Novel 2-phenylquinolin-7-yl-derived imidazo[1,5-a]pyrazines as potent insulin-like growth factor-I receptor (IGF-IR) inhibitors

Available online at www.sciencedirect.com

Bioorganic & Medicinal Chemistry 16 (2008) 1359–1375

Novel 2-phenylquinolin-7-yl-derived imidazo[1,5-a]pyrazines

as potent insulin-like growth factor-I receptor (IGF-IR) inhibitors

Mark J. Mulvihill,^*Qun-Sheng Ji, Heather R. Coate, Andrew Cooke, Hanqing Dong,

Lixin Feng, Kenneth Foreman, Maryland Rosenfeld-Franklin, Ayako Honda, Gilda Mak,

Kristen M. Mulvihill, Anthony I. Nigro, Matthew O’Connor, Caroline Pirrit,

Arno G. Steinig, Kam Siu, Kathryn M. Stolz, Yingchuan Sun,

Paula A. R. Tavares, Yan Yao and Neil W. Gibson

(OSI) Oncology, OSI Pharmaceuticals, Inc., 41 Pinelawn Road, Melville, NY 11747, USA

Received 1 August 2007; revised 16 October 2007; accepted 17 October 2007

Available online 23 October 2007

Abstract—A series of novel, potent quinolinyl-derived imidazo[1,5-a]pyrazine IGF-IR (IGF-1R) inhibitors—most notably, cis-3-(3-

azetidin-1-ylmethylcyclobutyl)-1-(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (AQIP)—is described. Synthetic details,

structure–activity relationships, and in vitro biological activity are reported for the series. Key in vitro and in vivo biological results

for AQIP are reported, including: inhibition of ligand-stimulated autophosphorylation of IGF-IR and downstream pathways in

3T3/huIGFIR cells; inhibition of proliferation and induction of DNA fragmentation in human tumor cell lines; a pharmacokinetic

proﬁle suitable for once-per-day oral dosing; antitumor activity in a 3T3/huIGFIR xenograft model; and eﬀects on insulin and glu-

cose levels.

1. Introduction

sias. The link between cancer and IGF signaling is sup-

ported by epidemiological studies showing an increased

relative risk for the development of colon, prostate,

breast, lung, and bladder cancers in individuals with cir-

culating IGF-I levels in the upper tertile of the normal

range.^10–15In addition, tumor IGF-IR expression corre-

lates with poor prognosis in renal cell carcinoma¹⁶and

increased IGF-II expression has been noted in a variety

of human tumors, including colorectal,^17,18ovarian,^19,20

lung,²¹and liver.²²Recent data from our research ef-

forts, as well as others, clearly indicate that an active

IGF-II autocrine loop plays an important role in colon

cancer progression.²³Reduction of IGF-IR activity has

been shown to induce apoptosis in tumors but only pro-

duce growth arrest in untransformed cells, suggesting a

potential basis for tumor selectivity in therapeutic appli-

cations.²⁴Inhibition of IGF-IR by various approaches,

including antisense,^25,26anti-IGF-IR antibodies,^27–30

dominant-negative IGF-IR,^31–33and small-molecule

inhibitors,^34–36has been shown to reduce tumor growth

in human tumor xenograft models.^37,38We report herein

a series of novel and potent quinolinyl-derived imi-

dazo[1,5-a]pyrazine IGF-IR inhibitors 2—most notably,

cis-3-(3-azetidin-1-ylmethylcyclobutyl)-1-(2-phenylquin-

olin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine 2f (AQIP)—

The insulin-like growth factor receptor, IGF-IR, is a tet-

rameric transmembrane tyrosine kinase which, when

activated by its cognate ligands insulin-like growth fac-

tor-I (IGF-I) and -II (IGF-II), has been implicated as

a key driver in certain forms of solid tumors and hema-

tologic neoplasias.^1–8The receptor is composed of two

subunits: an extracellular a subunit (responsible for li-

gand binding) and a b subunit (consisting of a trans-

membrane and cytoplasmic tyrosine kinase domain).

Ligand binding activates intrinsic tyrosine kinase activ-

ity, resulting in signaling cascades that progress through

the IRS-1/PI-3K/AKT/mTOR and Ras/Raf/MEK/ERK

pathways.⁹These downstream pathways ultimately

stimulate cellular proliferation, survival, transforma-

tion, metastasis, and angiogenesis. Increased expression

of the receptor and/or its ligands has been shown in a

broad range of solid tumors and hematologic neopla-

Keywords: Imidazopyrazines; Insulin-like growth factor-I receptor;

IGF-IR; IGF-1R; Tyrosine kinase inhibitor; Tumor growth inhibition;

Cancer.

*

Corresponding author. Tel.: +1 631 962 0787; fax: +1 631 845

5671; e-mail: mmulvihill@osip.com

doi:10.1016/j.bmc.2007.10.061

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M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

which was shown to aﬀect cell proliferation by inhibiting

Akt activation and, in turn, inducing apoptosis. Its

pharmacokinetic properties translated into robust anti-

tumor activity in a 3T3/huIGFIR xenograft model fol-

lowing once-a-day oral dosing.

line, which upon exposure to air underwent oxidation/

aromatization to 7-methyl-2-phenylquinoline (Scheme

1).⁴⁰While this air oxidation was a convenient and

greener method for small, 1-g scale synthesis, it was

advantageous to eﬀect the aromatization on a larger,

multigram scale by reﬂuxing with sulfur in methanol.

