Design, synthesis, and biological activity of pyridopyrimidine scaffolds as novel PI3K/mTOR dual inhibitors

Article

pubs.acs.org/jmc

Design, Synthesis, and Biological Activity of Pyridopyrimidine

Scaﬀolds as Novel PI3K/mTOR Dual Inhibitors

Thibault Saurat,^†,‡Frederic Buron,^†Nuno Rodrigues,^†Marie-Ludivine de Tauzia,^‡Lionel Colliandre,^†

́

Stephane Bourg,^†,‡Pascal Bonnet,^†Gerald Guillaumet,^†Mohamed Akssira,^§Anne Corlu,^∥

́

Christiane Guillouzo,^∥Pauline Berthier,^⊥Pascale Rio,^⊥Marie-Lise Jourdan,^⊥Helene Benedetti,^‡

́

̀

́

and Sylvain Routier*^,†

^†Institut de Chimie Organique et Analytique, Universite

́ ́

d’Orleans, UMR CNRS 7311, Rue de Chartres, BP 6759, 45067

Orlea

́

nsCedex 2, France

^‡Centre de Biophysique Molec

́

ulaire, CNRS Orlea

Equipe de Chimie Bioorganique & Analytique, URAC 22, Universite

Mohammedia, Morocco

́

ns, Rue Charles Sadron, 45071 Orlea

́

ns, France

§

́

Hassan II Mohammedia-Casablanca, BP 146, 28800

^∥Ho

̂

pital de Pontchaillou, Universite

́

de Rennes 1, INSERM, UMR-991, 65033 Rennes Cedex, France

^⊥Faculte

France

́

de Med

́

ecine, Centre Hospitalier Universitaire (CHU) Tours, INSERM U1069, 10 Boulevard Tonnelle, 37032 Tours Cedex,

́

S

* Supporting Information

ABSTRACT: The design, synthesis, and screening of dual

PI3K/mTOR inhibitors that gave nanomolar enzymatic and

cellular activities on both targets with an acceptable kinase

selectivity proﬁle are described. A docking study was

performed to understand the binding mode of the compounds

and to explain the diﬀerences in biological activity. In addition,

cellular eﬀects of the best dual inhibitors were determined on

six cancer cell lines and compared to those on a healthy diploid

cell line for cellular cytotoxicity. Two compounds are highly

potent on cancer cells in the submicromolar range without any

toxicity on healthy cells. A more detailed analysis of the cellular

eﬀect of these PI3K/mTOR dual inhibitors demonstrated that

they induce G1-phase cell cycle arrest in breast cancer cells and

trigger apoptosis. These compounds show an interesting kinase proﬁle as dual PI3K/mTOR tool compounds or as a chemical

series for further optimization to progress into in vivo experiments.

which plays a key role in protein synthesis, and 4E-BP1, an

actor in the translation initiation.¹¹

INTRODUCTION

■

The PI3K/Akt/mTOR pathway is associated with several

receptor tyrosine kinases (RTKs) and G-protein-coupled

receptors (GPCRs). PI3K (phosphoinositide 3-kinase), the

ﬁrst enzyme of the pathway, is a ubiquitous lipid kinase

involved in a large variety of diseases. It catalyzes the

transformation of phosphatidylinositol 4,5-diphosphate

(PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3).¹

Four PI3K isoforms²have been reported: PI3Kα in cancer,^3,4

PI3Kβ, which is more active in thrombosis-related phenom-

ena,⁵and PI3Kδ in allergies and PI3Kγ in inﬂammatory

responses.⁶

In cancer, tumors exhibit a high frequency of activating

mutations in the PI3K, Akt, and mTOR coding genes, leading

to an elevated pathway activity and resulting in extended

survival, growth, and angiogenesis of tumor cells.^12,13Activation

of the pathway is not vertical. It has been shown that mTOR

exerts a negative feedback loop on PI3K: mTOR once activated

will inhibit PI3K through IRS-1 (insulin receptor substrate 1).¹⁴

Furthermore, it has been observed in several studies that

external factors (growth factors, nutrient intake) can activate

Akt and mTOR, thereby bypassing PI3K and thus restarting

cancer genesis.^15,16

PI3Kα plays a signiﬁcant role in cancer-related diseases and

more speciﬁcally in insulin signaling.⁷⁻¹⁰In addition, cancers

are also associated with mTOR (mammalian target of

rapamycin), the third kinase of the signaling pathway studied

in the present research work, whose main targets are S6K1,

For these reasons, the need to synthesize compounds that

can inhibit the PI3K/Akt/mTOR pathway has been a great

Received: July 26, 2013

Published: December 17, 2013

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Figure 1. Examples of PI3K inhibitors.

motivation for research teams around the world.^17,18In 2011,

active drugs having an implication on PI3K, Akt, or mTOR

were reported in more than 380 clinical trials. Three drugs have

already been approved by the FDA (Food and Drug

Administration) for mTOR (Aﬁnitor, Torisel, and Zortress)

but none yet for Akt or PI3K protein kinases. According to the

National Cancer Institute, several drugs in phase I or II clinical

trials are claimed as speciﬁc PI3Kα inhibitors.¹⁹

Most of these PI3Kα inhibitors share similar structural and

chemical features, i.e., a central six-membered heterocycle

substituted by a morpholine and a (hetero)aromatic cycle

having a hydrogen bond donor group (Figure 1, ZSTK-414,

GDC-0941, GDC-0980, PI103). As mentioned in the pathway

functioning, due to the existence of a regulatory feedback loop,

an independent activation of mTOR or PI3K alone is not

suﬃcient to block the signaling pathway eﬃciently in cancer

cells.²⁰It is therefore crucial to consider the combined

inhibition of both enzymes.²¹To our knowledge, only a few

drugs that aﬀect PI3Kα and mTOR together are in an early

phase of clinical trials. NVP-BEZ-235, XL765, and an orally

available candidate, GDC-0980, are claimed to be dual PI3K/

mTOR inhibitors.²²

and mTOR as biological targets. In silico modeling studies were

used to support medicinal chemistry eﬀorts and proved to be

successful in explaining the SAR of the tested compounds.

Finally, the eﬀects of the seven best inhibitors were measured

on three cancer cell lines and normal ﬁbroblasts. Further

investigations were carried out for the two best compounds,

which were tested on three other cancer cell lines and were

shown to aﬀect PI3K and mTOR activities in these cells, to

block their cell cycle in the G1-phase, and to induce apoptosis.

RESULTS AND DISCUSSION

■

Synthesis and Kinase Assays. The derivatives 3a−g were

obtained after a two-step synthesis from 2,4-dichloropyrido-

[3,2-d]pyrimidine (1). S_NAr regioselectively occurred at C-4

using aliphatic primary or secondary amines in the presence of

Et₃N at room temperature and aﬀorded compounds 2a−g in

satisfying yields (Scheme 1, Table 1). In the second step, the 3-

a

Scheme 1

We recently developed a straightforward methodology able

to selectively functionalize 2,4-dichloropyridopyrimidine by two

successive S_NAr and Suzuki cross-coupling reactions.²³We thus

envisioned applying our method to the design of a dual PI3Kα/

mTOR inhibitor library. In this report, we report the synthesis

of newly formed pyrido[2,3-d]- and pyrido[3,2-d]pyrimidine

libraries I and II starting from a dichloro derivative of type III

or IV (Figure 2).

a

Reagents and conditions: (a) amine, Et₃N, THF, rt, 12 h; (b) 3-

HOC₆H₄B(OH)₂, K₂CO₃, Pd(PPh₃)₄, DME, MW, 150 °C, 1 h. For

yields see Table 1.

hydroxyphenyl group was introduced using a Suzuki cross-

coupling reaction under microwave irradiation to improve the

yields, which are very low otherwise. This palladium-catalyzed

reaction gave access to the attempted compounds in good to

excellent yields, except in the case of 3f (entry 6), which was

only detected as molecular traces.

After introducing an amine and a (het)aryl at C-2 or C-4, we

established a complete SAR study using PI3Kα, PI3Kδ, PI3Kγ,

The six ﬁnal products (3a−e and 3g) were evaluated for their

PI3K inhibition potency (Table 1). Morpholine seems essential

to strongly diminish PI3Kα activity, and the IC₅₀was measured

in the nanomolar range.²⁴Replacement of this core by other

cyclic amines causes a dramatic decrease in activity despite the

presence of electron-rich atoms.

Figure 2. Disubstituted pyridopyrimidine libraries I and II from

precursors III and IV.

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Table 1. Synthesis Yields and Biological Evaluation of Compounds 3a−g

a

b

Yields are indicated for isolated products. IC₅₀values are presented as the mean of a triplicate experiment. Standard deviations are reported in

Table S1 of the Supporting Information.

As oxygen seems to be essential for activity,²⁵we used

2(R),6(R)-dimethylmorpholine to obtain 3g (entry 7), but its

activity dropped to the micromolar range. Finally, 3a remains

the best compound, showing comparable IC₅₀values against

PI3Kα and PI3Kγ and a remarkable potency for the δ isoform

(IC₅₀= 3 nM).

Thus, we conserved the morpholino group to pursue our

investigation and further explore the SAR. For the next C-2

(het)Ar optimization (Scheme 2), we successfully achieved the

cross-coupling reactions using several commercially available

arylboronic acids or potassium aryltriﬂuoroborates (1.5 equiv)

under the conditions described above.

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a

cross-coupling reaction were mostly related to the puriﬁcation

step. Most of the reaction appeared to be complete during the

monitoring. Derivatives 4g and 4h (entries 8 and 9) were easily

obtained from a cross-coupling reaction between 2a and the

corresponding nitrophenylboronic acid, followed by reduction

of the nitro group using stannous chloride. In a complementary

single-step synthesis, we also prepared the mono- and

bisacetylated derivatives 5 and 6 from 4m in the same reaction

using an excess of Ac₂O in DMF under microwave irradiation

in 28% and 44% yields, respectively (entries 15 and 16).

In vitro activities toward PI3K kinase isoforms were

measured using the ﬁnal compounds 4a−m, 5, and 6, and

Scheme 2

a

Reagents and conditions: (a) (Het)ArB(OH)₂or (Het)ArBF₃K,

K₂CO₃, Pd(PPh₃)₄, DME, MW, 150 °C, 1 h. For yields see Table 2.

The desired products 4a−m were isolated in moderate to

excellent yields (Table 2). Variations in the eﬃciency of the

Table 2. Synthesis and Biological Evaluation of Derivatives 4

a

b

Yields are given for the isolated product. IC₅₀values are presented as the mean of a triplicate experiment. Standard deviations are reported in Table

c

d

e

f

S1 of the Supporting Information. Boronic acid was used. Potassium triﬂuoroborate was used. Overall yield of two steps. Compounds 5 (28%)

and 6 (44%) were obtained from 4m using Ac₂O (10.0 equiv) and DMF (120 °C, 30 min).

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the IC₅₀values were compared to that of 3a (entry 1). The best

position for the OH phenol group remains at the meta position

since its position shift to ortho or para leads to an overall

inactivation of the drugs. The hydrophilic character and the

ability of the drug to create a hydrogen bond in this region are

certainly crucial as proposed from docking results. Spacing the

OH group by addition of a methylene kneecap in the meta

position of the phenyl ring was well tolerated (4e vs 4f, entries

6 and 7), whereas methyl ethers 4c and 4d were also totally

inactive (entries 4 and 5).

Replacement of the phenyl hydroxyl group by a primary

amine or the phenol by a 3-pyridine or a 4-indazole (entries 8−

11) led to poorly active compounds 4i and 4j.²⁸Nevertheless,

with the 4-indazole-containing derivative 4k, we observed a

global increase of activity for PI3Ks and in particular for the α

isoform (entry 12).

It appears that 2-aminopyridine and -pyrimidine moieties

could be used as phenol isosters (entries 13 and 14). PI3Kα

and PI3Kδ were more aﬀected by the two novel lead

compounds. 4m, which inhibits PI3Kα with a remarkable

IC₅₀of 7 nM, is the most selective inhibitor ever designed in

the disubstituted pyridopyrimidine series. The activity of the 2-

aminopyrimidine-containing compound is due to the presence

of an acidic hydrogen atom since acetylated compound 5

remains slightly active and diacetylated 6 is fully inactive

(entries 15 and 16).²⁹

To pursue the SAR exploration, we next investigated

successively the eﬀect of reversing the C-4 morpholine and

the C-2 phenol groups on the pyrido[3,2-d]pyrimidine scaﬀold

and of the switch of the central core for the [2,3-d] regioisomer

(Scheme 3, Table 3).³⁰To obtain the expected derivatives, we

adapted the previously reported robust synthetic methods and

used the dichloro derivatives 1 and 10 as starting materials.

