N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy] -5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor

J. Med. Chem. 2006, 49, 6465-6488

6465

N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-

(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a Novel, Highly Selective,

Orally Available, Dual-Specific c-Src/Abl Kinase Inhibitor^†

Laurent F. Hennequin,*^,‡Jack Allen,^§Jason Breed,^§Jon Curwen,^§Michael Fennell,^§Tim P. Green,^§

Christine Lambert-van der Brempt,^‡Re´my Morgentin,^‡Richard A. Norman,^§Annie Olivier,^‡Ludovic Otterbein,^§

Patrick A. Ple´,^‡Nicolas Warin,^‡and Gerard Costello^§

Centre de Recherches, AstraZeneca, ZISE La Pompelle, B.P. 1050, 51689 Reims Cedex 2, France, and AstraZeneca Pharmaceuticals,

Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom

ReceiVed April 12, 2006

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer

progression. We report here a novel subseries of C-5-substituted anilinoquinazolines that display high affinity

and specificity for the tyrosine kinase domain of the c-Src and Abl enzymes. These compounds exhibit high

selectivity for SFKs over a panel of recombinant protein kinases, excellent pharmacokinetics, and in vivo

activity following oral dosing. N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-

(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) inhibits c-Src and Abl enzymes at low

nanomolar concentrations and is highly selective over a range of kinases. AZD0530 displays excellent

pharmacokinetic parameters in animal preclinically and in man (t_1/2) 40 h). AZD0530 is a potent inhibitor

of tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase

in survival in a highly aggressive, orthotopic model of human pancreatic cancer when dosed orally once

daily. AZD0530 is currently undergoing clinical evaluation in man.

Introduction

may enhance the antitumor efficacy of hormonal and cytotoxic

agents in preclinical models.^14,15

c-Src kinase is a nonreceptor tyrosine kinase that acts as a

signal transduction inhibitor that is a critical component of

multiple signaling pathways that control cell growth, prolifera-

tion, invasion, and apoptosis. While c-Src kinase is highly

regulated and active only at low levels in most normal cells,

studies have shown that c-Src kinase is upregulated in many

human tumor types.^1,2Recently emerging data support the

hypothesis that the predominant consequence of increased c-Src

activity in tumor cells is to reduce cell adhesion, facilitate

motility, and thereby promote an invasive phenotype. Conse-

quently, there is considerable interest in the inhibition of c-Src

kinase as a treatment for cancer and in particular as an anti-

invasion strategy. Tumor cell invasion is a feature common to

all malignant tumors and is a process that occurs throughout

the evolution of a cancer. There is evidence that c-Src kinase

activity is an important component of the invasive phenotype

in both early and advanced solid tumors. In early disease, c-Src

kinase plays a key role in the epithelium to mesenchymal

transition that marks the conversion of epithelial tumor cells to

a more invasive phenotype.³Increased c-Src kinase activity has

been linked with the disruption of E-cadherin-mediated cell-

cell adhesion^4-6and also impacts the assembly and turnover of

focal adhesions, which are critical for cell migration.^7,8Studies

on colon tumors have found that the highest level of c-Src kinase

activity occurs in metastatic tissue^9,10and that increased c-Src

kinase activity is an indicator of a poor prognosis.^11-13

Furthermore, emerging data suggest that c-Src kinase inhibition

As well as having a role in solid tumors, c-Src family kinases

might be involved in the progression of chronic myeloid and

acute lymphoid leukemias (CMLs and ALLs) that are positive

for the Philadelphia chromosome (Ph+). Studies have shown

that c-Src kinases have a function in imatinib-resistant CML

and ALL^16,17and that inhibitors of c-Src and Bcr-Abl kinases

have activity against both imatinib-sensitive and imatinib-

resistant cell lines.¹⁸c-Src kinase activity is also implicated in

metastatic bone disease, a characteristic of late-stage progression

of many solid tumor types, for example, breast¹⁹and prostate,²⁰

and of leukemias.^21,22In animal models, inhibition of c-Src

kinase has been shown to limit invasion of bone metastases and

destructive bone resorption.²³

c-Src kinase has been and still is one of the most studied

cellular protein tyrosine kinases, yet no inhibitor has reached

the market either for osteoporosis or for cancer by targeting

tumor growth, cell adhesion, or motility.²⁴However, several

classes of molecules have been studied preclinically for their

ability to inhibit c-Src and Abl kinases,^24-26of which the three

most advanced compounds (Figure 1) that are undergoing

clinical evaluation are the anilinoquinazoline AZD0530 [N-(5-

chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-

5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine,^27,28the thi-

azolecarboxamide BMS-354825,²⁹and the quinolinecarbonitrile

SKI-606.²⁴

AZD0530 (33) is a highly selective, orally available, dual-

specific c-Src/Abl kinase inhibitor which is in clinical develop-

ment for the treatment of a wide range of tumor types. On the

basis of current knowledge of c-Src kinase activity, 33 is likely

to have a therapeutic benefit as an anti-invasive agent, with

potential for activity in early and advanced solid tumors,

leukemia, and metastatic bone disease. In this paper, we describe

^†The atomic coordinates and structure factors (PDB ID code 2H8H)

have been deposited in the Protein Data Bank, Research Collaboratory for

Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://

www.rcsb.org/).

* Corresponding Author. Phone: 33 (0)3 26 61 68 49. Fax: 33 (0)3 26

61 68 42. E-mail: Laurent.hennequin@astrazeneca.com.

^‡AstraZeneca.

^§AstraZeneca Pharmaceuticals.

Published on Web 10/04/2006

6466 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

give 71. The selectivity observed for the C-5 position over C-7,

is most probably due to an internal coordination of the

magnesium atom with the C-4 and C-5 oxygen atoms, favoring

a concerted process. Selective protection of the N-3 position of

71 with the POM group led to 72, which was then substituted

at C-5 by the tetrahydropyran nucleus. The deprotection of the

C-7 methoxy group of 73 required stronger conditions, namely,

thiophenol and potassium carbonate in NMP at 195 °C to give

the C-7 hydroxyquinazolinone 74. Acylation of the C-7 position

allowed subsequent versatile modification of either the C-4 or

C-7 substituent to generate 11, 14-16, 18, 19, 25, 33, 41, 42,

and 80 after conventional deprotection and Mitsunobu reactions.

The 6′-chlorobenzodioxanamine³¹was prepared and coupled to

the quinazolinone 75 (Scheme 3) after in situ activation, as

described for the preparation of 77. Compound 120 was then

deprotected as described for 77 to give 121, which was

subsequently alkylated to give 41 and 42. The synthesis of the

C-5 piperidinyloxy derivatives is described in Scheme 4 and

utilizes the N-protected quinazolinones 72 and 83. Introduction

of the N-methylpiperidinyl moiety or its N-Boc-protected

equivalent at the C-5 position gave the key protected precursors

84-87, which were subsequently modified at positions C-4 and

C-7 to give 5, 6, 12, 13, 28, 29, 96, and 97. Deprotection under

acidic conditions of the BOC protecting group of 97 gave 24.

Reductive amination of the secondary piperidine 96 using

sodium borohydride triacetate and formaldehyde in acetic acid/

methanol gave the corresponding N-methyl derivative 27. The

series of C-5 isopropoxy derivatives was obtained as shown in

Scheme 5. The protected quinazolinone 83 was reacted with

2-propanol and subsequently deprotected using ammonia/

methanol in a one-pot process to give 99. Debenzylation

followed by acylation at C-7, chlorination at C-4, and displace-

ment of the chlorine by the 4-amino-5-fluoro-1,3-benzodioxole

or 4-amino-5-chloro-1,3-benzodioxole gave 101 and 102. Alky-

lation with 2-chloroethanol gave two chloroalkyl precursors, 103

and 104. Nucleophilic displacement of the aliphatic chlorine

atom of 103 and 104 by morpholine and N-acetylpiperazine led,

respectively, to 31 and 32. The difluoroquinazolinone 108,

prepared from the commercially available 3,5-difluoroaniline,

also proved to be a valuable intermediate in our strategies

(Scheme 6). Activation of the C-5 fluorine atom by the

quinazoline carbonyl group, together with the second fluorine

at the C-7 position allowed selective nucleophilic displacement

of the C-5 fluorine by morpholine to give 109. Subsequent

displacement of the C-7 fluorine atom by the (2-hydroxyethyl)-

pyrrolidine side chain required the formation of an intermediate

alkoxide to give 110. Chlorination followed by reaction with

the aminobenzodioxole gave 20. The C-5- and C-6-disubstituted

derivatives were synthesized as shown in Scheme 7. The

5-hydroxy functionality of 111 was generated from the 5-(ben-

zyloxy)-6-methoxybenzoquinazolone using TFA. Subsequently,

the N-3 position of 111 was selectively protected to give 112.

Reaction with tetrahydropyran-4-ol or N-methylpiperidin-4-ol

led to 113 and 114, respectively. These key intermediate

quinazolinones were subsequently processed through conven-

tional functionalization of the C-6 and C-4 positions to give 38

and 40.

Figure 1. Structures of AZD0530, BMS-354825, and SKI606.

the rationale and structure-activity relationships (SARs) leading

to the synthesis of 33.

Chemistry

The C-5-substituted anilinoquinazolines described in Table

1 were usually prepared by one of the two main routes as

described in Schemes 1-7. The general strategies to introduce

the C-5 and C-7 substituents on the quinazoline core were based

either on the selective dealkylation of the C-5 and/or C-7

methoxy of the (benzyloxy)quinazoline precursors 39, 43, 46,

58, 59, 61-64, 73, 88, 93, 99, and 113 or on the displacement

by alkoxide anions of the C-5 and/or C-7 fluorine atom of the

fluoroquinazoline intermediates 48, 52, 108, and 109. Displace-

ment of the C-5 fluorine atom of the commercially available

quinazolinone 48 by sodium methoxide led to 45 (Scheme 1).

Subsequent activation of the C-4 position by POCl₃, followed

by the nucleophilic displacement of the intermediate 4-chloro-

quinazoline with 2-chloro-5-methoxyaniline, led to 46. Depro-

tection of the C-5-methoxy group was achieved by heating with

pyridine/HCl to give 47. When the same sequence of reactions

was applied to the dimethoxy derivative 43, the last step led to

selective deprotection of the more reactive C-5 methoxy

substituent over the less reactive C-7 methoxy to give rise to

44. The phenols 44 and 47 were then treated under Mitsunobu

conditions with a range of alcohols to give 1, 2, and 4.

Alternatively, as shown with the synthesis of 52, the electron-

withdrawing property of the C-5 fluorine atom was used to

activate the C-4 position of the quinazoline 51 and facilitate

the introduction of the 4-amino-5-chloro-1,3-benzodioxole²⁷to

give 52. The C-5 fluorine atom, although less reactive in 52

than in 48, was subsequently displaced by primary or secondary

alkoxides in DMF at 80 °C to give 3 and 53. Cleavage of the

BOC protecting group of 53 under acidic conditions gave 54,

which was then methylated under reductive amination conditions

to give 26 (Scheme 1). To achieve a mild and selective

deprotection of the C-7 position after having introduced the

desired C-5 alkoxy substituent, we prepared the 5,7-bis-

(benzyloxy)anilinoquinazoline 59 from the isatin precursor 55

(Scheme 2). Treatment of 59 with pyridine/HCl gave the phenol

60, which was then reacted with a range of secondary alcohols

under Mitsunobu conditions to give 61-64 with excellent yields.

Deprotection of the C-7 benzyloxy protecting group with TFA

led to 65-68, and these were then reacted with either 4-(3-

hydroxypropyl)morpholine³⁰or 3-(4-methylpiperazin-1-yl)pro-

pan-1-ol to give 7-10 or 21, respectively. The [(5-chloro-1,3-

benzodioxolyl)amino]quinazolines were synthesized as described

in Schemes 3-6. The dimethoxyquinazolinone 43 was selec-

tively deprotected at the C-5 position using MgBr₂/pyridine to

Results and Discussion

For clarity of discussion, data on only a limited, representative

set of compounds are used to describe the structure-activity

relationships. Where trends are exemplified by a single pair of

compounds, it is to be understood that more examples exist to

support the SAR described.³²

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6467

6468 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

Scheme 1^a

^aReagents and conditions: (a) (i) POCl₃, DIPEA, DCE, reflux; (ii) aniline, ⁱPrOH, 80 °C; (b) pyridine hydrochloride; (c) R₂OH, PPh₃, DTAD, DCM; (d)

i

MeONa, THF, reflux; (e) tert-butyl-4-hydroxypiperidine-1-carboxylate, NaH, DMF; (f) PPh₃, CCl₄, DCE; (g) aniline, HCl(cat.), PrOH, 80 °C; (h) HCl,

Et₂O; (i) ROH, NaH, DMF, 80 °C; (j) HCHO, AcOH, MeOH, NaBH(OAc)₃.

Kinases are important biological targets, and tremendous

efforts have been made over the past few years to solve their

three-dimensional structures complexed with different classes

of inhibitors.³³This has supplied key information, not only on

the nature and topology of the ATP binding site, but also on

the conformation of their active and inactive forms, revealing

something of their complex and varied modes of activation.^34-39

Structural studies of kinase complexes have revealed the

existence of a pattern of clustered residues and conserved

hydrogen bond interactions surrounding the active site, providing

a basis for the understanding of the binding of ATP or

competitive inhibitors.^34,35The adenine moiety of ATP itself is

anchored in the active site by two such bonds as illustrated in

Figure 2. This hydrogen bond network has been widely exploited

Figure 2. Schematic representation of ATP bound to the Src kinase

domain.³⁶The binding site is represented by its molecular surface in

blue.⁷³Molecules are illustrated in stick representation with colored

elements. The adenine moiety is anchored by one donator and one

acceptor H bond with Ty340 and Met341, respectively. Crystal

structures of kinase-inhibitor complexes^43,50,51,55have shown that

another H bond could also be formed between an H bond donor (HBD)

and the backbone carbonyl of a residue equivalent to Met341. The

locations of the ATP binding site pockets exploited in drug design are

also shown.

to design purine-mimetic scaffolds and develop novel chemical

series of ATP competitive kinase inhibitors^27,40-50as shown in

many kinase-inhibitor complexes.^45-47,51-54Although hydrogen

bonding is essential for this mode of inhibition, the number of

protein-ligand hydrogen bonds is not correlated with potency.

Structural studies have also revealed the presence of other

pockets within the active site which play important roles in the

potency and selectivity of small-molecule inhibitors. One feature

frequently exploited in the design of kinase inhibitors is the

presence of a deep hydrophobic pocket adjacent to the adenine

binding site (Figure 2).^{41,42,44,46,47,52,56,57}This pocket is made

up of conserved and nonconserved residues and is not occupied

by ATP itself. While its nature remains mainly hydrophobic,

its size and shape vary noticeably between kinases and depend

on the activation state of the enzyme.^{36,37,39a,58-60}The residue

present at the entrance of this pocket, also known as the

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6469

Figure 3. Compound 33 (AZD0530) in complex with c-Src (inactivated

form). (a) Overlay of AZD0530 with ATP bound to c-Src. The

tetrahydropyran ring fits closely to the ribose ring. The chlorobenzo-

dioxole moiety of AZD0530 is buried in the hydrophobic pocket. Note

that this pocket is not fully occupied by the benzodioxole, in agreement

with the fact that this pocket is deeper in the inactivated (c-Src) than

in the activated (Lck) form. (b) Stereoview of AZD0530 in the ATP

binding site. The molecular surface of the binding site is colored

according to the hydrophobicity of the surrounding residues. Yellow

areas indicate hydrophobic regions, magenta hydrophilic, and blue and

red, respectively, positively and negatively charged residues. The

tetrahydropyran ring is seen to pack with a hydrophobic pocket in the

ribose binding site. The interactions are predominantly formed with

the glycine loop in the N-terminal domain (Leu273, Gly274) and with

Leu393 and Ser345 in the C-terminal domain.

Figure 4. A [(5′-chlorobenzodioxolyl)amino]quinazoline derivative

34²⁷docked in the ATP binding site of the Lck 3D structure used as a

model for activated c-Src.⁷⁴For clarity, the residue numbering of c-Src

has been used. (a) The quinazoline ring occupies the adenine binding

site. The H bond interaction between the quinazoline N1 and the

backbone NH of Met341 is represented by a black dotted line. The

chlorobenzodioxole moiety is buried in the hydrophobic pocket,

characterized by a Thr residue at its entry (Thr338). The C7 basic side

chain lies in the solvent. (b) Edge-on view showing how well the

chlorobenzodioxole fits into the hydrophobic pocket and is adequately

designed to be selective for the c-Src kinase family.

that would optimally fit the ribose pocket and provide additional

affinity for the enzyme active site. A wide range of substituents,

flexible or rigid, linear or cyclic, neutral or basic, proved to be

well tolerated at this C-5 position (Table 1). However, cyclic

substituents generally led to more potent enzyme inhibitors than

did acyclic, flexible substituents (Table 1; compare 4 and 5),

consistent with an accessible ribose pocket of limited dimen-

sions. Oxygen- and nitrogen-containing heterocycles such as

the tetrahydropyran-4-yloxy, tetrahydrofuran-3-yloxy, and the

(N-methylpiperidin-4-yl)oxy rings proved overall to be preferred

(Table 1, 5 and 7-10). Comparison of compounds 5 and 22

shows that these C-5 cyclic substituents can improve enzyme

affinity by ∼10-fold over a C-5 hydrogen. When we previously

applied this C-5 substitution strategy to the design of novel

anilinoquinazoline inhibitors of EGFR-TK (epidermal growth

factor receptor tyrosine kinase), we observed that a basic group

at C-5 was clearly beneficial to sustain good potency,⁶⁴probably

due to the presence of aspartic residues within the EGF kinase

ribose pocket. Unlike that, in c-Src the presence of a basic group

at C-5 does not affect potency (compare compounds 1, 4, and

2) despite the fact that the aspartic residues thought to play a

role in charge interactions with the EGFR-TK inhibitors are

conserved in c-Src (Asp404 in the conserved DFG motif and

Asp348). This suggests that the acidic side chains might be

orientated differently in these two enzymes and have a different

impact on the electrostatic surface surrounding the ribose pocket.

