Synthesis and biological evaluation of novel phosphatidylinositol 3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs of 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl] -1H-benzimidazole (ZSTK474)

Article abstract of DOI:10.1016/j.ejmech.2013.03.038

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H- benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH₂)₃NMe₂ analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.

Full text of DOI:10.1016/j.ejmech.2013.03.038

European Journal of Medicinal Chemistry 64 (2013) 137e147
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel phosphatidylinositol
3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs
of 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-
1H-benzimidazole (ZSTK474)
,
b
,
a
a
,
,b
Gordon W. Rewcastle a , Swarna A. Gamage , Jack U. Flanagan b, Jackie D. Kendall a
,
*
William A. Denny a b, Bruce C. Baguley a b, Christina M. Buchanan b c, Mindy Chao a,
Philip Kestell a, Sharada Kolekar c, Woo-Jeong Lee c, Claire L. Lill a, Alisha Malik c,
,
,
,
Ripudaman Singh a, Stephen M.F. Jamieson a b, Peter R. Shepherd b
,
,c
a Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New
Zealand
b Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
c Department of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-
(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more
soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-
O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combi-
nation with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in
a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable
pharmacokinetic profile.
Received 27 September 2012
Received in revised form
28 February 2013
Accepted 21 March 2013
Available online 6 April 2013
Keywords:
Anticancer
Antitumor
PI 3-kinase
Synthesis
ZSTK474
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
but all use phosphatidylinositol-4,5-diphosphate (PIP2) to produce
phosphatidylinositol-3,4,5-triphosphate (PIP3). The cellular levels
Phosphoinositide 3-kinases (PI3Ks) are a family of three distinct
classes (I, II and III) of lipid kinases that play key roles in cell and
of PIP3 are tightly controlled by phosphatases including PTEN
which dephosphorylates PIP3 back to PIP2 [8,9]. The importance of
this pathway in cancer is highlighted by the fact that defects in both
the kinase and phosphatase activities are commonly observed in
tumors, and there is now increasing evidence that a high propor-
tissue physiology [1e3]. The three class-Ia PI 3-kinases (p110
a/b/d)
and the sole class-Ib PI 3-kinase (p110 ) couple growth factor re-
g
ceptors and G-protein coupled receptors respectively to a wide
range of downstream pathways [4e6]. These enzymes have
different methods of activation and different kinetic properties [7],
tion of human cancers depend strongly on p110a for their survival
and resistance to therapy [8e15]. Therefore the targeting of PI3K
with small molecule inhibitors is one of the most promising new
approaches to cancer treatment, and a number of programs to
develop PI3K inhibitors are currently in progress [5,16e19], with
several inhibitors in clinical trial [20e26].
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-
1H-benzimidazole (ZSTK474) (1) (Fig. 1) is a potent ATP-competitive
pan-class I PI3K inhibitor, with high selectivity over other classes of
* Corresponding author. Auckland Cancer Society Research Centre, School of
Medical and Health Sciences, The University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand. Tel.: þ64 9 9236147; fax: þ64 9 3737502.
E-mail
addresses:
g.rewcastle@auckland.ac.nz,
grewcastle@orcon.net.nz
(G.W. Rewcastle).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.038

138
G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
Scheme 3 details an alternative (higher yielding) route to
alcohol 8 which was subsequently used for a larger scale prepara-
tion of amine 14. Thus, instead of proceeding through phenol 2,
whose prior synthesis involves a moderately yielding five-step
procedure [30,31], the synthesis of 8 was achieved directly in just
five steps starting from commercially available 2-amino-3-
nitrophenol (32). Alkylation of 32 gave alcohol 33 which was
converted to benzimidazole 34 by reduction and subsequent ring
closure with difluoroacetic acid. Protection of the alcohol group of
34 as its TBDMS derivative, followed by reaction with 2-chloro-4,6-
dimorpholino-1,3,5-triazine [33] in DMSO at 130 ꢀC gave 36 which
was then deprotected with TBAF to give 8 in 56% overall yield.
3. Results and discussion
3.1. Enzyme inhibition
Fig. 1. Structure of ZSTK474 (1) and substituted analogs.
All new compounds were tested for their enzyme activity
PI3K and protein kinases [27e29], and demonstrated antitumor ac-
tivity in vivo against human tumor xenografts [27,29].
against the p110
assay (Table 1).
a, b and d isoforms of PI3K using a lipid kinase
We recently performed a structure activity relationship (SAR)
study of this compound, and identified substitution at the 4- and 6-
positions of the benzimidazole ring as having significant effects on
the potency of substituted derivatives [30]. Electron donating sub-
stituents that increased the electron density on the benzimidazole 3-
nitrogen were found to be particularly efficacious, and this was
presumed to be due to enhanced H-bonding of the benzimidazole
nitrogen to a specific lysine amino group in the ATP binding site of
Compounds 6e17 explored a variety of solubilizing substituents
attached via a 4-oxygen linker with acidic carboxylate, neutral
alcohol, and basic amine substituents being investigated. The
oxyacetic acid derivative 6 did not show good enzyme or cellular
potency, and was not studied further. The alcohols 7e9 retained
good enzyme and very good cellular potency, but did not markedly
improve solubility. The basic amino compounds 10e17 also
retained good cellular potencies but had varying enzyme inhibitory
effects, with 14 displaying the best enzyme potency against all
three isoforms. Compounds 21e25 explored the use of a 4-nitrogen
linker, but all were less effective than the oxygen linked analogs.
Overall, the 4-O(CH2)3NMe2 analog 14 was the most active of
the basic amino compounds, and displayed good aqueous solubility
properties (25 mg/mL for the hydrochloride salt), and was therefore
selected for further evaluation.
PI3K (Lys802 in p110a, Lys805 in p110b, Lys833 in p110g and Lys779
in p110 ). 4-Substituents could also participate in H-bonding to the
d
lysine amino group, and the 4-OH derivative 2 was particularly
potent, although found to have poor pharmacokinetics, presumably
as a result of rapid glucuronidation. The more stable 4-OMe deriva-
tive 3 was less potent than 2, but showed greater selectivity for the
p110a isoform of PI3K. Amino substituents were also efficacious,
with the 4-NH2 derivative 4 having similar potency to 3, while the 4-
OMe, 6-NH2 derivative 5 was particularly potent. In vivo testing of 5
showed that it dramatically reducedcancer growth by 81%compared
to untreated controls, although it suffered from solubility issues [30].
In the present paper we investigate a wider range of benz-
imidazole 4-substituents, focusing particularly on oxygen or
nitrogen-linked solubilizing moieties that might overcome the
solubility problems seen with 5, while still retaining the ability to
assist in binding to the PI3K lysine amino group.
3.2. Cellular inhibition
The compounds were also evaluated using two human tumor
cells lines, NZOV9 (Y1021C mutation of p110
a enzyme) and NZB5
(wild-type p110 enzyme) and the data are shown in Table 1. The
a
growth inhibitory IC50 values for some compounds were compa-
rable with the IC50 values for isolated enzyme studies, while others
were considerably higher, suggesting variable efficiency in cellular
uptake. Growth inhibitory IC50 values for the NZOV9 line correlated
2. Chemistry
significantly to enzymatic IC50 values for p110
(Spearman rank correlation; R ¼ 0.60; p ¼ 0.011). However, NZOV9
IC50 values did not correlate significantly with p110 or p110 IC50
values, and NZB5 IC50 values did not correlate to IC50 values of any
of the enzyme assays. These results supported the hypothesis that
a IC50 values
The 4-oxygen linked derivatives (6e17) of Table 1 were prepared
by alkylation of phenol 2 [30,31] (Scheme 1). Compounds 6e9, 11,
12 and 15 were prepared directly from 2, but the one-step synthesis
of 14 occurred in too low a yield to be viable by this route.
Accordingly, 14 was prepared from alcohol 8, via mesylation and
subsequent reaction with dimethylamine. Primary amines 10 and
13 were prepared from 2 via the respective phthalimides 18 and 19,
while the amino-alcohols 16 and 17 were prepared via amine
addition to epoxide 20.
The 4-nitrogen linked derivatives (21e25) of Table 1 were pre-
pared from amine 4 [30,32] or its Boc derivative 26 [30] (Scheme 2).
Alkylation of 26 followed by Boc removal gave amine 21, while
amino-amide derivatives 22e25 were prepared via acylation of 4,
and subsequent reaction of intermediates 28e31. Thus primary
amines 22 and 24 were prepared by TFA deprotection of their Boc
derivatives 28 and 29, while tertiary amines 23 and 25 were pre-
pared by dimethylamine addition to chloroacetamide 29 or acryl-
amide 31 respectively.
b
d
the mutant p110a enzyme played a significant role in drug-induced
inhibition of cell growth.
