Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase

Article abstract of DOI:10.1016/j.bmcl.2012.11.076

Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.

Full text of DOI:10.1016/j.bmcl.2012.11.076

Bioorganic & Medicinal Chemistry Letters 23 (2013) 802–805
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines
as inhibitors of phosphoinositide 3-kinase
⇑
Michelle S. Miller, Jo-Anne Pinson, Zhaohua Zheng, Ian G. Jennings, Philip E. Thompson
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, 381 Royal Pde, Parkville, Victoria 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treat-
ment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the
treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benz-
imidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by
the traditional route results in a mixture of the two regioisomers. We have developed a straightforward
regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole
derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy
substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-
position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be
probed.
Received 18 September 2012
Revised 19 November 2012
Accepted 20 November 2012
Available online 1 December 2012
Keywords:
Phosphoinositide 3-kinase inhibitors
ZSTK474
Regioselective synthesis
Benzimidazole derivatives
Ó 2012 Elsevier Ltd. All rights reserved.
The phosphoinositide 3-kinase (PI3K) pathway is an important
cellular signalling pathway that functions to elevate levels of cell
growth and proliferation. It is one of the most frequently activated
pathways in cancer.1,2 There are four highly homologous isoforms,
potency.7 For example, 4-methoxy substitution alone results in a
3-fold increase in potency and a shift towards PI3K
as compared to ZSTK474.
a-selectivity
In a similar fashion, we were interested in investigating the
influence of 5- and 6-methoxy substituents on potency and selec-
tivity of ZSTK474 and analogues. The 2-difluoromethyl, 2-methyl
and 2-isopropyl derivatives 4a–c could be prepared readily
through sequential substitution of cyanuric chloride as detailed
in Scheme 1.7 However, this synthetic scheme was unsuitable for
the synthesis of 5- or 6-methoxy substituted benzimidazole deriv-
atives, as use of the 5-methoxybenzimidazoles as reagents resulted
in inseparable mixtures of the 5- and 6-methoxy regioisomers due
to tautomerism at the ring nitrogens.
This limitation has impeded others working on substituted
benzimidazoles relative to other variations such that relatively
few examples have been published. In certain cases it may be pos-
sible to separate the mixture of such 5- and 6-substituted regioi-
somers via chromatography as was shown in the case of triazine
based MAP kinase inhibitors and in other examples in the patent
literature.8–10 In our case, chromatographic separation couldn’t
be achieved readily, so a regioselective synthetic approach was
necessary.
designated PI3Ka, PI3Kb, PI3Kc and PI3Kd, each having a distinct
array of physiological functions. Activating mutations in PI3K
a
have been found in about a quarter of breast and endometrial can-
cers, identifying PI3K as an important target for novel cancer
therapeutics.3
Among a range of current clinical candidate drugs, ZSTK474 (4a)
is a potent, pan-PI3K inhibitor that selectively inhibits PI3K over
many other related kinases.4 It is currently in Phase I trials for
the treatment of advanced solid malignancies.5 Like the majority
of candidates it has comparable potency at the four Class I iso-
forms, which may lead to off-target effects that compromise ther-
apeutic utility. Certainly, there is a need for more isoform-selective
compounds to help delineate the roles played in cancer by individ-
ual isoforms.
The crystal structure of ZSTK474 bound to PI3Kd was released in
2010.6 Analysis of the binding mode of ZSTK474 to the p110d ATP
binding site showed that the difluoromethylbenzimidazole ring
projects into what has been described as an affinity pocket. It also
appeared likely that substitution of the benzimidazole ring might
lead to altered potency and selectivity. Indeed, a recent paper de-
tailed a limited number of 4-substituted and 4,6-disubstituted ana-
logues that were effective in improving both solubility and
A recent review has identified a number of approaches to regio-
selective syntheses of N1-substituted benzimidazoles.11 Palladium
or copper catalysed reactions can be used in either intra- or inter-
molecular amination and cyclisation reactions using amidines or
carbodiimides.12–16 More recently, Ma and Buchwald have re-
ported the development of a metal-catalysed cascade aryl amina-
tion and condensation process.17–19 These reactions proceed via
⇑
Corresponding author. Tel.: +61 3 9903 9672; fax: +61 3 9903 9582
E-mail address: philip.thompson@monash.edu (P.E. Thompson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.076