For the synthesis of the “inverso” derivative 9, 3-

(methoxymethoxy)phenyltriﬂoroborate potassium was pre-

pared to perform the Suzuki cross-coupling reaction.³¹The

palladium-catalyzed reaction occurred selectively using a near

stoichiometric amount of boronic acid in reﬂuxing toluene at

100 °C for 2 h with our classical catalytic system and yielded 7

in 45% yield. The C-2 S_NAr was next carried out with

morpholine and Et₃N in reﬂuxing THF and gave the desired

compound 8 in 68% yield. Finally, the MOM protective group

was removed with HCl (4 N in dioxane) at room temperature

to aﬀord 9 in 60% yield.

For the compound synthesis in the pyrido[2,3-d]pyrimidine

series, compound 10 was subjected to the reactions leading to

compounds 2 and 3 from 1. Derivatives of type 12 were

isolated in fair yields.

The derivative 9 showed an inhibition of PI3Kα (IC₅₀= 59

nM) which was still 3-fold less eﬃcient than that of 3a, whereas

against the γ and δ isoforms, dramatic decreases in activity were

observed (IC₅₀(PI3Kγ) = 1.06 μM, IC₅₀(PI3Kδ) = 0.186 μM).

The same general behavior was observed with pyrido[2,3-

d]pyrimidine compounds 12a−c; activities were partially lost

especially for the PI3Kα and PI3Kγ isoforms (vs the

corresponding compound 3a in the [3,2-d] series, Table 3).

Noteworthy, the preservation of the activity of compound 12c

against PI3Kδ is observed, leading to incipient selectivity

against other isoforms.

The last modiﬁcation we performed is entirely based on the

mTOR inhibitor discovery which mainly uses a urea to enhance

binding.³²Consequently, we prepared and evaluated ureas of

type 13. For this new proposal, the anilines 4g and 4h were

converted to the corresponding in situ formed isocyanates by

treatment with triphosgene in the presence of an excess of

DIPEA in THF at −78 °C, and the unstable intermediates were

immediately trapped with various amines to aﬀord products 13

with moderate to good yields (Scheme 4 and Table 4).

Regarding both the biological activity on PI3Kα and the

nature of the urea substituents, a small methyl is well tolerated

(entry 1), up to the size of a six-membered ring. To maintain

the kinase inhibition, urea is better positioned in the para

position of aryl ring A (Table 4, entry 3 vs entry 8), indicating

some additional interactions in the ATP binding pocket. In

addition, if R bears a supplementary donor or acceptor of

hydrogen bonds, the global activity increases.

For this novel function, a hydroxymethyl is preferred in the

meta position, whereas a simple hydroxyl is better tolerated in

the para position (entry 2 vs entry 3 and entry 5 vs entry 4). In

the pyridine series (entries 6 and 7), the m-nitrogen atom

interacts better with the biological targets. Except for 13c and

13h, the urea derivatives are more active on PI3Kα and PI3Kγ

than on the PI3Kδ isoform. The most selective compound is

indubitably 13e (entry 5), which exhibits a selective action on

the α isoform (5- and 10-fold more potent compared to the γ

and δ isoforms, respectively).

a

Scheme 3

Since mTOR and PI3K conserved a typical ATP site despite

their little kinase fold resemblance, we focused on the

inhibitory eﬀect of our compounds on mTOR. As it turns

out, all of our compounds displayed submicromolar activities

against both mTOR and PI3Kα (Table 5). Our dual inhibitors

are more eﬃcient at inhibiting PI3Kα than mTOR (entries 1−

8). However, our best PI3Kα inhibitors, 3a and 4n, displayed

very good nanomolar inhibitions against mTOR (entries 1 and

4).

Other derivatives such as 12a, 12b, 13e, and 13f have similar

activity ratios on the two kinases (entries 6, 7, 13, and 14).

Interestingly, the introduction of an aryl urea on ring B led to

compounds which are more active against mTOR than against

PI3Kα (entries 9−12). Compounds 13b and 13c in particular

displayed the strongest activities (IC₅₀= 1 and 5 nM,

a

Reagents and conditions: (a) 3-(MOM)C₆H₄BF₃K, K₂CO₃, Pd-

(PPh₃)₄, toluene, 100 °C, 2 h, 45%; (b) morpholine, Et₃N, THF,

reﬂux, 12 h, 68%; (c) HCl (4 N), EtOH, rt, 1 h, 60%; (d) morpholine,

Et₃N, THF, rt, 12 h, 80%; (e) (Het)ArB(OH)₂or (Het)ArBF₃K,

K₂CO₃, Pd(PPh₃)₄, DME, MW, 150 °C, 1 h. For exact structures and

yields see Table 3.

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Table 3. Synthesis and Biological Evaluation of Pyrido[2,3-d]pyrimidine-Type Compounds

a

b

Yields are indicated for isolated products. IC₅₀values are presented as the mean of a triplicate experiment. Standard deviations are reported in

Table S1 of the Supporting Information.

a

the side chain of the catalytic Lys802 was not reproduced for

two thienopyrimidine derivatives (PDB entries 3L16 and

3L17)²⁹and a pyrazolopyrimidine derivative (PDB entry

3IBE).³⁹

Scheme 4

After careful analysis of the active site, we established that

these ligands need a diﬀerent conformation of the ﬂexible

lateral chain of the catalytic Lys802 to correctly bind to our

docking model. The conformation of the corresponding lysine

observed in the structure of PI3Kγ in complex with the

pyrazolopyrimidine derivative (PDB entry 3IBE) was manually

assigned to Lys802 in the 3HHM structure. The use of this new

model enabled all interactions to be reproduced for each

docked inhibitor in the corresponding original structures. It was

thus used for all further docking experiments.

Next, our ligands were docked using the previously validated

model. The 10 best docking poses according to GlideScore³⁸

were conserved for each ligand. All the compounds adopted

orientations analogous to those of the cocrystallized inhibitors.

The binding is driven by one hydrogen bond created between

the oxygen atom of the morpholino group and the NH group

of Val851 in the hinge region of the active site (Figure 3). This

hydrogen bond is considered as the key interaction that confers

the binding capacity on the ligands. The strength of this

hydrogen bond (or its absence) explains why the activity of our

compounds decreases (or disappears) when the oxygen atom of

the morpholino group is modiﬁed (3a ≫ 3b ≫ 3c).

The binding of the morpholino group to the hinge region

leads to the orientation of the aryl group toward Asp810 and

the catalytic Lys802. The hydroxyl position on this aryl group

has a drastic inﬂuence on the activity of the compounds (Table

4). Docking of 3a shows that the hydroxyl function replaces a

conserved water molecule, as has already been shown in some

crystallographic structures of suﬃcient resolution (PDB entries

3L54, 3L08, and 3ML9).^22bThis hydroxyl function forms two

hydrogen bonds with the oxygen atom of the hydroxyl group of

Tyr836 and with one oxygen atom of the carboxyl group of

Asp810 (Figure 3). Moreover, the hydroxyl function of

compound 4e forms the same hydrogen bonds, slightly moving

the pyrido[3,2-d]pyrimidine moiety in the active site.

a

Reagents and conditions: (a) triphosgene, DIPEA, THF, −78 °C, 40

min, then RNH₂, DIPEA, THF, rt, 24 h. For exact structures and yields

see Table 4.

respectively) on mTOR, the last kinase in the pathway (entries

10 and 11).

Finally, we examined the selectivity proﬁle of 13c on a

diverse 50-kinase panel. The activity of the compound was

conﬁrmed on the PI3K family and mTOR, and the compound

appeared to be very selective toward other kinases with the

exception of CDK19 and CDK8 (Table 6).

Molecular Modeling. Docking studies were carried out to

investigate the binding mode of our compounds in the PI3Kα

isoform. Among numerous crystallographic structures of PI3K,

only three were available for the α isoform of human PI3K.

Two apo structures (PDB entries 2RD0 and 3HIZ)³³⁻³⁵and

one complex with the covalent inhibitor wortmannin (PDB

entry 3HHM)³³were used as structural models of PI3Kα in our

studies. Among all the crystallographic structures of PI3K, a

majority contain reversible inhibitors that provide useful

information on protein/ligand interactions found in their active

site. More importantly, some of these inhibitors are structurally

similar to our compounds. The strong sequence and structural

similarities between the diﬀerent classes and isoforms of PI3K

(especially in their active sites)³⁶suggest that the interactions

observed with these crystallographic inhibitors are likely to

occur for our compounds.

To select the best model for docking experiments,

wortmannin and seven PI3Kγ cocrystallized inhibitors (ligands

containing a pyrimidine 2-morpholino-4-heteroaryl or sub-

stituted 2-heteroaryl-4-morpholine motif) were docked in the

three previously selected structures. The use of the 3HHM

structure enabled the reproduction of the crystallographic

binding modes of the ligands. Nevertheless, the hydrogen bond

interaction seen in the original complex between the ligand and

The docking of the pyrido[2,3-d]pyrimidine isomer deriva-

tives (compounds 12a−c) does not show signiﬁcant diﬀerences

in the global binding mode compared to that of the previous

scaﬀold. Only a minor inﬂuence can be observed on the

conformation of the morpholino group due to the introduction

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Table 4. Synthesis Yields and Biological Evaluation of Ureas 13

a

b

Yields are given for isolated products. IC₅₀values are presented as the mean of a triplicate experiment. Standard deviations are reported in Table

S1 of the Supporting Information.

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Table 5. IC₅₀Values of the Best Inhibitors against PI3Kα and mTOR

a

IC₅₀values are presented as the mean of a triplicate experiment. Standard deviations are reported in Table S1 of the Supporting Information.

of a hydrogen atom in position 5 of the scaﬀold. This could

explain the lower activity of such compounds.

The predicted binding modes of the 4-urea derivatives are

similar to that of the cocrystallized substituted pyrazolopyr-

imidine inhibitor (PDB entry 3IBE), with the morpholino

group still forming one hydrogen bond with Val851 in the

hinge region. Nevertheless, some diﬀerences can be noticed.

The crystallographic complex shows that the urea function

forms three hydrogen bonds with the target. Each NH group of

the urea forms a hydrogen bond with the oxygen atom of the

carbonyl group of the side chain of Asp810, whereas the

carbonyl group of the urea forms one hydrogen bond with the

side chain of Lys802. In our docking poses, four hydrogen

bonds can be observed. Due to a slightly diﬀerent orientation of

Inversion of the morpholino and aryl groups (compound 8)

does not induce any changes in the observed interactions of

these groups but leads to a diﬀerent orientation of the scaﬀold

in the active site. The morpholino group still forms one

hydrogen bond with Val851 in the hinge region, and the aryl

group is still oriented toward Asp810 and the catalytic residue

Lys802. The diﬀerent orientation of the scaﬀold in the active

site can explain the lower activity of this compound.

Using urea derivatives, the binding site of PI3Kα was

explored beyond the catalytic Lys802 (compounds 13a−h).

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a

Table 6. Protein Kinase Selectivity of 13c in a Kinase Interaction Assay (KinomeScan)

kinase

AAK1

ctrl (%)

kinase

ctrl (%)

kinase

ctrl (%)

kinase

ctrl (%)

kinase

ctrl (%)

77

72

CDKL3

CDKL5

CIT

94

100

82

IRAK3

LZK

62

81

78

70

96

73

91

0

PCTK3

100

93

PIP5K1C

PIP5K2B

PIP5K2C

PKMYT1

PRP4

68

93

87

95

71

78

92

78

80

94

ACVR2A

ACVR2B

ANKK1

BIKE

PFTK1

98

MAP3K15

MAP3K4

MAST1

MEK5

PIK3C2B

PIK3C2G

PIK3CA

PIK3CB

PIK3CD

PIK3CG

PIK4CB

PIP5K1A

4.2

0.95

0

100

84

CSNK1A1L

EPHB6

ERBB3

ERK3

92

BUB1

87

85

0.15

5.4

2.5

81

RIPK1

CDC2L1

CDK11

CDK8

100

8.8

13

100

89

MKK7

RIPK4

ERK4

MTOR

NEK10

PCTK2

TIE1

ERN1

70

97

TNNI3K

YANK1

CDKL1

75

GAK

49

55

a

13c was screened at a 10 μM concentration on a 50-kinase interaction panel, and results for primary screen binding interactions are reported as “ctrl

(%)”, where lower numbers indicate stronger hits.

bond with this lysine (not shown), instead of the neighboring

Lys802. This interaction is not present in the crystallographic

structure because the conformation of the lysine in PI3Kγ

(PDB entry 3IBE) corresponding to Lys776 could not be

deﬁned, probably due to its ﬂexibility.

This study shows that our ligands adopt binding modes in

PI3Kα that are similar to those of known inhibitors in PI3Kγ.

Crystallographic studies of the complexes between PI3Kα and

our ligands should be considered to conﬁrm the predicted

binding modes. In particular, these studies would enable the

identiﬁcation of important residues in the PI3Kα active site

forming key interactions with the inhibitors (Lys776, Lys802,

Asp810). This information would lead to a better optimization

of our compounds.

Cellular Assays. Compounds which exhibited dual activity

on PI3Kα and mTOR with IC₅₀values lower than 100 nM

were selected for a primary cellular screening assay using six

cancer cell lines (Tables 7 and 8): hepatocellular colorectal and

mammary carcinoma cells and immortalized keratinocytes.

