEtNHCO
17 Me ClEtNHCO
18 Me ClEtNHCO
Me 19.44
Cl 19.00
Br 19.12
1693
81
156
1061
67
19
20
21
H
H
H
ClEtN(NO)CO Me 0.45
ClEtN(NO)CO Cl
ClEtN(NO)CO Br
0.56
0.45
47
22 Me ClEtN(NO)CO Me 17.65
23 Me ClEtN(NO)CO Cl 17.61
24 Me ClEtN(NO)CO Br 21.48
1139
204
294
a Torsion angles between ureido or nitrosoureido moieties and the
quinazoline ring. b IC50 values for inhibition of serum-stimulated growth
after a 24 h of cell starvation.
to a less planar structure, a geometry that may hinder its binding
in the ATP pocket of EGFR. Typical geometry-optimized
structures of 19 and 22 are shown in Figure 2, and values for
the calculated torsion angles between N3-H or N3-Me and the
aromatic H7, representing the deviation of the ureido function
from the quinazoline ring plane, are given in Table 2.
The nitrosourea 19 adopts an almost planar conformation with
nitrosoureas function forming an internal hydrogen bond and a
remarkably small torsion angle between N3-H and H7. The
formation of intramolecular hydrogen bonds in nitrosoureas is
now well documented.16-18 In contrast, in the N3-Me nitrosourea
22, the oxygen of the nitroso group is pointed away from the
methyl group and a torsion angle of 17.7° is observed between
the latter group and H7, leaving a ureido group markedly
deviated from the plane of the quinazoline ring. Similar results
were obtained in the urea series, the trend being a small torsion
angle (0.5-3°) for N3-H-containing ureas 13-15 and relatively
larger angles (∼19°) for N3-Me ureas 16-18. As shown in the
We surmised that carbamoylation of N-methylamino com-
pounds led to a decrease in EGFR TK inhibitory activity due
to the ability of the N3-Me group to impose a torsion angle
between the plane of the aromatic quinazoline bicycle and that
of the ureido function of the resulting molecules. This may lead

Combi-Targeting Synthesis of Nitrosoureas
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3547
Figure 3. Relationship between the IC50 (EGFR TK) of the 3′-Cl and
Br series and the torsion angles between the quinazoline bicycle and
the ureido and nitrosoureido moieties.
Figure 5. Predicted IC50 (EGFR TK) values vs the experimental IC50
(EGFR TK). (Pearson r ) 0.89, P (one-tailed) < 0.0001).
servations that increasing the angle between the carbonyl and
quinazoline planes could result in clashes with the receptor
prompted further investigation of this effect as a possible
explanation for the decreased activity of the N3-methylated
compounds. A partial least-squares linear regression model of
pIC50 (EGFR, M) was constructed in MOE using the MMFF94x
energies as descriptors. The total protein/ligand interaction
energy (EINT) was used along with the ligand bond stretch (ESTR
)
and total (dESTRAIN) strain energies to construct a QSAR model
for compounds 7-24. The resulting QSAR equation (given
below) has an r2 ) 0.80 and reflects the expected trend that
increasing the strain energy values (ESTR) and (dESTRAIN) will
lower the pIC50, while increasingly negative van der Waals
interaction energies (Evdw) will result in higher pIC50.
pIC50 (EGFR, M) ) 6.96 - 0.256Evdw - 0.256dEstrain
-
Figure 4. Compounds 7-24 overlaid in the EGFR TK ATP binding
pocket. The carbonyl oxygen in each compound is shown as a ball.
Structures with R ) CH3 are in yellow, while those of amines 7-12
and all others with R ) H are colored blue.
0.73468Estr
The plot of the predicted pIC50 versus the experimental values
in Figure 5 shows that the QSAR model does reflect the activity
with a general linear trend. The combination of this QSAR
equation and observations made about the optimized ligands in
the binding pocket suggest that binding of the N3-methylated
ureas implies a greater deformation of the 6-position side chain
than is required for the nonmethylated ureas. Although the
methylated compounds can be made to fit into the binding
pocket, this is only achieved at the cost of increasing the ligand
strain energy.
2.3. Tumor Selectivity. Overexpression of EGFR and/or its
closest related HER-2 receptor is correlated with unfavorable
prognosis in patients with prostate and breast cancers. Over-
expression of these receptors is also associated with reduced
sensitivity to chemotherapeutic agents.20-23 To test the potency
and selectivity of combi-molecules, we recently established a
model in which the ability to block serum-stimulated prolifera-
tion of the NIH3T3 cell line is compared with that of NIH3T3
cells transfected with HER-2 (NIH3T3/neu).24,25
In the current study, a statistically significant correlation was
observed between IC50 values for inhibition of EGFR TK and
selective blockade log[IC50(3T3/neu)/IC50(3T3)] of serum-
stimulated growth of HER-2-transfected cells by the various
compounds (Figure 6). These results indicate that IC50 values
for inhibition of EGFR TK represent good predictors for the
HER-2 selective potency of all the compounds analyzed herein,
including the nitrosoureas, in this type of assay. This property
may in principle be associated with the ability of the inhibitors
to block serum-stimulated phosphorylation of the HER-2 gene
product and subsequent growth signaling. However, in non-
growth-stimulated continuous exposure assays using the EGFR-
expressing human prostate carcinoma cells LNCaP, we found
Figure 3, compounds with the large torsion angle were less
active than those with small angles. As the range of IC50 values
for the 3′-X ) Me series was significantly different from those
of the halogenated compounds, they were not plotted.
2.2.3. Conformation of Amines, Ureas and Nitrosoureas
Docked into the EGFR ATP Binding Site. Analysis of X-ray
structures of the TK domain of EGFR cocrystallized with
4-anilinoquinazoline showed that the pocket is quite narrow
around the anilino and quinazoline ring systems but begins to
open up a few angstroms away from the 6- and 7-positions of
the quinazoline ring.19
A clear rationale for the geometry-dependent potency of the
carbamoylated derivatives is depicted in Figure 4 wherein an
overlay of all the refined compounds 7-24 in the EGFR TK
ATP binding pocket is presented. The carbonyl oxygen in each
compound is rendered as a ball for ease of visualization. The
color scheme reflects the substitution at the R-position. Struc-
tures with R ) CH3 are in yellow, while structures of amines
7-12 and all other structures with R ) H are colored blue.
Although all the structures may be docked and refined without
unreasonable energy clashes, the view in Figure 4 clearly shows
that the N3-methylated compounds have urea groups more out-
of-plane with respect to the quinazoline ring than the nonmeth-
ylated ones. Furthermore, as the carbonyl group rotates out-of-
plane with respect to the quinazoline ring, the carbonyl oxygen
begins to collide with the van der Waals interaction surface,
potentially increasing the van der Waals clash and decreasing
binding affinity.
