Preparation and evaluation of trisubstituted pyrimidines as phosphatidylinositol 3-kinase inhibitors. 3-Hydroxyphenol analogues and bioisosteric replacements

Article abstract of DOI:10.1016/j.bmc.2010.12.006

Two classes of trisubstituted pyrimidines related to PI-103 1 have been prepared and their inhibitory activities against phosphatidylinositol 3-kinase (PI3K) p110α were determined. From those with direct 6-aryl substitution compound 11a was the most potent inhibitor with an IC₅₀ value of 62 nM, and showed similar activity against other class 1a PI3K isoforms tested, p110β and p110γ. When a linking chain was introduced, as in the second exemplified class, compound 15f inhibited p110α with IC ₅₀ 142 nM, and showed greater selectivity towards p110α. Compounds of both classes showed promising inhibition of cellular proliferation in IGROV-1 ovarian cancer cells. Among compounds designed to replace the 3-phenolic motif with structural isosteres, analogues incorporating a 4-indazolyl group possessed enzyme and cellular activities comparable to the parent phenols.

Full text of DOI:10.1016/j.bmc.2010.12.006

Bioorganic & Medicinal Chemistry 19 (2011) 836–851
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preparation and evaluation of trisubstituted pyrimidines
as phosphatidylinositol 3-kinase inhibitors. 3-Hydroxyphenol analogues
and bioisosteric replacements
Jonathan M. Large a, Jane E. Torr a, Florence I. Raynaud a, Paul A. Clarke a, Angela Hayes a,
Francesca di Stefano a, Frederique Urban a, Stephen J. Shuttleworth b, Nahid Saghir b, Peter Sheldrake a,
Paul Workman a, Edward McDonald a,
⇑
a Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, Belmont, Surrey SM2 5NG, UK
b Piramed Pharma, 957 Buckingham Avenue, Slough, Berkshire SL1 4NL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Two classes of trisubstituted pyrimidines related to PI-103 1 have been prepared and their inhibitory
Received 3 August 2010
Revised 27 November 2010
Accepted 3 December 2010
Available online 9 December 2010
activities against phosphatidylinositol 3-kinase (PI3K) p110
6-aryl substitution compound 11a was the most potent inhibitor with an IC50 value of 62 nM, and showed
similar activity against other class 1a PI3K isoforms tested, p110b and p110 . When a linking chain was
introduced, as in the second exemplified class, compound 15f inhibited p110 with IC50 142 nM, and
showed greater selectivity towards p110 . Compounds of both classes showed promising inhibition of
cellular proliferation in IGROV-1 ovarian cancer cells. Among compounds designed to replace the 3-phe-
nolic motif with structural isosteres, analogues incorporating a 4-indazolyl group possessed enzyme and
cellular activities comparable to the parent phenols.
a were determined. From those with direct
c
a
a
Keywords:
PI3 kinase
Inhibitors
Cancer
Ó 2010 Elsevier Ltd. All rights reserved.
Pyrimidines
1. Introduction
The pyridofuropyrimidine PI-103 1 (Fig. 1) has been identified
as a useful and potent PI3K inhibitor and chemical tool com-
The phosphatidylinositol 3-kinase (PI3K) family of enzymes
occupies a central position in numerous important cellular signal-
ing pathways.1 Deregulation of activity of specific PI3K isoforms in
a variety of disease states including cancer,2 autoimmune disease3
and cardiovascular disease4 has been extensively studied. Further,
the lipid products derived from PI3K-catalysed phosphorylation at
the 3-position of phosphatidylinositols can interact with important
targets downstream of PI3K, notably AKT.1,5 The phosphorylated
form of AKT is also implicated in numerous aspects of cancer pro-
pound,24,25 with in vitro IC50 of 2 nM against PI3K p110
a and good
levels of cellular potency and selectivity over other classes of PI3K
isoforms. However, the compound is cleared rapidly from plasma
and has low aqueous solubility. In addition, synthetic approaches
to compounds of this type are not particularly amenable to rapid
analogue synthesis in the context of lead optimisation. Our goal
was therefore to modify the structure of 1 in order to generate
structurally simpler analogues with improved physical properties,
whilst maintaining potency and drug-likeness.26,27 In concept we
disconnected the bicyclic ring system of 1 at the 5-position of
the pyrimidine ring, and set out to prepare synthetically accessible
seco-compounds such as 2 and 3 (Fig. 1), where the morpholine
and phenol functionalities are conserved. Related compounds have
also been reported recently by other groups.28–31 Previous studies,
gression.6 The p110
a isoform (encoded by the PIK3CA gene) is
known to be over-expressed or activated by mutation in many hu-
man cancer types, while loss of the regulatory phosphatase PTEN
also leads to activation of the PI3K pathway.7–14 Thus a growing
body of direct and indirect evidence suggests that PI3K is an attrac-
tive target for anti-cancer drug discovery15–17 and the develop-
ment of novel small molecule inhibitors of PI3K could represent
a beneficial therapeutic intervention strategy in cancer treat-
ment.18–20 To this end, a number of compounds are entering or
have already begun early stage clinical trials.21–23
on the closely related PI3K p110c
isoform32 using the well-known
but non-selective PI3K inhibitor LY294002,33 have suggested that
the morpholine and phenol motifs form important binding interac-
tions in the PI3K catalytic site. We initially chose to target a small
set of compounds bearing a pyridyl substituent in the side chain at
the 2-position. For 3, variations of the linking atom X (nitrogen or
oxygen), the pyrimidine substitution pattern and the length of the
linking chain (1 or 2 carbon atoms) were included to study the im-
pact of these structural variations on biological activity. We also
⇑
Corresponding author. Tel.: +44 0 208 722 4294.
E-mail address: ted@icr.ac.uk (E. McDonald).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.006

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
837
O
N
O
N
O
N
A = C, B = N
A = N, B = C
or
5
N
A
O
N
N
X = O or NH
n = 1 or 2
N
OH
( )
OH
OH
X
B
N
N
n
R
R
Ar = 2-py, 3-py, 4-py
3
1
2
O
N
B
N
NH
H
H
N
N
N
A
SO2Me
aryl or
( )
heteroaryl
3-phenol
surrogate
X
n
R
4a
4b
4c
4
Figure 1. Structure of pyridofuropyrimidine 1 and proposed compound sets 2, 3 and 4a–4c.
HN
NH2
CN
(i), (ii)
O
N
O
N
OH
OH
OBn
(vii)
(v), (vi)
N
(iv)
5
6
OBn
N
N
Ar
N
OBn
OH
Ar
N
Ar
N
O
O
(iii)
9a-9e
CO2Me
10a-10e
11a-11e
Ar
7a-7e
Ar
a: Ar = 2-py
b: Ar = 3-py
c: Ar = 4-py
d: Ar = 4-ClC6H4
e: Ar = 3,4-Cl2C6H3
8a-8e
Scheme 1. Reagents and conditions: (i) NaH, BnBr, DMF, reflux; (ii) LiHMDS, HCl, 0 °C to rt; (iii) NaH, (MeO)2CO, THF, rt to reflux; (iv) nBuOH, 110 °C; (v) POCl3, PhNEt2,
i
110 °C; (vi) morpholine, Pr2NEt, dioxane, 80 °C; (vii) HBr, AcOH, reflux.
decided to prepare structures such as 4, designed to incorporate
bioisosteric replacements of the 3-phenolic motif in 3. Glucuroni-
dation of such functional groups can result in rapid and undesir-
able second pass metabolic clearance in vivo;34 indeed PI-103
has been shown to be metabolized in this way.24 We chose to eval-
uate 6-indolyl, 3-(methanesulfonylamino)phenyl and 4-indazolyl
bioisosteres (4a–4c, Fig. 1) in this study.
first with a small set of amines which contained a linking chain
and a pyridine motif, and second with morpholine (Scheme 2). Su-
zuki–Miyaura coupling with 3-hydroxyphenylboronic acid then
afforded the trisubstituted products 15a–15f. Due to the essen-
tially non-selective nature of the first amine displacement, 2-ami-
no-linked compounds 18a–18f were also easily available using the
same methodology. These desirable structures would allow us to
probe the effect of altering the pyrimidine regiochemistry on bio-
logical activity. A very similar route was also employed to prepare
the two O-linked congeners 19 and 20.40 This serves to highlight
the flexibility and scope of the approach.
2. Chemical synthesis
6-Aryl compounds29–31 were synthesised by means of well-
precedented condensation chemistry and standard manipulations
(Scheme 1). First, the known amidine 6 was prepared in two
steps35,36 from commercially available 3-cyanophenol 5; and a
small set of known aryl b-keto esters 8a–8e were prepared from
the corresponding ketones 7a–7e by deprotonation and quenching
with dimethylcarbonate.37 Condensation of these crude dicarbonyl
compounds with 6 gave pyrimidinones 9a–9e. These key interme-
diates were converted to the corresponding chlorides and then
reacted with morpholine, generating 10a–10e and, after deprotec-
tion, the desired phenolic targets 11a–11e. We included two
compounds (11d and 11e) that displayed non-pyridine aryl
substituents in this part of the study, to monitor the impact of
the pyridine nitrogen atoms on the desired biological activity.
Compounds 15a–15f containing a 6-amino linked substituent28
could be prepared from commercially available 2,4,6-trichloropyr-
imidine 12 by means of two sequential amine displacements;38,39
Comparison of 1H NMR spectroscopic data for intermediates
13a–13f/14a–14f (2-morpholines) and 16a–16f/17a–17f (4-mor-
pholines) (Scheme 2) showed that the chemical shift of the pyrim-
idine H-5 proton was broadly but not unambiguously diagnostic
for each series. Hence two different methods were employed to
confirm the regiochemical outcome of the amination steps. For
example, reaction of 21 and 22 (the precursors to 19 and 20, respec-
tively) with morpholine gave adducts 23 and 24, respectively
(Scheme 3). Examination of 13C NMR spectroscopic data showed four
distinct quaternary carbon signals for 23, but only three for the sym-
metrical pyrimidine 24. This verified the regiochemistry of the first
alkoxide substitution. In addition, reductive removal of the remain-
This proton always appeared at higher field for intermediates in the 2-morpholine
series (13a–f/14a–f) than those in the 4-morpholine series (16a–f/17a–f).39 See
Section 5.

838
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
O
N
O
Cl
N
(iii)
(ii)
N
N
N
N
N
N
( )
Ar n N
Cl
OH
( )
( )
H
Ar n N
Ar n N
Cl
5
H
H
Cl
13a-13f
14a-14f
15a-15f
a or d: Ar = 2-py
b or e: Ar = 3-py
c or f: Ar = 4-py
(i)
N
+
O
N
O
Cl
N
Cl
n = 1 or 2
Cl
N
12
N
N
(iii)
(ii)
5
N
( )
OH
Ar n N
H
N
Cl
( )
( )
Ar n N
N
Ar n N
H
N
Cl
H
16a-16f
17a-17f
18a-18f
O
N
O
N
(iv), (ii), (iii)
+
12
5
N
N
N
N
OH
N
OH
O
O
N
5
19
20
Scheme 2. Reagents and conditions: (i) Ar(CH2)nNH2, iPr2NEt, dioxane, 10 °C; (ii) morpholine, iPr2NEt, dioxane, 80 °C; (iii) PdCl2(dppf) (for 15a–15f) or Pd(Ph3P)4 (for
18a–18f), 3-HOC6H4B(OH)2, Na2CO3 (aq), DME, 80 °C; (iv) NaH, ArCH2OH, THF, 0 °C to rt.
ing chlorine atom in 14d and 17d gave 25 and 26 respectively. Anal-
O
N
O
N
ysis of these two products by 1H NMR spectroscopy revealed two
new aromatic doublets (coupling constant 6 Hz) in each case, estab-
lishing the position of the morpholine group. Combination of these
methods provided rigorous proof of the regiochemistry in each
chemical series.
(i)
N
N
N
N
N
N
O
Cl
O
N
Analogues bearing isosteric replacements for the 3-phenolic mo-
tif were prepared from intermediates 14d–14f or 17d–17f, respec-
tively, using modified Suzuki coupling protocols (Scheme 4). Thus
using the Bedford palladacycle 2941 as catalyst, reaction with the
requisite aryl or heteroaryl boronic acid or pinacol boronate ester
cleanly provided compounds 27a–27i and 28a–28i. The O-linked
congeners 30a–30c and 31a–31c were similarly prepared from 21
and 22 using the same protocol. We have described the use of palla-
dacycle 29 as an effective catalyst for Suzuki couplings involving a
closely related pyrimidine template.42 Here, the use of automated
microwave heating43 and shortened reaction times considerably
enhanced the efficiency and flexibility of the approach. This chem-
istry represents a useful sequential method for the preparation of
compounds of this type, given that access to both regioisomers of
the pyrimidine scaffold was again required.
O
21
O
23
O
N
N
(i)
N
N
N
N
O
N
Cl
O
N
N
O
22
24
O
O
N
H
N
H
(ii)
N
N
N
N
N
N
N
H
N
Cl
H
H
3. Results and discussion
J 6.0 Hz
14d
25
All the newly prepared compounds were assessed for in vitro
biological activity against PI3K p110a, using a radiometric scintil-
O
N
O
lation proximity assay.44 Under these assay conditions the IC50 va-
lue determined for PI-103 was 2 nm. We first tested compounds
bearing a 3-phenolic group (Table 1) and found, as expected, that
an unprotected phenol was required for optimal biological activity;
O-benzyl ethers 10a–10e showed no appreciable in vitro inhibition
of the enzyme (entries 1–5). On revealing the phenol motif, com-
pounds 11a–11e (entries 6–10) each possessed useful inhibitory
activity with 11a the most promising at 62 nM. We were also
encouraged to find that non-pyridine analogues 11d and 11e
N
(ii)
H
H
H
N
N
N
N
J 6.0 Hz
N
H
N
N
H
N
Cl
17d
26
Scheme 3. Reagents and conditions: (i) morpholine, 50 °C; (ii) H2, Pd/C, KOAc,
HOAc, rt.

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
839
O
N
O
N
O
N
O
N
(i)
(i)
N
N
N
N
N
N
N
N
N
N
Ar
Ar
O
Cl
O
R
N
H
Cl
N
H
R
14d-14f
27a-27i
21
30a-30c
O
O
O
N
O
N
N
N
N
(i)
(i)
N
N
N
N
N
Ar
Ar
O
N
Cl
O
N
R
N
H
N
Cl
N
H
N
R
17d-17f
28a-28i
22
31a-31c
NMe2
Ar = 2-py, 3-py, 4-py
R = surrogate A, B or C (Table 3)
Pd OCOCF3
PCy3
29
Scheme 4. Reagents and conditions: (i) 0.05–0.10 equiv 29, 2.2 equiv R-B(OH)2, 2.2 equiv Na2CO3 (aq), 1,2-DME, microwave heating, 150 °C, 30 min.
Table 1
comparable activity to the corresponding NH-linked analogues,
revealing that the hydrogen bond donating ability of the linking
atom is not a critical requirement for good inhibition. It also im-
plies that shorter linkers can be employed to generate useful levels
of activity, and that the orientation of the nitrogen atoms in the
pyrimidine scaffold may be important for some compounds.
We assessed the cancer cell growth inhibitory activity of the
more active compounds using a sulphorhodamine B assay45 (SRB)
in the IGROV-1 human ovarian cancer cell line which has a strongly
activated PI3 kinase pathway through a combination of loss of
PTEN and mutational activation of PIK3CA (Table 1). Where no link-
ing chain was present (entries 6–8), the 2-pyridyl analogue 11a
(entry 6) possessed the best activity in this series with a GI50 value
In vitro screening of trisubstituted pyrimidines against PI3K p110
a
Ara
n
p110
M)
a
IC50
SRB GI50 (lM)
b
b
Entry
Compound
No.
(l
c
c
c
c
c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
15a
15b
15c
15d
15e
15f
18a
18b
18c
18d
18e
18f
19
2-py
3-py
4-py
4-ClC6H4
3,4-Cl2C6H3
2-py
3-py
4-py
4-ClC6H4
3,4-Cl2C6H3
2-py
3-py
4-py
2-py
3-py
4-py
2-py
3-py
4-py
2-py
3-py
4-py
2-py
2-py
0
0
0
0
0
0
0
0
0
0
1
1
1
2
2
2
1
1
1
2
2
2
1
1
>10
>10
>10
>10
>10
0.062
0.26
0.14
0.69
0.20
0.90
0.72
1.0
0.25
0.26
0.14
1.0
0.88
0.86
0.18
0.25
0.23
0.11
0.86
0.37
0.63
1.2
c
c
c
c
c
for 50% growth inhibition of 0.37 lM, consistent with its potent
activity on PI3K. When compounds with a two-carbon linker were
considered (entries 14–16 and 20–22), both regioisomeric pyrimi-
dine series again showed similar levels of inhibition. The position
of the pyridine nitrogen was again not critical for good activity.
The O-linked congeners 19 and 20, which do not feature a hydro-
gen bond donor in the side chain, showed levels of cell growth
inhibition (entries 23 and 24) similar to those of NH compounds,
2.2
1.2
0.70
c
c
c
1.20
0.75
0.95
1.5
commensurate with their potencies as inhibitors of p110a .
Assessment of analogues containing structural surrogates of the
3-phenolic motif was carried out using the same two assay proto-
cols (Tables 2 and 3). The key SAR conclusions from these data are
that compounds in the 4-morpholinyl series (Table 3) were gener-
ally more potent than the corresponding 2-morpholinyl analogues
(Table 2); and that compounds incorporating the 6-indolyl or
3-MeSO2NH-phenyl substituents (surrogates A and B, respectively,
in Tables 2 and 3) were an order of magnitude less active than the
corresponding phenols.17,24 However compounds 27g–27i and
28g–28i with the 4-indazolyl group (surrogate C) had activities
comparable to the corresponding phenols. These data strongly sug-
gest that molecular interaction between the phenolic and 4-indaz-
20
2.5
a
b
c
py = pyridyl.
Mean of at least two separate measurements with typical variability <25%.
Not determined.
(entries 9 and 10) retained activity and so a broader exploration of
the SAR in this region might be fruitful. When a linking chain
containing a nitrogen heteroatom was introduced, inhibition was
generally greater when two additional linking carbon atoms were
present (entries 11–16 and 17–22). Encouragingly, both regioiso-
meric series of the pyrimidine scaffold provided useful levels of
in vitro activity, although no clear preference for the position of
the pyridine nitrogen was observed in either pyrimidine series.
We also wanted to probe the effect of changing the nature and
hydrogen bonding capability of the linking motif. When an oxygen
linking atom was introduced, compound 19 displayed better
inhibitory activity than 20 (entries 23–24), and both ethers have
olyl inhibitors and PI3K p110a includes H-bond donation that
cannot be mimicked effectively by either sulphonamido or 6-indo-
lyl analogues. The constrained geometries of the bicyclic com-
pounds then lead to the conclusion that the trajectory of the
postulated H-bond lies in the anti- rather than syn- direction with
respect to the pyrimidine moieties. The foregoing pattern of activ-
ity was broadly followed by the O-linked compounds, with

