Synthesis and biological evaluation of pyridazinone analogues as potential cardiac positron emission tomography tracers

2954

J. Med. Chem. 2008, 51, 2954–2970

Synthesis and Biological Evaluation of Pyridazinone Analogues as Potential Cardiac Positron

Emission Tomography Tracers

Ajay Purohit,^†,‡Heike Radeke,*^,†,‡Michael Azure,^‡Kelley Hanson,^‡Richard Benetti,^§Fran Su,^‡Padmaja Yalamanchili,^|

Ming Yu,^‡Megan Hayes,^‡Mary Guaraldi,^‡Mikhail Kagan,^‡Simon Robinson,^‡and David Casebier^‡

Research and DeVelopment, Bristol-Myers Squibb Medical Imaging, 331 Treble CoVe Road, North Billerica, Massachusetts 01862,

Boston UniVersity Medical School, 715 Albany Street, Boston, Massachusetts 02118, Lexicon Pharmaceuticals Inc., 350 Carter Road,

Princeton, New York 08540

ReceiVed NoVember 15, 2007

A series of ﬂuorinated pyridazinone derivatives with IC₅₀values ranging from 8 to 4000 nM for the

mitochondrial complex 1 (MC1) have been prepared. Structure–activity relationship (SAR) assessment

indicated preference of the ﬂuorine label to be incorporated on an alkyl side chain rather than directly on

the pyridazinone moiety. Tissue distribution studies of a series of analogues ([¹⁸F] 22–28) in Sprague-Dawley

(SD) rats identiﬁed [¹⁸F]27 as the most promising radiotracer with high uptake in cardiac tissue (3.41%ID/

g; 30 min post injection) in addition to favorable heart to nontarget organ distribution ratios. MicroPET

images of SD rats and nonhuman primates after [¹⁸F]27 administration allowed easy assessment of the

myocardium through 60 min with minimal lung or liver interference.

Introduction

Recent studies in the isolated rabbit heart with ¹²⁵I-iodorotenone

(2, Figure 1) indicated a superior extraction and retention than

^99mTc-sestamibi and ²⁰¹Tl.²⁰Rotenone (1), a natural product, is

widely used as an insecticide, acaricide, and miticide.^21,22Rotenone

is a potent inhibitor of mitochondrial complex 1 (MC1), which is

the ﬁrst enzyme of four electron transport complexes embedded

in the mitochondrial membrane.^23,24Because of the very high

weight percentage of mitochondria present in cardiomyocytes, MC1

may represent a new target for the development of cardiac PET

tracers.²⁵

Numerous classes of MC1 inhibitors have been reported in

addition to 1.^26–30Our laboratories were particularly interested

in compounds such as fenazaquin (3), S-chromone (4), tebufen-

pyrad (5), and pyridaben (6, Figure 2), which all share the same

binding site of the MC1 enzyme as rotenone.³¹In addition, MC1

inhibitors 3-6 are structurally similar as they all possess: (a) a

hydrophobic heterocyclic “headpiece”, for example a pyridazi-

none headpiece as 6, (b) a p-tert-butylphenyl moiety the “side

chain”, and (c) a heteroatom-containing linker, connecting (a)

and (b).

Nuclear myocardial perfusion imaging agents (MPIA)^ahave

been increasingly utilized in the assessment of coronary artery

disease (CAD).¹The most widely used MPIA are ²⁰¹Tl or

cationic ^99mTc complexes used with single-photon emission

computed tomography (SPECT) in the determination of myo-

cardial blood ﬂow and viability in patients.^2,3However, SPECT

imaging has inherent limitations such as accurate attenuation

correction, spatial resolution, and the ability to quantify myo-

cardial perfusion in absolute terms (milliliters per gram per

minute) compared to positron emission tomography (PET)

contrast agents.^4–6In addition, current SPECT tracers also

exhibit shortcomings in their pharmacokinetic properties such

as myocardial extraction, redistribution of the radiotracer to

nontarget tissue over time, and linearity of uptake at elevated

blood ﬂow (the “roll-off” phenomenon).^7–9Therefore, PET

imaging has become a clinically viable alternative in evalu-

ating myocardial blood ﬂow by use of positron-emitting

radionuclides.^10–14The most widely used PET ﬂow tracers in

the evaluation of myocardial perfusion are: ⁸²RbCl, ¹³NH₃, and

H₂¹⁵O.^15–19Unfortunately, their usage is limited by a short

physical half-life (<10 min) and, for ¹³NH₃and H₂¹⁵O, the

requirement of local cyclotron production. New PET tracers

should therefore have greater myocardial extraction and superior

linearity of uptake versus ﬂow to existing agents, retention

within the myocardium for the desired imaging window, and a

sufﬁcient half-life that single-dose commercial distribution

would be feasible.

Previous in vivo studies of [¹⁸F] labeled analogues 7 and 8

demonstrated rapid uptake of the radiotracer into the myocar-

dium in rats (Figure 3).^32,33However, both radiotracers showed

a signiﬁcant amount of washout after 60 min post injection from

the myocardium tissue. In this paper, we report our efforts

toward the development of a PET tracer based on MC1 inhibitor

6. Our goal is to incorporate PET radionuclide ¹⁸F into an

analogue of 6, maintain afﬁnity for MC1, and achieve good

uptake and retention in the myocardium.

Results and Discussion

* Corresponding author. Phone: (978)671-8151. Fax: (978)436-7500.

E-mail: heike.radeke@bms.com.

Authors contributed equally to this publication and are listed in

alphabetical order.

Structure–Activity Relationships. Previous structure–ac-

tivity relationship studies of pyridaben have shown a signiﬁcant

decrease in MC1 inhibitory activity when the t-butyl moiety

was replaced with an ethyl or methyl group demonstrating the

necessity of the t-butyl moiety in the pyridazinone headpiece.³⁴

Our initial efforts focused on replacement of the chlorine moiety

in the pyridazinone headpiece with other halogens such as

ﬂuorine to yield analogue 9 (Table 1). MC1 inhibitory activity

signiﬁcantly declined from an IC₅₀value of 8 nM of 6 to 279

†

‡

Research and Development, Bristol-Myers Squibb Medical Imaging.

Boston University Medical School.

Lexicon Pharmaceuticals Inc.

§

|

^aAbbreviations: MPIA, myocardial perfusion imaging agent; CAD,

coronary artery disease; SPECT, single-photon emission computed tomog-

raphy; PET, positron emission tomography; MC1, mitochondrial complex

1; TBDMS-Cl, tert-butyldimethylsilyl chloride; SMP, submitochondrial

particles.

10.1021/jm701443n CCC: $40.75

2008 American Chemical Society

Published on Web 04/19/2008

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2955

Table 2. Binding Afﬁnity (IC₅₀, Determined in Vitro) of Nonﬂuorinated

Pyridaben Derivatives Exploring the Linker Region

compound

X

n

IC₅₀(nM)

6

S

NH

O

1

2

8

4000

64

12

13

14

Figure 1. Natural product rotenone, a known MC1 inhibitor.

O

711

Table 3. Binding Afﬁnity (IC₅₀, Determined in Vitro) of Nonﬂuorinated

Pyridaben Derivatives Exploring the Side Chain Region

Figure 2. Various structure classes of known MC1 inhibitors.

Figure 3. [¹⁸F] labeled analogues of MC1 inhibitors fenazaquin (7)

and S-chromone (8).

Table 1. Binding Afﬁnity (IC₅₀, Determined in Vitro) of Known MC1

Inhibitors and Pyridaben Derivatives Exploring the Pyridazinone

Headpiece

A series of analogues were prepared to investigate the

heteroatom requirements in the linker as well as the optimal

chain length of the linker (Table 2). When the sulfur atom in

the linker of 6 was replaced with a nitrogen (12) or oxygen

(13) atom, MC1 inhibitory activity decreased ∼500 and 8 fold,

respectively. Although sulfur as the linking atom imparts the

greatest activity, the oxygen atom was preferred due to

anticipated greater metabolic stability.³⁵Extending the chain

of the linker of 13 by one carbon to generate analogue 14 was

detrimental to MC1 inhibitory activity. Therefore, compound

13 was chosen to represent the optimal compromise between

MC1 potency and metabolic stability. Further synthetic efforts

were directed toward modiﬁcation of the side chain of 13 for

future ﬂuorine incorporation (Table 3). Initially, removal of the

t-butyl moiety to yield compound 15 resulted in a >10 fold

decrease in MC1 inhibitory activity, indicating the requirement

of a para -substituted alkyl moiety in order to maintain biological

activity. Therefore, replacement of the t-butyl moiety in the side

chain with a n-butyl moiety as previously reported in the

preparation of ﬂuorinated analogues of 7 and 8 generated

compound 16 (IC₅₀) 17 nM), which was 2-fold more potent

than the parent analogue 13 (IC₅₀) 64 nM). Insertion of an

oxygen atom at the benzylic position in the four-atom linker of

16 to yield ﬁve-atom linker analogue 17 resulted in a decrease

in MC1 activity (IC₅₀) 27 nM). Interestingly, replacement of

the alkyl moiety of the side chain in analogue 16 with an aryl

substituent affording compound 18 resulted in a 2-fold decrease

in MC1 binding activity (IC₅₀) 31 nM). However, replacement

compound

R₁

IC₅₀(nM)

6

9

10

11

1

3

4

7

8

-Cl

-F

-CH₃

-CH₂F

8

279

21

250

16

90

52

11

9

nM for 9. Next, the chlorine moiety of 6 was replaced with a

methyl group to yield compound 10. The methyl substituent of

10 was used as an isostere for the chlorine atom of 6 but differed

electronically. Here, we observed only a 3-fold decrease in

activity yielding an IC₅₀value of 21 nM for 10 versus 8 nM

for 6. Unfortunately, attempts to incorporate a ﬂuorine atom

into compound 10 to yield compound 11 resulted in a signiﬁcant

decrease in MC1 activity. Simultaneously, known MC1 inhibi-

tors 1, 3, and 4 were screened for MC1 activity and found to

be less potent than 6, whereas analogues 7 and 8 were equivalent

in potency compared to 6.

2956 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Purohit et al.

Scheme 1. Alterations in the Pyridazinone Headpiece^a

Table 4. Binding Afﬁnity (IC₅₀, Determined in Vitro) of Fluorinated

Pyridaben Derivatives

compound

R₁

R₂

IC₅₀(nM)

22

23

24

25

26

27

28

-H

-D

-CH₂CH₂CH₂CH₂F

-OCH₂CH₂CH₂F

-CH₂CH₂CHFCH₃

-OCH(CH₂F)CH₂CH₃

-OCH₂CHFCH₃

-CH₂OCH₂CH₂F

-CH₂OCD₂CD₂F

15

47

10

16

8

11

28

of the aryl moiety in 18 with a naphthyl or quinoline group to

generate compounds 19 and 20 resulted in a 2- and 7-fold loss

of MC1 activity, respectively. Last, replacement of the aryl

moiety in the side chain of 6 with a n-decyl chain to yield

compound 21 resulted in complete loss of biological activity,

demonstrating the need of an aryl moiety in the side chain in

order to maintain MC1 activity.³⁶

^aReagents: (a) KF, K222, DMSO, 120 °C; (b) MeMgBr, diethyl ether,

0 °C; (c) 4-tert-butylbenzyl mercaptan, Cs₂CO₃, DMF, 65 °C; (d) NBS,

AIBN, CCl₄, reﬂux; (e) K₂CO₃, dioxane/water, 110 °C; (f) TBDMS-Cl,

imidazole, DMF, r.t.; (g) TBAF, THF, 0 °C; (h) MsCl, TMPDA, ACN,

0 °C.

Next, ﬂuorinated pyridaben analogues were prepared (Table

4) Analogue 22 possessed MC1 inhibition similar to nonﬂu-

orinated counterpart 16, whereas compound 23 exhibited ∼3-

fold decrease in activity versus analogue 22. Further SAR studies

of the most potent pyridaben analogue 22 were pursued.

Scheme 2. Variations in Chain Length and Hetero Atoms within

the Side Chain^a

According to the literature, radiotracers containing primary ¹⁸

F

labels may be metabolically deﬂuorinated in vivo at a faster

rate than secondary or aromatic ¹⁸F.^37,38However, primary

aliphatic ¹⁸F atoms, which are ꢀ to a heteroatom, e.g., [18F]FCH₂-

CH₂O-R, have been reported to be metabolized at a slower rate.

This has been termed the ꢀ-heteroatom effect.³⁹In this regard,

analogues 24–27 in Table 4 were prepared. Compound 24,

which contained a secondary ﬂuorine atom, exhibited equipotent

MC1 inhibitory activity compared to that of the parent com-

pound 16 with IC₅₀values of 10 nM versus 17 nM, respectively.

Introducing an oxygen atom in the side chain of compound 24

in order to take advantage of the ꢀ-heteroatom effect in addition

to the secondary nature of the ﬂuorine atom resulted in

compound 26 (IC₅₀) 8 nM). Compounds 25 and 27 also

contain primary ﬂuorine labels with an oxygen atom positioned

appropriately for increased metabolic stability, exhibiting potent

MC1 activities of 11 and 16 nM, respectively. Lastly, deuterium

atoms were substituted onto the carbon center bearing the

ﬂuorine atom of compound 27 in an attempt to further reduce

in vivo deﬂuorination. Researchers have observed an increased

metabolic stability of deuterated radiotracers due to the isotope

effect of deuterium on metabolism.⁴⁰The resulting analogue

28 exhibited a ∼2.5-fold decrease in MC1 activity compared

to that of the parent compound 27. Because analogues 22–28

all exhibit good MC1 inhibitory activity, further evaluations in

vivo were performed.

^aReagents: (a) 37, 38, or 39, Cs₂CO₃, DMF, 55-80 °C.

available 4-tert-butylbenzyl mercaptan in the presence of cesium

carbonate in DMF at 65 °C rendered ﬁnal compound 10,

whereas the synthesis of 11 required the introduction of a

ﬂuorine moiety in the pyridazinone headpiece. Bromination of

30 using N-bromosuccinimide in the presence of AIBN in

carbon tetrachloride under reﬂux condition afforded headpiece

31. Hydrolysis of the bromide moiety in 31 with potassium

carbonate in a mixture of dioxane and water at 110 °C

introduced the hydroxyl moiety in the headpiece, which was

protected with TBDMS-Cl to afford intermediate 33 in good

yield. Introduction of the side chain using standard alkylation

conditions (4-tert-butylbenzyl mercaptan, Cs₂CO₃, DMF, 65 °C)

yielded intermediate 34. Deprotection of the alcohol moiety in

headpiece 34 followed by subsequent mesylation afforded

ﬂuorination precursor 36. Fluorination of 36 using TBAF in

THF at 0 °C afforded ﬁnal compound 11.

Chemistry. The synthesis for pyridaben analogues 9–11,⁴¹

which describe modiﬁcations of the pyridazinone headpiece, are

shown in Scheme 1. Displacement of the chlorine atom in 6

using potassium ﬂuoride and kryptoﬁx222 at elevated temper-

ature in DMSO afforded ﬂuorinated analogue 9 in low yield.

Analogues 10 and 11 were prepared from common pyridazinone

headpiece 30,⁴¹which was obtained via methylation of 3,4-

dichloropyridazinone (29)⁴²with methylmagnesium bromide in

diethyl ether at 0 °C. Alkylation of 30 with commercially

Next, the length of the linker and heteroatom preference

within the linker of the side chain was investigated. In this

regard, analogues 12–14 were prepared as shown in Scheme 2.

Alkylation of headpiece 29 in the presence of cesium carbonate

in DMF at temperatures ranging from 55 to 80 °C with the

appropriate alcohol or amine (37–39) afforded the desired ﬁnal

compounds. Similar alkylation conditions were also utilized

when preparing compounds 16, 17, and 19 (Scheme 3a) to

investigate the substitution patterns of the phenyl moiety in the

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2957

Scheme 3. SAR Studies of the Side Chain Substitution Pattern^a

Scheme 5. Synthesis of Oxygen Containing Side Chains for

Pyridaben Analogues 23, 25-28^a

^aReagents: (a) 40, 41, 42, 43, or 44, Cs₂CO₃, DMF, 25-75 °C; (b) 45,

DIAD, PPh₃, THF, 0 °C.

Scheme 4. Synthesis of Fluorinated Pyridaben Analogues

22-27^a

^aReagents: (a) 1-bromo-3-propanol, K₂CO₃, DMF, 50 °C; (b) TBDMS-

Cl, imidazole, DMF, r.t.; (c) LAH, THF or ether, 0-25 °C; (d) 1-bromobu-

tane, Cs₂CO₃, DMF, r.t.; (e) 1-tert-butyldimethylsilyloxy-2-hydroxybutane,

DIAD, PPh₃, THF, 0 °C; (f) ethylene oxide, BF₃etherate, ether, DCM,

-10 °C.

alkylation conditions to couple the pyridazinone headpiece with

the appropriate side chain (Scheme 4a).

When pyridaben analogues 17 and 22–27 were prepared

according to the reaction sequences shown in Schemes 3 and

4, the required functionalized benzyl alcohols used as reaction

partners in Mitsunobu reactions or alkylations had to be

synthesized separately. Scheme 5 shows the preparation of side

chain precursors 41, 47, 49, and 51 required in the preparation

of ﬂuorinated pyridaben analogues 17, 23, 25, and 27.

^aReagents: (a) 46, Cs₂CO₃, DMF, 68 °C; (b) 47, 48, 49, 50, or 51, DIAD,

PPh₃, THF; (c) TBAF, THF, r.t.; (d) TsCl, DMAP, DIEA, DCM; (e) KF,

K222, ACN, 90 °C; (f) TsCl, pyridine, r.t.

