Bis(morpholino-l,3,5-triazine) derivatives: Potent adenosine 5′-triphosphate competitive phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibitors: Discovery of compound 26 (PKI-587), a highly efficacious dual inhibitor

Article abstract of DOI:10.1021/jm901830p

The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4, 6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic xenograft tumor models when administered intravenously. The structure-activity relationships and the in vitro and in vivo activity of analogues in this series are described.

Full text of DOI:10.1021/jm901830p

2636 J. Med. Chem. 2010, 53, 2636–2645
DOI: 10.1021/jm901830p
Bis(morpholino-1,3,5-triazine) Derivatives: Potent Adenosine 50-Triphosphate Competitive
Phosphatidylinositol-3-kinase/Mammalian Target of Rapamycin Inhibitors:
Discovery of Compound 26 (PKI-587), a Highly Efficacious Dual Inhibitor
Aranapakam M. Venkatesan,*,† Christoph M. Dehnhardt,† Efren Delos Santos,† Zecheng Chen,† Osvaldo Dos Santos,†
Semiramis Ayral-Kaloustian,† Gulnaz Khafizova,† Natasja Brooijmans,† Robert Mallon,‡ Irwin Hollander,‡ Larry Feldberg,‡
Judy Lucas,‡ Ker Yu,‡ James Gibbons,‡ Robert T. Abraham,‡ Inder Chaudhary,§ and Tarek S. Mansour†
†Chemical Sciences, ‡Oncology, and §Drug Metabolism, Wyeth Research, 401 N. Middletown Road, Pearl River, New York 10965
Received December 11, 2009
The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis,
and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable
therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized
to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]car-
bonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has
shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic
xenograft tumor models when administered intravenously. The structure-activity relationships and the in
vitro and in vivo activity of analogues in this series are described.
Introduction
levels of PIP3 with consequent deregulation of the PI3K/
Akt pathway signaling.1-6 This also contributes to onco
genesis.7,8,12 Therefore, inhibiting PI3KR is an attractive strat-
egy for cancer therapy. Furthermore, it has been demonstrated
that the PI3K pathway is paradoxically activated following
selective mTOR inhibition, thus providing a compelling ratio-
nale for developing dual PI3K/mTOR kinase inhibitors.13,14
Toward this end, several pharmaceutical companies
and academic institutions have been concentrating their
efforts on the development of small molecule PI3K/mTOR
inhibitors.2,15-17 Among the various small molecule inhi-
bitors, BEZ-235 1 (Figure 1) is the most advanced clini-
cal candidate.19a,b It inhibits PI3K and mTOR in an ATP-
competitive manner. This compound is also reported to
inhibit cancer cell proliferation, causing cell cycle arrest at
the G1 phase.19a,b Another compound that inhibits all class I
PI3K isoforms, but with significantly lower potency against
mTOR, is 2 GDC-0941.18 This compound was reported by
Genentech and has entered phase I clinical studies. Another
pan PI3K inhibitor that has gone into the clinic is Semafore
compound, SF1126.20 Highly selective mTOR inhibitors
based on a pyrazolopyrimidine scaffold such as 3a were
reported by Zask et al.21a,b and recently entered clinical
development. We also recently described a dual PI3K/mTOR
inhibitor 3 (PKI-402) based on a triazolopyrimidine scaffold
that has potent in vivo efficacy.21c
Phosphatidylinositol-3-kinases (PI3Ksa) are lipid kinases
that phosphorylate phosphatidylinositol diphosphate (PIP2)
to its corresponding phosphatidylinositol triphosphate (PIP3).
It has been recognized recently that the PI3K/Akt signaling
pathway is a key pathway in cell proliferation, growth, survival,
protein synthesis, and glucose metabolism.1-6 The product of
PI3K activity, namely, PIP3, acts as a second messenger
responsible for the activation of the downstream kinase Akt.1
PI3Ks are categorized as class IA, class IB, class II, and class III
enzymes, based on sequence homology and substrate prefer-
ences. The PI3K related kinases (PIKKs) include mTOR,
ATR, ATM, SMG1, and DNA-PK. PIKKs share a conserved
kinase domain with PI3Ks and regulate cellular response to
nutrient levels and environmental stress. The class IA subgroup
consists of PI3KR, PI3Kβ, and PI3Kδ isoforms. Among these
three isoforms, the gene encoding the PI3KR subunit, PIK3-
CA, is mutated and/or overexpressed in breast, ovarian, color-
ectal, brain (glioblastoma), and gastric cancers.6,9-11 Addi-
tionally, the PIP3 lipid phosphatase, phosphatase and tensin
homologue deleted on chromosome ten (PTEN), is frequently
inactivated in numerous late stage tumors, causing elevated
*To whom correspondence should be addressed. Phone: (845) 602-
4023. Fax (845) 602-5561. E-mail: venkata@wyeth.com or venkata699@
gmail.com.
a Abbreviations: PI3K, phosphoinositide 3-kinase; mTOR, mamma-
lian target of rapamycin; Akt, protein kinase B; PIP2, phosphatidylino-
sitol bisphosphate(4,5); PIP3, phosphatidylinositol triphosphate(3,4,5);
PTEN, phosphatase and tensin homologue; RTK, receptor tyrosine
kinase; PH, pleckstrin homology; mTORC1, mammalian target of
rapamycin complex 1; mTORC2, mammalian target of rapamycin
complex 2; PDK1, 3-phosphoinositide-dependent kinase 1; S, serine;
T, threonine; ATP, adenosine 50-triphosphate; Her2þ, human epider-
mal growth factor receptor 2þ; ELISA, enzyme-linked immunosorbent
assay; DELFIA, dissociation-enhanced lanthanide fluorescent.
In the present article, we describe the design, synthesis, and
identification of a highly potent bis(morpholino-1,3,5-
triazine) derivative 26 (PKI-587) as a PI3K/mTOR inhibitor.
Compound 26 decreases cell survival and proliferation and
increases apoptosis in both in vitro and in vivo models. It
shows compelling antitumor efficacy in subcutaneous and
orthotopic human xenograft tumor models when admini-
stered intravenously (iv) as a single agent.
r
pubs.acs.org/jmc
Published on Web 02/18/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2637
Figure 1. Previously known phosphatidylinositol-3-kinase inhibitors and selective mammalian target of rapamycin inhibitor.
Chemistry
permeability, and clogP data for analogues 8-30 are also
listed in Table 1.
The 2,4-bismorpholino-1,3,4-triazine derivatives 8-30,
which are exemplified in the present article, were prepared
starting from the commercially available cyanuric chloride 4,
as outlined in Scheme 1. Initially the two chlorines in cyanuric
chloride 4 were replaced with 2 equiv of morpholine 5 in the
presence of triethylamine at 0 °C to yield 6 in almost quanti-
tative yield. The third chlorine in compound 6 was displaced
by a 4-aminophenyl moiety using Suzuki coupling reaction
with 4-aminophenylboronic acid pinacol ester to yield 7.
