Identification of 4,5-dihydro-1 H -pyrazolo[4,3- h ]quinazoline Derivatives as a new class of orally and selective polo-like kinase 1 inhibitors

3532 J. Med. Chem. 2010, 53, 3532–3551

DOI: 10.1021/jm901713n

Identification of 4,5-Dihydro-1H-pyrazolo[4,3-h]quinazoline Derivatives as a New Class of Orally and

Selective Polo-Like Kinase 1 Inhibitors

Italo Beria,* Dario Ballinari, Jay Aaron Bertrand, Daniela Borghi, Roberto Tiberio Bossi, Maria Gabriella Brasca,

Paolo Cappella, Michele Caruso, Walter Ceccarelli, Antonella Ciavolella, Cinzia Cristiani, Valter Croci, Anna De Ponti,

Gabriele Fachin, Ronald Dale Ferguson,^†Jacqueline Lansen,^‡Jurgen Karl Moll, Enrico Pesenti, Helena Posteri, Rita Perego,

Maurizio Rocchetti,^‡Paola Storici,^§Daniele Volpi, and Barbara Valsasina

Nerviano Medical Sciences Srl, Oncology, Viale Pasteur 10, 20014 Nerviano, (Mi), Italy. ^†Present address: Merck Co. & Inc., 126 E.

Lincoln Avenue, Rahway, NJ 07065. ^‡Accelera, Nerviano Medical Sciences Srl, Viale Pasteur 10, 20014 Nerviano (MI ), Italy.

^§Present address: Sincrotrone Trieste S.C.p.A., Strada Statale 14, Area Science Park, 34149 Basovizza (TS), Italy

Received November 19, 2009

Polo-like kinase 1 (Plk1) is a fundamental regulator of mitotic progression whose overexpression is often

associated with oncogenesis and therefore is recognized as an attractive therapeutic target in the

treatment of proliferative diseases. Here we discuss the structure-activity relationship of the 4,5-

dihydro-1H-pyrazolo[4,3-h]quinazoline class of compounds that emerged from a high throughput

screening (HTS) campaign as potent inhibitors of Plk1 kinase. Furthermore, we describe the discovery

of 49, 8-{[2-methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide, as a highly potent and specific ATP mimetic inhibitor of Plk1

(IC₅₀= 0.007 μM) as well as its crystal structure in complex with the methylated Plk1_36-345construct.

Compound 49 was active in cell proliferation against different tumor cell lines with IC₅₀values in the

submicromolar range and active in vivo in the HCT116 xenograft model where it showed 82% tumor

growth inhibition after repeated oral administration.

Introduction

the best characterizedandisrecognizedtobeakey component

of the cell cycle control machinery with important roles in the

mitotic entry, centrosome duplication, bipolar mitotic spindle

formation, transition from metaphase to anaphase, cytoki-

nesis, and maintenance of genomic stability.^5-9Plk1 is often

overexpressed in many different tumor types including lung,

colon, prostate, ovary, breast, head and neck squamous cell

carcinoma, melanoma, and overexpression often correlates

with poor prognosis.^10-16In addition, interference with Plk1

activity and/or function by a variety of methods including

antisense oligonucleotides, small interfering RNA, and var-

ious dominant negatives results in tumor cell apoptosis in

culture and in vivo.^17-21Moreover, literature data report that

normal cells survive Plk depletion, whereas tumor cells do

not.²²All of this evidence supports Plk1 as a valid target for

anticancer drug development. Although depletion of Plk1 is

sufficient to induce G2/M cell cycle block, it is not clear

whether it is important or indeed beneficial to inhibit all of

the other Plk isoforms. In fact, less is known concerning the

physiological roles of Plk2 and Plk3 that, in contrast to Plk1,

are reported to be expressed in nonproliferating differentiated

post mitotic cells, like neurons, indicating a potentially better

safety profile for a Plk1 specific inhibitor.²³

Mitotic cell cycle progression is tightly regulated through

reversible covalent protein phosphorylation events coordi-

nated by regulatory kinases, including members of the Polo

subfamily.^1-4Among the four Plk^aisoforms (Plk1-4), Plk1 is

*To whom correspondence should be addressed. Phone: þ39

0331581516. Fax: þ39 03311581347. E-mail italo.beria@nervianoms.com.

^aAbbreviations: Plk, Polo-like kinase; ABL, Abelson protein-tyro-

sine kinase; AKT, signal transducer and activator of transcription or

protein kinase B; ALK, anaplastic lymphoma kinase; Aur, aurora;

CDC, cell division cycle; CDK, cyclin dependent kinase; CK, casein

kinase; CHK, Csk homologous kinase; EGFR, epidermal growth factor

receptor; ERK, extracellular signal-regulated kinase; FGFR, fibroblast

growth factor receptor; IGFR, insulin-like growth factor receptor;

GSK, glycogen synthase kinase; IKK2, IκB kinase; IR, insulin receptor;

LCK, lymphocyte-specific protein tyrosine kinase; MAP-KAPK2, mi-

togen activated proteinkinase-activatedprotein kinase-2; MET, high

affinity tyrosine kinase receptor for hepatocyte growth factor; HGF,

hepatocyte growth factor; NEK, NIMA-related kinase; NIMA, never in

mitosis gene A; NIM, nim1/cdr1 protein; PAK, p-21 activated kinase;

PKA, c-AMP-dependent kinase; PKC, protein kinase C; RET, receptor

tyrosine kinase, STLK, serine-threonine like kinase; SULU, protein

kinase 18; TRKA, catalytic receptor for the neurotrophin nerve growth

factor; NGF, neurotrophin nerve growth factor; VEGFR, vascular

endothelial growth factor receptor; AMP-PNP, adenylyl-imidodipho-

sphate tetralithium salt; NPM, nucleophosmin; TCTP, translationally

controlled tumor protein; ADME, adsorption, distribution, metabo-

lism, excretion; HLM human liver microsoms; P_app, apparent perme-

ability; PAMPA, parallel artificial membrane permeation assay; EDC,

N-(3-dimethylaminopropyl)-N⁰-ethylcarbodiimide; HOBT, 1-hydroxy-

Because of its essential role in cell proliferation there is a

high level of interest and an increasing effort to identify and

develop small-molecule inhibitors of Plks that has led to a

number of different compounds which are currently under

clinical investigation.^24-29

benzotriazole; HOBT NH₃, 1-hydroxybenzotriazole ammonium salt ;

3

(()-BINAP, (()-2,2⁰-bis(diphenylphosphino)-1,1⁰-binaphtalene; DMF-

DIPA, N,N-dimethylformamide diisopropyl acetal; DTBAD, di-tert-bu-

tylazodicarboxylate; LiN(TMS)₂, lithium bis(trimethylsilyl)amide; Pd₂-

(dba)₃, tris(dibenzylideneacetone)dipalladium(0); DMA, N,N-dimethyl-

acetamide.

In our effort to develop selective small-molecule Plk1

inhibitors, we identified a novel series of ATP-competitor

r

pubs.acs.org/jmc

Published on Web 04/16/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3533

Scheme 2. Synthesis of 1-Substituted 4,5-Dihydro-1H-pyrazolo-

[4,3-h]quinazoline derivatives 1a-c, 1e, 1f-i^a

Figure 1. HTS pyrazoloquinazoline hit 1a.

Scheme 1. Synthesis of Enaminone Intermediates 5a-d^a

^aConditions: (a) phenylguanidine carbonate, DMF, 110 ꢀC, 67-72%;

(b) 1.5 M KOH/EtOH, reflux, quant; (c) EDC, HOBt NH₃, DMF,

3

DIPEA, rt, 56-78%; (d) TFA, DCM, rt, 80%; (e) DTBAD, Ph₃P

polyimer-bound, alcohol R₁-OH (8a-d), THF, rt, 24-62%.

compounds 1a-c and 1e was obtained in two steps by hydro-

lysis of esters 7a-cor 7e, followed by a reaction with 1-hydroxy-

benzotriazole ammonium salt and EDC to introduce the pri-

mary amide function. Compound 1e was inturnusedasstarting

material to synthesize compounds 1f-i so as to complete the

series of 1-substituted pyrazole derivatives. Alkylation of 1e

with the suitable alcohols proceeded regioselectively at the

position 1 of pyrazole nucleus under Mitsunobu conditions

using polymer-bounded triphenyl phosphine to facilitate the

workup and purification of target compounds. As previously

described by Brasca and co-workers,³¹synthesis of derivatives

9-13 modified at the 3 position of the 4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline template (Figure 2) was performed

starting from ester 7a. Modifications of the 4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline scaffold at position 8 to afford

compounds 19-49 were introduced by Buchwald-Hartwig

coupling of the iodoamide 18 with suitable amines 50-76 or

85a-e using palladium(II) acetate as catalyst and (()-BINAP

as ligand (Scheme 3). Iodoamide 18 was in turn obtained in four

steps starting from enaminone 5a, following the recently

reported procedure (Figure 3).³¹Briefly, reaction of enaminone

5a with guanidine carbonate afforded intermediate 15, the

amino group was then converted into iodo derivative 16 via

diazonium salt, and finally the acid intermediate 17, obtained

from hydrolysis of ester 16, was converted into the desired

iodoamide 18. An alternative synthesis of a limited number of

derivatives (80 and 81) characterized by an amino or an amido

residue at the position 2⁰of the 8 anilino moiety was applied

(Scheme 4). According to this procedure, iodoester 16 was

coupled with the N-Boc-o-phenylenediamine under Buch-

wald-Hartwig conditions, then the protecting group was easily

removed with hydrochloric acid in dioxane, generating the

aminoester 78 that was coupled with acetyl chloride to yield

the corresponding amido esters 79. The final conversion of 79

into target amide 81 was achieved in a straightforward manner

with 30% NH₄OH in a sealed tube at 70 ꢀC. The same

procedure was applied to the ester intermediate 78 to obtain

the amide 80. Preparation of disubstituted aniline intermediates

85a-e is outlined in Schemes 5 and 6. Compounds 85a-b were

prepared starting from the commercially available anilines

82a-b. The amino group was first protected as an acetyl

derivative simply by reaction with acetic anhydride, then the

4-methylpiperazin-1-yl residue was introduced by subjecting

the bromo intermediates 83a-b to a Buchwald-Hartwig

coupling with 4-methylpiperazine using Pd₂(dba)₃as catalyst

and 2-dicyclohexylphosphino-2⁰-(N,N-dimethylamino)-biphenyl

^aConditions: (a) hydrazine R₁NHNH₂(6a-c), AcOH glacial, rt,

36-55%; (b) hydrazine hydrate, EtOH, reflux, 70%; (c) tritylchloride,

TEA, DCM, rt, 72%; (d) DMF-DIPA, DMF, 60 ꢀC, 54-71%.

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives and

herein we report their in vitro and in vivo characterization.³⁰

Starting from the unselective compound 1a (Figure 1) that was

fished out from a high throughput screening (HTS) campaign,

we developed compound 49, a highly potent (Plk1 IC₅₀

=

0.007 μM), selective, and orally bioavailable Plk1 inhibitor.

Compound 49 showed good selectivity in a panel of more than

40 protein kinases and with more than 30-fold selectivity with

respect to the Plk family members Plk2 and Plk3.

Structure-activity relationships (SAR) for this class to-

gether with data concerning the crystal structure of the

selective inhibitor 49 in complex with the Plk1 kinase domain

are reported below.

Chemistry

A versatile chemical strategy for the synthesis of the 4,

5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives involving

the synthesis of flexible intermediates 5a-d was established.

Enaminones 5a-d were prepared in 2/3 linear step process

starting from the diketoester 2 (Scheme 1).³¹Cyclization of the

diketoester 2 into the 1-substituted pyrazole derivatives 4a-c

was carried out in a regiospecific way by reacting suitable

hydrazines 6a-c in glacial acetic acid. Alternatively, reacting

2with hydrazine hydrate followed by unselective base catalyzed

alkylation of pyrazole derivative 3 with trityl chloride yielded a

mixture of 1 and 2-trityl regioisomers wherein the desired

intermediate 4d was easily isolated by crystallization. Reaction

of 7-oxo-tetrahydroindazole derivatives 4a-d with N,N-di-

methylformamide diisopropyl acetal yielded the versatile en-

aminones 5a-d. Further elaboration of intermediates 5a-d

to obtain the final compounds modified at positions 1, 3, or 8 of

the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline scaffold was

performed following the synthetic pathways reported in

Schemes 2-4 and Figure 2. Cyclization of intermediates

5a-d with phenyl guanidine carbonate at 110 ꢀC in N,N-

dimethylformamide allowed direct access to the 4,5-dihydro-

1H-pyrazolo[4,3-h]quinazoline scaffold decorated at position 8

with the desired anilino moiety (7a-d) (Scheme 2). Compound

7e was then obtained from 7d by acidic removal of the trityl

protecting group. Conversion of 7a-c and 7e into the final

3534 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

Scheme 3. Synthesis of 4,5-Dihydro-1H-pyrazolo[4,3-h]quinazoline Derivatives 19-49^a

^aConditions: (a) Pd(OAc)₂, (()-BINAP, K₂CO₃, amines (50-76; 85a-e), DMF, 80 ꢀC, 7-77%.

Scheme 4. Synthesis of 2⁰Anilino Derivatives 79 and 81^a

^aConditions: (a) Pd(OAc)₂, (()-BINAP, K₂CO₃, N-Boc-o-phenylenediamine, 61%; (b) 4N HCl/dioxane, DCM, rt, 96%; (c) acetyl chloride, DIPEA,

rt, 72%; (d) 30% NH₄OH, EtOH, sealed tube, 70 ꢀC, 17-64%.

palladium catalyzed Buchwald-Hartwig coupling conditions, in

the case of the less reactive 4-bromo-1-methoxy-2-nitro-benzene

83c, or by high temperature nucleophilic substitution condi-

tions for the more reactive 1-(2-chloro-6-nitro-phenyl)-etha-

none 83d. Final reduction of both nitro derivatives 84c-d was

carried out under catalytic palladium conditions, with hydro-

Figure 2. Compounds modified at the position 3.

gen in a Parr apparatus under 35 psi presssure generating 85c

and under atmospheric pressure with cyclohexene as hydrogen

source producing 85d. Amine intermediate 85e was prepared

subjecting the commercially available 1-(4-fluoro-2-hydroxy-

phenyl)-ethanone 86 to nucleophilic substitution with 4-methyl-

piperazine at high temperature (Scheme 6). Then, phenol deriva-

tive 87 was converted into the target aniline 85e by a one-pot

reaction with 2-bromo-2-methylpropionamide and NaOH in

N,N-dimethylacetamide via a Smiles rearrangement.³²

Figure 3. Synthesis of intermediate 18.

Results and Discussion

as ligand. Final removal of the amino protecting group under

acid conditions afforded intermediates 85a-b (Scheme 5). An

alternative approach was applied for the synthesis of 85c-d. The

first step (Scheme 5) foresaw the installation of the 4-methylpi-

perazin-1-yl residue by reacting 4-methylpiperazine under either

In the HTS campaign for the discovery of small-molecule

inhibitors of Plk1, compound 1a³¹belonging to the 4,5-dihydro-

1H-pyrazolo[4,3-h]quinazoline class emerged as an inter-

esting hit. Despite strong cross activity with others kinases,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3535

Scheme 5. Synthesis of Amino Intermediates 85a-d^a

^aConditions: (a) acetic anhydride, rt, 42-51%; (b) Pd₂(dba)₃, 1 M LiN(TMS)₂/THF, 4-methylpiperazine, 2-dicyclohexylphosphino-2⁰-(N,N-

dimethylamino)-biphenyl, reflux, 53-91%; (c) HCl conc, EtOH, reflux, 45%-quant; (d) 4-methylpiperazine, Pd(OAc)₂, K₃PO₄, 2-dicyclohexylphos-

phino-2⁰-(N,N-dimethylamino)-biphenyl, THF, reflux, 55%; (e) 4-methylpiperazine, 120 ꢀC sealed tube, 68%; (f ) cyclohexene, 10% Pd/C, HCl conc, THF/

H₂O/EtOH, 70 ꢀC, 95%; (g) 10% Pd/C, H₂, 35 psi, MeOH, 90%.

Scheme 6. Synthesis of Amino Intermediate 85e^a

Table 1. SAR: Variation of R₁Substitution^a

^aConditions: (a) 4-methylpiperazine, 130 ꢀC, quant; (b) NaOH,

DMA, 2-bromo-2-methylpropanamide, rt. then NaOH, 100 ꢀC, 30%.

e.g., Aur-A and CDK2/A, inhibitor 1a was selected for a

chemistry expansion aimed at defining the structure activity

relationship for this novel class and at improving both the

selectivity profile and the pharmacokinetic properties. In

particular, the effect of modifications at positions 1, 3, and 8

of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline scaffold will

be discussed in more depth.