2.2.4. Defining a Structure-Activity Relationship (SAR)
Based on Ligand-Receptor Interaction Energies. The ob-

3548 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Domarkas et al.
free amines are reversible inhibitors deprived of alkylating
functionalities. There thus remains the fact that the nitrosoureas
methylated at N1 (23 and 24), that can be hydrolyzed to the
amine inhibitor without the possibility of releasing toxic
isocyanates and that show reasonable stability, are the most
suitable for further development as useful drugs. The adverse
effects of their high torsion angles are compensated by the 3′-
halogens, which are less bulky than the methyl group of 22,
translating into 3-5-fold stronger inhibitory activity. Therefore,
taking into account the EGFR TK affinity, the degradation half-
life, the selectivity, and the antiproliferative potency, combi-
molecule 23, now referred to as JDA58, was considered to show
the optimal profile and was chosen for further investigation. It
has been recently selected by the National Cancer Institute (NCI)
of the United States for further development under NSC no.
741282. A complete study characterizing its degradation in cells
in Vitro and in ViVo, its mechanism of action, as well as its
potency in a xenograft model in SCID mice will be reported
shortly elsewhere.
Figure 6. Correlation between IC50 (EGFR TK) and selective serum-
stimulated growth inhibition of NIH3T3/neu-transfected cells (Pearson
r ) 0.80, P (one-tailed) < 0.0001).
3. Experimental Section
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 tetrameth-
ylsilane. Mass spectrometry was performed by the McGill Univer-
sity Mass Spectroscopy Center. Electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI) spectra were
performed on a Finnigan LC QDUO spectrometer. 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 basic alumina or
SiliCycle 40-63 µm ultrapure silica gel. Melting points were
determined in open capillary tubes on a Meltemp melting point
apparatus and were uncorrected.
Figure 7. Growth inhibitory effects of amine 12 and nitrosourea 24
on the LNCaP/EGFR prostate cancer cell line. Cells were exposed to
both drugs for 6 days, and growth inhibition measured using SRB assay.
Each point represents at least two independent experiments run in
triplicate.
that the antiproliferative potency of the stable nitrosourea 24
(IC50 (EGFR TK) ) 294 nM) was approximately 3-fold greater
than that of amine 12 (IC50 (EGFR TK) ) 5 nM) that it is
designed to release (see Figure 7). Also, it was more than 100-
fold more potent than the clinical drug bis(2-chloroethyl)-N-
nitrosourea (BCNU) (data not shown). The enhanced potency
of 24 may be imputed to its ability to not only degrade to another
inhibitor but also to release a DNA-alkylating species, as
previously demonstrated for 22.5
In summary, in the context of the combi-targeting principles,
the combi-nitrosoureas (TZ-I), like other combi-molecules, were
designed to degrade into an amine inhibitor (I) and the DNA-
damaging metastable chloroethyldiazonium (TZ) species. These
properties were previously demonstrated with FD137 (22),
which was a weak EGFR TK inhibitor and presented an
3.1. 4-Hydroxy-6-nitroquinazoline (2). 5-Nitroanthranilonitrile
1 (16.3 g, 100 mmol, 1.0 equiv) was added portionwise over 1 h
to a mildly refluxing formic acid solution (200 mL) containing
concentrated sulfuric acid (12 mL). After an additional 30 min,
the solution was cooled to 0 °C and poured on crushed ice. The
resulting precipitate was collected by filtration, washed with water,
and dried to give 4-hydroxyquinazoline 2 (17.9 g, 90% yield). Mp
1
283-285 °C; H NMR (400 MHz, DMSO-d6) δ 12.72 (br, 1H,
OH), 8.76 (d, 1H, J ) 2.8 Hz, H-5), 8.50 (dd, 1H, J ) 8.8 Hz,
J ) 2.8 Hz, H-7), 8.28 (s, 1H, H-2), 7.83 (d, 1H, J ) 8.8 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.
3.2. N4-(3-Methylphenyl)-4,6-quinazolinediamine (7). A mix-
ture of hydroxyquinazoline 2 (10.0 g, 52.4 mmol, 1.0 equiv) and
PCl5 (16.4 g, 78.5 mmol, 1.5 equiv) was melted and stirred for 2.5
h at 160 °C. The mixture was allowed to cool to room temperature,
and the resulting solid was well washed with petroleum ether,
filtered, and dried to give the 4-chloroquinazoline intermediate used
directly in the next step.
4-Chloroquinazoline (4.8 g, 22.7 mmol, 1.0 equiv) was suspended
in 2-propanol (25 mL), and 3-methylaniline (4.9 mL, 45.3 mmol,
2.0 equiv) was added. The resulting mixture was stirred at 60 °C
overnight, the precipitate collected by hot filtration, washed with
anomalously long half-life in serum-containing media (t1/2
)
41 h). On one hand, compounds 19-21 being primary ni-
trosoureas, although showing good EGFR TK inhibitory activi-
ties, have the potential to generate toxic isocyanates in a manner
similar to that of the clinical drug BCNU26 (Scheme 4). Their
instability in serum-containing medium (see Table 1) will
preclude their development as useful drugs. On the other hand,
Scheme 4. Decomposition of BCNU

Combi-Targeting Synthesis of Nitrosoureas
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3549
2-propanol, water, diethyl ether, and dried under vacuum to give 4
(4.4 g, 70% yield). Mp 248-250 °C; 1H NMR (400 MHz, DMSO-
d6) δ 10.35 (s, 1H, NH), 9.63 (s, 1H, H-5), 8.67 (s, 1H, H-2), 8.51
(dd, 1H, J ) 9.2 Hz, J ) 2.4 Hz, H-7), 7.88 (d, 1H, J ) 9.2 Hz,
H-8), 7.63 (d, 1H, J ) 8.0 Hz, H-4′), 7.61 (s, 1H, H-2′), 7.28 (t,
1H, J ) 7.8 Hz, H-5′), 7.69 (d, 1H, J ) 7.8 Hz, H-6′), 2.34 (s, 3H,
PhCH3).
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.
3.6. 4-[(3-Methylphenyl)amino]-6-(methylamino)quinazoline
(10). General Example of the Reductive Methylation Procedure.
Amine 7 (1.0 g, 4.0 mmol, 1.0 equiv) was dissolved in anhydrous
pyridine (20 mL), the solution was cooled to 0 °C in an ice bath,
and 2-chloroethylchloroformate (0.4 mL, 4.4 mmol, 1.1 equiv) was
added dropwise. Stirring continued at room temperature until
disappearance of starting material as monitored by TLC (0.5-1
h). Pyridine was removed under reduced pressure, and the solid
obtained was washed with 10% acetic acid solution and water until
neutrality and dried giving carbamate, used directly in the next step.