Compound 7 was reacted with various alkyl and aryl iso-
cyanates to yield 8-14, 17, 18, and 21. Alternatively, urea
derivatives 15, 16, 19, and 20 were obtained by reacting the
intermediate 7 with triphosgene/triethylamine and the corres-
ponding amines. In order to introduce amides bearing water
solubilizing amine functionality, intermediate 7 was reacted
with methyl 4-isocyanatobenzoate in dichloromethane at
room temperature to give 21 in good yield. The ester group
was hydrolyzed with 5 N NaOH in tetrahydrofuran/methanol
at reflux temperature to yield 22. The acid derivative 22 was
reacted with different amines in the presence of HOBt/EDCI
and triethylamine at room temperature. The final products
were purified either by flash column silica gel column chro-
matography or by preparative HPLC. All analogues 8-30
obtained by the above-mentioned route were analyzed for
purity by analytical HPLC on a prodigy ODS3 column
(150 mm ꢀ 4.6 mm) using acetonitrile/water mixture. The
purity of the newly synthesized compounds was analyzed
at 210-370 nm.
Several morpholine bearing fused pyrimidines such as
imidazolopyrimidines,25 pyrrolopyrimidines,26 and triazolo-
pyrimidines21c have been reported by us previously as PI3K/
mTOR dual inhibitors. In all these scaffolds, the morpholine
oxygen formed a pivotal hinge region hydrogen bond inter-
action with Val851 of the PI3KR catalytic domain18,21a-c
but also presented a metabolic liability via oxidation R to
the morpholine ring oxygen, causing loss of potency. Many
potent PI3Kenzyme inhibitors basedon the above-mentioned
scaffolds failed to show antitumor efficacy in in vivo models.
The most advanced compound 3 showed good efficacy in
MDA-361 xenograft model. However, advanced studies with
3 were halted because of its poor solubility.
Bearing these facts in mind, we decided to probe the other
scaffoldsthat resemble a pyrimidine core but have lower clogP
values than the triazolopyrimidine core in PKI-402. Among
the various heterocycles we synthesized, the 1,3,5-triazine
scaffold17 was very attractive for the following reasons:
(1) 1,3,5-Triazine is a monocyclic, symmetrical molecule
similar to the pyrimidine core with two nitrogens in the 1,3-
positions, and it has been used as a kinase inhibitor scaffold.17
Hence, we envisaged that 1,3,5-triazine could mimic the
pyrimidine scaffold and allow us to position the critical
substituents observed in our earlier series for optimum inter-
action with the binding site of PI3KR.
(2) The presence of three nitrogens in the 1,3,5-triazine core
can inherently impart polarity to the whole molecule, and the
clogP values of the designed compounds can be about 2 or
lower.
(3) We also envisioned also that a bismorpholino-1,3,5-
triazine scaffold would have an advantage under conditions
where one morpholine gets metabolically oxidized, still leav-
ing another morpholine to make the vital hinge region H bond
interaction in the PI3K catalytic domain.
However, we were aware that these modifications can solve
the clogP problem, and to an extent, the morpholine meta-
bolic liability might not address permeability and the micro-
somal stability problems. In order to explore the above-
mentioned points, compound 8 was designed and modeled
in the PI3Kγ homology model (Figure 2). The structural
Results and Discussion
Final compounds 8-30 were tested in vitro against PI3KR,
PI3Kγ, and mTOR. The IC50 values against PI3KR and
PI3Kγ enzymes were determined using a fluorescence polari-
zation format assay.22 The corresponding mTOR inhibition
for the newly synthesized compounds was determined using a
DELFIA format ELISA.23 PI3KR, PI3Kγ, andmTORaswell
as the cell proliferation (3 days growth inhibition) assay24 IC50
values in MDA-361 (breast, PI3K mutant [E545K]/Her2þ)
and PC3MM2 (prostate, PTEN deletion) human tumor cell
lines are shown in Table 1. The solubility (at pH 7.4), PAMPA

2638 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Scheme 1. General Methods To Prepare 8-30
Venkatesan et al.
similarity of the ATP-binding sites of PI3KR and PI3Kγ
isoforms enabled a homology model to be built based on an
in-house X-ray crystal structure of PI3Kγ in complex with a
related compound (3IBE.pdb).21a Overall sequence similarity
between PI3KR and PI3Kγ is 43% in the kinase catalytic
domain; however, the ATP-binding site is significantly more
conserved with 81% of the residues being identical. As
indicated in Figure 2, compound 8 docked in the homology
model forms the critical hinge region hydrogen-bond to
Val851 through a morpholine oxygen. The urea forms
two hydrogen-bonding interactions to Asp810 through both
urea-NH groups and one to the catalytic lysine (Lys802)
through the urea carbonyl group. Hence, compound 8
was prepared as depicted in Scheme 1. As can be seen from
Table 1, compound 8 was found to have potent PI3KR,
PI3Kγ, and mTOR inhibitory activity but only moderate
potency in MDA-361 and PC3MM2 cell proliferation assays.
This was attributed to the poor solubility and permeability of
compound 8.
However, this initial enzyme potency result encouraged us
to probe the structure-activity relationship more system-
atically to further optimize the cellular potency. There was
an observed drop in the potency against PI3KR and PI3Kγ
when the phenyl group in compound 8 was replaced with alkyl
groups such as methyl10, ethyl 9, or saturated heterocyclyl29.
However, the corresponding mTOR activity was not affected.
The structural basis for the observed difference in PI3KR/γ
and mTORactivityfor compounds9, 10, and29isnot entirely
clear because of a lack of detailed structural information on
the mTOR enzyme (Figure 3). However, three residue differ-
ences are observed in the loop that forms the top of the
binding site around the urea substituents. Asp805 in PI3KR
is a glutamic acid in mTOR, and this residue plays a critical
role in stabilizing the catalytic lysine (Lys802 in PI3KR,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2639
Table 1. In Vitro, Microsomal Stability, Solubility, and clogP Data for Analogues 8-30a
IC50 (nM)
compd
PI3K-R
PI3K-γ
mTOR
MDA-361
PC3-MM2
clogP
sol.b at pH 7.4
PAMPA at pH 7.4
8
3.0
27.0
814.5
789.0
52.5
438.0
219.5
528.0
14.5
21.5
23.0
34.0
28.5
65.0
85.0
48.5
20.0
8.0
3.9
10.0
11.3
3.9
6.3
6.7
9.3
1.9
1.8
1.7
1.2
1.2
2.3
2.5
1.5
0.7
0.5
1.4
1.6
0.8
0.7
460.0
0.6
56
NT
107
NT
2.5
1.2
NA
2.8
3.0
3.4
2.5
1.5
1.7
2.2
1.8
1.8
1.0
2.9
2.4
1.4
1.8
2.1
1.2
2.0
2.5
1.0
2.0
0.0
1.0
0.2
0.7
9
247.5
350.0
6.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
NT
NT
NT
0.0
NT
1.0
118.0
200.0
407.0
794.0
30.0
36.0
NT
193.0
364.0
293.0
5630.5
30.0
39.0
218
16.5
19.5
60.5
1.0
0.0
1.0
5.6
0.0
1.0
0.0
0.0
0.2
1.5
2.0
0.0
2.0
0.0
2.4
1.7
3.0
NT
92
0.0
0.0
0.5
1.4
45.0
38.0
115.0
816.0
19.0
3.0
43.0
21.0
45.0
28.0
24.0
11.0
13.0
13.1
8.3
11.0
12.5
6.0
1.0
1.0
NT
0.6
52.0
1.0
0.0
1.5
0.4
NT
0.1
7.0
0.0
0.5
0.4
9.0
5.4
10.0
4.0
0.0
0.1
14.0
33.5
31.0
100.0
9.0
0.7
0.6
10.0
10.0
7470.0
9.0
3.0
3.0
NT
NT
0.010
0.030
7.0
NT
1521.0
0.4
NT
9.8
18.3
a NT = not tested. PAMPA = permeability Pe ꢀ 10-6 cm/s. b Solubility is in μg/mL.