Commercially available methyl-4-hydroxybenzoate (76) was

used as a common intermediate to prepare side chain precursor

41, 47, and 49. Alkylation of 76 with 1-bromobutane in the

presence of cesium carbonate in DMF at room temperature

yielded alkylation product 75. Reduction of the ester moiety in

75 with LAH in ether at 0 °C afforded side chain precursor 41

in good yield. Side chain precursor 47 was prepared in a similar

fashion. Modiﬁed alkylation conditions (K₂CO₃, DMF, 50 °C)

using 1-bromo-3-propanol to afford alkylation product 81

followed by protection of the alcohol moiety with TBDMS-Cl

and imidazole in DMF at room temperature yielded 82.

Reduction of the ester moiety in 82 with LAH in ether at 0 °C

afforded functionalized benzyl alcohol 47. Side chain 49 was

prepared using Mitsunobu conditions to couple 76 with 1-tert-

butyldimethylsiloxy-2-hydoxybutane followed by reduction of

the ester moiety with LAH in ether at 0 °C to afford benzyl

alcohol 49. Commercially available methyl 4-(hydoxymethyl)

benzoate (70) was exposed to ethylene oxide in the presence

of a Lewis acid to afford intermediate 72. Protection of the

primary alcohol moiety in 72 with TBDMS-Cl and imidazole

in DMF to yield 86 followed by reduction of the ester moiety

in 86 with LAH in THF afforded ﬁnal side chain precursor 51.

The synthesis of the side chain precursor for the deuterated

side chain. Additional functionalized side chains were installed

using pyridazinone headpiece 3-hydroxy-4-chloropyridazinone

(56)⁴³and functionalized benzyl bromides in the presence of

cesium carbonate in DMF at 25 and 70 °C to afford analogues

15 and 18, respectively (Scheme 3b). Coupling of 56 with

functionalized benzyl alcohols via Mitsunobu conditions could

also be used to afford pyridaben analogues such as 20 and

intermediates 57–61 for ﬂuorinated pyridaben analogues 22–27

(Scheme 4). Reaction intermediates 57, 59, and 61 contained a

protected hydroxyl moiety at various positions within the side

chain. Deprotection of the alcohol moiety of these intermediates

with TBAF in THF led to pyridazinone intermediates 63, 65,

and 67. Formation of the toluenesulfonate ester of alcohols 58,

60, 63, 65, and 67 affords ﬂuorination precursors 64, 66, 69,

71, and 73. Fluorination of these sulfonates at 90 °C in

acetonitrile for time intervals ranging from 10 to 40 min

rendered ﬁnal pyridaben analogues 23–27. Final pyridaben

analogue 22 in this series was the only analogue prepared using

2958 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Scheme 6. Synthesis of All Carbon Side Chains 46 and 48^a

Purohit et al.

^aReagents: (a) butyryl chloride, DCM, r.t.; (b) i. oxalyl chloride, AlCl₃, DCM; ii. MeOH, 48 h. (c) TBDMS-Cl, imidazole, DMF, r.t.; (d) LAH, THF or

diethyl ether, 0 °C; (e) 3-butene-2-ol, Pd(OAc)₂, PPh₃, TEA.

Table 5. Biodistribution of [¹⁸F]Pyridaben Analogues in Sprague-Dawley Rats^a

heart uptake

30 min

tissue ratios^b

compounds

120 min

heart:blood

heart:lung

heart:liver

heart:femur

22

23

24

25

26

27

28

2.94 ( 0.24

1.31 ( 0.09

2.25 ( 0.04

1.05 ( 0.13

1.15 ( 0.36

3.41 ( 0.27

1.09 ( 0.06

2.99 ( 0.99

0.86 ( 0.43

2.14 ( 0.26

1.24 ( 0.06

1.14 ( 0.17

3.47 ( 0.37

1.60 ( 0.24

60.67

9.27

20.76

6.05

13.17

30.02

12.74

14.93

5.40

12.49

1.37

11.41

16.05

7.33

1.45

0.77

1.78

1.11

2.62

2.74

1.70

6.84

2.00

1.98

0.89

0.40

7.51

4.60

^aData are expressed as the %ID/g ( SD with three animals per data point. ^bRatios are taken from 30 min post injection data points.

Table 6. Radiochemical and Reaction Yield for the Synthesis of

¹⁸F]22-28

analogue 28 was prepared in an analogous fashion. The alcohol

70 was exposed to deuterated ethylene oxide in the presence of

a Lewis acid to afford intermediate 87. Subsequent protection

of the alcohol 87 (TBDMS-Cl, imidazole, DMF) yielded 88.

Reduction of the ester moiety in 88 with LAD in THF afforded

deuterated side chain precursor 52.

The preparation of side chain precursors 46 and 48, which

were used to prepare analogues 22 and 24, are shown in Scheme

6. Commercially available alcohol 77 was esteriﬁed using

butyryl chloride in DCM to afford intermediate 78. Introduction

of the benzyl ester moiety in 78 was accomplished via a two-

step procedure to afford intermediate 79 in good yield. Protec-

tion of the primary alcohol moiety in 79 with TBDMS-Cl and

imidazole in DMF and subsequent reduction of the benzyl ester

moiety with LAH in ether at 0 °C afforded side chain precursor

46, which was coupled with 29 to yield ﬁnal analogue 22. Side

chain precursor 48 was obtained from 4-bromomethylbenzoate

83 via a Heck coupling with 2-butene-2-ol, Pd(OAc)₂, PPh₃in

TEA to afford intermediate 84, followed by reduction of the

ketone moiety of 84 with LAH in THF.

[

compounds

RCP (%)

yield (%)

22

23

24

25

26

27

28

100

91.5

100

12.3

10.2

8.2

13.4

17.6

35

18.2

Scheme 7. Radiosynthesis of [¹⁸F]22^a

^a(a) K¹⁸F/K222, ACN, 90 °C, 30 min. Analogues [¹⁸F]23-28 were

prepared in an analogous fashion from the appropriate sulfonate precursor.

displacement with nucleophilic ¹⁸F, compared with secondary

sulfonate leaving groups, which often eliminate under ﬂuorina-

tion conditions to yield oleﬁnic byproducts. With this in mind,

the [¹⁸F]22–28 were prepared with chemical yields ranging from

8.2 to 35% (Table 6) by the reaction of their corresponding

tosylate precursors with kryptoﬁx222/K¹⁸F complex and potas-

sium carbonate in acetonitrile at 90 °C for 30 min followed by

preparative HPLC separation of the reaction mixture (Scheme

7). The appropriate fractions were concentrated and analyzed

for radiochemical yield and purity. The radioactivity of the ﬁnal

product was ∼25 mCi with radiochemical purity >99% when

prepared with 500 mCi [¹⁸F]ﬂuoride, with a total time of

synthesis of 90 min. The speciﬁc activity of the material ranged

In Vitro Experiments. The inhibitory activities of com-

pounds 1, 3, 4, and 6–28 for the MC1 enzyme were determined

by measuring the rate of NADH oxidation in the presence of

decyl ubiquinone at 340 nm with a UV–vis spectrophotometer

over 120 s.⁴⁴Submitochondrial particles (SMP) were isolated

from bovine heart mitochondria according to Lester and

Matsuno-Yagi et al.^45,46The SMP were used as a suspension,

and rotenone was run as a reference standard versus each

compound.

Radiosynthesis. Compounds 22–28 within this series were

radiolabeled with the PET isotope ¹⁸F. Nucleophilic radioﬂu-

orination methods require activated leaving groups such as

sulfonates (e.g., tosylates, mesylate) in order to generate

alkylﬂuoride containing radioligands.³⁵Primary sulfonates are

often the preferred leaving group because of their ease of

from 750 to 2000 Ci/mmol depending upon the quantity of ¹⁹

present in the system.

F

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2959

Table 7. Biodistribution of [¹⁸F]Pyridaben Analogues in Sprague-Dawley Rats

tissue distribution^a

compounds

22

lung

liver

femur

blood

0.01 ( 0.18^b

0.18 ( 0.03^c

0.24 ( 0.03^b

0.08 ( 0.00^c

0.18 ( 0.02^b

0.14 ( 0.08^c

0.79 ( 0.33^b

0.22 ( 0.02^c

0.10 ( 0.00^b

0.06 ( 0.01^c

0.21 ( 0.03^b

0.28 ( 0.04^c

0.15 ( 0.00^b

0.13 ( 0.01^c

2.03 ( 0.11^b

0.62 ( 0.08^c

1.70 ( 0.31^b

0.55 ( 0.05^c

1.27 ( 0.10^b

0.63 ( 0.16^c

0.98 ( 0.10^b

0.39 ( 0.01^c

0.44 ( 0.07^b

0.40 ( 0.04^c

1.25 ( 0.17^b

0.55 ( 0.12^c

0.64 ( 0.07^b

0.44 ( 0.04^c

0.43 ( 0.06^b

1.16 ( 0.12^c

0.65 ( 0.09^b

0.76 ( 0.71^c

1.14 ( 0.13^b

2.38 ( 0.28^c

1.23 ( 0.23^b

3.00 ( 0.43^c

2.91 ( 0.56^b

3.91 ( 0.78^c

0.45 ( 0.04^b

0.75 ( 0.08^c

0.23 ( 0.03^b

0.78 ( 0.07^c

0.11 ( 0.01^b

0.04 ( 0.01^c

0.15 ( 0.00^b

0.07 ( 0.01^c

1.15 ( 0.36^b

3.41 ( 0.27^c

0.18 ( 0.03^b

0.06 ( 0.00^c

0.09 ( 0.01^b

0.04 ( 0.00^c

0.11 ( 0.01^b

0.19 ( 0.01^c

0.08 ( 0.00^b

0.10 ( 0.01^c

28

27

26

25

24

23

^aData are expressed as the %ID/g ( SD with three animals per data point. ^bPercent ID/g determined after 30 min post injection. ^cPercent ID/g determined

after 120 min post injection.

In Vivo Studies. Biodistribution Study of [¹⁸F]Pyridaben-

Analogues 22–28 in Sprague-Dawley (SD) Rats. Biodistri-

bution studies were performed with [¹⁸F]pyridazinone deriva-

tives [¹⁸F]22–28 in SD rats (see Table 5).

as the radiotracers with high uptake in heart tissue (>2%ID/g)

in addition to exhibiting low in vivo deﬂuorination (<0.5%ID/

g). Heart:femur ratios of compounds [¹⁸F]22 and [¹⁸F]27

possessed the highest selectivity with ratios of 6.84 and 7.51,

respectively (Table 7) within this series of ﬂuorinated pyridaben

analogues. Although [¹⁸F]28 exhibited a 2-fold decrease of in

vivo deﬂuorination compared to [¹⁸F]27, the heart uptake was

only 1.09%ID/g, resulting in a heart:femur ratio of 4.60.

Analogously, [¹⁸F]24 exhibited high heart uptake (2.25%ID/g)

but also exhibited high femural uptake (1.13%ID/g), resulting

in a heart:femur ratio of 1.98. Stability toward in vivo

deﬂuorination of [¹⁸F]27 did not improve with the introduction

of deuterium atoms on the ﬂuorine bearing carbon (heart:femur

ratio 4.60).

These results suggest that although there is literature prece-

dence on strategies for minimization of in vivo deﬂuorination

of radiotracers in general, our compound series does not follow

these literature observations. This suggests that the phenomena

of in vivo deﬂuorination is highly substrate dependent as only

few records in the literature successfully address reducing in

vivo deﬂuorination using the above-mentioned strategies.⁴⁷In

addition, the metabolism of any compound may be species

dependent and therefore in vivo deﬂuorination may occur to a

more signiﬁcant extent in one species than the other.⁴⁸Never-

theless, this series does demonstrate that the alkyl portion of

the side chain can tolerate structural variation such as incorpora-

tion of heteroatoms and alternative placement of the ﬂuorine

label without profoundly affecting the MC1 activity. Tables 3

and 4 show that ﬁve out of the seven analogues synthesized

exhibit an MC1 inhibitory activity of less than 20 nM. However,

biodistribution studies of these compounds demonstrated sig-

niﬁcant differences in uptake of the individual radiotracers in

various tissues. Thus [¹⁸F]27 was chosen as the lead structure

in this series because it exhibited the highest uptake in the heart

at 30 min post injection (3.41%ID/g) with essentially no washout

over 120 min and superior target-to-background tissue ratios

compared to other compounds within this series. Comparison

studies with ^99mTc-Sestamibi demonstrated [¹⁸F]27 to be

superior in heart uptake, myocardium retention, and target-to-

background tissue ratios.^49,50Overall, these results suggest

[¹⁸F]27 to be a suitable agent for pursuing PET imaging studies.

High uptake of radiotracers [¹⁸F]22, [¹⁸F]24, and [¹⁸F]27 from

the blood pool into the myocardium was observed at 30 min

post injection (>2.0%ID/g; >20:1 heart:blood pool ratio). The

concentration of radiotracers [¹⁸F]22 and [¹⁸F]27 residing in

the heart remained constant from 30 to 120 min post injection,

and [¹⁸F]24 exhibited minimal wash-out at 120 min post

injection, at which point approximately 95% of the activity

present at 30 min post injection was still present.

The uptake of radiotracers [¹⁸F]22–28 in nontarget tissues

was measured as well. At 30 min post injection, radiotracers

[¹⁸F]22, [¹⁸F]24, and [¹⁸F]27 exhibited heart:lung tissue ratios

with >10 fold selectivity while maintaining high uptake in

myocardium tissue (>2.0%ID/g). However, [¹⁸F]27 exhibited

the best heart to lung ratio (16.05) while exhibiting the highest

uptake in myocardium tissue (3.41%ID/g) compared to [¹⁸F]22

(heart:lung ratio 14.93, 2.94%ID/g) and [¹⁸F]24 (heart:lung ratio

12.49, 2.25%ID/g) at 30 min post injection. Low uptake of the

radiotracer in lung tissue suggests an excellent signal-to-noise

ratio for future imaging studies with [¹⁸F]27. When measuring

the ratio of radiotracer in heart:liver tissue, [¹⁸F]27 showed a

selectivity of 2.74 fold for the myocardium, which is more

selective than [¹⁸F]22 (heart:liver ratio 1.45) and [¹⁸F]24 (heart:

liver ratio 1.78). Interestingly, the heart:liver ratio of [¹⁸F]26

was measured to be 2.62, which is comparable to [¹⁸F]27.

However the heart uptake of [¹⁸F]26 is only 1.15%ID/g at 30

min post injection.

Addressing the previous concerns regarding in vivo deﬂu-

orination and subsequent accumulation of the free ﬂuoride in

bone, compounds [¹⁸F]22, [¹⁸F]27, and [¹⁸F]28 exhibited

femural uptake of less than 0.5%ID/g after 30 min post injection

(Table 7). Introduction of an oxygen atom in the side chain of

[¹⁸F]22 to generate the ꢀ-heteroatom effect did not reduce in

vivo deﬂuorination (0.43%ID/g for [¹⁸F]22 vs 0.45%ID/g for

[¹⁸F]27) as previously suggested. On the other hand, introduction

of deuterium labels in [¹⁸F]27 to yield [¹⁸F]28 did result in a

2-fold decrease of in vivo deﬂuorination at 30 min post injection.

When the ﬂuorine label was secondary as in [¹⁸F]24, femur

uptake of the radiotracer at 30 min post injection was measured

to be 1.13%ID/g. Compounds [¹⁸F]25 and [¹⁸F]26, which also

contain a secondary ﬂuorine label in addition to the ꢀ-hetero-

atom, exhibited even higher uptake of free ﬂuoride in femur

than [¹⁸F]24. Compounds [¹⁸F]22 and [¹⁸F]27 were identiﬁed

Imaging Studies of [¹⁸F]27 in SD Rats and Nonhuman

Primates. Dynamic PET Imaging studies were carried out on

SD rats and nonhuman primates. The PET images obtained on

SD rats complemented the results obtained in the above tissue

distribution study. [¹⁸F]27 accumulated within minutes after the

2960 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Purohit et al.

and blood. No washout of the radiotracer was observed in the

myocardium at 120 min post injection.

Imaging studies of rats and nonhuman primates demonstrated

a high initial uptake of [¹⁸F]27 in the liver (more uptake was

seen in PET images with rats compared to nonhuman primates),

which cleared by 25 min post injection sufﬁciently to afford

high quality images of the heart. While some in vivo bone

uptake of [¹⁸F] was observed in rat images 30 min after [¹⁸F]27

administration, no in vivo [¹⁸F] bone accumulation was ob-

served in nonhuman primate PET images up to 120 min. This

observation strongly suggests that the in vivo deﬂuorination

observed in biodistribution and imaging studies of compounds

[¹⁸F]22–28 is a highly species-dependent phenomenon. Future

studies will therefore be directed toward examining the meta-

bolic proﬁle and potential utility of these compounds in other

animal species.

Figure 4. Images of a rat and nonhuman primate (NHP) heart at two

different time points using [¹⁸F]27.

injection of the rat in cardiac and liver tissue as is depicted in

Figure 4. Signiﬁcant uptake of the radiotracer is visible in the

images of the rat heart, which was maintained over a 120 min

imaging period with minimal loss of signal intensity. Sufﬁcient

clearance of compound [¹⁸F]27 from the liver was observed by

35–45 min post injection to obtain good myocardial images and

was complete by 55–60 min post injection. No interference from

lung uptake of the radiotracer was detected, as demonstrated

by the high quality images obtained from this study. However,

some in vivo bone uptake of [¹⁸F] could be detected in images

after 30 min post injection.