Oxidation of the 7-methyl group with selenium dioxide⁴¹

aﬀorded 2-phenylquinoline-7-carbaldehyde (4a) in a

42% three-step yield. A directed ortho-metalation of 2-

chloropyrazine with a preformed solution of LiTMP fol-

lowed by quenching with aldehyde 4a aﬀorded alcohol

5a in 76% yield.⁴²Mitsunobu reaction of alcohol 5a with

triphenylphosphine, phthalimide, and DIAD followed

by treatment with hydrazine aﬀorded amine 6a. While

this direct method worked on a smaller, 1-g scale, puri-

ﬁcation of the amino product from triphenylphosphine

oxide and reduced DIAD byproducts generated in the

Mitsunobu reaction proved cumbersome on a larger,

multigram scale. Optimization of the reaction by con-

verting alcohol 5a to its respective chloride via treatment

with thionyl chloride, followed by reaction with potas-

sium phthalimide, and then subsequent hydrazine-med-

iated phthalimide deprotection aﬀorded amine 6a in a

72% three-step yield. Reaction of cyclobutanecarbonyl

chloride with amine 6a in the presence of DIEA and

DMAP in methylene chloride aﬀorded amide 7a, which

2. Results and discussion

Identiﬁcation of initial key pharmacophoric interactions

as well as structural observations based upon ligand-

based SAR indicate that the 8-amino, the nitrogen and

oxygen acceptors at the 7-position of the imidazopyr-

azine core and oﬀ the meta position of the 1-phenyl moi-

ety, respectively, and a single atom spacer from the

oxygen acceptor to a phenyl are important for aﬃnity.³⁶

The 8-amino and N7-position comprise a canonical ki-

nase ATP-binding site hinge binding motif, interacting

with E1080 and M1082 in a mode paralleling that of

the pyridin-2-one in a recently published IGF-IR struc-

ture (PDB ID: 2OJ9).^35bAn X-ray co-crystal structure³⁹

of the insulin receptor (IR) with an earlier 3-benzyloxy-

phenyl-derived imidazopyrazine series led to eﬀorts fo-

cused on replacing the benzyloxyphenyl moiety with a

more constrained quinolinyl moiety. This X-ray struc-

ture shows that the methylene and ether oxygen are al-

most completely coplanar with the proximal phenyl

ring and that a modest hydrophobic cavity exists below

the proximal phenyl and benzylic carbon. The quinoli-

nyl modiﬁcation was postulated to maintain the key

pharmacophores, for example, the proximal and termi-

nal phenyl rings, as well as ﬁll the unoccupied hydro-

phobic pocket, replace a potentially metabolically

unstable benzylether moiety, and ultimately lock the

benzyloxyphenyl moiety into the preferred bioactive

conformation (Fig. 1).

43

upon treatment with neat POCl₃aﬀorded 8-chloroim-

idazopyrazine 8a in 67% yield. In order to reduce the

amount of POCl₃and facilitate the work-up, the reac-

tion was optimized by using acetonitrile as the solvent

and only 1.4 equiv of POCl₃in the presence of a cata-

lytic amount of DMF, improving the yield to 89%.

Treatment of 8-chloroimidazopyrazine 8a with ammo-

nia in isopropanol in a stainless steel Parr apparatus

aﬀorded the desired 8-aminoimidazopyrazine product

2a in 73% yield.

Initial proof of concept eﬀorts concentrated on synthe-

sizing the direct 2-phenylquinolinyl analog 2a of com-

pound 1. The synthesis began with the treatment of

commercially available 7-methylquinoline (3) with phen-

yllithium to aﬀord 7-methyl-2-phenyl-1,2-dihydroquino-

Inhibition of human IGF-IR was determined in 3T3/

huIGFIR transfected cells, a mouse 3T3 ﬁbroblast cell

line stably overexpressing human full-length IGF-IR.

3T3/huIGFIR cells form tumors in nude mice and,

therefore, represent an IGF-IR driven in vivo model.

hydrophobic back pocket

E1080

O

N

O

H

N

M1082

N

H

N

M1082

N

H

NH

N

H

K1033

N

1

2a

ribose-binding region

and progression to 2-

Figure 1. Model of key binding determinants of benzyloxyphenyl-derived imidazopyrazine IGF-1R inhibitor

phenylquinolinyl-derivative 2a.