Figure 3. Detailed of interactions of compound 9 with PI3K-R and
mTOR: overlay of the homology models of PI3KR (green ribbons
and carbons) and mTOR (magenta ribbons and carbons) with the
predicted binding mode of compound 9. Conserved residues are
Val851 (Val2240 in mTOR) and Lys802 (Lys2187) and are therefore
only labeled. Several residue differences near the urea-R groups (in
this case R = CH3) are Asp805-Glu2190, Gly804-His2189, and
Asn803-Gly2188.
Figure 2. Docking of compound 8 in PI3Kγ homology model.
Hydrogen bonds are shown by blue dashes. The morpholine forms
a hydrogen bond to the hinge region Val851. The urea forms three
hydrogen bonds to Asp810 and Lys802. The critical salt bridge
between the catalytic Lys802 and Asp805 is shown in purple.
of the phenyl group in compound 8 with 4-fluoro 11, 4-methyl
12, 4-chloro 13, and 2,4-difluorophenyl 14 led to compounds
with moderate PI3KR, PI3Kγ, and mTOR potency. It can also
be noted from Table 1 that substitution of the phenyl group with
a lipophilic group (examples 11, 12, 13, and 14) led to a decrease
in the potency against PI3KR. However, all these compounds,
with the exception of 29, had excellent potency against mTOR.
It appears from the different examples that PI3KR potency is
favored if the R group is an aryl substituent. Molecular model-
ing using the PI3Kγ homology model revealed that the sub-
stituents on the R groups are solvent exposed (Figure 4). Hence,
compounds 15 and 16, bearing polar entities such as -CH2OH
and -CH2CH2OH were prepared. This modification led to
improvement in both enzyme and cellular potencies. This could
Lys2187 in mTOR) through the formation of a salt bridge.
Substituted ureas of this type result in a concerted motion of
the catalytic lysine and the aspartic/glutamic acid,21a,b and it
is possible that mTOR with the longer side chain in the
glutamic acid, compared to the aspartic acid in PI3KR,
undergoes this motion more easily. The second noted residue
difference in this area is the different positioning of glycine. In
PI3KR, a glycine (Gly804) is directly adjacent to the aspartic
acid involved in the salt bridge, whereas in mTOR, a histidine
(His2189) separates the glutamic acid from the glycine
(Gly2188). Again, it is possible that the different position of
the glycine allows the mTOR loop to accommodate the hydro-
phobic groups coming off the urea more easily. Substitution

2640 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Venkatesan et al.
Figure 5. In vitro phosphoblot studies. MDA-361 tumor line was
exposed to 26 for 4 h.
Figure 4. Surface representation of the ATP binding site around
26, illustrating that substitutions coming off the urea extend into the
solvent.
473 (S473). Figure 5 also shows potent (IC50 < 10 nM)
suppression of Akt phosphorylation at S473 by26. Phophory-
lation of Akt kinase effector proteins GSK3 kinase, endothe-
lial nitric oxide synthase (ENOS), and prolinerich Akt sub-
strate (PRAS 40) was also suppressed by 26 at concentrations
less than 30 nM. The inhibition of mTOR containing TORC1
kinase activity by 26 was demonstrated by suppression of
p70S6K and 4EBP1 phosphorylation at concentrations less
than30nMby26. Compound 26did not affectthe overall Akt
content in MDA-361 cells at concentrations tested (Figure 5),
indicating pAkt suppression was not an artifact of compound
cytotoxicity.
Initial blood level studies in female nude mice were done
with compounds 24, 25, and26 via both oral (po) and iv routes
of administration. None of these three analogues had oral
exposure in nude mice. However, when 26 was administered
to nude mice at 25 mg/kg iv (in 5% dextrose [D5/W], 0.3%
lactic acid, pH 3.5), low plasma clearance (7 (mL/min)/kg)
compared to liver blood flow (90 (mL/min)/kg), high volume
of distribution (7.2 L/kg) compared to plasma volume
(0.7 L/kg), and long half-life, (14.4 h) were observed
(Table 3). Low clearance indicated minimal metabolism,
and high volume of distribution was suggestive of extensive
tissue distribution in nude mice. When compound 26 was
administered iv (single dose) at 25 mg/kg, it suppressed pAkt
(T308 and S473) for up to 36 h and induced cleaved PARP up
to 18 h in MDA-361 tumors staged in nude mice (Figure 6).
Compound 26 exhibiting potent antitumor efficacy against
MDA-361 tumors is also demonstrated in Figure 7. Com-
pound 26 administered iv at 20 mg/kg on an intermittent
regimen (days 1, 5, 9) caused regression of large staged
(∼900 mm3) tumors. This effect was more pronounced than
that observed for paclitaxel (Taxol) given at 60 mg/kg (single
dose, ip). In subsequent in vivo studies the minimum effi-
cacious dose (MED) of 26 was determined to be 3 mg/kg
against MDA-361 tumors and maximum tolerated single dose
(MTD) was determined to be 30 mg/kg.
Table 2. IC50 Values against Various PI3K Isoforms and Mutants
isoforms
PI3Kβ
PI3Kγ
PI3Kδ
H1047R
0.6
E545K
0.6
IC50 (nM)
6
8
6
be due to their marginally improved permeability. The PAMPA
permeability for compound 15 is 0.18 ꢀ 10-6 cm/s, and the
Caco2 data for compound 16 is 2.9 (A > B) and 7.2 (B > A).
Compounds 15 and 16 had poor human and nude mouse
microsomal stabilities, which precluded them from further
investigation. Heterocycles 18, 19, and 20 containing polar
nitrogen also gave potent compounds with slightly increased
human and nude mouse microsomal stabilities. However, these
analogues did not have a good solubility profile at pH 7.4.
Hence, compounds such as 24-30 bearing a basic amine moiety
were prepared. As can be seen from Table 1, except for
compound 29, compounds bearing a basic amine moiety with
an amide linkage exhibited excellent enzyme and cell potencies.
