Synthesis and biological evaluation of novel analogues of the pan class i phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(Difluoromethyl)-1-[4,6-di(4- morpholinyl)-1,3,5-triazin-2-yl]-1 H -benzimidazole (ZSTK474)

Article abstract of DOI:10.1021/jm200688y

A structure-activity relationship (SAR) study 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) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110β, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1 ^-/- mice, and at a dose of 50 mg/kg given by ip injection at a qd ?- 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.

Full text of DOI:10.1021/jm200688y

ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Novel Analogues
of the Pan Class I Phosphatidylinositol 3-Kinase (PI3K) Inhibitor
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-
1H-benzimidazole (ZSTK474)
||
Gordon W. Rewcastle,*,†,‡ Swarna A. Gamage,† Jack U. Flanagan,†,‡ Raphael Frederick,†,‡,
William A. Denny,†,‡ Bruce C. Baguley,†,‡ Philip Kestell,† Ripudaman Singh,† Jackie D. Kendall,†,‡
Elaine S. Marshall,† Claire L. Lill,† Woo-Jeong Lee,§ Sharada Kolekar,§ Christina M. Buchanan,‡,§
Stephen M. F. Jamieson,†,‡ and Peter R. Shepherd‡,§
†Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand
‡Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
§Department of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand
ABSTRACT: A structureꢀactivity relationship (SAR) study 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) iden-
tified substitution at the 4 and 6 positions of the benzimidazole
ring as having significant effects on the potency of substituted
derivatives. The 6-amino-4-methoxy analogue displayed a greater
than 1000-fold potency enhancement over the corresponding
6-aza-4-methoxy analogue against all three class Ia PI 3-kinase
enzymes (p110α, p110β, and p110δ) and also displayed signifi-
cant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo
against a U87MG human glioblastoma tumor xenograft model in Rag1ꢀ/ꢀ mice, and at a dose of 50 mg/kg given by ip injection
at a qd ꢁ 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.
’ INTRODUCTION
number of programs to develop PI3K inhibitors are currently in
progress,5,16ꢀ19 with several inhibitors in clinical trial.20ꢀ24
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazole (ZSTK474) (1, Figure 1) is a potent ATP-
competitive pan class I PI3K inhibitor, with high selectivity over
other classes of PI3K and protein kinases.25ꢀ29 It has demon-
strated antitumor activity in vivo against human tumor xeno-
grafts25,28ꢀ30 and is reported to be in phase I clinical trial,31
despite its poor aqueous solubility which has required the
development of an amorphous formulation containing a solid
dispersion of 1.32 The crystal structure of 1 in complex with
p110δ has been obtained33 and shows that the oxygen of one of
the morpholino groups is positioned as a hydrogen bond
acceptor from the hinge residue Val828, with the morpholino
ring adopting a chair conformation. The benzimidazole group
extends into the affinity pocket where its nitrogen acts as a
hydrogen bond acceptor for the primary amine of Lys779. The
difluoromethyl group points toward Pro758 in the upper wall of
the hydrophobic affinity pocket. The second morpholino group
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 tissue physiology.1ꢀ3 The three class-Ia PI 3-kinases
(p110α/β/δ) and the sole class-Ib PI 3-kinase (p110γ) couple
growth factor receptors and G-protein-coupled receptors, re-
spectively, to a wide range of downstream pathways.4ꢀ6 These
enzymes have different methods of activation and different
kinetic properties,7 but all use phosphatidylinositol 4,5-dipho-
sphate (PIP2) to produce phosphatidylinositol 3,4,5-tripho-
sphate (PIP3). The cellular levels 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. PTEN
is a tumor suppressor gene whose function is commonly lost in
tumors,8ꢀ10 while the PIK3CA gene that codes for p110α is com-
monly mutated in many cancers,11,12 and there is now increasing
evidence that a high proportion of human cancers depend strongly
on p110α for their survival and resistance to therapy.13ꢀ15 There-
fore, the targeting of PI3K with small molecule inhibitors is one
of the most promising new approaches to cancer treatment, and a
Received: May 30, 2011
Published: September 01, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Table 1. Inhibition of PI3K Isoforms and Cell Proliferation
for 2-Substituted Analogues of 1
Figure 1. Structure of 1.
PI3K IC50 a (nM)
cell line IC50 b (μM)
Scheme 1. Synthesis of 1a
compd
R
p110α p110β p110δ
NZB5c
NZOV9d
1e
CHF2
8.6
4.8
0.7
0.22
1.5
0.29
0.3
7af
7bg
7c
7d
7eg
7f
H
265
143
34
1540 142
Me
289
53
35
0.28
0.39
0.9
0.6
CH2F
CF3
13
0.35
0.7
202
>103
170
590
461
>103
39
70
CH2OH
CH2SMe
CH2SOMe
5470
41
0.75
0.083
0.40
0.25
0.2
5.5
0.086
0.49
0.44
0.2
7g
7h
7i
230
250
>103
383
35
CH2SO2Me 480
54
a Reagents and conditions: (i) K2CO3, DMF, room temp; (ii) morpho-
line, DMF or THF, room temp; (iii) NaH or K2CO3, DMF or DMSO,
120 °C.
SMe
2900
270
7j
SO2Me
203
76
10
2
a Compound concentration required to produce 50% inhibition of
phosphorylation in an in vitro lipid kinase assay with conditions as
previously described in ref 50. b Compound concentration required to
produce 50% inhibition of cell growth in an in vitro cell proliferation
assay with conditions as previously described in ref 69. c Cell line derived
from a medulloblastoma and expressing the wild-type gene for p110α.
d Cell line derived from a poorly differentiated endometrioid adenocar-
cinoma of the ovary and expressing a mutant p110α enzyme with a single
amino acid substitution in the kinase domain (Y1021C). e Reference 25.
f Reference 35. g Reference 47.
adopts a somewhat twisted chair conformation and projects out
of the ATP-binding pocket. This binding mode is flipped over
relative to that originally predicted in a computational model of 1
bound to p110γ.25
However, despite the large amount of data now available on 1,
apart from two bicyclic morpholino derivatives34 and some early
2-unsubstituted analogues,35 very little information regarding
related analogues is currently available outside of the patent
literature. In this paper we explore the structureꢀactivity rela-
tionships for this promising series, focusing initially on changes
to the benzimidazole portion of the molecule, with one of the
aims being the identification of suitable changes that might lead
to more soluble analogues.
The benzene ring substituted analogues 10aꢀy of Table 2
were prepared using method A of Scheme 1, via the monochlor-
otriazines 9 and the appropriately substituted 2-(difluoromethyl)-
benzimidazoles 8 (Scheme 3). The latter compounds were
normally prepared from the analogous o-phenylenediamines
and difluoroacetic acid, although compounds 8lꢀq and 8ad,
required for the synthesis of derivatives 10lꢀq, were prepared
from the analogous 4-carboxylic acid 8ah (Scheme 4).
It was again necessary to protect oxygen substituents as their
TBDMS ethers for the successful synthesis of the 4-hydroxy-
methyl derivative 10l and of the phenols 10a, 10t, and 10u
(Scheme 3). 4-Alkoxy derivatives 10dꢀg were subsequently
prepared by alkylation of phenol 10a. The 4-amino derivative
10i was prepared by reduction of the analogous nitro compound
10ae, while the 4-methylamino derivative 10j was prepared from
10i via alkylation of the Boc protected intermediate. The
6-amino-4-methoxy derivatives 10w, 10x, and 10y were similarly
prepared from the Boc protected precursor 10af by a combina-
tion of alkylation, reduction, and deprotection steps.
’ CHEMISTRY
The synthesis of 1 can be performed by two main routes
(Scheme 1). Method A involves the room temperature reaction
of 2-(difluoromethyl)benzimidazole (2)36 with 2,4-dichloro-
6-morpholino-1,3,5-triazine (3)37 to give the monochloro inter-
mediate 438 which is then reacted with morpholine to give 1.
Method B is a shorter procedure, involving direct reaction of 2
with 2-chloro-4,6-dimorpholino-1,3,5-triazine (5)39 to give 1,
although higher temperatures are required. In our hands method
B was found to be superior for the synthesis of 1, since in addition
to the shorter route it also gave a higher yield, although the same
was not always true for substituted benzimidazoles where method
A was often superior.
Both of the methods described in Scheme 1 were employed to
produce the 2-substituted analogues 7aꢀj of Table 1 (Scheme 2),
although in the case of the 2-hydroxymethyl derivative 7e, it was
necessary to use a TBDMS protecting group to prevent con-
current ether formation. Sulfoxide 7g and sulfones 7h and 7j
were most efficiently prepared by oxidation of the precursor
sulfides 7f and 7i.
The 4-methoxy-6-aza analogue 13 of Table 2 was prepared
from the imidazo[4,5-c]pyridine 11 via the intermediacy of
chloro derivative 12, although a higher temperature than normal
was needed for the first step because of the lower nucleophilicity
of 11 (Scheme 5). As with the earlier benzimidazoles, compound
11 was prepared from difluoroacetic acid by condensation with
the appropriate diaminopyridine40 (see Experimental Section).
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Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Synthesis of 2-Substituted Analogues 7a
Table 2. Inhibition of PI3K Isoforms and Cell Proliferation
for 4-, 5-, and 6-Substituted Analogues of 1
a
b
PI3K IC50
(nM)
cell line IC50
(μM)
compd
R
p110α p110β p110δ NZB5 NZOV9
10ac
10bd 4-OMe
4-OH
0.9
0.68
25
0.27
3.9
5.5
95
0.03
0.05
0.07
0.07
0.23
0.047
0.07
0.17
0.05
0.11
0.06
0.30
2.8
0.01
0.01
0.04
0.05
0.21
0.028
0.28
0.13
0.08
0.26
2.54
0.17
0.47
0.11
0.05
0.24
>20
2.7
10c
10d
10e
10f
4-OEt
4-OPr
4-OBu
2.7
104
518
16
28
2400 156
56 7.4
2100 430
4-OCHF2
4-OCHMe2
4-OSO2Me
4-NH2
26
a Reagents and conditions: (i) (for 7a and 7b) 5, NaH, DMF, 120 °C;
(ii) (for 7c, 7d, 7f, 7i, 7k) (a) 3, K2CO3, DMF, room temp; (b)
morpholine, THF, room temp or reflux; (iii) Bu4NF, THF, room temp;
(iv) aq NaIO4, EtOH, room temp; (v) aq KMnO4, HOAc, acetone,
room temp.
10g
10h
10ie
10j
364
294
4.8
570
41
158
0.85
67
4-NHMe
4-NMe2
210
5400
27
480
>103 539
10k
10l
4-CH2OH
68
12
65
The central ring pyrimidine analogues 16aꢀc, 19a, and
19b of Table 3 were prepared as shown in Scheme 6, with a
TIPS protecting group used to protect the 4-hydroxy group
of 16b. Thus, condensation of the appropriately substituted
2-(difluoromethyl)benzimidazoles 2, 8c, and 8ai with 2,4,
6-trichloropyrimidine (14) gave the analogous 2-substituted 4,
6-dichloropyrimidines 15a, 15c, 15d, which were then treated
with morpholine at reflux to give 2-(benzimidazolyl) derivatives
16a, 16c, and 16d. TIPS deprotection of 16d then gave 16b. The
4-(benzimidazolyl) isomers 19a and 19b were prepared by two
different routes. For the synthesis of 19a, 2-(difluoromethyl)-
benzimidazole (2) was condensed with 4,6-dichloro-2-morpho-
linopyrimidine (17) at 45 °C to give intermediate 18 which was
then reacted with morpholine at reflux to give 19a. For the
synthesis of 19b, benzimidazole 8c was reacted with 4,6-di-
chloro-2-(methylthio)pyrimidine 20 at room temperature to
give intermediate 21 which was reacted with morpholine at
room temperature to give intermediate 22. Oxidation of 22 gave
23 which gave 19b on treatment with morpholine at reflux.
10m 4-CO2Me
9.3
310
10n
10o
10p
10q
10r
10s
10t
10u
10v
10w
10x
10y
13
4-CONH2
>103
296
>103
61
>103 ∼103 0.76
4-CONHMe
4-CONMe2
4-CN
>103 ∼103 0.48
>103 >103
>103 66
0.73
>20
4,5-OCH2O-
4,5-(OMe)2
4-OH, 5-OMe
4-OMe, 5-OH
4,6-(OMe)2
4-OMe, 6-NH2
550
335
37
47
39
0.13
0.02
0.334
0.009
0.095
0.02
0.39
0.13
5.6
0.04
0.036
0.13
0.011
0.059
0.007
0.016
0.15
2.6
801
6.7
258
0.3
32
94
1.8
58
118
1.4
12.5
0.38
0.52
6.8
0.22
4-OMe, 6-NHMe 2.0
0.91
18.5
4-OMe, 6-NMe2
4-OMe, 6-aza
8.0
1290
1030 1600
a Compound concentration required to produce 50% inhibition of
phosphorylation in an in vitro lipid kinase assay (see Table 1 for details).
b Compound concentration required to produce 50% inhibition of cell
growth in an in vitro cell proliferation assay (see Table 1 for details).
c Reference 47. d Reference 48. e Reference 38.
’ RESULTS AND DISCUSSION
recently determined p110α X-ray crystal structure (PDB code
2rd0),46 where the top ranked and most populated predicted
binding mode made contacts to the active site analogous to those
observed in p110γ. The binding mode predicted in both the
p110γ and p110α ATP binding sites is in good agreement with
the crystal structure subsequently determined for the binding of
1 to p110δ.33 For example, our model showed hydrogen bonding
between one of the morpholine oxygens and Val882 and between
the benzimidazole 3-nitrogen and the amino groups of Lys833,
but in addition it indicated that an appropriate electron donating
substituent at the 4-position could probably participate in the
hydrogen bonding to the Lys833 amino group and that a similar
substituent at the 6-position might interact with the phenol group
Molecular Modeling. At the commencement of this work
there was no structural information available on the binding of 1
to PI3K, so to aid in the analysis of substituted analogues, we
developed a model of 1 bound to the crystal structure of
p110γ.41ꢀ43 To this end, 1 was docked within the p110γ binding
site (PDB code 2CHX) by means of the automated GOLD pro-
gram.44 In order to take into account protein flexibility, the
conformation with the highest score (GoldScore) was further
refined using the MINIMIZE module as implemented in SYBYL
version 7.3 (Tripos force field and GasteigerꢀH€uckel charges).45
This model (Figure 2) differed from one proposed at the time25
and was later supported by molecular docking studies with the
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Scheme 3. Synthesis of 4-, 5- and 6-Substituted Analogues 10a
a Reagents and conditions: (i) 3, K2CO3, DMF, room temp; (ii) morpholine, THF, room temp or reflux; (iii) Bu4NF, THF, room temp; (iv) RI, K2CO3,
DMF, room temp; (v) H2, Pd/C, MeOH, room temp; (vi) (a) Boc2O, dioxane, reflux; (b) NaH, MeI, DMF, room temp; (c) TFA, CH2Cl2, room temp;
(vii) TFA, CH2Cl2, room temp; (viii) NaH, MeI, DMF, room temp; (ix) (a) AcOCHO, CH2Cl2, room temp; (b) BH3.SMe2, (MeO)3B, THF, CH2Cl2,
room temp.
Scheme 4. Synthesis of Benzimidazoles 8 with 4-Carbon Substituentsa
a Reagents and conditions: (i) MeOH, H2SO4, reflux; (ii) LiAlH4, THF, reflux; (iii) TBDMSCl, pyridine, room temp; (iv) (a) SOCl2, reflux; (b) NH3,
dioxane, room temp; (v) (a) SOCl2, reflux; (b) MeNH3Cl, Et3N, dioxane, room temp; (vi) (a) SOCl2, reflux; (b) Me2NH2Cl, Et3N, dioxane, room
temp; (vii) SOCl2, reflux.
of Tyr867 at the back of the binding site. Finally, comparison of the
p110α binding model of 1 to the X-ray crystal structure of human
p110γ with ATP bound (PDB code 1e8x)41 indicated that sub-
stitution at the 2 position may be used to access the active site
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Scheme 5. Synthesis of 13a
a Reagents and conditions: (i) 3, K2CO3, t-BuOH, reflux; (ii) morpholine, room temp.
Table 3. Inhibition of PI3K Isoforms and Cell Proliferation
for Pyrimidine Analogues of 1
benzimidazole 3-nitrogen atom to the lysine amino group (Lys833
in p110γ is equivalent to Lys802 in p110α, Lys805 in p110β,
and Lys779 in p110δ), we started by exploring substituents at
that position.
The known47 4-OH analogue 10a showed exceptional enzyme
potency against all three isoforms studied (IC50 < 1 nM),
presumably because of tight specific binding of the OH to the
lysine amino group, although with the likely pharmacologic
liability of rapid glucuronic acid conjugation in an in vivo
situation. The 4-OMe and 4-OEt analogues (10b and 10c) were
also more active than 1 against the p110α-isoform, and although
the 4-OPr, 4-OBu, and 4-OCHF2 compounds (10dꢀf) retained
reasonable α-isoform potency, this was lost by the more bulky
4-OCHMe2 and 4-OSO2Me analogues 10g and 10h. This
suggests some bulk tolerance around the 4-position for small
cross-section substituents but not for those with branching close
to the benzimidazole group.
A similar pattern was observed with the 4-amino derivatives
10iꢀk, where the primary amino derivative 10i was more active
than the methylamino derivative 10j and significantly more
active than the dimethylamino derivative 10k.
Compounds 10lꢀq explored carbon linked subtituents, and
although the 4-CH2OH and 4-CO2Me derivatives 10l and 10m
showed reasonable activity, none of them displayed activity
comparable to that of oxygen linked substituents.
PI3K IC50 a (nM)
cell line IC50 b (μM)
compd
R
X, Y p110α p110β p110δ
NZB5
NZOV9
16ac
H
N, CH 30
72
10
0.12
0.10
0.06
0.42
0.43
0.26
0.11
0.03
0.3
16bd OH N, CH 1.5
16ce OMe N, CH 5.1
0.81
35
0.27
18
19a
H
CH, N 88
170
40
13
19b OMe CH, N
4
14
0.11
a Compound concentration required to produce 50% inhibition of
phosphorylation in an in vitro lipid kinase assay (see Table 1 for details).
b Compound concentration required to produce 50% inhibition of cell
growth in an in vitro cell proliferation assay (see Table 1 for details).
c Reference 38. d Reference 47. e Reference 49.
A variety of 4,5-disubstituted compounds (10rꢀu) were
relatively inactive, with the exception of the 4-OMe, 5-OH
analogue 10u, which was still less potent against the p110α
isoform than the 5-unsubstituted 4-OMe analogue 10b, indicat-
ing that substitution at the 5-position is sterically not preferred
for this isoform.
region traversed by the α and β nonhydrolyzed ATP phosphate
moieties. These possibilities were therefore explored in detail in
our SAR study of benzimidazole-substituted analogues of 1.
Enzyme Activity. All compounds were tested for their enzyme
activity against the p110α, p110β, and p110δ isoforms of PI3K
using a lipid kinase assay. Table 1 gives results for a series of
2-substituted derivatives (7aꢀj) compared to 1. The known35
unsubstituted compound 7a was a poor PI3K inhibitor, with IC50
> 140 nM across the isoforms, and the 2-Me and 2-CF3 analogues
(7b, 7d) were little better. The 2-CH2F compound 7c was
considerably more active, but apart from the 2-CH2SMe com-
pound 7f, which showed moderate activity, all of the other
substituted 2-alkyl analogues 7dꢀh were less active. The
2-SMe analogue 7i also showed poor activity, although the
2-SO2Me derivative 7j did show improved, but still only moderate,
activity. Therefore, since none of the 2-substituted analogues
investigated showed superior activity to that of the 2-difluor-
omethyl compound 1, this substituent group was employed in all
of the subsequently studied compounds.