From an in-house crystal structure of our best compound 33

complexed with c-Src, the tetrahydropyran ring fits tightly in

the ribose pocket (Figure 3). The interactions with the protein

are predominantly hydrophobic, and no H-bonding interaction

gatekeeper, is another key determinant for selective inhibition

within a kinase family.^44,61,62

We have previously reported that 2′-chloro-5′-methoxyanili-

noquinazolines substituted at the C-6 and C-7 positions of the

quinazoline ring (Table 1) display good inhibition of the c-Src

kinase enzyme.²⁷Interestingly, these inhibitors do not occupy

the ribose binding site. In this work we investigated a novel

series of anilinoquinazolines substituted at the C-5 position of

the quinazoline ring. The C-5 position allows substituents to

access the ATP ribose binding site and could thus provide

additional binding affinity for the enzyme. Unlike the selectivity

pocket, the ribose pocket is partially open to the solvent and

lined by a series of hydrophobic and hydrophilic residues (Figure

2). Modeling and docking of potential inhibitors into our 3D

model of c-Src suggested to us that heterocycles linked to the

position C-5 of the quinazoline would nicely fit the shape and

the chemical nature of the ribose pocket. Other work on

CDK1-2 (cyclin-dependent kinase) inhibitors had shown that

the ribose pocket could be occupied by a broad range of

substituents, including straight or branched alkyl chains, phenyl,

or cyclic amines.^50a,b,63,64

Despite extensive SAR studies aimed at modifying the

quinazoline core, little information was known about the possible

beneficial effects of C-5 substitution at the time of initiation of

this work. A thorough investigation of the SAR around the

substitution at the C-5 position of the quinazoline nucleus was

thus made in both the monocyclic 2′-chloro-5′-methoxyanili-

noquinazolines and the bicyclic [(chlorobenzodioxolyl)amino]-

quinazoline series with the aim of finding suitable substituents

6470 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

Scheme 2^a

^aReagents and conditions: (a) (ClCO)₂, 170 °C, 1 h; (b) NaOH, H₂O₂, H₂O, 70 °C; (c) Diazald, EtOH, NaOH, DCM, 0 °C; (d) formamidine acetate,

2-methoxyethanol, reflux, 2 h; (e) (i) POCl₃, DIPEA, DCE, 80 °C, 2 h; (ii) 2-chloro-5-methoxyaniline, ⁱPrOH, 80 °C, 30 min; (f) 2 equiv of HCl, pyridine,

reflux, 9 h; (g) Ph₃P, DTAD, R₁H, DCM; (h) TFA, 80 °C, 5 h; (i) Ph₃P, DTAD, ROH, DCM.

is observed between the ring oxygen and the ribose pocket. Due

to the presence of the bulky chlorobenzodioxole, the side chain

of Asp404 is indeed deflected opposite the hydrophobic pocket.

Increasing the degree of flexibility of the C-5 substituent as

illustrated by the larger and more flexible (N-methylpiperidin-

4-yl)methoxy substituent (compound 26) or conversely reducing

its flexibility as illustrated by the rigid and directly linked

morpholinyl substituent (compound 20) sustained a good level

of enzyme inhibition, although up to 12-fold lower than that of

the corresponding tetrahydropyran-4-yloxy or (N-methylpiperi-

din-4-yl)oxy equivalent (compare 26 and 3 and 20 and 16).

Branched alkyl chains, such as isopropoxy, gave potency

equivalent to that of the broad range of tolerated cyclic nuclei,

as shown by compounds 6-8, 10, 15, 24, 30, and 36. The

improvement in affinity achieved with the C-5 tetrahydropyran

is comparable in magnitude to that observed by interaction with

the protein through the C-7 position of the quinazoline as shown

by the comparison of 22 with 5 and 35.

although they were about 5-fold less potent than the monocyclic

2′-chloro-5′-methoxyanilines (compare 12 and 5). Expansion of

the benzodioxole ring size by one carbon atom as shown by

the benzodioxane nucleus retained a good level of c-Src enzyme

inhibition (comparison of compounds 12 and 13 and 14 and

17). Interestingly, this result differs significantly from that

observed in the C-6, C-7 quinazoline series where the benzo-

dioxane was 26-fold less active than the benzodioxole equiva-

lent.²⁷This possibly suggests that the C-5 substituent slightly

distorts the ATP site of the c-Src enzyme, thus slightly opening

up the entrance of the hydrophobic pocket to accommodate the

increased size of the benzodioxane ring. Further, introduction

of a chlorine atom at the C-6′ position of the 2,3-(methylene-

dioxy)aniline ring (see Table 1) (compare compounds 6, 11,

12, and 14-17), as observed in our previous work,²⁷also led

to a significant improvement in potency of up to 15-fold. The

chlorine atom is thought to add to the lipophilic interaction of

the compound with an alanine residue within the c-Src kinase

selectivity pocket (Ala403), thus increasing the binding affinity

(Figure 4). The chlorine substituent can be replaced by bromine

without loss in potency.⁶⁵Replacement by the smaller halogen

fluorine led to a slight but consistent reduction in potency

In the C-5 series, investigation of the substitution pattern of

the aniline also proved extremely important to help improve

the potency of our inhibitors. Bicyclic anilines such as the

benzodioxole (compounds 11, 12, and 14) were tolerated,

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6471

Scheme 3^a

^aReagents and conditions: (a) Diazald, EtOH, NaOH, DCM, 0 °C; (b) formamidine acetate, 2-methoxyethanol, reflux, 2 h; (c) MgBr₂, pyridine, reflux;

(d) NaH, ClPOM, 0 °C, DMF; (e) (i) Ph₃P, DTAD, 4-hydroxy-THP, DCM; (ii) MeOH, NH₃, overnight; (f) PhSH, K₂CO₃, NMP, 195 °C, 30 min; (g) Ac₂O,

i

catalytic pyridine, 80 °C, 30 min; (h) (i) POCl₃, DIPEA, DCE, 80 °C, 2 h; (ii) aniline, PrOH, 80 °C, 30 min; (i) MeOH, NH₃; (j) R₂Cl, K₂CO₃, DMF, 95

°C, 30 min; (k) Ph₃P, DTAD, R₂OH, DCM, 30 min; (l) (i) Ph₃P, CCl₄, DCE, 70 °C, 2 h; (ii) MeOH, NH₃, 2 h; (m) Ph₃P, DTAD, 2-pyrrolidin-1-ylethanol,

i

DCM; (n) ArNH₂, PrOH, reflux, 1.5 h; (o) HCHO, HCOOH, 100 °C.

(compare 31 and 32). A subsequent in-house crystal structure

of the benzodioxane derivative 17 in complex with c-Src has

provided interesting information. Compound 17 binds similarly

to the benzodioxole derivative 33 except that the benzodioxane

ring, which occupies the hydrophobic pocket, is rotated by about

180° compared to the chlorobenzodioxole ring. This particular

orientation may be necessary to relieve the steric bulk resulting

from the larger benzodioxane ring. Interestingly, in this orienta-

tion the ethylenedioxy bridge is no longer in the vicinity of the

gatekeeper residue (Thr338 in c-Src, Val916 in KDR), which

may explain the good potency to KDR and the reduced

selectivity.

We also noticed that the potency of the C-5-substituted series

was again increased by up to 25-fold by the introduction of an

electron-donating group (EDG) at the C-7 position of the

quinazoline nucleus (compare 1 and 5, 3 and 6, 23 and 24, and

3 and 28). In this C-7 position a large diversity of side chains

(basic, heterocyclic, heteroaromatic, neutral, etc.) were tolerated

by the enzyme with compounds showing low nanomolar

inhibition of c-Src (compounds 15, 18, 19, 23-25, and 27-

33). Moreover, this region of the molecule is exposed to the

solvent and thus proved to be ideal to anchor flexible side chains

bearing solubilizing groups to not only enhance the in vitro

potency but also optimize the physicochemical properties of this

series.^55,66Interestingly, the improvement in potency achieved

with the additional C-7 substituent proved to some extent to be

dependent on its relative basicity (compare 5 and 1, 7 and 2,

and 2 and 21). Steric hindrance near the quinazoline was

disfavored as indicated by the 10-25-fold reduction in potency

observed with the isopropoxy derivative 27 compared with

unbranched side chains, as illustrated by 6, 28, and 29. If a

second EDG is introduced at the C-6 position (ortho relative to

that at C-5), the activity of the resulting compound is reduced

by 17-100-fold compared with that of the C-5, C-7 isomer

(compare 5 and 38 and 21 and 40). This can probably be

explained by a conformational effect in which the relative peri

and ortho relations of the C-4, C-5, and C-6 substituents forces

the C-5 substituent to be oriented slightly out of the plane of

the quinazoline and thus induces unfavorable positioning or

steric contacts within the ribose pocket.

Kinase Selectivity. Selectivity for c-Src over other tyrosine

kinases was one of our major objectives to optimize the tolerance

profile of our inhibitors. As shown by the 2′-chloro-5′-

methoxyanilinoquinazoline 5, the C-5 series proved very selec-

6472 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

Scheme 4^a

^aReagents and conditions: (a) (i) 1-R₂-piperidin-4-ol, PPh₃, DTAD, DCM; (ii) NaOH or NH₃, MeOH; (b) PPh₃, CCl₄, DCE, 70 °C, 2 h; (c) aniline,

i

ⁱPrOH, reflux; (d) HCl, Et₂O; (e) (i) PPh₃, CCl₄, DCE, reflux, 2 h; (ii) aniline, PrOH, 80 °C, 1.5 h; (f) TFA, reflux, 6 h; (g) R₁OH, PPh₃, DTAD, DCM;

i

(h) Boc₂O, DMF; (i) (i) PrOH, PPh₃, DTAD, DCM; (ii) HCl, Et₂O; (j) NaBH(OAc)₃, HCHO, AcOH, MeOH.

tive for c-Src over VEGFR-2 (vascular endothelial growth factor

receptor 2, also known as KDR) (c-Src/KDR IC₅₀ratio >70-

fold). We had previously reported that in the binding to KDR

and c-Src of our C-6,C-7-disubstituted quinazoline-based inhibi-

tors²⁷the quinazoline ring is sandwiched between the N- and

C-terminal domains of the kinase and forms a number of

hydrophobic contacts (Figure 4). The exploitation of the

hydrophobic pocket had led us to design and develop the

selective (4-aminobenzodioxolyl)quinazoline series.²⁷In the

C-6,C-7-disubstituted quinazoline series, the 6′-H-benzodiox-

olamine fits particularly well into the hydrophobic pocket,

providing an excellent increase in selectivity over that of the

monocyclic 2′-chloro-5′-methoxyaniline, in particular versus

KDR.²⁷The increase in selectivity between c-Src and KDR

observed in the C-6, C-7 series had been rationalized by the

presence of a threonine at the entrance of the selectivity pocket

in c-Src (Thr338), which gives more favorable contacts with

the benzodioxole oxygens than the corresponding Val916

present in KDR.^27,67In contrast to what we reported in the C-6,

C-7 series,²⁷the 6′-H-benzodioxole aniline unexpectedly led to

a 5-10-fold reduction in selectivity for c-Src over KDR

compared with the monocyclic aniline as shown by the

comparison of 12 and 5 and 11 and 7. In the C-5 series, as

previously suggested for the c-Src enzyme, the size of this C-5

substituent probably distorts slightly the entrance of the

hydrophobic/selectivity pocket of KDR, thus minimizing the

differences between the two enzymes and leading to a reduced

selectivity. As a direct consequence of the benzodioxane ring

fitting KDR quite well in the C-5 quinazoline series, the

selectivity of these benzodioxane derivatives for c-Src over KDR

was reduced by 6-20-fold compared with that of the corre-

sponding benzodioxole (compare 14 and 17 and 12 and 13).

An improvement in selectivity was achieved in the C-5-

substituted benzodioxole series by the introduction of the 6′-

chlorine atom as shown by the comparison of compounds 14

and 16 and 11 and 15 (Figure 4). The chlorine lies in a

hydrophobic groove within the selectivity pocket, lined by an

alanine residue in c-Src (Ala403), whereas this residue is a

sterically and electronically less favorable cysteine in KDR.

Overall this series proved to deliver excellent selectivity for

c-Src inhibition over KDR inhibition as shown by the KDR/c-

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6473

Scheme 5^a

^aReagents and conditions: (a) MgBr₂, pyridine, reflux, 2 h; (b) NaH, ClPOM, 0 °C, DMF; (c) (i) Ph₃P, DTAD, ⁱPrOH, DCM; (ii) MeOH, NH₃, overnight;

(d) (i) NH₄+ CO₂^-, 10% Pd/C, DMF; (ii) Ac₂O, catalytic pyridine, 80 °C, 30 min; (e) (i) POCl₃, DIPEA, DCE, 80 °C; (ii) aniline, PrOH, 80 °C, 30 min;

i

(iii) MeOH, NH₃, 2 h; (f) DCE, DMF, K₂CO₃, 80 °C, 24 h; (g) Ph₃P, DTAD, 3-pyrrolidin-1-ylpropan-1-ol, DCM; (h) amine, K₂CO₃, KI, DMF, 80 °C.

Scheme 6^a

^aReagents and conditions: (a) (i) Chloral, NH₂OH, H₂O; (ii) H₂SO₄; (b) NaOH, H₂O₂; (c) Ph₃P, DEAD, MeOH, CH₂Cl₂; (d) formamidine acetate,

i

methoxyethanol, reflux; (e) morpholine, DMF; (f) 2-pyrrolidin-1-ylethanol, NaH, DMF; (g) (i) POCl₃, DIPEA; (ii) ArNH₂, PrOH, 80 °C.

Src IC₅₀ratios (62-fold to >7000-fold) of compounds 6, 15,

16, 18, 19, 23-25, and 28-33 (Table 1).

The excellent selectivity of these C-5-substituted anilino-

quinazolines for c-Src reported in Table 1 is not limited to KDR.

As shown in Table 4, this class of compounds proved to be

very selective against a range of kinases including cell cycle

kinases such as CDK2 and Aurora as well as antiproliferative

receptor and nonreceptor kinases such as EGFR-TK and MEK.

They also proved to be selective over Csk, the natural inactivator

of c-Src as demonstrated by compounds 15 and 33 (Csk/c-Src

IC₅₀ratios, respectively, >70-fold and 310-fold). However, this

series of compounds proved to possess significant activity versus

6474 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

Scheme 7^a

^aReagents and conditions: (a) TFA; (b) NaH, ClPOM, 0 °C, DMF; (c) (i) Ph₃P, DTAD, ROH, DCM; (ii) MeOH, NH₃, overnght; (d) Ph₃P, CCl₄, DCE,

70 °C, 2 h; (e) PhSH, K₂CO₃, NMP, 195 °C, 30 min; (f) aniline, HCl(cat.), IPA, reflux; (g) Ac₂O, catalytic pyridine, 80 °C, 30 min; (h) (i) Ph₃P, CCl₄, DCE,

70 °C, 2 h; (ii) MeOH, NH₃, overnight; (i) Ph₃P, DTAD, ROH, DCM.

other c-Src kinase family members and c-Src-related kinases

such as c-Yes and Lck as well as v-Abl (Table 4). The kinase

selectivity profile of 33 is representative of this series of

molecules and demonstrates potent dual-specific inhibition of

c-Src and Abl kinases. The functional redundancy demonstrated

in mouse gene knockout experiments among the ubiquitously

expressed SFKs c-Src, Yes, and Fyn coupled with emerging

evidence suggestive of similar mechanisms in cancer cells

suggests a pan SFK selective agent, such as AZD0530, might

be the profile required to ensure maximum inhibition of SFK

activity in cancer cells and tissues. It is anticipated a pan SFK

inhibitor could impact aspects of the immune cell function.

Although we have not seen evidence of this in preclinical studies

with AZD0530, active monitoring for immune cell and immune

function effects of the drug is being carried out in man.

In Vitro Cellular Activity. Inhibition of c-Src activity in

cells was evaluated in mouse NIH 3T3 cells transfected with

constitutively active human c-Src. The C-5-substituted anilino-

quinazoline series showed good permeability through cell

membranes (15, P_A-B> 1 × 10⁶cm/s; 33, P_A-B> 30 × 10⁶

cm/s), and consequently, an inhibition of c-Src at concentrations

below 10 nM in the enzyme assay translated into inhibition of

the c-Src-transfected NIH 3T3 cell proliferation at concentrations

below 100 nM. Inhibition of in vitro random cell motility

(chemokinesis) was measured by the ability of compounds to

prevent the migration of human lung tumor cells (A549 NSCLC)

suspended in an agarose microdroplet. In this assay the activity

of this series of molecules was consistently submicromolar and

again mirrored the potency found in the enzyme assay.

Compound 33 displayed IC₅₀values of 76 nM in the prolifera-

tion assay (c-Src-transfected NIH3T3 cells) and 140 nM in the

migration assays (A549 cells). The inhibition of migration

cannot be attributed to a direct antiproliferative effect on the

A549 cells in view of the weak antiproliferative activity of 33

on these cells (IC₅₀) 14 ( 1.5 µM (n ) 4)). Cellular activity

against Abl was evaluated in K562 cells (human line CML).

33 displayed in vitro antiproliferative IC₅₀values of 220 nM (n

) 3) in K562 (WT Ph+) cells.

Paxillin is a direct substrate of c-Src and is an adaptor/

scaffolding protein thought to be essential in linking newly

formed focal adhesions to the actin cytoskeleton. This link helps

provide the contractile forces required for cell motility. Mea-

surement of paxillin phosphorylation in tumor cells provides a

reliable in vitro mechanistic measure of c-Src activity, which

is linked to the adhesion/motility signaling pathway. Compounds

of this series displayed good activity in this assay,⁶⁸and in

particular 33 inhibited paxillin phosphorylation in vitro in A549

cells by 70% at a concentration of 1 µM (measured by Western

blot).

Moreover, the excellent selectivity observed for these com-

pounds at the kinase enzyme level was confirmed in cell assays

against selected targets with representatives of the series.⁶⁸

Physicochemical Properties and DMPK. As illustrated by

compounds 19 and 31, the solubility of almost neutral [(6′-

chlorobenzodioxolyl)amino]quinazolines is moderate at physi-

ological pH (Table 3). We had shown previously that the

solubility of anilinoquinazolines could be improved by the

introduction of a basic nitrogen.⁶⁶Introduction of basic side

chains at either the C-5 or the C-7 position of the quinazoline

core led to a very significant (>500-fold) increase in solubility

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6475

Table 2. Pharmacokinetic Parameters of Compounds 15, 18, 33, and 35

33

15

18

35

male

rat^a

female

rat^a

male

dog

male

dog

female

rat^b

male

dog

male

dog

rat^b

t

_1/2(h)

5-7

10

1.2

79

5-7

10

1

7-19

3.5

15

2.9

60

29

38

0.9

85

22

38

0.3

57

4.2

43

2.1

42

V_d(L/kg)

Cl [(L/h)/kg]

F (%)

11.6 ( 2.5

0.7 ( 0.1

>50

23.4

7.9

93

92

^aRat doses: po, 25 mg/kg; iv, 2 mg/kg. ^bRat doses: po, 20 mg/kg; iv, 2 mg/kg.

Table 3. Physicochemical Properties of Compounds 6, 15, 19, 31, 33,

and 35

Table 5. Mouse Total Plasma Levels

a

AUC_{0-24 h}

6

15

19 31 33 35

compd [(µg‚h)/mL] compd [(µg‚h)/mL] compd [(µg‚h)/mL]

molecular weight

polar surface area

no. of donor and acceptor H bonds 9

442

85

527 537 487 542 443

6

15

16

18

0.05

13

9

19

24

29

33

6

0.1

0.6

34

37

38

13

96

10

9

114 101 106 80

11

9

10 11

8

7

no. of rotatable bonds

5

8.2

2.9

8

31

12

a

pK_a

9.5 6.4 6.5 7.98 ∼9

^aAfter administration of a 20 mg/kg oral dose.

log D_7.4

2.3 >3.1 3.7 2.9

2

solubility (µM) at pH 7.4

>1000 1000 1.5

25

2

240^b324

f_u^c(%) in rat

16

17

1.7 2.5 13

7.5

f_u(%) in dog

f_u(%) in man

14

9

^apK_aof the basic side chain. ^bMeasured at pH 7. ^cFraction unbound to

plasma protein.

