Inhibitors of the TAM subfamily of tyrosine kinases: Synthesis and biological evaluation

European Journal of Medicinal Chemistry 61 (2013) 2e25

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Original article

Inhibitors of the TAM subfamily of tyrosine kinases: Synthesis and biological

evaluation

,

Rosa M. Suárez ^a, Franciane Chevot ^a, Andrea Cavagnino ^{b c}, Nicolas Saettel ^a, François Radvanyi ^d,

,

c

,

a,

*

Sandrine Piguel ^{a e}, Isabelle Bernard-Pierrot ^d, Véronique Stoven ^b

, Michel Legraverend

**

^aInstitut Curie, CNRS, UMR 176, Bât. 110-112, Centre Universitaire, 91405 Orsay, France

^bMines ParisTech, CBIO e Centre for Computational Biology, 35 rue Saint-Honoré, F-77300 Fontainebleau, France

^cInstitut Curie, INSERM U900, Paris F-75248, France

^dInstitut Curie, CNRS, UMR 144, F-75248 Paris 05, France

^eUniv Paris-Sud, Orsay F-91405, France

a r t i c l e i n f o

a b s t r a c t

Article history:

The TAM subfamily of Receptor Tyrosine Kinases (RTKs) contains three human proteins of therapeutical

interest, Axl, Mer, and Tyro3. Our goal was to design a type II inhibitor speciﬁc for this family, i.e. able to

interact with the allosteric pocket and with the hinge region of the kinase. We report the synthesis of

several series of purine analogues of BMS-777607. The structural diversity of the designed inhibitors was

expected to modify the interactions formed in the binding site and consequently to modulate their

selectivity proﬁles. The most potent inhibitor 6g exhibits K_ds of 39, 42, 65 and 200 nM against Axl, Mer,

Met and Tyro3 respectively. Analysis of the afﬁnity of 6g for active and inactive forms of Abl1, an RTK

protein that does not belong to the TAM subfamily, together with the binding modes of 6g predicted by

docking studies, indicates that 6g displays some selectivity for the TAM family and may act as a type II

inhibitor.

Received 19 April 2012

Received in revised form

29 May 2012

Accepted 3 June 2012

Available online 12 June 2012

Keywords:

Tyrosine kinase inhibitors

TAM subfamily

Molecular modelling

Docking

1. Introduction

three RTKs have emerged as potential oncology targets, due to their

over expression and/or involvement in several types of cancers [3].

The receptor tyrosine kinases (RTKs) are transmembrane

proteins which transduce signals from extracellular medium to the

cytoplasm. They regulate cellular functions such as growth, differ-

entiation or motility. Abnormal overexpression levels and/or

enhanced activities of RTKs have been associated to a variety of

human cancers, leading to a strong interest in the development of

inhibitors against these human cancers. There are 58 RTKs in the

human genome, which have been classiﬁed into 20 families based

on sequence similarities within the kinase domain and structural

similarities within their extracellular regions [1].

Mer (UniProt ID: Q12866) is expressed in at least 50% of acute

lymphoblastic leukemia (ALL), with a poor prognosis in 10e15% of

pediatric ALL cases while it is not expressed in normal human T-

and B-lymphocytes at any stage of development. Mer has therefore

a tumor speciﬁc pattern which makes this tyrosine kinase an

interesting therapeutic target. Axl (UniProt ID: P30530) is overex-

pressed in numerous tumor types such as chronic myelogenous

leukemia (CML), breast, brain, lung, pancreatic, prostate, esopha-

geal, and renal tumors and hence is also considered a potential

therapeutic target for the treatment of cancer. High levels of Axl

and of Gas6, as well as Mer have been found in NSCLC cell lines [4].

Tyro3 (UniProt ID: Q06418) is the least studied of the TAM receptors

[2]. Tyro3 is signiﬁcantly elevated in human primary melanoma

tissue samples and melanoma cell lines where it is an upstream

regulator of ITF. Tyro3 knockdown represses cellular proliferation,

colony formation and tumorigenesis in vivo of melanoma cells

indicating [5] that Tyro3 may serve as a therapeutic target for

melanoma. Kinases share a similar overall folding topology and

Mer, Axl and Tyro3 are the three human RTKs that belong to the

TAM subfamily, and that share the vitamine K-dependent ligands

Gas6 and Protein S [2]. Among RTKs, the TAM subfamily is deﬁned

by the presence of the KWIAIES signature motif. Recently, these

* Corresponding author. Tel.: þ33 01 69 86 30 85.

** Corresponding author. Mines ParisTech, CBIO

e Centre for Computational

enzymatic mechanism. They consist of a mainly

b-stranded N-

Biology, 35 rue Saint-Honoré, F-77300 Fontainebleau, France.

E-mail addresses: veronique.stoven@mines-paristech.fr

michel.legraverend@curie.fr (M. Legraverend).

terminal lobe, and a mainly helical C-terminal lobe and the two

terminal lobes are connected by a short strand named the hinge

(V.

Stoven),

http://dx.doi.org/10.1016/j.ejmech.2012.06.005

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

3

region. The ATP binding site is sandwiched between these two

lobes. In the active kinase, a characteristic DFG motif located

immediately before the activation loop, adopts the so-called DFG-in

conformation in which the D and F residues are oriented toward the

active site. In its active form, ATP binds to the active site and the

terminal phosphate group of ATP is transferred to the substrate.

The most common approach to conceive kinase inhibitors is to

design ATP competitive compounds, i.e. compounds that bind to

the active site when the kinase is in the DFG-in conformation. These

inhibitors are called type I inhibitors. Typically, type I inhibitors

form between one and three hydrogen bonds with the backbone

hinge aminoacid residues, and hydrophobic interactions with

a hydrophobic pocket belonging to the active site [6]. Although

a few examples of selective type I inhibitors have been reported, the

highly conserved nature of the ATP-binding region often results in

poor kinase selectivity.

The second broad class of kinase inhibitors, named type II

inhibitors, targets an inactive form of the kinase, in which the

protein adopts the DFG-out conformation [7,8]. In this conforma-

tion, a large conformational change of the conserved DFG motif and

of the activation loop creates a large hydrophobic pocket adjacent

to the active site, the allosteric site, into which type II inhibitors can

bind (Fig. 1). The phenylalanine side chain of the DFG motif blocks

the approach of ATP, and the kinase is trapped in an inactive

conformation. In addition to hydrophobic interactions within the

active site and allosteric pocket and to the stacking interaction with

the Phe side chain of the DFG motif, the majority of known type II

inhibitors make two pairs of conserved hydrogen bonds to their

respective kinase targets: one pair to an aminoacid of the hinge

region, and a second pair to the side chain carboxylate of

speciﬁcity, Sunitinib is approved by FDA for renal cell carcinoma as

well as gastrointestinal stromal tumor [13]. Similarly, Vandetanib is

approved for the treatment of unresectable locally advanced or

metastatic medullary thyroid cancer, although this drug displays

inhibitory activity on many kinases among the 109 kinases tested,

including mutants [14], and is also able to bind Axl, Mer and Tyro3

with moderate afﬁnities [14]. Another example of type I inhibitor

for a protein of the TAM subfamily is compound C-52, a weak

inhibitor of Mer. It has been co-crystallized with Mer (PDB ID:

3BPR), giving a good starting point for the rational design of more

potent type I inhibitors for this protein [15]. However, its selectivity

proﬁle within the TAM subfamily is unknown. A type I speciﬁc

inhibitor of Axl has been reported in the literature, as a dia-

minopyrimidine derivative (Fig. 2) and has been patented recently

[16]. Finally, using Mus musculus Sky (UniProt ID: P55144), the

murine orthologue of human Tyro3, a novel selective type I inhib-

itor for Tyro3 (Fig. 2) and with lower afﬁnity for Mer and Axl has

been recently reported. The X-ray structure of the kinase domain of

MmSky in complex with this spiroindoline-based molecule is

available at the PDB under the 3QUP entry [17].

As opposite to type I inhibitors, to our knowledge, only one

type II inhibitor has been reported to be active against Tyro3, Mer

and Axl: the BMS-777607 molecule, described by Schroeder [18]

(Fig. 2). This molecule also inhibits Met (UniProt ID: P08581),

a protein whose sequence does not contain the TAM signature.

However, Met presents strong sequence similarity with proteins

of the TAM subfamily (in the range of 40% sequence identity), and

Ron. This similarity between Met and proteins of the TAM

subfamily can be illustrated by the sequence alignment and by

the phylogenetic tree respectively shown in Figs. 1 and 2 of the

supplementary materials. X-ray crystal structure of the BMS-

777607/Met complex (PDB ID: 3F82) demonstrates that this

compound is a type II inhibitor. BMS-777607 is presently in Phase

I/II of clinical studies [2,18].

a conserved glutamate in the

a-C helix and to the backbone NH of

the aspartate of the DFG motif [6].

Several type I inhibitors for proteins of the TAM subfamily have

been reported in the literature (Fig. 2), although their selectivity

proﬁles are poor since they also target kinases outside of the TAM

subfamily. A complete review of available type I inhibitors is out of

the scope of this paper, but we will recall some of these studies. For

example, Sunitinib inhibits multiple RTKs such as platelet-derived

growth factor receptors (PDGFRs), vascular endothelium growth

factor receptors (VEGFRs), and Kit (also called Mast/stem cell

growth factor receptor) [11]. It also inhibits proteins from the TAM

subfamily such as Axl, Mer and Tyro3 [12]. Despite this lack of

At this point, it should be emphasized that given the high

sequence identity between the kinase domains of Axl, Mer, Tyro3,

the design of a speciﬁc inhibitor of either member of the TAM (or

even Met) subfamilies remains a challenge, although such inhibi-

tors would be of great interest to better understand the role of

these kinases. Optimization of type II inhibitors targeting mainly

the TAM family or Met is a strategy that could be adopted to design

inhibitors with even higher speciﬁcity.

In a context where only a few type II inhibitor are available

today for the Met family [19], the purpose of this study was to

synthesize and evaluate new type II inhibitors for this family of

RTKs. These inhibitors could then be optimized in a later stage of

the project.

We will ﬁrst present the strategy that was used to conceive

these inhibitors and detail their chemical synthesis. Then, the

results of inhibitory assays and biological tests will be presented.

Finally, structural analysis based on available X-Ray structures,

homology modelling and docking experiments will be used to

discuss the binding modes and selectivity of the tested

molecules.

2. Results and discussion

Fig. 1. Ribbon representations of the “active DFG-in form” (Panel A) and “in-active

DFG-out form” (Panel B) of a kinase domain. The activation loops are shown in green in

DFG-in conformation (A), and pink in DFG-out conformation (B). The phenylalanine

side chain of DFG motif is highlighted in blue, underlining its reorientation between

the two states. An ATP-competitive Src-Abl type I inhibitor (AP24283) is presented in

green spheres (A) while a Src-Abl type II inhibitor (AP24163) overhanging the allosteric

binging site is pictured with red spheres (B). (Figures generated using PyMol from X-

ray structures of Abl1; PDB ID: 3KF4 and 3KFA) [9,10]. (For interpretation of the

references to color in this ﬁgure legend, the reader is referred to the web version of this

article.)

2.1. Chemistry

Our goal was to design a type II inhibitor of the TAM subfamily

and therefore to build a structure able to interact both with the

allosteric pocket, on one side, and, with the hinge region of the

kinase on the other side. As a starting point of our project, we

focused on the Met inhibitor BMS-777607 which is also a potent

inhibitor of the TAM subfamily but more importantly is a type II

4

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

F

N

Br

F

HN

Cl

NH

N

NH

N

O HN

N

O

N

O

N

HN

N

H

N

HO

N

H

N

H

2

Sunitinib

Vandetanib

C-52

Pyrazolopyrimidine

F

(Zactima, ZD-6474)

PDB: 3GOE

PDB: 3BPR

PDB: 3TCP

Axl IC₅₀9 nM

Mer IC₅₀26 nM

Tyro3 IC₅₀49 nM

Axl K_d250 nM

Mer K_d1400 nM

Tyro3 K_d93 nM

Axl IC₅₀nd

Mer IC₅₀11.3

Tyro3 IC₅₀nd

Axl IC₅₀37 nM

Mer IC₅₀2.9 nM

Tyro3 IC₅₀48 nM

M

O

S

NH

H

N

F

O

NH

N

F

O

N

Cl

N

Cl

NH₂

N

F

(type II inhibitor)

N

O

N

H

HN

Diaminopyrimidine

BMS-777607

Spiroindoline

PDB: 3F82

PDB: 3QUP

Axl IC₅₀27 nM

Mer > 90% inhibition at 200 nM

Tyro3 > 50% inhibition at 200 nM

Axl IC₅₀1.1 nM

Mer IC₅₀14 nM

Tyro3 IC₅₀4.3 nM

Tyro3 IC₅₀476 nM

Fig. 2. Reported type I inhibitors of the TAM subfamily, with the exception of BMS-777607 (type II inhibitor).

inhibitor. Indeed, the X-ray crystal structure of the complex

between BMS-777607 and Met showed that the 2-aminopyridine

core (Fig. 2) displays two key hydrogen bonds with the hinge

backbone of the kinase, and the pyridone lateral chain interacts

with the allosteric pocket created by the rearrangement of the

protein in a DFG-out conformation [18]. Therefore, we synthesized

analogues of BMS-777607 by i) replacing the 2-aminopyridine

moiety by a purine ring that could also allow hydrogen bonds

with the hinge region ii) by keeping the lateral chain of BMS-

777607 or replacing it by another chain potentially interacting

with the allosteric site. The conservation of this lateral chain is of

utmost importance to target the inactive conformation of the TAM

subfamily. Introduction of structural diversity using various types

of connections to link the side chain to the purine scaffold was

expected to slightly modify the interactions formed between the

proteins and the inhibitors, and therefore to change their selectivity

proﬁles.

(6), an amino bonding (13/14) or a direct carbonecarbon bridge

(18), linked to a chain that mimics the chain found in BMS-777607

(Fig. 3). A ﬂuorine atom was maintained in the ortho position of the

ﬁrst phenyl group, both in the 6-oxo series 6 and in the 6-amino

series 14 since it was found important in several close tyrosine

kinase inhibitors [18,20]. A fourth series (26e29) is characterized

by an aniline at position 6 and an additional phenylbenzamide at

position 8. The synthesis of the targeted molecules (series 6, 13/14,

18, 26-29) was achieved by coupling various aryl carboxylic acids

(molecules 4a-i-CO₂H) with aminophenylpurine intermediates 3, 9,

10, 16 and 21 which were all synthesized from precursor 1 (6-

chloro-9-THP-purine), as described in Scheme 2 for intermediates

3, 9, 10, in Scheme 3 for intermediate 16, and in Scheme 4 for

intermediates 21aed.

The aryl carboxylic acid derivatives 4aee were commercially

available, while the 4fei derivatives were synthesized separately

following the experimental procedure described in the literature

[21] (Scheme 1). The carboxylic acids derivatives of 4fei were

synthesized from 2-hydroxynicotinic acid methyl ester, in the

Thus, three series of inhibitors have been synthesized, which

differ by the connection at the position 6, bearing an ether linkage

H

N

H

N

H

F

Ar

(F)

Ar

N

Ar

R

O

HN

6

7

9

1

2

N

8

NH

O

N

H

N

H

N

H

N

H

Ar

N

3

N

oxo-bridge series

amino-bridge series

C-C linkage series

(6)

(13/14)

(26-29)

(18)

Fig. 3. Proposed library for novel inhibitors of the TAM subfamily.

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

5

R

ii

i

iii

N

HO₂C

MeO₂C

HO₂C

OH

O

2-Hydroxynicotinic

acid

2-Hydroxynicotinic

acid methyl ester

MeO₂C-4f-i

HO₂C-4f-i

4f: R = m-F

4g: R = p-F

4h: R = p-CF₃

4i: R = m-CF₃

Scheme 1. Synthesis of 4fei carboxylic acid derivatives. (i) 1) SOCl₂, CH₂Cl₂, 75 ^ꢀC, 4 h; 2) MeOH, r.t., 12 h (92%); (ii) ArI, CuBr, 2-ethylester cyclohexanone, Cs₂CO₃, DMSO, 60 ^ꢀC, 18 h

(51e57%); (iii) LiOH (1N aq sol), THF, r.t., 3 h (58e74%).

presence of the corresponding aryl iodide, CuBr, 2-ethylester-

cyclohexanone and Cs₂CO₃in DMSO, according to the literature

[21]. Saponiﬁcation of the resulting methylesters was achieved in

THF and an aqueous 1N solution of LiOH, leading to 4fei-carboxylic

acids in short reaction time and good yields (58e74%).

This strategy was adopted because the method used by

Schroeder for the synthesis of BMS-777607 (Fig. 2) [18] for the

Ullmann-type arylation of the 2-pyridone with the carboxylic acid

in the 3 position (2-hydroxy-nicotinic acid) was assayed, but in our

hands, poor yields were obtained for the desired product 4fei-

CO₂H (for these free carboxylic acids and the corresponding

methyl esters).

In parallel, starting from the known precursor 6-chloro-9-

THP-purine 1 [22,23], the ﬁrst step differed in the four series

(6, 13/14, 18 or 26-29), depending on the kind of connection to be

reached. The tetrahydropyrane group was chosen as protecting

group for the purine N9 (1) due to its liability, envisaging the use

of mild conditions for the deprotection of the ﬁnal products.

Then, for series 6, nucleophilic substitution of the 6 chlorine

atom of 1 with 2-ﬂuoro-4-nitrophenol was followed by nitro

reduction by iron in aqueous ethanol (Scheme 2), leading to the

2.2. Tyrosine kinase inhibitory assays

The entire library of the synthesized molecules was evaluated

against ﬁve tyrosine kinases by the Kinomescan company (see

Experimental section) [27], namely Tyro3, Axl, Mer, Met, and Abl1.

Abl1 was included in these tests because it belongs to the RTK

family, but is not a member of the TAM subfamily. Moreover, type II

inhibitors have been well characterized, including Gleevec^Ò. Thus,

analysis of the inhibition proﬁles of the tested molecules against

these ﬁve kinases should allow to discuss their selectivities against

the TAM subfamily. The phosphorylated and the unphosphorylated

forms of Abl1 were tested. Indeed, phosphorylated-Abl1 is char-

acterized by a DFG-in conformation, while unphosphorylated-Abl1

being in a DFG-out conformation. In other words, inhibitor afﬁnity

for unphosphorylated-Abl1 and not for phosphorylated-Abl1 is an

indirect indication that studied inhibitors might be type II inhibi-

tors. The most active compounds at a concentration of 1 and 75

are presented on Table 1.

mM

The complete results of all tested molecules are given in Table 1

of the supplementary material. Some potent inhibitors of Axl, Mer,

Met and Tyro3 were identiﬁed (Tables 1 and 2). Furthermore, as

shown in Table 1, molecules 6g and 6f that present some inhibition

for the TAM subfamily also inhibit the activity of

unphosphorylated-Abl1. This indicates that we might indeed be in

the presence of type II inhibitors. In addition, some of the

compounds (6h, and 14h for example) are able to strongly inhibit

proteins from the TAM subfamily, their inhibition activity being

much lower against Abl1. This shows that the designed compounds

using our strategy may achieve speciﬁcity for the TAM subfamily. It

is interesting to notice that the presence of a triﬂuoromethyl group

at the para position of the terminal phenyl in 6h and 14h, strongly

decreased the inhibition of unphosphorylated Abl1 while the same

group in meta position (14i) gave a good inhibition of unphos-

phorylated Abl1, as in 6g, 6f, 14f, 14g or 13f where a small ﬂuorine

atom is present in the meta or para positions.

Interestingly, analysis of the inhibition activities within proteins

of the TAM subfamily shows that the inhibitors do not have the

same inhibition proﬁles, which illustrates that slight variations on

the molecular scaffold could achieve further selectivity within the

TAM subfamily. For example, only compounds carrying an

oxygenated linkage at position 6 of the purine showed Tyro3

inhibition (6g, 6f, 6h). Compounds with no bridge at position 6

purin-6-yloxyaniline

derivative

3.

For

series

13/14,

BuchwaldeHartwig coupling [24] of the 6-chloro-9-THP-purine 1

with (2-ﬂuoro)-4-nitroaniline was followed by nitro reduction

leading to the 6-aminophenylaminopurine derivatives 9 and 10

(Scheme 2). For series 18, the 6-nitrophenylpurine derivative 15

was obtained by a Suzuki coupling [25], between 1 and 3-

nitrophenyl boronic acid with Pd(PPh₃)₄, and K₂CO₃at 100 ^ꢀC

(Scheme 3). It was then reduced to 16 in the usual way.

Series 26e29 was obtained by aromatic nucleophilic substitu-

tion of the 6 chlorine atom of 1 with various aniline derivatives and

followed by iodination in position 8 of the purine moiety, leading to

intermediates 20aed which gave compounds 21aed after a Suzuki

coupling with 3-aminophenylboronic acid (Scheme 4).

The ﬁnal two steps of these convergent syntheses were the

connection (amide formation) between the various carbocyclic

acids 4aei-CO₂H and the different aminophenyl purine counter-

parts 3, 9,10, 16 and 21 followed by the purine deprotection. For the

coupling between the different amino purine derivatives (3, 9, 10,

16 and 21) with aryl carboxylic acids (both commercially available

4aee-COOH or compounds 4fei-COOH), the procedure of Okram

was used, employing HATU as the coupling reagent in the presence

of DIPEA as base, affording the corresponding products 5, 11, 12, 17

and 22e25 in high to excellent yield (71e99%) [8]. Finally these

intermediates were efﬁciently deprotected by treatment with tri-

ﬂuoroacetic acid at room temperature, leading to the desired pure

ﬁnal products (6, 13, 14, 18 and 26e29) in good yields after chro-

matography (65e98% yields; >95% purity by HPLC) [26].

(CeC bond, in 18aei) were inactive on Tyro3, Axl and Mer at 1

mM,

and marginally active at 75 M (see supplementary material,

m

Table 1). Replacement of the para ﬂuoro atom on the terminal

phenyl (6g) group by a meta ﬂuorine (6f) or a para-triﬂuoromethyl

group (6h) decreased the Tyro3 inhibition, but had almost no effect

on Axl, Mer and Met inhibition.

6

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

H

N

F

R₁

F

N

Ar

F

Ar

O

N

v

N

iv

N

H

Ar-CO₂H

Ar: 4c, 4f-h

N

R

N

THP

i

Cl

5c, 5f-h

6c, 6f-h

7

6

2 : R₁= NO₂

3 : R₁= NH₂

1

2

N

iii

8

N

THP

N

3

9

H

N

H

N

1

R₁

R

Ar

R

Ar

ii

O

HN

N

v

N

iv

N

THP

N

H

Ar-CO₂H

Ar: 4a-i

N

THP

7 : R = H, R₁= NO₂

9 : R = H, R₁= NH₂

11c-f, 11i: R = H

12a-i: R = F

13c-f, 13i: R = H

14a-i: R = F

iii

8 : R = F, R₁= NO₂

10 : R = F, R₁= NH₂

iii

a-i for 5, 6, 11-14 are as defined

below

Br

Ar:

N

CH₃

O

F

O

4a

4c

4e

4b

4d

N

F

N

CF₃

O

F

CF₃

4g

4f

4h

4i

Scheme 2. Synthesis of 6-phenoxy 6 and 6 aminophenyl 13, 14 derivatives. Reaction conditions : i) 2-ﬂuoro-4-nitrophenol, K₂CO₃, DMF, 100 ^ꢀC, 12 h (58%); ii) Pd(OAc)₂, X-phos, 4-

nitroaniline or 2-ﬂuoro-4-nitroaniline, t-BuOH/H₂O, 110 ^ꢀC, 15 h (91 or 94%); iii) iron powder, NH₄Cl, EtOH/H₂O, 90 ^ꢀC, (65e89%) 2 h; iv) carboxylic acid, HATU, CH₂Cl₂, DIPEA, r.t., 3 h

(71e99%); v) TFA, CH₃CN/H₂O, rt, 4 h (65e98%).

H

N

H

N

R

Ar

O

N

i

iii

iv

N

1

N

H

N

Ar-CO₂H

Ar: 4a-i

N

THP

17a-i

18a-i

15 : R = NO₂

16 : R = NH₂

ii

see Scheme 1 for 4a-i

Scheme 3. Synthesis of the 6-phenylpurine derivatives 18. Reaction conditions

:

i) Pd(PPh₃)₄, K₂CO₃, 3-nitrophenyl boronic acid, dioxane/H₂O, 100 ^ꢀC,

15 h (73%); ii) iron powder, NH₄Cl, EtOH/H₂O, 90 ^ꢀC, 2 h, 65e89%); iii) carboxylic acid, HATU, CH₂Cl₂, DIPEA, 0 ^ꢀC to r.t., 3 h (71e99%); iv) TFA, CH₃CN/H₂O, rt, 4h

(65e98%).

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

7

R

HN

NH₂

26f-h

N

v

N

i

iii

iv

27e-h

R'

1

28e-f, 28h

29e-h

Ar-CO₂H

Ar

O

N

Ar: 4a-c, 4e

HN

THP

19a-d R' = H

20a-d R' = I

21a-d

22f-h

23e-h

ii

24e-f, 24h

25e-h

19a, 20a : R = H

19b, 20b : R = m-CF₃

19c, 20c : R = m-F

19d, 20d : R = p-F

22f : R = H; Ar = 4a

24e : R = m-F; Ar = 4b

24f : R = m-F; Ar = 4a

24h : R = m-F; Ar = 4e

25e : R = p-F; Ar = 4b

25f : R = p-F; Ar = 4a

25g : R = p-F; Ar = 4c

25h : R = p-F; Ar = 4e

22g : R = H; Ar = 4c

R

22h : R = H; Ar = 4e

HN

23e : R = m-CF₃; Ar = 4b

23f : R = m-CF₃; Ar = 4a

23g : R = m-CF₃; Ar = 4c

23h : R = m-CF₃; Ar = 4e

N

Ar

O

H

HN

22f-h

26f-h

23e-h

27e-h

see Scheme 1 for 4a-c, 4e

24e-f, 24h

25e-h

28e-f, 28h

29e-h

R are identical in 19, 20, 21, 22-29

Scheme 4. Synthesis of derivatives 26e29. i) Pd(OAc)₂, X-phos, t-BuOH/H₂O are introduced into a preheated bath at 110 ^ꢀC for 1 min. Then, 1, aniline compound, K₂CO₃; 110 ^ꢀC for

15 h 84e97%); ii) LDA, THF, ꢁ78 ^ꢀC, 1 h, then I₂, ꢁ78 ^ꢀC, 1 h (48e84%); iii) Pd(PPh₃)₄, K₂CO₃, dioxane/water 4/1, 85 ^ꢀC, 5 min, then 3-aminophenylboronic acid, 100 ^ꢀC, 3 h (71e84%).

iv) HATU, DIPEA, CH₂Cl₂, 0 ^ꢀC to r.t., 12 h (29e92%); v) TFA, CH₃CN/H₂O, r.t., 3 h (39e99%).