Experimental Section

General Methods and Materials. All reagents used in synthesis

were commercial products and were used without further puriﬁca-

tion unless otherwise indicated. All solvents used were ACS or

1

HPLC grade. H NMR spectra were obtained in a Bruker DRX

600 MHz FTNMR spectrometer in CDCl₃unless otherwise

indicated. Chemical shifts are reported as δ values (parts per

million) relative to solvent peak. Coupling constants are reported

in Hz. The multiplicity is deﬁned by a s (singlet), d (doublet), t

(triplet), br (broad), or m (multiplet).

Observations made in the PET imaging studies of nonhuman

primates were similar to the PET imaging studies of rats. Rapid

accumulation of the radiotracer in cardiac tissue was observed

within minutes after the injection of the animal. Uptake of

[¹⁸F]27 in liver tissue was seen as well, although to a much

lesser extent than that in the PET images of rats. Direct

comparison among disparate species is possible via scaling to

quantiﬁed external activity sources placed beside the subject

animals in each study; normalization to these reference sources

gives comparable uptake values. Uptake levels of [¹⁸F]27 in

the myocardium allowed clear visualization of the right and left

ventricle walls. This signal intensity was maintained over a 120

min imaging period. At 35–45 min post injection, clearance of

the radiotracer from liver tissue was complete. As was observed

in the PET imaging studies of the rat, no interference from lung

uptake of [¹⁸F]27 was detected. However, unlike in the rat

imaging studies, no in vivo bone uptake of [¹⁸F]could be

detected in nonhuman primate images up to 120 min post

injection, conﬁrming a more favorable metabolic proﬁle in

higher species.

High-performance-liquid chromatography (HPLC) analysis and

puriﬁcation were performed on a Varian/PrepStar model SD-1 at

220 and 254 nm. The instrument was equipped with the ProStar/

Dynamax.24 software program on a Dell/Optiplex GX 1 computer

system. Water used in the preparation of the mobile phases was

puriﬁed using a Millipore/Milli-Q Gradient A10 system. Flash

chromatography was conducted using silica gel 230–400 mesh (60

Å), Merck. LCMS and TOF analysis were carried out on an Agilent

LC/MSD instrument (model G1969A), which is connected to an

1100 Series HPLC system. For radiolabeling studies, F-18 was

obtained from PETNET Pharmaceutical Services, Cummings Park,

Woburn, MA, and the synthesis was carried out in Wheaton vials

using a customized robotic synthesis platform. Biodistribution

studies were measured in an autogamma counter (Wallac Wizard

1480), while cardiac imaging was carried out using a microPET

camera (Focus220, CTI Molecular Imaging, Inc. Knoxville, TN).

All study protocols were approved by the Institutional Animal Care

and Use Committee.

Synthesis of 2-tert-Butyl-4-ﬂuoro-5-(4-tert-butylbenzyl)mer-

capto-3-(2H)-pyridazinone (9).⁴¹To a solution of KF (23.8 mg,

0.41 mmol) and kryptoﬁx222 (154 mg, 0.41 mmol) in DMSO (4.0

mL) was added pyridaben (6, 150 mg, 0.41 mmol). The reaction

mixture was heated at 120 °C. After 2.5 h, the reaction mixture

was cooled to room temperature and diluted with ethyl acetate (10

mL). The organic layer was separated, washed with water (40 mL),

brine (40 mL), dried over Mg₂SO₄, ﬁltered, and concentrated. The

crude brown oil was puriﬁed by HPLC (Phenomenex Luna C18(2)

column 10 µ, 21.2 mm × 250 mm; gradient: 0–90% B over 30

min at 20 mL/min; Mobile phase A ) water with 0.1% TFA

modiﬁer and B ) 90% acetonitrile in water with 0.1% TFA

modiﬁer) to obtain compound 9 (37 mg, 25% yield) as a ﬂuffy

Conclusion

A series of ﬂuorinated pyridaben derivatives with high afﬁnity

for MC1 have been prepared. SAR studies were focused on

addressing metabolic stability of the linker region of 6 as well

as the optimal location of the ﬂuorine label. Combined SAR

studies of 6 and 13 have shown that incorporation of a ﬂuorine

label in the pyridaben alkyl side chain such as compounds 22–28

exhibit more potent binding afﬁnities for the MC1 complex (IC₅₀

< 50 nM) than attachment of a ﬂuorine label directly onto the

pyridazinone ring (compounds 9 and 11, IC₅₀> 200 nM).

Introduction of an oxygen atom in the alkyl side chain or

changing the location of the ﬂuorine label within the alkyl side

chain did not disrupt MC1 inhibition (IC₅₀) 8–47 nM).

Biodistribution studies of [¹⁸F]22–28 in rats identiﬁed [¹⁸F]27

as the radiotracer with the highest absolute heart uptake at 30

min post injection (3.41%ID/g) in addition to the largest uptake

ratios of heart to nontarget organs such as liver, lung, femur,

1

white solid. H NMR (CDCl₃, 600 MHz) δ: 7.57 (d, 1H, J ) 7.5

Hz), 7.34 (d, 2H, J ) 8.3 Hz), 7.27 (d, 2H, J ) 8.3 Hz), 4.23 (s,

2H), 1.61 (s, 9H), 1.29 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz) δ:

155.2, 154.3, 153.4, 151.1, 133.4, 132.3, 128.4, 125.8, 65.9, 36.0,

34.5, 31.2, 27.7; ¹⁹F (CDCl_3,564 MHz) δ: –119.69 (m, 1F).

Synthesis of 2-tert-Butyl-4-methyl-5-chloro-3-(2H)-pyridazi-

none (30).⁴¹To a cooled (0 °C) solution of MeMgBr (6.02 mL of

3.0 M solution in ether, 18.1 mmol) was added dropwise a solution

of 29 (2.0 g, 2.27 mmol)⁴²dissolved in Et₂O (10 mL). The resulting

dark-red solution was stirred at 0 °C for 2 h and then quenched by

the dropwise addition of 5 N HCl (aq) (5 mL). The resulting yellow

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2961

solution was poured into a separatory funnel to separate the layers.

The aqueous layer was extracted with ethyl acetate (2 × 15 mL),

and the combined organic layers were washed with water (10 mL),

brine (10 mL), dried over Mg₂SO₄, ﬁltered, and concentrated. The

crude oil was puriﬁed by silica gel chromatography (hexanes:ether

gradient, 99:1 to 97:3) to obtain compound 30 (0.55 g, 31% yield)

stirred for 1.5 h at 65 °C before being ﬁltered at room temperature.

The ﬁltrate was diluted with ethyl acetate (20 mL), washed with

water (5 × 20 mL) and brine (20 mL), and then dried over Mg₂SO₄.

The crude oil obtained upon concentration in vacuo was puriﬁed

by silica gel chromatography (hexanes:diether gradient, 100 to 95:

5) to obtain 34 (280 mg, 78% yield). ¹H NMR (CDCl₃, 400 MHz)

δ: 7.84 (s, 1H), 7.23 (d, 2H, J ) 8.4 Hz), 7.13 (d, 2H, J ) 8.4 Hz),

4.52 (s, 2H), 4.34 (s, 2H), 1.69 (s, 9H), 1.27 (s, 9H), 0.9 (s, 9H),

0.02 (s, 6H); ¹³C NMR (CDCl₃, 100 MHz) δ: 159.4, 149.9, 145.7,

135.2, 132.6, 132.3, 128.5, 125.2, 65.5, 60.8, 35.7, 34.4, 31.2, 27.9,

25.8, –5.5; HRMS-TOF (m/z): [M + H]⁺calcd for C₂₆H₄₂N₂O₂SSi,

475.2809; found, 475.2813.

Synthesis of 2-tert-Butyl-4-hydroxymethyl-5-(4-tert-butylben-

zyl)mercapto-3-(2H)-pyridazinone (35). To a cooled (0 °C)

solution of compound 34 (0.12 g, 0.25 mmol) in THF (0.25 mL)

was added TBAF (0.5 mL of 1.0 M solution in THF, 0.5 mmol)

dropwise. After completion of addition the reaction mixture stirred

for 3 h followed by concentration in vacuo to obtain a crude oil.

Puriﬁcation by silica gel chromatography (hexanes:ethyl acetate

gradient, 100 to 92:8) of the crude material afforded compound 35

(73 mg, 80% yield) in good yield. ¹H NMR (CDCl₃, 600 MHz) δ:

7.71 (s, 1H), 7.35 (d, 2H, J ) 8.4 Hz), 7.26 (d, 2H, J ) 8.4 Hz),

4.64 (d, 2H, J ) 6 Hz), 4.36 (t, 1H, J ) 6 Hz), 4.17 (s, 2H), 1.61

(s, 9H), 1.30 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz) δ: 160.4, 151.1,

139.2, 136.0, 133.1, 132.1, 128.4, 125.8, 65.4, 59.3, 36.5, 34.5,

31.2, 27.8; HRMS-TOF (m/z): [M + H]⁺calcd for C₂₀H₂₈N₂O₂S,

361.1944; found, 361.1942.

1

as a thick oil. H NMR (CDCl₃, 600 MHz) δ: 7.52 (s, 1H), 2.21

(s, 3H), 1.60 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ: 157.4, 137.9,

135.4, 66.1, 27.7, 16.7.

Synthesis of 2-tert-Butyl-4-methyl-5-(4-tert-butylbenzyl)mer-

capto-3-(2H)-pyridazinone (10).⁴¹A suspension of compound 30

(64 mg, 0.32 mmol), 4-tert-butylbenzyl mercaptan (180.3 mg, 0.64

mmol), Cs₂CO₃(207.8 mg, 0.64 mmol) in DMF (1.5 mL) was

heated at 65 °C. After 2.5 h, the reaction mixture was diluted with

water (1 mL) and ethyl acetate (3 mL). The organic layer was

separated and washed with water (4 × 4 mL), brine (4 mL), dried

over Mg₂SO₄, ﬁltered, and concentrated. The crude oil was puriﬁed

by silica gel chromatography (hexanes:ethyl acetate gradient, 98:2

to 96:4) to obtain compound 10 as a white solid (60 mg, 55% yield).

¹H NMR (CDCl₃, 600 MHz) δ: 7.63 (s, 1H), 7.35 (d, 2H, J ) 8.4

Hz), 7.27 (d, 2H, J ) 8.4 Hz), 4.16 (s, 2H), 2.13 (s, 3H), 1.61 (s,

9H), 1.30 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz) δ: 160.2, 150.9,

138.8, 135.6, 132.5, 132.0, 128.4, 125.8, 64.8, 36.1, 34.5, 31.2,

27.9, 13.4.

Synthesis of 2-tert-Butyl-4-bromomethyl-5-chloro-3-(2H)-py-

ridazinone (31). A solution of compound 30 (0.4 g, 1.99 mmol),

N-bromosuccinimide (1.42 g, 7.99 mmol), and azobisisobutyronitrile

(AIBN, 16.40 mg, 0.1 mmol) in CCl₄(55 mL) was reﬂuxed for

16 h. The reaction mixture was cooled to room temperature and

ﬁltered. The ﬁltrate was concentrated in vacuo to obtain a crude

oil, which was dissolved in ethyl acetate (50 mL) and washed with

water (2×15 mL), brine (15 mL), and dried over Mg₂SO₄. A white

crystalline solid (0.48 g, 86% yield) was obtained upon concentra-

tion of the organic layer and was used in the next step without

further puriﬁcation; mp 99 °C. ¹H NMR (CDCl₃, 600 MHz) δ:

7.70 (s, 1H), 4.47 (s, 2H), 1.62 (s, 9H); ¹³C NMR (CDCl₃, 150

MHz) δ: 158.8, 137.4, 135.7, 134.3, 66.6, 28.0, 22.8; HRMS-TOF

(m/z): [M + H]⁺calcd for C₉H₁₂BrClN₂O, 278.9894; found,

278.9891.

Synthesis of 2-tert-Butyl-4-(methanesulfonyloxy)methyl-5-(4-

tert-butylbenzyl)mercapto-3-(2H)-pyridazinone (36). To a cooled

(0 °C) solution of compound 35 (11 mg, 0.03 mmol) in ACN (0.2

mL) and tetramethylenepropylenediamine (TMPDA, 5.96 mg, 0.045

mmol) was added MsCl (5.24 mg, 3.56 µL, 0.045 mmol). After 5

min, the reaction mixture was diluted with ethyl acetate (1 mL)

and water (0.5 mL). The organic layer was separated and washed

with water (0.5 mL) and brine (0.5 mL), dried over Mg₂SO₄,

ﬁltered, and then concentrated. Compound 36 was obtained as a

crude oil (14 mg, 100% yield), which was used in the next step

1

without further puriﬁcation. H NMR (CDCl₃, 400 MHz) δ: 7.70

(s, 1H), 7.24 (d, 2H, J ) 8.2 Hz), 7.12 (d, 2H, J ) 8.2 Hz), 5.06

(s, 2H), 4.39 (s, 2H), 2.93 (s, 3H), 1.68 (s, 9H), 1.26 (s, 9H); ¹³C

NMR (CDCl₃, 100 MHz) δ: 158.7, 149.9, 137.6, 136.6, 134.2,

131.9, 128.1, 125.0, 65.7, 64.9, 59.9, 35.7, 34.0, 30.8, 27.4; HRMS-

TOF (m/z): [M + H]⁺calcd for C₂₁H₃₀N₂O₄S₂, 439.1719; found,

439.1721.

Synthesis of 2-tert-Butyl-4-hydroxymethyl-5-chloro-3-(2H)-

pyridazinone (32). A suspension of compound 31 (0.48 g, 1.73

mmol), K₂CO₃(1.20 g, 8.7 mmol) in dioxane (3.2 mL), and water

(3.2 mL) was heated at 110 °C. After 4.5 h, the reaction mixture

was cooled to room temperature and diluted with DCM (20 mL).

The organic layer was separated and washed with water (10 mL),

brine (10 mL), and dried over Mg₂SO₄. Concentration of the organic

layer in vacuo afforded 32 (0.3 g, 80% yield) as a pale-yellow solid,

Synthesis of 2-tert-Butyl-4-(ﬂuoromethyl)-5-(4-tert-butylben-

zyl)mercapto-3-(2H)-pyridazinone (11). To a cooled (0 °C)

solution of compound 36 (14 mg, 0.03 mmol) dissolved in THF

(0.2 mL) was added TBAF (95.8 µL of 1.0 M solution in THF,

0.10 mmol) dropwise. After completion of addition the reaction

mixture was stirred for 40 min. Concentration of the reaction

mixture in vacuo afforded a crude oil, which was puriﬁed by

preparative silica gel chromatography (hexanes:ethyl acetate, 9:1)

1

which was used in the next step without further puriﬁcation. H

NMR (CD₃OD, 400 MHz) δ: 8.04 (s, 1H), 4.65 (s, 2H), 1.67 (s,

9H); ¹³C NMR (CDCl₃, 100 MHz) δ: 159.0, 143.4, 135.0, 67.8,

59.7, 28.0; HRMS-TOF (m/z): [M + H]⁺calcd for C₉H₁₃ClN₂O₂,

217.0734; found 217.0734.

1

Synthesis of 2-tert-Butyl-4-(tert-butyldimethylsilyloxy)methyl-

5-chloro-3-(2H)-pyridazinone (33). A solution of compound 32

(0.3 g, 1.38 mmol), tert-butyldimethylsilyl chloride (0.42 g, 2.76

mmol) and imidazole (0.19 g, 2.76 mmol) dissolved in DMF stirred

for 16 h. The reaction mixture was diluted with ethyl acetate (15

mL). The organic layer was separated, washed with water (5 × 20

mL), brine (20 mL), dried over Mg₂SO₄, ﬁltered, and concentrated.

The crude oil was puriﬁed by silica gel chromatography (hexanes:

diethyl ether gradient, 99:1 to 95:5) to obtain compound 33 (0.36

to obtain analogue 11 (7 mg, 50% yield) in moderated yield. H

NMR (CD₃OD, 400 MHz) δ: 7.76 (s, 1H), 7.25 (d, 2H, J ) 8.4

Hz), 7.10 (m, 2H), 5.22 (d, 2H, J ) 47.2 Hz), 4.26 (s, 2H), 1.68

(s, 9H), 1.26 (s, 9H); ¹³C NMR (CD₃OD, 100 MHz) δ: 161.1, 151.5,

144.0, 136.1, 133.4, 129.6, 126.3, 81.3 (79.7), 67.2, 36.9, 35.3, 31.6,

28.1; HRMS-TOF (m/z): [M + H]⁺calcd for C₂₀H₂₇FN₂OS,

363.1900; found, 363.1906.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-tert-butylbenzyl)amino-

3-(2H)-pyridazinone (12). A suspension of compound 29 (0.1 g,

0.45 mmol), 4-tert-butylbenzylamine (37, 0.15 g, 0.91 mmol), and

Cs₂CO₃(0.44 g, 1.36 mmol) in DMF (3 mL) was heated at 80 °C.

After 16 h, the reaction mixture was cooled to room temperature

and ﬁltered. The ﬁltrate was diluted with ethyl acetate (10 mL),

washed with water (2×5 mL) and brine (5 mL), dried over Mg₂SO₄,

ﬁltered, and then concentrated. The crude material was puriﬁed by

silica gel chromatography (hexanes:ethyl acetate gradient, 100 to

80:20) to obtain analogue 12 (78 mg, 50% yield) in moderate yield.

