Discovery of 3-[2-(imidazo[1,2- b ]pyridazin-3-yl)ethynyl]-4-methyl- N -{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of breakpoint cluster region-abelson (BCR-ABL) kinase including the T315I gatekeeper mutant

J. Med. Chem. 2010, 53, 4701–4719 4701

DOI: 10.1021/jm100395q

Discovery of 3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-yl)-

methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a Potent, Orally Active Pan-Inhibitor

of Breakpoint Cluster Region-Abelson (BCR-ABL) Kinase Including the T315I Gatekeeper Mutant

Wei-Sheng Huang,* Chester A. Metcalf, Raji Sundaramoorthi, Yihan Wang, Dong Zou, R. Mathew Thomas,

Xiaotian Zhu, Lisi Cai, David Wen, Shuangying Liu, Jan Romero, Jiwei Qi, Ingrid Chen, Geetha Banda, Scott P. Lentini,

Sasmita Das, Qihong Xu, Jeff Keats, Frank Wang, Scott Wardwell, Yaoyu Ning, Joseph T. Snodgrass, Marc I. Broudy,

Karin Russian, Tianjun Zhou, Lois Commodore, Narayana I. Narasimhan, Qurish K. Mohemmad, John Iuliucci,

Victor M. Rivera, David C. Dalgarno, Tomi K. Sawyer, Tim Clackson, and William C. Shakespeare

ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, Massachusetts 02139

Received March 29, 2010

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gate-

keeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-

guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key

structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side

chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited

the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC₅₀s, and potently

inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g

significantly prolonged survival of mice injected intravenously with BCR-ABL^T315Iexpressing Ba/F3 cells.

These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment

for CML, including patients refractory to all currently approved therapies.

Introduction

and blastic phases, with the percentage being 40-50% and

80%, respectively.⁴Mutations in the kinase domain of BCR-

ABL that impede effective inhibitor binding are the primary

mechanism of acquired imatinib resistance;⁵to date, at least 100

different point mutations have been identified in CML patients

who are resistant to this drug.

To overcome resistance in CML, a number of second gen-

eration inhibitors have been developed.⁶These include imatinib-

like inhibitors nilotinib⁷and bafetinib,⁸and dual SRC/ABL

inhibitors dasatinib⁹and bosutinib.¹⁰Both dasatinib and nilo-

tinib have been approved for the treatment of adults in all phases

of CML with resistance or intolerance to imatinib whereas

bafetinib and bosutinib are in late-stage clinical testing. Despite

the effectiveness of these second-generation compounds to

inhibit most imatinib resistant BCR-ABL mutants, subsets of

mutants remain resistant. In particular, a common characteristic

of these agents is their inability to inhibit the BCR-ABL^T315I

mutant at the “gatekeeper” position which represents ∼15-

20% of all clinically observed mutants.^10b,11,12

To address this unmet need, several programs targeting deve-

lopment of potent BCR-ABL^T315Iinhibitors have been estab-

lished and a number of reviews have detailed progress in this

area.¹³Briefly, compounds with notable BCR-ABL^T315Iinhibi-

tory activity include (Figure 1): PPY-A,¹⁴TG101113,¹⁵and dual

ABL/Aurora inhibitors tozasertib (MK-0457, VX-680),¹⁶danu-

sertib (PHA-739358),¹⁷AT-9283,¹⁸and KW-2449.¹⁹The dual

ABL/Aurora agents have advanced to clinical trials in pa-

tients with treatment-resistant CML, in general via intrave-

nous administration. While tozasertib has demonstrated some

activity as salvage therapy,¹⁶clinical development has been

halted due to toxicity concerns. Despite this progress, there

are presently no approved pharmaceutical therapies for CML

patients harboring the BCR-ABL^T315Imutation.

Chronic myeloid leukemia (CML^a), a hematological stem-

cell disorder, is characterized by the occurrence of the Philadel-

phia (Ph) chromosome, a truncated version of chromosome 22

resulting from the reciprocal translocation between chromo-

somes 9 and 22. This single mutagenic event leads to the juxta-

position of two genes, whose product is the fusion protein

BCR-ABL, a constitutively active tyrosine kinase that drives the

pathogenesis of CML.¹Development of tyrosine kinase inhibi-

tors targeting the BCR-ABL oncogene constitutes an effective

approach to treating CML, as demonstrated by the clinical suc-

cess of imatinib, a potent first generation BCR-ABL inhibitor.²

In patients with newly diagnosed CML in the chronic phase,

daily oral administration of imatinib has demonstrated 89%

major cytogenetic response rates and 82% complete cyto-

genetic response rates. These response rates translated into

impressive event-free survival (81%), freedom from progres-

sion (93%), and overall survival (86%) rates.³Despite the clear

benefits of imatinib, many patients eventually develop intrinsic

or acquired resistance to this first-line therapy. Resistance rates

were reported to be 3-4% per year in newly diagnosed chronic

phase CML and much higher with CML patients in accelerated

*To whom correspondence should be addressed. Phone: 617-621-

2341. Fax: 617-494-8144. E-mail: wei-sheng.huang@ariad.com.

^aAbbreviations: CML, chronic myeloid leukemia; BCR-ABL, break-

point cluster region-abelson; ADME, absorption distribution metabolism

excretion; Src/Abl, sarcoma/Abelson; DFG, aspartic acid-phenylalanine-

glycine; AUC, area under curve; PK, pharmacokinetic; Phþ, Philadelphia

chromosome-positive; FLT3, FMS-like tyrosine kinase 3; FGFR, fibro-

blast growth factor receptor; VEGFR, vascular endothelial growth factor

receptor; c-Kit, cytokine receptor; PDGF, platelet-derived growth factor;

CDK2, cyclic dependent kinase 2; EGFR, epidermal growth factor recep-

tor; FAK, focal adhesion kinase; IGF1R, insulin-like growth factor

receptor; JAK2, janus kinase 2; WT, wild type.

r

2010 American Chemical Society

Published on Web 06/01/2010

pubs.acs.org/jmc

4702 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Huang et al.

Figure 1. BCR-ABL^T315Ikinase inhibitors with disclosed chemical structures.

Figure 2. Key medicinal chemistry optimization leading to clinical candidate 20g.

It is generally believed that next-generation ABL inhibitors

capable of exerting a high level of disease control in CML should

incorporate potent activity not only against BCR-ABL^T315Ibut

also the full range of BCR-ABL kinase domain mutations as

well as the native enzyme itself. Such a “pan-BCR-ABL” inhibi-

tor would have the potential to treat disease resistant to all

current therapies and potentially to suppress the emergence of

resistance when deployed at earlier disease stages. Recently, we

described the biological characterization of AP24534 (20g), a

potent, orally available multitargeted kinase inhibitor.²⁰20g

is potently active against BCR-ABL^T315Iand all other tested

BCR-ABL variants and suppresses emergence of resistant

mutations in a cell-based screen, thereby fulfilling the criteria

for a pan-BCR-ABL inhibitor. In this report, we describe the

medicinal chemistry and structural design strategy that led to the

discovery of this molecule. Key anticipated design aspects

included template morphing, exploration of alternate hydro-

phobic linkers, and considerable SAR around the pendant

DFG-out phenyl ring (Figure 2).

with tetrachloroethene to yield 9-(trichloroethenyl)purine 3.²²

Exhaustive dechlorination of 3 with n-butyllithium followed

by quenching with methanol generated terminal alkyne 4.²²

Subsequent Sonogashira coupling²³of 4 with iodobenzamide 5

furnished inhibitors 6, albeit in low yields.

The syntheses of inhibitors 12 bearing C-CꢀC-C structural

motifs were based on a tandem Sonogashira coupling strategy.

Initially, two general methods (A and B) were explored employ-

ing either the heteroaryl bromides 7 or the iodobenzamides 5 as

coupling partners in the first Sonogashira reaction (Scheme 2).

In employing method B, the final coupling reaction between

ethynylbenzamides 11 and 7 often afforded low yields of desired

products with dimerization of 11 being the major competing

reaction. Method A therefore was adopted as the standard

approach to 12. In the second step involving cleavage of the

trimethylsilyl group (from 8 to 9, or 10 to 11), deprotection with

either tetrabutylammonium fluoride or potassium carbonate/

methanol worked equally well.

Scheme 3 illustrates the synthesis of inhibitors 12a-e

compiled in Table 2. Starting material N-(imidazo[1,2-a]pyridin-8-

yl)acetamide 13 was prepared by condensing 2,3-diaminopyridine

and chloroacetaldehyde diethylacetal followed by acetamida-

tion, according to literature methods.²⁴Selective bromination

of 13 furnished 7b, which was hydrolyzed under acidic con-

ditions to yield bromide 7a. Direct Sonogashira reaction of

7a with trimethylsilylacetylene failed to afford the desired

coupling product but the N,N-bis-Boc protected derivative 7a⁰

Inhibitor Synthesis

The synthesis of 9-(ethenyl)purine based inhibitors 1a-e

(Table 1) has been reported previously.^21aThe preparation

of 9-(ethynyl)purine based inhibitors 6a and 6b is depicted

in Scheme 1. 9-(Cyclopropylamino)purine 2, prepared by

S_NAr displacement of 6-chloropurine with cyclopropyl-

amine, was deprotonated with sodium hydride then reacted

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4703

Table 1. Early Structure-Activity Relationships (IC₅₀in nM)

^aMeasured in K562 cell line.

Scheme 1. Synthesis of 9-(Ethynyl)purine Based Inhibitors^a

^aReagents and conditions: (a) tetrachloroethylene, NaH, HMPA, 60 °C;(b) n-BuLi, THF, -78 °C; (c) 5 mol%Pd(PPh₃)₄, 2.6mol%CuI, Et₃N, Tol, 55 °C.

Scheme 2. General Methods for the Synthesis of Inhibitors

4704 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Scheme 3. Synthesis of Inhibitors 12a-e^a

Huang et al.

^aReagents and conditions: (a) Br₂, EtOH, rt; (b) trimethylsilylacetylene, 5 mol % Pd(PPh₃)₂Cl₂, 7.5 mol % CuI, (i-Pr)₂NEt, DMF, 80 °C; (c) TBAF,

THF, rt; (d) conc. HCl, EtOH, reflux; (e) (Boc)₂O, cat. DMAP, THF, rt; (f) 5 mol % Pd(PPh₃)₄, 7.5 mol % CuI, (i-Pr)₂NEt, DMF, rt; (g) TFA, CH₂Cl₂,

rt; (h) aq Me₂NH, NaBH₄; (i) 3-bromo-5-trifluoromethylaniline, 15 mol % 8-hydroxyquinoline, 15 mol % CuI, K₂CO₃, DMSO, 120 °C; (j) 3-iodo-4-

methylbenzoyl chloride, (i-Pr)₂NEt, THF, rt.

underwent smooth coupling. Subsequent desilylation furnished

terminal alkyne 9a, which was coupled with iodobenzamide 5c to

yield 12a⁰. Subsequent removal of the Boc protecting groups

furnished inhibitor 12a. For the synthesis of 12d, 2-imidazole-

carboxaldehyde was first reacted with dimethylamine under

reductive amination conditions to furnish 14. Compound 14

was then converted into iodobenzamide 5d similarly to the

preparation of 5c via a published two-step procedure employ-

ing a copper-catalyzed N-arylation with 3-bromo-5-(trifluoro-

methyl)aniline followed by amide formation with 3-iodobenzoyl

chloride.²⁵Subsequent Sonogashira coupling of 5d with 9c

(structure shown in Scheme 2) furnished inhibitor 12d. Com-

pound 12e was obtained via the same route as 12d using

4-imidazolecarboxaldehyde and pyrrolidine as starting materi-

als. Inhibitors 12b and 12c were constructed via the standard

three-step protocol outlined in general method A from 9b and

5c, or 9c and 5c, respectively.

The synthesis of inhibitors 19a-j (Table 3) followed general

method A via Sonogashira coupling of 9c with the appropriate

iodobenzamides. The preparation of 19a illustrates the general

approach (Scheme 4). Thus, 4-nitro-2-(trifluoromethyl)benzyl

bromide, prepared from bromination of 4-nitro-2-(trifluoro-

methyl)toluene with N-bromosuccinimide, underwent S_N2

displacement with N-methylpiperazine yielding 16a. Reduction

of the nitro group followed by amide bond formation provided

the requisite iodide 18a. Sonogashira coupling of 18a with 9c

furnished 19a. Analogues 19d-f and 19h-j were constructed

via similar routes but with the required commercially available

secondary amines as the nucleophile in the initial substitution

reaction. Inhibitors 19b, 19c, and 19g were prepared similarly

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4705

Table 2. SAR Based on Imidazo[1,2-a]pyridine Template (IC₅₀in nM)

from the coupling of 9c with 18b, 18c, and 18g, respectively. The

requisite benzoic acid precursor²⁶to 18b, the substituted aniline

precursor²⁷to 18g, and the iodobenzamide 18c²⁸were all pre-

pared according to published procedures.