Human diploid skin ﬁbroblasts were used as noncancer cells.

Figure 3. Representation of the predicted binding mode of compound

3a in the active site of PI3Kα. Hydrogen bonds are represented in

yellow-black dotted lines. For clarity, only polar hydrogens are

represented and residues 767−772 overlaying the ligands are hidden in

the ribbon representation (PDB reference codes 3HHM and 3IBE for

the positioning of Lys802).

the side chain of Asp810 in our model, the NH groups of the

urea of the docked ligands form two hydrogen bonds with the

same oxygen atom of the carbonyl group of the side chain of

Asp810. Moreover, the carbonyl group of the urea forms one

hydrogen bond with the amino groups of the side chains of

Lys802 (Figure 4). Lys776 is highlighted too because some

poses of our diﬀerent docked compounds form a hydrogen

Table 7. Cancer Cell Lines and Their PI3K Signaling

Pathway Status

pathway

cell line

Huh-7

human cell type

mutation overactivation

ref

hepatocellular

carcinoma

yes

39−41

Caco-2

HaCaT

MCF-7

colorectal

42, 43

44−46

47

adenocarcinoma

immortalized

keratinocyte

elevated Akt

activity

breast

adenocarcinoma

PI3KCA activated

MDA-MB-231

MDA-MB-468

normal

47

PTEN

over

First and foremost, our newly tested 2,4-disubstituted

pyridopyrimidine did not aﬀect the survival and proliferation

of human skin ﬁbroblasts. Regarding cancer and immortalized

cell lines, the most potent PI3Kα inhibitors 3a and 4m (Table

5, entries 1 and 4) act in the same concentration range as PI103

but do not appear to be the best products with respect to cell

toxicity. The results clearly show that the best in cellulo

inhibitory eﬀects were obtained with the best dual PI3K and

mTOR inhibitors and more particularly with the more potent

mTOR inhibitors 13b and 13c.

Figure 4. Representation of the predicted binding mode of compound

13c in the active site of PI3Kα. Hydrogen bonds are represented in

yellow-black dotted lines. For clarity, nonpolar H atoms are not

represented and residues 767−772 overlaying the ligands are hidden in

the ribbon representation (PDB reference codes 3HHM and 3IBE for

the positioning of Lys802).

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a

Table 8. In Cellulo Activity of the Best Inhibitors, IC₅₀(μM)

c

breast

entry

compd

Huh-7 (liver)

Caco-2 (colon)

HaCaT (skin)

no. of ﬁbroblasts

MCF-7

MDA-MB-231

MDA-MB-468

1

2

3

4

5

6

7

8

3a

2

3

>25

4m

13a

13b

13c

13e

13f

3

4

5

2.5

1

3

0.5

2

1

0.25

0.2

0.5

0.8

0.1

0.6

2

0.5

1

0.25

2

b

PI103

5

2

a

b

c

IC₅₀values are presented as the mean of a triplicate experiment. We used PI103 as the reference compound.³⁷Only 13b and 13c were tested on

three breast cancer cell lines.

Figure 5. Eﬀects of 13b and 13c on the AKT and mTORC1 activity in breast cancer cells by Western blotting.

Figure 6. Eﬀects of the best inhibitors 13b and 13c on the growth of breast cancer cell lines: left, distribution in the cell cycle; right, rate of apoptosis.

The best cellular eﬀects were measured with the dual

inhibitors 13b and 13c showing IC₅₀values around 50 nM on

PI3K and close to nanomolar IC₅₀values on mTOR (Table 8,

entries 4 and 5). For these two compounds, the micromolar

barrier is reached. For 13a, 13e, and 13f, the partial decrease of

PI3K or mTOR inhibition as compared to 13b and 13c

resulted in a cell survival enhancement.

Since the PI3K pathway is frequently activated in human

breast cancers and activation is associated with a poor prognosis

and resistance to treatment,⁴⁸the most cytotoxic derivatives

13b and 13c were further tested on three additional breast

cancer cell lines diﬀering in their level of PI3K pathway

activation (Table 7). A high activity of the best inhibitors 13b

and 13c was observed on all cell lines, as can be seen by the

IC₅₀values. The IC₅₀values of 13c were 200 and 250 nM for

MCF-7 and MDA-MB-231, respectively. The best inhibition

(IC₅₀= 100 nM) was obtained with 13c on MDA-MB-468, the

cell line harboring a PTEN mutation and thus an overactivation

of the PI3K pathway.

To ensure that 13b and 13c do inhibit PI3Kα and mTOR

inside the cells, we checked by Western blotting the activity of

the two major kinases of the pathway, Akt and mTORC1 (one

of the two protein complexes containing mTOR), after

treatment with inhibitors at the IC₅₀level (Figure 5).

mTORC1 activity was evaluated by quantifying the

phosphorylation level of its target, S6K1, in the MCF-7 and

MDA-MB-231 cell lines. mTORC1 was well inhibited in the

two cell lines with both inhibitors; the best reduction was

observed with 13b, which is the most active compound against

mTOR.

Akt activity was monitored by measuring its phosphorylation

level on Thr308 and Ser473 in MDA-MB-468, the cell line

expressing high levels of activated Akt. Both inhibitors reduced

p-AKT Ser473 (the mTORC2 target, the other protein

complex containing mTOR), while P-Akt Thr308 (the PDK1

target, a downstream kinase, in the PI3K pathway) was slightly

reduced by 13b only. These results are in accordance with the

results of enzymatic assays which show a more potent eﬃciency

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scale), and all coupling constant (J) values are in hertz. The following

abbreviations were used to explain the multiplicities: s (singlet), d

(doublet), t (triplet), q (quartet), m (multiplet), and dd (doublet of

doublets). Melting points are uncorrected. IR absorption spectra were

obtained on a Perkin-Elmer PARAGON 1000 PC, and the values are

reported in inverse centimeters. HRMS spectra were recorded on a

Bruker maXis mass spectrometer. Monitoring of the reactions was

performed using silica gel TLC plates (silica Merck 60 F₂₅₄). Spots

were visualized by UV light at 254 and 356 nm. Column

chromatography was performed using silica gel 60 (0.063−0.200

mm, Merck). Microwave irradiation was carried out in sealed 2−5 mL

vessels placed in a Biotage Initiator system using a standard

absorbance level (300 W maximum power). The temperatures were

measured externally by an IR probe that determined the temperature

on the surface of the vial and could be read directly from the

instrument screen. The reaction time was measured from when the

reaction mixture reached the stated temperature for temperature-

controlled experiments. Pressure was measured by a noninvasive

sensor integrated into the cavity lid.

The chromatographic purities of the ﬁnal compounds were

determined using an Agilent Technology 1260 Inﬁnity HPLC system

using a C18 column (Agilent ZORBAX Eclipse Plus, 3.5 μm, 4.6 mm

× 100 mm) operating at 30 °C. Elution was carried out using

acetonitrile containing 0.1% formic acid as mobile phase A and water

containing 0.1% formic acid as mobile phase B. Elution conditions: at

0 min, 10% phase A + 90% phase B; at 5 min, 50% phase A + 50%

phase B; at 7 min, 50% phase A + 50% phase B; at 7.1 min, 10% phase

A + 90% phase B; at 20 min, 10% phase A + 90% phase B. The ﬂow

rate of the mobile phase was 0.9 mL/min, and the injection volume of

the sample was 2 μL. Peaks were detected at 254 and 300 nm. The

purities of all the tested compounds were found to be >95% unless

otherwise stated.

for 13b as compared to 13c to inhibit PI3Kα and mTOR. A

discrepancy was observed with proliferation assay results since

13c appeared to be the most eﬃcient to reduce cell

proliferation. This could be explained by the poor sensitivity

of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-

lium bromide) assay.

As cytotoxicity may result from inhibition of proliferation

and/or an increase in apoptosis, we studied the eﬀects of 13b

and 13c on the distribution in the cell cycle and on apoptosis of

the three breast cancer cell lines by ﬂow cytometry.

The G2/M-phase was not aﬀected. In MCF-7 and MDA-

MB-468 cells, the S-phase was reduced while the G1-phase was

increased compared to the controls, implying that both

inhibitors blocked the cell cycle at the G1-phase (Figure 6).

Although the treatments changed the distribution of MDA-MB-

231 cells in the same way as they did for MCF-7 and MDA-

MB-468 cells, the changes were not statistically signiﬁcant. This

diﬀerential eﬀect on the cell lines may be related to the level of

PI3K pathway activation. Inhibitors are more eﬃcient in cells

harboring high levels of their target. Apoptosis was induced in

MCF-7 by 13b and 13c inhibitors as seen in Figure 6. It was

also increased in MDA-MB-231 and MDA-MB-468 but not

signiﬁcantly (data not shown).

In summary, the ureas 13b and 13c are the best candidates to

reduce proliferation of all the cancer cell lines tested. In breast

cancer cells, the inhibition of their targets PI3Kα and mTOR is

associated with an arrest of cells in the G1-phase along with an

induction of apoptosis.

General Procedure A for Amination at C-4 of the

Pyridopyrimidine by S_NAr. A solution of 1 or 10 (200 mg, 1.0

mmol, 1.0 equiv) in dry THF (20 mL) was cooled to 0 °C under an

inert atmosphere. A solution of amine (1.05 equiv) and triethylamine

(0.14 mL, 1.05 mmol, 1.05 equiv) in dry THF (5 mL) was added

slowly. The mixture was stirred at room temperature for 12 h. The

solvent was removed under reduced pressure. The crude product was

dissolved in CH₂Cl₂(30 mL), and the organic layer was washed with a

saturated solution of NaHCO₃(2 × 10 mL). The organic layer was

dried over MgSO₄and ﬁltered. The solvent was removed under

reduced pressure. The crude product was puriﬁed by ﬂash

chromatography.

General Procedure B for the Suzuki Cross-Coupling

Reaction. To a solution of 2 or 11 (1.0 equiv) in 1,2-dimethoxy-

ethane was added boronic acid or potassium aryltriﬂuoroborate (1.5

equiv). An aqueous solution (1 M) of potassium carbonate (3.0 equiv)

was then injected, and the mixture was degassed by argon bubbling for

15 min. Pd(PPh₃)₄(0.05 equiv) was added, and the mixture was

heated to 150 °C for 1 h under microwave irradiation. After cooling,

the solvent was removed in vacuo. The crude product was puriﬁed by

ﬂash chromatography.

General Procedure C for the Reduction of Nitro Derivatives.

To a solution of the nitro derivative (1 equiv) in ethanol was added

tin(II) chloride dihydrate (6.0 equiv). The mixture was reﬂuxed for 12

h. The solvent was removed under reduced pressure followed by the

addition of a solution of NaOH (1 M, 100 mL). The crude material

was extracted with EtOAc (3 × 60 mL), and the organic layers were

washed with water (20 mL). The combined organic layers were dried

over MgSO₄and ﬁltered. The solvent was removed under reduced

pressure. The crude product was obtained without puriﬁcation by ﬂash

chromatography.

General Procedure D for the Synthesis of Urea Derivatives.

A solution of triphosgene (65 mg, 0.22 mmol, 1.0 equiv) in dry THF

(10 mL) was cooled to −78 °C. A solution of 4h or 4i (3.0 equiv) and

N,N-diisopropylethylamine (DIPEA; 0.17 mL, 1.04 mmol, 4.8 equiv)

in dry THF (5 mL) was added dropwise. The mixture was stirred at

−78 °C for 5 min and then warmed to room temperature for 40 min.

A solution of amine (3.0 equiv) and DIPEA (85 μL, 0.52 mmol, 2.4

CONCLUSION

■

A new class of 2,4-disubsituted pyrido[3,2-d]pyrimidines and

2,4-disubsituted pyrido[2,3-d]pyrimidines were prepared fol-

lowing a two-step synthesis involving an S_NAr and a Suzuki-

type cross-coupling reaction from our original 2,4-dichloropyr-

idopyrimidine precursor. Biological activities were measured for

each novel compound on three PI3K isoforms. The best

candidates were additionally evaluated on mTOR to identify

their potential activity as dual PI3K/mTOR inhibitors. The new

library reported in this paper led to the discovery of seven

promising dual PI3Kα/mTOR inhibitors which exhibited in

vitro IC₅₀values of less than 100 nM on both kinases.

Structure−activity relationships are fully depicted, and molec-

ular modeling studies explain the interaction of active

compounds in the PI3Kα ATP binding site. In cellulo studies

demonstrated that in vitro PI3K and mTOR inhibitions are

related to cancer cell survival; a strong cytotoxic eﬀect was

observed. Indeed, the two urea derivatives which are the best

dual PI3K and mTOR inhibitors speciﬁcally aﬀect the survival

and proliferation of cancer cell lines below the micromolar

level. Further tests of the two lead compounds on breast cancer

cell lines demonstrated that they do act on their two targets

(PI3K and mTOR) inside the cells and reduce their

proliferation by blocking them in the G1-phase of their cell

cycle and by inducing apoptosis. Having in hand two lead

compounds which fully appear as pro-apoptotic drugs, further

eﬀorts are in progress to enhance their therapeutic eﬃcacy as

anticancer agents. A particular focus will be made on

bioavailability evaluation and ADME-tox studies.