¹H NMR (DMSO-d₆, 400 MHz) δ: 7.67 (s, 1H), 7.35 (d, 2H, J )

8 Hz), 7.25 (d, 2H, J ) 8 Hz), 4.48 (d, 2H, J ) 6.4 Hz), 3.30 (br,

1

g, 80% yield) as a white solid. H NMR (CDCl₃, 600 MHz) δ:

7.64 (s, 1H), 4.72 (s, 2H), 1.61 (s, 9H), 0.88 (s, 9H), 0.10 (s, 6H);

¹³C NMR (CDCl₃, 150 MHz) δ: 159.7, 137.7, 137.1, 134.6, 65.7,

56.9, 27.7, 25.8, 18.4, –5.3; HRMS-TOF (m/z): [M + H]⁺calcd

for C₁₅H₂₇ClN₂O₂Si, 331.1603; found, 331.1602.

Synthesis of 2-tert-Butyl-4-(tert-butyldimethylsilyloxy)methyl-

5-(4-tert-butylbenzyl)mercapto-3-(2H)-pyridazinone (34). 4-tert-

Butylbenzylmercaptan (0.4 g, 2.26 mmol) and Cs₂CO₃(0.74 g, 2.26

mmol) were added to a solution of compound 33 (0.25 g, 0.76

mmol) and dissolved in DMF (9 mL). The reaction mixture was

2962 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Purohit et al.

1H), 1.49 (s, 9H), 1.24 (s, 9H); ¹³C NMR (DMSO-d₆, 100 MHz)

δ: 157.4, 149.9, 144.1, 136.6, 127.0, 125.8, 125.0, 64.6, 45.1, 34.6,

31.5, 28.2; HRMS-TOF (m/z): [M + H]⁺calcd for C₁₉H₂₆ClN₃O,

348.1837; found, 348.1843.

room temperature. After 16 h, the reaction mixture was diluted with

ethyl acetate (30 mL) and water (30 mL). The organic layer was

separated and washed with water (4 × 30 mL), 1 M NaOH (aq)

(10 mL), and brine (30 mL), dried over Mg₂SO₄, ﬁltered, and then

concentrated. Compound 75 was obtained as a crude oil (1.20 g,

87% yield), which was used in the next step without further

Synthesis of 2-tert-Butyl-4-chloro-5-(4-tert-butylbenzyl)oxy-

3-(2H)-pyridazinone ( 13).⁴¹A suspension of compound 29 (0.1

g, 0.45 mmol), 4-tert-butylbenzylalcohol (38, 0.15 g, 0.91 mmol),

and cesium carbonate (0.29 g, 0.9 mmol) dissolved in DMF (3 mL)

was heated at 75 °C. After 16 h, the reaction mixture was cooled

to room temperature and ﬁltered. The ﬁltrate was diluted with ethyl

acetate (10 mL) and washed with water (2 × 5 mL) and brine (5

mL). The organic layer was dried over Mg₂SO₄and concentrated

in vacuo to obtain an oil. The crude material was puriﬁed by silica

gel chromatography (hexanes:ethyl acetate gradient, 100 to 94:6)

1

puriﬁcation. H NMR (CDCl₃, 600 MHz) δ: 7.98 (d, 2H, J ) 9

Hz), 6.90 (d, 2H, J ) 9 Hz), 4.02 (t, 2H, J ) 6.6 Hz), 3.90 (s, 3H),

1.79 (m, 2H), 1.50 (m, 2H), 0.99 (t, 3H, J ) 7.2 Hz); ¹³C NMR

(DMSO-d₆, 100 MHz) δ: 166.3, 163.0, 131.6, 122.1, 114.8, 67.9,

52.1, 31.0, 19.1, 14.0.

Synthesis of (4-Butoxyphenyl)-methanol (41). To a solution

of compound 75 (15 mg, 0.59 mmol) in ether at 0 °C was added

LAH (45 mg, 1.18 mmol). The resulting suspension was stirred

for 3 h before water (45 µL), 15% NaOH (aq) (45 µL), and water

(3 × 45 µL) were added sequentially. After completion of all the

additions, the reaction mixture stirred for an additional 15 min

before being ﬁltered and concentrated to obtain compound 41 (110

mg, 85% yield) as an oil. 41 was used in the next step without

further puriﬁcation. ¹H NMR (CDCl₃, 600 MHz) δ: 7.25 (d, 2H, J

) 7.8 Hz), 6.87 (d, 2H, J ) 7.8 Hz), 4.59 (s, 2H), 3.95 (t, 2H, J

) 7.8 Hz), 1.76 (m, 2H), 1.49 (m, 2H), 0.96 (t, 3H, J ) 7.8 Hz);

¹³C NMR (CDCl₃, 75 MHz) δ: 158.8, 132.9, 128.6, 114.5, 67.1,

65.1, 31.3, 19.2, 13.8.

1

to obtain analogue 13 (143 mg, 63% yield) in moderate yield. H

NMR (CDCl₃, 600 MHz) δ: 7.73 (s, 1H), 7.42 (d, 2H, J ) 8.4

Hz), 7.33 (d, 2H, J ) 8.4 Hz), 5.27 (s, 2H), 1.62 (s, 9H), 1.31 (s,

9H); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.0, 153.8, 151.9, 131.8,

126.9, 125.8, 125.1, 118.2, 71.8, 66.3, 34.6, 31.2, 27.8.

Synthesis of 2-tert-Butyl-4-chloro-5-(2-(4-tert-butylphenyl)-

ethyl)oxy-3-(2H)-pyridazinone (14). A suspension of compound

29 (0.08 g, 0.34 mmol), 2-(4-tert-butylphenyl) ethyl alcohol (39,

90 mg, 0.51 mmol), and Cs₂CO₃(166 mg, 0.51 mmol) in DMF (3

mL) was heated at 55 °C. After 16 h, the reaction mixture was

cooled to room temperature and ﬁltered. The ﬁltrate was diluted

with ethyl acetate (10 mL) and washed with water (2 × 5 mL) and

brine (5 mL). The organic layer was dried over Mg₂SO₄and

concentrated in vacuo to obtain an oil. The crude material was

puriﬁed by silica gel chromatography (hexanes:ethyl acetate, 90:

10) to obtain analogue 14 (28 mg, 23% yield) in moderate yield.

¹H NMR (CDCl₃, 400 MHz) δ: 8.16 (s, 1H), 7.31 (d, 2H, J ) 8

Hz), 7.24 (d, 2H, J ) 8 Hz), 4.1 (t, 2H, J ) 4 Hz), 3.0 (t, 2H, J )

4 Hz), 1.56 (s, 9H), 1.25 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ:

158.2, 154.4, 149.3, 134.7, 129.2, 126.4, 125.5, 115.5, 71.3, 65.7,

34.9, 34.5, 31.6, 27.9; HRMS-TOF (m/z): [M + H]⁺calcd for C₂₀-

H₂₇ClN₂O₂, 363.1833; found, 363.1836.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-butoxy-benzyloxy)-

3(2H)-pyridazinone (17). A suspension of compound 29 (100 mg,

0.45 mmol), compound 41 (51 mg, 0.28 mmol), and Cs₂CO₃(110

mg, 0.34 mmol) in DMF (5 mL) was heated at 75 °C. After 16 h,

an additional portion of compound 29 (0.05 g, 0.225 mmol)

and Cs₂CO₃(0.05 g, 0.17 mmol) was added. The reaction mixture

stirred at 75 °C for another 3 h before being diluted with water (10

mL). The aqueous layer was separated and extracted with DCM

(2 × 10 mL). All of the combined organic layers were washed

with water (4 × 10 mL) and brine (10 mL), dried over Mg₂SO₄,

ﬁltered, and then concentrated. The crude oil obtained was puri-

ﬁed by silica gel chromatography (hexanes:ethyl acetate gradient,

99:1 to 9:1) to afford analogue 17 (28 mg, 25% yield) in moderate

Synthesis of 2-tert-Butyl-4-chloro-5-benzyloxy-3-(2H)-py-

ridazinone (15).⁴¹A suspension of compound 56 (0.03 g, 0.15

mmol), benzyl bromide (43, 22.9 mg, 15.87 µL, 0.13 mmol), and

Cs₂CO₃(48.3 mg, 0.14 mmol) in DMF (1 mL) was stirred at room

temperature. After 2 h, the reaction mixture was concentrated in

vacuo to obtain a crude material, which was redissolved in a mixture

of ethyl acetate (5 mL) and water (2 mL). The organic layer was

separated and washed with water (2 × 2 mL), brine (2 mL), dried

over Mg₂SO₄, ﬁltered, and concentrated. The crude material was

puriﬁed by preparative silica gel chromatography (hexanes:ethyl

acetate, 4:1) to obtain analogue 15 (19 mg, 44% yield) as a viscous

1

yield. H NMR (DMSO-d₆, 400 MHz) δ: 8.26 (s, 1H), 7.37 (d,

2H, J ) 8.4 Hz), 6.95 (d, 2H, J ) 8.4 Hz), 5.35 (s, 2H), 3.95 (t,

2H, J ) 7.8 Hz), 1.67 (m, 2H), 1.55 (s, 9H), 1.41 (m, 2H), 0.92 (t,

3H, J ) 7.8 Hz); ¹³C (DMSO-d₆, 100 MHz) δ: 159.4, 158.3, 154.3,

130.2, 127.5, 126.7, 116.0, 115.0, 71.8, 67.6, 65.8, 31.1, 27.9, 19.1,

14.2; HRMS-TOF (m/z): [M + H]⁺C₁₉H₂₅ClN₂O₃: 364.1554, found

387.1446 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-biphenylmethyl)oxy-

3-(2H)-pyridazinone (18). A suspension of compound 56 (0.20 g,

1.01 mmol), 4-bromomethyl biphenyl (44, 0.25 g, 1.01 mmol), and

Cs₂CO₃(0.39 g, 1.2 mmol) in DMF (20 mL) was heated at 70 °C.

After 2 h, the reaction mixture was concentrated to obtain a crude

oil, which was redissolved in ethyl acetate (50 mL) and water (20

mL). The organic layer was separated and washed with water (2 ×

20 mL) and brine (20 mL), dried over Mg₂SO₄, ﬁltered, and then

concentrated. The crude material obtained was puriﬁed by prepara-

tive silica gel chromatography (hexanes:ethyl acetate, 4:1) to obtain

analogue 18 (0.11 g, 30% yield) as a thick oil. ¹H NMR (DMSO-

d₆, 400 MHz) δ: 8.20 (s, 1H), 7.70 (m, 4H), 7.47 (m, 5H), 5.50 (s,

2H), 1.57 (s, 9H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 158.3, 154.3,

140.8, 140.0, 135.0, 129.4, 128.9, 128.1, 127.4, 127.1, 126.7, 117.0,

71.6, 65.8, 27.9; HRMS-TOF (m/z): [M + H]⁺for C₂₁H₂₁ClN₂O₂,

368.1292; found, 391.1178 [M + Na]⁺.

1

oil. H NMR (CDCl_3,600 MHz) δ: 7.71 (s, 1H), 7.37 (m, 5H),

5.30 (s, 2H), 1.61 (s, 9H); ¹³C NMR (CDCl_3,150 MHz) δ: 159.0,

153.6, 134.8, 128.9, 128.7, 127.0, 125.1, 118.2, 71.8, 66.3, 27.8;

HRMS-TOF (m/z): [M + H]⁺calcd for C₁₅H₁₇ClN₂O₂, 292.0979;

found, 315.0874 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-butylbenzyl)oxy-3-

(2H)-pyridazinone (16). A suspension of compound 29 (0.1 g, 0.45

mmol), 4-butylbenzyl alcohol (40, 0.14 g, 0.90 mmol), and Cs₂CO₃

(0.29 g, 0.99 mmol) in DMF (2.0 mL) was stirred at 65 °C. After

16 h, the reaction mixture was cooled to room temperature and

diluted with ethyl acetate (10 mL). The organic layer was washed

with water (2 × 10 mL) and brine (10 mL), dried over Mg₂SO₄,

ﬁltered, and then concentrated. The crude oil was puriﬁed by silica

gel chromatography (hexanes:ethyl acetate gradient, 100 to 96:4)

Synthesis of 2-tert-Butyl-4-chloro-5-(2-naphthylmethyl)oxy-

3-(2H)-pyridazinone (19). A suspension of compound 29 (100 mg,

0.50 mmol), 2-naphthyl methanol (42, 118 mg. 0.75 mmol), and

Cs₂CO₃(240 mg, 0.75 mmol) in DMF (2 mL) was heated at 65

°C. After 16 h, the reaction mixture was concentrated to obtain a

crude oil, which was redissolved in ethyl acetate (10 mL). The

organic layer was washed with water (2 × 5 mL) and brine (5

mL), dried over Mg₂SO₄, ﬁltered, and then concentrated. The crude

oil was puriﬁed by silica gel chromatography (hexanes:ethyl acetate,

100 to 80:20) to obtain analogue 19 (35 mg, 25% yield) in moderate

1

to obtain analogue 16 (118 mg, 75% yield) as a viscous oil. H

NMR (CDCl_3,600 MHz) δ: 7.71 (s, 1H), 7.30 (d, 2H, J ) 8.4

Hz), 7.2 (d, 2H, J ) 8.4 Hz), 5.20 (s, 2H), 2.61 (t, 2H, J ) 7.6, 7.8

Hz), 1.62 (s, 9H), 1.59 (m, 2H), 1.34 (m, 2H), 0.91 (m, 3H); ¹³C

NMR (CDCl_3,150 MHz) δ: 159.2, 154, 143.8, 132.2, 129.1, 127.4,

125.4, 118.4, 72.2, 66.5, 35.5, 33.8, 28, 22.5,14.1; HRMS-TOF

(m/z): [M + H]⁺for C₁₉H₂₅ClN₂O₂, 349.1677; found, 349.1674.

Synthesis of Methyl-4-butoxybenzoate (75). A suspension of

methyl-4-hydroxybenzoate (76, 1.0 g, 6.57 mmol), 1-bromobutane

(0.9 g, 6.57 mmol), and Cs₂CO₃in DMF (15 mL) was stirred at

1

yield. H NMR (DMSO-d₆, 400 MHz) δ: 8.30 (s, 1H), 7.96 (m,

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2963

4H), 7.55 (m, 3H), 5.60 (s, 2H), 1.56 (s, 9H); ¹³C NMR (DMSO-

d₆, 100 MHz) δ: 157.9, 153.9, 133.0, 132.7, 128.5, 128.0, 127.7,

126.8, 126.6, 126.3, 125.5, 71.6, 65.4, 27.5; HRMS-TOF (m/z):

[M + H]⁺for C₁₉H₁₉ClN₂O₂, 342.1135; found, 365.1031 [M +

Na]⁺.

Synthesis of Methyl-4-[4-(tert-butyldimethylsilyloxy)butyl]-

benzoate (80). A solution of compound 79 (1.0 g, 4.8 mmol),

imidazole (0.5 g, 7.2 mmol), and tert-butyldimethylsilyl chloride

(1.08 g, 7.3 mmol) dissolved in DMF (10 mL) was stirred at room

temperature. After 2 h, the reaction mixture was diluted with ethyl

acetate and washed with water (5 × 20 mL) and saturated NaHCO₃

(aq) (2 × 20 mL). The organic layer was dried over Mg₂SO₄,

ﬁltered, and concentrated to give compound 80 as an oil (1.17 g,

Synthesis of 2-tert-Butyl-4-chloro-5-(6-quinolinylmethyl)oxy-

3-(2H)-pyridazinone (20). To a cooled (0 °C) solution of com-

pound 56 (49 mg, 0.24 mmol) in THF (2 mL) was added

6-quinolinyl methanol (45, 25 mg, 0.15 mmol), PPh₃(61 mg, 0.23

mmol), and diisopropylazodicarboxylate (DIAD, 48 mg, 0.23

mmol). After completion of addition, the reaction mixture was

stirred at 0 °C for 2 h and then concentrated to afford a yellow oil,

which was puriﬁed by preparative thin layer chromatography (silica

gel, hexanes:ethyl acetate, 1:1) to obtain analogue 20 (18 mg, 43%

yield) as a white crystalline solid. ¹H NMR (DMSO-d₆, 400 MHz)

δ: 8.90 (s, 1H), 8.40 (d, 1H, J ) 8 Hz), 8.30 (s, 1H), 8.08 (d, 1H,

J ) 7.8 Hz), 8.07 (s, 1H), 7.82 (m, 1H), 7.56 (m, 1H), 5.60 (s,

2H), 1.56 (s, 9H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 158.3, 154.3,

151.5, 147.9, 136.6, 134.1, 130.0, 129.4, 128.0, 127.3, 126.7, 122.4,

116.2, 71.6, 65.9, 27.9; HRMS-TOF (m/z): [M + H]⁺for

C₁₈H₁₈ClN₃O₂, 358.1846; found, 344.1160 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-decylsulfanyl-3-(2H)-py-

ridazinone (21). A suspension of compound 29 (100 mg, 0.45

mmol), decanethiol (158 mg, 0.90 mmol), and Cs₂CO₃(290 mg,

0.90 mmol) in DMF (1.5 mL) was stirred at room temperature for

3 h. The reaction mixture was diluted with water (1 mL) and ethyl

acetate (2 mL). The organic layer was separated and washed with

water (3 × 5 mL) and brine (5 mL), dried over Mg₂SO₄, ﬁltered,

and then concentrated. The crude oil was puriﬁed by silica gel

chromatography (hexanes:diethyl ether gradient, 100 to 96:4) to

obtain analogue 21 as a viscous oil (140 mg, 86% yield). ¹H NMR

(CDCl₃, 600 MHz) δ: 7.58 (s, 1H), 2.99 (t, 2H, J ) 7.2 Hz), 1.72

(m, 2H), 1.63 (s, 9H), 1.44 (m, 2H), 1.25 (br m, 12H), 0.8 (t, 3H,

J ) 7.8 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ: 156.0, 141.4, 129.9,

129.6, 66.1, 31.8, 31.1, 29.4, 29.3, 29.2, 29.1, 29.0, 28.6, 27.8, 22.6,

14.0; HRMS-TOF (m/z): [M + H]⁺for C₁₈H₃₁ClN₂OS, 358.1846;

found, 381.1741 [M + Na]⁺.