Results and Discussion

Compounds were evaluated for their kinase inhibitory acti-

vity against both native and mutant (T315I) ABL as previously

described.^21aTheir cellular activity was assessed using Ba/F3

cells transfected with native BCR-ABL or BCR-ABL^T315Iand

inhibition of parental, nontransfected Ba/F3 cells was used as

a control. Additionally, pharmacokinetic properties for active

compounds were evaluated early in rats to help guide series

selection.

In the synthesis of 19k-m, protection of the terminal piper-

azine nitrogens was necessary due to increased reactivity toward

aroyl chloride relative to the anilino NH in the amide formation

step; this is illustrated in Scheme 5. Iodobenzamide 18l was

prepared similarly to 18a using N-Boc-piperazine instead of

N-methylpiperazine as the starting nucleophile. Deprotection of

18l and subsequent alkylation with 1-bromo-2-fluoroethane

furnished 18k. In the preparation of 18m, a Boc group was

first introduced onto 16m, which was prepared via regio-

specific N-alkylation of the less sterically hindered NH of

2-methylpiperazine with 4-nitro-2-(trifluoromethyl)benzyl

bromide. Elaborated 16m⁰was then converted into iodide

18m in a manner similar to 18a. Sonagashira coupling of

Boc-protected iodides 18l or 18m with 9c and subsequent

deprotection furnished inhibitors 19l and 19m, respectively.

Synthesis of inhibitors 20a-p (Tables 4 and 5) followed the

Sonagashira coupling strategy generally outlined in method A.

All starting heteroaryl bromides were obtained from commercial

sources, and the corresponding iodobenzamides were prepared

from the requisite acids and anilines via chemistry discussed pre-

viously, with the exception of aniline precursor 17o (Scheme 6).

17o was synthesized via S_N2 substitution of 2-bromo-4-nitro-

benzyl bromide with N-methylpiperazine, followed by Suzuki

coupling of the corresponding bromide with cyclopropane-

boronic acid and subsequent reduction of the nitro group, as

shown in Scheme 6. The CH₂CH₂-linked analogue 24a (Table 6)

was accessed directly via hydrogenation of 20g and the trans-

CHdCH-linked inhibitor 24b (Table 6) was prepared via Heck

coupling of styrene 23 (Scheme 7). The latter was obtained from

a Stille coupling of 18a with tributyl(vinyl)tin as depicted in

Scheme 7.

Early Linker Exploration and Initial Template Morphing.

Recently, we described a novel series of potent, orally bioavail-

able ABL kinase inhibitors based on the 9-[aren(ethenyl)]purine

template which target the inactive, DFG-out conformation of

the ABL protein.²¹One subtype from this series, exemplified by

AP24163 (1a, Figure 2) and its des-methyl and des-cyclopropyl

analogues 1b and 1c (Table 1), demonstrated modest potency

against BCR-ABL^T315Iin both biochemical and cell-based

assays (IC₅₀s = 300-500 nM) in addition to potently inhibiting

native BCR-ABL.^21aIt is noteworthy that 1a-c inhibit

ABL^T315Ibecause the structurally similar nilotinib is completely

inactive against this mutant. When nilotinib is docked into the

model of ABL^T315Ikinase, there is an obvious steric clash bet-

ween the amino group on the pyrimidine ring and the larger

gatekeeper isoleucine.²⁹This steric repulsion, in combination

with a forfeited hydrogen bond between the amino group and

the hydroxyl side chain of threonine observed in the imatinib:

ABL cocrystal structure (PDB: 1IEP), severely impaired the

preferred ligand-protein interaction, impeding binding and

rendering both imatinib and nilotinib inactive toward the

ABL^T315Imutant.^29-32Such a clash is not observed in the model

of 1a when docked into the ATP-binding pocket of ABL^T315Idue

to the less sterically demanding vinyl linkage (Figure 4), which

may in part explain increased activity of 1a-c against ABL^T315I

The activity of the 9-[aren(ethenyl)]purine series toward

ABL^T315Istimulated further exploration of an alternate,

.

4706 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Table 3. B-Ring SAR around Inhibitor 19a (IC₅₀in nM)

Huang et al.

less sterically demanding two-carbon atom acetylenyl spacers

in the design of inhibitors against this mutant. Incorporation

of this rigid, rod-shaped linker should further reduce steric

repulsion with the bulk of the isoleucine side chain. As shown

in the ABL model (Figure 4), the corresponding 9-[(aren)-

ethynyl]purine analogue 1a⁰is predicted to bind to the protein

in a mode identical to 1a. All key ligand-protein interactions,

including hydrogen bonds, hydrophobic and van der Waals

(vdw) contacts are maintained and the acetylene linkage further

reduces steric clash with the larger Ile315 residue. This model

motivated us to prepare then test two matched sets of molecules

(1d vs 6a, 1e vs 6b, Table 1) against the ABL^T315Imutant.

Encouragingly, inhibitors bearing the acetylene linkage were

30-40-fold more potent in enzymatic assays and 5-10-fold

more potent in cell-based antiproliferation assays, relative to

their vinyl-linked analogues. Despite increased potency against

ABL^T315I, both 9-[(aren)ethynyl]purine compounds 6a and 6b

displayed poor pharmacokinetic profiles. When dosed orally in

which displayed higher AUC and C_maxlevels when evaluated

previously.^21aGiven the single structural modification between

6a and 1d (vinyl vs acetylenyl), we reasoned that it might be

very challenging to make orally active inhibitors based on

the 9-[(aren)ethynyl]purine template of 6a. Furthermore, in

contrast to the readily accessible 9-[(aren)ethenyl]purines via

Heck-type chemistry,³³preparation of the 9-[(aren)ethynyl]purine

core involved low yielding steps which made further SAR

exploration with this template impractical.²²

To overcome the liabilities associated with the 9-[(aren)-

ethynyl]purine-based inhibitors, we sought alternate ways to

exploit the potency gain exhibited by the acetylene linkage.

Instead of pursuing 1a⁰, the direct acetylene-linked analogue

of 1a (Figure 3), we targeted inhibitors in which the linker

connects the hinge binding template to the N-aryltoluamide

subunit via carbon atoms. Such C-CꢀC-C structural

motifs should be chemically and pharmacologically more

stable than the corresponding N-CꢀC-C moiety exhibited

by the ynamine-like 9-[(aren)ethynyl]purine series and are

easily accessed via well-known Sonogashira coupling reac-

tions.²³Thus, we implemented a template morphing strategy

rats, 6a exhibited low exposure (AUC_{0-24 h}/dose =11.8 ng h/

mL/mg/kg) and a short half-life (t_1/2=1.5 h). Note this oral

exposure is 2250-fold lower than 1d (compound 9i from ref 21a),

3

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4707

Scheme 4. Preparation of Inhibitor 19a and Chemical Structures of 18b, 18c, and 18g^a

^aReagents and conditions: (a) 1-methylpiperazine, Et₃N, CH₂Cl₂, rt; (b) Na₂S₂O₄, acetone/water, reflux; (c) 3-iodo-4-methylbenzoyl chloride,

(i-Pr)₂NEt, THF, rt; (d) 5 mol % Pd(PPh₃)₄, 7.5 mol % CuI, (i-Pr)₂NEt, DMF, rt.

Scheme 5. Synthesis of Inhibitors 19k-m^a

^aReagents and conditions: (a) TFA, CH₂Cl₂, rt; (b) 2-bromofluoroethane, NaHCO₃, DMF, 80 °C; (c) 5 mol % Pd(PPh₃)₄, 7.5 mol % CuI, (i-Pr)₂NEt,

DMF, rt; (d) (Boc)₂O, cat. DMAP, THF, rt; (e) (i) 10% Pd on carbon, 50 psi H₂, EtOH, rt, (ii) 3-iodo-4-methylbenzoyl chloride, (i-Pr)₂NEt, THF, rt.

initially targeting the 8-amino-imidazo[1,2-a]pyridine ring sys-

tem because 8-NH and N-1 of this template were predicted to

form paired hydrogen-bonds with the kinase hinge region. Rela-

tive to 1a-c, the prototype inhibitor 12a was 4-6-fold more

potent against ABL^T315Iin cells (IC₅₀=72 nM) while retaining

native ABL activity (Table 2), thus validating our template

morphing strategy and further highlighting the increased acti-

vity of the acetylene linkage toward ABL^T315I. Despite the

increased cellular potency, 12a exhibited low oral bioavailability

in rats (F = 5.5%) (Table 7). Likewise, the acetamide derivative

4708 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Table 4. Exploration of Alternate Hinge Binding Templates (IC₅₀in nM)

Huang et al.

^aAll data in this study were obtained from in-house assays. In a different enzymatic assay conducted by Reaction Biology Corporation,⁴³the IC₅₀s of

20g were determined to be 0.37 nM (ABL) and 2.0 nM (ABL^T315I), as previously reported.²⁰

12b retained potency but also exhibited undesirable rat PK.

Further attempts to cap the primary amine with the more stable

cyclopropyl group which had previously led to the orally

bioavailable compound 1a,^21aturned out to be unsuccessful.

At this point, we recognized the need to determine whether the

poor PK derived from C⁸substitution or the newly installed

Ar-CꢀC-Ar system itself. Previous experience with similar

series (e.g., 9-[(aren)ethynyl]purines) suggested that inhibi-

tors bearing free amino or acetamido moieties at the posi-

tion equivalent to C⁸of the current template often exhibited

undesirable PK. Thus, analogue 12c, devoid of any C⁸

substitution, was prepared and evaluated (Figure 3). Grati-

fyingly, this compound demonstrated good in vitro activity

(Table 2) with excellent oral exposure (AUC_{0-24 h}/dose =

5476 ng*h/mL/mg/kg) and good oral bioavailability (42%)

when tested in rats (Table 7). Furthermore, the cellular

potency of 12c and two other C⁸-unsubstituted analogues

12d-e correlated well with kinase activity, whereas inhibitors

12a and 12b exhibited larger disparities between both assays.

Therefore, on the basis of the overall improved proper-

ties exhibited by 12c, further SAR efforts focused on the

C⁸-unsubstituted 3-(arylacetylenyl)imidazo[1,2-a]pyridine che-

mical series.

In further optimization, we sought to increase molecular recog-

nition as a means to improve potency. To this end, inhibitor 19a

(Table 3), bearing a methylpiperazine moiety at C-4 on ring B,

was designed. The terminal piperazinyl nitrogen atom was

expected to be protonated at physiologic pH and the subsequent

quaternary salt predicted to form an H-bond with the carbonyl

oxygen atom of residue Ile360 in the activation loop of the

protein (Figure 5). Such ligand-protein interactions have been

previously observed in both imatinib:ABL (PDB: 1IEP) and

bafetinib:ABL (PDB: 2E2B) crystal structures;^34,35hence we

expected the increased molecular interaction would enhance

potency of the designed inhibitors against both native ABL and

ABL^T315I. Moreover, piperazine is a widely utilized solubilizing

group and its incorporation should improve cell permeability

and help reduce lipophilicity and protein binding. Relative to

12c, 19a demonstrated 2-fold increased activity against

the T315I mutated kinase and was approximately 20-fold

more potent at inhibiting the growth of Ba/F3 cells expres-

sing BCR-ABL^T315I(Table 3). Additionally, the presence

of 340 μM human serum albumin minimally shifted the

cellular potency of 19a against native ABL (4 vs 5.9 nM)

while a larger shift (8 to 51 nM) was observed with more

lipophilic 12c, suggesting that 19a might exhibit less protein

binding than 12c. Finally, 19a exhibited desirable pharma-

cokinetics when dosed orally in both rats and mice (Tables 7

and 8) and was subsequently identified as our first orally

active BCR-ABL^T315Iinhibitor in a pilot efficacy experiment

(vide infra).

Inhibitor Series Evolution. Despite a favorable PK profile,

12c exhibited lower cellular potency against ABL^T315I, re-

lative to 12a. Attempts to increase potency through incorpora-

tion of water-soluble amines onto the imidazole ring resulted

in only slightly increased cellular potency (12d-e, Table 2).