1

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

1361

N

Cl

a

b

O

R¹

N

R¹

N

H

OH

4a (R¹= Ph)

4b (R¹= H)

3

5a (R¹= Ph)

5b (R¹= H)

Cl

N

Cl

d

e

c

R¹

N

O

N

R¹

NH

NH₂

R²

6a (R¹= Ph)

6b (R¹= H)

7a (R¹= Ph, R²= cyclobutyl)

7b (R¹= H, R²= cyclobutyl)

7c (R¹= Ph, R²= methyl)

7d (R¹= Ph, R²= 3-methylenecyclobutyl)

R¹

N

Cl

NH₂

N

f

N

R²

8a (R¹= Ph, R²= cyclobutyl)

8b (R¹= H, R²= cyclobutyl)

8c (R¹= Ph, R²= methyl)

2a (R¹= Ph, R²= cyclobutyl)

2b (R¹= H, R²= cyclobutyl)

2c (R¹= Ph, R²= methyl)

8d (R¹= Ph, R²= 3-methylenecyclobutyl)

Scheme 1. Synthesis of imidazo[1,5-a]pyrazine-derived IGF-IR inhibitors 2a–c. Reagents and conditions: (a) compound 4a: i—PhLi, THF, 0 ꢁC; ii—

sulfur, MeOH, 70 ꢁC; iii—SeO₂, 160 ꢁC (42% three-step yield); (b) n-BuLi, tetramethylpiperidine, 2-chloropyrazine, THF, ꢀ78 ꢁC; (c) i—SOCl₂,

CHCl₃, rt; ii—potassium phthalimide; iii— NH₂NH₂, EtOH, rt; (d) 7a–c: R²COCl, DIEA, DMAP, CH₂Cl₂; 7d: 3-methylenecyclobutane carboxylic

acid, EDC, HOBT, DIEA, CH₂Cl₂; (e) POCl₃, DMF, CH₃CN, 55 ꢁC; (f) NH₃, i-PrOH, 110 ꢁC, 24 h.

The replacement of the 3-benzyloxyphenyl moiety of

compound 1 with 2-phenylquinolin-7-yl to aﬀord com-

pound 2a resulted in a 14-fold increase in cellular po-

tency (Table 1). To conﬁrm the key pharmacophores

and to test the binding model, we removed the terminal

phenyl ring, replaced the cyclobutyl ring with a smaller

methyl group, and also replaced the 8-amino group with

a hydroxyl group, synthesizing three key compounds,

2b–c and 9, respectively. Based on the binding model,

the space occupied by the terminal phenyl cannot be

fully occupied by hydrophobic collapse of the nearby

residues, suggesting compound 2b should be inactive.

In addition, the hinge interactions are considered critical

for aﬃnity. Compound 9 might retain activity, provided

tautomerization does not occur. Calculations using the

PM3 method in MOPAC suggest the amide tautomer

(predicted to be inactive) is favored over the aromatic

hydroxide depicted in Table 1. Compounds 2b–c and 9

were synthesized according to the procedures described

in Scheme 1 with two noted deviations. For compound

2c where R²= Me, acetyl chloride was used in the amide

formation step (compound 6a ! 7c) with the remaining

procedures paralleling that for compound 2a. Com-

pound 9 was synthesized by simply treating compound

8a with 6 N HCl in THF at 60 ꢁC. It was evident that

the terminal phenyl ring as well as the 8-amino group,

as exempliﬁed by compounds 2b and 9, respectively,

were both vital pharmacophores required for IGF-IR

inhibition. Truncation of the cyclobutyl moiety to

Table 1. 3T3/huIGFIR IC₅₀values for compounds 1, 2a–c, and 9

R¹

N

X

8

1

N

6

N

3

5

R²

Compound

X

R¹R²

IGF-IR cell IC₅₀(lM)

1

—

1.16

2a

2b

2c

9

NH₂Ph Cyclobutyl 0.086

NH₂Cyclobutyl >10.0

NH₂Ph Methyl 1.04

OH Ph Cyclobutyl >10.0

H

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M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

methyl (compound 2a ! 2c) aﬀorded a 12-fold loss in

potency, highlighting that critical mass was required at

the C3-position of the imidazopyrazine ring for signiﬁ-

cant IGF-IR inhibition. These data reinforced previous

SAR ﬁndings from the benzyloxyphenyl series and indi-

cates that multiple atoms in the ring make non-speciﬁc

contacts with the protein.³⁶

reductive amination with a variety of amines followed

by ammonolysis to aﬀord compounds 2j–o. Interest-

ingly, the reductive amination was highly stereoselective,

aﬀording predominately the cis-isomer with only a trace

of the trans-isomer (Scheme 2).

The aminomethylcyclobutyl derivatives 2d–i were more

potent than unsubstituted cyclobutyl 2a and the cis-

and trans-isomers displayed approximately the same po-

tency, suggesting that both stereoisomers were tolerated

and made roughly isoergic interactions with the lip of

the ATP-binding pocket and surrounding solvent (Table

2). A clear SAR unfolded around the cis-aminocyclobu-

tyl series 2j–o, where larger cyclic amines such as mor-

pholino (2o) were more potent against IGF-IR than

dimethylamino (2j) or azetidinyl (2k). In general, the

methylene-derived compounds 2d–i were more potent

than the aminocyclobutyl derivatives 2j–n, and both ser-

ies displayed an overall improvement in IGF-IR potency

and metabolic stability as well as a reduction in

CYP3A4 inhibition compared to prototype compound

2a.

