Novel nitrogen mustard-armed combi-molecules for the selective targeting of epidermal growth factor receptor overexperessing solid tumors: Discovery of an unusual structure-activity relationship

Article abstract of DOI:10.1021/jm070144p

To enhance the potency of "combi-molecules", we designed 6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional alkylator. The combi-molecules blocked EGFR TK with potency increasing with the basicity of the mustard moiety. They selectively kil

Full text of DOI:10.1021/jm070144p

J. Med. Chem. 2007, 50, 2605-2608
2605
Scheme 1. Stability of Triazenes Designed To Generate DNA
Cross-Linking Species
Novel Nitrogen Mustard-Armed Combi-Molecules
for the Selective Targeting of Epidermal Growth
Factor Receptor Overexperessing Solid Tumors:
Discovery of an Unusual Structure-Activity
Relationship
Zakaria Rachid,† Fouad Brahimi,† Qiyu Qiu,†
Christopher Williams,‡ Janet M. Hartley,§
John A. Hartley,§ and Bertrand J. Jean-Claude*,†
Cancer Drug Research Laboratory, Department of Medicine,
DiVision of Medical Oncology, McGill UniVersity Health Center/
Royal Victoria Hospital, Montreal, H3A 1A1, Quebec, Canada,
Chemical Computing Group Inc., 1010 Sherbrooke Street West,
Suite 910, Montreal, H3A 2R7, Que´bec, Canada, and
Drug-DNA Interactions Research Group, Department of Oncology,
UCL, Cancer Research UK, 91 Riding House Street,
London W1W 7BS, U.K.
Scheme 2. Decomposition of 6 under Physiological Conditions
ReceiVed February 7, 2007
Abstract: To enhance the potency of “combi-molecules”, we designed
6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional
alkylator. The combi-molecules blocked EGFR TK with potency
increasing with the basicity of the mustard moiety. They selectively
killed cells transfected with EGFR and were potent against the DU145
prostate cancer cells. Combi-molecule 6a blocked EGFR phosphory-
lation in an irreversible manner, induced DNA-cross-links, and arrested
the cells in mid-S.
be potent EGFR inhibitors.5 Thus, the syntheses of combi-
molecules of the triazene class capable of releasing chloro-
ethyldiazo species remained an unresolved issue.
The combi-targeting concept postulates that a molecule
termed “combi-molecule” designed to interact with the epider-
mal growth factor receptor (EGFR) on its own and allowed to
further degrade into another more stable inhibitor of the latter
receptor plus a DNA damaging species should be more potent
than combination of individual molecules involving EGFR
inhibition or DNA damage. Over the past 5 years, we demon-
strated the feasibility of this principle with triazene carrying a
methylating agent designed to alkylate guanine at the O-6 and
N-7 positions.1 Methylating combi-molecules showed significant
EGFR-DNA binary targeting potency. However, their anti-
proliferative activities were shown to be mitigated by DNA
repair enzymes.4 The major advantage of these triazene-based
molecules was their high affinity for the EGFR ATP binding
site (IC50 ) 0.05-0.1 µM). Therefore, their structures remained
a good platform for structural modification to enhance the
potency of the combi-targeting strategy. Accordingly, we
designed 1 (Scheme 1) to contain a chloroethyl group, which
upon hydrolysis of the triazene chain would lead to the formation
of the known cross-linking chlorethylating species 3. It is well-
known that this kind of compound can form DNA interstrand
cross-links and show significantly greater cytotoxic activity than
their methyltriazene counterparts.2-4 Unfortunately, all attempts
to synthesize such a molecule failed because of the instability
of the designed structure 1. It was rapidly converted to the
corresponding chloroethylaminoquinazolines 2 through rapid
loss of nitrogen at room temperature (Scheme 1). Therefore, it
could never be isolated. Fortunately, 2a and 2b were found to
Recently, to circumvent problems associated with the direct
attachment of the chloroethyl group to the N3 nitrogen of the
triazene ring, we designed 6 (Scheme 2) to contain a chloro-
ethylaminoethyl group that will confer a second alkylating
function to the released alkyldiazonium species (see TZ, Scheme
2). Thus, as per the combi-targeting principles,6 the combi-
molecule (TZ-I) was designed to generate a nitrogen mustard
analogue (TZ) capable of mimicking the alkylating properties
of mechlorethamine (5) (Scheme 2) and 4a, another inhibitor
of EGFR (I). Here, we report on the first combi-molecules of
the triazene class capable of releasing bifunctional alkylating
species. This study led to the discovery of a novel structure-
activity relationship based on the basicity of the central nitrogen
of the mustard moiety, which was elucidated by molecular
modeling. We also demonstrated the potent binary EGFR-DNA
targeting properties of these novel mixed mustard-triazene
combi-molecules.