1

75% yield). H NMR (CDCl₃, 600 MHz) δ: 0.05 (s, 6H), 0.90 (s,

9H), 1.49–1.74 (m, 4H), 2.65–2.70 (t, 2H, J ) 7.2 Hz), 3.59–3.64

(t, 2H, J ) 7.2 Hz), 3.89 (s, 3 H), 7.22–7.25 (m, 2 H), 7.93–7.96

(m, 2H); ¹³C NMR (CDCl₃, 150 MHz) δ: 167.1, 148.1, 129.6,

128.4, 127.7, 62.8, 51.9, 35.6, 32.3, 27.4, 25.9, 18.3, –5.3; HRMS-

TOF (m/z): [M + H]⁺for C₁₈H₃₀O₃Si, 322.1964; found, 345.1865

[M + Na]⁺.

Synthesis of (4-(4-(tert-Butyldimethylsilyloxy)butyl)phenyl)-

methanol (46). To a cooled (0 °C) solution of compound 80 (1.17

g, 3.6 mmol) in Et₂O (14 mL) was added portionwise solid LAH

(0.28 g, 7.2 mmol). After completion of addition, the reaction

mixture was stirred for 1 h. Sequential addition of water (0.28 mL),

15% NaOH (aq) (0.28 mL), and water (0.84 mL) resulted in a

cloudy solution, which stirred for an additional 15 min. The white

solid was removed via ﬁltration, and the ﬁltrate was dried over

Mg₂SO₄, ﬁltered, and concentrated. The crude material was puriﬁed

by silica gel chromatography (hexanes:ethyl acetate, 4:1) to afford

compound 46 (1.02 g, 96% yield) as a clear viscous liquid. ¹H

NMR (CDCl₃, 600 MHz) δ: 7.27 (d, 2H, J ) 6 Hz), 7.17 (d, 2H,

J ) 6 Hz), 4.60 (s, 2H), 3.60 (t, 2H, J ) 6 Hz), 2.60 (t, 2H. J )

6 Hz), 1.61 (m, 4H), 0.89 (s, 9H), 0.03 (s, 6H); ¹³C NMR (CDCl₃,

150 MHz) δ: 142.2, 138.2, 128.6, 127.1, 65.8, 65.3, 62.9, 35.3,

32.3, 27.6, 25.7, 18.3, –5.2.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(4-hydroxybutyl)ben-

zyloxy]-3-(2H)-pyridazinone (54). A suspension of compound 46

(0.41 g, 1.4 mmol), compound 29 (0.93 g, 4.2 mmol), and Cs₂CO₃

(1.37 g, 4.2 mmol) in DMF (11 mL) was heated at 68 °C. After

12 h, the reaction mixture was cooled down to room temperature

and ﬁltered. The ﬁltrate was diluted with ethyl acetate (20 mL)

and washed with water (5 × 25 mL) and brine (25 mL). The organic

layer was dried over Mg₂SO₄, ﬁltered, and concentrated. The crude

material was puriﬁed using silica gel chromatography (hexanes:

ethyl acetate, 9:1) to afford compound 53 (594 mg, 89% yield) as

Synthesis of 4-Phenylbutyl Butyrate (78). To a solution of

4-phenyl-1-butanol (77, 7.0 g, 47 mmol) dissolved in DCM (20

mL) was added a solution of butyryl chloride (4.79 g, 45 mmol) in

DCM (20 mL) dropwise. After completion of addition, the reaction

mixture stirred for 36 h. The reaction mixture was then concentrated

in vacuo to yield a crude oil, which was puriﬁed by silica gel

chromatography (hexanes:ethyl acetate, 3:1) to afford compound

1

an oil. H NMR (CDCl₃, 600 MHz) δ: 7.74 (s, 1 H), 7.33 (d, 2H,

J ) 8.4 Hz), 7.23 (d, 2H, J ) 8.4 Hz), 5.23 (s, 2H), 3.64 (t, 2H,

J ) 6.6 Hz), 2.65 (t, 2H, J ) 6.6 Hz), 1.64 (s, 9H), 0.90 (s, 9H),

0.05 (s, 6H); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.3, 154.0, 143.7,

132.3, 129.2, 127.4, 125.4, 118.4, 72.1, 66.5, 63.1, 35.6, 32.5, 28.0,

27.7, 26.1, 18.5.

1

78 (9.8 g, 94% yield) as a clear viscous liquid. H NMR (CDCl₃,

600 MHz) δ: 7.27 (m, 2H), 7.17 (m, 3H), 4.08 (t, 2H, J ) 7.2 Hz),

2.64 (t, 2H, J ) 7.2 Hz), 2.27 (t, 2H, J ) 7.8 Hz), 1.65 (m, 6H),

0.94 (t, 3H, J ) 7.8 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ: 173.7,

142.0, 128.3, 125.6, 64.0, 36.2, 35.4, 28.2, 27.7, 18.1, 13.6; HRMS-

TOF (m/z): [M + H]⁺for C₁₄H₂₀O₂, 220.1463; found, 243.1353

[M + Na]⁺.

To a solution of compound 53 (594 mg, 1.45 mmol) dissolved

in THF (3 mL) was added a solution of TBAF (2.9 mL of 1 M

solution in THF, 2.9 mmol). After 1 h, the reaction mixture was

concentrated under reduced pressure. The resulting crude material

was puriﬁed using silica gel chromatography (pentane:ethyl acetate,

1.8:1) to afford compound 54 (410 mg, 77% yield) in good yield.

¹H (CDCl₃, 600 MHz) δ: 1.61–1.64 (m, 11H), 1.67–1.74 (m, 2H),

2.68 (t, 2H, J ) 6.6 Hz), 3.68 (t, 2H, J ) 6.6 Hz), 5.23 (s, 2H),

7.23 (d, 2H, J ) 7.8 Hz), 7.33 (d, 2H, J ) 7.8 Hz), 7.74 (s, 1H);

¹³C (CDCl₃, 150 MHz) δ: 159.0, 153.7, 143.1, 132.2, 128.9, 127.2,

125.1, 118.2, 71.8, 66.3, 62.7, 35.3, 32.5, 27.8, 27.4; HRMS-TOF

(m/z): [M + H]⁺for C₁₉H₂₅ClN₂O₃, 364.1554; found, 387.1449

[M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-(4-p-toluenesulfony-

loxy butyl)benzyloxy]-3-(2H)-pyridazinone (55). A solution of

compound 54 (200 mg, 0.55 mmol), TsCl (125 mg, 0.66 mmol),

DMAP (80 mg, 0.66 mmol), and DIEA (85 mg, 0.66 mmol) in

DCM (2 mL) was stirred at room temperature. After 2 h, the

reaction mixture was diluted with ethyl acetate (10 mL) and washed

with 0.1 N HCl (aq) (3 mL) and brine (3 mL). The organic layer

was dried over Mg₂SO₄, ﬁltered, and concentrated. The resulting

crude oil was puriﬁed by silica gel chromatography (pentane:ethyl

acetate, 3:1) to provide compound 55 (197 mg, 69% yield). ¹H

(CDCl₃, 600 MHz) δ: 1.62–1.70 (m, 13H), 2.43 (s, 3H), 2.58 (t,

Synthesis of Methyl-4-(4-hydroxybutylbenzoate) (79). To a

cooled (0 °C) solution of AlCl₃(6.70 g, 0.05 mol) in DCM (100

mL) was added oxalyl chloride (6.4 g, 0.05 mol) dropwise. After

completion of addition, the mixture was stirred for 5 min before

the dropwise addition of a solution of compound 78 (9.80 g, 44

mmol) in DCM (50 mL). After completion of addition, the reaction

mixture was stirred at 0 °C for 4 h. The reaction mixture was poured

into a separatory funnel containing ice and brine. The organic layer

was separated and washed with brine (100 mL), dried over Mg₂SO₄,

ﬁltered, and then concentrated. Then 9.0 g of the resulting yellow

oil was suspended in methanol and stirred for 48 h. The reaction

mixture was concentrated in vacuo, and the resulting crude material

was puriﬁed by silica gel chromatography (hexanes:ethyl acetate,

2.5:1) to provide compound 79 (2.80 g, 31% yield) as a clear

1

viscous liquid. H NMR (CDCl₃, 600 MHz) δ: 7.93 (d, 2H, J )

7.8 Hz), 7.23 (d, 2H, J ) 7.8 Hz), 3.88 (s, 3H), 3.64 (t, 2H, J )

7.2 Hz), 2.67 (t, 2H, J ) 7.2 Hz), 1.66 (m, 2H), 1.56–1.61 (m, 2

H); ¹³C NMR (CDCl_3,150 MHz) δ: 167.3, 148.0, 129.8, 128.6,

62.8, 52.1, 35.8, 32.4, 27.6.

2964 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Purohit et al.

2H, J ) 7.2 Hz), 4.03 (t, 2H, J ) 6.0 Hz), 7.15 (d, 2H, J ) 7.4

Hz), 7.29–7.33 (m, 4H), 7.72 (s, 1H), 7.77 (d, 2H, J ) 6.6 Hz);

¹³C (CDCl₃, 150 MHz) δ: 159.0, 153.7, 144.7, 142.4, 133.1, 132.4,

129.8, 128.9, 127.8, 127.3, 125.1, 118.2, 71.8, 70.2, 66.3, 34.8,

28.3, 27.8, 26.9, 21.6; HRMS-TOF (m/z): [M + H]⁺for

C₁₈H₃₀O₃Si, 518.1642; found, 541.1529 [M + Na]⁺.

THF (3 mL) was added PPh₃(187 mg, 0.71 mmol), compound 56

(142 mg, 0.71 mmol), and DIAD (144 mg, 0.71 mmol). The reaction

mixture was stirred at 0 °C for 1 h and was then diluted with diethyl

ether (6 mL). The organic solution was washed with water (3 mL)

and brine (3 mL), dried over Mg₂SO₄, ﬁltered, and then concen-

trated. Silica gel chromatography (hexanes:ethyl acetate, 9:1) of

the crude material afforded compound 57 (106 mg, 31% yield).

¹H NMR (CDCl₃, 600 MHz) δ: 7.72 (s, 1H), 7.30 (d, 2H, J ) 6.6

Hz), 6.91 (d, 2H, J ) 6.6 Hz), 5.23 (s, 2 H), 4.06 (t, 2H, J ) 6.6

Hz), 3.79 (t, 2H, J ) 6.0 Hz), 1.97 (m, 2H), 1.62 (s, 9H), 0.87 (s,

9H), 0.03 (s, 6H); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.5, 159.0,

153.7, 128.9, 125.3, 118.3, 114.9, 71.8, 66.0, 64.6, 59.4, 32.3, 27.8,

25.9, 18.2; HRMS-TOF (m/z): [M + H]⁺for C₂₄H₃₇Cl N₂O₄Si,

481.2283; found, 481.2287.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(3-hydroxypropoxy)-

benzyloxy]-3-(2H)-pyridazinone (63). A solution of compound 57

(100 mg, 0.21 mmol) and TBAF (0.42 mL of 1 M solution in THF,

0.42 mmol) dissolved in THF (2 mL) was stirred at room

temperature. After 2 h, the reaction mixture was concentrated in

vacuo to yield a crude oil, which was puriﬁed by preparative thin

layer chromatography (silica gel, hexanes:ethyl acetate, 1:1) to

afford compound 63 (57.8 mg, 76% yield) in good yield. ¹H NMR

(CDCl₃, 600 MHz) δ: 7.71 (s, 1H), 7.31 (d, 2H, J ) 9.0 Hz), 6.92

(d, 2H, J ) 9.0 Hz), 5.30 (s, 2H), 4.13 (t, 2H, J ) 6.0 Hz), 3.86

(t, 2H, J ) 6.0 Hz), 2.04 (m, 2H), 1.62 (s, 9H) ; ¹³C NMR (CDCl₃,

150 MHz) δ: 159.2, 159.0, 153.7, 128.9, 127.0, 125.3, 118.3, 114.9,

71.8, 66.3, 65.5, 60.2, 31.9, 27.8; HRMS-TOF (m/z): [M + H]⁺

for C₁₈H₂₃Cl N₂O₄, 367.1419; found, 367.1422.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-(4-ﬂuorobutyl)benzyl)-

oxy-3-(2H)-pyridazinone (22). Compound 55 (57 mg, 0.10 mmol)

dissolved in ACN (1 mL) was added to a mixture of KF-

kryptoﬁx222 (1:1; 0.164 mmol) in ACN (1 mL). After completion

of addition, the reaction mixture was heated at 90 °C for 15 min.

The reaction mixture was concentrated in vacuo, and the resulting

crude oil was puriﬁed by silica gel chromatography (hexanes:ethyl

acetate, 4:1) to afford analogue 22 (28 mg, 70% yield) as an oil

that solidiﬁed upon standing. ¹H NMR (CDCl₃, 600 MHz) δ: 7.71

(s, 1H), 7.30 (d, 2H, J ) 8.4 Hz), 7.20 (d, 2H, J ) 8.4 Hz), 5.20

(s, 2H), 4.44 (dt, 2H, J ) 47.4, 6 Hz), 2.60 (t, 2H, J ) 7.2 Hz),

1.70 (m, 4H), 1.60 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.0,

153.0, 142.8, 132.3, 128.9, 127.2, 125.1, 118.2, 84.3 (83.3), 71.8,

66.3, 35.1, 29.9 (29.8), 27.8, 26.8; ¹⁹F (CDCl₃, 564 MHz) δ: -18.6

(m, 1F); HRMS-TOF (m/z): [M + H]⁺for C₁₉H₂₄ClFN₂O₂, 367.1583;

found, 367.1587.

Synthesis of Methyl-4-(3-hydroxy-propoxy)benzoate (81). A

suspension of 4-hydroxy methyl benzoate (76, 3.0 g, 0.02 mol),

3-bromo-1-propanol (4.17 g, 0.03 mol), and K₂CO₃(4.15 g, 0.03

mol) in DMF (40 mL) was heated at 50 °C. After 12 h, the reaction

mixture was diluted with ethyl acetate (50 mL) and washed with

0.1N HCl (aq) (2 × 15 mL), water (8 × 50 mL), and brine (50

mL). The organic layer was dried over Mg₂SO₄, ﬁltered, and then

concentrated to yield a crude oil, which was puriﬁed by silica gel

chromatography (hexanes:ethyl acetate, 1.68:1) to afford compound

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(3-(p-toluenesulfony-

loxy)propoxy)benzyloxy]-3-(2H)-pyridazinone (69). A solution

of compound 63 (40 mg, 0.11 mmol), TsCl (31 mg, 0.16 mmol),

DMAP (20 mg, 0.16 mmol), and DIEA (16.6 mg, 0.16 mmol)

dissolved in DCM (0.6 mL) was stirred at room temperature. After

1 h, the reaction mixture was concentrated in vacuo and the resulting

crude material was puriﬁed by preparatory thin layer chromatog-

raphy (silica gel, pentane:ethyl acetate, 3:2) to provide com-

pound 69 (18.6 mg, 33% yield) in moderate yield. ¹H NMR (CDCl₃,

600 MHz) δ: 7.73–7.75 (m, 3H), 7.29 (d, 2H, J ) 8.4 Hz), 7.23

(d, 2H, J ) 8.4 Hz), 6.78 (d, 2H, J ) 9.0 Hz), 5.22 (s, 2H), 4.23

(t, 2H, J ) 6.0 Hz), 3.95 (t, 2H, J ) 6.0 Hz), 2.37 (s, 3H), 2.11

(m, 2H), 1.62 (s, 9H); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.0, 158.9,

153.7, 144.8, 132.7, 129.8, 128.9, 127.8, 127.1, 125.1, 118.2, 114.7,

71.7, 66.8, 66.3, 63.1, 28.8, 27.8, 21.6; HRMS-TOF (m/z): [M +

H]⁺for C₂₅H₂₉Cl N₂O₆S, 520.1435; found, 543.1319 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(3-ﬂuoropropoxy)ben-

zyloxy]-3-(2H)-pyridazinone (23). To a solution of compound 69

(4.5 mg, 8.64 × 10^-3mmol) dissolved in ACN (0.25 mL) was

added a solution of KF (1.6 mg, 4.07 × 10^-2mmol) and

kryptoﬁx222 (15.0 mg, 4.07 × 10^-2mmol) in ACN (0.25 mL).

After completion of addition, the reaction vial was capped and

partially immersed in a 90 °C oil bath. The reaction mixture was

stirred for 40 min and was then concentrated under reduced pressure

to yield a crude oil. Puriﬁcation of the crude material by preparative

thin layer chromatography (silica gel, pentane:ethyl acetate, 3:2)

1

81 (1.25 g, 30% yield) as a white powder. H NMR (CDCl₃, 600

MHz) δ: 7.98 (m, 2 H), 6.91 (m, 2H), 4.17 (t, 2H, J ) 6.0 Hz),

3.87 (m, 5H), 2.04–2.08 (m, 2H); ¹³C NMR (CDCl₃, 150 MHz) δ:

166.8, 162.6, 11.5, 122.6, 114.0, 65.5, 59.8, 51.8, 31.8; HRMS-

TOF (m/z): [M + H]⁺for C₁₁H₁₄O₄, 210.0892; found, 233.0785

[M + Na]⁺.

