Synthesis and SAR of novel 4-morpholinopyrrolopyrimidine derivatives as potent phosphatidylinositol 3-kinase inhibitors

Article abstract of DOI:10.1021/jm901783v

Significant evidence suggests that deregulation of the PI3K/Akt pathway is important in tumor progression. Mechanisms include loss of function of the tumor suppressor PTEN and high frequency of mutation of the PI3K p110α isoform in human malignancies. This connection between PI3K and tumor genesis makes PI3K a promising target for cancer treatment. A series of 4- morpholinopyrrolopyrimidine derivatives were synthesized and evaluated as inhibitors of PI3Kα and mTOR, leading to the discovery of PI3Kα selective inhibitors (e.g., 9) and dual PI3Kα/mTOR kinase inhibitors (e.g., 46 and 48). PI3Kα/mTOR dual inhibitors demonstrated inhibition of tumor cell growth in vitro and in vivo and caused suppression of the pathway specific biomarkers [e.g., the phosphorylation of Akt at Thr308 (T308) and Ser473 (S473)] in the human breast cancer cell line MDA361. In addition, compound 46 demonstrated good in vivo efficacy in the MDA361 human breast tumor xenograft model.

Full text of DOI:10.1021/jm901783v

J. Med. Chem. 2010, 53, 3169–3182 3169
DOI: 10.1021/jm901783v
Synthesis and SAR of Novel 4-Morpholinopyrrolopyrimidine Derivatives as Potent
Phosphatidylinositol 3-Kinase Inhibitors
Zecheng Chen,*,† Aranapakam M. Venkatesan,† Christoph M. Dehnhardt,† Semiramis Ayral-Kaloustian,†
Natasja Brooijmans,† Robert Mallon,‡ Larry Feldberg,‡ Irwin Hollander,‡ Judy Lucas,‡ Ker Yu,‡ Fangming Kong,§ and
Tarek S. Mansour†
†Chemical Sciences, ‡Oncology, and §Analytical Quality Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, New York 10965
Received December 1, 2009
Significant evidence suggests that deregulation of the PI3K/Akt pathway is important in tumor
progression. Mechanisms include loss of function of the tumor suppressor PTEN and high frequency
of mutation of the PI3K p110R isoform in human malignancies. This connection between PI3K and
tumor genesis makes PI3K a promising target for cancer treatment. A series of 4-morpholinopyrrolo-
pyrimidine derivatives were synthesized and evaluated as inhibitors of PI3KR and mTOR, leading to the
discovery of PI3KR selective inhibitors (e.g., 9) and dual PI3KR/mTOR kinase inhibitors (e.g., 46 and
48). PI3KR/mTOR dual inhibitors demonstrated inhibition of tumor cell growth in vitro and in vivo and
caused suppression of the pathway specific biomarkers [e.g., the phosphorylation of Akt at Thr308
(T308) and Ser473 (S473)] in the human breast cancer cell line MDA361. In addition, compound 46
demonstrated good in vivo efficacy in the MDA361 human breast tumor xenograft model.
Introduction
one isoform, PI3Kγ, primarily activated by GPCRs. The pik3ca
gene, which encodes the catalytic subunit of PI3KR, is mutated
and overexpressed in a wide range of human cancers, including
breast, ovarian, colorectal, and brain tumors.4-7 In addition,
the activation of the PI3K pathway is negatively regulated by
dual phosphatase PTEN, and persistent activation of PI3K and
loss of PTEN function often coexist in various cancers. All these
factors provide strong evidence for the importance of the PI3K
pathway in cancer.8-11 Therefore, the significant connection
between PI3K, in particular PI3KR, with tumor genesis makes
PI3KR an attractive target for development of anticancer drugs.
To date, several small molecule PI3K inhibitors (Figure 1)
have been reported.12-23 LY294002 (1a)14 and Wortmannin
(1b)13 have been extensively studied as PI3K inhibitors; how-
ever, their toxicity and poor physicochemical properties
limited their potential therapeutic use.
Recently an imidazo[4,5-c]quinoline derivative, NVP-
BEZ235 (1c), was reported by Novartis as a pan-PI3K/mTOR
dual inhibitor.22,23 Compound 1c was shown to block the
activation of the PI3K pathway and potently inhibit cell
proliferation, causing G1 phase cell cycle arrest, and is currently
in phase I/II clinical trials as an anticancer agent. Compound
PI-103 (1d), a pyrido[30,20:4,5]furo[3,2-d]pyrimidine derivative,
was equipotent against PI3KR and PI3Kβ with an IC50 of
4 nM and was selective over other tested protein kinases.18
Genentech recently reported the thieno[3,2-d]pyrimidine deri-
vative GDC-0941 (1e), a potent and orally bioavailable inhi-
bitor of PI3K, in phase I clinical trials for the treatment of
cancer. Notably, compound 1e was equipotent against wild
type PI3KR and two common PI3KR mutants (E545K and
H1047R) with an IC50 of 3 nM.21 As part of our ongoing
research on a variety of fused-pyrimidine scaffolds,24-27 we
now report the synthesis and biological evaluation of novel
Phosphatidylinositol-3-kinases (PI3Ksa) are lipid kinases that
catalyze phosphorylation of the 3-hydroxy position of PIP2
(phosphatidylinositol 4,5-diphosphate) to PIP3 (phosphati-
dylinositol 3,4,5-triphosphate), an important second messenger
modulating activity of the PI3K downstream effectors Akt and
mTOR. The consequences of biological activation of Akt
include tumor progression, proliferation, survival, growth, in-
vasion, angiogenesis, and metastasis. Significant evidence sug-
gests that the PI3K/Akt pathway is deregulated in many human
cancers.1 PI3Ks are divided into three classes (I, II, and III)
based on differences in sequence homology, substrate prefer-
ence, and function.2,3 The class I PI3Ks are heterodimers
consisting of a catalytic subunit and a regulatory subunit. The
catalytic subunits of class I PI3Ks include four isoforms: p110R,
p110β, p110δ, and p110γ, encoded by four distinct genes termed
pik3ca, pik3cb, pik3cd, and pik3cg, respectively. The class I
PI3Ks are further divided into class IA and IB subgroups based
on different regulatory subunits and activation mechanisms.
Class IA includes three isoforms PI3KR, PI3Kβ, and PI3Kδ
activated by receptor tyrosine kinases (RTKs) and small
G-protein-coupled receptors (GPCRs), while class IB has only
*To whom correspondence should be addressed. Phone: 1-845-602-
8341. Fax: 1-845-602-5561. E-mail: chenz1@wyeth.com or chenzecheng@
hotmail.com.
a Abbreviations: PI3K, phosphatidylinositol 3-kinase; mTOR,
mammalian target of rapamycin; Akt, protein kinase B; PIP2, phospha-
tidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-tripho-
sphate; PTEN, phosphatase and tensin homologue; RTK, receptor
tyrosine kinase; GPCRs, G-protein-coupled receptors; PDK1, 3-phos-
phoinositide-dependent kinase 1; S, serine; T, threonine; ATP, adeno-
sine 50-triphosphate; Her2þ, human epidermal growth factor receptor
2þ; ELISA, enzyme-linked immunosorbent assay; DELFIA, dissocia-
tion-enhanced lanthanide fluorescent immunoassay.
r
2010 American Chemical Society
Published on Web 03/24/2010
pubs.acs.org/jmc

3170 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
Figure 1. Structures of PI3K inhibitors.
Scheme 1a
a Reagents and conditions: (a) morpholine (1.5 equiv), Et3N (3 equiv), CH2Cl2, room temp, 6 h; (b) ArB(OH)2 (1.5 equiv), Pd(Ph3P)4 (5 mol %),
DME, 2 N Na2CO3, 110 °C/30 min, microwave; (c) DMF DMA (excess), DMF, 110 °C/12-18 h; (d) 10% Pd/C, MeOH, room temp, 2-6 h; (e)
3
formaldehyde (2 equiv), amines (3 equiv), HOAc, 60 °C/6 h; (f ) TBSCl (1.2 equiv), imidazole (1.5 equiv), DMF, 80 °C/15 min, microwave; (g) CH3I
(1.2 equiv), NaH (2 equiv), THF, room temp, 2 h; (h) TFA, CH2Cl2, room temp, 6 h; (i) 4-piperidone (2.5 equiv), KOH (5 equiv), MeOH, 66 °C/15 h;
( j) 10% Pd/C, HCl, MeOH, 50 psi, room temp, 16 h; (k) ArCHO (1.5 equiv), ZnCl2 (1.5 equiv), NaBH3CN (1.5 equiv), MeOH, room temp, 5 h.
4-morpholinopyrrolopyrimidine derivatives as potent PI3KR
inhibitors.
conditions to provide the corresponding 2-aryl substituted
products. Reaction with 1,1-dimethoxy-N,N-dimethylmethan-
amine (DMF DMA) to form the corresponding enamine
intermediate followed by the reductive cyclization under cata-
lytic hydrogenation conditions gave the desired 5H-pyrrolo-
[3,2-d]pyrimidines 4-7. Mannich-type reaction of 4, by heating
with formaldehyde and different amines in acetic acid solution,
proceeded smoothly to yield compounds 8 and 9 with water-
solubilizing groups introduced at the C7 position. Protection of
the hydroxyl group with TBS in 4 followed by alkylation at the
3
Chemistry
The general synthetic route for the preparation of 4-mor-
pholinopyrrolo[3,2-d ]pyrimidine derivatives is shown in
Scheme 1. The starting material 2,4-dichloro-6-methyl-5-nitro-
pyrimidine (2) was treated with morpholine to give the corre-
sponding 4-morpholino substituted intermediate 3, which was
reacted with different boronic acids or esters under Suzuki

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3171
Scheme 2a
a Reagents and conditions: (a) POCl3, 120 °C/30 min, microwave; (b) morpholine (1.5 equiv), Et3N (3 equiv), EtOH, room temp; (c) ArB(OH)2
(1.5 equiv), Pd(Ph3P)4 (5 mol %), DME, 2 N Na2CO3, 150 °C/40 min, microwave; (d) 2-(dimethylamino)ethyl chloride (1.5 equiv), Cs2CO3 (3 equiv),
DMF, 80 °C/12 h; (e) 4-aminophenylboronic acid, pinacol ester (1.3 equiv), Pd(Ph3P)4 (5 mol %), DME, 2 M Na2CO3, 130 °C/30 min, microwave;
(f ) triphosgene (0.6 equiv), Et3N (3 equiv), amines (3-5 equiv), CH2Cl2, room temp, 2-6 h.
Scheme 3a
substitution with the morpholino group at the C4 position
provided intermediate 16. Suzuki coupling of 16 with different
boronic acids gave compounds 17 and 18. Alternatively,
alkylation of 16 with 2-(dimethylamino)ethyl chloride, using
Cs2CO3 as base, gave intermediate 19. Reaction of 19 with
different boronic acids provided 20 and 21. Suzuki reaction
of 19 with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
aniline provided 22, which was converted to urea derivatives
23-28 by reaction with triphosgene followed by appropriate
amines.
Conversion of intermediate 16 to the 4-pyridylurea ana-
logue 29 was achieved by a two-step sequence, described
above for the conversion of 19 to 23-28. Alkylation of 16
provided analogues 30 and 31, which were converted to the
corresponding 4-pyridylurea derivatives 32 and 33. Hydro-
lysis of the acetal group in 33, under acidic conditions, gave
the aldehyde 34. Reductive amination of 34, using different
amines, provided compounds 35-39. Alternatively, reduction
of the aldehyde group in 34, using NaBH4, gave the corre-
sponding alcohol analogue 40 (Scheme 3).
a Reagents and conditions: (a) 4-aminophenylboronic acid, pinacol
Suzuki reaction of the N7 substituted pyrrolo[2,3-d ]-
ester (1.3 equiv), Pd(Ph3P)4 (5 mol %), DME, 2 N Na2CO3, 130 °C/
30 min, microwave; (b) triphosgene (0.6 equiv), Et3N (3 equiv),
4-aminopyridine (5 equiv), CH2Cl2, room temp, 12 h; (c) CF3CH2I
(2 equiv for 30) or (CH3O)2CHBr (2 equiv for 31), Cs2CO3 (1.2 equiv),
DMF, 80 °C/12 h; (d) HCl, dioxane/H2O, 70 °C/12 h (e) amines
(6 equiv), ZnCl2 (2 equiv), NaBH3CN (2 equiv), MeOH, room temp,
12 h; (f ) NaBH4 (1.5 equiv), MeOH/THF, room temp, 2 h.
pyrimidine intermediates 30 and 41 gave the corresponding
anilines 42 and 43. Treatment of 42 and 43 with methyl
4-isocyanatobenzoate, followed by hydrolysis of the resulting
esters under basic conditions, gave the corresponding
4-ureidobenzoic acids 44 and 45. Finally, coupling of the acids
44 and 45 with different amines afforded the desired 4-ureido-
benzamide derivatives 46-52 (Scheme 4).
N5 position and removal of the TBS group afforded 10, which
underwent the Mannich-type reaction to give 11. Aldol-type
reaction of 6 with piperidin-4-one followed by catalytic hydro-
genation of the resulting double bond provided derivative 12.
Reductive amination of 12, using appropriate aldehydes and
NaCNBH3, gave compounds 13 and 14.
The general synthetic routes used for the preparation of
4-morpholinopyrrolo[2,3-d ]pyrimidine derivatives are out-
lined in Schemes 2-4. 7H-Pyrrolo[2,3-d]pyrimidine-2,4-diol
(15) was prepared, following a known procedure,28,29 by
condensing 6-aminouracil with chloroacetaldehyde. Chlori-
nation of 15 by heating with POCl3 followed by selective
Results and Discussion
All final compounds 4-14, 16-20, 23-29, 32-40, and
46-52 were tested for in vitro potency in a PI3KR fluorescence
polarization format assay25 and mTOR in a dissociation-
enhanced lanthanide fluorescent immunoassay (DELFIA) plat-
form enzyme-linked immunosorbent assay (ELISA).26 Com-
pounds that showed reasonable PI3KRpotency were selected for
further evaluation in the cell growth inhibition assays against
PC3 (prostate, PTEN mutant) and MDA-361 (breast, Her2þ/
PI3KCA [E545K] mutant) human tumor cell lines.25

