Design and synthesis of new stabilized combi-triazenes for targeting solid tumors expressing the epidermal growth factor receptor (EGFR) or its closest homologue HER2

Article abstract of DOI:10.1016/j.bmcl.2009.05.060

The monoalkyltriazene moiety lends itself well to the design of combi-molecules. However, due to its instability under physiological conditions, efforts were directed towards stabilizing it by grafting a hydrolysable carbamate onto the 3-position. The syn

Full text of DOI:10.1016/j.bmcl.2009.05.060

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5505–5509
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of new stabilized combi-triazenes for targeting solid
tumors expressing the epidermal growth factor receptor (EGFR) or its
closest homologue HER2
Zakaria Rachid a, Meaghan MacPhee a, Christopher Williams b, Margarita Todorova a,
Bertrand Jacques Jean-Claude a,
*
a Cancer Drug Research Laboratory, Division of Medical Oncology, Department of Medicine, McGill University/Royal Victoria Hospital, 687 Pine Avenue west Rm M-719, Montreal,
Québec, Canada H3A 1A1
b Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, Québec, Canada H3A 2R7
a r t i c l e i n f o
a b s t r a c t
Article history:
The monoalkyltriazene moiety lends itself well to the design of combi-molecules. However, due to its
instability under physiological conditions, efforts were directed towards stabilizing it by grafting a hydro-
lysable carbamate onto the 3-position. The synthesis and biological activities of these novel N-carbamyl
triazenes are described.
Received 15 January 2009
Revised 14 May 2009
Accepted 14 May 2009
Available online 20 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Combi-triazenes
EGFR
HER2
Quinazoline
Combi-molecules are agents designed to block multiple targets
in cancer cells. One such target the epidermal growth factor recep-
tor (EGFR) also known as erbB1 or HER1, is overexpressed in many
solid tumors and this is associated with aggressive tumor progres-
sion and reduced sensitivity to antitumor agents. Blockade of the
tyrosine kinase (TK) activity of EGFR and the erbB2 or HER2 gene
product p185neu induces significant antitumor activity in vivo
and IressaTM, an aminoquinazoline that binds to the ATP site of
EGFR has proven an extremely potent agent against many human
tumors in nude mice. This drug is now approved for the treatment
of lung cancer. A specific mutation in the EGFR site (deletion in
exon 19)1 is associated with the sensitivity of lung tumors to Ires-
saTM.2,3 However these tumors that initially responded to Iressa de-
velop resistance to the drug and this is believed to be associated
with the occurrence of an additional EGFR mutation in exon 20.4
In many cases, inhibition of EGFR does not suffice to eradicate
the tumor. Thus, we recently developed a novel class of molecules
termed ‘combi-molecules’ or TZ-I (Scheme 1) capable of not only
blocking EGFR-mediated signaling by binding to its ATP site (see
TZ-I-EGFR), but also damaging DNA (DNA-TZ-I). The combi-mole-
cules (TZ-I) require hydrolysis to generate the DNA damaging spe-
cies TZ and an additional EGFR inhibitor (see I, Scheme 1). Thus, the
1
TZ-I
TZ (DNAdamaging
I
(EGFR)
+
H2O
species)
EGFR
DNA
DNA-TZ-I
TZ-I-EGFR
Scheme 1.
degradation of this class of compounds is critical for their antitu-
mor activity. Importantly, we have also shown that these combi-
molecules induced sustained inhibition of EGFR which we believe
is due to their ability to alkylate the receptor, thereby inducing a
partially irreversible activity.5,6 It has now been shown that irre-
versible inhibitors such as CI1033 induce sustained inhibition of
the erb family and some of them are shown to be pan-erbB inhib-
itors.7 Recently, in order to delay the degradation of the comi-mol-
ecules, we designed a novel type of structures termed ‘cascade
release’ combi-molecules. Two such molecules RB248 and RB1079
(Scheme 2) are designed to loose their acetoxy group upon hydro-
lysis to give a methylol derivative that rapidly degrades to the cor-
responding monomethyltriazene TZ-I00 (Scheme 2).
The latter species is subsequently hydrolyzed to an alkyl diazo-
nium species and a free EGFR inhibitor I. When compared with the
rate of degradation of monoalkyltriazenes [e.g., BJ200010 (Scheme
2) or ZRBA111 (Scheme 4)], it was found that the addition of the
* Corresponding author. Tel.: +1 514 934 1934x35841; fax: +1 514 843 1475.
E-mail address: bertrandj.jean-claude@mcgill.ca (B.J. Jean-Claude).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.060