Compounds 10vꢀy and 13 explore 4,6-disubstitution. Since
our modeling suggested that an aza atom at the 6-postion of the
benzimidazole might form an H-bond to the hydroxyl group of a
tyrosine residue at the back of the ATP binding site (Tyr867 in
p110γ is equivalent to Tyr836 in p110α, Tyr839 in p110β, and
Tyr813 in p110δ), the 4-OMe-6-aza compound 13 was designed
to explore this possibility. However the dramatic loss of activity
of this compound compared to 4-OMe derivative 10b showed
that electronic effects play a much more important role in the
binding of these compounds, with the electron withdrawing effect
of the 6-aza atom being highly detrimental to good activity. Thus,
it appears that the hydrogen bonding interaction between the
benzimidazole 3-nitrogen and the lysine amino group (Lys802 in
p110α, Lys805 in p110β, Lys833 in p110γ, and Lys779 in
p110δ) represents a major factor in determining the potency
and activity of this class of inhibitors, and anything that reduces
this interaction should be avoided. Conversely, increasing the
Compounds 10aꢀy and 13 (Table 2) then explored the use of
different substituents on the benzene ring of the benzimidazole.
Since our modeling of 1 with p110γ suggested that an appropriate
substituent at the 4-position could complement the binding of the
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Scheme 6. Synthesis of Pyrimidinyl Analogues 16 and 19a
a Reagents and conditions: (i) NaH, DMF, room temp; (ii) morpholine, reflux; (iii) Bu4NF, THF, room temp; (iv) K2CO3, DMF, 45 °C; (v) K2CO3,
DMSO, room temp; (vi) morpholine, THF, room temp; (vii) aq KMnO4, HOAc, acetone, room temp; (viii) morpholine, THF, reflux.
the 4,6-dimethoxy derivative 10v is significantly more active than
13, although not as active as the sterically less hindered 6-amino
analogue 10w which was the most potent new compound against
the PI3K isoforms identified in this study. The N-methyl and N,
N-dimethyl analogues of 10w (10x and 10y) also displayed very
good potency, despite increased steric effects, thereby reinforcing
the strong preference for electron donating substituents. Our
belief that this electronic effect is due to increased electron
density on the benzimidazole 3-nitrogen, resulting in increased
binding to the lysine amino group, is in agreement with the fact
that the very high potency of the PI3K inhibitor 2,4-difluoro-
N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}-
benzenesulfonamide (GSK2126458) (24, Figure 6) is almost
certainly due to its strong charged interaction with the same lysine
amino group.22
With 2-CHF2 and 4-OH and 4-OMe established as suitable
benzimidazole substituents, Table 3 shows the results of a small
study of pyrimidine analogues of the central triazine ring
(compounds 16aꢀc, 19a,b). The 2-pyrimidine isomers 16aꢀc
showed only a slight reduction in potency compared to their
respective triazine analogues 1, 10a, and 10b, although a more
pronounced effect was seen with the 4-pyrimidine isomers 19a
and 19b where the unsubstituted benzimidazole analogue 19a
was significantly less potent than 1, although the 4-methoxy
derivative 19b displayed similar potency compared to 10b.
Figure 2. View of 1 docked in the ATP-binding site of PI3K p110γ.
electron density on the benzimidazole 3-nitrogen by the use of
electron donating substituents should be advantageous, and thus,
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Table 4. HTRF Assay Inhibition of PI3K Isoforms for 1, 10a,
10f, 10t, and 10w
Table 5. Inhibition of Cell Signaling in U87MG (PTEN Null)
Cells by 1, 10a, 10b, 10f, 10t, and 10w in Vitro
a
PI3K IC50
IC50 a (nM)
(nM)
compd
pAkt/PKB Ser473
pAkt/PKB Thr308
p110α
H1047Rb
p110α
E545Kc
1
111
1.3
55
32
compd
p110α
p110β
58
p110δ
p110γ
10a
10b
10f
10t
10w
2.9
28
1
8.9
0.53
4.7
70
38
0.65
34
2
83
6
25
12
0.12
3
10a
10f
10t
10w
3.5
>2,500d
0.83
8.5
427
583
4.8
418
378
8.8
56
52
32
44
158
0.95
49
1.0
3.1
27
1.7
a Compound concentration required to produce 50% inhibition of
phosphorylation in a cellular assay. 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 50
and 51.
a Compound concentration required to produce 50% inhibition of
phosphorylation in a PI3-kinase (human) HTRF assay. b Kinase domain
p110α mutant. Helical domain p110α mutant. d Solubility problems
c
encountered with this assay.
Table 6. Inhibition of Cell Signaling in HCT116+/+
(PIK3CA H1047R Mutant) Cells by 1, 10a, 10b, 10f, 10t, and
10w in Vitro
IC50 a (nM)
compd
pAkt/PKB Ser473
pAkt/PKB Thr308
1
97
78
10a
10b
10f
10t
10w
8.3
37
0.18
25
415
507
12
381
515
17
a Compound concentration required to produce 50% inhibition of
phosphorylation in a cellular assay. 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 50
and 51.
Figure 3. Correlation of NZOV9 cell line activity against p110α
enzyme activity.
significant correlation between logarithmic IC50 values for
NZOV9 cell line (r = 0.63; p < 0.0001) but not to those for
the NZB5 cell line (r = 0.29; p = 0.09). The two most potent
inhibitors of p110α (10a and 10w) were also the most potent
inhibitors of the NZOV9 cell line. These results strongly support
the hypothesis that proliferation of the NZOV9 line is driven by
an activated mutant p110α PI 3-kinase in these cells. On the
other hand the lower correlation found for the NZB5 cell line and
the occasional large differences in IC50 values between the two
cell lines (e.g., compounds 10o and 10x) suggest that prolifera-
tion of the NZB5 line is sensitive to factors additional to PI
3-kinase.
Effect on Cell Signaling. To determine whether the com-
pounds were capable of entering cells and attenuating signaling
downstream from PI 3-kinase, the effect for selective compounds
on phosphorylation of Akt/PKB was determined in cell lines
using previously described methods.50,51 U87MG and HCT116
cells were chosen for these experiments, as they are commonly
used in xenografts and have constitutive activation of the PI
3-kinase pathway due to PTEN deletion or activation of PIK3-
CA, respectively. We determined the IC50 for the inhibition of
phosphorylation at not only the most commonly measured
Compounds 10a, 10f, 10t, and 10w were additionally com-
pared to 1, against all four class I PI3K isoforms and two mutant
isoforms of p110α (H1047R and E545K) in a homogeneous
time resolved fluorescence (HTRF) assay (Table 4). Because of
the different assay conditions the PI3K IC50 values differ from
those in Tables 1 and 2 but confirm that 10a and 10w consistently
show potency superior to that of 1 against all PI3K enzyme
variants tested, including the two p110α mutant forms.
Cell Proliferation. The compounds were also evaluated in a
cellular assay by measuring IC50 values (drug concentrations
causing 50% inhibition of proliferation). Two early passage
human cancer cell lines were used. NZB5 was derived from a
patient with medulloblastoma and expressed the wild-type gene
for p110α, while NZOV9 was derived from a poorly differen-
tiated ovarian carcinoma and expressed a mutant p110α enzyme
with a single amino acid substitution in the kinase domain
(Y1021C). For all of the compounds in Tables 1ꢀ3, cell line
IC50 values for NZB5 and NZOV9 correlated significantly with
isolated enzyme IC50 values for p110α, as determined by Spear-
man rank order correlation (r = 0.53 and r = 0.40, respectively;
P < 0.0001 and P < 0.001, respectively). As shown in Figure 3, a
plot of those compounds having measurable values showed a
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Journal of Medicinal Chemistry
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Figure 4. Plasma pharmacokinetics of 1, 10a, and 10w after ip admin-
istration at 10 mg/kg to CD-1 mice.
Table 7. Plasma Pharmacokinetic Parameters for 1, 10a and
10w in Male CD-1 Micea
dose
(mg/
kg)
T1/
Cmax
Tmax
AUCINF
2
compd
route
(nM)
(h)
(nM h)
(h)
3
1
10
10
10
ip
ip
ip
2359
628
2
9657
1034
5219
1.52
1.55
1.48
10a
10w
0.25
3452
0.083
a Blood samples were centrifuged for 10 min at 6000 rpm to separate
plasma, which underwent protein precipitation in MeOH. Quantitative
analysis was carried out using multiple reaction monitoring and electro-
spray ionization. Pharmacokinetic parameters were determined by
noncompartmental analysis using WinNonlin 5.3 software (Pharsight,
Sunnyvale, CA, U.S.).
Figure 5. In vivo antitumor efficacy and bodyweight change following
treatment at MTD with 15 mg/kg 25, 40 mg/kg 1, and 50 mg/kg 10w in
a U87MG human glioblastoma xenograft model in Rag1ꢀ/ꢀ mice. All
treatments were administered qd ꢁ 10 by ip injection. (A) Relative
mean tumor volume and (B) bodyweight change in Rag1ꢀ/ꢀ mice
following the onset of drug treatment are shown. Bars represent the
mean and standard error of 5ꢀ7 mice per group.
Ser473 site but also for Thr 308, as this site is the one most
directly linked to activation of PI 3-kinase in the cell. In these
studies the rank order of IC50 against PI 3-kinase isoforms in vitro
matched with the rank order of IC50 against both Akt/PKB
phosphorylation sites in both cell lines (Tables 5 and 6). 10a and
10w showed the best inhibition of phosphorylation at both the
Ser473 and Thr308 sites on Akt/PKB and were selected for
further evaluation.
Pharmacokinetics. On the basis of both their effects on cell
signaling and exceptional potency against the isolated p110α and
p110δ enzymes, compounds 10a and 10w were selected for
pharmacokinetic evaluation in comparison with compound 1 in
CD-1 mice. The pharmacokinetic profile of these compounds at
a dose of 10 mg/kg by ip injection is shown in Figure 4 and
Table 7. Compound 1 reached a Cmax of 2359 nM 2 h after dosing
Table 8. Aqueous Solubility Measurements for 1, 10a, 10b,
10w, and 25a
compd
solubility (μg/mL)
1
0.61
0.32
0.08
4.2
10a
10b
10wb
25c
11.3
a Solubility was measured using an HPLC assay comparing the peak area
obtained for an aqueous solution compared with that from a standard
solution of the compound in DMSO. b Methanesulfonate salt.
c Dimethanesulfonate salt.
and, despite a short t1/2 of 1.5 h, reached an AUC of 9657 nM h
because of its delayed Tmax (Table 7). Compound 10w reached a
higher Cmax (3452 nM) than compound 1, but because of its
3
model in Rag1ꢀ/ꢀ mice. At a dose of 50 mg/kg given by ip
injection at a qdꢁ 10 dosing schedule, 10w reduced tumor
growth by 81.5% (18.5 ( 3.4% of control; P < 0.0001) by day 10,
compared to a 31.5% reduction by 25 at 15 mg/kg, and a 65.3%
reduction by compound 1 (34.7 ( 6.6% of control; P < 0.0001)
at 40 mg/kg (Figure 5A). All mice survived the 10-day study
duration. At MTD, 1 and 25 were well tolerated over the
treatment duration, but 10w was associated with delayed body-
weight loss (Figure 5B), thought to be a result of the poor
solubility properties of the compound as evidenced by the
presence of drug precipitate in the IP cavity of all mice on
post-mortem examination. The relative solubilities of 1, 10a,
10b, 10w, and 25 are shown in Table 8, and although 10w was
administered as the relatively soluble methanesulfonate salt, it
quicker Tmax and similar t1/2, its AUC (5219 nM h) was lower.
3
The pharmacokinetics of 10a were poor comparatively, with an
approximately 5-fold lower Cmax (628 nM) and AUC (1034
nM h) than 10w, reflecting the liability of the phenol group of
3
10a as a target for glucuronidation.
Antitumor Activity. As a result of the superior pharmacoki-
netic profile of 10w over 10a, the former was selected for
evaluation of in vivo antitumor activity alongside compound 1
and the pan PI3K/mTOR inhibitor 2-methyl-2-[4-[3-methyl-
2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]-
phenyl]propionitrile (NVP-BEZ235)20 (25, Figure 6) at MTD
in a PTEN-null U87MG human glioblastoma tumor xenograft
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A mixture of 2 (0.168 g, 1 mmol), 2-chloro-4,6-di(4-morpholinyl)-
1,3,5-triazine (5)39 (0.286 g, 1 mmol), and powdered K2CO3 (0.56 g, 4
mmol) in DMSO (5 mL) was heated and stirred at 130 °C for 3 h. After
cooling, the mixture was diluted with water and the white solid was
collected and dried to give 1 (0.35 g, 84% yield): mp (EtOH)
217ꢀ219 °C (lit.38 211ꢀ214 °C); 1H NMR (CDCl3) δ 8.33 (dd, J =
7.3, 1.4 Hz, 1H), 7.89 (dd, J = 7.2, 1.5 Hz, 1H), 7.56 (t, JHF = 53.6 Hz,
1H), 7.46ꢀ7.37 (m, 2H), 3.91ꢀ3.86 (m, 8H), 3.81ꢀ3.76 (m, 8H).
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimi-
dazole (7a). A solution of benzimidazole (6a) (0.59 g, 5 mmol) in
DMF (10 mL) was treated with NaH (250 mg, 60% in oil, 6.5 mmol),
and the mixture was stirred at room temperature for 30 min. Solid 5
(1.43 g, 5 mmol) in DMF (10 mL) was then added, and the resulting
mixture was heated at 90 °C overnight. After cooling, the reaction
mixture was diluted with water to give a solid which was collected
and recrystallized from EtOH to give 7a (1.46 g, 79% yield): mp
233ꢀ236 °C (lit.35 222ꢀ224 °C); 1H NMR (DMSO-d6) δ 9.15
(s, 1H), 8.39 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.41 (ddd, J =
8.3, 7.9, 1.2 Hz, 1H), 7.34 (ddd, J = 7.9, 7.8, 1.2 Hz, 1H), 3.91ꢀ3.75
(m, 8H), 3.73ꢀ3.64 (m, 8H).
Figure 6. Structures of 24 and 25.
was presumably rapidly converted to a less soluble hydrochloride
salt in vivo.
’ CONCLUSION
Molecular modeling suggestions that the addition of appro-
priate substituents at the 4- and 6-positions of the benzimidazole
ring of 1 could result in tighter binding derivatives were con-
firmed with the identification of 10a and 10w as very potent
analogues. Phenol 10a has poor pharmacokinetics, presumably as
a result of rapid glucuronidation, although more favorable drug
levels were achievable with 10w. On the basis of cell based studies
and the pharmacokinetic data, 10w was tested in a U87MG
human glioblastoma xenograft model, where it caused a signifi-
cant reduction in tumor growth at MTD. However, despite the
good pharmacokinetics and promising anticancer efficacy of
10w, its poor solubility properties prevented it from being well
tolerated in vivo. In conclusion, substitution at the 4 and 6
positions of the benzimidazole ring of 1 generates highly potent
PI3K inhibitors that are active in vivo, although more soluble
analogues are likely required before this class of compounds can
progress further.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-methyl-1H-
benzimidazole (7b). Similar to the synthesis of 7a, reaction of
2-methylbenzimidazole (6b) (0.66 g, 5 mmol) with 5 (1.43 g, 5 mmol)
gave 7b (1.39 g, 73% yield): mp (EtOH) 222ꢀ224 °C (lit.35
1
218ꢀ220 °C); H NMR (DMSO-d6) δ 8.18 (dd, J = 7.0, 1.7 Hz,
1H), 7.59 (dd, J = 6.9, 1.7 Hz, 1H), 7.31ꢀ7.63 (m, 2H), 3.81ꢀ3.76
(m, 8H), 3.71ꢀ3.65 (m, 8H), 2.50 (s, 3H).
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(fluoromethyl)-
1H-benzimidazole (7c). A mixture of 2-(fluoromethyl)-1H-benzi-
midazole (6c)52 (0.75 g, 5 mmol), 2,4-dichloro-6-(4-morpholinyl)-
1,3,5-triazine (3)37 (1.17 g, 5 mmol), and powdered K2CO3 (2.75 g,
20 mmol) in DMF (20 mL) was stirred at room temperature overnight.
After dilution with water, the solid precipitate was collected and
recrystallized from EtOH to give 1-[4-chloro-6-(4-morpholinyl)-1,3,5-
triazin-2-yl]-2-(fluoromethyl)-1H-benzimidazole (1.08 g, 62% yield);
mp 230ꢀ231 °C; 1H NMR (CDCl3) δ 8.42 (dd, J = 6.9, 2.0 Hz, 1H),
7.83 (dd, J = 6.8, 2.2 Hz, 1H), 7.44ꢀ7.37 (m, 2H), 5.99 (d, JHF = 47.0
Hz, 1H), 3.99ꢀ3.94 (m, 4H), 3.85ꢀ3.79 (m, 4H). Anal. Calcd for
C15H14ClFN6O: C, 51.7; H, 4.05; N, 24.1. Found: C, 51.8; H, 3.9;
N, 24.4%.
’ EXPERIMENTAL METHODS
Chemistry. Combustion analyses were performed by the Campbell
Microanalytical Laboratory, University of Otago, Dunedin, New Zeal-
and. Melting points were determined on an Electrothermal IA9100
melting point apparatus and are as read. 1H NMR spectra were obtained
on a Bruker Avance-400 spectrometer at 400 MHz referenced to Me4Si.
Low-resolution atmospheric pressure chemical ionization (APCI) mass
spectra were measured for organic solutions on a ThermoFinnigan
Surveyor MSQ mass spectrometer connected to a Gilson autosampler.
Thin-layer chromatography was carried out on aluminum-backed silica
gel plates (Merck 60 F254), with visualization of components by UV
light (254 nm). Column chromatography was carried out on silica gel
(Merck 230ꢀ400 mesh). Tested compounds were g95% pure, as
determined by combustion analysis or by HPLC conducted on an
Agilent 1100 system using a reversed-phase C8 column with diode array
detection.