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4709

Table 5. SAR around Inhibitor 20g (IC₅₀in nM)

Table 6. The Impact of Linker Hybridization on Potency (IC₅₀in nM)

compd

a-b

ABL kinase

T315I kinase

ABL (Ba/F3)

T315I (Ba/F3)

parental (Ba/F3)

20g

24a

24b

CꢀC

8.6

19

54

40

1.2

4.3

7.9

8.8

1219

5998

3241

CH₂CH₂

trans-CHdCH

317

536

295

211

Scheme 6. Synthesis of Aniline 17o^a

^aReagents and conditions: (a) 1-methylpiperazine, Et₃N, CH₂Cl₂, rt; (b) cyclopropaneboronic acid, 10 mol % Pd(OAc)₂, 20 mol % P(c-hexyl)₃;

K₃PO₄, Tol/H₂O, reflux; (c) Fe/HOAc, MeOH, reflux.

Encouraged by the overall properties of 19a, further SAR

around this series was explored. The reverse amide 19b

demonstrated similar potency, essentially preserving all ele-

ments of molecular recognition. Regioisomer 19c, with the

methylpiperazine meta, exhibited both decreased ABL^T315I

kinase and cellular potency (5- and 10-fold, respectively) but

was almost equipotent against native ABL. In our docking

model, the terminal piperazinyl nitrogen in 19c could also

form an H-bond to the protein albeit with a different residue

(Asp400), suggesting that the differential ABL^T315Iactivity

observed between 19a and 19c may derive from increased van

der Waals interactions of the piperazinyl ring on 19a with

residues Val289, Phe359, and Asp381 in addition to the Ile360.

SAR from other analogues further support this contention. The

requirement of the piperazinyl H-bond was clearly demon-

strated by the 12-fold decreased enzyme activity of the morph-

line analogue 19d against ABL^T315I. To the contrary, while

the terminal nitrogens of R-3-(dimethylamino)pyrrolidinyl

analogue 19e, homopiperazinyl analogue 19f and O-linked

piperidinyl analogue 19g were also predicted to form H-bonds

with the protein; they all exhibited decreased potency relative to

19a, supporting the optimal hydrophobic contact exhibited by

the piperazinyl analogues. Attempts to further increase potency

through terminal methyl group modifications (19h-m) were

4710 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Huang et al.

Figure 3. Generation of new lead 12c based on multipoint variations of 1a.

Scheme 7. Preparation of Inhibitor 24b^a

^aReagents and conditions: (a) 3 equiv tributyl(vinyl)tin, 2.7 mol % Pd₂(dba)₃, 20 mol % P(2-furyl)₃, DMF, rt; (b) 8 mol % Pd(OAc)₂, 16 mol %

P(o-tol)₃, (i-Pr)₂NEt, DMF, 110 °C.

activity was reduced by 2-4-fold although two compounds

(20a and 20d) demonstrated slightly increased cellular potency.

Simultaneously, we explored additional 6/5 fused aromatic ring

systems and established a goal to reduce the lipophilicity of 19a

(clogP = 6.69)³⁶through nitrogen atom incorporation. Three

pyridine ring permutations with CH replaced by nitrogen were

explored (each reduced the clogP by approximately 1 log unit).

Inhibitor 20e, with N occupying the H-bond donor C-8 posi-

tion, markedly reduced potency. However, both 20f and 20g

demonstrated increased enzyme and cellular potency relative to

19a. Compound 20g exhibited more favorable preliminary PK

than 20f (Table 7) and so its SAR was further explored.

SAR around 20g. To further explore SAR around the

advanced lead compound 20g, we re-examined distal pipera-

Figure 4. Model of 1a (green) and its “virtual” acetylene-linked

analogue 1a⁰(cyan) bound to ABL^T315I

.

zine nitrogen substitution and found that the SAR trends

disclosed in Table 3 were largely preserved with this new

template (Table 5). For example, 20h and 20i, both with larger

N-substituents exhibited similar potency to 20g. Not surpris-

ingly, deletion of the entire methylpiperazinyl moiety (20p)

markedly reduced ABL^T315Iactivity, with a decrease in potency

of 25- and 130-fold in enzymatic and cellular assays, respec-

tively. Next, the potency impact of the A-ring “flag-methyl”³⁷

group was briefly investigated. Chlorine substitution (20l)

was essentially equipotent in inhibiting proliferation of

BCR-ABL^T315Iexpressing Ba/F3 cells, whereas hydrogen

or fluorine substitution (20k, 20m) resulted in 3-4-fold

potency loss. Last, B-ring trifluoromethyl replacement with

either chlorine (20n) or cyclopropyl (20o) groups signifi-

cantly reduced cellular potency against mutant T315I but

only marginally affected activity against native ABL.

unremarkable, with no compound offering any advantage rela-

tive to the parent inhibitor 19a. Within this series (Table 3),

potency against native ABL was insensitive to small inhibitor

structural modifications, with most compounds exhibiting

activity within a narrow range relative to ABL^T315Iinhibition

where much greater sensitivity to minor structural perturba-

tions was observed.

Earlier success (Tables 2 and 3) with template morphing

from purine to imidazo[1,2-a]pyridine encouraged us to next

explore alternate heterocycles as hinge region binders. One

approach focused on templates that could potentially form

paired H-bonds with the protein; previously we had shown that

an exocyclic NH at C-8 on the imidazo[1,2-a]pyridine core

significantly increased potency (12a vs 12c, Table 2). Because of

the poor PK exhibited by this analogue (vide supra) and

insights from other internal programs, we targeted inhibitors

possessing endocyclic NHs as H-bond donors. Several diverse

heterocyclic templates were selected for investigation (20a-d,

Table 4), however, these inhibitors did not exhibit the expected

potency improvement over 19a. In general, ABL^T315Ikinase

To confirm the absolute requirement of the carbon-carbon

triple bond for the ABL^T315Ipotency of 20g, we systematically

added hydrogen back across this linker. As shown in Table 6,

both the double bond linked and fully saturated molecules

(24a and 24b) significantly reduced ABL^T315Iactivity.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4711

Table 7. Mean Pharmacokinetic Parameters Following Intravenous (iv) or Oral (po) Dosing in CD Rats^a

iv^b

po^c

d

DN AUC_{0-24 h}

(ng h/mL/mg/kg)

compd

CL (L/h/kg)

V_d(L/kg)

t_1/2(h)

C_max(ng/mL)

t_max(h)

t_1/2(h)

F (%)

3

12a

12c

19a

20a

20b

20c

20f

20g

20h

20i

0.36

0.08

0.75

1.25

2.04

2.44

2.90

0.65

1.02

1.53

1.18

0.37

8.2

1.0

15.8

9.1

151.9

5745.9

386.3

119

BQL^e

BQL

57.0

234.3

2346.4

148.7

71

1.0

6.0

5.2

21.6

24.7

14.3

5.5

42.4

29.0

15

11.0

19.6

15.9

17.7

14.7

9.7

10.2

10.9

5.4

32.7

5.0

3.5

BQL

57.8

4.0

6.0

4.0

6.0

5.0

11.0

22.3

3.4

16.7

18.2

53.0

22.3

79.2

62.3

10.2

8.9

278.5

518.0

145.3

673.8

1707.5

204.8

214.8

164.1

302.1

1184.7

13.1

7.7

3.5

9.9

20l

20m

16.7

3.1

20.2

11.6

5.9

^an = 3 animals per study. ^bDosed at 5 mg/kg. ^cDosed at 15 mg/kg. ^dDN AUC = AUC/dose. ^eBQL = below quantitation limit.

Figure 5. Model of inhibitor 19a bound to ABL^T315I

.

Figure 7. In vivo efficacy of inhibitors in a Ba/F3 BCR-ABL^T315I

survival model at dose level of 10 mg/kg (error bars represent SD).

Table 8. Mouse PK Parameters Following Oral Dosing^a

Elsewhere, we have reported the three-dimensional struc-

ture of the ABL^T315I:20g complex (PDB: 3IK3).²⁰The cry-

stal structure confirmed that 20g binds to the DFG-

out conformation of the protein (Figure 6), and the key

molecular recognition components are consistent with those

predicted by modeling shown in Figures 4 and 5. The coordi-

nates from this crystal structure were used to refine the

docking model for alternate linker analogues 24a and 24b

(Table 6), and the inhibitor-protein interactions were com-

pared. All three analogues bind to the protein in a similar

fashion, and all three linkers can evade steric clash from the

bulky gatekeeper isoleucine. One explanation for their dif-

fering potencies is that the least sterically demanding acet-

ylene linker makes more favorable van der Waals’ contact

with gatekeeper Ile315 and Phe382 of the DFG motif (the

dose

(mg/kg)

AUC_{0-24 h}

(ng h/mL)

C_max

(ng/mL)

compd

t_1/2(h)

3

19a

20f

20i

30

10

5

11253

2694

13710

6075

4713

2086

995

985.9

521.4

1327.2

441.1

416.3

209.9

122.7

2.9

2.7

2.4

2.9

3.2

20l

20g

^an=3 animals per study. 20h and 20m were evaluated in an abbreviated

assay in which plasma concentrations at 2, 6, and 24 h were determined

and the following results were obtained: for 20h, these concentrations

were 622.5, 387.4, and 8.8 ng/mL, respectively, when dosed at 30 mg/kg;

for 20m, 2475.8, 2098.7, and 163.1 ng/mL. For comparison, 20g demon-

strated concentrations of 416.3, 298.9, and 4.2 ng/mL.

˚

triple bond is 3.4-3.6 A away from Ile315 and also almost

bisected by the aromatic ring of Phe382 in the crystal

structure). Another potential factor contributing to the high

ABL^T135Ipotency of 20g is that the rigidity of the acetylene

linkage promotes an extended conformation of the unbound

inhibitor, which favors binding to ABL^T315Iin a DFG-out

mode. The more flexible linkers in 24a and 24b could bring

about a folded conformation in the unbound inhibitors;

transition to an extended conformation would be entropi-

cally less favorable and hence reduce potency.

Taken together, the SAR studies on 20g show that, as

with the preceding chemical series, the gatekeeper T315I

mutant is a more “stringent” target than native ABL. Minor

structural modifications to either the acetylene linker,

Figure 6. X-ray structure of ABL^T315Iin complex with inhibitor

20g (green) and model of 24a (brown) and 24b (cyan).

4712 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Huang et al.

might suggest it has greater potential in treating CNS Phþ

leukemia that often complicates late-stage CML.

In Vivo Efficacy. Several inhibitors possessing desirable in

vitro kinase and cellular potency and with favorable pharmaco-

kinetic profiles were evaluated for their antitumor activity in an

aggressive mouse model of CML driven by the T315I mutation.

In this model, Ba/F3 cells expressing BCR-ABL^T315Iwere

injected into the tail vein of SCID mice. After 3 days, the animals

were dosed orally with inhibitors at 10 mg/kg, once daily for 19

consecutive days. The level of increased overall survival of

treated mice relative to untreated control was used to assess

efficacy. Median survival of untreated mice in this model was

14 days. Among all six inhibitors tested, 20g demonstrated the

greatest increased overall survival (Figure 7). At the higher dose

of 30 mg/kg/day, 20g more than doubled the survival of animals

in this highly aggressive model (Figure 8) and was well tolerated

for the duration of study with no signs of overt toxicity.

Kinase and Mutant Selectivity Profile of 20g. To assess the

kinase selectivity profile of 20g beyond ABL, broad panel

kinase screening was conducted using an enzymatic assay as

previously reported.²⁰In brief, 20g was found to be a multi-

targeted inhibitor with activity against FLT3, FGFR, and

VEGFR family kinases in the single digit nanomolar range.

Like imatinib, nilotinib, and dasatinib, 20g also inhibits

c-Kit and PDGFRR/β. Many of these kinases are important

clinical targets in a variety of other malignancies and support

the potential testing of 20g more broadly against a range of

cancers. To the contrary, 20g does not inhibit Aurora kinases

and demonstrated over 500-fold selectivity relative to a num-

ber of kinases including CDK2/cyclin E, EGFR, FAK,

IGF1R, JAK2, and c-Met. The relatively broad kinase speci-

ficity profile of 20g can likely be attributed to the linear

ethynyl linkage, which permits binding to kinases with

hydrophobic residues at the gatekeeper position.

Figure 8. In vivo efficacy of 20g in a Ba/F3 BCR-ABL^T315Isurvival

model.

the methylpiperazinyl moiety, or the trifluoromethyl group

significantly affected ABL^T315Ipotency while modifica-

tions or even deletion of one of these binding elements

still yielded potent native ABL inhibitors. We conclude

that incorporation of multiple points of protein contact led

to the high potency of 20g against the refractory ABL

mutant T315I.