Another example of selectivity modulation is provided by

comparing 6c, where the pyridone motif is lacking, and 6g (or

6f). The former looses activity against Tyro3, Axl or Mer,

whereas inhibition of Met is preserved. The same observation

can be made for compounds 14aee (see supplementary

material).

K_ds were determined for the most active inhibitors against

proteins of the TAM family, namely 6g, 6f and 6h (Table 2). It is

interesting to notice the good K_dof 6g on Axl (39 nM), Mer (42 nM)

and Met (65 nM) and the somewhat increased K_don Tyro3

(200 nM).

2.3. In vitro cytotoxicity assays

The antiproliferative activities of the compounds were evalu-

ated at 10^ꢁ5M against four human tumor cell lines, including KB

(oral carcinoma), HT29 (colorectal carcinoma), MCF7 (breast

carcinoma) and PC3 (prostate adenocarcinoma) and one non

tumoral cell line, Vero cells (Kidney Vero cells). No cytotoxicity

could be detected at 10^ꢁ5M for all compounds listed on these ﬁve

cell lines. The growth inhibition of the cell lines was smaller than

10% at the concentration tested.

Furthermore, 6g and 6f bind preferentially the inactive non-

phosphorylated form of Abl1, as compared to the phosphory-

lated active enzyme (K_dof 6600 and 320 vs 40,000 and 3300,

respectively on the phosphorylated enzyme). Again, this

suggests that these inhibitors recognize preferentially the inac-

tive form of the enzyme, and therefore could be type II inhibi-

tors, although this could only be demonstrated by solving the X-

ray structure of the complex. However, analysis of the known

DFG-out structure of Met, together with modelling studies pre-

sented below seem to conﬁrm that we indeed designed type II

inhibitors.

A this point, one should note that K_ds shown in Table 2 were

determined on the phosphorylated forms of Axl, Mer and Met,

because the inactive (unphosphorylated) forms of these enzymes

were not available. As shown in Table 2 in the case of Abl1, a type II

inhibitor does present some afﬁnity for the active form of the

protein, although this afﬁnity is several orders of magnitude

weaker than for the inactive form. Therefore, the afﬁnities of the 6g,

6f and 6h molecules for proteins of the TAM subfamily in the

inactive form (unphosphorylated DFG-out conformation) are ex-

pected to be much stronger (i.e. smaller K_ds) than those reported in

Table 2.

2.4. Molecular modelling studies

A deeper structural interpretation of the above biological

results is required for future optimization of the designed

inhibitors. Therefore, we undertook

a series of molecular

modelling studies. Since no DFG-out structure is available in the

TAM subfamily, we ﬁrst built a model of structure for one of the

three human members of the TAM subfamily, namely Tyro3. We

modelled a DFG-out structure for Tyro3 because the context of

this study is to conceive type II inhibitors (see Experimental

section for a description of the modelling protocol and Fig. 1

in supplementary materials for sequence alignment). The

quality of the topology of this model, including bond lengths

and bond angles, was carefully checked, as detailed in the

Experimental section.

Among all the proteins for which a DFG-out structure in

complex with a type II inhibitor is available, Met is the closest one

to proteins of the TAM subfamily. This structure (PDB ID: 3F82)

corresponds to a Met DFG-out structure in complex with the BMS-

777607 inhibitor [18]. In addition, the inhibition tests presented in

the previous section included the Met protein. Therefore, we ﬁrst

compared the structure of the 3F82 complex to that of 6g docked in

8

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

Table 1

Active inhibitors of the TAM subfamily. Percent of the enzyme captured from the solid support in the presence of 1

m

M or 75 mM inhibitor.

Cmpd

Structure

% Binding afﬁnity at 1 or 75

Abl1 unphos. Abl1 phos.

75 75

mM

Axl

1

Mer

1

Met

1

Tyro3

1

75

99

75

H

N

F

N

O

F

6g

40

80

0

35

99

96

83

100

N

H

N

H

F

N

F

O

6f

75

73

4

18

93

99

89

97

51

98

20

94

N

H

N

H

F

N

O

CF₃

6h

0

35

0

88

99

84

99

92

99

0

92

N

H

N

H

N

F

O

F

O

6c

30

89

10

37

0

4

0

40

34

98

0

18

N

H

N

H

N

F

N

O

HN

CF₃

14h

18

22

0

45

88

33

66

53

90

0

10

N

H

N

H

N

F

N

F

O

HN

14f

38

41

4

0

81

90

75

81

76

86

0

4

N

H

N

H

F

N

O

HN

F

14g

84

78

18

8

45

57

39

55

6

22

0

N

H

N

H

F

N

CF₃

O

HN

14i

84

92

12

24

34

61

36

59

22

33

0

N

H

N

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

9

Table 1 (continued )

Cmpd

Structure

% Binding afﬁnity at 1 or 75

Abl1 unphos. Abl1 phos.

75

mM

Axl

1

Mer

1

Met

1

Tyro3

1

75

6

75

H

N

F

O

HN

13f

88

86

26

22

4

32

28

24

0

N

H

N

Sunitinib

Vandetanib

100

98

100

73

99

100

82

100

99

59

100

99

5

0

96

99

9

90

99

100

an empty DFG-out structure of Met. The 6g molecule was chosen

because, according to the biological results presented in the

previous section, it is one of the most active tested compounds

against the Met subfamily.

In the 3F82 structure, within the active site of Met, BMS-

777607 forms a double hydrogen bond with the backbone of

M1160 from the hinge region, and hydrophobic interactions

A672 (respectively equivalent to F1134, A1221 in Met). These

interactions are in agreement with the strong inhibitory activity

of BMS-777607 against Tyro3 (IC₅₀¼ 4.3 nM) [18], and they

indicate that Tyro3 could be inhibited by a type II inhibitor. At

this point, it should be recalled that there is a direct relation

between the K_dand the IC₅₀(see experimental section, kinase

assays).

with M1211, both interactions being also found in type

I

We docked all the synthesized and tested molecules (see

previous sections) in the DFG-out model of Tyro3, and analysed

the docking scores. We observed that all molecules active against

Tyro3 obtained best docking scores than any of the inactive

molecules. In other words, the top ranked molecules were active

against Tyro3 (Table 3). The complete docking scores for all the

tested molecules are given in Table 1 of the supplementary

materials.

inhibitors. In addition, it also binds to Met via several expected

interactions for type II inhibitors: a hydrogen bond with D1222

(of the DFG motif), hydrophobic stacking with F1223 (of the

DFG motif), and hydrophobic interactions in the allosteric site

with residues M1131, A1221, and F1134. A hydrogen bond is

also observed between the inhibitor and the side chain of

K1110 (Fig. 4AeC).

After removal of the BMS-777607 inhibitor from 3F82, we

docked the 6g molecule in the resulting empty DFG-out structure

of Met (see the Experimental section for details about the docking

protocol). Overall, the docked inhibitor occupies the same posi-

tion in the pocket than BMS-777607 does in 3F82. In addition,

most of the interactions observed between BMS-777607 and Met

in 3F82 are conserved in the complex formed by 6g docked in

Met. The 6g molecule binds to active site residues via the double

hydrogen bond with the M1160 backbone, and hydrophobic

interactions with M1211. It also binds via typical type II interac-

tions: the hydrogen bond with D1222, the hydrophobic stacking

with F1223, the hydrophobic interplay with the allosteric pocket

via M1131, A1221 and F1134 (see Fig. 4DeF). Therefore, the

docked complex is compatible with a type II binding mode for the

6g molecule.

Using the same docking protocol, we docked BMS-777607

(which inhibits Tyro3) in the DFG-out model of Tyro3. We

observed that the binding mode of BMS-777607 docked in Tyro3

is similar to its binding mode in the 3F82 structure of Met (see

Fig. 5AeC). In particular, the main interactions observed

between the docked BMS-777607 and Tyro3 are similar to the

key interactions in 3F82. In the active site, the inhibitor forms

a double hydrogen bond with M606 (of the hinge region,

equivalent to M1160 in Met), and a hydrophobic interaction with

L524 and L603 (comparable to the interaction with M1211 in

Met). In addition, the interactions typical of type II inhibitors are

also conserved: a double hydrogen bond with D673 (of the DFG

motif, equivalent to D1222 in Met), hydophobic stacking with

F674 (of the DFG motif, equivalent to F1223 in Met), and

hydrophobic interactions in the allosteric pocket with F575 and

These results validate the docking protocol used in this study

and allow further analysis of the docked complexes.

In particular, the complex formed by Tyro3 and the (docked)

6g molecule provides an example of how the active molecules

can bind to their target (Fig. 5DeF). In the active site, the purine

ring of 6g binds to M606 via a double hydrogen bond, and

a hydrophobic interaction is also formed with L524. In addition,

typical interactions for type II inhibitors are also observed in

this docked complex: hydrophobic stacking with F674, a double

hydrogen bond with D672, and a hydrophobic interaction in the

allosteric pocket with F575 and M571. Overall, the interactions

observed between 6g and Tyro3 are very similar to those

observed between BMS-777607 and Tyro3, and between BMS-

777607 or 6g and Met (Fig. 4). Therefore, the active molecules

synthesized in this study are indeed expected to be type II

inhibitors.

Table 2

K_dvalues of the most potent inhibitors, 6g, 6f and 6h. nd, not determined.

Kinase target/inhibitor

6g

6f

6h

K_d(nM)

Abl1-nonphosphorylated

Abl1-phosphorylated

Axl

Mer

Met

Tyro3

6600

40,000

39

42

65

200

320

3300

210

190

440

nd

170

260

150

2900

900

10

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

Fig. 4. Panels A and B: binding site of Met (in blue ribbon and in sticks for the side chains) with BMS-777607 (yellow sticks) in the 3F82 crystal structure, viewed from the

active site (Panel A) and from the allosteric site (Panel B). Panel C: 2D representation showing polar (black labelled residues) and hydrophobic (green labelled residues)

contacts in 3F82. Panels D and E: binding site of Met (in blue ribbon and in sticks for side chains) with the docked compound 6g (green sticks) viewed from the active site

(Panel D) and from the allosteric site (Panel E). Panel F: 2D representation of the interactions in the Met-6g docked complex. Figures generated using PyMol, 2D schemes

generated and modiﬁed from PoseView analysis) [28]. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this

article.)

Fig. 5. Panels A and B: schematic view of BMS-777607 (yellow sticks) docked in the Tyro3 model (in pink ribbon and in sticks for the side chains), viewed from the active site (Panel

A) and from the allosteric site (Panel B). Panel C: 2D representation of the polar (black labels) and hydrophobic (green labels) contacts in the docked Tyro3- BMS-777607 complex.

Panels D and E: compound 6g (green sticks) docked in Tyro3 viewed from the active site (panel C) and from the allosteric site (panel D). Panel F: 2D representation of polar (black

labels) and hydrophobic (green labels) contacts in the docked Tyro3-6g complex. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web

version of this article.)

Table 3

It is also interesting to analyse the docking results of inac-

tive molecules, since this could also guide the optimization of

the most active molecules (namely, 6g, 6f and 6h). The bio-

Top scored molecules according to the ChemGauss score

used in this study [29]. The best ranked molecules corre-

spond to the most active compounds according to the

logical tests showed that 6g strongly inhibits Tyro3, while 14g

does not, although these two molecules present only a single

and subtle structural difference: in 14g, an amino group is

bound to position 6 of the purine, while it is replaced by an

oxygen in 6g. In the docked complex of 14g, the conserved

hydrogen bond with M606 in the hinge region is lost because

of a rotation of the purine moiety (Fig. 6, Panels A, B and C).

This might explain why compound 14g is inactive against

Tyro3. It also suggests that docking could help to guide the

design of type II inhibitors able to present some selectivity

within the TAM subfamily.

experimental tests shown in Table 1.

Name

Docking score

6g

ꢁ133.6

ꢁ130.1

ꢁ129.4

ꢁ121.0

ꢁ115.7

ꢁ114.5

ꢁ111.4

ꢁ108.3

ꢁ106.6

ꢁ104.5

BMS-777607

6f

6h

14h

14g

14f

14i

13i

14a

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

11

Fig. 6. Panels A and B: schematic view of the 14g compound (blue sticks) docked in the Tyro3 model (in pink ribbon and in sticks for the side chains), viewed from the active site

(Panel A) and from the allosteric site (Panel B). Panel C: 2D representation of polar (in black) and hydrophobic (in green) contacts in the docked Tyro3-14g complex. (For inter-

pretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

3. Conclusions

4. Experimental section

We have synthesized a new series of purine derivatives, using

a convergent synthetic route, which allowed us to introduce efﬁ-

ciently structural diversity at several different points of the mole-

cule, and to access rapidly to a library of potential TAM inhibitors.

The best inhibitor 6g, exhibits K_ds of 39, 42, 65 and 200 nM against

Axl, Mer, Met and Tyro3 respectively. Much lower activity was

observed against unphosphorylated Abl1 (K_d¼ 6600 nM) and

phosphorylated Abl1 (K_d¼ 40,000 nM).

4.1. Chemistry

NMR spectra were recorded at 300 MHz (¹H NMR) or 75.3 MHz

(

¹³C NMR) with CDCl₃or DMSO-d₆as solvents and tetramethylsi-

lane as internal standard on a Bruker AC300 spectrometer. Chem-

ical shifts are expressed in ppm ( ) downﬁeld from TMS. J values are

d

expressed in Hertz (Hz). The following abbreviations are used for

the multiplicities: s, singlet; d, doublet; t, triplet; m, multiplet; b,

broad. MS spectra were recorded on a Waters ZQ2000 mass spec-

trometer with direct injection. HRMS spectra were performed by

the mass spectrometry service Imagif at the ICSN (Gif-sur-Yvette).

Melting points were recorded into a Stuart SMP30 melting point

apparatus unless otherwise stated and are uncorrected. A Koﬂer

Heizbank system was used in speciﬁed cases. Carboxylic acids 4a

(Janssen), 4b (Aldrich), 4c, 4e (Acros) and 4d (Alfa Aesar) are

commercially available. Carboxylic acids 4fei were synthesized

according to reported procedures [21]. Numbering of the purine

heterocycle is indicated on Scheme 2.

However, as mentioned in the result section, due to the acti-

vated form of Axl, Mer, Tyro3 and Met used in the kinase assays, the

activity of inhibitors like 6f, 6g and 6h may be underestimated,

since it is now well established that type II inhibitors bind prefer-

entially the DFG-out conformation of the enzyme [6,30] as Imatinib

(Gleevec) which exhibits a clear preference (>30 fold) for the non-

phosphorylated form of Abl1 [30]. The lower K_dof 6g and 6f for the

inactive unphosphorylated form (DFG-out) of Abl1 than for its

active phosphorylated form (DFG-in), suggests that 6g and 6f act as

type II inhibitors of Abl1. Furthermore, compound 6g is also active

against Met and Tyro3, and the docking studies into the known

DFG-out structure of Met, or the modelled DFG-out structure of

Tyro3 are also in agreement with a type II binding mode for this

molecule. This inhibitor is most active on Axl, Mer and Met, and

slightly less potent on Tyro3. In addition, the absence of cytotoxicity

against several tumor cell lines in culture makes this inhibitor

a good candidate for the growth inhibition of tumor cells that

would overexpress a gene belonging to the TAM subfamily.

Numerous selective protein kinase inhibitors are known to date,

but the design of speciﬁc inhibitors of each kinase still remains

a real challenge. In fact receptor tyrosine kinase inhibitors are often

less speciﬁc for the intended target, which may be of beneﬁt if the

additional targets contribute to oncogenesis and metastasis or

detrimental if additional adverse effects are elicited. In this context,

speciﬁc inhibitors remain highly desirable and useful, at least in

pre-clinical studies, in order to help delineate the individual

contributions of the different kinases to tumorigenesis and

metastasis [4]. A way to improve the selectivity of kinase inhibitors,

during the last few years, has been to design type II inhibitors,

which target an inactive form of the kinase and are therefore less

ATP competitive [31]. These inhibitors speciﬁcally exploit the DFG-

out conformation of the kinase, giving access to a deep hydrophobic

pocket which is less conserved than the ATP binding site and thus

open the possibility of creating more speciﬁc interactions with each

kinase [32,33].

4.2. HPLC chromatographies were performed on a Waters Alliance

apparatus equipped with a diode array and a XterraMS column

4.2.1. HPLC method I

A linear gradient of acetonitrile versus water was used ranging

from 98% of acetonitrile to 55% over 5 min then to 100% over 5 min

at a ﬂow rate of 1 mL/min.

4.2.2. HPLC method II

A linear gradient of acetonitrile versus water containing 0.1% of

TFA was used ranging from 98% of acetonitrile to 55% over 2 min

then to 5% over 4 min and ﬁnally, to 100% over 4 min at the ﬂow

1.4 mL/min.

4.3. 6-(2-Fluoro-4-nitrophenoxy)-9-(tetrahydro-2H-pyran-2-yl)-

9H-purine (2)

In a sealed tube purged with argon, 6-chloro-9-THP-purine 1

(100 mg, 0.42 mmol), 2-ﬂuoro-4-nitrophenol (79 mg, 0.50 mmol)

and K₂CO₃(174 mg, 1.26 mmol) were suspended into DMF (5 mL).

The reaction mixture was heated at 100 ^ꢀC during 12 h. The reaction

mixture was then allowed to reach room temperature, diluted in

EtOAc and washed with a small fraction of brine. The organic phase

was separated and dried over anhydrous MgSO₄. The solvent was

removed in vacuo, and the oily residue puriﬁed by column chro-

matography over silica gel, using EtOH/CH₂Cl₂mixtures (from 1 to

5%) as eluent. The product was obtained in 58% yield. M.p.

142e144 ^ꢀC. R_f(2% EtOH/CH₂Cl₂): 0.46. ¹H NMR (CDCl₃, 300 MHz)

In this context, we are presently developing new structural

modiﬁcations, necessary to improve the selectivity proﬁle of the

most promising molecules reported in this study. They will be

based on the ﬁrst structureeactvity relationships as well as

molecular modelling studies reported therein.

d

(ppm): 1.67e1.85 (m, 3H), 2.05e2.21 (m, 3H), 3.81 (dt, J ¼ 12.3 Hz,

12

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

1H), 4.21 (dd, J ¼ 12.3 Hz, 1H), 5.82 (dd, J ¼ 9.3 Hz, 1H), 7.54 (t,

J ¼ 9 Hz, 1H), 8.12e8.19 (m, 2H), 8.31 (s, 1H), 8.49 (s, 1H). ¹³C NMR

added slowly. The reaction mixture was becoming rapidly darker; it

was stirred at room temperature for 3 h. After that time, the

conversion showed to be complete by TLC. The reaction mixture

was acidiﬁed by addition of 1N HCl, till pH 2e3. The crude product

was diluted in EtOAc and washed with brine. The aqueous phases

were re-extracted with EtOAc (2 ꢂ 50 mL), and the combined

organic phases were dried over anhydrous MgSO₄. The crude

products were puriﬁed by column chromatography over silica gel,

eluting with EtOH/CH₂Cl₂mixtures (2%e10%), to afford the pure

corresponding carboxylic acids of the N-arylated-2-pyridones in

high yields (55e74%).

(CDCl₃, 75 MHz)

d (ppm): 22.7, 24.8, 31.8, 68.9, 82.5, 113.2, 120.4,

121.5,124.5,141.9,145.2,151.7,152.3,152.9,155.7,158.2. MS (EI) ES^þ:

360.2 ([M^þþH], 20).

4.4. General method A for the alkylation of 2-hydroxynicotinic acid

methyl ester (MeO₂C-4f-i) [21]

2-Hydroxynicotinic acid methyl ester (CAS number 10128-91-3)

(100e200 mg scale), the corresponding aryl iodide (0.95 equiv),

CuBr (10 mol%), 2-ethylester cyclohexanone (20 mol%) and Cs₂CO₃

(2.2 equiv) were suspended in DMSO (anhydrous, 2e3 mL) in

a sealed tube. The reaction was complete after about 18 h heating at

60 ^ꢀC. The mixture was diluted in EtOAc and washed with brine.

The aqueous phase was re-extracted once with EtOAc. The

combined organic phases were dried over anhydrous MgSO₄, and

the solids ﬁltered off. The solvent was removed in vacuo and the

resulting crude material puriﬁed by column chromatography over

silica gel, EtOH/CH₂Cl₂. The products were isolated in good yields

(53e58%).

4.5.1. 1-(3-Fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic

acid (HO₂C-4f)

Following the general method B described previously, the

desired product was isolated in 60% yield. R_f(7% EtOH/CH₂Cl₂): 0.6

¹H NMR (DMSO-d₆, 300 MHz)

d (ppm): 6.82 (bs, 1H), 7.83e7.92 (m,

3H), 8.06 (s,1H), 8.28 (d, J ¼ 3 Hz,1H), 8.50 (d, J ¼ 6 Hz,1H). ¹³C NMR

(DMSO-d₆, 75 MHz)

d (ppm): 108.3, 114.7, 116.2, 117.5, 123.2, 131.0,

140.4, 144.9, 146.4, 163.4, 164.5. MS (EI) ES^þ: 256 ([M^þþ Na], 100);

MS (EI) ES^ꢁ: 232 ([M^þ-H], 100).

4.4.1. Methyl 1-(3-ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-

carboxylate (MeO₂C-4f)

4.5.2. 1-(4-Fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic

acid (HO₂C-4g)

Following the general method A, the desired product was iso-

Following the general method B described previously, the

desired product was isolated in 74% yield. R_f(7% EtOH/CH₂Cl₂): 0.59

¹H NMR (DMSO-d₆, 300 MHz) d (ppm): 6.79 (dd, J ¼ 9.6 Hz, 1H),

7.43 (t, J ¼ 9 Hz, 2H), 7.63 (dd, J ¼ 9.6 Hz, 2H), 8.22 (dd, J ¼ 6.3 Hz,

lated in 55% yield. R_f(7% EtOH/CH₂Cl₂): 0.57. ¹H NMR (CDCl₃,

300 MHz)

d

(ppm): 3.91 (s, 3H), 6.35 (dd, J ¼ 9.6 Hz, 1H), 7.17 (dd,

J ¼ 9.3 Hz, 3H), 7.43e7.48 (m, 1H), 7.57 (dd, J ¼ 6.3 Hz, 1H), 8.25 (dd,

J ¼ 6.3 Hz, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d

(ppm): 52.4, 105.1,

1H), 8.49 (dd, J ¼ 9.3 Hz, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm):

114.6,116.1, 122.4, 130.6, 141.4, 142.1,145.4, 159.0, 160.9, 164.2, 165.5.

108.3, 116.0, 116.3, 117.4, 129.0, 129.2, 135.4, 145.2, 146.3, 160.4,

163.7, 164.6. MS (EI) ES^þ: 256 ([M^þþ Na], 100); MS (EI) ES^ꢁ: 232

([M^þ-H], 100).

4.4.2. Methyl 1-(4-ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-

carboxylate (MeO₂C-4g)

Following the general method A, the desired product was iso-

4.5.3. 2-Oxo-1-(4-(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-

carboxylic acid (HO₂C-4h)

lated in 58% yield. R_f(7% EtOH/CH₂Cl₂): 0.58. ¹H NMR (DMSO-d₆,

300 MHz)

2H), 7.33e7.38 (m, 2H), 7.57 (dd, J ¼ 6.3 Hz, 1H), 8.25 (dd, J ¼ 9.3 Hz,

1H). ¹³C NMR (DMSO-d₆, 75 MHz)

(ppm): 52.6, 105.5, 116.2, 116.5,

d

(ppm): 3.90 (s, 3H), 6.34 (t, J ¼ 6 Hz,1H), 7.17 (t, J ¼ 9 Hz,

Following the general method B described previously, the

desired product was isolated in 55% yield. R_f(7% EtOH/CH₂Cl₂): 0.45

d

¹H NMR (DMSO-d₆, 300 MHz)

d

(ppm): 6.82 (t, J ¼ 6 Hz, 1H), 7.82 (d,

122.0, 128.5, 128.6, 136.1, 143.3, 145.0, 160.8, 164.1.

J ¼ 6 Hz, 2H), 7.99 (d, J ¼ 9 Hz, 2H), 8.26 (d, J ¼ 6 Hz, 1H), 8.50 (d,

J ¼ 6 Hz, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 108.4, 117.7,

4.4.3. Methyl 2-oxo-1-(4-(triﬂuoromethyl)phenyl)-1,2-

dihydropyridine-3-carboxylate (MeO₂C-4h)

121.8, 126.4, 128.0, 129.6, 142.5, 144.6, 146.5, 163.3, 164.5. MS (EI)

ES^þ: 306 ([M^þþ Na],100); MS (EI) ES^ꢁ: 282 ([M^þ-H], 95), 238 ([M^þ-

CO₂H], 100).

Following the general method A, the desired product was iso-

lated in 54% yield. R_f(7% EtOH/CH₂Cl₂): 0.62 ¹H NMR (CDCl₃,

300 MHz)

d

(ppm): 3.91 (s, 3H), 6.35 (dd, J ¼ 9.6 Hz, 1H), 7.15e7.19

4.5.4. 2-Oxo-1-(3-(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-

carboxylic acid (HO₂C-4i)

Following the general method B described previously, the

desired product was isolated in 71% yield. R_f(7% EtOH/CH₂Cl₂): 0.57

(m, 3H), 7.43e7.51 (m, 1H), 7.57 (dd, J ¼ 6.3 Hz, 1H), 8.25 (dd,

J ¼ 6.3 Hz, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d

(ppm): 52.5, 105.3, 122.2,

125.4, 126.6, 127.3, 131.3, 141.9, 143.3, 145.7, 158.9, 165.5.