These analogues also had good microsomal stability in all three
species and exhibited good to moderate solubility. This dramatic
boost in the PI3KR enzyme potency by the amide substituent is
not very well understood and may be due to an increased
hydrogen bond accepting ability of the overall molecule. On
the basis of enhanced potency, solubility, microsomal stability,
and lack of Cyp inhibition, compound 26 was chosen for further
in vitro and in vivo evaluations. Compound 26 was evaluated
for its potency against class I PI3Ks (i.e., the β, γ, and δ
isoforms) and the most frequently occurring mutant forms of
PI3KR, notably the H1047R and E545K. The IC50 values are
shown in Table 2. These results show that compound 26 is a pan-
PI3K/mTOR inhibitor, with equivalent potency against mTOR
(IC50 = 1.4 nM, Table 1). This compound was also evalu-
ated against a panel of 236 other human protein kinases
at 10 μM, and it was found to be highly selective for PI3K
and mTOR.
Potent antitumor activity of compound 26 was also demon-
strated in an orthotopic version of the H1975 (non-small-cell
lung carcinoma, mutant EGFR [L858R, T790M]) xenograft
model (Figure 8). H1975 cells were injected into the pleural
cavity of nude mice, and the animals were treated with26 once
weekly at 25 mg/kg for 7 weeks. As can be seen from Figure 8,
all of the untreated control animals were dead by day 50,
compared to 90% survival in the group treated with 26.
In vitro, compound 26 showed good potency in cell growth
inhibition assays using MDA-361 and PC3-MM2 cell lines.
Tumor cell growth inhibition by 26 correlated with suppres-
sion of phosphorylation of PI3K/mTOR signaling pathway
proteins in MDA-361 tumor cells (Figure 5). Compound 26
prevented the phosphorylation of Akt (pAkt) at threonine 308
(T308; IC50 = 8 nM) (phosphoblot IC50 values were deter-
mined by densitometric scans of Western blots) and induced
cleaved PARP at 30 nM. Cleaved PARP is a marker for cells
undergoing apoptosis. Full activation of Akt kinase occurs
when the mTOR containing mammalian target of rapamycin
2 (TORC2) protein complex phosphorylates Akt at serine
Conclusion
We have shown that bis(morpholinotriazine) compounds
bearing bisarylureas are potent dual PI3K/mTOR inhibitors.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2641
Table 3. Pharmacokinetic Parameters of Compound 26 after a Single 25 mg/kg iv Dose in Female Nude Micea
C0 (ng/mL)
T1/2 (h)
AUClast (h ng/mL)
3
42593
AUC0-inf (h ng/mL)
3
57927
Clp ((mL/min)/kg)
7
Vss (L/kg)
MRT (h)
16.7
48523
14.4
7.2
a C0 = concentration at 0 min. T1/2 = half-life. AUC = area under the plasma concentration time curve. Vss = volume of distribution at steady state.
Clp = clearance;. MRT = mean resonance time.
Figure 6. Compound 26 in vivo biomarker profile at 25 mg/kg, iv,
single dose. Tumor tissue from the MDA-361 xenograft model was
examined for p-Akt suppression and induction of cleaved PARP at
different time points. The p70 pS6K and pS6 panels show suppres-
sion of phosphorylation of these mTOR effector proteins.
Figure 8. Orthotopic model for compound 26 in H1975 (non-
small-cell lung carcinoma). Compound 26 was given once weekly
for 7 weeks at 25 mg/kg. The Y axis represents % survival
(Kaplan-Meier plot).27
reported in parts per million (δ) relative to residual chloroform
(7.26 ppm), TMS (0 ppm), or dimethyl sulfoxide (2.49 ppm) as
an internal reference with coupling constants (J) reported in
hertz (Hz). The peak shapes are denoted as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad. Electro-
spray (ES) mass spectra were recorded in positive or negative
mode on a Micromass Platform spectrometer. Electron impact
(EI) and high-resolution mass spectra were obtained on a
Finnigen MAT-90 spectrometer. Combustion analyses were
obtained using Perkin-Elmer series II 2400 CHNS/O analyzer.
The purity of compounds 8-32 was determined by analytical
LC/MS using an Agilent 1100 LC equipped with an Agilent
MSD mass spectrometer and by analytical LC using an
X-Bridge BEH column, MeOH/H2O eluent at 1 mL/min flow
(containing 0.05% NH4OAc), 20 min gradient from 10%
MeOH to 90% ACN, monitored by UV absorption at
215 nm. The purity of all the above-mentioned compounds
was found to be >95%. Reverse phase HPLC purifications
were performed on a Gilson preparative HPLC system con-
trolled by Unipoint software, using a Phenomenex Gemini
100 mm ꢀ 30.0 mm column. Thin-layer chromatography
(TLC) was performed on Merck PLC prescored plates 60F254
.
Figure 7. Efficacy profile of compound 26 in MDA-361 xenograft
model. Shown is the tumor regression caused by 26 at 20 mg/kg, iv,
on days 1, 5, 9 and by paclitaxel (Taxol) at 60 mg/kg, ip (single dose).
Y axis represents tumor volume in mm3.
The terms “concentrated” and “evaporated” refer to removal of
solvents using a rotary evaporator at water aspirator pressure
with a bath temperature equal to or less than 40 °C. Unless
otherwise noted, reagents were obtained from commercial
sources and were used without further purification.
We have also discovered that the presence of an amide
functionality bearing water solubilizing groups boosted
potency, both in enzyme and in cell proliferation assays.
During our course of optimization, we identified compound
26, which has shown excellent efficacy especially in in vivo
xenograft models. Compound 26 is currently entering phase I
clinical trials as a single agent for iv administration.
Modeling. The homology model was built with Prime 1.6 with
3IBE21a as the template. Docking studies were performed using
Glide, version 4.5 initially and version 5.5 more recently. Dock-
ing studies were performed either without constraints or with a
hydrogen bonding constraint to the backbone-NH of Val851.
Figures 2, 3, and 4 were made using PyMOL: Prime, version 1.6
(2007) (Schrodinger, LLC, New York, NY); Glide, version 5.5
(2009) (Schrodinger, LLC, New York, NY); and PyMOL
Molecular Graphics System (2008) (W. L. DeLano; DeLano
Scientific LLC, Palo Alto, CA).
Experimental Section
General Methods. Melting points were determined in open
capillary tubes on a Mel-Temp melting point apparatus and are
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-
yl)phenyl]-3-phenylurea (8). Step 1: Preparation of 2-Chloro-
4,6-dimorpholin-4-yl[1,3,5]triazine (6). To a stirred solution of
cyanuric chloride (18.4 g, 10 mmol) in acetone (100 mL) and
1
uncorrected. H NMR spectra were determined with a Bruker
DPX-400 spectrometer at 400 MHz. Chemical shifts are

2642 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Venkatesan et al.
crushed ice (500 g), a mixture of triethylamine (30.0 g, excess)
and morpholine (17.4 g, 20 mmol) was added at -10 °C. After
the addition, the reaction mixture was stirred at room tempera-
ture for 1 h and diluted with 50 mL of water. Separated white
solid was filtered and washed with water. The white solid was
dried and filtered. The crude product was found to be pure
and taken to the next step without purification (25 g, 87% yield).