Pharmacokinetics. During this program, potent inhibitors

were continually evaluated for their pharmacokinetic (PK)

properties in rats immediately following assessment of their in

vitro activity (Table 7). In general, inhibitors bearing imida-

zole as the B ring appendage demonstrated higher dose

normalized AUC and C_maxthan those piperazine-containing

compounds (12c vs 19a). 12c had a much smaller volume of

distribution relative to 19a, consistent with a highly protein

bound species, which was confirmed by a potency shift in vitro

in the presence of human serum albumin (vide supra). The

exocyclic NH₂at C-8 on the imidazo[1,2-a]pyridine template

drastically reduced both AUC and C_maxof imidazole-bearing

inhibitors to the level of piperazine-containing compounds

(12c vs 12a and 19a).

Among those piperazine-bearing inhibitors possessing

alternate hinge binding templates, 20b and 20c exhibited

low oral absorption in rats. For the remaining inhibitors,

their dose normalized oral exposure in rats were ranked as

follows: 19a>20g>20a . 20f, and C_max20g>19a . 20a ≈

20f. Both 20g and 19a exhibited long half-lives and reduced

clearance relative to 20f. The latter exhibited the highest

clearance and hence lowest AUC. Five inhibitors (20g, 20h,

20i, 20l, 20m) bearing the same template, imidazo[1,2-b]-

pyridazine in general demonstrated good PK.

Elsewhere, we have reported the cellular activity of 20g

against a broad panel of clinically relevant BCR-ABL mutants,

confirming its pan-BCR-ABL activity.²⁰Briefly, 20g was active

against all tested mutants including, but not limited to, T315A,

F317L/V, Y253H, E255K/V, and F359V, mutations which

have been reported in patients failing on either dasatinib or

nilotinib.^11,12,40Additionally, in an accelerated mutagenesis

screen for resistance, 20g completely suppressed the outgrowth

of all mutants at a concentration of 40 nM. Together, these

results are consistent with the profile of a true pan-BCR-ABL

inhibitor, active against the native enzyme and all clinically

relevant mutants including T315I.

Mouse PK data for these inhibitors were collected as a

prerequisite to evaluating potency in in vivo efficacy models

(Table 8). In general, mouse PK exhibited similar trends to those

observed in rats. For 20g, PK parameters were determined orally

at different doses; good dose proportionality was observed.

The concentration of 20g in the brain was also measured to

evaluate its ability to penetrate the blood-brain barrier (BBB).

In a mouse PK study following a single oral 30 mg/kg dose, a

brain/plasma concentration ratio of 1.60 was determined at 6 h

postdosing, indicating high levels of BBB penetration. It should

be noted that among the three approved agents for CML

treatment, only dasatinib crosses the blood-brain barrier, with

human cerebrospinal fluid/plasma ratios ranging from 0.05 to

0.28.³⁸Among the inhibitors in clinical trials, bafetinib demon-

strated 10-fold lower brain levels compared to plasma in mice.

Despite the low levels, this concentration is sufficient for

bafetinib to inhibit the growth of Phþ leukemic cells in the

murine CNS.³⁹The higher level of BBB penetration for 20g

Conclusion

Starting from 1a, a lead compound that emerged from

our program developing DFG-out targeted ABL kinase inhi-

bitors based on the 9-[(aren)ethenyl]purine template, exten-

sive medicinal chemistry coupled with detailed structural

investigations has led to a novel series of kinase inhibitors

that potently inhibit native ABL and a broad panel of

clinically relevant mutants including T315I at the gatekeeper

position (Figure 2). Crystallographic studies confirmed that

this series binds to the inactive, “DFG-out” conformation of

the protein. One unusual structural feature of these potent

inhibitors is the inflexible acetylene linkage connecting

the heterocycle core serving as the hinge binder and the

N-arylbenzamide occupying the hydrophobic selectivity

pocket.⁴¹This rigid, less sterically demanding linker enables

binding to the enlarged Ile315 side chain of the T315I mutant

and correctly directs both inhibitor segments into well-defined

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4713

binding pockets making more productive interactions with

the protein. Another key structural element required for

achieving high potency is the hybrid N-arylbenzamide, which

allows cooperative molecular exploitation of both trifluoro-

methyl and piperazinylgroupstomaximize van der Waalsand

hydrogen-bonding interactions with the protein. In general,

the piperazine tail confers excellent cellular potency and

improved aqueous solubility with reduced plasma protein

binding, leading to favorable pharmacokinetic properties

following oral dosing in rats and mice. Inhibitor 20g exhibited

low nM cellular potency against cells lines expressing either

native BCR-ABL or BCR-ABL^T315Iand significantly pro-

longed survival in an aggressive mouse model of CML driven

by the T315I mutant. 20g also potently inhibited proliferation

of all clinically relevant ABL mutants in cell-based assays

characterizing it as the first pan-BCR-ABL inhibitor. On the

basis of these data, and its favorable overall ADME profile,

20g was nominated as a development candidate and is cur-

rently undergoing phase I clinical evaluation in patients with

refractory CML and other hematologic malignancies.⁴²

stream of argon for 30 min and then heated at 55 °C for 3 h. The

crude was chromatographed over silica gel eluting with hexanes-

EtOAc (2:8) to furnish a pale-yellow solid, which was fur-

ther washed with cold methanol to afford pure product

1

(0.029 g, 6% yield). H NMR (CDCl₃): δ 0.50 (m, 2H), 0.62

(m, 2H), 2.31 (s, 3H), 2.80 (s, 1H), 5.90 (s, 1H), 7.25 (m, 1H), 7.40

(d, J = 8.0 Hz, 1H), 7.70 (d J = 2 Hz, 1H), 7.80 (s, 1H), 8.05

(d, J = 1.9 Hz, 1H), 8.40 (d, J = 5.1 Hz, 1H), 8.50 (s, 1H), 8.61

(s, 1H), 8.65 (s, 1H). MS: m/z 479.1 [M þ H]^þ.

3-{2-[6-(Cyclopropylamino)purin-9-yl]ethynyl}-N-[5-(1,1-di-

methylethyl)isoxazol-3-yl]-4-methylbenzamide (6b). This was

prepared analogously to 6a using 5b^21aand 4. ¹H NMR

(CDCl₃): 0.80 δ (m, 2H), 1.00 (m, 2H), 1.70 (s, 9H), 2.61 (s, 3H),

3.10 (s, 1H), 6.50 (s, 1H), 7.05 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H),

8.00 (d, J = 2.0 Hz, 1H), 8.11 (s, 1H), 8.25 (d, J = 1.8 Hz, 1H),

8.70 (s, 1H), 9.25 (s, 1H). MS: m/z 457.0 [M þ H]^þ.

3-Bromoimidazo[1,2-a]pyridin-8-ylamine (7a). To a solution

of 7b (10.24 g, 40.30 mmol) in EtOH (500 mL) was added

concentrared aq HCl (51 mL). The suspension was refluxed

for 2 h. Upon cooling, the reaction mixture was basified under

stirring with 10 N NaOH until the pH reached ∼9. EtOH was

removed on a rotavap and the resulting suspension was parti-

tioned in CH₂Cl₂and H₂O. The organic layer was dried and

concentrated, giving the desried product as a brownish solid

Experimental Section

1

(7.66 g, 90%). H NMR (DMSO-d₆): δ 5.79 (br, 2H), 6.36 (d,

J = 7.4 Hz, 1H), 6.84 (dd, J = 6.9, 7.2 Hz, 1H), 7.57 (s, 1H), 7.62

(d, J = 7.4 Hz, 1H).

General Chemistry. All reagents and solvents were used as

received. ¹H NMR spectra were recorded on a Bruker ARX300

or a Bruker AVANCE400 spectrometer using TMS as internal

standard. MS spectra were recorded on a Waters Micromass ZQ

spectrometer. Elemental analyses were performed by Robertson

Microlit Laboratories in Madison, NJ. HPLC was performed

on an Agilent 1100 HPLC system. The purity of all SAR

compounds was determined to be g95% by reverse phase

HPLC (C-18 column, MeCN/H₂O with 0.1% CF₃CO₂H as

the mobile phase).

N-(3-Bromoimidazo[1,2-a]pyridin-8-yl)acetamide (7b). Bro-

mine was added dropwise to a solution of 8-acetamidoimidazo-

[1,2-a]pyridine²⁴(8.0 g, 45.7 mmol) in EtOH at 0 °C. The

solution was stirred at rt for 3 h, at which point HPLC indicated

complete conversion. After the volatile components were re-

moved on a rotavap, the residue was taken up in CH₂Cl₂and

then washed with saturated aq NaHSO₃and then 1N NaOH

until the pH reached 7. The organic layer was passed through

celite, dried over Na₂SO₄, and concentrated on a rotavap, giving

essentially pure material by NMR and HPLC. ¹H NMR

(DMSO-d₆): δ 2.22 (s, 3H), 7.06 (dd, J=7.2, 7.3 Hz, 1H), 7.74

(s, 1H), 8.05-8.08 (m, 2H), 10.06 (s, 1H).

N-(3-Bromoimidazo[1,2-a]pyridin-8-yl)imidodicarbonic Acid,

C,C⁰-Bis(1,1-dimethylethyl)ester (7a⁰). To a solution of 7a (5.75

g, 27.12 mmol) in THF (120 mL) was added Boc₂O (3 equiv) and

DMAP (0.1 equiv). After the mixture was stirred at rt for 18 h,

the solvent was removed and the residue was suspended in

EtOAc. Filtration gave a brownish solid, which was determined

by NMR to be pure product. The filtrate was concentrated

and purified by CombiFlash chromatography (30% EtOAc in

hexanes), giving the second crop of product. The combined yield

was 78%. ¹H NMR (DMSO-d₆): δ 1.38 (s, 18H), 7.15 (dd,

J = 7.0, 7.3 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.82 (s, 1H), 8.39

(d, J = 7.3 Hz, 1H).

General Procedure for the Preparation of Arylacetylenes 9:

3-ethynylimidazo[1,2-a]pyridine (9c). To a solution of 7c (5 g,

25.4 mmol) in MeCN (50 mL) was added Pd(PPh₃)₂Cl₂(0.445 g,

0.634 mmol), CuI (0.17 g, 0.89 mmol), dicyclohexylamine

(5.6 mL, 28 mmol), and ethynyltrimethylsilane (7.2 mL, 51 mmol).

The solution was purged with argon for 15 min, sealed, and

heated at 80 °C for 3 h, at which point HPLC indicated disap-

pearance of starting bromide. The solvents were concentrated and

to the residue was added H₂O and CH₂Cl₂(25 mL each). The

organic layer was separated, and the aqueous layer was repeatedly

extracted with CH₂Cl₂(3ꢁ20 mL). The combined extracts were

dried (Na₂SO₄) and concentrated (R_f= 0.47 in 50% hexanes in

EtOAc). The resulting residue was dissolved in THF (100 mL) and

treated with tetrabutyl ammonium fluoride monohydrate (8.3 g,

32 mmol) in H₂O (5 mL), and the mixture was stirred at rt for 2 h.

The solvents were concentrated, and the resulting residue was

partitioned between H₂O (25 mL) and CH₂Cl₂(150 mL). The

aqueous layer was extracted with CH₂Cl₂(2 ꢁ 30 mL). The

combined extracts were dried over Na₂SO₄and concentrated on

Cyclopropyl-(9-trichlorovinyl-9H-purin-6-yl)amine (3). A so-

lution of 6-(cyclopropylamino)-9H-purine (3 g, 17.1 mmol) in

anhydrous HMPA (156 mL) was added to NaH (1.5 g, 60%

dispersion in oil) under stirring until no effervescence was

observed. Tetrachloroethylene was then added dropwise, and

the mixture was heated at 60 °C overnight. Excess HMPA was

distilled off, and the resultant mixture was quenched with

methanol. The crude was extracted with EtOAc, washed with

water, and then dried over Na₂SO_4.The solvent was removed,

and the resultant residue was chromatographed over silica gel

eluting with hexanes-EtOAc to furnish the desired product

1

(0.65 g, 12% yield). H NMR (CDCl₃): δ 0.60 (m, 2H), 0.81

(m, 2H), 3.10 (m, 1H), 6.60 (br, 1H), 7.80 (s, 1H), 8.50 (s, 1H).