Compounds modified at position 1 of the 4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline template together with biochem-

ical activity against Plk1, CDK2/A, Aur-A kinases and in

vitro cell proliferation cytotoxicity on A2780 cell lines are

reported in Table 1. According to this data, substitutions at

position 1 of pyrazole ring did not improve the selectivity vs

CDK2/A or Aur-A kinases. In contrast, the introduction of

bulky groups in this position had dramatic effects on the Plk1

activity. Specifically, the compounds wherein the methyl

substituent was removed (1e) or replaced with the cyclohexyl

moiety (1f) showed a maintained or increased cytotoxi-

city (IC₅₀= 0.10 and 0.55 μM, respectively), but a

decreased Plk1 activity (Plk1 IC₅₀= 0.248 and 0.143 μM,

^aIC₅₀values are the average of n g 2.

modifications significantly decreased Plk1 activity. Even the

simple methyl amide derivative 10 showed a 60-fold loss of

activity in the Plk1 biochemical assay and 5-fold reduction in

cell proliferation potency with respect to the parent com-

pound 1a. The only derivative that maintained the progeni-

tor’s activity was the carboxylic acid 9, however, its selectivity

profile vs CDK2/A and Aur-A was not improved. Moreover,

cellular activity of 9 was decreased 5-fold with respect to 1a,

probably due to the reduced capability of the carboxylic acid

to cross the cellular membrane with respect to the primary

amide as confirmed by the low value in the Caco-2 perme-

ability assay of 9 with respect to 1a (P_app1a = 58.3 ꢀ 10^-6cm/s;

P_app9 = 2.7 ꢀ 10^-6cm/s).

respectively) with respect to progenitor 1a (Plk1 IC₅₀=

0.068 μM). Replacement of methyl with a small flexible

trifluoroethyl residue (1b) resulted in increased Plk1 activity

(IC₅₀= 0.006 μM) but decreased cellular activity (IC₅₀

=

2.75 μM) with respect to 1a. A decrease from 6- to more

than 100-fold in Plk1 biochemical activity was observed

with the increase of the substituent’s complexity (1c, 1 g-i).

On the basis of these data, the methyl group was identified

as the best residue at the position 1 of the 4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline core in terms of biochemical

and cellular activity.

Exploration of the 3 position was performed with the

synthesis of a set of compounds wherein the primary amide

residue of hit 1a was replaced with ethyl ester function (7a),

carboxylic acid residue (9), or withsecondary amides(10-13).

Activity results reported in Table 2 show that most of these

Exploration at position 8 of the scaffold was initiated by

evaluating the influence of substituents at positions 2⁰, 3⁰, or 4⁰

of the anilino residue. Analysis of the biochemical data

reported in Table 3 shows that in an homogeneous series,

3536 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Table 2. SAR: Variation of R₂Substituent^a

Beria et al.

IC₅₀, μM

compd

R₂

Plk1

0.068

>10

CDK2/A

Aur-A

A2780

1a

7a

9

NH₂

OEt

OH

0.002

0.251

0.003

0.019

0.081

3.900

0.574

0.050

>10

0.50

2.79

0.110

4.215

0.016

0.312

0.213

>10

2.54

2.63

10

11

12

13

NHMe

NHcyclopropyl

NHcyclopentyl

NHPh

>10

3.82

>10

^aIC₅₀values are the average of n g 2.

Table 3. SAR: Variation of R₃Substitution^a

compd

R₃

Plk1 (IC₅₀, μM) CDK2/A (IC₅₀, μM) CDK2/A/Plk1 ratio Aur-A (IC₅₀, μM) Aur-A/Plk1 ratio A2780 (IC₅₀, μM)

1a

19

20

21

22

23

24

25

26

27

28

29

30

H

0.068

0.432

0.051

0.872

0.346

0.100

0.197

0.042

0.135

0.256

0.488

0.002

0.306

0.006

0.003

1.263

0.002

0.041

0.005

0.004

0.209

0.005

0.014

0.030

0.708

0.118

0.050

>10

0.270

>10

0.176

0.740

>23

>196

0.310

>28

1.767

0.50

>10

o-CF₃

m-CF₃

p-CF₃

o-Ac

1.49

0.25

0.003

3.656

>10

m-Ac

p-Ac

0.020

0.009

0.09

0.04

0.94

1.05

0.05

3.49

1.68

1.96

0.116

0.607

0.158

0.128

0.589

14.452

1.164

o-OMe

m-OMe

p-OMe

o-NO₂

m-NO₂

p-NO₂

0.969

0.035

0.016

0.428

0.499

>10

>20

>10

<0.001

>10

^aIC₅₀values are the average of n g 2.

substitution at positions 3⁰and 4⁰of the phenyl ring was

inefficient to increase Plk1 selectivity, especially against

CDK2/A, as revealed by the substantially unmodified

CDK2A/Plk1 ratio or Aur-A/Plk1 ratio for the compound

pairs 20-21, 23-24, or 26-27 in comparison with 1a. A

unique behavior was shown by the nitro derivatives 29 and 30,

wherein despite a retention of activity on CDK2/A, a dra-

matic loss of activity was observed for Plk1 and Aur-A. As far

as the 2⁰substituted derivatives are concerned, the Plk1

activity was either maintained (25) or slightly decreased (19,

22, 28). However, more importantly, a general increase of

selectivity against both CDK2/A and Aur-A was evident. In

particular, the 2⁰-methoxy derivative 25 showed a marginal

gain in terms of selectivity vs both CDK2/A (CDK2/A/Plk1

ratio from 0.03 to 0.97) and vs Aur-A (Aur-A/Plk1 ratio from

0.74 to 14.45) and a maintained cytotoxic activity on A2780

with respect to 1a. An even more positive trend was shown by

the 2⁰-acetyl derivative 22, with an increased selectivity with

respect to 1a against both CDK2/A (CDK2/A/Plk1 ratio

from 0.03 to 3.65) and Aur-A (Aur-A/Plk1 ratio from 0.74

to >28) but with loss of cytotoxicity on A2780. The high

cytotoxicity shown by the unselective compounds 21, 23, 24,

and 27 (IC₅₀= 0.25, 0.09, 0.04, and 0.05 μM, respectively) in

comparison with 1a (IC₅₀= 0.5 μM) suggested that cellular

activity of these compounds could be due to the inhibition by

other kinases as, e.g., CDK2/A and Aur-A. The maintained

cytotoxicity and the improved Plk1 selectivity shown by

compound 25 in comparison with the unselective hit 1a

indicates that the ortho position is key for obtaining active

and selective Plk1 inhibitors. In light of these positive results, a

deeper investigationwas performed to mapthe chemical space

at position 2⁰of the anilino moiety with the synthesis of a set of

compounds differing in both electronic characteristics and

steric bulk (Table 4). The data confirmed 25 as the most

promising compound in terms of cellular activity on A2780

cell line, potency against Plk1 (IC₅₀= 0.042 μM), and

improved, but still low, selectivity vs Aur-A and CDK2/A

withrespecttoprogenitor 1a. Compound 25, however, didnot

show selectivity vs Plk2 and Plk3 isoforms. The lack of

selectivity against Plk2 and Plk3 was shared by all compounds

reported in Table 4, indicating that this position is probably

not suitable in achieving selectivity for Plk1 isoform; further-

more, all compounds, with the exception of 80, demonstrated

solubility at pH 7 below 30 μM. It is worth noting that

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3537

Table 4. SAR: Variation of R₃Substitution^a

IC₅₀, μM

CDK2/A

compd

R₃

Plk1

Plk2

Plk3

Aur-A

A2780

solubility at pH 7, μM

1a

19

22

25

31

32

33

34

35

36

37

38

39

40

41

42

43

44

80

81

H

CF₃

0.068

0.432

0.346

0.042

0.015

0.097

0.110

0.125

0.365

1.117

2.076

3.733

1.565

0.278

0.943

0.949

1.969

2.033

0.150

2.523

0.009

0.109

0.261

0.024

0.002

0.014

0.012

0.027

0.509

nd

0.014

0.095

0.103

0.013

0.006

0.016

0.011

0.037

0.146

nd

0.002

0.306

1.263

0.041

0.003

0.142

0.017

0.007

0.358

1.448

2.746

1.120

1.347

1.900

0.571

0.442

0.050

>10

0.607

0.605

>10

2.900

1.181

>10

2.497

>10

0.50

>10

0.94

3

4

Ac

2

OMe

Me

SMe

3

0.80

6.86

3

2

NHMe

F

i-Pr

7.54

0.67

23

3

>10

8.30

2

CO₂Me

CONH₂

SO₂NH₂

Ph

2

nd

<1

4

nd

>10

5.85

<1

190

16

OPh

2.666

nd

0.051

nd

benzyl

NHPh

benzoyl

SPh

nd

>10

0.007

1.037

>10

2.92

NH₂

NHAc

0.012

2.104

0.013

0.049

3.93

^aIC₅₀values are the average of n g 2. nd, not determined.

Table 5. SAR: Variation of R₃and R₄Substitution^a

IC₅₀, μM

CDK2/A

compd

R₃

R₄

Plk1

Plk2

0.009

Plk3

0.014

>10

2.292

>10

Aur-A

A2780

0.50

>10

0.762

solubility at pH 7, μM

1a

45

46

47

48

49

H

Ac

H

0.068

2.051

0.464

0.109

0.040

0.007

0.002

1.121

0.050

>10

3

170

3⁰-(4-methyl-piperazin-1-yl)

4⁰-(4-methyl-piperazin-1-yl)

5⁰methyl-piperazin-1-yl)

4⁰-(4-methyl-piperazin-1-yl)

5⁰-(4-methyl-piperazin-1-yl)

>10

Ac

>10

0.082

0.355

83

18

>10

OMe

0.082

0.238

0.066

0.450

0.146

0.009

>225

170

>10

^aIC₅₀values are the average of n g 2.

introduction of medium/large moieties (36-44 and 81) in the

2⁰position, with the exception of phenoxy derivative 40, was

detrimental not only for Plk1 inhibition (IC₅₀g 0.9 μM) but

also for the others kinases (IC₅₀> 0.4 μM) (Table 4). On the

contrary, small groups were well tolerated by Plk1 (19, 22, 25,

31-35, and 80) as confirmed by the only 6-fold loss of activity

on the enzyme for 19 (IC₅₀= 0.432 μM) and the 4-fold

increased activity shown by 31 (IC₅₀= 0.015 μM) when

compared with 1a (IC₅₀= 0.068 μM). A significant modifica-

tion in terms of potency and selectivity was achieved by the

introduction of an additional substituent on the anilino

moiety at position 8 (Table 5). Complete loss of biochemical

activity for all the kinases and loss of cellular proliferation

activity were obtained when the 4-methylpiperazino residue

was introduced at the position 3⁰of the 8-anilino substituent

(45). However, encouraging results were obtained by introdu-

cing the 4-methylpiperazino substituent at the position 4⁰of

the anilino moiety (46 and 48). These derivatives not only

maintained acceptable Plk1 activity but they also maintained

or even improved the selectivity toward CDK2/A and Aur-A.

In addition, improved selectivity against Plk2 and Plk3 was

also achieved (compare 46 and 48 with 22 and 25, re-

spectively). Particularly interesting were the data for 47 and

49, wherein the 4-methylpiperazino moiety was placed at the

5⁰position of the 8-anilino appendix. Derivative 49 proved to

be the best compound of the series, showing antiproliferative

activity in the low nanomolar range in the A2780 cell line

(IC₅₀= 0.009 μM), good activity on Plk1 (IC₅₀= 0.007 μM),

3538 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Table 6. Kinase Selective Profile of Compound 49

Beria et al.

and a high level of selectivity against Plk2, Plk3, CDK2/A,

and Aur-A (0.238, 0.450, 0.355, and >10 μM, respectively).

Furthermore, the introduction of 4-methylpiperazino residue

also proved to be beneficial to the compound’s solubility that,

in the case of 49, resulted more than 50-fold with respect to 1a.

The high level of selectivity of compound 49 was also con-

firmed using a larger panel of more than 40 kinases, giving

selectivity ratios that ranged from 16-fold to >1400-fold

(Table 6).

kinase

IC₅₀, ( μM)^a

kinase

IC₅₀, ( μM)^a

Plk1

CK2

0.007 ( 0.003

0.115 ( 0.045

0.160 ( 0.029

0.219 ( 0.092

0.238 ( 0.043

0.347 ( 0.127

CDK2/A

Plk3

0.355 ( 0.167

0.450 ( 0.101

0.587 ( 0.230

0.954 ( 0.317

1.904 ( 0.860

>10 (31 kinases)

CDK1/B

CDK5/p25

Plk2

VEGFR3

PDGFR

KIT

others^b

CDK2/E

^aData are reported as a mean ( standard deviation (n = 2). ^bc-ABL,

AKT1, ALK, Aur-A, Aur-B, CDC7, CDK7, CDK4/D1, CHK1,

EGFR1, ERK2, FGFR1, IGFR1, GSK3β, IKK2, IR, LCK, MAP-

KAPK2, MET, NEK-6, NIM, PAK4, PDK1, PKAR, PKCβ, P38R,

RET, STLK2, SULU1, TRKA, and VEGFR2.

A multiple construct approach was applied in order to

obtain the crystal structure of the Plk1 kinase domain in

complex with proprietary inhibitors. Specifically, out of 32

constructs that were cloned and tested for expression, 12 were

purified on a large scale and subjected to extensive crystal-

lization trials. However, initial efforts to obtain crystals of

these constructs, both in the presence and absence of inhibi-

tors, were unsuccessful. As a salvage approach, selected

constructs were methylated and subjected to subsequent

rounds of crystallization screening. In the end, this approach

proved to be successful and crystals were obtained of methy-

lated Plk1_36-345bound to the nonhydrolyzable ATP analogue

AMP-PNP. Interestingly, the crystal form that we obtained

independently is the same as the one subsequently reported in

the literature even though the crystallization conditions were

different.³³Remarkably, a zinc ion was observed in a crystal

contact while no zinc was intentionally added to the solution

providing these first crystals. A similar result concerning the

unexpected presence of zinc in the crystals was also reported

by Kothe and co-workers.³³Moreover, among several struc-

tures subsequently solved, a methylated lysine could be

unambiguously identified in only one structure (Lys257, data

not shown). By soaking crystals of the methylated-Plk1_36-345

:

AMP-PNP complex with compound 49, we were able to

replace the AMP-PNP with the inhibitor, as indicated by its

well-defined electron density in the structure.³⁴As expected,

49 binds in the cleft between the N- and C-terminal lobes with

Figure 4. Compound 49 bound to Plk1. Hydrogen bonds are

shown as red dashed lines.

Figure 5. Hydrogen bonding network that links compound 49 to the regulatory spine residues His105 and Phe195.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3539

the aminoquinazoline core, making three hydrogen bonds

with the main chain atoms of Glu131 and Cys133 in the hinge

region (Figure 4). The glycine-rich loop folds down tightly

over the inhibitor, with Leu59 sitting above the aromatic

group in position 8 and Cys67 above the pyrazolo ring. More

specifically, the aromatic group in position 8 is sandwiched

between Leu59 (above) and Arg136 (below) and the substit-

uents extend along the lower part of the hinge in opposite

directions. The inhibitor’s amide moiety points toward the

DFG motif of the enzyme (residues 194-196) and is part of an

extensive network of hydrogen bonds (Figure 5). The amide is

directly bound to the side chains of Asp194 and Lys82 as well

as to the main chain nitrogen of Asp194 through a bridging

water molecule (W3). Interestingly, a conserved structural

water molecule (W18)³⁵bridges together W3, the conserved

Glu101 of RC helix and Phe195 and His105, two residues

belonging to the regulatory spine of protein kinases.³⁶Not

suprisingly, the structural results, as elaborated below, are

consistent with our key SAR findings that include the im-

portance of the amide and the substituents on the aromatic

group in position 8. Specifically, the amide hydogen bonding

network and the shape of this region of the binding site can

explainthe decreasedactivity for compounds carryinganester

function (7a) or secondary amido residues (10-13) at position

3 of the template with respect to the primary amide derivative

(1a). The 2⁰-methoxy group of 49 is accommodated in a small

pocket close to the hinge residue Leu132, and this moiety is

believed to play a critical role in obtaining selectivity against

kinases bearing a residue bulkier than leucine in this position,

such as those present in Aurora-A and CDK2/A. It is worth

noting that a similar observation have been made for the

methoxy group of the inhibitor BI-2536.³⁷A contribution to

selectivity and to potency comes from the methylpiperazine

moietyatposition5⁰of the anilineresidue (compare22 with47

and 25 with 49). This substituent fits very wellin a pocket lined

mainly by residues of the kinase C-lobe, including Ser137 that

forms the “floor” of this pocket (Figure 6). The corresponding

amino acids in Aurora-A and CDK2/A are the larger Thr217

and Asp86, respectively. On the basis of the crystal structures

of Aurora-A and CDK2/A with compounds of the same

chemical class as 49 (proprietary data not shown), a clash

would be likely to occur between these residues and the

methylpiperazine substituent, explaining at least partially

the observed selectivity against these two enzymes. Moreover,

Glu140 of Plk1 is well positioned to establish a polar interac-

tion with one of the piperazine nitrogens (Figure 6). This,

together with the optimal fitting of the methylpiperazine into

its binding pocket, could explain the gain in potency given by

this substituent. Interestingly, the amino acid corresponding

to Glu140 of Plk1 is a histidine in both Plk2 and Plk3. Because

the chemical properties of histidine are likely to hamper an

interaction with a positively charged nitrogen, this feature

might be the basis of the selectivity displayed by compounds

47 and 49 for Plk1 with respect to Plk2 and Plk3. A similar

idea concerning the role of Plk1 residue Glu140 in the

selectivity versus Plk2 and Plk3 has been recently proposed

Figure 6. Binding pocket of the methylpiperazino substituent of 49,

shown as a Connolly surface. The indicated distance for the inter-

˚

action between Glu140 and 49 is in Angstroms.

Table 7. Antiproliferative Activity of Compound 49

cell line

origin

IC₅₀, ( μM)^a

cell line

A431

MCF7

origin

epithelial

mammary

IC₅₀, ( μM)^a

A2780

A2780/cis

ovary

0.009

0.011

0.057

0.014

0.016

0.022

0.044

0.025

0.066

0.078

0.010

0.044

0.004

0.073

0.005

0.008

0.003

0.005

ovary resistant to cis platinum

ovary resistant to adriamycin

ovary

A2780/ADR

A2780 1A9

A2780 1A9/PTX22

PANC-1

NCI-H929

OPM-2

NHDF

myeloma

ovary resistant to patlitaxel

pancreatic

human fibroblast

lymphoma

leukemia

KARPAS-299

Jurkat

KU812

MIA-PaCa-2

HCT-116

LoVo

colon

colon resistant to doxorubicin

HEL

erythroleukemia

LoVo/Dx

^aIC₅₀values are reported as the mean of 2-3 experiments with a coefficient of variation below 35%.