A mixture of the above chloro compound (0.75 g, 5 mmol) and
morpholine (4.35 g, 50 mmol) in THF (20 mL) was heated under reflux
for 1 h, cooled, and diluted with water to give 7c (0.39 g, 98% yield): mp
(EtOH) 229ꢀ231 °C; 1H NMR (CDCl3) δ 8.34 (dd, J = 7.4, 1.6 Hz,
1H), 7.82 (dd, J = 7.2, 1.7 Hz, 1H), 7.39ꢀ7.32 (m, 2H), 5.98 (d, JHF
=
47.3 Hz, 1H), 3.91ꢀ3.84 (m, 8H), 3.81ꢀ3.75 (m, 8H). Anal. Calcd for
C19H22FN7O2: C, 57.1; H, 5.55; N, 24.55. Found: C, 57.1; H, 5.3;
N, 24.8%.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(trifluoromethyl)-
1H-benzimidazole (7d). A mixture of 2-(trifluoromethyl)-1H-ben-
zimidazole (6d)52 (4.86 g, 26 mmol), 3 (5.11 g, 22 mmol), and
powdered K2CO3 (14 g, 0.1 mol) in DMF (100 mL) was stirred at
room temperature overnight. After dilution with water the solid was
collected by filtration and washed successively with water and then
EtOH to give 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(trifluoro-
methyl)-1H-benzimidazole (6.01 g, 71%): mp (EtOH) 239ꢀ241 °C;
1H NMR (CDCl3) δ 8.39 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H),
7.54 (ddd, J = 8.4, 7.8, 1.2 Hz, 1H), 7.46 (ddd, J = 8.3, 7.7, 1.2 Hz,
1H), 3.99ꢀ3.93 (m, 4H), 3.82ꢀ3.78 (m, 4H). Anal. Calcd for
C15H12ClF3N6O: C, 46.8; H, 3.1; N, 21.8. Found: C, 46.9; H, 3.3;
N, 22.1%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazole (1). A mixture of o-phenylenediamine
(5.41 g, 50 mmol) and difluoroacetic acid (9.6 g, 100 mmol) in 4 M
HCl (20 mL) was heated under reflux for 1 h and diluted with hot
water (50 mL). The solution was treated with charcoal and filtered
through Celite before being neutralized with aqueous NH3. The
resulting white precipitate was collected, washed with water, and dried
to give 2-(difluoromethyl)-1H-benzimidazole (2)36 (6.07 g, 72% yield):
mp 156ꢀ158 °C; 1H NMR (DMSO-d6) δ 13.28 (br, 1H), 7.76ꢀ
A mixture of the above chloro compound (0.386 g, 1 mmol) and
morpholine (0.435 g, 5 mmol) in THF (5 mL) was heated at 70 °C for
1 h. After cooling, the mixture was diluted with water to give a solid
7.68 (m, 1H), 7.61ꢀ7.54 (m, 1H), 7.36ꢀ7.26 (m, 2H), 7.26 (t, JHF
=
53.3 Hz, 1H).
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which was recrystallized from EtOH to give 7d (0.295 g, 68%): mp
197ꢀ200 °C; 1H NMR (CDCl3) δ 8.15 (d, J = 7.7 Hz, 1H), 7.90 (d, J =
7.8 Hz, 1H), 7.46 (ddd, J = 7.7, 7.3, 1.4 Hz, 1H), 7.41 (ddd, J = 7.7, 7.3,
1.3 Hz, 1H), 3.91ꢀ3.87 (m, 8H), 3.79ꢀ3.75 (m, 8H). Anal. Calcd for
C19H20F3N7O2: C, 52.4; H, 4.6; N, 22.5. Found: C, 52.1; H, 4.6;
N, 22.7%.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(hydroxy-
methyl)-1H-benzimidazole (7e). A mixture of 2-({[tert-butyl-
(dimethyl)silyl]oxy}methyl)-1H-benzimidazole (6e)53 (1.31 g, 5 mmol),
3 (1.175 g, 5 mmol), and powdered K2CO3 (5.5 g, 40 mmol) in DMF
(50 mL) was stirred at room temperature overnight and diluted with
water. The precipitate was collected by filtration and washed with water
and then with EtOH to give 2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-
1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (0.49 g,
20%): 1H NMR (CDCl3) δ 8.29ꢀ8.25 (m, 1H), 7.79ꢀ7.75 (m, 1H),
7.37ꢀ7.33 (m, 2H), 5.33 (s, 2H), 3.99ꢀ3.94 (m, 4H), 3.83ꢀ3.78
(m, 4H), 0.85 (s, 9H), 0.08 (s, 6H).
A mixture of the above chloro compound (0.31 g, 0.67 mmol) and
morpholine (0.59 g, 6.7 mmol) in THF (10 mL) was stirred at room
temperature for 1 h and diluted with water. Extraction with CH2Cl2 fol-
lowed by chromotography on silica, eluting with CH2Cl2/EtOAc (95:5),
gave 2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-1-[4,6-di(4-morpholinyl)-
1,3,5-triazin-2-yl]-1H-benzimidazole (7k) (0.25 g, 73%): 1H NMR
(CDCl3) δ 8.18ꢀ8.14 (m, 1H), 7.79ꢀ7.75 (m, 1H), 7.32ꢀ7.28
(m, 2H), 5.33 (s, 2H), 3.91ꢀ3.85 (m, 8H), 3.79ꢀ3.75 (m, 8H), 0.85
(s, 9H), 0.07 (s, 6H).
Reaction of the above crude silyl ether 7k with Bu4NF in THF gave 7e
(0.123 g, 63%): mp (EtOH) 271ꢀ273 °C (lit.47 208ꢀ210 °C dec); 1H
NMR (CDCl3) δ 12.49 (br, 1H), 7.54ꢀ7.49 (m, 2H), 7.19ꢀ7.14
(m, 2H), 5.48 (s, 2H), 3.70ꢀ3.65 (m, 8H), 3.60ꢀ3.54 (m, 8H).
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-[(methyl-
sulfanyl)methyl]-1H-benzimidazole (7f). A mixture of 2-[(methyl-
sulfanyl)methyl]-1H-benzimidazole (6f)54 (7.13 g, 40 mmol), 3 (9.40 g,
40 mmol), and powdered K2CO3 (22 g, 0.16 mol) in DMF (250 mL) was
stirred at room temperature for 2 h and diluted with water. The precipitate
was collected by filtration and washed successively with water and EtOH
to give 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-[(methylsulfanyl)-
methyl]-1H-benzimidazole (13.7 g, 91%): mp (CH2Cl2/MeOH) 213ꢀ
215 °C; 1H NMR (CDCl3) δ 8.39ꢀ8.34 (m, 1H), 7.75ꢀ7.70 (m, 1H),
7.40ꢀ7.33 (m, 2H), 4.41 (s, 2H), 4.00ꢀ3.91 (m, 4H), 3.86ꢀ3.78
(m, 4H), 2.14 (s, 3H). Anal. Calcd for C16H17ClN6OS: C, 51.0; H, 4.55;
N, 22.3. Found: C, 51.1; H, 4.85; N, 22.6%.
A mixture of the above chloro compound (3.769 g, 10 mmol) and
morpholine (4.4 g 50 mmol) in THF (250 mL) was heated under reflux
for 10 min, and the solvent was concentrated. Dilution with water gave 7f
(3.91 g, 91%): mp (CH2Cl2/MeOH) 195ꢀ197 °C; 1H NMR (CDCl3)
δ 8.27ꢀ8.23 (m, 1H), 7.75ꢀ7.71 (m, 1H), 7.34ꢀ7.29 (m, 2H), 4.14
(s, 2H), 3.93ꢀ3.85 (m, 8H), 3.82ꢀ3.76 (m, 8H), 2.16 (s, 3H). Anal.
Calcd for C20H25N7O2S: C, 56.2; H, 5.9 N, 22.9. Found: C, 56.5; H, 6.2;
N, 23.2%.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-[(methyl-
sulfinyl)methyl]-1H-benzimidazole (7g). A solution of 7f (0.43 g,
1 mmol) in EtOH (500 mL) was treated with a solution of NaIO4 (1.6 g,
7.5 mmol) in water (75 mL), and the mixture was stirred at room
temperature overnight. The solution was decolorized with aqueous
Na2SO3, and the EtOH was removed under vacuum. The residue was
diluted with water and extracted with EtOAc. Chromatography on
alumina, eluting with EtOAc, followed by recrystallization from MeOH
gave 7g (0.29 g, 65%): mp 208ꢀ210 °C; 1H NMR (CDCl3) δ
8.29ꢀ8.23 (m, 1H), 7.76ꢀ7.71 (m, 1H), 7.39ꢀ7.31 (m, 2H), 5.29
(dd, J = 12.6, 0.5 Hz, 1H), 4.66 (d, J = 12.6 Hz, 1H), 3.94ꢀ3.86 (m, 8H),
3.82ꢀ3.76 (m, 8H), 2.78 (s, 3H). Anal. Calcd for C20H25N7O3S: C,
54.2; H, 5.7; N, 22.1. Found: C, 54.4; H, 5.9; N, 22.4%.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-[(methyl-
sulfonyl)methyl]-1H-benzimidazole (7h). A solution of 7f (0.43 g,
1 mmol) in a mixture of acetone (130 mL) and HOAc (20 mL) was
treated with a solution of KMnO4 (1 g) in water (10 mL) at room
temperature. After 10 min the solution was decolorized with aqueous
Na2SO3 and the acetone was removed under vacuum. The residue was
diluted with aqueous NH3 to give a solid which was collected and dried.
Chromatography on silica, eluting with CH2Cl2/EtOAc (2:1), gave 7h
(0.426 g, 92%): mp (MeOH) 232ꢀ234 °C; 1H NMR (CDCl3) δ
8.28ꢀ8.23 (m, 1H), 7.78ꢀ7.73 (m, 1H), 7.40ꢀ7.33 (m, 2H), 5.47
(d, J = 0.4Hz, 2H), 3.92ꢀ3.85 (m, 8H), 3.81ꢀ3.75 (m, 8H), 3.09 (s, 3H).
Anal. Calcd for C20H25N7O4S:C, 52.3;H, 5.5; N, 21.3. Found:C, 52.5; H,
5.2; N, 21.6%.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(methyl-
sulfanyl)-1H-benzimidazole (7i). A mixture of 2-(methylsulfanyl)-
1H-benzimidazole (6i) (1.64 g, 10 mmol), 3 (2.35 g, 10 mmol), and
powdered K2CO3 (11 g, 80 mmol) in DMF (50 mL) was stirred at room
temperature for 1 h. The mixture was diluted with water and the
resulting precipitate was collected, washed with water and then EtOH,
and dried to give 3.56 g (98% yield) of 1-[4-chloro-6-(4-morpholinyl)-
1,3,5-triazin-2-yl]-2-(methylsulfanyl)-1H-benzimidazole: mp (CHCl3/
EtOH) 260ꢀ261 °C; 1HNMR(CDCl3) δ8.42 (br dd, J= 7.0, 1.9 Hz, 1H),
7.65 (br dd, J = 6.8, 1.9 Hz, 1H), 7.30 (m, 2H), 4.06 (m, 2H), 3.95
(m, 2H), 3.84 (m, 2H), 3.80 (m, 2H), 2.74 (s, 3H). Anal. Calcd for
C15H15ClN6OS: C, 49.65; H, 4.2; N, 23.2. Found: C, 49.8; H, 4.1;
N, 23.1%.
A solution of the above chloro compound (0.363 g, 1 mmol) and
morpholine (0.87 g, 10 mmol) in CHCl3 (50 mL) was stirred at room
temperature for 1 h. The solution was washed with water, and after being
dried (Na2SO4), the solvent was removed to give 7i (0.41 g, 100%
1
yield): mp (CHCl3/EtOH) 249ꢀ252 °C; H NMR (CDCl3) δ 8.30
(dd, J = 7.3, 1.5 Hz, 1H), 7.66 (dd, J = 7.4, 1.7 Hz, 1H), 7.29ꢀ7.21
(m, 2H), 4.01ꢀ3.83 (m, 8H), 3.81ꢀ3.75 (m, 8H), 2.72 (s, 3H). Anal.
Calcd for C19H23N7O2S: C, 55.2; H, 5.6; N, 23.7. Found: C, 55.1; H, 5.7;
N, 23.7%.
1-[4,6-Di(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(methyl-
sulfonyl)-1H-benzimidazole (7j). A solution of 7i (0.41 g, 1 mmol)
in a mixture of acetone (250 mL) and HOAc (35 mL) was treated with a
solution of KMnO4 (1 g) in water (50 mL), and the resulting mixture
was stirred for 15 min. After dilution with water and decolorization with
aqueous Na2SO3, the product was extracted into CHCl3, washed with
aqueous NH3, and dried (Na2SO4). Chromatography on alumina,
eluting with CH2Cl2, followed by recrystallization from MeOH gave
7j (0.112 g, 25% yield): mp (MeOH) 174ꢀ177 °C; 1H NMR (CDCl3)
δ 8.25 (br d, J = 8.4 Hz, 1H), 7.82 (br d, J = 8.0 Hz, 1H), 7.48 (ddd, J =
8.4, 7.3, 1.3 Hz, 1H), 7.48 (ddd, J = 8.0, 7.3, 1.3 Hz, 1H), 4.41ꢀ3.96
(m, 4H), 3.90ꢀ3.83 (m, 4H), 3.79ꢀ3.75 (m, 8H), 3.58 (s, 3H). Anal.
Calcd for C19H23N7O4S: C, 51.2; H, 5.2; N, 22.0. Found: C, 51.25; H, 5.25;
N, 22.05%.
2-(Difluoromethyl)-4-hydroxy-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazole (10a). 2-Amino-3-nitrophenol (7.71 g,
50 mmol) in EtOH (100 mL) was hydrogenated over 5% Pd on carbon,
and the solution was filtered through Celite and acidified with concentrated HCl.
The solvents were evaporated to dryness, and the residue was combined with
difluoroacetic acid (9.6 g, 100 mmol) in 4 M HCl (30 mL). The mixture was
heated under reflux for 3 h before being cooled and basified with aqueous
NH3. Extraction with EtOAc, followed by chromatography on silica, eluting with
CH2Cl2/EtOAc (7:3), gave 2-(difluoromethyl)-4-hydroxy-1H-benzimidazole
(8a) (7.68 g, 83%) as an oil: 1HNMR(CDCl3) δ9.80 (br, 1H), 9.46 (br, 1H),
7.28 (t, J = 8.0 Hz, 1H), 7.08 (t, JHF = 53.4 Hz, 1H), 7.05 (br, 1H), 6.88
(d, J = 7.9 Hz, 1H).
A mixture of 8a (7.68 g, 42 mmol) and TBDMSCl (10.7 g, 71 mmol)
in pyridine (100 mL) was stirred at room temperature overnight. The
pyridine was removed under vacuum, and the residue was diluted with
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water and extracted with CH2Cl2. Chromatography on alumina, eluting
with CH2Cl2, gave 4-(tert-butyldimethylsilyloxy)-2-(difluoromethyl)-
1H-benzimidazole (8aa) (11.9 g, 96%): mp (CH2Cl2/hexanes) 139ꢀ
141 °C; 1H NMR (CDCl3) δ 9.60 (br, 1H), 7.50ꢀ7.30 (m, 1H), 7.19
(t, J = 8.0 Hz, 1H), 6.89 (t, JHF = 53.9 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H),
1.04 (s, 9H), 0.29 (s, 6H).
A mixture of 8aa (1.49 g, 5 mmol), 3 (1.175 g, 5 mmol), and
powdered K2CO3 (5.5 g, 40 mmol) in acetone (40 mL) was stirred
at room temperature for 2 h and diluted with water. Extraction
with CH2Cl2 and chromatography on silica, eluting with CH2Cl2/
hexanes (3:1), gave 4-(tert-butyldimethylsilyloxy)-1-[4-chloro-6-
(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoromethyl)-1H-benzimida-
zole (9aa) (1.27 g, 51%): mp (hexanes) 143ꢀ145 °C; 1H NMR
(CDCl3) δ 7.99 (d, J = 8.4 Hz, 1H), 7.46 (t, JHF = 53.5 Hz, 1H), 7.32
(t, J = 8.2 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 3.98ꢀ3.93 (m, 3.5H),
3.89ꢀ3.86 (m, 0.5H), 3.84ꢀ3.78 (m, 3.5H), 3.76ꢀ3.73 (m, 0.5H), 1.05
(s, 9H), 0.29 (s, 6H). Anal. Calcd for C21H27ClF2N6O2Si: C, 50.75; H,
5.5; N, 16.9. Found: C, 50.7; H, 5.6; N, 17.0%.
A solution of 9aa (0.25 g, 0.5 mmol) and morpholine (0.44 g, 5
mmol) in THF (10 mL) was stirred at room temperature for 30 min and
diluted with water. Extraction with EtOAc and chromatography on silica,
eluting with CH2Cl2/hexanes (19:1), gave 4-{[tert-butyl(dimethyl)-
silyl]oxy}-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazole (10aa) (0.25 g, 91%): 1H NMR (CDCl3) δ 7.89
(dd, J = 8.3, 0.8 Hz, 1H), 7.45 (t, JHF = 53.6 Hz, 1H), 7.26 (t, J = 8.1 Hz,
1H), 6.84 (dd, J = 7.8, 0.8 Hz, 1H), 3.90ꢀ3.85 (m, 8H), 3.80ꢀ3.75
(m, 8H), 1.05 (s, 9H), 0.30 (s, 6H).
(t, J = 8.2 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 4.05 (s, 1H), 3.90ꢀ3.85
(m, 8H), 3.80ꢀ3.76 (m, 8H). Anal. Calcd for C20H23F2N7O3: C, 53.7;
H, 5.2; N, 21.9. Found: C, 53.75; H, 5.1; N, 22.05%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-ethoxy-1H-benzimidazole (10c). A solution of 2-amino-
3-ethoxynitrobenzene55 (1.64 g, 9 mmol) in MeOH (100 mL) was
hydrogenated over 5% Pd on carbon and filtered through Celite. The
solution was acidified with concentrated HCl, and the solvents were
removed under vacuum. The residue was combined with difluoroacetic
acid (1.73 g, 18 mmol) in 4 M HCl (10 mL), and the mixture was heated
under reflux for 3 h. After cooling, the solution was neutralized with
aqueous NH3 to give 2-difluoromethyl-4-ethoxy-1H-benzimidazole
(8c) (1.29 g, 68% yield): mp (aqueous MeOH) 185ꢀ187 °C; 1H
NMR (DMSO-d6) δ 13.30 (br, 1H), 7.20 (t, JHF = 53.3 Hz, 1H),
7.22ꢀ7.14 (m, 2H), 6.78 (br d, J = 7.5 Hz, 1H), 4.24 (q, J = 7.0 Hz, 2H),
1.41 (t, J = 7.0 Hz, 3H). Anal. Calcd for C10H10F2N2O: C, 56.6; H, 4.75;
N, 13.2. Found: C, 56.9; H, 4.8; N, 13.4%.
A mixture of 8c (0.85 g, 4 mmol), 3 (0.49 g, 4 mmol), and powdered
K2CO3 (4.4 g, 32 mmol) in DMF (25 mL) was stirred overnight and
then diluted with water. The resulting precipitate was collected by
filtration, washed with water and then with EtOH, and dried to give 1-[4-
chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoromethyl)-
4-ethoxy-1H-benzimidazole (9c) (1.48 g, 90%): mp (CHCl3/EtOH)
1
272ꢀ275 °C; H NMR (CDCl3) δ 7.88 (d, J = 8.4 Hz, 1H), 7.47 (t,
JHF = 53.4 Hz, 1H), 7.38 (t, J = 8.3 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 4.33
(q, J = 7.0 Hz, 2H), 3.99ꢀ3.93 (m, 4H), 3.84ꢀ3.78 (m, 4H), 1.56 (t, J =
7.0 Hz, 3H). Anal. Calcd for C17H17ClF2N6O2: C, 49.7; H, 4.2; N, 20.5.
Found: C, 49.8; H, 4.4; N, 20.6%.
A solution of 10aa (0.25 g, 0.46 mmol) in THF (10 mL) was treated
with an excess of Bu4NF (1 M in THF) at room temperature for 30 min,
and the solvent was removed under vacuum. Addition of water gave a
white solid which was collected and dried to give 10a (0.195 g, 98%): mp
(EtOH) 288ꢀ290 °C (lit.47 mp >250 °C); 1H NMR (CDCl3) δ 7.81
(d, J = 8.2 Hz, 1H), 7.55 (t, JHF = 53.6 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H),
6.90 (d, J = 8.0 Hz, 1H), 3.90ꢀ3.85 (m, 8H), 3.81ꢀ3.75 (m, 8H).