EXPERIMENTAL PART

■

General Information. ¹H NMR and ¹³C NMR spectra were

recorded on a Bruker DPX 250 or 400 MHz instrument using CDCl₃

or DMSO-d₆. The chemical shifts are reported in parts per million (δ

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equiv) in dry THF (5 mL) was added dropwise. The solution was

stirred at room temperature for 24 h. The reaction was stopped with a

saturated solution of NaHCO₃(10 mL). The crude material was

extracted with EtOAc (3 × 50 mL). The combined organic layers were

dried over MgSO₄and ﬁltered. The solvent was removed under

reduced pressure to aﬀord the ﬁnal product.

General Procedure E for the Cleavage of the MOM

Protecting Group. Into a solution of MOM-protected compound

(1.0 equiv) in dioxane was injected a solution of HCl in dioxane, 4.0 M

(6.0 equiv). The mixture was stirred at room temperature until

completion monitored by TLC. The solvent was then removed by

ﬁltration and the product washed with diethyl ether.

2-Chloro-4-morpholinopyrido[3,2-d]pyrimidine (2a). The

reaction was carried out as described for general procedure A using

1 and morpholine. Puriﬁcation by ﬂash chromatography on silica gel

(CH₂Cl₂/MeOH, 98/2) yielded 2a as a white solid (223 mg, 89%).

Mp: 174−176 °C. ¹H NMR (400 MHz, CDCl₃): δ 3.87 (t, J = 5.0 Hz,

4H), 4.57 (br s, 4H), 7.59 (dd, J = 4.2 Hz, J = 8.5 Hz, 1H) 8.02 (dd, J

= 1.7 Hz, J = 8.5 Hz, 1H), 8.67 (dd, J = 1.7 Hz, J = 4.2 Hz, 1H).

HRMS (EI/MS): m/z calcd for C₁₁H₁₁ClN₄O [M + H]⁺251.0621,

found 251.0700.

Puriﬁcation by ﬂash chromatography on silica gel (petroleum ether/

EtOAc, 15/85) yielded 2g as a white solid (243 mg, 87%). Mp: 158−

1

160 °C. H NMR (400 MHz, CDCl₃): δ 1.28 (s, 3H), 1.31 (s, 3H),

2.88 (m, 2H), 3.78 (dqd, J = 2.4 Hz, J = 6.2 Hz, J = 12.5 Hz, 2H), 5.35

(s, 1H), 6.19 (s, 1H), 7.59 (dd, J = 4.2 Hz, J = 8.5 Hz, 1H), 8.02 (dd, J

= 4.2 Hz, J = 8.5 Hz, 1H), 8.69 (dd, J = 1.7 Hz, J = 4.2 Hz, 1H).

HRMS (EI/MS): m/z calcd for C₁₃H₁₅ClN₄O [M + H]⁺279.0934,

found 279.1008.

2-(3-Hydroxyphenyl)-4-morpholinopyrido[3,2-d]pyrimidine

(3a). The reaction was carried out as described for general procedure

B using 2a (200 mg, 0.798 mmol) and 3-hydroxyphenylboronic acid

(132 mg, 0.958 mmol). Puriﬁcation by ﬂash chromatography on silica

gel (petroleum ether/EtOAc, 8/2) yielded 3a as a white solid (173 mg,

76%). Mp: 236−238 °C. ¹H NMR (400 MHz, (CD₃)₂SO): δ 3.81 (m,

4H), 4.51 (br s, 4H), 6.89 (m, 1H), 7.29 (dd, J = 4.0 Hz, J = 8.3 Hz,

1H), 7.80 (m, 1H), 7.89 (m, 2H), 8.17 (dd, J = 1.5 Hz, J = 8.3 Hz,

1H), 8.77 (d, J = 7.8 Hz, 1H), 9.54 (m, 1H). HRMS (EI/MS): m/z

calcd for C₁₇H₁₆N₄O₂[M + H]⁺309.1273, found 309.1364. Purity:

>96% by HPLC, t_R= 6.47 min.

2-(3-Hydroxyphenyl)-4-thiomorpholinopyrido[3,2-d]-

pyrimidine (3b). The reaction was carried out as described for

general procedure B using 2b (180 mg, 0.675 mmol) and 3-

hydroxyphenylboronic acid (124 mg, 0.810 mmol). Puriﬁcation by

ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 96/4) yielded 3b

2-Chloro-4-thiomorpholinopyrido[3,2-d]pyrimidine (2b).

The reaction was carried out as described for general procedure A

using 1 and thiomorpholine. Puriﬁcation by ﬂash chromatography on

silica gel (petroleum ether/EtOAc, 9/1) yielded 2b as a yellow solid

1

as a white solid (167 mg, 76%). Mp: 238−240 °C. H NMR (400

1

MHz, (CD₃)₂SO): δ 2.85 (br s, 4H), 4.75 (br s, 4H), 6.90 (m, 1H),

7.28 (t, J = 7.5 Hz, 1H), 7.78 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H), 7.88

(m, 2H), 8.17 (dd, J = 2.5 Hz, J = 10.0 Hz, 1H), 8.76 (dd, J = 2.5 Hz, J

= 5.0 Hz, 1H), 9.55 (s, 1H). HRMS (EI/MS): m/z calcd for

C₁₇H₁₆N₄OS [M + H]⁺325.1045, found 325.1113. Purity: >97% by

HPLC, t_R= 7.75 min.

(201 mg, 75%). Mp: 166−168 °C. NMR H (400 MHz, CDCl₃): δ

2.80 (t, J = 8.0 Hz, 4H), 4.71 (br s, 4H), 7.54 (dd, J = 4.1 Hz, J = 8.5

Hz, 1H), 8.00 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H), 8.63 (dd, J = 1.7 Hz, J

= 4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₁H₁₁ClN₄S [M +

H]⁺267.0393, found 267.0469.

2-Chloro-4-(1,1-dioxothiomorpholino)pyrido[3,2-d]-

pyrimidine (2c). The reaction was carried out as described for

general procedure A using 1 and thiomorpholine 1,1-dioxide.

Puriﬁcation by ﬂash chromatography on silica gel (CH₂Cl₂/MeOH,

2-(3-Hydroxyphenyl)-4-(1,1-dioxothiomorpholino)pyrido-

[3,2-d]pyrimidine (3c). The reaction was carried out as described for

general procedure B using 2c (160 mg, 0.536 mmol) and 3-

hydroxyphenylboronic acid (89 mg, 0.643 mmol). Puriﬁcation by ﬂash

chromatography on silica gel (CH₂Cl₂/MeOH, 98/2) yielded 3c as a

1

98/2) yielded 2c as a white solid (174 mg, 58%). Mp: 250 °C. H

NMR (400 MHz, (CD₃)₂SO): δ 3.40 (s, 4H), 4.82 (br s, 4H), 7.87

(dd, J = 4.2 Hz, J = 8.5 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.84 (d, J =

4.2 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₁H₁₁ClN₄O₂S [M +

H]⁺299.0291, found 299.0372.

1

white solid (162 mg, 85%). Mp: 219−221 °C. H NMR (400 MHz,

(CD₃)₂SO): δ 3.41 (br s, 4H), 4.90 (br s, 4H), 6.90 (d, J = 8.0 Hz,

1H), 7.31 (t, J = 8.0 Hz, 1H), 7.86 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H),

7.91 (m, 2H), 8.22 (dd, J = 1.6 Hz, J = 8.5 Hz, 1H), 8.81 (dd, J = 1.6

Hz, J = 4.1 Hz, 1H), 9.58 (s, 1H). HRMS (EI/MS): m/z calcd for

C₁₇H₁₆N₄O₃S [M + H]⁺357.0943, found 357.1018. Purity: >97% by

HPLC, t_R= 7.37 min.

2-Chloro-4-(4-methylpiperazin-1-yl)pyrido[3,2-d]pyrimidine

(2d). The reaction was carried out as described for general procedure

A using 1 and 1-methylpiperazine. Puriﬁcation by ﬂash chromatog-

raphy on silica gel (CH₂Cl₂/MeOH, 99/1) yielded 2d as a yellow solid

1

(224 mg, 85%). Mp: 111−113 °C. H NMR (400 MHz, CDCl₃): δ

2-(3-Hydroxyphenyl)-4-(4-methylpiperazin-1-yl)pyrido[3,2-

d]pyrimidine (3d). The reaction was carried out as described for

general procedure B using 2d (200 mg, 0.758 mmol) and 3-

hydroxyphenylboronic acid (126 mg, 0.910 mmol). Puriﬁcation by

ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 99/1) yielded 3d

2.35 (s, 3H), 2.59 (br s, 4H), 4.55 (br s, 4H), 7.58 (dd, J = 4.1 Hz, J =

8.5 Hz, 1H), 8.00 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H), 8.67 (dd, J = 1.7

Hz, J = 4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₂H₁₄ClN₅[M

+ H]⁺264.0938, found 264.1021.

1

as a yellow solid (211 mg, 87%). Mp: 174−176 °C. H NMR (400

2-Chloro-4-(4,4-diﬂuoropiperidin-1-yl)pyrido[3,2-d]-

pyrimidine (2e). The reaction was carried out as described for

general procedure A using 1 and 4,4-diﬂuoropiperidine. Puriﬁcation by

ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 99/1) yielded 2e

MHz, CDCl₃): δ 2.60 (s, 3H), 2.96 (br s, 4H), 4.90 (br s, 4H), 7.15

(d, J = 8.0 Hz, 1H), 7.35 (br, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.79 (dd, J

= 4.1 Hz, J = 8.5 Hz, 1H), 8.13 (m, 1H), 8.24 (dd, J = 1.6 Hz, J = 8.5

Hz, 1H), 8.34 (dd, J = 1.6 Hz, J = 4.1 Hz, 1H), 8.88 (m, 1H). HRMS

(EI/MS): m/z calcd for C₁₈H₁₉N₅O [M + H]⁺322.1590, found

322.1682 Purity: >96% by HPLC, t_R= 4.82 min.

1

as a yellow solid (228 mg, 80%). Mp: 136−138 °C. H NMR (400

MHz, CDCl₃): δ 2.18 (m, 4H), 4.62 (br s, 4H), 7.63 (dd, J = 4.1 Hz, J

= 8.5 Hz, 1H), 8.05 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H), 8.71 (dd, J = 1.7

Hz, J = 4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₂H₁₁ClF₂N₄

[M + H]⁺285.0640, found 285.0725.

4-(4,4-Diﬂuoropiperidin-1-yl)-2-(3-hydroxyphenyl)pyrido-

[3,2-d]pyrimidine (3e). The reaction was carried out as described for

general procedure B using 2e (200 mg, 0.703 mmol) and 3-

hydroxyphenylboronic acid (116 mg, 0.844 mmol). Puriﬁcation by

ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 99/1) yielded 3e

2-Chloro-4-((3-pyridylmethyl)amino)pyrido[3,2-d]-

pyrimidine (2f). The reaction was carried out as described for general

procedure A using 1 and 3-picolylamine. Puriﬁcation by ﬂash

chromatography on silica gel (CH₂Cl₂/MeOH, 99/1) yielded 2f as a

white solid (0.152 mg, 61%). Mp: 154−156 °C. ¹H NMR (400 MHz,

(CD₃)₂SO): δ 4.73 (d, J = 6.4 Hz, 2H), 7.35 (dd, J = 7.8 Hz, J = 4.8

Hz, 1H), 7.87 (m, 2H), 8.07 (dd, J = 8.5 Hz, J = 1.4 Hz, 1H), 8.49 (m,

1H), 8.62 (s, 1H), 8.82 (dd, J = 4.2 Hz, J = 1.3 Hz, 1H), 9.67 (t, J = 6.3

Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₃H₁₅ClN₄O [M + H]⁺

272.0621, found 279.0636.

1

as a yellow solid (147 mg, 61%). Mp: 175 °C. H NMR (400 MHz,

CDCl₃): δ 2.20 (m, 4H), 4.65 (s, 4H), 5.91 (br s, OH), 6.96 (d, J = 7.8

Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.62 (dd, J = 4.1 Hz, J = 8.5 Hz,

1H), 7.99 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 8.21 (dd, J = 2.6 Hz, J =

8.5 Hz, 1H), 8.70 (dd, J = 2.6 Hz, J = 4.1 Hz, 1H). HRMS (EI/MS):

m/z calcd for C₁₈H₁₆F₂N₄O [M + H]⁺343.1292, found 343.1364.

Purity: >96% by HPLC, t_R= 8.47 min.

2-Chloro-4-((2R,6R)-2,6-dimethylmorpholino)pyrido[3,2-d]-

pyrimidine (2g). The reaction was carried out as described for

general procedure A using 1 and (2R,6R)-2,6-dimethylmorpholine.