Ethyl carbamate (1.3 g, 4.0 mmol, 1.0 equiv) was dissolved in
anhydrous THF (150 mL) and cooled in an ice bath. LiAlH4 (0.6
g, 16.0 mmol, 4.0 equiv) was added by small doses, and mixture
was heated at reflux until disappearance of starting material as
monitored by TLC (1 h). The solution was cooled in an ice bath,
and the excess of hydrate was destroyed with 30% KOH (4 equiv)
solution. Insoluble materials were removed by filtration through
Celite, and the filtrate was concentrated under reduced pressure.
Chromatography on basic alumina eluting with ethyl acetate gave
6-methylamino-quinazoline 10 (0.37 g, 35% yield). Mp 115-117
A suspension of 4 (5.5 g, 20.0 mmol, 1.0 equiv) and palladium
(5% charcoal, 1.0 g, 9.4 mmol, 0.5 equiv) in methanol (100 mL)
was hydrogenated at 2 bar for 1 h at room temperature. Filtration,
evaporation, and chromatography on basic alumina with ethyl
acetate gave 7 (2.8 g, 55% yield). Mp 245-248 °C; 1H NMR (400
MHz, DMSO-d6) δ 9.22 (br s, 1H, NH), 8.31 (s, 1H, H-2), 7.66 (s,
1H, H-5), 7.65 (d, 1H, J ) 7.6 Hz, H-4′), 7.51 (d, 1H, J ) 8.4 Hz,
H-8), 7.35 (s, 1H, H-2′), 7.23-7.19 (m, 2H, H-5′,7), 6.86 (d, 1H,
J ) 7.6 Hz, H-6′), 5.54 (br s, 2H, NH2), 2.31 (s, 3H, PhCH3); 13
C
NMR (100 MHz, DMSO-d6) δ 156.6, 150.5, 147.8, 143.2, 140.5,
138.0, 129.3, 128.8, 124.3, 124.2, 122.9, 119.6, 117.4, 101.8, 22.2.
3.3. N-(2-Cyano-4-nitrophenyl)-N,N-dimethylformamide (3).
5-Nitroanthranilonitrile 1 (48.9 g, 300 mmol, 1.0 equiv) was
suspended in dimethylformamide dimethyl acetal (100 mL) and
the mixture was refluxed for 1.5 h. The resulting mixture was cooled
to room temperature and refrigerated overnight. The yellow
precipitate that formed was filtered, washed with ethyl ether, and
1
dried to give 3 (62.1 g, 95% yield). Mp 153-155 °C; H NMR
1
°C; H NMR (300 MHz, DMSO-d6) δ 9.25 (s, 1H, NH), 8.29 (s,
(400 MHz, DMSO-d6) δ 8.43 (s, 1H, H-3), 8.24 (s, 1H, NCHN),
8.23 (d, 1H, J ) 8.8 Hz, H-5), 7.34 (d, 1H, J ) 8.8 Hz, H-6), 3.15
(s, 3H, NCH3), 3.07 (s, 3H, NCH3); 13C NMR (100 MHz, DMSO-
d6) 161.0, 157.3, 140.7, 130.1, 129.3, 119.0, 117.5, 106.6, 41.2,
35.3.
1H, H-2), 7.65 (d, 1H, J ) 7.8 Hz, H-8), 7.59 (s,1H, H-5), 7.48 (d,
1H, J ) 7.8 Hz, H-7), 7.26-7.18 (m, 2H, H-5′,6′), 7.13 (d, 1H,
J ) 2.1 Hz, H-5′), 6.89 (d, 1H, J ) 7.2 Hz, H-4′), 6.24 (q, 1H,
J ) 5.4 Hz, CH3NH), 2.84 (d, 3H, J ) 5.4 Hz, CH3NH), 2.33 (s,
3H, PhCH3); 13C NMR (75 MHz, DMSO-d6) δ 156.6, 150.5, 149.0,
143.4, 140.3, 138.1, 129.1, 128.8, 124.5, 124.2, 123.4, 120.1, 117.3,
97.1, 31.0, 22.0.
3.4. N4-(3-Chlorophenyl)-4,6-quinazolinediamine (8). General
Example of the Coupling Procedure. A mixture of 3 (21.8 g,
100 mmol, 1.0 equiv) and 3-chloroaniline (110 mmol, 1.1 equiv)
was heated and stirred at reflux in acetic acid (100 mL) for 1 h.
The yellow precipitate that formed was filtered hot, washed with
hot acetic acid, diethyl ether, and dried to give the desired
nitroquinazoline 5 (25.5 g, 85% yield). Mp 278-281 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.40 (br s, 1H, NH), 9.57 (s, 1H, H-5),
8.71 (s, 1H, H-2), 8.49 (d, 1H, J ) 9.2 Hz, H-7), 8.02 (s, 1H,
H-2′), 7.88 (d, 1H, J ) 9.2 Hz, H-8), 7.80 (d, 1H, J ) 8.2, H-4′),
7.41 (t, 1H, J ) 8.2, H-5′), 7.19 (d, 1H, J ) 8.2, H-6′); 13C NMR
(75 MHz, DMSO-d6) δ 159.1, 158.0, 153.5, 145.2, 140.7, 133.4,
130.8, 130.2, 127.3, 124.6, 122.6, 121.5, 121.4, 115.1.
3.7. 4-[(3-Chlorophenyl)amino]-6-(methylamino)quinazoline
(11). 6-Methylaminoquinazoline 11 was prepared as described for
1
10: 0.42 g, 37%; mp 133-134 °C; H NMR (300 MHz, DMSO-
d6) δ 9.40 (s, 1H, NH), 8.37 (s, 1H, H-2), 8.06 (s,1H, H-5), 7.84
(d, 1H, J ) 8.0 Hz, H-6′), 7.53 (d, 1H, J ) 7.8 Hz, H-8), 7.38 (t,
1H, J ) 8.0 Hz, H-5′), 7.23 (d, 1H, J ) 7.8 Hz, H-7), 7.12-7.10
(m, 2H, H-4′,5), 6.31 (q, 1H, J ) 4.7 Hz, CH3NH), 2.85 (d, 3H,
J ) 4.7 Hz, CH3NH); 13C NMR (100 MHz, DMSO-d6) δ 156.2,
150.1, 149.1, 143.6, 142.0, 133.3, 130.6, 129.2, 124.5, 123.1, 121.7,
120.7, 117.3, 96.8, 31.0.