3172 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
Scheme 4a
a Reagents and conditions: (a) 4-aminophenylboronic acid, pinacol ester (1.3 equiv), Pd(Ph3P)4 (5 mol %), DME, 2 N Na2CO3, 130 °C/30 min,
microwave; (b) methyl 4-isocyanatobenzoate (1.2 equiv), CH2Cl2, room temp, 12 h; (c) 1 N NaOH (3 equiv), MeOH/THF, 70 °C/12 h; (d ) amines
(2 equiv), HOBt (1.5 equiv), EDCI (1.5 equiv), Et3N (2 equiv), THF, room temp, 12 h.
Table 1a
group resulted in about 100-fold loss in potency for 7. The
hydroxyl group in both 4 and 6 was found to be essential for
PI3KR activity, indicating that it should be involved in protein
binding as demonstrated in other fused pyrimidine series.21,24,25
Introduction of water solubilizing groups (-CH2NR1R2) at
the C7 position of 4resulted in about a 3-fold increase in PI3KR
potency for 8 and 9. Compound 9 showed very good selectivity
(171-fold) for PI3KR over mTOR. However, incorporating a
piperidine ring at the C7 position of 6 led to a 5-fold decrease in
IC50 (nM)
PI3KR potency for 12. Methyl substitution at the N5 position
compd
R1
R2
R3
PI3KR mTOR PC3
of the pyrrolo[3,2-d]pyrimidine core was not tolerated and
resulted in a significant loss in activity. The N-methyl substi-
tuted analogue 10 was about 30-fold less potent than the
corresponding NH analogue 4, while 11 was 200-fold less
potent than the corresponding compound 9.
In the meantime, a series of 4-morpholinopyrrolo[2,3-d ]-
pyrimidines were prepared by replacing the N5 of the imida-
zole ring in 1f with “CH”. The effects of different substituents
on the phenyl ring and the N7 position on enzyme and cell
potency are shown in Table 2.
As seen in Table 2, the 3-hydroxymethyl analogue 17 and
the 3-hydroxyl analogue 18 maintained PI3KR inhibitory
activity comparable to that of 1f. Introducing an amino side
chain at N7 of 17 led to about a 2-fold increase in PI3KR
potency for 20, while appending the same group on 18 led to a
slight decrease in PI3KR potency for 21. Both analogues 20
and 21 exhibited much weaker potency against mTOR. In
comparison with 1f, all four analogues in this series showed
comparable or improved cell potency against PC3. It was
found that this series of analogues showed good permeability,
as well as good solubility except for 18.
Recent studies showed that the phenolic group was found to
be a metabolic liability due to glucuronidation.21,26 Isosteres
for the phenolic group were explored to achieve a metaboli-
cally stable clinical candidate.21 In addition, our own research
teams have shown that incorporation of a urea appendage
instead of the phenolic group in the fused pyrimidine core not
only improved metabolic stability but also increased PI3KR
potency and cell potency.24,25 Therefore, a series of 4-ureido
1f
4
63
65
634
1625
1234
2075
ND
-CH2OH
-CH3
-OH
H
H
H
H
H
H
H
H
H
H
5
1506 5000
70 355
7500 430
6
1551
ND
7
-NH2
-CH2OH
-CH2OH
8
-CH2N(CH3)2
-CH2-pyrrolinyl
H
20
21
108
3600
>3160
>3160
9
10
11
12
13
14
-CH2OH Me
1996 >4000 ND
4207 >4000 ND
-CH2OH Me -CH2-pyrrolinyl
-OH
-OH
-OH
H
H
H
4-piperidinyl
1-benzyl-4-piperidinyl 340
354
5900
2000
3650
ND
ND
1652
1-(4-F-benzyl)-4-
piperidinyl
178
a The values are averages of at least two separate determinations with
a typical variation of less than (30%. ND: not determined.
The lead compound 1f, an imidazolopyrimidine derivative,
was previously prepared as a potent PI3K inhibitor.24 It
exhibited good PI3KR activity (IC50=63 nM) and moderate
cell potency against PC3 (IC50=1.2 μM), but this compound
had low solubility, poor metabolic stability, and poor expo-
sure. Replacement of the imidazole ring in 1f with a pyrrole
ring led to a series of 4-morpholinopyrrolo[3,2-d]pyrimidines
(Scheme 1). The effects on PI3KR inhibitory activity of
different substituents on the phenyl ring at C3 of the pyrrolo-
[3,2-d ]pyrimidine core are shown in Table 1. The 3-hydro-
xymethyl and 3-hydroxy analogues, 4 and 6, showed equal
potency against PI3KR, comparable to that of 1f. Removal of
the hydroxyl group in 4 led to a significant decrease in potency
for 5. Replacement of the 3-OH group in 6 with a 3-NH2

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3173
Table 2a
IC50 (nM)
mTOR
compd
R
R1
PI3KR
PC3
solubility ( μg/mL), pH 7.4
permeability (10-6 cm/s)
1f
63
80
43
42
76
634
205
1234
1211
580
1
38
1.4
2.3
0.9
1.9
2.0
17
18
20
21
-CH2OH
-OH
H
H
64
>800
>800
2
-CH2OH
-OH
-(CH2)2N(CH3)2
-(CH2)2N(CH3)2
691
1181
>100
>100
a The values are averages of at least two separate determinations with a typical variation of less than (30%.
Table 3a
IC50 (nM)
mTOR
compd
R/Ar
PI3KR
PC3
solubility (μg/mL), pH 7.4
stability T1/2 (min),b rat
1f
63
142
16
634
7
1234
882
137
100
189
ND
437
1
21
2
5
23
24
25
26
27
28
2-pyridyl
3-pyridyl
4-pyridyl
4-F-phenyl
Et
3
52
52
9
8
24
2
2
8
ND
>100
>100
ND
8
226
54
30
33
Me
9
a The values are averages of at least two separate determinations with a typical variation of less than (30%. ND: not determined. b Half-life of drug
when incubating with rat liver microsomes.
analogues were prepared by replacing the 3-hydroxyl group
on the phenyl ring, and their biological data are shown in
Table 3.
Since the 4-pyridylurea analogue25 showed the best invitro
profile in the series, we explored the effects of different
substitutions at the N7 position for further optimization.
The effects of the substitutions at N7 on PI3KR activity and
cellular activity are shown in Table 4. Compound 29, with no
substitution at N7, was less potent against PI3KR than 25
(bearing a dimethylaminoethyl group). Introducing cyclic
amino substituted ethyl goups at N7 resulted in a decrease
in PI3KR potency (35, 36, and 38), as well as cellular potency
when compared to 25. The same result was observed for
analogue 37, bearing an ethylenediaminoethyl group at the
N7 position. Compound 39, bearing a 2-piperazinylethyl
group at N7, exhibited PI3KR potency comparable to that
of 25 but showed 23-fold less in cell potency against PC3.
Replacement of the dimethylamino group in 25 with a hydro-
xy retained PI3KR potency as well as PC3 cell potency for 40.
The acetal analogue 33 was slightly less potent in PI3KR but
more potent in cell for PC3 compared to 25. In contrast to 33,
the aldehyde analogue 34 was more potent in enzyme for
PI3KR; however, it was 6-fold less potent in cell for PC3
compared to 25. Compound 32, with a triflouroethyl group at
N7, showed a comparable potencyagainst PI3KR and a better
PC3 potency relative to 25. In terms of pharmaceutical
Among the various pyridyl substituted ureas, the 4-pyridyl-
urea analogue25 showed the best PI3KR potency with anIC50
of 8 nM, which is 8-fold more potent than the lead compound
1f. In the meantime, mTOR potency was also dramatically
increased (IC50=2 nM for 25 versus 634 nM for 1f ). More
importantly, this compound exhibited a significant improve-
ment in cellular potency against PC3, which is 12-fold more
potent than 1f in the cell assay. For PI3KR enzyme potency,
the 3-pyridylurea analogue 24 was 2-fold less potent than
the 4-pyridylurea analogue 25, while the 2-pyridylurea ana-
logue 23 was 18-fold less potent than 25. The same trend
was observed in their PC3 cell potency, with the order
4-pyridyl > 3-pyridyl > 2-pyridyl. The F-substitued phenyl-
urea analogue 26 was 3-fold more potent against PI3KR than
1f; however, it was 3-fold less potent than the 4-pyridylurea
analogue 25. In comparison with the arylurea analogues, the
alkylurea analogues 27 and 28 were much less potent against
PI3KR. As for their properties, these urea analogues showed
better water solubility, yet no improvements were observed in
microsome stability in rat when compared to 1f.

3174 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
Table 4a
Figure 2. Modeling study of 40 (shown in turquoise for its skeleton)
docked in PI3KR homology model based on PI3Kγ crystal struc-
tures.30
analogues in this series showed improved stability in rat and
human liver microsomes, and compounds 46 and 48 showed
good stability in nude mouse liver microsomes with T1/2 > 30
min. The solubility of these compounds were poor at physio-
logical pH; however, it can be improved at pH 3.0 because of
the presence of the basic amino groups in their molecules, as
seen in examples for compounds 46-48.
Analogues 46 and 48 were then assayed in vivo for their
ability to suppress appropriate biomarkers. Inhibition of
PI3KR should result in suppression of the phosphorylation
of Akt, particularly at T308. As can be seen in Figure 3,
analogues 46 and 48 suppressed phosphorylation of Akt T308
in MDA361 breast tumor cells for up to 8 h when adminis-
tered 25 mpk, iv, in nude mouse. The actin (control protein)
signal was unaffected by 46 and 48. Both compounds also
inhibited phosphorylation of Akt S473 and S6K, substrates of
mTOR, indicating that they are PI3K/mTOR dual inhibitors.
Pharmacokinetic analysis showed that the blood concentra-
tions of compounds 46 and 48 at 8 h after a single 25 mpk iv
dose (vehicle: 5% dextrose/lactic acid, pH 3.5) were 1731 and
1683 ng/mL, respectively.
In vivo efficacy study of compound 46 was conducted in
nude mice bearing MDA361 human breast tumors (Figure 4).
Compound 46 was dosed at 50, 25, and 10 mg/kg, iv, once
daily for 5 days weekly (two rounds). Significant tumor
regression was observed in higher dose of 46 for 50 mpk and
no tumor regrowth until day32. Tumor growth inhibition was
also seen in the lower doses at 25 and 10 mpk.
a The values are averages of at least two separate determinations with
a typical variation of less than (30%. ND: not determined. b Half-life of
drug when incubating with rat liver microsomes.
properties, analogues 32 and 40 demonstrated improved
microsomal stability; however, both compounds showed poor
solubility at pH 7.4. Molecular modeling of analogue 40, as
shown in Figure 2, displays the crucial hydrogen bonding
interactions, including the morpholino oxygen binding to the
hinge region Val851 and the urea moiety binding to Asp810
and Lys802. It is observed that the pyridyl nitrogen is point-
ing away from the binding pocket toward the solvent
front. Hence, a series of 4-ureidobenzamide derivatives with
extended basic amino groups were prepared for further
optimization.
The assay results of 4-ureidobenzamide derivatives are
shown in Table 5. In general, the 4-ureidobenzamide ana-
logues possessed excellent enzyme potency againstPI3KR and
mTOR, as well as high cell potency against PC3 and
MDA361. Compound 46 was 11-fold more potent against
PI3KR and 9-fold more potent in cell against PC3 compared
to 32. Analogues 47-49 bearing different amide groups also
showed excellent PI3KR potency with IC50 values ranging
from 0.9 to 6 nM, as well as good cell potency against PC3
with IC50 values ranging from 17 to 45 nM. Replacement of
2,2,2-trifluoroethyl group with ethyl at N7, resulted in com-
pounds 50-52, which also showed excellent PI3KR potency
and good cell potency against PC3 and MDA361. Most
Conclusion
A series of 4-morpholinopyrrolopyrimidine derivatives
have been designed, synthesized, and evaluated as PI3K
inhibitors. Compound 9 was found to be a selective and
potent PI3KR inhibitor with an IC50 of 21 nM and selecti-
vity over mTOR. Replacement of the 3-hydroxymethyl
group with a 4-arylurea not only improved enzyme potency
against PI3KR and mTOR but also significantly increased cell
potency against PC3 (prostate) and MDA361 (breast) cancer