3.3. Inhibition of cell signaling
To determine whether compound 14 was capable of entering
cells and attenuating signaling downstream from PI 3-kinase, the
effect on phosphorylation of Akt/PKB was determined in HCT116
cells using previously described methods [34,35]. HCT116 cells
were chosen for this experiment as they are commonly used in
xenografts and have constitutive activation of the PI 3-kinase
pathway due to activation of PIK3CA. We determined the IC50 for
the inhibition of phosphorylation at not only the most commonly
measured Ser473 site but also for Thr308 as this site is the one most
directly linked to activation of PI 3-kinase in the cell. Compound 14

G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
139
Table 1
Enzyme and cellular inhibition.
a
a
a
b
b
Compound
R
p110
(nM)
a
IC50
p110
(nM)
b
IC50
p110
(nM)
d
IC50
NZB5 IC50
M)
NZOV9 IC50
(mM)
(
m
6
7
52
274
16
39
8.5
3.4
5.3
10
2.6
22
4.7
0.06
0.06
8
9
170
24
0.071
0.026
0.018
0.025
12
10
11
17
35
130
105
20
0.052
0.45
0.022
0.15
19
53
12
130
150
0.105
0.071
13
14
28
11
75
28
0.032
0.17
0.009
0.04
7.3
4.5
15
29
76
18
13
0.06
0.03
16
17
33
41
30
53
0.33
0.11
5.5
0.054
0.025
21
22
174
119
73
15
52
0.20
0.14
0.11
0.06
740
23
24
379
855
1040
3015
99
0.05
0.44
0.05
0.12
210
25
486
1260
112
0.29
0.14
a
In vitro lipid kinase assay. IC50 values are the mean of duplicate or triplicate measurements.
In vitro cell proliferation assay. IC50 values are the mean of duplicate or triplicate measurements.
b
was found to inhibit phosphorylation at both sites, showing that it
3.4. Pharmacokinetics and antitumor efficacy
was capable of entering cells and attenuating downstream
signaling, with a potency comparable to that of compounds 1, and 3
[30] (Table 2), although not as potent as compound 5 with the
additional 6-amino group.
The plasma pharmacokinetic profile of 14 was determined in
C57 mice following a dose of 10 mg/kg by i.p. injection (Table 3). A
peak plasma concentration of 1938 nM was reached 15 min after

140
G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
Scheme 1. Reagents and conditions: (i) RCH2X, K2CO3, DMF, rt or 50e60 ꢀC; (ii) MsCl, Et3N, THF, 0 ꢀC; (iii) 40% aq. Me2NH, THF, rt; (iv) N-(bromoalkyl)phthalimide, K2CO3, DMF,
50 ꢀC; (v) NH2NH2$H2O, CH2Cl2, THF, rt; (vi) epibromohydrin, K2CO3, DMF, 50 ꢀC; (vii) (for 16) 40% aq. Me2NH, THF, rt; (viii) (for 17) morpholine, THF, MeOH, rt.
dosing, after which the compound was cleared with a half-life of
1.56 h, generating an AUCINF of 2586 nM h. The antitumor efficacy of
14 was determined in a U87MG xenograft model in Rag1ꢁ/ꢁ mice
alongside compounds 1 and NVP-BEZ235 (37) (Fig. 2). Each com-
pound was administered by i.p. injection at its maximally tolerated
dose at a qdx14 dosing schedule. At a dose of 60 mg/kg, 14 delayed
tumor growth relative to controls, but was less effective than both 1
Scheme 2. Reagents and conditions: (i) NaH, Me2N(CH2)2Cl, DMF, 0 ꢀC to rt; (ii) TFA,
CH2Cl2, rt; (iii) (for 28 and 30) BocNH(CH2)nCO2H, EDCI, DMAP, DMF, rt; (iv) (for 29)
ClCH2COCl, K2CO3, CH2Cl2, rt; (v) (for 31) CH2]CHCOCl, DIPEA CH2Cl2, 0 ꢀC to rt; (vi)
(for 23) 40% aq. Me2NH, EtOH, reflux; (vii) (for 25) 40% aq. Me2NH, EtOH, rt.
Scheme 3. Reagents and conditions: (i) HO(CH2)3Cl, K2CO3, acetone, reflux; (ii) H2, Pd/
C, MeOH; (iii) F2CHCO2H, 4 N HCl, reflux; (iv) TBDMSCl, pyridine, rt; (v) 2-chloro-4,6-
dimorpholino-1,3,5-triazine, K2CO3, DMSO, 130 ꢀC; (vi) TBAF, THF, rt.

G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
141
Table 2
A
Inhibition of cell signaling in HCT116þ/þ (PIK3CA H1047R mutant) cells by 1, 3, 5 and
14 in vitro.
1200
Control
15 mg/kg 37
40 mg/kg 1
60 mg/kg 14
a
a
Compound
pAkt/PKB Ser473 IC50 (nM)
pAkt/PKB Thr308 IC50 (nM)
800
400
0
1b
3b
5b
14
97
37
12
36
78
25
17
74
a
Compound concentration required to produce 50% inhibition of phosphoryla-
tion in a cellular assay.
b
Data from Ref. [30]. Cells were exposed to inhibitor dissolved in DMSO for
15 min before stimulation with 500 nM insulin for 5 min. Protein isolation and
immunoblotting for phospho-Akt/PKB was carried out according to the methods
previously described in Refs. [34,35].
0
5
10
15
Time from Start of Treatment (days)
B
20
10
0
Control
Table 3
15 mg/kg 37
Mouse plasma pharmacokinetic parameters for 14.
40 mg/kg
1
Compound Dose (mg/kg) Route Cmax (nM) Tmax (h) AUCINF (nM h) T1/2 (h)
60 mg/kg 14
14
10
i.p.
1938
0.25
2586
1.56
-10
-20
and 37 (Fig. 3A). Tumor growth inhibition relative to controls was
calculated on day 7 with 14 inhibiting tumor growth by 56.3 ꢂ 2.6%
(P < 0.001), 1 by 67.6 ꢂ 2.7% (P < 0.001) and 37 by 60.3 ꢂ 6.0%
(P < 0.001). Each treatment was well tolerated for the majority of
animals, with only mild bodyweight loss averaging between 5 and
7% of pre-treatment size observed for the 3 treatment groups
(Fig. 3B). However, significant bodyweight loss (19%) resulting in
mortality was observed with 1 mouse treated with 1, while an
unexpected mortality with no preceding signs of toxicity or body-
weight loss occurred with 14.
0
5
10
15
Time from Start of Treatment (days)
Fig. 3. Average tumor volume (A) and bodyweight loss (B) in Rag1ꢁ/ꢁ mice with
U87MG tumors treated with 15 mg/kg 37, 40 mg/kg 1 or 60 mg/kg 14. Bars represent
the mean and standard error of 5e7 mice per group.
Lys802 was also predicted to interact with oxygen atoms at the
benzimidazole 4-position. The 6-amino moiety of 5 was predicted
to form interactions with Asp754 and 877 in the same region.
RMSD values for the ZSTK474 core of the predicted binding modes
3.5. Modeling of binding
compared to that bound in p110d was not improved from com-
A binding mode for ZSTK474 (1) was predicted in the p110
a
pound 1 with the 4 and 6 substitutions of compounds 2e5 (com-
pound 2 RMSD 0.25, compound 3 RMSD 0.67, compound 4 RMSD
0.75 and compound 5 RMSD 0.48).
kinase catalytic site where the benzimidazole moiety was bound in
the affinity pocket as previously reported [30]. A possible interac-
tion between the benzimidazole 3-nitrogen atom and Lys802 was
identified, and one morpholine unit formed what we consider to be
an essential interaction with the back bone amide of Val851 [30].
This predicted binding mode was in good agreement with that
Fig. 4 shows the 4-methoxy derivative 2 (magenta stick) docked
into the kinase active site of p110a, with both the oxygen atom of the
methoxy group and the benzimidazole 3-nitrogen atom forming H-
observed in the X-ray crystal structure of the p110
d isoform (PDB
2wxl) [36]. Further, when superimposed on ZSTK474 bound in the
p110
d
active site, the conformation generated upon docking into
had an RMSD of 0.23 A, with most variation seen in the non-
ꢀ
p110a
essential morpholine group. Similar binding modes were found for
compounds 2e5 when active site flexibility was introduced at the
side chains of residues Lys720, Lys776, Lys802, Asp805, Asp933
along with a soft potential interaction model at residues Leu807,
Asp810, Tyr836 and Ile848. Binding models that predicted a Lys802
interaction with the benzimidazole 3-nitrogen were not necessarily
the top scoring binding poses, and under the conditions used
Fig. 4. Compound 2 (magenta stick) docked into the kinase active site of p110
idues Lys802 and Asp933 defining the benzimidazole binding site are shown in ball
and stick. The positions of these residues in the p110 structure 2rd0 are shown in
a. Res-
a
yellow, while the final positions associated with the flexible receptor docking of 2 are
shown in green. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
Fig. 2. Structure of NVP-BEZ235 (37).

142
G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
bond interactions with Lys802. The results presented here indicate
that the better inhibitory activity of O-linked solubilizing groups
(compounds 6e17) may result from the maintenance of an interac-
tion with Lys802, while the less well tolerated amine and amide
linked groups (compounds are 21e25) are likely to disrupt this.