3.8. 4-[(3-Bromophenyl)amino]-6-(methylamino)quinazoline
(12). 6-Methylaminoquinazoline 12 was prepared as described for
10: 0.43 g, 33%; mp 141-144 °C; H NMR (400 MHz, DMSO-
6-Nitroquinazoline 5 (8.0 g, 26.6 mmol, 1.0 equiv) and iron (10.0
g, 180 mmol, 7.0 equiv) were suspended in aqueous ethanol (700
mL, 70% v/v) containing acetic acid (24 mL, 360 mmol, 14 equiv)
and heated at reflux until disappearance of starting material. The
reaction mixture was cooled to room temperature and alkalinized
by addition of concentrated ammonia (150 mL). Insoluble material
was removed by filtration through Celite, and the filtrate was
evaporated under reduced pressure. The resulting solid was washed
with 10% solution of K2CO3 and water until neutrality and dried
in a desiccator over CaSO4 to give amine 8 (5.8 g, 80% yield). Mp
1
d6) δ 9.39 (s, 1H, NH), 8.36 (s, 1H, H-2), 8.16 (t, 1H, J ) 1.8 Hz,
H-2′); 7.90 (md, 1H, J ) 8.0 Hz, H-6′), 7.52 (d, 1H, J ) 9.2 Hz,
H-8), 7.32 (t, 1H, J ) 8.0 Hz, H-5′), 7.25-7.18 (m, 2H, H-7,4′),
7.11 (d, 1H, J ) 2.4 Hz, H-5), 6.33 (q, 1H, J ) 5.2 Hz, CH3NH),
2.84 (d, 3H, J ) 5.2 Hz, CH3NH); 13C NMR (75 MHz, DMSO-d6)
δ 156.2, 150.1, 149.1, 143.5, 142.2, 130.9, 129.2, 126.0, 124.5,
124.5, 121.8, 121.1, 117.3, 96.8, 31.0.
3.9. Standard Procedure for Preparation of 2-Chloroethyl
Ureas (13-18). Amines 7-12 (1.0 mmol, 1.0 equiv) were dissolved
in anhydrous THF (5 mL), and the solution was cooled to 0 °C in
an ice bath. 2-Chloroethylisocyanate (130 µL, 1.5 mmol, 1.5 equiv)
was added dropwise, and the reaction mixture was stirred until
complete disappearance of the starting material. Thereafter, the
solvent was evaporated, and the resulting solid was washed with
10% K2CO3, water, methylene chloride, and dried to give ureas
13-18 as off-white solids.
3.10. 1-(2-Chloroethyl)-3-{[4-(3-methylphenyl)amino]quinazo-
lin-6-yl}urea (13): 0.34 g, 96%; mp 260-263 °C (dec); 1H NMR
(400 MHz, DMSO-d6) δ 9.59 (s, 1H, Ar-NH-Ar); 8.90 (s, 1H,
CONH-aryl), 8.43 (s, 1H, H-2), 8.34 (d, 1H, J ) 2.3 Hz, H-5),
7.83 (dd, 1H, J ) 8.8 Hz, J ) 2.3 Hz, H-7), 7.67 (d, 1H, J ) 8.8
Hz, H-8), 7.60 (s, 1H, H-2′), 7.59 (d, 1H, J ) 7.6 Hz, H-6′), 7.23
(t, 1H, J ) 7.6 Hz, H-5′), 6.89 (d, 1H, J ) 7.6 Hz, H-4′), 6.64 (t,
1H, J ) 6.0 Hz, ClCH2CH2NH), 3.69 (t, 2H, J ) 6.0 Hz, ClCH2-
CH2), 3.47 (q, 2H, J ) 6.0 Hz, ClCH2CH2), 2.32 (s, 3H, PhCH3);
1
235-237 °C; H NMR (400 MHz, DMSO-d6) δ 10.5 (br s, 1H,
NH), 8.56 (s, 1H, H-2), 8.00 (br s, 1H, H-2′), 7.74 (dd, 1H, J )
8.0, J ) 0.9 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, J ) 0.9 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.
3.5. N4-(3-Bromophenyl)-4,6-quinazolinediamine (9). 6-Ami-
noquinazoline 9 was prepared as described for 8. 6-Nitro-4-(3-
bromophenyl)aminoquinazoline (6): 5.9 g, 86%; mp 267-270 °C;
1H 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 Hz,
J ) 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 Hz, 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.
N4-(3-Bromophenyl)-4,6-quinazolinediamine (9): 11.4 g, 83%; mp
204-206 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H, NH),

3550 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Domarkas et al.
13C NMR (100 MHz, DMSO-d6) δ 157.9, 155.7, 153.0, 145.8,
140.0, 138.8, 138.1, 128.9, 128.7, 126.9, 124.9, 123.5, 120.3, 116.3,
110.3, 45.2, 42.1, 22.1; MS (APCI+) m/z 356.0 (MH+ with 35Cl),
358.0 (MH+ with 37Cl), 277.2 (M - ClEtNH - H, 10.7); HRMS
(FAB+) m/z calcd for C16H19N5OCl, 356.127 813, found 356.127
670 (M + H)+.
3.11. 1-(2-Chloroethyl)-3-{[4-(3-chlorophenyl)amino]quinazo-
lin-6-yl}urea (14): 0.35 g, 94%; mp 254-256 °C (dec); 1H NMR
(400 MHz, DMSO-d6) δ 9.98 (s, br, 1H, Ar-NH-Ar), 9.03 (s,
1H, CONH), 8.55 (s, 1H, H-2), 8.42 (d, 1H, J ) 1.8 Hz, H-5),
8.02 (s, 1H, H-2′), 7.85 (dd, 1H, J ) 9.0 Hz, J ) 1.8 Hz, H-7),
7.77 (d, 1H, J ) 8.0 Hz, H-4′), 7.72 (d, 1H, J ) 9.0 Hz, H-8), 7.39
(t, 1H, J ) 8.0 Hz, H-5′), 7.15 (d, 1H, J ) 8.0 Hz, H-6′), 6.69 (t,
1H, J ) 5.9 Hz, ClCH2CH2NH), 3.69 (t, 2H, J ) 5.9 Hz, ClCH2-
CH2), 3.23 (q, 2H, J ) 5.9 Hz, ClCH2CH2); 13C NMR (100 MHz,
DMSO-d6) δ 157.6, 155.6, 152.8, 146.1, 141.8, 139.0, 133.3, 130.7,
129.0, 127.0, 123.5, 121.9, 121.0, 111.4, 110.0, 45.2, 42.1; MS
(ESI+) m/z 376.1 (MH+ with 35Cl), 378.1 (MH+ with 37Cl), 342.1
(M - Cl + H, 27.0), 146.8 (M - ClEtNH - H, 46.0); HRMS
(FAB+) m/z calcd for C17H16N5OCl2, 376.073 191, found 376.073
120 (M + H)+.
