Synthesis of pyrimidinopyridine-triazene conjugates targeted to abl tyrosine kinase

Article abstract of DOI:10.1016/S0960-894X(03)00553-5

The synthesis and abl tyrosine kinase inhibitory activities of alkyltriazenes conjugated to phenylaminopyrimidines are described. Significant abl inhibitory activities were observed only when a benzamido spacer was inserted between the 1,2,3-triazene chai

Full text of DOI:10.1016/S0960-894X(03)00553-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3297–3300
Synthesis of Pyrimidinopyridine–Triazene Conjugates Targeted
to abl Tyrosine Kinase
Zakaria Rachid, Athanasia Katsoulas,
Fouad Brahimi and Bertrand Jacques Jean-Claude*
Cancer Drug Research Laboratory, Division of Medical Oncology, Department of Medicine,
´
McGill University/Royal Victoria Hospital, 687 Pine Avenue West Rm M-719, Montreal, Quebec, Canada H3A 1A1
Received 24 March 2003; revised 13 May 2003; accepted 2 June 2003
Abstract—The synthesis and abl tyrosine kinase inhibitory activities of alkyltriazenes conjugated to phenylaminopyrimidines are
described. Significant abl inhibitory activities were observed only when a benzamido spacer was inserted between the 1,2,3-triazene
chain and the 2-phenyaminopyridopyrimidine moiety.
# 2003 Elsevier Ltd. All rights reserved.
The alkyltriazenes represent one of the most potent
classes of cytotoxic drugs used in the clinic in the
chemotherapy of many cancers including melanoma,
gliomas and leukemias.1,2 Dimethyltriazenes have
superior stability when compared with monoalkyl-
triazenes.3 They require metabolic activation to gen-
erate the cytotoxic monoalkyltriazene.4,5 As outlined
in Scheme 1, the clinical drug dacarbazine has been
shown to be oxidized in vivo to its corresponding
hydroxymethyl metabolite which following loss of
formaldehyde gives the potent methyl-
triazenylimidazole-4-carboxamide (MTIC), a mono-
alkyltriazene that is further hydrolyzed to
methyldiazonium species and 5-aminoimidazole-4-car-
boxamide.6,7 Recently, the cyclic triazene temozolo-
Recently, we developed a novel strategy termed ‘Combi-
Targeting’ that seeks to synthesize chimeric molecules
designed to simultaneously block tyrosine kinase-medi-
ated growth signaling while inducing cytotoxic DNA
damage.10À13 More importantly, the resulting molecules
also termed ‘combi-molecules’ were designed to degrade
to another inhibitor of the same tyrosine kinase under
physiological conditions. This was expected to culmi-
nate into selective and sustained antiproliferative activ-
ity in cells whose growth depend on the targeted
tyrosine kinase. Here, we report the first attempt to
apply these principles to the targeting of bcr-abl, a tyr-
osine kinase proven to be the cause of chronic myelo-
genous leukemia (CML).14,15
a
mide (TEM),
a stable prodrug of MTIC was
developed that does not require metabolic oxidation
to generate the cytotoxic species.8 TEM is now used
in the clinic in the therapy of many malignancies
including melanoma and brain tumours.2,9 However,
the toxicity of these compounds which is believed to
be associated with their lack of tumor selectivity, is a
major deterrent for their use in cancer therapy.
Despite the significant potency of triazenes in the
clinic, little effort is directed at ameliorating their
tumor selectivity.
The bcr-abl oncoprotein is a 210KDa fusion protein that
is believed to play an anti-apoptotic role and blockade of
its abl kinase activity has now been shown to translate
into significant antitumor activity in both mouse models
and in the clinic.14À16 STI571 or GleevecTM 16
, one of the
most potent in vitro and in vivo inhibitors of abl
*Corresponding author. Tel.:+1-514-934-1934x35841; fax:+514-843-
1475; e-mail: jacques.jeanclaude@staff.mcgill.ca
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00553-5