The synthesis of 4a proceeded as described by Roth et al.7-10
and that of amines 11, 13, and 16 as in Scheme 3. Briefly, 7
obtained from the treatment of 2-bromoethylamine hydrobro-
mide with benzyl chloroformate was mixed with 2-(methylami-
no)ethanol or N-benzylethanolamine in the presence of potas-
sium iodide in DMF to provide 14. The latter amino alcohol
was chlorinated with POCl3 to afford 15. Hydrolysis of 15 in 6
N HCl gave the desired amines 16. The synthesis of aniline
derivatives proceeded as described in Scheme 3. Amines 8 were
treated with ethylene oxide in the presence of zirconium
tetrachloride to give amino alcohols 9, which were chlorinated
with POCl3 to give 10. The amino group of 10 was unveiled
by hydrolysis in 6 N HCl to provide the desired amines 11.
Amino alcohol 12 was prepared in a similar fashion using
propylene oxide instead of ethylene oxide to generate the amino
* To whom correspondence should be addressed. Phone: +1 514 934
1934, ext 35841. Fax: +1 514 843 1475. E-mail: bertrandj.jean-
claude@mcgill.ca.
† McGill University.
‡ Chemical Computing Group Inc.
§ Cancer Research UK.
10.1021/jm070144p CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/02/2007

2606 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Letters
Table 1. EGFR Tyrosine Kinase (TK) Inhibition and Antiproliferative
Scheme 3a
Data for “Combi-Molecules” of the Mustard Class
IC50 (µM)
inhibition
inhibition
of serum-
stimulated
growth
inhibition
in DU145
cellsb
of serum-
stimulated
growth in
NIH3T3b
EGFRa
enzyme
assay
growth in
compd
NIH3T3 HER14b
4a
6a
6b
6c
6d
1713
18
5
0.2
34.14
2.6
10.55
7.95
4.7
37.23
11.4
7.6
38.38
27.18
15.19
9.068
27.56
39.50
21.53
3.19
1.31
2.93
6.224
11.95
1.67
0.0044
0.071
3.96
2.2
0.037
0.97
5.75
a Polysubstrate (L-glutamic acid/L-tyrosine, 4:1) phosphorylation by
EGFR was detected using an antiantiphosphotyrosine antibody. b Cell
growth was measured using SRB assay.14 Values are the average from at
least two independent experiments. Variation was generally (5%.
a Reagents and conditions: (i) DMF, Et3N, 0 °C; (ii) KI, DMF,
RNHC2H5OH or ArNH2, rt; (iii) ethylene oxide, ZrCl4/CH2Cl2, rt; or
propylene oxide, anhydrous EtOH; (iv) POCl3 at 0 °C , then refluxing; (v)
6 N HCl, heat.
Scheme 4a
Figure 1. Linear correlation between log(IC50) of 6a-d, 17, and 18
and the pKa values.