3.12. 1-(2-Chloroethyl)-3-{[4-(3-bromophenyl)amino]quinazo-
lin-6-yl}urea (15): 0.39 g, 94%; mp 268-271 °C (dec); 1H NMR
(400 MHz, DMSO-d6) δ 9.80 (s, 1H, Ar-NH-Ar), 8.96 (s, 1H,
CONH), 8.51 (s, 1H, H-2), 8.39 (s, 1H, H-5), 8.16 (s, 1H, H-2′),
7.82-7.85 (m, 2H, H-6′,7), 7.71 (d, 1H, J ) 9.2 Hz, H-8), 7.32 (t,
1H, J ) 8.0 Hz, H-5′), 7.26 (d, 1H, J ) 8.0 Hz, H-4′), 6.65 (t, 1H,
J ) 5.8 Hz, ClCH2CH2NH), 3.69 (t, 2H, J ) 5.8 Hz, ClCH2CH2),
3.47 (q, 2H, J ) 5.8 Hz, ClCH2CH2); 13C NMR (100 MHz, DMSO-
d6) δ 157.7, 155.6, 152.6, 145.3, 141.8, 139.1, 131.0, 128.5, 127.2,
126.6, 125.0, 121.8, 121.6, 116.3, 110.0, 45.2, 42.1; MS (APCI)
m/z 420.0 (MH+ with 79Br, 64.8), 422.0 (MH+ with 81Br, 79.3),
341.0 (M - ClEtNH - H, with 79Br, 10.0), 343.1 (M - ClEtNH
- H, with 81Br, 14.5); HRMS (FAB+) m/z calcd for C17H16N5-
OClBr, 420.022 674, found 420.022 780 (M + H)+.
3.13. 3-(2-Chloroethyl)-1-methyl-1-{[4-(3-methylphenyl)ami-
no]quinazolin-6-yl}urea (16): 0.35 g, 95%; mp 262-265 °C (dec);
1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H, Ar-NH-Ar), 8.56
(s, 1H, H-2), 8.45 (s, 1H, H-5), 7.75 (s, 2H, H-7,8), 7.66 (d, 1H,
J ) 8.0 Hz, H-6′), 7.64 (s, 1H, H-2′), 7.26 (t, 1H, J ) 8.0 Hz,
H-5′), 6.94 (d, 1H, J ) 8.0 Hz, H-4′), 6.54 (t, 1H, J ) 5.6 Hz,
ClCH2CH2NH), 3.57 (t, 2H, J ) 6.8 Hz, ClCH2CH2), 3.32 (q, 2H,
J ) 6.8 Hz, ClCH2CH2), 3.28 (s, 3H, NCH3), 2.33 (s, 3H, PhCH3);
13C NMR (100 MHz, DMSO-d6) δ 158.0, 157.9, 154.9, 148.2,
142.3, 139.6, 138.3, 133.3, 129.3, 129.0, 125.2, 123.4, 120.5, 120.2,
116.2, 44.2, 43.1, 38.0, 22.1; MS (ESI+) m/z 370.2 (MH+ with
35Cl), 372.2 (MH+ with 37Cl), 265.2 (M - ClEtNHCO + H);
HRMS (FAB+) m/z calcd for C19H21N5OCl, 370.143 463, found
370.143 450 (M + H)+.
3.14. 3-(2-Chloroethyl)-1-methyl-1-{[4-(3-chlorophenyl)ami-
no]quinazolin-6-yl}urea (17): 0.36 g, 93%; mp 232-235 °C (dec);
1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H, Ar-NH-Ar), 8.64
(s, 1H, H-2), 8.44 (s, 1H, H-5), 8.11 (s, 1H, H-2′), 7.84 (d, 1H,
J ) 8.2 Hz, H-4′), 7.79 (s, 2H, H-7,8), 7.41 (t, 1H, J ) 8.2 Hz,
H-5′), 7.16 (d, 1H, J ) 8.2 Hz, H-6′), 6.57 (t, 1H, J ) 5.4 Hz,
ClCH2CH2NH), 3.58 (t, 2H, J ) 6.8 Hz, ClCH2CH2), 3.23 (q, 2H,
J ) 5.4 Hz, ClCH2CH2), 3.28 (s, 3H, NCH3); 13C NMR (100 MHz,
DMSO-d6) δ 157.8, 157.3, 154.6, 148.3, 142.5, 141.4, 133.4, 130.8,
129.4, 123.8, 121.9, 120.9, 120.4, 116.1, 44.2, 43.1, 38.0; MS
(ESI+) m/z 390.2 (MH+ with 35Cl), 392.2 (MH+ with 37Cl), 356.2
(M - Cl + H with 35Cl, 54.3), 358.2 (M - Cl + H with 37Cl,
14.8), 285.4 (M - ClEtNHCO + H, 27.5); HRMS (FAB+) m/z
calcd for C18H18N5OCl2, 390.088 841, found 390.088 960 (M +
H)+.
3.15. 3-(2-Chloroethyl)-1-methyl-1-{[4-(3-bromophenyl)ami-
no]quinazolin-6-yl}urea (18): 0.41 g, 94%; mp 253-255 °C (dec);
1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H, Ar-NH-Ar), 8.64
(s, 1H, H-2), 8.44 (s, 1H, H-2′), 8.22 (t, 1H, J ) 2.0 Hz, H-5),
7.91 (d, 1H, J ) 8.0 Hz, H-6′), 7.79-7.78 (m, 2H, H-7,8), 7.35 (t,
1H, J ) 8.0 Hz, H-5′), 7.29 (d, 1H, J ) 8.0 Hz, H-4′), 6.57 (t, 1H,
J ) 5.4 Hz, ClCH2CH2NH), 3.58 (t, 2H, J ) 6.5 Hz, ClCH2CH2),
3.32 (q, 2H, J ) 6.5 Hz, ClCH2CH2), 3.28 (s, 3H, NCH3); 13C
NMR (100 MHz, DMSO-d6) δ 157.8, 157.3, 154.6, 148.3, 141.5,
133.5, 131.1, 129.4, 126.7, 124.7, 121.9, 121.2, 120.4, 116.1, 44.2,
43.1, 38.0; MS (APCI) m/z 434.1 (MH+ with 79Br, 74), 436.1 (MH+
with 81Br, 100), 329.3 (M - ClEtNHCO + H, with 79Br), 331.1
(M - ClEtNHCO + H, with 81Br); HRMS (FAB+) m/z calcd for
C18H18N5OClBr, 434.038 324, found 434.038 210 (M + H)+.
3.16. Standard Procedure for Preparation of Nitrosoureas
(19-24). Ureas 13-18 (0.5 mmol, 1.0 equiv) were suspended in
anhydrous acetonitrile (3 mL) containing acetic acid (43 µL, 0.75
mmol, 1.0 equiv), and the resulting solution was cooled to 0 °C in
an ice bath. Nitrosonium tetrafluoroborate (88.5 mg, 0.75 mmol,
1.5 equiv) was added, and the reaction mixture was stirred at 0 °C
until disappearance of starting material as monitored by TLC (3-4
h in the case of desmethylureas or overnight for N3-Me ureas).