¹H NMR (DMSO-d₆, 300 MHz)

d

(ppm): 6.81 (t, J ¼ 6 Hz, 1H),

4.4.4. Methyl 2-oxo-1-(3-(triﬂuoromethyl)phenyl)-1,2-

dihydropyridine-3-carboxylate (MeO₂C-4i)

7.80e7.95 (m, 3H), 8.06 (s, 1H), 8.28 (dd, J ¼ 6.3 Hz, 1H), 8.50 (dd,

J ¼ 9.3 Hz, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 108.3, 117.6,

Following the general method A, the desired product was iso-

124.2, 126.2, 129.9, 130.6, 131.2, 139.7, 144.9, 146.4, 163.4, 164.6. MS

(EI) ES^þ: 306 ([M^þþ Na], 100); MS (EI) ES^ꢁ: 282 ([M^þ-H], 95), 238

([M^þ-CO₂H], 100).

lated in 53% yield. R_f(7% EtOH/CH₂Cl₂): 0.60 ¹H NMR (CDCl₃,

300 MHz)

d

(ppm): 3.91 (s, 3H), 6.35 (dd, J ¼ 9.6 Hz, 1H), 7.15e7.19

(m, 3H), 7.43e7.51 (m, 1H), 7.57 (dd, J ¼ 6.3 Hz, 1H), 8.25 (dd,

J ¼ 6.3 Hz, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d

(ppm): 52.5, 105.3, 122.0,

4.5.5. N-(4-nitrophenyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-

6-amine (7)

123.9, 125.8, 130.0, 130.2, 131.9, 140.7, 142.2, 145.8, 165.5.

The source of palladium, Pd(OAc)₂(19 mg, 0.08 mmol), and the

ligand, X-phos [2-dicyclohexylphosphino-2⁰,4⁰,6⁰-triisopropylbi-

phenyl] (80 mg, 0.17 mmol), were mixed into a sealed tube with

a deoxygenated mixture of t-BuOH/H₂O (3 þ 1 mL), and introduced

into a preheated bath at 110 ^ꢀC for 1 min. Afterwards, the 6-Cl-9-

THP-purine 1 (200 mg, 0.84 mmol), 4-nitroaniline (174 mg,

1.26 mmol) and K₂CO₃(290 mg, 2.09 mmol) were added to the

reaction mixture and it was additionally heated at 110 ^ꢀC for 15 h.

4.5. General method B for the saponiﬁcation of methyl esters

MeO₂C-4fei

The methyl 2-oxo-1-aryl-1,2-dihydropyridine-3-carboxylates

(MeO₂C-4fei) were deprotected to the corresponding carboxylic

acids. The methyl ester compounds (500 mg scale) were suspended

in THF (15 mL) and an aqueous 1N solution of LiOH (1.5 equiv) was

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

13

The reaction mixture was allowed to reach room temperature and

diluted into EtOAc. It was ﬁltered through a short pad of celite,

which was washed with EtOAc. The ﬁltrate was extracted with

brine and the organic phase dried over anhydrous MgSO₄. The

solids were ﬁltered off and the solvent removed in vacuo. The

residue was puriﬁed by column chromatography over silica gel,

using as eluent EtOH/CH₂Cl₂mixtures (from 2% to 8%), obtaining

the product as a yellow powder (261 mg, 91% isolated yield). M.p.

>300 ^ꢀC. R_f(2% EtOH/CH₂Cl₂): 0.19. ¹H NMR (CDCl₃, 300 MHz)

d (ppm): 1.67e1.78 (m, 3H), 2.04e2.17 (m, 3H), 3.79 (bt,

J ¼ 9 Hz, 1H), 4.18 (bd, J ¼ 12 Hz, 1H), 5.73 (dd, J ¼ 9.3 Hz, 1H), 6.73

(d, J ¼ 9 Hz, 2H), 7.48 (d, J ¼ 9 Hz, 1H), 7.53 (bs, 1H), 8.03 (s, 1H), 8.48

(s,1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.9, 24.9, 31.9, 68.8, 81.9,

115.6, 119.9, 123.1, 129.6, 138.0, 143.2, 148.7, 152.8, 153.2. MS (EI)

ES^þ: 311 ([M^þþ H], 100), 333 ([M^þþ Na], 42).

4.6.3. 2-Fluoro-N1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)

benzene-1,4-diamine (10)

d

(ppm): 1.69e1.80 (m, 3H), 2.05e2.17 (m, 3H), 3.80 (dt, J ¼ 12. 3 Hz,

According to procedure C in 89% yield. Brownish solid. M.p.

1H), 4.20 (dd, J ¼ 12.3 Hz, 1H), 5.78 (dd, J ¼ 12.3 Hz, 1H), 8.06 (d,

J ¼ 9 Hz, 2H), 8.13 (s, 1H), 8.19 (bs, 1H), 8.26 (d, J ¼ 9 Hz, 2H), 8.64 (s,

130e132 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.22; ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.66e1.77 (m, 3H), 2.07e2.11 (m, 3H), 3.78 (dd, 1H), 4.19

1H); ¹³C NMR (CDCl₃, 75 MHz)

d

(ppm): 22.8, 24.9, 31.9, 68.9, 82.1,

(bd, J ¼ 12 Hz, 1H), 5.74 (d, J ¼ 15 Hz, 1H), 6.49 (bs, 1H), 6.52 (s, 1H),

118.8, 120.8, 125.2, 139.5, 142.5, 144.9, 149.6, 151.2, 152.6. MS (EI)

7.49 (bs, 1H), 7.98 (t, J ¼ 9 Hz, 1H), 8.04 (s, 1H), 8.49 (s, 1H). ¹³C NMR

ES^þ: 341 ([MþH], 30).

(CDCl₃, 75 MHz) d (ppm): 22.9, 24.9, 31.9, 68.8, 81.9, 102.5, 110.7,

117.0, 120.3, 125.4, 138.3, 144.4, 148.9, 152.0, 152.7, 157.1. MS (EI)

4.5.6. N-(2-ﬂuoro-4-nitrophenyl)-9-(tetrahydro-2H-pyran-2-yl)-

9H-purin-6-amine (8)

ES^þ: 329 ([M^þþ H], 55), 351 ([M^þþ Na], 100).

Following the same experimental procedure than for the

synthesis of the compound 7, the BuchwaldeHartwig coupling of

the 6-chloro-THP-purine 1 (400 mg, 1.68 mmol) with 2-ﬂuoro-4-

nitroaniline (393 mg, 2.52 mmol) led to the desired product 4 in

94% isolated yield (567 mg), after puriﬁcation of the crude material

by column chromatography over silica gel using EtOH/CH₂Cl₂

mixtures as eluent. M.p. 176 ^ꢀC. R_f(4% EtOH/CH₂Cl₂): 0.47. ¹H NMR

4.6.4. 3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)aniline (16)

According to procedure C in 81% yield. Light yellow solid. M.p.

180e182 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.37 ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.69e1.75 (m, 3H), 2.08e2.14 (m, 3H), 3.78e3.82 (m, 1H),

4.18e4.22 (bd, J ¼ 15 Hz, 1H), 5.85 (bd, J ¼ 9 Hz, 1H), 6.86 (d,

J ¼ 9 Hz,1H), 7.35 (t, J ¼ 6 Hz,1H), 8.09 (bs,1H), 8.20 (d, J ¼ 6 Hz,1H),

8.33 (s, 1H), 9.00 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8,

(CDCl₃, 300 MHz)

d

(ppm): 1.70e1.89 (m, 3H), 2.06e2.15 (m, 3H),

24.8, 31.8, 68.9, 81.9, 115.8, 117.8, 120.4, 129.6, 131.1, 136.5, 142.0,

146.8, 151.6, 152.3, 155.13. MS (EI) ES^þ: 296 ([M^þþ H], 95), 318

([M^þþ Na], 65).

3.81 (dt, J ¼ 12.3 Hz, 1H), 4.21 (d, J ¼ 12 Hz, 1H), 5.78 (dd, J ¼ 9.3 Hz,

1H), 8.06 (dd, J ¼ 12.3 Hz, 1H), 8.13 (bs, 1H), 8.16 (s, 1H), 8.66 (s, 1H),

9.17 (t, J ¼ 9 Hz, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9,

31.9, 68.9, 82.2, 111.0, 119.8, 120.9, 121.3, 134.0, 139.9, 141.5, 149.7,

4.7. 6-(3-nitrophenyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine

(15) [25]

149.7, 150.7, 152.3, 152.5. MS (EI) ES^ꢁ: 357 ([M^þ-H], 100).

4.6. General procedure C for the preparation of 3, 9, 10 and 16 (NO₂

reduction)

A sealed tube containing a suspension of the 6-chloro-9-THP

purine 1 (300 mg, 1.26 mmol) and Pd(PPh₃)₄(145 mg, 0.13 mmol)

in a degassed mixture of dioxane/H₂O (4 þ 1 mL), was preheated at

85 ^ꢀC for 5 min. Next, K₂CO₃(520 mg, 3.77 mmol) and 3-

nitrophenyl boronic acid (315 mg, 1.88 mmol), were added to the

mixture and the reaction was additionally heated at 100 ^ꢀC in the

sealed tube for 15 h. Afterwards, the crude material was allowed to

reach room temperature. It was diluted in EtOAc and ﬁltered

through a short pad of celite, washing the solids with EtOAc. The

ﬁltrate was washed with brine and the organic phase separated and

dried over anhydrous MgSO₄. After ﬁltration of the solids and

evaporation in vacuo, the resulting oily residue was puriﬁed by

column chromatography over silica gel, using EtOH/CH₂Cl₂

mixtures (from 0% to 3%) as eluent. The puriﬁcation gave the

desired pure product in high yield (299 mg, 73% yield). Light yellow

solid. M.p. 190e192 ^ꢀC. R_f(2% EtOH/CH₂Cl₂): 0.41; ¹H NMR (CDCl₃,

To a suspension of the corresponding nitro compound (2, 7, or 8,

100 mg scale) in a mixture of EtOH/H₂O (3 þ 1 mL), iron (325 mesh

powder, 2.5 equiv) and NH₄Cl (6 equiv) were added. The reaction

mixture was heated at 90 ^ꢀC in a round bottom ﬂask equipped with

a reﬂux system. After 2 h, the reaction mixture had become black

and the conversion showed to be complete by TLC, giving the

desired compound as the major product. The resulting mixture was

ﬁltered through a short pad of celite to remove the metal residues,

and the solids were carefully washed several times with EtOAc and

with a ﬁnal fraction of EtOH. The ﬁltrates were concentrated in

vacuo and the residue was directly puriﬁed by column chroma-

tography, using EtOH/CH₂Cl₂mixtures (from 2% to 10%) as eluent.

This puriﬁcation led to the pure desired products in good to high

yields (65e89% yields).

300 MHz) d (ppm): 1.64e1.87 (m, 3H), 2.05e2.23 (m, 3H), 3.84 (dd,

J ¼ 12.3 Hz, 1H), 4.22 (dd, J ¼ 12.3 Hz, 1H), 5.88 (dd, J ¼ 9.3 Hz, 1H),

7.74 (dd, J ¼ 9.6 Hz, 1H), 8.37 (dd, J ¼ 9.3 Hz,1H), 8.41 (s, 1H), 9.07 (s,

1H), 9.22 (d, J ¼ 9 Hz, 1H), 9.72 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

4.6.1. 3-Fluoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

yloxy)aniline (3)

According to procedure C in 69% yield. Slightly coloured white

d (ppm): 22.8, 24.9, 31.9, 69.0, 82.2, 124.7, 125.3, 129.6, 131.3, 135.5,

solid. M.p. (decomposition) 120 ^ꢀC. R_f(6% EtOH/CH₂Cl₂): 0.40. ¹H

137.4, 142.9, 148.7, 151.9, 152.1, 152.4. MS (EI) ES^þ: 326 ([M^þþ H],

NMR (CDCl₃, 300 MHz)

d (ppm): 1.68e1.75 (m, 3H), 2.08e2.14 (m,

78), 242 ([M^þþH-THP], 100).

3H), 3.71e3.79 (m, 1H), 4.17e4.22 (m, 1H), 5.79 (dd, J ¼ 9.3 Hz, 1H),

6.47e6.56 (m, 2H), 7.08 (t, J ¼ 9 Hz, 1H), 8.24 (s, 1H), 8.52 (s, 1H). ¹³C

4.8. General procedure D for the reaction of phenylaminopurines

with phenyl carboxylic acids

NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9, 31.8, 68.8, 82.2, 103.5,

110.7, 121.3, 124.2, 131.1, 140.9, 145.7, 152.2, 153.3, 156.5, 160.0. MS

(EI) ES^þ: 352 ([M^þþ Na], 65), 330 ([M^þþ H], 100).

An experimental procedure previously described, that employs

HATU as the coupling reagent [8] was used for the reaction between

the four different amino purine derivatives (3, 9, 10 and 16) with

several aryl carboxylic acids (both commercially available 4aee or

compounds 4fei). The corresponding amino purine derivative

(50 mg scale) was dissolved in 3 mL of anhydrous DMF or CH₂Cl₂

4.6.2. N1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)benzene-

1,4-diamine (9)

According to procedure C in 82% yield. Brownish solid. M.p.

210e212 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.18. ¹H NMR (CDCl₃, 300 MHz)

14

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

under argon, and the corresponding commercial or previously

synthesized arylcarboxilic acid (1.15 equiv) and HATU (1.2 equiv)

were added. The mixture was cooled at 0 ^ꢀC (ice/water bath) and

DIPEA (1.2 equiv) was added dropwise via a syringe. The reaction

mixture was allowed to reach room temperature, and the reaction

was already complete after 3 h. The crude product was concen-

trated in vacuo and redissolved in EtOAc. It was washed twice with

saturated aqueous NH₄Cl and once with brine. The organic phase

was dried over anhydrous MgSO₄. The solids were ﬁltered off and

the solvent was removed in vacuo. This crude material was puriﬁed

by column chromatography over silica gel, using EtOH/CH₂Cl₂

mixtures (0%e3% ethanol) as eluent, giving the desired products in

high yields (71e99%).

(dd, J ¼ 9.6 Hz, 1H), 7.23-7.29 (m, 1H), 7.38 (bd, J ¼ 9Hz, 1H), 7.60 (d,

J ¼ 9 Hz, 2H), 7.60-7.64 (m, 1H), 7.87 (d, J ¼ 9 Hz, 2H), 7.96 (dd,

J ¼ 12.3 Hz, 1H), 8.25 (s, 1H), 8.51 (s, 1H), 8.77 (dd, J ¼ 9.3 Hz, 1H),

11.87 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9, 31.9,

68.9, 82.3, 107.6, 109.2, 116.1, 121.2, 122.4, 123.8, 127.1, 127.2, 135.7,

137.1, 141.0, 141.1, 145.5, 152.1, 152.4, 155.9, 159.5, 161.1, 162.2. MS

(EI) ES^þ: 617 ([M^þþ Na], 100), 595 ([M^þþ H], 2), 533 ([M^þþNa-

THPþH), 20).

4.8.5. 2-Fluoro-N-(4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)benzamide (11c)

According to procedure D. Yield 82%. Oil. R_f(5% EtOH/CH₂Cl₂):

0.4. ¹H NMR (CDCl₃, 300 MHz)

d (ppm): 1.68e1.79 (m, 3H),

2.04e2.13 (m, 3H), 3.80 (t, J ¼ 9 Hz, 1H), 4.19 (d, J ¼ 12 Hz, 1H), 5.76

(d, J ¼ 9 Hz, 1H), 7.19 (dd, J ¼ 12.9 Hz, 1H), 7.32 (t, J ¼ 6 Hz, 1H), 7.51

(m, 1H), 7.69 (d, J ¼ 9 Hz, 1H), 7.85 (d, J ¼ 9 Hz, 1H), 8.00 (s, 1H), 8.09

(s, 1H), 8.18 (dd, J ¼ 9.6 Hz, 1H), 8.48 (d, J ¼ 15 Hz, 1H), 8.55 (s, 1H).

4.8.1. 2-Fluoro-N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6-yloxy)phenyl)benzamide (5c)

According to procedure D. Yield 82%. Brownish solid. M.p.

232e234 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.48. ¹H NMR (CDCl₃, 300 MHz)

¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9, 31.9, 68.9, 81.9, 116.2,

d

(ppm): 1.69e1.80 (m, 3H), 2.09e2.15 (m, 3H), 3.81 (dd, J ¼ 12.9 Hz,

120.0,121.0,121.3,121.4,125.1,132.2,133.4,133.7,135.5,138.4,148.8,

152.1, 153.0, 158.7, 161.2, 162.0. MS (EI) ES^þ: 455 ([M^þþ Na], 100),

371 ([M^þþ Na-THP þ H], 65).

1H), 4.20 (d, J ¼ 12 Hz, 1H), 5.81 (dd, J ¼ 9.3 Hz, 1H), 7.17e7.24 (m,

2H), 7.32e7.36 (m, 2H), 7.52e7.59 (m, 1H), 7.89 (dd, J ¼ 12.3 Hz, 1H),

8.19 (dt, J ¼ 6.3 Hz, 1H), 8.29 (s, 1H), 8.52 (s, 1H), 8.58 (bs, 1H). ¹³C

NMR (CDCl₃, 75 MHz)

d

(ppm): 22.8, 24.9, 31.9, 68.9, 82.3, 109.6,

4.8.6. 2-Fluoro-N-(4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)pyridine-3-carboxamide (11d)

116.1, 116.4, 121.0, 124.0, 125.3, 132.3, 134.2, 135.9, 136.5, 141.2, 152.1,

152.6, 156.0, 158.8, 159.4, 161.3, 162.0. MS (EI) ES^þ: 474 ([M^þþ Na],

100), 452 ([M^þþ H], 2), 390 ([M^þþ Na-THP þ H), 45).

According to procedure D. Yield 88%. Brownish solid. M.p.

156e158 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.67. ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.68e1.78 (m, 3H), 2.05e2.13 (m, 3H), 3.81 (dd, J ¼ 12.9 Hz,

4.8.2. N-(3-fluoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

yloxy)phenyl)-1-(3-ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-

carboxamide (5f)

1H), 4.19 (d, J ¼ 12 Hz, 1H), 5.76 (d, J ¼ 9 Hz, 1H), 7.41e7.45 (m, 1H),

7.70 (d, J ¼ 9 Hz, 2H), 7.85 (d, J ¼ 9 Hz, 1H), 8.08 (s, 1H), 8.39 (d,

J ¼ 3 Hz, 1H), 8.56 (dd, J ¼ 9.6 Hz, 1H), 8.68 (dt, J ¼ 9.3 Hz, 1H). MS

(EI) ES^þ: 456 ([M^þþ Na], 100), 434 ([M^þþ H], 2), 372

([M^þþ H þ Na-THP], 50).

According to procedure D. Yellowish solid. Yield 92%. M.p.

>250 ^ꢀC. R_f(7% EtOH/CH₂Cl₂): 0.39 ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.69e1.79 (m, 3H), 2.08e2.17 (m, 3H), 3.80 (dt, J ¼ 12.3 Hz,

1H), 4.19 (bd, J ¼ 12 Hz, 1H), 5.80 (dd, J ¼ 9.3 Hz, 1H), 6.62 (t,

J ¼ 6 Hz, 1H), 7.19e7.28 (m, 4H), 7.38 (dd, J ¼ 9.3 Hz, 1H), 7.53e7.59

(m, 1H), 7.63 (dd, J ¼ 6.3 Hz, 1H), 7.96 (dd, J ¼ 12.3 Hz, 1H), 8.25 (s,

1H), 8.51 (s, 1H), 8.76 (dd, J ¼ 6.3 Hz, 1H), 11.93 (s, 1H). ¹³C NMR

4.8.7. 2-Methoxy-N-(4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-

6-ylamino)phenyl)pyridine-3-carboxamide (11e)

According to procedure D. Yield 89%. White solid. M.p.

(decomposition) 170 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.33 ¹H NMR (CDCl₃,

(CDCl₃, 75 MHz)

d

(ppm): 22.8, 24.9, 31.9, 68.9, 82.3, 107.4, 109.2,

300 MHz) d (ppm): 1.68e1.84 (m, 3H), 2.09e2.13 (m, 3H),

109.5, 114.4, 114.7, 116.1, 116.7, 121.2, 122.3, 122.4, 123.8, 131.2, 135.4,

135.6, 137.1, 141.1, 141.3, 145.3, 152.1, 152.4, 159.6, 161.3, 162.2. MS

(EI) ES^þ: 567 ([M^þþ Na], 100), 545 ([M^þþ H], 2), 483 ([M^þþ Na-

THP þ H), 35).

3.81e3.96 (m, 1H), 4.18e4.21 (m, 1H), 4.21 (s, 1H), 5.76 (d, J ¼ 9 Hz,

1H), 7.13 (m, 1H), 7.71 (d, J ¼ 6 Hz, 2H), 7.80 (t, J ¼ 9 Hz, 3H), 8.07 (s,

1H), 8.33 (bs, 1H), 8.55 (s, 1H), 8.61 (d, J ¼ 6 Hz, 1H), 9.84 (s, 1H). MS

(EI) ES^þ: 468 ([M^þþ Na], 100), 384 ([M^þþ Na-THP þ H], 45); ES^ꢁ:

444 ([M^þ-H], 100).

4.8.3. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

yloxy)phenyl)-1-(4-ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-

carboxamide (5g)

4.8.8. 1-(3-ﬂuorophenyl)-2-oxo-N-(4-(9-(tetrahydro-2H-pyran-2-

yl)-9H-purin-6-ylamino)phenyl)-1,2-dihydropyridine-3-

carboxamide (11f)

According to procedure D. White solid. Yield 99%. M.p.

(decomposition) > 220 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.41 ¹H NMR

According to procedure D. Yield 91%. R_f(5% EtOH/CH₂Cl₂): 0.35.

(CDCl₃, 300 MHz)

d

(ppm): 1.69e1.79 (m, 3H), 2.08e2.14 (m, 3H),

¹H NMR (CDCl₃, 300 MHz)

d (ppm): 1.67e1.78 (m, 3H), 1.96e2.17

3.80 (t, J ¼ 9 Hz, 1H), 4.20 (bd, J ¼ 12 Hz, 1H), 5.80 (dd, J ¼ 12.3 Hz,

1H), 6.61 (dd, J ¼ 9.6 Hz, 1H), 7.24e7.30 (m, 3H), 7.36e7.42 (m, 3H),

7.62 (dd, J ¼ 6.3 Hz, 1H), 7.95 (dd, J ¼ 12.3 Hz, 1H), 8.25 (s, 1H), 8.51

(s, 1H), 8.75 (dd, J ¼ 6.3 Hz, 1H), 11.96 (s, 1H). ¹³C NMR (CDCl₃,

(m, 3H), 3.76e3.80 (m, 1H), 4.19 (d, J ¼ 9 Hz, 1H), 5.74 (dd,

J ¼ 9.3 Hz,1H), 6.61 (dd, J ¼ 9.6 Hz,1H), 7.21e7.24 (m, 3H), 7.55e7.61

(m, 2H), 7.76 (bs, 5H), 8.06 (s, 1H), 8.54 (s, 1H), 8.75 (dd, J ¼ 9.3 Hz,

1H), 11.76 (s, 1H). MS (EI) ES^þ: 548 ([M^þþ Na], 100), 526 ([M^þþ H],

10), 464 ([M^þþ Na-THP þ H], 48).

75 MHz)

d (ppm): 22.8, 24.9, 31.9, 68.9, 82.3, 107.3, 109.5, 116.1,

116.7, 117.0, 121.2, 122.1, 123.8, 128.4, 128.5, 135.9, 137.3, 141.1, 141.8,

145.2, 152.1, 152.4, 159.6, 161.4, 162.5, 174.9. MS (EI) ES^þ: 567

([M^þþ Na], 80), 483 ([M^þþ Na-THP þ H), 45).

4.8.9. 2-Oxo-N-(4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)-1-(3-(triﬂuoromethyl)phenyl)-1,2-

dihydropyridine-3-carboxamide (11i)

4.8.4. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

yloxy)phenyl)-2-oxo-1-(4-(triﬂuoromethyl)phenyl)-1,2-

dihydropyridine-3-carboxamide (5h)

According to procedure D. Yield 84%. Yellow solid. M.p.

210e230 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.38 ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.64e1.78 (m, 3H), 2.08e2.12 (m, 3H), 3.76e3.83 (m, 1H),

According to procedure D. Yield 71%. Slightly coloured solid

4.19 (d, J ¼ 12 Hz, 1H), 5.75 (d, J ¼ 9 Hz, 1H), 6.64 (dd, J ¼ 9.6 Hz, 1H),

7.58e7.64 (m, 4H), 7.76 (bs, 4H), 7.87 (d, J ¼ 9 Hz, 2H), 8.06 (s, 1H),

8.54 (s, 1H), 8.77 (dd, J ¼ 6.3 Hz, 1H), 11.70 (s, 1H). MS (EI) ES^þ: 598

([M^þþ Na], 100), 576 ([M^þþ H], 30), 514 ([M^þþ Na-THPþH], 45).

(beige) M.p. > 260 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.40; ¹H NMR (CDCl₃,

300 MHz)

d (ppm): 1.69e1.80 (m, 3H), 2.11e2.15 (m, 3H), 3.80 (t,

J ¼ 9 Hz, 1H), 4.20 (bd, J ¼ 12 Hz, 1H), 5.80 (bd, J ¼ 12 Hz, 1H), 6.66

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

15

4.8.10. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)-2-methoxybenzamide (12a)

4.8.15. 4-Fluoro-N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6-ylamino)phenyl)benzamide (12f)

According to procedure D. Yield 82%. White solid. M.p.

According to procedure D. Yield 93%. Yellow solid. M.p.

166e168 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.36 ¹H NMR (CDCl₃, 300 MHz)

(decomposition) > 250 ^ꢀC (Koﬂer). R_f(5% EtOH/CH₂Cl₂): 0.34. ¹H

d

(ppm): 1.67e1.78 (m, 3H), 2.09e2.17 (m, 3H), 3.71e3.83 (m, 1H),

NMR (CDCl₃, 300 MHz) d (ppm): 1.68e1.78 (m, 3H), 2.09e2.12 (m,

4.07 (s, 3H Me), 4.17e4.21 (m, 1H), 5.75 (d, J ¼ 9 Hz, 1H),

7.02e7.05 (d, J ¼ 9 Hz, 1H), 7.11e7.16 (m, 1H), 7.20e7.28 (m, 1H),

7.50 (m, 1H), 7.81 (bs, 1H), 7.93 (d, J ¼ 12 Hz, 1H), 8.08 (s, 1H), 8.27

(d, J ¼ 6 Hz, 1H), 8.52 (m, 1H), 8.55 (s, 1H), 9.87 (s, 1H). ¹³C NMR

3H), 3.76e3.83 (m, 1H), 4.17e4.21 (m, 1H), 5.76 (d, J ¼ 9 Hz, 1H),

6.60 (dd, J ¼ 9.6 Hz, 1H), 7.25e7.30 (m, 2H), 7.39e7.43 (m, 2H), 7.61

(d, J ¼ 6 Hz, 1H), 7.71 (bs, 1H), 7.99 (d, J ¼ 12 Hz), 8.07 (s, 1H),

8.51e8.55 (m, 1H), 8.55 (bs, 1H), 8.74 (d, J ¼ 6 Hz, 1H), 11.89 (s, 1H).