form of interaction with their cognate ATP site. In an effort to
elucidate this structure-activity relationship, we undertook a
molecular modeling study whereby all the combi-molecules
were modeled into the ATP site of EGFR using the MOI711
software package. The X-ray structure of a 4-anilinoquinazoline
inhibitor bound to EGFR (PDBa code 1m17)12 was used as a
template to manually construct starting structures for 6a-d, 17,
and 18 in the EGFR pocket. The 4-anilinoquinazoline moiety
of 6a-d, 17, and 18 was assumed to bind in the same mode as
the 4-anilinoquinazoline moiety in the PDB structure. The water
molecule bridging N1 to the receptor was retained during the
modeling process, as it was crucial for obtaining more precise
refined geometries. Each starting structure was minimized to a
gradient of 0.001 kcal/A in the presence of the fixed protein
using the MMFF94x force field as implemented in MOE. A
systematic conformational search was performed on each ligand,
and the resulting conformations were reminimized in the EGFR
pocket with the MMFF94x force field to a gradient of 0.001
kcal/Å. The conformation with the lowest combined strain and
receptor interaction energy was chosen as the docked pose. It
was found that 6-substituted structures possessed the appropriate
length to permit the orientation of the central nitrogen of the
bifunctional moiety toward Asp776, suggesting that, as depicted
in Figure 2, upon protonation it may exert an electrostatic
interaction with the carboxylate of Asp776 (Figure 3).
a Reagents and conditions: (vi) NOBF4, 0 °C, CH3CN; (vii) Et3N, Et2O,
corresponding amine.
alcohol 12, which was chlorinated and hydrolyzed to give the
amine 13. Following the synthesis of their corresponding amines,
the bifunctional combi-molecules were synthesized as outlined
in Scheme 4. Diazotization of the aminoquinazoline 4a using
NOBF4 in dry acetonitrile followed by addition of the amine
11, 13, or 16 and neutralization with triethylamine gave 6a-d
(Scheme 2) and 17 and 18 (Scheme 4) as pure solids after
purification by column chromatography on basic alumina. The
1
structures of the combi-molecules were confirmed by H and
13C NMR and mass spectrometry, and their purity was confirmed
by elemental analysis (see Supporting Information).
Having demonstrated the feasibility of “combi-triazenes”
containing a pro-nitrogen mustard, we then determined whether
this structural modification that introduced a more bulky side
chain to the 6-position of the quinazoline ring negatively
affected EGFR inhibitory activities. Interestingly, the IC50 for
the different compounds varied between 0.0044 and 3.96 µM
(Table 1).
The most potent agents were compounds 6a and 6b with the
most basic monoalkyl side chain (IC50 ) 0.0044-0.071 µM).
When the nitrogen of the bifunctional moiety was directly
attached to a phenyl ring, EGFR inhibitory activity was
considerably depleted. Thus, to rationalize these results, we first
correlated the IC50 values with the basicity of the corresponding
amine side chains.
A linear correlation (R ) 0.83) was obtained between the
relative pKa of the amines (determined as that of amines related
to chloroethylamino moiety (see Supporting Information)) and
log(IC50) for inhibiting the EGFR TK activity by compounds
6a-d, 17, and 18 (Figure 1). This indicated that the central
nitrogen of the bifunctional moiety might be involved in some
Thus, increased basicity of the nitrogen as in 6a and 6b may
account for the superior potency of the latter compounds when
compared with the combi-molecules carrying aniline mustards
(6c-d, 18). It is known that the basicity of nitrogen mustards
correlate with their reactivity. Thus, we investigated whether
the most basic 6a could react with the receptor, thereby inducing
a Abbreviations: PDB, protein data bank; MOE, molecular operating
environment; MMFF94X, molecular mechanics force field 94X; ERCC1,
excision repair cross-complementing, group 1; rt, room temperature.

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2607
Figure 6. (A) Formation of fluorescent aminoquinazoline 4a from 6a.
A microplate spectrofluorometer was used to detect the released 4a
from 6a added to media with 10% of FBS and incubated for 4 h at
37 °C (excitation 270 nm, emission 451 nm). (B) DNA interstrand cross-
linking expressed as % decrease in comet tail moment following
irradiation of DU145 cells treated for 2 h with 6a or 5.
Figure 2. Requirements for a good binder.
Figure 3. Molecular modeling in the EGFR pocket of the basic ligands
6a and 6b.
Figure 7. Cell cycle effect of 6a, 5, and Iressa on DU145. Cells were
treated with the indicated concentrations of each drug for 24 h. Cells
were stained with PI and analyzed by flow cytometry.