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Z. Rachid et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5505–5509
H3C
H3C
HN
N
X
HN
N
X
1
2
N
3
N
N
O
N
N
N
AcO-
N
N
O
fast
iminium ion
TZ-I
X=Br RB24
X=Cl RB107
-HCHO
fast
H3C
HN
X
N
HO
N
N
N
N
H
TZ-I"
O
H
H2O
fast
R1
R1
HN
X
HN
N
X
N
N
N
N
N
N
O
-CO2
H
N
N
R2OH
O
R2
N
TZ-I'''
N3-carbamyltriazenes
HN
X
H2N
N
+
H3C
N
N
TZ
N
I
Scheme 2.
acetoxymethyl group to N3 only delayed the degradation of the
monomethyltriazene by approximately 5 min. We further at-
tempted to increase the bioavailability of combi-molecules by
designing stable 3,3-dimethyltriazene prodrugs to be metaboli-
cally activated. These types of molecules failed to generate ade-
quate levels of monoalkyltriazenes in vivo.12
Here, we report on a novel approach that seeks to mask the
monoalkyltriazene with carbamates whose hydrolysis can be rate
limiting. The stability of the various carbamates (vinyl, chloroalkyl
and acetoxymethyl, p-nitrophenyl carbamates) was modulated by
altering the leaving group designed to depart following water addi-
tion on the carbonyl moiety (see N3-carbamyltriazene, Scheme 2).
The synthesis of the 3-carbamyl triazenes (see general structure
TZ-I0, Scheme 2) proceeded as described in Scheme 3. With the pur-
pose of identifying a leaving group that would confer optimal sta-
bility to the combi-molecule, we designed three types of
carbamates: (a) three with an electron-deficient leaving group
[e.g., vinyl (4 and 5) or p-nitrophenyl (10), (b) two with a degrad-
able chloroalkyl function (6 and 9) and (c) one with an acetoxy-
methyl moiety (8).
nitrophenyl chloroformate and 1-chloroethyl chloroformate. The
combi-molecule 8 was obtained by treatment of the methyltria-
zene 3 with chloromethyl chloroformate and pyridine in acetoni-
trile at cold temperature to give 6. Exchange of the Cl atom using
potassium iodide in dry acetone, gave the corresponding iodom-
ethyloxycarbonyl-methyltriazene 7.19 The latter intermediate
was treated with silver acetate in situ to provide 8 as a pure solid
after purification by preparative TLC alumina plates. The structures
of all the combi-molecules were confirmed by 1H, 13C NMR and
mass spectrometry (ESI). The stability of the resulting structures
was studied by measuring the rate of evolvement of blue fluores-
cence (Exct 340, Emission 451 nm) from drugs dissolved and kept
in serum-containing media at 37 °C. As outlined in Scheme 2 and
previously demonstrated,20,21 the ultimate degradation product
of each of the synthesized drug is an aminoquinazoline FD105
(1) that fluoresces in the blue. The chloromethylcarbamate ZRL1
decomposed at the same rate as RB107 and was the least stable
of the series. The most stable compound was ZRL2 (5) containing
a vinyl carbamate. ZRS1 (8) and ZRL5 (10) had approximately the
same level of stability. ZRL3 was less stable than its 1-chloroethyl
counterpart.
Compound 1 was obtained as described by Tsou et al.13–17 Diaz-
otization of aminoquinazolines 1 using NOBF4 in acetonitrile, fol-
lowed by addition of N,N-dimethyl ethylenediamine or
methylamine gave 2 and 311,18 as pure solids after neutralization
with triethylamine and purification by column chromatography
on basic alumina. Compounds 4 and 5 were synthesized by treat-
ment of 2 and 3 with the commercially available vinyl chlorofor-
mate. By the same procedure, we obtained 9 and 10 using p-
These results in toto can be rationalized in light of the chemistry
of the carbamates. ZRL3 (9) being a 1-chloroethyl-1-substituted
carbamate may perhaps form a more stable oxonium ion interme-
diate than its ZRL4 (6) counterpart, thereby leading to a faster rate
of hydrolysis. However, the case of ZRL1 (4), a vinyl carbamate
which is twofold less stable than its counterpart ZRL2 (5) (the most
stable of the series) deserves special attention. Although being