(CDCl₃, 75 MHz)

d

(ppm): 22.8, 24.9, 31.9, 56.3, 68.8, 82.0, 108.2,

¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9, 31.9, 68.8, 81.9, 107.3,

111.5, 115.7, 120.5, 121.4, 121.7, 122.6, 122.8, 122.9, 132.4, 133.4,

134.5, 138.7, 149.1, 152.0, 152.8, 157.2, 163.2. MS (EI) ES^þ: 463

([M^þþ H], 8), 485 ([M^þþ Na], 100); ES^ꢁ: 461 ([M^þ-H], 100).

108.1, 115.9, 116.7, 117.0, 120.6, 122.3, 123.1, 128.4, 128.5, 134.3, 135.9,

138.6, 141.5, 145.0, 149.1, 151.9, 152.8, 161.2, 162.5. MS (EI) ES^þ: 544

([M^þþ H], 100).

4.8.11. 4-Bromo-N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6-ylamino)phenyl)-3-methylbenzamide (12b)

4.8.16. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)-1-(4-ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-

carboxamide (12g)

According to procedure D. Slightly brownish solid. Yield 80%.

M.p. >250 ^ꢀC (Koﬂer). R_f(5% EtOH/CH₂Cl₂): 0.33. ¹H NMR (CDCl₃,

According to procedure D. Yield 87%. Yellow solid. M.p.

300 MHz)

d (ppm): 1.65e1.78 (m, 3H), 2.09e2.17 (m, 3H), 2.46 (s,

(decomposition) 166 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.32. ¹H NMR (CDCl₃,

3H), 3.80 (dd, J ¼ 12.9 Hz, 1H), 4.19 (bd, J ¼ 12 Hz, 1H), 5.75 (dd,

J ¼ 9.3 Hz, 1H THP), 7.24 d, J ¼ 9 Hz, 1H), 7.52 (bd, J ¼ 9 Hz, 1H), 7.62

(d, J ¼ 6 Hz, 1H), 7.75 (d, J ¼ 12 Hz, 2H), 7.85 (dd, J ¼ 12.3 Hz, 1H),

8.01 (bs, 1H), 8.09 (s, 1H), 8.55 (s, 1H), 8.60 (d, J ¼ 9 Hz, 1H). ¹³C NMR

300 MHz) d (ppm): 1.68e1.78 (m, 3H), 2.09e2.17 (m, 3H), 3.76e3.84

(m, 1H), 4.17e4.21 (m, 1H), 5.76 (d, J ¼ 9 Hz,1H), 6.62 (dd, J ¼ 9.6 Hz,

1H), 7.21e7.26 (m, 2H), 7.53e7.61 (m, 2H), 7.72 (bs, 1H), 7.99 (d,

J ¼ 12 Hz, 1H), 8.07 (s, 1H), 8.52e8.55 (m, 2H), 8.55 (bs, 1H), 8.75

(CDCl₃, 75 MHz)

d

(ppm): 23.0, 24.9, 31.9, 68.9, 82.0, 107.8, 108.2,

(bd, J ¼ 6 Hz, 1H), 11.85 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm):

115.6, 120.6, 122.4, 123.6, 125.6, 129.1, 129.4, 132.8, 133.7, 138.8,

149.1, 151.9, 152.7, 164.9. MS (EI) ES^þ: 547 ([M^þþ Na], 90), 549

(100); ES^ꢁ: 523 ([M^þ-H], 100).

22.8, 24.9, 31.9, 68.9, 82.0, 100.0, 107.4, 107.8, 108.1, 114.8, 115.9,

120.6, 122.2, 122.2, 122.4, 123.0, 131.1, 131.2, 134.3, 138.7, 141.2, 145.1,

149.1, 151.9, 152.8, 154.7, 161.1, 162.2. MS (EI) ES^þ: 566 ([M^þþ Na],

100), 544 ([M^þþ H], 20); ES^ꢁ: 542 ([M^þ-H], 65).

4.8.12. 2-Fluoro-N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6-ylamino)phenyl)benzamide (12c)

4.8.17. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)-2-oxo-1-(4-(triﬂuoromethyl)phenyl)-1,2-

dihydropyridine-3-carboxamide (12h)

According to procedure D. Yield 86%. White solid. M.p.

(decomposition) 170 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.32. ¹H NMR (CDCl₃,

300 MHz)

d

(ppm): 1.68e1.79 (m, 3H), 2.09e2.17 (m, 3H),

According to procedure D. Yield 82%. White solid. M.p.

3.77e3.84 (m, 1H), 4.18e4.21 (m, 1H), 5.76 (d, J ¼ 12 Hz, 1H),

7.16e7.35 (m, 3H), 7.52 (bs, 1H), 7.89 (bt, J ¼ 12 Hz, 2H), 8.02 (s, 1H),

8.15 (m, 1H), 8.56 (s, 1H), 8.49e8.62 (m, 2H). ¹³C NMR (CDCl₃,

(decomposition) > 250 ^ꢀC (Koﬂer). R_f(5% EtOH/CH₂Cl₂): 0.35. ¹H

NMR (CDCl₃, 300 MHz)

d (ppm): 1.68e1.78 (m, 3H), 2.09e2.12 (m,

3H), 3.76e3.83 (m, 1H), 4.17e4.21 (m, 1H), 5.76 (d, J ¼ 9 Hz, 1H),

6.65 (dd, J ¼ 9.6 Hz, 1H), 7.24e7.27 (m, 1H), 7.58e7.60 (m, 3H), 7.72

(bs, 1H), 7.86 (d, J ¼ 9 Hz, 2H), 8.07 (d, J ¼ 15 Hz, 1H), 8.08 (s, 1H),

8.52e8.55 (m, 1H), 8.55 (bs, 1H), 8.76 (d, J ¼ 6 Hz, 1H), 11.80 (s, 1H).

75 MHz)

d (ppm): 22.8, 24.9, 31.9, 68.9, 82.0, 108.2, 115.8, 116.2,

120.6, 121.0, 122.5, 123.6, 125.2, 132.3, 133.6, 134.0, 138.8, 149.1,

151.5, 152.3, 154.7, 158.7, 161.2.

¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9, 31.9, 68.9, 82.0, 107.6,

4.8.13. 2-Fluoro-N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6 ylamino)phenyl)pyridine-3-carboxamide (12d)

108.1, 115.8, 120.6, 122.3, 122.5, 123.1, 127.1, 127.2, 134.3, 138.7, 140.8,

142.8, 145.2, 149.1, 151.9, 152.8, 161.0, 162.2. MS (EI) ES^þ: 616

([M^þþ Na], 100), 594 ([M^þþ H], 20); ES^ꢁ: 592 ([M^þ-H], 100).

According to procedure D. Yield 89%. Slightly coloured solid

(beige). M.p. (decomposition) 110 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.24;

¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.72e1.79 (m, 3H), 2.10e2.17

4.8.18. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)-2-oxo-1-(3-(triﬂuoromethyl)phenyl)-1,2-

dihydropyridine-3-carboxamide (12i)

(m, 3H), 3.77e3.84 (m, 1H), 4.18e4.22 (m, 1H), 5.76 (d, J ¼ 9 Hz,

1H), 7.25 (m, 1H), 7.44 (bs, 1H), 7.79 (s, 1H), 7.90 (d, J ¼ 12 Hz,

1H), 8.10 (s, 1H), 8.40 (bs, 1H), 8.57 (s, 1H), 8.61e8.70 (m, 3H). MS

(EI) ES^þ: 452 ([M^þþ H], 10), 474 ([M^þþ Na], 100); ES^ꢁ: 450 ([M^þ-

H], 100).

According to procedure D. Yellow solid. Yield 98%. M.p.

(decomposition) > 250 ^ꢀC (Koﬂer). R_f(5% EtOH/CH₂Cl₂): 0.35 ¹H

NMR (CDCl₃, 300 MHz)

d (ppm): 1.67e1.85 (m, 3H), 2.09e2.17 (m,

3H), 3.76e3.83 (m, 1H), 4.17e4.21 (m, 1H), 5.75 (d, J ¼ 12 Hz, 1H),

6.64 (t, J ¼ 6 Hz, 1H), 7.27 (m, 1H), 7.61e7.63 (m, 1H), 7.68 (bs, 1H),

7.72e7.76 (m, 2H), 7.82 (m, 1H), 7.98 (d, J ¼ 12 Hz, 1H), 8.08 (s, 1H),

8.52e8.55 (m, 1H), 8.55 (bs, 1H), 8.76 (d, J ¼ 6 Hz, 1H), 11.79 (s, 1H).

MS (EI) ES^þ: 616 ([M^þþ Na], 100), 594 ([M^þþ H], 15); ES^ꢁ: 592

([M^þ-H], 100).

4.8.14. N-(3-ﬂuoro-4-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

ylamino)phenyl)-2-methoxypyridine-3-carboxamide (12e)

According to procedure D. Yield 83%. White solid. M.p.

166e168 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.34. ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.68e1.79 (m, 3H), 2.09e2.17 (m, 3H), 3.69e3.84 (m,

1H), 4.18e4.20 (m, 1H), 4.21 (s, 3H Me), 5.77 (d, J ¼ 9 Hz, 1H), 7.13

(dd, J ¼ 6.3 Hz, 1H), 7.26 (m, 1H), 7.79 (bs, 1H), 7.92 (bd, J ¼ 12 Hz,

1H), 8.09 (s, 1H), 8.33 (d, J ¼ 3 Hz, 1H), 8.56-8.61 (m, 1H), 8.61 (s,

4.9. General method E for the THP deprotection of 9-N-THP-purines

1H), 9.89 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9,

The precursors (50 mg scale) were suspended into a mixture of

CH₃CN/H₂O (2 þ 2 mL) and triﬂuoroacetic acid (1 mL/mmol of s.m.)

was added slowly at 0 ^ꢀC via a syringe. The reaction mixture was

allowed to reach room temperature. After 4 h [23], the crude was

concentrated till dryness and directly puriﬁed through a short pad

31.9, 54.5, 68.9, 82.0, 108.1, 115.8, 116.0, 118.2, 120.6, 122.5, 123.2,

134.0, 138.7, 141.8, 149.1, 150.1, 151.9, 152.7, 154.8, 160.2, 161.7.

MS (EI) ES^þ: 464 ([M^þþ H], 18), 486 ([M^þþ Na], 100); ES^ꢁ: 462

([M^þ-H], 85).

16

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

of silica gel, using mixtures EtOH/H₂O (from 3% to 10%) as eluent.

The desired ﬁnal products were isolated as pure compounds with

high recovery after chromatography.

349 ([M^þþ H], 85); EI^ꢁ: 347 ([M^þ-H], 100). HRMS-ESI (m/z) calcd

for C₁₈H₁₄FN₆O (M þ 1) 349.1213, found: 349.1213. HPLC purity

(method I) 100%.

4.9.1. N-(4-(9H-purin-6-yloxy)-3-ﬂuorophenyl)-2-ﬂuorobenzamide

(6c)

4.9.6. N-(4-(9H-purin-6-ylamino)phenyl)-2-ﬂuoropyridine-3-

carboxamide (13d)

According to method E, 6c was obtained as a slightly coloured

solid (beige). Yield 65%. R_f(7% EtOH/CH₂Cl₂): 0.18. M.p.

According to method E. Light yellow solid. Yield 96%. R_f(7%

EtOH/CH₂Cl₂): 0.11. M.p. 228.2e230.5 ^ꢀC. ¹H NMR (DMSO-d₆,

159.8e162.3 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

d

(ppm): 7.58 (dd,

300 MHz)

d

(ppm): 7.52 (dt, J ¼ 6.3 Hz, 1H), 7.70 (d, J ¼ 9 Hz, 2H),

J ¼ 9.6 Hz, 1H), 7.77 (bs, 2H), 8.02 (bs, 2H), 8.67 (bs, 1H), 8.98

7.89 (d, J ¼ 9 Hz, 2H), 8.26 (dt, J ¼ 9.3 Hz, 1H), 8.40 (s, 1H), 8.41

(s, 1H), 9.12 (bs, 1H), 10.54 (s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

(bs, 1H), 8.46 (s, 1H), 10.13 (bs, 1H), 10.58 (s, 1H). ¹³C NMR (DMSO-

d

(ppm): 108.5, 108.6, 116.4, 116.7, 116.8, 118.5, 124.8, 125.0, 125.1,

d₆, 75 MHz) d (ppm): 117.5, 120.0, 120.5, 120.6, 121.9, 122.6, 122.9,

130.3, 133.2, 135.5, 144.5, 151.7, 155.4, 156.3, 158.0. MS (EI) EI^þ:

390 ([M^þþ Na], 100), 368 ([M^þþ H], 30). HRMS-ESI (m/z) calcd

for C₁₈H₁₂F₂N₅O₂(M þ 1) 368.0959, found: 368.0958. HPLC purity

(method I) 100%.

134.9, 136.0, 141.6, 142.0, 149.7, 151.0, 151.0, 151.2, 163.8. MS (EI)

EI^þ: 372 ([M^þþ Na], 5), 350 ([M^þþ H], 100). HPLC purity

(method I) 98%.

4.9.7. N-(4-(9H-purin-6-ylamino)phenyl)-2-methoxypyridine-3-

carboxamide (13e)

4.9.2. N-(4-(9H-purin-6-yloxy)-3-ﬂuorophenyl)-1-(3-

ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (6f)

According to method E. Yield 89%. Yellow solid. R_f(8% EtOH/

6f was obtained according to method E. Yield 82%. M.p. > 250 ^ꢀC

CH₂Cl₂): 0.16. M.p. 325.2e335.5 ^ꢀC (decomposition). ¹H NMR

(Koﬂer). ¹H NMR (DMSO-d₆, 300 MHz)

d

(ppm): 6.75 (dd, J ¼ 9.6 Hz,

(DMSO-d₆, 300 MHz)

d

(ppm): 4.00 (s, 3H), 7.15 (t, J ¼ 6 Hz, 2H), 7.68

1H), 7.41e7.47 (m, 4H), 7.55e7.65 (m, 2H), 7.99 (dd, J ¼ 12.3 Hz, 1H),

8.16 (dd, J ¼ 6.3 Hz, 1H), 8.44 (s, 1H), 8.55 (bs, 1H), 8.61 (dd,

J ¼ 6.3 Hz, 1H), 12.06 (s, 1H), 13.69 (bs, 1H NH₉purine). ¹³C NMR

(d, J ¼ 9 Hz, 2H), 7.93 (bd, J ¼ 9 Hz, 2H), 8.07 (dd, J ¼ 6.3 Hz,1H), 8.27

(s, 1H), 8.32e8.36 (m, 2H), 9.78 (s, 1H), 10.15 (s, 1H), 13.16 (bs, 1H

NH9-purine). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 54.3, 117.6,

(DMSO-d₆, 75 MHz)

d

(ppm): 107.7, 108.6, 108.8, 115.7, 116.7, 117.1,

117.7, 119.8,120.6, 120.7,121.2, 136.3, 139.7, 139.8, 140.4, 149.3,149.4,

152.3, 160.2, 160.3. MS (EI) EI^þ: 384 ([M^þþ Na], 85), 362 ([M^þþ H],

100). HPLC purity (method I) 99%.

120.6,123.5,125.1,131.6,135.5,137.4,138.2,141.7,143.8,144.5,145.5,

145.6, 151.7, 162.0, 162.8. MS (EI) EI^þ: 483 ([M^þþ Na], 100), 461

([M^þþ H], 2); EI^ꢁ: 459 ([M^þ-H], 100). HRMS-ESI (m/z) calcd for

C₂₃H₁₅F₂N₆O₃(M þ 1) 461.1174, found: 461.1157. HPLC purity

(method I) 99%.

4.9.8. N-(4-(9H-purin-6-ylamino)phenyl)-1-(3-ﬂuorophenyl)-2-

oxo-1,2-dihydropyridine-3-carboxamide (13f)

According to method E. Yield 68%. Yellow solid. R_f(7% EtOH/

4.9.3. N-(4-(9H-purin-6-yloxy)-3-ﬂuorophenyl)-1-(4-

CH₂Cl₂): 0.14. M.p.: 323.5e328.1 ^ꢀC (decomposition). ¹H NMR

ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (6g)

According to method E, 6g was obtained as white solid. Yield

92%. R_f(6% EtOH/CH₂Cl₂): 0.17. M.p. 232.2e234.8 ^ꢀC. ¹H NMR

(DMSO-d₆, 300 MHz)

d

(ppm): 6.73 (dd, J ¼ 9.6 Hz, 1H), 7.39e7.44

(m, 2H), 7.54e7.62 (m, 2H), 7.67 (bd, J ¼ 9 Hz, 2H), 7.91 (bd,

J ¼ 9 Hz, 2H), 8.12 (dd, J ¼ 9.3 Hz, 1H), 8.30 (s, 1H), 8.38 (s, 1H), 8.59

(dd, J ¼ 9.3 Hz, 1H), 9.86 (s, 1H), 11.84 (s, 1H). ¹³C NMR (DMSO-d₆,

(DMSO-d₆, 300 MHz)

d

(ppm): 6.74 (dd, J ¼ 9.6 Hz, 1H), 7.40e7.46

(m, 4H), 7.60e7.63 (m, 2H), 7.98 (d, J ¼ 15 Hz, 1H), 8.14 (d,

75 MHz) d (ppm): 107.3, 115.4, 116.7, 118.8, 120.3, 120.4, 121.0, 121.6,

J ¼ 6 Hz, 1H), 8.44 (s, 1H), 8.55 (s, 1H), 8.60 (d, J ¼ 6 Hz, 1H), 12.10 (s,

123.8, 131.5, 133.7, 137.1, 141.0, 143.4, 143.9, 145.2, 151.4, 151.8, 152.0,

161.8, 162.1. MS (EI) EI^þ: 464 ([M^þþ Na], 80), 442 ([M^þþ H], 100);

EI^ꢁ: 440 ([M^þ-H], 100). HPLC purity (method I) 97.5%.

1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 54.1, 107.5, 108.7, 116.3,

116.6, 116.7, 129.8, 144.8, 145.6, 151.6. MS (EI) EI^þ: HRMS-ESI (m/z)

calcd for C₂₃H₁₅F₂N₆O₃(M þ 1) 461.1174; found: 461.1168. HPLC

purity (method I) 99%.

4.9.9. N-(4-(9H-purin-6-ylamino)phenyl)-2-oxo-1-(3-

(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-carboxamide (13i)

According to method E. Yield 98%. Yellow solid. R_f(7% EtOH/

CH₂Cl₂): 0.18. M.p. 288.5e292.5 ^ꢀC (decomposition). ¹H NMR

4.9.4. N-(4-(9H-purin-6-yloxy)-3-ﬂuorophenyl)-2-oxo-1-(4-

(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-carboxamide (6h)

According to method E, 6h was obtained as a slightly coloured

solid (beige). Yield 98%. R_f(7% EtOH/CH₂Cl₂): 0.22. M.p.

(DMSO-d₆, 300 MHz)

d

(ppm): 6.74 (t, J ¼ 6 Hz, 1H), 6.98 (s, 1H), 7.15

(s,1H), 7.32 (s,1H), 7.67 (d, J ¼ 9 Hz,1H), 7.83e7.92 (m, 4H), 8.04 (bs,

1H), 8.16 (dd, J ¼ 6.3 Hz, 1H), 8.29 (s, 1H), 8.37 (s, 1H), 8.60 (dd,

J ¼ 9.3 Hz, 1H), 9.82 (s, 1H), 11.79 (s, 1H). ¹³C NMR (DMSO-d₆,

276.5e279.9 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

d (ppm): 6.78 (dd,

J ¼ 9.6 Hz, 1H), 7.46 (bs, 2H), 7.83 (d, J ¼ 9 Hz, 2H), 7.99 (d, J ¼ 9 Hz,

3H), 8.20 (dd, J ¼ 9.3 Hz, 1H), 8.43 (s, 1H), 8.52 (bs, 1H), 8.62 (dd,

75 MHz) d (ppm): 107.7, 118.9, 120.2, 120.3, 121.1, 121.6, 124.8, 126.1,

J ¼ 6.3 Hz, 1H), 12.02 (s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d

(ppm):

126.4, 130.3, 131.0, 132.2, 133.3,136.3, 140.7, 141.1, 143.9, 144.1, 145.2,

151.5, 152.3, 162.4. MS (EI) EI^þ: 514 ([M^þþ Na], 55), 492 ([M^þþ H],

100); EI^ꢁ: 490 ([M^þ-H], 100). HPLC purity (method I) 100%.

107.8, 108.6, 108.7, 116.7, 117.4, 121.2, 125.0, 127.1, 127.1, 128.7, 130.0,

143.7, 143.9, 144.1, 143.9, 145.6, 145.8, 151.7, 161.9, 162.4. MS (EI) EI^þ:

533 ([M^þþ Na], 50), 511 ([M^þþ H], 2); EI^ꢁ: 509 ([M^þ-H], 100).

HRMS-ESI (m/z) calcd for C₂₄H₁₅F₄N₆O₃(M þ 1) 511.1142, found:

511.1151. HPLC purity (method I) 100%.

4.9.10. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-2-

methoxybenzamide (14a)

According to method E. Yield 94%. Slightly coloured white solid.

4.9.5. N-(4-(9H-purin-6-ylamino)phenyl)-2-ﬂuorobenzamide (13c)

According to method E. Slightly coloured white solid. Yield 94%.

R_f(8% EtOH/CH₂Cl₂): 0.18 m.p.: 219.7e223.5 ^ꢀC 1H NMR (DMSO-d₆,

R_f(8% EtOH/CH₂Cl₂): 0.19. M.p. 275.6e278.9 ^ꢀC. ¹H NMR (DMSO-d₆,

300 MHz)

d

(ppm): 3.90 (s, 3H), 7.08 (dd, J ¼ 9.6 Hz, 1H), 7.19 (d,

J ¼ 6Hz, 1H), 7.49 (t, J ¼ 9 Hz, 2H), 7.63 (d, J ¼ 9 Hz, 2H), 7.81 (d,

300 MHz)

1H), 7.68 (d, J ¼ 6 Hz, 3H), 7.91 (d, J ¼ 9 Hz, 2H), 8.30 (s, 1H), 8.38 (s,

1H), 9.81 (s,1H),10.37 (s,1H). ¹³C NMR (DMSO-d₆, 75 MHz)

(ppm):

d (ppm): 7.12 (s, 1H), 7.29e7.38 (m, 2H), 7.57e7.59 (m,

J ¼ 15 Hz, 1H), 8.26 (d, J ¼ 3 Hz, 2H), 9.39 (bs, 1H), 10.30 (s, 1H). ¹³C

NMR (DMSO-d₆, 75 MHz)

d (ppm): 56.2, 107.7, 112.5, 112.6, 115.7,

d

118.6, 120.9, 120.9, 125.0, 125.1, 127.8, 130.0, 132.9, 141.0, 152.0,

152.3, 157.0, 165.4. MS (EI) ES^þ: 379 ([M^þþ H], 12), 401 ([M^þþ Na],

100). HPLC purity (method I) 100%.

116.4, 116.7, 120.2, 120.2, 121.2, 121.5, 125.1, 125.4, 130.6, 132.9, 134.1,

136.3, 140.7, 151.5, 152.3, 162.3. MS (EI) EI^þ: 371 ([M^þþ Na], 100),

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

17

4.9.11. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-4-bromo-3-

methylbenzamide (14b)

1H), 8.58 (bs, 1H), 9.33 (s, 1H), 12.00 (s, 1H), 13.14 (bs, 1H NH9-pur).

¹³C NMR (DMSO-d₆, 75 MHz)

(ppm): 107.5, 107.7, 107.7, 114.4,

d

According to method E. Yield 85%. Light yellow solid. R_f(8%

115.5,115.6,115.9,116.5,120.8,122.5, 123.6, 128.4, 131.4,140.5, 141.8,

144.1, 144.2, 144.3, 145.4, 145.4, 152.4, 161.9, 162.2. MS (EI) ES^þ: 460

([M^þþ H], 12), 482 ([M^þþ Na], 100). HPLC purity (method I) 91.2%.

EtOH/CH₂Cl₂): 0.19. M.p. 323.2e325.4 ^ꢀC. ¹H NMR (DMSO-d₆,

300 MHz)

d

(ppm): 2.45 (s, 3H), 7.56 (d, J ¼ 9 Hz, 1H), 7.66e7.86 (m,

4H), 7.95 (s, 1H), 8.36 (d, J ¼ 3Hz, 2H), 9.68 (bs, 1H), 10.50 (s, 1H). ¹³C

NMR (DMSO-d₆, 75 MHz)

d

(ppm): 22.9, 107.7, 108.3, 116.3, 116.4,

4.9.17. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-2-oxo-1-(4-

(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-carboxamide (14h)

According to method E. Yellow solid. Yield 72%. R_f(8% EtOH/

CH₂Cl₂): 0.19. M.p. 316.8e318.9 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

117.6, 121.8, 127.4 (CH), 127.5, 130.6, 132.6, 134.2, 138.0, 141.6, 151.5,

151.3, 155.1, 165.2, 165.3. MS (EI) ES^þ: 441 ([M^þþ H], 100). HPLC

purity (method A) 97.5%. HPLC purity (method I) 97.5%.

d

(ppm): 6.77 (t, J ¼ 6 Hz, 1H), 7.32 (d, J ¼ 9 Hz, 1H), 7.68 (dd,

4.9.12. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-2-

ﬂuorobenzamide (14c)

J ¼ 9.6 Hz, 1H), 7.81 (d, J ¼ 9 Hz, 2H), 7.89 (d, J ¼ 12 Hz, 1H), 7.98 (d,

J ¼ 9 Hz, 2H), 8.16 (bd, J ¼ 3 Hz, 1H), 8.26 (s, 2H), 8.61 (bd, J ¼ 6 Hz,

1H), 9.37 (bs, 1H), 11.96 (s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

According to method E. Yield 88%. White solid. R_f(8% EtOH/

CH₂Cl₂): 0.19. M.p.: 288.6e289.4 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

d

(ppm): 107.8, 107.9, 108.0, 115.6, 115.8, 120.6, 120.9, 127.5, 127.1,

d

(ppm): 7.33e7.41 (m, 2H), 7.46 (d, J ¼ 6 Hz, 1H), 7.57e7.78 (m, 4H),

8.23 (bs, 2H), 9.36 (s, 1H), 10.62 (s, 1H), 13.13 (bs, 1H NH9-purine).