Table 4. Kinase Selectivity Profile of Compounds 15 and 33^a

15

33

no. of

tests

no. of

tests

enzyme

c-Src

VEGFR-2

Csk

c-Yes

Lck

v-Abl

c-Kit

Flt-1

Flt-4

IC₅₀(µM)

<0.004

4

3

0.0027 ( 0.0005

16

4

3

2

3

2

2.5 ( 0.8

20.9 ( 4.1

0.84 ( 0.31

0.004 ( 0.001

<0.004

0.03

0.27 ( 0.3

0.003 ( 0.001

Figure 5. Correlation between volume of distribution V_d(L/kg) and

pK_a.

0.007 ( 0.001

nt

0.2

>33

>10

0.9 ( 0.1

37

2

1

>100

with regard to their ADME properties. Of the two main

subseries, the C-5 tetrahydropyranyl one (compounds 15 and

16) always demonstrated significantly higher oral exposure than

the C-5 piperidine one (compounds 6, 24, and 29) despite the

excellent Lipinski properties⁶⁹and moderate pK_aof the latter

(compound 6, Table 3). The difference between the two C-5

subseries was suggested to be due to reduced permeability and

increased efflux properties of the C-5 N-methylpiperidine series.

In the C-5 tetrahydropyranyl series, absorption is good and

excellent exposure is achieved even when basic side chains are

present at C-7 as shown by compounds 15 (pK_a) 9.5) and 18

(pK_a) 9.4). The C-5-substituted series compares very favorably

from an ADME properties point of view with the C-6,C-7-

disubstituted series we previously reported (comparison of 15

and 18 and 34 and 37). As shown in Table 2, anilinoquinazolines

bearing basic functions can display large volumes of distribution

(V_d) which tend to lead to long half-lives (15, 18, 35) when

combined with moderate to low clearance.⁶⁶Similarly to what

was observed in the C6,C-7-disubstituted anilinoquinazolines,

we have shown that in this C-5, C-7 series, the modulation of

the pK_aof the side chain (at C-5 or C-7) correlated generally

well with the V_d(Figure 5) for compounds displaying moderate

clearance and quite similar unbound fractions in plasma.

This in turn permitted a relatively precise control of the t_1/2

of the final molecule. Moreover, we identified that the pK_aof

the side chain was also playing an important role with regard

to the affinity for the hERG receptor of our compounds. Very

basic compounds proved to have more affinity for this receptor

than less basic or neutral ones. With its moderate pK_a(7.9),

compound 33 displayed very low affinity for the hERG channel.

>10

EGFR

FGFR-1

MEK

2.59

>10

nt

14

Aurora-3

CDK-2

PDGFR-â

PDGFR-R

MAPKK

8.9

>10

nt

1

2

>10

1

3

2

1

10

>5

10

14

^aIC₅₀> 10 µM for the following enzymes: GSK3b, Chk, JNK, PDK,

PKA, PKCa, PI3K.

when measured at pH 7.4 by virtue of protonation of the basic

moiety. Even with moderately basic C-7 substituents such as

(N-methylpiperazinyl)ethoxy (compound 33, pK_a) 7.9), the

solubility increased very significantly compared with that of

their neutral counterparts (comparison of compounds 33 and

19). Moreover, the basic function led to a 5-15-fold increase

in the fraction unbound in the plasma of these derivatives as

illustrated by compounds 6, 15, and 33 over 19 and 31.

To design compounds to incorporate good oral bioavailability

amenable for chronic oral administration, we paid particular

attention to the molecular properties that have been reported to

affect absorption such as the total polar surface area (PSA),

molecular weight (MW), H-bond donor-acceptor (HBDA)

properties, and number of rotatable bonds.⁶⁹Our C-5 derivatives

were designed to possess moderate PSA and a minimum number

of rotatable bonds. This approach led us to design compounds

with excellent exposure as shown by the total area under the

curve (AUC_{0-24 h}) in mice following oral administration (Table

5). However, all the C-5-substituted subseries are not equivalent

6476 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

33 in the NIH3T3 model, this suggested that 33 should also

prevent formation of metastasis in vivo. We also demonstrated

that AZD0530 prevents the formation of lymph node metastasis

in a bladder model (NBT-II) and liver metastasis in an orthotopic

L3.6 human pancreatic tumor model.^68,72Moreover, in an

orthotopic model of human pancreas (BxPC-3 cells), athymic

mice treated with 33, given orally once daily at doses of 25

(mg/kg)/d for more than 50 days, demonstrated a significant

38% (p < 0.05) increase in survival compared with control

animals which may, in part, be attributable to an anti-invasive

activity in this aggressive orthotopic tumor model.⁸⁵

Conclusions

C-5-substituted (benzodioxolylamino)quinazolines are potent

and dual-specific inhibitors of c-Src and Abl kinases. Their

enzyme inhibition profiles translate very well into activity in

cells in vitro in terms of inhibition of both proliferation and

cell migration. The presence of a moderately basic side chain

at the C-7 position confers excellent physicochemical properties,

in particular good aqueous solubility and moderate binding to

plasma proteins. The C-5 tetrahydropyranyl series possesses

good pharmacokinetic properties and demonstrates appropriate

exposure following oral administration to rodents and dogs.

These properties, combined with good intrinsic potencies, led

to good in vivo activity in a range of preclinical models. The

full exploitation of our knowledge of the shape, size, and

properties of the different binding sites of c-Src and KDR and

of the best fit substituents led to the design of 33 (AZD0530).

33 displays potent c-Src enzyme inhibition (IC₅₀) 2.7 nM)

and excellent selectivity for c-Src over KDR (7740-fold). In

view of its good physicochemical properties, selectivity profile,

pharmacokinetics, and activity in preclinical models, compound

33 was selected for further development and is currently

undergoing phase I clinical evaluation in patients with advanced

cancers.

Figure 6. Inhibition of an Src-transfected 3T3 tumor xenograft

following daily oral administration of AZD0530.

When tested in animal models over a range of doses, 33 did

not display modification of the ECG nor prolongation of the

QT interval of ECGs.⁶⁵Compound 33 represented a good

compromise among moderate pK_a, leading to excellent bio-

availability and general ADME properties in preclinical species

(Table 2), low affinity for hERG, and good physicochemical

properties. The pharmacokinetic profiles of 33 observed in rat

and dog (Table 2) were subsequently confirmed in man and

proved consistent with once daily oral dosing with a mean

terminal half-life at steady-state of 40 h (range 32-47) and low

interpatient variability.⁷⁰

In Vivo Activity. Mouse NIH 3T3 fibroblasts engineered to

overexpress a deregulated, constitutively active form of human

c-Src have a phenotype altered from that of the wild-type

parental line. In vivo, the transfected cell line grows subcutane-

ously to form tumors in athymic rats and mice, while the wild-

type 3T3 cells do not. Implanted subcutaneously in athymic rats,

the transfected lines develop large tumors in approximately 3

weeks. Animals treated with 15 orally once daily at doses of

10 (mg/kg)/d for 18 days show 90% inhibition of tumor volume,

while animals treated with 33 orally (Figure 6) once daily at

doses of 6 (mg/kg)/d for 18 days show >95% inhibition of

tumor volume and tumor weight at the end of the experiment.

The activity observed in this model is unlikely to be due to

a direct inhibition of tumor angiogenesis. As expected from its

very weak activity against VEGFR-2, 33 does not inhibit the

in vitro VEGF- or FGF-stimulated growth of HUVECs (VEGF-

HUVEC and FGF-HUVEC IC₅₀> 5 µM (n ) 4)) and was

shown to be inactive in in vivo angiogenesis assays.⁶⁵Moreover,

inhibition of paxillin phosphorylation at tyrosine Y31 as well

as FAK (focal adhesion kinase) tyrosine phosphorylation

demonstrated in vitro was confirmed in vivo. More than 80%

inhibition of both paxillin and FAK phosphorylation has been

demonstrated in Calu-6 xenograft tumors 6 h post last dose

following oral administration of 20 (mg/kg)/d of 33.⁶⁸

Experimental Section

All experiments were carried out under an inert atmosphere and

at room temperature unless otherwise stated. Flash chromatography

was carried out on Merck Kieselgel 50 (Art. 9385). The purities of

compounds for biological testing were assessed by analytical HPLC

on a Hichrom S5ODS1 Spherisorb column system set to run

isocratically with 60-70% MeOH + 0.2% CF₃COOH in water as

eluent. Purification by preparative HPLC/MS was performed on a

Waters LC/MS system using a Waters Symmetry column (C18, 5

µm, 19 mm diameter, 100 mm length) with a mixture of water

(containing 1% acetic acid) and acetonitrile (gradient from 5% to

100%) as solvent. NMR spectra were obtained on a JEOL JNM

EX 400 (400 MHz) spectrometer and Bruker Avance 500 (500

MHz) spectrometer. Chemical shifts are expressed in δ (ppm) units,

and peak multiplicities are expressed as follows: s, singlet; d,

doublet; dd, doublet of doublets; t, triplet; br s, broad singlet; m,

multiplet. Mass spectrometry was carried out on an analytical

Waters LC/MS system with positive and negative ion data collected

automatically. NMR and mass spectra were run on isolated

intermediates and final products and were consistent with the

proposed structures. For the microanalysis, all the adducts men-

tioned were measured: water was assayed by the Karl-Fisher

method using a Mettler DL 18, the HCl content was determined

on a Metrohm 686 by titration using silver nitrate solution, and

organic adducts were measured by ¹H NMR. The following

abbreviations have been used: ADDP, 1,1′-(azodicarbonyl)dipip-

eridine; Boc, tert-butoxycarbonyl; DEAD, diethyl azodicarboxylate;

DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DPPA,

diphenylphosphoryl azide; Gold’s reagent, [3-(dimethylamino)-2-

azaprop-2-en-1-ylidene]dimethylammonium chloride; NaHMDS,

sodium bis(trimethylsilyl)amide; POM, (pivaloyloxy)methyl; TFA,

In this xenograft model 33 is more active than our previously

reported 2′-chloro,5′-methoxyanilinoquinazoline derivative 35

(M475271) and delivers complete inhibition of tumor growth

at 1/10 of the dose required by 35 to achieve the same effect.²⁷

c-Src is overexpressed in pancreatic adenocarcinomas, and

we have previously reported that 35 prevented the formation

of metastasis in an orthotopic L3.6pl human pancreatic tumor

model. Untreated animals developed liver metastasis within a

few weeks, whereas complete inhibition of liver metastasis

formation was achieved with once daily oral administration of

25 (mg/kg)/d of 35.⁷¹Combined with the superior activity of

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6477

trifluoracetic acid; DCM, dichloromethane; THF, tetrahydrofuran;

DIPEA, N,N-diisopropylethylamine; NMP, N-methylpyrrolidone.

N-(2-Chloro-5-methoxyphenyl)-5-[(1-methylpiperidin-4-yl)ox-

y]quinazolin-4-amine (1). To a mixture of 47 (150 mg, 0.5 mmol),

triphenylphosphine (210 mg, 0.8 mmol), and 1-methylpiperidin-

4-ol (69 mg, 0.6 mmol) in dichloromethane (4 mL) at room

temperature, DTAD (184 mg, 0.8 mmol) was added slowly. The

mixture was stirred for 2 h at room temperature, the solvent

evaporated, and the residue purified by flash chromatography using

a mixture of dichloromethane/ethyl acetate (80:20) to remove the

impurities and then using a mixture of dichloromethane/methanol

(98:2). After evaporation of the solvent, 1 was dissolved in diethyl

ether and treated with a 5 N solution of HCl gas in diethyl ether

(100 µL) to give 100 mg of 1 as a hydrochloride (50%): ¹H NMR

(CDCl₃) (free base) δ 2.0-2.1 (m, 2H), 2.1-2.2 (m, 2H), 2.25(s,

3H), 2.75-2.85 (m, 2H), 3.8 (s, 3H), 4.5-4.6 (m, 1H), 6.6 (dd,

1H), 6.9 (d, 1H), 7.25 (d, 1H), 7.4 (d, 1H), 7.6 (dd, 1H), 8.1 (d,

1H), 8.6 (s, 1H); MS-ESI m/z 399 and 401 [MH⁺]. Anal.

(C₂₁H₂₃N₄O₂Cl‚H₂O‚2HCl) C, H, N.

dichloromethane (5:95); the resulting precipitate was eliminated by

filtration, and the filtrate was evaporated down to give 5 as a free

base: ¹H NMR (CDCl₃) (free base) δ 2.20-2.50 (m, 5H), 2.84

(m, 2H), 3.78 (s, 3H), 3.86 (s, 3H), 4.55 (m, 1H), 6.48 (d, 1H),

6.59 (dd, 1H), 6.80 (d, 1H), 7.25 (d, 1H), 8.51 (s, 1H), 9.70 (br s,

1H); MS-ESI m/z 443 and 445 [MH]⁺.

N-(2-Chloro-5-methoxyphenyl)-7-(3-morpholin-4-ylpropoxy)-

5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (7). To a

mixture of 65 (100 mg, 0.25 mmol), triphenylphosphine (105 mg,

0.4 mmol), and 3-morpholin-4-ylpropan-1-ol (44 mg, 0.3 mmol)

in dichloromethane (5 mL) at 0 °C was slowly added DTAD (92

mg, 0.4 mmol). The mixture was stirred for 2 h at room temperature

and was purified by SiO₂chromatography using a mixture of

dichloromethane/methanol (98:2) as eluent to remove the impurities

and then a mixture of dichloromethane-methanol/ammonia (97:

3) to give 60 mg of 7 (46%) as a white solid: ¹H NMR (DMSO-d₆

and CF₃CO₂D) δ 1.9-2.0 (m, 2H), 2.05-2.1 (m, 2H), 2.2-2.3

(m, 2H), 3.1-3.2 (m, 2H), 3.3-3.4 (m, 2H), 3.6-3.7 (m, 4H), 3.7-

3.8 (m, 2H), 3.85 (s, 3H), 3.95-4.15 (m, 4H), 4.25-4.35 (m, 2H),

5.1-5.2 (m, 1H), 6.9 (d, 1H), 7.02 (dd, 1H), 7.1 (d, 1H), 7.53-

7.55 (m, 2H), 8.87 (s, 1H); MS-ESI m/z 529 and 531 [MH]⁺. Anal.

(C₂₇H₃₃ClN₄O₅) C, H, N.

A similar procedure was used to prepare 8, 9, 10, and 21.

N-1,3-Benzodioxol-4-yl-7-(3-pyrrolidin-1-ylpropoxy)-5-(tet-

rahydro-2H-pyran-4-yloxy)quinazolin-4-amine (11). To a mix-

ture of 78 (114 mg, 0.3 mmol), triphenylphosphine (126 mg, 0.48

mmol), and 3-pyrrolidin-1-ylpropan-1-ol (47 mg, 3.6 mmol) in

dichloromethane (5 mL) at 0 °C was slowly added DTAD (110

mg, 0.48 mmol). The mixture was stirred for 2 h at room

temperature and was purified by SiO₂chromatography using a

mixture of 7 N ammonia solution in methanol/dichloromethane (4:

96) as eluent to give 93 mg of 11 (63%): ¹H NMR (DMSO-d₆) δ

1.7-1.73 (m, 4H), 1.8-1.9 (m, 2H), 1.9-2.0 (m, 2H), 2.1-2.2

(m, 2H), 2.4-2.5 (m, 4H), 2.6 (t, 2H), 3.5-3.6 (m, 2H), 3.88-

3.93 (m, 2H), 4.18 (t, 2H), 4.95-5.05 (m, 1H), 6.1(s, 2H), 6.72 (d,

1H), 6.8 (d, 1H), 6.85-6.95 (m, 2H), 8.08 (d, 1H), 8.48 (s, 1H),

9.83 (s, 1H); MS-ESI m/z 493 [MH]⁺. Anal. (C₂₇H₃₂N₄O₅‚0.1H₂O)

C, H, N.

A similar procedure was used to prepare 2.

N-(5-Chloro-1,3-benzodioxol-4-yl)-5-[(1-methylpiperidin-4-

yl)oxy]quinazolin-4-amine (3). To a suspension of 60% sodium

hydride (100 mg, 2.4 mmol) in DMF (4 mL) was added 1-meth-

ylpiperidin-4-ol (50 mg, 0.42 mmol) under nitrogen at room

temperature. The reaction mixture was stirred for 10 min, and then

52 (100 mg, 0.28 mmol) was added. The mixture was heated to 80

°C and stirred for 6 h. After cooling, the solution was poured

dropwise into water (20 mL), extracted twice with ethyl acetate,

dried over magnesium sulfate, and concentrated. The crude was

purified by SiO₂chromatography eluting with methanol/dichlo-

romethane/ethyl acetate (2:48:50) and then with a solution of 7 N

ammonia in methanol/dichloromethane/ethyl acetate (3:47:50) to

give 54 mg of 3 (46%) after evaporation of the solvent: ¹H NMR

(CDCl₃) δ 2.02 (m, 2H), 2.17 (m, 2H), 2.25-2.40 (m, 5H), 2.70

(m, 2H), 4.64 (m, 1H), 6.01 (s, 2H), 6.69 (d, 1H), 6.87 (d, 1H),

6.94 (d, 1H), 7.42 (d, 1H), 7.60 (t, 1H), 8.56 (s, 1H), 9.47 (s, 1H);

MS-ESI m/z 413 and 415 [MH]⁺. Anal. (C₂₁H₂₁ClN₄O₃‚0.8H₂O)

C, H, N.

A similar procedure was used to prepare 14, 15, 16, 19, 25, and

33.

N-(2-Chloro-5-methoxyphenyl)-7-methoxy-5-(3-morpholin-4-

ylpropoxy)quinazolin-4-amine (4). To a mixture of 44 (200 mg,

0.6 mmol), triphenylphosphine (240 mg, 0.9 mmol), and 3-mor-

pholin-4-ylpropan-1-ol (130 mg, 0.9 mmol) in dichloromethane (3

mL) was added portionwise DTAD (210 mg, 0.9 mmol) under

nitrogen at room temperature. The solution was stirred for 1 h and

then poured onto a column of silica gel eluting with pure

dichloromethane to remove impurities and then with a solution of

7 N ammonia in methanol/dichloromethane (1:99). Evaporation of

the solvent gave a solid which was triturated in diethyl ether, filtered

off, and washed with diethyl ether to give 130 mg of 4 (48%): ¹H

NMR (DMSO-d₆) δ 2.05 (m, 2H), 2.29 (m, 4H), 2.42 (t, 2H), 3.50

(t, 4H), 3.79 (s, 3H), 3.91 (s, 3H), 4.45 (t, 2H), 6.76 (dd, 1H), 6.82

(d, 1H), 6.85 (d, 1H), 7.46 (d, 1H), 8.30 (d, 1H), 8.52 (s, 1H),

10.09 (s, 1H); MS-ESI m/z 459 and 461 [MH]⁺. Anal. (C₂₃H₂₇-

ClN₄O₄) C, H, N.