4-((2R,6R)-2,6-Dimethylmorpholino)-2-(3-hydroxyphenyl)-

pyrido[3,2-d]pyrimidine (3g). The reaction was carried out as

described for general procedure B using 2g (200 mg, 0.718 mmol) and

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3-hydroxyphenylboronic acid (119 mg, 0.861 mmol). Puriﬁcation by

ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 98/2) yielded 3g

as a white solid (205 mg, 85%). Mp: 219 °C. H NMR (400 MHz,

phenylboronic acid (146 mg, 0.958 mmol). Puriﬁcation by ﬂash

chromatography on silica gel (petroleum ether/EtOAc, 8/2) yielded 4f

as a white solid (183 mg, 71%). Mp: 221−223 °C. H NMR (400

1

MHz, CDCl₃): δ 3.93 (m, 4H), 4.60 (br s, 4H), 4.77 (s, 2H), 7.46 (d, J

= 8.1 Hz, 2H), 7.60 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H), 8.18 (dd, J = 1.7

Hz, J = 8.5 Hz, 1H), 8.47 (d, J = 8.2 Hz, 2H), 8.67 (dd, J = 1.7 Hz, J =

4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₈H₁₈N₄O₂[M + H]⁺

323.1430, found 323.1512. Purity: >99% by HPLC, t_R= 6.10 min.

2-(3-Aminophenyl)-4-morpholinopyrido[3,2-d]pyrimidine

(4g). The reaction was carried out as described for general procedure

B using 2a (200 mg, 0.798 mmol, 1.0 equiv) and 3-nitrophenylboronic

acid (161 mg, 0.958 mmol, 1.2 equiv). Puriﬁcation by ﬂash

chromatography on silica gel (petroleum ether/EtOAc, 85/15) yielded

the nitro derivative as a yellow solid (191 mg, 71%). Mp: 193−195 °C.

¹H NMR (400 MHz, (CD₃)₂SO): δ 3.84 (m, 4H), 4.56 (br s, 4H),

7.83 (m, 2H), 8.27 (dd, J = 1.4 Hz, J = 8.5 Hz, 1H), 8.36 (dd, J = 4.0

Hz, J = 8.5 Hz, 1H), 8.81 (dd, J = 1.4 Hz, J = 4.0 Hz, 1H), 8.85 (d, J =

7.8 Hz, 1H), 9.16 (s, 1H). HRMS (EI/MS): m/z calcd for

C₁₇H₁₅N₅O₃[M + H]⁺338.1175, found 338.1250. Then the reduction

of the nitro group was carried out as described for general procedure C

using the nitro derivative (180 mg, 0.534 mmol, 1.0 equiv). The crude

product was obtained without puriﬁcation by ﬂash chromatography to

aﬀord 4g as a white solid (131 mg, 80%). Mp: 182−184 °C. ¹H NMR

(400 MHz, (CD₃)₂SO): δ 3.82 (m, 4H), 4.51 (br s, 4H), 5.45 (br,

2H), 6.70 (dd, J = 1.4 Hz, J = 7.8 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H),

7.63 (d, J = 7.7 Hz, 1H), 7.72 (s, 1H), 7.77 (dd, J = 4.1 Hz, J = 8.5 Hz,

1H), 8.15 (dd, J = 1.4 Hz, J = 8.5 Hz, 1H), 8.74 (dd, J = 1.4 Hz, J = 4.0

Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₇H₁₇N₅O [M + H]⁺

308.1433, found 308.1507. Purity: >99% by HPLC, t_R= 5.97 min.

2-(4-Aminophenyl)-4-morpholinopyrido[3,2-d]pyrimidine

4h. The reaction was carried out as described for general procedure B

using 2a (200 mg, 0.798 mmol, 1.0 equiv) and 4-nitrophenylboronic

acid (161 mg, 0.958 mmol, 1.2 equiv). Puriﬁcation by ﬂash

chromatography on silica gel (CH₂Cl₂/petroleum ether, 8/2) yielded

the nitro derivative as a yellow solid (234 mg, 87%). Mp: 211−213 °C.

¹H NMR (400 MHz, CDCl₃): δ 3.82 (m, 4H), 4.63 (br s, 4H), 7.66

CDCl₃): δ 1.28 (s, 3H), 1.30 (s, 3H), 2.91 (dd, J = 10.9 Hz, J = 12.9

Hz, 2H), 3.83 (m, 2H), 5.70 (m, 2H), 6.35 (m, 1H), 6.90 (m, 1H),

7.27 (t, J = 7.9 Hz, 1H), 7.51 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H), 7.93 (d,

J = 7.8 Hz, 1H), 7.98 (s, 1H), 8.17 (dd, J = 1.6 Hz, J = 8.5 Hz, 1H),

8.63 (dd, J = 1.6 Hz, J = 4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for

C₁₉H₂₀N₄O₂[M + H]⁺337.1586, found 337.1661. Purity: >96% by

HPLC, t_R= 7.50 min.

2-(2-Hydroxyphenyl)-4-morpholinopyrido[3,2-d]pyrimidine

(4a). The reaction was carried out as described for general procedure

B using 2a (200 mg, 0.798 mmol) and 2-hydroxyphenylboronic acid

(132 mg, 0.958 mmol). Puriﬁcation by ﬂash chromatography on silica

gel (petroleum ether/EtOAc, 7/3) yielded 4a as a yellow solid (215

mg, 87%). Mp: 173−175 °C. ¹H NMR (400 MHz, CDCl₃): δ 3.94 (t,

J = 5.0 Hz, 4H), 4.63 (br s, 4H), 6.90 (dd, J = 7.2 Hz, J = 8.0 Hz, 1H),

7.02 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 7.2 Hz, 1H), 7.61 (dd, J = 4.0 Hz,

J = 4.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.41 (dd, J = 2.5 Hz, J = 10.0

Hz, 1H), 8.66 (dd, J = 2.5 Hz, J = 5.0 Hz, 1H), 14.3 (s, 1H). HRMS

(EI/MS): m/z calcd for C₁₇H₁₆N₄O₂[M + H]⁺309.1273, found

309.1363. Purity: >98% by HPLC, t_R= 11.84 min.

2-(4-Hydroxyphenyl)-4-morpholinopyrido[3,2-d]pyrimidine

(4b). The reaction was carried out as described for general procedure

B using 2a (200 mg, 0.798 mmol) and 4-hydroxyphenylboronic acid

(132 mg, 0.958 mmol). Puriﬁcation by ﬂash chromatography on silica

gel (petroleum ether/EtOAc, 7/3) yielded 4b as a white solid (182

mg, 74%). Mp: 252−254 °C. ¹H NMR (400 MHz, (CD₃)₂SO): δ 3.81

(t, J = 5.2 Hz, 4H), 4.48 (br s, 4H), 6.80 (m, 2H), 7.74 (m, 1H), 8.25

(m, 1H), 8.28 (m, 2H), 8.70 (m, 1H). HRMS (EI/MS): m/z calcd for

C₁₇H₁₆N₄O₂[M + H]⁺309.1273, found 309.1352. Purity: >98% by

HPLC, t_R= 6.25 min.

2-(3-Methoxyphenyl)-4-morpholinopyrido[3,2-d]pyrimidine

(4c). The reaction was carried out as described for general procedure B

using 2a (200 mg, 0.798 mmol) and 3-methoxyphenylboronic acid

(146 mg, 0.957 mmol). Puriﬁcation by ﬂash chromatography on silica

gel (petroleum ether/EtOAc, 2/8) yielded 4c as a white solid (219 mg,

85%). Mp: 118−120 °C. ¹H NMR (400 MHz, CDCl₃): δ 3.93 (s, 4H),

3.95 (s, 3H), 4.60 (br s, 4H), 7.03 (dd, J = 2.6 Hz, J = 8.1 Hz, 1H),

7.40 (tJ = 7.9 Hz,, 1H), 7.61 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H), 8.08 (dd,

J = 5.1 Hz, J = 13.3 Hz, 2H), 8.19 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H),

8.68 (dd, J = 1.7 Hz, J = 4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for

C₁₈H₁₈N₄O₂[M + H]⁺323.1616, found 323.1504. Purity: >97% by

HPLC, t_R= 7.97 min.

2-(4-Methoxyphenyl)-4-morpholinopyrido[3,2-d]pyrimidine

(4d). The reaction was carried out as described for general procedure

B using 2a (200 mg, 0.798 mmol) and 4-methoxyphenylboronic acid

(146 mg, 0.958 mmol). Puriﬁcation by ﬂash chromatography on silica

gel (petroleum ether/EtOAc, 7/3) yielded 4d as a white solid (196

mg, 76%). Mp: 178−180 °C¹H NMR (400 MHz, CDCl₃): δ 3.88 (s,

3H), 3.92 (t, J = 5.0 Hz, 4H), 4.58 (m, 4H), 6.99 (d, J = 9.0 Hz, 2H),

7.58 (dd, J = 4.2 Hz, J = 8.5 Hz, 1H), 8.14 (dd, J = 1.7 Hz, J = 8.5 Hz,

1H), 8.45 (d, J = 9.0 Hz, 2H), 8.63 (dd, J = 1.7 Hz, J = 4.2 Hz, 1H).

HRMS (EI/MS): m/z calcd for C₁₈H₁₈N₄O₂[M + H]⁺323.1616,

found 323.1504. Purity: >97% by HPLC, t_R= 7.13 min.

2-(3-(Hydroxymethyl)phenyl)-4-morpholinopyrido[3,2-d]-

pyrimidine (4e). The reaction was carried out as described for

general procedure B using 2a (200 mg, 0.798 mmol) and 3-

(hydroxymethyl)phenylboronic acid (146 mg, 0.958 mmol). Puriﬁca-

tion by ﬂash chromatography on silica gel (petroleum ether/EtOAc,

8/2) yielded 4e as a yellow solid (226 mg, 88%). Mp: 119−121 °C. ¹H

NMR (400 MHz, CDCl₃): δ 3.92 (t, J = 6.0 Hz, 4H), 4.59 (br s, 4H),

4.78 (s, 2H), 7.45 (m, 2H), 7.60 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 7.65

(m, 1H), 8.17 (m, 1H), 8.38 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 8.45 (m,

1H), 8.66 (dd, J = 2.0 Hz, J = 4.0 Hz, 1H). HRMS (EI/MS): m/z

calcd for C₁₈H₁₈N₄O₂[M + H]⁺323.1430, found 323.1498. Purity:

>97% by HPLC, t_R= 6.17 min.

(dd, J = 4.1 Hz, J = 8.5 Hz, 1H), 8.20 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H),

8.32 (d, J = 8.9 Hz, 2H), 8.66 (d, J = 8.0 Hz, 2H), 8.73 (dd, J = 1.7 Hz,

J = 4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₇H₁₅N₅O₃[M +

H]⁺338.1175, found 338.1257. Then the reduction of the nitro group

was carried out as described for general procedure C using the nitro

derivative (600 mg, 1.78 mmol, 1.0 equiv) The crude product was

obtained without puriﬁcation by ﬂash chromatography to aﬀord 4h as

a white solid (443 mg, 80%). Mp: 221−223 °C. ¹H NMR (400 MHz,

CDCl₃): δ 1.62 (br, 2H), 3.93 (m, 4H), 4.56 (br s, 4H), 6.75 (d, J =

8.7 Hz, 2H), 7.56 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H), 8.12 (dd, J = 1.6

Hz, J = 8.5 Hz, 1H), 8.33 (d, J = 8.7 Hz, 2H), 8.61 (dd, J = 1.7 Hz, J =

4.1 Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₇H₁₇N₅O [M + H]⁺

308.1433, found 308.1509. Purity: >96% by HPLC, t_R= 6.29 min.

4-Morpholino-2-(3-pyridyl)pyrido[3,2-d]pyrimidine (4i). The

reaction was carried out as described for general procedure B using 2a

(200 mg, 0.798 mmol) and 3-pyridineboronic acid (118 mg, 0.958

mmol). Puriﬁcation by ﬂash chromatography on silica gel (CH₂Cl₂/

MeOH, 99/1) yielded 4i as a yellow solid (178 mg, 76%). Mp: 159−

161 °C. ¹H NMR (400 MHz, CDCl₃): δ 3.92 (t, J = 6.0 Hz, 4H), 4.63

(br s, 4H), 7.61−7.89 (m, 4H), 8.20 (dd, J = 1.75 Hz, J = 8.5 Hz, 1H),

8.31 (dd, J = 1.5 Hz, J = 6.25 Hz, 1H), 8.73 (dd, J = 1.5 Hz, J = 6.25

Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₆H₁₅N₅O [M + H]⁺

294.1277, found 294.1359. Purity: >95% by HPLC, t_R= 6.48 min.