In an attempt to increase IGF-IR potency and improve

solubility, extension of the molecule toward the solvent

exposed ribose-binding region of the protein from C3 of

the cyclobutyl moiety with substituted-amino and ami-

nomethyl groups was pursued.⁴⁴Key 3-methylene-

cyclobutane intermediate 8d was targeted, which allowed

for the synthesis of both series. Coupling of 3-methyl-

enecyclobutane carboxylic acid with amine 6a in the

presence of EDC/HOBT aﬀorded amide 7d in 88% yield,

which was subsequently cyclized to imidazopyrazine 8d

in 93% yield with POCl₃and DMF in acetonitrile

(Scheme 1). Hydroboration of 8-chloroimidazopyrazine

8d with 9-BBN followed by oxidative cleavage aﬀorded

a 5:1 ratio of cis/trans-hydroxymethylcyclobutyl iso-

mers. When aq NaOH/H₂O₂was used in the work-up

to cleave the carbon–boron bond, the yield was ca.

60% due to the displacement of 8-chloro by hydroxy.

A milder reagent, sodium perborate,⁴⁵was employed

and the yield was greatly improved to 95%. This cis/

trans-mixture was treated with ammonia in a Parr reac-

tor at 110 ꢁC followed by tosylation of the primary alco-

hol to aﬀord the cis/trans-mixture of tosylates, 10a and

10b, respectively. At this stage, the isomers were readily

separable by silica gel chromatography and both were

structurally elucidated by 2D NMR NOE experiments.

Displacement of tosylate in compounds 10a and 10b

with various amines aﬀorded compounds 2d–i. To fur-

ther explore tethering out to the solvent exposed ri-

bose-binding region of the protein with a methylene

truncated version of aminomethyl derivatives 2d–i, ami-

no groups were directly linked to C3 of the cyclobutyl

ring. Eﬀorts began with the conversion of compound

8d to cyclobutanone derivative 11 via osmium tetraox-

ide-mediated dihydroxylation of the exocyclic oleﬁnic

moiety followed by sodium periodate oxidative cleavage

of the resulting diol. Cyclobutanone 11 was subjected to

Compound 2f (AQIP) met the stringent in vitro criteria

of CYP3A4 inhibition >20 lM, metabolic stability as

measured by predicted extraction ratios (ERs) of <0.60

in both human (hu) and mouse (mu) microsomes, and

IGF-IR cellular potency <50 nM, and was progressed

to further in vitro and in vivo studies. AQIP, a potent

inhibitor of human IGF-IR with a cellular IC₅₀of

20 nM (Table 3), has favorable microsomal stability

across species as represented by the predicted extraction

ratios (ERs) of 60.70 in human, dog, rat, and mouse,

and displays minimal inhibition against a panel of 28 ki-

nases when assayed at ATP concentrations approximat-

ing the respective K_mvalue for each form of the enzyme

unless otherwise noted (Table 4). The low selectivity be-

tween IR and IGF-IR biochemical activity is unsurpris-

ing given that the kinase domains of IR and IGF-IR are

nearly identical in and around the ATP binding pock-

et.⁴⁶When the inhibition of insulin-stimulated p-IR in

HepG2 human hepatoma cells was compared with

p-IGF-IR inhibition in 3T3/huIGFIR cells, AQIP was

approximately 5-fold more selective toward human

Ph

N

Ph

c

Ph

N

NH₂

Cl

b

d

N

a

N

2j-o

N

2d-i

N

O

OTs

10a (cis

10b (trans

)

8d

11

)

Scheme 2. Synthesis of compounds 2d–o from intermediate 8d. Reagents and conditions: (a) i—9-BBN, THF, 0 ꢁC ! rt (62%); ii—NH₃, i-PrOH,

110 ꢁC, 24 h (51%); iii—Ts₂O, pyridine, CH₂Cl₂, ꢀ40 ꢁC ! rt (53%); (b) Amine, THF, 50 ꢁC; (c) i—NMO, cat. K₂OsO₄, THF/H₂O, rt; ii—NaIO₄,

THF/H₂O (3:1), 0 ꢁC ! rt (88% two-step yield); (d) i—Na(OAc)₃BH, THF, amine, DCE, rt; ii—NH₃, i-PrOH, 110 ꢁC, 24 h.

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

1363

Table 2. 3T3/huIGFIR and CYP3A4 IC₅₀values and microsomal stability for compounds 2a and 2d–o

N

NH₂

N

R³

n

2d-o

Compound

n

cis/trans-R³

IGF-IR cell IC₅₀(nM)

(ER) microsomal stability mu/hu^a

CYP3A4 IC₅₀(lM)

2a

2d

2e

2f

—

1

0

—

86

19

0.88/0.87

0.59/0.32

0.64/0.75

0.52/0.28

0.71/0.22

0.51/0.61

0.61/0.76

0.66/0.48

0.61/0.40

0.47/0.55

0.68/0.55

0.88/0.88

0.68/0.69

3.4

15

cis-Dimethylamino

trans-Dimethylamino

cis-Azetidinyl

21

20

15

>20

17

2g

2h

2i

trans-Azetidinyl

cis-Morpholino

trans-Morpholino

cis-Dimethylamino

cis-Azetidinyl

42

18

>20

19

17

2j

148

147

77

2k

2l

>20

17

cis-Pyrrolidinyl

cis-Piperidinyl

cis-Thiomorpholino

cis-Morpholino

2m

2n

2o

47

7.5

7.8

12

130

18

^aER, extraction ratio; mu, murine; hu, human.