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-methoxy-1H-benzimidazole (10b). 2-Amino-3-methoxy-
nitrobenzene55 (15.10 g, 0.09 mol) was hydrogenated over palladium on
carbon in MeOH, and the solution was filtered through Celite into a
methanolic HCl solution. The solvent was removed under vacuum, and
the resulting hydrochloride salt was combined with difluoroacetic acid
(19.2 g, 0.18 mol) and 4 M HCl (100 mL). The mixture was heated
under reflux for 3 h, diluted with water, decolorized with charcoal, and
filtered through Celite. Neutralization with aqueous ammonia gave
2-difluoromethyl-4-methoxy-1H-benzimidazole56 (8b) (15.2 g, 84%)
A mixture of 9c (0.205 g, 0.5 mmol) and morpholine (0.44 g, 0.5
mmol) in CHCl3 (25 mL) was stirred at room temperature for 30 min
and washed with water. The solvent was then evaporated to dryness to
give 10c (0.201 g, 98%); mp (CHCl3/EtOH) 225ꢀ228 °C; 1H NMR
(CDCl3) δ 7.87 (d, J = 8.4 Hz, 1H), 7.47 (t, JHF = 53.5 Hz, 1H), 7.32
(t, J = 8.2 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 4.33 (q, J = 7.0 Hz, 2H),
3.90ꢀ3.85 (m, 8H), 3.80ꢀ3.76 (m, 8H), 1.55 (t, J = 7.0 Hz, 3H). Anal.
Calcd for C21H25F2N7O3: C, 54.7; H, 5.5; N, 21.25. Found: C, 54.85; H,
5.7; N, 21.5%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-propoxy-1H-benzimidazole (10d). A mixture of 10a (108
mg, 0.25 mmol), 1-iodopropane (0.47 mg, 0.28 mmol), and powdered
K2CO3 (0.40 g, 2.9 mmol) in DMF (5 mL) was stirred at room
temperature overnight and diluted with water to give 10d (102 mg,
86%): mp (CH2Cl2/MeOH) 192ꢀ194 °C; 1H NMR (CDCl3) δ 7.87
(d, J = 8.3 Hz, 1H), 7.47 (t, JHF = 53.5 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H),
6.81 (d, J = 8.0 Hz, 1H), 4.21 (t, J = 6.9 Hz, 2H), 3.90ꢀ3.85 (m, 8H),
3.80ꢀ3.76 (m, 8H), 1.97 (sextet, J = 7.2 Hz, 2H), 1.08 (t, J = 7.4 Hz,
3H). Anal. Calcd for C22H27F2N7O3: C, 55.6; H, 5.7; N, 20.6. Found: C,
55.8; H, 5.8; N, 20.8%.
4-Butoxy-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-
triazin-2-yl]-1H-benzimidazole (10e). A mixture of 10a (108 mg,
0.25 mmol), 1-iodobutane (51 mg, 0.28 mmol), and powdered K2CO3
(0.40 g, 2.9 mmol) in DMF (5 mL) was stirred at room temperature
overnightand diluted with water to give10e (95mg, 69%): mp (CH2Cl2/
MeOH) 211ꢀ213 °C; 1H NMR (CDCl3) δ 7.86 (d, J = 8.1 Hz, 1H),
7.46 (t, JHF = 53.5 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H), 6.81 (d, J = 8.0 Hz,
1H), 4.26 (t, J = 6.8 Hz, 2H), 3.90ꢀ3.85 (m, 8H), 3.80ꢀ3.76 (m, 8H),
1.93 (m, 2H), 1.55 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). Anal. Calcd for
C23H29F2N7O3: C, 56.4; H, 6.0; N, 20.0. Found: C, 56.7; H, 5.9;
N, 20.2%.
1
as a solid: H NMR (CDCl3) (tautomeric mixture) δ 9.95ꢀ9.70 (m,
exchangeable with D2O, 1H), 7.44 (br d, J = 7.9 Hz, 0.4H), 7.31ꢀ7.24
(m, 1H), 7.12 (br d, J = 8.0 Hz, 0.5H), 6.89 (t, JHF = 53.8 Hz, 1H),
6.82ꢀ6.74 (m, 1H), 4.03 and 3.98 (2s, 3H).
A mixture of 8b (3.96 g, 20 mmol), 3 (4.70 g, 20 mmol), and
powdered K2CO3 (22 g, 80 mmol) in DMF (150 mL) was stirred rapidly
for 3 h and then diluted with water. The resulting precipitate was
collected by filtration, washed with water and then with EtOH, and oven-
dried to give 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoro-
methyl)-4-methoxy-1H-benzimidazole (9b) (6.82 g, 86%): mp
1
(CHCl3/EtOH) 263ꢀ266 °C; H NMR (CDCl3) δ 7.99 (d, J = 8.4
Hz, 1H), 7.48 (t, JHF = 53.4 Hz, 1H), 7.40 (t, J = 8.3 Hz, 1H), 6.86 (d, J =
8.1 Hz, 1H), 4.05 (s, 3H), 3.99ꢀ3.93 (m, 4H), 3.84ꢀ3.78 (m, 4H).
Anal. Calcd for C16H15ClF2N6O2: C, 48.4; H, 3.8; N, 21.2. Found: C,
48.3; H, 3.8; N, 21.1%.
A suspension of 9b (0.199 g, 0.5 mmol) in morpholine (0.87 g, 10
mmol) was heated at 70 °C for 1 h and diluted with water. The resulting
solid was collected by filtration and recrystallized from CH2Cl2/EtOH
to give 10b (0.188 g, 84%): mp 270ꢀ273 °C (lit.48 > 250 °C); 1H NMR
(CDCl3) δ 7.88 (d, J = 8.4 Hz, 1H), 7.47 (t, JHF = 53.5 Hz, 1H), 7.35
4-(Difluoromethoxy)-2-(difluoromethyl)-1-[4,6-di(4-mor-
pholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (10f). A mix-
ture of 10a (228 mg, 0.52 mmol) and dry powdered K2CO3 (1.8 g,
13.6 mmol) in DMF (5 mL) was stirred at 20 °C for 30 min. Then
sodium chloro(difluoro)acetate (396 mg, 2.6 mmol) was added and the
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mixture was stirred at 80 °C for 20 h. The reaction mixture was cooled to
room temperature and diluted with water. The resulting precipitate was
collected by filtration, washed with water, and dried. Chromatography
on silica, eluting with CH2Cl2/EtOAc (9:1), gave 10f (105 mg, 41%):
mp (CH2Cl2/hexanes) 231ꢀ233 °C; 1H NMR (CDCl3) δ 8.14 (dd, J =
8.4, 0.8 Hz, 1H), 7.49 (t, JHF = 53.3 Hz, 1H), 7.32 (t, JHF = 74.6 Hz, 1H),
7.17 (dd, J = 8.0, 0.3 Hz, 1H), 3.89ꢀ3.87 (m, 8H), 3.80ꢀ3.77 (m, 8H).
Anal. Calcd for C20H21F4N7O3: C, 49.7; H, 4.4; N, 20.3. Found: C, 49.7;
H, 4.4; 20.3%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-isopropoxy-1H-benzimidazole (10g). A mixture of 10a
(108 mg, 0.25 mmol), 2-iodopropane (0.47 mg, 0.28 mmol), and
powdered K2CO3 (0.40 g, 2.9 mmol) in DMF (5 mL) was stirred at
20 °C overnight and diluted with water to give 10g (121 mg, 100%): mp
(CH2Cl2/MeOH) 218ꢀ220 °C; 1H NMR (CDCl3) δ 7.85 (d, J = 8.2
Hz, 1H), 7.46 (t, JHF = 53.5 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H), 6.82 (d, J =
8.0 Hz, 1H), 4.96 (septet, J = 6.1 Hz, 1H), 3.90ꢀ3.85 (m, 8H),
3.80ꢀ3.76 (m, 8H), 1.47 (d, J = 6.1 Hz, 6H). Anal. Calcd for
C22H27F2N7O3: C, 55.6; H, 5.7; N, 20.6. Found: C, 55.6; H, 5.85;
N, 20.7%.
(m, 8H), 3.51 (s, 3H). Anal. Calcd for C20H23ClF2N7O5S: C, 47.0; H,
4.5; N, 19.2. Found: C, 46.6; H, 4.6; N 19.0%.
4-Amino-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,
5-triazin-2-yl]-1H-benzimidazole (10i). A mixture of 1,2-diamino-
3-nitrobenzene (5 g, 33 mmol) and difluoroacetic acid (15 g, 0.156 mol)
in 4 M HCl (75 mL) was heated under reflux for 4 days and cooled to
give2-(difluoromethyl)-4-nitro-1H-benzimidazole (8ae)57 (4.74 g, 68%):
1H NMR (DMSO-d6) δ 14.00 (br, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.23
(d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.1 Hz, 1H), 7.31 (t, JHF = 52.7 Hz, 1H).
A mixture of 8ae (1.07 g, 5 mmol), 3 (1.29 g, 5.5 mmol), and
powdered K2CO3 (6.5 g, 47 mmol) in THF (50 mL) was stirred at room
temperature overnight and then diluted with water to give a solid, which
was collected and dried. Chromatography on silica, eluting with
CH2Cl2/EtOAc (19:1), gave 1-[4-chloro-6-(4-morpholinyl)-1,3,5-tria-
zin-2-yl]-2-(difluoromethyl)-4-nitro-1H-benzimidazole (9ae) (0.90 g,
1
44%): mp (CH2Cl2/EtOH) 236ꢀ238 °C; H NMR (CDCl3) δ 8.83
(dd, J = 8.4, 1.0 Hz, 1H), 8.24 (dd, J = 8.1, 1.0 Hz, 1H), 7.62 (t, J = 8.3 Hz,
1H), 7.50 (t, JHF = 53.0 Hz, 1H), 4.02ꢀ3.99 (m, 2H), 3.97ꢀ3.94
(m, 2H), 3.86ꢀ3.81 (m, 4H). Anal. Calcd for C15H12ClF2N7O3: C, 43.75;
H, 2.9; N, 23.8. Found: C, 44.0; H, 3.1; N, 24.0%.
A mixture of 9ae (0.90 g, 2.2 mmol) and morpholine (0.95 g, 11
mmol) in THF (20 mL) was stirred at room temperature for 1 h and
diluted with water to give 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-
1,3,5-triazin-2-yl]-4-nitro-1H-benzimidazole (10ae) (1.01 g, 99%): mp
(CH2Cl2/EtOH) 292ꢀ295 °C; 1H NMR (CDCl3) δ 8.66 (dd, J = 8.4,
0.9 Hz, 1H), 8.21 (dd, J = 8.1, 0.9 Hz, 1H), 7.55 (t, J = 8.2 Hz, 1H), 7.51
(t, JHF = 53.1 Hz, 1H), 3.90ꢀ3.85 (m, 8H), 3.80ꢀ3.76 (m, 8H). Anal.
Calcd for C19H20F2N8O4: C, 49.35; H, 4.4; N, 24.2. Found: C, 49.55; H,
4.5; N, 24.4%.
A suspension of 10ae (0.925 g, 2 mmol) in MeOH (100 mL) was
hydrogenated over 5% Pd on C to give a solution, which was filtered
through Celite to remove the catalyst. Removal of the solvent, followed
by chromatography on alumina, eluting with CH2Cl2/EtOAc (19:1),
gave 10i (0.79 g, 91%): mp (MeOH) 229ꢀ231 °C (lit.38 mp 214ꢀ
216 °C); 1H NMR (DMSO-d6) δ 7.69 (t, JHF = 53.0 Hz, 1H), 7.44 (dd, J =
8.2, 0.8 Hz, 1H), 7.13 (t, J = 8.1 Hz, 1H), 6.55 (dd, J = 7.8, 0.8 Hz, 1H),
5.59 (br s, 2H), 3.81ꢀ3.77 (m, 8H), 3.70ꢀ3.65 (m, 8H). Anal. Calcd for
C19H22F2N8O2: C, 52.8; H, 5.1; N, 25.9. Found: C, 52.9; H, 5.3;
N, 26.2%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazol-4-yl Methanesulfonate (10h). A solu-
tion of 2-amino-3-nitrophenol (1.54 g, 1 mmol) in pyridine (20 mL) was
cooled to 0 °C, and methanesulfonyl chloride (1.73 g, 1.5 mmol) was
added with stirring. The temperature was allowed to rise to room
temperature as stirring was continued overnight. Water was added to
give a solid which was collected and dried. The solid was dissolved in
CH2Cl2 and eluted through a short column of alumina to remove polar
impurities. Removal of the solvent gave 2-amino-3-nitrophenyl metha-
nesulfonate (2.06 g, 89% yield): mp (CH2Cl2/MeOH) 128ꢀ130 °C;
1H NMR (CDCl3) δ 8.12 (dd, J = 8.8, 1.4 Hz, 1H), 7.50 (dd, J = 7.9, 1.3
Hz, 1H), 6.73 (dd, J = 8.6, 8.1 Hz, 1H), 6.35 (br, 2H), 3.27 (s, 3H). Anal.
Calcd for C7H8N2O5S: C, 36.2; H, 3.5; N, 12.1. Found: C, 36.5; H, 3.7;
N, 12.1%.
A solution of the above compound (1.16 g, 5 mmol) in MeOH
(50 mL) was hydrogenated over 5% Pd on carbon, and after filtration
through Celite, the solution was acidified with concentrated HCl. The
solvent was evaporated to dryness, and the residue was combined with
difluoroacetic acid (0.96 g, 10 mmol) in 4 M HCl (20 mL). The mixture
was heated under reflux for 4 h, cooled, and neutralized with aqueous
NH3. Extraction with EtOAc gave 2-(difluoromethyl)-1H-benzimida-
zol-4-yl methanesulfonate (8h) (0.71 g, 54% yield): mp (MeOH/
i-Pr2O) 160ꢀ162 °C; 1H NMR (DMSO-d6) δ 13.76 (br, 1H), 7.65ꢀ7.59
(m, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.31 (t, JHF = 53.1 Hz, 1H), 7.28 (d, J =
7.7 Hz, 1 H), 3.53 (s, 3H). Anal. Calcd for C9H8F2N2O3S: C, 41.2; H,
3.1. Found: C, 41.1; H, 3.3%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-(methylamino)-1H-benzimidazole (10j). A mixture of
10i (0.23 g, 0.53 mmol) and di-tert-butyl dicarbonate (0.35 g, 1.6 mmol)
in dioxane (50 mL) was heated under reflux for 2 days. Dilution with
water gave a white solid which was collected and dried. Chromatography
on alumina, eluting with CH2Cl2, gave tert-butyl 2-(difluoromethyl)-
1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-ylcar-
1
bamate (0.21 g, 55% yield): mp (CH2Cl2/MeOH) 248ꢀ251 °C; H
A mixture of 8h (1.57 g, 6 mmol), 3 (1.41 g, 6 mmol), and powdered
K2CO3 (6.6 g, 48 mmol) in acetone (50 mL) was stirred at room
temperature for 3 h and then diluted with water. Extraction with EtOAc,
followed by chromatography on silica, eluting with CH2Cl2/EtOAc
(9:1), gave 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoro-
methyl)-1H-benzimidazol-4-yl methanesulfonate (9h) (1.70 g, 61%
yield): mp (CH2Cl2/EtOH) 208ꢀ210 °C; 1H NMR (CDCl3) δ 8.38
(dd, J = 8.4, 0.8 Hz, 1H), 7.52 (t, JHF = 53.2 Hz, 1H), 7.50 (t, J = 8.2 Hz,
1H), 7.41 (dd, J = 8.1, 0.7 Hz, 1H), 4.10ꢀ3.97 (m, 2H), 3.95ꢀ3.92
(m, 2H), 3.85ꢀ3.80 (m, 4H), 3.50 (s, 3H). Anal. Calcd for C16H15-
ClF2N6O4S: C, 41.7; H, 3.3; N, 13.2. Found: C, 42.0; H, 3.3; N 13.4%.
A mixture of 9h (0.23 g, 0.5 mmol) and morpholine (0.22 g, 6 mmol)
in THF (20 mL) was stirred at room temperature for 15 min and diluted
with water to give a solid which was recrystallized from CHCl3/EtOH to
NMR (CDCl3) δ 8.05 (d, J = 8.0 Hz, 1H), 7.90 (dd, J = 8.3, 0.6 Hz, 1H),
7.80 (br s, exchangeable with D2O, 1H), 7.56 (t, JHF = 53.6 Hz, 1H), 7.38
(t, J = 8.2 Hz, 1H), 3.89ꢀ3.85 (m, 8H), 3.80ꢀ3.75 (m, 8H), 1.55
(s, 9H). Anal. Calcd for C24H30F2N8O9: C, 54.2; H, 5.7; N, 21.0. Found:
C, 54.0; H, 5.7; N, 21.3%.
The above carbamate (0.20 g, 0.38 mmol) in DMF (20 mL) was
treated with NaH (60%, 24 mg, 6 mmol), and the mixture was stirred at
room temperature for 15 min before iodomethane (82 mg, 0.58 mmol)
was added. The resulting mixture was stirred for 30 min, and water
was added. The solid was collected and dried. Chromatography on
silica, eluting with CH2Cl2/EtOAc (9:1), gave tert-butyl 2-(difluoromethyl)-
1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazol-4-yl(methyl)-
carbamate (0.14 g, 67%): 1HNMR(CDCl3) δ8.17 (dd, J=8.3, 0.9 Hz, 1H),
7.51 (t, JHF = 49.2 Hz, 1H), 7.37 (t, J = 8.1 Hz, 1H), 7.90 (dd, J = 8.3, 0.6 Hz,
1H), 7.26 (m, 1H), 3.90ꢀ3.85 (m, 8H), 3.81ꢀ3.76 (m, 8H), 3.45 (s, 3H),
1.42 (s, 9H).
1
give 10h (0.25 g, 97% yield): mp 249ꢀ251 °C; H NMR (CDCl3) δ
8.27 (dd, J = 8.3, 0.9 Hz, 1H), 7.50 (t, JHF = 53.3 Hz, 1H), 7.44 (t, J = 8.2
Hz, 1H), 7.37 (dd, J = 8.0, 0.9 Hz, 1H), 3.89ꢀ3.85 (m, 8H), 3.80ꢀ3.76
7116
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Journal of Medicinal Chemistry
ARTICLE
The above carbamate (0.14 g, 0.26 mmol) was treated with TFA in
CH2Cl2 to give 10j (0.11 g, 99%): mp (CH2Cl2/MeOH) 285ꢀ288 °C;
1H NMR (CDCl3) δ 7.56 (d, J = 8.3 Hz, 1H), 7.53 (t, JHF = 53.8 Hz, 1H),
7.28 (t, J = 8.1 Hz, 1H), 6.47 (d, J = 7.9 Hz, 1H), 5.06 (br d, J = 4.3 Hz,
1H), 3.89ꢀ3.84 (m, 8H), 3.79ꢀ3.75 (m, 8H), 3.00 (d, J = 4.5 Hz, 3H).
Anal. Calcd for C20H24F2N8O2: C, 53.8; H, 5.4; N, 25.1. Found: C,
53.9; H, 5.5; N, 25.3%.
2-(Difluoromethyl)-4-(dimethylamino)-1-[4,6-di(4-mor-
pholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (10k). N1,
N1-dimethyl-2-nitro-1,3-benzenediamine58 (1.09 g, 6 mmol) in MeOH
(50 mL) was hydrogenated over Pd on carbon, and the solution was
filtered through Celite and acidified with concentrated HCl. The solvent
was evaporated to dryness, and the residue was combined with
difluoroacetic acid (3 g, 31 mmol) in 4 M HCl (20 mL). The mixture
was heated under reflux for 3 h, clarified with charcoal, cooled and
neutralized with aqueous NH3. Extraction with EtOAc, followed by
chromatography on silica, eluting with CH2Cl2/EtOAc (9:1), gave
2-(difluoromethyl)-4-(dimethylamino)-1H-benzimidazole (8k) (0.77
g, 61% yield): mp (CH2Cl2/hexanes) 100ꢀ101 °C; 1H NMR
(CDCl3) δ 9.49 (br, 1H), 7.22 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0
Hz, 1H), 6.86 (t, JHF = 53.9 Hz, 1H), 6.54 (d, J = 7.9 Hz, 1H), 3.21
(s, 6H). Anal. Calcd for C10H11F2N3: C, 56.9; H, 5.25; N, 19.9. Found:
C, 57.0; H, 5.3; N, 20.1%.