The reaction mixture was poured into 100 mL of ice-cooled water/
ethyl acetate 1/1 solution, and the pH was adjusted to 5-6 by
careful addition of 5% sodium bicarbonate solution. The water phase
was extracted twice with ethyl acetate, and the organic extracts
were washed with brine, dried over MgSO4, and concentrated under
reduced pressure without heating. Chromatography on silica gel
eluting with a mixture of ethyl acetate/petroleum ether gave the
expected nitrosoureas with moderate yield.
3.17. 1-(2-Chloroethyl)-1-nitroso-3-{[4-(3-methylphenyl)ami-
no]quinazolin-6-yl}urea (19): 0.092 g, 48%; mp 135-137 °C
1
(dec); H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H, Ar-NH-
Ar), 9.79 (s, 1H, CONH-aryl), 8.74 (s, 1H, H-5), 8.54 (s, 1H, H-2),
8.03 (d, 1H, J ) 8.8 Hz, H-7), 7.80 (d, 1H, J ) 8.8 Hz, H-8), 7.61
(s, br, 2H, H-2′,6′), 7.26 (t, 1H, J ) 7.8 Hz, H-5′), 6.94 (d, 1H,
J ) 7.8 Hz, H-4′), 4.22 (t, 2H, J ) 6.4 Hz, ClCH2CH2), 3.73 (t,
2H, J ) 6.4 Hz, ClCH2CH2), 2.33 (s, 3H, PhCH3); 13C NMR (100
MHz, DMSO-d6) δ 158.2, 154.3, 152.0, 147.4, 139.7, 138.2, 135.9,
129.3, 128.9, 128.8, 125.1, 123.6, 120.4, 115.9, 115.5, 41.3, 22.1
(1C overlay with DMSO-d6); MS (ESI+) m/z 384.8 (MH+ with
35Cl, 15.7), 386.8 (MH+ with 37Cl, 4.2), 277.2 (M - ClEtNNO -
H, 100.0); HRMS (FAB+) m/z calcd for C18H18N6O2Cl, 385.117
977, found 385.118 090 (M + H)+.
3.18. 1-(2-Chloroethyl)-1-nitroso-3-{[4-(3-chlorophenyl)ami-
no]quinazolin-6-yl}urea (20): 0.103 g, 51%; mp 141-143 °C
1
(dec); H NMR (300 MHz, DMSO-d6) δ 11.05 (s, 1H, Ar-NH-
Ar), 9.97 (s, 1H, CONH), 8.76 (d, 1H, J ) 2.1 Hz, H-5), 8.62 (s,
1H, H-2), 8.07 (dd, 1H, J ) 8.7 Hz, J ) 2.1 Hz, H-7), 8.05 (s, 1H,
H-2′), 7.79-7.85 (m, 2H, H-4′,8), 7.40 (t, 1H, J ) 7.9 Hz, H-5′),
7.16 (d, 1H, J ) 7.9 Hz, H-6′), 4.23 (t, 2H, J ) 6.2 Hz, ClCH2CH2),
3.73 (t, 2H, J ) 6.2 Hz, ClCH2CH2); 13C NMR (75 MHz, DMSO-
d6) δ 158.0, 154.1, 152.1, 147.5, 141.5, 136.2, 133.4, 130.7, 129.5,
128.9, 123.8, 122.2, 121.2, 115.9, 115.2 (2C overlay with DMSO-
d6); MS (ESI+) m/z 404.8 (MH+ with 35Cl), 406.8 (MH+ with 37Cl),
297.2 (M - ClEtNNO - H, with 35Cl), 299.2 (M - ClEtNNO -
H, with 37Cl); HRMS (FAB+) m/z calcd for C17H15N6O2Cl2,
405.063 354, found 405.063 230 (M + H)+.
3.19. 1-(2-Chloroethyl)-1-nitroso-3-{[4-(3-bromophenyl)ami-
no]quinazolin-6-yl}urea (21): 0.108 g, 48%; mp 144-146 °C
1
(dec); H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H, Ar-NH-
Ar), 9.97 (s, 1H, CONH), 8.75 (d, 1H, J ) 1.8 Hz, H-5), 8.61 (s,
1H, H-2), 8.16 (s, 1H, H-2′), 8.06 (dd, 1H, J ) 9.2 Hz, J ) 1.8
Hz, H-7), 7.82-7.87 (m, 2H, H-6′,8), 7.34 (t, 1H, J ) 7.8 Hz,
H-5′), 7.29 (d, 1H, J ) 7.8 Hz, H-4′), 4.23 (t, 2H, J ) 6.4 Hz,
ClCH2CH2), 3.73 (t, 2H, J ) 6.4 Hz, ClCH2CH2); 13C NMR (100
MHz, DMSO-d6) δ 158.1, 154.2, 152.1, 147.7, 141.7, 136.3, 131.1,
129.6, 129.0, 126.7, 125.0, 121.8, 121.6, 116.0, 115.2, 41.4 (1C
overlay with DMSO-d6); MS (ESI+) m/z 450.9 (MH+ with 79Br),
452.9 (MH+ with 81Br), 341.1 (M - ClEtNNOCO - H, with 79Br),
343.1 (M - ClEtNNOCO - H, with 81Br); HRMS (FAB+) m/z
calcd for C17H15N6O2ClBr, 449.012 838, found 449.012 890 (M +
H)+.
3.20. 1-(2-Chloroethyl)-1-nitroso-3-methyl-3-{[4-(3-meth-
ylphenyl)amino]quinazolin-6-yl}urea (22): 0.100 g, 50%; mp
155-157 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H,
Ar-NH-Ar), 8.54 (s, 1H, H-2), 8.53 (d, 1H, J ) 2.2 Hz, H-5),
7.81 (dd, 1H, J ) 8.6 Hz, J ) 2.2 Hz, H-7), 7.73 (d, 1H, J ) 8.6

Combi-Targeting Synthesis of Nitrosoureas
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3551
Hz, H-8), 7.61-7.63 (m, 2H, H-2′,6′), 7.26 (t, 1H, J ) 7.4 Hz,
H-5′), 6.95 (d, 1H, J ) 7.4 Hz, H-4′), 4.06 (t, 2H, J ) 6.2 Hz,
ClCH2CH2), 3.70 (t, 2H, J ) 6.2 Hz, ClCH2CH2), 3.59 (s, 3H,
NCH3), 2.33 (s, 3H, PhCH3); 13C NMR (100 MHz, DMSO-d6) δ
158.1, 155.4, 148.4, 142.3, 139.4, 138.3, 132.5, 129.5, 129.0, 125.4,
123.5, 120.5, 120.2, 115.9, 42.9, 22.0 (2C overlay with DMSO-
d6); MS (ESI-) m/z 369.9 (M-1 with 35Cl, 25.4), 398.9 (M-1 with
37Cl, 6.5), 262.3 (M - ClEtNNO - H, with 35Cl, 100.0), 263.2
(M - ClEtNNO - H, with 37Cl, 29.6); HRMS (FAB+) m/z calcd
for C19H20N6O2Cl, 399.133 627, found 399.135 550 (M + H)+.