N-(2-Chloro-5-methoxyphenyl)-7-methoxy-5-[(1-methylpip-

eridin-4-yl)oxy]quinazolin-4-amine (5). To a mixture of 90 (675

mg, 2.2 mmol), 2-chloro-5-methoxyaniline hydrochloride (510 mg,

2.6 mmol) in propan-2-ol (5 mL) was added a 5 N solution of HCl

gas in propan-2-ol (36 µL, 0.2 mmol), and the resulting mixture

was heated at 80 °C for 1.5 h. After cooling, the formed precipitate

was filtered off and washed with propan-2-ol and then diethyl ether

to give 1 g of 5 as a dihydrochloride salt (93%). ¹H NMR (DMSO-

d₆) δ 2.15-2.35 (m, 2H), 2.40 (m, 2H), 2.76 (d, 3H), 3.12 (m,

2H), 3.55 (m, 2H), 3.80 (s, 3H), 3.98 (s, 3H), 5.08 (m, 0.75H),

5.21 (m, 0.25H), 6.95 (dd, 0.75H), 7.02 (m, 1H), 7.09 (d, 0.25H),

7.16 (d, 0.75H), 7.47 (d, 0.25H), 7.55 (m, 1H), 7.79 (d, 0.75H),

8.76 (s, 0.25H), 8.81 (s, 0.75H), 10.12 (br s, 0.25H), 10.29 (br s,

0.75H), 10.77 (br s, 0.75H), 11.08 (br s, 0.25H); MS-ESI m/z 429

[MH]⁺. Anal. (C₂₂H₂₅ClN₄O₃‚2.35HCl‚0.28C₃H₈O) C, H, N.

The free base was generated by dissolving the dihydrochloride

salt of the compound in a mixture of 7 N ammonia in methanol/

N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-

yl)ethoxy]-5-(t etrahydro-2H-pyran-4-yloxy)quinazolin-4-amine

(33, AZD0530). 79 (190 mg, 0.46 mmol) was reacted with 2-(4-

methylpiperazin-1-yl)ethanol (79 mg, 0.55 mmol) to give 180 mg

of 33 (AZD0530) (73%): ¹H NMR (CDCl₃) δ 1.9-2.05 (m, 2H),

2.2-2.3 (m, 2H), 2.31 (s, 3H), 2.4-2.7 (m, 8H), 2.87 (m, 2H),

3.6-3.7 (m, 2H), 3.95-4.05 (m, 2H), 4.25 (m, 2H), 4.7-4.8 (m,

1H), 6.05 (s, 2H), 6.55 (d, 1H), 6.72 (d, 1H), 6.83 (d, 1H), 6.97 (d,

1H), 8.52 (s, 1H), 9.26 (s, 1H); MS-ESI m/z 542 and 544 [MH]⁺.

Anal. (C₂₇H₃₂N₅O₅Cl‚0.15H₂O) C, H, N.

N-1,3-Benzodioxol-4-yl-7-methoxy-5-[(1-methylpiperidin-4-

yl)oxy]quinazolin-4-amine (12). A mixture of 90 (100 mg, 0.33

mmol), 1,3-benzodioxol-4-amine (50 mg, 0.36 mmol), and a 5 N

solution of HCl gas in propan-2-ol (70 µL, 0.34 mmol) in propan-

2-ol (2 mL) was heated at 80 °C for 1.5 h. After cooling, the formed

precipitate was filtered off and washed with propan-2-ol and then

diethyl ether to give 120 mg of 12 as a dihydrochloride salt

(75%): ¹H NMR (CDCl₃) (free base) δ 2.05 (m, 2H), 2.26 (m,

2H), 2.33 (s, 3H), 2.84 (m, 2H), 3.92 (s, 3H), 4.55 (m, 1H), 6.02

(s, 2H), 6.50 (d, 1H), 6.66 (d, 1H), 6.84 (d, 1H), 6.90 (t, 1H), 8.00

(d, 1H), 8.58 (s, 1H), 9.72 (br s, 1H); MS-ESI m/z 409 [MH]⁺.

Anal. (C₂₂H₂₄N₄O₄‚2.3HCl‚2.2H₂O) C, H, N.

A similar procedure was used to prepare 6 and 13.

N-(2,3-Dihydro-1,4-benzodioxin-5-yl)-7-(2-pyrrolidin-1-

ylethoxy)-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine

(17). A mixture of 82 (95 mg, 0.25 mmol) and 2,3-dihydro-1,4-

benzodioxin-5-amine hydrochloride (52 mg, 0.28 mmol) in 2-pro-

panol (3 mL) was heated under reflux for 90 min. After cooling,

the precipitate was filtered to give 84 mg of 17 (60%) as a

hydrochloride: ¹H NMR (DMSO-d₆and CF₃CO₂D) δ 1.9-2.0 (m,

4H), 2.0-2.1 (m, 2H), 2.1-2.2 (m, 2H), 3.1-3.2 (m, 2H), 3.5-

6478 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

3.6 (m, 2H), 3.6-3.8 (m, 4H), 3.95-4.05 (m, 2H), 4.35-4.4 (m,

2H), 4.45-4.5 (m, 2H), 4.5-4.6 (m, 2H), 5.15-5.25 (m, 1H), 6.86

(dd, 1H), 6.95 (dd, 1H), 6.99 (d, 1H), 7.2 (d, 1H), 7.99 (d, 1H),

8.94 (d, 1H); MS-ESI m/z 493 [MH]⁺. Anal. (C₂₇H₃₂N₄O₅‚2HCl‚

2.7H₂O) C, H, N.

dichloromethane (1 mL) at room temperature, and the solution was

stirred for 1 h. A 2 N solution of HCl gas in diethyl ether (3 mL)

was added to the mixture, then the resulting mixture was stirred

for 1.5 h. Then the mixture was diluted with diethyl ether (1 mL),

and the precipitate was filtered and taken up in a mixture of a 7 N

solution of ammonia in methanol/dichloromethane (1:9). The

resulting solid was removed by filtration and the filtrate concentrated

to give 74 mg of 28 (56%): ¹H NMR (CDCl₃) δ 2.05 (m, 2H),

2.21 (m, 2H), 2.3-2.5 (m, 5H), 2.74 (m, 2H), 4.34 (ddd, 2H), 4.63

(m, 1H), 4.83 (ddd, 2H), 6.06 (s, 2H), 6.59 (d, 1H), 6.73 (d, 1H),

6.81 (d, 1H), 6.98 (d, 1H), 8.53 (s, 1H), 9.28 (br s, 1H); MS-ESI

m/z 475 and 477 [MH]⁺. Anal. (C₂₃H₂₄ClFN₄O₄‚0.6H₂O) C, H, N.

N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[(1-methylpiperidin-4-yl-

)methoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-

amine (18). A solution of 80 (250 mg, 0.4 mmol) in a mixture of

formic acid (5 mL) and 37% aqueous formaldehyde solution (0.5

mL) was heated at 100 °C for 2 h. The volatiles were removed

under vacuum, and the residue was made alkaline by addition of a

6 N solution of ammonia in methanol, dissolved in dichloromethane,

and purified by SiO₂chromatography using a mixture of 6 N

ammonia in methanol/dichloromethane (4:96) as eluent to give 100

mg of 18 (50%): ¹H NMR (CDCl₃) δ 1.4-1.5 (m, 2H), 1.75-

1.85 (m, 2H), 1.9-2.05 (m, 3H), 2.2-2.3 (m, 2H), 2.29 (s, 3H),

2.9-3.0 (m, 2H), 3.6-3.7 (m, 2H), 3.95 (d, 2H), 4.0-4.1 (m, 4H),

4.7-4.8 (m, 1H), 6.05 (s, 2H), 6.5 (d, 1H), 6.7 (d, 1H), 6.8 (d,

1H), 6.98 (d, 1H), 8.5 (s, 1H), 9.25 (s, 1H)). MS-ESI m/z 527 and

529 [MH]⁺. Anal. (C₂₇H₃₁N₄O₅Cl) C, H, N.

N-(5-Chloro-1,3-benzodioxol-4-yl)-5-morpholin-4-yl-7-(2-pyr-

rolidin-1-ylethoxy)quinazolin-4-amine (20). A mixture of 110

(260 mg, 0.75 mmol), phosphorus oxychloride (84 µL, 0.9 mmol),

and N,N-diisopropylethylamine (340 µL, 2 mmol) in 1,2-dichlo-

roethane (20 mL) was heated to 75 °C for 2 h. The solvent was

evaporated under vacuum, the crude intermediate was reacted with

(5-chloro-1,3-benzodioxol-4-yl)amine (140 mg, 0.8 mmol) in

2-propanol (4 mL), and the reaction mixture was heated at 80 °C

for 1 h. The volatiles were removed under vacuum, and the residue

was made alkaline by addition of a solution of 6 N ammonia in

methanol, filtered, and purified by SiO₂chromatography using a

mixture of 6 N ammonia in methanol/dichloromethane (3:97) as

eluent to give after evaporation and trituration in diethyl ether 35

mg of 20 (10%): ¹H NMR (CDCl₃) δ 1.8-1.9 (m, 4H), 2.6-2.7

(m, 4H), 2.98 (t, 2H), 3.0-3.1 (m, 2H), 3.1-3.2 (m, 2H), 4.75-

4.85 (m, 2H), 3.95-4.05 (m, 2H), 4.25 (t, 2H), 6.04 (s, 2H), 6.75

(d, 1H), 6.9-7.1 (3d, 3H), 8.52 (s, 1H), 11.4 (s, 1H); MS-ESI m/z

498 and 500 [MH]⁺.

A similar procedure was used to prepare 29 and 96.

N-(5-Chloro-1,3-benzodioxol-4-yl)-5-isopropoxy-7-(3-pyrro-

lidin-1-ylpropoxy)quinazolin-4-amine (30). To a mixture of 102

(112 mg, 0.3 mmol), triphenylphosphine (126 mg, 0.48 mmol), and

3-pyrrolidin-1-ylpropan-1-ol (46 mg, 0.36 mmol) in dichlo-

romethane (5 mL) at room temperature was added DTAD (110

mg, 0.48 mmol). After the resulting mixture was stirred for 2 h,

the solvent was evaporated and the residue purified by preparative

LC/MS using a gradient of ammonium carbonate and acetonitrile.

After evaporation, the residue was dissolved in dichloromethane,

dried (MgSO₄), and evaporated to give 120 mg of 30 (84%): ¹H

NMR (CDCl₃) δ 1.5 (d, 2H), 1.8-1.9 (m, 4H), 2.0-2.1 (m, 2H),

2.5-2.6 (m, 4H), 2.6-2.7 (m, 2H), 4.12 (t, 2H), 4.8 (q, 1H), 6.0

(s, 2H), 6.5 (d, 1H), 6.7 (d, 1H), 6.8 (d, 1H), 6.95 (d, 1H), 8.5 (s,

1H), 9.4 (s, 1H); MS-ESI m/z 485 and 487 [MH]⁺. Anal. (C₂₅H₂₉-

ClN₄O₄‚2.3H₂O‚2.3HCl) C, H, N.

7-[2-(4-Acetylpiperazin-1-yl)ethoxy]-N-(5-fluoro-1,3-benzo-

dioxol-4-yl)-5-isopropoxyquinazolin-4-amine (32). A mixture of

103 (28 g, 67 mmol), potassium iodide (22 g, 133 mmol), and

N-acetylpiperazine (25.7 g, 200 mmol) in DMF (120 mL) was

heated at 95 °C under nitrogen for 5 h. After cooling, the reaction

mixture was concentrated and the residue taken up in dichlo-

romethane. Solids were removed by filtration, and a 7 N solution

of ammonia in methanol was added. The solvent was evaporated

off and the crude material purified by SiO₂chromatography eluting

with a gradient of methanol/dichloromethane (4:96 up to 10:90).

After evaporation of the solvent, the solid was washed with diethyl

ether and filtered off to give 28 g of 32 (83%): ¹H NMR (CDCl₃)

δ 1.52 (d, 6H), 2.10 (s, 3H), 2.58 (quint, 4H), 2.88 (t, 2H), 3.51

(dd, 2H), 3.66 (dd, 2H), 4.23 (t, 2H), 4.81 (quint, 1H), 6.05 (s,

2H), 6.52 (d, 1H), 6.66 (m, 2H), 6.81 (d, 1H), 8.53 (s, 1H), 9.29

(br s, 1H); MS-ESI m/z 512 [MH]⁺. Anal. (C₂₆H₃₀FN₅O₅) C, H,

N.

N-(5-Chloro-1,3-benzodioxol-4-yl)-7-methoxy-5-(piperidin-4-

yloxy)quinazolin-4-amine (24). 97 (170 mg, 0.32 mmol) was

stirred in a 2 N solution of HCl gas in diethyl ether (15 mL) at

room temperature for 1 h. The mixture was diluted with diethyl

ether, and the precipitate was filtered off, washed with diethyl ether,

and dried under vacuum to give 134 mg of 24 as a dihydrochloride

salt (89%): ¹H NMR (DMSO-d₆) δ 2.2-2.4 (m, 4H), 3.12 (m,

2H), 3.34 (m, 2H), 4.0 (s, 3H), 5.17 (m, 1H), 6.17 (s, 2H), 7.05

(m, 2H), 7.15 (d, 1H), 7.18 (d, 1H), 8.82 (s, 1H), 9.11 (br s, 1H),

9.33 (br s, 1H), 10.09 (br s, 1H); MS-ESI m/z 429 and 431 [MH]⁺.

Anal. (free base) (C₂₁H₂₁ClN₄O₄‚1.45H₂O) C, H, N.

A similar procedure was used to prepare 31.

N-(2-Chloro-5-methoxyphenyl)-6-methoxy-5-[(1-methylpip-

eridin-4-yl)oxy]quinazolin-4-amine (38). A mixture of 115 (65

mg, 0.21 mmol), (2-chloro-5-methoxyphenyl)amine hydrochloride

(49 mg, 0.25 mmol), and a catalytic amount of HCl (from a 6 N

HCl solution in propan-2-ol) in propan-2-ol (2 mL) was heated

under reflux for 1 h. After cooling, the precipitate was filtered off

and washed with propan-2-ol, ethyl acetate, and diethyl ether to

give 69 mg of 38 (65%) as a dihydrochloride: ¹H NMR (free base)

(CDCl₃) δ 1.9-2.2 (m, 6H), 2.25 (s, 3H), 2.8-3.0 (m, 2H), 3.85

(s, 3H), 3.99 (s, 3H), 4.40-4.55 (m, 1H), 6.65 (dd, 1H), 7.30 (d,

1H), 7.54 (d, 1H), 7.67 (d, 1H), 8.48 (d, 1H), 8.61 (s, 1H), 10.43

(s, 1H); MS-ESI m/z 429 and 431 [MH]⁺.

N-(2-Chloro-5-methoxyphenyl)-5,7-dimethoxyquinazolin-4-

amine (39). A mixture of 43 (2 g, 9.7 mmol), phosphorus

oxychloride (0.1 mL, 10.7 mmol), and DIPEA (4.2 mL, 27.7 mmol)

in 1,2-dichloroethane (20 mL) was refluxed for 2 h under nitrogen.

The dark solution was concentrated, and then propan-2-ol (20 mL)

was added to the crude followed by 2-chloro-5-methoxyaniline (1.85

g, 11.7 mmol). The mixture was heated at 80 °C for 1 h and 30

min. After cooling, the precipitate was filtered off and washed with

propan-2-ol and then diethyl ether to give 2.8 g of 39 as a

hydrochloride salt (76%): ¹H NMR (DMSO-d₆) δ 3.85 (s, 3H),

4.00 (s, 3H), 4.16 (s, 3H), 7.00 (m, 3H), 7.56 (d, 1H), 7.60 (d,

1H), 8.8 (s, 1H), 10.90 (s, 1H); MS-ESI m/z 346 and 348 [MH]⁺.

A similar procedure was used to prepare 23.

N-(5-Chloro-1,3-benzodioxol-4-yl)-7-isopropoxy-5-[(1-meth-

ylpiperidin-4-yl)oxy]quinazolin-4-amine (27). To a mixture of 96

(0.125 g, 0.27 mmol), formaldehyde (42 µL, 0.55 mmol), and acetic

acid (19 µL, 0.33 mmol) in a 2:5 mixture of methanol/dichlo-

romethane (7 mL) was added portionwise sodium triacetoxyboro-

hydride (0.09 g, 0.41 mmol). The reaction mixture was stirred for

1 h at room temperature and then concentrated. The residue was

taken up in ethyl acetate and washed with a 1 N aqueous solution

of sodium hydroxide. The aqueous phase was extracted with ethyl

acetate, and the organics were combined, washed with brine, and

dried (MgSO₄). Evaporation of the solvent gave 110 mg of 27 as

a white foam (87%): ¹H NMR (CDCl₃) δ 1.42 (d, 6H), 2.03 (m,

2H), 2.25 (m, 2H), 2.35 (m, 5H), 2.75 (m, 2H), 4.66 (m, 1H), 4.72

(quint.), 6.05 (s, 2H), 6.48 (d, 1H), 6.72 (d, 1H), 6.82 (d, 1H), 6.97

(d, 1H), 8.52 (s, 1H), 9.27 (s, 1H); MS-ESI m/z 471 and 473 [MH]⁺.

A similar procedure was used to prepare 26.

N-(5-Chloro-1,3-benzodioxol-4-yl)-7-(2-fluoroethoxy)-5-[(1-

methylpiperidin-4-yl)oxy]quinazolin-4-amine (28). To a mixture

of 89 (120 mg, 0.28 mmol), triphenylphosphine (147 mg, 0.56

mmol), and 2-fluoroethanol (25 µL, 0.42 mmol) in dichloromethane

(2 mL) was added a solution of DTAD (130 mg, 0.56 mmol) in

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6479

N-(2-Chloro-5-methoxyphenyl)-6-[3-(4-methylpiperazin-1-yl)-

propoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine

(40). A mixture of 119 (90 mg, 0.21 mmol), (2-chloro-5-methox-

yphenyl)amine hydrochloride (50 mg, 0.26 mmol), and a catalytic

amount of HCl (from a 6 N HCl solution in propan-2-ol) in propan-

2-ol (3 mL) was heated under reflux for 1 h. After cooling, the

precipitate was filtered off and washed with propan-2-ol, ethyl

acetate, and diethyl ether to give 90 mg of 40 (68%) as a

dihydrochloride: ¹H NMR (free base) (CDCl₃) δ 1.9-2.2 (m, 6H),

2.49 (s, 3H), 2.6-2.9 (m, 10H), 3.3-3.4 (m, 2H), 3.90 (s, 3H),

4.0-4.1 (m, 2H), 4.23 (t, 2H), 4.5-4.6 (m, 1H), 6.65 (dd, 1H),

7.34 (d, 1H), 7.54 (d, 1H), 7.67 (d, 1H), 8.54 (d, 1H), 8.63 (s, 1H),

10.42 (s, 1H); MS-ESI m/z 542 and 544 [MH]⁺.

5,7-Dimethoxyquinazolin-4(3H)-one (43). A mixture of 70 (16

g, 76 mmol) and formamidine acetate (24 g, 230 mmol) in

2-methoxyethanol (330 mL) was heated at reflux for 2 h. After

cooling, the solution was concentrated, and the residue was taken

up in water (100 mL). The formed white solid was filtered off,

washed twice with water, and dried under vacuum at 50 °C in the

presence of phosphorus pentoxide to give 14.5 g of 43 (94%): ¹H

NMR (DMSO-d₆) δ 3.82 (s, 3H), 3.90 (s, 3H), 6.54 (d, 1H), 6.66

(d, 1H), 7.93 (s, 1H), 11.78 (br s, 1H); MS-ESI m/z 207 [MH]⁺.