A mixture of 8k (0.422 g, 2 mmol), 3 (0.564 g, 2.4 mmol), and
powdered K2CO3 (2.8 g, 20 mmol) in THF (20 mL) was stirred at room
temperature for 3 days and then diluted with water. Extraction with
CH2Cl2 followed by chromatography on silica, eluting with CH2Cl2/
EtOAc (49:1), gave 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-
2-(difluoromethyl)-4-(dimethylamino)-1H-benzimidazole (9k) (0.50 g,
61%): mp (CH2Cl2/MeOH) 200ꢀ201 °C; 1H NMR (CDCl3) δ 7.82
(dd, J = 8.3, 0.7 Hz, 1H), 7.48 (t, JHF = 53.7 Hz, 1H), 7.31 (t, J = 8.2 Hz,
1H), 6.94 (d, J = 7.8 Hz, 1H), 3.98ꢀ3.94 (m, 4H), 3.83ꢀ3.78 (m, 4H),
3.24 (s, 6H). Anal. Calcd for C17H18ClF2N7O: C, 49.8; H, 4.4; N, 23.9.
Found: C, 50.4; H, 4.4; N, 24.0%.
A mixture of 9k (0.10 g, 0.25 mmol) and morpholine (0.22 g, 2.5
mmol) in THF (10 mL) was stirred at room temperature for 30 min and
diluted with water to give 10k (97 mg, 84%): mp (CH2Cl2/MeOH)
231ꢀ233 °C; 1H NMR (CDCl3) δ 7.74 (dd, J = 8.3, 0.6 Hz, 1H), 7.46
(t, JHF = 53.7 Hz, 1H), 7.27 (t, J = 8.1 Hz, 1H), 6.61 (d, J = 8.0 Hz, 1H),
3.89ꢀ3.85 (m, 8H), 3.79ꢀ3.75 (m, 8H), 3.23 (s, 6H). Anal. Calcd for
C21H26F2N8O2: C, 54.8; H, 5.7; N, 24.3. Found: C, 54.8; H, 5.7;
N, 24.1%.
(CDCl3) δ 7.52 (d, J = 7.6 Hz, 1H), 7.32ꢀ7.27 (m, 2H), 7.25 (t, JHF
=
53.3 Hz, 1H), 5.19 (br s, 1H), 4.87 (s, 2H), 4.05 (br s, 1H).
A mixture of 8l (300 mg, 1.51 mmol) and TBDMSCl (456 mg,
3.2 mmol) in pyridine (5 mL) was stirred at room temperature for 3 h.
The mixture was diluted with water and extracted into EtOAc. After the
mixture was washed with water and dried with Na2SO4, the solvent
was removed. Chromatography of the residue on silica, eluting
with CH2Cl2/MeOH (99:1), gave 4-({[tert-butyl(dimethyl)silyl]
oxy}methyl)-2-(difluoromethyl)-1H-benzimidazole (8ad) (443 mg,
94%): 1H NMR (CDCl3) δ 10.22 (br, 1H), 7.73 (d, J = 8.2 Hz, 1H),
7.26 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 7.3 Hz, 1H), 6.89 (t, JHF = 54.0 Hz,
1H), 5.10 (s, 2H), 0.97 (s, 9H), 0.14 (s, 6H).
A mixture of 8ad (295 mg, 0.94 mmol), 3 (332 mg, 1.5 equiv), and
K2CO3 (389 mg, 3 equiv) in dry THF (10 mL) was stirred under reflux
for 20 h. The mixture was poured into water (100 mL), and the result-
ing precipitate was collected by filtration and dried. Chromatography
on silica, eluting with CH2Cl2/hexanes (3:1), gave 4-({[tert-butyl-
(dimethyl)silyl]oxy}methyl)-1-[4-chloro-6-(4-morpholinyl)-1,3,5-tria-
zin-2-yl]-2-(difluoromethyl)-1H-benzimidazole (9ad) (225 mg, 47%):
1H NMR (CDCl3) δ 8.29 (dd, J = 8.2, 0.6 Hz, 1H), 7.60 (dd, J = 7.6, 1.0
Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.56 (t, JHF = 53.5 Hz, 1H), 5.31
(s, 2H), 4.00ꢀ3.95 (m, 4H), 3.85ꢀ3.80(m, 4H), 0.99(s, 9H), 0.15(s, 6H).
A mixture of 9ad (220 mg, 0.43 mmol) and morpholine (0.5 mL,
excess) in THF (5 mL) was stirred at 20 °C for 18 h, and the mixture was
poured into water. The resulting precipitate was collected by filtration,
washed with water, and oven-dried to give 4-({[tert-butyl(dimethyl)-
silyl]oxy}methyl)-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazole (10ad) (241 mg, 100%): 1H NMR (CDCl3) δ
8.19 (d, J = 7.8 Hz, 1H), 7.56 (dd, J = 7.7, 1.1 Hz, 1H), 7.55 (t, JHF = 48.9
Hz, 1H), 7.45ꢀ7.41 (m, 1H). 5.32 (s, 2H), 3.88ꢀ3.82 (m, 8H),
3.80ꢀ3.77 (m, 8H), 0.99 (s, 9H), 0.16 (s, 6H).
To a solution of 10ad (241 mg, 0.43 mmol) in THF (10 mL) was
added TBAF (1 M in THF, 3 mL), and the reaction mixture was stirred
at 20 °C for 1 h and diluted with water. Chromatography of the resulting
precipitate on silica, eluting with CH2Cl2/MeOH (9:1), gave 10l (102
mg, 53%): mp (CH2Cl2/hexanes) 266ꢀ270 °C; 1H NMR (CDCl3) δ
8.24 (d, J = 7.6 Hz, 1 H), 7.55 (t, J = 49.9, 1 H), 7.40 (d, J = 8.4 Hz, 1 H),
7.33 (d, J = 6.6 Hz, 1 H), 5.17 (d, J = 6.3 Hz, 2 H), 3.89ꢀ3.87 (m, 8 H),
3.80ꢀ3.78 (m, 8 H), 3.25 (t, J = 6.4 Hz, 1 H). Anal. Calcd for
C20H23F2N7O3: C, 53.7; H; 5.2; N, 21.9. Found: C, 53.7; H, 5.2;
N, 22.0%.
Methyl 2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,
5-triazin-2-yl]-1H-benzimidazole-4-carboxylate (10m). A mix-
ture of 8ah (223 mg, 1.05 mmol), 2.2-dimethoxypropane, and concen-
tratedH2SO4 (1 mL) inMeOH (20 mL) was heatedunder reflux for 16 h,
and the solvent was concentrated. The residue was basified with aqueous
NH3 and extracted with CH2Cl2. After the mixture was dried with
Na2SO4, the solvent was evaporated to dryness, and the resulting
residue was chromatographed on silica, eluting with CH2Cl2 followed by
CH2Cl2/MeOH (99.5:0.5) to give methyl 2-(difluoromethyl)-1H-benzi-
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-(hydroxymethyl)-1H-benzimidazole (10l). A mixture of
2,3-diaminobenzoic acid 3.42 g, 18.13 mmol) and difluoroacetic acid
(6.96 g, 4.6 mL, 72.5 mmol) in 4 M HCl (20 mL) was refluxed for 16 h
and cooled to room temperature. The reaction mixture was diluted with
water (20 mL) and basified with aqueous NH3. The resulting clear
solution was filtered and neutralized with aqueous HOAc to produce a
precipitate, which was collected and dried to give 2-(difluoromethyl)-
1H-benzimidazole-4-carboxylic acid (8ah) (3.7 g, 96% yield): mp (aq
MeOH) 280ꢀ282 °C; 1H NMR (DMSO-d6) δ 13.39ꢀ13.10 (br, 2H),
8.01 (d, J = 8.0 Hz, 1H), 7.93 (dd, J = 7.6, 0.7 Hz, 1H), 7.40 (t, J = 7.7 Hz,
1H), 7.25 (JHF = 53.3 Hz, 1H). Anal. Calcd for C9H6F2N2O2: C, 51.0;
H; 2.9; F, 17.9; N, 13.2. Found: C, 50.9; H, 3.0, F, 18.2; N, 13.3%.
To a solution of 8ah (1.0 g, 4.71 mmol) in dry THF (20 mL) at 0 °C
was added LiAlH4 (268 mg, 7.05 mmol) in portions. The mixture was
stirred at this temperature for 5 min and then heated under reflux for 20
min. After the mixture was cooled to 20 °C, another portion of LiAlH4
(200 mg) was added, and the resulting mixture was heated under reflux
for 4 h. The mixture was cooled to 20 °C and slightly acidified by the
careful addition of dilute HCl. The mixture was extracted into EtOAc
and dried (Na2SO4). Removal of the solvents gave 2-(difluoromethyl)-
1
midazole-4-carboxylate (8m) (237 mg 100%): H NMR (CDCl3) δ
10.74 (br s, 1H), 8.04 (t, J = 7.7 Hz, 2H), 7.40 (t, J = 7.9 Hz, 1H), 6.90
(t, JHF = 53.7 Hz, 1H), 4.03 (s, 3H).
A mixture of 8m (237 mg, 1.05 mmol), 3 (296 mg, 1.26 mmol), and
powdered dry K2CO3 (290 mg, 2.15 mmol) in THF (20 mL) was stirred
at 20 °C for 16 h. The solvent was evaporated to dryness and the residue
was chromatographed on silica, eluting with CH2Cl2/EtOAc (9:1), to
give methyl 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoro-
methyl)-1H-benzimidazole-4-carboxylate (9m) (358 mg, 80%): mp
1
(CH2Cl2ꢀhexanes) 233ꢀ235 °C; H NMR (CDCl3) δ 8.69 (dd, J =
8.4, 1.1 Hz, 1H), 8.09 (dd, J = 7.7, 1.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H),
7.50 (t, JHF = 53.2 Hz, 1H), 4.05 (s, 3H), 4.00ꢀ3.95 (m, 4H), 3.84ꢀ3.80
(m, 4H). Anal. Calcd for C17H15ClF2N6O3: C, 48.1; H, 3.6; N, 19.8.
Found: C, 48.1; H, 3.4; N, 19.9%.
1
4-(hydroxymethyl)-1H-benzimidazole (8l) (306 mg, 33%): H NMR
7117
dx.doi.org/10.1021/jm200688y |J. Med. Chem. 2011, 54, 7105–7126

Journal of Medicinal Chemistry
ARTICLE
To a suspension of 9m 10 (337 mg, 0.79 mmol) in THF (30 mL) was
added morpholine (350 mg, 4 mmol), and the reaction mixture was
stirred at room temperature for 20 h. The solvent was concentrated, and
the residue was diluted with water. The resulting precipitate was
collected by filtration, washed with water and hexanes, and recrystallized
4-carboxamide (8p) in 28% yield: 1H NMR (CDCl3) δ 10.62 (br, 1H),
7.92 (d, J = 7.7 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.33 (dd, J = 8.1, 7.6 Hz,
1H), 6.85 (t, JHF =53.8 Hz, 1H), 3.25 (s, 6H).
Reaction of 8p with 3 as above gave 1-[4-chloro-6-(4-morpholinyl)-
1,3,5-triazin-2-yl]-2-(difluoromethyl)-N,N-dimethyl-1H-benzimida-
zole-4-carboxamide (9p) in 58% yield: 1H NMR (CDCl3) δ 8.47 (dd, J =
7.9, 1.6 Hz, 1 H), 7.48 (t, J = 53.3 Hz, 1 H, CHF2), 7.61ꢀ7.48 m, 2 H),
4.00ꢀ3.94 (m, 4 H), 3.84ꢀ3.80 (m, 4 H), 3.23 (s, 3 H), 2.92 (s, 3 H).
Reaction of 9p with morpholine gave 10p in 100% yield: mp
1
from CH2Cl2/hexanes to give 10m (361 mg, 96%): mp >290 °C; H
NMR (CDCl3) δ 8.55 (dd, J = 8.37, 1.14 Hz, 1H), 8.06 (dd, J = 7.64,
1.09 Hz, 1H), 7.50 (t, JHF = 53.3 Hz, 1H), 7.49 (t, J = Hz, 1H), 4.06
(s, 3H), 3.89ꢀ3.87 (m, 8H), 3.79ꢀ3.77 (m, 8H). Anal. Calcd for
C21H23F2N7O4: C, 53.1; H, 4.6; N, 20.6. Found: C, 53.0; H, 4.6;
N, 20.7%.
1
(CH2Cl2/hexanes) 262ꢀ266 °C; H NMR (CDCl3) δ 8.35 (quintet,
J = 3.6 Hz, 1H), 7.49 (t, JHF = 53.4 Hz, 1H), 7.47ꢀ7.45 (m, 2H),
3.89ꢀ3.87 (m, 8H), 3.80ꢀ3.78 (m, 8H), 3.23 (s, 3H), 2.93 (s, 3H).
Anal. Calcd for C21H24F2N8O3: C, 54.1; H, 5.4; N, 22.9. Found: C, 54.3;
H, 5.5; N, 23.0%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazole-4-carboxamide (10n). A mixture of
8ah (1.0 g, 4.7 mmol), thionyl chloride (10 mL), and a trace of DMF
was refluxed for 1 h and cooled to 20 °C, and the excess thionyl chloride
was removed under vacuum. The resulting residue was suspended in 1,4-
dioxane (10 mL) and cooled in ice. NH3 was bubbled through the
suspension, and the resulting mixture was stirred at room temperature
for 72 h and poured into water (50 mL). The resulting precipitate was
collected by filtration, washed with water, and dried to give 2-(difluoro-
methyl)-1H-benzimidazole-4-carboxamide (8n) (543 mg, 56% yield):
mp (H2O) 238ꢀ241 °C; 1H NMR (DMSO-d6) δ 13.85 and 13.03 (2 br
s, 1H), 7.89ꢀ7.25 (m, 6H). Anal. Calcd for C9H7F2N3O: C, 51.2; H,
3.3; N, 19.9. Found: C, 51.3; H, 3.5; H, 19.4%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-1H-benzimidazole-4-carbonitrile (10q). A mixture of 8n
(204 mg, 0.97 mmol) and thionyl chloride (1 mL) was refluxed for 20 h
and cooled to 20 °C, and the excess thionyl chloride was removed under
vacuum. The residue was treated with water, and the mixture was filtered
to remove an insoluble solid. The filtrate was neutralized with aqueous
NH3 and extracted into EtOAc. The solution was dried (Na2SO4), and
the solvent was removed. The resulting residue was chromatographed
on silica, eluting with CH2Cl2/EtOAc (17:3), to give 2-(difluoro-
methyl)-1H-benzimidazole-4-carbonitrile (8q) (101 mg, 54% yield):
Reaction of 8n and 3 as before gave 1-[4-chloro-6-(4-morpholinyl)-
1,3,5-triazin-2-yl]-2-(difluoromethyl)-1H-benzimidazole-4-carboxamide
(9n) in 78% yield: 1H NMR (DMSO-d6) δ 8.70 (br, 1H), 8.56 (dd, J =
1
mp (CH2Cl2/hexanes) 180ꢀ182 °C; H NMR (DMSO-d6) δ 10.03
(br, 1H), 7.97 (d, J =8.1 Hz, 1H), 7.82(dd, J = 7.5, 0.7Hz, 1H), 7.45 (t, J=
7.9, Hz, 1H), 7.36 (t, JHF = 53.1 Hz, 1H). Anal. Calcd for C9H5F2N3:
C, 56.0; H, 2.6, N, 21.8: Found: C, 56.2; H, 2.7; N, 21.9%.
8.4, 1.1 Hz, 1H), 8.09 (dd, J =7.6, 1.1Hz, 1H), 7.96 (br, 1H), 7.73(t, JHF
=
52.3 Hz, 1H), 7.70ꢀ7.67 (m, 1H), 3.90ꢀ3.86 (m, 4H), 3.78ꢀ3.72
A mixture of 8q (100 mg, 0.52 mmol), 3 (182 mg, 0.78 mmol), and
dry K2CO3 (214 mg, 1.56 mmol) in THF (10 mL) was refluxed for 20 h.
The resulting mixture was diluted with water and extracted into CH2Cl2.
Chromatography on silica, eluting with CH2Cl2/EtOAc (9:1), gave
1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoromethyl)-
1H-benzimidazole-4-carbonitrile (9q) (108 mg, 54% yield): mp (CH2Cl2/
hexanes) 215ꢀ218 °C; 1H NMR (CDCl3) δ 8.72 (dd, J = 8.5, 1.0 Hz,
1H), 7.78 (dd, J = 7.6, 1.0 Hz, 1H), 7.49 (t, JHF = 53.0 Hz, 1H), 7.57 (dd,
J = 8.4, 7.4 Hz, 1H), 4.01ꢀ3.99 (m, 2H), 3.96ꢀ3.94 (m, 2H), 3.85ꢀ3.81
(m, 4H). Anal. Calcd for C16H12ClF2N2O: C, 49.1; H, 3.1: N, 25.0.
Found: C, 49.3; H, 3.2; N, 25.1%.
A mixture of 9q (100 mg, 0.26 mmol) and morpholine (0.25 mL,
excess) in THF (10 mL) was stirred at room temperature for 3 h and
then diluted with water. The resulting precipitate was collected and
dried. Recrystallization from CH2Cl2/hexanes gave 10q (112 mg, 97%
yield): mp >300 °C; 1H NMR (DMSO-d6) δ 8.66 (dd, J = 8.4, 0.8 Hz,
1H), 7.97 (dd, J = 7.6, 0.9 Hz, 1H), 7.67 (dd, J = 8.3, 7.8 Hz, 1H), 7.76
(t, JHF = 52.4 Hz, 1 H), 3.81ꢀ3.0 (br m, 8H), 3.69 (br, 8H). Anal. Calcd
for C20H20F2N8O2: C, 54.3; H, 4.6; N, 25.33. Found: C, 54.5; H, 4.6;
N, 25.5%.
7-(Difluoromethyl)-6-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-6H-[1,3]dioxolo[4,5-e]benzimidazole (10r). A mixture of
6-bromo-4,5-dinitro-1,3-benzodioxole59 (2.25 g, 7.7 mmol) and Et3N
(1.2 g, excess) in MeOH (100 mL) was hydrogenated over 5% Pd on
carbon, and the mixture was filtered through Celite into methanolic HCl.
The solvent was evaporated to dryness, and the residue was combined
with difluoroacetic acid (3.7 g, 39 mmol) in 4 M HCl (20 mL). The
mixture was heated under reflux for 30 min before being cooled and
neutralized with aqueous NH3. The product was extracted into EtOAc
and dried. Chromatography on silica, eluting with CH2Cl2/EtOAc
(4:1), gave 7-(difluoromethyl)-6H-[1,3]dioxolo[4,5-e]benzimidazole
(8r) (1.23 g, 75%): mp (i-Pr2O) 149ꢀ150 °C; 1H NMR (DMSO-d6)
δ 13.33 (br, 1H), 7.22 (t, JHF = 53.2 Hz, 1H), 7.14ꢀ7.05 (m, 1H), 7.03
(d, J = 8.4 Hz, 1H), 6.11 (s, 2H). Anal. Calcd for C9H6F2N2O2: C, 50.95;
H, 2.85; N, 13.2. Found: C, 51.1; H, 2.9; N, 13.2%.
(m, 4H).