Cyclopropyl-(9-ethynyl-9H-purin-6-yl)amine (4). To the solu-

tion of 3 (0.5 g, 1.65 mmol) in THF (17 mL) was added n-BuLi

(2.6 mL, 6.56 mmol) at -78 °C. The reaction mixture was stirred

at this temperature for one additional hour and then carefully

quenched with methanol. The solvent was stripped off, and the

resultant residue was partitioned between EtOAc and water. After

drying and concentration, the crude was chromatographed over

silica gel eluting with hexanes-EtOAc (6:4) to furnish the desired

product (0.1 g, 30% yield). ¹H NMR (CDCl₃): δ 0.50 (m, 2H),

0.72 (m, 2H), 2.80 (m, 1H), 3.10 (s, 1H), 5.90 (s,1H), 7.80 (s, 1H),

8.30 (s, 1H). MS: m/z 200.0 [M þ H]^þ.

N-{3-[2-[(Dimethylamino)methyl]-1H-imidazol-1-yl]-5-(trifluo-

romethyl)phenyl}-3-iodo-4-methylbenzamide(5d).This was prepared

analogously to 5c via a published procedure²⁵using 14⁴⁴instead of

1

imidazole. H NMR (CDCl₃): δ 2.21 (s, 9H), 3.30 (s, 2H), 7.10

(s, 2H), 7.55 (m, 4H), 7.65 (s, 2H). MS: m/ztalic> 529.0 [M þ H]^þ.

3-{2-[6-(Cyclopropylamino)purin-9-yl]ethynyl}-4-methyl-N-

[4-(trifluoromethyl)-pyridin-2-yl]-benzamide (6a). Iodobenza-

mide 5a^21a(0.44 g, 1.1 mmol) and alkyne 4 (0.2 g, 1.0 mmol)

was dissolved in a mixture of toluene and triethylamine (10 mL,

1:2). To this was added Pd(PPh₃)₄(0.062 g, 0.05 mmol) and CuI

(0.0051 g, 0.026 mmol). The resulting solution was degassed by a

4714 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Huang et al.

a rotavap. The resulting residue was purified by combiflash on silica

gel using hexanes-EtOAc. The desired product was eluted with

50% hexanes in EtOAc and isolated as an off-white solid in 84%

yield. ¹H NMR (DMSO-d₆): δ 5.08 (s, 1H), 7.13 (ddd, J = 0.9, 6.8,

6.9 Hz, 1H), 7.42 (ddd, J=1.2,6.8,6.9Hz,1H),7.71(d,J=6.9Hz,

1H), 7.96 (s, 1H), 8.46 (d, J =6.8Hz, 1H). MS: m/z 200 [M þ H]^þ.

N-(3-Ethynylimidazo[1,2-a]pyridin-8-yl)imidodicarbonic Acid,

C,C⁰-Bis(1,1-dimethylethyl)ester (9a). This was made from 7a

in CCl₄(40 mL) was refluxed under N₂for 16 h, at which point

HPLC indicated ca. 50% conversion. More NBS (10 mmol) and

AIBN (0.6 mmol) was added, and the mixture was refluxed for

another 14 h. HPLC indicated ca. 80% conversion. The reaction

mixture was cooled down, and the solid was filtered off and washed

with EtOAc. The combined filtrate was washed with aq NaHCO₃,

dried over Na₂SO₄, filtered, concentrated on rotavap, and further

dried under vacuum. ¹H NMR indicated the ratio of desired

product 2-(bromomethyl)-5-nitrobenzotrifluoride to unreacted

2-methyl-5-nitrobenzotrifluoride was 75:25, based on the integra-

tion of CH₂Br (4.68 ppm in CDCl₃) and CH₃(2.63 ppm). This

material was not purified but used directly in the next step.

To a solution of crude 2-(bromomethyl)-5-nitrobenzotri-

fluoride (13.33 mmol, 75% pure) in CH₂Cl₂(10 mL) was added

Et₃N (1.4 mL, 10 mmol) and 1-methylpiperazine (1.1 mL, 10 mmol).

After stirring for 3 h at rt, aq NaHCO₃was added, and the

mixture was extracted with CH₂Cl₂. The combined organic layer

was dried over Na₂SO₄, filtered, concentrated, and the resulting

residue was purified by silica gel chromatography (eluent: 10%

MeOH in CH₂Cl₂), giving the product as a pale-yellow oil (72%,

1

via bis-Boc protection. H NMR (DMSO-d₆): δ 1.34 (s, 18H),

5.11 (s, 1H), 7.13 (dd, J = 6.8, 7.3 Hz, 1H), 7.39 (dd, J = 0.9, 7.3

Hz, 1H), 7.95 (s, 1H), 8.46 (dd, J = 0.9, 6.8 Hz, 1H).

N-(3-Ethynylimidazo[1,2-a]pyridin-8-yl)acetamide (9b). ¹H NMR

(DMSO-d₆): δ 2.23 (s, 3H), 5.08 (s, 1H), 7.06 (dd, J = 6.9, 7.4 Hz,

1H), 7.94 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 8.16 (dd, J = 0.9, 6.7 Hz,

1H), 10.10 (s, 1H).

3-Ethynylimidazo[1,2-b]pyridazine (9d). ¹H NMR (DMSO-

d₆): δ 4.93 (s, 1H), 7.35 (dd, J = 4.4, 9.2 Hz, 1H), 8.11 (s, 1H),

8.21 (dd, J = 1.6, 9.2 Hz, 1H), 8.65 (dd, J = 1.6, 4.4 Hz, 1H).

General Procedure for the Preparation of Inhibitors 12: 3-[2-(8-

Aminoimidazo[1,2-a]pyridin-3-yl)ethynyl]-N-[3-(1H-imidazol-1-

yl)-5-(trifluoromethyl)phenyl]-4-methylbenzamide (12a). Alkyne

9a (130 mol %), iodobenzamide 5c (0.2 mmol), Pd[(PPh₃)₄]

(5 mol %), and CuI (7.5 mmol %) was placed in a vial with rubber

septum. The mixture underwent 3 cycles of vacuum/filling with N₂.

DMF (1.5 mL) and N,N-diisopropylethylamine (150 mol %) was

then added. The mixture was stirred at rt for 16 h and then

quenched with H₂O. EtOAc and more water were added for

extraction. The combined organic layer was dried (over Na₂SO₄),

filtered, concentrated, and the resulting residue was purified by

silica gel chromatography (eluent: 5% MeOH in CH₂Cl₂), giving

the 12a⁰as an off-white solid. Standard deprotection to cleave both

Boc groups with TFA furnished 12a. ¹H NMR (DMSO-d₆): δ 2.74

(s,3H),5.85(br,2H),6.60(d,J= 7.4 Hz, 1H), 7.06 (dd, J= 7.0, 7.1

Hz, 1H), 7.42 (s, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.86-8.10 (m, 5H),

8.38 (s, 1H), 8.40 (d, J = 1.3 Hz, 1H), 8.52 (s, 1H), 8.65 (s, 1H),

10.92 (s, 1H). MS: m/z 500.9 [M þ H]^þ.

1

2.21 g). H NMR (CDCl₃): δ 2.46 (s, 3H), 2.66 (m, 8H), 3.84

(s, 2H), 8.10 (d, J = 8.4 Hz, 1H), 8.32 (dd, J = 2.3, 8.4 Hz, 1H),

8.58 (d, J = 2.3 Hz, 1H).

4-[(4-Methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)aniline

(17a). A suspension of 16a (1.23 g, 4 mmol) and sodium

hydrosulfite (7.0 g, 85% pure from Aldrich, 40 mmol) in acetone

and water (1:1, 20 mL) was refluxed for 3 h. Upon cooling, the

volatile components (mainly acetone) were removed on rotavap,

and the resulting mixturewas subjected tofiltration. The solid was

thoroughly washed with EtOAc. The combined filtrate was

extracted with n-BuOH (4ꢁ), and the combined organic layer

was washed with saturated aq NaHCO₃, dried (Na₂SO₄), filtered,

concentrated, and the resulting residue was purified by silica gel

chromatography (eluent: 5% MeOH in CH₂Cl₂, MeOH was

presaturated with ammonia gas), giving the product as a pale-

yellow solid (65%, 0.71 g). ¹H NMR: (CDCl₃): δ 2.25 (s, 3H), 2.44

(m, 8H), 3.46 (s, 2H), 3.84 (br, 2H), 6.75 (dd, J = 2.3, 8.3 Hz, 1H),

6.88 (d, J = 2.4 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H).

3-{2-[8-(Acetylamino)imidazo-[1,2-a]pyridin-3-yl]ethynyl}-N-[3-

(1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-4-methylbenza-

mide (12b). This was made from coupling of 9b and 5c. ¹H NMR

(DMSO-d₆): δ 2.10 (s, 3H), 2.70 (s, 3H), 7.22 (dd, J = 6.6, 7.4

Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.71-8.47 (m, 11H), 10.22

(s, 1H), 10.83 (s, 1H). MS: m/z 542.9 [M þ H]^þ.

3-Iodo-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(tri-

fluoromethyl)phenyl}-benzamide (18a). 3-Iodo-4-methylbenzoyl

chloride (0.48 g, 1.7 mmol), prepared from the reaction of 3-iodo-4-

methylbenzoic acid and SOCl₂, was added to a solution of 17a

(0.47 g, 1.7 mmol), N,N-diisopropylethylamine (0.26 g, 2.0 mmol),

and a catalytic amount of DMAP in THF (10 mL). After stirring at

rt for 2 h, the reaction was quenched with water. EtOAc was added

and the layers separated. The combined organic layers were con-

centrated to dryness and purified by silica gel chromatography

(eluent: 5% MeOH in CH₂Cl₂, MeOH was presaturated with

ammonia gas), giving the desired product as an off-white solid

(57%, 0.51 g). ¹H NMR: (acetone-d₆): δ 2.07 (s, 3H), 2.26 (m, 8H),

2.37 (s, 3H), 3.50 (s, 2H), 7.36 (d, J= 7.9 Hz, 1H), 7.67 (d, J=8.5Hz,

1H), 7.84 (dd, J = 1.8, 8.5 Hz, 1H), 7.92 (dd, J = 1.9, 8.5 Hz, 1H),

8.11 (d, J = 2.2 Hz, 1H), 8.33 (d, J = 1.8 Hz, 1H), 9.66 (s, 1H).

N-(3-Iodo-4-methylphenyl)-4-[(4-methyl-1-piperazinyl)methyl]-3-

(trifluoromethyl)-benzamide (18b). N-(4-(4-Methylpiperazin-

1-yl)methyl)-3- (trifluoromethyl benzoic acid²⁶(1.0 g, 2.67 mmol)

and 3-iodo-4-methyl aniline (0.68 g, 2.90 mmol) were dissolved in

DMF (14 mL). To this, HATU (1.5 g, 4.00 mmol) and DIPEA

(1.5 mL) were added and the mixture was stirred overnight. The

solvent was removed under vacuum, and the crude residue was

chromatographed over silica gel eluting initially with EtOAc and

then with CH₂Cl₂-MeOH (95:5), giving the desired product

N-[3-(1H-Imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[2-(imi-

dazo-[1,2-a]pyridin-3-yl)ethynyl]-4-methylbenzamide (12c). This

was made from coupling of 9c and 5c. ¹H NMR (DMSO-d₆): δ

2.64 (s, 3H), 7.21 (dd, J = 6.6, 6.7 Hz, 1H), 7.68 (dd, J = 7.0, 7.1

Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.72-7.88 (m, 3H), 7.97 (dd,

J = 1.8, 8.0 Hz, 1H), 8.10 (s, 1H), 8.24 (s, 1H), 8.31 (dd, J = 1.7,

8.0 Hz, 1H), 8.36 (overlapped singlet, 2H), 8.64 (d, J = 6.6 Hz,

1H), 10.76 (s, 1H). MS: m/z 486.0 [M þ H]^þ.