Table 3. In vitro proﬁle of AQIP

IGF-IR than human IR. The increased selectivity in a

cellular format suggests that undetermined components,

likely exterior to the kinase domains, inﬂuence the selec-

tivity ratio of these compounds.

IGF-IR IC₅₀(lM)

Enzyme 0.035 0.002

Cell (3T3/huIGFIR) 0.020 0.001

IGF-IR vs IR cellular selectivity

CYP3A4 IC₅₀(lM)

Microsomal stability (ER) Human

5·

>20

AQIP was tested for inhibition of ligand-stimulated

IGF-IR tyrosine kinase activity in intact 3T3/huIGFIR

and MCF-7 (data not shown) cells, where it dose-

responsively inhibited IGF-I-stimulated phosphoryla-

tion of IGF-IR, pAKT, pERK1/2, and p-p70S6K

(Fig. 2). Accordingly, in cell-based functional assays,

AQIP inhibited IGF-I induced proliferation of 3T3/

0.28 0.06

0.52 0.03

0.70 0.06

0.42 0.03

Mouse

Rat

Dog

Table 4. Kinase selectivity of AQIP

Kinase

IC₅₀(lM)

Kinase

IC₅₀(lM)

0

-

0

0.01 0.1

1

AQIP (μM)

IGF-IR

IR

Abl

0.035^a

0.076^a

>10

9.6^a

>10

4.3^a

>10

Rock2

RON

>5

5.9^a

>10

>30

>10

+

IGF-I (100 ng/mL)

p-IGF-IR

SRC

MEK1

EGFR

FES

AKT1

PDK1

Total IGF-IR

FGFR3

FYN

FAK

PKA

PKCa

p-AKT (Ser473)

p-ERK 1/2

FLT3

KDR

Lck

P70S6K

Aurora-A

CDK1/cyclin B

CDK2/cyclin A

CDK2/cyclin E

JNK3

p-p70S6K (Thr389)

PDGFRb

c-Raf

MAPK2

IKKb

GAPDH

CK2

Figure 2. Inhibition of ligand-stimulated IGF-IR autophosphoryla-

tion and downstream pathways in 3T3/huIGFIR cells by AQIP.

^aAssayed at 100 lM ATP concentration.

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M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

huIGFIR cells with an IC₅₀value of 0.05 lM. The anti-

proliferative activity observed in 3T3/huIGFIR cells was

extended to a number of tumor cell lines cultured in reg-

ular media containing 10% FCS with IC₅₀values rang-

ing from 0.27 to 2.98 lM. Furthermore, 10 lM AQIP

induced DNA fragmentation in those cells in the pres-

ence of 10% FCS (Table 5). Taken together, these data

clearly demonstrate that AQIP eﬀectively blocks IGF-

IR activation and downstream pAKT and pERK1/2 sig-

naling in tumor cells, which in turn inhibits cellular pro-

liferation and survival.

Table 6 (reported values for the rat and dog are means

of 3 animals). The oral bioavailability was signiﬁcantly

(p < 0.05) lower in rats than in mice or dogs, and

although lower bioavailability might be expected from

the higher ER in the rat, it is likely that incomplete

absorption is a major contributory factor. The volume

of distribution increased markedly in the dog compared

to the mouse and the rat, and correspondingly the oral

exposure is much lower in this species.

The favorable pharmacokinetic proﬁle displayed by

AQIP in the mouse, with high oral bioavailability and

exposure, led to its evaluation in antitumor eﬃcacy

studies. To evaluate antitumor eﬃcacy of AQIP, a

3T3/huIGFIR xenograft mouse model was employed

and AQIP was administered orally once daily for 14

consecutive days. In this model, 92% and >100% tumor

growth inhibition (TGI) were achieved with 25 and

75 mg/kg of AQIP, respectively (p 6 0.001 for both

groups vs control). As is common with molecular tar-

geted agents versus classical cytotoxic agents, antitumor

eﬀects at the lower 25 mg/kg daily dose are more cyto-

static and, therefore, eﬃcacy is limited to the dosing per-

iod; once drug treatment is completed, tumors grow out

at similar rates as vehicle treated tumors. Additionally,

once tumor volumes reach the predetermined target size

of 400%, they continue to grow out at a rate similar to

control animals. However, in this model, at the higher

dose, we observed approximately 50% maximal tumor

regression (p 6 0.001 vs control) during the dosing per-

iod and a 15-day growth delay compared to the control

group to reach a target tumor volume of 400% (Fig. 3),

indicating a dose–response. Overall, at this higher dose,

we observed less than a 10% body weight loss (data not

shown) during the treatment period suggesting that

administration of 75 mg/kg daily is relatively well

tolerated.

The pharmacokinetic properties of AQIP were deter-

mined following intravenous (iv) and oral gavage (po)

administrations in female CD-1 mice, female Sprague

Dawley rats, and male Beagle dogs (Table 6). AQIP

was dosed at 5, 1, and 2.5 mg/kg iv in saline adjusted

to pH 2 with 0.01 mol/L hydrochloric acid and at 25,

50, and 5 mg/kg po in 25 mM tartaric acid in mice, rats,

and dogs, respectively (three animals per timepoint).