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3175
Table 5a
IC50 (nM)
compd
PI3KR
mTOR
MDA361
PC3
T1/2b (Rat)
T1/2b (Human)
sol.,c pH 7.4
sol.,c pH 3.0
32
46
47
48
49
50
51
52
10
1
53
<3.0
10.3
6.7
82
>30
29
ND
>30
>30
>30
ND
1
0
0
0
1
1
0
1
ND
>100
68
0.9
1.4
2.4
6.0
1.8
2.7
0.8
0.6
0.4
1.7
1.7
0.5
1.4
0.9
13.0
23.0
17.0
45
>30
27
>100
ND
ND
11
>30
>30
15
5.5
4.5
26.5
17.0
27.0
>30
>30
>30
ND
ND
4.5
>30
a The values are averages of at least two separate determinations with a typical variation of less than (30%. ND: not determined. b Half-life of drug
when incubating with liver microsomes of the species shown. c Solubility in μg/mL.
exhibited excellent cell potency with single digit nanomolar
IC50 value in the tumor cell (MDA361) growth inhibition
assays. Among them, compounds 46 and 48 were found to
have good blood levels at 8 h after iv administration. In vivo
biomarker studies showed that both compounds 46 and 48
suppressed the formation of pAkt (T308), pAkt (S473), and
pS6K up to 8 h when administered at 25 mpk, iv, in the
MDA361 xenograft model. On the basis of the above results,
compound 46 was selected for in vivo efficacy studies in which
Figure 3. In vivo biomarker studies 8 h after compounds were
it demonstrated in vivo antitumor efficacy in the MDA361
administered at 25 mpk (iv) to MDA361 tumor bearing nude mice.
xenograft model. Evaluating the antitumor efficacy of 46 in
other tumor models is in progress.
Experimental Section
General. All solvents and reagents were used as received. 1H
NMR spectra were recorded with a Bruker DRX400 spectro-
meter; Chemical shifts are reported in parts per million (δ) using
tetramethylsilane as the internal standard with coupling con-
stants (J ) reported in hertz (Hz). The peak shapes are denoted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad. Mass spectra (MS) and high-resolution mass spectra
(HRMS) were measured with an Agilent TOF 2 spectrometer.
The purity of final compounds was determined by analytical
HPLC using a Prodigy ODS3 column (150 mm ꢀ 4.6 mm).
Conditions were as follows: ACN/H2O eluent at 1 mL/min flow
(containing 0.05% TFA) at 40 °C, 20 min, gradient 5% ACN to
95% ACN, monitored by UV absorption at 215 nm. All final
compounds were found to have g95% purity unless otherwise
specified. Reversed-phase HPLC (preparative HPLC) purifi-
cations were performed on a Gilson preparative HPLC system
controlled by Unipoint software using a Phenomenex Gemini
column (100 mm ꢀ 30 mm). Thin-layer chromatography (TLC)
was performed on TLC silica gel 60F254 aluminum sheets. The
terms “concentrated” and “evaporated” refer to removal of sol-
vents using a rotary evaporator at water aspirator pressure with
a bath temperature equal to or less than 40 °C.
4-(2-Chloro-6-methyl-5-nitropyrimidin-4-yl)morpholine (3).
To a stirred solution of 2,4-dichloro-6-methyl-5-nitropyrimi-
dine (5.0 g, 24.15 mmol) in CH2Cl2 (50 mL) was added a solution
Figure 4. Antitumor efficacy of 46 in the MDA361 xenograft
model.
cell lines. By use of molecular modeling, 4-ureidobenzamide
derivatives were designed and introduced, most of which