5.1.2. 2-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)ethanol (7)
A mixture of 2 (85 mg, 0.0196 mmol), 2-iodoethanol (37 mg,
0.22 mmol), and powdered K2CO3 (83 mg, 0.6 mmol) in 10 mL of
DMF was stirred at room temperature overnight, and then diluted
with water. The product was collected by filtration, dried, and pu-
rified by chromatography on silica eluting with EtOAc to give 7
(47 mg, 50% yield): mp (EtOAc) 243e246 ꢀC; 1H NMR (DMSO-d6)
4. Conclusion
d
7.89 (d, J ¼ 8.1 Hz, 1H), 7.70 (t, JHF ¼ 52.8 Hz, 1H), 7.38 (t, J ¼ 8.2 Hz,
The pharmacokinetic parameters of 14, in particular T1/2 and
to a lesser degree, Cmax, are comparable with those reported for
compounds 1 and 5 in CD-1 mice [30]. However, the AUCINF of 14
was approximately half that of 5 and a quarter that of 1. There-
fore, despite the potency of 14 against the PI3K isoforms and its
improved solubility relative to 1, it demonstrated less potency
than 1 in a U87MG xenograft model as a result of its unfavorable
pharmacokinetic profile. Further work is therefore ongoing to
identify compounds that display both good aqueous solubility
and improved PK properties that will result in a better in vivo
profile.
1H), 6.95 (d, J ¼ 7.9 Hz, 1H), 4.93 (t, J ¼ 5.5 Hz, exchangeable with
D2O, 1H), 4.24 (t, J ¼ 5.0 Hz, 2H), 3.84e3.79 (m, 10H), 3.69 (m, 8H);
13C NMR (DMSO-d6)
d 164.4 (C), 161.4 (C), 151.1 (C), 144.2 (t,
JCF ¼ 26.7 Hz, C-2), 134.5 (C), 131.6 (C), 126.8 (CH), 108.6 (t,
JCF ¼ 237.4 Hz, CHF2), 108.0 (C), 106.5 (C), 70.4 (CH2), 68.9 (CH2),
59.6 (CH2), 43.6 (CH2). Anal. Calcd for C21H25F2N7O4: C, 52.8; H, 5.3;
N, 20.5. Found: C, 52.9; H, 5.2; N, 20.5%.
5.1.3. 3-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-1-propanol (8)
5.1.3.1. Method A (Scheme 1). A mixture of 2 (0.36 g, 0.83 mmol), 3-
chloropropanol (0.40 g, 4.2 mmol), powdered K2CO3 (0.58 g,
4.2 mmol) and NaI (0.63 g, 4.2 mmol) in DMF (5 mL) was heated at
50e60 ꢀC for 24 h, and diluted with water. The product was
extracted with CH2Cl2 and chromatographed on silica, eluting with
CH2Cl2eMeOH (96:4), and recrystallized from CH2Cl2/EtOH to give
5. Experimental
5.1. Chemistry
Elemental analyses were performed by the Microchemical
Laboratory, University of Otago, Dunedin, New Zealand. Melting
points were determined on an Electrothermal IA9100 melting point
apparatus and are as read. NMR spectra were obtained on a Bruker
Avance-400 spectrometer at 400 MHz for proton spectra and
100 MHz for carbon spectra, referenced to Me4Si. Low-resolution
atmospheric pressure chemical ionization (APCI) mass spectra
were measured for organic solutions on a ThermoFinnigan Sur-
veyor MSQ mass spectrometer, connected to a Gilson autosampler.
High-resolution electron impact (HREIMS) and fast atom
bombardment (HRFABMS) mass spectra were determined on a
Varian VG-70SE mass spectrometer at nominal 5000 resolution.
Thin-layer chromatography was carried out on aluminum-backed
silica gel plates (Merck 60 F254), with visualization of compo-
nents by UV light (254 nm). Column chromatography was carried
out on silica gel, (Merck 230e400 mesh) unless otherwise stated.
Tested compounds were ꢃ95% purity, as determined by combus-
tion analysis, or by HPLC conducted on an Agilent 1100 system,
using a reversed-phase C8 column with diode array detection.
8 (200 mg, 49% yield): mp 200e203 ꢀC; 1H NMR (CDCl3)
d 7.93 (d,
J ¼ 8.0 Hz,1 H), 7.48 (t, JHF ¼ 53.5 Hz,1H), 7.33 (t, J ¼ 8.2 Hz,1H), 6.91
(d, J ¼ 7.8 Hz, 1 H), 4.48 (t, J ¼ 5.9 Hz, 2H), 3.97 (q, J ¼ 5.6 Hz, 2H),
3.88e3.86 (m, 8H), 3.79e3.77 (m, 8 H), 3.12 (t, J ¼ 6.0 Hz,
exchangeable with D2O, 1 H), 2.16e2.10 (m, 2H); 13C NMR (CDCl3)
d
164.5 (C), 161.5 (C), 151.0, 144.1 (t, JCF ¼ 26.8 Hz, C-2), 134. 8 (C),
132.4 (C), 126.1 (CH), 108.8 (CH), 108.6 (CH), 108.0 (t, JCF ¼ 240.2 Hz,
CHF2), 66.5 (CH2), 66.1 (CH2), 58.8 (CH2), 43.6 (CH2), 43.4 (CH2), 31.8
(CH2). Anal. Calcd for C22H27F2N7O4; C, 57.8; H, 5.5; N, 20.0. Found:
C, 54.0; H, 5.6; N, 20.2%.
5.1.4. 3-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-1-propanol (8)
5.1.4.1. Method B (Scheme 3). A mixture of 2-amino-3-nitrophenol
(32) (7.2 g, 46.7 mmol), 3-chloropropanol (6.6 g, 1.5 equiv) and dry
powdered K2CO3 (19.3 g, 3 equiv) in acetone (50 mL) was heated
and stirred under reflux for 20 h. The solvent was evaporated to
dryness and the residue was diluted with water and extracted into
EtOAc and dried (Na2SO4). Evaporation of the solvent and chro-
matography of the residue on silica, eluting with CH2Cl2eEtOAc
(4:1), gave 10.0 g (100% yield) of 2-(2-amino-3-nitrophenoxy)
propanol (33) as a yellow solid: mp (CH2Cl2/hexanes) 72e74 ꢀC; 1H
5.1.1. ({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazol-4-yl}oxy)acetic acid (6)
A mixture of 2-(difluoromethyl)-4-hydroxy-1-[4,6-di(4-morpho
linyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (2) [30,31] (275 mg,
0.63 mmol) and dry K2CO3 (800 mg) in DMF (3 mL) was stirred at
20 ꢀC for 30 min. A premixed suspension of K2CO3 (1.0 g) and
bromoacetic acid (1 mL) in DMF (3 mL) was then added. The re-
action mixture was stirred at 50 ꢀC for 24 h and then diluted with
water. After filtration through a pad of celite, the filtrate was
acidified with conc. HCl. The precipitate was collected by filtration,
washed with water, and dried. Two recrystallizations from
CH2Cl2/EtOH gave 6 (190 mg, 60% yield): mp 305e308 ꢀC; 1H NMR
NMR (CDCl3)
d
7.71 (dd, J ¼ 8.9, 1.2 Hz, 1H), 6.92 (d, J ¼ 7.7 Hz, 1H),
6.60 (dd, J ¼ 8.9, 7.8 Hz, 1H), 6.42 (s, exchangeable with D2O, 2H),
4.20 (t, J ¼ 6.1 Hz, 2H), 3.89 (q, J ¼ 5.7 Hz, 2H), 2.12 (pentet,
J ¼ 6.1 Hz, 2H), 1.48 (t, J ¼ 5.0 Hz, exchangeable with D2O, 1H). Anal.
Calcd for C9H12N2O4: C, 50.9; H, 5.7; N, 13.3. Found: C, 51.0; H, 5.7;
N, 13.3%.