Blood samples were obtained by cardiac puncture from

mice and via cannulated veins from rats and dogs, and

plasma samples were quantitatively analyzed by

HPLC–MS/MS. The PK parameters were calculated

by non-compartmental modeling using the median

(n = 3) concentration at each timepoint for the mouse

and concentration–time proﬁles from individual animals

(n = 3) for the rat and dog, and are summarized in

Table 5. Inhibition of proliferation and induction of DNA fragmen-

tation in human tumor cell lines by AQIP in normal culture media

containing 10% FCS

Cell line

IC₅₀(lM) for inhibition

of cell proliferation

Fold induction of DNA

fragmentation over

control at 10 lM

H292

1.95

0.69

2.98

1.26

0.27

0.93

51·

16·

2.7·

46·

4·

Colo-699

H358

HT29

With the tyrosine kinase domains of the insulin-like

growth factor-I and insulin receptors sharing 84% se-

quence identity,⁴⁶and only modest (5-fold) in vitro cel-

GEO

Colo-205

0.8·

1600

Dosing period

1400

Table 6. Pharmacokinetic properties of AQIP in mouse, rat, and dog

1200

1000

800

Species

Mouse

Rat

Dog

Route

Intravenous

5

Dose (mg/kg)

Cl (mL/min/kg)

V_ss(L/kg)

t_1/2(h)

1

2.5

30

24

10

5.1

10

14

19.9

50

19.0

600

Route

Dose (mg/kg)

Oral

25

Control

400

50

1.6

5

25 mg/kg

C_max(lM)

C_24h(lM)

AUC_0–1(ng h/mL)

Oral bioavailability (%F)

3.4

0.4

0.17

0.09

3010

94

200

0.9

37429

38

75 mg/kg

25582

100

0

1

11

21

31

Note. Cl, clearance; V_ss, volume of distribution at steady state; t_1/2

,

Day

elimination half-life; C_max, maximum measured plasma concentration;

C_24h, plasma concentration at 24 h; and AUC_0–1, area under the

concentration–time curve extrapolated to inﬁnity.

Figure 3. Antitumor activity of AQIP in 3T3/huIGFIR xenograft

model.

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

Table 8. Plasma insulin levels 4 h post-ﬁnal dose with AQIP

1365

lular h-IGF-IR versus h-IR selectivity observed with

AQIP (Table 3), we were compelled to evaluate the

in vivo eﬀects of AQIP on insulin receptor inhibition

via measuring insulin and glucose levels in mice. The ini-

tial study began with measuring blood glucose levels in

non-fasted mice following 14 consecutive daily oral

doses of 25, 50, or 100 mg/kg of AQIP (Table 7). The

25 and 50 mg/kg doses of AQIP caused little alteration

in blood glucose; however, the 100 mg/kg dose caused

a signiﬁcant increase in blood glucose within 4 h of the

ﬁnal dose, with levels remaining elevated in some of

the mice for 24–48 h after dosing was discontinued (data

not shown). To evaluate the eﬀects of AQIP on glucose

clearance, a glucose tolerance test was conducted in mice

treated with vehicle alone or with AQIP at oral doses of

25, 50, or 100 mg/kg for three consecutive days (Fig. 4).

Upon administration of the glucose load, there was a

dose-dependent glucose intolerance in the 50 and

100 mg/kg treated groups compared to the control. At

2 h post-glucose challenge, 44% glucose elevation in

mice dosed with the highest dose (100 mg/kg) of AQIP

compared to the control animals was observed, whereas

the 25 mg/kg dose had no signiﬁcant eﬀect on glucose

clearance. With respect to insulin, mice treated with

AQIP for 3 days also had a dose-dependent increase in

circulating insulin levels, 4 h post-dosing (Table 8). Ta-

ken together, these data indicate that following a 14 con-

secutive daily oral dosing regimen, 25 mg/kg of AQIP

aﬀords signiﬁcant tumor growth inhibition with minimal

eﬀects on blood glucose.

Dose (mg/kg)

Insulin levels (ng/mL)

Vehicle control

25

0.94 0.5

13.1 2.8

14.7 4.7

27.0 2.3

50

100

3. Conclusions

In summary, cis-3-(3-azetidin-1-ylmethylcyclobutyl)-1-

(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine

(AQIP, compound 2f) was identiﬁed as a novel, highly

potent insulin-like growth factor-I receptor inhibitor.

AQIP exhibits strong cellular potency against IGF-IR

(3T3/huIGFIR cells), modest 5-fold IR selectivity

(HepG2 cells), and superior selectivity against a panel

of 28 kinases which led to in vitro inhibition of prolifer-

ation and induction of apoptosis. The pharmacokinetics

of AQIP indicated its suitability for once-per-day oral

dosing in mouse xenograft eﬃcacy studies, where signif-

icant tumor growth inhibition was demonstrated at the

25 mg/kg dose and tumor regression at the 75 mg/kg

dose. Despite a dose-dependent elevation in insulin, a

minimal eﬀect on blood glucose was observed at the

25 mg/kg dose, suggesting that homeostatic insulin-re-

lease mechanisms can compensate for a partial inhibi-

tion of IR and maintain glucose uptake in vivo. Taken

together, these data demonstrate an advance in the ﬁeld

of small-molecule IGF-IR inhibitors, meriting further

evaluation of this class of compounds as anticancer

agents.