2-(1H-Indazol-6-yl)-4-morpholinopyrido[3,2-d]pyrimidine

(4j). The reaction was carried out as described for general procedure B

using 2a (200 mg, 0.798 mmol) and potassium 1H-indazole-6-

triﬂuoroborate (155 mg, 0.958 mmol). Puriﬁcation by ﬂash

chromatography on silica gel (petroleum ether/EtOAc, 8/2) yielded

1

4j as a white solid (119 mg, 45%). Mp: 236−238 °C. H NMR (400

MHz, CDCl₃): δ 3.97 (s, 4H), 4.56 (br s, 4H), 7.55 (d, J = 8.1 Hz,

2H), 7.53 (m, 1H), 8.20 (d, J = 8.8 Hz, 2H), 8.62 (d, J = 8.1 Hz, 2H),

8.67 (m, 1H), 8.97 (s, 1H), 10.31 (bd, 1H). HRMS (EI/MS): m/z

calcd for C₁₈H₁₆N₆O [M + H]⁺333.1386, found 333.1451. Purity:

>98% by HPLC, t_R= 6.26 min.

2-(4-(Hydroxymethyl)phenyl)-4-morpholinopyrido[3,2-d]-

pyrimidine (4f). The reaction was carried out as described for general

procedure B using 2a (200 mg, 0.798 mmol) and 4-(hydroxymethyl)-

625

dx.doi.org/10.1021/jm401138v | J. Med. Chem. 2014, 57, 613−631

Journal of Medicinal Chemistry

Article

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2-(1H-Indazol-4-yl)-4-morpholinopyrido[3,2-d]pyrimidine

(4k). The reaction was carried out as described for general procedure

B using 2a (200 mg, 0.798 mmol) and potassium 1H-indazole-4-

triﬂuoroborate (191 mg, 0.957 mmol). Puriﬁcation by ﬂash

chromatography on silica gel (CH₂Cl₂/MeOH, 98/2) yielded 4k as

a white solid (129 mg, 49%). Mp: 212−214 °C. ¹H NMR (400 MHz,

(CD₃)₂SO): δ 3.77−3.91 (m, 4H), 4.54 (br s, 4H), 7.50 (t, J = 7.8 Hz,

1H), 7.71 (d, J = 8.2 Hz, 1H), 7.84 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H),

8.29 (d, J = 7.2 Hz, 1H), 8.35 (dd, J = 1.6 Hz, J = 8.5 Hz, 1H), 8.80

(dd, J = 1.6 Hz, J = 4.1 Hz, 1H), 9.01 (s, 1H), 13.22 (s, 1H). HRMS

(EI/MS): m/z calcd for C₁₈H₁₆N₆O [M + H]⁺333.1458, found

333.1460. Purity: >99% by HPLC, t_R= 6.80 min.

189−191 °C. H NMR (400 MHz, CDCl₃): δ 3.56 (s, 3H), 5.28 (s,

2H), 6.98 (dd, J = 1.7 Hz, J = 7.2 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H),

7.56 (dd, J = 4.2 Hz, J = 8.7 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.96

(dd, J = 1.6 Hz, J = 8.7 Hz, 1H), 8.63 (dd, J = 1.6 Hz, J = 4.2 Hz, 1H).

HRMS (EI-MS): m/z calcd for C₁₅H₁₂ClN₃O₂[M + H]⁺302.1352,

found 302.1358.

4-(3-Hydroxyphenyl)-2-morpholinopyrido[3,2-d]pyrimidine

(9). To a solution of 7 (150 mg, 0.497 mmol, 1.0 equiv) in dry THF

were added successively morpholine (70 μL, 0.746 mmol, 1.5 equiv)

and triethylamine (103 μL, 0.746 mmol, 1.5 equiv). The mixture was

reﬂuxed for 12 h. The solvent was removed under reduced pressure,

and the crude material was dissolved in CH₂Cl₂(30 mL). The organic

layer was washed with water (2 × 10 mL). The combined organic

layers were dried over MgSO₄. Puriﬁcation by ﬂash chromatography

on silica gel (petroleum ether/EtOAc, 8/2) yielded the protected 8 as

a yellow solid (120 mg, 68%). Direct deprotection of the

methoxymethoxy function was directly carried out as described ifor

general procedure E (using EtOH as the solvent) to aﬀord 9 as a

2-(2-Aminopyridyl)-4-morpholinopyrido[3,2-d]pyrimidine

(4l). The reaction was carried out as described for general procedure B

using 2a (200 mg, 0.798 mmol) and potassium 2-aminopyridine-5-

triﬂuoroborate (211 mg, 0.958 mmol). Puriﬁcation by ﬂash

chromatography on silica gel (CH₂Cl₂/MeOH, 96/4) yielded 4l as a

1

yellow solid (71 mg, 23%). Mp: 242−244 °C. H NMR (400 MHz,

1

CDCl₃): δ 3.92 (m, 4H), 4.61 (br s, 4H), 4.73 (br s, 2H), 6.57 (dd, J =

8.6 Hz, J = 0.8 Hz, 1H), 7.57 (dd, J = 8.5 Hz, J = 4.1 Hz, 1H), 8.10

(dd, J = 8.5 Hz, J = 1.7 Hz, 1H), 8.53 (dd, J = 8.6 Hz, J = 2.3 Hz, 1H),

8.63 (dd, J = 4.1 Hz, J = 1.7 Hz, 1H), 9.19 (dd, J = 2.3 Hz, J = 0.7 Hz,

1H) . HRMS (EI/MS): m/z calcd for C₁₆H₁₆N₆O [M + H]⁺309.1458,

found 309.1460. Purity: >97% by HPLC, t_R= 5.76 min.

yellow solid (63 mg, 60%). Mp: 162−164 °C. H NMR (400 MHz,

CDCl₃): δ 3.86 (m, 4H), 4.04 (m, 4H), 6.41 (s, 1H), 7.01 (dd, J = 1.7

Hz, J = 7.2 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H), 7.56 (dd, J = 4.1 Hz, J =

8.6 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.96 (dd, J = 1.6 Hz, J = 8.7 Hz,

1H), 8.63 (dd, J = 1.6 Hz, J = 4.1 Hz, 1H). HRMS (EI/MS): m/z

calcd for C₁₇H₁₆N₄O₂[M + H]⁺309.1352, found 309.1358. Purity:

>99% by HPLC, t_R= 8.59 min.

2-(2-Aminopyrimidinyl)-4-morpholinopyrido[3,2-d]-

pyrimidine (4m). The reaction was carried out as described for

general procedure B using 2a (200 mg, 0.798 mmol) and potassium 2-

aminopyrimidine-5- triﬂuoroborate (212 mg, 0.958 mmol). Puriﬁca-

tion by ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 96/4)

2-(3-Hydroxyphenyl)-4-morpholinopyrido[2,3-d]pyrimidine

(12a). The reaction was carried out as described for general procedure

B using 11 (160 mg, 0.638 mmol) and 3-hydroxyphenylboronic acid

(89 mg, 0.643 mmol). Puriﬁcation by ﬂash chromatography on silica

gel (CH₂Cl₂/MeOH, 99/1) yielded 12a as a white solid (118 mg,

60%). Mp: > 260 °C. ¹H NMR (250 MHz, (CD₃)₂SO): δ 3.83 (d, J =

4.5 Hz, 4H), 3.89 (d, J = 4.4 Hz, 4H), 6.93 (s, 1H), 7.31 (d, J = 8.0 Hz,

1H), 7.47 (dd, J = 4.3 Hz, J = 8.3 Hz, 1H), 7.94 (d, J = 7.6 Hz, 2H),

8.47 (dd, J = 1.7 Hz, J = 8.3 Hz, 1H), 9.01 (dd, J = 1.7 Hz, J = 4.2 Hz,

1H), 9.59 (s, 1H). HRMS (EI/MS): m/z calcd for C₁₇H₁₆N₄O₂[M +

H]⁺309.1273, found 309.1364. Purity: >97% by HPLC, t_R= 5.82 min.

2-(3-(Hydroxymethyl)phenyl)-4-morpholinopyrido[2,3-d]-

pyrimidine (12b). The reaction was carried out as described for

general procedure B using 11 (200 mg, 0.798 mmol) and 3-

(hydroxymethyl)phenylboronic acid (89 mg, 0.643 mmol). Puriﬁca-

tion by ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 98/2)

yielded 12b as a white solid (167 mg, 65%). Mp: > 260 °C. ¹H NMR

(400 MHz, (CD₃)₂SO): δ 3.87−3.78 (m, 4H), 3.97−3.87 (m, 4H),

4.62 (d, J = 3.4 Hz, 2H), 5.32 (s, 1H), 7.47 (dd, J = 3.8 Hz, J = 7.2 Hz,

3H), 8.37 (s, 1H), 8.51−8.45 (m, 2H), 9.02 (dd, J = 1.7 Hz, J = 4.2

Hz, 1H). HRMS (EI/MS): m/z calcd for C₁₈H₁₈N₄O₂[M + H]⁺

323.1508, found 323.1514. Purity: >95% by HPLC, t_R= 5.62 min.

2-(2-Aminopyrimidinyl)-4-morpholinopyrido[2,3-d]-

pyrimidine (12c). The reaction was carried out as described for

general procedure B using 11 (200 mg, 0.798 mmol) and potassium 2-

aminopyrimidine-5- triﬂuoroborate (89 mg, 0.643 mmol). Puriﬁcation

by ﬂash chromatography on silica gel (CH₂Cl₂/MeOH, 98/2) yielded

1

yielded 4m as a white solid (178 mg, 72%). Mp: 260 °C. H NMR

(400 MHz, CDCl₃): δ 3.92 (m, 4H), 4.58 (br s, 4H), 5.30 (m, 2H),

7.59 (dd, J = 4.0 Hz, J = 8.5 Hz, 1H), 8.11 (d, J = 4.0 Hz, J = 8.5 Hz,

1H), 8.65 (d, J = 4.0 Hz, 1H), 9.33 (s, 2H). HRMS (EI/MS): m/z

calcd for C₁₅H₁₅N₇O [M + H]⁺310.1338, found 310.1403. Purity:

>98% by HPLC, t_R= 5.75 min.

4-Morpholino-2-(2-(acetylamino)pyrimidinyl)pyrido[3,2-d]-

pyrimidine (5). To a solution of 4m (125 mg, 0.404 mmol, 1.0 equiv)

in dimethylformamide (DMF; 2 mL) was added acetic anhydride

(0.38 mL, 4.04 mmol, 10.0 equiv). The mixture was heated at 120 °C

for 30 min under microwave irradiation. After cooling, the crude

material was then diluted in water (30 mL) and extracted with EtOAc

(5 mL). The combined organic layers were washed with water (2 × 30

mL) and dried over MgSO₄. The solvent was removed in vacuo, and

puriﬁcation by ﬂash chromatography on silica gel (CH₂Cl₂/MeOH,

1

98/2) yielded 5 as a white solid (40 mg, 28%). Mp: 260−262 °C. H

NMR (400 MHz, CDCl₃): δ 2.56 (d, J = 4.7 Hz, 3H), 3.92 (d, J = 3.9

Hz, 4H), 4.60 (br s, 4H), 7.63 (m, 1H), 8.15 (s, 1H), 8.52 (s, 1H),

8.69 (s, 1H), 9.57 (d, J = 5.2 Hz, 2H). HRMS (EI/MS): m/z calcd for

C₁₇H₁₇N₇O₂[M + H]⁺352.1444, found 352.1505. Purity: >99% by

HPLC, t_R= 6.69 min.

4-Morpholino-2-(2-(diacetylamino)pyrimidinyl))pyrido[3,2-

d]pyrimidine 6. Compound 6 was isolated during the transformation

of 4n into 5 (vide supra) during the puriﬁcation by ﬂash

chromatography on silica gel as a white solid (70 mg, 44%). Mp:

1

12c as a white solid (170 mg, 69%). Mp: > 260 °C. H NMR (400

MHz, (CD₃)₂SO): δ 3.81 (m, 4H), 3.89 (m, 4H), 7.24 (s, 2H), 7.42

(dd, J = 4.3 Hz, J = 8.2 Hz, 1H), 8.43 (dd, J = 1.5 Hz, J = 8.2 Hz, 1H),

8.99−8.93 (dd, J = 1.5 Hz, J = 4.3 Hz, 1H), 9.20 (s, 2H). HRMS (EI/

MS): m/z calcd for C₁₅H₁₅N₇O [M + H]⁺310.1416, found 310.1423.

Purity: >99% by HPLC, t_R= 4.87 min.

2-(4-(1-Methyl-3-phenylurea))-4-morpholinopyrido[3,2-d]-

pyrimidine (13a). The reaction was carried out as described for

general procedure D using 4h (200 mg, 0.651 mmol) and

methylamine (22 μL, 0.651 mmol). The crude product was obtained

without puriﬁcation by ﬂash chromatography to aﬀord 13a as a yellow

solid (50 mg, 21%). Mp: 223−225 °C. ¹H NMR (400 MHz,

(CD₃)₂CO): δ 2.83 (br s, 3H), 3.87 (m, 4H), 4.57 (br s, 4H), 5.68 (br

s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.58 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H),

8.00 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H), 8.14 (br s, 1H), 8.30 (d, J = 8.8

Hz, 2H), 8.57 (dd, J = 1.7 Hz, J = 4.1 Hz, 1H). HRMS (EI/MS): m/z

calcd for C₁₉H₂₀N₆O₂[M + H]⁺365.1648, found 365.1717. Purity:

>96% by HPLC, t_R= 6.19 min.