3.23.2. Drug Treatment. Compounds were delivered dissolved
in DMSO and subsequently diluted in sterile DMEM media
containing 10% fetal bovine serum (FBS) (Life Technologies,
Burlington, Canada) prior to addition to cell culture medium. The
DMSO concentration never exceeded 0.2% (v/v).
3.23.3. Degradation. The study of the conversion of the
nitrosoureas to their corresponding aminoquinazolines was per-
formed by spectrofluorimetry as the latter amines were fluorescent
(absorption 290 nm, emission 450 nm). Briefly, 125 mM of the
nitrosoureas was added to RPMI-1640 with 10% FBS and incubated
for 4 h at 37 °C in a microplate spectrofluorometer (SpectraMax
Gemini flourescence reader, Molecular Device, CA). The data are
acquired and analyzed by SoftMaxPro (Molecular Device, CA).
3.21. 1-(2-Chloroethyl)-1-nitroso-3-methyl-3-{[4-(3-chloro-
phenyl)amino]quinazolin-6-yl}urea (23): 0.113 g, 54%; mp 140-
1
142 °C (dec); H NMR (400 MHz, CDCl3) δ 9.76 (s, 1H, Ar-
NH-Ar), 8.85 (s, 1H, H-2), 8.53 (d, 1H, J ) 1.8 Hz, H-5), 8.07
(s, 1H, H-2′), 7.85 (dd, 1H, J ) 9.0 Hz, J ) 1.8 Hz, H-7), 7.75-
7.81 (m, 2H, H-4′,8), 7.41 (t, 1H, J ) 8.1 Hz, H-5′), 7.17 (d, 1H,
J ) 8.1 Hz, H-6′), 4.07 (t, 2H, J ) 6.2 Hz, ClCH2CH2), 3.71 (t,
2H, J ) 6.2 Hz, ClCH2CH2), 3.60 (s, 3H, NCH3); 13C NMR
(CDCl3) δ 157.3, 154.9, 147.5, 142.9, 139.2, 134.8, 132.2, 130.3,
130.2, 125.0, 121.9, 119.8, 118.5, 115.3, 42.4, 41.9, 40.9; MS
(ESI-) m/z 416.8 (M - 1 with 35Cl, 30.2), 418.9 (M - 1 with
37Cl, 17.3), 282.2 (M - ClEtNNOCO with 35Cl, 100.0), 284.2
(M - ClEtNNOCO with 37Cl, 31.7); HRMS (FAB+) m/z calcd
for C18H17N6O2Cl2, 419.079 004, found 419.079 000 (M + H)+.
3.22. 1-(2-Chloroethyl)-1-nitroso-3-methyl-3-{[4-(3-bromo-
phenyl)amino]quinazolin-6-yl}urea (24): 0.114 g, 49%; mp 139-
141 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H, Ar-
NH-Ar), 8.64 (s, 1H, H-2), 8.54 (d, 1H, J ) 2.0 Hz, H-5), 8.18 (t,
1H, J ) 2.0 Hz, H-2′), 7.83-7.87 (m, 2H, H-6′,7), 7.77 (d, 1H,
J ) 8.8 Hz, H- 8), 7.35 (t, 1H, J ) 8.2 Hz, H-5′), 7.30 (d, 1H,
J ) 8.2 Hz, H-4′), 4.07 (t, 2H, J ) 6.2 Hz, ClCH2CH2), 3.70 (t,
2H, J ) 6.2 Hz, ClCH2CH2), 3.60 (s, 3H, NCH3); 13C NMR (100
MHz, DMSO-d6) δ 157.8, 155.2, 155.0, 148.5, 142.5, 141.2, 132.8,
131.1, 129.7, 126.9, 124.8, 121.9, 121.3, 120.4, 115.8, 42.9, 41.0;
MS (APCI) m/z 462.8 (MH+ with 79Br), 464.8 (MH+ with 81Br),
432.2 (M - NO, with 79Br), 434.2 (M - NO, with 81Br), 328.2
(M - ClEtNNOCO, with 79Br), 330.2 (M - ClEtNNOCO, with
81Br); HRMS (FAB+) m/z calcd for C18H17N6O2ClBr, 463.028 488,
found 463.028 640 (M + H)+.
Acknowledgment. We thank the U.S. Department of
Defense (Award: DAMD17-03-0152) for financial support. We
are also grateful to the Cancer Research Society Inc. (Montreal,
Canada) for its continuous support of the development of the
combi-targeting concept.
Supporting Information Available: NMR spectra, HRMS data,
and HPLC chromatograms. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Brahimi, F.; Matheson, S. L.; Dudouit, F.; McNamee, J. P.; Tari, A.
M.; Jean-Claude, B. J. Inhibition of epidermal growth factor receptor-
mediated signaling by “combi-triazene” BJ2000, a new probe for
combi-targeting postulates. J. Pharmacol. Exp. Ther. 2002, 303, 238-
246.
(2) Matheson, S. L.; McNamee, J.; Jean-Claude, B. J. Design of a
chimeric 3-methyl-1,2,3-triazene with mixed receptor tyrosine kinase
and DNA damaging properties: a novel tumor targeting strategy. J.
Pharmacol. Exp. Ther. 2001, 296, 832-40.
(3) Matheson, S. L.; Mzengeza, S.; Jean-Claude, B. J. Synthesis of a
1-[4-(m-tolyl)amino-6-quinazolinyl-3-[14C]-methyl triazene: a ra-
diolabeled probe for the combi-targeting concept. J. Labelled Compds.
Radiopharm. 2003, 46, 729-735.
(4) Matheson, S. L.; McNamee, J.; Jean-Claude, B. J. Differential
responses of EGFR/AGT-expressing cells to the “combi-triazene”
SMA41. Cancer Chemother. Pharmacol. 2003, 51, 11-20.
(5) Qiu, Q.; Dudouit, F.; Banerjee, R.; McNamee, J.; Jean-Claude, B. J.
Inhibition of cell signaling by the combi-nitrosourea FD137 in the
androgen independent DU145 prostate cancer cell line. Prostate 2004,
59, 13-21.
(6) Qiu, Q.; Dudouit, F.; Matheson, S. L.; Brahimi, F.; Banerjee, R.;
McNamee, J.; Jean-Claude, B. J. The combi-targeting concept: a
novel 3,3-disubstituted nitrosourea with EGFR tyrosine kinase
inhibitory properties. Cancer Chemother. Pharmacol. 2003, 51, 1-10.