3176 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
of morpholine (2.1 mL, 24.15 mmol) in CH2Cl2 (20 mL),
followed by the addition of triethylamine (6.7 mL, 48.3 mmol)
at 0 °C. The resulting mixture was stirred at room temperature
overnight and diluted with CH2Cl2. The organic solution was
washed with water and brine and dried over MgSO4. The solvent
was evaporated, and the residue was purified by flash chroma-
tography to give the title compound as a yellow solid (6.17 g,
99% yield). H NMR (CDCl3, 400 MHz) δ 2.46 (s, 3H), 3.57
(t, 4H, J=4.58 Hz), 3.76 (t, 4H, J=4.8 Hz). MS (ESI): m/z 259
[M þ H].
2-(3-Methylphenyl)-4-morpholin-4-yl-5H-pyrrolo[3,2-d ]pyri-
midine (5). Compound 5 was isolated as an off-white solid (1.4%
yield), as a byproduct in the preparation of 4. 1H NMR (DMSO-
d6, 400 MHz) δ 2.39 (s, 3H), 3.80 (s, 8H), 6.53 (dd, 1H, J=3.3, 1.8
Hz), 7.20 (d, 1H, J=7.8 Hz), 7.32 (t, 1H, J=7.8 Hz), 7.59 (t, 1H,
J=2.8 Hz), 8.17 (d, 1H, J=7.8 Hz), 8.20 (s, 1H), 11.46 (s, 1H).
MS (ESI): m/z 295 [M þ H]. HRMS calcd for C17H18N4O [M þ
H] 295.1553, obsd 295.1552. HPLC purity 95%.
1
3-(4-Morpholin-4-yl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)aniline
(7). Compound 7 was prepared from 3 to give a yellow solid
(53% yield), according to the procedure described for 6 using
3-(4-Morpholin-4-yl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)phenol
(6). Step 1. To a stirred solution of 4-(2-chloro-6-methyl-5-
nitropyrimidin-4-yl)morpholine (3) (400 mg, 1.55 mmol) in
8 mL of 1,2-dimethoxymethane (DME) were added 3-benzyloxy-
phenylboronic acid (533 mg, 2.34 mmol), Pd(Ph3)4 (90mg,5mol%),
and 2 N Na2CO3 aqueous solution (6 mL). The resulting mixture
was heated at 110 °C for 30 min in a microwave oven. The
reaction mixture was cooled to room temperature, and the resul-
tant solid was filtered off and washed with THF. The resulting
filtrate was diluted with EtOAc, washed with brine, and dried
over MgSO4. The solvent was evaporated, and the residue
was purified by flash chromatography to give 4-{2-[3-(benzyl-
oxy)phenyl]-6-methyl-5-nitropyrimidin-4-yl}morpholine as a yel-
low solid (600 mg, 95% yield). MS (ESI): m/z 407 [M þ H].
Step 2. A mixture of 4-{2-[3-(benzyloxy)phenyl]-6-methyl-5-
nitropyrimidin-4-yl}morpholine (600 mg, 1.48 mmol) and 20
mL of N,N-dimethylformamide dimethyl acetal (DMF-DMA)
was heated at 110 °C overnight. The reaction mixture was cooled
to room temperature and concentrated in vacuo. The residue
was diluted with EtOAc and filtered through a short silica gel
column. The filtrate was concentrated, and the residue was
triturated with diethyl ether. The resulting red solid was col-
lected by filtration to give (E)-2-{2-[3-(benzyloxy)phenyl]-
6-morpholin-4-yl-5-nitropyrimidin-4-yl}-N,N-dimethylethen-
amine (641 mg, 94% yield). MS (ESI): m/z 462 [M þ H].
Step 3. To a solution of (E)-2-{2-[3-(benzyloxy)phenyl]-6-
morpholin-4-yl-5-nitropyrimidin-4-yl}-N,N-dimethylethen-
amine (350 mg, 0.76 mmol) in 50 mL of methanol was added
40 mg of 10% Pd/C as catalyst. The resulting mixture was
shaken under hydrogen (H2, 50 psi) at room temperature for 2 h.
The reaction mixture was filtered through a pad of Celite. The
filtration was concentrated in vacuo, and the residue was
purified by flash chromatography (EtOAc/hexanes=80:20) to
give 2-[3-(benzyloxy)phenyl]-4-morpholin-4-yl-5H-pyrrolo[3,2-
d ]pyrimidine as an off-white solid (249 mg, 85% yield). MS
(ESI): m/z 387.2 [M þ H].
1
3-nitrophenylboronic acid. H NMR (DMSO-d6, 400 MHz) δ
3.85 (t, 4H, J=4.8 Hz), 4.12 (t, 4H, J=4.8 Hz), 6.63 (d, 1H, J=
2.5 Hz), 6.91 (d, 1H, J=7.8 Hz), 7.31 (t, 1H, J=7.8 Hz), 7.42 (d,
1H, J=7.8 Hz), 7.49 (s, 1H), 7.89 (t, 1H, J=2.5 Hz), 12.50 (s,
1H). MS (ESI): m/z 296 [M þ H]. HRMS calcd for C16H17N5O
[M þ H] 296.1506, obsd 296.1506. HPLC purity 95%.
{3-[4-Morpholin-4-yl-7-(pyrrolidin-1-ylmethyl)-5H-pyrrolo-
[3,2-d ]pyrimidin-2-yl]phenyl}methanol (9). To a stirred solution
of [3-(4-morpholin-4-yl-5H-pyrrolo[3,2-d ]pyrimidin-2-yl)phe-
nyl]methanol (4) (19 mg, 0.06 mmol) in acetic acid (80% in
water, 1 mL) was added formaldehyde (37% in water, 19 mg,
0.24 mmol), followed by addition of pyrrolidine (13 mg, 0.18
mmol). The resulting mixture was heated at 60 °C for 6 h and
cooled to room temperature. The reaction mixture was concen-
trated, and the residue was subjected to HPLC separation to
give the title compound 9 as an off-white solid (12 mg, 52%
yield). 1H NMR (DMSO-d6, 400 MHz) δ 1.67 (m, 4H), 2.54 (m,
4H), 3.80 (m, 8H), 3.82 (s, 2H), 4.58 (br, 2H), 5.23 (br, 1H), 7.33
(d, 1H, J=7.6 Hz), 7.39 (t, 1H, J=7.6 Hz), 7.47 (s, 1H), 8.28 (d,
1H, J=7.6 Hz), 8.36 (s, 1H), 11.33 (s, 1H). MS (ESI): m/z 394
[M þ H]. HRMS calcd for C22H27N5O2 [M þ H] 394.2238, obsd
394.2237. HPLC purity 95%.
3-{7-[(Dimethylamino)methyl]-4-morpholin-4-yl-5H-pyrrolo-
[3,2-d]pyrimidin-2-yl}phenyl)methanol (8). Compound 8 was
prepared from 4 to give an off-white solid (64% yield), accord-
1
ing to the procedure described for 9, using dimethylamine. H
NMR (DMSO-d6, 400 MHz) δ 2.19 (s, 6H), 3.66 (s, 2H), 3.81 (m,
8H), 4.58 (br, 2H), 5.23 (br, 1H), 7.33 (d, 1H, J=7.6 Hz), 7.39 (t,
1H, J=7.6 Hz), 7.47 (s, 1H), 8.29 (d, 1H, J=7.6 Hz), 8.37 (s, 1H),
11.37 (s, 1H). MS (ESI): m/z 368 [M þ H]. HRMS calcd
for C20H25N5O2 [M þ H] 368.2081, obsd 368.2078. HPLC
purity 95%.
[3-(5-Methyl-4-morpholin-4-yl-5H-pyrrolo[3,2-d ]pyrimidin-2-
yl)phenyl]methanol (10). Step 1. To a solution of [3-(4-morpho-
lin-4-yl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)phenyl]methanol (4)
(1.469 g, 4.74 mmol) in DMF (5 mL) were added imidazole
(0.483 g, 7.10 mmol) and tert-butyldimethylsilyl chloride
(0.857 g, 5.69 mmol). The resulting mixture was heated at
80 °C for 15 min in microwave oven and cooled to room
temperature. The mixture was poured onto 20 mL of water
and extracted with EtOAc. The combined organic phases were
washed with water and brine and dried over MgSO4. The solvent
was evaporated, and the residue was purified by flash chroma-
tography (EtOAc/hexanes = 1:1) to give 2-[3-({[tert-butyl-
(dimethyl)silyl]oxy}methyl)phenyl]-4-morpholin-4-yl-5H-pyrro-
lo[3,2-d ]pyrimidine as a white solid (1.949 g, 97% yield). MS
(ESI): m/z 425 [M þ H].
Step 4. To a solution of 2-[3-(benzyloxy)phenyl]-4-morpho-
lin-4-yl-5H-pyrrolo[3,2-d]pyrimidine (249 mg, 0.64 mmol) in
20 mL of methanol were added 10% Pd/C (40 mg) and acetic
acid (1 mL). The resulting mixture was shaken under hydrogen
(H2, 50 psi) at room temperature overnight. The reaction
mixture was filtered through a pad of Celite, and the filtrate
was concentrated. The residue was purified by flash column
chromatography (EtOAc/hexanes=80:20) to give 6 as an off-
white solid (180 mg, 95% yield). 1H NMR (DMSO-d6, 400
MHz) δ 3.82-3.77 (m, 8H), 6.52 (d, 1H, J=3.0 Hz), 6.77 (dd,
1H, J=8.1, 2.8 Hz), 7.21 (t, 1H, J=7.8 Hz), 7.58 (t, 1H, J=3.3
Hz), 7.84-7.80 (m, 2H), 9.36 (s, 1H), 11.44 (s, 1H). MS (ESI):
m/z 297 [M þ H]. HRMS calcd for C16H16N4O2 [M þ H]
297.1346, obsd 297.1348. HPLC purity 98.9%.
Step 2. To a solution of 2-[3-({[tert-butyl(dimethyl)silyl]-
oxy}methyl)phenyl]-4-morpholin-4-yl-5H-pyrrolo[3,2-d ]pyri-
midine (424 mg, 1.0 mmol) in THF (5 mL) was added NaH (60%
in mineral oil, 80 mg, 2.0 mmol) at room temperature. After the
mixture was stirred for 10 min, iodomethane (170 mg, 1.2 mmol)
was added to the reaction mixture, and the resulting mixture was
stirred at room temperature for 2 h. The reaction was quenched
by addition of 2 mL of saturated aqueous ammonium chloride
solution, followed by addition of 10 mL of water. The mixture
was extracted with EtOAc, and combined organic phases were
washed with water and brine and dried over MgSO4. The solvent
was evaporated, and the residue was dissolved in 10 mL of
[3-(4-Morpholin-4-yl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)phenyl]-
methanol (4). Compound 4 was prepared from 3 to give an off-
white solid (41% yield), according to the procedure described for
1
6 (steps 1-3), using 3-(hydroxymethyl)phenylboronic acid. H
NMR (DMSO-d6, 400 MHz) δ 3.80 (s, 8H), 4.57 (d, 2H, J=5.5
Hz), 5.23 (d, 1H, J=5.8 Hz), 6.54 (dd, 1H, J=3.3, 1.5 Hz), 7.33
(d, 1H, J=7.6 Hz), 7.38 (t, 1H, J=7.6 Hz), 7.59 (t, 1H, J=3.3
Hz), 8.25 (d, 1H, J=7.6 Hz), 8.36 (s, 1H), 11.45 (s, 1H). MS
(ESI): m/z 311 [M þ H]. HRMS calcd for C17H18N4O2 [M þ H]
311.1502, obsd 311.1499. HPLC purity 97.6%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3177
CH2Cl2. To this solution was added dropwise trifluoroacetic
acid (TFA, 2 mL) at room temperature. The resulting mixture
was stirred at room temperature for 3 h and concentrated. The
residue was treated with 1 N NaOH aqueous solution (10 mL)
and extracted with CH2Cl2. The combined organic extracts were
washed with water and brine and dried over MgSO4. The solvent
was evaporated, and the residue was purified by flash chroma-
tography to give 10 as an off-white solid (252 mg, 78% yield). 1H
NMR (DMSO-d6, 400 MHz) δ 3.40 (t, 4H, J=4.8 Hz), 3.85 (t,
4H, J=4.8 Hz), 3.99 (s, 3H), 4.58 (d, 2H, J=5.8 Hz), 5.26 (t, 1H,
J=5.5 Hz), 6.60 (d, 1H, J=2.8 Hz), 7.35 (d, 1H, J=7.6 Hz), 7.41
(t, 1H, J=7.8 Hz), 7.67 (d, 1H, J=3.0 Hz), 8.27 (d, 1H, J=7.8
Hz), 8.38 (s, 1H). MS (ESI): m/z 325 [M þ H]. HRMS calcd for
C18H20N4O2 [M þ H] 325.1659, obsd 325.1663. HPLC purity
98.4%.
{3-[5-Methyl-4-morpholin-4-yl-7-(pyrrolidin-1-ylmethyl)-5H-
pyrrolo[3,2-d ]pyrimidin-2-yl]phenyl}methanol (11). Compound
11 was prepared from 10 to give an off-white solid (26% yield),
according to the procedure described for 9, using pyrrolidine.
1H NMR (DMSO-d6, 400 MHz) δ 1.68 (m, 4H), 2.54 (m, 4H),
3.40 (t, 4H, J=4.5 Hz), 3.80 (s, 2H), 3.85 (t, 4H, J=4.5 Hz), 3.92
(s, 3H), 4.59 (d, 2H, J=5.3 Hz), 5.23 (t, 1H, J=5.3 Hz), 7.35 (d,
1H, J=7.6 Hz), 7.4 (t, 1H, J=7.6 Hz), 7.55 (s, 1H), 8.3 (d, 1H,
J=7.6 Hz), 8.4 (s, 1H). MS (ESI): m/z 408 [M þ H]. HRMS
calcd for C23H29N5O2 [M þ H] 408.2394, obsd 408.2390. HPLC
purity 97.0%.
488 [M þ H]. HRMS calcd for C28H30FN5O2 [M þ H] 488.2456,
obsd 488.2455. HPLC purity 96.1%.
3-[7-(1-benzylpiperidin-4-yl)-4-morpholin-4-yl-5H-pyrrolo[3,2-d]-
pyrimidin-2-yl]phenol (13). Compound 13 was prepared from 12 to
give an off-white solid (TFA salt, 57% yield), according to the
procedure described for 14, using benzaldehyde. 1H NMR
(DMSO-d6, 400 MHz) δ 1.93 (q, 2H, J=12.5 Hz), 2.27 (d, 2H,
J=12.5 Hz), 3.09 (br, 2H), 3.12 (t, 1H, J=11.7 Hz), 3.52 (d, 2H, J=
11.7 Hz), 3.81 (t, 4H, J=4.5 Hz), 3.95 (br, 4H), 4.40 (s, 2H), 6.95
(br, 1H), 7.33 (t, 1H, J=7.4 Hz), 7.60-7.47 (m, 6H), 7.65 (br, 2H),
9.97 (br, 1H). MS (ESI): m/z 470 [M þ H]. HRMS calcd for
C28H31N5O2 [M þ H] 470.2551, obsd 470.2548. HPLC purity
97.7%.
[3-(4-Morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-2-yl)phenyl]-
methanol (17). Step 1. 7H-Pyrrolo[2,3-d ]pyrimidine-2,4-diol
(15). To a suspended solution of 6-aminouracil (12.7 g, 100
mmol) and sodium acetate (8.2 g, 100 mmol) in H2O (100 mL)
at a temperature of 70-75 °C was added a solution of
chloroacetaldehyde (50% in water, 23.6 g, 150 mmol). The
resulting reaction mixture was stirred at 80 °C for 20 min and
then cooled to room temperature. The resulting solid was
collected by filtration, washed with water and acetone, and
dried in vacuo to give 15 as brown solid (14.74 g, 98% yield).
1H NMR (DMSO-d6, 400 MHz) δ 6.22 (s, 1H), 6.56 (s, 1H),
10.46 (s, 1H), 11.08 (br, 1H), 11.43 (br, 1H). MS (ESI,
negative): m/z 150 [M - H].
3-(4-Morpholin-4-yl-7-piperidin-4-yl-5H-pyrrolo[3,2-d ]pyri-
midin-2-yl)phenol (12). Step 1. To a solution of 2-[3-(benzyl-
oxy)phenyl]-4-morpholin-4-yl-5H-pyrrolo[3,2-d]pyrimidine (see
preparation of 6) (500 mg, 1.29 mmol) in methanol (5 mL) was
added KOH (362 mg, 6.45 mmol, 5 equiv) and 4-piperidine
monohydrate hydrochloride (495 mg, 3.23 mmol, 2.5 equiv).
The resulting solutionwas heated at 66 °C overnight. The mixture
was cooled to room temperature and concentrated. The residue
was subjected to HPLC separation to give 2-[3-(benzyloxy)-
phenyl]-4-morpholin-4-yl-7-(1,2,3,6-tetrahydropyridin-4-yl)-5H-
pyrrolo[3,2-d ]pyrimidine as a yellow solid (300 mg, 50% yield).
MS (ESI): m/z 468 [M þ H].
Step 2. 2,4-Dichloro-7H-pyrrolo[2,3-d ]pyrimidine (15a). To a
20 mL vial were added 7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (15) (2.5
g, 16.6 mmol), POCl3 (10 mL, 107 mmol), and N,N-dimethylani-
line (1 mL, 7.9 mmol). The resulting mixture was heated at
120 °C for 30 min in a microwave oven. The reaction mixture
was cooled to room temperature and poured onto ice (about
200 g). The resulting solid was filtered and washed with water
to give dichloro 15a as a brown solid (1.323 g, 43% yield).
1H NMR (DMSO-d6, 400 MHz) δ 6.67 (m, 1H), 7.74 (m, 1H),
12.78 (br, 1H). MS (ESI): m/z 188 [M þ H].
Step 3. 2-Chloro-4-morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimi-
dine (16). To a solution of 2,4-dichloro-7H-pyrrolo[2,3-d]-
pyrimidine (1.38 g, 7.4 mmol) in CH2Cl2 (30 mL) were added
morpholine (0.96 mL, 11 mmol) and Et3N (2.1 mL, 15 mmol).
The mixture was stirred at room temperature overnight. The
resulting solid was filtered and washed with EtOH and water to
give 16 as a yellow solid (1.19 g, 68%). 1H NMR (DMSO-d6, 400
MHz) δ 3.72 (t, 4H, J=5.0 Hz), 3.84 (t, 4H, J=5.0 Hz), 6.67 (dd,
1H, J=3.3, 1.3 Hz), 7.21(dd, 1H, J=3.8, 2.3 Hz), 11.91 (br, 1H).
MS (ESI): m/z 239 [M þ H].
Step 2. To a solution of 2-[3-(benzyloxy)phenyl]-4-morpho-
lin-4-yl-7-(1,2,3,6-tetrahydropyridin-4-yl)-5H-pyrrolo[3,2-d ]-
pyrimidine (250 mg, 0.53 mmol) in methanol (20 mL) was added
10% Pd/C (50 mg) and concentrated HCl (30%, 0.2 mL). The
resulting mixture was shaken under hydrogen (H2, 50 psi) at
room temperature overnight. The reaction mixture was filtered
through a pad of Celite. The filtration was concentrated, and the
residue was purified by HPLC to give 12 as an off-white solid
(200 mg, 99% yield). 1H NMR (DMSO-d6, 400 MHz) δ 1.79 (qd,
2H, J=13.6, 3.5 Hz), 2.14 (d, 2H, J=13.3 Hz), 3.01 (q, 2H, J=
12.1 Hz), 3.33 (t, 1H, J=11.8 Hz), 3.45 (d, 2H, J=12.1 Hz), 3.84
(t, 4H, J=4.8 Hz), 4.13 (t, 4H, J=4.8 Hz), 7.11 (dd, 1H, J=8.1,
2.0 Hz), 7.45 (t, 1H, J=7.8 Hz), 7.51 (t, 1H, J=2.0 Hz), 7.54 (d,
1H, J=7.8 Hz), 7.78 (d, 1H, J=3.0 Hz), 8.48 (d, 1H, J=10.3 Hz),
8.64 (d, 1H, J=10.6 Hz), 12.46 (d, 1H, J=3.0 Hz). MS (ESI): m/z
380 [M þ H]. HRMS calcd for C21H25N5O2 [M þ H] 380.2081,
obsd 380.2088. HPLC purity 95%.
3-{7-[1-(4-Fluorobenzyl)piperidin-4-yl]-4-morpholin-4-yl-5H-
pyrrolo[3,2-d ]pyrimidin-2-yl}phenol (14). To a solution of 3-(4-
morpholin-4-yl-7-piperidin-4-yl-5H-pyrrolo[3,2-d]pyrimidin-2-
yl)phenol (12) (22 mg, 0.058 mmol) in methanol (1 mL) was
added 4-fluorobenzaldehyde (22 mg, 0.177 mmol), followed by
addition of ZnCl2 (24 mg, 0.174 mmol) and NaCNBH3 (11 mg,
0.174 mmol). The resulting mixture was stirred at room tem-
perature overnight. The solvent was evaporated, and the residue
was subjected to HPLC separation to give 14 as an off-white
solid (TFA salt, 17.2 mg, 49% yield). 1H NMR (DMSO-d6, 400
MHz) δ 1.93 (q, 2H, J=12.6 Hz), 2.27 (d, 2H, J=12.6 Hz), 3.07
(br, 2H), 3.22 (t, 1H, J=11.6 Hz), 3.52 (d, 2H, J=11.6 Hz), 3.81
(t, 4H, J=4.5 Hz), 3.95 (br, 4H), 4.40 (s, 2H), 6.95 (s, 1H), 7.31 (t,
1H, J=7.4 Hz), 7.35 (t, 2H, J=8.6 Hz), 7.54 (br, 1H), 7.63 (dd,
2H, J=8.6, 5.4 Hz), 7.68 (br, 2H), 9.98 (br, 1H). MS (ESI): m/z
Step 4. [3-(4-Morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-2-
yl)phenyl]methanol (17). To a 10 mL vial were added 2-chloro-
4-morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidine (16) (150 mg,
0.63 mmol), 3-hydroxymethylphenylboronic acid (144 mg,
0.94 mmol), Pd(PPh3)4 (36 mg, 5 mol %), 1,2-dimethoxyethane
(DME, 2.5 mL), and 2 M Na2CO3 aqueous solution (1.5 mL).
The resulting mixture was heated at 120 °C for 1 h in a microwave
oven. The reaction mixture was cooled to room temperature and
diluted with EtOAc. The aqueous phase was extracted with
EtOAc, and the combined organic phases were washed with brine
and dried over MgSO4. The solvent was evaporated, and the
residue was subjected to HPLC separation to give 17 as an off-
white solid (98 mg, 50% yield). 1H NMR (DMSO-d6, 400 MHz) δ
3.78 (t, 4H, J=4.8 Hz), 3.95 (t, 4H, J=4.8 Hz), 4.58 (d, 2H, J=5.8
Hz), 5.24 (t, 1H, J=5.8 Hz), 6.66 (d, 1H, J=3.5 Hz), 7.24 (dd, 1H,
J=3.5, 2.8 Hz), 7.35 (d, 1H, J=7.3 Hz), 7.40 (t, 1H, J=7.8 Hz),
8.24 (d, 1H, J=7.8 Hz), 8.35 (s, 1H), 11.80 (s, 1H). MS (ESI): m/z
311 [M þ H]. HRMS calcd for C17H18N4O2 [M þ H] 311.1502,
obsd 311.1501. HPLC purity 95%.
3-(4-Morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-2-yl)phenol
(18). Following the same procedure as for the preparation of 17,
Suzuki coupling of 2-chloro-4-morpholin-4-yl-7H-pyrrolo[2,3-
d]pyrimidine (16) (150 mg, 0.63 mmol) with 3-hydroxyphenyl-
boronic acid (130 mg, 0.94 mmol) gave 18 as a yellow solid