(M þ H) 286.7
the title compound was isolated as a white solid (65 mg, 33%
yield). (M þ H) 476.4. Purity by analytical HPLC 98.2%.
(Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient acetonitrile
in water, trifluoroacetic acid, detector wavelengths, 215 and
254 nM.) 1H NMR (DMSO-d6) δ 3.6 (m, 8H), 3.7 (m, 8H), 2.5 (s,
3H), 7.1 (d, J=9.6 Hz, 2H), 7.3 (d, J=8.3 Hz, 2H), 7.6 (d, J=8.6
Hz, 2H), 8.2 (d, J=9.6 Hz, 2H), 8.6 (s, 1H), 8.9 (s, 1H) ppm.
Preparation of 1-(4-Chlorophenyl)-3-[4-(4,6-dimorpholin-4-yl-
1,3,5-triazin-2-yl)phenyl]urea (13). Starting from 4-(4,6-dimor-
pholin-4-yl-1,3,5-triazin-2-yl)aniline (0.140 g 0.40 mmol) and
4-chlorophenyl isocyanate (94 mg, 0.61 mmol) and following the
procedure outlined for the preparation of 8, the title compound
was isolated as a white solid (60 mg, 30% yield). (M þ H) 496.3.
Purity by analytical HPLC 96.8%. (Prodigy ODS3, 0.46 cm ꢀ
15 cm, 20 min gradient acetonitrile in water, trifluoroacetic acid,
detector wavelengths, 215 and 254 nM.) 1H NMR (DMSO-d6) δ
3.6 (m, 8H), 3.7 (m, 8H), 7.1 (m, 2H), 7.4 (m, J=8.1 Hz, 2H),
7.6 (d, J=8.8 Hz, 2H), 8.2 (d, J=8.3 Hz, 2H), 8.8 (s, 1H), 9.0
(s, 1H) ppm.
Step 2: Preparation of 4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-
2-yl)aniline (7). A mixture of 2-chloro-4,6-dimorpholin-4-yl-
[1,3,5]triazine (1.4 g, 4.9 mmol), a catalytic amount of tetrakis-
(triphenylphosphine)palladium(0) (70 mg, 0.061 mmol), sodium
carbonate solution 2 M (3 mL), 4-aminophneylboronic acid
pinacol ester (1.6 g, 7.3 mmol), and DME (100 mL) was refluxed
for 24 h. The solvent was evaporated, and the residue was
dissolved in methylene chloride and filtered through Celite.
The filtrate was washed with water (200 mL), and the organic
layer was dried with magnesium sulfate. This was filtered, and
the solvent was evaporated. The residue was purified by SiO2
column chromatography and eluted with ethyl acetate/hexanes
(1:1) to give of 4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)ani-
line (1.40 g, 83% yield) as an amorphous solid. (M þ H) 343.
Step 3: Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-
triazin-2-yl)phenyl]-3-phenylurea (8). To a stirred mixture of
4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)aniline (140 mg, 0.40
mmol) and a catalytic amount of dimethylaminopyridine
(DMAP) in methylene chloride (100 mL) was added a small
excess of phenyl isocyanate (59.5 mg, 0.50 mmol). The mixture
was stirred at room temperature for 48 h. The reaction mixture
was concentrated to half of its original volume, and the sepa-
rated precipitate was collected by filtration and washed with
methanol (15 mL) and then with diethyl ether. In some cases the
crude product obtained was purified by SiO2 column chromato-
graphy by eluting it with 10% ethyl acetate/hexane (128 mg,
68% yield). White solid. 462.3 (M þ H). Purity by analytical
HPLC 99.3%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min
gradient acetonitrile in water, trifluoroacetic acid, detector
wavelengths, 215 and 254 nM.) 1H NMR (DMSO-d6) δ 3.6
(m, 8H), 3.7 (m, 8H), 7.0 (t, J=7.0 Hz, 1H), 7.3 (t, J=7.3 Hz,
2H), 7.5 (d, J=8.1 Hz, 2H), 7.6 (d, J=8.8 Hz, 2H), 8.3 (d, J=8.6
Hz, 2H), 8.7 (s, 1H), 9.0 (s, 1H) ppm.
Preparation of (2,4-Difluorophenyl)-3-[4-(4,6-dimorpholin-4-
yl-1,3,5-triazin-2-yl)phenyl]urea (14). Starting from 4-(4,6-di-
morpholin-4-yl-1,3,5-triazin-2-yl)aniline (0.105 g 0.30 mmol)
and 2,4-difluorophenyl isocyanate (71 mg, 0.45 mmol) and
following the procedure outlined in example 8, the title com-
pound was isolated as a white solid (40 mg, 27% yield). (M þ H)
498.4. Purity by analytical HPLC 97.5%. (Prodigy ODS3,
0.46 cm ꢀ 15 cm, 20 min gradient acetonitrile in water, trifluoro-
1
acetic acid, detector wavelengths, 215 and 254 nm.) H NMR
(DMSO-d6) δ 3.65 (m, 8H), 3.8 (b, 8H), 7.04 (m, 1H), 7.32 (m,
1H), 7.54 (d, J=8.6 Hz, 2H), 8.07 (m, 1H), 8.1 (d, J=8.2 Hz,
2H), 8.57 (s, 1H), 9.32 (s, 1H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-[4-(1-hydroxyethyl)phenyl]urea (16). To a stirred mix-
ture of 4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)aniline (0.140,
0.40 mmol) in methylene chloride at 0 °C was added triphosgene
(0.25, 0.84 mmol) and Et3N (3 mL). The reaction mixture was
stirred for 20 min at 0 °C. Then 1-(4-aminophenyl)ethanol
(0.10 g, 0.73 mmol) was added to the mixture. The reaction
mixture was stirred for about 16 h at room temperature. The
solvent was removed. The residue was dissolved in DMSO and
placed in an HPLC instrument, with acetonitrile buffer TFA to
give [4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]-3-[4-(1-
hydroxyethyl)phenyl]urea (48 mg, 24% yield). (M þ H) 506.4.