M. S. Miller et al. / Bioorg. Med. Chem. Lett. 23 (2013) 802–805
803
cyclisation process.20,21 Alternatively, an appropriate o-nitroaniline
can be used, with the free amine group functionalised, followed by
a reduction and cyclisation to form the benzimidazole.22 To our
knowledge, there are no reports in the literature of these methods
being applied to benzimidazole-substituted triazines.
We have developed a synthetic approach starting with appro-
priate 4- and 5-methoxy-2-nitroanilines for the synthesis of corre-
sponding methoxy-substituted analogues of ZSTK474 derivatives.
This efficient and general synthesis can be achieved under rela-
tively mild conditions, provides regiospecific access to substituted
benzimidazole–triazines and allows for further diversification
through the 2-position of the benzimidazole (Scheme 2). It has al-
lowed us to elucidate the specific influence of these substituents on
PI3K affinity and isoform selectivity.
The precursor 2-chloro-4,6-dimorpholino-1,3,5-triazine 57 was
subjected to Buchwald–Hartwig amination conditions with 4-
methoxy-2-nitroaniline or 5-methoxy-2-nitroaniline to yield the
corresponding substituted triazines 6a and 6b in 85% and 77%
yields, respectively. The nitro group was reduced with SnCl2 under
mild, non-acidic conditions.23 Due to differences in solubility of the
two precursors, the use of ethanol was preferable for the 4-meth-
oxy-2-nitroaniline derivative, giving a yield of 91% of 7a while
ethyl acetate gave enhanced reaction time and yield of 83% for
the 5-methoxy-2-nitroaniline derivative 7b. SnCl2 reduction under
acidic conditions was trialled initially, but it was found that the
morpholine group was labile.
Quite specific conditions were required to form the desired
benzimidazole products, 9a–f. In the first instance, where the
phenylenediamine derivative 7a was refluxed with difluoroacetic
acid and a catalytic amount of polyphosphoric acid, a 60:40 mix-
ture of the 6- and 5-methoxy derivatives 9a and 9b resulted, a con-
sequence of acid-catalysed isomerisation of the diamine species.
Alternatively, refluxing 7a or 7b under non-acidic conditions with
Scheme 1. Reagents and conditions: (a) morpholine, triethylamine, acetone,
ꢀ20 °C; (b) 2-substituted benzimidazole, K2CO3, DMF, room temp; (c) morpholine,
K2CO3, DMF, MW 140 °C; (d) morpholine, triethylamine, acetone, 0 °C to room
temp; (e) 2-substituted benzimidazole, Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane,
MW 150 °C, 40 min.
o-aminoanilides as intermediates, before a condensation reaction
produces the desired benzimidazole product. Metal catalysts can
also be used to amidate o-halonitroarenes, followed by a reductive
Scheme 2. Reagents and conditions: (a) morpholine, triethylamine, acetone, 0 °C to room temp; (b) 4- or 5-methoxy-2-nitroaniline, Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane,
MW 150 °C; (c) SnCl2ꢁ2H2O, ethanol or ethyl acetate, N2, 70 °C; (d) acid chloride, DIPEA, DCM, room temp; (e) acetic acid, xylenes, 130 °C.