Anal. (C₁₀H₁₀N₂O₃‚0.1H₂O) C, H, N.

tert-Butyl 4-[(4-Oxo-3,4-dihydroquinazolin-5-yl)oxy]piperi-

dine-1-carboxylate (49). To a suspension of NaH (60% dispersion

in mineral oil) (110 mg, 2.7 mmol) in DMF (2 mL) was added

tert-butyl 4-hydroxypiperidine-1-carboxylate (330 mg, 1.64 mmol).

After the resulting mixture was stirred for 15 min at room

temperature, 48 (180 mg, 1.1 mmol) was added to the mixture,

which was stirred overnight at room temperature. To complete the

reaction, NaH (60% dispersion in mineral oil) (66 mg, 1.6 mmol)

and then tert-butyl 4-hydroxypiperidine-1-carboxylate (220 mg, 1.1

mmol) were added, and the reaction temperature was raised to 50

°C for 1 h. After cooling, the reaction mixture was poured into

water (20 mL), and the pH was adjusted to 5 with acetic acid. The

precipitate was filtered off, washed with water, and dried under

vacuum at 50 °C in the presence of phosphorus pentoxide to give

300 mg of 49 as a white solid (79%): ¹H NMR (CDCl₃) δ 1.47 (s,

9H), 1.94 (m, 2H), 3.55 (m, 2H), 3.69 (m, 2H), 4.70 (quint, 1H),

6.96 (d, 1H), 7.33 (d, 1H), 7.65 (t, 1H), 7.95 (s, 1H); MS-ESI m/z

346 [MH]⁺.

N-(5-Chloro-1,3-benzodioxol-4-yl)-5-fluoroquinazolin-4-

amine (52). A mixture of 51 (400 mg, 2.2 mmol), (5-chloro-1,3-

benzodioxol-4-yl)amine (410 mg, 2.4 mmol), and a catalytic amount

of a 5 N solution of HCl gas in propan-2-ol was heated in propan-

2-ol (4 mL) at 80 °C for 1.5 h. After cooling, the formed precipitate

was filtered off and washed with propan-2-ol and then diethyl ether

to give 730 mg of 52 as a hydrochloride salt (94%): ¹H NMR

(DMSO-d₆) δ 6.14 (s, 2H), 7.04 (d, 1H), 7.11 (d, 1H), 7.71 (dd,

1H), 7.84 (d, 1H), 8.11 (td, 1H), 8.86 (s, 1H), 10.3 (br s, 1H);

MS-ESI m/z 318 and 320 [MH]⁺.

tert-Butyl 4-[({4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]quin-

azolin-5-yl}oxy)methyl]piperidine-1-carboxylate (53). To a sus-

pension of NaH (60% dispersion in oil) (100 mg, 2.4 mmol) in

DMF (4 mL) was added tert-butyl 4-(hydroxymethyl)piperidine-

1-carboxylate (90 mg, 0.42 mmol) under nitrogen at room tem-

perature. The reaction mixture was stirred for 10 min, and then 52

(100 mg, 0.28 mmol) was added. The mixture was stirred for 4 h

at 80 °C. After cooling, the solution was poured dropwise into water

(20 mL), and the precipitate was filtered off, washed with water,

and dried overnight at 50 °C under vacuum in the presence of

phosphorus pentoxide to give 140 mg of 53 (97%), which was used

in the next step without further purification: ¹H NMR (CDCl₃) δ

1.30 (m, 2H), 1.45 (s, 9H), 1.92 (m, 2H), 2.20 (m, 1H), 2.78 (m,

2H), 4.11 (d, 2H), 4.20 (m, 2H), 6.05 (s, 2H), 6.73 (d, 1H), 6.89

(d, 1H), 6.98 (d, 1H), 7.49 (d, 1H), 7.65 (t, 1H), 8.61 (s, 1H), 9.33

(s, 1H); MS-ESI m/z 513 and 515 [MH]⁺.

N-(5-Chloro-1,3-benzodioxol-4-yl)-5-(piperidin-4-ylmethoxy)-

quinazolin-4-amine (54). 53 (140 mg, 0.27 mmol) was stirred in

a 2 N solution of HCl gas in diethyl ether (20 mL) at room

temperature for 3 h. The mixture was diluted with diethyl ether,

and the precipitate was filtered off, washed with diethyl ether, and

dried under vacuum to give 134 mg of 54 as a dihydrochloride salt

(89%), which was used in the next step without further purifica-

tion: ¹H NMR (DMSO-d₆) δ 1.53 (m, 2H), 1.96 (m, 2H), 2.39 (m,

1H), 2.88 (m, 2H), 3.35 (m, 2H), 4.42 (d, 2H), 6.15 (s, 2H), 7.07

(d, 1H), 7.15 (d, 1H), 7.53 (m, 2H), 8.07 (t, 1H), 8.80-9.00 (m,

3H), 10.57 (br s, 1H); MS-ESI m/z 513 [MH]⁺.

A similar procedure was used to prepare 58.

4-[(2-Chloro-5-methoxyphenyl)amino]-7-methoxyquinazolin-

5-ol (44). A mixture of 39 (2.5 g, 6.5 mmol) and pyridine

hydrochloride (760 mg, 6.5 mmol) in pyridine (50 mL) was refluxed

overnight. The solution was cooled and concentrated. The residue

was taken up in water (50 mL) and made alkaline to pH 11 by

addition of 30% NH₄OH. The solid was filtered off, washed with

water, triturated in dichloromethane and then diethyl ether, and dried

under vacuum at 50 °C in the presence of phosphorus pentoxide to

give 2.14 g of 44 (98%), which was used in the next step without

further purification: ¹H NMR (DMSO-d₆and CF₃CO₂D) δ 3.84

(s, 3H), 3.95 (s, 3H), 6.81 (m, 2H), 6.98 (dd, 1H), 7.53 (d, 1H),

7.87 (d, 1H), 8.88 (s, 1H); MS-ESI m/z 332 and 334 [MH]⁺.

5-Methoxyquinazolin-4(3H)-one (45). A mixture of 48 (1.64

g, 10 mmol) and sodium methoxide (1.2 g, 30 mmol) in THF (20

mL) was refluxed overnight. The solvent was evaporated under

vacuum, and the residue was made acidic (pH 5) by addition of 2

N hydrochloric acid. The precipitate formed was filtered, washed

thoroughly with water and diethyl ether, and dried to give 1.2 g of

45 (68%): ¹H NMR (DMSO-d₆) δ 3.85 (s, 3H), 7,0 (d, 1H), 7.15

(d, 1H), 7.7 (t, 1H), 7.95 (s,1H); MS-ESI m/z 177 [MH⁺]. Anal.

(C₉H₈N₂O₂) C, H, N.

N-(2-Chloro-5-methoxyphenyl)-5-methoxyquinazolin-4-

amine (46). A mixture of 45 (2.1 g, 12 mmol), phosphorus

oxychloride (1.23 mL, 13.2 mmol), and DIPEA (5.2 mL, 30 mmol)

in 1,2-dichloroethane (20 mL) was heated to 75 °C for 2 h. The

solvent was evaporated under vacuum, the crude intermediate was

reacted with 2-chloro-5-methoxyaniline (1.9 g, 12 mmol) in propan-

2-ol (20 mL) containing a catalytic amount of a 5 N solution of

HCl gas in 2-propanol (330 µL, 2 mmol), and the reaction mixture

was heated at 80 °C for 30 min. After cooling, the precipitate formed

was filtered, washed with propan-2-ol and diethyl ether, and dried

to give 3.4 g of 46 as a hydrochloride (81%): ¹H NMR (DMSO-

d₆) δ 3.8 (s, 3H), 4.17 (s, 3H), 7.02 (dd, 1H), 7.43 (d, 1H), 7.54-

7.58 (m, 3H), 8.07 (t, 1H), 8.89 (s, 1H), 11.18 (br s, 1H); MS-ESI

m/z 316 and 318 [MH⁺]. Anal. (C₁₆H₁₄N₃O₂Cl‚1.5HCl‚1.5H₂O) C,

H, N, Cl.

4-[(2-Chloro-5-methoxyphenyl)amino]quinazolin-5-ol (47). A

mixture of 46 (3.3 g, 9.4 mmol) and pyridine hydrochloride (1.1 g,

10 mmol) in pyridine (30 mL) was heated under reflux overnight.

Pyridine was evaporated, and the residue was made alkaline by

addition of 30% NH₄OH. The precipitate formed was washed with

water and diethyl ether and dried to give 1.4 g of 47 (50%): ¹H

NMR (DMSO-d₆) δ 3.8 (s, 3H), 7.02 (dd, 1 H), 7.25 (dd, 1H),

7.30 (d, 1H), 7.57 (d, 1H), 7.82 (d, 1H), 7.92 (t, 1H), 8.94 (s, 1H);

MS-ESI m/z 302 and 304 [MH⁺]. Anal. (C₁₅H₁₂N₃O₂Cl‚0.3H₂O)

C, H, N.

4,6-Bis(benzyloxy)-1H-indole-2,3-dione (55). Using a procedure

similar to that described by Newmam,⁷⁶[3,5-bis(benzyloxy)phenyl]-

amine⁷⁵(10 g, 29 mmol) gave 8.8 g of 55 (84%): ¹H NMR

(DMSO-d₆) δ 5.25 (s, 2H), 5.27 (s, 2H), 6.13 (s, 1H), 6.42 (s, 1H),

7.25-7.60 (m, 10H); MS-ESI m/z 382 [MNa]⁺.

2-Amino-4,6-bis(benzyloxy)benzoic Acid (56). Using a proce-

dure similar to that described by Newmam,⁷⁶55 (14.3 g, 40 mmol)

gave 8 g of 56 (57%): ¹H NMR (DMSO-d₆) δ 5.06 (s, 2H), 5.14

(s, 2H), 6.01 (d, 1H), 6.05 (d, 1H), 7.25-7.60 (m, 10H); MS-ESI

m/z 372 [MNa]⁺.

Methyl 2-Amino-4,6-bis(benzyloxy)benzoate (57). 57 was

prepared using the method described by Lombardi.⁷⁷In a new

capped flask was dissolved Diazald (11.5 g, 53 mmol) in ethanol

(75 mL) in which nitrogen was bubbling. From the top of it Teflon

tubing was connected to a second flask and plunged into a solution

of 56 (8 g, 23 mmol) in dichloromethane (170 mL) at 0 °C to

6480 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

produce bubbling. To the Diazald solution was cautiously added a

concentrated aqueous solution of sodium hydroxide (30 mL) while

an important nitrogen flow rate was maintained. At the end of

addition, the dichloromethane solution was stirred for 30 min and

then concentrated. The obtained red solid was washed with diethyl

ether and filtered off, giving 7.7 g of 57 (91%) as a white solid:

¹H NMR (DMSO-d₆) δ 3.74 (s, 3H), 5.08 (s, 2H), 5.12 (s, 2H),

5.99 (d, 1H), 6.04 (d, 1H), 6.24 (br s, 2H), 7.25-7.55 (m, 10H);

MS-ESI m/z 386 [MNa]⁺.

5,7-Bis(benzyloxy)-N-(2-chloro-5-methoxyphenyl)quinazolin-

4-amine (59). To a mixture of 58 (5.7 g, 16 mmol) and DIPEA

(7.25 mL; 42 mmol) in 1,2-dichloroethane (120 mL) was added

dropwise POCl₃(1.8 mL, 20 mmol). The solution was then heated

at 80 °C for 2 h. The solvent was evaporated, and the crude

4-chloroquinazoline was reacted with 2-chloro-5-methoxyaniline

hydrochloride (3.1 g, 16 mmol) in propan-2-ol (50 mL). The mixture

was heated at 80 °C for 30 min. After cooling, the precipitate was

filtered off and dried under vacuum to give 6.34 g of 59 as a

hydrochloride salt (71%): ¹H NMR (DMSO-d₆) δ 3.81 (s, 3H),

5.31 (s, 2H), 5.64 (s, 2H), 6.98 (dd, 1H), 7.01 (d, 1H), 7.12 (d,

1H), 7.4-7.6 (m, 9H), 7.58 (d, 2H), 7.68 (d, 1H), 8.8 (s, 1H); MS-

ESI m/z 498 and 500 [MH]⁺.

7-(Benzyloxy)-4-[(2-chloro-5-methoxyphenyl)amino]quinazo-

lin-5-ol (60). A mixture of 59‚HCl (4.35 g, 8.1 mmol) and pyridine

hydrochloride (941 mg, 8.1 mmol) in pyridine (90 mL) was heated

at reflux for 9 h. After evaporation, the residue was taken up in

water (90 mL) to give after filtration and drying 3.14 g of 60

(95%): ¹H NMR (DMSO-d₆and CF₃CO₂D) δ 3.85 (s, 3H), 5.3 (s,

2H), 6.85 (s, 2H), 7.01 (dd, 1H), 7.4-7.6(m, 6H), 7.8 (d, 1H), 8.85

(s, 1H); MS-ESI m/z 407 and 409 [MH]⁺.

7-(Benzyloxy)-N-(2-chloro-5-methoxyphenyl)-5-(tetrahydro-

2H-pyran-4-yloxy)quinazolin-4-amine (61). To a mixture of 60

(1.22 g, 3 mmol), triphenylphosphine (1.26 g, 4.8 mmol), and

tetrahydro-2H-pyran-4-ol (350 µL, 3.6 mmol) in dichloromethane

(30 mL) cooled in an ice bath at 0 °C was slowly added DTAD

(1.1 g, 4.8 mmol). The mixture was stirred for 2 h at room

temperature. The crude was purified by SiO₂chromatography and

eluted with a mixture of dichloromethane/ethyl acetate (9:1 up to

8:2) to give 800 mg of 61 (55%) as a white solid: ¹H NMR

(DMSO-d₆) δ 1.75-1.85 (m, 2H), 2.1-2.2 (m, 2H), 3.45-3.55

(m, 2H), 3.85-3.95 (m, 2H), 3.8 (s, 3H), 5.0-5.1 (m, 1H), 5.3 (s,

2H), 6.8 (dd, 1H), 6.95 (d, 1H), 7.02 (d, 1H), 7.3-7.6 (m, 6H), 8.1

(d, 1H), 8.5 (s, 1H), 9.86 (s, 1H); MS-ESI m/z 491 and 493[MH]⁺.

A similar procedure was used to prepare 62-64.

94 g of 71 (98%) as a white solid: ¹H NMR (DMSO-d₆) δ 3.86 (s,

3H), 6.45 (d, 1H), 6.64 (d, 1H), 8.08 (s, 1H); MS-ESI m/z 193

[MH]⁺.

A similar procedure was used to prepare 98.

tert-Butyl [(5-Hydroxy-7-methoxy-4-oxoquinazolin-3(4H)-yl)-

methyl]carbonate (72). To a suspension of NaH (60% suspension

in oil) (44 g, 1.1 mol) in DMF (1 L) cooled at 0 °C was slowly

added solid 71 (93 g, 480 mmol). The mixture was then stirred at

room temperature for 1 h and cooled again in an ice bath before

dropwise addition of chloromethyl pivalate (99 mL, 1.4 mol). After

being stirred at 0 °C for 1 h, the reaction mixture was poured into

a solution of acetic acid (230 mL) in water (3 L). The precipitate

was filtered and taken up in dichloromethane. The solution was

washed with brine, dried (MgSO₄), and evaporated to give 121 g

of 72 (83%): ¹H NMR (CDCl₃) δ 1.2 (s, 9H), 3.85 (s, 3H), 5.85

(s, 2H), 6.48 (d, 1H), 6.65 (d, 1H), 8.12 (s, 1H); MS-ESI m/z 307

[MH]⁺.

A similar procedure was used to prepare 83.

7-Methoxy-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4(3H)-

one (73). To a mixture of 72 (120 g, 390 mmol), triphenylphosphine

(164 g, 620 mmol), and tetrahydro-2H-pyran-4-ol (44 g, 430 mmol)

in dichloromethane (1.5 L) at 0 °C was slowly added DTAD (143

g, 620 mmol). The mixture was then stirred at room temperature

for 1 h and evaporated. The residue was taken up in a 6 N methanol/

ammonia solution and the resulting mixture stirred at room

temperature overnight. The precipitate was filtered, washed with

methanol and dichloromethane, and dried under vacuum to give

94 g of 73 (88%) as a beige solid: ¹H NMR (DMSO-d₆) δ 1.6-

1.7 (m, 2H), 1.9-2.0 (m, 2H), 3.5-3.6 (m, 2H), 3.86 (s, 3H), 3.85-

3.95 (m, 2H), 4.7-4.8 (m, 1H), 6.63 (d, 1H), 6.68 (d, 1H), 7.92 (s,

1H); MS-ESI m/z 277 [MH]⁺. Anal. (C₁₄H₁₆N₂O₄‚0.4H₂O) C, H,

N.

7-Hydroxy-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4(3H)-

one (74). A mixture of 73 (94 g, 340 mmol), potassium carbonate

(69 g, 500 mmol), and benzenethiol (51 mL, 500 mmol) in NMP

(400 mL) was heated at 195 °C for 35 min. The solvent was

evaporated and the residue acidified at pH 5 with 6 N hydrochloric

acid. The precipitate formed was filtered, washed thoroughly with

water and dichloromethane, and dried to give 98 g of 74 (100%):

¹H NMR (DMSO-d₆) δ 1.6-1.7 (m, 2H), 1.9-2.0 (m, 2H), 3.4-

3.5 (m, 2H), 3.85-3.95 (m, 2H), 4.6-4.7 (m, 1H), 6.55 (d, 1H),

6.60 (d, 1H), 8.1 (s, 1H); MS-ESI m/z 263 [MH]⁺. Anal.

(C₁₃H₁₄N₂O₄‚0.2H₂O) C, H, N.

4-Oxo-5-(tetrahydro-2H-pyran-4-yloxy)-3,4-dihydroquinazo-

lin-7-yl Acetate (75). A mixture of 74 (90 g, 340 mmol), acetic

anhydride (600 mL), and pyridine (900 mL) was heated at 120 °C

for 2 h. After evaporation the residue was taken up in a mixture of

methanol (400 mL) and water (400 mL) and the resulting mixture

stirred for 45 min at room temperature. After evaporation to dryness

the crude material was purified by SiO₂chromatography eluting

with a mixture of dichloromethane/methanol (98:2 up to 96:4) as

eluent to give 58.5 g of 75 (57%): ¹H NMR (DMSO-d₆) δ 1.6-

1.7 (m, 2H), 1.9-2.0 (m, 2H), 2.29 (s, 3H), 3.45-3.55 (m, 2H),

3.85-3.95 (m, 2H), 4.65-4.75 (m, 1H), 6.91 (d, 1H), 6.93 (d, 1H),

7.97 (s, 1H); MS-ESI m/z 305 [MH]⁺. Anal. (C₁₅H₁₆N₂O₅‚0.2H₂O)

C, H, N.