Purity by analytical HPLC 95.5%. (Prodigy ODS3, 0.46 cm ꢀ
15 cm, 20 min gradient acetonitrile in water, trifluoroacetic acid,
detector wavelengths, 215 and 254 nm.) 1H NMR (DMSO-d6) δ
2.66 (t, J=7.1 Hz, 2H), 3.43 (b, 1H), 3.57 (t, J=7.6, 2H), 3.66 (m,
8H), 3.81 (b, 8H), 7.13 (d, J=8.1 Hz, 2H), 7.36 (d, J=8.3 Hz,
2H), 7.54 (d, J=8.8 Hz 2H), 8.27 (d, J=8.26 Hz 2H), 8.63 (s,
1H), 8.95 (s, 1H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-[4-(hydroxymethyl)phenyl]urea (15). Starting from
4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)aniline (0.140, 0.40
mmol) and 4-aminophenylmethanol (100 mg, 0.81 mmol) and
following the procedure described in example 16, 1-[4-(4,6-
dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]-3-[4-(hydroxymethyl)-
phenyl]urea was isolated by HPLC purification (58 mg, 26%
yield). (M þ H) (ESI) m/z=492.3. Purity by analytical HPLC
95.8%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient
acetonitrile in water, trifluoroacetic acid, detector wavelengths,
215 and 254 nm.) 1H NMR (DMSO-d6) δ 3.43 (b, 1H), 3.66 (m,
8H), 3.81 (b, 8H), 4.43 (s, 2H), 7.23 (d, J=8.8 2H), 7.41 (d, J=
8.3 Hz, 2H), 7.55 (d, J=8.6 Hz, 2H), 8.27 (d, J=8.8 Hz, 2H), 8.7 (s,
1H), 8.98 (s, 1H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-ethylurea (9). Starting from 4-(4,6-dimorpholin-4-yl-
1,3,5-triazin-2-yl)aniline (0.130 g 0.38 mmol) and ethyl iso-
cyanate (260 mg, 10-fold excess) and following the procedure
outlined for compound 8, the title compound was isolated
as a white solid (38 mg, 25% yield). (M þ H) 414.3. Purity
by analytical HPLC 97.8%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm,
20 min gradient acetonitrile in water, trifluoroacetic acid,
detector wavelengths, 215 and 254 nM.) 1H NMR (DMSO-d6)
δ 3.6 (m, 8H), 3.7 (m, 8H), 3.75 (t, J=6.8 Hz, 3H), 3.8 (q, J=6.8
Hz, 2H), 7.6 (d, 2H, J=8.3 Hz), 8.2 (d, 2H, J=8.3 Hz), 8.9
(broad s, 2H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-(4-fluorophenyl)urea (11). Starting from 4-(4,6-di-
morpholin-4-yl-1,3,5-triazin-2-yl)aniline (0.140 g 0.40 mmol)
and 4-fluorophenyl isocyanate (83 mg, 0.61 mmol) and follow-
ing the procedure outlined in example 8, 65 mg (33% yield) of
the title product was isolated as white solid. (M þ H) 480.3.
Purity by analytical HPLC 97.9%. (Prodigy ODS3, 0.46 cm ꢀ
15 cm, 20 min gradient acetonitrile in water, trifluoroacetic acid,
detector wavelengths, 215 and 254 nM.) 1H NMR (DMSO-d6) δ
3.6 (m, 8H), 3.7 (m 8H), 7.2 (t, J=8.3 Hz, 2H), 7.4 (m, 2H),
7.5 (d, J=9.3 Hz, 2H), 8.3 (d, J=9.1 Hz, 2H), 8.8 (s, 1H), 9.0
(s, 1H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-thiophen-2-ylurea (17). Starting from 4-(4,6-dimor-
pholin-4-yl-1,3,5-triazin-2-yl)aniline (60 mg, 0.17 mmol) and
2-thienyl isocyanate (18 mg, 0.14 mmol) and following the
procedure as outlined in example 8, the title compound was
isolated as a gray solid after SiO2 column chromatography by
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-(4-methylphenyl)urea (12). Starting from 4-(4,6-di-
morpholin-4-yl-1,3,5-triazin-2-yl)aniline (0.140 g 0.40 mmol)
and 4-methylphenyl isocyanate (74 mg, 0.56 mmol) and follow-
ing the procedure outlined for the preparation of compound 8,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2643
eluting with 5% ethyl acetate/methanol. (12 mg, 14% yield).
(M þ H)=468.3. Purity by analytical HPLC 96.9%. (Prodigy
ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient acetonitrile in water,
trifluoroacetic acid, detector wavelengths, 215 and 254 nm.) 1H
NMR (DMSO-d6) δ 3.66 (m, 8H), 3.81 (b, 8H), 6.58 (dd, J=1.3
Hz, J=3.8 Hz, 1H), 6.82 (dd, J=3.8 Hz, J=5.8 Hz, 1H), 6.9 (dd,
J=1.3 Hz, J=5.8 Hz, 1H), 7.55 (d, J=8.6 Hz, 2H), 8.28 (d, J=
8.6 Hz 2H), 9.05 (s, 1H), 9.68 (s, 1H), ppm.
Preparation of 4-[3-{4-(4,6-Dimorpholino-1,3,5-triazin-2yl)-
phenyl}ureido]benzoic Acid (22). To a stirred mixture of methyl
4-(3-(4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)ureido)-
benzoate (1.4 g, 2.69 mmol), THF (10 mL), MeOH (5 mL), and
H2O (2.5 mL) was added LiOH H2O (339 mg, 8.07 mmol). The
3
mixture was then heated under reflux for 8 h and concentrated.
H2O (5 mL) was added, and the mixture was then acidified with
2 N HCl. The solid was filtered, washed with H2O, and dried to
give the product as a tan solid, which was taken to next step
without purification (1.3 g, 96% yield). MS (ESI) m/z=506.2.
Purity by analytical HPLC 98.4%. (Prodigy ODS3, 0.46 cm ꢀ
15 cm, 20 min gradient acetonitrile in water, trifluoroacetic acid,
detector wavelengths, 215 and 254 nm.) 1H NMR (DMSO-d6) δ
3.6 (m, 8H), 3.7 (m, 8H), 7.56-7.59 (m, 4H), 7.9 (d, J=8.6 Hz,
2H), 8.3 (d, J=7.6 Hz, 2H), 9.31-9.34 (d, J=10.8 Hz, 2H) ppm.
Preparation of of N-[2-(Dimethylamino)ethyl]-4-({[4-(4,6-di-
morpholin-4-yl-1,3,5-triazin-2-yl)phenyl]carbamoyl}amino-
benzamide (24). A solution of 4-[3-{4-(4,6-dimorpholino-
1,3,5-triazin-2yl)phenyl}ureido]benzoic acid (150 mg,
0.297 mmol), Hunig’s base (303 μL, 1.782 mmol), and
HBTU (563 mg, 1.485 mmol) in 2 mL of NMP was stirred
for 1 h at room temperature. N0,N0-Dimethylethane-1,2-
diamine (130 uL, 1.188 mmol) was added, and then the
mixture was stirred overnight. At the end, CH2Cl2 (40 mL)
was added and washing was done with saturated NaHCO3
and H2O. The mixture was concentrated and purified by
silica gel chromatography, with CH2Cl2/methanol/7N NH3
in MeOH (10:1:0.22) to give the product as a white solid
(98 mg, 57% yield). MS (ESI) m/z = 576.4, MS (ESI) m/z
288.7. HRMS: calcd for C29H37N9O4 þ Hþ, 576.3041;
found 576.3036. Purity by analytical HPLC 98.4%.
(Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient aceto-
nitrile in water, trifluoroacetic acid, detector wavelengths,
215 and 254 nm.) 1H NMR (DMSO-d6) δ 3.6 (m, 8H), 3.7 (m,
8H), 2.5 (s, 6H), 2.8 (d, J=5.3 Hz, 2H), 3.2 (q, J=5.3 Hz,
2H), 7.55-7.58 (m, 4H), 7.8 (d, J=9.1 Hz, 2H), 8.2 (d, J=8.8
Hz, 2H), 8.6 (m, 1H), 9.6 (s, 1H), 9.7 (broad s,1H) ppm.