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Z. Rachid et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3297–3300
Scheme 1. Metabolic activation of dacarbazine.
kinase has now been approved by the Food and Drug
Administration for the therapy of CML. Unfortu-
nately, clinical studies with GleevecTM showed rapid
development of resistance in CML patients.16 To cir-
cumvent this problem, the combination of this potent
c-abl inhibitor with cytotoxic agents has become an
actively explored strategy.17,18 Here we report the first
attempt to combine these two major mechanisms of
antitumour actions into single molecules termed:
‘combi-molecules’.
Scheme 2. (i) CH3CN, NOBF4, À5 ꢀC; (ii) Et2O, Et3N, alkylamine or
1/10mixture of aqueous methylamine (40%)/aqueous formaldehyde
(37%); (iii) p-nitrobenzoylchloride, pyridine, DMAP, rt overnight;
(iv) Fe, EtOH, H2O, AcOH, refluxing 4 h.
The design of our combi-molecules was first based on
appending the triazene tail to the 2-phenylaminopyri-
midinopyridine moiety (see square on the structure of
STI571) since derivatives of the latter group retained
significant bcr-abl kinase inhibitory activities.19 Thus,
the synthesis of the phenylaminopyrimidines 1 and 2
(Scheme 2) proceeded according to strategy described
by Zimmermann et al.20 Triazenes directly branched
onto the 2-phenylaminopyrimidopyridine moiety were
obtained by diazotization of 1 or 2 with nitrosonium
tetrafluoborate in acetonitrile followed by addition of
the desired amine and neutralization with triethylamine
to give 3 and 4. Compound 11 was synthesized by
addition of a 1/10mixture of aqueous methylamine
(40%)/aqueous formaldehyde (37%).
ing peaks at 315 and 365 nm. Interestingly, the bcr-abl
kinase inhibitory activities of the latter compounds
increased by more than 10-fold when compared with
those of 1, 3 and 4 (see Table 1).
These results can easily be rationalized in light of pre-
vious structure–activity relationships and X-ray struc-
tures of STI571 or its variants21,22 co-crystallized with
c-abl that showed a hydrogen bonding role for the
benzamido moiety. Indeed, recent studies showed that
Table 1. Cytotoxicity and v-abl tyrosine kinase inhibitory activities
for compounds 1–8 and 9–12
The abl inhibitory activities of these compounds were
tested in an enzyme immunosorbent assay (ELISA)
using poly(l-glutamic acid-l-tyrosine, 4/1)(PGT) as a
substrate for isolated v-abl kinase.20 In this assay,
STI571 showed an IC50=0.04 mM. This assay permits a
short 10-min drug exposure, thereby avoiding sub-
stantial degradation of the triazenes prior to binding to
the ATP site of abl kinase. Compounds 3 and 4 with the
triazenes directly attached to the phenylaminopyr-
imidine ring showed IC50 values in range 8–10 mM. In
view of the rather weak activities of these triazenes, we
decided to insert the benzamide spacer to our con-
jugates as in 9–12. This was achieved by treating amines
1 and 2 with p-nitrobenzoyl chloride to give 5 and 6
which were reduced with Fe in ethanol to provide 7 and
8. Diazotization of these amines (7–8) with nitrosonium
tetrafluoborate in acetonitrile followed by addition of
the desired amine and neutralization with triethylamine
gave triazene conjugates 9–12. While 1027 and 12 were
stable molecules, compounds 9 and 11 had half-lives of
16 and approximately 30min, respectively, as deter-
mined by spectrophotometric monitoring of disappear-
Compd
Inhibition of
abl kinase
IC50, mMa
IC50, mMb
K562 cells
Alkylating
activityc
 10À4
STI571
TEM
1
0.04
>100
>1021.69
8.6
0.11
156.1
ND
13
3
5.39
4
7
.11
1010
4.89
9.88
2
16.18
2.164
3.151
17.35
8
9
10
11
12
>1068
0.903
2
2
15
1.4
0.1946
1.057
0.8496
ND, not determined.
aIC50 values were determined by the v-abl kinase binding assay.20
Results are means of two separate experiments.
bIC50 values were determined by the MTT assay24 after 72 h of con-
tinuous drug exposure. Values are means of two separate experiments.
cValues are expressed by OD (540nm)/mM of drug) measured following
addition of triethylamine/acetone (v/v) to solutions of drug (0.1 mM)
+NBP pre-incubated in at pH7.4 TRIS buffer solution for 2 h at 37ꢀC.26