Figure 7. Flow cytometry analysis of 49 in A2780 cells. Cells were treated for 24 h with different doses of 49 (0.05 μM, 0.5 μM and 5 μM). DNA

profile of untreated (C) and Nocodazole (N) treated cells are also reported.

3540 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

based on a homology model of Plk1 bound to a thiophene

inhibitor.³⁸

growth in vitro across a wide range of solid as well as

hematological tumor cell lines. Compound 49 was also active

in resistant cell lines like A2780 human ovarian adenocarci-

noma cell line, which is resistant to cis-platinum (A2780/cis

IC₅₀= 0.011 μM), A2780 human ovary adenocarcinoma cell

On the basis of its favorable biochemical profile together

with high potency in A2780 cell lines (IC₅₀= 9 nM) and good

solubility (170 μM at pH 7), compound 49 was selected for

further in vitro and in vivo testing. In vitro antiproliferative

activity of 49 was evaluated in a broad panel of cell lines

(Table 7). Compound 49 proved to be a potent inhibitor of cell

line ,which is resistant to adriamycin (A2780/ADR IC₅₀

=

0.057 μM), or the A2780 human ovary adenocarcinoma

resistant to paclitaxel (A2780 1A9/PTX22 IC₅₀= 0.016

μM), displaying comparable activity to that found in the

parental cell line (A2780 IC₅₀= 0.009 μM and A2780 1A9

IC₅₀= 0.014 μM, respectively). The same behavior was

observed in the LoVo human colon adenocarcinoma cell line

resistant to doxorubicin (LoVo/Dx IC₅₀= 0.078 μM) com-

pared to the parental line (LoVo IC₅₀= 0.066 μM). The

mechanism of action of compound 49 was checked by flow

cytometry analysis evaluating the percentage of cells with a

4N DNA content in A2780 cells. After 24 h treatment a clear

G2/M block was observed by fluorescence activated cell

sorting (FACS) (Figure 7). Analysis of cell cycle markers on

A2780 cells at different dosages of 49 was also evaluated by

Western blot. An increase in pSer10 histone H3 and pThr199

NPM in agreement with a mitotic block mechanism was

observed in a dose-dependent manner (Figure 8). The specific

Plk1 mechanism of action of 49 was reinforced by the clear

down-regulation of phosphorylation of Ser46 in TCTP, a

reported substrate of Plk1.³⁹Apoptosis induction was eval-

uated in two different cell lines, A2780 and HCT116 cells,

which were treated attwo differentdoses of49(0.5 μM and 1.5

μM) for 24 h followed by growth of cells for additional 24 h in

the absence of compound. A G2/M cell cycle block was shown

by flow cytometry after 24 h compound treatment, while after

Figure 8. Western blot analysis of 49 on A2780 cells. Cells were

treated with 49 at two different doses (0.1 and 0.2 μM) for 24 h.

Shown are untreated (C) and Nocodazole (N) treated cells. The Plk

specific marker (pSer46 TCTP) as well as mitotic markers (pSer10

Histone H3 and pThr199 NPM) were evaluated. Total TCTP was

used as loading control.

Figure 9. Flow cytometry and Western blot analysis of A2780 (A) or HCT116 (B) cells treated for 24 h with 0.5 μM or 1.5 μM of 49 and after

additional 24 h in the absence of compound. Expression of GAPDH was used as loading control.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3541

Figure 10. Aberrant monopolar spindles upon inhibition of Plk1. Immunofluorescence pictures of representative U2OS cells during mitosis.

Control (A), siRNA targeting Plk1 (NM005030, sense strand 5⁰-CGAGCUGCUUAAUGACGAG) as assessed 72 h after transfection (B), and

treatment with 0.3 μM of compound 49 for 72 h (C). Blue, DNA; red, pSer10 histone H3; green, R-tubulin. Images were taken with an objective

40ꢀ by using a confocal laser mounted on a Zeiss microscopy (Radiance 2000, Biorad).

Table 8. In Vitro ADME Parameters of Compound 49

permeability

metabolic stability

Cl_int(L/h/kg) rat

solubility 10%

Tween 80 (mg/mL)

Caco-2

(P_app10^-6cm/s)

PAMPA^a

(P_app10^-6cm/s)

Cl_int(L/h/kg)

HLM^b(1 μM)

hepatocyte (1 μM)

2.7

29.4

50.0

12.5

0.6

^aParallel artificial membrane permeability. ^bHuman liver microsomes.

Table 9. Mean ( Standard Deviation Pharmacokinetic Parameters of Compound 49 Following iv Bolus Single Dose and Oral Single Dose in Harlan

nu/nu Mice^a

in vivo ADME parameters

PK data (iv), dose^b: 10 mg/kg

PK data (po), dose^b: 10 mg/kg

C

_max( μM)

AUC_¥( μM h)

6.51 ( 0.60

CL (L/h/kg)

3.55 ( 0.43

Vss (L/kg)

2.43 ( 0.31

t

(h)

C_max( μM)

t_max(h)

0.42 ( 0.14

AUC_¥( μM h)

1.36 ( 0.17

t

(h)

1/2

F^c(%)

13.7

3

1/2

6.07 ( 1.27

0.73 ( 0.04 0.85 ( 0.16

0.86 ( 0.17

^an = 3 animals per study. ^bDosed as HCl in situ salt/glucosate. ^cBioavailability.

an additional 24 h of recovery in the absence of compound, a

clear caspase-3 induction was observed. Also a high percent-

age of cellswithin the subG1 population, whichis indicative of

massive apoptosis, was observed at all tested doses. These

data suggest that the compound acts by an initial prolonged

mitotic block, followed by an induction of apoptosis through

activation of caspases (Figure 9A,B).

To access the phenotype of cells treated with 49, U2OS cells

were treated either with 49 or transfected with siRNA specific

for Plk1 and assessed for markers for the mitotic spindle

(R-tubulin), DNA, and pSer10 histone H3, using confocal

microscopy. Clearly, the presence of aberrant, predominantly

monopolar spindles was observed in both siRNA transfected

cells and in 49 treated cells, which is in line with the expected

Figure 11. Compound 49 shows antitumor activity (TGI = 82%)

in rodent BALB/c nu/nu mice carrying subcutaneous HCT116

human colon adenocarcinoma. Mice were treated with either vehi-

cle or 49 by oral administration (schedule 90 mg/kg 1, 2 daily ꢀ 4

cycles). Data are represented as mean ( SEM.

mechanism of action of inhibitng Plk-1, while normal bipolar

spindles were present in control cells (Figure 10).

In addition, in vitro physicochemical ADME properties

were evaluated showing that compound 49 had an excellent

metabolic stability, both in rat (Cl_int= 12.5 L/h/kg at 1 μM)

and in human liver hepatocytes (Cl_int= 0.6 L/h/kg) (Table 8).

Inhibitor 49 also displayed high permeability in PAMPA as

well as in Caco-2 cell models (P_app10^-6cm/s = 50 and 29.4,

respectively), suggesting a possible oral bioavailability for the

compound. In vivo pharmacokinetic properties were assessed

in rodent species following intravenous (iv) and oral (po)

dosing (Table 9). In mice, after iv dosing, 49 showed medium/

high clearance (3.55 L/h/kg) and a volume of distribution

about three times the mouse total body water (2.43 L/kg),

indicating a good tissue distribution. Plasma levels were

detectable up to 6 h post dosing, with a half-life of 0.7 h and

compatible to that found after iv administration and with a

modest but acceptable oral bioavailability (F = 13.7%).

In vivo efficacy of compound 49 was evaluated in male

BALB/c nu/nu mice subcutaneously xenografted with

HCT116 colon adenocarcinoma cells. The compound was

administered by oral route at 90 mg/kg, day 1-2 on a weekly

schedule ꢀ 4 cycles showing good antitumor activity (TGI_max

81%) and good tolerability (Figure 11).

Conclusion

In summary, we have reported the discovery of a new

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline class of ATP

competitive Plk1 inhibitors. Optimization of the HTS hit

an AUC of 6.51 μM h. After po dosing, the t_maxof compound

3

49 was found to be reached at 0.4 h, with a terminal half-life

3542 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

compound 1a led to 8-{[2-methoxy-5-(4-methylpiperazin-1-

yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-

quinazoline-3-carboxamide inhibitor 49. The crystal struc-

ture of methylated Plk1_36-345construct in complex with

several inhibitors together with the SAR information

acquired in the optimization of the class have been fruitful

to identify key structural components necessary to achieve

Plk1 activity and kinase selectivity. This resulted in com-

pound 49, which is active on Plk1 in the low nM range,

selective vs Plk2, Plk3, and a panel of 39 unrelated Plk

kinases, with high in vitro potency across a wide range of

solid as well as hematological tumor cell lines. The com-

pound has shown modest but acceptable oral bioavailability

resulting in a plasma exposure sufficient to achieve good

tumor growth inhibition in subcutaneous HCT-116 tumor

xenograft and it was well tolerated after repeated oral

administration.

hydrate (2.1 mL, 67.4 mmol) was added and the solution stirred at

reflux for one day. The solvent was evaporated and the residue

redissolved with DCM (20 mL). The organic layer was washed with

water (20 mL), dried over anhydrous sodium sulfate, and evapo-

rated to dryness. The crude residue was triturated with diethyl ether

1

and filtered to give the title compound (6.0 g, 70%). H NMR

(DMSO-d₆) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 2.03-2.09 (m, 2H),

2.52 (t, J = 6.1 Hz, 2H), 2.91 (t, J = 6.1 Hz, 2H), 4.29 (q, J = 7.1

Hz, 2H), 14.41 (bs, 1H). LCMS (ESI) m/z 209 (M þ H)^þ.

Ethyl 7-Oxo-1-(2,2,2-trifluoroethyl)-4,5,6,7-tetrahydro-1H-

indazole-3-carboxylate (4b). To a solution of 2 (10.0 g, 41,62

mmol) in glacial acetic acid (15 mL), trifluoroethylhydrazine

(70% in water, 6.3 mL, 50.0 mmol) was added. The mixture was

stirred at room temperature for 6 h. The solvent was evaporated

and the crude redissolved in water (20 mL), made basic with

30% NH₄OH, and extracted with DCM (2 ꢀ 50 mL). The

combined organic layers were then dried over anhydrous so-

dium sulfate and evaporated to dryness. The crude residue was

triturated with diethyl ether and filtered to give the title com-

pound (6.64 g, 55%). ¹H NMR (DMSO-d₆) δ ppm 1.31 (t, J =

7.1 Hz, 3H), 2.05-2.09 (m, 2H), 2.59 (dd, J = 6.1, 7.3 Hz, 2H),

2.95 (t, J = 6.1 Hz, 2H), 4.31 (q, J = 7.1 Hz, 2H), 5.48 (q, J =

8.8 Hz, 2H). LCMS (ESI) m/z 291 (M þ H)^þ. HRMS (ESI) calcd

for C₁₂H₁₃F₃N₂O₃þ H^þ291.0951, found 291.0941.

Experimental Section

1. Chemistry. All solvents and reagents, unless otherwise

stated, were high grade, commercially available, and used with-

out further purification. All experiments dealing with moisture-

sensitive compounds were conducted under dry nitrogen or

argon. Organic solutions were evaporated using a Heidolph

WB 2001 rotary evaporator at 15-20 mmHg. Thin-layer chro-

matography was performed on Merck silica gel 60 F₂₅₄pre-

coated plates. Column chromatography was conducted either

under medium pressure on silica (Merck silica gel 40-63 μm) or

on prepacked silica gel cartridges (Biotage). Components were

visualized by UV light (λ: 254 nm) and by iodine vapor. NMR

spectra were recorded in DMSO-d₆or CDCl₃on a Varian Inova

400 spectrometer operating at 400.50 MHz for ¹H and on a

Varian Inova 500 spectrometer operating at 499.75 MHz for ¹H

and at 125.67 MHz for ¹³C. Residual solvent signal was used as

reference (δ = 2.50 or 7.27 ppm for ¹H and δ = 39.5 ppm for ¹³C).

Chemical shifts (δ) are reported in parts per million (ppm) and

coupling constants (J) in Hz. The following abbreviations areused

for multiplicities: s = singlet, bs = broad signal, d = doublet, t =

triplet, m = multiplet, dd = doublet of doublets. Standard two-

dimensional sequences provided by Varian (gradient-enhanced

HSQC and HMBC) were used to assign carbons.

Electrospray (ESI) mass spectra were obtained on a Finnigan

LCQ ion trap. Unless otherwise specified, all final compounds

were homogeneous (purity of not less than 95%), as determined

by high-performance liquid chromatography (HPLC). HPLC-

UV-MS analyses, used to assess compound purity, were carried

out combining the ion trap MS instrument with HPLC system

SSP4000 (Thermo Separation Products) equipped with an

autosampler LC Pal (CTC Analytics) and UV6000LP diode

array detector (UV detection 215-400 nm). Instrument control,

data acquisition and processing were performed with the Xca-

libur 1.2 software (Finnigan). HPLC chromatography was run

at room temperature, and 1 mL/min flow rate, using a Waters X

Terra RP 18 column (4.6 mm ꢀ 50 mm; 3.5 μm). Mobile phase A

was ammonium acetate 5 mM buffer (pH 5.5 with acetic acid):

acetonitrile 90:10, and mobile phase B was ammonium acetate

5 mM buffer (pH 5.5 with acetic acid):acetonitrile 10:90; the

gradient was from 0 to 100% B in 7 min then hold 100% B for 2

min before requilibration. ESI(þ) high resolution mass spectra

(HRMS) were obtained on a Waters Q-Tof Ultima directly

connected with micro HPLC 1100 Agilent as previously de-

scribed.⁴⁰

Ethyl 7-Oxo-1-phenyl-4,5,6,7-tetrahydro-1H-indazole-3-car-

boxylate (4c). By employment of the above-described proce-

dure, starting from 2 and using the suitable substituted

hydrazine, the compound 4c was prepared. Yield, 36%. ¹H

NMR (DMSO-d₆) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.08-2.15

(m, 2H), 2.56 (dd, J = 5.7, 7.5 Hz, 2H), 3.03 (t, J = 6.1 Hz, 2H),

4.34 (q, J = 7.1 Hz, 2H), 7.51 (s, 5H). LCMS (ESI) m/z 285 (M þ

H)^þ; HRMS (ESI) calcd for C₁₆H₁₆N₂O₃þ H^þ285.1234, found

285.1236.

Ethyl 7-Oxo-1-trityl-4,5,6,7-tetrahydro-1H-indazole-3-car-

boxylate (4d). To a solution of 3 (2.1 g, 10.00 mmol) in DCM

(100 mL), triethylamine (1.59 mL, 11.00 mmol) and triphenyl-

methyl chloride (3.2 g, 11.00 mmol) were added. The solution

was stirred at room temperature for 6 h, then DCM (50 mL) was

added and finally washed with water (30 mL). The combined

organic layers were dried over anhydrous sodium sulfate and

evaporated to dryness. The crude residue was triturated with

diethyl ether and filtered to give the title compound (3.2 g, 72%).

¹H NMR (DMSO-d₆) δ ppm 1.25 (t, J = 7.0 Hz, 3H), 1.90-1.97

(m, 2H), 2.18 (dd, J = 5.5, 7.5 Hz, 2H), 3.00 (t, J = 6.1 Hz, 2H),

4.26 (q, J = 7.0 Hz, 2H), 6.90-7.35 (m, 15H). LCMS (ESI) m/_þz

468 (M þ NH₄)^þ. HRMS (ESI) calcd for C₂₉H₂₆N₂O₃þ NH₄

468.2282, found 468.2289.

Ethyl 6-[(Dimethylamino)methylidene]-7-oxo-1-(2,2,2-trifluoro-

ethyl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (5b). To a solu-

tion of 4b (10.0 g, 34.45 mmol) in DMF (100 mL) N,N-dimethyl-

formamide diisopropyl acetal (12.4 mL, 86.17 mmol) was added. The

reaction mixture was stirred at 60 ꢀC for 8 h. The solvent was then

evaporated in vacuo and the product crystallized from EtOH to give

1

the title compound (16.9 g, 71%). H NMR (DMSO-d₆) δ ppm

1.30 (t, J=7.1Hz, 3H), 2.85(t, J= 6.3 Hz, 2H), 2.93 (t, J=6.3Hz,

2H), 3.14 (s, 6H), 4.30 (q, J = 7.1 Hz, 2H), 5.60 (q, J = 9.0 Hz, 2H),

7.55 (s, 1H). LCMS (ESI) m/z 346 (M þ H)^þ.

Compounds 5c and 5d. By employment of the above-described

procedure, starting from 4c and 4d, the compounds 5c and 5d

were prepared.

Ethyl 6-[(Dimethylamino)methylidene]-7-oxo-1-phenyl-4,5,6,

7-tetrahydro-1H-indazole-3-carboxylate (5c). Yield, 60%. ¹H NMR

(DMSO-d₆) δ ppm 1.32 (t, J = 7.1 Hz, 3H), 2.83 (t, J = 6.3 Hz,

2H), 2.95 (t, J = 6.3 Hz, 2H), 3.12 (s, 6H), 4.30 (q, J = 7.1 Hz, 2H),

7.40 (s, 1H), 7.51 (s, 5H). LCMS (ESI) m/z 340 (M þ H)^þ.