840
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
Table 2
In vitro screening of trisubstituted pyrimidines 27a–27i and 30a–30c against PI3K p110
a
O
N
N
N
Ar
X
surrogate
Surrogateb
n
Ara
X
n
p110a
IC50
(l
M)
SRB GI50 (lM)
c
c
Compound No.
27a
27b
27c
27d
27e
27f
27g
27h
27i
2-py
3-py
4-py
2-py
3-py
4-py
2-py
3-py
4-py
2-py
2-py
2-py
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
2
2
2
2
2
2
2
2
2
1
1
1
A
A
A
B
B
B
C
C
C
A
B
C
> 10
> 10
> 10
> 10
> 10
> 10
1.3
0.66
0.77
> 10
> 10
0.50
12.0
6.0
14.5
15.0
11.6
15.0
7.8
2.9
2.7
6.0
16.0
1.9
30a
30b
30c
O
O
a
b
c
py = pyridyl.
A = 6-indole; B = 3-(methanesulfonylamino)phenyl; C = 4-indazole (see Fig. 1 for structures).
Mean of at least two separate measurements with typical variability <25%.
Table 3
In vitro screening of trisubstituted pyrimidines 28a–28i and 31a–31c against PI3K p110
a
O
N
N
Ar
X
N
surrogate
Surrogateb
n
Ara
X
n
p110a
IC50
(l
M)
SRB GI50 (lM)
c
c
Compound No.
28a
28b
28c
28d
28e
28f
28g
28h
28i
2-py
3-py
4-py
2-py
3-py
4-py
2-py
3-py
4-py
2-py
2-py
2-py
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
2
2
2
2
2
2
2
2
2
1
1
1
A
A
A
B
B
B
C
C
C
A
B
C
8.9
7.2
>10
>10
>10
9.1
3.6
4.2
3.0
11.5
16.2
6.2
2.7
2.9
1.5
9.0
1.1
0.82
0.44
5.2
6.2
1.35
31a
31b
31c
O
O
13.5
2.3
a
b
c
py = pyridyl.
A = 6-indole; B = 3-(methanesulfonylamino)phenyl; C = 4-indazole (see Fig. 1 for structures).
Mean of at least two separate measurements with typical variability <25%.
indazoles 30c and 31c only slightly less active than the parent phe-
nols 19 and 20. In the cell growth inhibition assay, again the 6-
indolyl and 3-MeSO2NH analogues were significantly less active
than the corresponding phenol parents but the indazoles showed
good levels of cancer cell growth inhibitory activity with GI50s gen-
erally only twofold greater than the parent phenols. It is interesting
to note that several surrogates listed in Tables 2 and 3 are weak
The cellular activities described above might be a consequence
of activity of these compounds against more than one target. A
small number of the most active phenolic compounds were there-
fore assessed for selectivity against the class 1a PI3K isoforms
p110b and p110c (Table 4). When no linking chain was present
(entries 1–3), compounds 11a–11c could not effectively discrimi-
nate between the three isoforms in vitro. However, compounds
15d–15f (entries 5–7) showed 5–10-fold selectivity for the
inhibitors of p110a but nevertheless show moderate levels of cell
growth inhibition, possibly due to inhibition of non-target
p110a target enzyme, particularly over p110b. Such levels of selec-
enzymes.
tivity are very interesting for structurally simple and easily pre-
pared compounds. On changing the pyrimidine scaffold in 15d to
its regioisomeric counterpart in 18d (entry 8), a different selectiv-
ity profile resulted. It is also interesting to note the subtle changes
in selectivity when nature and length of the linking chain was
In summary, indazolyl analogues are inhibitors of p110a and of
IGROV-1 human ovarian cancer cell proliferation with activities
comparable to the parent phenols and therefore offer an attractive
opportunity for further optimisation.

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
841
Table 4
Table 5
Inhibitory activities of key trisubstituted pyrimidines against three PI3K p110
isoforms
Metabolic stabilities of trisubstituted pyrimidines in
mouse liver microsomes
a
a
a
Entry Compound
No.
p110
a
IC50
p110b IC50
M)
p110
c
IC50
Compound No.
% Metabolised at
15 min 30 min
(
lM)
(
l
(l
M)
1
2
3
4
5
6
7
8
9
11a
11b
11c
15a
15d
15e
15f
18d
19
0.062
0.26
0.14
0.90
0.25
0.26
0.14
0.18
0.11
0.10
0.44
0.20
2.3
2.6
1.9
1.6
5.2
0.37
0.044
0.34
0.15
7.6
0.83
0.96
0.74
0.38
0.73
11a
15f
27g
27h
27i
28c
28f
28i
30c
39
54
58
66
37
38
33
49
44
57
71
84
98
68
60
53
74
66
a
Mean of at least two separate measurements with typical variability <25%.
compared to the parent phenol 15f. It is therefore clear that further
structural modifications will be necessary to confer high levels of
metabolic stability.
altered. Compound 15a (entry 4), although not among the most ac-
tive against the primary oncogenic target p110 , was able to dis-
criminate against p110 . The O-linked congener 19 (entry 9)
a
c
Finally we conducted pharmacodynamic biomarker analysis
experiments in the PI3K pathway activated IGROV-1 ovarian can-
cer cells, utilising key compounds 11a and 15f and the known
PI3K inhibitor LY29400233 (Fig. 3). All compounds were found to
inhibit constitutive phosphorylation of AKT at Ser473 following 24
and 48-h exposure to 1 and 5 times the cellular GI50 determined
for 96-h exposure. These biomarker data are consistent with cancer
cell growth inhibition through blockade of the PI3K pathway. Inhi-
bition of signaling through the PI3K/AKT pathway has previously
been demonstrated to decrease expression of cyclin D1 protein.24
In agreement with this observation, all three compounds also re-
duced cyclin D1 protein levels at the same two time points. Fur-
ther, cell cycle analysis experiments with 11a and 15f (Fig. 4)
revealed a robust G1 arrest with loss of cells from S phase. This
profile is similar to that of PI-10344 and the results are consistent
showed a very similar profile, but with enhanced activities against
all three isoforms. These useful observations should aid the design
of further inhibitors against any of the three class 1a PI3K enzymes
with improved overall characteristics. Taking into account all the
foregoing results, we considered that the 4-pyridyl side chain ana-
logues 15f and 18f (entries 16 and 22) showed the best overall pro-
file of activity.
The effect of the structural replacements on metabolic stability
was examined by conducting mouse microsomal incubation stud-
ies on the most biochemically active phenols 11a and 15f, several
key indazoles and also on examples of the less active indoles and
sulfonamides (28c and 28f, respectively). As expected for phenols34
compounds 11a and 15f, like PI-103,24 were rapidly metabolised,
with glucuronidation an important pathway in each case (Fig. 2).
However, the results (Table 5) show that the surrogate compounds
are also metabolised at rates similar to the representative phenols
with only marginal reductions for analogues 27i, 28f, 28c and 30c
with the interpretation that intracellular inhibition of the p110
target is important for the biological activities observed above.
a
Figure 2. Metabolite identification of compounds 11a and 15f by MS analysis (O = oxidation, gluc = glucoronide).

842
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
cycle analysis of 11a and 15f confirmed that these compounds
inhibited the PI3K pathway in genetically relevant cancer cells,
consistent with their growth inhibitory effects.
5. Experimental
5.1. General synthetic chemistry remarks
Commercially available starting materials, reagents and dry
solvents were used as supplied. Flash chromatography was per-
formed using Merck Silica Gel 60 (0.025–0.04 mm). Ion exchange
chromatography was performed using acidic Isolute flash SCX-II
cartridges. 1H NMR spectra were recorded on a Bruker Avance
dpx250 at 250 MHz or a Bruker Avance-500 at 500 MHz. 13C spec-
tra were recorded on a Bruker Avance-500 at 125 MHz. Samples
were prepared as solutions in either CDCl3 or DMSO-d6 and refer-
enced to the appropriate internal non-deuterated solvent peak or
tetramethylsilane. Chemical shifts were recorded in ppm (d) down-
field of tetramethylsilane. Coupling constants were recorded in Hz
to the nearest 0.5 ppm. HPLC–MS spectra were recorded on a
Waters Micromass LCT with a Waters Alliance 2795 separations
module, using a Phenomenex Gemini C18 column (5
mm) and either a 6 min (a) or 10 min (b) gradient (MeOH/0.1% for-
mic acid), with nominal mass, LC injection, positive ionisation and
l
, 50 ꢀ 4.6
Figure 3. Effect of 11a and 15f applied at onefold and fivefold GI50 concentrations
on levels of phospho-AKT (at Ser473) and cyclin D1 in IGROV-1 cells at 24 h (a) and
48 h (b) time points.
an injection volume of 2 lL (a) or 3 lL (b). High-resolution mass
4. Conclusions
spectra were obtained to within 5 ppm accuracy using the same
instrumental set-up and LC injection with a 10 min gradient
(MeOH and 0.1% formic acid), positive ionisation and an injection
In summary, we have prepared two series of 2,4,6-trisubsti-
tuted pyrimidines and demonstrated that they are structurally
simple and useful inhibitors of the PI3K p110
a validated and important cancer target. Compounds such as 11a
possessed good inhibitory activity and also showed similar activity
volume of 4
mo-Finnigan Surveyor HPLC system at 30 °C, using a Phenomenex
Gemini C18 column (5
lL. Analytical HPLC spectra were recorded on a Ther-
a enzyme, which is
l
, 50 ꢀ 4.6 mm) and a 6 min (a) or 10 min
(b) gradient of 10?90% MeOH/0.1% formic acid, visualising at
254 nm. Compounds 634,35 and 8a–8e36 were prepared according
to the reported procedures. All compounds were characterised by
1H NMR spectroscopy and low/high-resolution mass spectrome-
try—those that were biologically tested were also assessed for pur-
ity by HPLC.
against other PI3K isoforms tested, namely p110b and p110c.
When a linking chain was introduced, in compounds like 15f, po-
tency was maintained and an interesting discrimination between
PI3K isoforms was observed. The most active compounds were also
able to inhibit growth at sub-micromolar concentrations of IGROV-
1 human ovarian cancer cells, which have an activated PI3 kinase
pathway. Isosteric replacement of the 3-phenolic motif led to three
further sets of compounds, with 4-indazolyl analogues displaying
comparably potent enzyme inhibition and antiproliferative active
in IGROV-1 cells comparable to the parent phenols. All three surro-
gate types had metabolic stabilities similar to those of parent phe-
nols, indicating that further optimisation of pharmacokinetic
properties will be necessary. Pharmacodynamic biomarker and cell
5.2. Compound preparation and characterisation
5.2.1. General procedure 1: condensation of 6 with 8a–8e
5.2.1.1. 2-(3-(Benzyloxy)phenyl)-6-(pyridin-2-yl)pyrimidin-4-ol
(9a). A solution of the amidine 6 (1.1 equiv, 276 mg, 1.23 mmol)
and the b-ketoester 8a (1 equiv, 200 mg, 1.12 mmol) in nBuOH
(3 mL) was stirred at 110 °C for 18 h. During this time a solid
Figure 4. Cell cycle analysis of compounds 11a and 15f applied at one- and fivefold GI50 concentrations to IGROV-1 cells for 24 h.