1

225−227 °C. H NMR (400 MHz, CDCl₃): δ 2.34 (s, 6H), 3.93 (m,

4H), 4.64 (br s, 4H), 7.68 (dd, J = 4.1 Hz, J = 8.5 Hz, 1H), 8.20 (dd, J

= 1.5 Hz, J = 8.5 Hz, 1H), 8.75 (dd, J = 1.5 Hz, J = 4.0 Hz, 1H), 9.79

(s, 2H). HRMS (EI/MS): m/z calcd for C₁₉H₁₉N₇O₃[M + H]⁺

394.1549, found 394.1618. Purity: >98% by HPLC, t_R= 9.26 min.

2-Chloro-4-(3-(methoxymethoxy)phenyl)pyrido[3,2-d]-

pyrimidine (7). To a solution of 1 (300 mg, 1.5 mmol, 1.0 equiv) in

dry toluene were added potassium 3-(methoxymethoxy)-

phenyltriﬂuoroborate (256 mg, 1.58 mmol, 1.05 equiv), potassium

carbonate (311 mg, 2.25 mmol), and Pd(PPh₃)₄(87 mg, 0.08 mmol,

0.05 equiv). The mixture was stirred at 100 °C for 2 h. The solvent

was then removed under reduced pressure, and the crude material was

dissolved in CH₂Cl₂(30 mL). The organic layer was washed with

water (2 × 10 mL). The combined organic layers were dried over

MgSO₄. Puriﬁcation by ﬂash chromatography on silica gel (petroleum

ether/EtOAc, 8/2) yielded 7 as a yellow solid (204 mg, 45%). Mp:

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2-(4-(1-(3-(Hydroxymethyl)phenyl)-3-phenylurea))-4-

morpholinopyrido[3,2-d]pyrimidine (13b). The reaction was

carried out as described for general procedure D using 4h (100 mg,

0.325 mmol) and (3-aminophenyl)methanol (40 mg, 0.325 mmol).

The crude product was obtained without puriﬁcation by ﬂash

chromatography to aﬀord 13b as a brown solid (59 mg, 40%). Mp:

244−246 °C. ¹H NMR (400 MHz, (CD₃)₂SO): δ 3.83 (m, 4H), 4.49

(br s, 6H), 5.19 (br s, 1H), 6.94 (d, J = 6.2 Hz, 1H), 7.24 (s, 1H), 7.34

(d, J = 7.1 Hz, 1H), 7.46 (s, 1H), 7.60 (d, J = 7.8 Hz, 2H), 7.79 (s,

1H), 8.16 (d, J = 8.1 Hz, 1H), 8.39 (d, J = 7.7 Hz, 2H), 8.73 (br s,

2H), 8.92 (s, 1H). HRMS (EI/MS): m/z calcd for C₂₅H₂₄N₆O₃[M +

H]⁺457.1477, found 457.1985. Purity: >95% by HPLC, t_R= 6.98 min.

2-(4-(1-(4-(Hydroxymethyl)phenyl)-3-phenylurea))-4-

morpholinopyrido[3,2-d]pyrimidine (13c). The reaction was

carried out as described for general procedure D using 4h (65 mg,

0.211 mmol) and (4-aminophenyl)methanol (26 mg, 0.211 mmol).

The crude product was obtained without puriﬁcation by ﬂash

chromatography to aﬀord 13c as a yellow solid (85 mg, 89%). Mp:

8.25−8.10 (m, 2H), 8.38 (d, J = 8.7 Hz, 2H), 8.65 (d, J = 1.7 Hz, 1H),

8.73 (dd, J = 1.6 Hz, J = 4.0 Hz, 1H), 9.61 (s, 1H). HRMS (EI/MS):

m/z calcd for C₂₃H₂₁N₇O₂[M + H]⁺428.1757, found 428.1831.

Purity: >95% by HPLC, t_R= 5.77 min.

2-(3-(1-(3-(Hydroxymethyl)phenyl)-3-phenylurea))-4-

morpholinopyrido[3,2-d]pyrimidine (13h). The reaction was

carried out as described for general procedure D using 4g (65 mg,

0.22 mmol) and (3-aminophenyl)methanol (80 mg, 0.22 mmol). The

crude product was obtained without puriﬁcation by ﬂash chromatog-

1

raphy to aﬀord 13h as a yellow solid (48 mg, 47%). Mp: 230 °C. H

NMR (400 MHz, (CD₃)₂SO): δ 3.83 (m, 4H), 4.42 (d, J = 5.6 Hz,

2H), 4.53 (br s, 4H), 5.05 (t, J = 5.6 Hz, 1H), 7.23 (d, J = 8.5 Hz, 2H),

7.42 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.81 (dd, J = 4.1 Hz,

J = 8.5 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 8.20 (dd, J = 1.5 Hz, J = 8.5

Hz, 1H), 8.43 (s, 1H), 8.60 (s, 1H) 8.77 (m, 1H), 8.85 (s, 1H). HRMS

(EI/MS): m/z calcd for C₂₅H₂₄N₆O₃[M + H]⁺457.1910, found

457.1988. Purity: >95% by HPLC, t_R= 7.05 min.

Molecular Modeling. Hardware and Software. All molecular

modeling studies were performed with Schrodinger Molecular

Modeling Suite 2010.⁴⁹Maestro was the interface piloting the diverse

modules. Glide³⁸was used to dock ligands. Calculations were run on a

Linux station: Intel Core i7 CPU 950 at 3.07 GHz.

Structure Preparation. The three structures of PI3Kα apo or in

complex with wortmannin were retrieved from the Protein Data

Bank.³³Subunit A of each structure, corresponding to the catalytic

subunit, was conserved. Structures were aligned and superimposed on

their backbone using the Protein Structure Alignment tool of

Schrodinger Molecular Modeling Suite 2010. Bond orders and

connections of wortmannin were manually corrected, and the ligand

was transposed in the two apo structures. Next all three complexes

were prepared using the Protein Preparation Wizard workﬂow of

Schrodinger Molecular Modeling Suite 2010. The proteins were

subsequently preprocessed (hydrogen atoms added, incomplete

residues ﬁlled), and an exhaustive sampling was done regarding

hydrogen bond assignment. Finally, the hydrogen atoms of the

complexes were reﬁned by a minimization stage with a constraint to

converge to a structure with an RMSD of 0.3 Å (OPLS2005 force

ﬁeld) to remove steric clashes. The seven cocrystallized inhibitors used

as references were extracted from the structures 2X6K, 3LS8, 3L13,

3L16, 3L17, 3DBS, and 3IBE. On the basis of the 3IBE structure of

PI3Kγ, the lateral chain of Lys802 in 3HHM was moved by manually

modifying the dihedral angles of the residue. The resulting protein

model was ﬁnally reﬁned by a minimization stage with a constraint to

converge to a structure with an RMSD of 0.3 Å (OPLS2005 force

ﬁeld). All docked ligands were built within Marvin.⁵⁰Three-

dimensional structures were generated using Corina.⁵¹Next 3D

structures were submitted to the LigPrep module of Schrodinger

Molecular Modeling Suite 2010. All ionization states (via the Ionizer

module) and tautomers were taken into account at pH 7.0 2.0. The

resulting structures were used as the starting point for all docking

simulations.

1

220−222 °C. H NMR (400 MHz, (CD₃)₂SO): δ 3.34 (s, 2H), 3.83

(m, 4H), 4.51 (br s, 4H), 5.2 (br s, 1H)7.22 (d, J = 8.5 Hz, 2H), 7.45

(d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.78 (dd, J = 4.1 Hz, J =

8.5 Hz, 1H), 8.15 (dd, J = 1.6 Hz, J = 8.5 Hz, 1H), 8.37 (d, J = 8.8 Hz,

2H), 8.73 (dd, J = 1.6 Hz, J = 4.1 Hz, 1H), 9.22 (s, 1H), 9.45 (s, 1H).

HRMS (EI/MS): m/z calcd for C₂₅H₂₄N₆O₃[M + H]⁺457.1477,

found 457.1995. Purity: >96% by HPLC, t_R= 6.86 min.

2 - ( 4 - ( 1 - ( 3 - H y d r o x y p h e n y l ) - 3 - p h e n y l u r e a ) ) - 4 -

morpholinopyrido[3,2-d]pyrimidine (13d). The reaction was

carried out as described for general procedure D using 4h (90 mg,

0.293 mmol) and 3-aminophenol (32 mg, 0.293 mmol). The crude

product was obtained without puriﬁcation by ﬂash chromatography to

aﬀord 13d as a brown solid (58 mg, 45%). Mp: 221−223 °C. ¹H NMR

(400 MHz, (CD₃)₂SO): δ 3.83 (d, J = 4.6 Hz, 4H), 4.51 (br s, 4H),

6.39 (dd, J = 2.1 Hz, J = 8.0 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 7.06

(m, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.78 (dd, J = 4.1 Hz, J = 8.5 Hz,

1H), 8.16 (dd, J = 1.5 Hz, J = 8.5 Hz, 1H), 8.39 (d, J = 8.7 Hz, 2H),

8.63 (s, 1H), 8.73 (dd, J = 1.5 Hz, J = 4.0 Hz, 1H), 8.88 (s, 1H), 9.34

(s, 1H). HRMS (EI/MS): m/z calcd for C₂₄H₂₂N₆O₃[M + H]⁺

443.1753, found 443.1827. Purity: >95% by HPLC, t_R= 7.09 min.

2 - ( 4 - ( 1 - ( 4 - H y d r o x y p h e n y l ) - 3 - p h e n y l u r e a ) ) - 4 -

morpholinopyrido[3,2-d]pyrimidine (13e). The reaction was

carried out as described for general procedure D using 4h (120 mg,

0.390 mmol) and 3-aminophenol (43 mg, 0.390 mmol). The crude

product was obtained without puriﬁcation by ﬂash chromatography to

1

aﬀord 13e as a brown solid (100 mg, 58%). Mp: 239−241 °C. H

NMR (250 MHz, (CD₃)₂SO): δ 3.82 (m, 4H), 4.51 (br s, 4H), 6.70

(m, 2H), 7.24 (m, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.77 (dd, J = 4.1 Hz,

J = 8.5 Hz, 1H), 8.15 (dd, J = 1.7 Hz, J = 8.5 Hz, 1H), 8.38 (m, 3H),

8.72 (dd, J = 1.7 Hz, J = 4.1 Hz, 1H), 8.82 (s, 1H), 9.09 (s, 1H).

HRMS (EI/MS): m/z calcd for C₂₄H₂₂N₆O₃[M + H]⁺443.1753,

found 443.1826. Purity: >96% by HPLC, t_R= 6.88 min.

4-Morpholino-2-(4-(1-(4-pyridinyl)-3-phenylurea))pyrido-

[3,2-d] pyrimidine (13f). The reaction was carried out as described

for general procedure D using 4h (110 mg, 0.358 mmol) and 3-

aminopyridine (34 mg, 0.358 mmol). The crude product was obtained

without puriﬁcation by ﬂash chromatography to aﬀord 13f as a white

solid (109 mg, 71%). Mp: > 260 °C. ¹H NMR (400 MHz,

(CD₃)₂SO): δ 3.84 (s, 4H), 4.53 (br s, 4H), 7.48 (d, J = 5.0 Hz,

1H), 7.65 (dd, J = 3.5 Hz, J = 8.0 Hz, 2H), 7.79 (dd, J = 4.0 Hz, J = 8.4

Hz, 1H), 7.97 (m, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.38 (m, 3H), 8.80−

8.71 (m, 2H), 9.73 (br s, 1H), 10.1 (br s, 1H). HRMS (EI/MS): m/z

calcd for C₂₃H₂₁N₇O₂[M + H]⁺428.1757, found 428.1828. Purity:

>96% by HPLC, t_R= 5.67 min.

4-Morpholino-2-(4-(1-(3-pyridinyl)-3-phenylurea))pyrido-

[3,2-d]pyrimidine (13g). The reaction was carried out as described

for general procedure D using 4h (115 mg, 0.374 mmol) and 3-

aminopyridine (35 mg, 0.374 mmol). The crude product was obtained

without puriﬁcation by ﬂash chromatography to aﬀord 13g as a white

solid (110 mg, 69%). Mp: 249−251 °C. ¹H NMR (400 MHz,

(CD₃)₂SO): δ 3.97−3.65 (m, 4H), 4.51 (br s, 4H), 7.33 (d, J = 4.7 Hz,

1H), 7.62 (d, J = 8.6 Hz, 2H), 7.78 (m, 1H), 7.97 (d, J = 7.4 Hz, 1H),

Docking Parameters. The receptor grids were generated using

wortmannin as the centroid of the enclosing box, allowing the docking

of ligands with a length of 21 Å. Docking calculations were performed

with the standard precision of Glide. Ligand ﬂexibility was taken into

account, and the option of ring conformation sampling was activated.