Figure 4. Inhibition of EGFR phosphorylation by 6a in DU145 cell
line. Serum-starved cells were preincubated for 2 h with the indicated
concentrations of 6a prior to stimulation with EGF for 10 min. Equal
amounts of cell lysates were analyzed by Western blot using an
antiphosphotyrosine antibody.
It is known that the basicity of nitrogen mustards correlate
with their cytotoxic activity. This effect was not observed here
because the control drug 5 that has similar basicity when
compared with 6a was approximately 3-fold less potent than
the latter against the DU145 cells. Likewise, its potency was
not markedly superior to that of the less basic aniline mustards
6c,d. Interestingly, 17, whose structure is similar to that of 6a
but deprived of the chloroethylamino moiety, was more than
10-fold less potent than the latter, indicating that the chloroethyl
function confers significant antiproliferative advantage.
Having shown that the novel combi-molecules could achieve
selective killing of solid tumor cells, it was critical to determine
its mode action. UV fluorescence spectroscopy and HPLC
analysis showed that the most potent compound 6a decomposes
with a t1/2 of 7 min to generate 4a (Figure 6A).
Figure 5. Reverse EGFR phosphorylation in the presence of 6a in
DU145. Duplicate sets of cells were treated with the drug (2 µM) for
2 h. One set of cells was then stimulated with EGF. The other set of
cells was stimulated with EGF after two washouts at 2 h intervals and
an additional 4 h recovery. Cell lysates were analyzed by Western blot
using antiphosphotyrosine or anti-EGFR antibodies.
irreversible inhibition of the latter. Using the human prostate
cancer cell line DU145, we showed that 6a could block EGFR
phosphorylation in a dose-dependent manner (Figure 4).
When the cells were exposed to 6a for 2 h followed by
multiple washouts, inhibition of phosphorylation was still
maintained 8 h post-treatment. In contrast, in nondrug treated
cells, EGFR phosphorylation was significantly recovered. Thus,
6a blocked EGFR phosphorylation with an at least partially
irreversible mechanism (Figure 5). This is in agreement with
previous study that showed a partially irreversible inhibition
for 17 and similar combitriazenes.13
To determine whether the potency of the combi-molecules
translates into selective targeting of cells expressing EGFR, we
examined their antiproliferative activity in an isogenic pair of
NIH3T3 cells, one of which is transfected with the EGFR gene
and the established human prostate cancer cell line DU145. The
results showed that selective targeting of the combi-molecules
was directly proportional to the strength of the EGFR inhibitory
potency of the combi-molecules, with the most selective
agent being 6a (20-fold selectivity for the EGFR transfectant)
(Table 1).
Because of their short half-lives, we were not able to detect
the released nitrogen mustards. However, the single cell gel
electrophoresis (comet) assay15 showed that 6a was capable of
inducing marked dose-dependent levels of DNA cross-links,
with potency almost in the same range as that of 5, indicating
that the DNA cross-linking species may have been generated
by these molecules (Figure 6B).
More importantly, at 10 µM, combi-molecule 6a induced
significant cell cycle arrest in mid-S, with cell cycle phase
distribution profile similar to that of 5 (IC50 ) 7.6 µM). This
indicates that in addition to its EGFR inhibitory function, its
DNA damaging properties play an antiproliferative role and it
also induces sufficient levels of DNA damage to block cell cycle
progression. It is also noted that the EGFR inhibitor Iressa used
as a control in this study only induced cell cycle arrest in G1.
This suggests that the cell cycle distribution profile of 6a may
be a combination of cells arrested in G1 and in mid-S. Indeed,
the proportion of cells arrested in G1 by 6a was superior to
that observed for 5 (Figure 7).
In summary, this work has allowed us to circumvent the
problem associated with the instability of chloroethyltriazene

2608 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Letters
(6) Rachid, Z.; Brahimi, F.; Katsoulas, A.; Teo, N.; Jean-Claude, B. J.
The combi-targeting concept: chemical dissection of the dual
targeting propereties of a series of combi-triazenes. J. Med. Chem.