N-{3-[2-[(Dimethylamino)methyl]-1H-imidazol-1-yl]-5-(triflu-

oromethyl)phenyl}-3-[2-(imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-me-

thylbenzamide (12d). This was made from coupling of 9c and 5d

in 59% yield. ¹H NMR (CDCl₃): δ 2.41 (s, 6H), 2.50 (s, 3H), 3.75

(s, 2H), 6.90 (m, 1H), 7.00(s, 1H), 7.11 (s, 1H), 7.30 (d, J = 6.1

Hz, 2H), 7.60 (m, 1H), 7.71 (s, 1H), 7.83 (m, 1H), 8.11 (s, 2H),

8.33(m, 3H), 9.60 (s, 1H). MS: m/z 543.9 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-{3-[4-(1-pyr-

rolidinylmethyl)-1H-imidazol-1-yl]-5-(trifluoromethyl)phenyl}-

benzamide (12e). This was made analogously to 12d using pyrro-

lidine instead of dimethylamine in the first step of the reaction

sequence depicted in Scheme 3. ¹H NMR (DMSO-d₆): δ 2.20 (m,

4H), 2.51 (s, 3H), 3.55 (m, 6H), 7.40(m, 1H), 7.70(m, 1H), 7.83 (d,

J= 8.2 Hz, 1H), 8.0 (d, J= 8.5 Hz, 1H), 8.11 (s, 1H), 8.20 (m, 2H),

8.30 (s, 1H), 8.44(s, H), 8.50 (d, J = 1.6 Hz, 1H), 8.74 (m, 2H), 8.90

(d, J = 6.6 Hz, 1H), 11.1 (s, 1H). MS: m/z 568.9 [M þ H]^þ.

1-Methyl-4-{[4-nitro-2-(trifluoromethyl)phenyl]methyl}pipe-

razine (16a). A suspension of 2-methyl-5-nitrobenzotrifluoride

(3.90 g, 19 mmol), N-bromosuccinimide (NBS, 3.56 g, 20 mmol),

and 2,2⁰-azobis(2-methylpropionitrile) (AIBN, 94 mg, 0.6 mmol)

1

(0.98 g, 70%). H NMR (CDCl₃): δ 2.20 (s, 3H), 2.21 (s, 3H),

2.50 (m, 8H), 3.76 (s, 2H), 7.15 (d, J = 8.2 Hz, 1H), 7.50 (m, 1H)

7.73 (br, 1H), 7.90(s, 2H), 8.00 (s, 2H). MS: m/z 518.0 [M þ H]^þ.

3-Iodo-4-methyl-N-[4-(1-methylpiperidin-4-yloxy)-3-(trifluoro-

methyl)phenyl]benz-amide (18g). This was made from the reac-

tion of 3-iodo-4-methylbenzoyl chloride with 4-(1-methylpiperi-

din-4-yloxy)-3-(trifluoromethyl)aniline,²⁷as described in the

synthesis of 18a. MS: m/z 518.9 [M þ H]^þ.

Article

N-{4-[(4-(2-Fluoroethyl)piperazin-1-yl)methyl]-3-(trifluoro-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4715

1H), 7.72 (d, J = 9.4 Hz, 1H), 7.75 (d, J = 9.1 Hz, 1H), 7.94 (dd,

J = 1.7, 8.0 Hz, 1H), 8.08 (s, 1H), 8.09 (d, J = 9.1 Hz, 1H), 8.23

(d, J = 1.9 Hz, 1H), 8.27 (d, J = 1.6 Hz, 1H), 8.63 (d, J = 6.7 Hz,

1H), 10.55 (s, 1H). MS: m/z 532.1 [M þ H]^þ.

methyl)phenyl}-3-iodo-4-methylbenzamide (18k). Iodobenzamide

18l was prepared analogously to 18a using N-Boc-piperazine

instead of N-methylpiperazine in the first step of the reaction

sequence depicted in Scheme 4. 18l (0.5 g) was then treated with

TFA (10 mL) for 3 h. Excess solvent was removed, and the

resultant salt was dissolved in DMF (4 mL). 2-Bromofluoroethane

(0.15 mL) and NaHCO₃(0.4 g) was then added, and the resulting

mixture was heated at 80 °C for overnight. Solvent was evapo-

rated; the residue was taken up in EtOAc and washed with water.

The combined layer was dried over Na₂SO₄and concentra-

ted under vacuum. The residue was then flash chromatographed

over silica gel eluting with CH₂Cl₂-MeOH (95:5) to furnish the

desired product (0.25 g). ¹H NMR (CDCl₃): δ 2.41 (m, 4H), 2.51

(s, 3H), 2.50 (m, 6H), 3.82 (s, 2H), 4.50 (m, 1H), 4.69 (m, 1H), 7.25

(m, 1H), 7.80 (m, 5H), 8.20 (s, 1H). MS: m/z 518.9 [M þ H]^þ.

N-{3-Cyclopropyl-4-[(4-methylpiperazin-1-yl)methyl]phenyl}-

3-iodo-4-methyl-benzamide (18o). To a solution of 2-bromo-4-

nitrobenzyl bromide (1.56 g, 3.4 mmol) in CH₂Cl₂(10 mL) was

added Et₃N (0.48 mL, 3.4 mmol) and N-methylpiperazine (0.38 mL,

3.4 mmol) at 0 °C. The reaction mixture was stirred for 3 h at

rt before aq NaHCO₃was added. The resulting solution was

extracted with CH₂Cl₂. The combined organic layer was dried

over Na₂SO₄, filtered, concentrated, and the resulting residue

was purified by silica gel chromatography (eluent: 10% MeOH

in CH₂Cl₂), giving the product 21as a pale-yellow oil (75%, 0.80

g). ¹H NMR (DMSO-d₆): δ 2.16 (s, 3H), 2.34 (m, 4H), 2.46 (m,

4H), 3.62 (s, 2H), 7.75 (d, J = 8.5 Hz, 1H), 8.23 (dd, J = 2.3, 8.5

Hz, 1H), 8.39 (d, J = 2.3 Hz, 1H).

N-{3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methylphenyl}-

4-[(4-methyl-1-piperazinyl)methyl]-3-(trifluoromethyl)benzamide

(19b). This was made from the coupling of 9c and 18b in 20% yield.

¹H NMR (DMSO-d₆): δ 2.10 (s, 3H), 2.11 (s, 3H), 2.50 (m, 8H),

3.76 (s, 2H), 7.01(m, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.30 (m, 1H),

7.52 (m, 2H), 7.75(d, J = 8.2 Hz, 1H), 7.90 (s, 2H), 8.10 (m, 2H),

8.35 (d, J = 6.6 Hz, 1H), 10.20 (s, 1H). MS: m/z 532.4 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-{3-[(4-me-

thylpiperazin-1-yl)methyl]-5-(trifluoromethyl)phenyl}benzamide

(19c). This was made from the coupling of 9c and 18c²⁸in 68%

yield; characterized as HCl salt. ¹H NMR (D₂O): δ 2.35 (s, 3H),

2.41 (s, 3H), 2.64 (m, 8H), 3.71 (s, 2H), 7.30 (m, 1H), 7.52 (m,

3H), 7.90 (d, J = 9.0 Hz, 1H), 8.05 (dd, J = 1.8 Hz, 1H), 8.20

(m, 2H), 8.32 (s, 1H), 8.51(d, J = 1.6 Hz, 1H), 8.80 (d, J = 6.7

Hz, 1H), 10.60 (s, 1H). MS: m/z 532.0 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-[4-(4-mor-

pholinylmethyl)-3-(trifluoromethyl)phenyl]benzamide (19d). This

was made analogously to 19a using morpholine instead of

N-methylpiperazine in the first step S_N2 displacement of the

reaction sequence depicted in Scheme 4. ¹H NMR (acetone-d₆):

δ 2.46 (t, J = 4.5 Hz, 4H), 2.65 (s, 3H), 3.65 (m, 4H), 7.17 (t, J =

6.8 Hz, 1H), 7.46 (dd, J = 7.3, 7.7 Hz, 1H), 7.52 (d, J = 8.1 Hz,

1H), 7.72 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.97 (dd,

J = 1.7, 8.0 Hz, 1H), 8.10 (s, 1H), 8.11 (d, J = 8.1 Hz, 1H), 8.24

(d, J = 1.5 Hz, 1H), 8.29 (s, 1H), 8.67 (d, J = 4.3 Hz, 1H), 9.83

(s, 1H). MS: m/z 518.8 [M þ H]^þ.

A solution of 21 (0.943 g, 3.0 mmol), cyclopropaneboronic

acid (0.773 g, 9.0 mmol), K₃PO₄(2.87 g, 13.5 mmol), Pd(OAc)₂

(67 mg, 0.3 mmol), and tricyclohexylphosphine (0.168 g,

0.6 mmol) in toluene (15 mL) and water (3 mL) was degassed

with N₂and then heated at reflux overnight. Extraction followed

by silica gel column chromatography (eluent: 5% NH₃presatu-

rated MeOH in CH₂Cl₂) gave the desired product 22 (0.797 g,

96%). ¹H NMR (CDCl₃): δ 0.77 (m, 2H), 1.05 (m, 1H), 2.18 (m,

1H), 2.28 (s, 3H), 2.45 (m, 4H), 2.52 (m, 4H), 3.71 (s, 2H), 7.53

(d, J = 8.4 Hz, 1H), 7.33 (d, J = 2.4 Hz, 1H), 7.98 (dd, J = 2.3,

8.4 Hz, 1H).

N-{4-[[(3S)-3-(Dimethylamino)-1-pyrrolidinyl]methyl]-3-(tri-

fluoromethyl)phenyl}-3-[2-(imidazo[1,2-a]pyridin-3-yl)ethynyl]-

1

4-methylbenzamide (19e). H NMR (DMSO-d₆): δ 1.63 (m, 1H),

1.86 (m, 1H), 2.09 (s, 6H), 2.33 (m, 2H), 2.55 (m, 2H), 2.57 (s, 3H),

2.79 (m, 1H), 3.61 (s, 2H), 7.12 (t, J = 6.8 Hz, 1H), 7.40 (dd, J = 7.3,

7.7 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 9.1 Hz, 1H), 7.72

(d, J = 9.4 Hz, 1H), 7.89 (dd, J = 1.7, 8.0 Hz, 1H), 7.96 (d, J =

9.1 Hz, 1H), 7.99 (s, 1H), 8.11 (d, J = 1.9 Hz, 1H), 8.19 (d, J =

1.6 Hz, 1H), 8.58 (d, J = 6.7 Hz, 1H), 10.49 (s, 1H). MS: m/z 546.1

[M þ H]^þ.

Substituted ntrobenzene 22 was completely converted into

corresponding aniline 17o after heating in MeOH at reflux for 3 h

in the presence of reducing agent Fe/HOAc. The solvent was

removed and the residue was partitioned between aq NaHCO₃

and EtOAc. Extraction, combination of organic layer, drying over

Na₂SO₄, and concentration afforded essentially pure material

(by HPLC). This was reacted with 3-iodo-4-methylbenzoyl chlo-

ride to furnish desired amide product 18o in a similar fashion to

N-{4-[(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)methyl]-

3-(trifluoromethyl)-phenyl}-3-[2-(imidazo[1,2-a]pyridin-3-yl)ethy-

nyl]-4-methylbenzamide (19f). ¹H NMR (DMSO-d₆): δ 1.80 (m,

2H), 2.27 (s, 3H), 2.63 (s, 3H), 2.64 (m, 8H), 3.74 (s, 2H), 7.20 (t,

J = 6.8 Hz, 1H), 7.47 (dd, J = 7.3, 7.7 Hz, 1H), 7.57 (d, J = 8.2

Hz, 1H), 7.75 (d, J = 9.1 Hz, 1H), 7.80 (d, J = 9.4 Hz, 1H), 7.96

(dd, J = 1.7, 8.0 Hz, 1H), 8.03 (d, J = 9.1 Hz, 1H), 8.06 (s, 1H),

8.18 (d, J = 1.9 Hz, 1H), 8.27 (d, J = 1.6 Hz, 1H), 8.66 (d, J =

6.7 Hz, 1H), 10.56 (s, 1H). MS: m/z 546.1 [M þ H]^þ.

1

the preparation of 18a. H NMR (DMSO-d₆): δ 0.60 (m, 2H),

0.94 (m, 2H), 2.20 (m, 1H), 2.48 (s, 3H), 2.50 (m, 8H), 3.65 (s,

2H), 7.18 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 7.48 (d, J = 8.4 Hz,

1H), 7.65 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 8.40

(s, 1H), 10.12 (s, 1H).

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-{4-[(1-

methyl-4-piperidinyl)oxy]-3-(trifluoromethyl)phenyl}benzamide

(19g). ¹H NMR (CD₃OD): δ 1.93 (m, 2H), 2.01 (m, 2H), 2.33 (s,

3H), 2.50 (m, 2H), 2.64 (s, 3H), 2.70 (m, 2H), 4.65 (m, 1H), 7.20

(d, J = 8.2 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 8.0 Hz,

1H), 7.52 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.88 (dd,

J = 2.0, 8.1 Hz, 2H), 7.93 (s, 1H), 8.00 (d, J = 2.5 Hz, 1H), 8.18

(d, J = 1.9 Hz, 1H), 8.56 (d, J = 6.8 Hz, 1H).