3178 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
1
(130 mg, 70% yield). H NMR (DMSO-d6, 400 MHz) δ 3.78
1-(4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-pyridin-3-ylurea (24). Com-
pound 24 was prepared from 22 to give an off-white solid (45%
yield), according to the procedure described for 23, using
3-aminopyridine. MS (ESI): m/z 487 [M þ H]. HRMS calcd
for C26H30N8O2 [M þ H] 487.2564, obsd 487.2564. HPLC
purity 96.0%.
1-(4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-pyridin-4-ylurea (25). Com-
pound 25 was prepared from 22 to give an off-white solid (62%
yield), according to the procedure described for 23, using
4-aminopyridine. 1H NMR (CD3OD, 400 MHz) δ 2.34 (s,
6H), 2.83 (t, 2H, J=6.8 Hz), 3.85 (t, 4H, J=5.0 Hz), 4.00 (t,
4H, J=5.3 Hz), 4.42 (t, 2H, J=7.1 Hz), 6.60 (d, 1H, J=3.5 Hz),
7.16 (d, 1H, J=3.5 Hz), 7.52 (t, 2H, J=1.8 Hz), 7.54 (d, 2H, J=
1.8 Hz), 8.32 (dd, 2H, J=4.8, 1.5 Hz), 8.39 (d, 2H, J=8.3 Hz).
MS (ESI): m/z 487 [M þ H]. HRMS calcd for C26H30N8O2 [M þ
H] 487.2564, obsd 487.2564. HPLC purity 95%.
1-(4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-(4-fluorophenyl)urea (26).
Compound 26 was prepared from 22 to give an off-white solid
(33% yield), according to the procedure described for 23, using
4-fluoroaniline. MS (ESI): m/z 504 [M þ H]. HRMS calcd
for C27H30FN7O2 [M þ H] 504.2518, obsd 504.2515. HPLC
purity 95%.
1-(4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-ethylurea (27). Compound
27 was prepared from 22 to give a yellow solid (41% yield),
according to the procedure described for 23, using ethylamine.
1H NMR (DMSO-d6, 400 MHz) δ 1.06 (t, 3H, J=7.1 Hz), 2.88
(s, 6H), 3.12 (q, 2H, J=7.1 Hz), 3.61 (br, 2H), 3.77 (t, 4H, J=4.5
Hz), 3.94 (t, 4H, J=4.5 Hz), 4.62 (t, 2H, J=6.1 Hz), 6.27 (br,
1H), 6.75 (d, 1H, J=3.6 Hz), 7.33 (d, 1H, J=3.6 Hz), 7.49 (d, 2H,
J=8.7 Hz), 8.28 (d, 2H, J=8.7 Hz), 8.71 (s, 1H), 9.65 (br, 1H).
MS (ESI): m/z 438 [M þ H]. HRMS calcd for C23H31N7O2 [M þ
H] 438.2612, obsd 438.2609. HPLC purity 97.2%.
1-(4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-methylurea (28). Compound
28 was prepared from 22 to give a yellow solid (34% yield),
according to the procedure described for 23, using methylamine.
1H NMR (DMSO-d6, 400 MHz) δ 2.66 (s, 3H), 2.88 (s, 6H), 3.61
(br, 2H), 3.77 (t, 4H, J=4.5 Hz), 3.94 (t, 4H, J=4.5 Hz), 4.61 (t,
2H, J=6.1 Hz), 6.12 (br, 1H), 6.75 (d, 1H, J=3.6 Hz), 7.33 (d,
1H, J=3.6 Hz), 7.50 (d, 2H, J=8.7 Hz), 8.28 (d, 2H, J=8.7 Hz),
8.78 (s, 1H), 9.60 (br, 1H). MS (ESI): m/z 424 [M þ H]. HRMS
calcd for C22H29N7O2 [M þ H] 424.2456, obsd 424.2453. HPLC
purity 98.2%.
1-[4-(4-Morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)phe-
nyl]-3-pyridin-4-ylurea (29). To a 10 mL vial were charged
4-isocyantophenylboronic acid, pinacol ester (368 mg, 1.5
mmol), 4-aminopyridine (188 mg, 2.0 mmol), Et3N (0.28 mL,
2.0 mmol), and DME (3 mL). The mixture was stirred at room
temperature for 5 h, and to the mixture were then added
2-chloro-4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidine (16)
(238 mg, 1.0 mmol), Na2CO3 aqueous solution (2 M, 2 mL),
and Pd(PPh3)4 (58 mg, 5 mol %). The resulting mixture was
heated at 120 °C for 30 min in microwave oven. The reaction
mixture was cooled to room temperature and diluted with
EtOAc. The aqueous phase was extracted with EtOAc, and the
combined organic phases were washed with brine and dried
over MgSO4. The solvent was evaporated, and the residue was
subjected to HPLC separation to give 29 as a yellow solid (66
mg, 16% yield). 1H NMR (DMSO-d6, 400 MHz) δ 3.78 (t, 4H,
J=4.5 Hz), 3.95 (t, 4H, J=5.0 Hz), 6.66 (m, 1H), 7.23 (m, 1H),
7.63 (d, 2H, J=8.6 Hz), 7.96 (d, 2H, J=7.3 Hz), 8.34 (d, 2H, J=
8.6 Hz), 8.62 (d, 2H, J=7.6 Hz), 9.96 (s, 1H), 10.84 (s, 1H),
11.79 (s, 1H). MS(ESI): m/z 416 [M þ H]. HRMS calcd
for C22H21N7O2 [M þ H] 416.1829, obsd 416.1830. HPLC
purity 95%.
(t, 4H, J=5.0 Hz), 3.93 (t, 4H, J=5.0 Hz), 6.65 (d, 1H, J=3.3
Hz), 6.80 (dd, 1H, J=8.3, 2.5 Hz), 7.23 (dd, 2H, J=7.8, 4.5 Hz),
7.80 (m, 2H), 9.40 (s, 1H), 11.77 (s, 1H). MS (ESI): m/z 297 [M þ
H]. HRMS calcd for C16H16N4O2 [M þ H] 297.1346, obsd
297.1347. HPLC purity 95.3%.
2-Chloro-4-morpholin-4-yl-7-[2-(dimethylamino)ethyl]-7H-pyr-
rolo[2,3-d ]pyrimidine (19). To a solution of 2-chloro-4-morpho-
lin-4-yl-7H-pyrrolo[2,3-d ]pyrimidine (16) (154 mg, 0.65 mmol)
in DMF (5 mL) were added 2-(dimethylamino)ethyl chloride
hydrochloride (140 mg, 0.97 mmol) and Cs2CO3 (635 mg, 1.95
mmol). The resulting mixture was heated at 80 °C under
nitrogen overnight and cooled to room temperature. Water
was added, and the mixture was extracted with EtOAc. The
combined extracts were washed with water and brine and dried
over MgSO4. The solvent was evaporated to give 19 as a yellow
syrup (169 mg, 84% yield), which was used in next step without
further purification. 1H NMR (CDCl3, 300 MHz) δ 2.27 (s, 6H),
2.68 (t, 2H, J=6.4 Hz), 3.82 (t, 4H, J=4.9 Hz), 3.94 (t, 4H, J=5.3
Hz), 4.25 (t, 2H, J=6.4 Hz), 6.43 (d, 1H, J=3.8 Hz), 7.03 (d, 1H,
J=3.8 Hz). MS (ESI): m/z 310 [M þ H].
(3-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl}phenyl)methanol (20). Following the same
procedure as for the preparation of 17, Suzuki coupling of
2-chloro-4-morpholin-4-yl-7-[2-(dimethylamino)ethyl]-7H-pyr-
rolo[2,3-d ]pyrimidine (19) (80 mg, 0.26 mmol) and 3-hydroxy-
methylphenylboronic acid (58 mg, 0.38 mmol) gave 20 as an off-
white solid (96 mg, 88% yield). 1H NMR (CD3OD, 300 MHz) δ
3.01 (s, 6H), 3.79-3.71 (m, 2H), 3.94 (t, 4H, J=4.9 Hz), 4.11 (t,
4H, J=5.3 Hz), 4.76 (s, 2H), 4.87 (m, 2H), 7.09 (d, 1H, J=3.8
Hz), 7.66-7.56 (m, 3H), 8.07 (d, 1H, J=7.5 Hz), 8.17 (s, 1H).
MS (ESI): m/z 382 [M þ H]. HRMS calcd for C21H27N5O2 [M þ
H] 382.22386, obsd 382.2235. HPLC purity 97.5%.
3-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl}phenol (21). Following the same procedure
as for the preparation of 17, Suzuki coupling of 2-chloro-4-
morpholin-4-yl-7-[2-(dimethylamino)ethyl]-7H-pyrrolo[2,3-d]-
pyrimidine (19) (80 mg, 0.26 mmol) and 3-hydroxyphenylboronic
acid (54 mg, 0.38 mmol) gave 21 as an off-white solid (81.9 mg,
78% yield). 1H NMR (CD3OD, 300 MHz) δ 3.01 (s, 6H), 3.74 (t,
2H, J=6.0 Hz), 3.90 (t, 4H, J=5.3 Hz), 4.06 (t, 4H, J=5.3 Hz),
4.79 (t, 2H, J=6.0 Hz), 6.96 (d, 1H, J=3.8 Hz), 7.0 (dd, 1H, J=
7.9, 2.3 Hz), 7.37 (t, 1H, J=7.9 Hz), 7.50 (d, 1H, J=3.8 Hz),
7.68-7.62 (m, 2H). MS (ESI): m/z 368 [M þ H]. HRMS calcd for
C20H25N5O2 [M þ H] 368.2081, obsd 368.2082. HPLC purity
98.8%.
4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl}aniline (22). Following the same procedure
as for the preparation of 17, Suzuki coupling of 2-chloro-4-
morpholin-4-yl-7-[2-(dimethylamino)ethyl]-7H-pyrrolo[2,3-d]-
pyrimidine (19) (261 mg, 0.84 mmol) and 4-aminophenylboronic
acid pinacol ester (277 mg, 1.27 mmol) gave 22 as a yellow oil (278
mg, 90% yield). 1H NMR (CDCl3, 300 MHz) δ 2.32 (s, 6H), 2.76
(t, 2H, J=6.4 Hz), 3.86 (t, 4H, J=5.3 Hz), 4.0 (t, 4H, J=5.3 Hz),
4.37 (t, 2H, J=6.4Hz), 6.42 (d, 1H, J=3.8Hz), 6.72 (d, 2H, J=8.7
Hz), 6.98 (d, 1H, J=3.8 Hz), 8.30 (d, 2H, J=8.7 Hz). MS (ESI):m/
z 367 [M þ H].
1-(4-{7-[2-(Dimethylamino)ethyl]-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-pyridin-2-ylurea (23). To a
solution of 4-{7-[2-(dimethylamino)ethyl]-4-morpholin-4-yl-
7H-pyrrolo[2,3-d ]pyrimidin-2-yl}aniline (22) (22 mg, 0.06
mmol) in CHCl3 (1 mL) were added Et3N (25 μL, 0.18 mmol)
and triphosgene (18 mg, 0.06 mmol). The mixture was stirred at
room temperature for 15 min, and 2-aminopyridine (17 mg, 0.18
mmol) was added. The mixture was stirred at room temperature
overnight. The solvent was evaporated, and the residue was
subjected to HPLC separation to give 23 as an off-white solid
(15 mg, 51% yield). MS (ESI): m/z 487 [M þ H]. HRMS calcd
for C26H30N8O2 [M þ H] 487.2564, obsd 487.2561. HPLC
purity 99.0%.