For each ligand, postdocking minimization was performed on 100

poses, and the 10 best poses were kept and analyzed.

Biological Assays. In Vitro Kinase Assay on PI3K. The activity of

mTOR was evaluated using a puriﬁed protein (ref PV4753 from

Invitrogen). The TR-FRET mTOR kinase LANCE Ultra assay kit

(Perkin-Elmer) is a ﬂuorescence-based immunoassay for the detection

of the phosphorylation of a synthetic peptide, 4EBP1f, a substrate of

mTOR. It takes place in 384-well plates and can be divided into two

phases: a kinase reaction phase and a phosphorylated peptide

detection phase.

In the kinase reaction phase, the following components were mixed

in the same well: 5 μL of a solution containing 20 μM ATP and 2 μM

PIP2 diluted in kinase assay buﬀer (50 mM HEPES, pH 7.5, 100 mM

NaCl, 0.03% CHAPS, 1 mM EGTA, 3 mM MgCl₂, 2 mM DTT), 2.5

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μL of kinase at the optimal concentration (222 ng/mL) diluted in

kinase assay buﬀer, and 2.5 μL of serial dilutions of inhibitors diluted

in 4% DMSO and kinase assay buﬀer. For each inhibitor

concentration, the assay was run in triplicate. The reaction was then

incubated for 60 min at room temperature.

For the ADP detection phase, 5 μL of a detection solution

containing europium-labeled anti-ADP antibody (6 nM), Alexa Fluor

647-labeled ADP tracer (12 nM), and EDTA (30 mM, to stop the

kinase reaction) diluted in TR-FRET dilution buﬀer (provided by the

manufacturer) was added to the assay well. After 30 min of incubation

at room temperature, the 384-well plates were read in a Victor V plate

reader conﬁgured for HTRF (Perkin-Elmer). Excitation was

performed at 340 nm, and emission was measured at 665 and 615

nm. The inhibition curve was then plotted as emission ratio 665 nm/

615 nm versus inhibitor concentration.

kinase assays. The liganded beads were blocked with excess biotin and

washed with blocking buﬀer (SeaBlock (Pierce), 1% BSA, 0.05%

Tween 20, 1 mM DTT) to remove unbound ligand and to reduce

nonspeciﬁc phage binding. Binding reactions were assembled by

combining kinases, liganded aﬃnity beads, and test compounds in 1×

binding buﬀer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM

DTT).Test compounds were prepared as 40× stocks in 100% DMSO

and directly diluted into the assay. All reactions were performed in

polypropylene 384-well plates in a ﬁnal volume of 0.04 mL. The assay

plates were incubated at room temperature with shaking for 1 h, and

the aﬃnity beads were washed with wash buﬀer (1× PBS, 0.05%

Tween 20). The beads were then resuspended in elution buﬀer (1×

PBS, 0.05% Tween 20, 0.5 μM nonbiotinylated aﬃnity ligand) and

incubated at room temperature with shaking for 30 min. The kinase

concentration in the eluates was measured by qPCR.

To calculate substrate conversion values (conversion (%) of ATP in

ADP) from the raw TR-FRET emission ratios, an ATP-ADP titration

curve was performed with a total nucleotide concentration of 10 μM.

The resulting TR-FRET emission ratio was plotted against the

conversion of ATP to ADP. The data of this curve were ﬁtted to a

three-parameter hyperbolic model with the following equation

obtained from the GraphPad Prism software:

The compound(s) were screened at the concentration(s) requested,

and the results for primary screen binding interactions are reported as

“ctrl (%)”, where lower numbers indicate stronger hits in the matrix:

ctrl (%) calculation

test compound signal − positive control signal

negative control signal − positive control signal

=

× 100

Y = C + A(1 − (X/(B + X)))

Cell Culture and Survival Assay. Skin diploid ﬁbroblast cells were

provided by BIOPREDIC International Co. (Rennes, France). HuH7,

CaCo-2, and HaCaT cell lines were obtained from the ECACC

collection. Cells were grown according to ECACC recommendations.

The toxicity test of the compounds on these cells was as follows: A

total of 4 × 10³cells/well were seeded in 96-well plates. Twenty-four

hours after cell seeding, the cells were exposed to increasing

concentrations of the compounds (0.1, 0.3, 0.9, 2.7, 8.3, and 25

μM). After 48 h of treatment, the cells were washed in PBS and ﬁxed

in cooled 90% ethanol/5% acetic acid for 20 min. Then the nuclei

were stained with Hoechst 3342 (Sigma). Image acquisition and

analysis were performed using a Cellomics ArrayScan VTI/HCS

reader (Thermo Scientiﬁc).

Breast Cancer Cell Proliferation (MTT Assay). Cells were seeded at

1.5 × 10⁴cells/cm²in 24-well plates. After 24 h, the cells were grown

for 72 h with inhibitors used over a 10⁻⁹to 10 μM concentration

range. Cell proliferation was assessed with the MTT assay. Brieﬂy, the

cells were incubated with 400 μL of MTT (0.5 mg/mL in culture

medium) for 1 h at 37 ◦C. The medium was then aspirated. Formazan

crystals were dissolved with 400 μL dimethyl sulfoxide for 5 min. The

absorbance of each well was read at 570 nm in a Spectramax 190

spectrophotometer (Molecular Devices Corp., Sunnyvale, CA). The

assay was performed in three wells per condition. Absorbance is

expressed as a percentage of the control (vehicle).

Cell Cycle Analysis. Cells were grown in the presence of inhibitors

for 24 h. Breast cancer cells were treated with the Coulter DNA-Prep

reagents kit (Beckman Coulter, France) as previously described.⁵²

Flow cytometry analyses were performed using a Coulter Epics Elite

ESP ﬂow cytometer (Beckman Coulter). Cell cycle distribution was

analyzed with the Multicycle-AV software (Phoenix Flow Systems, San

Diego, CA).

Quantiﬁcation of Apoptotic Cells. Cells were grown in the

presence of inhibitors for 48 h. Apoptosis was assessed by ﬂow

cytometry with a method adapted to adherent cells, which is based on

the selective thermal denaturation of apoptotic DNA and detection of

denatured DNA with a monoclonal antibody against single-stranded

DNA (anti-ssDNA Mab, F7−26, from AbCys, Paris, France). The cells

were treated as previously described.⁵²Data were analyzed with the

Expo32 software (Beckman Coulter, France).

The conversion corresponding to each TR-FRET ratio was calculated

using the following equation:

(C + A − ratio)

conversion (%) = B

ratio − C

The conversion was then plotted versus the concentration of inhibitor.

The amount of inhibitor required to elicit a 50% change in the %

conversion of ATP into ADP (the EC50) was then determined. This

EC₅₀corresponds to the IC₅₀value of the inhibitor.

In Vitro Kinase Assay on mTOR. The activity of mTOR was

evaluated using a puriﬁed protein (ref PV4753 from Invitrogen). The

TR-FRET mTOR kinase LANCE Ultra assay kit (Perkin-Elmer) is a

ﬂuorescence-based immunoassay for the detection of the phosphor-

ylation of a synthetic peptide from 4EBP1, a substrate of mTOR. It

takes place in 384-well plates and can be divided into two phases: a

kinase reaction phase and a phosphorylated peptide detection phase.

In the kinase reaction phase, the following components were mixed

in the same well: 5 μL of a solution containing 10 μM ATP and 50 nM

peptide (covalently linked to an acceptor ﬂuorophore, ULight) diluted

in kinase assay buﬀer (50 nM HEPES, pH 7.5, 0.1% Tween 20, 1 mM

EGTA, 10 mM MgCl₂, 3 mM MnCl₂, 2 mM DTT), 2.5 mL of kinase

at the optimal concentration (3 ng/μL) diluted in kinase assay buﬀer,

and 5 μL of serial dilutions of inhibitors diluted in 4% DMSO and

kinase assay buﬀer. For each inhibitor concentration, the assay was run

in triplicate. The reaction was then incubated for 2 h in a solution

containing 32 mM EDTA.

For the detection phase, 5 μL of a detection solution containing

europium-labeled antiphosphopeptide antibody (2 nM ﬁnal concen-

tration) diluted in dilution buﬀer (provided by the manufacturer) was

added to the assay well. After 1 h of incubation at room temperature,

the 384-well plates were read in a Victor V plate reader conﬁgured for

HTRF (Perkin-Elmer). Excitation of europium was performed at 340

nm, and emission of UV light was measured at 665 nm. The inhibition

curve was then plotted as the intensity of emission at 665 nm versus

the logarithm of the inhibitor concentration.

Kinase Assays. For most assays, kinase-tagged T7 phage strains

were grown in parallel in 24-well blocks in an Escherichia coli host

derived from the BL21 strain. E. coli were grown to the log phase and

infected with T7 phage from a frozen stock (multiplicity of infection

0.4) and incubated with shaking at 32 °C until lysis (90−150 min).

The lysates were centrifuged (6000g) and ﬁltered (0.2 μm) to remove

cell debris. The remaining kinases were produced in HEK-293 cells

and subsequently tagged with DNA for qPCR detection. Streptavidin-

coated magnetic beads were treated with biotinylated small-molecule

ligands for 30 min at room temperature to generate aﬃnity resins for

Western Blot Analysis. Cells were grown in the presence of

inhibitors for 48 h. Cellular lysates (PBS/5% SDS lysis buﬀer

containing protease inhibitors and phosphatase inhibitors) were

subjected to SDS−polyacrylamide (10%) gel electrophoresis and

transferred onto nitrocellulose membranes. Immunoblotting was done

using rabbit monoclonal antibodies (Cell Signaling Technology,

Ozyme, France) against Akt phosphorylated on Thr 308 (ref 4056)

or Ser 473 (ref 4060) and total Akt (ref 4685) and against S6K1

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Journal of Medicinal Chemistry

Article

phosphorylated on Thr 389 (ref 9234) and total S6K1 (ref 2708).

Detection was performed with the enhanced chemiluminescence

reagent (ECL Western blotting detection reagents, Interchim, France).

Bands were quantitated using the Multi Gauge software (Fujiﬁlm).

Statistical Analysis. Statistical analysis was performed using the

GraphPad Prism software. Data are presented as the mean SD from

at least three independent experiments, and diﬀerences are considered

signiﬁcant when P < 0.05 as determined by the nonparametric

unpaired Student t test.

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ASSOCIATED CONTENT

■

S

* Supporting Information

Additional characterizations for compounds 2−13, ¹H and ¹³

C

NMR spectra for compounds 2−13, and standard deviations

for IC₅₀(Table S1). This material is available free of charge via

the Internet at http://pubs.acs.org.

Accession Codes

Crystal structure PDB IDs for docking model construction used

in this paper: 3HHM, 3IEB.

AUTHOR INFORMATION

Corresponding Author

*Phone: 33 238 494 853. Fax: 33 238 417 281. E-mail: sylvain.

routier@univ-orleans.fr.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the Ligue contre le Cancer (Comites

́

des Deux

e, de l’Ille et Vilaine, du Loir-et-Cher, de

opole

ion Centre (CrikMapAkt program)

Sev

̀

res, du Finister

Loire Atlantique, du Loiret, de la Vienne), the Cancer

Grand Ouest, and the Reg

̀

́

̂

́

for their ﬁnancial support. The ICOA synthesis team thanks Dr.

A. Chartier and N. Percina for the analytical methods to

determine the purity of each compound. We thank Dr. L.

Meijer, Dr. S. Ruchaud, Dr. O. Lozach, and Dr. S. Bach (CNRS

Roscoﬀ) for CDK, DYRK1A, CK1, and GSK3 kinase

evaluations. Additionally, we thank Dr. Le Guevel and Dr. M.

́

Ravache from the technical ImPACcell platform support

(Biogenouest and Biosit).

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ABBREVIATIONS USED

■

EtOAc, ethyl acetate; nM, nanomolar; μM, micromolar; μL,

microliter; vs, versus; ATR, attenuated total reﬂection; mg,

milligram; satd, saturated; IS, ion spray; RTKs, several receptor

tyrosine kinases; GPCRs, G-protein-coupled receptors; PI3K,

phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4,5-

diphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate;

mTOR, mammalian target of rapamycin; IRS-1, insulin

receptor substrate 1; SAR, structure−activity relationship;

MW, microwave; Ac₂O, acetic anhydride; DMF, dimethylfor-

mamide; EtOH, ethanol; rﬂx, reﬂux; MOM, methyl methyl

ether; DYRK, dual-speciﬁcity tyrosine-phosphorylation-regu-

lated kinase; CK, cyclin kinase; GSK, glycogen synthase kinase;

FDA, Food and Drug Administration

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