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
843
precipitate formed. After cooling to room temperature, the solid
was filtered off and air-dried to provide 9a (190 mg, 48%) as a pale
yellow solid; dH (500 MHz, DMSO-d6) 12.89 (1H, br), 8.74 (1H, d, br,
J 4.5), 8.47 (1H, d, br, J 8.0), 8.03 (1H, td, J 8.0, 2.0), 7.94 (1H, s, br),
7.90 (1H, d, J 7.0), 7.54 (1H, ddd, J 7.5, 5.0, 1.0), 7.51–7.49 (1H, m),
7.50 (2H, t, J 8.0), 7.42 (2H, t, J 8.0), 7.36 (1H, t, br, J 7.5), 7.25 (1H,
dd, J 8.0, 2.0), 7.23 (1H, s), 5.26 (2H, s); HPLC–MSa Rt 5.26 min;
(C22H17N3O2): m/z (ESI) 356 ([M+H]+, 100%); C22H17N3O2 requires
356.1399, found: [M+H]+, 356.1396.
concentrated in vacuo to give crude product. Purification by pre-
parative TLC (hexane/EtOAc, 1:1) gave 10a (28 mg, 47% for two
steps) as a colourless solid; Rf 0.40 (hexane/EtOAc, 1:1); dH
(500 MHz, CDCl3) 8.70 (1H, ddd, J 5.0, 1.5, 1.0), 8.64 (1H, dt, J 8.0,
1.5), 8.19–8.16 (2H, m), 7.90 (1H, td, J 2.0), 7.61 (1H, s), 7.52 (1H,
d, br, J 7.5), 7.44–7.36 (5H, m), 7.11 (1H, ddd, J 8.0, 3.0, 1.0), 5.21
(2H, s), 3.86 (8H, s, br); HPLC–MSa Rt 5.86 min; (C26H24N4O2): m/
z (ESI) 425 ([M+H]+, 100%); C26H24N4O2 requires 425.1978, found:
[M+H]+, 425.1974; HPLCb Rt 9.13 min, area 99.5%.
5.2.1.2. 2-(3-(Benzyloxy)phenyl)-6-(pyridin-3-yl)pyrimidin-4-ol
(9b). Prepared from compounds 6 and 8b according to general
procedure 1; pale yellow solid, 48% yield; dH (500 MHz, DMSO-
d6) 12.90 (1H, br), 9.34 (1H, d, J 2.0), 8.81 (1H, dd, J 4.5, 1.5), 8.61
(1H, dt, J 8.0, 2.0), 7.92 (1H, s, br), 7.88 (1H, d, br, J 8.0), 7.55 (1H,
dd, J 8.0, 4.5), 7.51–7.48 (1H, m), 7.50 (2H, t, J 8.0), 7.42 (2H, t, J
8.0), 7.35 (1H, t, br, J 8.0), 7.25 (1H, dd, J 8.0, 2.0), 7.05 (1H, s),
5.24 (2H, s); HPLC–MSa Rt 5.00 min; (C22H17N3O2): m/z (ESI) 356
([M+H]+, 100%); C22H17N3O2 requires 356.1399, found: [M+H]+,
356.1401.
5.2.2.2. 4-(2-(3-(Benzyloxy)phenyl)-6-(pyridin-3-yl)pyrimidin-
4-yl)morpholine (10b). Prepared from compound 9b according
to general procedure 2; colourless solid, 47% yield for two steps;
Rf 0.15 (hexane/EtOAc, 1:1); dH (500 MHz, CDCl3) 9.28 (1H, d, br,
J 2.0), 8.72 (1H, dd, J 5.0, 1.5), 8.38 (1H, dt, J 8.0, 2.0), 8.16 (1H,
s), 8.16–8.12 (1H, m), 7.45–7.26 (7H, m), 7.05–7.01 (1H, m), 6.76
(1H, s), 5.12 (2H, s), 3.82–3.72 (8H, m); HPLC–MSa Rt 5.67 min;
(C26H24N4O2): m/z (ESI) 425 ([M+H]+, 100%); C26H24N4O2 requires
425.1978, found: [M+H]+, 425.1981; HPLCb Rt 9.89 min, area 95.9%.
5.2.2.3. 4-(2-(3-(Benzyloxy)phenyl)-6-(pyridin-4-yl)pyrimidin-
4-yl)morpholine (10c). Prepared from compound 9c according
to general procedure 2; colourless solid, 40% yield for two steps;
Rf 0.21 (hexane/EtOAc, 1:1); dH (250 MHz, CDCl3) 8.70 (2H, d, J
5.0), 8.07–8.04 (2H, m), 7.99 (2H, d, J 5.0), 7.44–7.24 (6H, m),
7.04 (1H, ddd, J 8.0, 2.5, 1.5), 6.80 (1H, s), 5.12 (2H, s), 3.81–3.74
(8H, m); HPLC–MSa Rt 5.67 min; (C26H24N4O2): m/z (ESI) 425
([M+H]+, 100%); C26H24N4O2 requires 425.1978, found: [M+H]+,
425.1964; HPLCb Rt 9.85 min, area 93.4%.
5.2.1.3. 2-(3-(Benzyloxy)phenyl)-6-(pyridin-4-yl)pyrimidin-4-ol
(9c). Prepared from compounds 6 and 8c according to general
procedure 1; colourless solid, 44% yield; dH (500 MHz, DMSO-d6)
12.49 (1H, br), 8.67 (2H, d, J 6.0), 8.08 (2H, d, J 6.0), 7.92 (1H, s,
br), 7.88 (1H, d, br, J 8.0), 7.51–7.49 (1H, m), 7.50 (2H, t, J 8.0),
7.43 (2H, t, J 8.0), 7.35 (1H, t, br, J 7.5), 7.25 (1H, dd, J 8.0, 2.0),
7.09 (1H, s), 5.24 (2H, s); HPLC–MSa Rt 4.92 min; (C22H17N3O2):
m/z (ESI) 356 ([M+H]+, 100%); C22H17N3O2 requires 356.1399,
found: [M+H]+, 356.1382.
5.2.2.4. 4-(2-(3-(Benzyloxy)phenyl)-6-(4-chlorophenyl)pyrimi-
din-4-yl)morpholine (10d). Prepared from compound 9d accord-
ing to general procedure 2; colourless solid, 29% yield for two
steps; Rf 0.68 (hexane/EtOAc, 1:1); dH (250 MHz, CDCl3) 8.08–
8.04 (2H, m), 7.99 (2H, d, J 8.5), 7.39 (2H, d, J 8.5), 7.39–7.26 (6H,
m), 7.04–7.00 (1H, m), 6.71 (1H, s), 5.12 (2H, s), 3.82–3.72 (8H,
m); HPLC–MSb Rt 9.25 min; (C27H24Cl1N3O2): m/z (ESI) 458
([35M+H]+, 100%), 460 ([37M+H]+, 33); C27H24Cl1N3O2 requires
458.1635, found: [35M+H]+, 458.1626; HPLCb Rt 9.45 min, area
96.7%.
5.2.1.4. 2-(3-(Benzyloxy)phenyl)-6-(4-chlorophenyl)pyrimidin-
4-ol (9d). Prepared from compounds 6 and 8d according to gen-
eral procedure 1; pale yellow solid, 15% yield; dH (250 MHz,
DMSO-d6) 8.21 (2H, d, J 8.5), 7.91 (1H, d, br, J 8.0), 7.87 (1H, s,
br), 7.59 (2H, d, J 8.5), 7.53–7.36 (5H, m), 7.26 (1H, dd, J 8.0, 2.0),
6.97 (1H, s), 5.24 (2H, s); HPLC–MSb Rt 9.27 min; (C23H17Cl1N2O2):
m/z (ESI) 389 ([35M+H]+, 100%), 391 ([37M+H]+, 32); C23H17Cl1N2O2
requires 389.1057, found: [35M+H]+, 389.1052.
5.2.1.5. 2-(3-(Benzyloxy)phenyl)-6-(3,4-dichlorophenyl)pyrimi-
din-4-ol (9e). Prepared from compounds 6 and 8e according to
general procedure 1; pale yellow solid, 30% yield; dH (250 MHz,
DMSO-d6) 8.47 (1H, d, J 2.0), 8.23 (1H, dd, J 8.5, 2.0), 7.95 (1H, d,
br, J 8.0), 7.89 (1H, s), 7.85 (1H, d, J 8.5), 7.59–7.41 (6H, m), 7.31
(1H, dd, J 8.0, 2.0), 7.12 (1H, s), 5.30 (2H, s); HPLC–MSb Rt
9.85 min; (C23H18Cl2N2O2): m/z (ESI) 423 ([35,35M+H]+, 100%), 425
([37,35M+H]+, 53), 427 ([37,37M+H]+, 13); C23H18Cl2N2O2 requires
423.0667, found: [35,35M+H]+, 423.0671.
5.2.2.5. 4-(2-(3-(Benzyloxy)phenyl)-6-(3,4-dichlorophenyl)pyr-
imidin-4-yl)morpholine (10e). Prepared from compound 9e
according to general procedure 2; colourless solid, 26% yield for
two steps; Rf 0.63 (hexane/EtOAc, 1:1); dH (250 MHz, CDCl3) 8.15
(1H, d, J 2.0), 8.07–8.04 (2H, m), 7.89 (1H, dd, J 8.5, 2.0), 7.48
(1H, d, J 8.5), 7.51–7.26 (6H, m), 7.05–7.00 (1H, ddd, J 8.0, 4.0,
1.0), 6.69 (1H, s), 5.12 (2H, s), 3.80–3.70 (8H, m); HPLC–MSa Rt
9.99 min; (C27H23Cl2N3O2): m/z (ESI) 496 ([37,37M+H]+, 11%), 494
([37,35M+H]+, 60), 492 ([35,35M+H]+, 100); C27H23Cl2N3O2 requires
492.1246, found: [35,35M+H]+, 492.1241; HPLCb Rt 9.38 min, area
99.8%.
5.2.2. General procedure 2: conversion of 9a–9e to chlorides and
nucleophilic displacement
5.2.2.1. 4-(2-(3-(Benzyloxy)phenyl)-6-(pyridin-2-yl)pyrimidin-
4-yl)morpholine (10a). Compound 9a (50 mg, 0.14 mmol) was
dissolved in POCl3 (1 mL) and N,N-diethylaniline (1 equiv,
0.14 mmol, 0.02 mL) was added and the mixture stirred overnight
at 110 °C. After cooling the excess POCl3 was removed in vacuo, ice
water (2 ml) added to the residue. After extraction with CHCl3
(2 ꢀ 2 mL), the combined extracts were dried (MgSO4) and concen-
trated in vacuo. The residue was dissolved in dioxane (1 mL) and
diisopropylethylamine (0.5 mL) and morpholine (5 equiv,
0.70 mmol, 0.07 mL) and stirred at 85 °C overnight. The solvents
were removed in vacuo and the residue partitioned between water
(2 mL) and EtOAc (2 mL)—the organic layer was separated and the
aqueous layer was further extracted with EtOAc (2 mL) and CHCl3
(2 mL). The combined organic extracts were dried (Na2SO4) and
5.2.3. General procedure 3: deprotection of 10a–10e
5.2.3.1. 3-(4-Morpholino-6-(pyridin-2-yl)pyrimidin-2-yl)phenol
(11a). A solution of compound 10a (5 mg, 0.012 mmol) in HBr
(48% in acetic acid, 0.5 mL) was stirred at reflux for 3 h. The sol-
vents were then removed in vacuo and the residue dried at high
vacuum in the presence of sodium hydroxide to give 11a (3 mg,
61%) as a yellow solid; dH (250 MHz, DMSO-d6) 9.58 (1H, s), 9.17
(1H, d, J 8.0), 8.94–8.92 (1H, m), 8.10–8.03 (1H, m), 7.86–7.81
(2H, m), 7.50 (1H, s), 7.23 (1H, t, J 8.0), 6.85 (1H, dd, J 8.0, 1.5),
3.80–3.67 (8H, m); HPLC–MSa Rt 3.90 min; (C19H18N4O2): m/z
(ESI) 335 ([M+H]+, 100%); C19H18N4O2 requires 335.1508, found:
[M+H]+, 335.1501; HPLCa Rt 5.65 min, area 97.0%.

844
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
5.2.3.2. 3-(4-Morpholino-6-(pyridin-3-yl)pyrimidin-2-yl)phenol
(11b). Prepared from compound 10b according to general proce-
dure 3; pale brown solid, 70% yield; dH (250 MHz, DMSO-d6) 8.81
(1H, s), 8.62 (1H, d, J 8.0), 8.18 (1H, t, J 8.0), 8.00–7.90 (2H, m),
7.72–7.68 (2H, m), 8.34 (1H, t, J 7.5), 6.93 (1H, d, J 7.5), 3.83–3.79
(8H, m); HPLC–MSb Rt 4.14 min; (C19H18N4O2): m/z (ESI) 335
([M+H]+, 100%); C19H18N4O2 requires 335.1508, found: [M+H]+,
335.1517; HPLCa Rt 5.86 min, area 93.4%.
5.2.4.2. 4,6-Dichloro-N-(pyridin-3-ylmethyl)pyrimidin-2-amine
(16b) and 2,6-dichloro-N-(pyridin-3-ylmethyl)pyrimidin-4-
amine (13b). Prepared from compound 12 according to general
procedure 4; 16b (19% yield) and 13b (45% yield) were obtained
as off-white solids.
Compound 16b: Rf 0.54 (EtOAc); dH (250 MHz, CDCl3) 8.55 (1H,
d, J 2.0), 8.47 (1H, dd, J 5.0, 1.5), 7.61 (1H, dd, J 8.0, 2.0), 7.21 (1H,
dd, J 7.5, 4.5), 6.59 (1H, s), 6.06 (1H, br), 4.59 (2H, d, J 6.0); HPLC–
MSb Rt 4.20 min; (C10H8Cl2N4): m/z (ESI) 259 ([37,37M+H]+, 3%), 257
([37,35M+H]+, 70), 255 ([35,35M+H]+, 100); C10H8Cl2N4 requires
255.0204, found: [35,35M+H]+, 255.0200.
5.2.3.3. 3-(4-Morpholino-6-(pyridin-4-yl)pyrimidin-2-yl)phenol
(11c). Prepared from compound 10c according to general proce-
dure 3; pale brown solid, 58% yield; dH (250 MHz, DMSO-d6) 8.70
(2H, br), 7.96–7.91 (2H, m), 7.59 (1H, s), 7.32 (1H, t, J 8.0), 6.92
(1H, ddd, J 7.5, 2.0, 1.0), 3.89–3.86 (4H, m) 3.80–3.75 (4H, m);
HPLC–MSa Rt 3.72 min; (C19H18N4O2): m/z (ESI) 335 ([M+H]+,
100%); C19H18N4O2 requires 335.1508, found: [M+H]+, 335.1495;
HPLCa Rt 5.46 min, area 91.5%.
Compound 13b: Rf 0.30 (EtOAc); dH (250 MHz, CDCl3) 8.53–8.47
(2H, m), 7.62 (1H, d, J 7.5), 7.28–7.25 (1H, m), 6.26 (1H, s), 5.95
(1H, s, br), 4.58 (2H, s, br); HPLC–MSb Rt 2.95 min; (C10H8Cl2N4):
m/z (ESI) 259 ([37,37M+H]+, 2%), 257 ([37,35M+H]+, 46), 255
([35,35M+H]+, 100); C10H8Cl2N4 requires 255.0204, found:
[
35,35M+H]+, 255.0211.
5.2.3.4.
3-(4-(4-Chlorophenyl)-6-morpholinopyrimidin-2-yl)
5.2.4.3. 4,6-Dichloro-N-(pyridin-4-ylmethyl)pyrimidin-2-amine
(16c) and 2,6-dichloro-N-(pyridin-4-ylmethyl)pyrimidin-4-
amine (13c). Prepared from compound 12 according to general
procedure 4; 16c (21% yield) and 13c (45% yield) were obtained
as pale yellow solids.
phenol (11d). Prepared from compound 10d according to general
procedure 3; yellow solid, 72% yield; dH (250 MHz, DMSO-d6) 8.33
(2H, d, J 8.5), 7.96–7.89 (2H, m), 7.61 (2H, d, J 8.5), 7.37–7.27 (2H,
m), 6.90 (1H, ddd, J 8.5, 2.5, 1.0), 3.85–3.80 (4H, m), 3.77–3.72 (4H,
m); HPLC–MSa Rt 5.19 min; (C20H18Cl1N3O2): m/z (ESI) 368
([35M+H]+, 100%), 370 ([37M+H]+, 53); C20H18Cl1N3O2 requires
368.1166, found:[35M+H]+, 368.1156;HPLCa Rt 4.31 min, area 93.6%.
Compound 16c: Rf 0.47 (EtOAc); dH (250 MHz, CDCl3) 8.49 (2H,
d, J 6.0), 7.17 (2H, d, J 6.0), 6.60 (1H, s), 6.24 (1H, s, br), 4.61 (2H, d, J
6.5); HPLC–MSa Rt 2.46 min; (C10H8Cl2N4): m/z (ESI) 259
([37,37M+H]+, 4%), 257 ([37,35M+H]+, 51), 255 ([35,35M+H]+, 100);
5.2.3.5.
3-(4-(3,4-Dichlorophenyl)-6-morpholinopyrimidin-2-
C
10H8Cl2N4 requires 255.0204, found: [35,35M+H]+, 255.0203.
yl)phenol (11e). Prepared from compound 10e according to gen-
eral procedure 3; yellow solid, 65% yield; dH (250 MHz, DMSO-d6)
8.56 (1H, d, J 2.0), 8.33 (1H, br, d, J 8.0), 7.92–7.89 (2H, m), 7.82
(1H, d, J 8.0), 7.41 (1H, s), 7.31 (1H, t, J 8.0), 6.90 (1H, ddd, J 8.0,
2.5, 1.0), 3.87–3.83 (4H, m), 3.77–3.74 (4H, m); HPLC–MSb Rt
8.80 min; (C20H17Cl2N3O2): m/z (ESI) 402 ([35,35M+H]+, 100%), 404
([37,35M+H]+, 53), 406 ([37,37M+H]+, 13); C20H17Cl2N3O2 requires
402.0776, found: [35,35M+H]+, 402.0768; HPLCa Rt 5.70 min, area
91.6%.
Compound 13c: Rf 0.27 (EtOAc); dH (250 MHz, CDCl3) 8.62 (2H,
d, J 6.0), 7.23 (2H, d, J 6.0), 6.31 (1H, s), 4.62 (2H, s, br); HPLC–MSa
Rt 1.80 min; (C10H8Cl2N4): m/z (ESI) 259 ([37,37M+H]+, 3%), 257
([37,35M+H]+, 49), 255 ([35,35M+H]+, 100); C10H8Cl2N4 requires
255.0204, found: [35,35M+H]+, 255.0204.
5.2.4.4.
4,6-Dichloro-N-(2-(pyridin-2-yl)ethyl)pyrimidin-2-
amine (16d) and 2,6-dichloro-N-(2-(pyridin-2-yl)ethyl)pyrimi-
din-4-amine (13d). Prepared from compound 12 according to
general procedure 4; 16d (19% yield) and 13d (44% yield) were ob-
tained as pale yellow solids.
5.2.4. General procedure 4: treatment of 2,4,6-trichloropyrimi-
dine 12 with amines
Compound 16d: Rf 0.64 (CHCl3/MeOH, 9:1); dH (250 MHz,
CDCl3) 8.48 (1H, d, J 6.0), 7.54 (1H, td, J 7.5, 2.0), 7.11–7.06 (2H,
m), 6.50 (1H, s), 6.20 (1H, br), 3.80 (2H, q, J 6.0), 3.01 (2H, t, J
6.0); HPLC–MSa Rt 3.77 min; (C10H8Cl2N4): m/z (ESI) 273
([37,37M+H]+, 10%), 271 ([37,35M+H]+, 72), 269 ([35,35M+H]+, 100);
5.2.4.1. 4,6-Dichloro-N-(pyridin-2-ylmethyl)pyrimidin-2-amine
(16a) and 2,6-dichloro-N-(pyridin-2-ylmethyl)pyrimidin-4-
amine (13a). A solution of 2,4,6-trichloropyrimidine 12 (1.00 g,
5.45 mmol) in dioxane (15 mL) at 10 °C was treated with diisopro-
pylethylamine (1.1 equiv, 6.00 mmol, 1.04 mL) and dropwise with
2-aminomethylpyridine (1.1 equiv, 6.00 mmol, 0.62 mL) and stir-
red for 2 h at room temperature. TLC analysis (EtOAc/hexane,
3:1) showed conversion to 2 products. The dioxane was evaporated
in vacuo, and the residue partitioned between H2O (15 mL) and
CHCl3 (15 mL). The organic layer was separated and the aqueous
layer further extracted with CHCl3 (2 ꢀ 10 mL). The combined ex-
tracts were dried (Na2SO4) and concentrated in vacuo. Purification
by column chromatography (same eluent) gave 16a (280 mg, 20%)
and 13a (575 mg, 41%) as pale yellow solids.
Compound 16a: Rf 0.69 (EtOAc/hexane, 3:1); dH (250 MHz,
CDCl3) 8.49 (1H, d, J 4.5), 7.61 (1H, td, J 7.5, 1.5), 7.23 (1H, d, J
7.5), 7.14 (1H, dd, J 7.5, 5.0), 6.71 (1H, br), 6.56 (1H, s), 4.66 (2H,
d, J 5.0); HPLC–MSa Rt 3.44 min; (C10H8Cl2N4): m/z (ESI) 259
([37,37M+H]+, 10), 257 ([37,35M+H]+, 56), 255 ([35,35M+H]+, 100);
C10H8Cl2N4 requires 255.0204, found: [35,35M+H]+, 255.0201.
Compound 13a: Rf 0.39 (EtOAc/hexane, 3:1); dH (250 MHz,
CDCl3) 8.49 (1H, d, J 4.5), 7.66 (1H, td, J 7.5, 1.5), 7.25–7.17 (2H,
m), 6.85 (1H, br), 6.36 (1H, s, br), 4.66 (2H, s, br); HPLC–MSb Rt
5.15 min; (C10H8Cl2N4): m/z (ESI) 259 ([37,37M+H]+, 4%), 257
([37,35M+H]+, 52), 255 ([35,35M+H]+, 100); C10H8Cl2N4 requires
255.0204, found: [35,35M+H]+, 255.0202.
C
10H8Cl2N4 requires 269.0361, found: [35,35M+H]+, 269.0359.
Compound 13d: Rf 0.45 (CHCl3/MeOH, 9:1); dH (250 MHz,
CDCl3) 8.55 (1H, d, J 4.0), 7.66 (1H, td, J 7.5, 2.0), 7.23–7.19 (2H,
m), 6.66 (1H, br), 6.31 (1H, s), 3.92–3.85 (2H, m), 3.12 (2H, t, J
6.0); HPLC–MSa Rt .88 min; (C10H8Cl2N4): m/z (ESI) 273
([37,37M+H]+, 10%), 271 ([37,35M+H]+, 43), 269 ([35,35M+H]+, 100);
C
10H8Cl2N4 requires 269.0361, found: [35,35M+H]+, 269.0361.
5.2.4.5.
4,6-Dichloro-N-(2-(pyridin-3-yl)ethyl)pyrimidin-2-
amine (16e) and 2,6-dichloro-N-(2-(pyridin-3-yl)ethyl)pyrimi-
din-4-amine (13e). Prepared from compound 12 according to
general procedure 4; 16e (21% yield) and 13e (37% yield) were ob-
tained as pale yellow solids.
Compound 16e: Rf 0.45 (CHCl3/MeOH, 9:1); dH (250 MHz,
CDCl3) 8.43–8.41 (2H, m), 7.50 (1H, dt, J 7.5, 2.0), 7.20 (1H, dd, J
7.5, 5.0), 6.55 (1H, s), 5.48 (1H, br), 3.64 (2H, q, J 7.0), 2.85 (2H,
q, J 7.0); HPLC–MSa Rt 3.77 min; (C10H8Cl2N4): m/z (ESI) 273
([37,37M+H]+, 10%), 271 ([37,35M+H]+, 72), 269 ([35,35M+H]+, 100);
C
10H8Cl2N4 requires 269.0361, found: [35,35M+H]+, 269.0359.
Compound 13e: Rf 0.33 (CHCl3/MeOH, 9:1); dH (250 MHz,
CDCl3) 8.41–8.37 (2H, m), 7.48 (1H, dt, J 7.5, 1.5), 7.20 (1H, dd, J
7.5, 5.5), 6.20 (1H, s), 5.75 (1H, br), 3.63–3.61 (2H, m), 2.88 (2H,