2003, 46, 4313-4321.
(7) 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. 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.
(8) 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.
(9) 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[5,4-d]-
pyrimidines designed as inhibitors of the epidermal growth factor
receptor. J. Med. Chem. 1997, 40, 1820-1826.
(10) Roth, G. A.; Tai, J. J. A new synthesis of aryl substituted quinazolin-
4(1H)-ones. J. Heterocycl. Chem. 1996, 33, 2051-2053.
(11) MOE software (version 2006.07) available from Chemical Computing
Group Inc., 1010 Sherbrooke Street West, Montreal, Quebec, Canada;
www.chemcomp.com.
(12) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the
epidermal growth factor receptor kinase domain alone and in complex
with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 2002, 277,
46265-46272.
(13) Brahimi, F.; Rachid, Z.; McNamee, J. P.; Alaoui-jamali, M. A.; Tari,
A. M.; Jean-Claude, B. J. Mechanism of action of a novel “combi-
triazene” engineered to possess a polar functional group on the
alkylating moiety: evidence for enhancement of potency. Biochem.
Pharmacol. 2005, 70 (4), 511-519.
(14) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; Mc Mahon, J.;
Vistica, D. New colorimetric cytotoxicity assay for anticancer-drug
screening. J. Natl. Cancer Inst. 1990, 82, 1107-1112.
(15) Spanswick, V. J.; Hartley, J. M.; Ward, T. H.; Hartley, J. A.
Measurement of Drug-Induced DNA Interstrand Crosslinking Using
the Single Cell Gel Electrophoresis (Comet) Assay. In Cytotoxic Drug
Resistance Mechanisms; Brown, R., Boger-Brown, U., Eds.; Methods
in Molecular Medicine, Vol. 28; Humana Press: Totowa, NJ, 1999;
pp 143-154.
moiety when linked to the quinazoline ring. Using the 1,2,3-
triazene as a carrier of the bulky mustard has allowed us to
overcome steric hindrance of the chloroethyaminoethyl group.
However, the most critical elements for the enhanced potency
of the combi-molecules remain the mustard nitrogen that, as
suggested by molecular modeling, may interact with the ATP
site through a protonated nitrogen and its chloroethyl function
that confers the ability to induce DNA cross-links, irreversible
EGFR inhibition, and cell cycle arrest in mid-S. Thus, this study
has permitted the development of a unique molecule with
multiple antiproliferative mechanisms. A study describing its
synergistic mechanism of action (i.e., down-regulation of the
DNA repair protein ERCC1 through EGFR inhibitory function
and delayed DNA repair) will be reported elsewhere.
Acknowledgment. We thank the Canadian Institute of
Health (Grant FRN49440) for financial support. We are also
grateful to the National Cancer Institute.
Supporting Information Available: Experimental section,
NMR, mass, and elemental analysis data, and details of the
modeling programs. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) 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 tumour targeting strategy.
J. Pharmacol. Exp. Ther. 2001, 296, 832-840.
(2) Ma, J.; Murphy, M.; O’Dwyer, P. J.; Berman, E.; Reed, K.; Gallo,
J. M. Biochemical changes associated with a multidrug-resistant
phenotype of a human glioma cell line with temozolomide-acquired
resistance. Biochem. Pharmacol. 2002, 63, 1219-1228.
(3) Pepponi, R.; Griaziani, G.; Falcinelli, S.; Vernole, P.; Levati, L.;
Lacal, P. M.; Pagani, E.; Bonmassar, E.; Jiricny, J.; D’Arti, S. hMSH3
overexpression and cellular response to cytotoxic anticancer agents.
Carcinogenesis 2001, 22, 1131-1137.
(4) Yin, J. H.; Yang, D. I.; Chou, H.; Thompson, E. M.; Xu, J.; Hsu, C.
Y. Inducible nitric oxide synthase neutralizes carbamoylating potential
of 1,3-bis(2-chloroethyl)-1-nitrosourea in C6 glioma cells. J. Phar-
macol. Exp. Ther. 2001, 297, 308-315.
(5) 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.
JM070144P