¹³C NMR (DMSO-d₆, 75 MHz)

(ppm): 107.6, 107.7, 107.8, 115.4,

128.6, 128.7, 129.6, 141.0, 143.8, 143.9, 144.0, 145.5, 152.1, 152.3,

161.8, 162.3. MS (EI) EI^þ: 510 ([M^þþ H], 55), 532 ([M^þþ Na], 100).

HPLC purity (method I) 92.1%.

d

115.6, 116.7, 117.0, 122.6, 125.1, 127.8, 130.0, 130.3, 133.2, 140.3, 151.6,

152.6, 163.4. MS (EI) ES^þ: 367 ([M^þþ H], 35), 389 ([M^þþ Na], 100).

HPLC purity (method I) 100%.

4.9.18. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-2-oxo-1-(3-

(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-carboxamide (14i)

According to method E. Yield 81%. Yellow solid. R_f(8% EtOH/

CH₂Cl₂): 0.23. M.p. 307.4e309.2 ^ꢀC. ¹H NMR (DMSO-d₆,

4.9.13. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-2-

ﬂuoropyridine-3-carboxamide (14d)

300 MHz)

d

(ppm): 6.75 (dd, J ¼ 9.6 Hz, 1H), 7.36 (d, J ¼ 6 Hz,

According to method E. Yield 97%. White solid. R_f(8% EtOH/

1H), 7.69 (t, J ¼ 9 Hz, 1H), 7.83e7.93 (m, 4H), 8.04 (s, 1H), 8.16

CH₂Cl₂): 0.17. M.p. 298.5e299.6 ^ꢀC (decomposition). ¹H NMR

(dd, J ¼ 6.3 Hz, 1H), 8.34 (s, 2H), 8.60 (dd, J ¼ 6.3 Hz, 1H), 11.97 (s,

(DMSO-d₆, 300 MHz)

d

(ppm): 7.45 (d, J ¼ 9 Hz, 1H), 7.54 (dd,

1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 107.7, 108.0, 108.1,

J ¼ 6.3 Hz, 1H), 7.68e7.80 (m, 2H), 8.29 (m, 2H), 8.31 (s, 1H), 8.42 (d,

115.9, 116.1, 117.6, 120.7, 124.8, 125.8, 127.7, 130.7, 131.9, 136.7,

141.6, 144.1, 144.2, 145.4, 145.5, 151.3, 151.9, 161.9, 162.4. MS (EI)

EI^þ: 509.9 ([M^þþ H], 35), 532 ([M^þþ Na], 100). HPLC purity

(method I) 97.2%.

J ¼ 3 Hz, 1H), 9.52 (bs, 1H), 10.79 (s, 1H). ¹³C NMR (DMSO-d₆,

75 MHz) d (ppm): 107.6, 107.7, 115.8, 115.9, 118.1, 119.4, 122.7, 122.8,

122.9,127.5, 127.7, 141.6,150.0,151.9,152.0, 152.0, 161.9. MS (EI) ES^þ:

368 ([M^þþ H], 25), 390 ([M^þþ Na], 100). HPLC purity (method I)

100%.

4.9.19. 2-Methoxy-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-

6-yl)phenyl)benzamide (17a)

4.9.14. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-2-

methoxypyridine-3-carboxamide (14e)

According to procedure D. Yield 85%. R_f(5% EtOH/CH₂Cl₂): 0.54

¹H NMR (CDCl₃, 300 MHz)

d (ppm): 1.70e1.80 (m, 3H), 2.10e2.16

According to method E. Yield 95%. White solid. R_f(8% EtOH/

(m, 3H), 3.68e3.71 (m, 1H), 3.83 (dd, J ¼ 12.9 Hz, 1H), 4.10 (s, 3H),

5.86 (d, J ¼ 9 Hz, 1H), 7.04e7.16 (m, 3H), 7.46e7.60 (m, 3H), 8.10 (d,

J ¼ 9 Hz, 1H), 8.28 (dd, J ¼ 9 Hz, 2H), 8.38 (s, 1H), 8.60 (d, J ¼ 6 Hz,

1H), 8.83 (s, 1H), 9.04 (s, 1H), 10.00 (s, 1H). MS (EI) ES^þ: 452

([M^þþ Na], 95), 430 ([M^þþ H], 2); ES^ꢁ: 344 ([M^þ-THP], 100).

CH₂Cl₂): 0.19. M.p. 207.3e210.5 ^ꢀC (decomposition). ¹H NMR

(DMSO-d₆, 300 MHz)

d

(ppm): 4.00 (s, 3H), 7.17 (t, J ¼ 6 Hz, 1H), 7.50

(t, J ¼ 9 Hz, 1H), 7.68 (dd, J ¼ 9.6 Hz, 1H), 7.83 (d, J ¼ 15 Hz, 1H), 8.07

(d, J ¼ 6 Hz, 1H), 8.36 (bs, 3H), 9.73 (bs, 1H), 10.42 (s, 1H). ¹³C NMR

(DMSO-d₆, 75 MHz)

d (ppm): 54.3, 55.3, 107.6, 107.7, 115.8, 115.9,

117.6, 117.7, 119.4, 127.7, 139.7, 141.9, 149.6, 151.5, 151.9, 160.2, 160.3,

163.9. MS (EI) EI^þ: 380 ([M^þþ H], 100). HPLC purity (method I)

100%.

4.9.20. 4-Bromo-3-methyl-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-

9H-purin-6-yl)phenyl)benzamide (17b)

According to procedure D. Yield 99%. White solid. M.p.

214e216 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.54 ¹H NMR (CDCl₃, 300 MHz)

4.9.15. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-1-(3-

d (ppm): 1.70e1.82 (m, 3H), 2.10e2.17 (m, 3H), 2.46 (s, 3H), 3.82 (dd,

ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (14f)

According to method E. Yield 84%. Yellow solid. R_f(8% EtOH/

CH₂Cl₂): 0.21. M.p. 308.8e309.8 ^ꢀC (decomposition). ¹H NMR

J ¼ 12.9 Hz, 1H), 4.21 (dd, J ¼ 12.3 Hz, 1H), 5.86 (dd, J ¼ 9.3 Hz, 1H),

7.55e7.63 (m, 3H), 7.76 (bs, 1H), 8.14 (s, 1H), 8.17 (bs, 1H), 8.34 (s,

1H), 8.60 (d, J ¼ 9 Hz, 1H), 8.80 (t, J ¼ 3 Hz, 1H), 9.02 (s, 1H). ¹³C NMR

(DMSO-d₆, 300 MHz)

d

(ppm): 6.73 (t, J ¼ 6 Hz, 1H), 7.35 (d, J ¼ 6 Hz,

(CDCl₃, 75 MHz) d (ppm): 22.8, 23.0, 24.9, 31.9, 68.9, 82.0, 120.9,

2H), 7.43 (dd, J ¼ 9.6 Hz, 2H), 7.61 (m, 1H), 7.68 (m, 1H), 7.91 (d,

J ¼ 12 Hz, 1H), 8.12 (d, J ¼ 6 Hz, 1H), 8.30 (s, 2H), 8.59 (d, J ¼ 6 Hz,

1H), 9.48 (bs, 1H), 12.06 (s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

122.9, 125.7, 126.0, 129.0, 129.5, 129.7, 131.1, 132.7, 133.9, 136.4,

138.3, 138.7, 142.3, 151.8, 152.4, 154.1, 165.1. MS (EI) ES^þ: 492

([M^þþ H], 100); ES^ꢁ: 490 ([M^þ-H], 100).

d

(ppm): 107.3, 107.7, 107.8, 108.0, 115.8, 116.2, 116.4, 118.0, 118.2,

120.7,128.2,129.7,136.5,136.7,141.3,144.5,144.7,145.3,145.5,151.9,

152.0, 162.1, 162.3. MS (EI) EI^þ: 460 ([M^þþ H], 85), 482 ([M^þþ Na],

100). HPLC purity (method I) 97.9%.

4.9.21. 2-Fluoro-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

yl)phenyl)benzamide (17c)

According to procedure D. Yield 94%. R_f(5% EtOH/CH₂Cl₂):

0.70 ¹H NMR (CDCl₃, 300 MHz)

d (ppm): 1.70e1.86 (m, 3H),

4.9.16. N-(4-(9H-purin-6-ylamino)-3-ﬂuorophenyl)-1-(4-

2.09e2.21 (m, 3H), 3.82 (dd, J ¼ 12.9 Hz, 1H), 4.22 (bd, J ¼ 9 Hz,

1H), 5.87 (dd, J ¼ 9.3 Hz, 1H), 7.15e7.21 (m, 2H), 7.29e7.34 (m,

1H), 7.52e7.62 (m, 2H), 7.98e8.04 (m, 1H), 8.11e8.21 (m, 2H),

8.41 (s, 1H), 8.60 (d, J ¼ 9 Hz, 1H), 8.70 (d, J ¼ 15 Hz, 1H), 8.83 (s,

1H), 9.05 (s, 1H). MS (EI) ES^þ: 440 ([M^þþ Na], 25), 356 (100);

ES^ꢁ: 332 ([M^þ-THP], 100).

ﬂuorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (14g)

According to method E. Yield 70%. Yellow solid. R_f(8% EtOH/

CH₂Cl₂): 0.18. M.p. 314.0e320.9 ^ꢀC (decomposition). ¹H NMR

(DMSO-d₆, 300 MHz)

d (ppm): 6.74 (s, 1H), 7.33-7.42 (m, 4H), 7.58

(bdd, J ¼ 12.9 Hz, 3H), 7.89 (bd, J ¼ 12 Hz, 1H), 8.13 (bs, 1H), 8.25 (s,

18

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

4.9.22. 2-Fluoro-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-

yl)phenyl)pyridine-3-carboxamide (17d)

(CDCl₃, 75 MHz) d (ppm): 22.8, 24.9, 31.8, 68.9, 81.9, 107.5, 121.4,

122.8, 122.9, 125.9, 127.0, 127.3, 129.3, 131.2, 136.4, 138.7, 140.7,

142.2, 142.9, 145.3, 151.7, 152.3, 154.5, 161.2, 162.2. MS (EI) ES^þ: 583

([M^þþ Na], 100), 561 ([M^þþ H], 2), 499 ([M^þþ Na-THPþH), 45).

According to procedure D. Yield 96%. R_f(5% EtOH/CH₂Cl₂): 0.60

¹H NMR (CDCl₃, 300 MHz)

d (ppm): 1.70e1.80 (m, 3H), 2.10e2.17

(m, 3H), 3.82 (dd, J ¼ 12.9 Hz, 1H), 4.22 (d, J ¼ 9 Hz, 1H), 5.87 (d,

J ¼ 12 Hz, 1H), 7.43 (m, 1H), 7.60 (dd, J ¼ 9.6 Hz, 1H), 8.06 (bd,

J ¼ 9 Hz, 1H), 8.37 (bs, 2H), 8.67 (bd, J ¼ 9 Hz, 2H), 8.71e8.79 (m,

4.9.27. 2-Oxo-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)

phenyl)-1-(3-(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-

carboxamide (17i)

1H), 8.92 (s, 1H), 9.04 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): MS

(EI) ES^þ: 441 ([M^þþ Na], 100), 419 ([M^þþ H], 2); ES^ꢁ: 333 ([M^þ-

According to procedure D. Yield 89%. Slightly coloured solid

THP], 100).

(beige). M.p. 200e202 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.47; ¹H NMR

(CDCl₃, 300 MHz)

d (ppm): 1.69e1.91 (m, 3H), 2.08e2.14 (m, 3H),

4.9.23. 2-Methoxy-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-

6-yl)phenyl)pyridine-3-carboxamide (17e)

3.82 (dd, J ¼ 12.9 Hz, 1H), 4.20 (bd, J ¼ 12 Hz, 1H), 5.85 (dd,

J ¼ 12.3 Hz, 1H), 6.63 (dd, J ¼ 9.6 Hz, 1H), 7.54e7.79 (m, 6H), 8.02 (d,

J ¼ 9 Hz, 1H), 8.33 (s, 1H), 8.58 (d, J ¼ 9 Hz, 1H), 8.79 (dd, J ¼ 6.3 Hz,

1H), 8.99 (s, 1H), 9.03 (s, 1H), 11.87 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

According to procedure D. Yield 86%. White solid. M.p.

150e152 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.50; ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.71e1.80 (m, 3H), 2.10e2.17 (m, 3H), 3.15e3.19 (m, 1H),

d

(ppm): MS (EI) ES^þ: 583 ([M^þþ Na], 100), 561 ([M^þþ H], 5), 499

3.84 (dd, J ¼ 12.9 Hz, 1H), 4.24 (s, 3H), 5.88 (d, J ¼ 9 Hz, 1H), 7.13 (dd,

J ¼ 9.6 Hz, 1H), 7.59 (t, J ¼ 9 Hz, 1H), 8.08 (d, J ¼ 9 Hz, 1H), 8.34 (bd,

J ¼ 3 Hz, 1H), 8.38 (s, 1H), 8.63 (dd, J ¼ 9.3 Hz, 2H), 8.88 (s, 1H), 9.04

([M^þþ Na-THP þ H), 15).

4.9.28. N-(3-(9H-purin-6-yl)phenyl)-2-methoxybenzamide (18a)

According to method E. Yield 65%. White solid. R_f(8% EtOH/

CH₂Cl₂): 0.34. M.p. 189.5e192.1 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

(s, 1H), 10.01 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d (ppm): 22.8, 24.9,

31.8, 55.8, 68.9, 82.0, 116.2, 118.2, 121.3, 123.3, 126.2, 129.5, 131.2,

136.4, 138.4, 141.9, 142.4, 150.0, 152.4, 154.3, 160.3, 162.0. MS (EI)

ES^þ: 453 ([M^þþ Na], 100), 431 ([M^þþ H], 3); ES^ꢁ: 429 ([M^þ-H], 25),

313 ([M^þ-H-THP-OMe], 100).

d

(ppm): 3.92 (s, 3H), 7.08 (t, J ¼ 6 Hz, ¹H), 7.20 (d, J ¼ 9 Hz, 1H),

7.49e7.58 (m, 2H), 7.66 (d, J ¼ 6 Hz, 1H), 7.90 (d, J ¼ 9 Hz, 1H), 8.66

(bs, 2H), 8.97 (s, 1H), 9.10 (bs, 1H), 10.34 (s, 1H). ¹³C NMR (DMSO-d₆,

75 MHz) d (ppm): 56.2, 112.4, 112.5, 120.6, 120.7, 120.9, 121.7, 125.3,

4.9.24. 1-(3-Fluorophenyl)-2-oxo-N-(3-(9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-6-yl)phenyl)-1,2-dihydropyridine-3-carboxamide

(17f)

125.8, 129.6, 130.0, 132.2, 136.5, 139.8, 145.5, 152.3, 157.0, 153.7. MS

(EI) EI^þ: 346 ([M^þþ H], 15), 368 ([M^þþ Na], 100); MS (EI) EI^ꢁ: 344

([M^þ-H], 100). HRMS-ESI (m/z) calcd for C₁₉H₁₆N₅O₂(M þ 1)

346.1304, found: 346.1295. HPLC purity (method I) 97.8%.

According to procedure D. Yield 91%. White solid. M.p.

196e198 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.68; ¹H NMR (CDCl₃, 300 MHz)

d

(ppm): 1.69e1.81 (m, 3H), 2.09e2.15 (m, 3H), 3.78e3.86 (m, 1H),

4.9.29. N-(3-(9H-purin-6-yl)phenyl)-4-bromo-3-methylbenzamide

(18b)

4.19e4.22 (m, 1H), 5.85 (d, J ¼ 9 Hz, 1H), 6.59 (t, J ¼ 6 Hz, 1H),

7.24e7.29 (m, 2H), 7.40e7.45 (m, 2H), 7.50e7.61 (m, 1H), 8.01 (m,

1H), 8.34 (s,1H), 8.57 (d, J ¼ 6 Hz,1H), 8.77 (d, J ¼ 6 Hz,1H), 8.99 (bs,

According to method E. White solid. Yield 94%. R_f(8% EtOH/

CH₂Cl₂): 0.23. M.p. 323.1e324.7 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

1H), 9.03 (s, 1H), 11.96 (s, 1H). ¹³C NMR (CDCl₃, 75 MHz)

d

(ppm):

d

(ppm): 2.45 (s, 3H), 7.58 (dd, J ¼ 9.6 Hz, 1H), 7.77 (s, 2H), 8.01 (s,

2H), 8.66 (s, 2H), 8.97 (s, 1H), 9.10 (s, 1H), 10.53 (s, 1H), 13.70 (bs, 1H

NH9-purine). ¹³C NMR (DMSO-d₆, 75 MHz)

(ppm): 22.9, 121.6,

22.8, 24.9, 31.8, 68.9, 81.9, 107.2, 116.7, 117.0, 121.4, 122.5, 123.0,

125.9, 128.4, 128.6, 129.3, 131.2, 136.3, 138.7, 141.4, 142.2, 145.1, 151.7,

152.3, 154.5, 161.5, 162.5, 164.4, 208.4. MS (EI) ES^þ: 533 ([M^þþ Na],

85), 511 ([M^þþ H], 5).

d

122.4, 123.5, 126.1, 127.4, 128.1, 129.3, 129.8, 130.6, 132.6, 134.6,

136.7, 139.8, 145.5, 152.8, 152.3, 154.5, 165.4. MS (EI) EI^þ: 408

([M^þþ H], 25), 430 ([M^þþ Na], 100); MS (EI) EI^ꢁ: 406 ([M^þ-H], 85).

H], 85). HRMS-ESI (m/z) calcd for C₁₉H₁₅BrN₅O (M þ 1) 408.0460,

found: 408.0451. HPLC purity (method II) 100%.

4.9.25. 1-(4-Fluorophenyl)-2-oxo-N-(3-(9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-6-yl)phenyl)-1,2-dihydropyridine-3-carboxamide

(17g)

According to procedure D. Yield 94%. White solid. M.p.

4.9.30. N-(3-(9H-purin-6-yl)phenyl)-2-ﬂuorobenzamide (18c)

According to method E. White solid. Yield 98%. R_f(8% EtOH/

CH₂Cl₂): 0.23. M.p. 275.0e282.5 ^ꢀC (decomposition) ¹H NMR

(decomposition) > 200 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.68; ¹H NMR

(CDCl₃, 300 MHz)

d (ppm): 1.69e1.81 (m, 3H), 2.09e2.15 (m, 3H),

3.78e3.86 (m, 1H), 4.19e4.22 (m, 1H), 5.85 (d, J ¼ 9 Hz, 1H), 6.59 (t,

J ¼ 6 Hz, 1H), 7.24e7.29 (m, 2H), 7.40e7.45 (m, 2H), 7.50e7.61 (m,

1H), 8.01 (m, 1H), 8.34 (s, 1H), 8.57 (d, J ¼ 6 Hz, 1H), 8.77 (d, J ¼ 6 Hz,

1H), 8.99 (bs, 1H), 9.03 (s, 1H), 11.96 (s, 1H). ¹³C NMR (CDCl₃,

(DMSO-d₆, 300 MHz) d (ppm): 7.32e7.39 (m, 2H), 7.58 (dd,

J ¼ 9.6 Hz, 2H), 7.71 (dt, J ¼ 6.3 Hz, 1H), 7.93 (d, J ¼ 6 Hz, 1H), 8.65 (s,

2H), 8.97 (s, 1H), 9.09 (bs, 1H), 10.68 (s, 1H), 13.64 (bs, 1H NH9-

purine). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 116.6, 116.7, 120.9,

75 MHz)

d

(ppm): 22.8, 24.9, 31.8, 68.9, 81.9,107.2,116.7,117.0,121.4,

121.9, 122.9, 125.1, 125.8, 129.7, 130.3, 130.4, 132.9, 136.5, 139.5,

145.5, 152.3, 153.0, 154.1, 163.6. MS (EI) EI^þ: 334 ([M^þþ H], 15), 356

([M^þþ Na], 100); MS (EI) EI^ꢁ: 332 ([M^þ-H], 100). HRMS-ESI (m/z)

calcd for C₁₉H₁₃FN₅O (M þ 1) 334.1104, found: 334.1104. HPLC

purity (method I) 100%.

122.5, 123.0, 125.9, 128.4, 128.6, 129.3, 131.2, 136.3, 138.7, 141.4,

142.2, 145.1, 151.7, 152.3, 154.5, 161.5, 162.5, 164.4, 208.4. MS (EI)

ES^þ: 533 ([M^þþ Na], 85), 511 ([M^þþ H], 5).

4.9.26. 2-Oxo-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)

phenyl)-1-(4-(triﬂuoromethyl)phenyl)-1,2-dihydropyridine-3-

carboxamide (17h)

4.9.31. N-(3-(9H-purin-6-yl)phenyl)-2-ﬂuoropyridine-3-

carboxamide (18d)

According to procedure D. Yield 87%. White solid. M.p.

According to method E. Yield 98%. Yellow solid. R_f(8% EtOH/

190e192 ^ꢀC. R_f(5% EtOH/CH₂Cl₂): 0.46; ¹H NMR (CDCl₃, 300 MHz)

CH₂Cl₂): 0.13. M.p. 268.5e272.3 ^ꢀC. ¹H NMR (DMSO-d₆, 300 MHz)

d

(ppm): 1.71e1.81 (m, 3H), 2.11e2.15 (m, 3H), 3.82 (dd, J ¼ 12.9 Hz,

d

(ppm): 7.52 (dd, J ¼ 6.3 Hz, 1H), 7.60 (t, J ¼ 9 Hz, 1H), 7.92 (d,

1H), 4.20 (bd, J ¼ 9 Hz, 1H), 5.86 (dd, J ¼ 6.3 Hz, 1H), 6.62 (t, J ¼ 6 Hz,

1H), 7.54 (dd, J ¼ 9.6 Hz, 2H), 7.60 (d, J ¼ 6 Hz, 2H), 7.87 (d, J ¼ 9 Hz,

2H), 8.03 (d, J ¼ 6 Hz, 1H), 8.33 (s, 1H), 8.58 (d, J ¼ 9 Hz, 1H), 8.79

(dd, J ¼ 9.3 Hz, 1H), 8.99 (s, 1H), 9.03 (s, 1H), 11.87 (s, 1H). ¹³C NMR

J ¼ 9 Hz, 1H), 8.28 (t, J ¼ 9 Hz, 1H), 8.40 (d, J ¼ 3 Hz, 1H), 8.63 (s, 1H),

8.63e8.67 (m, 1H), 8.96 (s, 1H), 9.09 (s, 1H), 10.85 (s, 1H), 13.69 (bs,

1H NH9-purine). ¹³C NMR (DMSO-d₆, 75 MHz)

d

(ppm): 119.6,

120.9, 121.8, 122.5, 122.9, 126.0, 129.7, 137.0, 139.2, 141.9, 145.5,

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

19

149.8, 150.0, 150.1, 152.2, 152.3, 152.8. MS (EI) EI^þ: 335 ([M^þþ H],

35), 357 ([M^þþ Na], 100); MS (EI) EI^ꢁ: 333 ([M^þ-H], 100). HRMS-ESI

(m/z) calcd for C₁₇H₁₂FN₆O (M þ 1) 335.1057, found: 335.1048. HPLC

purity (method I) 100%.

300 MHz)

d

(ppm): 6.78 (dd, J ¼ 9.6 Hz,1H), 7.59 (dd, J ¼ 9.6 Hz,1H),

7.85 (dd, J ¼ 9.6 Hz, 1H), 7.94 (dd, J ¼ 9.6 Hz, 2H), 8.03 (bd, J ¼ 9 Hz,

1H), 8.08 (bs, 1H), 8.21 (dd, J ¼ 6.3 Hz, 1H), 8.64 (dd, J ¼ 9.3 Hz, 2H),

8.67 (s, 1H), 8.96 (s, 1H), 9.06 (bs, 1H), 12.07 (s, 1H), 13.64 (bs, 1H

NH9-purine). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm): 107.7, 120.6,

4.9.32. N-(3-(9H-purin-6-yl)phenyl)-2-methoxypyridine-3-

carboxamide (18e)

121.6,121.9,122.4,124.1,122.7,126.2, 129.7,130.2, 131.0,132.2,133.3,

138.0, 141.8, 144.0, 144.2, 145.6, 152.2, 154.4, 154.7, 163.8. MS (EI)

EI^þ: 499 ([M^þþ Na], 100). HRMS-ESI (m/z) calcd for C₂₄H₁₆F₃N₆O₂

(M þ 1) 477.1287, found: 477.1289. HPLC purity (method I) 98%.

According to method E. Yield 94%. Slightly coloured beige solid.

R_f(8% EtOH/CH₂Cl₂): 0.24. M.p. 233.5e241.3 ^ꢀC. ¹H NMR (DMSO-d₆,

300 MHz)

d

(ppm): 4.00 (s, 3H), 7.17 (dd, J ¼ 9.3 Hz, 1H), 7.58 (t,

J ¼ 9 Hz, 1H), 7.92 (d, J ¼ 6 Hz, 1H), 8.08 (d, J ¼ 6 Hz, 1H), 8.35 (dd,

J ¼ 6.3 Hz, 1H), 8.66 (bs, 2H), 8.97 (s, 1H), 9.12 (bs, 1H), 10.45 (s, 1H),

4.10. General procedure F of palladium-anilination of 6-chloro-9-

THP-purine [24]

13.67 (bs, 1H NH9-purine). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm):

54.2, 117.7, 119.6, 120.8, 121.6, 122.8, 125.9, 129.5, 138.2, 139.5, 139.6,

145.3, 149.4, 152.2, 152.3, 160.3, 160.5, 163.9. MS (EI) EI^þ: 347

([M^þþ H], 55), 369 ([M^þþ Na], 100). HRMS-ESI (m/z) calcd for

C₁₈H₁₅N₆O₂(M þ 1) 347.1256, found: 347.1250. HPLC purity

(method I) 95.8%.