Synthesis of Methyl-4-(3-(tert-butyldimethylsilyloxy)propoxy)-

benzoate (82). A solution of compound 81 (300 mg, 1.4 mmol),

tert-butyldimethylsilyl chloride (317 mg, 2.1 mmol), and imidazole

(146 mg, 2.1 mmol) in DMF (4 mL) was stirred at room

temperature. After 2 h, the reaction mixture was diluted with ethyl

acetate (20 mL) and washed with 0.1 N HCl (aq) (2 × 5 mL),

water (4 × 10 mL), and brine (10 mL) and then dried over Mg₂SO₄,

ﬁltered, and concentrated. The resulting crude material was puriﬁed

using silica gel chromatography (hexanes:ethyl acetate, 9.5:1) to

afford compound 82 (413 mg, 91% yield) in high yield. ¹H NMR

(CDCl₃, 600 MHz) δ: 7.97 (d, 2H, J ) 8.4 Hz), 6.90 (d, 2H, J )

9.0 Hz), 4.11 (t, 2H, J ) 6.0 Hz), 3.87 (s, 3H), 3.79 (t, 2H, J ) 6.0

Hz), 1.97–2.01 (m, 2H), 0.87 (s, 9H), 0.03 (s, 6H); ¹³C NMR

(CDCl₃, 150 MHz) δ: 166.9, 162.9, 131.5, 122.4, 114.0, 64.6, 59.2,

51.7, 32.3, 25.8, 18.3; HRMS-TOF (m/z): [M + H]⁺for C₁₇-

H₂₈O₄Si, 324.1757; found, 347.1651 [M + Na]⁺.

Synthesis of (4-(3-(tert-Butyldimethylsilyloxy)propoxy) ben-

zyl alcohol (47). To a cooled (0 °C) solution of compound 82 (396

mg, 1.22 mmol) in Et₂O (10 mL) was added portionwise solid LAH

(93 mg, 2.44 mmol). After completion of addition, the reaction

mixture was stirred for 2 h. Sequential addition of water (0.093

mL), 15% NaOH (aq) (0.093 mL), and water (0.28 mL) resulted

in a cloudy solution. The white solid was removed by ﬁltration,

and the ﬁltrate was dried over Mg₂SO₄, ﬁltered, and concentrated

to yield compound 47 (291 mg, 80% yield). 47 was used in the

next step without further puriﬁcation. ¹H NMR (CDCl₃, 600 MHz)

δ: 7.26 (m, 2H), 6.88 (m, 2H), 4.60 (s, 2H), 4.05 (t, 2H, J ) 6.6

Hz), 3.79 (t, 2H, J ) 6.6 Hz), 1.97 (m, 2H), 0.88 (s, 9H), 0.04 (s,

6H); ¹³C NMR (CDCl₃, 150 MHz) δ: 158.7, 132.9, 128.6, 114.5,

65.1, 64.5, 59.5, 32.4, 25.9, 18.3; HRMS-TOF (m/z): [M + H]⁺

for C₁₆H₂₈O₃Si, 297.1880; found, 297.1884.

1

provided analogue 23 (0.8 mg, 25% yield) in moderate yield. H

NMR (CDCl₃, 400 MHz) δ: 8.20 (s, 1H), 7.39 (d, 2H, J ) 8.4

Hz), 6.98 (d, 2H, J ) 8.4 Hz), 5.36 (s, 2H), 4.59 (m, 2H), 4.07 (t,

2H, J ) 6.4 Hz), 4.09–4.11 (m, 2H), 2.09 (m, 2H, J ) 25.6 Hz),

1.59 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ: 158.7, 157.9, 153.9,

129.9, 127.4, 126.3, 114.6, 81.6 (80.0), 71.4, 65.4, 63.6, 63.5, 29.7,

27.5; ¹⁹F NMR (CDCl_3,564.6 MHz) δ: –222.66 (m, 1F); HRMS-

TOF (m/z): [M + H]⁺for C₁₈H₂₂ClFN₂O₃, 387.1481; found,

387.1486 [M + NH₄]⁺.

Synthesis of Methyl-4-(3-oxobutyl)benzoate (84). To a solution

of methyl-4-bromobenzoate (83, 1.0 g, 4.65 mmol) in TEA (13

mL) was added 3-buten-2-ol (1 mL, 11.63 mmol), Pd(OAc)₂(104

mg, 0.465 mmol), and PPh₃(244 mg, 0.93 mmol). The reaction

mixture was stirred at 75 °C overnight. The reaction mixture was

cooled to room temperature and concentrated in vacuo to yield a

crude oil. The crude material was partitioned between water (20

mL) and ethyl acetate (20 mL). The organic layer was separated

Synthesis of 2-tert-Butyl-4-chloro-5-(4-(3-(tert-butyl-dimeth-

ylsilyloxy)propoxy)benzyloxy)-3-(2H)-pyridazinone (57). To a

cooled (0 °C) solution of compound 47 (211 mg, 0.71 mmol) in

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2965

and washed with water (20 mL) and brine (20 mL), dried over

Na₂SO₄, ﬁltered, and then concentrated. The crude product was

puriﬁed by silica gel chromatography (hexane:ethyl acetate gradient,

5:1 to 3:1) to obtain compound 84 (250 mg, 26% yield) in moderate

yield. H NMR (CDCl_3,600 MHz) δ: 7.95 (d, 2H, J ) 8.4 Hz),

7.25 (d, 2H, J ) 8.4 Hz), 3.90 (s, 3H), 2.95 (t, 2H, J ) 7.5 Hz),

2.77 (t, 2H, J ) 7.7 Hz), 2.14 (s, 3H).

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(3-hydroxybutyl)ben-

zyloxy]-3-(2H)-pyridazinone (58). To a solution of compound 84

(505 mg, 2.45 mmol) in THF (19 mL) at 0 °C was added LAH

(12.2 mL of 1 M THF solution, 12.24 mmol) dropwise. After

completion of addition the reaction mixture was stirred at room

temperature. After 1 h, water (183 µL), 15% NaOH (aq) (183 µL),

and water (548 µL) was added sequentially. The reaction mixture

was stirred for an additional 15 min before being ﬁltered. The ﬁltrate

was concentrated under reduced pressure to obtain compound 48

(314 mg, 71% yield), which was used in the next step without

further puriﬁcation.

was puriﬁed by silica gel chromatography (hexanes:ethyl acetate

gradient, 100:0 to 94:6) to obtain 1-tert-butyldimethylsilyloxy-2-

1

hydroxybutane (1 g, 45% yield). H NMR (CDCl₃, 600 MHz) δ:

3.60 (m, 1H), 3.50 (m, 1H), 3.40 (m, 1H), 2.40 (s, 1H), 1.44 (m,

2H), 0.99 (m, 3H), 0.90 (s, 9H), 0.06 (s, 6H).

1

A solution of methyl-4-hydroxybenzoate (76, 1.10 g, 7.34 mmol),

1-tert-butyldimethylsilyloxy-2-hydroxybutane (0.75 g, 3.67 mmol),

and PPh₃(1.97 g, 7.34 mmol) dissolved in THF (8 mL) was cooled

to 0 °C. DIAD (1.49 g, 7.34 mmol) was added dropwise The

reaction mixture was stirred for 2 h before being concentrated in

vacuo. The crude material was puriﬁed using silica gel chroma-

tography (hexanes:diethyl ether gradient, 100 to 99:1) to obtain

compound 85 (1.0 g, 83% yield) as a viscous oil. ¹H NMR (CDCl₃,

600 MHz) δ: 7.90 (m, 2H), 6.90 (m, 2H), 4.30 (m, 1H), 3.90 (s,

3H), 3.70 (2H), 1.78 (m, 1H), 1.70 (m, 1H), 0.90 (t, 3H, J ) 7.8

Hz), 0.89 (s, 9H), 0.05 (s, 3H), 0.01 (s, 3H). ¹³C NMR (CDCl₃,

150 MHz) δ: 166.8, 162.8, 131.5, 122.3, 115.2, 80.0, 64.5, 51.7,

25.8, 24.1, 18.2, 9.5, –5.3.

To a solution of compound 56 (234 mg, 1.16 mmol) in THF (45

mL) was added compound 48 (312 mg, 1.73 mmol), PPh₃(454

mg, 1.73 mmol), and DIAD (335 µL, 1.73 mmol). The reaction

mixture was stirred at room temperature overnight. The reaction

mixture was concentrated in vacuo, and the crude material was

puriﬁed by silica gel chromatography (hexanes:ethyl acetate gradi-

ent, 4:1 to 100% ethyl acetate) to obtain compound 58 (200 mg,

Synthesis of 4-(1-tert-Butyldimethylsilyloxy-but-2-oxy) ben-

zyl alcohol (49). To a solution of compound 76 (1 g, 2.95 mmol)

in ether (15 mL) at 0 °C was added LAH (336 mg, 8.8 mmol).

After completion of addition, the reaction mixture was stirred for

1.5 h. Addition of water (0.336 mL), 15% NaOH (aq) (0.336 mL),

and water (1.00 mL) in succession quenched the reaction mixture.

The resulting cloudy mixture was stirred for an additional 20 min

before being ﬁltered. The ﬁltrate was dried over Mg₂SO₄, ﬁltered,

and concentrated to yield compound 49 (0.50 g, 54% yield) as a

1

48% yield) as a clear oil. H NMR (CDCl₃, 600 MHz) δ: 7.73 (s,

1H), 7.32 (d, 2H, J ) 8.0 Hz), 7.24 (d, 2H, J ) 8.0 Hz), 5.30 (s,

1H), 5.27 (s, 2H), 3.83 (m, 1H), 2.80–2.76 (m, 1H), 2.71–2.66 (m,

1H), 1.63 (s, 9H), 1.23 (d, 3H, J ) 6.2 Hz); ¹³C NMR (CDCl₃,

150 MHz) δ: 159.3, 153.9, 143.2, 132.5, 129.2, 127.6, 125.4, 118.5,

73.4, 67.6, 66.6, 40.9, 32.0, 28.1, 23.9; HRMS calcd for

C₉H₂₅ClN₂O₃, 365.1626; found, 365.1624.

1

white solid. H NMR (CDCl₃, 600 MHz) δ: 7.24 (d, 2H, J ) 8.4

Hz), 6.92 (d, 2H, J ) 8.4 Hz), 4.60 (d, 2H, J ) 5.4 Hz), 4.20 (m,

1H), 3.76 (dd, 1H, J ) 5.4, 10.8 Hz), 3.66 (dd, 1H, J ) 5.4, 10.8

Hz), 1.74 (m, 1H), 1.65 (m, 1H), 1.50 (t, 1H, J ) 5.4 Hz), 0.90 (t,

3H, J ) 7.8 Hz), 0.89 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H); ¹³C NMR

(CDCl₃, 150 MHz) δ: 158.5, 133.0, 128.4, 116.1, 80.1, 65.0, 64.5,

25.8, 24.1, 18.2, 9.5, –5.3; HRMS-TOF (m/z): [M + H]⁺for

C₁₇H₃₀O₃Si, 310.1964; found 333.1867 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(3-ﬂuorobutyl)benzy-

loxy]-3-(2H)-pyridazinone (24). To a solution of compound 58

(200 mg, 0.55 mmol) in pyridine (10 mL) was added TsCl (209

mg, 1.10 mmol). The reaction mixture was stirred at room

temperature overnight. The reaction mixture was diluted with ethyl

acetate (30 mL) and washed repeatedly with 5% CuSO₄(200 mL)

until a light-blue aqueous solution was maintained. The organic

layer was then dried over Na₂SO₄, ﬁltered, and concentrated. The

crude material was puriﬁed by silica gel chromatography (hexanes:

ethyl acetate gradient, 3:1 to 100% ethyl acetate) to recover the

unreacted starting material (90 mg) and compound 64 as a clear

Synthesis of 2-tert-Butyl-4-chloro-5-(4-(1-hydroxy-but-2-oxy)-

benzyl)oxy-3-(2H)-pyridazinone (65). To a cooled (0 °C) solution

of compound 56 (0.48 g, 2.42 mmol) dissolved in THF (40 mL)

was added compound 49 (500 mg, 1.61 mmol), PPh₃(633 mg, 2.42

mmol), and DIAD (488 mg, 2.42 mmol). After completion of

addition, the reaction mixture was stirred for 2 h and then

concentrated in vacuo to obtain a crude oil. Puriﬁcation of the crude

material by silica gel chromatography (hexanes:ethyl acetate

gradient, 100 to 94:6) afforded compound 59 (330 mg, 41% yield)

1

oil (74 mg, 47% yield based on recovered starting material). H

1

NMR (CDCl₃, 600 MHz) δ: 7.80 (d, 2H, J ) 8.3 Hz), 7.72 (s,

1H), 7.33 (d, 2H, J ) 8.0 Hz), 7.30 (d, 2H, J ) 8.1 Hz), 7.13 (d,

2H, J ) 8.1 Hz), 5.27 (s, 2H), 4.66 (m, 1H), 2.65 (m, 1H), 2.54

(m, 1H), 2.45 (s, 3H), 1.94 (m, 1H), 1.81 (m, 1H), 1.63 (s, 9H),

1.26 (s, 3H).

as an oil. H NMR (CDCl₃, 600 MHz) δ: 7.72 (s, 1H), 7.20 (d,

2H, J ) 9 Hz), 6.90 (d, 2H, J ) 8.4 Hz), 5.20 (s, 2H), 4.20 (m,

1H, J ) 5.4 Hz), 3.75 (dd, 1H, J ) 6 Hz), 3.68 (dd, 1H, J) 6 Hz),

1.75 (m, 1H), 1.65 (m, 1H), 1.60 (s, 9H), 0.99 (m, 3H), 0.85 (s,

9H), 0.02 (s, 3H), –0.01 (s, 3H); ¹³C NMR (CDCl₃, 600 MHz) δ:

159.6, 159.3, 154.0, 129.0, 126.9, 125.0, 118.5, 116.5, 80.3, 72.1,

66.5, 64.8, 28.1, 26.0, 24.4, 18.4, 9.6, –5.3.

To a solution of compound 64 (18.2 mg, 0.035 mmol) in ACN

(400 µL) was KF (4.1 mg, 0.070 mmol) and kryptoﬁx222 (26.4 mg,

0.070 mmol). The reaction mixture was stirred at 90 °C. After 20 min,

the reaction was cooled to room temperature and concentrated under

reduced pressure. The crude material was puriﬁed by preparative thin

layer chromatography (silica gel, hexanes:ethyl acetate, 4:1) to obtain

To a solution of compound 59 (0.3 g, 0.6 mmol) in THF (2 mL)

was added TBAF (1.8 mL of 1.0 M solution in THF, 1.8 mmol)

dropwise. After completion of addition, the reaction mixture was

stirred for 1.5 h before being concentrated in vacuo. The crude oil

was puriﬁed by silica gel chromatography (hexanes:ethyl acetate

gradient, 100 to 4:1) to obtain compound 65 (185 mg, 80% yield)

1

analogue 24 (5 mg, 39% yield). H NMR (CDCl₃, 600 MHz) δ:

7.70 (s, 1H), 7.34 (d, 2H, J ) 7.9 Hz), 7.24 (d, 2H, J ) 8.0 Hz),

5.28 (s, 2H), 4.71–4.60 (m, 2H), 2.84–2.80 (m, 1H), 2.73–2.69 (m,

1H), 2.02–1.93 (m, 1H), 1.87–1.77 (m, 1H), 1.63 (s, 9H), 1.35 (m,

3H, J ) 6.2, 23.9 Hz);¹³C NMR (CDCl₃, 150 MHz) δ: 159.1, 153.8,

142.4, 132.5, 129.0, 127.4, 125.2, 118.3, 90.4 (89.3), 71.9, 66.3,

38.5 (38.4), 31.1 (31.0), 27.9, 21.1 (21.0);¹⁹F NMR (CDCl₃, 564

MHz) δ: –174.7 (1F, m); HRMS-TOF (m/z): [M + H]⁺for

C₁₉H₂₃ClFN₂O₂, 367.1583; found, 367.1582.

1

in good yield. H NMR (CDCl₃, 600 MHz) δ: 7.74 (s, 1H), 7.30

(d, 2H, J ) 8.4 Hz), 6.90 (d, 2H, J ) 8.4 Hz), 5.20 (s, 2H), 4.30

(m, 1H), 3.81–3.77 (br m, 2H), 1.84 (br m, 1H), 1.72 (m, 2H),

1.64 (s, 9H), 0.98 (m, 3H); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.2,

158.9, 153.9, 129.2, 127.5, 125.4, 116.6, 80.4, 71.9, 66.5, 64.2,

28.0, 23.5, 9.7; HRMS-TOF (m/z): [M + H]⁺for C₁₉H₂₅ClN₂O₄,

380.1503; found, 403.1394 [M + Na]⁺.

Synthesis of Methyl-4-(1-tert-butyldimethylsilyloxy-but-2-oxy)-

benzoate (85). To a solution of 1,2-butanediol (1 g, 11.09 mmol)

in DMF (8 mL) was added TBDMS-Cl (2.5 g, 16.64 mmol) and

imidazole (1.88 g, 27.7 mmol). The reaction mixture was stirred

for 10 h before being diluted with DCM (20 mL). The organic

layer was washed with water (4 × 20 mL) and brine (20 mL),

dried over Mg₂SO₄, ﬁltered, and then concentrated. The crude oil

Synthesis of 2-tert-Butyl-4-chloro-5-(4-(1-tosyloxy-but-2-oxy)-

benzyl)oxy-3-(2H)-pyridazinone (71). A solution of compound 65

(50 mg, 0.13 mmol), TsCl (75 mg, 0.39 mmol), DMAP (48 mg,

0.39 mmol), and DIEA (50 mg, 0.39 mmol) in DCM (2 mL) was

stirred at room temperature. After 35 min, the reaction mixture was

diluted with water (1 mL). The organic layer was separated and

washed with water (1 mL) and brine (1 mL), dried over Mg₂SO₄,

2966 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Purohit et al.