Ethyl 6-[(Dimethylamino)methylidene]-7-oxo-1-trityl-4,5,6,7-

tetrahydro-1H-indazole-3-carboxylate (5d). Yield, 54%. ¹H

NMR (DMSO-d₆) δ ppm 1.26 (t, J = 7.1 Hz, 3 H), 2.74-2.79

(m, 2 H), 2.85-2.90 (m, 2 H), 3.10 (s, 6H), 4.24 (q, J = 7.1 Hz,

2 H), 6.90-7.30 (m, 15 H), 7.43 (s, 1 H). LCMS (ESI) m/z 506

Synthesis of compounds 1a, 2, 4a, 5a, 7a, 9-13, 15-18 was

previously described,³¹compounds 4a-c, 8a-d, 14a-d, and50-76

are commercially available.

Ethyl 7-Oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (3).

To a solution of ethyl (3-ethoxy-2-oxocyclohex-3-en-1-yl)-

(oxo)acetate 2 (10.0 g, 41.62 mmol) in ethanol (100 mL), hydrazine

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3543

(M þ H)^þ. HRMS (ESI) calcd for C₃₂H₃₁N₃O₃þ H^þ506.2444,

found 506.2439.

Compounds 1c and 1e. By employment of the above-described

procedure, starting from 7c and 7e, the compounds 1c and 1e

were prepared.

Ethyl 8-(Phenylamino)-1-(2,2,2-trifluoroethyl)-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxylate (7b). Phenyl guanidine

carbonate (2.13 g, 13.31 mmol) was added to a suspension of 5b

(3.58 g, 10.81 mmol) in DMF (30 mL) and stirred at 110 ꢀC for 6 h.

After the mixture was cooled, the solvent was evaporated and the

residue was dissolved in DCM and washed with water. The

organic layer was then dried over sodium sulfate and concen-

trated. The residue was triturated with diethyl ether and filtered to

give 7b as a white solid (4.51 g, 67%). ¹H NMR (DMSO-d₆) δppm

1.35 (t, J = 7.1 Hz, 3H), 2.89 (t, J = 7.1 Hz, 2H), 3.04 (t, J = 7.1

Hz, 2H), 4.38 (q, J = 7.1 Hz, 2H), 5.55 (q, J = 8.8 Hz, 2H),

6.89-6.94 (m 1H), 7.24-7.29 (m, 2H), 7.79-7.83 (m, 2H). LCMS

(ESI) m/z 418 (M þ H)^þ. HRMS(ESI) calcdforC₂₀H₁₈F₃N₅O₂þ

H^þ418.1486, found 418.1472.

1-Phenyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]-

quinazoline-3-carboxamide (1c). Yield, 85%. ¹H NMR (DMSO-d₆)

δ ppm 2.84-2.93 (m, 2H), 3.00-3.06 (m, 2H), 6.70-6.77 (m, 1H),

6.84-6.92 (m, 2H), 6.98-7.03 (m, 2H), 7.37 (bs, 1H), 7.50-7.63

(m, 5H), 7.63 (bs, 1H), 8.45 (s, 1H), 9.33 (s, 1H). ¹³C NMR

(DMSO-d₆) δ ppm 163.7, 158.8, 157.5, 151.9, 143.4, 140.6, 140.4,

137.4, 3 ꢀ 129.1, 2 ꢀ 128.3, 126.5, 2 ꢀ 125.2, 120.1, 118.9, 2 ꢀ 117.2,

28.9, 19.1. LCMS (ESI) m/z 383 (M þ H)^þ. HRMS (ESI) calcd for

C₂₂H₁₈N₆O þ H^þ383.1615, found 383.1626.

8-(Phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-

1

3-carboxamide (1e). Yield, 56%. H NMR (DMSO-d₆) δ ppm

2.84 (t, J = 8.0 Hz, 2H), 3.00 (t, J = 8.0 Hz, 2H), 6.93 (m, 1H),

7.25-7.34 (m, 2H), 7.34 (bs, 1H), 7.47 (bs, 1H), 7.85-7.90 (m,

2H), 8.38 (s, 1H), 13.91 (bs, 1H). ¹³C NMR (DMSO-d₆) δ ppm

163.4, 159.7, 156.9, 152.9, 141.3, 140.8, 138.4, 2 ꢀ 128.3, 122.8,

120.8, 118.8, 2 ꢀ 118.2, 23.7, 18.9. HRMS (ESI) calcd for

C₁₆H₁₄N₆O þ H^þ307.1302, found 307.1306.

Compounds 7c and 7d. By employment of the above-described

procedure, starting from 5c and 5d, the compounds 7c and 7d

were prepared.

Ethyl

1-Phenyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxylate (7c). Yield, 62%. ¹H NMR

(DMSO-d₆) δ ppm 1.34 (t, J = 7.1 Hz, 3H), 2.93 (t, J = 7.3

Hz, 2H), 3.04 (t, J = 7.3 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 6.75

(t, J = 7.19 Hz, 1H), 6.72-6.77 (m, 1H), 6.87-6.92 (m, 2H),

6.98-7.02 (m, 2H), 7.54-7.62 (m, 5H), 8.47 (s, 1H), 9.36 (s, 1H).

LCMS (ESI) m/z 412 (M þ H)^þ. HRMS (ESI) calcd for

C₂₄H₂₁N₅O₂þ H^þ412.1768, found 412.1763.

1-Cyclohexyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]-

quinazoline-3-carboxamide (1f ). Amixtureof1e (46 mg, 0.15 mmol)

in THF (4 mL) was treated with triphenylphosphine polimer-

bound (0.2 g, 0.6 mmol), di-tert-butylazadicarboxylate (80 mg,

0.45 mmol), and cyclohexanol (60 μL, 0.6 mmol) for 1 h at room

temperature. The resin was filtered off, and the solution was con-

centrated. Crystallization from diethyl ether gave the title com-

pound (14 mg, 24%). ¹H NMR (DMSO-d₆) δ ppm 1.10-1.96 (m,

10H), 2.79 (t, J = 8.0 Hz, 2H), 2.97 (t, J = 8.0 Hz, 2H), 5.50-5.59

(m, 1H), 6.97-7.02 (m 1H), 7.23 (bs, 1H), 7.28-7.34 (m, 2H), 7.34

(bs, 1H), 7.61-7.65 (m, 2H), 8.40 (s, 1H), 9.42 (s, 1H). ¹³C NMR

(DMSO-d₆) δ ppm 164.1, 159.0, 157.0, 153.1, 140.8, 140.4, 135.3, 2

ꢀ 128.6, 124.3, 121.4, 2 ꢀ 119.2, 118.9, 57.8, 2 ꢀ 32.1, 3 ꢀ 24.3, 23.7,

18.3. LCMS (ESI) m/z 389 (M þ H)^þ.HRMS (ESI) calcd for

C₂₂H₂₄N₆O þ H^þ389.2084, found 389.2084.

Ethyl 8-(Phenylamino)-1-trityl-4,5-dihydro-1H-pyrazolo[4,3-h]-

quinazoline-3-carboxylate (7d). Yield, 22%. ¹H NMR (DMSO-d₆)

δppm 1.28 (t, J= 7.1 Hz, 3H), 2.69 (t, J= 6.7 Hz, 2H), 3.01 (t, J=

6.7 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 6.83 (m, 1H), 6.96-7.06 (m,

8H), 7.14 (m, 2H), 7.19-7.28 (m, 9H), 7.41 (bs, 1H), 8.18 (s, 1H).

LCMS (ESI) m/z 578 (M þ H)^þ. HRMS (ESI) calcd for

C₃₇H₃₁N₅O₂þ H^þ578.2551, found 578.2545.

Ethyl 8-(Phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quina-

zoline-3-carboxylate (7e). Compound 7d (1.5 g, 2.66 mmol) in

DCM (50 mL) was treated with trifluoroacetic acid (5 mL). The

resulting mixture was stirred at room temperature for 1 h. DCM

(40 mL) was added, and the organic phase was washed with

saturated solution of sodium hydrogen carbonate, then with

brine, dried over sodium sulfate, and concentrated. By crystal-

lization from diisopropyl ether, the title compound was ob-

tained (0.7 g, 80%). ¹H NMR (DMSO-d₆) δ ppm 1.33 (t, J = 7.1

Hz, 3H), 2.87 (t, J = 7.4 Hz, 2H), 2.99 (t, J = 7.4 Hz, 2H), 4.33

(q, J = 7.1 Hz, 2H), 6.90-6.95 (m, 1H), 7.25-7.30 (m, 2H),

7.85-7.90 (m, 2H), 8.40 (s, 1H), 9.53 (bs, 1H), 14.17 (bs, 1H).

LCMS (ESI) m/z 336 (M þ H)^þ. HRMS (ESI) calcd for

C₁₈H₁₇N₅O₂þ H^þ336.1455, found 336.1454.

Compounds 1g-1i. By employment of the above-described

procedure, starting from 1e and using the suitable alcohol, the

compounds 1g-1i were prepared.

8-(Phenylamino)-1-(propan-2-yl)-4,5-dihydro-1H-pyrazolo[4,3-h]-

quinazoline-3-carboxamide (1g). Yield, 48%. ¹H NMR (DMSO-d₆)

δppm 1.48 (d, J= 6.6 Hz, 6H), 2.79 (t, J= 7.2 Hz, 2H), 2.97 (t, J=

7.2 Hz, 2H), 5.97 (m, 1H), 6.95-7.00(m,1H),7.26(bs,1H),7.31(m,

2H), 7.36 (bs, 1H), 7.65-7.69 (m, 2H), 8.40 (s, 1H), 9.48 (s, 1H).

LCMS (ESI) m/z 349 (M þ H)^þ. HRMS (ESI) calcd for

C₁₉H₂₀N₆O þ H^þ349.1771, found 349.178.

1-(1-Methylpiperidin-4-yl)-8-(phenylamino)-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (1h). Yield, 62%. ¹H

NMR (DMSO-d₆) δ ppm 1.85-1.97 (m, 4H), 2.10-2.14 (m,

2H), 2.18 (s, 3H), 2.76-2.82 (m, 4H), 2.96 (t, J = 7.8 Hz, 2H),

5.43-5.54 (m, 1H), 7.00-7.03 (m, 1H), 7.25 (bs, 1H), 7.31-7.35

(m, 2H), 7.36 (bs, 1H), 7.59-7.61 (m, 2H), 8.40 (s, 1H), 9.42 (s,

1H). LCMS (ESI) m/z 404 (M þ H)^þ. HRMS (ESI) calcd for

C₂₂H₂₅N₇O þ H^þ404.2193, found 404.2211.

8-(Phenylamino)-1-(2,2,2-trifluoroethyl)-4,5-dihydro-1H-pyra-

zolo[4,3-h]quinazoline-3-carboxamide (1b). Compound 7b (10.86 g,

26.04 mmol) was suspended in anhydrous EtOH (250 mL) and

treated with 1.5 M KOH in 95% EtOH (60 mL, 90 mmol). The

resulted suspension was refluxed for 2 h until an HPLC control

revealed the disapparance of the starting material. The resulting

precipitate was collected by filtration to give 10.68 g of potassium

1-methyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazo-

line-3-carboxylate (96%), which was used as such, without any

purification. A suspension of this intermediate (197 mg, 0.46 mmol)

in anhydrous DMF (5 mL) was treated with N,N-diisopropylethy-

lamine (0.12 mL, 0.69 mmol), EDC (131 mg, 0.69 mmol) and

8-(Phenylamino)-1-[2-(piperidin-1-yl)ethyl]-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (1i). Yield, 60%. ¹H

NMR (DMSO-d₆) δ ppm 1.20-1.26 (m, 6H), 2.18-2.24 (m,

4H), 2.62 (t, J = 6.6 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H), 2.97 (t,

J = 7.6 Hz, 2H), 4.89 (t, J = 6.6 Hz, 2H), 6.94-6.99 (m, 1H),

7.25 (bs, 1H), 7.28-7.33 (m, 2H), 7.41 (bs, 1H), 7.63-7.67 (m,

2H),8.40 (s, 1H), 9.44 (s, 1H). ¹³C NMR (DMSO-d₆) δ ppm

164.1, 159.2, 153.1, 156.8, 141.5, 140.8, 136.9, 2 ꢀ 128.1, 124.5,

121.1, 2 ꢀ 119.3, 118.8, 58.6, 2 ꢀ 53.8, 48.8, 2 ꢀ 25.4, 23.9, 23.7,

18.9. LCMS (ESI) m/z 418 (M þ H)^þ. HRMS (ESI) calcd for

C₂₃H₂₇N₇O þ H^þ418.235, found 418.2363.

HOBt NH₃(105 mg, 0.69 mmol). The reaction was kept at room

3

temperature overnight and then was diluted with water and the

resulting precipitate was collected by filtration to afford 1b (148 mg,

78%). ¹H NMR (DMSO-d₆) δ ppm 2.85 (t, J = 8.0 Hz, 2H), 3.02

(t, J = 8.0 Hz, 2H), 5.79 (q, ³J_HF= 8.9 Hz, 2H), 6.98-7.02 (m,

1H), 7.28-7.32 (m, 2H), 7.42 (bs, 1H), 7.46 (bs, 1H), 7.57-7.61 (m,

2H), 8.44 (s, 1H), 9.58 (bs, 1H). ¹³C NMR (DMSO-d₆) δ ppm

163.2, 159.0, 157.4, 151.5, 143.3, 140.0, 137.2, 2 ꢀ 128.8, 124.9,

123.6 (q, ¹J_CF= 280 Hz), 121.7, 2 ꢀ 119.4, 118.6, 50.6, 23.6, 18.6.

LCMS (ESI) m/z 389 (M þ H)^þ.

8-[(2-Acetylphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (22). Palladium acetate Pd(OAc)₂

(20 mg, 0.09 mmol), (()-BINAP (55 mg, 0.09 mmol), and DMF

(5 mL) were charged in a round-bottom flask flushed with argon.

The flask was evacuated and backfilled with argon. The mixture

was stirred under argon for 30 min and added to a mixture of 18

3544 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

(319 mg, 0.9 mmol), 2-acetylaniline (0.162 mL, 1.35 mmol), and

potassium carbonate (1.24 g, 8.97 mmol) in DMF (10 mL). The

resulting mixture was stirred at 80 ꢀC for 4 h under argon. After

cooling to room temperature, the reaction mixture was filtered on a

pad of celite. The solvent was concentrated, the crude solid was

purified by flash chromatography on silica gel (eluant, DCM/EtOH

90/10) to afford 153 mg (47%) of the title compound; mp 287 ꢀC.

¹H NMR (DMSO-d₆) δ ppm 2.69 (s, 3H), 2.86 (t, J = 8.0 Hz, 2H),

3.01 (t, J = 8.0 Hz,, 2H), 4.34 (s, 3H), 7.09 (ddd, J = 8.0, 7.2, 1.1

Hz, 1H), 7.27 (bs, 1H), 7.49 (bs, 1H), 7.64 (ddd, J= 8.5, 7.2, 1.1 Hz,

1H), 8.07 (dd, J = 8.0, 1.1 Hz, 1H) 8.50 (s, 1H), 8.73 (dd, J = 8.5,

1.1 Hz, 1H), 11.59 (s, 1H). ¹³C NMR (DMSO-d₆) δ ppm 203.1,

164.0, 158.2, 157.1, 153.2, 141.9, 141.0, 136.1, 134.7, 132.7, 124.8,

121.5, 120.8, 120.2, 118.7, 39.4, 28.7, 24.1, 18.7. LCMS (ESI) m/z

363 (M þ H)^þ. HRMS (ESI) calcd for C₁₉H₁₈N₆O₂þ H^þ

363.1564, found: 363.155.

1H), 8.38 (s, 1H). ¹³C NMR (DMSO-d₆) δ 164.4, 159.6, 156.2,

153.4, 150.5, 141.4, 136.6, 129.2, 124.9, 122.3, 120.2, 119.6, 119.4,

110.0, 54.6, 38.5, 23.2, 18.0. LCMS (ESI) m/z 351 (M þ H)^þ.;

HRMS (ESI) calcd for C₁₈H₁₈N₆O₂þ H^þ351.1564, found

351.1559.

8-[(3-Methoxyphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (26). Yield, 25%; mp 298 ꢀC. ¹H

NMR (DMSO-d₆) δ ppm 2.81 (t, J = 7.4 Hz, 2H), 2.98 (t, J = 7.4

Hz, 2H), 3.74 (s, 3H), 4.34 (s, 3H), 6.54 (d, J = 8.3 Hz, 1H), 7.20

(dd, J = 8.3, 8.0 Hz, 1H), 7.25 (bs, 1H), 7.33 (d, J = 8.0 Hz, 1H),

7.35 (bs, 1H), 7.45 (bs, 1H), 8.41 (s, 1H), 9.48 (s, 1H). ¹³C NMR

(DMSO-d₆) δ ppm 164.7, 160.7, 159.7, 157.0, 153.8, 142.7, 141.8,

137.2, 125.5, 119.9, 129.3, 106.3, 104,9, 104.9, 54.8, 39.3, 23.5, 18.8.

LCMS (ESI) m/z 351 (M þ H)^þ. HRMS (ESI) calcd for

C₁₈H₁₈N₆O₂þ H^þ351.1564, found 351.1566.

8-[(4-Methoxyphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (27). Yield, 62%; mp 234 ꢀC. ¹H

NMR (DMSO-d₆) δ ppm 2.79 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.2

Hz, 2H), 3.73 (s, 3H), 4.30 (s, 3H), 6.87-6.91 (m, 2H), 7.25 (bs,

Compounds 19-21, 23-49. By employment of the above-

described procedure, starting from 18 and using the suitable

substituted aniline, the compounds 19-21, 23-49 were prepared.