Article
2-Chloro-4-morpholin-4-yl-7-(2,2,2-trifluoroethyl)-7H-pyr-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3179
and sodium carbonate aqueous solution (2M, 4 mL). The result-
ing mixture was heated at 130 °C for 30 min in a microwave oven.
The reaction mixture was cooled to room temperature and diluted
with EtOAc. The aqueous phase was extracted with EtOAc, and
the combined organic solution was concentrated. The residue was
purified by flash chromatography to give 4-[7-(2,2-dimethoxy-
ethyl)-4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]aniline
as a brown oil (760 mg, 97% yield). MS (ESI): m/z 384 [M þ H].
Step 2. To a solution of 4-[7-(2,2-dimethoxyethyl)-4-morpho-
lin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]aniline (766 mg, 2 mmol)
in CHCl3 (10 mL) were added Et3N (0.55 mL, 3.9 mmol) and
triphosgene (594 mg, 2 mmol). The mixture was stirred at room
temperature for 15 min before a solution of 4-aminopyridine
(564 mg, 6 mmol) in THF (10 mL) was added. The mixture was
heated at 50 °C overnight. The solvent was evaporated, and the
residue was subjected to HPLC separation to give 33 as a yellow
solid (350 mg, 35% yield). 1H NMR (DMSO-d6, 400 MHz) δ 3.31
(s,6H), 3.78(t, 4H, J=4.8Hz), 3.95(t, 4H, J=4.8Hz), 4.35 (d, 2H,
J=5.3Hz), 4.78(t, 1H, J=5.3Hz), 6.67(d, 1H, J=3.5Hz), 7.27 (d,
1H, J=3.5 Hz), 7.52 (d, 2H, J=6.5 Hz), 7.57 (d, 2H, J=8.8 Hz),
8.35 (d, 2H, J=8.8 Hz), 8.40 (d, 2H, J=6.5 Hz), 9.15 (s, 1H),
9.32 (s, 1H). MS (ESI): m/z 504 [M þ H]. HRMS calcd
for C26H29N7O4 [M þ H] 504.2354, obsd 504.2358. HPLC
purity 95%.
rolo[2,3-d ]pyrimidine (30). To a solution of 2-chloro-4-morpho-
lin-4-yl-7H-pyrrolo[2,3-d ]pyrimidine (16) (340 mg, 1.4 mmol) in
DMF (5 mL) were added 1,1,1-trifluoro-2-iodoethane (0.28 mL,
2.8 mmol) and Cs2CO3 (559 mg, 1.7 mmol). The resulting
mixture was heated at 80 °C under nitrogen overnight and
cooled to room temperature. The reaction mixture was
quenched with water and extracted with EtOAc. The combined
extracts were washed with water and brine and dried over
MgSO4. The solvent was evaporated to give 30 as a light-yellow
solid (199 mg, 43% yield), which was used in the next step
without further purification. 1H NMR (DMSO-d6, 400 MHz) δ
3.72 (t, 4H, J=5.0 Hz), 3.86 (t, 4H, J=5.0 Hz), 5.05 (q, 2H, J=
9.3 Hz), 6.84 (d, 1H, J=3.5 Hz), 7.34 (d, 1H, J=3.8 Hz). MS
(ESI): m/z 321 [M þ H].
4-[7-(2,2,2-Trifluoroethyl)-4-morpholin-4-yl-7H-pyrrolo[2,3-d ]-
pyrimidin-2-yl]aniline (42). To a 10 mL vial were added 2-chloro-
4-morpholin-4-yl-7-(2,2,2-trifluoroethyl)-7H-pyrrolo[2,3-d]pyri-
midine (30) (294 mg, 0.9 mmol), 4-aminophenylboronic acid
pinacol ester (302 mg, 1.4 mmol), Pd(PPh3)4 (53 mg, 5 mol %),
1,2-dimethoxyethane (DME, 3 mL), and Na2CO3 aqueous
solution (2 M, 2 mL). The resulting mixture was heated at
130 °C for 30 min in a microwave oven. The reaction mixture
was cooled to room temperature and diluted with EtOAc. The
aqueous phase was extracted with EtOAc, and the combined
organic phases were washed with brine and dried over MgSO4.
The solvent was evaporated, and the residue was purified by
flash chromatography to give 42 as a brown oil (286 mg, 83%
yield). 1H NMR (DMSO-d6, 400 MHz) δ 3.77 (t, 4H, J=5.0 Hz),
3.91 (t, 4H, J=4.8 Hz), 5.12 (q, 2H, J=9.3 Hz), 5.44 (s, 2H), 6.61
(d, 2H, J=9.3 Hz), 6.74 (d, 1H, J=4.0 Hz), 7.25 (d, 1H, J=4.0
Hz), 8.12 (d, 2H, J=9.1 Hz). MS (ESI): m/z 378 [M þ H].
1-{4-[4-Morpholin-4-yl-7-(2,2,2-trifluoroethyl)-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (32). To a solu-
tion of 4-[7-(2,2,2-trifluoroethyl)-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl]aniline (42) (25 mg, 0.066 mmol) in CHCl3
(1 mL) were added Et3N (28 μL, 0.2 mmol) and triphosgene
(20 mg, 0.066 mmol). The mixture was stirred at room tempera-
ture for 15 min before a solution of 4-aminopyridine (19 mg, 0.2
mmol) in THF (1 mL) was added. The mixture was stirred at
room temperature overnight. The solvent was evaporated, and
the residue was subjected to HPLC separation to give 32 as an
off-white solid (24.5 mg, 61% yield). 1H NMR (DMSO-d6, 300
MHz) δ 3.78 (t, 4H, J=5.3 Hz), 3.97 (t, 4H, J=5.3 Hz), 5.18 (q,
2H, J=9.8 Hz), 6.82 (d, 1H, J=3.8 Hz), 7.35 (d, 1H, J=3.8 Hz),
7.63 (d, 2H, J=8.7 Hz), 7.94 (d, 2H, J=7.2 Hz), 8.41 (d, 2H, J=
8.7 Hz), 8.61 (d, 2H, J=7.2 Hz), 9.85 (s, 1H), 10.64 (s, 1H). MS
(ESI): m/z 498 [M þ H]. HRMS calcd for C24H22F3N7O2 [M þ
H] 498.1860, obsd 498.1860. HPLC purity 97.6%.
2-Chloro-7-(2,2-dimethoxyethyl)-4-morpholin-4-yl-7H-pyr-
rolo[2,3-d ]pyrimidine (31). To a solution of 2-chloro-4-morpho-
lin-4-yl-7H-pyrrolo[2,3-d ]pyrimidine (16) (650 mg, 2.7 mmol) in
DMF (10 mL) were added 2-bromo-1,1-dimethoxyethane
(0.65 mL, 5.4 mmol) and Cs2CO3 (1.067 g, 3.3 mmol). The
resulting mixture was heated at 80 °C under nitrogen overnight
and cooled to room temperature. Water was added, and the
mixture was extracted with EtOAc. The combined extracts were
washed with water and brine and dried over MgSO4. The solvent
was evaporated to give 31 as a light-yellow solid (665 mg, 75%
yield), which was used in the next step without further purifica-
tion. 1H NMR (DMSO-d6, 400 MHz) δ 3.27 (s, 6H), 3.72 (t, 4H,
J=5.0 Hz), 3.84 (t, 4H, J=5.3 Hz), 4.20 (d, 2H, J=5.5 Hz), 4.67
(t, 1H, J=5.3 Hz), 6.7 (d, 1H, J=4.0 Hz), 7.26 (d, 1H, J=3.8
Hz). MS (ESI): m/z 327 [M þ H].
1-{4-[4-Morpholin-4-yl-7-(2-oxoethyl)-7H-pyrrolo[2,3-d]pyri-
midin-2-yl]phenyl}-3-pyridin-4-ylurea (34).
A mixture of
1-{4-[7-(2,2-dimethoxyethyl)-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (33) (300
mg, 0.6 mmol), dioxane (3 mL), and 6 M HCl (3 mL) was
heated at 70 °C for 3 h and cooled to room temperature. The
mixture was concentrated in vacuo, and the residue was
triturated with EtOAc. The resulting solid was collected by
filtration and washed with EtOAc to give 34 as an off-white
solid (479 mg, 85% yield). 1H NMR (DMSO-d6, 400 MHz) δ
3.79 (t, 4H, J=4.5 Hz), 3.96 (t, 4H, J=4.5 Hz), 5.21(s, 2H),
6.74 (d, 1H, J=3.6 Hz), 7.25 (d, 1H, J=3.6 Hz), 7.60 (d, 2H,
J=8.6 Hz), 7.94 (d, 2H, J=6.7 Hz), 8.35 (d, 2H, J=8.6 Hz),
8.61 (d, 2H, J=6.7 Hz), 9.72 (s, 1H), 9.81 (s, 1H), 10.62 (s,
1H). MS (ESI): m/z 458 [M þ H]. HPLC purity 95%.
1-{4-[4-Morpholin-4-yl-7-(2-pyrrolidin-1-ylethyl)-7H-pyr-
rolo[2,3-d ]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (35). To a
solution of 1-{4-[4-morpholin-4-yl-7-(2-oxoethyl)-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (34) (24 mg,
0.05 mmol) in MeOH (2 mL) were added pyrrolidine (22 mg,
0.3 mmol), ZnCl2 (14 mg, 0.1 mmol), and NaBH3CN (6 mg, 0.1
mmol). The resulting mixture was stirred at room temperature
for 2 h, and 0.5 mL of NaOH (1 M in water) was added. The
solvent was evaporated, and the residue was subjected to HPLC
separation to give 35 as an off-white solid (9.2 mg, 25% yield).
MS (ESI): m/z 513 [M þ H]. HRMS calcd for C28H32N8O2 [M þ
H] 513.2721, obsd 513.2718. HPLC purity 95%.
1-{4-[4-Morpholin-4-yl-7-(2-piperidin-1-ylethyl)-7H-pyrrolo-
[2,3-d]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (36). Com-
pound 36 was prepared from 34 to give an off-white solid
(27% yield), according to the procedure described for 35,
using piperidine. MS (ESI): m/z 527 [M þ H]. HRMS calcd
for C29H34N8O2 [M þ H] 527.2880, obsd 527.2877. HPLC
purity 95.7%.
1-{4-[7-(2-{[2-(Dimethylamino)ethyl]amino}ethyl)-4-morpho-
lin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-2-yl]phenyl}-3-pyridin-4-yl-
urea (37). Compound 37 was prepared from 34 to give an off-
white solid (37% yield), according to the procedure described
for 35, using N,N-dimethylethylenediamine. 1H NMR (DMSO-
d6, 400 MHz) δ 2.81 (s, 6H), 3.33 (m, 2H), 3.42 (m, 2H), 3.54 (t,
2H, J=6.1 Hz), 3.78 (t, 4H, J=4.5 Hz), 3.96 (m, 4H), 4.58 (d, 2H,
J=6.3 Hz), 6.77 (d, 1H, J=3.6 Hz), 7.32 (d, 1H, J=3.6 Hz), 7.66
(d, 2H, J=8.7 Hz), 7.98 (d, 2H, J=6.8 Hz), 8.42 (d, 2H, J=8.7
Hz), 8.63 (d, 2H, J=6.8 Hz), 10.46 (s, 1H), 11.38 (s, 1H). MS
(ESI): m/z 530 [M þ H]. HRMS calcd for C28H35N9O2 [M þ H]
530.2986, obsd 530.2976. HPLC purity 95%.
1-{4-[7-(2,2-Dimethoxyethyl)-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (33). Step 1. To
a 20 mL vial were added 2-chloro-7-(2,2-dimethoxyethyl)-4-mor-
pholin-4-yl-7H-pyrrolo[2,3-d]pyrimidine (31) (665 mg, 2 mmol),
4-aminophenylboronic acid pinacol ester (670 mg, 3 mmol), Pd-
(PPh3)4 (118 mg, 5 mol %), 1,2-dimethoxyethane (DME, 6 mL),