4-[(2-Chloro-5-methoxyphenyl)amino]-5-(tetrahydro-2H-py-

ran-4-yloxy)qui nazolin-7-ol (65). A solution of 61 (780 mg, 1.6

mmol) in trifluoroacetic acid (5 mL) was heated at 80 °C for 5 h.

After evaporation the residue was made alkaline using a 7 N

solution of ammonia in methanol, and dichloromethane was added.

The solution was filtered and purified by SiO₂chromatography

using a mixture of dichloromethane/methanol (96:4) as eluent to

give 470 mg of 65 (86%) as a white solid: ¹H NMR (DMSO-d₆)

δ 1.75-1.85 (m, 2H), 2.1-2.2 (m, 2H), 3.45-3.55 (m, 2H), 3.8

(s, 3H), 3.85-3.95 (m, 2H), 4.9-5.0 (m, 1H), 6.7 (d, 1H), 6.81

(dd, 1H), 7.5 (d, 1H), 8.1 (d, 1H), 8.4 (s, 1H), 9.8 (s, 1H); MS-ESI

m/z 401 and 403 [MH]⁺.

A similar procedure was used to prepare 66-68.

4-(1,3-Benzodioxol-4-ylamino)-5-(tetrahydro-2H-pyran-4-yloxy)-

quinazolin-7-yl Acetate (76). To a mixture of 75 (3.04 g, 10 mmol)

and DIEA (4.34 mL, 25 mmol) in 1,2-dichloroethane (60 mL) was

added dropwise POCl₃(1.08 mL, 11 mmol). The solution was then

heated at 80 °C for 2 h. The solvent was evaporated, the

intermediate 4-chloroquinazoline was reacted immediately with 2,3-

(methylenedioxy)aniline (1.5 g, 11 mmol) in propan-2-ol (20 mL),

and the reaction mixture was heated at 80 °C for 1 h. After cooling,

the precipitate was washed with 2-propanol and diethyl ether,

filtered, and dried under vacuum to give 3.6 g of 76 as a

hydrochloride (78%): ¹H NMR (DMSO-d₆and CF₃CO₂D) δ 1.9-

2.0 (m, 2H), 2.1-2.2 (m, 2H), 2.38 (s, 3H), 3.5-3.6 (m, 2H), 3.9-

4.0 (m, 2H), 5.05-5.15 (m, 1H), 6.17 (s, 2H), 6.9-7.0 (m, 2H),

7.32 (d, 1H), 7.50 (d, 1H), 7.60 (dd, 1H), 8.99 (s, 1H); MS-ESI

m/z 424 [MH]⁺. Anal. (free base) (C₂₂H₂₁N₃O₆) C, H, N.

Methyl 2-Amino-4,6-dimethoxybenzoate (70). 70 was prepared

using the method described by Lombardi.⁷⁷69⁷⁸(15.9 g, 81 mmol)

was reacted with diazomethane generated from Diazald (31 g, 145

mmol) to give 16.2 g of 70 (95%): ¹H NMR (DMSO-d₆) δ 3.72

(m, 9H), 5.76 (d, 1H), 5.91 (d, 1H), 6.15 (br s, 2H); MS-ESI m/z

234 [MNa]⁺.

5-Hydroxy-7-methoxyquinazolin-4(3H)-one (71). To a mixture

of 43 (103 g, 500 mmol) in pyridine (1 L) was slowly added MgBr₂

(92 g, 0.5 mol) at room temperature. The reaction mixture was

then heated at reflux temperature for 1.5 h. Pyridine was then

evaporated, the residue taken up with a mixture of water (1 L) and

acetic acid (200 mL), and the resulting mixture stirred for 15 min.

The precipitate formed was filtered off, washed thoroughly with

water and diethyl ether, and dried at 60 °C under vacuum to give

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6481

A similar procedure was used to prepare 77.

1H), 5.89 (s, 2H), 6.50 (d, 1H), 6.71 (d, 1H), 8.18 (s, 1H); MS-

ESI m/z 404 [MH]⁺.

4-(1,3-Benzodioxol-4-ylamino)-5-(tetrahydro-2H-pyran-4-yloxy)-

quinazolin-7-ol (78). A mixture of 76‚HCl (4 g, 7.6 mmol) in a 6

N methanol/ammonia solution (50 mL) was stirred at room

temperature for 6 h. The solvent was evaporated and the residue

thoroughly washed with water, dissolved in dichloromethane and

methanol, and rapidly purified by SiO₂chromatography using a

mixture of dichloromethane/methanol (95:5) as eluent to give 2.3

g of 78 (80%): ¹H NMR (DMSO-d₆) δ 1.8-1.88 (m, 2H), 2.1-

2.2 (m, 2H), 3.5-3.6 (m, 2H), 3.9-3.95 (m, 2H), 4.9-5.0 (m, 1H),

6.11 (s, 2H), 6.66 (d, 1H), 6.71-6.76 (m, 2H), 6.87 (t, 1H), 8.04

(d, 1H), 8.4 (s, 1H), 9.8 (s, 1H); MS-ESI m/z 382 [MH]⁺.

A similar procedure was used to prepare 79.

tert-Butyl 4-({[4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-5-

(tetrahydro-2H-pyran-4-yloxy)quinazolin-7-yl]oxy}methyl)-

piperidine-1-carboxylate (80). A mixture of 79 (410 mg, 1 mmol),

tert-butyl 4-((((4-methylphenyl)sulfonyl)oxy)methyl)piperidine-1-

carboxylate (410 mg, 3.3 mmol) and cesium fluoride (460 mg, 3

mmol) in DMF (5 mL) was heated at 70 °C for 4 h. The volatiles

were removed under vacuum, and the residue was diluted with water

and then extracted with ethyl acetate The organic layer was washed

with saturated aqueous sodium hydrogen carbonate solution, water,

and brine, dried (MgSO₄), evaporated. After evaporation of the

solvents, the residue was purified by SiO₂chromatography using a

mixture of dichloromethane/methanol (98:2) as eluent to give 500

mg of 80 (81%): ¹H NMR (CDCl₃) δ 1.3-1.4 (m, 2H), 1.4 (m,

9H), 1.75-1.85 (m, 2H), 1.9-2.05 (m, 3H), 2.2-2.3 (m, 2H), 2.7-

2.8 (m, 2H), 3.6-3.7 (m, 2H), 3.95 (d, 2H), 4.0-4.1 (m, 2H), 4.1-

4.25 (m, 2H), 4.7-4.8 (m, 1H), 6.05 (s, 2H), 6.5 (d, 1H), 6.7 (d,

1H), 6.8 (d, 1H), 6.98 (d, 1H), 8.5 (s, 1H), 9.28 (s, 1H); MS-ESI

m/z 613 and 615 [MH]⁺.

4-Chloro-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-7-ol (81).

A mixture of 75 (1.92 g, 6.3 mmol), triphenylphosphine (3.3 g, 13

mmol), and carbon tetrachloride (1.83 mL, 19 mmol) in 1,2-

dichloroethane (150 mL) was heated at 70 °C for 2 h. The solvent

was evaporated and the residue purified by SiO₂chromatography

using a mixture of dichloromethane/ethyl acetate (90:10) for elution

to give 2 g of 4-chloro-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-

7-yl acetate (66%). This intermediate was taken up in a 6 N solution

of ammonia in methanol and the resulting mixture stirred at room

temperature for 2 h to give a precipitate that was filtered and dried

under vacuum to give 860 mg of 81 (78%): ¹H NMR (DMSO-d₆)

δ 1.7-1.8 (m, 2H), 2.0-2.1 (m, 2H), 3.55-3.65 (m, 2H), 3.85-

3.95 (m, 2H), 4.8-4.9 (m, 1H), 6.83 (2d, 2H), 8.71 (s, 1H); MS-

ESI m/z 281 and 283 [MH]⁺.

4-Chloro-7-(2-pyrrolidin-1-ylethoxy)-5-(tetrahydro-2H-pyran-

4-yloxy)quinazoline (82). To a mixture 81 (750 mg, 2.7 mmol),

triphenylphosphine (1.14 g, 4.3 mmol), and 2-pyrrolidin-1-ylethanol

(372 mg, 3.3 mmol) in dichloromethane (20 mL) at room temper-

ature was added DTAD (990 mg, 4.3 mmol). After the resulting

mixture was stirred for 30 min, the solvent was evaporated and the

residue purified by SiO₂chromatography using a mixture of

dichloromethane-6 N methanol/ammonia solution (97:3) as eluent

to give 900 mg of 82 (88%): ¹H NMR (CDCl₃) δ 1.8-1.9 (m,

4H), 1.9-2.0 (m, 2H), 2.1-2.2 (m, 2H), 2.6-2.7 (m, 4H), 3.0 (t,

2H), 3.65-3.75 (m, 2H), 4.0-4.1 (m, 2H), 4.25 (t, 2H), 4.7-4.8

(m, 1H), 6.7 (d, 1H), 6.96 (d, 1H), 8.81 (s, 1H); MS-ESI m/z 378

and 380 [MH]⁺.

[7-Methoxy-5-[(1-methylpiperidin-4-yl)oxy]-4-oxoquinazolin-

3(4H)-yl]methyl pivalate (1.75 g, 4.3 mmol) was stirred in a 7 N

solution of ammonia in methanol (100 mL) at room temperature

overnight. The mixture was concentrated and taken up in diethyl

ether. The formed precipitate was filtered off, washed with diethyl

ether, and dried under vacuum at 50 °C to give 930 mg of 84

(75%): ¹H NMR (DMSO-d₆) δ 1.70 (m, 2H), 1.90 (m, 2H), 2.18

(s, 3H), 2.20 (m, 2H), 2.65 (m, 2H), 3.82 (s, 3H), 4.48 (m, 1H),

6.57 (d, 1H), 6.66 (d, 1H), 7.89 (s, 1H); MS-ESI m/z 290 [MH]⁺.

7-(Benzyloxy)-5-[(1-methylpiperidin-4-yl)oxy]quinazolin-4(3H)-

one (85). To a mixture of 83 (6 g, 15.7 mmol), triphenylphosphine

(6.2 g, 23.5 mmol), and 1-methylpiperidin-4-ol (2.2 g, 19 mmol)

in dichloromethane (100 mL) was added dropwise a solution of

DTAD (5.4 g, 23.5 mmol) in dichloromethane (20 mL) under

nitrogen at 0 °C. The solution was stirred for 1 h at room

temperature and then poured onto a column of silica gel eluting

with methanol/dichloromethane/ethyl acetate (5:45:50) to remove

impurities and then with a solution of 7 N ammonia in methanol/

dichloromethane/ethyl acetate (5:45:50). Evaporation of the solvent

gave the intermediate, which was dissolved and stirred overnight

in a 7 N solution of ammonia in methanol (240 mL) at room

temperature. The mixture was concentrated and taken up in diethyl

ether. The precipitate was filtered off, washed with diethyl ether,

and dried under vacuum at 50 °C to give 3.7 g of 85 (65%): ¹H

NMR (CDCl₃) δ 2.00 (m, 4H), 2.29 (s, 3H), 2.35 (m, 2H), 2.73

(m, 2H), 4.46 (m, 1H), 5.13 (s, 1H), 6.56 (d, 1H), 6.82 (d, 1H),

7.3-7.55 (m, 5H), 7.91 (s, 1H), 11.2 (br s, 1H); MS-ESI m/z 366

[MH]⁺.

tert-Butyl 4-{[7-(Benzyloxy)-4-oxo-3,4-dihydroquinazolin-5-

yl]oxy}piperidine-1-carboxylate (87). To a mixture of 83 (1.95

g, 5.1 mmol), triphenylphosphine (2 g, 7.6 mmol), and tert-butyl

4-hydroxypiperidine-1-carboxylate (1.23 g, 6.1 mmol) in dichlo-

romethane (15 mL) was added portionwise DTAD (0.88 g, 7.6

mmol) under nitrogen at 15 °C. The solution was stirred for 1 h at

room temperature and then concentrated. The crude was taken up

in methanol (25 mL), and NaOH pellets (360 mg, 8.9 mmol) were

added to the solution. The reaction mixture was stirred for 30 min

until the NaOH pellets dissolved. The solvent was evaporated, and

the crude product was purified on silica gel eluting with methanol/

dichloromethane/ethyl acetate (1:49:50 up to 5:45:50). Evaporation

of the solvent gave 1.4 g of 87 (62%): ¹H NMR (CDCl₃) δ 1.48

(s, 9H), 1.93 (m, 4H), 3.54 (m, 2H), 3.71 (m, 2H), 4.65 (m, 1H),

5.17 (s, 2H), 6.59 (d, 1H), 6.87 (d, 1H), 7.35-7.55 (m, 5H), 7.92

(s, 1H), 10.56 (br s, 1H); MS-ESI m/z 452 [MH]⁺.

7-(Benzyloxy)-N-(5-chloro-1,3-benzodioxol-4-yl)-5-[(1-meth-

ylpiperidin-4-yl)oxy]quinazolin-4-amine (88). A mixture of 85

(3.7 g, 10 mmol), triphenylphosphine (5.3 g, 20 mmol), and carbon

tetrachloride (9.65 mL, 100 mmol) in 1,2-dichloroethane (100 mL)

was heated at 70 °C for 2 h under nitrogen. The dark solution was

concentrated, and then propan-2-ol (20 mL) was added to the crude

followed by 6-chloro-2,3-(methylenedioxy)aniline (1.9 g, 11 mmol)

and a 5 N solution of HCl gas in propan-2-ol (2.1 mL, 10.5 mmol).

The mixture was heated at 80 °C for 30 min. The solution was

concentrated, taken up in a mixture of 7 N solution of ammonia in

methanol/dichloromethane (5:95). The resulting precipitate was

eliminated by filtration, and the filtrate was evaporated down and

purified by SiO₂chromatography eluting with methanol/ethyl

acetate/dichloromethane (3:50:47) to give 4.2 g of 88 (81%): ¹H

NMR (CDCl₃) δ 2.04 (m, 2H), 2.19 (m, 2H), 2.31 (m, 5H), 2.74

(m, 2H), 4.59 (m, 1H), 5.18 (s, 2H), 6.06 (s, 2H), 6.60 (d, 1H),

6.73 (d, 1H), 6.94 (d, 1H), 6.98 (d, 1H), 7.32-7.55 (m, 5H), 8.53

(s, 1H), 9.28 (s, 1H); MS-ESI m/z 519 and 521 [MH]⁺. Anal.

(C₂₈H₂₇ClN₄O₄) C, H, N.

7-Methoxy-5-[(1-methylpiperidin-4-yl)oxy]quinazolin-4(3H)-

one (84). To a mixture of 72 (1.5 g, 4.9 mmol), triphenylphosphine

(1.9 g, 7.3 mmol), and 1-methylpiperidin-4-ol (675 mg, 5.9 mmol)

in dichloromethane (20 mL) was added dropwise a solution of

DTAD (1.7 g, 7.3 mmol) in dichloromethane (5 mL) under nitrogen

at 0 °C. The solution was stirred for 1 h at room temperature and

then poured onto a column of silica gel eluting with methanol/

dichloromethane/ethyl acetate (5:45:50) to remove impurities and

then with a 7 N solution of ammonia in methanol/dichloromethane/

ethyl acetate (5:45:50). Evaporation of the solvent gave 1.75 g of

[7-methoxy-5-[(1-methylpiperidin-4-yl)oxy]-4-oxoquinazolin-3(4H)-

yl]methyl pivalate (89%): ¹H NMR (CDCl₃) δ 1.20 (s, 9H), 2.04

(m, 4H), 2.25-2.55 (m, 5H), 3.86 (m, 2H), 3.89 (s, 3H), 4.46 (m,

4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-5-[(1-methylpiperi-

din-4-yl)oxy]quinazolin-7-ol (89). 88 (1.5 g, 2.9 mmol) was heated

under reflux in TFA (15 mL) for 6 h. After cooling, the solvent

was evaporated off, and water was added to the residue. The

solution was made alkaline by adding portionwise sodium bicarbon-

ate powder until pH 9 and extracted three times with ethyl acetate.

6482 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

The combined organic phases were washed with brine, dried over

magnesium sulfate, and concentrated. The crude material was

purified by SiO₂chromatography eluting with methanol/ethyl

acetate/dichloromethane (5:47.5:47.5 up to 10:45:45) to give 800

mg of 89 (64%): ¹H NMR (CDCl₃) δ 1.98 (m, 2H), 2.11 (m, 2H),

2.2-2.4 (m, 5H), 2.69 (m, 2H), 4.47 (m, 1H), 6.06 (s, 2H), 6.49

(d, 1H), 6.70 (d, 1H), 6.84 (d, 1H), 6.94 (d, 1H), 8.42 (s, 1H), 9.35

(s, 1H); MS-ESI m/z 429 and 431 [MH]⁺. Anal. (C₂₁H₂₁ClN₄O₄‚

0.6H₂O‚0.1C₄H₈O₂‚0.05CH₂Cl₂) C, H, N.

4-Chloro-7-methoxy-5-[(1-methylpiperazin-4-yl)oxy]quinazo-

line (90). A mixture of 84 (7 g, 24 mmol), triphenylphosphine (12.7

g, 48 mmol), and carbon tetrachloride (7 mL, 72 mmol) in 1,2-

dichloroethane (100 mL) was heated at 70 °C for 2 h under nitrogen.

After cooling, the solvent was evaporated off, and the crude was

purified by SiO₂chromatography eluting with methanol/dichlo-

romethane/ethyl acetate (5:45:50) to remove impurities and then

with a 7 N solution of ammonia in methanol/dichloromethane/ethyl

acetate (5:45:50) to give 5.3 g of 90 (71%): ¹H NMR (CDCl₃) δ

2.09 (m, 4H), 2.34 (s, 3H), 2.44 (m, 2H), 2.73 (m, 2H), 3.95 (s,

3H), 4.58 (m, 1H), 6.60 (d, 1H), 6.94 (d, 1H), 8.81 (s, 1H); MS-

ESI m/z 308 and 310 [MH]⁺. Anal. (C₁₅H₁₈ClN₃O₂‚0.55H₂O‚

0.01C₄H₁₀O‚0.04CH₂Cl₂) C, H, N.

A similar procedure was used to prepare 50, 51, 86, 91, and 92.

tert-Butyl 4-({7-(Benzyloxy)-4-[(5-chloro-1,3-benzodioxol-4-

yl)amino]quinazolin-5-yl}oxy)piperidine-1-carboxylate (93). A

mixture of 87 (1.9 g, 4.1 mmol), (5-chloro-1,3-benzodioxol-4-yl)-

amine (77 mg, 4.5 mmol), and a 5 N HCl solution in propan-2-ol

(36 µL, 0.2 mmol) in propan-2-ol (20 mL) was heated at 50 °C for

30 min. After cooling, the precipitate formed was filtered off and

washed with propan-2-ol and then diethyl ether to give 2.4 g of 93

as a hydrochloride salt (92%): ¹H NMR (DMSO-d₆) δ 1.4 (s, 9H),

1.9 (m, 2H), 2.0 (m, 2H), 3.1 (m, 2H), 3.9 (m, 2H), 5.1 (m, 1H),

5.35 (s, 2H), 6.1 (s, 2H), 7.0 (m, 2H), 7.1 (d, 1H), 7.2 (s, 1H),

7.3-7.6 (m, 5H), 8.75 (s, 1H), 10.1 (br s, 1H); MS-ESI m/z 605

and 607 [MH]⁺. Anal. (free base) (C₃₂H₃₃ClN₄O₆) C, H, N.