1-Methyl-8-{[2-(trifluoromethyl)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (19). Yield, 10%. ¹H

NMR (DMSO-d₆) δ ppm 2.79 (t, J = 7.2 Hz, 2H), 2.96 (t, J =

7.2 Hz, 2H), 4.03 (s, 3H), 7.22 (bs, 1H), 7.41 (t, J = 7.4 Hz, 1H),

7.43 (bs, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.74 (d, J = 7.4 Hz, 2H),

8.32 (s, 1H), 8.73 (s, 1H). ¹³C NMR (DMSO-d₆) δ ppm 163.9,

160.5, 157.3, 152.9, 141.0, 137.9, 136.2, 132.9, 129.4, 126.5,

125.4, 124.4, 123.9 (q, ¹J_CF= 270 Hz, 120.0, 119.1, 38.2, 23.9,

18.8). LCMS (ESI) m/z 389 (M þ H)^þ. HRMS (ESI) calcd for

C₁₈H₁₅F₃N₆O þ H^þ389.1332, found 389.132.

1H), 7.45 (s, 1H), 7.55-7.60 (m, 2H), 8.34 (s, 1H), 9.29 (s, 1H). ¹³

C

NMR (DMSO-d₆) δ ppm 164.0, 159.2, 156.6, 154.4, 152.7, 140.9,

136.4, 133.4, 124.3 2 ꢀ 120.7, 118.1, 2 ꢀ 113.2, 54.9, 39.3, 23.8, 18.7.

LCMS (ESI) m/z 351 (M þ H)^þ. HRMS (ESI) calcd for

C₁₈H₁₈N₆O₂þ H^þ351.1564, found 351.1557.

1-Methyl-8-[(2-nitrophenyl)amino]-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (28). Yield, 74%. ¹H NMR

(DMSO-d₆) δ ppm 2.84 (t, J = 7.7 Hz, 2H), 3.00 (t, J = 7.70 Hz,

2H), 4.21 (s, 3 H), 7.25 (m, 1H), 7.27 (bs, 1H), 7.47 (bs, 1H), 7.73

(m, 1 H), 8.08 (m, 1H), 8.16 (m, 2H), 8.44 (s, 1H), 10.02 (s, 1 H).

¹³C NMR (DMSO-d₆) δ ppm 163.9, 158.0, 156.9, 152.9, 140.9,

139.7, 135.9, 134.9, 134.3, 124.9, 124.6, 123.6, 122.3, 120.8, 39.0,

23.4, 18.5. LCMS (ESI) m/z 366 (M þ H)^þ. HRMS (ESI) calcd

for C₁₇H₁₅N₇O₃þ H^þ366.1309, found 366.1295.

1-Methyl-8-{[3-(trifluoromethyl)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (20). Yield, 25%. ¹H

NMR (DMSO-d₆) δ ppm 2.84 (t, J = 7.2 Hz, 2H), 3.00 (t, J =

7.2 Hz, 2H), 4.32 (s, 3H), 7.26 (bs, 1H), 7.28 (d, J = 8.6 Hz, 1H),

7.46 (bs, 1H), 7.53 (t, J= 8.6 Hz, 1H), 7.92 (d, J= 8.6 Hz, 1H), 8.19

(s, 1H), 8.47 (s, 1H), 9.87 (s, 1H). ¹³C NMR (DMSO-d₆) δ 163.9,

158.6, 156.7, 152.9, 141.6, 141.1, 136.4, 130.3, 129.3, 125.5 (q,

¹J_CF= 270 Hz), 124.9, 122.0, 119.9, 116.6, 114.2, 39.2, 24.1, 18.2.

LCMS (ESI) m/z 389 (M þ H)^þ.

1-Methyl-8-[(3-nitrophenyl)amino]-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (29). Yield, 64%. ¹H NMR

(DMSO-d₆) δ ppm 2.85 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.6 Hz,

2H), 4.36 (s, 3H), 7.27 (bs, 1H), 7.48 (bs, 1H), 7.59 (t, J = 8.2

Hz, 1H), 7.80 (dd, J = 8.2, 1.9 Hz, 1H), 8.02 (dd, J = 8.2, 1.0

Hz, 1H), 8.51 (s, 1H), 8.82 (dd, J = 1.9, 1.0 Hz, 1H), 10.08 (s,

1H). ¹³C NMR (DMSO-d₆) δ ppm 163.8, 158.6, 156.8, 153.2,

148.4, 142.0, 140.8, 136.2, 129.3, 124.7, 123.8, 120.3, 115.0,

111.6, 39.2, 23.5, 18.3. LCMS (ESI) m/z 366 (M þ H)^þ. HRMS

(ESI) calcd for C₁₇H₁₅N₇O₃þ H^þ366.1309, found 366.1294.

1-Methyl-8-[(4-nitrophenyl)amino]-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (30). Yield, 63%. ¹H NMR

(DMSO-d₆) δ ppm 2.87 (t, J = 7.3 Hz, 2H), 3.01 (t, J = 7.3 Hz,

2H), 4.37 (s, 3H), 7.30 (bs, 1H), 7.54 (bs, 1H), 7.97-8.00 (m,

2H), 7.22-7.25 (m, 2H), 8.54 (s, 1H), 10.39 (s, 1H). LCMS (ESI)

m/z 366 (M þ H)^þ. HRMS (ESI) calcd for C₁₇H₁₅N₇O₃þ H^þ

366.1309, found 366.1303.

1-Methyl-8-{[4-(trifluoromethyl)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (21). Yield, 9.5%.

¹H NMR (DMSO-d₆) δ ppm 2.84 (t, J = 7.2 Hz, 2H), 3.00 (t,

J = 7.2 Hz, 2H), 4.35 (s, 3H), 7.26 (bs, 1H), 7.48 (bs, 1H), 7.65 (m,

2H), 7.95 (m, 2H), 8.48 (s, 1H), 9.97 (s, 1H). ¹³C NMR (DMSO-d₆)

δ ppm 164.2, 158.9, 157.1, 153.3, 144.7, 141.3, 136.6, 2 ꢀ 125.7,

124.9, 124.5 (q, ¹J_CF= 270 Hz), 121.5, 120.5, 2 ꢀ 118.1, 39.8, 23.9,

18.6. LCMS (ESI) m/z 389 (M þ H)^þ. HRMS (ESI) calcd for

C₁₈H₁₅F₃N₆O þ H^þ389.1332, found 389.1317.

8-[(3-Acetylphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (23). Yield, 8%; ¹H NMR

(DMSO-d₆) δ ppm 2.57 (s, 3H), 2.83 (t, J = 7.2 Hz, 2H), 2.99

(t, J = 7.2 Hz, 2H), 4.33 (s, 3H), 7.26 (bs, 1H), 7.46 (t, J =

7.8 Hz, 1H), 7.47 (bs, 1H), 7.59 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H),

7.94 (ddd, J = 7.8, 2.2, 1.0 Hz, 1H), 8.32 (t, J = 2.2, 1.6 Hz,

1H), 8.44 (s, 1H), 9.73 (s, 1H). ¹³C NMR (DMSO-d₆) δ ppm

198.3, 163.4, 158.8, 156.8, 153.0, 141.0, 140.9, 137.6, 136.3,

129.0, 124.6, 123.6, 121.7, 119.5, 118.0, 39.5, 26.7, 24.0, 18.9.

LCMS (ESI) m/z 363 (M þ H)^þ. HRMS (ESI) calcd for

C₁₉H₁₈N₆O₂þ H^þ363.1564, found 363.1548.

1-Methyl-8-[(2-methylphenyl)amino]-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (31). Yield, 7%. ¹H NMR

(DMSO-d₆) δ ppm 2.24 (s, 3H), 2.78 (t, J = 7.3 Hz, 2H), 2.96

(t, J = 7.3 Hz, 2H), 4.11 (s, 3H), 7.05 (m, 1H), 7.17 (m, 1H), 7.21

(bs, 1H), 7.22 (bs, 1H), 7.22 (m, 1H), 7.42 (bs, 1H), 7.50 (m, 1H),

8.31 (s, 1H), 8.73 (s, 1H). LCMS (ESI) m/z 335 (M þ H)^þ. HRMS

(ESI) calcd for C₁₈H₁₈N₆O þ H^þ335.1615, found 335.1614.

1-Methyl-8-{[2-(methylsulfanyl)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (32). Yield, 52%; mp

8-[(4-Acetylphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (24). Yield, 8%. ¹H NMR

(DMSO-d₆) δ ppm 2.52 (s, 3H), 2.85 (t, J = 7.4 Hz, 2H), 3.00

(t, J = 7.4 Hz, 2H), 4.37 (s, 3H), 7.26 (bs, 1H), 7.48 (bs, 1H),

7.85-7.90 (m, 2H), 7.91-7.95 (m, 2H), 8.49 (s, 1H), 9.99 (s, 1H).

LCMS (ESI) m/z 363 (M þ H)^þ. HRMS (ESI) calcd for

C₁₉H₁₈N₆O₂þ H^þ363.1564, found 363.1559.

1

215 ꢀC. H NMR (DMSO-d₆) δ ppm 2.39 (s, 3H), 2.79 (t, J =

7.3 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 4.12 (s, 3H), 7.15-7.25 (m,

2H), 7.22 (bs, 1H), 7.36-7.40 (m, 1H), 7.43 (bs, 1H), 7.60-7.63

(m, 1H), 8.32 (s, 1H), 8.63 (bs, 1H). ¹³C NMR (DMSO-d₆) δ ppm

163.7, 159.8, 157.1, 152.9, 140.9, 137.5, 136.1, 133.3, 127.3, 125.3,

125.2, 125.2, 124.1, 118.4, 39.3, 23.8, 19.3, 15.1. LCMS (ESI) m/z

367 (M þ H)^þ. HRMS (ESI) calcd for C₁₈H₁₈N₆OS þ H^þ

367.1336, found 367.1346.

8-[(2-Methoxyphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (25). Yield, 77%; mp 256 ꢀC. ¹H

NMR (DMSO-d₆) δ ppm 2.81 (t, J = 7.7 Hz, 2H), 2.98 (t, J = 7.7

Hz, 2H), 3.85 (s, 3H), 4.25 (s, 3H), 6.92-6.99 (m, 1H), 7.00-7.09

(m, 2H), 7.24 (br s, 1H), 7.45 (br s, 1H), 8.01-8.05 (m, 1H), 8.13 (s,

1-Methyl-8-{[2-(methylamino)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (33). Yield, 30%; mp

256 ꢀC. ¹H NMR (DMSO-d₆) δ ppm 2.71 (d, J = 5 Hz, 3H), 2.76

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3545

(t, J = 7.8 Hz, 2H), 2.95 (t, J = 7.8 Hz, 2H), 4.09 (s, 3H), 5.05 (q,

J = 5 Hz, 1H), 6.56-6.61 (m, 2H), 7.01-7.06 (m, 1H), 7.21 (bs,

1H), 7.24-7.27 (m, 1H), 7.42 (bs, 1H), 8.28 (s, 1H), 8.40 (s, 1H).

¹³C NMR (DMSO-d₆) δ 164.1, 160.6, 156.7, 153.0, 144.8, 141.2,

136.6, 125.5, 125.5, 125.4, 124.2, 117.9, 114.9, 109.4, 38.7, 29.5,

23.7, 18.9. LCMS (ESI) m/z 350 (M þ H)^þ. HRMS (ESI) calcd

for C₁₈H₁₉N₇O þ H^þ350.1724, found: 350.1714.

m/z 413 (M þ H)^þ. HRMS (ESI) calcd for C₂₃H₂₀N₆O₂þ H^þ

413.172, found 413.1711.

8-[(2-Benzylphenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (41). Yield, 33%. ¹H NMR

(DMSO-d₆) δ ppm 2.76 (t, J = 7.3 Hz, 2H), 2.95 (t, J = 7.3 Hz,

2H), 4.02 (s, 2H), 4.06 (s, 3H), 7.07-7.25 (m, 9H), 7.42 (bs, 1H),

7.53 (dd, J = 7.9, 0.8 Hz, 1H), 8.28 (s, 1H), 8.69 (bs, 1H). LCMS

(ESI) m/z411 (M þ H)^þ. HRMS(ESI) calcdforC₂₄H₂₂N₆Oþ H^þ

411.1928, found 411.1931.

8-[(2-Fluorophenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (34). Yield, 13%. ¹H NMR

(DMSO-d₆) δ ppm 2.80 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.2

Hz, 2H), 4.16 (s, 3H), 7.10-7.27(m, 3H), 7.23 (bs, 1H), 7.44 (bs,

1H), 7.70-7.76 (m, 1H), 8.36 (s, 1H), 9.08 (bs, 1H). ¹³C NMR

1-Methyl-8-{[2-(phenylamino)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (42). Yield, 22%. H

1

NMR (DMSO-d₆) δ ppm 2.79 (t, J = 7.3 Hz, 2H), 2.97 (t, J =

7.3 Hz, 2H), 4.21 (s, 3H), 6.73-6.78 (m, 1H), 6.85-6.90 (m, 2H),

6.99-7.07 (m, 2H), 7.13-7.19 (m, 2H), 7.23 (bs, 1H), 7.25-7.28

(m, 1H), 7.44 (bs, 1H), 7.82-7.87 (m, 1H), 8.35 (s, 1H), 8.44 (bs,

1H). ¹³C NMR (DMSO-d₆) δ 163.6, 159.4, 156.8, 152.8, 144.5,

140.8, 136.2, 135.0, 131.8, 2 ꢀ 128.8, 124.3, 124.1, 123.9, 122.0,

120.6, 118.9, 118.5, 2 ꢀ 115.9, 39.0, 23.8, 18.7. LCMS (ESI) m/z

412 (M þ H)^þ. HRMS (ESI) calcd for C₂₃H₂₁N₇O þ H^þ

412.188, found: 412.1867.

1

(DMSO-d₆) δ 164.3, 159.6, 157.0, 155.8 (d, J_CF= 160 Hz),

153.1, 141.2, 136.4, 128.0, 125.3, 124.6, 124.4, 123.7, 119.0, 115.1,

38.9, 23.8, 18.6. LCMS (ESI) m/z 339 (M þ H)^þ. HRMS (ESI)

calcd for C₁₇H₁₅FN₆O þ H^þ339.1364, found 339.1348.

1-Methyl-8-{[2-(propan-2-yl)phenyl]amino}-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (35). Yield, 17%. ¹H

NMR (DMSO-d₆) δ ppm 1.13 (d, J = 7.0 Hz, 6H), 2.76 (t, J =

7.8 Hz, 2H), 2.95 (t, J = 7.8 Hz, 2H), 3.25 (m, 1H), 4.05 (s, 3H),

7.15-7.22 (m, 2H), 7.20 (bs, 1H), 7.30-7.36 (m, 2H), 7.41 (bs,

1H), 8.26 (s, 1H), 8.74 (bs, 1H). ¹³C NMR (DMSO-d₆) δ 163.8,

161.1, 157.1, 152.9, 144.3, 140.9, 136.9, 136.3, 127.3, 125.5,

125.5, 125.2, 124.2, 117.7, 38.7, 26.8, 23.8, 2 ꢀ 22.7, 18.7.

LCMS (ESI) m/z 363 (M þ H)^þ. HRMS (ESI) calcd for

C₂₀H₂₂N₆O þ H^þ363.1928, found 363.1913.

1-Methyl-8-{[2-(phenylcarbonyl)phenyl]amino}-4,5-dihydro-

1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (43). Yield, 55%. ¹H

NMR (DMSO-d₆) δ ppm 2.79 (t, J = 7.3 Hz, 2H), 2.98 (t, J = 7.3

Hz, 2H), 4.26 (s, 3H), 7.14 (ddd, J = 8.4, 7.8, 1.1 Hz, 1H), 7.26 (bs,

1H), 7.45-7.66 (m, 8H), 8.23 (dd, J = 8.4, 0.7 Hz, 1H), 8.27 (s,

1H), 10.27 (s, 1H). LCMS (ESI) m/z 425 (M þ H)^þ. HRMS (ESI)

calcd for C₂₄H₂₀N₆O₂þ H^þ425.172, found 425.1715.

Methyl 2-[(3-Carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazolin-8-yl)amino]benzoate (36). Yield, 18%. ¹H

NMR (DMSO-d₆) δ ppm 2.86 (t, J = 7.9 Hz, 2H), 3.01 (t, J = 7.9

Hz, 2H), 3.87 (s, 3H), 4.33 (s, 3H), 7.07 (dd, J = 8.7, 7.9 Hz, 1H),

7.27 (bs, 1H), 7.48 (bs, 1H), 7.64 (dd, J = 8.2, 7.9 Hz, 1H), 7.99 (d,

J = 8.2 Hz, 1H), 8.50 (s, 1H), 8.67 (d, J = 8.7 Hz, 1H), 10.73 (s,

1H). LCMS (ESI) m/z 379 (M þ H)^þ. HRMS (ESI) calcd for

C₁₉H₁₈N₆O₃þ H^þ379.1513, found 379.1517.

1-Methyl-8-{[2-(phenylsulfanyl)phenyl]amino}-4,5-dihydro-

1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (44). Yield, 44%. ¹H

NMR (DMSO-d₆) δ ppm 2.80 (t, J = 7.3 Hz, 2H), 2.97 (t, J =

7.3 Hz, 2H), 4.18 (s, 3H), 7.13 (td, J = 7.8, 1.5 Hz, 1H), 7.18-7.23

(m, 3H), 7.24(bs, 1H), 7.26-7.32 (m, 2H), 7.41-7.45(m, 2H), 7.46

(bs, 1H), 8.00 (dd, J = 8.7, 1.5 Hz, 1H), 8.35 (s, 1H), 8.64 (s, 1H).

¹³C NMR (DMSO-d₆) δ 163.8, 159.1, 156.9, 153.1, 141.0, 140.6,

136.1, 135.7, 134.1, 2 ꢀ 129.4, 129.1, 2 ꢀ 128.7, 126.7, 125.7, 124.5,

124.4, 123.2, 119.5, 39.1, 23.9, 18.6. LCMS (ESI) m/z 429 (M þ

H)^þ. HRMS (ESI) calcd for C₂₃H₂₀N₆OS þ H^þ429.1492, found

429.1505.