Z. Rachid et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5505–5509
5507
HN
Cl
HN
Cl
N
H
N
H2N
i
N
N
R
N
N
N
1
2 R = N,N-dimethylaminoethyl
FD105
3 R = CH3
ii
HN
N
Cl
HN
Cl
N
H
N
N
N
N
O
H3C
N
N
R1
N
O
3
iii
iv
N
4 R1 = N,N-dimethylaminoethyl
5 R1 = CH3
HN
Cl
N
N
O
N
H3C
N
6
N
HN
N
Cl
O
v
N
N
O
Cl
N
HN
Cl
H3C
R2
N
O
N
N
O
N
H3C
N
O
9 R2 = 1-chloroethyl
10 R2 = p-nitrophenyl
N
7
I
vi
HN
N
Cl
N
N
N
H3C
N
O
O
8
O
O
CH3
Scheme 3. Reagents and conditions: (i) CH3CN/NOBF4/ꢀ5 °C/ether then addition of correspondent amine, 0 °C; (ii) vinyl chloroformate, ACN/Pyr/0 °C; (iv) p-nitrophenyl
chloroformate or 1-chloroethylcholroformate, ACN/Pyr/0 °C; (iii) chloromethyl chloroformate, ACN/Pyr/0 °C; (v) acetone anhydrous/KI (1.1 equiv) at room temperature; (vi)
silver acetate.
showed a level of potency similar to that of RB107 and was the
most potent of the series. To rationalize these results, molecular
3'
Cl
+
+
N
HN
N
modeling was used to compare ZRL1 with its closest analogue
ZRL2, a vinyl carbamate containing a methyl group in lieu of the
dimethylethylamino group and that is more than three times less
potent than ZRL1 (Fig. 1). The experimental structure of the tyro-
sine-kinase domain of the EGFR complexed with a 4-anilinoqui-
nazoline inhibitor was downloaded from the PDB (PDB code
1M17)22 and used as the target structure in this study. The 1M17
PDB file was loaded into the MOE software package version
2008.10.23 All crystallographic waters were deleted except for
one water molecule near the N3 nitrogen in the quinazoline ring.
This water is conserved in both the complex (1M17) and the apo
(PDB code 1M14) PDB structures. It forms a hydrogen bond with
the N3 quinazoline nitrogen in the bound complex. The proton-
ation state of the system was set using the Protonate3D function
in MOE, which resulted in the expected deprotonation of the sol-
vent-exposed ASP 776 residue near the opening of the 1M17 bind-
ing pocket. ZRL1 was constructed in the binding pocket using the
X-ray ligand as a template, followed by minimization of the new
ligand in the field of the fixed protein atoms. The MMFF94s force-
field was used with the Born salvation model.
N
O
3
N
1
N
Cl
HN
N
O
H
N
N
N
N
2
H
N
H
N
O
O
H
H
O
H
H
C
HN
N
N
HN
N
N
N
ZRBA1
Scheme 4.
electron deficient, the vinyl group does not present the extension
of conjugation offered by the p-nitrophenyl of 10 nor the ability
to lose a chloro through formation of aldehyde as in 6 and 9. How-
ever, as depicted in Scheme 4, in ZRL1, the hydrolysis of the carba-
mate may be assisted by the neighboring dimethylamino group
that catalyzes the hydrolysis of the carbamate group, thereby
accelerating its rate of decomposition into ZRBA1.3
The EGFR tyrosine kinase (TK) inhibitory potency of the series
did not correlate with stability. Because of their bulkiness, none
of the structures were expected to show greater EGFR TK inhibitory
potency than RB107 or IressaTM. However, surprisingly, ZRL1
The results showed that both compounds retain the essential
quinazoline interactions with the receptor—namely the H-bond
between N1 and M_769 and the water-mediated interaction be-
tween N3 and T_766—but only ZRL1 can form an interaction with
ASP_776. This additional interaction significantly increases its