Preparation of 4-(3-(4-(4,6-Dimorpholino-1,3,5-triazin-2-
yl)phenyl)ureido)-N-methylbenzamide (23). A solution of
4-(3-(4-(4,6-dimorpholino-1,3,5-triazin-2yl)phenyl)ureido)ben-
zoic acid (150 mg, 0.297 mmol), Hunig’s base (303 μL, 1.782
mmol), and HBTU (563 mg, 1.485 mmol) in 2 mL of NMP was
reacted according to example 24 with methylamine (594 μL, 2 M
solution in THF) to give the product as a white solid (118 mg,
77% yield). MS (ESI) m/z = 519.3. HRMS: calcd for
C26H30N8O4 þ Hþ, 519.2463; found 519.2456. Purity by analy-
tical HPLC 95.7%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min
gradient acetonitrile in water, trifluoroacetic acid, detector
wavelengths, 215 and 254 nm.) 1H NMR (DMSO-d6) δ 3.6
(m, 8H), 3.7 (m, 8H), 2.81 (d, J=3.8 Hz, 3H), 7.51-7.57 (m, 4H),
7.8 (d, J = 7.3 Hz, 2H), 8.3 (d, J=7.6 Hz, 2H), 8.9 (s, 1H), 9.9 (s,
1H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-pyridin-3-ylurea (18). Starting from 4-(4,6-dimor-
pholin-4-yl-1,3,5-triazin-2-yl)aniline (0.08 g 0.23 mmol) and
3-pyridyl isocyanate (30 mg, 0.25 mmol) and following the
procedure as outlined in example 8, the title compound was
isolated as a white solid. The product was purified by SiO2
column chromatography by eluting it with 10% methanol/ethyl
acetate. (60 mg, 56% yield). (M þ H) 463.2. Purity by analytical
HPLC 98.2%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min
gradient acetonitrile in water, trifluoroacetic acid, detector
1
wavelengths, 215 and 254 nm.) H NMR (DMSO-d6) δ 3.66
(m, 8H), 3.82 (b, 8H), 7.33 (m, 1H), 7.56 (d, J=8.6 Hz, 2H), 7.96
(d, J=8.8 Hz, 1H), 8.21 (d, J=5.3 Hz, 1H), 8.58 (d, J=8.3 Hz,
2H), 8.61 (d, J=2.5 Hz 1H), 8.91 (s, 1H), 9.13 (s, 1H), ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-4-pyridin-4-ylurea (19). To a mixture of 4-(4,6-dimor-
pholin-4-yl-1,3,5-triazin-2-yl)aniline (0.20 g 0.40 mmol) in
methylene chloride (80 mL) at 0 °C was added triphosgene
(0.25 mg, 0.84 mmol) and triethylamine (3 mL). The mixture was
stirred for 20 min at 0 °C, and 4-aminopyridine (0.10 g 0.83
mmol) was added to the reaction mixture. The mixture was
stirred for another 2 h at room temperature. The solvent was
evaporated and the residue was submitted to HPLC using
acetonitrile/TFA as mobile phase to give 1-[4-(4,6-dimo-
phorlin-4-yl-1,3,5-triazin-2-yl)phenyl]-3-pyridin-4-ylurea (98.2
mg 36% yield). (M þ H) 463.4. Purity by analytical HPLC
97.9%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient
acetonitrile in water, trifluoroacetic acid, detector wavelengths,
1
215 and 254 nm.) H NMR (DMSO-d6) δ 3.51 (b, 8H), 3.64
(b, 8H), 7.60 (d, J=8.6 Hz, 2H), 7.92 (d, J=7.3 Hz, 2H), 8.32 (d,
J = 8.8 Hz, 2H), 8.58 (d, J = 7.6 Hz, 2H), 9.79 (s,1H), 10.54
(s, 1H) ppm.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-pyridazin-4-ylurea (20). To a mixture of 4-(4,6-dimor-
pholin-4-yl-1,3,5-triazin-2-yl)aniline (50 mg 0.15 mmol) in
methylene chloride (80 mL) at 0 °C was added triphosgene
(26 mg, 0.088 mmol) and triethylamine (0.2 mL). The mixture
was stirred for 20 min at 0 °C. Pyridazine-4-amine (42 mg
0.48 mmol) was added to the reaction mixture, and the mixture
was stirred for another 2 h at room temperature. The solvent was
evaporated and the residue was submitted to the HPLC using
acetonitrile/NH3 as mobile phase to give 17 mg (25% yield)
of 1-[4-(4,6-dimophorlin-4-yl-1,3,5-triazin-2-yl)phenyl]-3-pyri-
dazin-4-ylurea. (M þ H) 464.2. Purity by analytical HPLC
95.6%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient
acetonitrile in water, trifluoroacetic acid, detector wavelengths,
215 and 254 nm.) 1H NMR (DMSO-d6) δ 3.6 (m, 8H), 3.7 (m,
8H), 7.5 (d, J=9.4 Hz 1H), 7.81-7.84 (m, 2H), 8.3 (d, J=8.3 Hz,
2H), 8.9 (d, J=6.4 Hz, 2H), 9.1 (m, 1H), 9.4 (s, 1H) ppm.
Preparation of Methyl 4-(3-(4-(4,6-Dimorpholino-1,3,5-tria-
zin-2-yl)phenyl)ureido)benzoate (21). Starting from 4-(4,6-di-
morpholin-4-yl-1,3,5-triazin-2-yl)aniline (342 mg, 1.0 mmol)
and methyl 4-isocyanatobenzoate (177 mg, 1 mmol) and follow-
ing the procedure as outlined in example 8, the title compound
was isolated as a gray solid (480 mg, 92%) after SiO2 column
chromatography by eluting with 50% ethyl acetate/hexane.
(M þ H) 520.3. Purity by analytical HPLC 95.3%. (Prodigy
ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient acetonitrile in water,
trifluoroacetic acid, detector wavelengths, 215 and 254 nm.) 1H
NMR (DMSO-d6) δ 3.6 (m, 8H), 3.7 (m, 8H), 3.8 (s, 3H),
7.57-7.62 (m, 2H), 7.8 (d, J=7.6 Hz, 4H), 8.3 (d, J=9.1 Hz,
2H), 9.11-9.15 (d, J=12.6 Hz, 2H) ppm.