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
845
t, J 7.0); HPLC–MSa Rt 2.69 min; (C10H8Cl2N4): m/z (ESI) 273
3.65–3.62 (10H, m), 2.98 (2H, t, J 6.5); HPLC–MSb Rt 3.70 min;
(C15H18Cl1N5O1): m/z (ESI) 320 ([35M+H]+, 100%), 322 ([37M+H]+,
29); C15H18Cl1N5O1 requires 320.1278, found: [35M+H]+, 320.1269.
([37,37M+H]+, 5%), 271 ([37,35M+H]+, 45), 269 ([35,35M+H]+, 100);
C
10H8Cl2N4 requires 269.0361, found: [35,35M+H]+, 269.0356.
5.2.4.6. 4,6-Dichloro-N-(2-(pyridin-4-yl)ethyl)pyrimidin-2-ami-
ne (16f) and 2,6-dichloro-N-(2-(pyridin-4-yl)ethyl)pyrimidin-4-
amine (13f). Prepared from compound 12 according to general
procedure 4; 16f (27% yield) and 13f (29% yield) were obtained
as pale yellow solids.
Compound 16f: Rf 0.47 (CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3)
8.46 (2H, dd, J 4.5, 1.5), 7.09 (2H, dd, J 4.5, 1.5), 6.55 (1H, s), 5.54
(1H, br), 3.66 (2H, q, J 7.0), 2.84 (2H, t, J 7.0); HPLC–MSa Rt
2.42 min; (C10H8Cl2N4): m/z (ESI) 273 ([37,37M+H]+, 5%), 271
([37,35M+H]+, 45), 269 ([35,35M+H]+, 100); C10H8Cl2N4 requires
269.0361, found: [35,35M+H]+, 269.0357.
5.2.5.5. 6-Chloro-2-morpholino-N-(2-(pyridin-3-yl)ethyl)pyrim-
idin-4-amine (14e). Prepared from compound 13e according to
general procedure 5; pale yellow solid, 51% yield; Rf 0.08 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.43 (1H, d, J 1.5), 8.39 (1H, dd,
br, J 5.5, 2.0), 7.45 (1H, dt, J 8.0, 2.0), 7.18 (1H, dd, J 8.0, 5.0), 5.62
(1H, s), 4.73 (1H, br), 3.66 (8H, br s), 3.52 (2H, br q J 6.5), 2.83
(2H, t J 7.0); HPLC–MSb Rt 3.53 min; (C15H18Cl1N5O1): m/z (ESI)
320 ([35M+H]+, 100%), 322 ([37M+H]+, 30); C15H18Cl1N5O1 requires
320.1278, found: [35M+H]+, 320.1270.
5.2.5.6. 6-Chloro-2-morpholino-N-(2-(pyridin-4-yl)ethyl)pyrim-
idin-4-amine (14f). Prepared from compound 13f according to
general procedure 5; pale brown solid, 51% yield; Rf 0.10 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.58 (2H, d, J 6.0), 7.24 (2H, d, J
6.0), 5.74 (1H, s), 4.88 (1H, br), 3.80–3.63 (10H, m), 2.98 (2H, t, J
7.0); HPLC–MSb Rt 3.21 min; (C15H18Cl1N5O1): m/z (ESI) 320
([35M+H]+, 100%), 322 ([37M+H]+, 15); C15H18Cl1N5O1 requires
320.1278, found: [35M+H]+, 320.1277.
Compound 13f: Rf 0.35 (CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3)
8.47 (2H, d, J 6.0), 7.08 (2H, d, J 6.0), 6.20 (1H, s), 5.28 (1H, br), 3.62
(2H, s, br), 2.87 (2H, t, J 7.0); HPLC–MSa Rt 2.45 min; (C10H8Cl2N4):
m/z (ESI) 273 ([37,37M+H]+, 10%), 271 ([37,35M+H]+, 45), 269
([35,35M+H]+, 100); C10H8Cl2N4 requires 269.0361, found:
[
35,35M+H]+, 269.0362.
5.2.5. General procedure 5: reaction of 13a–13f with
morpholine
5.2.5.1. 6-Chloro-2-morpholino-N-(pyridin-2-ylmethyl)pyrimi-
5.2.6. General procedure 6: reaction of 16a–16f with
morpholine
din-4-amine (14a).
A
solution of compound 13a (50 mg,
5.2.6.1. 4-Chloro-6-morpholino-N-(pyridin-2-ylmethyl)pyrimi-
0.19 mmol) in dioxane (1 mL) was treated with diisopropylethyl-
amine (1.5 equiv, 0.29 mmol, 0.05 mL) and morpholine (1.8 equiv,
0.35 mmol, 0.03 mL) and heated to 70 °C for 8 h. TLC analysis
(CHCl3/MeOH, 96:4) showed complete conversion. The dioxane
was evaporated in vacuo, and the residue partitioned between
H2O (5 ml) and CHCl3 (5 ml). The organic layer was separated
and the aqueous layer further extracted with CHCl3 (2 ꢀ 2 mL).
The combined extracts were dried (Na2SO4) and concentrated in
vacuo. Purification by column chromatography (same eluent) gave
14a (39 mg, 65%) as a colourless solid; Rf 0.60 (CHCl3/MeOH, 9:1);
dH (250 MHz, CDCl3) 8.49 (1H, d, J 4.5), 7.60 (1H, td, J 8.0, 2.0), 7.21
(1H, d, J 7.5), (1H, dd, J 7.5, 5.0), 5.85 (1H, br), 5.75 (1H, s), 4.56 (2H,
din-2-amine (17a). A solution of compound 16a (100 mg,
0.39 mmol) in dioxane (1 mL) at room temperature was treated
with diisopropylethylamine (1.5 equiv, 0.59 mmol, 0.10 mL) and
morpholine (1.5 equiv, 0.59 mmol, 0.05 mL) and stirred at room
temperature for 2 h. TLC analysis (EtOAc) then showed complete
conversion. The dioxane was evaporated in vacuo, and the residue
partitioned between H2O (5 mL) and CHCl3 (5 mL). The organic
layer was separated and the aqueous layer further extracted with
CHCl3 (2 ꢀ 2 mL). The combined extracts were dried (Na2SO4)
and concentrated in vacuo. Purification by column chromatogra-
phy (same eluent) gave 17a (101 mg, 83%) as a colourless solid;
Rf 0.33 (EtOAc); dH (250 MHz, CDCl3) 8.47 (1H, d, J 4.0), 7.56 (1H,
td, J 7.5, 2.0), 7.21 (1H, d, J 7.5), 7.09 (1H, dd, J 7.5, 5.0), 6.07 (1H,
br), 5.81 (1H, s), 4.62 (2H, d, J 5.5), 3.67–3.60 (4H, m), 3.46–3.42
(4H, m); HPLC–MSa Rt 2.48 min; (C14H16Cl1N5O1): m/z (ESI) 306
([35M+H]+, 100%), 308 ([37M+H]+, 32); C14H16Cl1N5O1 requires
306.1122, found: [35M+H]+, 306.1124.
d,
J
5.0), 3.68–3.60 (8H, m); HPLC–MSb Rt 4.09 min;
(C14H16Cl1N5O1): m/z (ESI) 306 ([35M+H]+, 100%), 308 ([37M+H]+,
29); C14H16Cl1N5O1 requires 306.1122, found: [35M+H]+, 306.1122.
5.2.5.2. 6-Chloro-2-morpholino-N-(pyridin-3-ylmethyl)pyrimi-
din-4-amine (14b). Prepared from compound 13b according to
general procedure 5; colourless solid, 76% yield; Rf 0.32 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.51 (1H, d, J 2.0), 8.46 (1H, dd,
J 5.0, 1.5), 7.57 (1H, dd, J 8.0, 2.0), 7.20 (1H, dd, J 8.0, 5.0), 5.68
(1H, s), 5.03 (1H, br), 4.49 (2H, d, J 6.0), 3.68–3.60 (8H, m);
HPLC–MSa Rt 2.63 min; (C14H16Cl1N5O1): m/z (ESI) 306 ([35M+H]+,
100%), 308 ([37M+H]+, 33); C14H16Cl1N5O1 requires 306.1122,
found: [35M+H]+, 306.1132.
5.2.6.2. 4-Chloro-6-morpholino-N-(pyridin-3-ylmethyl)pyrimi-
din-2-amine (17b). Prepared from compound 16b according to
general procedure 6; colourless solid, 33% yield; Rf 0.47 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.52 (1H, s), 8.43 (1H, d, J 4.0),
7.58 (1H, dt, J 8.0, 1.5), 7.17 (1H, dd, J 8.0, 5.0), 5.83 (1H, s), 5.61
(1H, br), 4.52 (2H, d, J 6.0), 3.65–3.62 (4H, m), 3.46–3.42 (4H, m);
HPLC–MSb Rt 3.21 min; (C14H16Cl1N5O1): m/z (ESI) 306 ([35M+H]+,
100%), 308 ([37M+H]+, 30); C14H16Cl1N5O1 requires 306.1122,
found: [35M+H]+, 306.1120.
5.2.5.3. 6-Chloro-2-morpholino-N-(pyridin-4-ylmethyl)pyrimi-
din-4-amine (14c). Prepared from compound 13c according to
general procedure 5; pale yellow solid, 58% yield; Rf 0.43 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.52 (2H, d, J 6.0), 7.33 (2H, d, J
6.0), 5.72 (1H, s), 5.36 (1H, br), 4.57 (2H, d, J 6.0), 3.65–3.55 (8H,
m); HPLC–MSb Rt 2.87 min; (C14H16Cl1N5O1): m/z (ESI) 306
([35M+H]+, 100%), 308 ([37M+H]+, 28); C14H16Cl1N5O1 requires
306.1122, found: [35M+H]+, 306.1131.
5.2.6.3. 4-Chloro-6-morpholino-N-(pyridin-4-ylmethyl)pyrimi-
din-2-amine (17c). Prepared from compound 16c according to
general procedure 6; pale yellow solid, 51% yield; Rf 0.43 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.50 (2H, d, J 6.0), 7.32 (2H, d, J
6.0), 5.86 (1H, s), 5.52 (1H, br), 4.57 (2H, d, J 6.0), 3.64–3.60 (4H,
m), 3.43–3.39 (4H, m); HPLC–MSb Rt 2.81 min; (C14H16Cl1N5O1):
m/z (ESI) 306 ([35M+H]+, 100%), 308 ([37M+H]+, 29); C14H16Cl1N5O1
requires 306.1122, found: [35M+H]+, 306.1130.
5.2.5.4. 6-Chloro-2-morpholino-N-(2-(pyridin-2-yl)ethyl)pyrim-
idin-4-amine (14d). Prepared from compound 13d according to
general procedure 5; pale yellow solid, 70% yield; Rf 0.18 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.48 (1H, dt, J 5.0, 1.5), 7.55
(1H, td, J 7.5, 2.0), 7.12–7.07 (2H, m), 5.64 (1H, s), 5.39 (1H, br),
5.2.6.4. 4-Chloro-6-morpholino-N-(2-(pyridin-2-yl)ethyl)pyrim-
idin-2-amine (17d). Prepared from compound 16d according to
general procedure 6; pale yellow solid, 58% yield; Rf 0.13 (CHCl3/