Table 7. Blood glucose in mice following 14 consecutive daily doses of

AQIP

4. Experimental

Dose (po, qd)

Vehicle

25 mg/kg 50 mg/kg 100 mg/kg

4.1. General experimental procedures

Blood glucose

(mg/dL)

138 16 134 29

163 18

18%

242 106

75%

Unless otherwise noted, all materials/reagents were ob-

tained from commercial suppliers and used without fur-

ther puriﬁcation. Reactions were monitored by thin

layer chromatography (TLC) on silica gel 60F₂₅₄

(0.2 mm) precoated aluminum foil and visualized using

UV light. Flash chromatography was performed with

silica gel (400–230 mesh). Preparatory TLC was per-

% Change in

blood glucose

—

ꢀ2.9%

450

400

350

300

250

200

150

100

50

˚

formed on Whatman LK6F Silica Gel 60 A size 20 ·

20 cm plates with a thickness of 1000 lm. Hydromatrix

(diatomaceous earth) was purchased from Varian.

Mass-directed HPLC puriﬁcation of compounds was

performed on a Waters system composed of the follow-

ing: 2767 Sample Manager, 2525 Binary Gradient Mod-

ule, 600 Controller, 2487 Dual k Absorbance Detector,

Micromass ZQ for mass ionization, Phenomenex Luna

˚

C18(2) 5 l 100 A, 150 · 21.2 mm column with mobile

phases of 0.1% Formic Acid Acetonitrile (A) and 0.1%

Formic Acid in HPLC water (B). The ﬂow rate is

20 mL/min, run time of 13 min, and a gradient proﬁle

of 0.00 min 2% A, 2.10 min 10% A, 8.00 min 50% A,

12.0 min 99% A, 12.8 min 2% A. LC–MS data were col-

lected on either OpenLynx or ZQ3. OpenLynx is an Agi-

lent 1100 HPLC equipped with a Gilson Auto injector

and Waters Micromass ZQ for ionization. ZQ3 is an

Agilent 1100 HPLC equipped with an HP Series 1100

Control

25 mg/kg

50 mg/kg

100 mg/kg

0

0.25

0.5

Time (hr)

Figure 4. Glucose tolerance test for AQIP in conventional CD-1 mice.

1

2

1366

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

auto injector and Waters Micromass ZQ for ionization.

Both systems use the Xterra MS C18, 5 l particle size,

4.6 · 50 mm with a mobile phase of Acetonitrile (A)

and 0.01% Formic Acid in HPLC water (B). The ﬂow

rate is 1.3 mL/min, run time of 5 min, and a gradient

proﬁle of 0.00 min 5% A, 3.00 min 90% A, 3.50 min

90% A, 4.00 min 5% A, 5.00 min 5% A. High resolution

mass spectra (HRMS) were obtained by UPLC–TOF–

MS on a QStar Pulsar mass spectrometer (Applied Bio-

systems) using positive turbo ion-spray ionization mode

and a scan range of m/z = 140–730 and were accurate to

5 ppm. The UPLC system used a Waters BEH C18,

1.7 lm, 50 · 2.1 mm column heated to 50 ꢁC condi-

tioned with a mobile phase of 10% acetonitrile in

10 mM aq ammonium acetate (A) and acetonitrile (B)

and a gradient proﬁle of 0.0 min 0% B, 0.5 min 0% B,

2.3 min 90% B, 2.4 min 90% B, 2.5 min 0% B, 3.0 min

0% B. The ﬂow rate was 0.8 mL/min. Samples were

approximately 5 or 10 lg/mL of each compound in mo-

bile phase A, each also containing an internal standard

for mass calibration. ¹H NMR (400 MHz) and ¹³C

NMR (100.6 MHz) spectra were recorded on Bruker

or Varian instruments at ambient temperature with

TMS or the residual solvent peak as the internal stan-

dard. The line positions or multiples are given in ppm

(d) and the coupling constants (J) are given as absolute

values in Hertz (Hz). All melting points were determined

with a Mel-Temp II apparatus and are uncorrected. Ele-

mental analyses were obtained by Atlantic Microlab,

Inc., Norcross, GA.

added to the reaction ﬂask. The suspension was ﬁltered

and the ﬁltrate was concentrated in vacuo to a dark

brown solid. The compound was puriﬁed by silica gel

chromatography eluting with 20% EtOAc/Hexanes to

aﬀord 26.6 g of 2-phenylquinoline-7-carbaldehyde

(50% yield). Mp 112.5–114 ꢁC. ¹H NMR (CDCl₃) d

7.48–7.58 (m, 3H), 7.91 (d, 1H, J = 8.8 Hz), 7.99–8.02

(m, 2H), 8.18–8.20 (m, 2H), 8.26 (d, 1H, J = 8.8 Hz),

8.62 (s, 1H), 10.25 (s, 1H). ¹³C NMR (CDCl₃) d 120.9,

122.6, 127.3 (2C), 128.4, 128.8 (2C), 129.7, 130.5,

135.5, 136.4, 137.0, 138.5, 147.6, 158.1, 191.9. MS

(ES+): m/z 234.12 (100) [MH⁺]. HPLC: t_R= 3.6 min

(OpenLynx, polar_5 min). Elemental Analysis calcd

for C₁₆H₁₁NO: C, 82.38; H, 4.75; N, 6.00. Found: C,

82.03; H, 4.59; N, 5.95.