Reaction of 9n with morpholine gave 10n in 82% yield: mp (H2O)
>295 °C; 1H NMR (CDCl3) δ 9.36 (s, 1 H), 8.50 (dd, J = 8.3, 1.0 Hz,
1H), 8.29 (dd, J = 7.6, 1.0 Hz, 1H), 7.55 (t, JHF = 53.3 Hz, 1H), 7.77 (t, J =
8.0 Hz, 1H), 5.92 (s, 1H), 3.90ꢀ3.87 (m, 8H), 3.80ꢀ3.79 (m, 8H).
Anal. Calcd for C20H22F2N8O3: C, 52.2; H, 4.8, N, 24.3; F, 8.3%. Found:
C, 52.0; H, 4.8; N, 24.4; F, 8.4%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-N-methyl-1H-benzimidazole-4-carboxamide (10o). Si-
milar to the above, treatment of 8ah with thionyl chloride followed by
addition of dry methylamonium chloride and Et3N in 1,4-dioxane gave
2-(difluoromethyl)-N-methyl-1H-benzimidazole-4-carboxamide (8o)
1
in 75% yield: H NMR (DMSO-d6) δ 13.81 (br, 1H), 9.20 (br, 1H),
7.93 (d, J = 6.9 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 7.48ꢀ7.22 (m, 2H), 2.94
(s, 3H).
Reaction of 8o with 3 as before gave 1-[4-chloro-6-(4-morpholinyl)-
1,3,5-triazin-2-yl]-2-(difluoromethyl)-N-methyl-1H-benzimidazole-4-carbo-
xamide (9o) in 32% yield: mp (CH2Cl2/hexanes) 277ꢀ281; 1H NMR
(CDCl3) δ 9.29 (d, J = 3.4 Hz, 1 H), 8.56 (dd, J = 8.4, 1.1 Hz, 1 H), 8.35
(dd, J = 7.7, 1.1 Hz, 1 H), 7.58 (t, JHF = 53.2 Hz, 1H), 7.60 (t, J = 8.0 Hz,
1H), 4.00 (t, J = 4.9 Hz, 2H), 3.96 (t, J = 4.9 Hz, 2H), 3.86ꢀ3.81
(m, 4H), 3.11 (d, J = 4.8 Hz, 3H). Anal. Calcd for C17H16F2N7O2: C,
48.2; H, 3.8; N, 23.1. Found: C, 48.2; H, 3.9; N, 23.2%.
Reaction of 9o with morpholine gave 10o in 99% yield: mp (CH2Cl2/
hexanes) 291ꢀ293 °C; 1H NMR (CDCl3) δ 9.43 (q, J = 4.5 Hz, 1H,
NH), 8.45 (dd, J = 8.4, 1.0 Hz, 1H), 8.31 (dd, J = 7.6, 1.1 Hz, 1H), 7.55
(t, JHF = 53.3 Hz, 1H), 7.54 (dd, J = 10.2, 5.6 Hz, 1H), 3.90ꢀ3.87
(m, 8H), 3.80ꢀ3.79 (m, 8H), 3.15 (d, J = 4.8 Hz, 3H). Anal. Calcd for
C21H24F2N8O3: C, 53.2; H, 5.1; N, 23.6. Found: C, 53.1; H, 5.1;
N, 23.7%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-N,N-dimethyl-1H-benzimidazole-4-carboxamide (10p).
Similar to the above, treatment of 8ah with thionyl chloride fol-
lowed by addition of dry dimethylammonium chloride and Et3N in
1,4-dioxane gave 2-(difluoromethyl)-N,N-dimethyl-1H-benzimidazole-
7118
dx.doi.org/10.1021/jm200688y |J. Med. Chem. 2011, 54, 7105–7126

Journal of Medicinal Chemistry
ARTICLE
A mixture of 8r (1.06 g, 5 mmol), 3 (1.17 g, 5 mmol), and powdered
K2CO3 (3.5 g, 25 mmol) in DMF (50 mL) was stirred at room
temperature for 30 min and then diluted with water. The crude product
was collected by filtration, washed with water, and then with EtOH.
Recrystallization from CHCl3/EtOH gave 6-[4-chloro-6-(4-mor-
pholinyl)-1,3,5-triazin-2-yl]-7-(difluoromethyl)-6H-[1,3]dioxolo[4,5-
e]benzimidazole (9r) (1.65 g, 80%); mp >300 °C; 1H NMR (CDCl3) δ
7.95 (d, J = 8.8 Hz, 1H), 7.50 (t, JHF = 53.3 Hz, 1H), 7.04 (d, J = 8.8 Hz,
1H), 6.16 (s, 2H), 3.99ꢀ3.96 (m, 2H), 3.95ꢀ3.92 (m, 2H), 3.84ꢀ3.79
(m, 4H). Anal. Calcd for C16H13ClF2N6O3: C, 46.8; H, 3.2; N, 20.5.
Found: C, 47.1; H, 3.3; N, 20.5%.
benzenesulfonate (2.9 g, 90% yield): mp (H2O) 140ꢀ142 °C; 1H NMR
(CDCl3) δ 10.13 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H), 7.94 (dd, J = 8.5, 1.7
Hz, 2H), 7.76ꢀ7.71 (m, 1H), 7.62ꢀ7.58 (m, 2H), 7.07 (d, J = 9.3 Hz,
1H), 3.75 (s, 3H). Anal. Calcd for C13H12N2O6S: C, 48.2; H, 3.7; N, 8.6.
Found: C, 48.3; H, 3.9; N, 8.7%.
A solution of the above compound (4.0 g, 12.33 mmol) in aqueous 2
N NaOH (20 mL) and EtOH (20 mL) was refluxed for 30 min, and the
EtOH was removed. The resulting aqueous solution was neutralized
with concentrated HCl to give 2-amino-6-methoxy-3-nitrophenol (2.04
g, 90%) yield: 1H NMR (CDCl3) δ 7.79 (d, J = 9.6 Hz, 1H), 6.36 (d, J =
9.65 Hz, 1H), 6.14 (br, 2H), 5.52 (s, 1H), 3.96 (s, 3H).
A mixture of 9r (0.206 g, 0.5 mmol) and morpholine (0.22 g, 2.5
mmol) in THF (20 mL) was heated at 50 °C for 30 min and cooled.
Dilution with water gave 10r (0.23 g, 99%): mp (CH2Cl2/MeOH) >
300 °C; 1H NMR (CDCl3) δ 7.83 (d, J = 8.7 Hz, 1H), 7.49 (t, JHF = 53.4
Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.13 (s, 2H), 3.88ꢀ3.84 (m, 8H),
3.81ꢀ3.76 (m, 8H). Anal. Calcd for C20H21F2N7O4: C, 52.1; H, 4.6; N,
21.25. Found: C, 52.2; H, 4.7; N, 21.5%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4,5-dimethoxy-1H-benzimidazole (10s). A solution of 3,
4-dimethoxy-2-nitroaniline60 (0.92 g, 4.6 mmol) in MeOH (100 mL) was
hydrogenated over Pd on carbon, and the solution was filtered through
Celite and acidified with concentrated HCl. The solvent was evaporated
to dryness and the residue was combined with difluoroacetic acid (0.89
g, 2 equiv) in 4 M HCl (10 mL). The mixture was heated under reflux for
2 h, cooled, and neutralized with aqueous NH3. Extraction with EtOAc,
followed by chromatography on silica, eluting with CH2Cl2/EtOAc
(4:1), gave 2-(difluoromethyl)-4,5-dimethoxy-1H-benzimidazole (8s)
(0.97 g, 92% yield): mp (i-Pr2O) 110ꢀ111 °C; 1H NMR (DMSO-d6) δ
13.13 (br, 1H), 7.30ꢀ7.20 (m, 1H), 7.20 (t, JHF = 53.2 Hz, 1H), 7.09 (d,
J = 8.8 Hz, 1H), 4.08ꢀ3.99 (m, 3H), 3.82 (s, 3H). Anal. Calcd For
C10H10F2N2O2: C, 52.6; H, 4.4; N, 12.3. Found: C, 52.9; H, 4.5; N,
12.25%.
The above compound (2.0 g, 0.87 mmol) was hydrogenated over Pd/
C in MeOH. This was filtered into a solution of concentrated HCl
(3 mL) in MeOH (10 mL) and evaporated to dryness. The resulting
residue was refluxed in 4 N HCl (20 mL) and difluoroacetic acid (5 mL)
for 20 h. The reaction mixture was diluted with H2O, neutralized with
aqueous NH3, and stirred overnight. The resulting precipitate was
collected, washed with H2O, and recrystallized from aqueous MeOH
to give 2-(difluoromethyl)-4-hydroxy-5-methoxy-1H-benzimidazole
(8t) (2.29 g, 98% yield), which was used directly.
A mixture of 8t (2.29 g, 10.6 mmol) and TBDMSCl (4.79 g, 3 equiv)
in pyridine (10 mL) was stirred at 20 °C for 4 days, diluted with water,
extracted in EtOAc (3 ꢁ 50 mL), and dried (Na2SO4), and the solvent
was evaporated. The residue was chromatographed on silica, eluting with
CH2Cl2/EtOAc (9:1) to give a viscous oil, which was stirred in hexanes
to give 4-{[tert-butyl(dimethyl)silyl]oxy}-2-(difluoromethyl)-5-meth-
1
oxy-1H-benzimidazole (8ab) (1.96 g, 56% yield) as a solid: H NMR
(CDCl3) δ 9.56 and 9.20 (2 br, 1H, NH), 7.38 (d, J = 8.8 Hz, 1H),
7.06ꢀ7.99 (m, 1H), 6.84 (t, JHF = 54.0 Hz, 1H), 3.88 and 3.85 (2s, 3H),
1.08 and 1.06 (2s, 9H), 0.24 and 0.22 (2s, 6H).
Reaction of 8ab with 3 gave 4-{[tert-butyl(dimethyl)silyl]oxy}-1-
[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoromethyl)-5-methoxy-
1H-benzimidazole (9ab) which was used without further purification.
Reaction with morpholine in THF gave 4-{[tert-butyl(dimethyl)-
silyl]oxy}-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-
yl]-5-methoxy-1H-benzimidazole (10ab) in 68% yield over two steps:
1H NMR (CDCl3) δ 7.83 (d, J = 8.9 Hz, 1H), 7.45 (t, JHF = 53.6 Hz,
1H), 7.07 (d, J = 8.9 Hz, 1H), 3.89ꢀ3.86 (m, 8H), 3.87 (s, 3H),
3.78ꢀ3.77 (m, 8H), 1.06 (s, 9H), 0.26 (s, 6H).
A mixture of 8s (0.57 g g, 2.5 mmol), 3 (0.59 g, 2.5 mmol), and
powdered K2CO3 (1.7 g, 12.5 mmol) in DMF (35 mL) was stirred at
room temperature for 30 min and then diluted with water. The crude
product was collected by filtration, washed with water and then with
EtOH, and dried. Chromatography on silica, eluting with CH2Cl2/
EtOAc (9:1), gave 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-
2-(difluoromethyl)-4,5-dimethoxy-1H-benzimidazole (9s) (0.71 g,
Deprotection of 10ab with TBAF in THF gave 10t in 92% yield: mp
(CH2Cl2/MeOH) 294ꢀ300 °C; 1H NMR (DMSO-d6) δ 9.67 (br, 1H),
7. 70 (d, J = 8.0 Hz, 1H), 7.70 t, JHF = 53.0 Hz, 1H), 7.18 (d, J = 9.0 Hz,
1H), 3.82 (s, 3H), 3.81ꢀ3.79 (m, 8H), 3.69 (br, 8H). Anal. Calcd for
C20H23F2N7O4: C, 51.8; H, 5.0; N, 21.2. Found: C, 52.1; H, 5.0;
N, 21.2%
1
67%): mp (CH2Cl2/EtOH) 209ꢀ211 °C; H NMR (CDCl3) δ 8.02
(d, J = 9.0 Hz, 1H), 7.49 (t, JHF = 53.4 Hz, 1H), 7.13 (d, J = 9.0 Hz, 1H),
4.35 (s, 3H), 3.99ꢀ3.92 (m, 4H), 3.94 (s, 3H), 3.84ꢀ3.78 (m, 4H).
Anal. Calcd For C17H17ClF2N6O3: C, 47.8; H, 4.0; N, 19.7. Found: C,
47.8; H, 3.9; N, 19.9%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-5-hydroxy-4-methoxy-1H-benzimidazole (10u). A solu-
tion of 4-(benzyloxy)-3-methoxy-2-nitrobenzaldehyde62 (2.87 g, 10 mmol)
in a mixture of acetone (110 mL) and DMSO (45 mL) was combined
with a solution of sulfamic acid (1.65 g, 17 mmol) in water (33 mL), and
the resulting mixture was cooled to 0 °C. A solution of NaClO2 (1.75 g,
19 mmol) in water (75 mL) was added slowly with stirring, and after a
further 30 min at 0 °C, the mixture was diluted with water and extracted
with EtOAc. Removal of the solvent gave 4-(benzyloxy)-3-methoxy-2-
nitrobenzoic acid63 (2.93 g, 97%): 1H NMR (DMSO-d6) δ 13.75
(br, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.51 (br d, J = 8.4 Hz, 2H),
7.47ꢀ7.41 (m, 3H), 7.40ꢀ7.35 (m, 1H), 5.33 (s, 2H), 3.84 (s, 3H).
A mixture of the above crude acid (1 g, 3.3 mmol) and DPPA (1.0 g, 4
mmol) in t-BuOH (2.5 g, 33 mmol) was heated under reflux for 1 h.
After cooling, the mixture was diluted with water, and bascified with aq.
NH3 to give a solid, which was collected and dried. Chromatography on
silica, eluting with CH2Cl2/hexanes (3:1), gave tert-butyl 4-(benzyloxy)-
3-methoxy-2-nitrophenylcarbamate (0.68 g, 55%): mp (aqueous MeOH)
94ꢀ96 °C; 1H NMR (CDCl3) δ 7.67 (d, J = 9.3 Hz, 1H), 7.43ꢀ7.31
A mixture of 9s (0.213 g, 5 mmol) and morpholine (0.217 g, 25
mmol) in THF (10 mL) was heated to 50 °C for 30 min and diluted with
water to give 10s (0.23 g, 96%): mp (CH2Cl2/MeOH) 242ꢀ245 °C; 1H
NMR (CDCl3) δ 7.92 (d, J = 9.0 Hz, 1H), 7.49 (t, JHF = 53.5 Hz, 1H),
7.11 (d, J = 9.0 Hz, 1H), 4.34 (s, 3H), 3.93 (s, 3H), 3.88ꢀ3.85 (m, 8H),
3.79ꢀ3.76 (m, 8H). Anal. Calcd for C21H25F2N7O4: C, 52.8; H, 5.3; N,
20.5. Found: C, 52.9; H, 5.3; N, 20.5%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-hydroxy-5-methoxy-1H-benzimidazole (10t). 3-Meth-
oxy-6-nitro-2-[(phenylsulfonyl)oxy]benzoic acid61 (3.5 g, 9.9 mmol) in
SOCl2 (10 mL) with a trace of DMF was refluxed for 1 h and cooled to
20 °C, and the excess SOCl2 was removed under vacuum. The resulting
solid residue was dissolved in acetone (15 mL) and added slowly to a
solution of NaN3 (2 g, 30.8 mmol) in H2O (10 mL) at 0 °C. The
reaction mixture was stirred for 20 h and cooled to 0 °C, and the
resulting precipitate was collected by filtration. The solid was suspended
in aqueous HOAc (72%) (10 mL), and the mixture was refluxed for 2 h,
cooled, and diluted with H2O. The resulting precipitate was filtered,
washed with water and dried to give 2-amino-6-methoxy-3-nitrophenyl
7119
dx.doi.org/10.1021/jm200688y |J. Med. Chem. 2011, 54, 7105–7126

Journal of Medicinal Chemistry
ARTICLE
(m, 5H), 7.13 (br, 1H), 7.06 (d, J = 9.3 Hz, 1H), 5.12 (s, 2H), 4.00
(s, 3H), 1.49 (s, 9H). Anal. Calcd for C19H22N2O6: C, 60.95; H, 5.9; N,
7.5. Found: C, 61.0; H, 5.8; N, 7.6%.
dryness. The residue was dissolved in dry acetone (65 mL) and
combined with a solution of NaN3 (13 g) in water (25 mL) with rapid
stirring. After 10 min the mixture was poured into water and the pre-
cipitate was collected and washed with water. The solid was suspended
in a mixture of acetic acid (400 mL) and water (40 mL), and the resulting
mixture was heated slowly to reflux and maintained at that temperature
for 2 h. After cooling, the mixture was neutralized with aqueous NH3 to
give 3,5-dimethoxy-2-nitroaniline (6.06 g, 87%): mp (aqueous i-PrOH)
112ꢀ114 °C; 1H NMR (CDCl3) δ 5.58 (d, J = 2.5 Hz, 1H), 5.79 (d, J =
2.5 Hz, 1H), 5.52 (br, 2H), 3.86 (s, 3H), 3.80 (s, 3H). Anal. Calcd for
C8H10N2O4: C, 48.5; H, 5.1; N, 14.1. Found: C, 48.65; H, 5.2; N, 14.3%.
A solution of the above compound (1.98 g, 10 mmol) in MeOH
(100 mL) was hydrogenated over 5% Pd on carbon and filtered through
Celite into a mixture of MeOH and concentrated HCl. The solvents
were removed under vacuum and the residue was combined with
difluoracetic acid (1.92 g, 20 mmol) in 4 M HCl (20 mL). The mixture
was heated under reflux for 2 h, cooled, and neutralized with aqueous
NH3. Extraction with EtOAc, followed by chromatography on silica,
eluting with CH2Cl2/EtOAc (4:1), gave 2-(difluoromethyl)-4,6-di-
methoxy-1H-benzimidazole (8v) (1.83 g, 80%): mp (aqueous MeOH)
165ꢀ167 °C; 1H NMR (DMSO-d6) δ 13.12 (br, 1H), 7.14 (t, JHF = 53.4
Hz, 1H), 6.65ꢀ6.57 (m, 1H), 6.44ꢀ6.38 (m, 1H), 3.91 (m, 3H), 3.79 (s,
3H). Anal. Calcd for C10H10F2N2O2: C, 52.6; H, 4.4; N, 12.3. Found: C,
52.85; H, 4.45; N, 12.3%.
A mixture of 2-(difluoromethyl)-4,6-dimethoxy-1H-benzimidazole
(8v) (0.57 g g, 2.5 mmol), 3 (0.59 g, 2.5 mmol), and powdered
K2CO3 (1.7 g, 12.5 mmol) in DMF (35 mL) was stirred at room
temperature for 30 min and then diluted with water. The crude product
was collected by filtration and washed with water and then with EtOH to
give 1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoromethyl)-
4,6-dimethoxy-1H-benzimidazole (9v) (0.79 g, 74%): mp (CH2Cl2/
EtOH) 261ꢀ263 °C; 1H NMR (CDCl3) δ 7.58 (d, J = 2.1 Hz, 1H), 7.40
(t, JHF = 53.6 Hz, 1H), 6.49 (d, J = 2.1 Hz, 1H), 4.01 (s, 3H), 3.99ꢀ3.93
(m, 4H), 3.89 (s, 3H), 3.83ꢀ3.78 (m, 4H). Anal. Calcd for
C17H17ClF2N6O3: C, 47.8; H, 4.0; N, 19.7. Found: C, 47.8; H, 3.9;
N, 19.9%.
A mixture of the above chloro compound (0.213 g, 5 mmol) and
morpholine (0.217 g, 25 mmol) in THF (10 mL) was heated to 50 °C
for 30 min and diluted with water to give 10v (0.215 g, 90%): mp
(CH2Cl2/MeOH) 296ꢀ299 °C; 1H NMR (CDCl3) δ 7.49 (d, J = 2.2
Hz, 1H), 7.44 (t, JHF = 53.7 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 4.02
(s, 3H), 3.92ꢀ3.87 (m, 8H), 3.86 (s, 3H), 3.80ꢀ3.77 (m, 8H). Anal.
Calcd for C21H25F2N7O4: C, 52.8; H, 5.3; N, 20.5. Found: C, 52.95; H,
5.3; N, 20.5%.