846
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
MeOH, 9:1); dH (250 MHz, CDCl3) 8.47 (1H, d, J 5.0), 7.52 (1H, td, J
7.5, 2.0), 7.09–7.04 (2H, m), 5.77 (1H, s), 5.38 (1H, br t, J 5.0), 3.78–
3.65 (6H, m), 3.57–3.46 (4H, m), 2.98 (2H, t, J 6.5); HPLC–MSb Rt
3.09 min; (C15H18Cl1N5O1): m/z (ESI) 320 ([35M+H]+, 100%), 322
([37M+H]+, 28); C15H18Cl1N5O1 requires 320.1278, found:
5.2.7.4. 3-(2-Morpholino-6-(2-(pyridin-2-yl)ethylamino)pyrimi-
din-4-yl)phenol (15d). Prepared from compound 14d according
to general procedure 7. Purification by preparative TLC (CHCl3/
MeOH, 95:5) was followed by further purification by 2 g scx car-
tridge, eluting with MeOH, then 1 M NH3 in MeOH; this gave 15d
(55% yield) as an off-white solid; Rf 0.33 CHCl3/MeOH, 9:1); dH
(250 MHz, CDCl3) 8.61 (1H, app dq, J 5.0, 1.0), 7.67 (1H, td, J 8.0,
2.0), 7.46 (2H, app t, J 8.0), 7.30–7.19 (3H, m), 6.91 (1H, ddd, J
8.0, 2.0, 1.0), 6.00 (1H, s), 5.25 (1H, br t, J 5.5), 3.84–3.72 (10H,
m), 3.14 (2H, t, J 6.5); HPLC–MSb Rt 2.60 min; (C21H23N5O2): m/z
(ESI) 378 ([M+H]+, 100%); C21H23N5O2 requires 378.1930, found:
[M+H]+, 378.1940; HPLCa Rt 3.40 min, area 92.2%.
[
35M+H]+, 320.1272.
5.2.6.5. 4-Chloro-6-morpholino-N-(2-(pyridin-3-yl)ethyl)pyrim-
idin-2-amine (17e). Prepared from compound 16e according to
general procedure 6; pale yellow solid, 50% yield; Rf 0.08 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.41–8.38 (2H, m), 7.45 (1H, dt,
J 7.5, 1.5), 7.15 (1H, dd, J 7.5, 5.0), 5.80 (1H, s), 5.18 (1H, br),
3.74–3.66 (4H, m), 3.56 (2H, q, J 7.0), 3.50–3.41 (4H, m), 2.82
(2H, t, J 7.0); HPLC–MSb Rt 3.12 min; (C15H18Cl1N5O1): m/z (ESI)
320 ([35M+H]+, 100%), 322 ([37M+H]+, 30); C15H18Cl1N5O1 requires
320.1278, found: [35M+H]+, 320.1277.
5.2.7.5. 3-(2-Morpholino-6-(2-(pyridin-3-yl)ethylamino)pyrimi-
din-4-yl)phenol (15e). Prepared from compound 14e according
to general procedure 7; pale yellow solid, 52% yield; Rf 0.38
CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3) 8.42–8.40 (2H, m), 7.48
(1H, br d, J 8.0), 7.39–7.34 (2H, m), 7.21–7.14 (2H, m), 6.82 (1H,
dd, J 7.0, 1.5), 5.92 (1H, s), 4.75 (1H, br t, J 6.0), 3.75–3.65 (8H,
m), 3.55 (2H, q, J 6.5), 2.81 (2H, t, J 6.5); HPLC–MSb Rt 2.49 min;
(C21H23N5O2): m/z (ESI) 378 ([M+H]+, 100%); C21H23N5O2 requires
378.1930, found: [M+H]+, 378.1924; HPLCa Rt 3.26 min, area 95.5%.
5.2.6.6. 4-Chloro-6-morpholino-N-(2-(pyridin-4-yl)ethyl)pyrim-
idin-2-amine (17f). Prepared from compound 16f according to
general procedure 6; pale yellow solid, 39% yield; Rf 0.10 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.45 (2H, d, J 6.0), 7.11 (2H, d, J
6.0), 5.82 (1H, s), 4.98 (1H, br), 3.70–3.66 (4H, m), 3.59 (2H, q, J
7.0), 3.49 (4H, m), 2.83 (2H, t, J 7.0); HPLC–MSb Rt 2.87 min;
(C15H18Cl1N5O1): m/z (ESI) 320 ([35M+H]+, 100%), 322 ([37M+H]+,
35); C15H18Cl1N5O1 requires 320.1278, found: [35M+H]+, 320.1285.
5.2.7.6. 3-(2-Morpholino-6-(2-(pyridin-4-yl)ethylamino)pyrimi-
din-4-yl)phenol (15f). Prepared from compound 14f according to
general procedure 7; pale yellow solid, 43% yield; Rf 0.29 CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.45 (2H, d, br, J 4.5), 7.39–7.37
(2H, m), 7.19 (1H, t, J 8.0), 7.09 (2H, d, J 6.0), 6.83 (1H, ddd, J 8.0,
2.0, 1.0), 5.96 (1H, s), 4.70 (1H, t, J 6.0), 3.78–3.74 (4H, m), 3.70–
3.67 (4H, m), 3.56 (2H, q, J 6.5), 2.86 (2H, t, J 6.5); HPLC–MSb Rt
2.34 min; (C21H23N5O2): m/z (ESI) 378 ([M+H]+, 100%);
5.2.7. General procedure 7: Suzuki coupling of 14a–14f and 17a–
17f
5.2.7.1. 3-(2-Morpholino-6-(pyridin-2-ylmethylamino)pyrimi-
din-4-yl)phenol (15a). A mixture of compound 14a (20 mg,
0.065 mmol) and PdCl2(dppf) (5 mol %, 2.5 mg) in DME (0.5 mL)
was stirred at room temperature for 15 min. 3-hydroxyphenylbo-
ronic acid (3 equiv, 0.195 mmol, 27 mg), Na2CO3 (2.5 equiv,
0.16 mmol, 17 mg) and five drops of water were added and the
mixture heated at 80 °C for 18 h. The solvents were then removed
in vacuo and the residue partitioned between water (3 mL) and
chloroform (4 mL). The organic phase was dried (Na2SO4) and con-
centrated in vacuo. Purification by preparative TLC (CHCl3/MeOH,
95:5) gave 15a (6 mg, 25%) as an off-white solid; Rf 0.37 CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.50 (1H, d, J 4.5), 7.62 (1H, td,
J 8.0, 2.0), 7.35–7.29 and 7.20–7.15 (5H, 2 ꢀ m), 6.83 (1H, br d, J
8.0), 5.94 (1H, s), 5.74 (1H, br), 4.64 (2H, d, J 5.5), 3.66–3.62 (8H,
m); HPLC–MSb Rt 3.10 min; (C20H21N5O2): m/z (ESI) 364 ([M+H]+,
100%); C20H21N5O2 requires 364.1774, found: [M+H]+, 364.1779;
HPLCa Rt 3.73 min, area 92.2%.
C
21H23N5O2 requires 378.1930, found: [M+H]+, 378.1932; HPLCa
Rt 2.78 min, area 90.1%.
5.2.7.7. 3-(6-Morpholino-2-(pyridin-2-ylmethylamino)pyrimi-
din-4-yl)phenol (18a). Prepared from compound 17a according
to general procedure 7. Purification by prep TLC (CHCl3/MeOH,
9:1) was followed by further purification by scx cartridge, eluting
with MeOH, then 1 M NH3 in MeOH; this gave 18a (20% yield) as
a pale yellow solid; Rf 0.33 (CHCl3/MeOH, 9:1); dH (250 MHz,
CDCl3) 8.55 (1H, d, J 4.0), 7.64 (1H, td, J 8.0, 2.0), 7.42–7.36 (3H,
m), 7.33 (1H, d, J 8.0), 7.22 (1H, t, J 8.0), 6.89 (1H, ddd, J 8.0, 2.0,
1.0), 6.20 (1H, s), 6.09 (1H, br), 4.76 (2H, d, J 5.5), 3.74–3.71 (4H,
m), 3.58–3.54 (4H, m); HPLC–MSb Rt 3.08 min; (C20H21N5O2): m/z
(ESI) 364 ([M+H]+, 100%); C20H21N5O2 requires 364.1774, found:
[M+H]+, 364.1763; HPLCa Rt 3.66 min, area 96.8%.
5.2.7.2. 3-(2-Morpholino-6-(pyridin-3-ylmethylamino)pyrimi-
din-4-yl)phenol (15b). Prepared from compound 14b according
to general procedure 7; pale yellow solid, 25% yield; Rf 0.27
CHCl3/MeOH, 9:1); dH (250 MHz, CD3OD) 8.45 (1H, s), 8.30 (1H,
d, J 4.0), 7.74 (1H, d, J 8.0), 7.32–7.26 (3H, m), 7.12 (1H, t, J 8.0),
6.73 (1H, ddd, J 8.0, 2.5, 1.0), 6.18 (1H, s), 4.53 (2H, s), 3.67–3.64
(4H, m), 3.60–3.57 (4H, m); HPLC–MSb Rt 2.50 min;
(C20H21N5O2): m/z (ESI) 364 ([M+H]+, 100%); C20H21N5O2 requires
364.1774, found: [M+H]+, 364.1786; HPLCa Rt 3.24 min, area
>99.9%.
5.2.7.8. 3-(6-Morpholino-2-(pyridin-3-ylmethylamino)pyrimi-
din-4-yl)phenol (18b). Prepared from compound 17b according
to general procedure 7; pale yellow solid, 42% yield; Rf 0.25
(CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3) 8.73 (1H, br s), 8.51
(1H, d, J 3.5), 7.73 (1H, d, J 8.0), 7.45 (1H, br, q, J 1.5), 7.37–7.24
(3H, m), 7.28 (1H, t, J 8.0), 6.93 (1H, ddd, J 8.0, 2.5, 1.0), 6.29 (1H,
s), 5.66 (1H, br), 4.63 (2H, d, J 6.0), 3.79–3.75 (4H, m), 3.63–3.59
(4H, m); HPLC–MSb Rt 2.49 min; (C20H21N5O2): m/z (ESI) 364
([M+H]+, 100%); C20H21N5O2 requires 364.1774, found: [M+H]+,
364.1765; HPLCa Rt 3.19 min, area 92.3%.
5.2.7.3. 3-(2-Morpholino-6-(pyridin-4-ylmethylamino)pyrimi-
din-4-yl)phenol (15c). Prepared from compound 14c according
to general procedure 7; off-white solid, 40% yield; Rf 0.25 CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.49 (2H, d, J 5.0), 7.40–7.36
and 7.23–7.18 (5H, 2 ꢀ m), 6.82 (1H, ddd, J 8.0, 2.5, 1.0), 6.06
(1H, s), 5.07 (1H, br t, J 6.0), 4.56 (2H, d, J 6.0), 3.71–3.69 (4H, m),
3.67–3.62 (4H, m); HPLC–MSb Rt 2.20 min; (C20H21N5O2): m/z
(ESI) 364 ([M+H]+, 100%); C20H21N5O2 requires 364.1774, found:
[M+H]+, 364.1768; HPLCa Rt 2.02 min, area 92.4%.
5.2.7.9. 3-(6-Morpholino-2-(pyridin-4-ylmethylamino)pyrimi-
din-4-yl)phenol (18c). Prepared from compound 17c according
to general procedure 7; pale yellow solid, 31% yield; Rf 0.26
(CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3) 8.41 (2H, d, J 6.0), 7.39
(1H, br, t, J 2.0), 7.25–7.20 (2H, m), 7.15 (2H, d, J 6.0), 6.80 (1H,
ddd, J 7.5, 2.5, 1.0), 6.18 (1H, s), 5.94 (1H, br), 4.48 (2H, d, J 5.5),
3.67–3.63 (4H, m), 3.51–3.47 (4H, m); HPLC–MSb Rt 2.14 min;

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
847
(C20H21N5O2): m/z (ESI) 364 ([M+H]+, 100%); C20H21N5O2 requires
364.1774, found: [M+H]+, 364.1758; HPLCa Rt 1.67 min, area 95.9%.
3.53–3.50 (4H, m); HPLC–MSb Rt 7.16 min; (C14H15Cl1N4O2): m/z
(ESI) 307 ([35M+H]+, 100%), 309 ([37M+H]+, 29); C14H15Cl1N4O2 re-
quires 307.0962, found: [35M+H]+, 307.0958.
5.2.7.10. 3-(6-Morpholino-2-(2-(pyridin-2-yl)ethylamino)pyr-
imidin-4-yl)phenol (18d). Prepared from compound 17d accord-
ing to general procedure 7; pale yellow solid, 14% yield; Rf 0.52
(EtOAc/MeOH, 9:1); dH (250 MHz, CDCl3) 8.58 (1H, ddd, J 4.0, 2.0,
1.0), 7.64 (1H, td, J 8.0, 2.0), 7.56–7.52 (1H, m), 7.38–7.23 (3H,
m), 7.16 (1H, ddd, J 7.5, 5.0, 1.0), 6.95 (1H, ddd, J 8.0, 2.5, 1.0),
6.25 (1H, s), 3.89 (2H, q, J 7.0), 3.83–3.80 (4H, m), 3.71–3.67 (4H,
m), 3.16 (2H, t, J 7.0); HPLC–MSb Rt 2.75 min; (C21H23N5O2): m/z
(ESI) 378 ([M+H]+, 100%); C21H23N5O2 requires 378.1930, found:
[M+H]+, 378.1935; HPLCa Rt 3.42 min, area 99.2%.
Compound 22: Rf 0.63 (CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3)
8.51 (1H, d, J 4.5), 7.63 (1H, td, J 8.0, 2.0), 7.31 (1H, d, J 8.0), 7.16
(1H, dd, J 7.0, 5.5), 6.08 (1H, s), 5.40 (2H, s), 3.68–3.59 (8H, m);
HPLC–MSb Rt 5.21 min; (C14H15Cl1N4O2): m/z (ESI) 307 ([35M+H]+,
100%), 309 ([37M+H]+, 29); C14H15Cl1N4O2 requires 307.0962,
found: [35M+H]+, 307.0953.
5.2.7.14. 3-(2-Morpholino-6-(pyridin-2-ylmethoxy)pyrimidin-4-
yl)phenol (19). Prepared from compound 21 according to general
procedure 7. Purification by preparative TLC (CHCl3/MeOH, 95:5)
was followed by further purification by 2 g scx cartridge, eluting
with MeOH, then 1 M NH3 in MeOH; this gave 19 (13 mg, 43%)
as a colourless solid; Rf 0.44 CHCl3/MeOH, 9:1); dH (250 MHz,
CDCl3) 8.54 (1H, d, J 4.5), 7.67 (1H, td, J 7.5, 2.0), 7.42–7.38 and
7.24–7.17 (5H, 2 ꢀ m), 6.85 (1H, ddd, J 8.0, 2.5, 1.0), 6.39 (1H, s),
5.45 (2H, s), 3.70–3.61 (8H, m); HPLC–MSb Rt 7.11 min;
(C20H20N4O3): m/z (ESI) 365 ([M+H]+, 100%); C20H20N4O3 requires
365.1614, found: [M+H]+, 365.1608; HPLCa Rt 5.97 min, area 94.6%.
5.2.7.11. 3-(6-Morpholino-2-(2-(pyridin-3-yl)ethylamino)pyr-
imidin-4-yl)phenol (18e). Prepared from compound 17e accord-
ing to general procedure 7; pale yellow solid, 23% yield; Rf 0.44
(EtOAc/MeOH, 9:1); dH (250 MHz, CDCl3) 8.44 (1H, br s), 8.37
(1H, dd, J 5.0, 1.0), 7.46 (1H, dt, J 8.0, 2.0), 7.37 (1H, s), 7.27–7.09
(3H, m), 6.80 (1H, ddd, J 8.0, 2.5, 1.0), 6.15 (1H, s), 5.28 (1H, br),
3.72–3.68 (4H, m), 3.59–3.53 (6H, m), 2.82 (2H, t, J 7.0); HPLC–
MSb Rt 2.60 min; (C21H23N5O2): m/z (ESI) 378 ([M+H]+, 100%);
C21H23N5O2 requires 378.1930, found: [M+H]+, 378.1928; HPLCa
Rt 3.35 min, area 94.3%.
5.2.7.15. 3-(6-Morpholino-2-(pyridin-2-ylmethoxy)pyrimidin-4-
yl)phenol (20). Prepared from compound 22 according to general
procedure 7; pale yellow solid, 49% yield; Rf 0.46 (CHCl3/MeOH,
9:1); dH (250 MHz, CDCl3) 8.50 (1H, d, J 4.5), 7.62 (1H, td, J 8.0,
1.5), 7.51–7.47 (2H, m), 7.34 (1H, d, J 8.0), 7.18 (1H, t, J 8.0),
7.16–7.11 (1H, m), 6.86 (1H, ddd, J 8.0, 2.5, 1.0), 6.85 (1H, s), 5.52
(2H, s), 3.70–3.65 (4H, m), 3.58–3.54 (4H, m); HPLC–MSb Rt
4.85 min; (C20H20N4O3): m/z (ESI) 365 ([M+H]+, 100%);
5.2.7.12. 3-(6-Morpholino-2-(2-(pyridin-4-yl)ethylamino)pyr-
imidin-4-yl)phenol (18f). Prepared from compound 17f accord-
ing to general procedure 7; pale yellow solid, 28% yield; Rf 0.40
(EtOAc/MeOH, 9:1); dH (250 MHz, CDCl3) 8.33 (2H, d, J 6.0), 7.36
(1H, s), 7.00 (2H, d, J 6.0), 7.24–7.10 (2H, m), 6.81–6.72 (1H, br),
6.13 (1H, s), 5.40 (1H, br), 3.69 (4H, t, J 5.0), 3.56–3.50 (6H, m),
2.76 (2H, t, J 7.0); HPLC–MSb Rt 2.37 min; (C21H23N5O2): m/z (ESI)
378 ([M+H]+, 100%); C21H23N5O2 requires 378.1930, found:
[M+H]+, 378.1934; HPLCa Rt 3.15 min, area 90.8%.
C
21H23N5O2 requires 365.1614, found: [M+H]+, 365.1605; HPLCa
Rt 4.84 min, area 97.6%.
5.2.7.16. 4,40-(6-(Pyridin-2-ylmethoxy)pyrimidine-2,4-diyl)dim-
orpholine (23). Compound 21 (306 mg, 1.0 mmol) was stirred in
morpholine (0.5 mL) and heated to 50 °C. After 4 h, the reaction
mixture was poured into water and the products extracted into
ethyl acetate. The organic layers were rinsed (brine) and dried
(Na2SO4) and evaporated to give 23 (327 mg, 92%) as a colourless
solid; Rf 0.88 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3) 8.56 (1H,
d, J 5.0), 7.66 (1H, td, J 7.5, 1.5), 7.40 (1H, d, J 7.5), 7.18 (1H, dd, J
7.5, 5.0), 5.45 (2H, s), 5.40 (1H, s), 3.74 (4H, t, J 4.5), 3.68 (8H,
br), 3.51 (4H, t, J 5.0); dC (125 MHz, CDCl3) 170.6 (C), 165.2 (C),
160.9 (C), 157.8 (C), 149.1 (CH), 136.5 (CH), 122.3 (CH), 121.1
(CH), 76.2 (CH), 67.8 (CH2), 66.8 (CH2), 66.6 (CH2), 44.7 (CH2),
44.4 (CH2); HPLC–MSb Rt 5.93 min; (C18H23N5O3): m/z (ESI) 358
([M+H]+, 100%); C18H23N5O3 requires 358.1879, found: [M+H]+,
358.1882.
5.2.7.13. Preparation of 4-(4-chloro-6-(pyridin-2-ylmeth-
oxy)pyrimidin-2-yl)morpholine 21 and 4-(6-chloro-2-(pyridin-
2-ylmethoxy)pyrimidin-4-yl)morpholine 22. 248 mg of NaH
(60% in mineral oil, 10.36 mmol) was added to a solution of 2-pyr-
idinemethanol (0.90 equiv, 9.81 mmol, 0.95 mL) in THF (20 ml) at
room temperature and stirred for 30 min. After cooling to –78 °C,
2,4,6-trichloropyrimidine 12 (2 g, 10.90 mmol) was added drop-
wise and the reaction allowed to warm to room temperature and
stirred for 3 h. Saturated aqueous NH4Cl (20 mL) was added and
the mixture extracted with EtOAc (20 mL). The organic phase
was dried (MgSO4) and evaporated in vacuo. Column chromatogra-
phy (EtOAc/DCM, 1:1) gave a mixture of two products (491 mg,
18%) as a pale yellow solid and in a ratio of 2.4:1, with the 4-isomer
tentatively assigned as the major component; Rf 0.81 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 8.66–8.61 (2H, m, 1 ꢀ major,
1 ꢀ minor), 7.75 (1H, td, J 8.0, 2.0, 1 ꢀ major), 7.74 (1H, td, J 8.0,
2.0, 1 ꢀ minor), 7.53 (1H, d, J 8.0, 1 ꢀ minor), 7.44 (1H, d, J 8.0,
1 ꢀ major), 7.32–7.24 (1H, 2H, m, 1 ꢀ major, 1 ꢀ minor), 7.09
(1H, s, 1 ꢀ minor), 6.84 (1H, s, 1 ꢀ major), 5.59 (2H, s, 2 ꢀ minor),
5.58 (2H, s, 2 ꢀ major); HPLC–MSa Rt major, 3.99 min; minor,
3.87 min; (C10H8Cl2N4): m/z (ESI) 260 ([37,37M+H]+, 5%), 258
([37,35M+H]+, 35), 256 ([35,35M+H]+, 100).
This mixture (100 mg, 0.39 mmol) was treated with diisopro-
pylethylamine (2.5 equiv, 0.98 mmol, 0.17 mL) and morpholine
(3.5 equiv, 1.37 mmol, 0.12 mL) in dioxane (1 mL) according to
general procedure 5. Purification by preparative TLC (CHCl3/MeOH,
95:5) gave 21 (60 mg, 50%) and 22 (31 mg, 26%) as colourless
solids.
Compound 21: Rf 0.80 (CHCl3/MeOH, 9:1); dH (250 MHz, CDCl3)
8.49 (1H, d, J 5.0), 7.63 (1H, td, J 8.0, 2.0), 7.44 (1H, d, J 8.0), 7.14
(1H, dd, J 8.0, 5.0), 6.11 (1H, s), 5.41 (2H, s), 3.68–3.64 (4H, m),
5.2.7.17. 4,40-(2-(Pyridin-2-ylmethoxy)pyrimidine-4,6-diyl)dim-
orpholine (24). Prepared from compound 22 using the same pro-
cedure as for 23; compound 24 was obtained as a colourless solid
(91% yield); Rf 0.75 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3) 8.51
(1H, d, J 4.5), 7.62 (1H, td, J 7.5, 1.5), 7.46 (1H, d, J 8.0), 7.13 (1H,
dd, J 6.5, 4.5), 5.43 (2H, s), 5.23 (1H, s), 3.69 (8H, t, J 5.0), 3.48
(8H, t, J 5.0); dC (125 MHz, CDCl3) 165.3 (C), 164.1 (C), 158.1 (C),
148.8 (CH), 136.5 (CH), 122.1 (CH), 121.0 (CH), 75.9 (CH), 68.9
(CH2), 66.5 (CH2), 44.7 (CH2); HPLC–MSb Rt 4.37 min;
(C18H23N5O3): m/z (ESI) 358 ([M+H]+, 100%); C18H23N5O3 requires
358.1879, found: [M+H]+, 358.1873.
5.2.7.18.
2-Morpholino-N-(2-(pyridin-2-yl)ethyl)pyrimidin-4-
amine (25). Compound 14d (50 mg, 0.16 mmol), 5% palladium
on carbon (15 mg, 0.007 mmol, 4 mol %) and potassium acetate
(100 mg, 1.02 mmol, 6 equiv) were stirred in AcOH (3 mL) under
an atmosphere of H2 for 18 h. The reaction mixture was added to