8-[(2-Carbamoylphenyl)amino]-1-methyl-4,5-dihydro-1H-pyra-

zolo[4,3-h]quinazoline-3-carboxamide (37). Yield, 25%. ¹H NMR

(DMSO-d₆) δ ppm 2.84 (t, J = 7.3 Hz, 2H), 3.00 (t, J = 7.3 Hz,

2H), 4.33 (s, 3H), 7.01 (m, 1H), 7.26 (bs, 1H), 7.47 (bs, 1H), 7.50

(m, 1H), 7.62 (bs, 1H), 7.78 (dd, J = 7.8, 1.5 Hz, 1H), 8.19 (bs,

8-{[2-Acetyl-3-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (45).

Yield, 36%. ¹H NMR (DMSO-d₆) δ ppm 2.22 (s, 3H), 2.40-2.48

(m, 4H), 2.56 (s, 3H), 2.80 (t, J = 7.3 Hz, 2H), 2.89-2.94 (m, 4H),

2.97 (t, J = 7.3 Hz, 2H), 4.22 (s, 3H), 6.94 (d, J = 8.8 Hz, 1H),

7.24 (bs, 1H), 7.39 (dd, J =8.8, 7.8Hz), 7.45 (bs, 1H), 7.70(d, J =

7.8 Hz, 1H), 8.35 (s, 1H), 9.03 (s, 1H). ¹³C NMR (DMSO-d₆) δ

ppm 206.2, 164.7, 158.9, 156.7, 153.3, 152.1, 141.1, 137.2, 136.2,

130.7, 127.7, 124.6, 119.5, 117.8, 114.1, 2 ꢀ 54.5, 2 ꢀ 52.5, 45.4,

39.2, 30.9, 24.1, 18.8. LCMS (ESI) m/z 461 (M þ H)^þ. HRMS

(ESI) calcd for C₂₄H₂₈N₈O₂þ H^þ461.2408, found 461.2417.

8-{[2-Acetyl-4-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (46).

1H), 8.45 (s, 1H), 8.61 (dd, J = 8.5, 0.7 Hz, 1H), 11.45 (s, 1H). ¹³

C

NMR (DMSO-d₆) δ ppm 171.5, 163.8, 158.5, 156.8, 152.6, 141.3,

140.9, 136.1, 131.6, 128.7, 124.5, 119.9, 119.8, 119.0, 118.9, 39.4,

23.5, 17.4. LCMS (ESI) m/z 364 (M þ H)^þ. HRMS (ESI) calcd

for C₁₈H₁₇N₇O₂þ H^þ364.1516, found 364.1516.

1-Methyl-8-[(2-sulfamoylphenyl)amino]-4,5-dihydro-1H-pyra-

zolo[4,3-h]quinazoline-3-carboxamide (38). Yield, 22%. ¹H NMR

(DMSO-d₆) δ ppm 2.86 (t, J = 7.3 Hz, 2H), 3.01 (t, J = 7.3 Hz,

2H), 4.29 (s, 3H), 7.14 (ddd, J= 8.0, 7.4, 1.0 Hz, 1H), 7.26 (bs, 1H),

7.48 (bs, 1H), 7.56 (ddd, J = 8.4, 7.4, 1.6 Hz, 1H), 7.84 (dd, J =

8.0, 1.6 Hz, 1H), 8.46 (dd, J=8.4, 1.0Hz, 1H), 8.48(s, 1H). LCMS

(ESI) m/z 400 (M þ H)^þ. HRMS (ESI) calcd for C₁₇H₁₇N₇O₃S þ

H^þ400.1186, found 400.1188.

1

Yield, 50%; mp 242 ꢀC. H NMR (DMSO-d₆) δ ppm 2.26 (s,

3H), 2.47-2.55 (signal obscured by DMSO, 4H), 2.65 (s, 3H),

2.82 (t, J = 7.7 Hz, 2H), 2.99 (t, J = 7.7 Hz, 2H), 3.12-3.19 (m,

4H), 4.31 (s, 3H), 7.25 (br s, 1H), 7.32 (dd, J = 9.1, 2.7 Hz, 1H),

7.43 (d, J = 2.7 Hz, 1H), 7.47 (bs, 1H), 8.42 (s, 1H), 8.48 (d, J =

9.1 Hz, 1H), 11.03 (s, 1H). ¹³C NMR (DMSO-d₆) δ ppm 203.2,

163.9, 158.2, 157.0, 153.2, 145.3, 140.7, 136.3, 133.8, 124.6,

123.4, 122.5, 120.6, 119.9, 117.8, 2 ꢀ 54.5, 2 ꢀ 48.4, 45.7, 39.3,

28.6, 24.0, 18.6. LCMS (ESI) m/z 461 (M þ H)^þ. HRMS (ESI)

calcd for C₂₄H₂₈N₈O₂þ H^þ461.2408, found 461.2411.

8-(Biphenyl-2-ylamino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]-

quinazoline-3-carboxamide (39). Yield, 30%. ¹H NMR (DMSO-d₆)

δ ppm 2.73 (t, J = 7.2 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H), 4.10 (s,

3H), 7.22 (bs, 1H), 7.23-7.43 (m, 9H), 7.71 (dd, J = 8.0, 1.1 Hz,

1H), 8.23 (s, 1H), 8.38 (bs, 1H). ¹³C NMR (DMSO-d₆) δ ppm

163.9, 160.1, 157.2, 152.7, 141.1, 139.7, 137.0, 136.6, 136.4, 130.2,

2 ꢀ 128.8, 2 ꢀ 128.2, 128.2, 126.8, 126.1, 124.8, 124.3, 118.2, 38.9,

23.9, 18.5. LCMS (ESI) m/z 397 (M þ H)^þ. HRMS (ESI) calcd

for C₂₃H₂₀N₆O þ H^þ397.1771, found 397.1778.

1-Methyl-8-[(2-phenoxyphenyl)amino]-4,5-dihydro-1H-pyra-

zolo[4,3-h]quinazoline-3-carboxamide (40). Yield, 62%.; ¹H

NMR (DMSO-d₆) δ ppm 2.79 (t, J = 7.3 Hz, 2H), 2.96 (t, J =

7.3 Hz, 2H), 4.23 (s, 3H), 6.93-6.99 (m, 3H), 7.06-7.11 (m, 2H),

7.18 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.24 (bs, 1H), 7.31-7.36 (m,

2H), 7.44 (bs, 1H), 8.01 (dd, J = 7.9, 1.6 Hz, 1H), 8.33 (s, 1H),

8.50 (s, 1H). ¹³C NMR (DMSO-d₆) δ 163.8, 158.7, 157.4, 157.1,

152.8, 147.8, 140.8, 135.9, 131.3, 2 ꢀ 130.3, 124.4, 124.0, 124.0,

123.9, 123.9, 119.5, 118.9, 2 ꢀ 118.1, 39.7, 24.3, 19.3. LCMS (ESI)

8-{[2-Acetyl-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (47).

Yield, 45%. ¹H NMR (DMSO-d₆) δ ppm 2.23 (s, 3H),

2.42-2.47 (m, 4H), 2.54 (s, 3H), 2.85 (t, J = 7.3 Hz, 2H), 3.00

(t, J = 7.7 Hz, 2H), 3.35-3.40 (m, 4H), 4.33 (s, 3H), 6.60 (dd,

J = 9.1, 2.7 Hz, 1H), 7.27 (bs, 1H), 7.48 (bs, 1H), 7.85 (d, J = 9.1

Hz, 1H), 8.30 (d, J = 2.7 Hz, 1H), 8.52 (s, 1H), 12.12 (s, 1H). ¹³

C

NMR (DMSO-_d6) δ ppm 200.0, 164.8, 158.4, 156.1, 155.1,

153.6, 144.4, 141.1, 136.2, 133.9, 125.3, 120.7, 111.9, 105.4,

3546 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

100.6, 2 ꢀ 54.2, 2 ꢀ 46.0, 45.3, 38.9, 27.4, 24.0, 18.2. LCMS

(ESI) m/z 461 (M þ H)^þ. HRMS (ESI) calcd for C₂₄H₂₈N₈O₂þ

H^þ461.2408, found 461.2394.

3H), 2.07 (s, 3H), 2.83 (t, J = 7.4 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H),

4.18 (s, 3H), 4.28 (q, J = 7.1 Hz, 1H), 7.05-7.11 (m, 1H),

7.14-7.20 (m, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 7.7

Hz, 1H), 8.37 (s, 1 H), 8.58 (bs, 1H), 9.62 (bs, 1 H). HRMS (ESI)

calcd for C₂₁H₂₂N₆O₃þ H^þ336.1567, found: 336.1582.

8-{[2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (48).

Yield, 11%; mp 127 ꢀC. ¹H NMR (DMSO-d₆) δ ppm 2.30 (br s,

3H), 2.52-2.62 (m, 4H), 2.77 (t, J = 7.7 Hz, 2H), 2.96 t, J =

7.7 Hz, 2H), 3.10-3.20 (m, 4H), 3.80 (s, 3H), 4.22 (s, 3H), 6.50

(dd, J = 8.8, 2.5 Hz, 1H), 6.63 (d, J = 2.5 Hz, 1H), 7.23 (br s,

1H), 7.43 (s, 1H), 7.65 (d, J = 8.8 Hz, 1H), 8.00 (s, 1H), 8.28 (s,

1H). ¹³C NMR (DMSO-d₆) δ ppm 164.9, 160.0, 157.1, 153.3,

152.4, 149.0, 141.4, 136.8, 124.9, 123.7, 121.0, 118.4, 106.3,

100.0, 55.7, 2 ꢀ 54.5, 2 ꢀ 48.9, 45.8, 39.2, 24.3, 18.9. LCMS

(ESI) m/z 449 (M þ H)^þ. HRMS (ESI) calcd for C₂₃H₂₈N₈O₂þ

H^þ449.2408, found 449.2398.

8-[(2-Aminophenyl)amino]-1-methyl-4,5-dihydro-1H-pyrazolo-

[4,3-h]quinazoline-3-carboxamide (80). To a suspension of 78

(65 mg, 0.18 mmol) in EtOH (10 mL) 30% NH₄OH (20 mL)

was added. The mixture was maintained at 70 ꢀC under stirring

for 12 h in a sealed bottle. The solvent was then evaporated to

dryness and the crude was purified by reverse phase chromatog-

raphy to give 80 (10 mg, 17%) of the final compound. ¹H NMR

(DMSO-d₆) δ ppm 2.7 (t, J = 7.3 Hz, 2H), 2.96 (t, J = 7.7 Hz,

2H), 4.15 (s, 3H), 4.84 (bs, 2H), 6.57 (td, J = 7.9, 1.5 Hz, 1H),

6.74 (dd, J = 7.9, 1.5 Hz, 1H), 6.88 (td, J = 7.9, 1.5 Hz, 1H), 7.21

(bs, 1 H), 7.32 (dd, J = 7.9, 1.5 Hz, 1H), 7.42 (bs, 1H), 8.29 (s,

1H), 8.47 (s, 1H). ¹³C NMR (DMSO-d₆) δ 164.2, 160.3, 155.9,

153.3, 142.7, 140.9, 136.5, 125.3, 125.2, 125.1, 124.4, 124.2, 117.9,

115.7, 38.3, 23.8, 18.7. LCMS (ESI) m/z 336 (M þ H)^þ. HRMS

(ESI) calcd for C₁₇H₁₇N₇O þ H^þ336.1567, found 336.1582.

8-{[2-(Acetylamino)phenyl]amino}-1-methyl-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxamide (81). By employment

of the above-described procedure, starting from 79, the com-

8-{[2-Methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-

4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (49).

1

Yield, 25%; mp 133 ꢀC. H NMR (DMSO-d₆) δ ppm 2.22 (s,

3H), 2.43-2.49 (m, 4H), 2.80 (t, J = 7.7 Hz, 2H), 2.98 (t, J =

7.7 Hz, 2H), 2.99-3.03 (m, 4H), 3.78 (s, 3H), 4.26 (s, 3H), 6.58

(dd, J = 8.9, 2.9 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H), 7.25 (bs.,

1H), 7.45 (bs, 1H), 7.72 (d, J = 2.9 Hz, 1H), 8.06 (s, 1H), 8.39 (s,

1H). ¹³C NMR (DMSO-d₆) δ ppm 163.6, 158.9, 157.2, 152.9,

145.7, 143.8, 140.9, 136.1, 129.0, 124.4, 118.9, 111.3, 110.7,

110.1, 56.2, 2 ꢀ 54.7, 2 ꢀ 49.9, 45.9, 39.2, 23.8, 18.8. LCMS

(ESI) m/z 449 (M þ H)^þ. HRMS (ESI) calcd for C₂₃H₂₈N₈O₂þ

H^þ449.2408, found 449.24.

1

pound 81 was prepared. Yield, 64%. H NMR (DMSO-d₆) δ

ppm: 2.07 (s, 3H), 2.83 (t, J = 7.4 Hz, 2H), 2.97 (t, J = 7.4 Hz,

2H), 4.16 (s, 3H), 7.06-7.12 (m, 1H), 7.14-7.20 (m, 1H), 7.23

(bs, 1H), 7.44 (bs, 1H), 7.47 (dd, J = 8.2, 1.2 Hz, 1H), 7.80 (dd,

J = 7.9, 1.2 Hz, 1H), 8.35 (s, 1H), 8.56 (bs, 1 H), 9.62 (bs, 1H).

LCMS (ESI) m/z 378 (M þ H)^þ. HRMS (ESI) calcd for

C₁₉H₁₉N₇O₂þ H^þ378.1673, found: 378.1679.

Ethyl 8-({2-[(tert-Butoxycarbonyl)amino]phenyl}amino)-1-

methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate

(77). Palladium acetate [Pd(OAc)₂] (101 mg, 0.45 mmol), (()-

BINAP (280 mg, 0.45 mmol), and DMF (65 mL) were charged to a

round-bottom flask flushed with argon. The flask was evacuated

and backfilled with argon. The mixture was stirred under argon for

30 min and added to a mixture of N-Boc-o-phenylendiamine (2.6 g,

12.52 mmol), compound 16 (1.6 g, 4.16 mmol), and potassium

carbonate (5.74 g, 41.53 mmol) in DMF (50 mL). The resulting

mixture was stirred at 70 ꢀC for 6 h under argon. After cooling to

room temperature, the reaction mixture was filtered on a pad of

celite. The solvent was concentrated, the crude solid was purified by

flash chromatography on silica gel (eluant, hexane/ethyl acetate 60/

40) to afford the title compound (1.18 g, 61%). ¹H NMR (DMSO-

d₆) δ ppm 1.30 (t, J = 7.1 Hz, 3H), 1.44 (s, 9H), 2.83 (t, J = 7.3 Hz,

2H), 2.96 (t, J = 7.7 Hz, 2H), 4.18 (s, 3H), 4.28 (q, J = 7.7 Hz, 2H),

7.06-7.15 (m, 2 H), 7.47-7.52 (m, 1H), 7.68-7.71 (m, 1H), 8.36 (s,

1H), 8.64 (s, 1H), 8.68 (bs, 1H). LCMS (ESI) m/z 465 (M þ H)^þ.

HRMS (ESI) calcd for C₂₄H₂₈N₆O₄þ H^þ465.2245, found

465.2238.

N-(4-Bromo-2-methoxyphenyl)acetamide (83a). A mixture of

4-bromo-2-methoxy-phenylamine 82a (2.00 g, 9.90 mmol) in

acetic anhydride (2 mL, 21.15 mmol) was stirred at room

temperature overnight. The solvent was evaporated to dryness,

and the solid was tritured with diethyl ether and filtered to give

the title compound (1.26 g, 51%). ¹H NMR (DMSO-d₆) δ ppm

2.07 (s, 3H), 3.84 (s, 3H), 7.07 (dd, J = 8.5, 2.2 Hz, 1H), 7.20 (d,

J = 2.2 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 9.17 (bs, 1H). LCMS

(ESI) m/z 245 (M þ H)^þ.

N-(2-Acetyl-4-bromophenyl)acetamide (83b). By employment

of the above-described procedure, starting from 82b, the com-

pound 83b was prepared. Yield, 42%. ¹H NMR (DMSO-d₆) δ

ppm 2.10 (s, 3H), 2.60 (s, 3H), 7.75 (dd, J = 8.9, 2.4 Hz, 1H),

8.04 (d, J = 2.4 Hz, 1H), 8.11 (d, J = 8.9 Hz, 1H), 10.94 (s, 1 H).

LCMS (ESI) m/z 257 (M þ H)^þ.

N-[2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl]acetamide (84a).

Pd₂(dba)₃(47 mg, 0.05 mmol), 2-dicyclohexylphosphino-2⁰-(N,N-

dimethylamino)-biphenyl (39 mg, 0.02 mmol), and 83a (1.26 g,

5.16 mmol) in THF (5 mL) were charged in a round-bottom

flask flushed with argon. The flask was evacuated and backfilled

with argon. LiN(TMS)₂solution (1 M in THF, 11.5 mL) and

4-methylpiperazine (0.67 mL, 6.2 mmol) were added, and the

reaction refluxed for 3 h. The reaction mixture was then allowed

to cool to room temperature and concentrated. The crude solid was

purified by flash chromatography on silica gel (eluant, DCM/

MeOH 90/10) to afford compound 84a (1.26 g, 91%). ¹H NMR

(DMSO-d₆) δ ppm 2.01 (s, 3H), 2.26 (bs, 3H), 2.46-2.54 (signal

oscured by DMSO, 4H), 3.07-3.15 (m, 4H), 3.80 (s, 3H), 6.42 (dd,

J = 8.7, 2.5 Hz, 1H), 6.58 (d, J = 2.5 Hz, 1H), 7.58 (d, J = 8.66,

1H), 8.88 (s, 1H). LCMS (ESI) m/z 264 (M þ H)^þ.