4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-5-(piperidin-4-ylox-

y)quinazolin-7-ol (94). A solution of 93 (2.8 g, 4.3 mmol) in TFA

(28 mL) was refluxed under nitrogen for 6 h. After cooling, the

solvent was evaporated off, and the crude was taken up in water.

The pH was adjusted to 10 by adding a 1 N solution of NaOH.

The mixture was then stirred at room temperature for 1 h, and the

precipitate was filtered off, washed with water, and dried under

vacuum at 50 °C in the presence of phosphorus pentoxide to give

1.4 g of 94 (78%), which was used in the next step without further

purification: ¹H NMR (DMSO-d₆) δ 1.71 (m, 2H), 2.06 (m, 2H),

2.69 (m, 2H), 2.95 (m, 2H), 4.80 (m, 1H), 6.08 (s, 2H), 6.63 (d,

1H), 6.69 (d, 1H), 6.91 (d, 1H), 7.05 (d, 1H), 8.26 (s, 1H), 9.22 (s,

1H); MS-ESI m/z 415 and 417 [MH]⁺.

tert-Butyl 4-({4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-7-hy-

droxyquinazolin-5-yl}oxy)piperidine-1-carboxylate (95). A mix-

ture of 94 (1.4 g, 3.4 mmol) and Boc₂O (74 mg, 3.4 mmol) in

DMF (14 mL) was stirred at room temperature under nitrogen for

2 h, then the solvent was removed under vacuum, and the residue

was dissolved in ethyl acetate, washed with water and brine, and

dried over magnesium sulfate. The crude was purified by SiO₂

chromatography eluting with a methanol/dichloromethane mixture

(0:100 up to 4:96) to give 1.2 g of 95 (68%): ¹H NMR (CDCl₃) δ

1.47 (s, 9H), 1.78 (m, 2H), 2.09 (m, 2H), 3.06 (m, 2H), 3.86 (m,

2H), 4.59 (m, 1H), 6.03 (s, 2H), 6.54 (s, 1H), 6.72 (d, 1H), 6.96

(m, 2H), 8.42 (s, 1H), 9.38 (s, 1H); MS-ESI m/z 415 and 417

[MH]⁺. Anal. (C₂₅H₂₇ClN₄O₆‚0.1H₂O) C, H, N.

tion: ¹H NMR (DMSO-d₆) δ 1.42 (s, 9H), 1.95 (m, 2H), 2.10 (m,

2H), 3.08 (m, 2H), 3.88 (m, 2H), 4.00 (s, 3H), 5.09 (m, 1H), 6.14

(s, 2H), 6.97 (d, 1H), 7.05 (d, 1H), 7.13 (d, 1H), 7.16 (d, 1H), 8.78

(s, 1H), 10.11 (br s, 1H); MS-ESI m/z 529 and 531 [MH]⁺.

7-(Benzyloxy)-5-isopropoxyquinazolin-4(3H)-one (99). To a

mixture of 83 (30 g, 78 mmol), triphenylphosphine (33 g, 125

mmol), and 2-propanol (7.3 mL, 94 mmol) in dichloromethane (350

mL) was added portionwise DTAD (29 g, 125 mmol) over 30 min

under nitrogen at 0 °C. The solution was stirred for 1 h at room

temperature and then concentrated. The residue was dissolved and

stirred overnight in a 7 N solution of ammonia in methanol (450

mL) at room temperature, then concentrated, and treated with diethyl

ether. The resulting precipitate was filtered, washed with diethyl

ether, and purified by chromatography using methanol/dichlo-

romethane as eluent (2:98 up to 5:95) to give 24 g of 99 (97%):

¹H NMR (DMSO-d₆) δ 1.29 (d, 6H), 4.66 (quint, 1H), 5.23 (s,

2H), 6.62 (d, 1H), 6.75 (d, 1H), 7.3-7.6 (m, 5H), 7.89 (s, 1H);

MS-ESI m/z 311 [MH]⁺.

5-Isopropoxy-4-oxo-3,4-dihydroquinazolin-7-yl Acetate (100).

To a solution of 99 (24 g, 77 mmol) in DMF (300 mL) were added

successively Pd/C (10%) (2.8 g) and ammonium formate (48 g,

768 mmol). The mixture was stirred for 2 h at room temperature.

The solution was passed through a pad of Celite, and the filtrate

was concentrated. The solid was taken up in water (250 mL), filtered

off, washed twice with water and diethyl ether, and dried under

vacuum at 50 °C in the presence of phosphorus pentoxide to give

the intermediate 7-hydroxy-5-isopropoxyquinazolin-4(3H)-one as

a white solid (16 g). A mixture of 7-hydroxy-5-isopropoxyquinazo-

lin-4(3H)-one (15.9 g, 72 mmol), acetic anhydride (34 mL), and

pyridine (0.62 mL, 7.2 mmol) was heated at 70 °C for 30 min.

After evaporation the residue was taken up in water (200 mL) and

the resulting mixture stirred for 20 min at 80 °C. The precipitate

was filtered and dried on phosphorus pentoxide under vacuum to

give 17.8 g of 100 (94%): ¹H NMR (DMSO-d₆) δ 1.32 (d, 6H),

2.31 (s, 3H), 4.66 (quint., 1H), 6.86 (d, 1H), 6.91 (d, 1H), 7.93 (s,

1H); MS-ESI m/z 263 [MH]⁺.

4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-5-isopropoxyquinazo-

lin-7-ol (102). To a mixture of 100 (22 g, 84 mmol) and DIPEA

(38 mL, 218 mmol) in 1,2-dichloroethane (600 mL) was added

dropwise POCl₃(9.4 mL, 101 mmol). The solution was then heated

at 80 °C for 2 h. The solvent was evaporated, the intermediate

4-chloroquinazoline was reacted immediately with (5-chloro-1,3-

benzodioxol-4-yl)amine (15.2 g, 85 mmol) in propan-2-ol (200 mL),

and the reaction mixture was heated at 80 °C for 1.5 h. After

cooling, propan-2-ol was evaporated, and the residue was stirred

in a 6 N solution of ammonia in methanol (25 mL) for 30 min at

room temperature. Methanol was removed under vacuum, and after

addition of dichloromethane the organic phase was filtered and

purified by SiO₂chromatography using a mixture of dichlo-

romethane-6 N methanol/ammonia (99:1 to 93:7) as eluent to give

29 g of 102 (92%): ¹H NMR (DMSO-d₆) δ 1.44 (d, 6H), 4.91

(quint., 1H), 6.07 (s, 2H), 6.64 (m, 2H), 6.91 (d, 1H), 7.05 (d, 1H),

8.26 (d, 1H), 9.26 (s, 1H); MS-ESI m/z 374 and 376 [MH]⁺.

A similar procedure was used to prepare 101.

N-(5-Fluoro-1,3-benzodioxol-4-yl)-7-(2-chloroethoxy)-5-iso-

propoxyquinazolin-4-amine (103). A mixture of 101 (32 g, 90

mmol) and potassium carbonate (22 g, 161 mmol) in 1,2-

dichloroethane (450 mL) and DMF (225 mL) was heated at 85 °C

under nitrogen for 24 h. After cooling, the solid was removed by

filtration and after concentration of the filtrate. The crude was

purified by SiO₂chromatography eluting with methanol/dichlo-

romethane (0:100 up to 2:98) to give 29 g of 103 (78%): ¹H NMR

(CDCl₃) δ 1.52 (d, 6H), 3.89 (t, 2H), 4.36 (t, 2H), 4.82 (quint,

1H), 6.05 (s, 2H), 6.55 (d, 1H), 6.66 (m, 2H), 6.79 (d, 1H), 8.54

(s, 1H), 9.30 (br s, 1H); MS-ESI m/z 420 and 422 [MH]⁺.

A similar procedure was used to prepare 104.

tert-Butyl 4-({4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-7-

methoxyquinazolin-5-yl}oxy)piperidine-1-carboxylate (97). A

mixture of 91 (140 mg, 0.33 mmol), (5-chloro-1,3-benzodioxol-4-

yl)amine (60 mg, 0.36 mmol), and a catalytic amount of a 5 N

solution of HCl gas in propan-2-ol was heated in propan-2-ol (2

mL) at 80 °C for 1.5 h. After cooling, the solution was concentrated

to give a residue which was treated with diethyl ether, and the

resulting precipitate was filtered and washed with ethyl acetate and

then diethyl ether to give 170 mg of 97 as a hydrochloride salt

(92%), which was used in the next step without further purifica-

4,6-Difluoro-2,3-indolinedione (105). To a reactor were intro-

duced Na₂SO₄‚10H₂O (3.5 kg), water (3.5 L), and chloral (132 mL,

1.36 mol). In another flask, 3,5-difluoroaniline (160 g, 1.24 mol)

in water (1.3 L) was converted into its hydrochloride by adding

concentrated HCl (104 mL, 1.24 mol), and the solution obtained

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6483

was added to the reactor. Then an aqueous solution of NH₂OH‚

HCl (258.5 g, 3.72 mol) in water (1.25 L) was added rapidly. The

mixture was heated at 120 °C for 2 h. After cooling, the precipitate

was filtered, washed with water, and dried. To a flask containing

concentrated H₂SO₄(700 mL) was added portionwise the previous

intermediate (177 g) under stirring. The reaction mixture was heated

at 80 °C for 10 min. After cooling, it was poured on a mixture of

water (4 L)/ice (3 kg). The solid was filtered, washed with water

and diethyl ether, and dried to give 157 g of 105 (72%): ¹H NMR

(DMSO-d₆) δ 6.6 (dd, 1H), 6.9 (dd, 1H); MS-ESI m/z 182 [M -

H]. Anal. (C₈H₃NO₂F₂) C, H, N.

2-Amino-4,6-difluorobenzoic Acid (106). To a large beaker

containing a solution of NaOH pellets (424 g, 10.6 mol) in water

(1.3 L) was added 105 (157 g, 0.856 mol). The mixture was heated

at 60-70 °C, and 30% H₂O₂(260 mL) was added dropwise with

stirring. After strong gas evolution occurred, the mixture was heated

under stirring for 30 min. The mixture was cooled to 0 °C and

acidified at pH 4 by addition of 12 N HCl (770 mL). The mixture

was extracted with ethyl acetate. The organic phase was washed,

dried (MgSO₄), and evaporated to give 118 g of 106 as a solid

(80%): ¹H NMR (DMSO-d₆) δ 6.25 (dd, 1H), 6.35 (dd, 1H); MS-

ESI m/z 172 [M - H]. Anal. (C₇H₅NO₂F₂) C, H, N.

Methyl 2-Amino-4,6-difluorobenzoate (107). To a mixture of

104 (118 g, 0.68 mol), triphenylphosphine (196 g, 0.75 mol), and

methanol (39 mL, 0.95 mol) in dichloromethane (1.5 L) cooled in

ice was added dropwise DEAD (118 mL, 0.75 mol). The mixture

was stirred for 1 h at room temperature. The organic phase was

purified by chromatography on SiO₂and eluted with dichlo-

romethane to give after evaporation 111 g of 107 (87%) as a white

solid: ¹H NMR (DMSO-d₆) δ 3.9 (s, 3H), 5.9 (m, 2H), 6.1(m,

2H); MS-ESI m/z 188 [MH⁺]. Anal. (C₈H₇NO₂F₂) C, H, N.

5,7-Difluoroquinazolin-4(3H)-one (108). A mixture of 107 (82

g, 440 mmol) and formamidine acetate (137 g, 1320 mmol) in

2-methoxyethanol (1.5 L) was heated at reflux temperature for 10

h. The solvent was evaporated and the residue taken up in water.

The precipitate was filtered, washed with water and Et₂O, purified

by SiO₂chromatography, and eluted using a mixture of dichlo-

romethane/methanol (95:5) to give 61 g of 108 (76%): ¹H NMR

(DMSO-d₆) δ 7.3 (m, 2H), 8.12 (s,1H); MS-ESI m/z 183 [MH⁺].

Anal. (C₈H₄N₂OF₂) C, H, N.

7-Fluoro-5-morpholin-4-ylquinazolin-4(3H)-one (109). A mix-

ture of 108 (910 mg, 5 mmol) and morpholine (900 µL) in DMF

(20 mL) was heated at 100 °C for 1 h. The volatiles were removed

under vacuum, and the residue was made alkaline by addition of 6

N methanol/ammonia solution and again evaporated. The solid

formed was washed with water and diethyl ether to give 850 mg

of 109 (69%): ¹H NMR (DMSO-d₆) δ 2.95-3.05 (m, 4H), 3.7-

3.8 (m, 4H), 6.8 (dd, 1H), 6.9 (dd, 1H), 8.0 (s, 1H); MS-ESI m/z

250 [MH]⁺.

5-Morpholin-4-yl-7-(2-pyrrolidin-1-ylethoxy)quinazolin-4(3H)-

one (110). To an ice-cold solution of 3-pyrrolidin-1-ylpropan-1-ol

(0.7 mL, 6 mmol) in DMF (15 mL) was added under stirring NaH

(60% suspension in oil) (600 mg, 1.5 mmol). The mixture was

stirred at room temperature for 15 min, and 109 (750 mg, 3 mmol)

was added as a solid. The mixture was heated at 90 °C for 4 h.

The volatiles were removed under vacuum, after addition of acetic

acid (900 µL, 15 mmol)) the residue was dissolved in a mixture of

dichloromethane/methanol and filtered to remove impurities, and

the organic phase was purified by SiO₂chromatography using a

mixture of dichloromethane-6 N methanol/ammonia (95:5) as

eluent to give after evaporation 500 mg of 110 (50%): ¹H NMR

(DMSO-d₆) δ 1.6-1.7 (m, 4H), 2.8-2.9 (m, 2H), 2.95-3.05 (m,

4H), 3.7-3.8 (m, 4H), 4.2 (t, 2H), 6.45 (d, 1H), 6.7 (d, 1H), 8.0 (s,

1H), 11.6 (s, 1H); MS-ESI m/z 345 [MH]⁺.

5-Hydroxy-6-methoxyquinazolin-4(3H)-one (111). A suspen-

sion of 5-(benzyloxy)-6-methoxyquinazolin-4(3H)-one⁷⁸(5 g, 17.7

mmol) in a mixture of TFA (50 mL) and water (100 mL) was stirred

at room temperature for 30 min, and then the obtained solution

was concentrated off. The crude was taken up in water (100 mL),

and the pH was adjusted to 8 by adding solid sodium bicarbonate.

The formed precipitate was filtered off, washed with water, and

dried under vacuum at 50 °C in the presence of phosphorus

pentoxide, giving 3.6 g of 111 as a white solid (quantitative), which

was used in the next step without further purification: ¹H NMR

(DMSO-d₆) δ 3.85 (s, 3H), 7.13 (d, 1H), 7.53 (d, 1H), 7.99 (s,

1H), 11.89 (br s, 1H)), 12.2 (br s, 1H); MS-ESI m/z 193 [MH]⁺.

(5-Hydroxy-6-methoxy-4-oxoquinazolin-3(4H)-yl)methyl Piv-

alate (112). Solid 111 (3.6 g, 19 mmol) was slowly added to a

suspension of NaH (60% suspension in oil) (1.6 g, 40 mmol) in

DMF (36 mL) cooled at 0 °C and under a nitrogen atmosphere.

The mixture was then stirred at room temperature for 1 h and cooled

again in an ice bath before dropwise addition of chloromethyl

pivalate (4.1 mL, 28.5 mmol). After being stirred at room

temperature for 1.5 h, the reaction mixture was poured into a

solution of acetic acid (2 mL) in water (180 mL). The precipitate

was filtered off and taken up in dichloromethane; the solution was

dried (MgSO₄) and evaporated to give 4.6 g of 112 (79%): ¹H

NMR (CDCl₃) δ 1.2 (s, 9H), 3.97 (s, 3H), 5.90 (s, 2H), 7.21 (d,

1H), 7.36 (d, 1H), 8.06 (s, 1H), 11.49 (s, 1H); MS-ESI m/z 307

[MH]⁺.

6-Methoxy-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4(3H)-

one (113). To a mixture of 112 (3 g, 10 mmol), triphenylphosphine

(4.2 g, 16 mmol), and tetrahydro-2H-pyran-4-ol (1.2 mL, 13 mmol)

in dichloromethane (50 mL) cooled at 0 °C was added portionwise

DTAD (3.6 g, 16 mmol). The mixture was then stirred at room

temperature for 1 h and evaporated. The residue was taken up in a

7 N solution of ammonia in methanol and the resulting mixture

stirred at room temperature for 7 h. The mixture was concentrated

and purified by SiO₂chromatography using a mixture of ethyl

acetate/dichloromethane/methanol (50:48:2 up to 50:45:5) as eluent

to give 2.3 g of 113 (83%): ¹H NMR (DMSO-d₆) δ 1.6-1.8 (m,

2H), 1.8-2.0 (m, 2H), 3.5-3.6 (m, 2H), 3.85-4.0 (m, 5H), 4.29

(m, 1H), 7.41 (d, 1H), 7.59 (d, 1H), 7.88 (s, 1H); MS-ESI m/z 277

[MH]⁺.

6-Methoxy-5-[(1-methylpiperidin-4-yl)oxy]quinazolin-4(3H)-

one (114). To a mixture of 112 (1.55 g, 5 mmol), triphenylphos-

phine (2 g, 7.6 mmol), and 1-methylpiperidin-4-ol (757 mg, 6.6

mmol) in dichloromethane (15 mL) cooled at 10 °C was added

portionwise DTAD (1.75 g, 7.6 mmol). The mixture was then stirred

at room temperature for 1 h and then poured onto a SiO₂column.

Purification was performed using a mixture of ethyl acetate/

dichloromethane/methanol (50:50:0 up to 50:45:5) and then with a

7 N solution of ammonia in methanol/ethyl acetate/dichloromethane

(5:50:45) to give the intermediate [6-methoxy-5-[(1-methylpiperi-

din-4-yl)oxy]-4-oxoquinazolin-3(4H)-yl]methyl pivalate, which was

taken up in a 7 N solution of ammonia in methanol, and the resulting

mixture was stirred at room temperature over the weekend. The

mixture was concentrated and then triturated in diethyl ether. The

formed precipitate was filtered off to give 920 mg of 114 (63%):

¹H NMR (DMSO-d₆) δ 1.7-1.9 (m, 4H), 1.9-2.0 (m, 2H), 2.15

(s, 3H), 2.65-2.75 (m, 2H), 3.85 (s, 3H), 4.08 (m, 1H), 7.39 (d,

1H), 7.57 (d, 1H), 7.87 (s, 1H); MS-ESI m/z 290 [MH]⁺.