ﬁltered, and then concentrated. The crude oil was puriﬁed by silica

gel chromatography (hexanes:ethyl acetate gradient, 90:10 to 70:

30) to obtain compound 71 (54 mg, 77% yield) as a viscous

colorless oil. ¹H NMR (CDCl₃, 600 MHz) δ: 7.75 (s, 1H), 7.74 (s,

2H), 7.30 (m, 4H), 6.84 (d, 2H, J ) 9 Hz), 5.20 (s, 2H), 4.38 (m,

1H), 4.15 (m, 2H), 2.44 (s, 3H), 1.72 (m, 2H), 1.60 (s, 9H), 0.95

(t, 3H, J ) 7.2 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.2, 158.5,

153.9, 145.1, 133.0, 130.0, 129.0, 128.1, 127.2, 125.4, 118.5, 116.5,

71.9, 70.2, 66.6, 28.1, 24.2, 21.8, 9.4; HRMS-TOF (m/z): [M +

H]⁺for C₂₆H₃₁ClN₂O₆S, 534.1591; found, 557.1490 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-(4-(1-ﬂuoro-but-2-oxy)-

benzyl)oxy-3-(2H)-pyridazinone (25). To a solution of compound

71(28 mg, 52.4 µmol) dissolved in ACN (0.5 mL) was added a

solution of KF (4.5 mg, 78.6 µmol) and kryptoﬁx222 (29.6 mg,

78.6 µmol) in 0.5 mL ACN. The ﬂask was ﬁtted with a reﬂux

condenser, and the solution was then immersed in an oil bath

preheated at 90 °C. The reaction mixture was stirred for 90 min,

after which all the volatiles were removed under reduced pressure.

The crude oil was puriﬁed by preparative thin layer chromatography

(silica gel, hexanes:ethyl acetate, 7:3) to obtain compound 25 (13

mg, 65% yield) in good yield. ¹H NMR (CDCl₃, 600 MHz) δ: 7.72

(s, 1H), 7.30 (m, 2H), 6.90 (m, 2H), 5.23 (s, 2H), 4.54 (dd, 2H, J

) 4.8, 46.2 Hz), 4.40 (m, 1H), 1.74 (m, 2H), 1.60 (s, 9H), 1.0 (t,

3H, J ) 7.2 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ: 159.0, 158.7,

153.7, 129.0, 127.5, 125.2, 118.3, 116.4, 83.9 (82.7), 78.0, 71.1,

66.3, 27.8, 23.2, 9.5. ¹⁹F NMR (CDCl₃, 564 MHz) δ: –228 (1F,

m); HRMS-TOF (m/z): [M + H]⁺for C₁₉H₂₄ClFN₂O₃, 382.1459;

found, 405.1350 [M + Na]⁺.

Synthesis of Toluene-4-sulfonic acid-2-[4-(1-tert-butyl-5-

chloro-6-oxo-1,6-dihydro-pyridazin-4-yloxymethyl)-phenoxy]-1-

methyl-ethyl ester (66). To a solution of compound 50³³(269 mg,

1.48 mmol) and compound 56 (250 mg, 1.23 mmol) dissolved in

anhydrous THF (18.5 mL) was added PPh₃(485 mg, 1.85 mmol)

and DIAD (0.358 mL, 1.85 mmol). After completion of addition,

the reaction mixture stirred for 20 h before being diluted with water.

The aqueous layer was separated and extracted with ethyl acetate

(3 × 50 mL). All combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated to provide an oil. The crude material was

puriﬁed using silica gel chromatography (pentane:ethyl acetate, 1:1)

to provide compound 60 (234 mg, 51% yield) in moderate yield.

¹H NMR (CDCl₃, 600 MHz) δ: 7.71 (s, 1H), 7.33 (d, 2H, J ) 8.7

Hz), 6.94 (d, 2H, J ) 8.7 Hz), 5.24 (s, 2H), 4.19 (m, 1H), 3.95

(dd, 1H, J ) 9.2, 3.1 Hz), 3.81 (dd, 1H, J ) 9.2, 7.7 Hz), 1.62 (s,

9H) 1.29 (d, 3H, J ) 6.4 Hz).

gel thin layer chromatography plate (pentane:ethyl acetate, 4:1) to

isolate unreacted starting material (5.8 mg, 0.011 mmol) in addition

to analogue 26 (12.5 mg, 41% yield based on recovered starting

material). ¹H NMR (CDCl₃600 MHz) δ: 7.73 (s, 1H) 7.34 (d, 2H,

J ) 8.6 Hz), 6.95 (d, 2H, J ) 8.6 Hz), 5.25 (s, 2H), 5.06–4.96 (m,

1H), 4.06 (2H, m), 1.63 (s, 9H) 1.47 (dd, 3H, J ) 6.4, 23.6 Hz);

¹³C NMR (DMSO-d₆, 150 MHz) δ: 158.4, 157.8, 153.9, 129.8,

127.6, 126.2, 115.5, 114.6, 89.0 (88.0), 71.2, 70.4 (70.3), 65.3, 27.4,

16.9 (16.8); ¹⁹F(DMSO-d₆, 564 MHz) δ: –178.20 (1F, m); HRMS-

TOF (m/z): [M + H]⁺C₁₈H₂₂ClFN₂O₃, 369.1375; found, 369.1370.

Synthesis of Methyl-4-(2-hydroxyethoxymethyl)benzoate (72).

A solution of methyl 4-hydroxymethylbenzoate (70, 2.50 g, 0.015

mol) in DCM (30 mL) was charged into a two-neck round-bottom

ﬂask that was equipped with a Dewar condenser and cooled at –10

°C in a salt/ice bath. Ethylene oxide (1.10 mL) was added to the

cooled stirring solution dropwise, followed by the addition of boron

triﬂuoride etherate (0.51 mL). The reaction mixture was stirred for

45 min and then warmed to room temperature for 30 min to distill

out any excess of ethylene oxide in the reaction mixture. The

reaction mixture was then diluted with brine (50 mL). The aqueous

layer was extracted with dichloromethane (3 × 50 mL). The organic

layers were combined, dried over Na₂SO₄, ﬁltered, and concentrated

to provide an oil. The crude material was puriﬁed using silica gel

chromatography (pentane:ethyl acetate, 4:1) to provide compound

72 (537 mg, 17% yield) in low yield. ¹H NMR (CDCl₃, 600 MHz)

δ: 8.36 (d, 2H, J ) 8.4 Hz), 7.41 (d, 2H, J ) 8.5 Hz), 4.62 (s, 3H),

3.92 (s, 2H), 3.78 (m, 2H), 3.63 (m, 2H); ¹³C NMR (CDCl₃, 150

MHz) δ: 167.1, 143.5, 130.0, 129.8, 127.5, 72.9, 72.0 62.1, 52.3.

Synthesis of 4-[2-(tert-Butyldimethylsilanyloxy)ethoxymethyl]-

benzoic acid methyl ester (86). To a solution of the compound

72 (544.5 mg, 2.59 mmol) in DMF (26 mL) was added imidazole

(264 mg, 3.89 mmol) and TBDMS-Cl (586 mg, 3.89 mmol). The

reaction mixture was stirred at room temperature overnight, then

quenched with water. The aqueous layer was extracted with ethyl

acetate (3 × 100 mL). All combined organic layers were dried over

Na₂SO₄, ﬁltered, and concentrated. The crude material was puriﬁed

using silica gel chromatography (pentane:ethyl acetate, 4:1) to

provide compound 86 (677.5 mg, 84% yield) in good yield. ¹H

NMR (CDCl₃, 600 MHz) δ: 8.01 (d, 2H, J ) 8.3 Hz), 7.42 (d, 2H,

J ) 8.4 Hz), 4.63 (s, 2H), 3.91 (s, 2H), 3.82 (t, 2H, J ) 5.0 Hz),

3.58 (t, 2H, J ) 5.1 Hz), 0.91 (s, 9H), 0.07 (s, 6H); ¹³C NMR

(CDCl₃,150 MHz) δ: 166.5, 143.5, 129.2, 128.8, 126.5, 72.1, 71.6,

62.3, 51.5, 25.4, 17.9, –5.8.

Synthesisof{4-[2-(tert-Butyldimethylsilanyloxy)ethoxymethyl]-

phenyl}methanol (51). To a solution of compound 86 (670 mg,

2.18 mmol) dissolved in THF (22 mL) was added LAH (2.18 mL

of 1.0 M solution in THF, 2.18 mmol) dropwise. After completion

of addition the reaction mixture was stirred at room temperature

for 3 h. The reaction mixture was diluted with water, and the

aqueous layer was extracted with ethyl acetate (3 × 50 mL). The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated to provide compound 51 as an oil (587 mg, 91% yield),

which was used in the next step without any further puriﬁcation.

¹H NMR (CDCl₃, 600 MHz) δ: 7.34 (s, 4H), 4.68 (s, 2H), 4.57 (s,

2H), 3.80 (t, 2H, J ) 5.2 Hz), 3.56 (t, 2H, J ) 5.3 Hz), 1.69 (br,

s, 1H), 0.90 (s, 9H), 0.07 (s, 6H); ¹³C NMR (CDCl₃, 150 MHz) δ:

140.4, 138.3, 128.0, 127.2, 73.2, 71.9, 65.4, 63.0, 26.2, 18.6, –5.0.

Synthesisof 2-tert-Butyl-5-{4-[2-(tert-butyldimethylsilanyloxy)-

ethoxymethyl]benzyloxy}-4-chloro-3-(2H)-pyridazinone (61). To

a solution of compound 51 (437 mg, 1.48 mmol) and compound

56 (250 mg, 1.23 mmol) dissolved in THF (12 mL) was added

PPh₃(485 mg, 1.85 mmol) and DIAD (0.358 mL, 1.85 mmol).

After completion of addition, the reaction mixture was stirred at

room temperature for 20 h and then diluted with water (20 mL).

The aqueous layer was separated and extracted with ethyl acetate

(3 × 50 mL). The combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated to provide an oil. The crude material was

puriﬁed using silica gel chromatography (pentane:ethyl acetate, 4:1)

To a solution of compound 60 (200 mg, 0.55 mmol) dissolved

in DCM (6.0 mL) was added TsCl (125 mg, 0.66 mmol), DMAP

(100 mg, 0.82 mmol), and TEA (0.0914 mL, 0.66 mmol). The

reaction mixture stirred for 22 h, before being diluted with water.

The aqueous layer was separated and extracted with ethyl acetate

(3 × 20 mL). All combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated to provide an oil. The crude material was

puriﬁed using silica gel chromatography (pentane:ethyl acetate, 70:

30) to provide compound 66 (166 mg, 58% yield) in good yield.

¹H NMR (CDCl₃, 600 MHz) δ: 7.80 (d, 2H, J ) 8.3 Hz), 7.72 (s,

1H), 7.32 (d, 2H, J ) 7.9 Hz), 7.29 (d, 2H, J ) 8.7 Hz), 6.74 (d,

2H, J ) 8.7 Hz), 5.22 (s, 2H), 4.19 (m, 1H), 4.02 (dd, 1H, J )

10.4, 6.0 Hz), 3.93 (dd, 1H, J ) 10.4, 4.5 Hz), 2.44 (s, 3H), 1.63

(s, 9H) 1.42 (d, 3H, J ) 6.5 Hz); ¹³C NMR (CDCl₃, 150 MHz) δ:

158.9, 158.3, 153.6, 144.6, 133.8, 129.6, 128.8, 127.8, 127.4, 125.1,

118.0, 114.7, 76.8, 71.5, 69.7, 66.2, 27.7, 21.5, 17.6; HRMS-TOF

(m/z): [M + H]⁺for C₂₅H₂₉ClN₂O₆S, 521.1507; found, 521.1505.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(2-ﬂuoropropoxy)ben-

zyloy]-3-(2H)-pyridazinone (26). To a solution of compound 66

(50 mg, 0.10 mmol) in ACN (1.0 mL) was added KF (11.2 mg,

0.19 mmol) and kryptoﬁx222 (72.4 mg, 0.19 mmol). After

completion of addition, the reaction mixture was heated at 90 °C.

After 40 min, the reaction mixture was cooled to room temperature

and diluted with water (1.0 mL). The aqueous layer was separated

and extracted with ethyl acetate (3 × 5 mL). The combined organic

layers were dried over Na₂SO₄, ﬁltered, and concentrated to provide

an oil. The crude material was puriﬁed using a preparative silica

1

to provide compound 61 (528 mg, 89% yield) in good yield. H

NMR (CDCl₃, 600 MHz) δ: 7.70 (s, 1H), 7.38 (m, 4H), 5.30 (s,

2H), 4.58 (s, 2H), 3.80 (t, 2H, J ) 5.4 Hz), 3.57 (t, 2H, J ) 5.4

Pyridazinone Analogues as Cardiac PET Tracers

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2967

Hz), 1.63 (br s, 9H), 0.90 (s, 9H), 0.07 (s, 6H); ¹³C NMR (CDCl₃,

150 MHz) δ: 159.0, 153.7, 138.8, 134.4, 128.3, 127.3, 125.1, 118.5,

72.8, 71.7, 71.6, 66.4, 61.9, 29.7, 27.9, 25.6, –5.1; HRMS-TOF

(m/z): [M + H]⁺for C₂₄H₃₇ClN₂O₄Si, 481.2283; found, 481.2282.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(2-hydroxyethoxym-

ethyl)benzyloxy]-3-(2H)-pyridazinone (67). To a solution of

compound 61 (528 mg, 1.09 mmol) dissolved in THF (11 mL) was

added a solution of TBAF (1.65 mL of 1.0 M solution in THF,

1.65 mmol) dropwise. After completion of addition, the reaction

was stirred at room temperature for 1 h and then quenched with

water (10 mL). The aqueous layer was separated and extracted with

ethyl acetate (3 × 20 mL). The combined organic layers were dried

over Na₂SO₄, ﬁltered, and concentrated to provide an oil. The crude

material was puriﬁed using silica gel chromatography (hexanes:

ethyl acetate, 4:1) to provide compound 67 (311 mg, 78% yield).

¹H NMR (CDCl₃, 600 MHz) δ: 7.70 (s, 1H), 7.38 (m, 4H), 5.30

(s, 2H), 4.56 (s, 2H), 3.76 (t, 2H, J ) 4.9 Hz), 3.60 (t, 2H, J ) 4.8

Hz), 2.00 (br s, 1H), 1.61 (br s, 9H); ¹³C NMR (CDCl₃, 150 MHz)

δ: 159.0, 153.6 138.8, 134.4, 128.2, 127.2, 125.1, 118.3, 72.8, 71.6,

71.6, 66.4, 61.9, 27.8; HRMS-TOF (m/z): [M + H]⁺for C₁₈-

H₂₃ClN₂O₄, 367.1419; found, 367.1419.

Synthesis of Toluene-4-sulfonic acid-2-[4-(1-tert-butyl-5-

chloro-6-oxo-1,6-dihydro-pyridazin-4-yloxymethyl)-benzyloxy]-

ethyl ester (73). To a solution of compound 67 (200 mg, 0.55

mmol) dissolved in DCM (5.50 mL) was added TsCl (125 mg,

0.66 mmol), DMAP (100 mg, 0.82 mmol), and TEA (0.091 mL,

0.66 mmol). The reaction mixture was stirred at room temperature

for 22 h and then was diluted with water (10 mL). The aqueous

layer was separated and extracted with ethyl acetate (3 × 20 mL).

All combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated to provide an oil. The crude material was puriﬁed using

silica gel chromatography (pentane:ethyl acetate, 3:2) to provide

compound 73 (232 mg, 82% yield) in good yield. ¹H NMR (CDCl₃,

600 MHz) δ: 7.79 (d, 2H, J ) 8.3 Hz), 7.71 (s, 1H), 7.38 (d, 2H,

J ) 8.2 Hz), 7.32 (m, 4H), 5.30 (s, 2H), 4.50 (s, 2H), 4.21 (m,

2H), 3.69 (m, 2H), 2.43 (s, 3H), 1.63 (br s, 9H); ¹³C NMR (CDCl₃,

150 MHz) δ: 159.0, 153.7, 144.8, 138.8, 134.4, 133.1, 129.8, 128.1,

128.0, 127.2, 125.1, 118.4, 72.8, 71.7, 69.2, 67.8, 66.4, 27.9, 21.6;

HRMS-TOF (m/z): [M + H]⁺for C₂₅H₂₉ClN₂O₆, 521.1507; found,

521.1503.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(2-ﬂuoro-ethoxymethyl)-

benzyloxy]-3-(2H) -pyridazinone (27). To a solution of compound

73 (50 mg, 0.10 mmol) in ACN (1.0 mL) was added KF (11.2 mg,

0.19 mmol) and kryptoﬁx222 (72.4 mg, 0.19 mmol). After

completion of addition, the reaction mixture was heated at 90 °C.

After 10 min, the reaction mixture was cooled to room temperature

and diluted with water (1 mL). The aqueous layer was separated

and extracted with ethyl acetate (3 × 5 mL). All combined organic

layers were dried over Na₂SO₄, ﬁltered, and concentrated to provide

an oil. The crude material was puriﬁed using silica gel chroma-

tography (pentane:ethyl acetate, 4:1) to provide analogue 27 (28

mg, 79% yield) in good yield. ¹H NMR (DMSO-d₆, 600 MHz) δ:

8.22 (s, 1H), 7.45 (d, 2H, J ) 8.2 Hz), 7.39 (d, 2H, J ) 8.2 Hz),

5.42 (s, 2H), 4.60 (m, 1H), 4.54 (s, 2H), 4.52 (m, 1H), 3.71 (m,

1H), 3.66 (m, 1H), 1.57 (s, 9H); ¹³C NMR (DMSO-d₆, 150 MHz)

δ: 157.8, 153.8, 138.6, 134.6, 127.8, 127.7, 126.2, 115.6, 83.5

(82.4), 71.6, 71.2, 69.1 (69.0), 65.3, 27.4; ¹⁹F NMR (DMSO-d₆,

564 MHz) δ: –221.74 (1F, m); HRMS-TOF (m/z): [M + H]⁺for

C₁₈H₂₂ClFN₂O₃, 369.1375; found, 369.1377.