Ethyl 8-[(2-aminophenyl)amino]-1-methyl-4,5-dihydro-1H-

pyrazolo[4,3-h]quinazoline-3-carboxylate (78). To a solution of 77

(0.85 g, 1.83 mmol) in DCM (50 mL) HCl 4 N in dioxane (30 mL)

was added. The solution was stirred at room temperature for 2 h

and then the solvent removed in vacuo. The residue was crystal-

lized from diethyl ether to give compound 78 (0.70 g, 96%) of the

title compound. ¹H NMR (DMSO-d₆) δ ppm (as free base) 1.30

(t, J = 7.1 Hz, 3H), 2.80 (t, J = 7.3 Hz, 2H), 2.95 (t, J = 7.7 Hz,

2H), 4.17 (s, 3 H), 4.28 (q, J = 7.1 Hz, 2 H), 4.83 (bs, 2 H), 6.56 (td,

J = 7.9, 1.5 Hz, 1H), 6.74 (dd, J = 7.9, 1.5 Hz, 1H), 6.88 (td, J =

7.9, 1.5 Hz, 1H), 7.30 (dd, J= 7.9, 1.5Hz, 1H), 8.31(s, 1H), 8.51(s,

1H). LCMS (ESI) m/z 365 (M þ H)^þ. HRMS (ESI) calcd for

C₁₉H₂₀N₆O₂þ H^þ365.172, found 365.1713.

Ethyl 8-{[2-(Acetylamino)phenyl]amino}-1-methyl-4,5-dihydro-

1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (79). To a solution

of 78 (0.3 g, 0.75 mmol) and DIPEA (0.51 mL, 2.93 mmol) in DCM

(30 mL) under nitrogen acetyl chloride (0.11 mL, 1.49 mmol)

dissolved in DCM (2 mL) was added. After 1 h the reaction

mixture was concentrated and the residue was diluted with DCM,

washed with NaHCO₃(2 ꢀ 30 mL) and water (1 ꢀ 20 mL), dried

over anhydrous Na₂SO₄, and concentrated. The crude residue was

diluted with diethyl ether and decanted, to give 0.22 g (72%) of the

final compound. ¹H NMR (DMSO-d₆) δ ppm 1.30 (t, J = 7.1 Hz,

N-[2-Acetyl-4-(4-methylpiperazin-1-yl)phenyl]acetamide (84b).

By employment of the above-described procedure, starting from

83b, the compound 84b was prepared. Yield, 53%. H NMR

1

(DMSO-d₆) δ ppm 2.10 (s, 3H), 2.27 (s, 3H), 2.60 (s, 3H),

2.43-2.57 (m, 4H), 3.07-3.17 (m, 4H), 7.11 (dd, J = 8.7, 2.6

Hz, 1H), 7.43 (d, J = 2.6 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 10.94

(s, 1H). LCMS (ESI) m/z 276 (M þ H)^þ.

1-(4-Methoxy-3-nitrophenyl)-4-methylpiperazine (84c). Pd(OAc)₂

(85 mg, 0.38 mmol), 2-dicyclohexylphosphino-2⁰-(N,N-dimethyl-

amino)-biphenyl (225 mg, 0.57 mmol), K₃PO₄(2.26 g, 10.68 mmol),

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3547

and 4-bromo-1-methoxy-2-nitro-benzene 83c (1.77 g, 7.63 mmol)

in THF (50 mL) were charged in a round-bottom flask flushed

with argon. The flask was evacuated and backfilled with argon.

4-Methylpiperazine (1.0 mL, 9.15 mmol) was added, and the

reaction mixture was refluxed for 72 h. The reaction mixture was

then allowed to cool to room temperature and concentrated. The

crude solid was purified by flash chromatography on silica gel

(eluant, DCM/EtOH 90/10) to afford the title compound (1.0 g,

was treated with 4-methylpiperazine (5 mL) at 130 ꢀC for 3 h. The

solvent was evaporated under vacuum to yield give 6.7 g of the

desired compound in a quantitative yield. ¹H NMR (DMSO-d₆)

δ ppm 2.20 (s, 3H), 2.37-2.41 (m, 4H), 2.47 (s, 3H), 3.33-3.38

(m, 4H), 6.26 (d, J = 2.6 Hz, 1H), 6.52 (dd, J = 9.1, 2.6 Hz, 1H),

7.66(d, J=9.1 Hz, 1H), 12.73(s, 1H). LCMS(ESI)m/z 235 (M þ

H)^þ. HRMS (ESI) calcd for C₁₃H₁₈N₂O₂þ H^þ235.1441, found

235.1431.

1

1-[2-Amino-4-(4-methyl-piperazin-1-yl)-phenyl]-ethanone (85e).

To a solution of 87 (5.2 g, 22.20 mmol) in DMA (50 mL), NaOH

(2.7 g, 67.50 mmol) was added. The mixture was stirred at room

temperature for 1 h, then 2-bromo-2-methylpropanamide (11.1 g,

66.7 mmol) was added and the mixture was stirred at room

temperature overnight. NaOH (8.01 g, 200 mmol) was added,

and the resulting mixture was stirred at 100 ꢀC for 2 h, then water

(50 mL) was added, and the mixture was stirred at 100 ꢀC for 1 h.

After cooling to room temperature, the mixture was concen-

trated, diluted with DCM, and washed with water. The organic

phase was dried over sodium sulfate, the solvent allowed under

reduced pressure, and the crude solid was purified by flash

chromatography on silica gel (eluant: DCM/EtOH, 95/5) to

55%). H NMR (DMSO-d₆) δ ppm 2.22 (s, 3H), 2.42-2.48 (m,

4H), 3.07-3-13 (m, 4H), 3.83 (s, 3H), 7.22 (part of ABX sytem,

J= 9.3 Hz, 1H), 7.26 (part of ABX sytem, J=9.3,2.9Hz,1H),7.35

(d, J = 2.9 Hz, 1H). LCMS (ESI) m/z 252 (M þ H)^þ. HRMS (ESI)

calcd for C₁₂H₁₇N₃O₃þ H^þ252.1343, found 252.1342.

1-[2-(4-Methylpiperazin-1-yl)-6-nitrophenyl]ethanone (84d). In a

sealed tube, 1-(2-chloro-6-nitro-phenyl)-ethanone 83d (300 mg,

1.5 mmol) and 4-methylpiperazine (12.0 mL) were heated for

40 h at 120 ꢀC. The solvent was removed under reduced pressure,

and the residue was dissolved in DCM. The solution was washed

twice with water, and the organic phase was dried over anhydrous

sodium sulfate. The crude was purified by flash chromatography

1

(eluant, acetone/MeOH 75:25), affording 84 (272 mg, 68%). H

1

afford 85e (1.51 g, 30%). H NMR (DMSO-d₆) δ ppm 2.21 (s,

NMR (DMSO-d₆) δ ppm 2.21 (s, 3H), 2.35-2.44 (m, 4H),

2.86-2.91 (m, 4H), 7.66 (t, J = 8.7 Hz, 1H), 7.76 (dd, J = 8.7,

0.8 Hz, 1H), 7.91 (dd, J = 8.7, 0.8 Hz, 1H). LCMS (ESI) m/z 264

(M þ H)^þ. HRMS (ESI) calcd for C₁₃H₁₇N₃O₃þ H^þ264.1343,

found 264.133.

3H), 2.36 (s, 3H), 2.37-2.43 (m, 4H), 3.19-3.25 (m, 4H), 6.08 (d,

J = 2.8Hz, 1H), 6.21 (dd, J =9.1, 2.8Hz, 1H), 7.06 (bs, 2H), 7.52

(d, J = 9.1 Hz, 1H). LCMS (ESI) m/z 234 (M þ H)^þ.

2. Registry Numbers (RN). Methylhydrazine (RN, 60-34-4),

hydrazine monohydrate (RN, 7803-57-8), phenylhydrazine

(RN, 100-63-0), 2,2,2-trifluoroethylhydrazine soln 70% in water

(RN, 5042-30-8), N,N-dimethylformamide diisopropyl acetal

(RN, 18503-89-4), phenylguanidine carbonate salt (RN, 14018-

90-7), cyclohexanol (RN, 108-93-0), 4-hydroxy-1-methylpiper-

idine (RN, 106-52-5), 1-(2-hydroxyethyl)-piperidine (RN,

3040-44-6), triphenylphosphine polymer bound (RN, 39319-

11-4), methylamine (RN, 74-89-5), cyclopentylamine (RN,

1003-03-8), phenylamine (RN, 62-53-3), guanidine carbonate

salt (RN, 593-85-1), 2-aminobenzotrifluoride (RN, 88-17-5),

3-aminobenzotrifluoride (RN, 89-16-8), 4-aminobenzotrifluoride

(RN, 455-14-1), 2-aminoacetophenone (RN, 551-93-9), 3-ami-

noacetophenone (RN, 99-03-6), 4-aminoacetophenone (RN, 99-

92-3), o-anisidine (RN, 90-04-0), m-anisidine (RN, 536-90-3),

p-anisidine (RN, 104-94-9), o-nitroaniline (RN, 88-74-4),

m-nitroaniline (RN, 99-09-2), p-nitroaniline (RN, 100-01-6),

o-toluidine (RN, 95-53-4), 2-(methylthio)aniline (RN, 2987-

53-3), N-methyl-1,2-phenylenediamine (RN, 4760-44-3), 2-fluor-

oaniline (RN, 348-54-9), 2-isopropylaniline (RN, 643-28-7),

methylantranilate (RN, 134-20-3), 2-aminobenzamide (RN,

88-68-6), 2-aminobenzenesulfonamide (RN, 3306-62-5), 2-ami-

nobiphenyl (RN, 90-41-5), 2-aminodiphenyl ether (RN, 2688-

84-8), 2-benzylaniline (RN, 28059-64-5), N-phenyl-2-phenylene-

diamine (RN, 534-85-0), 2-aminodiphenylsulfide (RN, 1134-

94-7), 2-aminobenzophenone (RN, 2835-77-0), 4-bromo-2-

methoxyaniline (RN, 59557-91-4), 5-bromo-2-methoxyaniline

(RN, 6358-77-6), 4⁰-fluoro-2⁰-hydroxyacetophenone (RN, 1481-

27-2), 1-(2-amino-5-bromophenyl)ethanone (RN, 29124-56-9),

1-(2-amino-6-chlorophenyl)ethanone (RN, 20895-91-4).

2-Methoxy-4-(4-methylpiperazin-1-yl)aniline (85a). A solution of

84a (2.3 g, 8.73 mmol) in EtOH (52 mL) was treated with HCl 37%

(26 mL). After 1 h under reflux, the mixture was concentrated and

tritured with hexane to give in quantitative yield, 2.5 g of the title

compound as dihydrochloride salt. ¹H NMR (DMSO-d₆) δ ppm

2.82 (bs, 3H), 3.08-3.16 (m, 4 H), 3.46-3.53 (m, 2H), 3.85-3.92

(m, 2H), 3.90 (s, 3H), 6.60 (dd, J= 8.8, 2.6 Hz, 1H), 6.78 (d, J=2.6

Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 9.74 (bs, 3H) 10.79 (bs, 1H).

LCMS (ESI) m/z 222 (M þ H)^þ. HRMS (ESI) calcd for

C₁₂H₁₉N₃O þ H^þ222.1601, found 222.1596.

1-[2-Amino-4-(4-methylpiperazin-1-yl)phenyl]ethanone (85b).

By employment of the above-described procedure, starting from

84b, the compound 85b was prepared. Yield, 45%. H NMR

1

(DMSO-d₆) δ ppm 2.53 (s, 3H), 2.82 (bs, 3H), 2.92-3.03 (m,

4H), 3.08-3.25 (m, 4H), 3.58-3.66 (m, 4H), 6.85 (d, J = 8.5 Hz,

1H), 7.15 (dd, J = 8.5, 2.5 Hz, 1H), 7.27 (d, J = 2.5 Hz, 1H),

10.40 (bs, 1H). LCMS (ESI) m/z 234 (M þ H)^þ. HRMS (ESI)

calcd for C₁₃H₁₉N₃O þ H^þ234.1601, found 234.1602.

2-Methoxy-5-(4-methylpiperazin-1-yl)aniline (85c). A solu-

tion of 84c (1.0 g, 3.97 mmol) in MeOH (100 mL) in the presence

of Pd/C 10% (150 mg) was hydrogenated at 35 psi for 2 h. The

mixture was filtered over a pad of celite, and the solution was

concentrated to afford the title compound (0.8 g, 90%). ¹H

NMR (DMSO-d₆) δ ppm 2.20 (s, 3H), 2.39-2.43 (m, 4H),

2.91-2.96 (m, 4H), 3.67 (s, 3H), 4.54 (bs, 2H), 6.08 (dd, J =

8.7, 2.9 Hz, 1H), 6.29 (d, J = 2.9 Hz, 1H), 6.63 (d, J = 8.7 Hz,

1H). LCMS (ESI) m/z 222 (M þ H)^þ. HRMS (ESI) calcd for

C₁₂H₁₉N₃O þ H^þ222.1601, found 222.1609.

1-[2-Amino-6-(4-methylpiperazin-1-yl)phenyl]ethanone (85d).

To a solution of 84d (0.270 g, 1.02 mmol) in a mixture

(1:1:1.5:2.5) of cyclohexene:THF:H₂O:EtOH (12 mL), Pd/C 10%

(328 mg), and two drops of HCl 37% were added. The mixture was

heated at 70 ꢀC for 3 h. The Pd was filtered over a pad of celite, and

the solvents were removed from the filtrate under reduced pressure.

The crude was purified by flash chromatography (eluant, DCM/

MeOH/7 N NH₃in MeOH 9/1/1), to give compound 85d as orange

oil (225 mg, 95%). ¹H NMR (DMSO-d₆) δ ppm 2.51 (s, 3H), 2.84

(bs, 3H), 2.94-3.05 (m, 2H), 3.13-3.23 (m, 4H), 3.41-3.49 (m,

2H), 6.32 (dd, J = 7.9, 0.8 Hz, 1H), 6.49 (dd, J = 8.3, 0.8 Hz, 1H),

7.09 (dd, dd, J = 8.3, 7.9 Hz, 1H), 9.95 (bs, 1H). LCMS (ESI) m/z

234 (M þ H)^þ. HRMS (ESI) calcd forC₁₃H₁₉N₃O þ H^þ234.1601,

found 234.1605.

3. Protein Production and Characterization. Plk1_36-345has

been expressed in H5 insect cells as GST tag protein. The tag has

been removed by enzymatic digestion with PreScission Protease

(GE), and the protein has been purified by ion exchange column.

Plk1 has then been submitted to limited lysine methylation using

a reductive methylation kit (Jena Biosciences). After methyla-

tion, the protein has been gel filtered to remove excess of

reagents. Level of methylation has been assessed by LC/MS

analysis on a 1100 Agilent instrument using a Vydac C-4 column

˚

(2.1 mm ꢀ 250 mm, 5 μm, poresize 300 A) Positive ion ESI mass

spectra were obtained using 1946 single quadrupole mass spec-

trometer (Agilent) with an orthogonal ESI.

4. Crystallography Methods. In the final optimized crystal-

lization conditions, methylated-Plk1_36-345at a concentration of

20 mg/mL was incubated with 10 mM AMP-PNP and 10 mM

1-[2-Hydroxy-4-(4-methylpiperazin-1-yl)phenyl]ethanone (87).

1-(4-Fluoro-2-hydroxy-phenyl)-ethanone 86 (4.5 g, 29.22 mmol)

3548 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

MgCl₂prior to crystallization via the vapor diffusion method.

Crystallization was done by mixing 0.5 μL of protein mixture

with 0.5 μL of a reservoir solution consisting of 1.2 M Na/K

tartrate and 0.1 M MES buffer at pH 6.0. After one week at 4 ꢀC,

Zn acetate was added to the drops at a final concentration of

about 25 mM, and the drops were immediately seeded using

crystals of the methylated-Plk1_36-345:AMP-PNP complex.

Crystals grew in a few days. One crystal was soaked for three

hours in a solution containing 1.2 M Na/K tartrate, 0.1 M MES

pH 6.0, 20% glycerol, and 1 mM compound 49 and flash-cooled

in liquid nitrogen. Diffraction data were collected at beamline

ID14-EH3 at the European Synchrotron Radiation Facility

(Grenoble, France). Data were processed with MOSFLM⁴¹and

programs of the CCP4 suite.⁴²The structure has been solved by

molecular replacement using MOLREP⁴³and an in-house avail-

able structure of PLK1_36-345in complex with AMP-PNP as a

search model. Crystals belong to space group P3₂21 and contain

one molecule in the asymmetric unit. The structure was refined with

Refmac5,⁴⁴water molecules were added with Arp,⁴⁵and model

building was done with Coot.⁴⁶The final model has R_factor= 0.210

and R_free= 0.298. All structural figures were generated with PyMol

(http://pymol.sourceforge.net/).

5. Kinase Assays. The inhibitory activity of putative kinase

inhibitors and the potency of selected compounds were deter-

mined using a transphosphorylation assay. Specific peptide or

protein substrates were transphosphorylated by their specific

serine-threonine or tyrosine kinase, in the presence of ATP

traced with ³³P-γ-ATP, at optimized buffer and cofactors con-

ditions. At the end of the phosphorylation reaction, more than

98% unlabeled ATP and radioactive ATP was captured by

adding an excess of the ion exchange dowex resin; the resin then

settles down to the bottom of the reaction plate by gravity.