A solution of 33 (4.3 g, 20.26 mmol) in MeOH (50 mL) was hy-
drogenated over 10% Pd on C and then filtered into a solution of
5 mL conc. HCl in MeOH (50 mL). After removal of the solvents, the
resulting residue was dissolved in a mixture of 4 N HCl (60 mL) and
difluoroacetic acid (5 mL), and the resulting mixture was heated
under reflux for 6 h. After cooling to 20 ꢀC the mixture was diluted
with water (100 mL), neutralized with aq. NH3, extracted into EtOAc
(5 ꢄ 40 mL), and dried (Na2SO4). Evaporation of the solvent gave
crude 2-{[2-(difluoromethyl)-1H-benzimidazol-4-yl]oxy}ethanol
(34) which was combined with TBDMSCl (6.1 g, 2 equiv) in pyridine
(15 mL) and stirred at room temperature for 3 h before being
poured into water (150 mL), extracted with EtOAc, and dried
(DMSO-d6)
d 13.0 (br s, exchangeable with D2O, 1H), 7.91 (d,
J ¼ 8.4 Hz, 1H), 7.70 (t, JHF ¼ 52.8 Hz,1H), 7.37 (t, J ¼ 8.2 Hz, 1H), 6.86
(d, J ¼ 8.1 Hz, 1H), 4.96 (s, 2H), 3.82e3.80 (m, 8H), 3.69 (m, 8H); 13
C
NMR (DMSO-d6)
d 170.2 (C), 164. 4 (C), 161.3 (C), 150.2 (C), 144.3 (t,
JCF ¼ 26.7 Hz, C-2), 134.6 (C), 131.4 (C), 126.6 (CH), 108.6 (t,
JCF ¼ 237.2 Hz, CHF2), 108.2 (CH), 106.8 (CH), 65.9 (CH2), 65.6 (CH2),
43.6 (CH2). Anal. Calcd for C21H23F2N8O5$0.25H2O: C, 50.9; H, 4.8;
N, 19.8. Found: C, 50.8; H, 4.5; N, 19.9%.

G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
143
(Na2SO4). Evaporation of the solvents and the chromatography of
the residue on silica, eluting with CH2Cl2eEtOAc (9:1) gave of 4-(2-
{[tert-butyl(dimethyl)silyl]oxy}propoxy)-2-(difluoromethyl)-1H-
benzimidazole (35) (5.79 g, 84% yield): mp (CH2Cl2/hexanes) 133e
To a solution of 18 (292 mg, 0.25 mmol) in CH2Cl2 (15 mL) and
THF (10 mL) was added hydrazine monohydrate (2 mL), and the
resulting mixture was stirred at room temperature for 24 h. After
removal of the solvent under vacuum, the residue was diluted with
water, and the resulting precipitate was collected by filtration, and
dried. Chromatography on neutral alumina eluting with CH2Cl2/
MeOH (95:5) gave 10 (133 mg, 58% yield): mp (CH2Cl2/hexanes)
135 ꢀC; 1H NMR (DMSO-d6) (tautomeric mixture)
d 13.40 and 13.23
(s, exchangeable with D2O, 1H), 7.36e7.05 (m, 3H), 6.86 and 6.75
(2d, J ¼ 7.8 Hz and 7.4 Hz,1H), 4.27 and 4.22 (2t, J ¼ 6.3 Hz, 2H), 3.87
and 3.81 (2t, J ¼ 6.1 Hz, 1H), 2.05e1.95 (m, 2H), 0.85 and 0.84 (2s,
9H), 0.02 and 0.01 (2s, 6H).
A mixture of 35 (684 mg, 1.92 mmol), dry powdered K2CO3
(530 mg, 3.84 mmol) and 2-chloro-4,6-dimorpholino-1,3,5-triazine
[33] (285 mg, 3.84 mmol) in DMSO (5 mL) was heated at 130 ꢀC for
3 h. The cooled reaction mixture was diluted with water, and the
resulting precipitate was filtered, washed with water, and recrys-
tallized from CH2Cl2/EtOH to give 4-(3-{[tert-butyl(dimethyl)silyl]
oxy}propoxy)-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazole (36) (971 mg, 83% yield): 1H NMR
201e203 ꢀC; 1H NMR (CDCl3)
d
7.90 (d, J ¼ 8.4 Hz, 1H), 7.49 (t,
JHF ¼ 53.5 Hz,1H), 7.33 (t, J ¼ 8.2 Hz,1H), 6.84 (d, J ¼ 7.8 Hz,1H), 4.31
(t, J ¼ 5.2 Hz, 2H), 3.88e3.87 (m, 8H), 3.79e3.77 (m, 8H) 3.25 (t,
J ¼ 5.2 Hz, 2H); 13C NMR (CDCl3)
d 165.2 (C), 162.2 (C), 151.6 (C),
144.6 (t, JCF ¼ 26.8 Hz, C-2), 135.4 (C), 132.60 (C), 126.7 (CH),108.7 (t,
JCF ¼ 239.9 Hz, CHF2), 108.5 (CH), 106.5 (CH), 71.3 (CH2), 66.8 (CH2),
44.1 (CH2), 41.5 (CH2). Anal. Calcd for C21H26F2N8O3$0.5H2O: C,
52.0; H, 5.6; N, 23.1. Found: C, 52.2; H, 5.4; N, 22.1%.
5.1.7. 2-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-N,N-dimethylethanamine
(11)
(DMSO-d6)
d
7.88 (d, J ¼ 8.2 Hz, 1H), 7.48 (t, JHF ¼ 53.5 Hz, 1H), 7.34
(t, J ¼ 8.2 Hz, 1H), 6.87 (d, J ¼ 8.0 Hz, 1H), 4.38 (t, J ¼ 6.6 Hz, 2H),
3.88e3.84 (m, 6H), 3.81e3.78 (m, 4H) 2.16 (pentet, J ¼ 6.3 Hz, 2H),
0.91 (s, 9H), 0.62 (s, 3H), 0.02 (s, 3H).
A mixture of 2 (301 mg, 0.69 mmol), NaI (1 g), and K2CO3
(956 mg, 6.9 mmol) in DMF (5 mL) was stirred at 20 ꢀC for 5 min
when a yellow suspension was obtained. An excess of 2-N,N-
dimethylaminoethyl chloride hydrochloride (1 g) was added, and
the mixture was stirred at 50 ꢀC for 3 days. The reaction mixture
was diluted with water, extracted with CH2Cl2, and dried (Na2SO4).
After solvent removal, the residue was chromatographed on silica
eluting with CH2Cl2/MeOH (9:1) containing 1% aq. NH3. Further
chromatography on alumina eluting with CH2Cl2/MeOH (94:6) gave
11 (19 mg, 5% yield): mp (CH2Cl2/hexanes) 190e192 ꢀC; 1H NMR
To a solution of 36 (809 mg, 1.35 mmol) in THF (10 mL) was
added TBAF (1 M, 2.5 mL) and the reaction mixture was stirred for
30 min. The mixture was then diluted with H2O (150 mL) and
stirred for 1 h. The resulting precipitate was filtered, washed with
water and chromatographed on silica eluting with CH2Cl2eMeOH
(96:4) to give 8 (504 mg, 81% yield), identical with material pre-
pared in Method A.
5.1.5. 3-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-1,2-propanediol (9)
To a solution of 2 (208 mg, 0.48 mmol) in DMF (5 mL) was added
powdered K2CO3 (331 mg, 2.4 mmol) and 3-chloro-1,2-propanediol
(1 mL). The stirred mixture was heated at 50 ꢀC for 48 h and then
diluted with water. The resulting precipitate was collected by
filtration, washed with water, and dried. Recrystallization from
CH2Cl2/MeOH gave 9 (150 mg, 62% yield): mp 229e231 ꢀC; 1H NMR
(CDCl3)
d
7.89 (d, J ¼ 8.1 Hz, 1H), 7.46 (t, JHF ¼ 53.5 Hz, 1H), 7.32 (t,
J ¼ 8.2 Hz, 1H), 6.84 (d, J ¼ 7.9 Hz, 1H), 4.39 (t, J ¼ 6.3 Hz, 2H), 3.88e
3.87 (m, 8H), 3.79e3.77 (m, 8H), 2.91 (t, J ¼ 6.3 Hz, 2H), 2.38 (s, 6H);
13C NMR (CDCl3)
d 164.5 (C), 162.1 (C), 149.4 (C), 144.4 (t,
JCF ¼ 26.8 Hz, C-2), 135.4 (C), 132.4 (C), 131.8 (C), 127.0 (CH), 110.0
(CH), 108.5 (t, JCF ¼ 240.1 Hz, CHF2), 107.9 (CH), 66.8 (CH2), 65.1
(CH2), 64.9 (CH2), 57.1 (CH2), 44.4 (CH3), 44.2 (CH2), 44.1 (CH2);
HRMS (FABþ) Calcd for C23H30F2N8O3 [MHþ]: m/z 505.2487. Found:
505.2479. Anal. Calcd for C23H30F2N8O3$0.5H2O: C, 53.8; H, 6.1; N,
21.8. Found: C, 53.7; H, 5.8; N, 21.5%.
(DMSO-d6)
d
7.88 (d, J ¼ 8.2 Hz, 1H), 7.70 (t, JHF ¼ 52.8 Hz, 1H), 7.38
(t, J ¼ 8.2 Hz, 1H), 6.94 (d, J ¼ 8.0 Hz, 1H), 5.05 (d, J ¼ 5.2 Hz, 1H),
4.71 (t, J ¼ 5.7 Hz, 1H), 4.23 (dd, J ¼ 10.0, 4.2 Hz, 1H), 4.10 (dd,
J ¼ 10.0, 6.2 Hz, 1H), 3.91 (m, 1H), 3.81e3.79 (m, 8H), 3.69 (br s, 8H),
5.1.8. 2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-
yl]-4-[2-(4-morpholinyl)ethoxy]-1H-benzimidazole (12)
3.51 (t, J ¼ 6.0 Hz, 2H); 13C NMR (DMSO-d6)
d 164.3 (C), 161.3 (C),
151.2 (C), 144.2 (t, JCF ¼ 26.6 Hz, C-2), 134.5 (C), 131.6 (C), 126.8 (CH),
108.6 (t, JCF ¼ 237.1 Hz, CHF2), 106.5 (CH), 106.2 (CH), 70.5 (CH2),
70.0 (CH), 65.8 (CH2), 62.7 (CH2), 43.6 (CH2). Anal. Calcd for
A mixture of 2 (215 mg, 0.49 mmol), K2CO3 (683 mg, 4.9 mmol),
and 4-(2-chloroethyl)morpholine hydrochloride (455 mg, 2.5 mmol)
in DMF (5 mL) was stirred at 50 ꢀC for 2 h. The mixture was diluted
with water and the resulting precipitate was collected by filtration,
washed with water, and dried. Recrystallization from CH2Cl2/EtOH
C
22H27F2N7O5$0.25H2O: C, 51.6; H, 5.4; N, 19.2. Found: C, 51.6; H,
5.4; N, 19.0%.