6-Amino-2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,
5-triazin-2-yl]-4-methoxy-1H-benzimidazole (10w). A mixture
of 2,3-diamino-5-nitroanisole65 (1.10 g, 6 mmol) and difluoroacetic acid
(2.31 g, 24 mmol) in polyphosphoric acid (PPA) (50 g) was heated at
130 °C in an oil bath for 1 h. The hot solution was poured into water and,
with cooling, the pH was adjusted to neutral with aqueous NH3 to
give 2-(difluoromethyl)-4-methoxy-6-nitro-1H-benzimidazole (1.33 g,
91%): mp (EtOH/H2O) 192ꢀ194 °C; 1H NMR (DMSO-d6) δ 14.18
(br, exchangeable with D2O, 1H), 8.18 (br, 1H), 7.65 (dd, J = 1.4 Hz,
1H), 7.30 (t, JHF = 52.9 Hz, 1H), 4.07 (s, 3H). Anal. Calcd for
C9H7F2N3O3: C, 44.45; H, 2.9; N, 17.3. Found: C, 44.75; H, 3.0;
N, 17.3%.
Treatment of the above carbamate with TFA in CH2Cl2 gave
1
4-(benzyloxy)-3-methoxy-2-nitroaniline: mp (i-Pr2O) 70ꢀ71 °C; H
NMR (CDCl3) δ 7.42ꢀ7.35 (m, 5H), 6.94 (d, J = 9.0 Hz, 1H), 6.42
(d, J = 9.0 Hz, 1H), 5.03 (s, 2H), 4.48 (br, 1H), 4.00 (s, 3H). Anal. Calcd
for C14H14N2O4: C, 61.3; H, 5.1; N, 10.2. Found: C, 61.4; H, 5.2; N,
10.15%.
A solution of the above compound (1.10 g, 4 mmol) in MeOH
(100 mL) was hydrogenated over Pd on carbon, and the mixture was
filtered through Celite into a mixture of MeOH and concentrated HCl.
The solution was evaporated to dryness, and the residue was combined
with difluoroacetic acid (3.84 g, 40 mmol) and 4 M HCl (20 mL) and
heated under reflux for 2 h. After cooling, the mixture was bascified with
aqueous NH3 and extracted with EtOAc. Chromatography on silica,
eluting with CH2Cl2/EtOAc (7:3), gave 2-(difluoromethyl)-5-hydroxy-
1
4-methoxy-1H-benzimidazole (8u) (0.7 g, 82%) as an oil: H NMR
(DMSO-d6) δ 13.10 (br, 1H), 9.18 and 8.61 (2 br, 1H), 7.35ꢀ6.96
(m, 2H), 6.87 (d, J = 8.5 Hz, 1H), 4.07 and 3.89 (2 br, 3H).
The above crude 8u (0.7 g, 3.3 mmol) was combined with TBDMSCl
(0.84 g, 5.6 mmol) in pyridine (10 mL), and the resulting mixture was
stirred at room temperature overnight. After removal of the pyridine
under vacuum, the residue was diluted with water and extracted with
CH2Cl2. Chromatography on silica, eluting with CH2Cl2/EtOAc (95:5),
gave 5-{[tert-butyl(dimethyl)silyl]oxy}-2-(difluoromethyl)-4-methoxy-
1H-benzimidazole (8ac) (0.335 g, 31%) as an oil: 1H NMR (CDCl3) δ
10.5ꢀ9.0 (br 1H), 7.29ꢀ7.20 (m, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.86
(t, JHF = 53.9 Hz, 1H), 4.05 (br, 3H), 1.03 (s, 9H), 0.20 (s, 6H).
A mixture of crude 8ac (0.33 g, 1 mmol), 3 (0.23 g, 1 mmol), and
powdered K2CO3 (0.56 g, 4 mmol) in THF (20 mL) was stirred at room
temperature for 5 days and diluted with water. The product was
extracted in to CH2Cl2 and dried. Chromatography on silica, eluting
with CH2Cl2/EtOAc (97:3), gave 5-{[tert-butyl(dimethyl)silyl]oxy}-
1-[4-chloro-6-(4-morpholinyl)-1,3,5-triazin-2-yl]-2-(difluoromethyl)-
4-methoxy-1H-benzimidazole (9ac) (0.26 g, 49%): mp (hexanes)
1
140ꢀ142 °C; H NMR (CDCl3) δ 7.93 (d, J = 8.9 Hz, 1H), 7.48
(t, JHF = 53.5 Hz, 1H), 7.03 (d, J = 8.9 Hz, 1H), 4.26 (s, 3H), 3.99ꢀ3.92
(m, 4H), 3.84ꢀ3.78 (m, 4H), 1.03 (s, 9H), 0.19 (s, 6H). Anal. Calcd for
C22H29ClF2N6O3Si: C, 50.1; H, 5.6; N, 15.95. Found: C, 50.15; H, 5.6;
N, 16.1%.
A mixture of 9ac (0.21 g, 0.40 mmol) and morpholine (0.35 g, 4.0
mmol) in THF (10 mL) was stirred at room temperature for 30 min and
diluted with water to give 5-{[tert-butyl(dimethyl)silyl]oxy}-2-(difluoro-
methyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-4-methoxy-1H-ben-
zimidazole (10ac) (0.22 g, 97%): mp (hexanes) 212ꢀ214 °C; 1H NMR
(CDCl3) δ7.83(d, J=8.9 Hz, 1H), 7.48 (t, JHF =53.6 Hz, 1H), 6.99 (d, J=
8.9 Hz, 1H), 4.25 (s, 3H), 3.88ꢀ3.84 (m, 8H), 3.80ꢀ3.76 (m, 8H), 1.03
(s, 9H), 0.19 (s, 6H). Anal. Calcd for C26H37F2N7O4Si: C, 54.1; H, 6.5;
N, 17.0. Found: C, 54.1; H, 6.6; N, 17.1%.
A solution of the above compound (0.14 g, 0.35 mmol) in THF
(5 mL) was treated with a slight excess of Bu4NF in THF at room
temperature for 5 min. The solvent was removed under vacuum and the
residue was diluted with water to give a solid, which was collected by
filtration and recrystallized from MeOH, to give 10u (76 mg, 68%): mp
(MeOH) 253ꢀ256 °C; 1H NMR (CDCl3) δ 7.87 (d, J = 8.9 Hz, 1H),
7.49 (t, JHF = 53.5 Hz, 1H), 7.07 (d, J = 8.9 Hz, 1H), 5.66 (s, 1H), 4.42 (s,
3H), 3.88ꢀ3.84 (m, 8H), 3.79ꢀ3.75 (m, 8H). Anal. Calcd for
C20H23F2N7O4: C, 51.8; H, 5.0; N, 21.2. Found: C, 51.9; H, 4.9;
N, 21.0%.
A solution of the above compound (1.22 g, 5 mmol) in MeOH
(50 mL) was hydrogenated over 10% Pd on carbon (50 mg). After
filtration to remove the catalyst, the solution was evaporated to dryness.
The residue was combined with di-tert-butyl dicarbonate (3.2 g, 15
mmol) in dioxane (20 mL), and the mixture was heated under reflux for
5 h. The solvent was removed under vacuum, and the residue was
dissolved in MeOH (30 mL) containing aqueous NaOH (2 M, 12.5 mL,
5 equiv). The mixture was stirred at room temperature for 1 h,
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4,6-dimethoxy-1H-benzimidazole (10v). A solution of 3,
5-dimethoxy-2-nitrobenzoic acid64 (7.95 g, 35 mmol) in SOCl2 (100 mL)
was heated under reflux for 2 h, and the solvent was evaporated to
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Journal of Medicinal Chemistry
ARTICLE
neutralized with HOAc, and evaporated to dryness. The residue was
extracted with EtOAc, washed with NaHCO3 solution, and dried over
Na2SO4. Chromatography on silica, eluting with CH2Cl2/EtOAc (9:1),
gave 1.54 g (98% yield) of tert-butyl 2-(difluoromethyl)-4-methoxy-1H-
benzimidazol-6-ylcarbamate (8af): mp (i-Pr2O) 189ꢀ191 °C; 1H NMR
(DMSO-d6) δ 13.0 (br, exchangeable with D2O, 1H), 9.31 (br s,
exchangeable with D2O, 1 H), 7.42 (br s, 1H), 7.15 (t, JHF = 53.4 Hz,
1H), 6.90 (br, 1H), 3.90 (s, 3H), 1.49 (s, 9H). Anal. Calcd for
C14H17F2N3O3: C, 53.7; H, 5.5; N, 13.4. Found: C, 53.9; H, 5.6;
N, 13.4%.
A mixture of 8af (0.47 g, 1.5 mmol), 3 (0.35 g, 1.5 mmol), and
powdered K2CO3 (0.83 g, 6 mmol) in DMF (10 mL) was stirred at room
temperature for 30 min. The reaction mixture was then diluted with
water. The resulting precipitate was collected, washed with water and
then MeOH, and dried to give tert-butyl 1-[4-chloro-6-(4-morpholinyl)-
1,3,5-triazin-2-yl]-2-(difluoromethyl)-4-methoxy-1H-benzimidazol-
6-ylcarbamate (9af) (0.45 g, 59% yield): mp (CH2Cl2/MeOH) > 300 °C;
1H NMR (CDCl3) δ 8.45 (d, J = 0.6 Hz, 1H), 7.57 (t, JHF = 53.6 Hz,
1H), 6.67 (br, exchangeable with D2O, 1H), 6.63 (d, J = 0.9 Hz, 1H),
4.11 (m, 2H), 4.02 (s, 3H), 3.97 (m, 2H), 3.88 (m, 2H), 3.82 (m, 2H),
1.52 (s, 9H). Anal. Calcd for C21H24ClF2N7O4: C, 49.3; H, 4.7; N,
19.15. Found: C, 49.4; H, 4.8; N, 19.2%.
2-(Difluoromethyl)-6-(dimethylamino)-1-(4,6-dimorpho-
lino-1,3,5-triazin-2-yl)-4-methoxy-1H-benzimidazole (10y).
A solution of 10x (0.163 g, 0.34 mmol) in CH2Cl2 (30 mL) was treated
with an excess of acetic formic anhydride at room temperature for 3 h.
Dilution with water and recrystallization from CH2Cl2/MeOH
gave 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-
4-methoxy-1H-benzimidazol-6-yl(methyl)formamide (0.16 g, 93%):
mp 255ꢀ257 °C; 1H NMR (CDCl3) δ 8.54 (s, 1H), 7.77 (d, J = 1.9 Hz,
1H), 7.46 (t, JHF = 53.4 Hz, 1H), 6.63 (d, J = 1.9 Hz, 1H), 4.07 (s, 3H),
3.89ꢀ3.85 (m, 8H), 3.79ꢀ3.76 (m, 8H), 3.38 (s, 3H). Anal. Calcd for
C22H26F2N8O4: C, 52.4; H, 5.2; N, 22.2. Found: C, 52.4; H, 5.5;
N, 22.3%.
A suspension of the above compound (151 mg, 0.3 mmol) and
(MeO)3B (186 mg, 1.8 mmol) in THF (100 mL) was treated with
BH3 SMe2 (136 mg, 1.8 mmol), and the mixture was diluted with
3
CH2Cl2 until a clear solution was obtained. The solution was stirred at
room temperature overnight, and MeOH (1 mL) was added. The
solvents were evaporated to dryness, and water was added. The solid
was collected, washed with aqueous NaHCO3 and dried. Chromatog-
raphy on silica, eluting with CH2Cl2/EtOAc (3:2), gave 10y (116 mg,
79%); mp (CH2Cl2/MeOH) 280ꢀ282 °C; 1H NMR (CDCl3) δ 7.42
(t, JHF = 53.8 Hz, 1H), 7.27 (d, J = 2.1 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H),
4.03 (s, 3H), 3.92ꢀ3.85 (m, 8H), 3.78ꢀ3.74 (m, 8H), 3.02 (s, 6H).
Anal. Calcd for C22H28F2N8O3: C, 53.9; H, 5.75; N, 22.8. Found: C,
54.15; H, 5.8; N, 22.9%.
A mixture of 9af (205 mg, 0.4 mmol) and morpholine (0.35 g, 4.0
mmol) in THF (20 mL) was stirred at room temperature for 1 h and
diluted with water to give tert-butyl 2-(difluoromethyl)-1-[4,6-di(4-
morpholinyl)-1,3,5-triazin-2-yl]-4-methoxy-1H-benzimidazol-6-ylcar-
bamate (10af) (0.22 g, 98%): 1H NMR (CDCl3) δ 8.63 (br s, 1H), 7.48
(t, JHF = 53.6 Hz, 1H), 6.60 (br s, 1H), 6.36 (d, J = 1.6 Hz, 1H), 3.99
(s, 3H), 3.98ꢀ3.91 (m, 4H), 3.90ꢀ3.84 (m, 4H), 3.83ꢀ3.75 (m, 8H),
1.51 (s, 9H).
2-(Difluoromethyl)-3-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-7-methoxy-3H-imidazo[4,5-c]pyridine (13). A solution of
4-amino-3-methoxy-5-nitropyridine40 (1.08 g, 6.4 mmol) in MeOH
(50 mL) was hydrogenated over 5% Pd on C, and after filtration to
remove the catalyst the solution was acidified with concentrated HCl.
The solvent was removed under vacuum, and the residue was combined
with difluoroacetic acid (3.1 g, 32 mmol) in PPA (40 g). The mixture was
stirred at 100 °C for 1 h and poured into water. The solution was
neutralized with aqueous NH3 and extracted with EtOAc to give
2-(difluoromethyl)-7-methoxy-3H-imidazo[4,5-c]pyridine (11) (0.87
g, 68% yield): mp (EtOH) 234ꢀ238 °C; 1H NMR (CDCl3) δ
14.0ꢀ12.0 (br, 1H), 8.69 (s, 1H), 6.04 (s, 1H), 7.22 (t, JHF = 53.3
Hz, 1H), 4.06 (s, 3H). Anal. Calcd for C8H7F2N3O: C, 48.25; H, 3.5; N,
21.1. Found: C, 48.15; H, 3.4; N, 20.9%.
A mixture of 11 (0.21 g, 1 mmol), 3 (0.32 g, 1.37 mmol), and
powdered K2CO3 (0.69 g, 5 mmol) in t-BuOH (25 mL) was heated
under reflux for 5 h and cooled to room temperature. Morpholine (0.87
g, 10 mmol) was added, and the resulting mixture was stirred at room
temperature for 1 h before being diluted with water. Extraction with
CH2Cl2, followed by chromatography on alumina, eluting with CH2Cl2/
EtOAc (3:1), gave 2-(difluoromethyl)-3-[4,6-di(4-morpholinyl)-1,3,
5-triazin-2-yl]-7-methoxy-3H-imidazo[4,5-c]pyridine (13) (58 mg, 13%
yield): mp (CH2Cl2/MeOH) 262ꢀ264 °C; 1H NMR (CDCl3) δ 9.88
(d, J = 0.8 Hz, 1H), 8.72 (d, J = 1.4 Hz, 1H), 6.92 (t, JHF = 54.5 Hz, 1H),
4.22 (s, 3H), 3.96ꢀ3.87 (m, 8H), 3.82ꢀ3.76 (m, 8H). Anal. Calcd for
C19H22F2N8O3: C, 50.9; H, 4.9; N, 25.0. Found: C, 51.2; H, 4.9;
N, 25.0%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-2-pyrimidinyl]-
1H-benzimidazole (16a). A solution of 2 (0.84 g, 5 mmol) in DMF
(25 mL) was treated with 60% NaH (0.24 g, 6 mmol), and the mixture
was stirred at room temperature for 30 min. 2,4,6-Trichloropyrimidine
(14) (0.92 g, 5 mmol) was added, and the resulting mixture was stirred
for 2 h. Dilution with water gave a solid which was collected and dried.
Chromatography on silica, eluting with CH2Cl2/hexanes (4:1), gave
mixed fractions which were recrystallized from CH2Cl2ꢀhexanes to give
1-(4,6-dichloro-2-pyrimidinyl)-2-(difluoromethyl)-1H-benzimidazole38
(15a) (0.54 g, 34%): mp 131ꢀ132 °C; 1H NMR (CDCl3) δ 8.48 (br d, J
= 8.3 Hz, 1H), 7.92 (br d, J = 7.6 Hz, 1H), 7.67 (t, JHF = 53.5 Hz, 1H),
7.52 (td, J = 8.3, 1.3 Hz, 1H), 7.46 (td, J = 7.9, 1.3 Hz, 1H), 7.35 (s, 1H).
Treatment of 10af with TFA in CH2Cl2, followed by chromatography
on silica, eluting with CH2Cl2/EtOAc (3:2), gave 10w (88% yield): mp
1
(MeOH) 277ꢀ280 °C; H NMR (CDCl3) δ 7.39 (t, JHF = 53.7 Hz,
1H), 7.18 (d, J = 1.8 Hz, 1H), 6.21 (d, J = 1.8 Hz, 1H), 3.98 (s, 3H),
3.88ꢀ3.84 (m, 8H), 3.82 (m, exchangeable with D2O, 2H), 3.78ꢀ3.75
(m, 8H). Anal. Calcd for C20H24F2N8O3: C, 51.9; H, 5.2; N, 24.2.
Found: C, 52.1; H, 5.3; N, 24.5%.
Methanesulfonate: mp (MeOH/EtOAc) 260ꢀ264 °C; 1H NMR
(DMSO-d6) δ 10.50ꢀ8.00 (br, exchangeable with D2O, 2H), 7.71 (d, J
= 1.2 Hz, 1H), 7.67 (t, JHF = 52.9 Hz, 1H), 6.77 (d, J = 1.5 Hz, 1H), 3.97
(s, 3H), 3.83ꢀ3.78 (m, 8H), 3.73ꢀ3.67 (m, 8H), 2.34 (s, 3H). Anal.
Calcd for C21H28F2N8O6S: C, 45.2; H, 5.05; N, 20.1. Found: C, 45.4; H,
5.1; N, 20.4%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-methoxy-6-(methylamino)-1H-benzimidazole (10x).
A solution of 10af (0.25 g, 0.44 mmol) in DMF (20 mL) at room
temperature was treated with 60% NaH in oil (27 mg, 0.67 mmol) to
give a red solution. Iodomethane (86 mg, 0.55 mmol) was added, and
after 5 min the reaction mixture was diluted with water to give tert-butyl
2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-
4-methoxy-1H-benzimidazol-6-yl(methyl)carbamate (10ag) (0.25
g, 97%): 1H NMR (CDCl3) δ 7.89 (br s, 1H), 7.46 (t, JHF = 53.5 Hz,
1H), 6.73 (d, J = 1.8 Hz, 1H), 4.03 (s, 3H), 3.89ꢀ3.84 (m, 8H),
3.79ꢀ3.75 (m, 8H), 3.32 (s, 3H), 1.46 (s, 9H).
A solution of 10ag (0.17 g, 0.3 mmol) in CH2Cl2 (10 mL) was treated
with TFA (1 mL), and the mixture was stirred at room temperature for 4
h and diluted with water. The mixture was basified with aqueous NH3,
and the organic layer was separated and dried. Removal of the solvent
gave 10x (0.14 g, 99%): mp (CH2Cl2/MeOH) 277 °C dec; 1H NMR
(CDCl3) δ7.41 (t, JHF =53.8 Hz, 1H), 7.14 (d, J =1.9 Hz, 1H), 6.13 (d, J=
1.9 Hz, 1H), 3.98 (s, 3H), 3.91ꢀ3.84 (m, 9H), 3.78ꢀ3.75 (m, 8H), 2.87
(s, 3H). Anal. Calcd for C21H26F2N8O3: C, 52.9; H, 5.5; N, 23.5. Found:
C, 53.0; H, 5.5; N, 23.8%.