Synthesis of Methyl-4-[2-hydroxy-d₄-ethoxymethyl]benzoate

(87). A solution of methyl 4-hydroxymethylbenzoate (70, 2.5 g,

15 mmol) in DCM (30 mL) was added to a two-neck round-bottom

ﬂask, which was equipped with a Dewar condenser and cooled at

–10 °C in a salt/ice bath. d₄-Ethylene oxide (1.10 mL) was added

to the cooled stirring solution dropwise followed by the addition

of boron triﬂuoride etherate (0.51 mL). The reaction mixture was

stirred for 45 min and then warmed to room temperature for 30

min to distill out any excess of d₄-ethylene oxide in the reaction

mixture. The reaction mixture was then diluted with brine (50 mL).

The aqueous layer was extracted with dichloromethane (3 × 50

mL). The organic layers were combined, dried over Na₂SO₄, ﬁltered,

and concentrated to provide an oil. The crude material was puriﬁed

using silica gel chromatography (pentane:ethyl acetate, 4:1) to

provide compound 87 (520 mg, 16% yield) in low yield. ¹H NMR

(CDCl₃, 600 MHz) δ: 8.02 (d, 2H, J ) 8.2 Hz), 7.41 (d, 2H, J )

8.1 Hz), 4.62 (s, 2H), 3.92 (s, 3H); ¹³C NMR (CDCl₃, 150 MHz)

δ: 167.1, 143.5, 130.8, 129.9, 127.5, 72.8, 52.4.

Synthesis of Methyl-4-[2-(tert-butyldimethylsilanyloxy)-d₄-

ethoxymethyl]benzoate (88). To a solution of compound 87 (500

mg, 2.33 mmol) in DMF (23 mL) was added TBDMS-Cl (528 mg,

3.50 mmol) and imidazole (238 mg, 3.50 mmol). The reaction was

stirred at room temperature overnight and then diluted with water

(30 mL) and extracted with ethyl acetate (2×50 mL). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated

in Vacuo to obtain a crude oil. The oil was puriﬁed bypassage

through a thick pad of silica gel (pentane:ethyl acetate, 3:1) to obtain

the compound 88 as a clear oil (602 mg, 79% yield). ¹H NMR

(CDCl₃, 600 MHz) δ: 8.00 (d, 2H, J ) 8.3 Hz), 7.40 (d, 2H, J )

8.5 Hz), 4.62 (s, 2H), 3.90 (s, 3H), 0.90 (s, 9H), 0.06 (s, 6H); ¹³

C

NMR (CDCl₃,150 MHz) δ: 167.2, 144.2, 129.9, 129.5, 127.3, 72.8,

52.3, 26.1, 18.6, –5.8.

Synthesis of {4-[2-(tert-Butyldimethylsilanyloxy)-d₄-ethoxy-

d₂-methyl]phenyl}methanol (52). To a solution of compound 88

(610 mg, 1.86 mmol) in THF (19 mL) at 0 °C was added LAD

(1.9 mL of 1 M solution in THF, 1.86 mmol) dropwise. After

completion, the reaction was stirred at room temperature for 3.5 h

before being diluted with water and extracted with ethyl acetate (2

× 50 mL). The combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated to obtain compound 52 as a clear oil (482

mg, 86% yield). The material was used in the next step without

1

further puriﬁcation. H NMR (600 MHz, CDCl₃) δ: 7.33 (s, 4H),

4.56 (s, 2H), 0.89 (s, 9H), 0.06 (s, 6H); ¹³C NMR (CDCl₃, 150

MHz) δ: 140.3, 138.4, 128.1, 127.3, 73.1, 29.9, 26.2, 18.6, –5.0.

Synthesis of 2-tert-Butyl-4-chloro-5-{4-[2-(tert-butyldimeth-

ylsilanyloxy)-d₄-ethoxy-d₂-methyl]benzyloxy}-3-(2H)-pyridazi-

none (62). To a solution of compound 56 (212 mg, 1.05 mmol) in

THF (15 mL) was added compound 52 (475 mg, 1.57 mmol), PPh₃

(412 mg, 1.57 mmol), and DIAD (304 µL, 1.57 mmol). The reaction

mixture was stirred at room temperature for 2 h. After concentration

of the reaction mixture in vacuo, the crude material was puriﬁed

by silica gel chromatography (pentane:ethyl acetate, 9:1) to obtain

compound 62 as a clear oil (336 mg, 66% yield). ¹H NMR (CDCl₃,

600 MHz) δ: 7.70 (s, 1H), 7.39 (m, 4H), 4.58 (s, 2H), 1.63 (s,

9H), 0.90 (s, 9H), 0.07 (s, 6H); ¹³C NMR (CDCl₃, 150 MHz) δ:

158.5, 153.2, 139.0, 133.4, 127.5, 126.7, 124.6, 117.8, 72.2, 65.8,

27.3, 25.4, 17.8, –5.1; HRMS-TOF (m/z): [M + H]⁺for C₂₄H₃₁-

D₆ClN₂O₄Si, 486.2588; found, 509.2480 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(2-hydroxy-d₄-ethoxy-

d₂-methyl)benzyloxy]-3-(2H)-pyridazinone (68). To a solution of

compound 62 (330 mg, 0.68 mmol) in THF (7 mL) was added

TBAF (1 mL of 1 M solution in THF, 1.02 mmol) dropwise. The

reaction mixture was stirred at room temperature for 2 h followed

by solvent removal under reduced pressure to yield a crude oil.

The crude material was dissolved in DCM and passed through a

thick pad of silica (100% ethyl acetate) to obtain compound 68

(250 mg, 99% yield) containing a minor percentage of the

corresponding silanol. The material was used in the next step

1

without further puriﬁcation. H NMR (CDCl₃, 600 MHz) δ: 7.72

(s, 1H), 7.41 (s, 4H), 4.59 (s, 2H), 1.64 (s, 9H); ¹³C NMR (CDCl₃,

150 MHz) δ: 159.2, 153.9, 139.5, 134.5, 128.5, 127.5, 125.3, 118.6,

73.0, 66.6, 28.1; HRMS-TOF (m/z): [M + H]⁺for C₁₈H₁₇-

D₆ClN₂O₄, 395.1609; found, 395.1615 [M + Na]⁺.

Synthesis of 2-tert-butyl-4-chloro 5-[4-(2-p-toluenesulfony-

loxy-d₄-ethoxy-d₂-methyl)benzyl]oxy-3-(2H)-pyridazinone (74).

To a solution of compound 68 (250 mg, 0.67 mmol) in DCM (7

mL) was added TsCl (153 mg, 0.81 mmol), DMAP (98 mg, 0.81

mmol), and TEA (140 µL, 1.01 mmol). The reaction was stirred at

room temperature overnight and was then concentrated under

reduced pressure. The crude material was puriﬁed by silica gel

chromatography (hexanes:ethyl acetate gradient, 2:1 to 100% ethyl

acetate) to recover the unreacted starting material 68 (9 mg) and

the product 74 (261 mg, 77% yield based on recovered starting

2968 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10

Purohit et al.

1

material) as a clear oil. H NMR (CDCl₃, 600 MHz) δ: 7.76 (d,

acetate pH 7.5, 1.5 mM adenosine triphosphate (ATP), and 10 mM

MgCl₂. The samples were stored at -80 °C.

2H, J ) 8.3 Hz), 7.73 (s, 1H), 7.36 (d, 2H, J ) 8.1 Hz), 7.29 (m,

4H), 4.47 (s, 2H), 2.40 (s, 3H), 1.61 (s, 9H); ¹³C NMR (CDCl₃,

150 MHz) δ: 159.0, 153.8, 145.0, 138.5, 134.4, 133.1, 129.9, 128.1,

128.0, 127.3, 125.2, 118.1, 72.7, 71.0, 37.0, 63.4, 28.0, 21.7;

HRMS-TOF (m/z): [M + H]⁺for C₂₅H₂₃D₆ClN₂O₆S, 526.1811;

found, 549.1705 [M + Na]⁺.

Synthesis of 2-tert-Butyl-4-chloro-5-[4-(2-ﬂuoro-d₄-ethoxy-

d₂-methyl)-benzyloy]-3-(2H)-pyridazinone (28). To a solution of

compound 74 (14 mg, 0.03 mmol) in ACN (300 µL) was added

KF (3.1 mg, 0.05 mmol) and kryptoﬁx222 (20 mg, 0.05 mmol).

The reaction mixture was stirred at 90 °C for 10 min. The reaction

mixture was then cooled to room temperature and concentrated

under reduced pressure. The crude material was puriﬁed by

preparative TLC (hexanes:ethyl acetate, 2:1) to obtain analogue 28

(6.2 mg, 62% yield) as an oil. ¹H NMR (CDCl₃, 600 MHz) δ:

7.70 (s, 1H), 7.40 (s, 4H), 4.61 (s, 2H), 1.63 (s, 9H); ¹³C NMR

(CDCl₃, 150 MHz) δ: 158.5, 153.1, 138.2, 133.8, 127.7, 126.8,

124.6, 117.8, 72.4, 65.9, 27.3; ¹⁹F NMR (CDCl₃, 564 MHz) δ:

–225.2 (m, 1F).

General Procedure for the Radiosynthesis of F-18 Labeled

Pyridazinone Analogues 22–28. A 500 mCi lot of aqueous F-18

was made by the ¹⁸O(p,n)¹⁸F reaction (PETNET Pharmaceutical

Services, Cummings Park, Woburn, MA) and applied to a previ-

ously activated MP1 anion exchange resin (BioRad) cartridge. This

cartridge was placed into an elution loop contained within a

remotely controlled radiosynthesis system. The radioactivity was

eluted from the cartridge and collected into a silanized 25 mL pear-

shaped ﬂask by the addition of 1 mL of a solution prepared as

follows: K₂CO₃(15 mg) was dissolved in deionized water (1 mL),

and kryptoﬁx222 (90 mg) was dissolved in ACN (4 mL); the two

solutions were combined, and the appropriate aliquot was used for

elution of the column. The eluate from the MP1 anion exchange

resin was then concentrated to dryness by applying a gentle stream

of heated He and a slight vacuum. ACN (0.5 mL) was added, and

the solvent was again removed by the same vacuum heated He

procedure to ensure removal of all water. The material was

reconstituted with ACN (0.5 mL). The ¹⁸F solution with the

remaining constituents (K, CO₃^2-, K222) was transferred to a

conical bottomed 5 mL Wheaton vial containing 3.0 mg of the

desired tosylate precursor 55, 64, 66, 69, 71, 73, or 74 (3.0 mg)

dissolved in ACN (0.5 mL). The vial and contents were heated at

90 °C for 30 min. The reaction solution was transferred to a 25

mL pear-shaped ﬂask and diluted with water (18.5 mL). The

resultant mixture was passed through a Sep Pak C18 cartridge, then

rinsed with water (5 mL). The Sep Pak C18 cartridge was then

eluted with ACN (3 mL) and the column dried with N_2(g). The

collected acetonitrile fraction was puriﬁed via HPLC (Phenomenex

LUNA C-18 column 250 mm × 10 mm, 5 µm particle size, 100 Å

pore, mobile phases A: 90/10 H₂O/CH₃CN; B: CH₃CN, both

containing 0.1%TFA as stabilizer; gradient: 0–100/15 sustained at

100% B to 20 min, ﬂow rate 2.5 mL/min) ¹⁸F analogues 22–28

eluted from the column and were collected as single fractions (Table

6). The solvent was then removed via rotary evaporation. Upon

drying, the contents of the ﬂask were reconstituted with a 10%

ethanol solution. The ﬁnal product yield was ∼25 mCi, with a

radiosynthesis and puriﬁcation time of 90 min. Aliquots of this

material were used for biodistribution and PET imaging studies in

rats.

Bovine submitochondrial particles (SMPs) were prepared from

mitochondria as described by Matsuno-Yagi et al.⁴⁵Isolated bovine

heart mitochondria were sonicated in batches of 15 mL for 1 min

with a digital Branson soniﬁer (Branson, Danbury, CT) at 70%

maximum output in an ice bath. The sonicated suspension was

centrifuged at 16000g for 10 min, and the supernatant was

centrifuged at 150000g for 45 min at 4 °C. The submitochondrial

pellet was resuspended in buffer containing 0.25 M sucrose, 10

mM Tris-acetate, pH 7.5. The protein concentration was determined

using the BioRad Protein Assay Kit (BioRad Life Science Research,

Hercules CA), and the samples were stored at –80 °C, at a

concentration of 20 mg/mL.

Submitochondrial Particle (Bovine) Catalytic Activity and

Compound Inhibition Assay. The procedure for determining the

catalytic activity of submitochondrial particles was adapted from

Satoh et al.⁴⁶NADH-DB reductase activity was measured using a

stirred cuvette in a spectrophotometer (Hewlett-Packard, Houston

TX) at 37 °C, as the rate of NADH oxidation at 340 nm (ꢀ ) 5.4

mM^-1× cm^-1) for 120 s. The ﬁnal volume of the reaction was

2.5 mL, containing 50 mM K₂HPO₄(pH 7.4), 0.4 µM Antimycin

A, and 2 mM KCN. The ﬁnal SMP concentration was 45 µg/mL.

The enzyme reaction was initiated by the addition of 100 µM decyl

ubiquinone (DB) and 50 µM NADH. Inhibitors at varying

concentrations were preincubated with the reaction mixture contain-

ing SMPs for 4 min prior to initiation of the reaction. The IC₅₀

value was determined as the concentration of the inhibitor required

for 50% inhibition of NADH oxidation. The IC₅₀value was

calculated using GraphPad Prism Version 4 (GraphPad, San Diego,

CA).

Tissue Biodistribution of [¹⁸F]22–28 in rats (300–400 g male

Sprague–Dawley rats). Tissue biodistribution of [¹⁸F]22–28 was

examined in sodium pentobarbital (50 mg/kg, ip) anesthetized

Sprague–Dawley rats. After anesthesia, the left femoral vein of rats

was canulated and [¹⁸F]22–28 at a dose of about 15 µCi in 0.3 mL

10% ethanol saline per rat was injected intravenously. At 30 or

120 min after the injections, rats (n ) 3/each time point) were

euthanized in a CO₂chamber and tissue samples of blood, heart,

lung, liver, spleen, kidney, femur, muscle, and brain were collected,

weighed, and counted in an autogamma counter (Wallac Wizard

1480, PerkinElmer Life and Analytical Sciences, Shelton, CT) for

radioactivity. The radioactivity measurements were decay corrected

and the net injected dose was calulcated by substracting residual

activities in the syringe and venous catheter. The tissue biodistri-

bution of [¹⁸F]22–28 was expressed as % injected dose per gram

of tissue (%ID/g) (Table 7).

Cardiac Imaging with [¹⁸F]27 in Rats (300–400 g Male

Sprague–Dawley rats) and Nonhuman Primate (Male and Fe-

male Rhesus Monkeys, 4–6 kg, Covenace). Rats were anesthetized

with pentobarbital (50 mg/kg ip) and the left femoral vein was

canulated for imaging agent injection. Nonhuman primates were

anesthetized with acepromazine (0.3 mg/kg, i.m) and ketamine (10

mg/kg, i.m) in addition to being orally intubated and maintained

with isoﬂurane 0.4–1.5%). The saphenous vein was canulated for

drug injection. Cardiac imaging was performed using a microPET

camera (Focus220, CTI Molecular Imaging, Inc. Knoxville, TN),

which provides 95 transaxial slices in 22 cm operational ﬁeld of

view. After the animal was positioned for cardiac imaging, about

1 mCi of [¹⁸F]27 was injected intravenously into the rat, whereas

3 mCi of [¹⁸F]27 was injected into the nonhuman primate via the

canulated saphenous vein. Image acquisition was started 5 min post

injection and performed for the next 2 h in dynamic mode (10 min

× 12 frames). The images were reconstructed using OSEM2D

algorithm with 256 × 256 matrix at zoom 2, no attenuation

correction. Image visualization, ROI placement, and quantiﬁcation

were performed using ASIPRO software developed by the

manufacturer.

Preparation of Submitochondrial Particles from Bovine

Hearts. Bovine heart mitochondria were prepared as described by

Lester et al.⁴⁴A brief description of the procedure follows: bovine

heart was minced, and 200 g of ground heart tissue was suspended

in 400 mL of 0.25 M sucrose, 0.01 M Tris-Cl, 1 mM Tris-succinate,

and 0.2 mM ethylenediamine tetra-acetic acid (EDTA) and ho-

mogenized in a Waring blender. The homogenate was centrifuged

for 20 min at 1200g, and the supernatant was centrifuged for 15

min at 26000g, resulting in a mitochondrial pellet. The protein

concentration of the mitochondrial samples as measured by a

BioRad Protein Assay Kit (BioRad Life Science Research, Hercules,

CA) was adjusted to 20 mg/mL using 0.25 M sucrose, 10 mM Tris-

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JM701443N

Article Doi

Synthesis and biological evaluation of pyridazinone analogues as potential cardiac positron emission tomography tracers

DOI: 10.1021/jm701443n

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