Z. Rachid et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3297–3300
3299
in addition to hydrogen bonds between Met 318 amide
backbone and the pyridine nitrogen, between the pyr-
imidine NH and the Thr 315 OH group, the amide bond
of the benzamido moiety was involved in a donor–
acceptor interaction wherein the oxygen of Glu 286
formed a hydrogen bond with the benzamide NH and
the oxygen of the latter accepted a hydrogen bond from
the amide NH of Asp 381. The monoalkyltriazenes were
designed with the hope that the benzamide hydrogen
could be replaced by the hydrogen of the non-con-
jugated tautomeric form as depicted in Figure 1. The
lack of abl kinase inhibitory activity may be due to the
notorious instability of this tautomer, the structure of
which resembles the transition state for protolysis of
monoalkyltriazenes.23
Figure 2. Overlay of STI571 with dimethyltriazene conjugate 10.
As outlined in Scheme 1, dialkyltriazenes like dacarb-
azine are known to be metabolized by oxidation of their
methyl group to generate a methylol intermediate that is
rapidly converted to the cytotoxic monoalkyltriazene.
The latter ultimately decomposes to an aromatic amine
and an alkylating agent.6,7 Similarly, our dimethyl-
triazene 10 will require metabolic oxidation to generate
its putative metabolite 11, the precursor of an alkylating
species. Indeed, alkylating test using the p-nitro-
benzylpyridine (NBP) assay,25 showed that in contrast
to dimethyltriazene 10, that is devoid of alkylating
activity, hydroxymethyltriazene 11 strongly alkylated
NBP (Table 1). The translation of these properties into
cytotoxic activity was studied in K562 promyelocytic
leukemia cells. Triazenes 3 and 4 showed significant
cytotoxic activities; however, this must not be rational-
ized on the basis of their bcr-abl inhibitory activities in
these cells as their IC50 values for abl TK inhibition
were extremely high (8.6–10 mM). In contrast, dime-
thyltriazene 10 that does not generate methyldiazonium
owes its strong cytotoxic effect solely to its abl kinase
inhibitory activity. Interestingly, the hydroxy-
methyltriazene analogue 11, despite being a 5-fold
weaker inhibitor of abl kinase than 10 was only 2-fold
less cytotoxic than the latter. This may well be due to
the combination of effects associated with the abl inhib-
itory activity and its ability to alkylate DNA. Indeed,
it was found that compound 11 damage DNA in K562
cells (data not shown). Detailed mechanism of the cyto-
toxic effects of 10 and 11 are reported elsewhere.
Furthermore, the contribution of the piperazine ring to
the potency of STI571 is now known. It is located in a
partially hydrophobic pocket forming van der Waals
interactions with Val 289, Phe 359 and Asp 381.22 Since
these interactions would be unavailable in our con-
jugates, we did not expect our benzamide-containing
conjugates to exhibit bcr-abl inhibitory activities super-
ior or in the same range as STI571. However, surpris-
ingly the dimethyltriazene showed activity in the
submicromolar range (Table 1). More importantly, the
hydroxymethyl derivative that differs from the dime-
thyltriazenes by only a hydroxy group was 5-fold less
active. This may be explained by the disfavored orien-
tation of the polar hydroxy group towards the partially
hydrophobic pocket of abl.
To rationalize these results, we overlaid the MM2 opti-
mized structures with the X-ray determined conforma-
tion23 of c-abl-bound STI571, using the Chem3D
software package. (Fig. 2) The model showed that of
all the triazenes (3–4, 9–12), the dimethyltriazene 10
adopted the conformation found the closest to that of
STI571. The dimethyl group appears in an orientation
where it may form, like the piperazinyl ring STI571,
van der Waals interactions with the partially hydro-
phobic pocket of c-abl. The presence of the polar
hydroxy group of 11 may decrease such interactions
leading, as observed, to a lesser affinity for the ATP
site. It is noteworthy that while the activity of 11 was
5-fold less than that of 10, it was 5–10-fold more
potent than derivatives 1–4 (devoid of the benzamido
spacer) and amines 7–8.
From the current studies, we have identified a potent
abl inhibitor of the dimethyltriazene class that does not
generate alkylating species in vitro and synthesized its
putative metabolite 11 that showed significant bcr-abl
tyrosine kinase inhibitory activity and alkylating activ-
ity comparable with that of temozolomide, a prodrug of
the dacarbazine-derived monoalkyltriazene.7 Further
studies are required in vivo to demonstrate the meta-
bolic conversion of 10 to 11 and to establish the phar-
macological advantages of these novel chimeric
molecular approach.
Acknowledgements
Figure 1. Predicted interactions of the non-conjugated tautomers of
the monoalkyltriazenes with the active site of c-abl (dotted lines indi-
cate possible hydrogen bonds).
We thank the Leukemia Research Fund of Canada for
financial support.

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Burnette, P. K.; Loughram, T.; Tepperberg, J.; Jove, R.;
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27. Experimental data for combi-molecule 10. Amine 8 (100
mg, 0.25 mmol) was dissolved in dry acetonitrile (10 mL) and
after cooling to À5 ꢀC, a solution of nitrosonium tetra-
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added. The clear solution was stirred for 1 h at À5 ꢀC and a
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m/z 452.9 (MH+), 380(M-dimethyltriazene, 76.7); 1H NMR
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8.97 (s, 1H, ArH), 8.66 (d, 1H, J=4.8 Hz, ArH), 8.49–8.44 (m,
2H, ArH), 8.05 (d, 1H, J=2.0Hz, ArH), 7.93 (d, 2H, J=8.8 Hz,
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(d, 2H, J=8.4 Hz, ArH), 3.52 (br s, 3H, N(Me)2), 3.18 (br s, 3H,
N(Me)2), 2.21 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) d
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