3180 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
1-(4-{7-[2-(4-Methylpiperazin-1-yl)ethyl]-4-morpholin-4-yl-
7H-pyrrolo[2,3-d ]pyrimidin-2-yl}phenyl)-3-pyridin-4-ylurea (38).
Compound 38 was prepared from 34 to give an off-white solid
(42% yield), according to the procedure described for 35, using
1-methylpiperazine. 1H NMR (DMSO-d6, 400 MHz) δ 2.74 (s,
3H), 2.95(t, 2H, J=6.0 Hz), 3.30 (br, 8H), 3.78(t, 4H, J=4.5 Hz),
3.95(t, 4H, J=4.5Hz), 4.42 (t, 2H, J=6.0Hz), 6.68(d, 1H, J=3.6
Hz), 7.35 (d, 1H, J=3.6 Hz), 7.66 (d, 2H, J=8.7 Hz), 7.98 (d, 2H,
J=7.0 Hz), 8.38 (d, 2H, J=8.7 Hz), 8.63 (d, 2H, J=7.0 Hz), 10.46
(s, 1H), 11.41 (s, 1H). MS (ESI): m/z 542 [M þ H]. HRMS calcd
for C29H35N9O2 [M þ H] 542.2986, obsd 542.2981. HPLC purity
96.5%.
1-{4-[4-Morpholin-4-yl-7-(2-piperazin-1-ylethyl)-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (39). Com-
pound 39 was prepared from 34 to give an off-white solid
(39% yield), according to the procedure described for 35,
using piperazine. 1H NMR (DMSO-d6, 400 MHz) δ 2.90 (br,
4H), 2.99 (br, 2H), 3.07 (br, 4H), 3.78 (t, 4H, J=4.5 Hz), 3.95
(t, 4H, J=4.5 Hz), 4.43 (t, 2H, J=6.0 Hz), 6.69 (d, 1H, J=3.6
Hz), 7.35 (d, 1H, J=3.6 Hz), 7.66 (d, 2H, J=8.8 Hz), 7.98 (d,
2H, J=7.0 Hz), 8.38 (d, 2H, J=8.8 Hz), 8.62 (d, 2H, J=7.0
Hz), 10.40 (s, 1H), 11.40 (s, 1H). MS (ESI): m/z 528 [M þ H].
HRMS calcd for C28H33N9O2 [M þ H] 528.2830, obsd
528.2828. HPLC purity 95.5%.
1-{4-[7-(2-Hydroxyethyl)-4-morpholin-4-yl-7H-pyrrolo[2,3-d ]-
pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (40). To a stirred mix-
ture of 1-{4-[4-morpholin-4-yl-7-(2-oxoethyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]phenyl}-3-pyridin-4-ylurea (34) (215 mg, 0.47
mmol), MeOH (4 mL), and THF (4 mL) was added NaBH4
(27 mg, 0.7 mmol). The resulting mixture was stirred at room
temperature for 30 min, and 2 mL of NaOH (1 M in water) was
added. The mixture was concentrated, and the residue was
subjected to HPLC separation to give 40 as an off-white solid
(165 mg, 76% yield). 1H NMR (DMSO-d6, 400 MHz) δ 3.78 (t,
6H, J=5.0 Hz), 3.94 (t, 4H, J=5.0 Hz), 4.29 (t, 2H, J=5.5 Hz),
4.96 (t, 1H, J=5.5 Hz), 6.65 (d, 1H, J=3.8 Hz), 7.29 (d, 1H, J=
3.8 Hz), 7.45 (d, 2H, J=6.3 Hz), 7.56 (d, 2H, J=8.6 Hz), 8.34 (d,
2H, J=8.6 Hz), 8.37 (d, 2H, J=6.3 Hz), 9.06 (s, 1H), 9.13 (s, 1H).
MS (ESI): m/z 460 [M þ H]. HRMS calcd for C24H25N7O3 [M þ
H] 460.2092, obsd 460.2092. HPLC purity 98.1%.
0.12 mmol). The resulting mixture was stirred at room tempera-
ture overnight and concentrated. The residue was subjected to
HPLC separation to give 46 as an off-white solid (TFA salt, 38.6
mg, 87% yield). 1H NMR (DMSO-d6, 400 MHz) δ 2.83 (br, 6H),
3.02 (s, 3H), 3.35 (q, 2H, J=5.8 Hz), 3.79 (br, 6H), 3.97 (br, 4H),
5.19 (q, 2H, J=8.6 Hz), 6.82 (d, 1H, J=3.8 Hz), 7.35 (d, 1H, J=
3.8 Hz), 7.47 (d, 2H, J=8.3 Hz), 7.57 (t, 4H, J=8.3 Hz), 8.36 (d,
2H, J=8.8 Hz), 9.48 (s, 1H), 9.54 (s, 1H), 10.03 (br, 1H). MS
(ESI): m/z 625 [M þ H]. HRMS calcd for C31H35F3N8O3 [M þ
H] 625.2857, obsd 625.2857. HPLC purity 96.7%.
N-[2-(Dimethylamino)ethyl]-4-[({4-[4-morpholin-4-yl-7-(2,2,
2-trifluoroethyl)-7H-pyrrolo[2,3-d ]pyrimidin-2-yl]phenyl}carba-
moyl)amino]benzamide (47). Compound 47 was prepared from
44 to give an off-white solid (TFA salt, 99% yield), according to
the procedure described for 46, using N,N-dimethylethylenedi-
amine. 1H NMR (DMSO-d6, 400 MHz) δ 2.83 (s, 3H), 2.84 (s,
3H), 3.26 (q, 2H, J=5.8 Hz), 3.62 (q, 2H, J=5.8 Hz), 3.78 (t, 4H,
J=4.8 Hz), 3.96 (t, 4H, J=4.8 Hz), 5.18 (q, 2H, J=8.6 Hz), 6.81
(d, 1H, J=3.8 Hz), 7.34 (d, 1H, J=3.8 Hz), 7.57 (dd, 4H, J=8.6,
1.8 Hz), 7.87 (d, 2H, J=8.6 Hz), 8.35 (d, 2H, J=8.6 Hz), 8.67 (t,
1H, J=5.8 Hz), 9.51 (s, 1H), 9.59 (s, 1H), 9.94 (br, 1H). MS
(ESI): m/z 611 [M þ H]. HRMS calcd for C30H33F3N8O3 [M þ
H] 611.2700, obsd 611.2700. HPLC purity 96.8%.
1-{4-[(4-Methylpiperazin-1-yl)carbonyl]phenyl}-3-{4-[4-mor-
pholin-4-yl-7-(2,2,2-trifluoroethyl)-7H-pyrrolo[2,3-d ]pyrimidin-
2-yl]phenyl}urea (48). Compound 48 was prepared from 44 to
give an off-white solid (TFA salt, 97% yield), according to the
1
procedure described for 46, using 1-methylpiperazine. H NMR
(DMSO-d6, 400MHz) δ2.78(s, 3/2H), 2.79(s, 3/2H), 3.07(m, 2H),
3.40 (m, 4H), 3.78 (t, 4H, J=4.8 Hz), 3.96 (t, 4H, J=4.8Hz), 5.18(q,
2H, J=8.6Hz),6.81(d,1H,J=3.5 Hz), 7.34 (d, 1H, J=3.5 Hz), 7.34
(d, 1H, J=3.5 Hz), 7.42 (d, 2H, J=8.6 Hz), 7.57 (d, 4H, J=8.6 Hz),
8.35 (d, 2H, J=8.6 Hz), 9.42 (s, 1H), 9.50 (s, 1H), 10.94 (s, 1H). MS
(ESI): m/z 623 [M þ H]. HRMS calcd for C31H33F3N8O3 [M þ H]
623.2700, obsd 623.2700. HPLC purity 95%.
1-(4-{[4-(Dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-{4-
[4-morpholin-4-yl-7-(2,2,2-trifluoroethyl)-7H-pyrrolo[2,3-d ]-
pyrimidin-2-yl]phenyl}urea (49). Compound 49 was prepared
from 44 to give an off-white solid (HCl salt, 68% yield),
according to the procedure described for 46, using 4-dimethyl-
1
4-({[4-(7-(2,2,2-Trifluoroethyl)-4-morpholin-4-yl-7H-pyrrolo-
[2,3-d ]pyrimidin-2-yl)phenyl]carbamoyl}amino)benzoic Acid
(44). Step 1. To a solution of 4-[7-(2,2,2-trifluoroethyl)-4-mor-
pholin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl]aniline (42) (479
mg, 1.3 mmol) in CH2Cl2 (10 mL) was added methyl 4-isocya-
natobenzoate (269 mg, 1.5 mmol), and the resulting mixture was
stirred at room temperature overnight. The resulting solid was
collected by filtration and washed with CH2Cl2 to give methyl
4-[({4-[4-morpholin-4-yl-7-(2,2,2-trifluoroethyl)-7H-pyrrolo-
[2,3-d]pyrimidin-2-yl]phenyl}carbamoyl)amino]benzoate as an
off-white solid (539 mg, 77% yield). MS (ESI): m/z 555 [M þ H].
Step 2. To a solution of methyl 4-[({4-[4-morpholin-4-yl-
7-(2,2,2-trifluoroethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]phenyl}-
carbamoyl)amino]benzoate (500 mg, 0.9 mmol) in MeOH
(30 mL) and THF (10 mL) was added 1 N NaOH aqueous
solution (2.7 mL), and the mixture was heated at 70 °C over-
night. The mixture was cooled to room temperature and con-
centrated. The residue was treated water and acidified to pH
4-5 by addition of 1 N HCl, and the resulting solid was collected
by filtration, washed with water, and dried in air to give 44 as
an off-white solid (486 mg, 100% yield). MS (ESI): m/z 541
[M þ H].
aminopiperidine. H NMR (DMSO-d6, 400 MHz) δ 1.61 (m,
2H), 2.04 (br, 2H), 2.72 (s, 3H), 2.74 (s, 3H), 3.43 (m, 1H), 3.78 (t,
4H, J=4.5 Hz), 3.96 (t, 4H, J=4.5 Hz), 5.18 (q, 2H, J=8.8 Hz),
6.81 (d, 1H, J=3.8 Hz), 7.33 (d, 1H, J=3.8 Hz), 7.38 (d, 2H, J=
8.8 Hz), 7.55 (d, 2H, J=8.8 Hz), 7.57 (d, 2H, J=8.8 Hz), 8.35 (d,
2H, J=8.8 Hz), 9.31 (s, 1H), 9.35 (s, 1H), 10.33 (s, 1H). MS
(ESI): m/z 651 [M þ H]. HRMS calcd for C33H37F3N8O3 [M þ
H] 651.3013, obsd 651.3013. HPLC purity 95%.
4-(2-Chloro-7-ethyl-7H-pyrrolo[2,3-d ]pyrimidin-4-yl)morpho-
line (41). Compound 41 was prepared from 16 to give an off-
white solid, according to the procedure described for 30, using
iodoethane. MS (ESI): m/z 267 [M þ H].
4-(7-Ethyl-4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-
aniline (43). Compound 43 was prepared from 41 to give an off-
white solid, according to the procedure described for 42, using
4-aminophenylboronic acid pinacol ester. MS (ESI): m/z 324
[M þ H].
4-({[4-(7-Ethyl-4-morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-
2-yl)phenyl]carbamoyl}amino)benzoic Acid (45). Step 1. To a
solution of 4-(7-ethyl-4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyri-
midin-2-yl)aniline (43) (1.72 g, 5.3 mmol) in CH2Cl2 (50 mL) was
added methyl 4-isocyanatobenzoate (1.13 g, 6.4 mmol), and the
resulting mixture was stirred at room temperature overnight. The
resulting solid was collected by filtration and washed with CH2Cl2
to give methyl 4-({[4-(7-ethyl-4-morpholin-4-yl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl)phenyl]carbamoyl}amino)benzoate as an off-
white solid (1.81 g, 68% yield). MS (ESI): m/z 501 [M þ H].
Step 2. To a solution of methyl 4-({[4-(7-ethyl-4-morpholin-4-
yl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)phenyl]carbamoyl}amino)-
benzoate (1.81 g, 3.6 mmol) in MeOH (50 mL) and THF (20 mL)
N-[2-(Dimethylamino)ethyl]-N-methyl-4-[({4-[4-morpholin-4-
yl-7-(2,2,2-trifluoroethyl)-7H-pyrrolo[2,3-d ]pyrimidin-2-yl]phe-
nyl}carbamoyl)amino]benzamide (46). To a solution of 4-
({[4-(7-(2,2,2-trifluoroethyl)-4-morpholin-4-yl-7H-pyrrolo[2,3-
d]pyrimidin-2-yl)phenyl]carbamoyl}amino)benzoic acid (44)
(32 mg, 0.06 mmol) in THF (2 mL) were added N,N,N0-
trimethylethylenediamine (12 mg, 0.12 mmol), Et3N (12 mg,
0.12 mmol), HOBt (16 mg, 0.12 mmol), and EDCI (23 mg,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3181
was added 1 N NaOH aqueous solution (18 mL), and the
mixture was heated at 70 °C for 3 h. The mixture was cooled
to room temperature and concentrated. The residue was treated
with water and acidified to pH 3-4 by addition of 1 N HCl. The
resulting solid was collected by filtration, washed with water,
and dried in air to give 45 as an off-white solid (1.65 g, 94%
yield). MS (ESI): m/z 487 [M þ H].
(7) 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. Colorectal cancer: mutations in a signaling
pathway. Nature 2005, 436, 792.
(8) Cully, M.; You, H.; Levine, A. J.; Mak, T. W. Beyond PTEN
mutations: the PI3K pathway as an integrator of multiple inputs
during tumorigenesis. Nat. Rev. Cancer 2006, 6, 184–192.
(9) Salmena, L.; Carracedo, A.; Pandolfi, P. P. Tenets of PTEN tumor
suppression. Cell 2008, 133, 403–414.
N-[2-(Dimethylamino)ethyl]-4-({[4-(7-ethyl-4-morpholin-4-yl-
7H-pyrrolo[2,3-d ]pyrimidin-2-yl)phenyl]carbamoyl}amino)benz-
amide (50). Compound 50 was prepared from 45 to give an off-
white solid (54% yield), according to the procedure described
for 46, using N,N-dimethylethylenediamine. 1H NMR (DMSO-
d6, 400 MHz) δ 1.40 (t, 3H, J=7.3 Hz), 2.17 (s, 6H), 2.39 (t, 2H,
J=6.8 Hz), 3.33 (m, 2H), 3.77 (t, 4H, J=4.8 Hz), 3.94 (t, 4H, J=
4.8 Hz), 4.27 (q, 2H, J=7.3 Hz), 6.66 (d, 1H, J=3.8 Hz), 7.33 (d,
1H, J=3.8 Hz), 7.53 (d, 2H, J=8.6 Hz), 7.56 (d, 2H, J=8.6 Hz),
7.79 (d, 2H, J=8.6 Hz), 8.23 (t, 1H, J=5.5 Hz), 8.34 (d, 2H, J=
8.6 Hz), 8.98 (br, 2H). MS (ESI): m/z 557 [M þ H]. HRMS calcd
for C30H36N8O3 [M þ H] 557.2983, obsd 557.2983. HPLC
purity 98.6%.
(10) Sulis, M. L.; Parsons, R. PTEN: from pathology to biology. Trends
Cell Biol. 2003, 13, 478–483.
(11) Li, J.; Yen, C.; Liaw, D.; Podsypanina, K.; Bose, S.; Wang, S. I.;
Puc, J.; Miliaresis, C.; Rodgers, L.; McCombie, R.; Bigner, S. H.;
Giovanella, B. C.; Ittmann, M.; Tycko, B.; Hibshoosh, H.; Wigler,
M. H.; Parsons, R. PTEN, a putative protein tyrosine phosphatase
gene mutated in human brain, breast, and prostate cancer. Science
1997, 275, 1943–1947.
(12) Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi,
T.; Okada, M.; Ohta, M.; Tsukamoto, S.; Parker, P.; Workman, P.;
Waterfield, M. Synthesis and biological evaluation of 4-morpho-
lino-2-phenylquinazolines and related derivatives as novel PI3
kinase p110R inhibitors. Bioorg. Med. Chem. 2006, 14, 6847–6858.
(13) Schultz, R. M.; Merriman, R. L.; Andis, S. L.; Bonjouklian, R.;
Grindey, G. B.; Rutherford, P. G.; Gallegos, A.; Massey, K.;
Powis, G. In vitro and in vivo antitumor activity of the phospha-
tidylinositol-3-kinase inhibitor, wortmannin. Anticancer Res. 1995,
15, 1135–1139.
(14) Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F. A specific
inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-
phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 1994,
269, 5241–5248.
(15) Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills, G. B.
Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat.
Rev. Drug Discovery 2005, 4, 988–1004.
1-[4-(7-Ethyl-4-morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-2-
yl)phenyl]-3-{4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}urea
(51). Compound 51 was prepared from 45 to give an off-white
solid (35% yield), according to the procedure described for 46,
1
using 1-methylpiperazine. H NMR (DMSO-d6, 400 MHz) δ
1.40 (t, 3H, J=7.1 Hz), 2.20 (s, 3H), 2.31 (br, 4H), 3.49 (br, 4H),
3.77 (t, 4H, J=5.0 Hz), 3.94 (t, 4H, J=5.0 Hz), 4.27 (q, 2H, J=
7.1 Hz), 6.66 (d, 1H, J=3.8 Hz), 7.32 (d, 1H, J=3.8 Hz), 7.34 (d,
2H, J=8.8 Hz), 7.53 (d, 2H, J=8.8 Hz), 7.56 (d, 2H, J=8.8 Hz),
8.34 (d, 2H, J=8.8 Hz), 8.93 (s, 1H), 8.94 (s, 1H). MS (ESI): m/z
569 [M þ H]. HRMS calcd for C31H36N8O3 [M þ H] 569.2983,
obsd 569.2983. HPLC purity 99.6%.
(16) Drees, B. E.; Mills, G. B.; Rommel, C.; Prestwich, G. D. Thera-
peutic potential of phosphoinositide 3-kinase inhibitors. Expert
Opin. Ther. Pat. 2004, 14, 703–732.
1-[4-(7-Ethyl-4-morpholin-4-yl-7H-pyrrolo[2,3-d ]pyrimidin-2-
yl)phenyl]-3-[4-(piperazin-1-ylcarbonyl)phenyl]urea (52). Com-
pound 52 was prepared from 45 to give an off-white solid
(46% yield), according to the procedure described for 46, using
piperazine. 1H NMR (DMSO-d6, 400 MHz) δ 1.40 (t, 3H, J=7.1
Hz), 2.68 (br, 4H), 3.41 (br, 4H), 3.78 (t, 4H, J=5.0 Hz), 3.94 (t, 4H,
J=5.0 Hz), 4.27 (q, 2H, J=7.1 Hz), 6.66 (d, 1H, J=3.5 Hz), 7.32 (d,
1H, J=3.5 Hz), 7.33 (d, 2H, J=8.6 Hz), 7.53 (d, 2H, J=8.6 Hz),
7.56 (d, 2H, J=8.6 Hz), 8.34 (d, 2H, J=8.6 Hz), 8.98 (s, 1H), 9.01 (s,
1H). MS (ESI): m/z 555 [M þ H]. HRMS calcd for C30H34N8O3
[M þ H] 555.2827, obsd 555.2830. HPLC purity 95.2%.
(17) Norman, B. H.; Shih, C; Toth, J. E.; Ray, J. E.; Dodge, J. A.;
Johnson, D. W.; Rutherford, P. G.; Schultz, R. M.; Worzalla, J. F.;
Vlahos, C. J . Studies on the mechanism of phosphatidylinositol 3-
kinase inhibition by wortmannin and related analogs. J. Med.
Chem. 1996, 39, 1106–1111.
(18) 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. Synthesis and
biological evaluation of pyrido[30,20:4,5]furo[3,2-d]pyrimidine
derivatives as novel PI3 kinase p110R inhibitors. Bioorg. Med.
Chem. Lett. 2007, 17, 2438–2442.
(19) Yaguchi, S.; Fukui, Y.; Koshimizu, I.; Yoshimi, H.; Matsuno, T.;
Gouda, H.; Hirono, S.; Yamazaki, K.; Yamori, T. Antitumor
activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibi-
tor. J. Natl. Cancer Inst. 2006, 98, 545–556.
(20) Kong, D.; Yamori, T. ZSTK474 is an ATP-competitive inhibitor of
class I phosphatidylinositol 3 kinase isoforms. Cancer Sci. 2007, 98,
1638–1642.
(21) 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.; Kugendradas, 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.; Wan, N. C.; Wiesmann, C.; Workman, P.; Zhyvoloup, A.;
Zvelebil, M. J.; Shuttleworth, S. J. The identification of 2-(1H-
indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-mor-
pholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent,
selective, orally bioavailable inhibitor of class I PI3 kinase for the
treatment of cancer. J. Med. Chem. 2008, 51, 5522–5532.
(22) Stauffer, F.; Maira, S. M.; Furet, P.; Garcia-Echeverria, C.
Imidazo[4,5-c]quinolines as inhibitors of the PI3K/PKB-pathway.
Bioorg. Med. Chem. Lett. 2008, 18, 1027–1030.
(23) Maira, S.; Stauffer, F.; Brueggen, J.; Furet, P.; Schnell, C.; Fritsch,
C.; Brachmann, S.; Chene, P.; De Pover, A.; Schoemaker, K.;
Fabbro, D.; Gabriel, D.; Simonen, M.; Murphy, L.; Finan, P.;
Sellers, W.; Garcia-Echeverria, C. Identification and characteriza-
tion of NVP-BEZ235, a new orally available dual phosphatidyli-
nositol 3-kinase/mammalian target of rapamycin inhibitor with
potent in vivo antitumor activity. Mol. Cancer Ther. 2008, 7, 1851–
1863.
Acknowledgment. We thank Wyeth Chemical Technolo-
gies Department for compound identification and the phar-
maceutical profiling results. Wealso thank Dr. Derek Cole for
his helpful suggestions regarding this manuscript.
References
(1) Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3-kinase-
AKT pathway in human cancer. Nat. Rev. Cancer 2002, 2, 489–501.
(2) Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso,
R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D.
Synthesis and function of 3-phosphorylated inositol lipids. Annu.
Rev. Biochem. 2001, 70, 535–602.
(3) Vanhaesebroeck, B.; Leevers, S. J.; Panayotou, G.; Waterfield,
M. D. Phosphoinositide 3-kinases: a conserved family of signal
transducers. Trends Biochem. Sci. 1997, 22, 267–272.
(4) Shayesteh, L.; Lu, Y.; Kuo, W. L.; Baldocchi, R.; Godfrey, T.;
Collins, C.; Pinkel, D.; Powell, B.; Mills, G. B.; Gray, J. W.
PIK3CA is implicated as an oncogene in ovarian cancer. Nat.
Genet. 1999, 21, 99–102.
(5) 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. V.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.; Velculescu,
V. E. High frequency of mutations of the PIK3CA gene in human
cancers. Science 2004, 304, 554.
(6) Campbell, I. G.; Russell, S. E.; David; Choong, D. Y.; Montgomery,
K. G.; Ciavarella, M. L.; Hooi, C. S.; Cristiano, B. E.; Pearson,
R. B.; Wayne, A.; Phillips, W. A. Mutation of the PIK3CA gene in
ovarian and breast cancer. Cancer Res. 2004, 64, 7678–7681.
(24) Venkatesan, A. M.; Dehnhardt, C. M.; Chen, Z.; Delos Santos, E.;
Santos, O.; Bursavich, M.; Gilbert, A. M.; Ellingboe, J. W.; Ayral-
Kaloustian, S.; Brooijmans, B.; Mallon, R.; Hollander, I.;