gave 12 (193 mg, 73% yield): mp 228e230 ꢀC; 1H NMR (CDCl3)
d 7.90
5.1.6. 2-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)ethanamine (10)
A mixture of 2 (281 mg, 0.65 mmol) and powdered K2CO3
(449 mg, 3.25 mmol) in dry DMF (5 mL) was stirred at 20 ꢀC for
30 min, and then N-(2-bromoethyl)phthalimide (330 mg) was
added. The resulting mixture was heated and stirred at 50 ꢀC for
(dd, J ¼ 8.3, 0.5 Hz, 1H), 7.47 (t, JHF ¼ 53.5 Hz, 1H), 7.32 (t, J ¼ 8.2 Hz,
1H), 6.83 (d, J ¼ 7.7 Hz,1H), 4.41 (t, J ¼ 6.1 Hz, 2H), 3.88e3.86 (m, 8H),
3.96e3.74 (m, 12H), 2.95 (t, J ¼ 6.1 Hz, 2H), 2.62 (br t, J ¼ 4.6 Hz, 4H);
13C NMR (CDCl3) 165.2 (C), 162.2 (C), 151.4 (C), 144.7 (t, JCF ¼ 27.0 Hz,
C-2), 135.4 (C), 132.6 (C), 126.7 (CH), 108.7 (t, JCF ¼ 240.1 Hz, CHF2),
108.6 (CH), 106.5 (CH), 67.0 (CH2), 66.8 (CH2), 66.3 (CH2), 57.6 (CH2),
54.2 (CH2), 44.2 (CH2), 44.1 (CH2). Anal. Calcd for C25H32F2N8O4: C,
54.9; H, 5.9; N, 20.5. Found: C, 55.0; H, 6.0; N, 20.4%.
24 h, and
a second batch of N-(2-bromoethyl) phthalimide
(330 mg) was added. After an additional 24 h the reaction mixture
was diluted with water, and the resulting precipitate was collected
by filtration, washed with water, and dried. Recrystallization from
CH2Cl2/EtOH gave 2-[2-({2-(difluoromethyl)-1-[4,6-di(4-morpho
linyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl}oxy)ethyl]-1H-iso-
indole-1,3(2H)-dione (18) (366 mg, 93% yield): mp 250e252 ꢀC; 1H
5.1.9. 3-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-1-propanamine (13)
Similarly to the synthesis of 10, reaction of 2 with N-(3-
bromopropyl)phthalimide gave 2-[3-({2-(difluoromethyl)-1-[4,6-
di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl}oxy)
propyl]-1H-isoindole-1,3(2H)-dione (19) in 96% yield: mp (CH2Cl2/
NMR (CDCl3)
d
7.90 (d, J ¼ 4 Hz, 1H), 7.87e7.84 (m, 2H), 7.73e7.70
(m, 2H), 7.44 (t, JHF ¼ 53.5 Hz, 1H), 7.31 (t, J ¼ 8.2 Hz, 1H), 6.90 (d,
J ¼ 7.8 Hz, 1H), 4.61 (t, J ¼ 6.6 Hz, 2H), 4.22 (t, J ¼ 6.6 Hz, 2H), 3.87e
3.86 (m, 8H), 3.79e3.76 (m, 8H).
EtOH) 212e215 ꢀC; 1H NMR (CDCl3)
7.80 (m, 2H), 7.71e7.67 (m, 2H), 7.40 (t, JHF ¼ 53.6 Hz, 1H), 7.30 (t,
d
7.87 (d, J ¼ 8.1 Hz, 1H), 7.85e

144
G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
J ¼ 8.2 Hz,1H), 6.80 (d, J ¼ 7.8 Hz,1H), 4.37 (t, J ¼ 6.5 Hz, 2H), 3.96 (t,
J ¼ 6.7 Hz, 2H), 3.88e3.86 (m, 8H), 3.79e3.76 (m, 8H), 2.35 (pentet,
J ¼ 6.6 Hz, 2H).
for 30 min and epibromohydrin (0.5 mL) was added. The resulting
mixture was heated at 50 ꢀC for 30 min, cooled, and diluted with
water. The resulting precipitate was filtered, washed with water,
and dried to give 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-
1,3,5-triazin-2-yl]-4-(2-oxiranylmethoxy)-1H-benzimidazole (20)
Deprotection of 19 with hydrazine monohydrate (as for 10) gave
13 (28% yield): mp (CH2Cl2/hexanes) 218e221 ꢀC; 1H NMR (CDCl3)
d
7.91 (d, J ¼ 8.1 Hz, 1H), 7.50 (t, JHF ¼ 53.6 Hz, 1H), 7.32 (t, J ¼ 8.2 Hz,
(341 mg, 97% yield): 1H NMR (CDCl3)
d
7.92 (d, J ¼ 7.9 Hz, 1H), 7.48
1H), 6.87 (d, J ¼ 7.9 Hz, 1H), 4.40 (t, J ¼ 6.1 Hz, 2H), 3.88e3.86 (m,
4H), 3.79e3.76 (m, 4H), 3.15 (t, J ¼ 6.5 Hz, 2H), 2.19 (pentet,
(t, JHF ¼ 53.4 Hz, 1H), 7.33 (t, J ¼ 8.1 Hz, 1H), 6.89 (d, J ¼ 7.9 Hz, 1H),
4.51 (dd, J ¼ 11.5, 3.8 Hz, 1H), 4.35 (dd, J ¼ 11.5, 5.4 Hz, 1H), 3.88e
3.87 (m, 8H), 3.79e3.77 (m, 8H), 3.52e3.48 (m, 1H), 2.93 (t,
J ¼ 4.6 Hz, 1H), 2.80 (dd, J ¼ 4.9, 2.6 Hz, 1H).
J ¼ 6.2 Hz, 2H); 13C NMR (CDCl3)
d 165.1 (C), 162.1 (C), 144.7 (t,
JCF ¼ 26.8 Hz, C-2), 151.4 (C), 135.3 (C), 132.6 (C), 126.8 (CH), 108.9
(CH), 108.7 (t, JCF ¼ 240.1 Hz, CHF2), 107.6 (CH), 66.8 (CH2), 67.6
(CH2), 44.2 (CH2), 44.1 (CH2), 38.7 (CH2), 30.8 (CH2); HRMS (EIþ)
Reaction of the above epoxide 20 with excess 40% aqueous
dimethylamine in THF (10 mL) and MeOH (10 mL) at room tem-
perature gave 16 (96% yield): mp (CH2Cl2/hexanes) 197e200 ꢀC; 1H
Calcd for
491.2316.
C
22H29F2N8O3 [MHþ]: m/z 491.22325. Found: m/z
NMR (CDCl3)
d
7.91 (d, J ¼ 7.9 Hz, 1H), 7.48 (t, JHF ¼ 53.5 Hz, 1H), 7.33
(t, J ¼ 8.3 Hz, 1H), 6.90 (d, J ¼ 7.6 Hz, 1H), 4.33e4.20 (m, 1H), 4.30 (t,
J ¼ 4.9 Hz, 2H), 3.89e3.86 (m, 8H), 3.79e3.77 (m, 8H), 2.61e2.49
5.1.10. 3-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-N,N-dimethyl-1-
propanamine (14)
(m, 2H), 2.33 (s, 6H); 13C NMR (CDCl3)
d 165.2 (C), 162.2 (C), 151.5
(C), 144.6 (t, JCF ¼ 26.8 Hz, C-2), 135.3 (C), 132.5 (C), 126.8 (CH), 108.7
(CH), 108.6 (t, JCF ¼ 240.0 Hz, CHF2), 107.2 (CH), 66.8 (CH), 72.4
(CH2), 66.8 (CH2), 62.1 (CH2), 44.9 (CH3), 44.1 (CH2). Anal. Calcd for
To a solution of 8 (150 mg, 0.31 mmol) and Et3N (0.5 mL) in THF
(15 mL) at 0 ꢀC, was added methanesulfonyl chloride (0.37 mmol).