848
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
saturated NaHCO3 (3 mL), then DCM (2 mL) was added; the organic
layer was collected, rinsed (brine), dried (Na2SO4) and concen-
trated to give a yellow oil. The residue was filtered through a plug
of Celite to give 25 (30 mg, 68%) as a colourless solid; Rf 0.23
(CHCl3/MeOH 9:1); dH (500 MHz, CD3OD) 8.46 (1H, d, J 6.0), 7.74
(1H, td, J 6.5, 2.0), 7.67 (1H, d, J 6.5), 7.30 (1H, d, J 7.0), 7.26 (1H,
ddd, J 7.0, 5.0, 1.0), 5.80 (1H, d, J 6.0), 3.70–3.74 (6H, m), 3.64–
3.67 (4H, m), 3.06 (2H, t, J 7.0); dC (500 MHz, CD3OD) 161.0 (C),
159.2 (C), 158.1 (C), 152.3 (CH), 147.1 (CH), 136.9 (CH), 122.3
(CH), 120.3 (CH), 75.9 (CH), 65.2 (CH2), 43.0 (CH2), 35.9 (CH2)1
HPLC–MSa Rt 0.68 min; (C15H19N5O1): m/z (ESI) 286 ([M+H]+,
100%); C15H19N5O1 requires 286.1662, found: [M+H]+, 286.1668.
9:1); dH (500 MHz, CD3OD) 8.40 (1H, s), 8.35 (1H, d, J 4.0), 8.04
(1H, s), 7.68 (1H, d, J 8.0), 7.60 (1H, dd, J 8.5, 1.5) 7.57 (1H, d, J
8.5), 7.32 (1H, dd, J 7.5, 5.0), 7.27 (1H, d, J 3.0), 6.46 (1H, dd, J
3.0, 0.5), 6.24 (1H, s), 3.84–3.82 (4H, m), 3.78–3.76 (4H, m), 3.66
(2H, t,
J 7.0), 2.96 (2H, t, J 7.0); HPLC–MS Rt 2.85 min;
(C22H24N6O1) m/z (ESI) 401 ([M+H]+, 100%); C22H24N6O1 requires
401.2090 for [M+H]+, found: 401.2098; HPLCb Rt 3.88 min, purity
>99%.
5.2.8.3.
6-(1H-indol-6-yl)-2-morpholino-N-(2-(pyridin-4-yl)
ethyl)pyrimidin-4-amine (27c). Prepared from compound 14f
according to general procedure 8. Purification by method C gave
27c (41% yield) as a pale yellow solid; mp >230 °C; Rf 0.43
(CHCl3/MeOH, 9:1); dH (500 MHz, CD3OD) 8.43 (2H, dd, J 4.5, 1.5),
8.07 (1H, br s), 7.60 (1H, dd, J 8.5, 1.5), 7.56 (1H, d, J 8.5), 7.34
(2H, dd, J 4.5, 1.5), 7.31 (1H, d, J 3.0), 6.46 (1H, d, J 3.0), 6.28 (1H,
s), 3.72 (2H, t, J 7.0) 3.84–3.82 (4H, m), 3.78–3.76 (4H, m) 3.00
(2H, t, J 7.0); HPLC–MS Rt 2.66 min; (C22H24N6O1) m/z (ESI) 401
([M+H]+, 100%); C22H24N6O1 requires 401.2090 for [M+H]+, found:
401.2083; HPLCb Rt 3.60 min, purity 99%.
5.2.7.19.
4-Morpholino-N-(2-(pyridin-2-yl)ethyl)pyrimidin-2-
amine (26). From compound 17d, and using the same procedure
as for 25; compound 26 was obtained as a colourless solid (78%
yield); Rf 0.19 (CHCl3/MeOH 9:1); dH (500 MHz, DMSO-d6, 330 K)
8.49 (1H, d, J 6.0), 7.82 (1H, d, J 6.0), 7.67 (1H, td, J 7.5, 2.0), 7.24
(1H, d, J 7.5), 7.19 (1H, dd, J 7.5, 5.0), 6.33 (1H, br), 5.99 (1H, d, J
6.0), 3.65–3.63 (4H, m), 3.62–3.58 (2H, m), 3.51–3.49 (4H, m),
2.96 (2H, t, J 7.5); dC (500 MHz, CD3OD) 164.2 (C), 162.8 (C),
161.0 (C), 156.6 (CH), 149.8 (CH), 138.5 (CH), 125.2 (CH), 123.0
(CH), 94.1 (CH), 67.7 (CH2), 45.3 (CH2), 42.1 (CH2), 38.8 (CH2);
HPLC–MSa Rt 0.63 min; (C15H19N5O1): m/z (ESI) 286 ([M+H]+,
100%); C15H19N5O1 requires 286.1668, found: [M+H]+, 286.1671.
5.2.8.4. N-(3-(2-Morpholino-6-(2-(pyridin-2-yl)ethylamino)pyr-
imidin-4-yl)phenyl)methanesulfonamide (27d). Prepared from
compound 14d according to general procedure 8. Purification by
method A, followed by method B (CHCl3/MeOH, 9:1) gave 27d
(31% yield) as an off-white solid; mp 155–158 °C; Rf 0.47 (CHCl3/
MeOH, 9:1); dH (500 MHz, CD3OD) 8.46 (1H, d, J 4.0), 7.90 (1H, t,
J 2.0), 7.74 (1H, td, J 7.5, 2.0), 7.70 (1H, d, J 8.0), 7.38 (1H, t, J 8.0),
7.33–7.29 (2H, m), 7.25 (1H, dd, J 7.5, 1.5), 6.22 (1H, s), 3.81–3.73
(10H, m), 3.09 (2H, t, J 7.0), 2.97 (3H, s, CH3); HPLC–MSb Rt
2.68 min; (C22H26N6O3S1) m/z (ESI) 455 ([M+H]+, 100%);
5.2.8. General procedure 8: Suzuki coupling of intermediates
14d–14f, 17d–17f, 21 and 22 with aryl or heteroaryl boronic
acids or esters
The chloropyrimidine (1.0 equiv), boronic acid or ester
(2.2 equiv), Bedford catalyst 29 (0.05 equiv) and 2 M aqueous
Na2CO3 (2.2 equiv) were dissolved in 1,2-DME. The solution was
degassed and backfilled with nitrogen, then stirred with micro-
wave heating at 150 °C for 30 min. The reaction mixture was
C
22H26N6O3S1 requires 455.1865 for [M+H]+, found: 455.1885;
HPLCb Rt 3.62 min, purity 94%.
cooled, absorbed onto
a
plug of silica (100 mg) and eluted
5.2.8.5. N-(3-(2-Morpholino-6-(2-(pyridin-3-yl)ethylamino)pyr-
imidin-4-yl)phenyl)methanesulfonamide (27e). Prepared from
compound 14e according to general procedure 8. Purification by
method A, followed by method B (CHCl3/MeOH, 9:1) gave 27e
(27% yield) as an off-white solid; mp 208–211 °C; Rf 0.43 (CHCl3/
MeOH, 9:1); dH (500 MHz, CDCl3) 8.51 (1H, d, J 2.0), 8.50 (1H, dd,
J 5.0, 2.0), 7.81 (1H, br t, J 2.0), 7.77 (1H, dt, J 8.0, 2.0), 7.55 (1H,
dt, J 8.0, 2.0), 7.41 (1H, t, J 8.0), 7.33 (1H, ddd, J 8.0, 2.0, 1.0), 7.26
(1H, dd, J 7.5, 5.0), 6.11 (1H, s), 4.79 (1H, br t, J 6.5), 3.86–3.84
(4H, m), 3.79–3.77 (4H, m), 3.68 (2H, dt, J 7.0, 6.5), 3.01 (3H, s),
2.95 (2H, t, J 7.0); HPLC–MS Rt 2.55 min; (C22H26N6O3S1) m/z
(ESI) 455 ([M+H]+, 100%); C22H26N6O3S1 requires 455.1865 for
[M+H]+, found: 455.1871; HPLCb Rt 3.47 min, purity 97%.
(CHCl3/MeOH, 9:1). The crude product was obtained by evaporat-
ing the filtrate in vacuo and purified by one of the following
methods:
Method A: The crude compound was dissolved in methanol and
purified using an SCX-2 ion exchange column, eluting first with
MeOH, then 2 M NH3 in MeOH.
Method B: Purification by preparative TLC using the specified
eluent.
Method C: Recrystallisation from MeOH/CHCl3/hexane, 1:1:4.
5.2.8.1. 6-(1H-indol-6-yl)-2-morpholino-N-(2-(pyridin-2-yl)ethyl)
pyrimidin-4-amine (27a). Prepared from compound 14d accord-
ing to general procedure 8, using 14d (50 mg, 0.16 mmol), 29
(0.05 equiv, 4.9 mg), indole 6-boronic acid (2.2 equiv, 0.34 mmol,
55 mg), 2 M Na2CO3 (2.2 equiv, 0.34 mmol, 0.17 mL) and DME
(1 mL). Purification by method A, followed by method B (EtOAc/
MeOH, 9:1) gave 27a (10 mg, 16%) as an off-white solid; mp
204–208 °C; Rf 0.68 (CHCl3/MeOH, 9:1); dH (500 MHz, CD3OD)
8.45 (1H, d, J 4.5), 8.05 (1H, s), 7.70 (1H, td, J 8.0, 2.0), 7.61 (1H,
dd, J 8.5, 1.0), 7.57 (1H, d, J 8.5), 7.29 (1H, d, J 8.0), 7.27 (1H, d, J
3.0), 7.22 (1H, dd, J 6.5, 4.5), 6.46 (1H, dd, J 3.0, 0.5), 6.25 (1H, s),
3.84–3.82 (4H, m), 3.78–3.74 (6H, m), 3.09 (2H, t, J 7.0); HPLC–
MS Rt 2.89 min; (C22H24N6O1) m/z (ESI) 401 ([M+H]+, 100%);
5.2.8.6. N-(3-(2-Morpholino-6-(2-(pyridin-4-yl)ethylamino)pyr-
imidin-4-yl)phenyl)methanesulfonamide (27f). Prepared from
compound 14f according to general procedure 8. Purification by
method B (EtOAc/MeOH, 9:1), followed by method C gave 27f
(6% yield) as an off-white solid; mp 134–136 °C; Rf 0.43 (CHCl3/
MeOH, 9:1; dH (500 MHz, CDCl3) 8.57 (2H, d, J 5.0), 7.81 (1H, s),
7.77 (1H, d, J 8.0), 7.43 (1H, t, J 8.0), 7.32 (1H, d, J 8.0), 7.18 (2H,
d, J 5.0), 6.09 (1H, s), 3.89–3.87 (4H, m), 3.81–3.80 (4H, m), 3.73
(2H, dt, J 7.0, 6.5), 3.04 (3H, s), 2.27 (2H, t, J 7.0); HPLC–MS Rt
2.28 min; (C22H26N6O3S1) m/z (ESI) 455 ([M+H]+, 100%);
C
22H24N6O1 requires 401.2090 for [M+H]+, found: 401.2107; HPLCb
Rt 3.80 min, purity 96%.
C
22H26N6O3S1 requires 455.1865 for [M+H]+, found: 455.1873;
HPLCa Rt 1.81 min, purity >99%.
5.2.8.2.
6-(1H-indol-6-yl)-2-morpholino-N-(2-(pyridin-3-yl)
5.2.8.7. 6-(1H-Indazol-4-yl)-2-morpholino-N-(2-(pyridin-2-yl)
ethyl)pyrimidin-4-amine (27g). Prepared from compound 14d
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1) gave 27g (19% yield) as a pale yellow solid;
mp 120–130 °C; Rf 0.37 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3),
ethyl)pyrimidin-4-amine (27b). Prepared from compound 14e
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1) followed by method C gave 27b (25% yield)
as a pale brown solid; mp 217–218 °C; Rf 0.53 (CHCl3/MeOH,