Preparation of 1-[4-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-
phenyl]-3-{4-[(4-methylpiperazine-1-yl)carbonyl]phenyl}urea Hy-
drochloride (25). A stirred solution of 4-(3-(4-(4,6-dimorpho-
lino-1,3,5-triazin-2yl)phenyl)ureido)benzoic acid (150 mg, 0.297
mmol), Hunig’s base (303 μL, 1.782 mmol), and HBTU (563 mg,
1.485 mmol) in 2 mL of NMP was reacted according to example
24 with 1-methylpiperazine (132 μL, 1.188 mmol) to give
1-(4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)-3-(4-(4-methyl-
piperazine-1-carbonyl)phenyl)urea as a white solid (95 mg, 54%
yield). MS (ESI) m/z = 588.4 and 294.7. HRMS: calcd for
C30H37N9O4 þ Hþ, 588.3041; found (ESI, [M þ H]þ obsd),
588.3033. Purity by analytical HPLC 98.2%. (Prodigy ODS3, 0.46
cm ꢀ 15 cm, 20 min gradient acetonitrile in water, trifluoroacetic
acid, detector wavelengths, 215 and 254 nm.) 1H NMR (DMSO-d6)
δ 2.2 (s, 3H), 2.5 (m, 4H), 3.3 (m, 4H), 3.6 (m, 8H), 3.7 (m, 8H), 7.3
(d, J=8.3 Hz, 2H), 7.51-7.58(m, 4H), 8.3(d, J=9.0 Hz, 2H), 8.9 (s,
1H), 9.1 (s, 1H) ppm. To 1-(4-(4,6-dimorpholino-1,3,5-tria-
zin-2-yl)phenyl)-3-(4-(4-methylpiperazine-1-carbonyl)phenyl)urea

2644 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Venkatesan et al.
(70 mg, 0.119 mmol) and MeOH (1 mL) was added 4 N HCl in
dioxane (1 mL), and the mixture was stirred for 3 h. The solid was
filtered and washed with ether to give the product as a white solid
(74 mg, 100% yield).
for nudemouse microsomeassays, andDr. LiDiand Susan Li
for human microsome assays.
References
Preparation of 1-(4-{[4-(Dimethylamino)piperidin-1-yl]car-
bonyl}phenyl)-3-[4-(4,6-dimorpholin-4- yl-1,3,5-triazin-2-yl)phe-
nyl]urea (26). A solution of 4-(3-(4-(4,6-dimorpholino-1,3,5-
triazin-2yl)phenyl)ureido)benzoic acid (50 mg, 0.099 mmol),
Hunig’s base (103 μL, 0.594 mmol), and HBTU (188 mg,
0.495 mmol) in 2 mL of NMP was reacted according to example
24 with N,N-dimethylpiperidin-4-amine (51 mg, 0.396 mmol).
The solvent was evaporated and purified by HPLC to give the
product (30.6 mg, 52% yield). MS (ESI) m/z=616.7. HRMS:
calcd for C32H41N9O4 þ Hþ, 616.335 43; found (ESI-FTMS, [M
þ H]þ), 616.334 24. Purity by analytical HPLC 99.3%. (Prodigy
ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient acetonitrile in water,
trifluoroacetic acid, detector wavelengths, 215 and 254 nm.) 1H
NMR (DMSO-d6) δ 1.29-1.36 (m, 6H), 2.6 (m, 4H), 2.9
(m,1H), 3.3 (m, 4H), 3.6 (m, 8H), 3.7 (m, 8H), 7.3 (d, J=8.3
Hz, 2H), 7.51-7.57 (m, 4H), 8.3 (d, J=8.3 Hz 2H), 8.9 (s, 1H),
9.0 (s, 1H) ppm. Anal. Calcd for C32H41N9O4: C 62.42%, H
6.71%, N 20.47%. Found: C 62.34%, H 6.67%, N 20.39%.
Preparation of 1-[4-(4,6-Dimorpholin-4-yl-1,3,5-triazin-2-yl)-
phenyl]-3-{4-[(4-pyrrolidin-1-ylpiperidin-1-yl)carbonyl]phenyl}-
urea (27). Compound 27 was prepared by reacting 4-(3-(4-(4,6-
dimorpholino-1,3,5-triazin-2yl)phenyl)ureido)benzoic acid and
4-pyrrolidi-1-ylpiperidine. (M þ H) m/z=642.3. HRMS: calcd
for C34H43N9O4 þ Hþ, 642.3510; found (ESI-FTMS, [M þ
H]þ), 642.3491. Purity by analytical HPLC 97.2%. (Prodigy
ODS3, 0.46 cm ꢀ 15 cm, 20 min gradient acetonitrile in water,
trifluoroacetic acid, detector wavelengths, 215 and 254 nm.) 1H
NMR (DMSO) δ 1.62 (m, 2H), 1.86 (m, 2H), 1.98 (m, 2H), 2.08
(m, 2H), 2.94 (brs, 2H), 3.07 ((m, 2H), 3.38 (m, 1H), 3.48 (m,
2H), 3.72-4.01 (m, 17H), 7.35 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.8
Hz, 2H), 7.57 (d, J=8.8 Hz, 2H), 8.28 (d, J=8.8 Hz, 2H), 9.33 (s,
1H), 9.37 (s, 1H), 10.43 (m,1H) ppm.
Preparation of 1-(4-(1,40-Bipiperidine-10-carbonyl)phenyl)-
3-(4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)urea (28). A
solution of 4-(3-(4-(4,6-dimorpholino-1,3,5-triazin-2yl)phenyl)-
ureido)benzoic acid (50 mg, 0.099 mmol), Hunig’s base (103 μL,
0.594 mmol), and HBTU (188 mg, 0.495 mmol) in 2 mL of NMP
was reacted according to example 24 with 1,40-bipiperidine (67
mg, 0.396 mmol). The solvent was evaporated and purified by
HPLC to give the product as a white solid (39 mg, 60% yield).
MS (ESI) m/z=656.8.
Preparation of N-(2-(Dimethylamino)ethyl)-4-(3-(4-(4,6-di-
morpholino-1,3,5-triazin-2-yl)phenyl)ureido)-N-methylbenz-
amide Hydrochloride (30). A stirred solution of 4-(3-(4-(4,6-
dimorpholino-1,3,5-triazin-2yl)phenyl)ureido)benzoic acid (150
mg, 0.297 mmol), Hunig’s base (303 uL, 1.782 mmol), and HBTU
(563 mg, 1.485 mmol) in 2 mL of NMP was reacted according to
example 24 with N1,N1,N2-trimethylethane-1,2-diamine (154 μL,
1.188 mmol) to give N-(2-(dimethylamino)ethyl)-4-(3-(4-(4,6-
dimorpholino-1,3,5-triazin-2-yl)phenyl)ureido)-N-methylbenz-
amide as a white solid (88 mg, 50% yield). MS (ESI) m/z=590.2.
To N-(2-(dimethylamino)ethyl)-4-(3-(4-(4,6-dimorpholino-
1,3,5-triazin-2-yl)phenyl)ureido)-N-methylbenzamide (55 mg,
0.127 mmol) and MeOH (1 mL) was added 4 N HCl in dioxane
(1 mL), and the mixture was stirred for 3 h. The solid was filtered
and washed with ether to give the product as a white solid (70
mg, yield = 88%). HRMS: calcd for C30H39N9O4 þ Hþ,
590.3198; found (ESI-FTMS, [M þ H]þ), 590.3194. Purity by
analytical HPLC 99.8%. (Prodigy ODS3, 0.46 cm ꢀ 15 cm, 20
min gradient acetonitrile in water, trifluoroacetic acid, detector
wavelengths, 215 and 254 nm.)
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