In a ﬂamed sealed tube, Pd(OAc)₂(19 mg, 0.08 mmol), and X-

phos (80 mg, 0.17 mmol) were introduced in a deoxygenated

mixture of t-BuOH/H₂O (3/1 mL). The tube was heated at 110 ^ꢀC for

1 min. Afterwards, 1 (200 mg, 0.84 mmol), the aniline compound

(1.26 mmol) and K₂CO₃(290 mg, 2.09 mmol) were added to the

reaction mixture and heated at 110 ^ꢀC for 15 h. At room tempera-

ture, the reaction mixture was diluted into EtOAc and ﬁltered

through a short pad of celite. The ﬁltrate was extracted with brine

and the organic phase dried over anhydrous MgSO₄and ﬁltered off.

After concentration in vacuo, the residue was puriﬁed by column

chromatography over silica gel, using EtOH: CH₂Cl₂mixtures as

eluent.

4.9.33. N-(3-(9H-purin-6-yl)phenyl)-1-(3-ﬂuorophenyl)-2-oxo-

1,2-dihydropyridine-3-carboxamide (18f)

According to method E, 18f was obtained in 85% yield. R_f(8%

EtOH/CH₂Cl₂): 0.28. M.p. 315.5e316.7 ^ꢀC. ¹H NMR (DMSO-d₆,

300 MHz)

d

(ppm): 6.74 (dd, J ¼ 9.6 Hz, 1H), 7.43 (t, J ¼ 9 Hz, 2H),

7.55e7.65 (m, 3H), 8.03 (d, J ¼ 6 Hz, 1H), 8.13 (dd, J ¼ 6.3 Hz, 1H),

8.61 (dt, J ¼ 6.3 Hz 2H), 8.67 (s,1H), 8.96 (s,1H), 9.02 (s, 1H),12.15 (s,

1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d

(ppm): 107.7, 116.4, 120.9, 120.9,

4.10.1. N-Phenyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

(19a)

121.5,121.6,122.5, 125.5,129.7,129.8,129.9,136.7,137.7, 144.1,144.5,

145.3, 152.3, 154.4, 154.5, 162.0. MS (EI) EI^þ: 427 ([M^þþ H], 25), 449

([M^þþ Na], 100); MS (EI) EI^ꢁ: 425 ([M^þ-H], 100). HRMS-ESI (m/z)

calcd for C₂₃H₁₆FN₆O₂(M þ 1) 427.1319, found: 427.1316. HPLC

purity (method I) 96.9%.

Following the general procedure F, 19a was obtained in 100%

yield. ¹H NMR (300 MHz, CDCl₃)

d 8.55 (s, 1H), 8.07 (s, 1H), 7.79 (t,

J ¼ 7.7 Hz, 3H), 7.39 (t, J ¼ 7.9 Hz,1H), 7.12 (t, J ¼ 7.4 Hz,1H), 5.75 (dd,

J ¼ 10.0, 2.7 Hz, 1H), 4.15 (dd, J ¼ 19.1, 10.4 Hz, 1H), 3.93e3.66 (m,

1H), 1.99 (m 3H), 1.68 (m 3H). ¹³C NMR (75 MHz, CDCl₃)

d 153.0,

4.9.34. N-(3-(9H-purin-6-yl)phenyl)-1-(4-ﬂuorophenyl)-2-oxo-

1,2-dihydropyridine-3-carboxamide (18g)

152.3, 148.9, 138.6, 138.4, 129.1, 123.6, 120.3, 81.9, 68.9, 31.9, 24.9,

22.8. MS (ES^þ) m/z (%): 296(100) [M þ H]^þ, 297(20) [M-H]^þ,

318(20) [M þ Na]^þ, 319(5) [M þ Na]^þ.

According to method E, 18g was obtained in 61% yield. R_f(8%

EtOH/CH₂Cl₂): 0.23. M.p. 314.7e319.2 ^ꢀC (decomposition) ¹H NMR

(DMSO-d₆, 300 MHz)

d

(ppm): 6.74 (dd, J ¼ 9.6 Hz, 1H), 7.42 (t,

4.10.2. 9-(Tetrahydro-2H-pyran-2-yl)-N-(3-(triﬂuoromethyl)

phenyl)-9H-purin-6-amine (19b)

J ¼ 9 Hz, 2H), 7.56e7.62 (m, 3H), 7.98 (d, J ¼ 9 Hz, 1H), 8.08 (d,

J ¼ 6 Hz, 1H), 8.61 (m, 3H), 8.94 (s, 1H), 9.07 (s, 1H NH), 12.16 (s, 1H

CH), 13.66 (bs, 1H NH9-purine). ¹³C NMR (DMSO-d₆, 75 MHz)

Following the general procedure F, 19b was obtained in 97%

yield. ¹H NMR (300 MHz, CDCl₃)

d

8.59 (s, 1H), 8.26 (d, J ¼ 18.2 Hz,

d

(ppm): 107.0, 115.9, 116.2, 120.4, 121.9, 124.9, 129.2, 129.4, 130.0,

2H), 8.08 (s, 1H), 7.98 (d, J ¼ 8.2 Hz, 1H), 7.47 (dd, J ¼ 10.2, 5.6 Hz,

1H), 7.34 (d, J ¼ 7.7 Hz, 1H), 7.00 (s, OH), 5.77 (dd, J ¼ 10.2, 2.5 Hz,

1H), 4.27e4.15 (m, 1H), 3.80 (td, J ¼ 11.6, 2.9 Hz, 1H), 2.25e1.95 (m,

3H), 1.90e1.56 (m, 6H), 1.33e1.22 (m, 3H), 0.96 (d, J ¼ 6.7 Hz, 1H).

136.3,136.4,138.5,143.9,144.8,151.6,151.7,153.5,160.2,161.3, 161.9,

163.5. MS (EI) EI^þ: 427 ([M^þþ H], 2), 449 ([M^þþ Na], 100). HRMS-

ESI (m/z) calcd for C₂₃H₁₆FN₆O₂(M þ 1) 427.1319, found: 427.1318.

HPLC purity (method I) 100%.

¹³C NMR (75 MHz, CDCl₃)

d 152.8, 151.9, 149.1, 145.9, 139.5, 138.8,

129.4, 123.1, 120.3, 119.7, 116.7, 82.0, 68.9, 31.9, 24.8, 22.8. MS

4.9.35. N-(3-(9H-purin-6-yl)phenyl)-2-oxo-1-(4-(triﬂuoromethyl)

phenyl)-1,2-dihydropyridine-3-carboxamide (18h)

(ES^þ) m/z (%): 364(100) [M þ H]^þ, 365(15) [M þ H]^þ.

According to method E, 18h was obtained in 81% yield. R_f(5%

4.10.3. N-(3-ﬂuorophenyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6-amine (19c)

EtOH/CH₂Cl₂): 0.13. M.p.: 315.5e317.4 ^ꢀC. ¹H NMR (DMSO-d₆,

300 MHz)

d

(ppm): 6.77 (t, J ¼ 6 Hz, 1H), 7.58 (dd, J ¼ 9.6 Hz, 1H),

Following the general procedure F 19c was obtained in 87%

7.81 (d, J ¼ 6 Hz, 2H), 7.96e8.01 (m, 3H), 8.14 (dd, J ¼ 6.3 Hz, 1H),

yield. ¹H NMR (300 MHz, CDCl₃)

d

8.57 (s, 1H), 8.25 (d, J ¼ 14.7 Hz,

8.57 (d, J ¼ 9 Hz, 1H), 8.61 (d, J ¼ 3 Hz, 1H), 8.63 (s, 1H), 8.94 (s, 1H),

1H), 8.06 (s, 1H), 7.90 (dt, J ¼ 11.4, 2.1 Hz, 1H), 7.40e7.33 (m, 1H),

7.31e7.22 (m, 1H), 6.77 (td, J ¼ 8.2, 1.6 Hz, 1H), 5.74 (dd, J ¼ 10.0,

2.5 Hz, 1H), 4.21e4.07 (m, 1H), 3.77 (td, J ¼ 11.4, 2.7 Hz, 1H),

2.22e1.93 (m, 3H), 1.84e1.55 (m, 5H). ¹³C NMR (75 MHz, CDCl₃)

8.98 (bs, 1H), 12.05 (s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz)

d (ppm):

108.0, 120.9, 121.1, 121.4, 122.5, 125.5, 127.1, 127.1, 128.7, 130.0, 130.2,

137.6, 143.7, 143.9, 145.2, 145.5, 152.3, 161.9, 162.4. MS (EI) EI^þ: 499

([M^þþ Na], 100), 477 ([M^þþ H], 5). HRMS-ESI (m/z) calcd for

C₂₄H₁₆F₃N₆O₂(M þ 1) 477.1297, found: 477.1274. HPLC purity

(method I) 100%.

d

164.6, 161.4, 152.8, 151.9, 149.0, 140.5, 140.4, 138.7, 130.0, 129.9,

120.3, 115.3, 110.0, 109.7, 107.6, 107.3, 82.0, 68.8, 31.8, 24.9, 22.8.

MS (ES^þ) m/z (%): 314 (100) [M þ H]^þ.

4.9.36. N-(3-(9H-purin-6-yl)phenyl)-2-oxo-1-(3-(triﬂuoromethyl)

phenyl)-1,2-dihydropyridine-3-carboxamide (18i)

4.10.4. N-(4-ﬂuorophenyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-

purin-6-amine (19d)

According to method E, 18i was obtained in 78% yield. R_f(5%

Following the general procedure F 19d was obtained in 84%

EtOH/CH₂Cl₂): 0.14. M.p.: 301.5e306.6 ^ꢀC. ¹H NMR (DMSO-d₆,

yield. ¹H NMR (300 MHz, CDCl₃)

d 8.49 (s, 1H), 8.38 (s, 1H), 8.01 (s,

20

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

1H), 7.76e7.63 (m, 2H), 7.03 (t, J ¼ 8.7, 2H), 5.72 (dd, J ¼ 10.2, 2.4 Hz,

1H), 4.13 (dd, J ¼ 13.3, 9.7 Hz, 1H), 3.76 (td, J ¼ 11.5, 2.7 Hz, 1H),

2.15e1.94 (m, 3H), 1.71 (ddd, J ¼ 26.3, 11.7, 8.0 Hz, 3H). ¹³C NMR

m/z (%): 440(100) [M þ H]^þ, 441(20) [M þ H]^þ, 356(100) [M-

THP]^þ, 357(20) [M-THP]^þ.

(75 MHz, CDCl₃)

d

160.7, 157.6, 153.3e152.3 (m), 148.9, 138.6e138.2

4.12. General procedure H for the Suzuki reaction with 8-iodo-9-

(tetrahydro-2H-pyran-2-yl)-9H-purin compounds [34]

(d), 134.6, 122.6e122.14 (m), 120.0, 116.1e115.1 (m), 81.9, 68.8, 31.9,

24.9, 22.8. MS (ES^þ) m/z (%): 314(100) [M þ H]^þ.

In a ﬂamed sealed tube under argon, the appropriate compound

(20aed) (1eq), tetrakis(triphenylphosphine)palladium (10 mol%)

and potassium carbonate (3eq) in a mixture of dioxane/water 4/1

were warmed at 85 ^ꢀC for 5 min. Then 3-aminophenylboronic acid

(1.5equiv) was added and the reaction mixture stirred at 100 ^ꢀC till

complete disappearance of the starting material on TLC plate. The

crude material was puriﬁed by column chromatography over silica

gel, using cyclohexane/ethyl acetate as eluent to afford the desired

products 21aed.

4.11. General procedure G for the 8-iodination of 6-chloro-9-

(tetrahydro-2H-pyran-2-yl)-9H-purine 1 [22,23]

In a dry ﬂask, a solution of lithium amide (LDA) was prepared in situ

from n-butyllithium (n-BuLi,1.6 M in hexane) and diisopropylamine in

freshly distilled THF (Na/benzophenone). The mixture was stirred for

30 min at ꢁ78 ^ꢀC under positive pressure of argon. A solution of the

appropriate purine in freshly distilled THF was then added dropwise

and stirred over 1 h with LDA at ꢁ78 ^ꢀC. A solution of the electrophile

(I₂) in THF was then added dropwise (unless mentioned otherwise)

and the resulting mixture was stirred over 1 h while maintaining the

temperature at ꢁ78 ^ꢀC, before it was warmed to room temperature.

Water was added and the mixture extracted twice with AcOEt. The

organic layers were combined, dried over MgSO₄and evaporated

under vacuum. The crude material was puriﬁed by ﬂash chromatog-

raphy on silica gel eluting with a gradient of DCM/EtOH (0e1 %).

4.12.1. 8-(3-Aminophenyl)-N-phenyl-9-(tetrahydro-2H-pyran-2-

yl)-9H-purin-6-amine (21a)

Following the general procedure H from 20a, 21a was obtained

in 84% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.57 (s, 1H), 7.95 (s, 1H),

7.80 (d, J ¼ 7.8 Hz, 2H), 7.33 (dt, J ¼ 23.7, 6.7 Hz, 4H), 7.20e7.01 (m,

3H), 6.85 (d, J ¼ 7.4 Hz, 1H), 5.54 (d, J ¼ 10.0 Hz, 1H), 4.20 (t,

J ¼ 17.8 Hz, 1H), 4.01 (s, 2H), 3.66 (t, J ¼ 11.4 Hz, 1H), 3.16 (dd,

J ¼ 22.5, 10.4 Hz, 1H), 2.06e1.49 (m, 6H). ¹³C NMR (75 MHz,

4.11.1. 8-Iodo-N-phenyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-

6-amine (20a)

CDCl₃) d 152.4, 152.1, 152.0, 151.0, 147.0, 138.9, 130.9, 129.8, 129.0,

123.3, 120.2, 120.1, 119.5, 117.0, 115.9, 84.3, 68.9, 28.6, 24.7, 23.5.

MS (ES^þ) m/z (%): 409(30) [M þ Na]^þ, 387(100) [M þ H]^þ,

353(100) [M-THP]^þ.

Following the general procedure G from 19a, 20a was obtained

in 70% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.49 (s, 1H), 7.79 (d,

J ¼ 7.6 Hz, 2H), 7.64 (s, 1H), 7.40 (t, J ¼ 8.0 Hz, 2H), 7.13 (t, J ¼ 7.4 Hz,

1H), 5.65 (dd, J ¼ 11.2, 2.4 Hz, 1H), 4.25 (d, J ¼ 11.9 Hz, 1H), 3.76 (dd,

J ¼ 12.8, 10.8 Hz, 1H), 3.15 (ddd, J ¼ 24.3, 12.7, 4.0 Hz, 1H), 2.14 (s,

1H), 1.90 (d, J ¼ 12.2 Hz, 2H), 1.83e1.71 (m, 3H), 1.65 (d, J ¼ 14.4 Hz,

1H). MS (ES^þ) m/z (%): 422 (100)[M þ H]^þ, 338 (25))[M-THP]^þ.

4.12.2. 8-(3-Aminophenyl)-9-(tetrahydro-2H-pyran-2-yl)-N-(3-

(triﬂuoromethyl)phenyl)-9H-purin-6-amine (21b)

Following the general procedure H from 20b, 21b was obtained

in 75% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.61 (s, 1H), 8.20 (s, 1H),

7.98 (s, 2H), 7.47 (t, J ¼ 7.3 Hz, 1H), 7.39e7.24 (m, 3H), 6.88 (d,

J ¼ 7.3 Hz, 1H), 5.56 (d, J ¼ 10.7 Hz, 1H), 4.25 (d, J ¼ 9.8 Hz, 1H),

3.98 (s, 2H), 3.68 (t, J ¼ 11.6 Hz, 1H), 3.17 (d, J ¼ 11.6 Hz, 1H),

4.11.2. 8-Iodo-9-(tetrahydro-2H-pyran-2-yl)-N-(3-

(triﬂuoromethyl)phenyl)-9H-purin-6-amine (20b)

Following the general procedure G from 19b, 20b was obtained

1.97e1.52 (m, 5H). ¹³C NMR (75 MHz, CDCl₃)

d 152.6, 152.3, 151.6,

in 48% yield. ¹H NMR (300 MHz, CDCl₃)

d

8.53 (s, 1H), 8.22 (s, 1H),

151.2, 147.0, 139.6, 130.8, 129.8, 129.5, 122.7, 120.4, 119.5, 117.1,

116.4, 115.9, 84.3, 68.9, 28.6, 24.7, 23.4. MS (ES^þ) m/z (%): 455

(100)[M þ H]^þ.

7.95 (d, J ¼ 8.2 Hz, 1H), 7.71 (s, 1H), 7.50 (t, J ¼ 7.9 Hz, 1H), 7.37 (d,

J ¼ 7.7 Hz,1H), 5.67 (dd, J ¼ 11.2, 2.4 Hz,1H), 4.25 (d, J ¼ 11.7 Hz,1H),

3.78 (td, J ¼ 11.8, 2.7 Hz, 1H), 3.15 (ddd, J ¼ 24.2, 12.8, 4.2 Hz, 1H),

2.17 (d, J ¼ 10.8 Hz,1H),1.97e1.73 (m, 4H). ¹³C NMR (CDCl₃, 75 MHz)

4.12.3. 8-(3-Aminophenyl)-N-(3-ﬂuorophenyl)-9-(tetrahydro-2H-

pyran-2-yl)-9H-purin-6-amine (21c)

d

152.4, 151.0, 150.4, 139.1, 131.3, 129.5, 123.6, 122.9, 120.0, 116.7,

116.6, 99.0, 86.8, 69.3, 29.1, 24.7, 23.4.

Following the general procedure H from 20c, 21c was obtained

in 71% yield. Mp 208 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, CDCl₃)

d 8.60 (s,

4.11.3. N-(3-ﬂuorophenyl)-8-iodo-9-(tetrahydro-2H-pyran-2-yl)-

9H-purin-6-amine (20c)

1H), 7.98e7.82 (m, 2H), 7.43e7.22 (m, 4H), 6.82 (dd, J ¼ 32.5, 7.0 Hz,

2H), 5.55 (d, J ¼ 10.9 Hz, 1H), 4.25 (d, J ¼ 10.8 Hz, 1H), 3.96 (s, 2H),

3.67 (t, J ¼ 11.7 Hz,1H), 3.17 (dd, J ¼ 24.5,12.0 Hz,1H), 2.06e1.67 (m,

Following the general procedure G from 19c, 20c was obtained

in 84% yield. ¹H NMR (300 MHz, CDCl₃)

d

8.50 (s, 1H), 7.89 (d,

4H), 1.60 (m, 2H). ¹³C NMR (75 MHz, CDCl₃)

d 164.8, 161.5, 152.5,

J ¼ 11.1 Hz, 1H), 7.66 (s, 1H), 7.41e7.20 (m, 3H), 6.79 (d, J ¼ 7.7 Hz,

1H), 5.64 (d, J ¼ 11.1 Hz, 1H), 4.24 (d, J ¼ 10.8 Hz, 1H), 3.75 (t,

J ¼ 11.5 Hz, 1H), 3.26e3.03 (m, 1H), 1.99e1.59 (m, 5H). ¹³C NMR

152.3, 151.6, 151.1, 147.0, 140.6, 130.8, 130.1, 129.8, 120.4, 119.5,

117.0, 115.8, 115.0, 109.9, 109.6, 107.4, 107.0, 84.3, 68.9, 28.6, 24.7,

23.4. MS (ES^ꢁ) m/z (%): 403(80) [M ꢁ H]^ꢁ, 404(30) [M-H]^ꢁ.

(75 MHz, CDCl₃) d 164.7, 161.5, 152.4, 150.9, 150.4, 140.1, 140.0, 130.2,

130.0, 123.6, 115.2, 110.3, 110.0, 107.6, 107.2, 98.9, 86.8, 77.5, 77.0,

4.12.4. 8-(3-Aminophenyl)-N-(4-ﬂuorophenyl)-9-(tetrahydro-2H-

pyran-2-yl)-9H-purin-6-amine (21d)

76.6, 69.3, 29.0, 24.7, 23.4. MS (ES^þ) m/z (%): 440(100) [M þ H]^þ.

Following the general procedure H from 20d, 21d was obtained

4.11.4. N-(4-ﬂuorophenyl)-8-iodo-9-(tetrahydro-2H-pyran-2-yl)-

9H-purin-6-amine (20d)

in 84% yield. ¹H NMR (300 MHz, CDCl₃)

d

8.55 (d, J ¼ 7.7 Hz, 1H),

7.61e7.53 (m, 2H), 7.49 (dd, J ¼ 7.6, 2.8 Hz, 3H), 7.34 (t, J ¼ 7.8 Hz,

1H), 7.21e7.03 (m, 4H), 6.91e6.85 (m, 1H), 5.56 (t, J ¼ 11.2, 2.1 Hz,

1H), 4.26 (d, J ¼ 8.6 Hz, 1H), 3.75e3.62 (m, 1H), 3.31e3.04 (m,

Following the general procedure G from 19d, 20d was obtained

in 84% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.44 (s, 1H), 7.71 (d,

J ¼ 4.5 Hz, 2H), 7.54 (s, 1H), 7.26 (s, 1H), 7.07 (t, J ¼ 7.4 Hz, 2H), 5.63

(d, J ¼ 11.0 Hz, 1H), 4.23 (d, J ¼ 10.2 Hz, 1H), 3.75 (t, J ¼ 11.4 Hz, 1H),

3.25e3.04 (m, 1H), 2.24e2.09 (m, 1H), 2.02e1.59 (m, 5H). ¹³C NMR

1H), 2.04e1.62 (m, 5H). ¹³C NMR (75 MHz, CDCl₃)

d 162.0, 161.6,

158.8, 152.4, 151.7, 151.2, 139.5, 138.2, 134.1, 134.0, 132.3, 131.5,

131.1, 130.6, 129.6, 129.4, 129.3, 125.9, 125.8, 125.2, 122.8, 122.5,

122.3, 121.8, 121.1, 121.0, 120.6, 120.4, 119.5, 116.5, 116.4, 116.1,

84.3, 68.9, 28.7, 24.7, 23.4. MS (ES^ꢁ) m/z (%): 403 (100) [M-H]^ꢁ.

(75 MHz, CDCl₃)

d 160.8, 157.6, 152.6, 150.8, 150.7, 134.3, 123.4,

122.2, 122.1, 115.9, 115.6, 98.4, 86.8, 69.3, 29.1, 24.7, 23.4. MS (ES^þ)

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

21

4.12.5. 2-Methoxy-N-(3-(6-(phenylamino)-9-(tetrahydro-2H-

pyran-2-yl)-9H-purin-8-yl)phenyl)benzamide (22f)

1H), 4.09 (s, 4H), 3.76 (t, J ¼ 12.2 Hz, 1H), 3.15 (d, J ¼ 11.7 Hz, 1H),

1.94 (s, 1H), 1.82 (t, J ¼ 18.0 Hz, 2H), 1.65 (d, J ¼ 22.3 Hz, 2H), 1.43 (s,

Following the general procedure D from 21a, 22f was obtained

2H). ¹³C NMR (75 MHz, CDCl₃)

d 163.5, 157.3, 152.4, 152.0, 151.6,

in 74% yield. ¹H NMR (300 MHz, CDCl₃)

d

10.00 (s, 1H), 8.60 (s, 1H),

151.2, 139.5, 139.0, 133.6, 132.7, 130.6, 129.6, 129.5, 125.1, 122.7,

122.6, 121.8, 121.7, 121.4, 120.4, 116.5, 111.6, 84.3, 77.2, 69.0, 56.3,

28.7, 24.7, 23.4. MS (ES^ꢁ) m/z (%): 587 (100) [M-H]^ꢁ, 588 (30) [M-

H]^ꢁ. HPLC purity (method I) 98%.

8.32 (d, J ¼ 7.5 Hz, 1H), 8.10e7.96 (m, 2H), 7.88e7.74 (m, 3H), 7.55

(dd, J ¼ 15.5, 8.3 Hz, 3H), 7.39 (t, J ¼ 7.2 Hz, 2H), 7.12 (dt, J ¼ 17.0,

8.0 Hz, 3H), 5.63 (d, J ¼ 11.0 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃)

d

163.48, 157.3, 152.6, 152.0, 151.5, 151.0, 139.0, 138.8, 133.6, 132.7,

130.7, 129.6, 129.1, 125.1, 123.4, 122.5, 121.8, 121.6, 121.4, 120.3, 120.1,

111.6, 100.0, 84.3, 77.5, 77.0, 76.6, 68.9, 56.3, 30.2, 28.8, 26.9, 24.7,

23.4. MS (ES^þ) m/z (%): 543(45) [M þ Na]^þ, 521(50) [M þ H]^þ,

437(100) [M-THP]^þ. HPLC purity (method I): 100%.

4.12.10. 2-Fluoro-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-6-(3-

(triﬂuoromethyl)phenylamino)-9H-purin-8-yl)phenyl)benzamide

(23g)

Following the general procedure D from 21b, 23g was obtained

in 73% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.68e8.56 (m, 2H),

4.12.6. 2-Fluoro-N-(3-(6-(phenylamino)-9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-8-yl)phenyl)benzamide (22g)

8.26e8.15 (m, 3H), 8.00 (d, J ¼ 7.9 Hz, 1H), 7.96e7.86 (m, 2H), 7.66

(d, J ¼ 7.7 Hz, 1H), 7.56 (dd, J ¼ 10.5, 5.0 Hz, 2H), 7.48 (t, J ¼ 7.9 Hz,

1H), 7.35 (t, J ¼ 7.6 Hz, 2H), 7.24e7.16 (m, 1H), 5.65 (d, J ¼ 9.5 Hz,

1H), 4.29 (d, J ¼ 9.3 Hz, 1H), 3.76 (t, J ¼ 11.8 Hz,1H), 3.11 (dd, J ¼ 16.1,

7.5 Hz, 1H), 2.05 (d, J ¼ 16.4 Hz, 1H), 1.78 (d, J ¼ 12.6 Hz, 4H), 1.56

Following the general procedure D from 21a, 22g was obtained

in 68% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.65 (s, 1H), 8.59 (s, 1H),

8.20 (t, J ¼ 7.5 Hz, 1H), 8.14 (s, 1H), 7.93 (d, J ¼ 7.3 Hz, 1H), 7.82 (d,

J ¼ 7.5 Hz, 2H), 7.74 (s, 1H), 7.66 (d, J ¼ 6.8 Hz, 1H), 7.57 (t, J ¼ 6.8 Hz,

2H), 7.44e7.31 (m, 3H), 7.20 (d, J ¼ 10.9 Hz, 1H), 7.10 (t, J ¼ 6.8 Hz,

1H), 5.64 (d, J ¼ 10.9 Hz, 1H), 4.29 (d, J ¼ 10.6 Hz, 1H), 3.75 (t,

J ¼ 11.9 Hz, 1H), 3.20e3.02 (m, 1H), 1.90e1.72 (m, 3H), 1.66e1.50

(dd, J ¼ 17.1, 10.0 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)

d 161.5, 152.4,

151.7,151.3,139.5,138.3,134.2,132.4,131.6,130.8,129.7,129.5,125.8,

125.3, 122.8, 122.6, 121.8, 120.4, 116.6, 116.1, 84.3, 77.2, 69.0, 28.7,

24.7, 23.4. MS (ES^ꢁ) m/z (%): 575 (100) [M ꢁ H]^ꢁ, 491 (40) [M-THP]^ꢁ.