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
849
8.65 (1H, s), 8.58 (1H, d, J 5.0), 7.62 (1H, td, J 7.5, 1.5), 7.59 (1H, d, J
7.5), 7.53 (1H, d, J 7.5), 7.43 (1H, t, J 7.5), 7.19 (1H, d, J 7.5), 7.16 (1H,
dd, J 7.5, 5.0), 6.23 (1H, s), 5.39 (1H, br t, J 5.0), 3.90–3.83 (6H, m),
3.81–3.79 (4H, m), 3.13 (2 H, t, J 6.5); HPLC–MS Rt 2.31 min;
(C22H23N7O1) m/z (ESI) 402 ([M+H]+, 100%); C22H23N7O1 requires
402.2042 for [M+H]+, found: 402.2042; HPLCb Rt 3.52 min, purity
96%.
8.5), 7.38–7.37 (3H, m), 6.52 (1H, s), 6.51 (1H, dd, J 3.0, 0.5),
3.80–3.74 (10H), 3.04 (2H, t,
J 7.0); HPLC–MS Rt 2.76 min;
(C22H24N6O1) m/z (ESI) 401 ([M+H]+, 100%); C22H24N6O1 requires
401.2090 for [M+H]+, found: 401.2108; HPLCb Rt 3.86 min, purity
>99%.
5.2.8.13.
N-(3-(6-Morpholino-2-(2-(pyridin-2-yl)ethylamino)
pyrimidin-4-yl)phenyl)methanesulfonamide (28d). Prepared
from compound 17d according to general procedure 8. Purification
by method A gave 28d (75% yield) as an off-white solid; mp 90–
95 °C; Rf 0.36 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3) 8.54 (1H,
d, J 5.0), 7.76 (1H, br), 7.64 (1H, d, J 6.5), 7.59 (1H, td, J 7.5, 2.0),
7.35 (1H, t, J 7.5), 7.31 (1H, d, J 8.0), 7.19 (1H, d, J 7.5), 7.12 (1H,
dd, J 7.5, 5.0), 6.21 (1H, s), 5.60 (1H, br), 3.85 (2H, td, J 6.5, 6.0),
3.63 (4H, t, J 5.0), 3.52 (4H, t, J 4.5), 3.12 (2H, t, J 7.0), 2.98 (3H,
s); HPLC–MS Rt 2.53 min; (C22H26N6O3S1) m/z (ESI) 455 ([M+H]+,
100%); C22H26N6O3S1 requires 455.1865 for [M+H]+, found:
455.1870; HPLCb Rt 3.59 min, purity 98%.
5.2.8.8. 6-(1H-Indazol-4-yl)-2-morpholino-N-(2-(pyridin-3-yl)
ethyl)pyrimidin-4-amine (27h). Prepared from compound 14e
according to general procedure 8. Purification by method A gave
27h (16% yield) as an off-white solid; mp 100–105 °C; Rf 0.24
(CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3) 8.65 (1H, s), 8.54 (1H, d,
J 1.5), 8.52 (1H, dd, J 5.0, 1.5), 7.58–7.54 (2H, m), 7.53 (1H, d, J
8.5), 7.41 (1H, dd, J 8.0, 7.5), 7.25 (1H, dd, J 7.5, 5.0), 6.20 (1H, s),
4.87 (1H, t, J 5.5), 3.90–3.88 (4H, m), 3.81–3.79 (4H, m), 3.71–
3.67 (2H, m), 2.97 (2H, t,
J 7.0); HPLC–MS Rt 2.56 min;
(C22H23N7O1) m/z (ESI) 402 ([M+H]+, 100%); C22H23N7O1 requires
402.2042 for [M+H]+, found: 402.2059; HPLCb Rt 3.39 min, purity
97%.
5.2.8.14.
N-(3-(6-Morpholino-2-(2-(pyridin-3-yl)ethylamino)
(28e). Prepared
pyrimidin-4-yl)phenyl)methanesulfonamide
5.2.8.9. 6-(1H-Indazol-4-yl)-2-morpholino-N-(2-(pyridin-4-yl)
ethyl)pyrimidin-4-amine (27i). Prepared from compound 14f
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1), followed by method C gave 27i (95% yield) as
a pale brown solid; mp 108–115 °C; Rf 0.23 (CHCl3/MeOH, 9:1);
dH (500 MHz, CD3OD), 8.65 (1H, s, br), 8.55 (2H, d, J 6.0), 7.58
(1H, d, J 7.5), 7.55 (1H, d, J 8.5), 7.49 (1H, dd, J 8.5, 7.5), 7.17 (2H,
d, J 6.0), 6.21 (1H, s, br), 4.69 (1H, s, br), 3.83–3.82 (4H, t, J 5.0),
3.76 (4H, t, J 5.0), 3.70 (2H, dt, J 7.0, 6.5), 2.95 (2H, t, J 7.0);
HPLC–MS Rt 2.41 min; (C22H23N7O1) m/z (ESI) 402 ([M+H]+,
from compound 17e according to general procedure 8. Purification
by method B (CHCl3/MeOH, 9:1) gave 28e (95% yield) as a yellow
solid; mp 130–138 °C; Rf 0.35 (CHCl3/MeOH, 9:1); dH (500 MHz
CDCl3) 8.55 (1H, s), 8.47 (1H, d, J 5.0, 1.5), 7.72 (1H, s), 7.65 (1H,
d, J 7.0), 7.58 (1H, d, J 8.0), 7.39 (1H, t, J 8.0), 7.33 (1H, d, J 8.0),
7.23 (1H, d, J 8.0, 5.0), 6.25 (1H, s), 3.80–3.78 (4H, m), 3.72 (2H,
dt, J 7.0, 6.5), 3.66–3.63 (4H, m), 3.02 (3H, s), 2.95 (2H, t, J 7.0);
HPLC–MS Rt 2.42 min; (C22H26N6O3S1) m/z (ESI) 455 ([M+H]+,
100%);
C
22H26N6O3S1 requires 455.1865 for [M+H]+, found:
455.1877; HPLCb Rt 3.45 min, purity 98%.
100%);
C
22H23N7O1 requires 402.2042 for [M+H]+, found:
402.2049; HPLCb Rt 3.18 min, purity 96%.
5.2.8.15.
N-(3-(6-Morpholino-2-(2-(pyridin-4-yl)ethyl-
amino)pyrimidin-4-yl)phenyl)methanesulfonamide (28f). Pre-
pared from compound 17f according to general procedure 8.
Purification by method B (EtOAc/MeOH, 9:1) gave 28f (47% yield)
as a pale yellow solid; mp 145–152 °C; Rf 0.36 (CHCl3/MeOH,
9:1); dH (500 MHz, CDCl3) 9.87 (1H, br), 8.45 (2H, d, J 4.5), 8.31
(1H, s), 7.92 (1H, br), 7.76 (1H, d, J 7.5), 7.41 (1H, t, J 8.0), 7.30–
7.27 (3H, m), 6.73 (1H, s), 3.68–3.66 (4H, m), 3.61–3.56 (6H, m),
5.2.8.10.
4-(1H-Indol-6-yl)-6-morpholino-N-(2-(pyridin-2-yl)
ethyl)pyrimidin-2-amine (28a). Prepared from compound 17d
according to general procedure 8. Purification by method A gave
28a (73% yield) as a yellow solid; mp 91–98 °C; Rf 0.35 (CHCl3/
MeOH, 9:1); dH (250 MHz, CDCl3) 9.78 (1H, br), 8.49 (1H, d, J 4.5),
8.19 (1H, s), 7.61 (1H, d, J 8.0), 7.58 (1H, d, J 8.0), 7.52 (1H, td, J
7.5, 1.5), 7.15 (1H, br), 7.12 (1H, d, J 7.5), 7.07 (1H, dd, J 7.0, 4.5),
6.47 (1H, s), 6.29 (1H, br), 3.86–3.83 (2H, m), 3.76–3.74 (4H, m),
3.62–3.60 (4H, m), 3.09 (2H, t, J 7.0); HPLC–MS Rt 3.30 min;
(C22H24N6O1) m/z (ESI) 401 ([M+H]+, 100%); C22H24N6O1 requires
401.2090 for [M+H]+, found: 401.2085; HPLCb Rt 4.27 min, purity
>99%.
3.00 (3H, s), 2.90 (2H, t,
J 7.0); HPLC–MS Rt 2.28 min;
(C22H26N6O3S1) m/z (ESI) 455 ([M+H]+, 100%); C22H26N6O3S1 re-
quires 455.1865 for [M+H]+, found: 455.1880; HPLCb Rt 3.62 min,
purity 98%.
5.2.8.16. 4-(1H-Indazol-4-yl)-6-morpholino-N-(2-(pyridin-2-yl)
ethyl)pyrimidin-2-amine (28g). Prepared from compound 17d
according to general procedure 8. Purification by method B
(EtOAc/MeOH, 9:1) gave 28g (16% yield) as a colourless solid; mp
170–180 °C; Rf 0.33 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3),
8.61 (1H, s), 8.57 (1H, d, J 4.5), 7.61–7.58 (2H, m), 7.52 (1H, d, J
8.0), 7.43 (1H, t, J 8.0), 7.21 (1H, d, J 8.0) 7.13 (1H, dd, J 7.5, 5.0),
6.37 (1H, s, br), 5.46 (1H, s, br, NH), 3.93 (dt, J 7.0, 6.5), 3.81–3.79
(4H, m), 3.68–3.66 (4H, m), 3.15 (2H, t, J 7.0); HPLC–MSb Rt
2.57 min; (C22H23N7O1) m/z (ESI) 402 ([M+H]+, 100%); C22H23N7O1
requires 402.2042 for [M+H]+, found: 402.2057; HPLCb Rt
3.44 min, purity 98%.
5.2.8.11.
4-(1H-indol-6-yl)-6-morpholino-N-(2-(pyridin-3-yl)
ethyl)pyrimidin-2-amine (28b). Prepared from compound 17e
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1) gave 28b (91% yield) as an off-white solid;
mp 188–194 °C; Rf 0.26 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3)
9.22 (1H, br s), 8.49 (1H, s), 8.44 (1H, dd, J 5.0, 1.5), 8.17 (1H, s),
7.65 (1H, d, J 8.5), 7.61 (1H, d, J 8.5), 7.47 (1H, d, J 7.5), 7.20 (1H,
t, J 3.0), 7.16 (1H, dd, J 7.5, 5.0), 6.52 (1H, br), 6.35 (1H, s), 3.79–
3.77 (4H, m), 3.64–3.60 (6H, m), 2.87 (2H, t, J 7.0); HPLC–MS Rt
3.03 min; (C22H24N6O1) m/z (ESI) 401 ([M+H]+, 100%); C22H24N6O1
requires 401.2090 for [M+H]+, found: 401.2100; HPLCb Rt
4.08 min, purity 97%.
5.2.8.17. 4-(1H-Indazol-4-yl)-6-morpholino-N-(2-(pyridin-3-yl)
ethyl)pyrimidin-2-amine (28h). Prepared from compound 17e
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1) gave 28h (63% yield) as an off-white solid;
mp 105–110 °C; Rf 0.26 (CHCl3/MeOH, 9:1); dH (500 MHz, CD3OD),
8.52 (1H, s), 8.52 (1H, d, J 1.5), 8.47 (1H, dd, J 4.5, 1.5), 7.72 (1H, d, J
8.0), 7.61 (1H, d, J 8.0), 7.58 (1H, d, J 7.0), 7.46–7.43 (1H, dd, J 8.0,
7.0) 7.32 (1H, dd, J 8.0, 4.5), 6.46 (1H, s), 5.4 (1H, s, br), 3.76–3.74
5.2.8.12.
4-(1H-Indol-6-yl)-6-morpholino-N-(2-(pyridin-4-yl)
ethyl)pyrimidin-2-amine (28c). Prepared from compound 17f
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1) gave 28c (87% yield) as an orange solid; mp
133–135 °C; Rf 0.22 (CHCl3/MeOH, 9:1); dH (500 MHz, CD3OD)
8.44 (2H, d, J 6.0), 7.96 (1H, s), 7.64 (1H, d, J 8.5), 7.54 (1H, d, J