The reaction mixture was stirred at 0 ꢀC for 45 min, and then 3 mL of
a 40% aqueous solution of dimethylamine was added. The resulting
mixture was stirred at room temperature for 24 h. The solvent was
removed under vacuum, the residue was diluted with water, and the
resulting precipitate was collected, washed with water, and dried.
Chromatography on silica eluting with CH2Cl2/MeOH (95:5) con-
taining 1% aq. NH3 gave 14 (130 mg, 81% yield): mp (CH2Cl2/hex-
C24H32F2N8O4.0.25H2O: C, 53.4; H, 5.9; N, 20.7. Found: C, 53.5; H,
6.1; N, 20.8%.
5.1.13. 1-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-3-(4-morpholinyl)-2-
propanol (17)
Morpholine (0.26 g, 3 mmol) was added to a solution of 20
(300 mg, 0.61 mmol) in THF (10 mL) and MeOH (10 mL), and the
resulting mixture was stirred at room temperature for 16 h before
being evaporated to dryness. The residue was triturated with water,
and the resulting precipitate was collected, dried, and recrystallized
from CH2Cl2/hexanes to give 17 (353 mg, 100% yield): mp 173e
anes) 191e193 ꢀC; 1H NMR (CDCl3)
d
7.88 (d, J ¼ 8.1 Hz, 1H), 7.47 (t,
JHF ¼ 57.8 Hz,1H), 7.32 (t, J ¼ 8.3 Hz,1H), 4.33 (t, J ¼ 6.7 Hz, 2H), 3.88e
3.87 (m, 8H), 3.79e3.77 (m, 8H), 2.56 (br, 2H), 2.32 (br s, 6H), 1.16
(pentet, J ¼ 6.4 Hz, 2H); 13C NMR (CDCl3)
d 165.2 (C), 162.2 (C), 151.7
(C),144.5 (t, JCF ¼ 27.0 Hz, C-2),135.4 (C),132.6 (C),126.72 (CH),108.8
(t, JCF ¼ 240.0 Hz, CHF2), 108.2 (CH), 106.3 (CH), 67.6 (CH2), 66.8
(CH2), 56.5 (CH2), 45.7 (CH3), 44.1 (CH2), 27.5 (CH2). HRMS (FABþ)
Calcd for C24H32F2N8O3 [MHþ]: m/z 519.2644. Found: 519.2644.
175 ꢀC; 1H NMR (CDCl3)
JHF ¼ 53.4 Hz, 1H), 7.33 (t, J ¼ 8.1 Hz, 1H), 6.90 (d, J ¼ 7.6 Hz, 1H),
4.35e4.26 (m, 3H), 3.89e3.86 (m, 8H), 3.79e3.77 (m, 8H), 3.74e
3.71 (m, 4H), 2.69e2.60 (m, 4H), 2.55e2.49 (m, 2H); 13C NMR
d
7.91 (dd, J ¼ 8.4, 0.6 Hz, 1H), 7.48 (t,
Hydrochloride: 1H NMR (DMSO-d6)
d 9.97 (br s, exchangeable
with D2O, 1H), 7.92 (d, J ¼ 8.1 Hz, 1H), 7.70 (t, JHF ¼ 52.8 Hz, 1H), 7.41
(t, J ¼ 8.2 Hz, 1H), 6.98 (d, J ¼ 7.9 Hz, 1H), 4.33 (t, J ¼ 6.1 Hz, 2H),
3.82e3.80 (m, 8H); 3.69 (m, 8H), 3.25 (m, 2H), 2.81 (s, 6H), 2.27e
(CDCl3)
d
165.2 (C), 162.2 (C), 151.4 (C), 144.7 (t, JCF ¼ 26.8 Hz, C-2),
135.4 (C), 132.6 (C), 126.8 (CH), 108.9 (CH), 108.6 (t, JCF ¼ 240.1 Hz,
CHF2), 107.3 (CH), 72.3 (CH2), 67.2 (CH2), 66.8 (CH), 66.1 (CH2), 61.3
(CH2), 54.1 (CH2), 44.1 (CH2). Anal. Calcd for C26H34F2N8O5: C, 54.2;
H, 5.9; N, 19.4. Found: C, 54.4; H, 5.9; N, 19.5%.
2.20 (m, 2H); 13C NMR (DMSO-d6)
d 164.3 (C), 161.3 (C), 150.5 (C),
144.1 (t, JCF ¼ 26.7 Hz, C-2), 134.5 (C), 131.6 (C), 126.8 (CH), 108.5 (t,
JCF ¼ 237.0 Hz, CHF2), 108.4 (CH), 107.0 (CH), 66.0 (CH2), 66.8 (CH2),
54.2 (CH2), 43.6 (CH2), 42.2 (CH3), 24.0 (CH2). Anal. Calcd for
5.1.14. N1-{2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}-N2,N2-dimethyl-1,2-
ethanediamine (21)
C
24H32F2N8O.31.2 HCl H2O: C, 49.7; H, 6.1; Cl, 7.3; N, 19.3. Found: C,
49.6; H, 6.0; Cl, 7.4; N, 19.1%.
tert-Butyl 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-ylcarbamate (26) [30] (149 mg,
0.3 mmol) was suspended in dry DMF (2 mL) at 0 ꢀC, and NaH
(64 mg 2.7 mmol) was added. The reaction mixture was stirred for
5.1.11. 2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-[2-(4-morpholinyl)propoxy]-1H-benzimidazole (15)
Similarly to the synthesis of 12, reaction of 2 with 4-(3-
bromopropyl)morpholine hydrobromide gave 15 (29% yield); mp
30 min, and
a solution of N,N-dimethylaminoethyl chloride
(CH2Cl2/EtOH) 188e190 ꢀC; 1H NMR (CDCl3)
d
7.88 (d, J ¼ 8.3 Hz,1H),
(generated from 5 g amine hydrochloride and aq. K2CO3) in dry
benzene was added. The mixture was stirred at room temperature
for 24 h, and was then quenched with H2O and extracted with
EtOAc. The organic fraction was dried (Na2SO4) and evaporated to
dryness. Chromatography of the residue on silica eluting with
CH2Cl2/MeOH (95:5) gave crude tert-butyl 2-(difluoromethyl)-1-
[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl
[2-(dimethylamino)ethyl] carbamate (27) (111 mg, 61% yield),
which was treated with TFA (5 mL) in CH2Cl2 (5 mL) for 48 h. The
reaction mixture was evaporated to dryness at 20 ꢀC, and after
being neutralized with aqueous NH3, the product was purified by
chromatography on silica eluting with CH2Cl2/MeOH (9:1) to give a
white solid, which was recrystallized from CH2Cl2/hexanes to give
7.47 (t, JHF ¼ 53.5 Hz,1H), 7.32 (t, J ¼ 7.9 Hz,1H), 6.85 (d, J ¼ 8.0 Hz,1H),
4.34 (t, J ¼ 8.0 Hz, 2H), 3.90e3.87 (m, 8H), 3.79e3.76 (m, 8H), 3.73e
3.71 (m, 4H), 2.60e2.58 (m, 2H), 2.49 (m, 4H), 2.15e2.10 (m, 2H); 13
C
NMR (CDCl3)
d
165.2 (C),162.2 (C),151.7 (C),144.6 (t, JCF ¼ 26.8 Hz, C-
2),135.4 (C),132.6 (C),126.7 (CH),108.8 (t, JCF ¼ 240.1 Hz, CHF2),108.3
(CH), 106.4 (CH), 67.5 (CH2), 67.1 (CH2), 66.8 (CH2), 55.6 (CH2), 53.8
(CH2), 44.1 (CH2), 26.3 (CH2). Anal. Calcd for C26H34F2N8O4: C, 55.7; H,
6.1: N, 20.0. Found: C, 55.7; H, 6.1; N, 20.1%.
5.1.12. 1-({2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}oxy)-3-(dimethylamino)-2-
propanol (16)
A mixture of 2 (288 mg, 0.66 mmol) and powdered K2CO3
(454 mg, 2.4 mmol) in DMF (5 mL) was stirred at room temperature
21 (25 mg, 18% yield): mp 237e239 ꢀC; 1H NMR (CDCl3)
J ¼ 8.2 Hz, 1H), 7.52 (t, JHF ¼ 53.7 Hz, 1H), 7.20e7.23 (m, 1H), 6.48 (d,
d 7.55 (d,

G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
145
J ¼ 7.8 Hz, 1H), 5.33 (t, J ¼ 5.1 Hz, exchangeable with D2O, 1H), 3.87
(m, 8H), 3.79e3.76 (m, 8H), 3.38 (q, J ¼ 5.6 Hz, 2H), 2.62 (t,
J ¼ 6.2 Hz, 2H), 2.30 (s, 6H); HRMS (EIþ) Calcd for C23H31F2N9O2: m/
z 503.2573. Found: m/z 503.2575.