HPLC purity (method I): 99%.

(m, 2H). ¹³C NMR (75 MHz, CDCl₃)

d 161.5, 158.8, 152.6, 152.1,

151.3, 151.0, 138.8, 138.3, 134.2, 134.1, 132.4, 131.0, 129.7, 129.1,

125.8, 125.3, 123.4, 122.4, 121.8, 121.1, 120.9, 120.3, 120.2, 116.4,

116.1, 84.2, 68.9, 28.8, 24.7, 23.4. MS (ES^ꢁ) m/z (%): 507(100) [M-

H]^ꢁ. HPLC purity (method I) 100%.

4.12.11. 2-Methoxy-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-6-(3-

(triﬂuoromethyl)phenylamino)-9H-purin-8-yl)phenyl)nicotinamide

(23h)

Following the general procedure D from 21b, 23h was obtained

4.12.7. 2-Methoxy-N-(3-(6-(phenylamino)-9-(tetrahydro-2H-

pyran-2-yl)-9H-purin-8-yl)phenyl)nicotinamide (22h)

in 62% yield. ¹H NMR (300 MHz, CDCl₃)

d 10.03 (s, 1H), 8.64 (s, 2H),

8.37 (s, 1H), 8.26 (s, 1H), 8.15 (s, 1H), 7.98 (dd, J ¼ 17.7, 8.6 Hz, 3H),

7.61 (d, J ¼ 14.9 Hz, 2H), 7.49 (d, J ¼ 8.4 Hz, 1H), 7.35 (d, J ¼ 7.2 Hz,

1H), 7.16 (s, 1H), 5.66 (d, J ¼ 11.2 Hz, 1H), 4.29 (s, 1H), 4.24 (s, 3H),

3.78 (t, J ¼ 11.7 Hz, 1H), 3.15 (d, J ¼ 11.4 Hz, 1H), 2.05 (s, 1H), 1.85 (s,

Following the general procedure D from 21a, 22h was obtained

in 39% yield. Mp 245 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, CDCl₃)

d 10.01

(s, 1H), 8.62 (d, J ¼ 12.3 Hz, 2H), 8.35 (s, 1H), 8.09 (s, 1H), 8.00 (d,

J ¼ 7.5 Hz, 1H), 7.83 (d, J ¼ 7.4 Hz, 2H), 7.76 (s, 1H), 7.59 (dd, J ¼ 14.9,

7.2 Hz, 2H), 7.39 (t, J ¼ 7.2 Hz, 2H), 7.19e7.05 (m, 2H), 5.63 (d,

J ¼ 11.0 Hz, 1H), 4.34e4.14 (m, 4H), 3.75 (t, J ¼ 11.5 Hz, 1H), 3.12 (q,

J ¼ 11.3 Hz, 1H), 2.02 (s, 1H), 1.73 (dd, J ¼ 52.2, 28.3 Hz, 7H). ¹³C NMR

4H), 1.63 (s, 2H). ¹³C NMR (75 MHz, CDCl₃)

d 162.1, 160.3, 152.4,

151.9,151.7,151.3,150.3,142.0,139.5,138.6,130.7,129.6,129.5,125.5,

122.7, 122.6, 121.7, 120.4, 118.3, 116.5, 116.0, 84.3, 77.2, 69.0, 54.5,

28.7, 24.7, 23.4. MS (ES^ꢁ) m/z (%): 588 (100) [M-H]^ꢁ. HPLC purity

(method I): 98%.

(75 MHz, CDCl₃) d 165.3, 162.1, 160.3, 152.6, 152.1, 151.4, 151.0, 150.3,

142.0, 138.8, 138.6, 130.9, 129.7, 129.1, 125.5, 123.4, 122.5, 121.7,

120.3, 120.1, 118.3, 116.0, 84.2, 68.9, 54.5, 28.8, 24.7, 23.4. MS (ES^þ)

m/z (%): 544(90) [M þ Na]^þ, 522(40) [M þ H]^þ, 438(100) [M-THP]^þ.

HPLC purity (method I): 94%.

4.12.12. 4-Bromo-N-(3-(6-(3-ﬂuorophenylamino)-9-(tetrahydro-

2H-pyran-2-yl)-9H-purin-8-yl)phenyl)-3-methylbenzamide (24e)

Following the general procedure D from 21c, 24e was ob-

tained in 39% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.60 (s, 1H), 8.25

4.12.8. 4-Bromo-3-methyl-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-6-

(3-(triﬂuoromethyl)phenylamino)-9H-purin-8-yl)phenyl)

benzamide (23e)

(d, J ¼ 19.6 Hz, 2H), 8.08 (s, 1H), 7.92 (d, J ¼ 11.1 Hz, 1H),

7.87e7.77 (m, 2H), 7.67e7.51 (m, 4H), 7.43 (d, J ¼ 8.1 Hz, 1H), 6.78

(t, J ¼ 8.2 Hz, 1H), 5.63 (d, J ¼ 10.5 Hz, 1H), 4.26 (d, J ¼ 8.4 Hz,

1H), 3.74 (t, J ¼ 11.8 Hz, 1H), 3.12 (q, J ¼ 23.7, 12.0 Hz, 1H),

2.47 (s, 3H), 1.88e1.73 (m, 2H), 1.71e1.54 (m, 3H). ¹³C NMR

Following the general procedure D from 21b, 23e was obtained

in 77% yield. ¹H NMR (300 MHz, CDCl₃)

d 8.59 (s, 1H), 8.18 (s, 1H),

8.15e8.03 (m, 3H), 7.95 (d, J ¼ 8.6 Hz, 1H), 7.80e7.76 (m, 1H),

7.69e7.40 (m, 6H), 7.31 (d, J ¼ 7.6 Hz, 1H), 5.62 (d, J ¼ 11.2 Hz,

1H), 4.25 (d, J ¼ 9.6 Hz, 1H), 3.73 (t, J ¼ 11.4 Hz, 1H), 3.22e3.00

(m, 1H), 2.49 (s, 3H), 2.09e1.94 (m, 1H), 1.71e1.49 (m, 3H). ¹³C

(75 MHz, CDCl₃) d 169.9, 168.9, 165.7, 161.8, 157.5, 156.3, 155.8,

146.6, 146.5, 144.7, 137.5, 135.0, 134.9, 134.6, 129.7, 127.3, 125.8,

123.4, 121.6, 117.3, 114.3, 112.7, 112.3, 61.1, 33.3, 23.4, 19.4, 18.1.

MS (ES^þ) m/z (%) 601 (80) [M þ H]^þ. HPLC purity (method I):

90%.

NMR (75 MHz, CDCl₃)

d 165.2, 152.4, 151.7, 151.3, 139.5, 138.9,

138.3, 133.6, 132.9, 130.6, 129.5, 129.4, 125.7, 122.7, 122.5, 121.6,

120.5, 119.6, 116.5, 84.3, 69.0, 28.7, 24.7, 23.4, 23.1. HPLC purity

(method I) 100%.

4.12.13. N-(3-(6-(3-ﬂuorophenylamino)-9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-8-yl)phenyl)-2-methoxybenzamide (24f)

Following the general procedure D from 21c, 24f was obtained

4.12.9. 2-Methoxy-N-(3-(9-(tetrahydro-2H-pyran-2-yl)-6-(3-

(triﬂuoromethyl)phenylamino)-9H-purin-8-yl)phenyl)benzamide

(23f)

in 52% yield. ¹H NMR (300 MHz, CDCl₃)

d 10.00 (s, 1H), 8.63 (s, 1H),

8.32 (dd, J ¼ 7.8, 1.4 Hz, 1H), 8.10 (s, 1H), 8.03e7.89 (m, 3H),

7.65e7.51 (m, 3H), 7.42 (d, J ¼ 8.1 Hz, 1H), 7.37e7.26 (m, 1H), 7.17

(t, J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 8.3 Hz, 1H), 6.80 (t, J ¼ 8.4 Hz, 1H),

5.65 (d, J ¼ 11.1 Hz, 1H), 4.35e4.24 (m, 1H), 4.09 (s, 3H), 3.77 (t,

J ¼ 11.8 Hz, 1H), 3.15 (qd, J ¼ 12.6, 3.6 Hz, 1H), 2.12e2.01 (m, 1H),

1.92e1.75 (m, 2H), 1.71e1.55 (m, 2H). MS (ES^þ) m/z (%): 561 (100)

[M þ Na]^þ, 539 (40) [M þ H]^þ. HPLC purity (method I): 96%.

Following the general procedure D from 21b, 23f was obtained

in 92% yield. ¹H NMR (300 MHz, CDCl₃)

d 9.99 (s, 1H), 8.63 (s, 1H),

8.32 (d, J ¼ 7.1 Hz, 1H), 8.24 (s, 1H), 8.11 (s, 1H), 8.03e7.87 (m, 3H),

7.65e7.40 (m, 4H), 7.33 (d, J ¼ 6.3 Hz, 1H), 7.18 (d, J ¼ 7.1 Hz, 1H),

7.07 (d, J ¼ 7.7 Hz, 1H), 5.64 (d, J ¼ 11.0 Hz, 1H), 4.28 (d, J ¼ 10.6 Hz,

22

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

4.12.14. N-(3-(6-(3-ﬂuorophenylamino)-9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-8-yl)phenyl)-2- methoxynicotinamide (24h)

Following the general procedure D from 21c, 24h was obtained

7.18e7.02 (m, 3H), 5.62 (dd, J ¼ 11.2, 2.0 Hz, 1H), 4.27 (d, J ¼ 11.4 Hz,

1H), 4.21 (s, 3H), 3.74 (t, J ¼ 11.1 Hz, 1H), 3.11 (qd, J ¼ 12.8, 3.9 Hz,

1H), 2.04 (d, J ¼ 14.2 Hz, 1H), 1.81e1.53 (m, 4H). ¹³C NMR

in 52% yield. ¹H NMR (300 MHz, CDCl₃)

d

10.03 (s, 1H), 8.64 (t,

(75 MHz, CDCl₃) d 162.2, 160.7, 160.4, 157.5, 152.7, 152.1, 151.6,

J ¼ 3.8 Hz, 2H), 8.37 (dd, J ¼ 4.8, 2.0 Hz, 1H), 8.13 (s, 1H), 8.06e7.92

(m, 2H), 7.81 (s, 1H), 7.67e7.55 (m, 2H), 7.17 (dd, J ¼ 7.6, 4.8 Hz, 2H),

6.82 (t, J ¼ 8.3 Hz, 1H), 5.65 (dd, J ¼ 11.5, 2.4 Hz, 1H), 4.24 (s, 3H),

3.77 (t, J ¼ 11.1 Hz, 1H), 3.16 (q, J ¼ 24.6, 12.8 Hz, 1H), 2.07 (d,

J ¼ 11.6 Hz, 2H), 1.94e1.72 (m, 4H). ¹³C NMR (75 MHz, CDCl₃)

151.1, 150.4, 142.1, 138.7, 134.9, 130.9, 129.8, 125.6, 122.6, 122.2,

122.1, 121.8, 120.3, 118.4, 116.1, 116.0, 115.7, 84.4, 69.1, 54.6, 30.3,

28.9, 24.8, 23.6. MS (ES^þ) m/z (%): 562 (100) [M þ H]^þ, 563 (40)

[M þ H]^þ, 456 (60) [M-THP]^þ, 457 (15) [M þ THP]^þ. HPLC purity

(method I): 96%.

d

169.9, 161.8, 157.3, 155.1, 144.7, 140.9, 137.5, 134.9, 129.7, 128.1,

127.2, 125.8, 123.4, 120.4, 120.1, 117.3, 108.2, 99.9, 61.18, 33.3, 23.4,

19.4, 18.1. MS (ES^ꢁ) m/z (%): 538 (30) [M ꢁ H]^ꢁ, 454 (100) [M-

THP]^ꢁ. HPLC purity (method I): 94%.

4.12.19. 2-Methoxy-N-(3-(6-(phenylamino)-9H-purin-8-yl)phenyl)

benzamide (26f)

Following the general method E from 22f, 26f was obtained as

a solid in 88% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz,

4.12.15. 4-Bromo-N-(3-(6-(4-ﬂuorophenylamino)-9-(tetrahydro-

2H-pyran-2-yl)-9H-purin-8-yl)phenyl)-3-methylbenzamide (25e)

Following the general procedure D from 21d, 25e was obtained

DMSO-d₆) d 10.35 (s, 1H), 10.05 (s, 1H), 8.75 (s, 1H), 8.45 (s, 1H),

8.03e7.83 (m, 3H), 7.81e7.64 (m, 2H), 7.62e7.46 (m, 2H), 7.37 (t,

J ¼ 7.8 Hz, 2H), 7.22 (d, J ¼ 8.3 Hz, 1H), 7.14e6.83 (m, 2H), 3.94

in 29% yield. ¹H NMR (300 MHz, CDCl₃)

d

8.53 (d, J ¼ 6.2 Hz, 1H),

(s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

d 164.6, 158.4, 157.9, 156.5,

8.19 (s, 1H), 8.08 (s, 1H), 7.89 (s, 1H), 7.80e7.69 (m, 4H), 7.66e7.50

(m, 5H), 7.05 (t, J ¼ 8.7 Hz, 2H), 5.61 (dd, J ¼ 11.2, 2.0 Hz, 1H),

4.24 (d, J ¼ 7.6 Hz, 1H), 3.73 (t, J ¼ 11.2 Hz, 1H), 3.10 (qd, J ¼ 12.7,

4.0 Hz, 1H), 2.56e2.32 (m, 3H), 2.01 (s, 3H), 1.21e0.82 (m, 3H). ¹³C

152.1, 150.9, 150.6, 150.1, 139.5, 139.2, 132.2, 129.8, 129.6, 129.4,

128.5, 124.5, 122.9, 122.0, 121.0, 120.5, 118.1, 112.0, 55.9. MS

(ES^þ) m/z (%): 437 (100) [M

þ

H]^þ. HRMS (EI) calcd. for

C₂₅H₂₀N₆O₂: 437.1726, found: 437.1717 (M^þ) HPLC purity

(method II): 100%.

NMR (75 MHz, CDCl₃) d 165.7, 165.4, 160.6, 157.3, 152.5, 152.0, 151.3,

151.0, 138.7, 138.5, 134.7, 133.7, 132.7, 132.1, 132.0, 132.0, 131.6, 130.6,

129.6, 129.4, 129.2, 128.6, 128.4, 125.8, 125.5, 122.3, 122.0, 121.9,

121.7, 120.1, 115.8, 115.5, 84.2, 68.9, 38.6, 30.2, 26.9, 23.0. MS (ES^ꢁ)

m/z (%): 599 (100) [M-H]^ꢁ, 601 (100) [M-H]^ꢁ, 514 (30) [M-THP]^ꢁ,

516 (30) [M-THP]^ꢁ. HPLC purity (method I) : 70%.

4.12.20. 2-Fluoro-N-(3-(6-(phenylamino)-9H-purin-8-yl)phenyl)

benzamide (26g)

Following the general method E from 22g, 26g was obtained as

a yellow solid in 84% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR

(300 MHz, DMSO-d₆) d 13.70 (s, 1H), 10.65 (s, 1H), 9.89 (s, 1H), 8.75

4.12.16. N-(3-(6-(4-ﬂuorophenylamino)-9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-8-yl)phenyl)-2-methoxybenzamide (25f)

(s, 1H), 8.42 (s, 1H), 7.98 (d, J ¼ 7.9 Hz, 2H), 7.92 (d, J ¼ 7.9 Hz, 1H),

7.72 (t, J ¼ 7.7 Hz, 2H), 7.58 (dd, J ¼ 16.8, 8.8 Hz, 2H), 7.44e7.29 (m,

4H), 7.05 (t, J ¼ 7.2 Hz, 1H), 3.85 (s, 3H). ¹³C NMR (75 MHz, DMSO-

Following the general procedure D from 21d, 25f was obtained

in 29% yield. ¹H NMR (300 MHz, CDCl₃)

d

9.99 (s, 1H), 8.57 (s, 1H),

d₆) d 162.9, 160.6, 157.3, 151.6, 139.6, 139.2, 132.7, 132.6, 130.1, 129.9,

8.32 (d, J ¼ 7.8 Hz, 1H), 8.10 (s, 1H), 7.97 (d, J ¼ 7.5 Hz, 1H), 7.90 (s,

1H), 7.77 (dd, J ¼ 8.9, 4.7 Hz, 2H), 7.63e7.47 (m, 3H), 7.16 (t,

J ¼ 7.5 Hz, 1H), 7.07 (t, J ¼ 8.6 Hz, 3H), 5.64 (d, J ¼ 11.1 Hz, 1H), 4.28

(d, J ¼ 9.7 Hz, 1H), 4.08 (s, 2H), 3.76 (t, J ¼ 11.7 Hz, 1H), 3.24e3.06

(m, 1H), 1.94e1.76 (m, 2H), 1.62 (dd, J ¼ 23.0, 12.7 Hz, 3H). ¹³C

129.4, 128.4, 124.8, 124.6, 122.5, 122.2, 121.9, 120.7, 118.3, 116.3,

116.0. MS (ES^þ) m/z (%): 447 (30) [M þ Na]^þ, 425 (100) [M þ H]^þ.

HPLC purity (method II): 98%.

4.12.21. 2-Methoxy-N-(3-(6-(phenylamino)-9H-purin-8-yl)phenyl)

nicotinamide (26h)

NMR (75 MHz, CDCl₃)

d 163.6, 160.7, 157.5, 157.4, 152.7, 152.1, 151.7,

151.1,139.1,134.9,134.9, 133.7,132.8,130.8,129.7,125.2,122.6,122.2,

122.1, 121.9, 121.7, 121.5, 120.2, 115.9, 115.6, 111.7, 84.3, 69.1, 56.4,

31.1, 28.9, 24.8, 23.5. MS (ES^ꢁ) m/z (%): 537 (100) [M-H]^ꢁ, 453 (30)

[M-THP]^ꢁ. HPLC purity (method I): 100%.

Following the general method E from 22h, 26h was obtained

in 66% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-d₆)

d

13.69 (s, 1H), 10.42 (s, 1H), 9.88 (s, 1H), 8.71 (s, 1H), 8.48e8.32

(m, 2H), 8.17e8.06 (m, 1H), 7.95 (dd, J ¼ 17.3, 7.8 Hz, 4H), 7.74

(d, J ¼ 7.9 Hz, 1H), 7.56 (t, J ¼ 7.9 Hz, 1H), 7.35 (t, J ¼ 7.8 Hz, 2H),

7.19 (dd, J ¼ 7.2, 5.1 Hz, 1H), 7.05 (t, J ¼ 7.3 Hz, 2H), 3.97 (d,

J ¼ 3.53 Hz, 4H), 3.42 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆)

4.12.17. 2-Fluoro-N-(3-(6-(4-ﬂuorophenylamino)-9-(tetrahydro-

2H-pyran-2-yl)-9H-purin-8- yl)phenyl)benzamide (25g)

Following the general procedure D from 21d, 25g was obtained

d 200.4, 178.7, 163.3, 159.9, 151.7, 149.1, 139.7, 139.3, 139.2, 130.1,

in 57% yield.¹H NMR (300 MHz, CDCl₃)

d

8.64 (d, J ¼ 15.8 Hz, 1H),

129.5, 128.4, 122.5, 122.2, 121.9, 120.7, 119.0, 118.3, 117.3, 53.8. MS

(ES^þ) m/z (%): 460 (100) [M þ Na]^þ, 438 (90) [M þ H]^þ. HRMS (EI)

calcd. for C₂₄H₁₉N₇O₂(M^þ): 438.1660, found: 438.1678. HPLC

purity (method II): 100%.

8.58 (s, 1H), 8.27e8.15 (m, 2H), 8.00 (s, 1H), 7.94 (d, J ¼ 7.6 Hz, 1H),

7.80 (dd, J ¼ 8.9, 4.8 Hz, 2H), 7.67 (d, J ¼ 7.3 Hz, 1H), 7.59 (t,

J ¼ 7.4 Hz, 2H), 7.36 (t, J ¼ 7.6 Hz, 1H), 7.23 (dd, J ¼ 12.6, 8.5 Hz, 1H),

7.10 (t, J ¼ 8.6 Hz, 2H), 5.66 (d, J ¼ 11.3 Hz, 1H), 4.30 (d, J ¼ 8.7 Hz,

1H), 3.77 (t, J ¼ 12.1 Hz, 1H), 3.13 (d, J ¼ 11.9 Hz, 1H), 2.04 (s, 1H),

4.12.22. 4-Bromo-3-methyl-N-(3-(6-(3-(triﬂuoromethyl)

phenylamino)-9H-purin-8-yl)phenyl)benzamide (27e)

1.94e1.61 (m, 4H). ¹³C NMR (CDCl₃, 75 MHz)

d 161.5, 160.6, 157.4,

152.6,152.0,151.3,151.0,138.2,134.8,134.2,134.1,132.4,130.8,129.7,

125.8, 125.3, 122.5, 122.1, 122.0, 121.8, 121.1, 120.9, 120.0, 116.4, 116.1,

115.8, 115.5, 84.2, 68.9, 28.7, 24.7, 23.4. MS (ES^þ) m/z (%): 525 (50)

[M þ H]^þ, 441 (30) [M-THP]^þ. HPLC purity (method I): 90%.

Following the general method E from 23e, 27e was obtained in

94% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-d₆)

d

10.53 (s,1H), 10.30 (s,1H), 8.79 (s, 1H), 8.52 (d, J ¼ 8.3 Hz, 2H), 8.26

(d, J ¼ 8.1 Hz, 1H), 8.07e7.88 (m, 2H), 7.80 (d, J ¼ 8.3 Hz, 3H), 7.58

(dd, J ¼ 7.8, 4.3 Hz, 2H), 7.38 (d, J ¼ 7.6 Hz, 1H), 2.46 (s, 3H). ¹³C NMR

4.12.18. N-(3-(6-(4-ﬂuorophenylamino)-9-(tetrahydro-2H-pyran-

2-yl)-9H-purin-8-yl)phenyl)-2-methoxynicotinamide (25h)

Following the general procedure D from 21d, 25h was obtained

(75 MHz, DMSO-d₆) d 164.8, 152.5, 151.4, 150.7, 150.1, 140.6, 139.5,

137.6, 133.9, 132.3, 130.2, 129.8, 129.6, 129.5, 129.4, 129.0, 127.9,

127.0, 126.1, 124.0, 122.7, 122.5, 122.3, 119.2, 118.6, 116.5, 22.5. MS

(ES^þ) m/z (%): 567 (100) [M þ H]^þ, 569 (100) [M þ H]^þ. HRMS (EI)

calcd. for C₂₆H₁₈BrF₃N₆O (M^þ) : 567.0756, found: 567.0737. HPLC

purity (method II): 100%.

in 60% yield. ¹H NMR (300 MHz, CDCl₃)

d 10.00 (s, 1H), 8.62 (dd,

J ¼ 7.6, 1.9 Hz, 1H), 8.56 (s, 1H), 8.35 (dd, J ¼ 4.8, 1.9 Hz, 1H), 8.11 (s,

1H), 7.96 (d, J ¼ 8.0 Hz, 1H), 7.80e7.71 (m, 3H), 7.68e7.52 (m, 2H),

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

23

4.12.23. 2-Methoxy-N-(3-(6-(3-(triﬂuoromethyl)phenylamino)-

9H-purin-8-yl)phenyl)benzamide (27f)

130.1, 129.9, 129.8, 129.7, 129.4, 124.5, 122.1, 120.5, 118.2, 116.4, 112.1,

109.1, 108.8, 107.4, 107.1, 55.9. MS (ES^þ) m/z (%): 477 (20) [M þ H]^þ,

455 (100) [M þ H]^þ. HRMS (EI) calcd. for C₂₅H₁₉FN₆O₂(M^þ) :

455.1632, found: 455.1623. HPLC purity (method II): 100%.

Following the general method E from 23f, 27f was obtained as

a solid in 81% yield. M.p. 242 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-

d₆)

d

10.34 (s, 2H), 8.76 (s, 1H), 8.52 (d, J ¼ 7.8 Hz, 2H), 8.27 (d,

J ¼ 8.2 Hz, 1H), 7.92 (d, J ¼ 7.5 Hz, 1H), 7.71 (t, J ¼ 8.3 Hz, 2H),

7.63e7.49 (m, 3H), 7.38 (d, J ¼ 7.5 Hz, 1H), 7.22 (d, J ¼ 8.2 Hz, 1H),

7.10 (t, J ¼ 7.2 Hz, 1H), 3.95 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

4.12.28. N-(3-(6-(3-ﬂuorophenylamino)-9H-purin-8-yl)phenyl)-2-

methoxynicotinamide (28h)

Following the general method E from 24h, 28h was obtained as

d

164.6, 156.6, 152.5, 151.4, 150.7, 150.2, 140.6, 139.5, 132.3, 129.9,

a solid in 40% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz,

129.7, 129.6, 129.4, 129.4, 129.0, 126.1, 124.5, 124.0, 122.5, 122.1,

120.6, 118.6, 118.6, 118.3, 116.6, 116.6, 116.5, 112.1, 55.9. MS (ES^þ) m/z

(%): 527 (15) [M þ Na]^þ, 505 (100) [M þ H]^þ. HPLC purity (method

II): 100%.