850
J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
(4H, m), 3.70–3.67 (2H, m), 3.66–3.64 (4H, m), 2.97 (2H, t, J 7.0);
HPLC–MS Rt 2.46 min; (C22H23N7O1) m/z (ESI) 402 ([M+H]+,
5.2.8.23. N-(3-(6-Morpholino-2-(pyridin-2-ylmethoxy)pyrimi-
din-4-yl)phenyl)methanesulfonamide (31b). Prepared from
compound 22 according to general procedure 8. Purification by
method B (EtOAc/MeOH, 9:1) gave 31b (55% yield) as a pale yellow
solid; mp 80–86 °C; Rf 0.59 (CHCl3/MeOH, 9:1); dH (500 MHz,
CDCl3) 8.57 (1H, d, J 4.5), 7.85 (1H, s), 7.74 (1H, br), 7.69 (1H, t, J
8.0), 7.55 (1H, d, J 8.0), 7.42–7.41 (2H, m), 7.20 (1H, dd, J 7.5,
5.0), 6.57 (1H, s), 5.59 (2H, s), 3.78–3.76 (4H, m), 3.68–3.66 (4H,
m), 3.01 (3H, s); HPLC–MS Rt 5.13 min; (C21H23N5O4S1) m/z (ESI)
442 ([M+H]+, 100%); C21H23N5O4S1 requires 442.1549 for [M+H]+,
found: 442.1554; HPLCb Rt 5.63 min, purity 99%.
100%);
C
22H23N7O1 requires 402.2042 for [M+H]+, found:
402.2061; HPLCb Rt 3.44 min, purity 98%.
5.2.8.18. 4-(1H-Indazol-4-yl)-6-morpholino-N-(2-(pyridin-4-yl)
ethyl)pyrimidin-2-amine (28i). Prepared from compound 17f
according to general procedure 8. Purification B (CHCl3/MeOH,
9:1), followed by method C gave 28i (38% yield) as a pale yellow
solid; mp 182–185 °C; Rf 0.28 (CHCl3/MeOH, 9:1); dH (500 MHz,
CDCl3) 8.60 (1H, s), 8.52 (2H, d, J 5.0), 7.60 (1H, d, J 7.0), 7.54
(1H, d, J 8.0), 7.45 (1H, dd, J 8.0, 7.0), 7.19 (2H, d, J 5.0), 6.41 (1H,
s), 3.82–3.77 (6H, m), 3.67–3.65 (4H, m), 2.98 (2H, t, J 7.0);
HPLC–MS Rt 2.31 min; (C22H23N7O1) m/z (ESI) 402 ([M+H]+,
5.2.8.24. 4-(6-(1H-Indazol-4-yl)-2-(pyridin-2-ylmethoxy)pyrim-
idin-4-yl)morpholine (31c). Prepared from compound 22
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1), followed by method C gave 31c (89% yield)
as a yellow solid; mp 205–209 °C; Rf 0.27 (CHCl3/MeOH, 9:1); dH
(500 MHz, CDCl3) 10.27 (1H, s, br), 8.61 (1H, d, J 4.5), 8.48 (1H,
s), 7.70 (1H, td, J 8.0, 1.5), 7.68 (1H, d, J 7.0), 7.59–7.57 (2H, m),
7.45 (1H, dd, J 8.0, 7.5), 7.21 (1H, dd, J 7.0, 5.0), 6.71 (1H, s, br),
5.65 (2H, s), 3.80–3.78 (4H, m), 3.72–3.70 (4H, m); HPLC–MS Rt
5.24 min; (C21H20N6O2) m/z (ESI) 389 ([M+H]+, 100%); C21H20N6O2
requires 389.1726 for [M+H]+, found: 389.1735; HPLCb Rt
6.00 min, purity 99%.
100%);
C
22H23N7O1 requires 402.2042 for [M+H]+, found:
402.2059; HPLCb Rt 3.22 min, purity 97%.
5.2.8.19. 4-(4-(1H-Indol-6-yl)-6-(pyridin-2-ylmethoxy)pyrimi-
din-2-yl)morpholine (30a). Prepared from compound 21 accord-
ing to general procedure 8. Purification by method C gave 30a (28%
yield) as a pale yellow solid; m.p. 189–190 °C; Rf 0.63 (CHCl3/
MeOH, 9:1); dH (500 MHz, CD3OD) 8.53 (1H, d, J 5.5), 8.17 (1H, s),
7.86 (1H, td, J 7.5, 2.0), 7.72 (1H, dd, J 8.5, 1.5), 7.60 (1H, d, J 8.5),
7.56 (1H, d, J 7.5), 7.36 (1H, dd, J 7.5, 5.5), 7.33 (1H, d, J 3.5), 6.69
(1H, s), 6.48 (1H, dd, J 3.0), 5.51 (2H, s), 3.84–3.71 (4H, m), 3.32–
3.32 (4H, m); HPLC–MS Rt 7.73 min; (C22H21N5O2) m/z (ESI) 388
([M+H]+, 100%); C22H21N5O2 requires 388.1873 for [M+H]+, found:
388.1764; HPLCa Rt 4.96 min, purity >99%.
5.3. PI3K biochemical screening
Compound inhibition of PI3K was determined in a radiometric
(scintillation proximity) assay44 using purified, recombinant hu-
5.2.8.20. N-(3-(2-Morpholino-6-(pyridin-2-ylmethoxy)pyrimi-
din-4-yl)phenyl)methanesulfonamide (30b). Prepared from
compound 21 according to general procedure 8. Purification by
method B (CHCl3/MeOH, 9:1) gave 30b (12% yield) as a colourless
solid; mp 206–210 °C; Rf 0.67 (CHCl3/MeOH, 9:1); dH (500 MHz,
DMSO-d6) 9.87 (1H, br s), 8.56 (1H, d, J 5.0), 7.96 (1H, t, J 2.0),
7.82 (1H, td, J 7.5, 2.0), 7.78 (1H, d, J 8.0), 7.48 (1H, d, J 8.0), 7.43
(1H, t, J 7.9), 7.35–7.32 (2H, m), 6.69 (1H, s), 5.47 (2H, s), 3.74–
3.72 (4H, m), 3.65–3.63 (4H, m), 3.01 (3H, s); HPLC–MS Rt
6.78 min; (C21H23N5O4S1) m/z (ESI) 442 ([M+H]+, 100%);
man enzyme and ATP at a concentration of 1 lM. All compounds
were serially diluted in 100% DMSO (final concentration = 4%).
The kinase reaction was incubated for 1 h at room temperature
and the reaction was terminated by the addition of PBS. IC50 values
were subsequently determined using a sigmoidal dose–response
curve fit (variable slope) and represent the means of at least two
experiments.
5.4. SRB cellular proliferation assay
C
21H23N5O4S1 requires 442.1549 for [M+H]+, found: 442.1529;
HPLCb Rt 7.44 min, purity 99%.
The IGROV-1 human ovarian cancer cell line was grown in
DMEM (Invitrogen, Paisley, UK) supplemented with 10% FCS
(PAA, Somerset, UK). Cell number was measured using the sulfo-
rhodamine B assay45 (SRB). Ten thousand cells per well were
seeded in a 96-well plate 48 h before treatment. After an incuba-
tion time of 96 h, cells were fixed and stained with SRB. This was
then solubilised with 10 mM Tris. The absorbance was read at
540 nm.
5.2.8.21. 4-(4-(1H-Indazol-4-yl)-6-(pyridin-2-ylmethoxy)pyrim-
idin-2-yl)morpholine (30c). Prepared from compound 21
according to general procedure 8. Purification by method B
(CHCl3/MeOH, 9:1), followed by method C gave 30c (55% yield)
as an off-white solid; mp 170–172 °C; Rf 0.33 (CHCl3/MeOH, 9:1);
dH (500 MHz, CDCl3), 10.20 (1H, br), 8.67 (1H, s, br), 8.62 (1H, d, J
4.0), 7.73 (1H, td, J 7.5, 1.5), 7.68 (1H, dd, J 7.0, 0.5), 7.60 (1H, d, J
8.5), 7.50–7.46 (2H, m), 7.25 (1H, dd, J 7.0, 5.0), 6.69 (1H, s), 5.59
(2H, s), 3.90 (4H, t, J 4.5), 3.78 (4H, t, J 5.0); HPLC–MS Rt
7.20 min; (C21H20N6O2) m/z (ESI) 389 ([M+H]+, 100%); C21H20N6O2
requires 389.1726 for [M+H]+, found: 389.1734; HPLCb Rt
7.78 min, purity 99%.
5.5. Pharmacodynamic marker analysis
After treating cells as described in the figures, protein extracts
were prepared by washing the cells once in ice-cold phosphate-
buffered saline and then re-suspended in Cell Lysis Buffer (Cell
Signalling Technologies, Hertfordshire, UK), supplemented with
protease inhibitors (Boehinger/Roche, Mannheim, Germany). Pro-
tein concentration for each lysate was determined using the BCA
Protein Assay method (Pierce, Rockford, USA). Equal amounts of
protein were analysed by western blotting. Polyclonal antibodies
against phospho-Ser473-AKT and AKT were obtained from Cell Sig-
nalling Technologies (Hertfordshire, UK), that for Cyclin-D1 was
obtained from NeoMarkers (Lab Vision Corporation, California,
USA), and GAPDH was obtained from Chemicon International (Cal-
ifornia, USA). All antibodies were used as recommended by the
manufacturer and visualised using the ECL PLUS western blot
detection kit (Amersham Biosciences, Buckinghamshire, UK).
5.2.8.22. 4-(6-(1H-Indol-6-yl)-2-(pyridin-2-ylmethoxy)pyrimi-
din-4-yl)morpholine (31a). Prepared from compound 22 accord-
ing to general procedure 8. Purification by method B (EtOAc/MeOH,
9:1) gave 31a (52% yield) as a pale brown solid; mp 186–190 °C; Rf
0.61 (CHCl3/MeOH, 9:1); dH (500 MHz, CDCl3) 8.59 (1H, d, J 5.0),
8.39 (1H, br), 8.68 (1H, s), 7.70–7.67 (3H, m), 7.59 (1H, d, J 8.0),
7.30 (1H, t, J 3.0), 7.19 (1H, dd, J 7.0, 5.5), 6.67 (1H, s), 6.58 (1H,
br), 5.64 (2H, s), 3.79–3.77 (4H, m), 3.69–3.67 (4H, m); HPLC–MS
Rt 5.04 min; (C22H21N5O2) m/z (ESI) 388 ([M+H]+, 100%);
C
22H21N5O2 requires 388.1873 for [M+H]+, found: 388.1769; HPLCb
Rt 5.58 min, purity >99%.

J. M. Large et al. / Bioorg. Med. Chem. 19 (2011) 836–851
851
11. Kang, S.; Bader, A. G.; Vogt, P. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 802.
5.6. Flow cytometry
12. Parsons, D. W.; Wang, T.-L.; Samuels, Y.; Bardelli, A.; Cummins, J. M.; DeLong,
L.; Silliman, N.; Ptak, J.; Szabo, S.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.;
Vogelstein, B.; Lengauer, C.; Velculescu, V. E. Nature 2005, 436, 792.
13. Yuan, T. L.; Cantley, L. C. Oncogene 2008, 27, 5497.
14. Guillard, S.; Clarke, P. A.; te Poole, R.; Mohri, Z.; Bjerke, L.; Valenti, M.; Raynaud,
F.; Eccles, S. A.; Workman, P. Cell Cycle 2009, 8, 443.
15. Drees, B. A.; Mills, G. B.; Rommel, C.; Prestwich, G. D. Expert Opin. Ther. Patents
2004, 14, 703.
16. Crabbe, T.; Welham, M. J.; Ward, S. G. Trends Biochem. Sci. 2007, 32, 450.
17. Workman, P. Biochem. Soc. Trans. 2004, 32, 393.
For cell cycle analysis, 1000 cells were harvested and fixed in
70% ethanol, then stained with 10 lg/mL propidium iodide (Molec-
ular Probes, Eugene, Oregon, USA) for 24 h, and analysed on a Beck-
man Coulter EPICS Elite ESP.24 The data were quantitated using
WINMIDI version 2.8 and Cylchred software.
5.7. Microsomal incubation experiments
18. Workman, P.; Clarke, P. A.; Guillard, S.; Raynaud, F. I. Nat. Biotechnol. 2006, 24,
794.
19. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.;
Chuckowree, I. S.; Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes,
A.; Hancox, T. C.; Kugendadras, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.;
Patel, S.; Pergl-Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.;
Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann,
C.; Workman, P.; Zhyvoloup, A.; Zvelebil, M. J.; Shuttleworth, S. J. J. Med. Chem.
2008, 51, 5522.
20. Raynaud, F. I.; Eccles, S. A.; Patel, S.; Alix, S.; Box, G.; Chuckowree, I.; Folkes, A.;
Gowna, S.; De Haven Brandon, A.; Di Stefano, F.; Hayes, A.; Henley, A. T.;
Lensun, L.; Pergl-Wilson, G.; Robson, A.; Saghir, N.; Zhyvoloup, A.; McDonald,
E.; Sheldrake, P.; Shuttleworth, S.; Valenti, M.; Wan, N. C.; Clarke, P. A.;
Workman, P. Mol. Cancer Ther. 2009, 8, 1725.
21. Workman, P.; Clarke, P. A.; Raynaud, F. I.; van Montfort, R. L. M. Cancer Res.
2010, 70, 2146.
22. Shuttleworth, S.; Silva, F.; Tomassi, C.; Cecil, A.; Hill, T.; Rogers, H.; Townsend,
P. In Progress in Medicinal Chemistry; Lawton, G., Witty, D. R., Eds.; Elsevier B. V.,
2010; Vol. 48, pp 81–131.
Compounds (10 mM) were incubated with male CD1 mouse li-
ver microsomes containing 1 mg/mL protein (Tebu-Bio, Peterbor-
ough, UK) in the presence of NADPH (1 mM), MgCl2 (3 mM) and
UDP-glucuronic acid (2.5 mM) in phosphate buffered saline
(10 mM, pH 7.4) at 37 °C. All reagents were supplied by Sigma Al-
drich (Gillingham, UK). Incubations were conducted for 0, 15 and
30 min. Control incubations were generated by omitting either
cofactors or microsomal protein. Incubations were terminated by
the addition of three volumes of methanol containing an analytical
internal standard followed by centrifugation at 13000g for 5 min at
4 °C. The percentage of compound remaining was determined after
analysis by LC–MS.
A Supelcosil Discovery C18 column (5 cm ꢀ 4.6 mm ID, 5
lm;
23. Maira, S.-M.; Stauffer, F.; Schnell, C.; García-Echeverría, C. Biochem. Soc. Trans.
2009, 37, 265.
Supelco, Gillingham, UK) was used for chromatographic separa-
tion. The mobile phase consisted of methanol and 0.1% formic acid
in water. After an initial 0.5 min isocratic period, methanol was in-
creased from 10% to 90% over 6 min, held at 90% for 3.5 min, re-
turned to 10% over 0.5 min, and equilibrated for 3.5 min. The
flow rate was 1 mL/min and the sample injection volume was
24. Raynaud, F. I.; Eccles, S.; Clarke, P. A.; Hayes, A.; Nutley, B.; Alix, S.; Henley, A.;
Di-Stefano, F.; Ahmad, Z.; Guillard, S.; Bjerke, L. M.; Kelland, L.; Valenti, M.;
Patterson, L.; Gowan, S.; de Haven Brandon, A.; Hayakawa, M.; Kaizawa, H.;
Koizumi, T.; Ohishi, T.; Patel, S.; Saghir, N.; Parker, P.; Waterfield, M.;
Workman, P. Cancer Res. 2007, 67, 5840.
25. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Yamano, M.;
Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Workman, P.; Waterfield, M.
D.; Parker, P. Bioorg. Med. Chem. Lett. 2007, 17, 2438.
26. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46, 3.
27. Teague, S. J.; Davis, A. M.; Leeson, P. D.; Oprea, T. Angew. Chem., Int. Ed. 1999, 38,
3743.
28. Nuss, J. M.; Pecchi, S.; Renhowe, P. A. PCT Int. Appl. WO2004048365, 2004;
Chem. Abstr. 141, 38627.
29. Bailey, J. P.; Giles, M. B.; Pass, M. PCT Int. Appl. WO2006005914, 2006; Chem.
Abstr. 144, 150379.
30. Pass, M. PCT Int. Appl. WO2006005915, 2006; Chem. Abstr. 144, 128997.
31. Pass, M. PCT Int. Appl. WO2006005918, 2006; Chem. Abstr. 144, 128996.
32. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann,
M. P.; Williams, R. L. Mol. Cell 2000, 6, 909.
33. Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F. J. Biol. Chem. 1994,
269, 5241.
25
l
L.
An LCQ ion trap mass spectrometer coupled to a SpectraSYSTEM
P4000 pump and an AS3000 autosampler (ThermoFinnigan, Rad-
lett, UK) was used for analysis. The mass spectrometer was
equipped with an electrospray source and operated in positive
mode. The capillary temperature and spray voltage were operated
at 250 °C and 4.5 kV respectively. Sheath and auxiliary gases were
set to 80 and 20 arbitrary units, respectively. Spectra were ac-
quired in full scan mode over the m/z range 50 to 850.
Acknowledgements
This work was supported by Cancer Research UK [CUK] pro-
gramme Grant Nos. C309/A2187 and C2536/A5708. Paul Workman
is a Cancer Research UK Life Fellow. The authors thank Dr. Amin
Mirza, Mr. Meirion Richards and Mr. Andrew Dick for their assis-
tance with NMR and mass spectrometry.
34. For a review see: Magdalou, J.; Fournel-Gigleux, S.; Testa, B.; Ouzzine, M. In The
Practice of Medicinal Chemistry; Wermuth, C. G., Ed.; Elsevier Academic Press:
Oxford, 2003; pp 517–543.
35. Astles, P. C.; Brown, T. J.; Harris, N. V.; Harper, M. F.; McCarthy, C.; Porter, B.;
Smith, C.; Walsh, R. J. A. Eur. J. Med. Chem. 1997, 32, 515.
36. Thurkauf, A.; Hutchison, A.; Peterson, J.; Cornfield, L.; Meade, R.; Huston, K.;
Harris, K.; Ross, P. C.; Gerber, K.; Ramabhadran, T. V. J. Med. Chem. 1995, 38,
2251.
37. Eid, C. N., Jr.; Konopelski, J. P. Tetrahedron 1991, 47, 975.
38. D’Atri, G.; Gomarasca, P.; Resnati, G.; Tronconi, G.; Scolastico, C.; Sirtori, C. R. J.
Med. Chem. 1984, 27, 1621.
39. Montebugnoli, D.; Bravo, P.; Brenna, E.; Mioskowski, C.; Panzeri, W.; Viani, F.;
Volonterio, A.; Wagner, A.; Zanda, M. Tetrahedron 2003, 59, 7147.
40. Suzuki, M.; Ohdoi, K.; Kato, K.; Matsumoto, H.; Toyama, K.; Kitahara, M.;
Yotsumoto, T. PCT Int. Appl. WO9965881, 1999; Chem. Abstr. 132, 49974.
41. Bedford, R. B.; Cazin, C. S. J.; Coles, S. J.; Gelbrich, T.; Horton, P. N.; Hursthouse,
M. B.; Light, M. E. Organometallics 2003, 22, 987.
References and notes
1. Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso, R.; Driscoll, P. C.;
Woscholski, R.; Parker, P. J.; Waterfield, M. D. Annu. Rev. Biochem. 2001, 70, 535.
2. Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489.
3. Patel, R. K.; Mohan, C. Immunol. Res. 2005, 31, 47.
4. Oudit, G. Y.; Sun, H.; Kerfant, B.-G.; Crackower, M. A.; Penninger, J. M.; Backx, P.
H. J. Mol. Cell. Cardiol. 2004, 37, 449.
5. Cantley, L. C. Science 2002, 296, 1655.
42. Large, J. M.; Clarke, M.; Williamson, D. M.; McDonald, E.; Collins, I. Synlett 2006,
861.
43. For a review see Kappe, C. O.; Stadler, A. Microwaves in Organic and Medicinal
Chemistry; Wiley-VCH, 2005.
44. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Okada, M.;
Ohta, M.; Tsukamoto, S.; Parker, P.; Workman, P.; Waterfield, M. Bioorg. Med.
Chem. 2006, 14, 6847.
45. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.;
Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991,
83, 757.
6. Lawlor, M. A.; Alessi, D. R. J. Cell Sci. 2001, 114, 2903.
7. Shayesteh, L.; Lu, Y.; Kuo, W.-L.; Baldocchi, R.; Godfrey, T.; Collins, C.; Pinkel, D.;
Powell, B.; Mills, G. B.; Gray, J. W. Nat. Genet. 1999, 21, 99.
8. Ma, Y.-Y.; Wei, S.-J.; Lin, Y.-C.; Lung, J.-C.; Chang, T.-C.; Whang-Peng, J.; Liu, J.
M.; Yang, D.-M.; Yang, W. K.; Schen, C.-Y. Oncogene 2000, 19, 2739.
9. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.;
Gazdar, A.; Powell, S. M.; Riggins, G. J.; Willson, J. K.; Markowitz, S.; Kinzler, K.
W.; Vogelstein, B.; Velculescu, V. E. Science 2004, 304, 554.
10. Campbell, I. G.; Russell, S. E.; Choong, D. Y.; Montgomery, K. G.; Ciavarella, M.
L.; Hooi, C. S.; Cristiano, B. E.; Pearson, R. B.; Phillips, W. A. Cancer Res. 2004, 64,
7678.