66.2 (CH2), 63.6 (CH2), 45.6 (CH3), 43.6 (CH2), 43.5 (CH2). Anal. Calcd
for C23H29F2N9O3: C, 53.3; H, 5.7; N, 24.4. Found: C, 53.5; H, 5.6; N,
24.5%.
5.1.17. 3-Amino-N-{2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-
1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl}propanamide (24)
Similarly to the synthesis of 22, EDCI coupling of 4 and N-(tert-
5.1.15. 2-Amino-N-{2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-
1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl}acetamide (22)
A mixture of amine 4 [30,32] (202 mg, 0.48 mmol), [(tert-
butoxycarbonyl)amino]acetic acid (164 mg, 0.96 mmol), DMAP
(230 mg, 1.9 mmol), and EDCI (360 mg, 1.9 mmol) in DMF (5 mL)
was stirred at room temperature for 6 h, and additional [(tert-
butoxycarbonyl)amino]acetic acid (82 mg, 0.24 mmol) was added.
After an additional 2 h the reaction mixture was diluted with water,
and the resulting precipitate was collected by filtration, washed
with water, and dried. Recrystallization from CH2Cl2/EtOH gave
tert-butyl 2-({2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}amino)-2-oxoethylcarbamate
butoxycarbonyl)-b-alanine gave tert-butyl 3-({2-(difluoromethyl)-
1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-
yl}amino)-3-oxopropylcarbamate (30) (85% yield): mp (CH2Cl2/
EtOH) 217e219 ꢀC; 1H NMR (DMSO-d6)
d 9.96 (s, exchangeable with
D2O, 1H), 8.10 (d, J ¼ 7.7 Hz, 1H), 8.01 (dd, J ¼ 8.3, 0.7 Hz, 1H), 7.74 (t,
JHF ¼ 52.65,1H), 7.41 (t, J ¼ 8.2 Hz,1H), 6.79 (br s, exchangeable with
D2O, 1H), 3.83e3.80 (m, 8H), 3.69 (m, 8H), 3.28e3.23 (m, 2H), 2.68
(t, J ¼ 7.0 Hz, 2H), 1.38 (s, 9H). Anal. Calcd for C27H35F2N9O5: C, 53.7;
H, 5.8; N, 20.9. Found: C, 53.7; H, 5.9; N, 21.1%.
Deprotection of 30 with TFA in CH2Cl2 gave 24 as the tri-
(28) (238 mg, 86% yield): mp 238 ꢀC; 1H NMR (DMSO-d6)
d 9.79 (s,
fluoroacetate salt (86% yield): mp (MeOH/EtOAc) 237e240 ꢀC; 1H
1H), 8.10 (d, J ¼ 7.7 Hz, 1H), 8.01 (dd, J ¼ 8.3, 0.7 Hz, 1H), 7.74 (t,
JHF ¼ 52.7 Hz, 1H), 7.44 (t, J ¼ 8.2 Hz, 1H), 7.33 (t, J ¼ 4.8 Hz, 1H), 3.87
(d, J ¼ 6.1 Hz, 2H), 3.82e3.80 (m, 8H), 3.69 (m, 8H), 1.42 (s, 9H).
Anal. Calcd for C26H33F2N9O5: C, 53.0; H, 5.6; N, 21.4. Found: C, 52.7;
H, 5.8; N, 21.1%.
NMR (DMSO-d6) d 10.31 (s, exchangeable with D2O, 1H), 8.09 (d,
J ¼ 7.8 Hz, 1H), 8.04 (d, J ¼ 8.2 Hz, 1H), 7.75 (t, J ¼ 52.6 Hz, 1H), 7.69
(br s, exchangeable with D2O, 2H), 7.45 (t, J ¼ 8.2 Hz, 1H), 3.83e3.80
(m, 8H), 3.69 (m, 8H), 3.13 (t, J ¼ 6.6 Hz, 2H), 2.91 (t, J ¼ 6.2 Hz, 2H);
13C NMR (DMSO-d6)
d 169.3 (C), 164.3 (C), 161.3 (C), 144.8 (t,
A mixture of the above carbamate (28) (205 mg, 0.35 mmol) and
TFA (6 mL) in CH2Cl2 (10 mL) was stirred at room temperature for
16 h before being evaporated to dryness. The residue was triturated
with MeOH/EtOAc and the resulting precipitate was collected,
washed with EtOAc, and dried to give 22 as the trifluoroacetate salt
JCF ¼ 27.9 Hz, C-2), 133.3 (C), 132.7 (C), 130.4 (C), 126.2 (CH), 115.2
(CH), 110.9 (CH), 108.5 (t, JCF ¼ 237.5 Hz, CHF2) 65.8 (CH2), 43.7
(CH2), 35.0 (CH2), 33.3 (CH2). Anal. Calcd for C24H28F5N9O5: C, 46.7;
H, 4.6; N, 20.4. Found: C, 46.7; H, 4.6; N, 20.7%.
(195 mg, 92% yield): mp 218e221 ꢀC; 1H NMR (DMSO-d6)
d
10.65 (s,
5.1.18. N-{2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}-3-(dimethylamino)
propanamide (25)
exchangeable with D2O, 1H), 8.01 (br s, exchangeable with D2O,
2H), 8.13 (d, J ¼ 8.07 Hz, 1H), 8.07 (d, J ¼ 8.28 Hz, 1H), 8.07 (d,
J ¼ 8.3 Hz,1H), 7.76 (t, JHF ¼ 52.6 Hz, 1H), 7.49 (t, J ¼ 8.2 Hz,1H), 3.99
(s, 2H), 3.83e3.81 (m, 8H), 3.70 (m, 8H); 13C NMR (DMSO-d6)
To a mixture of 4 (198 mg, 0.45 mmol) and DIPEA (1 mL) in
CH2Cl2 (10 mL) at 0 ꢀC was added acryloyl chloride (1 mL). The
reaction mixture was allowed to warm to 20 ꢀC and then stirred for
an additional 2 h. The mixture was diluted with water, extracted
with CH2Cl2, and dried (Na2SO4). The solvents were removed and
the residue was recrystallized from CH2Cl2/EtOH to give N-{2-
(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-
benzimidazol-4-yl}acrylamide (31) (168 mg, 76% yield): mp 246e
d
165.8 (C), 162.3 (C), 161.2 (C), 145.0 (t, JCF ¼ 27.1 Hz, C-2), 133.4 (C),
132.6 (C), 129.8 (C), 126.4 (CH), 114.8 (CH), 11.4 (CH), 108.5 (t,
JCF ¼ 237.1 Hz, CHF2), 65.8 (CH2), 43.6 (CH2), 41.2 (CH2). Anal. Calcd
for C23H26F5N9O5$H2O, C, 44.6, H, 4.5, N, 20.3. Found: C, 44.8; H, 4.5,
N, 20.3%.
5.1.16. N-{2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazol-4-yl}-2-(dimethylamino)acetamide
(23)
249 ꢀC; 1H NMR (DMSO-d6)
d 10.27 (s, exchangeable with D2O, 1H),
8.19 (d, J ¼ 8.2 Hz, 1H), 8.04 (dd, J ¼ 8.3, 0.6 Hz, 1H), 7.75 (t,
JHF ¼ 52.6 Hz, 1H), 7.45 (t, J ¼ 8.2 Hz, 1H), 6.90 (dd, J ¼ 17.0, 10.2 Hz,
1H), 6.31 (dd, J ¼ 17.0, 2.0 Hz, 1H), 5.78 (dd, J ¼ 10.2, 2.0 Hz, 1H),
3.82e3.81 (m, 8H), 3.70 (m, 8H).
To a suspension of 4 (158 mg, 0.37 mmol) and K2CO3 (200 mg,
2.9 mmol) in CH2Cl2 (10 mL) was added chloroacetyl chloride
(1 mL), and the mixture was stirred for 4 h. The mixture was diluted
with water, extracted with CH2Cl2, and dried (Na2SO4). The solvent
was removed and the residue was recrystallized from CH2Cl2/
hexanes to give 2-chloro-N-{2-(difluoromethyl)-1-[4,6-di(4-
morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl}acetamide
(29) (188 mg, 100% yield): mp 287e289 ꢀC, 1H NMR (DMSO-d6)
Reaction of acrylamide 31 with 40% aq. dimethylamine in EtOH
(5 mL) at room temperature, followed by chromatography on
neutral alumina, eluting with CH2Cl2/EtOAc (4:1), gave 25 (35%
yield): mp (CH2Cl2/MeOH) 228e230 ꢀC; 1H NMR (CDCl3)
d 12.06 (s,
exchangeable with D2O,1H), 8.34 (d, J ¼ 7.9 Hz,1H), 7.94 (dd, J ¼ 8.4,
0.7 Hz, 1H), 7.52 (t, JHF ¼ 56.8 Hz, 1H), 7.40 (t, J ¼ 8.2 Hz, 1H), 3.88e
3.87 (br, 8H), 3.79 (br, 8H), 2.74 (t, J ¼ 5.9 Hz, 2H), 2.60 (t, J ¼ 5.9 Hz,
d
10.39 (s, exchangeable with D2O, 1H), 8.12 (d, J ¼ 7.9 Hz, 1H), 8.06
(dd, J ¼ 8.3, 0.7 Hz, 1H), 7.75 (t, JHF ¼ 52.6 Hz, 1H), 7.46 (t, J ¼ 8.2 Hz,
2H), 2.46 (s, 6H); 13C NMR (CDCl3)
d 171.0 (C), 164.5 (C), 161.6 (C),
1H) 4.53 (s, 2H), 3.83e3.80 (m, 8H), 3.70 (m, 8H). Anal. Calcd for
143.8 (t, JCF ¼ 26.7 Hz, C-2), 133.1 (C), 132.3 (C), 131.3 (C), 126.3 (CH),
113.0 (CH), 109.8 (CH), 107.9 (t, JCF ¼ 239.4 Hz, CHF2), 66.2 (CH2),
54.3 (CH2), 44.0 (CH3), 43.6 (CH2), 43.5 (CH2), 33.7 (CH2). Anal. Calcd
for C24H31F2N9O3: C, 54.2; H, 5.9; N, 23.7. Found: C, 54.3; H, 5.8; N,
24.0%.