General Procedure for the Preparation of Inhibitors 19 and 20:

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-{4-[(4-me-

thylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide

(19a). Alkyne 9c (37 mg, 0.26 mmol), iodobenzamide 18a

(103.4 mg, 0.2 mmol), Pd[(PPh₃)₄] (11.6 mg, 5 mol %), and

CuI (2.9 mg, 7.5 mmol%) was placed in a vial with a rubber

septum. After the mixture underwent 3 cycles of vacuum/filling

with N₂, DMF (1.5 mL) and N,N-diisopropylethylamine (53 μL,

0.3 mmol) was added. The mixture was then stirred at rt for 16 h.

Water was added to quench the reaction, and EtOAc was added

for extraction. The combined organic layer was dried (Na₂SO₄),

filtered, concentrated, and the resulting residue was purified by

silica gel chromatography (eluent: 5% MeOH in CH₂Cl₂,

MeOH was presaturated with ammonia gas), giving the titled

compound as an off-white solid (53%, 56 mg). ¹H NMR

(DMSO-d₆): δ 2.19 (s, 3H), 2.50 (m, 8H), 2.62 (s, 3H), 3.61 (s,

2H), 7.20 (t, J = 6.8 Hz, 1H), 7.46 (m, 1H), 7.56 (d, J = 8.2 Hz,

N-{4-[(4-Ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phe-

nyl}-3-[2-(imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methylbenzamide

(19h). ¹H NMR (DMSO-d₆): 1.10 (br, 3H), 2.42 (s, 3H), 2.60 (m,

4H), 2.71 (m, 6H), 3.81 (s, 2H), 7.20 (m, 1H), 7.54 (m, 1H), 7.64

(d, J = 8.0 Hz, 1H), 7.72 (m, 2H), 7.91 (d, J = 8.0 Hz, 1H), 8.15

(m, 1H), 8.22 (m, 1H), 8.31 (m, 2H), 8.60 (d, J = 6.6 Hz, 1H),

10.6 (s, 1H). MS: m/z 546.0 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-{4-[[4-(1-me-

thyl)ethylperazin-1-yl]methyl]-3-(trifluoromethyl)phenyl}benza-

1

mide (19i). H NMR (DMSO-d₆): δ 1.01 (d, J = 6.8 Hz, 6H),

2.39 (m, 8H), 2.65 (s, 3H), 2.98 (m, 1H), 3.60 (s, 2H), 7.19 (t, J =

6.8 Hz, 1H), 7.47 (dd, J = 7.3, 7.7 Hz, 1H), 7.56 (d, J = 8.2 Hz,

1H), 7.74 (d, J = 9.1 Hz, 1H), 7.80 (d, J = 9.4 Hz, 1H), 7.95

4716 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12

Huang et al.

(dd, J = 1.7, 8.0 Hz, 1H), 8.02 (d, J = 9.1 Hz, 1H), 8.05 (s, 1H),

8.18 (d, J = 1.9 Hz, 1H), 8.27 (d, J = 1.6 Hz, 1H), 8.65 (d, J =

6.7 Hz, 1H), 10.55 (s, 1H). MS: m/z 560.2 [M þ H]^þ.

4-Methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoro-

methyl)phenyl}-3-[2-(3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxa-

zin-7-yl)ethynyl]benzamide (20d). ¹H NMR (DMSO-d₆): δ 2.16

(s, 3H), 2.34 (m, 4H), 2.39 (m, 4H), 2.55 (s, 3H), 3.57 (s, 2H), 4.70

(d, J = 6.3 Hz, 2H), 7.51 (d, J = 8.2 Hz, 1H), 7.56 (d, J = 1.8 Hz,

1H), 7.71 (d, J = 8.5 Hz, 1H), 7.92 (dd, J = 1.7, 8.0 Hz, 1H), 8.06

(dd, J = 1.7, 8.5 Hz, 1H), 8.15 (d, J = 1.6 Hz, 1H), 8.16 (d, J = 1.9

Hz, 1H), 8.20 (d, J = 2.0 Hz, 1H), 10.52 (s, 1H), 11.51 (s, 1H). MS:

m/z 564.1 [M þ H]^þ.

N-{4-[(4-(2-Hydroxyethyl)piperazin-1-yl)methyl]-3-(trifluoro-

methyl)phenyl}-3-[2-(imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-me-

thylbenzamide (19j). ¹H NMR (MeOH-d₄): δ 2.41 (m, 6H), 2.55

(s, 3H), 2.80 (m, 4H), 3.62 (m, 2H), 3.83 (s, 2H), 7.20 (m, 1H),

7.41 (m, 2H), 7.65 (d, J = 9.1 Hz, 1H) 7.77 (d, J = 9.1 Hz, 1H),

7.81 (m, 3H), 8.05 (d, J = 1.9 Hz, 1H), 8.11 (d, J = 1.9 Hz, 1H),

8.55 (d, J = 6.7 Hz, 1H). MS: m/z 562.2 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyrimidin-3-yl)ethynyl]-4-methyl-N-{4-

[(4-Methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}ben-

zamide (20e). ¹H NMR (DMSO-d₆): δ 2.16 (s, 3H), 2.34 (m, 4H),

2.40 (m, 4H), 2.62 (s, 3H), 3.57 (s, 2H), 7.31 (dd, J = 4.2, 6.8 Hz,

1H), 7.57 (d, J =8.2 Hz, 1H), 7.72 (d, J=8.6 Hz, 1H), 7.95 (dd, J=

1.9, 8.0 Hz, 1H), 8.07 (dd, J=2.0, 8.4 Hz, 1H), 8.22 (d, J=2.0 Hz,

1H), 8.23 (s, 1H), 8.28 (d, J=1.8 Hz, 1H), 8.67 (dd, J=2.0, 4.1

Hz, 1H), 9.06 (dd, J=2.0, 6.8 Hz, 1H), 10.54 (s, 1H). MS: m/z

533.2 [M þ H]^þ.

N-{4-[(4-(2-Fluoroethyl)piperazin-1-yl)methyl]-3-(trifluoro-

methyl)phenyl}-3-[2-(imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-me-

thylbenzamide (19k). This was made from coupling of 9c and 18k

in 81% yield. ¹H NMR (DMSO-d₆): δ 2.31 (m, 4H), 2.41 (s, 3H),

2.50 (m, 6H), 3.72 (s, 2H), 4.53 (m, 1H), 4.70 (m, 1H), 7.30 (m,

1H), 7.55 (m, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.81 (m, 2H), 8.10 (d,

J = 8.1 Hz, 1H), 8.22 (m, 2H), 8.31 (s, 1H), 8.35 (s, 1H), 8.70 (d,

J = 6.6 Hz, 1H), 10.3 (s, 1H). MS: m/z 564.2 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-[4-(1-pipe-

razinylmethyl)-3-(trifluoromethyl)phenyl]benzamide (19l). This

was made from coupling of 9c and 18l followed by standard

de-Boc with TFA. ¹H NMR (DMSO-d₆): δ 2.33 (m, 4H), 2.62 (s,

3H), 2.72 (t, J = 4.5 Hz, 4H), 3.54 (s, 2H), 7.20 (t, J = 6.7 Hz, 1H),

7.47 (dd, J = 6.9, 8.9 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.73 (d,

J = 3.9 Hz, 1H), 7.76 (d, J = 4.6 Hz, 1H), 7.94 (d, J= 7.9 Hz, 1H),

8.07 (d, J= 5.1 Hz, 1H), 8.08 (s, 1H), 8.22 (s, 1H), 8.27 (s, 1H), 8.63

(d, J = 6.6 Hz, 1H), 10.54 (s, 1H). MS: m/z 518.1 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyridin-3-yl)ethynyl]-4-methyl-N-{4-[(3-

methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benza-

mide (19m). Substituted N-benzylpiperazine 16m was pre-

pared analogously to 16a using 2-methylpiperazine instead of

N-methylpiperazine as starting material. Coventional Boc pro-

tection (Boc₂O, DMAP) of 16m gave 16m⁰in 69% yield. This

was converted to 18m similarly to the synthesis of 18a from

16a except that the reduction of NO₂to NH₂was done via

hydrogenation on Pd/C in 66% yield. The coupling of 9c and

18m followed by standard de-Boc with TFA furnished desired

product, which was purified by reverse phase prep-HPLC

(MeCN/H₂O, 0.1% TFA) as TFA salt. ¹H NMR (DMSO-d₆):

δ 1.19 (d, J = 6.9 Hz, 3H), 2.15 (t, J = 11.2 Hz, 1H), 2.34 (t,

J = 11.3 Hz, 1H), 2.62 (s, 3H), 2.84 (dd, J = 10.9, 11.7 Hz, 2H),

3.02 (d, J = 9.2 Hz, 1H), 3.28 (d, J = 10.4 Hz, 2H), 3.66 (s, 2H),

7.20 (dd, J = 6.5, 6.7 Hz, 1H), 7.48 (dd, J = 6.9, 8.1 Hz, 1H), 7.56

(d, J = 8.1 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 8.1 Hz,

1H), 7.94 (d, J=7.9Hz, 1H), 8.10(s, 1H), 8.12(d, J= 8.6 Hz, 1H),

8.25 (d, J = 9.1 Hz, 2H), 8.50 (br, 1H), 8.63 (d, J = 6.4 Hz, 1H),

8.93 (br, 1H), 10.59 (s, 1H). MS: m/z 532.2 [M þ H]^þ.

3-[2-(Imidazo[1,2-a]pyrazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-

methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benza-

mide (20f). ¹H NMR (DMSO-d₆): δ 2.10 (m, 4H), 2.17 (s, 3H),

2.40 (m, 7H), 3.71 (s, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.65 (d,

J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz,

1H), 8.10 (s, 2H), 8.25 (d, J = 8.0 Hz, 1H), 8.31 (s, 1H), 8.55 (d,

J = 4.4 Hz, 1H), 9.10 (s, 1H), 10.50 (s, 1H). MS: m/z 533.1

[M þ H]^þ.

3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-

[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}ben-

zamide (20g). ¹H NMR (DMSO-d₆): δ 2.15 (s, 3H), 2.33 (m, 4H),

2.39 (m, 4H), 2.61 (s, 3H), 3.56 (s, 2H), 7.39 (dd, J = 4.5, 9.2 Hz,

1H), 7.55 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.95 (dd,

J = 1.9, 8.0 Hz, 1H), 8.06 (dd, J = 1.9, 8.5 Hz, 1H), 8.21 (d, J =

1.7 Hz, 2H), 8.23 (s, 1H), 8.25 (dd, J = 1.5, 9.2 Hz, 1H), 8.72 (dd,

J = 1.5, 4.4 Hz, 1H), 10.54 (s, 1H). MS: m/z 533.3 [M þ H]^þ.

N-{4-[(4-Ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phe-

nyl}-3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methylbenza-

mide (20h). Purified as HCl salts. ¹H NMR (D₂O): δ 1.32 (t, J =

7.2 Hz, 3H), 2.38 (s, 3H), 3.27 (q, J = 7.2 Hz, 2H), 3.28 (m, 4H),

3.48 (m, 4H), 4.15 (s, 2H), 7.26 (d, J = 8.0 Hz, 1H), 7.39 (dd, J =

4.2, 9.6 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.0 Hz,

1H), 7.70 (d, J = 8.8 Hz, 1H), 7.88 (d, J = 9.2 Hz, 2H), 8.02 (s,

1H), 8.09 (d, J=9.6 Hz, 1H), 8.67 (d, J=4.4 Hz, 1H).

N-{4-[(4-(2-Hydroxyethyl)piperazin-1-yl)methyl]-3-(trifluoro-

methyl)phenyl}-3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-me-

thylbenzamide (20i). Purified as HCl salt. ¹H NMR (D₂O): δ 1.25

(s, 3H), 3.44 (s, 2H), 3.61 (m, 4H), 3.71 (m, 4H), 3.94 (s, 2H), 4.44

(s, 2H), 7.11 (d, J = 7.6 Hz, 1H), 7.54-7.59 (m, 3H), 7.70 (d, J =

8.0 Hz, 1H), 7.74 (s, 1H), 7.88 (s, 1H), 8.02 (s, 1H), 8.10 (d,

J = 9.2 Hz, 1H), 8.70 (d, J = 2.0 Hz, 1H).