3182 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Chen et al.
Feldberg, L.; Lucas, J.; Yu, K.; Gibbons, J.; Abraham, R.;
Mansour, T. S. Novel imidazolopyrimidines as dual PI3-Kinase/
mTOR Inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 653–656.
(25) Dehnhardt, C. M.; Venkatesan, A. M.; Delos Santos, E.; Chen, Z.;
Santos, O.; Ayral-Kaloustian, S.; Brooijmans, B.; Mallon,
R.; Hollander, I.; Feldberg, L.; Lucas, J.; Yu, K.; Gibbons, J.;
Abraham, R.; Mansour, T. S. Lead optimization of N-3-substi-
tuted-7-morpholino-triazolopyrimidines as dual PI3K/mTor inhi-
bitors: discovery of PKI-402. J. Med. Chem. 2010, 53, 798–810.
(26) Zask, A.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Richard, D. J.;
Nowak, P.; Malwitz, D. J.; Brooijmans, N.; Bard, J.; Svenson, K.;
Lucas, J.; Toral-Barza, L.; Zhang, W. G.; Hollander, I.; Gibbons,
J. J.; Abraham, R. T.; Ayral-Kaloustian, S.; Mansour, T. S.; Yu, K.
ATP-competitive inhibitors of the mammalian target of rapamy-
cin: design and synthesis of highly potent and selective pyrazolo-
pyrimidines. J. Med. Chem. 2009, 52, 5013–5016.
American Chemical Society, Washington, DC, August 16-20, 2009;
American Chemical Society: Washington, DC, 2009; MEDI-363.
(28) Noell, C. W.; Robins, R. K. Aromaticity in heterocyclic systems. II.
The application of n.m.r. in a study of the synthesis and structure of
certain imidazo[1,2-c]pyrimidines and related pyrrolo[2,3-
d]pyrimidines. J. Heterocycl. Chem. 1964, 1, 34–41.
(29) Senda, S.; Hirota, K. Pyrimidine derivatives and related com-
pounds. XXII. Synthesis and pharmacological properties of
7-deazaxanthine derivatives. Chem. Pharm. Bull. 1974, 22, 1459–
1467.
(30) The homology model was built with Prime 1.6 (Prime, version 1.6;
Schrodinger, LLC: New York, 2007) using an in-house X-ray structure
of PI3K-g in complex with a related pyrazolopyrimidine (3IBE.pdb) as
the template. Docking studies were performed using Glide, version 4.5,
initially, and version 5.5 (Glide, version 5.5; Schrodinger, LLC: New
York, 2009) more recently. Docking studies were either performed
without constraints or with a hydrogen bonding constraint to the
backbone-NH of Val851. Figure 2 was made using PyMOL (DeLano,
W. L. The PyMOL Molecular Graphics System; DeLano Scientific
LLC: Palo Alto, CA, 2008).
(27) Chen, Z.; Venkatesan, A. M.; Dehndardt, C. M.; Ayral-Kaloustian,
S.; Mansour, T.; Brooijmans, N.; Mallon, R.; Hollander, I.; Yu, K.
Discovery of Novel Pyrrolo[3,2-d]pyrimidine Derivatives as PI3-
Kinase Inhibitors. Abstracts of Papers, 238th National Meeting of the