DMSO-d₆) d 10.42 (s, 1H), 10.17 (s, 1H), 8.72 (s, 1H), 8.48 (s, 1H), 8.37

(dd, J ¼ 4.9, 1.9 Hz, 1H), 8.15e8.02 (m, 2H), 7.92 (d, J ¼ 7.8 Hz, 1H),

7.76 (dd, J ¼ 21.3, 8.6 Hz, 2H), 7.56 (t, J ¼ 7.9 Hz, 1H), 7.37 (dd,

J ¼ 15.4, 8.1 Hz, 1H), 7.19 (dd, J ¼ 7.4, 5.0 Hz, 1H), 6.85 (dd, J ¼ 8.5,

6.2 Hz, 1H), 4.03 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

d 163.7, 163.4,

4.12.24. 2-Fluoro-N-(3-(6-(3-(triﬂuoromethyl)phenylamino)-9H-

purin-8-yl)phenyl)benzamide (27g)

160.5, 159.9, 152.5, 151.5, 150.8, 149.9, 149.1, 141.7, 141.5, 139.3, 139.2,

130.0, 129.9, 129.5, 122.3, 122.1, 118.9, 118.3, 117.3,116.2, 108.8, 108.6,

107.3, 106.9, 53.8. MS (ES^þ) m/z (%): 456 (100) [M þ H]^þ. HRMS (EI)

calcd. for C₂₄H₁₈FN₇O₂(Mþ): 456.1577, found: 456.1584. HPLC

purity (method II): 94%.

Following the general method E from 23g, 27g was obtained as

a solid in 90% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz,

DMSO-d₆)

d 10.65 (s, 1H), 10.34 (s, 1H), 8.78 (s, 1H), 8.51 (d,

J ¼ 5.4 Hz, 2H), 8.25 (d, J ¼ 8.4 Hz, 1H), 7.93 (d, J ¼ 7.7 Hz, 1H),

7.80e7.51 (m, 6H), 7.38 (q, J ¼ 7.9 Hz, 4H), 4.60 (s, 1H). ¹³C NMR

4.12.29. 4-Bromo-N-(3-(6-(4-ﬂuorophenylamino)-9H-purin-8-yl)

phenyl)-3-methylbenzamide (29e)

(75 MHz, DMSO-d₆)

d 203.4, 164.8, 143.8, 140.6, 139.5, 139.4, 137.6,

133.9, 132.3, 132.0, 130.1, 129.82, 129.6, 129.4, 127.9, 127.0, 124.0,

122.7, 122.3, 119.2, 118.6, 116.5, 22.5. MS (ES^þ) m/z (%): 493 (100)

[M þ H]^þ. HRMS (EI) calcd. for C₂₅H₁₈F₃N₇O₂(M^þ): 493.1400,

found: 493.1382. HPLC purity (method II): 100%.

Following the general method E from 25e, 29e was obtained in

39% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-d₆)

d

10.51 (s, 1H), 9.93 (s, 1H), 8.76 (s, 1H), 8.39 (s, 1H), 8.04e7.90 (m,

4H), 7.77 (q, J ¼ 8.3 Hz, 3H), 7.56 (t, J ¼ 7.9 Hz, 1H), 7.19 (t, J ¼ 8.9 Hz,

2H), 2.46 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

164.8, 159.4, 156.3,

d

4.12.25. 2-Methoxy-N-(3-(6-(3-(triﬂuoromethyl)phenylamino)-

9H-purin-8-yl)phenyl) nicotinamide (27h)

151.5, 139.5, 137.6, 135.9, 133.9, 132.3, 130.2, 129.9, 129.4, 127.9,

127.0, 122.6, 122.6, 122.5, 122.2, 119.1, 115.1, 114.8, 100.2, 22.5. MS

(ES^þ) m/z (%): 517 (70) [M þ H]^þ, 519 (70) [M þ H]^þ. HRMS (EI)

calcd. for C₂₅H₁₈F₃N₇O₂(M^ꢁ): 515.0631, found: 515.0615. HPLC

purity (method II): 81%.

Following the general method E from 23h, 27h was obtained as

a solid in 94% yield. M.p. 240e242 ^ꢀC (Koﬂer). ¹H NMR (300 MHz,

DMSO-d₆)

d 10.42 (s, 1H), 10.33 (s, 1H), 8.74 (s, 1H), 8.51 (d,

J ¼ 7.4 Hz, 2H), 8.37 (dd, J ¼ 4.9, 1.8 Hz, 1H), 8.27 (d, J ¼ 8.5 Hz, 1H),

8.16e8.07 (m, 1H), 7.94 (d, J ¼ 7.5 Hz, 1H), 7.74 (d, J ¼ 8.1 Hz, 1H),

7.58 (dd, J ¼ 13.3, 7.8 Hz, 2H), 7.38 (d, J ¼ 7.3 Hz,1H), 7.19 (dd, J ¼ 7.3,

4.12.30. N-(3-(6-(4-ﬂuorophenylamino)-9H-purin-8-yl)phenyl)-2-

methoxybenzamide (29f)

5.0 Hz, 1H), 4.03 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

d

163.3, 159.9,

Following the general method E from 25f, 29f was obtained in

158.5, 158.0, 152.5, 151.4, 150.7, 150.0, 149.1, 140.6, 139.3, 139.2,

130.0, 129.6, 129.5, 129.0, 126.1, 124.0, 122.5, 122.3, 122.1, 118.9,

118.6, 118.3, 117.3, 116.6, 116.5, 54.9, 53.8. MS (ES^ꢁ) m/z (%): 504

(100) [M-H]^ꢁ. HRMS (EI) calcd. for C₂₅H₁₈F₃N₇O₂(M^ꢁ): 504.1396,

found: 504.1394. HPLC purity (method II): 100%.

92% yield. M.p. 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-d₆)

d

10.34 (s, 1H), 10.10 (s, 1H), 8.74 (s, 1H), 8.42 (s, 1H), 8.06e7.84 (m,

3H), 7.70 (t, J ¼ 8.1 Hz, 2H), 7.54 (t, J ¼ 8.1 Hz, 2H), 7.21 (t, J ¼ 7.7 Hz,

3H), 7.10 (t, J ¼ 7.4 Hz,1H), 3.94 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

d

164.6, 156.6, 156.4, 152.1, 151.1, 150.8, 149.9, 139.5, 135.7, 132.3,

129.9, 129.7, 129.4, 124.5, 122.9, 122.8, 122.0, 120.6, 118.2, 115.2,

114.9, 112.1, 55.9. MS (ES^þ) m/z (%): 455 (100) [M þ H]^þ. HRMS

(EI) calcd. for C₂₅H₁₉FN₆O₂(M^þ): 455.1632, found: 455.1619. HPLC

purity (method II): 92%.

4.12.26. 4-Bromo-N-(3-(6-(3-ﬂuorophenylamino)-9H-purin-8-yl)

phenyl)-3-methylbenzamide (28e)

Following the general method E from 24e, 28e was obtained as

a solid in 99% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz,

DMSO-d₆)

d

13.76 (s,1H),10.52 (s,1H),10.12 (s,1H), 8.77 (s,1H), 8.46

4.12.31. 2-Fluoro-N-(3-(6-(4-ﬂuorophenylamino)-9H-purin-8-yl)

phenyl)benzamide (29g)

(s, 1H), 8.07 (d, J ¼ 12.3 Hz, 1H), 7.99 (s, 1H), 7.92 (d, J ¼ 7.7 Hz, 1H),

7.78 (dd, J ¼ 12.3, 8.5 Hz, 4H), 7.56 (t, J ¼ 7.8 Hz, 1H), 7.36 (d,

J ¼ 7.8 Hz, 1H), 6.86 (d, J ¼ 7.6 Hz, 1H), 2.46 (s, 3H). ¹³C NMR

Following the general method E from 25g, 29g was obtained in

73% yield. M.p. > 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-d₆)

(75 MHz, DMSO-d₆)

d

165.8, 163.7, 160.5, 152.0, 150.5, 139.7, 137.7,

d 10.64 (s, 1H), 10.05 (s, 1H), 8.75 (s, 1H), 8.40 (s, 1H), 8.02e7.89 (m,

134.5,132.3,130.1,129.5,128.3,128.1,127.0,124.9,122.9,122.3,119.1,

116.5, 20.9. MS (ES^ꢁ) m/z (%): 515 (100) [M-H]^ꢁ, 517 (100) [M-H]^ꢁ.

HPLC purity (method II): 94%.

3H), 7.79e7.52 (m, 4H), 7.37 (dd, J ¼ 15.1, 7.7 Hz, 2H), 7.19 (t,

J ¼ 8.9 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆)

d 163.04, 160.67,

159.65, 157.38, 152.20, 151.30, 150.94, 149.86, 139.39, 135.82, 132.86,

132.77, 130.00, 129.59, 124.89, 124.74, 124.69, 122.93, 122.83, 122.37,

122.09, 118.42, 116.45, 116.16, 115.28, 114.98. MS (ES^þ) m/z (%): 443

(100) [M þ H]^þ. HRMS (EI) calcd. for C₂₄H₁₆F₂N₆O₂(M^þ) : 443.1432,

found: 443.1427. HPLC purity (method II): 100%.

4.12.27. N-(3-(6-(3-ﬂuorophenylamino)-9H-purin-8-yl)phenyl)-2-

methoxybenzamide (28f)

Following the general method E from 24f, 28f was obtained as

a solid in 77% yield. M.p. 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-

d₆)

d

10.34 (s, 1H), 10.21 (s, 1H), 8.75 (s, 1H), 8.49 (s, 1H), 8.05 (d,

4.12.32. N-(3-(6-(4-ﬂuorophenylamino)-9H-purin-8-yl)phenyl)-2-

methoxynicotinamide (29h)

J ¼ 12.2 Hz, 1H), 7.91 (d, J ¼ 7.8 Hz, 1H), 7.79 (d, J ¼ 8.6 Hz, 1H), 7.70

(dd, J ¼ 13.1, 5.5 Hz, 2H), 7.61e7.50 (m, 2H), 7.38 (dd, J ¼ 15.4, 8.2 Hz,

1H), 7.22 (d, J ¼ 8.3 Hz, 1H), 7.10 (t, J ¼ 7.5 Hz, 1H), 6.87 (dd, J ¼ 9.6,

Following the general method E from 25h, 29h was obtained as

a solid in 82% yield. M.p. 250 ^ꢀC (Koﬂer). ¹H NMR (300 MHz, DMSO-

7.3 Hz, 1H), 3.94 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

d

164.7, 163.7,

d₆)

d 10.43 (s, 1H), 10.19 (s, 1H), 8.73 (s, 1H), 8.45 (s, 1H), 8.36 (dd,

160.5,158.0, 156.6, 152.3, 151.1,150.6, 150.2, 141.4,141.3,139.5,132.3,

J ¼ 4.8, 1.5 Hz, 1H), 8.10 (dd, J ¼ 7.3, 1.5 Hz, 1H), 7.98e7.87 (m, 3H),

24

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

7.73 (d, J ¼ 8.1 Hz, 1H), 7.57 (t, J ¼ 7.9 Hz, 1H), 7.20 (dt, J ¼ 9.0, 6.9 Hz,

3H), 4.02 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆)

163.4, 159.9, 151.8,

150.7, 150.2, 149.1, 139.3, 135.4, 129.8, 129.5, 123.1, 123.0, 122.2,

122.1, 118.9, 118.2, 117.3, 115.3, 115.0, 53.8. MS (ES^þ) m/z (%): 456

(100) [M þ H]^þ. HRMS (EI) calcd. for C₂₄H₁₈FN₇O₂(M^þ): 456.1584,

found: 456.1578. HPLC purity (method II): 91%.

compounds in 1ꢂ binding buffer (20% SeaBlock, 0.17ꢂ PBS, 0.05%

Tween 20, 6 mM DTT). Test compounds were prepared as 40ꢂ 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

(1ꢂ PBS, 0.05% Tween 20). The beads were then re-suspended in

d

4.13. Molecular modelling and docking

elution buffer (1ꢂ PBS, 0.05% Tween 20, 0.5

mM non-biotinylated

afﬁnity ligand) and incubated at room temperature with shaking

for 30 min. The kinase concentration in the eluates was measured by

qPCR.

Experimental crystallographic coordinates of human Met (PDB

ID: 3F82, resolution 2.50 Å) and Kit (PDB ID: 1T46, resolution

1.60 Å) without water molecules were used as templates to obtain

the Tyro3 modelled structure. The homology modelling of human

Tyro3 was performed using the software MODELLER [35] with an

automated protocol for optimization and minimization of the

models.

The sequence alignement of Met, Kit and Tyro 3 was accom-

plished with BlastP [36] utilizing BLOSUM62 matrix algorithm [37],

T-Coffee and Expresso [38,39] and resulting in Tyro3-Met identity

of 44% and similary of 60%, and identity of 31% and similarity of 43%

Compounds that bind the kinase active site and directly (steri-

cally) or indirectly (allosterically) prevent kinase binding to the

immobilized ligand, will reduce the amount of kinase captured on

the solid support. Conversely, test molecules that do not bind the

kinase have no effect on the amount of kinase captured on the solid

support. Screening “hits” are identiﬁed by measuring the amount of

kinase captured in test versus control samples by using a quanti-

tative, precise and ultra-sensitive qPCR method that detects the

associated DNA label. In a similar manner, dissociation constants

(K_ds) for test compoundekinase interactions are calculated by

measuring the amount of kinase captured on the solid support as

a function of the test compound concentration. The values of K_dand

IC₅₀are related by the ChengePrusoff equation: K_d¼ (IC₅₀)/

(1 þ ([S]/[K_m])), where K_mis the Michaelis constant of the enzyme,

and [S] is the substrate’s concentration [44].

for Tyro3-Kit comparison. Although Met presents

a higher

sequence similarity with Tyro3, three solvent exposed loops

involving respectively 17, 5 and 4 residues, are absent in the 3F82

structure, whereas they are present in the 1T46 structure of Kit.

Therefore, we used both proteins in order to increase the number of

residues in the Tyro3 sequence for which a template structure was

available. The resulting sequence alignment presented only a small

gap corresponding to a 5 residue loop in insertion in the Tyro3

sequence (Fig. 2 in supplementary materials).

4.15. Cell culture and cell proliferation assays

The model with the best MODELLER target function was further

analysed according to its Ramachandran plot, QMEAN server

analysis [40], MolProbity [41] in order to check its overall topo-

logical quality. This model was retained to perform docking

experiments.

Docking experiments were performed with FRED [42] using

Chemgauss3 scoring function [29]. A conformational database of

listed compounds was generated with OMEGA [43] and subse-

quently used as input to FRED. OMEGA generates high quality 3D

conformers with low energies. Bond lengths and angles remain

ﬁxed, but rotation around rotatable torsion angles is allowed. The

required receptor ﬁle was built with FRED_Receptor using multiple

molecular probes detection. No constraint was used in order not to

restrict the pose selection. The best ranked pose for each compound

was retained and analysed in order to clarify the interactions

involved in the ligand binding.

Cytotoxicity assays were carried out in Gif sur Yvette, France at

the Imagif/CNRS/ICSN platform.

The human cell lines were originated from ATCC, except when

otherwise stated. The human cell line KB (mouth epidermoid

carcinoma) and Vero (epithelial monkey kidney) were grown in

MEM medium supplemented with 10% fetal calf serum (InVi-

trogen), in the presence of 100 UI/mL penicilline, 100 g/mL

streptomycine and 1.5

g/mL fungizone in 75 cm²ﬂask under 5%

D-

m

CO₂, whereas HT29 (colon adenocarcinoma), MCF7 (breast adeno-

carcinoma) from Matthias Kassac (Bonn) and PC-3 (prostate

adenocarcinoma) were grown in RPMI medium.

Cells were plated in 96-well tissue culture plates in 200

ml

medium and treated 24 h later with compounds dissolved in DMSO

using a Biomek 3000 (Beckman). Controls received the same

volume of DMSO (1% ﬁnal volume). After 72 h exposure MTS

reagent (Celltiter 96Aqueous One solution, Promega) was added

and incubated for 3 h at 37 ^ꢀC: the absorbance was monitored at

490 nm and results expressed as the inhibition of cell proliferation

calculated as the ratio [(1 ꢁ (OD490 treated/OD490 control)) ꢂ 100]

in triplicate experiments.

4.14. Kinase assays

Kinase assays were carried out at Kinomescan, a division of Dis-

coveRex, 11180 Roselle St. Suite D, San Diego, CA 92121, as follows.

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 log-phase and infected with T7

phage from a frozen stock (multiplicity of infection ¼ 0.4) and

incubated with shaking at 32 ^ꢀC until lysis (90e150 min). The lysates

Acknowledgements

This study was supported by the Centre National de la Recher-

che Scientiﬁque, the Institut Curie, the INCa and the Ligue Nationale

Contre le Cancer (to FR and IBB, accredited team).

were centrifuged (6000 ꢂ g) and ﬁltered (0.2

m

m) to remove cell

The authors are thankful to Institut Curie for a post-doctoral

fellowship for Rosa Suárez and French Ministry of Research for

a doctoral fellowship for Franciane Chevot. Andrea Cavagnino was

supported by INCa (Translational research 2011 program). The

authors thank Mariane Bombled for performing mass spectra.

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 mole-

cule ligands for 30 min at room temperature to generate afﬁnity

resins for 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 non-speciﬁc phage binding. Binding reactions were assem-

bled by combining kinases, liganded afﬁnity beads, and test

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in

theonlineversion, athttp://dx.doi.org/10.1016/j.ejmech.2012.06.005.

R.M. Suárez et al. / European Journal of Medicinal Chemistry 61 (2013) 2e25

[19] J. Porter, Expert Opin. Ther. Patents 20 (2010) 159e177.

25

References

[20] D.K. Williams, X.-T. Chen, C. Tarby, R. Kaltenbach, Z.-W. Cai, J.S. Tokarski, Y. An,

J.S. Sack, B. Wautlet, J. Gullo-Brown, B.J. Henley, R. Jeyaseelan, K. Kellar,

V. Manne, G.L. Trainor, L.J. Lombardo, J. Fargnoli, R.M. Borzilleri, Bioorg. Med.

Chem. Lett. 20 (2010) 2998e3002.

[1] D.R. Robinson, Y.-M. Wu, S.-F. Lin, Oncogene 19 (2000) 5548e5557.

[2] R.M.A. Linger, A.K. Keating, H.S. Earp, D.K. Graham, Adv. Canc. Res. (2008)

35e83.

[3] D.K. Graham, T.L. Dawson, D.L. Mullaney, H.R. Snodgrass, H.S. Earp, Cell

Growth Differ. 5 (1994) 647e657.

[4] R.M. Linger, A.K. Keating, H.S. Earp, D.K. Graham, Expert Opin. Ther. Targets 14

(2010) 1073e1090.

[5] S. Zhu, H. Wurdak, Y. Wang, A. Galkin, H. Tao, J. Li, C.A. Lyssiotis, F. Yan, B.P. Tu,

L. Miraglia, J. Walker, F. Sun, A. Orth, P.G. Schultz, X. Wu, Proc. Natl. Acad. Sci.

U.S.A. 106 (2009) 17025e17030.

[21] X. Lv, W. Bao, J. Org. Chem. 72 (2007) 3863e3867.

[22] N. Ibrahim, F. Chevot, M. Legraverend, Tet. Lett. 52 (2011) 305e307.

[23] M.J. Robins, E.F. Godefroi, T. E. C, L.R. Lewis, A. Jackson, J. Am. Chem. Soc. 83

(1961) 2574e2579.

[24] B.P. Fors, P. Krattiger, E. Strieter, S.L. Buchwald, Org. Lett. 10 (2008)

3505e3508.

[25] P. Capek, M. Vrábel, Z. Hasník, R. Pohl, M. Hocek, Synthesis (2006) 3515e3526.

[26] T. Borrmann, A. Abdelrahman, R. Volpini, C. Lambertucci, E. Alksnis,

S. Gorzalka, M. Knospe, A.C. Schiedel, G. Cristalli, C.E. Müller, J. Med. Chem. 52

(2009) 5974e5989.

[27] M.A. Fabian, W.H. Biggs, D.K. Treiber, C.E. Atteridge, M.D. Azimioara,

M.G. Benedetti, T.A. Carter, P. Ciceri, P.T. Edeen, M. Floyd, J.M. Ford, M. Galvin,

J.L. Gerlach, R.M. Grotzfeld, S. Herrgard, D.E. Insko, M.A. Insko, A.G. Lai, J.-

M. Lélias, S.A. Mehta, Z.V. Milanov, A.M. Velasco, L.M. Wodicka, H.K. Patel,

P.P. Zarrinkar, D.J. Lockhart, Nat. Biotechnol. 23 (2005) 329e336.

[28] K. Stierand, M. Rarey, ChemMedChem 2 (2007) 853e860.

[29] M.R. McGann, H.R. Almond, A. Nicholls, J.A. Grant, F.K. Brown, Biopolymers 68

(2003) 76e90.

[30] L.M. Wodicka, P. Ciceri, M.I. Davis, J.P. Hunt, M. Floyd, S. Salerno, X.H. Hua,

J.M. Ford, R.C. Armstrong, P.P. Zarrinkar, D.K. Treiber, Chem. Biol. 17 (2010)

1241e1249.

[31] D. Mucs, R.A. Bryce, P. Bonnet, J. Comput. Aided Mol. Des. 25 (2011) 569e581.

[32] JJ.-L. Liao, J. Med. Chem. 50 (2007) 409e424.

[33] F. Milletti, J.C. Hermann, ACS Med. Chem. Lett. 3 (2012) 383e386.

[34] L. Vandromme, M. Legraverend, S. Kreimerman, O. Lozach, L. Meijer,

D.S. Grierson, Bioorg. Med. Chem. 15 (2007) 130e141.

[35] A. Fiser, A. Sali, in: J.A.R.M.S. Charles, W. Carter (Eds.), Macromolecular Crys-

tallography, Part D, vol. 374, Academic Press, 2003, pp. 461e491.

[36] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, J. Mol. Biol. 215

(1990) 403e410.

[6] Y. Liu, N.S. Gray, Nat. Chem. Biol. 2 (2006) 358e364.

[7] H.G. Choi, P. Ren, F.J. Adrian, F. Sun, H.S. Lee, X. Wang, Q. Ding, G. Zhang, Y. Xie,

J. Zhang, Y. Liu, T. Tuntland, M. Warmuth, P.W. Manley, J. Mestan, N.S. Gray,

T. Sim, J. Med. Chem. 53 (2010) 5439e5448.

[8] B. Okram, A. Nagle, F.J. Adri$n, C. Lee, P. Ren, X. Wang, T. Sim, Y. Xie, X. Wang,

G. Xia, G. Spraggon, M. Warmuth, Y. Liu, N.S. Gray, Chem. Biol. 13 (2006)

779e786.

[9] W.L. Delano, DeLano Scientiﬁc LLC, San Carlos, CA, USA. http://www.pymol.

org.

[10] T. Zhou, L. Commodore, W.-S. Huang, Y. Wang, T.K. Sawyer, W.C. Shakespeare,

T. Clackson, X. Zhu, D.C. Dalgarno, Chem. Biol. Drug Design 75 (2010) 18e28.

[11] P. Bhargava, M.O. Robinson, Curr. Oncol. Rep. 13 (2011) 103e111.

[12] A. Mollard, S.L. Warner, L.T. Call, M.L. Wade, J.J. Bearss, A. Verma, S. Sharma,

H. Vankayalapati, D.J. Bearss, ACS Med. Chem. Lett. 2 (2011) 907e912.

[13] J.M.L. Ebos, C.R. Lee, W. Cruz-Munoz, G.A. Bjarnason, J.G. Christensen,

R.S. Kerbel, Cancer Cell 15 (2009) 232e239.

[14] M.W. Karaman, S. Herrgard, D.K. Treiber, P. Gallant, C.E. Atteridge,

B.T. Campbell, K.W. Chan, P. Ciceri, M.I. Davis, P.T. Edeen, R. Faraoni, M. Floyd,

J.P. Hunt, D.J. Lockhart, Z.V. Milanov, M.J. Morrison, G. Pallares, H.K. Patel,

S. Pritchard, L.M. Wodicka, P.P. Zarrinkar, Nat. Biotechnol. 26 (2008) 127e132.

[15] X. Huang, P. Finerty Jr., J.R. Walker, C. Butler-Cole, M. Vedadi, M. Schapira,

S.A. Parker, B.E. Turk, D.A. Thompson, S. Dhe-Paganon, J. Struct. Biol. 165

(2009) 88e96.

[37] S.R. Eddy, Nat. Biotechnol. 22 (2004) 1035e1036.

[16] A. Ullrich, P. Knyazev, Y. Zhang, K. György, L. Örﬁ, I. Szabadkai, in: WO 2009/

127417 A1, DE, 2009, pp. 1e201.

[38] F. Armougom, S.B. Moretti, O. Poirot, S.P. Audic, P. Dumas, B. Schaeli,

V. Keduas, C. Notredame, Nucleic Acids Res. 34 (2006) W604eW608.

[39] C.D. Notredame, D.G. Higgins, J. Heringa, J. Mol. Biol. 302 (2000) 205e217.

[40] P. Benkert, M. Künzli, T. Schwede, Nucleic Acids Res. 37 (2009) W510eW514.

[41] I.W. Davis, A. Leaver-Fay, V.B. Chen, J.N. Block, G.J. Kapral, X. Wang,

L.W. Murray, W.B. Arendall, J. Snoeyink, J.S. Richardson, D.C. Richardson,

Nucleic Acids Res. 35 (2007) W375eW383.

[17] N.A. Powell, J.T. Kohrt, K.J. Filipski, M. Kaufman, D. Sheehan, J.E. Edmunds,

A. Delaney, Y. Wang, F. Bourbonais, D.-Y. Lee, F. Schwende, F. Sun,

P. McConnell, C. Catana, H. Chen, J. Ohren, L.A. Perrin, Bioorg. Med. Chem. Lett.

22 (2012) 190e193.

[18] G.M. Schroeder, Y. An, Z.-W. Cai, X.-T. Chen, C. Clark, L.A.M. Cornelius, J. Dai,

J. Gullo-Brown, A. Gupta, B. Henley, J.T. Hunt, R. Jeyaseelan, A. Kamath, K. Kim,

J. Lippy, L.J. Lombardo, V. Manne, S. Oppenheimer, J.S. Sack, R.J. Schmidt,

G. Shen, K. Stefanski, J.S. Tokarski, G.L. Trainor, B.S. Wautlet, D. Wei,

D.K. Williams, Y. Zhang, Y. Zhang, J. Fargnoli, R.M. Borzilleri, J. Med. Chem. 52

(2009) 1251e1254.

[42] M. McGann, J. Chem, Inf. Model. 51 (2011) 578e596.

[43] OEChem, Version 1.7.4, OpenEye Scientiﬁc Software, Inc., Santa Fe, NM, USA,

2010.

[44] Y. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099e3108.

Article Doi

Inhibitors of the TAM subfamily of tyrosine kinases: Synthesis and biological evaluation

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