4-Methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoro-

methyl)phenyl}-3-[2-(1H-pyrrolo[2,3-b]pyridin-5-yl)ethynyl]ben-

zamide (20a). ¹H NMR (DMSO-d₆): δ 2.17 (s, 3H), 2.33 (m, 4H),

2.40 (m, 4H), 2.58 (s, 3H), 3.57 (s, 2H), 6.52 (dd, J = 1.8, 3.4 Hz,

1H), 7.52 (d, J = 8.0 Hz, 1H), 7.57 (dd, J = 2.8, 3.2 Hz, 1H), 7.71

(d, J = 8.4 Hz, 1H), 7.90 (dd, J = 1.8, 7.8 Hz, 1H), 8.07 (dd, J =

1.8, 7.8 Hz, 1H), 8.17 (d, J= 1.8Hz, 1H), 8.22(m, 2H), 8.45(d, J=

2.0 Hz, 1H), 10.51 (s, 3H), 11.93 (s, 3H). MS: m/z 531.8 [M þ H]^þ.

3-[2-(3H-Imidazo[4,5-b]pyridin-6-yl)ethynyl]-4-methyl-N-{4-

[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benza-

mide (20b). ¹H NMR (CD₂Cl₂): δ 2.27 (s, 3H), 2.47 (m, 8H), 2.55

(s, 3H), 3.63 (s, 2H), 7.36 (d, J = 8.1 Hz, 1H), 7.61 (m, 1H), 7.75

(d, J = 8.5 Hz, 1H), 7.79 (m, 1H), 7.82 (dd, J = 1.8, 8.0 Hz, 1H),

7.94 (d, J = 8.8 Hz, 1H), 8.07 (dd, J = 1.8, 8.9 Hz, 1H), 8.09 (s,

1H), 8.30 (s, 1H), 8.56 (d, J = 1.6 Hz, 1H), 8.88 (br, 1H).

4-Methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoro-

methyl)phenyl}-3-[2-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyri-

din-6-yl)ethynyl]benzamide (20c). ¹H NMR (DMSO-d₆): δ 2.17

(s, 3H), 2.37 (m, 8H), 2.55 (s, 3H), 3.57 (s, 2H), 7.38 (d, J = 1.4

Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.90

(dd, J = 1.8, 8.0 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 8.14 (d, J =

1.7 Hz, 2H), 8.21 (d, J = 1.8 Hz, 1H), 10.50 (s, 1H), 11.03 (s, 1H),

11.61 (s, 1H). MS: m/z 549.2 [M þ H]^þ.

3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-[4-(1-

piperazinylmethyl)-3-(trifluoromethyl)phenyl}benzamide (20j).

¹H NMR (DMSO-d₆): δ 2.38 (m, 4H), 2.66 (s, 3H), 2.75 (m,

4H), 3.61 (s, 2H), 7.40 (dd, J = 4.4, 9.2 Hz, 1H), 7.58 (d, J = 8.2

Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.97 (dd, J = 1.9, 8.0 Hz, 1H),

8.09 (dd, J = 2.0, 8.4 Hz, 1H), 8.15-8.31 (m, 4H), 8.77 (dd, J =

1.5, 4.4 Hz, 1H), 10.59 (s, 1H). MS: m/z 519.0 [M þ H]^þ.

3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-N-{4-[(4-methyl-

piperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (20k).

¹H NMR (DMSO-d₆): δ 2.17 (s, 3H), 2.37 (m, 8H), 3.57 (s, 2H),

7.40 (dd, J = 4.5, 9.2 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.72 (d,

J = 8.6 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 8.04 (dd, J = 1.2, 9.4

Hz, 1H), 8.07 (d, J = 9.3 Hz, 1H), 8.22 (m, 1H), 8.23 (s, 1H), 8.25

(d, J = 1.6 Hz, 1H), 8.28 (d, J = 1.6 Hz, 1H), 8.73 (dd, J = 1.5,

4.4 Hz, 1H), 10.63 (s, 1H). MS: m/z 519.2 [M þ H]^þ.

4-Chloro-3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-N-{4-[(4-

methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benza-

mide (20l). ¹H NMR (DMSO-d₆): δ 2.15 (s, 3H), 2.50 (m, 4H),

2.84 (m, 4H), 3.63 (s, 2H), 7.44 (dd, J = 4.5, 9.2 Hz, 1H), 7.73

(d, J = 8.6 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 8.03 (dd, J = 2.2,

8.5 Hz, 1H), 8.09 (d, J=8.5 Hz, 1H), 8.20 (d, J=2.0 Hz, 1H),

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4717

8.28 (s, 1H), 8.29 (dd, J = 1.5, 9.2 Hz, 1H), 8.35 (d, J = 2.1 Hz,

1H), 8.74 (dd, J = 1.4, 4.5 Hz, 1H), 10.68 (s, 1H). MS: m/z

553.3 [M þ H]^þ.

7.49 (d, J = 16.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.10 (t, J =

4.5 Hz, 1H), 3.76 (s, 2H), 2.87 (br, 6H), 2.70 (br, 2H), 2.56 (s,

3H), 1.56 (s, 3H). MS: m/z 535.4 [M þ H]^þ.

4-Fluoro-3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-N-{4-[(4-me-

thylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide

(20m). ¹H NMR (DMSO-d₆): δ 2.20 (s, 3H), 2.50 (m, 8H), 3.59 (s,

2H), 7.43 (dd, J = 4.5, 9.2 Hz, 1H), 7.59 (dd, J = 8.9, 9.1 Hz, 1H),

7.73 (d, J = 8.7 Hz, 1H), 8.05-8.14 (m, 2H), 8.21 (d, J = 2.0 Hz,

1H), 8.29 (s, 1H), 8.30 (m, 1H), 8.35 (dd, J = 2.2, 6.8 Hz, 1H), 8.75

(dd, J = 1.5, 4.4 Hz, 1H), 10.63 (s, 1H). MS: m/z 537.3 [M þ H]^þ.

N-{3-Chloro-4-[(4-methylpiperazin-1-yl)methyl]phenyl}-3-[2-(imi-

Modeling Studies. The binding site model used for Abl dock-

ing was based on the X-ray coordinates of Abl bound with

˚

imatinib (PDB: 1IEP). Protein side chains within a 5 A radius of

the bound ligand were allowed to relax to optimize binding

interactions using the Induced Fit protocol in the Schrodinger

package.⁴⁵Docking studies were performed with Glide in the

Schrodinger modeling package.⁴⁵The solution with the best

docking score was chosen and used for analysis.

1

dazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methylbenzamide (20n). H

Biological Characterization of Compounds. In vitro kinase

assays were carried out in-house as previously described.^21a

This assay configuration differed from that used in our pre-

vious publication on 20g,²⁰which was a commercial assay from

Reaction Biology Corporation.⁴³Cellular proliferation assays

were performed as previously described,^21aindependently of

the data set previously reported for 20g.²⁰Pharmacokinetic

analyses^21aand in vivo efficacy studies²⁰were carried out

according to previously published procedures.

NMR (DMSO-d₆): δ 2.17 (s, 3H), 2.33 (m, 4H), 2.44 (m, 4H),

2.62 (s, 3H), 3.53 (s, 2H), 7.40 (dd, J = 4.5, 9.2 Hz, 1H), 7.44 (d,

J = 8.4 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 2.1, 9.4

Hz, 1H), 7.94 (dd, J = 1.9, 8.0 Hz, 1H), 7.98 (d, J = 2.0 Hz, 1H),

8.20 (d, J = 1.8 Hz, 1H), 8.24 (s, 1H), 8.27 (dd, J = 1.5, 9.2 Hz,

1H), 8.73 (dd, J = 1.5, 4.4 Hz, 1H), 10.43 (s, 1H).

N-{3-Cyclopropyl-4-[(4-methylpiperazin-1-yl)methyl]phenyl}-

3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methylbenzamide

(20o). ¹H NMR (DMSO-d₆): δ 0.60 (m, 2H), 0.94 (m, 2H), 2.20

(s, 3H), 2.25 (m, 1H), 2.27-2.50 (m, 8H), 2.60 (s, 3H), 3.56 (s, 2H),

7.20(d, J=8.3Hz, 1H), 7.34(d,J= 1.9 Hz, 1H), 7.39 (dd, J=4.4,

9.2 Hz, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.91

(dd, J=1.7, 8.2 Hz, 1H), 8.18 (d, J=1.6 Hz, 1H), 8.23 (s, 1H), 8.26

(dd, J=1.4, 9.2 Hz, 1H), 8.73 (dd, J=1.3, 4.4Hz, 1H), 10.16(s, 1H).

3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-[3-(tri-

fluoromethyl)phenyl] Benzamide (20p). ¹H NMR (CDCl₃): δ 8.52

(d, J = 5.2 Hz, 1 H), 8.22 (s, 1H), 8.10 (s, 1H), 8.07 (d, J = 2.4

Hz, 1H), 8.04 (d, J = 9.2 Hz, 1H), 8.00 (s, 1H), 7.93 (d, J = 8.0

Hz, 1H), 7.84 (dd, J = 1.9, 8.0 Hz, 1 H), 7.51 (t, J = 8.1 Hz, 1H),

7.42 (m, 2H), 7.18 (dd, J = 4.8, 5.0 Hz, 1H), 2.65 (s, 3H).

3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethyl]-4-methyl-N-{4-[(4-me-

thylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide

(24a). A tri-HCl salt of 20g (400 mg) was dissolved in water

(10 mL) and then subjected to hydrogenation under 28 psi for

72 h using 10% wet Pd/C (100 mg) as the catalyst. The catalyst

was then removed by filtration, and the filtrate was basified to

pH ∼9 with 2 M aq NaOH. Extraction with CH₂Cl₂and silica

gel column chromatography (eluent: 5% NH₃presaturated

MeOH in CH₂Cl₂) followed by recrystallization from EtOH

gave the desired product as an off-white solid (240 mg, 70%). ¹H

NMR (CDCl₃): δ 8.36 (d, J = 2.9 Hz, 1H), 7.95 (m, 2H), 7.89 (s,

1H), 7.74 (m, 2H), 7.61 (d, J = 7.9 Hz, 1H), 7.53 (s, 1H), 7.47 (s,

1H), 7.28 (d, J = 9.2 Hz), 7.02 (m, 1H), 3.69 (s, 2H), 3.34 (t, J =

7.8 Hz, 2H), 3.15 (t, J = 7.8 Hz, 2H), 2.67 (m, 6H), 2.48 (m, 2H),

2.42 (s, 3H), 1.27 (s, 3H). MS: m/z 537.4 [M þ H]^þ.

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3-[2-(Imidazo[1,2-b]pyridazin-3-yl)vinyl]-4-methyl-N-{4-[(4-me-

thylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide

(24b). A solution of 18a (189.3 mg, 0.366 mmol), tributyl(vinyl)-

tin (464.1 mg, 1.464 mmol), Pd₂(dba)₃CHCl₃(10 mg, 0.01 mmol),

3

and tri(2-furyl)phosphine (18 mg, 0.074 mmol) in dry DMF

(4 mL) was degassed with N₂and then stirred at rt for 15 h. The

solvent was evaporated, and the residue was dissolved in EtOAc.

Washing with satd aq Na₂CO₃followed by evaporation of

organic solvents and silica gel column chromatography

(eluent: 10% MeOH in CH₂Cl₂) gave substituted styrene 23 as

a white solid (146 mg, 96%).

A suspension of 23 (146 mg, 0.35 mmol), 7d (133 mg, 0.67

mmol), Pd(OAc)₂(6.4 mg, 0.028 mmol), P(o-tol)₃(17.4 mg,

0.057 mmol), and (i-Pr)₂NEt (0.29 mL, 1.39 mmol) in DMF

(3 mL) was degassed with N₂and then heated at 110 °C for 15 h.

Upon cooling, the reaction mixture was dilute with CH₂Cl₂.

After washing with water and evaporation of organic solvents,

the residue was purified by silica gel column chromatography

(eluent: 5% NH₃presaturated MeOH in CH₂Cl₂), giving a dark-

brown solid. Recrystallization from CHCl₃/ether afforded an

off-white solid (50 mg, 25%). ¹H NMR (CDCl₃): δ 8.46 (d, J =

3.0 Hz, 1H), 8.20 (d, J = 1.3 Hz, 1H), 8.06 (s, 1H), 8.04 (m, 1H),

8.02-7.97 (m, 2H), 7.93-7.86 (m, 2H), 7.69 (d, J = 8.4 Hz, 1H),

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Article Doi

Discovery of 3-[2-(imidazo[1,2- b ]pyridazin-3-yl)ethynyl]-4-methyl- N -{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of breakpoint cluster region-abelson (BCR-ABL) kinase including the T315I gatekeeper mutant

DOI: 10.1021/jm100395q

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