804
M. S. Miller et al. / Bioorg. Med. Chem. Lett. 23 (2013) 802–805
Table 1
Inhibition of PI3K isoforms
Compound
R
OCH3 position
IC50 (lM)
PI3K
0.006
>5
0.375
0.290
9.2
0.823
0.431
>10
1.05
a
PI3Kb
0.006
>10
0.214
0.523
>10
1.79
0.419
>10
0.726
PI3K
0.038
>10
>10
1.86
>10
>10
2.53
c
PI3Kd
4a
9a
9b
4b
9c
9d
4c
9e
9f
CHF2
CHF2
CHF2
CH3
CH3
CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
—
5
6
—
5
6
—
5
6
0.003
>1
0.110
0.187
2.77
0.458
0.128
1.12
>10
>10
0.423
IC50 values are the average of three independent experiments.
acetaldehyde in DMF in the presence of sodium metabisulfite did
give the desired regioselective 2-methyl products 9c and d.24 How-
ever, a 2-desmethyl side-product was also present which made
purification very difficult, resulting in low yields. It was thought
to arise from thermal degradation or polymerisation of acetalde-
hyde during the reaction. Stirring 7a and acetaldehyde at room
temperature in the presence of sodium sulphate as a dehydrating
agent gave no reaction after 48 h.25
Finally, success was obtained using a two-step approach. Mono-
acylation of 7a and 7b with difluoroacetylchloride was high yield-
ing and gave the regioisomerically pure acetamides 8a and 8b,
respectively. Cyclisation was completed by refluxing overnight in
a mixture of acetic acid and xylenes to give 9a and 9b. The corre-
sponding 2-methyl (9c, d) and 2-isopropyl derivatives (9e, f) were
also obtained using acetyl chloride and isobutyryl chloride, respec-
tively, illustrating the applicability of this method to the introduc-
tion of various substituents in the 2-position.
With the six methoxy-substituted derivatives 9a–f and their
unsubstituted counterparts 4a–c in hand, they were then tested
for activity against each of the four Class I PI3K isoforms. The re-
sults are summarized in Table 1.
The inclusion of a methoxy substituent at the 6- or 5-position of
ZSTK474 results in analogues manyfold less potent than ZSTK474
(4a) itself. The 6-methoxy substituted analogue 9b was at least
30-fold less potent against the PI3K isoforms and in fact activity
was abolished against PI3K
even less favoured with compound 9a unable to achieve 50% inhi-
bition below 1 M at any of the isoforms. This emphasizes the dif-
c. Substitution at the 5-position was
l
ference compared to the 4-methoxy substituted analogue reported
by Rewcastle et al. which showed improved potency.7 It is also
consistent with their results, which revealed that the 4,5-dime-
thoxy derivative was significantly less active than both the 4-
methoxy and the 4,6-dimethoxy derivatives.7
Interestingly, the 2-position benzimidazole substituent influ-
enced the magnitude of potency lost when 5- or 6-methoxy sub-
stituents were introduced. Both the 2-methyl and 2-isopropyl
substituted analogues of ZSTK474 are themselves somewhat less
active than ZSTK474, but in these cases the drop in potency upon
addition of a 6-methoxy substituent as in 9d and 9f was more mar-
Figure 1. Compounds docked in the ATP-binding site of p110d (2WXL PDB). (a)
Comparison of the docked pose of 9a (cyan) with the crystal structure binding mode
of ZSTK474 (4a) (green); (b) comparison of the docked pose of 9b (magenta) and
ZSTK474 (4a) (green).
ginal at around 2- to 3-fold, except for PI3K
be inhibited.
c which again failed to
the expected pose (Fig. 1b). Compound 9a, however, fails to as-
sume the expected pose, instead the 5-methoxybenzimidazole
group is facing the solvent exposed region, the second morpholine
is in the affinity pocket and the triazine is rotated through the
plane (Fig. 1a). The relative potency of the 6- and 5-methoxy sub-
stituents appears to be accounted for, however, as 6-methoxybenz-
imidazole derivatives can form a hydrogen bond with Y813 that
the 5-methoxy derivatives cannot. The lost activity at PI3Kc im-
plies that this isoform is in some way more sensitive to
substitution.
Molecular docking was used to study the binding of 5- and 6-
methoxy derivatives compared to the crystallographically deter-
mined pose of ZSTK474 in PI3Kd (PDB code 2WXL).6 In ZSTK474,
one morpholinyl oxygen atom forms a hydrogen bond with the
backbone amide of V828 in the hinge region of PI3K; the benzimid-
azole substituent extends into the affinity pocket and the second
morpholine group faces the solvent exposed region. Docking sug-
gests that the 6-methoxy substitution should not be as disfavoured
as the biochemical data suggests, as compound 9b is able to adopt

M. S. Miller et al. / Bioorg. Med. Chem. Lett. 23 (2013) 802–805
805
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Acknowledgments
This work was funded by a project Grant from the National
Health and Medical Research Council (Grant no.: 545943). M.S.
Miller was supported by an Australian Postgraduate Award schol-
arship and a Cooperative Research Centre for Cancer Therapeutics
top-up scholarship.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
11.076.
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