4-Chloro-6-methoxy-5-[(1-methylpiperidin-4-yl)oxy]quinazo-

line (115). A mixture of 112 (300 mg, 1 mmol), triphenylphosphine

(544 mg, 2 mmol), and carbon tetrachloride (300 µL, 3 mmol) in

1,2-dichloroethane (13 mL) was heated at 70 °C for 2.5 h. The

solution was allowed to cool to room temperature and then poured

onto a SiO₂column. Purification was performed using a mixture

of ethyl acetate/dichloromethane/methanol (50:50:0 up to 50:45:5)

as eluent and then with a 7 N solution of ammonia in methanol/

ethyl acetate/dichloromethane (5:50:45) to give 216 mg of 115

(68%): ¹H NMR (CDCl₃) δ 1.8-2.1 (m, 6H), 2.25 (s, 3H), 2.8-

2.95 (m, 2H), 3.97 (s, 3H), 4.38 (m, 1H), 7.67 (d, 1H), 7.81 (d,

1H), 8.81 (s, 1H); MS-ESI m/z 308 and 310 [MH]⁺.

6-Hydroxy-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4(3H)-

one (116). A mixture of 113 (1.9 g, 6.9 mmol), potassium carbonate

(1.4 g, 10 mmol), and benzenethiol (1 mL, 10 mmol) in NMP (20

mL) was heated at 200 °C for 30 min. The solvent was evaporated,

and the residue was dissolved in a mixture of dichloromethane (25

mL), methanol (1 mL), and acetic acid (2 mL) and then poured

onto a SiO₂column. The crude was purified using a mixture of

ethyl acetate/dichloromethane/methanol (50:48:2 up to 50:45:5) as

6484 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22

Hennequin et al.

eluent to give 1.65 g of 116 (91%): ¹H NMR (DMSO-d₆) δ 1.7-

1.9 (m, 4H), 3.2-3.4 (m, 2H), 3.85-3.95 (m, 2H), 4.30 (m, 1H),

7.29 (d, 1H), 7.35 (d, 1H), 7.83 (s, 1H), 9.2-9.6 (br s, 1H), 11.5-

12.0 (br s, 1H); MS-ESI m/z 263 [MH]⁺.

by the Abelson murine leukemia virus under the control of a T7

expression system (New England BioLabs, Inc., Ipswich, MA) was

diluted to 40 U/mL in enzyme dilution buffer (100 mM HEPES, 2

mM DTT, 0.2 mM sodium orthovanadate, 0.02% BSA). The

HEPES was discarded from the substrate plates, and the following

additions were made in order, 25 µL/well compound dilution (water

in the case of +ve and -ve controls), 25 µL/well of ATP/MgCl₂

or MgCl₂(-ve controls) alone, and finally 50 µL/well of Abl kinase

in dilution buffer to start the reaction. The final reaction concentra-

tions were 20 U/mL Abl kinase, 20 mM MgCl₂, and 3 µM ATP

(determined as the K_mfor ATP). The reaction time allowed was

22 min at room temperature on a plate shaker. The assay was

stopped by washing the plates four times with PBST (150 µL/well).

Detection of the resultant tyrosine phosphorylation was facilitated

by the addition of an anti-phosphotyrosine monoclonal antibody

conjugated to alkaline phosphatase (anti-pY/HRP, Santa Cruz

Biotechnology Inc., California); this was diluted 1:5000 in PBST/

B/O (PBST + 0.5% BSA + 0.1 mM sodium orthovanadate), added

at 100 µL/well, and incubated for 1 h. The plates were again washed

(7×). One tablet of the HRP substrate 3,3′,5,5′-tetramethylbenzidine

(TMB; Sigma-Aldrich) was dissolved in 100 µL of DMSO and

added per 10 mL of phosphate/citrate buffer with sodium perborate

(supplied as soluble capsules, Sigma-Aldrich). TMB substrate

solution (100 µL/well) was added. After 5 min of color development

the reaction was stopped by the addition of 50 µL/well of 0.8 M

H₂SO₄, and the positive control wells gave an A_{450 nm}of ca. 1.2-

1.5. Control and blank wells were included on all plates, containing

compound diluent and MgCl₂solution with and without ATP,

respectively, to determine the dynamic range of the assay. The

curves were plotted, and the IC₅₀values for compound enzyme

inhibition were interpolated using KC3 Kineticalc software (Bio-

Tek Instruments) following subtraction of the blank values.

(iii) In Vitro KDR Kinase Inhibition Test. This assay

determines the ability of test compounds to inhibit KDR kinase

activity. The method is as reported previously.⁴⁸

(iv) Other in Vitro Kinase Inhibition Test. Flt-4 tyrosine kinase

was obtained from ProQinase GmbH (Freiburg, Germany) and c-Kit

tyrosine kinase from Upstate Biotechnology, Inc. (Lake Placid, NY).

Each additional kinase used was generated as a cell lysate, following

infection of insect cells with recombinant baculoviruses containing

kinase domains. All enzyme assays were run at, or just below, the

respective K_mfor ATP (0.2-30 µmol/L). The inhibitory activity

of the compounds was determined against a range of recombinant

tyrosine kinases [CSK, c-Yes, LCK, Flt-1, Flt-4, c-Kit, PDGFR-R,

PDGFR-â, FGFR1, Abl, epidermal EGFR, and Aur-B] using ELISA

methodology described previously.⁴⁸Selectivity versus CDK2

serine/threonine kinase was examined using scintillation proximity

assays with a retinoblastoma substrate and [γ-³³P]ATP.^66bActivity

versus the dual-specificity kinase MAPK kinase (MEK) was

determined with a MAPK substrate, [γ-³³P]ATP, and paper capture/

scintillation counting.^66bMicrocal Origin software (v. 3.78, Microcal

Software, Inc., Northhampton, MA) was used to interpolate IC₅₀

values by nonlinear regression.

4-Oxo-5-(tetrahydro-2H-pyran-4-yloxy)-3,4-dihydroquinazo-

lin-6-yl Acetate (117). A mixture of 116 (700 mg, 2.7 mmol), acetic

anhydride (10 mL), and pyridine (700 µL) was heated at 100 °C

for 1 h. After evaporation the residue was taken up in a mixture of

methanol (9 mL) and water (9 mL) and the resulting mixture stirred

for 1 h at room temperature. The formed precipitate was filtered

off, and methanol evaporation gave a second crop after filtration.

Both precipitates were combined to give 540 mg of 117 (65%):

¹H NMR (DMSO-d₆) δ 1.55-1.75 (m, 2H), 1.8-1.9 (m, 2H), 2.31

(s, 3H), 3.2-3.4 (m, 2H), 3.75-3.85 (m, 2H), 4.25 (m, 1H), 7.41

(d, 1H), 7.57 (d, 1H), 8.01 (s, 1H), 11.9-12-5 (br s, 1H); MS-

ESI m/z 305 [MH]⁺.

4-Chloro-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-6-ol (118).

A mixture of 117 (540 mg, 1.8 mmol), triphenylphosphine (930

mg, 3.5 mmol), and carbon tetrachloride (515 µL, 5.3 mmol) in

1,2-dichloroethane (24 mL) was heated at 70 °C for 2.5 h. The

solvent was evaporated, and the residue was taken up in a 7 N

solution of ammonia in methanol (20 mL). The solution was stirred

at room temperature for 1 h, and then the solvent was evaporated

off. The crude was purified by SiO₂chromatography using a mixture

of ethyl acetate/dichloromethane (0:100 up to 50:50) as eluent to

give 1.12 g of a mixture of 118 and 2 equiv of triphenylphosphine

oxide (corrected 74%). 118 was used in the next step without further

purification: ¹H NMR (CDCl₃) δ 1.85-2.05 (m, 4H), 3.30-3.45

(m, 2H), 3.95-4.10 (m, 2H), 4.4-4.5 (m, 1H), 7.75 (d, 1H), 7.80

(d, 1H), 7.96 (s, 1H), 8.88 (s, 1H); MS-ESI m/z 281 and 283 [MH]⁺.

4-Chloro-6-[3-(4-methylpiperazin-1-yl)propoxy]-5-(tetrahydro-

2H-pyran-4-yloxy)quinazoline (119). To a mixture of impure 118

(1.12 g), triphenylphosphine (527 mg, 2 mmol), and 3-(4-meth-

ylpiperazin-1-yl)propan-1-ol (254 mg, 1.6 mmol) in dichlo-

romethane (10 mL) was added DTAD (462 mg, 2 mmol) at room

temperature. After being stirred for 30 min, the solution was poured

onto a SiO₂column. Chromatography was performed using a

mixture of ethyl acetate/dichloromethane/methanol (50:50:0 up to

50:45:5) as eluent and then with a 7 N solution of ammonia in

methanol/ethyl acetate/dichloromethane (5:50:45) to give 510 mg

of 119 containing 30 mol % 3-(4-methylpiperazin-1-yl)propan-1-

ol (corrected 81%). 119 was used in the next step without further

purification: ¹H NMR (CDCl₃) δ 1.8-2.2 (m, 6H), 2.31 (s, 3H),

2.4-2.7 (m, 10H), 3.3-3.4 (m, 2H), 4.0-4.1 (m, 2H), 4.24 (t, 2H),

4.6-4.7 (m, 1H), 7.74 (d, 1H), 7.85 (d, 1H), 8.86 (s, 1H); MS-ESI

m/z 421 and 423 [MH]⁺.

Biological Evaluation. IC₅₀values reported are means of at least

three to five measurements.

(i) In vitro Src Kinase Inhibition Test. This assay determines

the ability of test compounds to inhibit c-Src kinase activity. The

method is as reported previously.²⁷

(ii) In vitro Abl Kinase Inhibition Test. This assay determines

the ability of test compounds to inhibit Abl kinase activity. A poly-

(Glu, Ala, Tyr) 6:3:1 random copolymer (Sigma-Aldrich, Poole,

U.K.) was used as the tyrosine-containing substrate. It was stored

as a 2 mg/mL stock solution in PBS at -20 °C and diluted 1:1000

with PBS to coat 96-well plates (100 µL/well). The substrate was

plated the day before an assay, and the plates were covered with

adhesive seals and stored overnight at 4 °C. On the day of the assay

the substrate solution was discarded, and the plates were then

incubated with 120 µL/well of 5% BSA in PBS/A for 10 min. The

plates were then washed once with PBST (PBS containing 0.05%

v/v Tween 20) and then incubated with 50 mM HEPES (pH 7.4)

at 100 µL/well until the next stage. Test compounds were dissolved

in DMSO at 10 mM. A dilution series was then made in doubly

distilled H₂O to give solutions at 4 times the final required reaction

concentrations. Solutions of 12 µM ATP in 80 mM MgCl₂and 80

mM MgCl₂alone (for -ve controls) were prepared. Abl protein

tyrosine kinase (PTK) is a truncated form (45 kDa) of v-Abl PTK

and is identical to the normal c-Abl PTK. Abl isolated from a strain

of E. coli cells carrying the Abl kinase catalytic domain encoded

(v) In Vitro c-Src 3T3 Proliferation Assay. This assay

determines the ability of test compounds to inhibit the proliferation

in culture of mouse NIH3T3 fibroblast cells transfected to over-

express active c-Src kinase. The method is as reported previously.²⁷

(vi) In Vitro K562 Proliferation Assay. These assays determine

the ability of test compounds to inhibit the proliferation of cells in

culture. The cells used were K-562, a human chronic myelogenous

leukemia (CML) cell line, ATCC No. CCl-243. The K562 suspen-

sion cell line was maintained at between 10⁵and 10⁶cells/mL in

phenol red free RPMI 1640 medium (Invitrogen) supplemented with

1% L-glutamine, 10% fetal bovine serum (Sigma), and 50 mM

HEPES.

For assay the cells were plated into 96-well plates at 5000 cells/

90 µL of the indicated growth medium per well. One extra plate

designated the “predose plate” was also made at this time. After 2

h the cells were dosed with 10 µL/well of compound solution. (The

compounds were stored in stock concentrations of 10mM in 100%

DMSO.) The dilutions ensure that the final concentration of DMSO

AZD0530, a Dual-Specific c-SRC/Abl Kinase Inhibitor

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6485

in each assay well is 0.1% and that the final compound concentra-

tion in the assay wells ranges between 10 and 0.00 15µM. MTS

reagent (20 µL) (no. G1111/2, Promega Corp., Madison, WI) was

then added to the predose plate, and after a 2 h incubation the plate

was read (as indicated below). After a further 72 h 20 µL of MTS

reagent was added to all the remaining wells. After a 2 h incubation,

25 µL of 10% SDS was added to the wells. Then the plates were

read at A_{490 nm}. Only the inner 60 wells of the 96-well plates were

used in this assay to minimize edge effects. The outer wells were

filled with sterile PBS during the course of the assay to help prevent

any evaporation. The average absorbance of the predose plate was

subtracted from the later plate readings in determining the results.

(vii) In Vitro A549 Microdroplet Migration (Chemokinesis)

Assay. This assay determines the ability of test compounds to inhibit

the random motility (chemokinesis) of A549 cells (human epithelial

lung carcinoma cells, ATCC CCL 185). The method is as reported

previously.²⁷

(viii) Pharmacokinetics. Pharmacokinetics were determined in

mouse, rat, and dog following single intravenous or oral doses

(doses described in Tables 2 and 6) of the compound. For the iv

studies, the compounds were formulated in a mixture of 25% (w/

v) (hydroxypropyl)-â-cyclodextrin/Sorenson’s phosphate buffer (pH

5.5). For the oral studies, the compounds were formulated as a

solution in either 1% polysorbate or 0.1 M citrate buffer (pH 3).

(ix) Rat Xenografts. This assay determines the ability of the

test compound to inhibit the growth of c-Src-transfected 3T3 cells

implanted subcutaneously following once daily oral administration.

The method is as reported previously.²⁷

(x) BxPC-3 Orthotopic Model. Female athymic nude NMRI

ν/ν mice (Bioservices, The Netherlands) were orthotopically

inoculated with human pancreatic tumor cells (BxPC-3) at day 0.

The mice were treated by gavage (25 mg/kg of AZD0530 daily).

The first oral treatment was administered 1 h prior to inoculation

of the human pancreatic (BxPC-3) tumor cells and treatment

continued thereafter until sacrifice. The primary outcome for

AZD0530 activity was prolongation of survival, evaluated by two

separate means. First, the percentage T/C (survival) was calculated

by dividing the median day of sacrifice in the treated group “T”

by the median day of sacrifice in the control group “C” and

multiplying that number by 100. In this model, a T/C value >130%

indicates a significant prolongation of survival when compared with

the survival of the vehicle-treated control group. Second, the effect

of AZD0530 was evaluated by conducting a Kaplan-Meier analysis

with the cutoff level of significance (log-rank statistics) set at a p

value of <0.05.

Crystallography. Protein and crystals were prepared following

Xu et al.⁷⁹with minor modifications in crystallization. Crystals were

grown by sitting drop vapor diffusion at 15 °C, combining 1.5 µL

of reservoir solution (50 mM PIPES (pH 6.5), 10 mM DTT, 100

mM sodium chloride, and 4-9% PEG4000 (w/v)) with 1.5 µL of

protein-inhibitor solution (made by mixing 10 µL of 10 mg/mL

protein with 1.4 µL of 20 mM inhibitor dissolved in 10 mM PIPES

(pH 6.5) and 20% DMSO, adjusted so that pH > 4.0). Crystals

appeared within 48 h and were cryoprotected by being dipped into

50 mM PIPES (pH 6.5), 20% (w/v) PEG4000, 0.1 M sodium

chloride, 10 mM DTT, and 22.5% glycerol before being cooled to

100 K. The crystals belong to space group P2₁2₁2₁with unit cell

dimensions a ) 49.81 Å, b ) 72.47 Å, and c ) 171.57 Å, with

one molecule in the asymmetric unit. Diffraction data were recorded

at the ESRF (Grenoble), beamline ID14-4, on an ADSC Quantum4R

CCD detector. Data were integrated with the program MOSFLM.⁸³

CCP4 programs⁸⁴were used for subsequent data analysis. The

structure was determined by molecular replacement using the

published structure of c-Src.⁷⁹Model building used QUANTA2000

(Accelrys), and refinement was to R ) 20.5% (R_free) 27.6%). The

final statistics are given in Table 6. Thr523 and Glu524 were

modeled as Ala because of poor side chain density. The coordinates

are deposited in the Protein Data Bank with accession code 2H8H.

Table 6. Data Collection and Refinement Statistics

space group

P2₁2₁2₁

cell constants (a, b, c, Å)

resolution range (Å)

completeness (%)

no. of unique reflns

multiplicity

49.81, 72.47, 171.57

50.00-2.20

95.49

29365

3.4

8.3

20.4

R_merge^a(%)

R^b(%)

R_free^c(%)

27.2

rms deviation from ideal values

bond lengths (Å)

bond angles (deg)

average B value (Å²)

protein main chain atoms

all protein atoms

ligand

0.013

1.4

36.9

37.7

36.0

38.8

solvent

c

^aR_merge) ∑_hkl[(∑_i|I_i- I |)/∑_iI_i]. ^bR ) ∑_hkl||F_o| - |F_c||/∑_hkl|F_o|. R_free

is the cross-validatifon R factor computed for a test set of 5% of the unique

reflections.

Acknowledgment. We acknowledge the excellent technical

expertise of the following scientists: Vivien Jacobs, Karen

Malbon, Lindsey Millard, Robin Whittaker, Jacques Pelleter,

Patrice Koza, Alain Bertrandie, Dominique Boucherot, Myriam

Didelot, Francoise Magnien, Marie-Jeanne Pasquet, Michel

Vautier, Christian Delvare, John Swales, Tim Smith, Delphine

Dorison-Duval, Harj Bansal, Steve Brook, Peter McLachlan,

Wenqing Xu, and Richard Pauptit.

Solubility Measurements. The thermodynamic solubility of a

research compound is measured under standard conditions. A known

amount of compound is stirred in 0.1 M pH 7.4 phosphate buffer

at constant temperature (25 °C) for 24 h. The supernatant is then

separated from undissolved material by double centrifugation and

subsequently analyzed and quantified against a standard of known

concentration in DMSO using generic HPLC-UV methodology

coupled with mass spectral peak identification.

Molecular Modeling. To be consistent with our enzymatic assay,

which targets the activated form of c-Src kinase, initial modeling

and docking studies of the kinase domain were performed using

the crystal structure of activated Lck as a surrogate for Src.⁷⁴Indeed,

our in-house crystal structures of c-Src contain the SH2, SH3, and

kinase domains in an inactivated form, as do published structures

of c-Src. They differ from the closely related kinase Lck, which is

published as the isolated and activated kinase domain.⁷⁹Notable

structural differences have been observed in the size of the

hydrophobic pocket, which is deeper in the inactive form than in

the active one,³⁶although the shape of the sugar pocket is not

significantly different. These observations supported the choice of

the Lck structure as a model for activated c-Src. Our inhibitors

were built using Quanta, and the charges were assigned by the

Quanta charge template method.^80,81These inhibitors were docked

manually into the ATP binding site, and the most relevant solutions

were then energy minimized with the CHARMm force field to

relieve possible unfavorable contacts.⁸²

Supporting Information Available: Microanalysis data, ad-

ditional experimental details, and spectroscopic data for compounds

2, 6, 8-10, 13-16, 19, 21, 23, 25, 26, 29, 31, 50, 51, 58, 62-64,

66, 68, 77, 79, 83, 86, 91, 92, 96, 98, 101, and 104. This material

is available free of charge via the Internet at http://pubs.acs.org.

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JM060434Q

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N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy] -5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor

DOI: 10.1021/jm060434q

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