(7) Meden, H.; Kuhn, W. Overexpression of the oncogene c-erbB-2
(HER-2, neu) in ovarian cancer: a new prognostic factor. Eur. J.
Obstet. Gynecol. Reprod. Biol. 1997, 71, 173-179.
(8) 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.
(9) Xinmei, C.; Yeung, T. K.; Wang, Z. Enhanced drug resistance in
cells coexpressing erb2 with EGF receptor or erb3. Biochem. Biophys.
Res. Commun. 2000, 277, 757-763.
(10) 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.
3.23. Molecular Modeling. 3.23.1. PDB Structure Processing
and Pocket Analysis 3.23.1.a. Alignment. The structures of the
TK domain of EGFR, unbound and in complex with 4-anilino-
quinazoline inhibitor, were downloaded from the PDB and aligned
using the sequence and structural alignment application pro-Align
in the 2004.03 version of the MOE software. The application default
settings were used. The aligned structures had a high residue identity
of 98.6% and a backbone aligned rmsd of 0.3849 A.
3.23.1.b. Ligand Refinement in the Pocket. The ligand
coordinates from the ligand-bound X-ray structure were used as a
template to manually construct compounds 7-24 in the receptor
pocket. A water molecule located near N1 in the quinazoline ring
was also included in the calculations as it was crucial in order to
retain more precise refined geometries. Each structure was mini-
mized to a gradient of 0.01 kcal/A mol in the presence of the fixed
receptor atoms using the MMFF94x force field as implemented in
MOE. The default force field settings were used. Each of the refined
starting structures was then subjected to a systematic conformational
search using the Csearch systematic conformational search routine
in the MOE software. Each of the resulting conformations was
refined by minimization in the fixed receptor pocket using the
MMFF94x force field. The conformation with the lowest combined
strain and receptor interaction energy was chosen as the representa-
tive bound pose for each compound and used for calculation of
the van der Waals ligand/receptor interaction energy and the ligand
strain energies. Dihedral angles between the quinazoline and ureido
planes were read from Gaussian 98W software geometry-optimized
structures. The Becke-style three-parameter density functional
theory (B3LYP) method with the 3-21G basic set was used, and
energy minima were confirmed by frequency calculations done with
the same method and basic set.
(11) Roth, G. A.; Tai, J. J. A new synthesis of aryl substituted quinazolin-
4(1H)-ones. J. Heterocycl. Chem. 1996, 33, 2051-2053.
(12) Morley, J. S.; Simson, J. C. E. The chemistry of simple heterocyclic
systems. Part I. Reactions of 6- and 7-nitro-4-hydroxyquinazoline
and their derivatives. J. Chem. Soc. 1948, 360-366.
(13) 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-[(phenylmethyl)amino]- and
4-(phenylamino)quinazolines as potent adenosine 5′-triphosphate
binding site inhibitors of the tyrosine kinase domain of the epidermal
growth factor receptor. J. Med. Chem. 1995, 38, 3482-3487.

3552 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Domarkas et al.
(14) Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B.
C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.;
Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.; Wissner,
A. 6-Substituted-4-(3-bromophenylamino)quinazolines as putatives
irreversible inhibitors of the epidermal growth factor receptor (EGFR)
and human epidermal growth factor receptor (HER-2) tyrosine kinases
with enhanced antitumor activity. J. Med. Chem. 2001, 44, 2719-
2734.
(20) Tanner, B.; Kreutz, W.; Meinert, R.; Knapstein, P. G.; Becker, R.
Prognostic significance of c-erbB2 mRNA in ovarian carcinoma.
Gynecol. Oncol. 1996, 62, 268-277.
(21) Yen, L.; Zengrong, N.; You, X. L.; Richard, S.; Langton-Webster,
B. C.; Aloui-Jamali, M. A. Regulation of cellular response to
cisplatin-induced DNA damage and DNA repair in cells overex-
pressing p185neu is dependent on the ras signaling pathway.
Oncogene 1999, 1827-1835.
(22) Hengstler, J. G.; Lange, J.; Kett, A.; Gornhofer, N.; Meinert, R.;
Arand, M.; Knapstein, P. G.; Becker, R.; Oesch, F.; Tanner, B.
Contribution of c-erbB-2 and topoisomerase II alpha to chemoresis-
tance in ovarian cancer. Cancer Res. 1999, 59, 3206-3214.
(23) Slamon, D. J.; Godolphin, W.; Jones, L. A.; et al. Studies of the
HER-2/neu proto-oncogene in human breast and ovarian cancer.
Science 1989, 244, 707-712.
(24) Banerjee, R.; Rachid, Z.; McNamee, J.; Jean-Claude, B. J. Synthesis
of a prodrug designed to release multiple inhibitors of the epidermal
growth factor receptor tyrosine kinase and an alkylating agent: a
novel tumor targeting concept. J. Med. Chem. 2003, 46, 5546-5551.
(25) Rachid, Z.; Brahimi, F.; Domarkas, J.; Jean-Claude, B. J. Synthesis
of half-mustard combi-molecules with fluorescence properties: cor-
relation with EGFR status. Bioorg. Med. Chem. Lett. 2005, 15, 1135-
1138.
(15) Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.; McMichael,
A.; Showalter, H. D. H.; Fry, D. W.; Kraker, A. J.; Denny, W. A.
Tyrosine kinase inhibitors. 8. An unusually steep structure-activity
relationship for analogues of 4-(3-bromoanilino)-6,7-dimethoxy-
quinazoline (PD 153035), a potent inhibitor of the epidermal growth
factor receptor. J. Med. Chem. 1996, 39, 267-276.
(16) Lown, J. W.; Chauhan, M. S. Synthesis of specifically 15N- and 13C-
labeled antitumor (2-haloethyl)nitrosoureas. The study of their
conformations in solution by nitrogen-15 and carbon-13 nuclear
magnetic resonance and evidence for stereoelectronic control in their
aqueous decomposition. J. Org. Chem. 1981, 46, 5309-5321.
(17) Butler, R. N.; Lambe, T. M.; Tobin, J. C.; Scott, F. L. Stable
heterocyclic primary nitroso-amines. J. Chem. Soc., Perkin Trans. 1
1973, 1357-1361.
(18) Smith, H. W.; Camerman, A. Molecular structure of 1-(2-chloroethyl)-
3-(trans-4-methylcyclohexyl)-1-nitrosourea. J. Med. Chem. 1978, 21,
468-471.
(19) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the
epidermal growth factor receptor kinase domain alone and in complex
with 4-anilinoquinazoline inhibitor. J. Biol. Chem. 2002, 277, 46265-
46272.
(26) Colvin, M.; Brundrett, R. Chemical decomposition of chloroethyl-
nitrosoureas. Nitrosoureas Current Status and New DeVelopments;
Prestayako, A. W., et al., Eds.; Academic Press: 1981, 43-49.
JM0600390