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Anal. Calcd for C12H6Cl2F2N4: C, 45.7; H, 1.9; N, 17.8. Found: C, 46.0;
H, 2.0; N, 17.7%.
= 53.3 Hz, 1H), 6.84 (s, 1H), 3.91ꢀ3.84 (m, 4H), 3.81ꢀ3.77 (m, 4H).
Anal. Calcd for C16H14ClF2N5O: C, 52.5; H, 3.9; N, 19.15. Found: C,
52.8; H, 3.8; N, 19.4%.
A solution of the above compound (0.33 g, 1.05 mmol) in morpho-
line (0.91 g, 10.5 mmol) was heated under reflux for 1 h, cooled, and
diluted with water to give 16a (0.47 g 100%): mp (EtOH) 205ꢀ208 °C
(lit.38 mp 201ꢀ202 °C); 1H NMR (CDCl3) δ 8.22 (br d, J = 8.2 Hz,
1H), 7.89 (br d, J = 7.1 Hz, 1H), 7.51 (t, JHF = 53.6 Hz, 1H), 7.43ꢀ7.35
(m, 2H), 5.51 (s, 1H), 3.84ꢀ3.80 (m, 8H), 3.65ꢀ3.61 (m, 8H). Anal.
Calcd for C20H22F2N6O2: C, 57.7; H, 5.3; N, 20.2. Found: C, 57.9; H,
5.3; N, 20.1%.
A solution of 18 (0.16 g, 0.44 mmol) in morpholine (10 mL) was
heated under reflux for 30 min and diluted with water to give 19a (0.175
1
g, 96%): mp (aqueous MeOH) 172ꢀ174 °C; H NMR (CDCl3) δ
7.92ꢀ7.88 (m, 1H), 7.67ꢀ7.62 (m, 1H), 7.42ꢀ7.36 (m, 2H), 7.24
(t, JHF = 53.5 Hz, 1H), 6.09 (s, 1H), 3.83ꢀ3.76 (m, 12H), 3.68ꢀ3.63
(m, 4H). Anal. Calcd for C20H22F2N6O2: C, 57.7; H, 5.3. Found: C,
57.3; H, 5.3%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-
2-yl]-4-hydroxy-1H-benzimidazole (16b). A mixture of 8a (7.0 g,
38 mmol), imidazole (50 mL), and TIPSCl (15 g, 78 mmol) in DMF was
stirred at room temperature for 2 h and diluted with water. Extraction
with EtOAc and chromatography on silica, eluting with hexanes/EtOAc
(9:1), gave 2-(difluoromethyl)-4-[(triisopropylsilyl)oxy]-1H-benzimi-
2-(Difluoromethyl)-1-[2,6-di(4-morpholinyl)-4-pyrimidinyl]-
4-methoxy-1H-benzimidazole (19b). A mixture of 4,6-dichloro-
2-(methylsulfanyl)pyrimidine (20) (7.72 g, 0.04 mol), 8b (7.93 g, 0.04
mol), and powdered K2CO3 (22 g, 0.26 mol) in DMSO (100 mL)
was stirred at room temperature for 3 days and diluted with water. The
solid was collected, washed with water, and dried. Chromatography on
silica, eluting with CH2Cl2-EtOAc (95:5), gave 1-[6-chloro-2-(methy-
lsulfanyl)-4-pyrimidinyl]-2-(difluoromethyl)-4-methoxy-1H-benzimi-
dazole (21) (5.91 g, 41% yield): mp (i-Pr2O/hexanes) 120ꢀ121 °C; 1H
NMR (CDCl3) δ 7.40 (t, J = 8.2 Hz, 1H), 7.32 (s, 1H), 7.26 (dd, J = 8.4,
0.7 Hz, 1H), 7.18 (t, JHF = 53.3 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.07
(s, 3H), 2.62 (s, 3H). Anal. Calcd for C14H11ClF2N4OS: C, 47.1; H, 3.1;
N, 15.7. Found: C, 47.3; H, 3.4; N, 15.7%.
1
dazole (8ai) (10.85 g, 84%): mp (hexanes) 120ꢀ121 °C; H NMR
(DMSO-d6) δ 13.21 (br, 1H), 7.24 (t, JHF = 53.2 Hz, 1H), 7.19ꢀ7.13
(m, 2H), 6.73ꢀ6.68 (m, 1H), 1.36 (septet, J = 7.5 Hz, 3H), 1.08 (d, J =
7.5 Hz, 18H). Anal. Calcd for C17H26F2N2OSi: C, 60.0; H, 7.7; N, 8.2.
Found: C, 59.8; H, 7.5; N, 8.3%.
A mixture of 8ai (3.405 g, 10 mmol), 14 (2.75 g, 15 mmol), and
powdered K2CO3 (5.5 g, 40 mmol) in THF (25 mL) was stirred at room
temperature for 1 week and filtered to remove all solids. Morpholine (8.7
g, 0.1 mol) was added to the solution, and the resulting mixture was
heated under reflux for 8 h and diluted with water. The product was
extracted with CH2Cl2, and after drying, the solvent was removed and
the crude residue of 16d was dissolved in THF and treated with a slight
excess of 1 M Bu4NF in THF. Dilution with water gave a solid which was
collected and dried. Chromatography on silica, eluting with CH2Cl2/
EtOAc (3:1), gave 16b (2.30 g, 53%): mp (CH2Cl2/EtOH) 272ꢀ
275 °C (lit.47 mp >250 °C); 1H NMR (DMSO-d6) δ 10.16 (br, 1H),
7.65 (t, JHF = 53.0 Hz, 1H), 7.62 (dd, J = 8.3, 0.7 Hz, 1H), 7.22 (t, J = 8.1
Hz, 1H), 6.72 (dd, J = 7.9, 0.7 Hz, 1H), 5.97 (s, 1H), 3.72ꢀ3.67 (m, 8H),
3.65ꢀ3.60 (m, 8H).
Further elution with CH2Cl2ꢀEtOAc (9:1) gave the bis-addi-
tion byproduct 2-(difluoromethyl)-1-[6-[2-(difluoromethyl)-4-meth-
oxy-1H-benzimidazol-1-yl]-2-(methylsulfanyl)-4-pyrimidinyl]-4-meth-
1
oxy-1H-benzimidazole (4.16 g, 20% yield): H NMR (CDCl3) δ 7.64
(s, 1H), 7.45ꢀ7.38 (m, 4H), 7.26 (t, JHF = 53.3 Hz, 2H), 6.87 (dd, J =
6.9, 2.0 Hz, 2H), 4.07 (s, 6H), 2.62 (s, 3H).
A mixture of 21 (3.57 g, 10 mmol) and morpholine (2.18 g, 25 mmol)
in THF (50 mL) was stirred at room temperature for 30 min
and diluted with water to give 2-(difluoromethyl)-4-methoxy-1-
[2-(methylsulfanyl)-6-(4-morpholinyl)-4-pyrimidinyl]-1H-benzimidazole
(22) (3.85 g, 94% yield): mp (MeOH) 169ꢀ171 °C; 1H NMR (CDCl3)
δ 7.32 (t, J = 8.2 Hz, 1H), 7.22 (dd, J = 8.4, 0.7 Hz, 1H), 7.18 (t, JHF
=
53.4 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H), 6.42 (s, 1H), 4.06 (s, 3H), 3.82
(m, 4H), 3.71 (m, 4H), 2.54 (s, 3H). Anal. Calcd for C18H19F2N5O2S:
C, 53.1; H, 4.7; N, 17.2. Found: C, 53.1; H, 4.7; N, 17.3%.
2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-2-pyrimidinyl]-
4-methoxy-1H-benzimidazole (16c). A mixture of 8b (0.396 g,
2 mmol), 14 (0.37 g, 2 mmol), and powdered K2CO3 (1.1 g, 8 mmol) in
DMSO (10 mL) was stirred at room temperature for 3 h and diluted
with water. The solid was dried and chromatographed on silica, eluting
first with CH2Cl2/hexanes (4:1) and then with CH2Cl2, to give 1-(4,
6-dichloro-2-pyrimidinyl)-2-(difluoromethyl)-4-methoxy-1H-benzimidazole49
(15c) (0.475g, 69%):mp(i-Pr2O) 158ꢀ159 °C; 1HNMR(CDCl3) δ8.01
(dd, J= 8.4, 0.6 Hz, 1H), 7.59 (t, JHF = 53.5 Hz, 1H), 7.41 (t, J =8.3 Hz, 1H),
7.33 (s, 1H), 6.87 (d, J = 7.9 Hz, 1H), 4.06 (s, 3H). Anal. Calcd for
C13H8Cl2F2N4O: C, 45.2; H, 2.3; N, 16.2. Found: C, 45.3; H, 2.55; N, 16.3%.
A solution of 15c (0.172 g, 0.5 mmol) in morpholine (10 mL) was
heated under reflux for 30 min, cooled, and diluted with water to give
16c (0.21 g, 94%): mp (MeOH) 209ꢀ211 °C (lit.49 mp 203ꢀ205 °C);
A solution of 22 (2.04 g, 5 mmol) in a mixture of acetone (500 mL)
and acetic acid (50 mL) was combined with a solution of KMnO4 (5 g)
in water (100 mL), and the resulting mixture was stirred at room
temperature for 1 h. Dilution with water and decolorization with
NaHSO3 gave 2-(difluoromethyl)-4-methoxy-1-[2-(methylsulfonyl)-
6-(4-morpholinyl)-4-pyrimidinyl]-1H-benzimidazole (23) (1.80 g,
1
82% yield) as a white solid: mp (MeOH) 190ꢀ191 °C; H NMR
(CDCl3) δ 7.38 (t, J = 8.2 Hz, 1H), 7.26 (dd, J = 8.4, 0.7 Hz, 1H), 7.13 (t,
JHF = 53.2 Hz, 1H), 6.86 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 4.07 (s, 6H),
3.85 (m, 4H), 3.31 (s, 3H). Anal. Calcd for C18H19F2N5O4S: C, 49.2; H,
4.4; N, 15.9. Found: C, 49.4; H, 4.25; N, 15.9%.
1H NMR (CDCl3) δ 7.78 (dd, J = 8.4, 0.6 Hz, 1H), 7.42 (t, JHF
=
A mixture of 23 (0.439 g, 1 mmol) and morpholine (0.87 g, 10 mmol)
in THF (20 mL) was heated under reflux for 3 h before being cooled, and
diluted with water, to give 19b (0.41 g, 92% yield): mp (CH2Cl2/
MeOH) 180ꢀ182 °C; 1H NMR (CDCl3) δ 7.30 (t, J = 8.9 Hz, 1H),
7.22 (dd, J = 8.3, 0.8 Hz, 1H), 7.15 (t, JHF = 54.1 Hz, 1H), 6.78 (dd, J =
7.9, 0.7 Hz, 1H), 6.09 (s, 1H), 4.05 (s, 3H), 3.82ꢀ3.74 (m, 12H),
3.66ꢀ3.62 (m, 4H). Anal. Calcd for C21H24F2N6O3: C, 56.5; H, 5.4; N,
18.8. Found: C, 56.3; H, 5.7; N, 19.1%.
Molecular Modeling. Molecular modeling studies were carried
out using SYBYL software, version 7.3, running on a Linux work-
station.45 Compound 1 was built using the Sketch module and opti-
mized using the Tripos force field. The protein structures were prepared
for docking using SYBYL7.3, and this included stripping all waters and
adding hydrogens consistent with the changes proposed after analysis of
the structure with Molprobity.67 Docking simulations were performed
53.5 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 5.50
(s, 1H), 4.05 (s, 3H), 3.83ꢀ3.80 (m, 8H), 3.64ꢀ3.61 (m, 8H). Anal.
Calcd for C21H24F2N6O3: C, 56.5; H, 5.4; N, 18.8. Found: C, 56.7; H,
5.4; N, 19.0%.
2-(Difluoromethyl)-1-[2,6-di(4-morpholinyl)-4-pyrimidinyl]-
1H-benzimidazole (19a). A mixture of 2 (0.336 g, 2 mmol), 4-(4,6-
dichloro-2-pyrimidinyl)morpholine (17) (0.367 g, 2 mmol), and pow-
dered K2CO3 (2 g, 15 mmol) in DMF (10 mL) was heated at 45 °C
overnight and diluted with water. The precipitate was collected by
filtration and dried. Chromatography on silica, eluting with CH2Cl2/
EtOAc (49:1), gave 1-[6-chloro-2-(4-morpholinyl)-4-pyrimidinyl]-
2-(difluoromethyl)-1H-benzimidazole66 (18) (0.17 g, 23%): mp
1
(MeOH) 159ꢀ161 °C; H NMR (CDCl3) δ 7.92 (dd, J = 7.9, 1.5
Hz, 1H), 7.67 (dd, J = 7.2, 1.6 Hz, 1H), 7.49ꢀ7.41 (m, 2H), 7.19 (t, JHF
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on human p110γ (PDB code 2CHX) and human p110α (2rd0) using
the automated GOLD program.44 Twenty conformations were gener-
ated. In order to take into account protein flexibility, the conformation
with the best score (GoldScore) was further refined using the MINI-
MIZE module. The minimization process used the Powell method with
the Tripos force field (dielectric constant 1r) to reach a final conver-
noncompartmental analysis using WinNonlin 5.3 software (Pharsight,
Sunnyvale, CA, U.S.).
Tumor Xenografts. Age-matched specific pathogen-free female
Rag1ꢀ/ꢀ mice were subcutaneously inoculated on the right flank with
5 ꢁ 106 U87MG cells in phosphate buffered saline (PBS). Tumor volume
(mm3) was calculated using the formula (Lw2)(π/6) (where L is the
longest tumor diameter and w is the perpendicular diameter). Dosing
began when tumors were well established, averaging approximately 7
mm in diameter. Compound 10w was administered as a suspension in
D5W, 25 was administered in 10% EtOH and 1 in 10% DMSO, 15%
Cremophor EL, 15% EtOH, and 60% saline. All drugs were dosed by ip
injection at MTD as the free base equivalent at a dosing volume of
10 mL/kg. Tumor volume and animal body weight were measured daily.
Mice were culled if they developed signs of toxicity or if bodyweight loss
exceeded 20% of starting weight. All animal experiments followed
protocols approved by the Animal Ethics Committee of The University
of Auckland.
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 μL 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.
gence of 0.01 kcal molꢀ1 68
.
Enzyme Assays. The class Ia PI3K lipid kinase assays were
performed using recombinant PI 3-kinase and phosphatidylinositol
as a substrate and [33P]ATP tracer with a final ATP concentration of
10 μM, as described previously.50,69 For the HTRF assay p85α/p110δ
was obtained from Invitrogen while all other isoforms were produce in
house by coexpressing full length human p85α with the indicated human
full length catalytic subunit containing a His tag at the amino terminus to
allow purification. IC50 values were evaluated using the PI 3-kinase
(human) HTRF assay (Millipore no. 33-016). The PI 3-kinases
were titrated and used at a concentration between their EC65 and
EC80. The protocol was carried out according to the manufacturer’s
recommendations, with the final mixture being incubated for 2 h prior to
reading in a BioTek Synergy 2 plate reader equipped with excitation filter
(360/40) and emission filters (620/40 and 665/7). IC50 data are
normally the average of at least two determinations.
Cell Proliferation Assay. The early passage cell lines used in this
study were NZB5, a cell line derived from a medulloblastoma and
expressing the wild-type gene for p110α, and NZOV9 a cell line derived
from a poorly differentiated endometrioid adenocarcinoma of the ovary
and expressing a mutant p110α enzyme with a single amino acid sub-
stitution in the kinase domain (Y1021C). The derivation of the latter
line has been described previously.70 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.69 IC50 data are normally
the average of at least two determinations.
’ AUTHOR INFORMATION
Inhibition of Cell Signaling. HCT116 and U87MG cells were
grown in MEM α supplemented with 10% (v/v) FBS (fetal bovine
serum), 100 units/mL penicillin, and 100 μg/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 medium.
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 were carried
out according to the methods previously described, with antibodies from
Cell Signaling Technology (Ser473 catalogue no. 9271, Thr308 catalo-
gue no. 9275).50
Pharmacokinetics. Age-matched specific pathogen-free male CD-
1 mice were administered a single 10 mg/kg dose of 1 in 5% DMSO, 10%
Cremophor EL, 10% EtOH, 75% saline, 10a in 20% DMSO, 70% H2O,
10% NaOH or 10w in 10% DMSO, 40% PEG-400, 50% D5W (5%
dextrose). Mice were culled at multiple time points after dosing, and
blood was removed by cardiac puncture into EDTA collection tubes
(Becton Dickinson, Auckland, New Zealand). Blood samples were
centrifuged for 10 min at 6000 rpm to separate plasma, which underwent
protein precipitation in MeOH. Quantitative analysis was carried out on
an Agilent 6460 triple quadrupole LCꢀMS/MS (Agilent Technologies,
Forest Hill, Victoria, Australia) using multiple reaction monitoring and
electrospray ionization. An Agilent Zorbax SB-C18 column (2.1 mm ꢁ
50 mm, 5 μm) column was used for chromatographic separation with a
mobile phase gradient of 20ꢀ100% MeOH in 0.1% formic acid and
5 mM ammonium formate at a flow rate of 0.4 mL/min. Quantitation
was achieved against a calibration curve from 10 to 10 000 nM,
and quality controls were included at 65, 650, and 6500 nM. A MeOH
slug was run between each sample to prevent contamination from
previous samples. Pharmacokinetic parameters were determined by
Corresponding Author
*Phone: (+649) 923 6147. Fax: (+649) 3737502. E-mail: g.rewcastle@
auckland.ac.nz.
Present Addresses
Drug Design and Discovery Center (D3C), University of
Namur, FUNDP, 61 Rue de Bruxelles, 5000 Namur, Belgium.
’ ACKNOWLEDGMENT
We thank Shannon Black and Maruta Boyd 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 Biodiscovery, and Pathway Therapeutics Inc.
’ ABBREVIATIONS USED
AKT, v-akt murine thymoma viral oncogene homologue 1; ATP,
adenosine triphosphate; AUC, area under the curve; AUCINF, area
under the curve to time infinity;Boc, tert-butoxycarbonyl; Cmax
,
maximum concentration; D5W, 5% dextrose in water; DMF, N,
N-dimethylformamide; DMSO, dimethylsulfoxide; DPPA, diphenyl-
phosphorylazide; E545K, somatic mutation replacing glutamic
acid by lysine at position 545 of p110α; EtOAc, ethyl acetate;
EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum;
H1047R, somatic mutation replacing histidine by arginine at
position 1047 of p110α; HOAc, acetic acid; HTRF, homogeneous
time-resolved fluorescence; ip, intraperitoneal; MEM, minimum
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ARTICLE
essential medium; mTOR, mammalian target of rapamycin; PBS,
phosphate buffered saline; PEG, polyethylene glycol; PFK, per-
fluorokerosene; PIK3CA, phosphoinositide 3-kinase, catalytic, α
polypeptide; PIP2, phosphatidylinositol 4,5-biphosphate; PIP3,
phosphatidylinositol 3,4,5-triphosphate; PI3K, phosphoinositide
3-kinase; PKB, protein kinase B;pPKB, phosphoprotein kinase B;
PTEN, phosphatase and tensin homologue gene; PPA, poly-
phosphoric acid; qd, every day; SAR, structureꢀactivity relation-
ship; t1/2, biological half-life; TBAF, tetrabutylammonium fluoride;
TBDMS, tert-butyldimethylsilyl; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; TIPS, triisopropylsilyl; Tmax, time to maximum
plasma concentration; Y1021C, somatic mutation replacing tyr-
osine by cysteine at position 1021 of p110α
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