C
21H23ClF2N8O3: C, 49.6; H, 4.6; N, 22.0. Found: C, 49.5; H, 4.6; N,
21.9%.
A mixture of chloroacetamide 29 (206 mg, 0.40 mmol) and 40%
aq. dimethylamine (2 mL) in ethanol (10 mL) was heated under
reflux for 2 h. The solvent was removed and the residue was
recrystallized from CH2Cl2/EtOH to give 23 (171 mg, 83% yield): mp
5.2. Enzyme assays
259e261 ꢀC; 1H NMR (DMSO-d6)
d 9.96 (s, exchangeable with D2O,
1H), 8.25 (d, J ¼ 8.0 Hz, 1H), 8.01 (dd, J ¼ 8.4, 0.7 Hz, 1H), 7.74 (t,
Compounds were evaluated for their ability to inhibit the Class I
PI 3-kinase enzymes p110a/p85, p110b/p85 and p110d/p85. The
JHF ¼ 52.7 Hz, 1H), 7.45 (t, J ¼ 8.2 Hz, 1H), 3.82e3.80 (m, 8H), 3.70
(m, 8H), 3.19 (s, 2H), 2.37 (s, 6H); 13C NMR (CDCl3)
d
169.1 (C), 164.5
Class Ia PI3K lipid kinase assays were performed using recombinant
(C), 161.5 (C), 144.0 (t, JCF ¼ 26.8 Hz, C-2) 133.2 (C), 132.1 (C), 130.0
PI 3-kinase and phosphatidylinositol as a substrate and [33P]-ATP
(C), 126.3 (CH), 112.9 (CH), 110.3 (CH), 106.8 (t, JCF ¼ 239.0 Hz, CHF2),
tracer with a final ATP concentration of 10
mM, as described

146
G.W. Rewcastle et al. / European Journal of Medicinal Chemistry 64 (2013) 137e147
previously [34,37]. IC50 data is normally the average of at least two
determinations.
with Holm-Sidak multiple comparison analysis using SigmaPlot
11.0 (Systat Software Inc., Chicago, IL, USA). Mice were culled if they
developed moderate signs of toxicity or if bodyweight loss excee-
ded 20% of starting weight. All animal experiments followed pro-
tocols approved by the Animal Ethics Committee of The University
of Auckland.
5.3. Cell proliferation assay
The compounds were evaluated in cellular assays by comparing
NZB5, a cell line derived from a medulloblastoma and expressing
the wild-type gene for p110
poorly differentiated endometrioid adenocarcinoma of the ovary
and expressing a mutant p110 enzyme with a single amino acid
substitution in the kinase domain (Y1021C). The derivation of the
latter line has been described previously [38]. Cells were cultured in
the presence of drugs in a 5-day assay as previously described and
proliferation was measured by incorporation of [3H]-thymidine at
the end of the incubation period [37]. The IC50 values (concentra-
tions for 50% inhibition relative to controls were averages of at least
two determinations).
a, and NZOV9 a cell line derived from a
5.7. Molecular modeling
a
The protein was prepared for docking as previously described
[39,40]. Ligands were prepared using the SKETCHER module of
SYBYL8.0.3 with optimisation by MAXIMIN2 using the MMFF94s
force field with MMFF94 charges and a distance dependent
dielectric function with a dielectric constant of 80, and converged
ꢀ
to 0.05 kcal/mol*A. Ligands were docked into an 18 A cavity
centered on Ile800 using GOLD [41] (v5.0.1) with search efficiency
set at 200%. The Chemscore scoring function with kinase specific
modification was used, and 10 dockings were performed with all
poses kept. Residues Lys720, Lys776, Lys802, Asp805, Asp933 were
treated as flexible and side chain conformations restricted to library
entries, while residues Leu807, Asp810, Tyr836 and Ile848 were
assigned to an alternative soft potential model using the 4-8 2-4
option available in GOLD.
5.4. Inhibition of cell signaling
HCT116 cells were grown in MEM alpha supplemented with
10% (v/v) FBS (fetal bovine serum), 100 units/mL penicillin and
100
mg/mL streptomycin (all from Invitrogen). For inhibition
studies, cells were seeded in 12-well plates and grown for 1 day
before overnight starvation in serum-free media. Cells were then
exposed to varying concentrations of inhibitor dissolved in DMSO
or DMSO alone (final concentration of DMSO in media 0.1%) for
15 min before stimulation with 500 nM insulin for 5 min. Protein
isolation and immunoblotting for phospho-PKB was carried out
according to the methods previously described, with antibodies
from Cell Signaling Technology (Ser473 catalogue# 9271, Thr308
catalogue# 9275) [34].
5.8. Aqueous solubility determinations
The solid compound sample was mixed with water (enough to
make a 2 mM solution) in an Eppendorf tube, and the suspension
was sonicated for 15 min and then centrifuged at 13,000 rpm for
6 min. An aliquot of the clear supernatant was diluted 2-fold with
water, and then centrifuged again at 13,000 rpm for 6 min. A 100 mL
aliquot of the clear supernatant was injected into the HPLC and the
peak area measured. The solubility was calculated by comparing
the peak area obtained with that from a standard solution of the
compound in DMSO (after allowing for varying dilution factors and
injection volumes). HPLC was conducted on an Agilent 1100 system
using an Altima C18 column with a gradient elution from an organic
phase of 80% v/v acetonitrile and water (40%), and an aqueous
mobile phase of 45 mM ammonium formate solution (pH 3.5)
(60%), to 100% organic phase.
5.5. Pharmacokinetics
Age-matched specific pathogen-free male C57 mice were
administered a single 10 mg/kg dose of 14 in 10% EtOH and were
culled at multiple time-points after dosing. Blood was removed by
cardiac puncture into EDTA collection tubes (Becton Dickinson,
Auckland, New Zealand) and centrifuged for 10 min at 6000 rpm to
separate plasma, which underwent protein precipitation in MeOH.
Quantitative analysis was carried out as described previously [30].
Pharmacokinetic parameters were determined by noncompart
mental analysis using Phoenix WinNonlin 6.2 (Pharsight, Sunnyvale,
CA, USA).
Acknowledgments
We thank Shannon Black, Maruta Boyd and Lydia Liew for the
NMR spectra, and Sisira Kumara for HPLC purity and solubility
determinations. This work was funded by the Auckland Division of
the Cancer Society of New Zealand, the Health Research Council of
New Zealand, the Maurice Wilkins Centre for Molecular Bio-
discovery, and Pathway Therapeutics Inc.
5.6. Antitumor efficacy
Age-matched specific pathogen-free female Rag1ꢁ/ꢁ mice were
subcutaneously inoculated on the right flank with 5 ꢄ 106 U87MG
cells in phosphate buffered saline (PBS). Tumor diameter was
measured by callipers to calculate tumor volume (mm3) using the
Appendix A. Supplementary data
formula (L ꢄ w2) ꢄ
p/6 (where; L ¼ longest tumor diameter and
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.03.038.
w ¼ perpendicular diameter). Dosing began 3e4 weeks after
inoculation when tumors were well established, averaging
approximately 8 mm in diameter. The dosing vehicle for compound
14 was D5W (5% dextrose), while 1 was administered in 10% DMSO,
15% Cremophor EL, 15% EtOH, 60% saline and 37 was administered
in 10% EtOH. All drugs were dosed by i.p. injection as the free base
equivalent at a dosing volume of 10 mL/kg. Tumor volume and
animal bodyweight were measured every 2e3 days. Tumor growth
inhibition was calculated as the difference between the average
tumor volume of treatment mice vs control mice at the final control
measurement expressed as a percentage of tumor volume in con-
trol mice. Statistical significance was determined by 1-way ANOVA
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