Supernatant, containing the phosphorylated substrate, was

subsequently withdrawn and transferred into a counting plate,

followed by evaluation by β-counting. Inhibitory potency eva-

luation for all the tested kinases was performed at 25 ꢀC using a

60 min end-point assay where the concentrations of ATP and

substrates were kept equal to 2 ꢀ RK_mand saturated (>5 ꢀ

RK_m), respectively.⁴⁷

Plk1 Assay. Inhibition of Plk1 kinase activity was assessed

using a Dowex resin capture technique. In this assay, 85 μM

RCasein (Sigma) was phosphorylated by 3 nM Plk1 (2-345) in

the presence of ATP (40 μM) traced with ³³P-γ-ATP in kinase

buffer (50 mM Hepes pH = 7.9, 10 mM beta glycerylphosphate,

1 mM DTT, 3 μM NaVO₃, 10 mM MgCl₂, 0.2 mg/mL BSA) for

60 min. By addition of an acidic suspension of Dowex resin

(SIGMA, custom-prepared resin Dowex 1 ꢀ 8200-400 mesh

equilibrated in 150 mM sodium formate, pH = 3.0) the un-

reacted ATP was captured and separated from the supernatant

which contained the phosphorylated substrate: this was then

transferred into a new plate for radioactivity counting. The

assay is run in a robotized format on 384-well plates.

Plk2 Assay. Inhibition of Plk2 kinase activity was assessed

using a Dowex resin capture technique. In this assay, 175 μM

RCasein (Sigma) was phosphorylated by 5 nM Plk2 (2-375) in

the presence of ATP (200 μM) traced with ³³P-γ-ATP in kinase

buffer (50 mM Hepes pH = 7.9, 10 mM beta glycerylphosphate,

1 mM DTT, 3 μM NaVO₃, 5 mM MgCl₂, 0.2 mg/mL BSA) for

60 min. By addition of an acidic suspension of Dowex resins

(SIGMA, custom-prepared resin Dowex 1 ꢀ 8200-400 mesh

equilibrated in 150 mM sodium formate, pH = 3.0) the un-

reacted ATP is captured and separated from the supernatant

which contained the phosphorylated substrate: this was then

transferred into a new plate for radioactivity counting. The

assay is run in a robotized format on 384-well plates.

1 mM DTT, 3 μM NaVO₃, 5 mM MgCl₂, 0.2 mg/mL BSA) for

60 min at room temperature. By addition of an acidic suspension

of Dowex resin (SIGMA, custom-prepared resin Dowex 1 ꢀ 8

200-400 mesh equilibrated in 150 mM sodium formate, pH =

3.0), the unreacted ATP was captured and separated from the

supernatant which contained the phosphorylated substrate: this

was then transferred onto a new plate for radioactivity counting.

The assay is run in a robotized format on 384-well plates.

6. Cell Cycle Analysis. Floating cells in the medium (usually

mitotic or in apoptosis) or released during PBS washes were

collected, added to adherent cells, detached from the plastic by

mild treatment with trypsin, and fixed in 70% ethanol/PBS. Cells

were washed with PBS to remove fixative and stained with 25 μg/

mL propidium iodide, 5 μg/mL RNase, and 0.125 μg/mL Nonidet

P40. Cells were kept at room temperature for 60 min in the dark

and were analyzed using a flow cytometry BD FACSCalibur

system (BD Biosciences). DNA content analysis was performed

on g10000 gated cells and DNA histograms were analyzed using

ModFit LT (Verity Software House, Topsham, ME).

7. Western Blot Analysis. Cells were lysed in 125 mmol/L Tris-

HCl (pH = 6.8) and 2% SDS and heated for 10 min at 95 ꢀC.

Total proteins (20 μg), as determined by BCA protein assay

(Pierce, Rockford, IL), were separated by 4-12% SDS-PAGE.

Immunoblot analysis was done using the following antibodies:

anti-TCTP (BD Transduction Laboratories, Franklin Lakes,

NJ), antiphosphorylated TCTP Ser46 (custom-made by Zymed,

San Francisco, CA), antiphosphorylated histone H3 Ser10

(Upstate Biotechnology, Lake Placid, NY), anti-histone H3

(Abcam, Cambridge, United Kingdom), antiphosphorylated

NPM Thr199 (Cell Signaling, Danvers, MA), anti-caspase-3 (Cell

Signaling Technology, Inc.) and anti-GAPDH (FL-335) (Santa

Cruz Biotechnology, Inc.). Immunoblot was carried out according

to standard methods. Amersham ECL (GE Healthcare) was used

for detection.

8. Immunohistochemisty and siRNA. Immunohistochemisty

and siRNA experiments were performed as previously de-

scribed.^48,49

9. Cell Culture. A2780 human ovarian cancer cells and their

resistant variants, A431 epidermoid carcinoma, OPM-2 and

NCI-H929 myeloma, KARPAS-299 and Jurkat lymphoma

cells, KU812 leukemia, and HEL erythroleukemia lines were

cultured in RPMI1640 supplemented with 10% fetal bovine

serum, HCT116 colon adenocarcinoma in McCoy’s, LoVo

colon adenocarcinoma, and its doxorubicin resistant derivative

line in HAM’s F12, Mia-PaCa-2, and Panc-1 pancreatic ade-

nocarcinoma in DMEM supplemented with 10% fetal bovine

serum, MCF7 in EMEM plus 10% fetal bovine serum, and 1%

NEAA, NHDF in fibroblast basal medium kit supplemented

with the appropriate growth factors (PromoCell), 1 ng/mL

bFGF, and 10% fetal bovine serum. A2780, A2780/ADR,

A2780/cis, A431, HCT116, LoVo, MCF7, Mia-PaCa-2 lines

were obtained from the ECACC, KARPAS-299, and OPM-2

from the DSMZ collection, A2780 1A9, and A2780 1A9/PTX22

were kind gifts from Professor R. Giavazzi (Mario Negri

Institute, Milan), NHDF were purchased from PromoCell, the

resistant variant LoVo/DX was generated growing its parent

cell line in the presence of 100 ng/mL doxorubicin, all other lines

were obtained from ATCC collection.

10. Inhibition of Cell Proliferation. Cells were seeded into 384-

well white plates at a density of 3000 cells/well for adherent and

5000 cells/well for nonadherent lines in complete growth med-

ium and treated with compounds dissolved in 0.1% DMSO 24 h

after the seeding. The cells were incubated at 37 ꢀC and 5% CO₂

and after 72 h of treatment the plates were processed using

CellTiter-Glo assay (Promega) following the manufacturer’s

instruction. CellTiter-Glo is a homogeneous method based on

the quantification of the ATP present as an indicator of meta-

bolically active cells. ATP was measured using a system based on

luciferase and ^D-luciferin resulting in light generation. The

luminescent signal is proportional to the number of cells present

Plk3 Assays. Inhibition of Plk3 kinase activity was assessed

using a Dowex resin capture technique. In this assay, 110 μM

RCasein (Sigma) was phosphorylated by 0.5 nM Plk3 (2-355) in

the presence of ATP (50 μM) traced with ³³P-γ-ATP in kinase

buffer (50 mM Hepes pH = 7.9, 10 mM beta glycerylphosphate,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3549

in culture. Briefly 25 μL/well reagent solution was added and

mixed to each well, and after 5 min, the microplates were read in

a luminometer. The concentration required to inhibit cell

growth by 50% (IC₅₀) was calculated from point-to-point curves

using Assay Explorer (MDL) software.

11. High-Throughput Solubility. Nominal 250 μM com-

pounds suspensions/solutions in aqueous ammonium acetate

buffer at pH = 7 were prepared on a Multiscreen-GV, 0.2 μm

filter plate (Millipore, Billerica, MA). These solutions were

stored for 24 h at room temperature to presaturate the mem-

brane filter and to reach a “pseudothermodynamic solubility”.

Then a premixed solution of acetonitrile/water (40/60 v/v) was

stirred, filtered, and diluted (25-fold). The so obtained nominal

10 μM solutions were then analyzed simultaneously by LC-MS/

MS with standard solutions for quantification.

Human Colon Adenocarcinoma Cells (Caco-2). Caco-2 cells

(American Type Culture Collection, Rockville, MD) monolayers

on 24-transwell plates were preincubated at 37 ꢀC and pH = 7.4

with HBSS containing 25 mM Hepes (Gibco BRL). Permeability

studies were performed in two transport directions, apical to

basolateral (absorption) and basolateral to apical (secretion), add-

ing fresh donor solution containing 10 μM of test compound in

HBSS on the donor side while placing compound-free HBSS on the

receiver side. The 24-transwell plates were placed on a heated plate

shaker. After 120 min, the solutions from the receivers and donors

chambers were collected and aliquots were analyzed by LC-MS/

MS. The Permeability coefficients (P_app) were calculated using the

following the equation:

P_appðcm=sÞ ¼ ðdCV_rÞ=ðdt AC_oÞ

12. Metabolic Stability in Human Liver Microsomes (HLM).

HLM (BD Gentest, Woburn, MA) at a concentration of 0.8 mg/

mL, were preincubated with 1 mM test compound for 5 min at

37 ꢀC in 0.1 M phosphate buffer, pH = 7.4, and then the

reactions were initiated by adding warmed NADPH (1 mM).

After 0, 5, 10, and 30 min incubations, the reactions were

stopped with a double volume of ice-cold acetonitrile. The

samples were centrifuged for 20 min at 1500 rpm, and the

amount of compound remaining was determined with LC-

MS/MS. The in vitro Cl_intwas calculated and then scaling

factors have been used to estimate the in vivo Cl_intaccording

to the following equation:

where: C_ois the initial concentration, dC/dt is the flux across the

monolayer, V_ris the volume of the receiver compartment, and A is

the membrane surface area.

15. In Vivo pharmacokinetics Nude Mouse. The pharmacoki-

netic profile of the compounds was investigated in overnight

fasted male nude mice following a single dose given intrave-

nously (iv) or orally (po). The compound was formulated as in

situ hydrochloride salt in glucosate solution as vehicle. A total of

six mice were treated (three for each leg). Blood samples of each

mouse were collected from the saphenous vein at predose, 0.083,

0.5, 1, 6, and 24 h postdosing following iv dosing, and at

predose, 0.25, 0.5, 1, 6, and 24 h following oral dosing. Samples

were centrifuged at 10000 rpm for 3 min at 4 ꢀC, and the plasma

was stored at -80 ꢀC until analysis. Samples were analyzed by

LC/MS/MS technique.

Cl_intinvivo¼ ð0:693=t₁₌₂Þðincubationvolume=mgHLMÞ

ðmgHLM=ðgliverÞ=kgbodyweightÞ

Pharmacokinetic Noncompartmental Data Analysis. The

pharmacokinetic parameters were derived by noncompartmen-

tal methods using the WinNonlin software program. The high-

est concentration C_maxand the time to peak t_maxwere read as the

coordinates of the highest observed concentration. The terminal

half-life (t_1/2,z) was calculated by the formula t_1/2,z= ln(2)/λz,

where λz is the slope of the terminal linear phase of natural-log

concentrations vs time curve. The choice of the points on the

terminal phase was based on visual inspection of the data. The

area under the plasma level vs time curve, AUC_¥, was calculated

by the linear trapezoidal rule up to the last detectable concen-

tration C(t_z) and beyond that time by extrapolation from C(t_z)

assuming monoexponential decay, using the following formula:

AUC = AUC(0 - t_z) þ C(t_z)/λ_z. The following formulas were

applied for the estimate of plasma clearance (CL) and volume of

distribution at steady state (V_ss). CL = dose/AUC_¥; MRT =

where mg HLM/g of liver = 45 mg and g liver/kg body weight

(human) = 20 g.

13. Intrinsic Clearance Determination in Rat Hepatocytes

(RH). Cl_intdeterminations in rat hepatocytes (prepared in-

house) suspensions were performed in Leibovitz’s medium, with

1.0 ꢀ 10⁶viable cells/mL and 1 mM test compound, at 37 ꢀC.

The reactions were stopped at 0, 10, 30, 60, and 90 min with a

double volume of ice-cold acetonitrile. The samples were cen-

trifuged at 700g for 20 min and then the amount of compound

remaining was determined with LC-MS/MS. The in vitro Cl_int

was calculated, and then scaling factors were used to estimate

the in vivo Cl_intaccording to the following equation:

Cl_intinvivo¼ ð0:693=t₁₌₂ÞðincubationvolumeofRHÞ

ðnumberofRH=gliverÞ=ðgliver=kgbodyweightÞ

AUMC/AUC_¥; V_ss= CL MRT; AUMC = AUMC(0 - t_z) þ

where number of RH/g of liver = 1 ꢀ 10 ⁸and g liver/kg body

3

2

C(t_z) t_z/λ_zþ C(t_z)/λ_z, with AUMC(0 - t_z) calculated using the

weight (rat) = 44 g.

linear trapezoidal rule on C t vs t plots. The oral bioavailability

(expressed as percent) was estimated by the ratio of dose-

normalized AUC_¥values after oral and iv dose.

3

14. Cell Permeability. Parallel Artificial Membrane Perme-

ability Assay (PAMPA). A 96-well filtration plate, MAIP N45

(Millipore, Billerica, MA), was used as support for 4 μL of the

membrane forming solution and as acceptor plate, filled with

200 μL of blank buffer solution, HBSS pH = 7.4 with HEPES

(Gibco BRL). The membrane forming solution consisted of

15% soy lecithin in n-dodecane. Then 200 μL of donor solution,

containing the compound at 10 μM, in HBSS pH 7.4 with

HEPES, were placed in a 96-well donor plate (p-ION, Woburn,

MA) and then the acceptor plate was placed on top of the donor

plate. The obtained “sandwich” was incubated at 37 ꢀC for 4 h.

After incubation, acceptor, donor, and initial donor solution

(reference) were analyzed by LC-MS/MS. Compound apparent

Permeability (P_app) is calculated using the following formula:

16. LC-MS/MS Analyses. Analyses were performed with an

LC-MS/MS system which consisted of an 1100 binary pump

(Agilent Technologies, Santa Clara, CA), an HTS-PAL auto-

sampler (CTC Analytics AG, Zwingen, Switzerland), and an

API 2000 mass spectrometer (AB/MDS-SCIEX, Concord, ON,

Canada). Chromatography was performed on a SB-C8 guard-

column, 5 μM, 12.5-4.6 mm (Agilent Technologies, Santa

Clara, CA), eluted at a flow rate of 0.6 mL/min. Mobile phase

A was 95% ammonium formate 10 mM, pH 4.0 and 5%

acetonitrile, while mobile phase B was 5% ammonium formate

10 mM, pH 4.0 þ 95% acetonitrile. Compound elution was

obtained at a flow rate of 0.6 mL/min with a step gradient from 0

to 100% B at time zero. Detection was done by using a Turbo

IonSpray source in the multiple-reaction monitoring MS/MS

mode.

P_appðcm=sÞ ¼ f - ln½1 - C_rðtÞ=C_eqꢁg=½Að1=V_dþ 1=V_rÞtꢁ

where A is the filter area, V_dis the donor volume, V_ris the

receiver volume, t is the incubation time (s), C_r(t) is the con-

centration in the receiver at time t, C_d(t) is the concentration in

the donor at time t, and C_eqis the equilibrium concentration

[C_d(t) (V_dþ C_r(t))V_r]/(V_dþ V_r).

17. Evaluation of Antitumor Efficacy. BALB/c nu/nu male

mice from Harlan Italy, 6 weeks old weighing 21-30 g, were

maintained in cages with paper filter covers, food, sterilized

bedding, and acidified water. HCT116 cells (from American

3550 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Beria et al.

Type Culture Collection) were inoculated subcutaneously. The

treatments started when the tumors were measurable; mean

tumor weight for all the groups was 0.170 g and compound 49

was suspendend in methocel. Treatments were administered

orally once a day at day 13, 14, 17, 18, 22, 23, 27, and 28. In

the control group, animals were treated with the vehicle with the

same scheduling. The tumor growth was evaluated every three

days. Tumour dimension was regularly measured by calipers

during the experiments, and the tumor mass was calculated as

described.⁵⁰

(10) Wolf, G.; Elez, R.; Doermer, A.; Holtrich, U.; Ackermann, H.;

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Acknowledgment. The authors are grateful to the group of

assay development of Nerviano Medical Sciences and to

Fulvia Roletto for the biochemical assay on Plk2 and Plk3.

We thank Walter Moretti and Dania Tesei for their skilled

synthetic chemistry support, Daniele Pezzetta, Cristina Cioc-

ca for ADME characterization and solubilily data, Marina

Fasolini for her support in the crystallographic screening,

Stefania Re Depaolini, Simona Rizzi for constructs produc-

tion, Francesco Sola and Maria Laura Giorgini for immuno-

fluorescence images and for apoptosis evaluation, Francesco

Colotta, Daniele Donati, Eduard Felder, Arturo Galvani,

Antonella Isacchi, Nicola Mongelli, Mario Varasi for discus-

sion and support.

(15) Knecht, R.; Elez, R.; Oechler, M.; Solbach, C.; von Ilberg, C.;

Strebhardt, K. Prognostic Significance of Polo-like Kinase (PLK)

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Note Added after ASAP Publication. This paper was pub-

lished on April 16, 2010 with a production error in the Results

and Discussion section. The revised version was published on

April 21, 2010.

Supporting Information Available: X-ray crystallographic

studies of compound 49. This material is available free of charge

via the Internet at http://pubs.acs.org.

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Article Doi

Identification of 4,5-dihydro-1 H -pyrazolo[4,3- h ]quinazoline Derivatives as a new class of orally and selective polo-like kinase 1 inhibitors

DOI: 10.1021/jm901713n

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Article abstract of DOI:10.1021/jm901713n

Full text of DOI:10.1021/jm901713n

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