5508
Z. Rachid et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5505–5509
Table 1
Rate of formation of FD105 from and biological properties of combi-triazenes
Compounds EGFR TK Inhibitiona Rate of formation
Growth inhibition IC50
l
Mb NIH3T3neu
,
IC50
,
lM
SEM
of FD105 ꢁ10ꢀ3
Iressa
RB107
0.033
0.24b
0.54 0.019
ND
7.2
7.0
2.9
5.6
5.1
4.7
4.6
4, ZRL1
5, ZRL2
9, ZRL3
8, ZRS1
6, ZRL4
10, ZRL5
0.18 0.08
0.72 0.21
0.50 0.11
0.63 0.23
0.39 0.22
0.77 0.23
2.28 0.17
2.37 0.18
2.07 026.
1.50 0.16
4.94 0.82
3.79 0.18
a
IC50 for inhibition of the phosphorylation of poly(
L
-glutamic acid-
L
-tyrosine)
and data are averages of two independent experiments run in triplicates.
b
IC50 for growth inhibition of NIH3T3 cells transfected with the erbB2 gene.
600
ZRL1
ZRL2
400
200
0
0.000 6.250 12.500 25.000 50.000 100.000
Concentration (µM)
Figure 2. Comparison between tail moments induced by ZRL1 and ZRL2 in human
MDA-MB468 breast cancer cells.
between the DNA damaging potency of ZRL2 and ZRL1 is showed
in Figure 2.
To compare the effect of the different agents on cell growth,
we chose the NIH3T3neu cell line, a HER2 transfectant that has
already been shown to be extremely sensitive to EGFR inhibi-
tors and a direct correlation was established between EGFR
TK IC50 values for EGFR inhibitors and growth inhibitory po-
tency against the latter cell line.25 Our positive control Iressa
being a strong inhibitor of EGFR, showed the greatest potency
of all combi-triazenes tested. The drug with the closest potency
level when compared with IressaTM was ZRS1 that despite being
an almost 20-fold weaker EGFR inhibitor showed an IC50 value
only threefold lower than that of IressaTM against NIH3T3neu.
The purpose of this work being to identify a stabilizing strategy
for combi-triazenes, the results suggest that based on its stabil-
ity, its EGFR inhibitory property and its strongest growth inhib-
itory effect in the series, ZRS1 can be considered the optimized
structure.
This study conclusively demonstrated that combi-molecules
stabilized by grafting a carbamate to the N3 of the triazene chain
can achieve greater stability when compared with our previously
reported carboxymethyl-N3 strategy8,9 exemplified by 5. More
importantly, we showed that stability could be achieved without
any significant loss in the binary EGFR-DNA targeting property of
the identified lead molecule ZRS1 (8). Further studies are ongoing
to demonstrate their and potency in vivo.
Figure 1. The 2D protein–ligand interaction diagram of ZRL2 (a) compared with
ZRL1 (b); (c) A Gaussian-Connolly surface is drawn around the binding pocket, and
the residue ASP_776 is labeled. The position of the 4-anilinoquinazoline core is
essentially unchanged from the X-ray structure, while the N,N-dimethylaminoethyl
is of sufficient length to interact with ASP_776 (indicated by the dotted line).
EGFR TK inhibitory potency when compared with ZRL2 and the
other analogues (see Table 1).
A complete analysis of EGFR-DNA targeting properties of the
molecules in a human cell line is reported elsewhere.24 Neverthe-
less it is worth mentioning that all compounds were capable of
damaging DNA, except the most stable drug ZRL2, which did not
induce a dose-dependent increase in DNA damage. A comparison
Acknowledgment
We thank the Canadian Institutes of Health Research (CHIR) for
financial support (Grant No. FRN 49440).

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