4.2.2. (3-Chloropyrazin-2-yl)-(2-phenylquinolin-7-yl)meth-

anol (5a). A solution of 2,2,6,6-tetramethylpiperidine

(0.820 mL, 4.86 mmol) in dry THF (15 mL) at ꢀ78 ꢁC

was charged with n-BuLi (2.5 M in hexanes; 1.95 mL,

4.88 mmol). The reaction mixture was warmed to

0 ꢁC for 15 min and then recooled to ꢀ78 ꢁC. After

5 min,

a solution of 2-chloropyrazine (0.370 mL,

4.14 mmol) in THF (0.5 mL) was added. After 25

min, a solution of 2-phenylquinoline-7-carbaldehyde

(890 mg, 3.82 mmol) in dry THF (7 mL) was added over

a period of 5 min. The mixture was stirred at ꢀ78 ꢁC for

2 h, warmed to 0 ꢁC for 30 min, and then quenched by

the addition of citric acid (0.25 M aqueous solution,

15 mL). The mixture was extracted with EtOAc

(4 · 30 mL) and the combined organic extracts were

washed with water, NaHCO₃, brine, dried over MgSO₄,

ﬁltered, and concentrated in vacuo. The crude material

was puriﬁed by silica gel chromatography eluting with

CH₂Cl₂to 10% EtOAc in CH₂Cl₂to obtain the desired

compound as an oﬀ-white foam (1.01 g, 76% yield). Mp

4.2. Speciﬁc procedures

4.2.1. 2-Phenylquinoline-7-carbaldehyde (4a). 7-Methyl-

quinoline (130 g, 0.910 mol) was dissolved in 1 L of

dry THF and cooled to below ꢀ5 ꢁC. Phenyllithium

(500 mL, 1.00 mol, 2 M solution in dibutylether) was

added dropwise over 30 min while keeping the tempera-

ture below 0 ꢁC. The mixture was kept at 0 ꢁC for

30 min and then allowed to warm to rt for 2.5 h. The

mixture was quenched with methanol (200 mL), then

water (100 mL) was added, and the organic solvents

were removed in vacuo. The product was extracted into

EtOAc (3 · 300 mL), the combined organic layers were

washed with brine (200 mL), dried over Na₂SO₄, and

then concentrated in vacuo to yield 207 g (100% yield)

of 1,2-dihydro-7-methyl-2-phenylquinoline, which was

advanced to the next step without further puriﬁcation.

A mixture of 1,2-dihydro-7-methyl-2-phenylquinoline

(201 g, 0.910 mol), sulfur (32.0 g, 1.00 mol), and MeOH

(100 mL) was heated at reﬂux for 16 h. Additional

MeOH (1 L) was added to the mixture followed by char-

coal (15.0 g). The mixture was heated to reﬂux and ﬁl-

tered through Celite. The ﬁltrate was concentrated to

approximately 400 mL and cooled to 0 ꢁC to give a crys-

talline precipitate which was isolated by ﬁltration

(108 g). The ﬁltrate was again concentrated in vacuo

and a second crop was isolated by ﬁltration (60.0 g).

The solids were combined to aﬀord 168 g of 7-methyl-

2-phenylquinoline (84% yield). A mixture of 7-methyl-

2-phenylquinoline (50.0 g, 0.230 mol) and SeO₂(38.0 g,

0.340 mol) was heated to 160 ꢁC for 20 h. The melt

was cooled to 100 ꢁC and DCE (100 mL) was carefully

142–143 ꢁC. ¹H NMR (CDCl₃)

d 4.81 (d, 1H,

J = 8.0 Hz), 6.25 (d, 1H, J = 8.0 Hz), 7.44–7.48 (m,

1H), 7.52 (dddd, 2H, J = 6.8, 6.8, 0.4, 0.4 Hz), 7.58

(dd, 1H, J = 8.4, 2.0 Hz), 7.82 (d, 1H, J = 8.0 Hz), 7.87

(d, 1H, J = 8.4 Hz), 8.07 (br s, 1H), 8.12 (ddd, 2H,

J = 7.2, 0.8, 0.8 Hz), 8.20 (d, 1H, J = 8.8 Hz), 8.41 (d,

1H, J = 2.4 Hz), 8.62 (d, 1H, J = 2.4 Hz). ¹³C NMR

(CDCl₃) d 72.1, 119.5, 125.7, 126.8, 127.6 (2C), 128.0,

128.3, 128.8 (2C), 129.4, 136.6, 139.4, 141.3, 141.9,

143.3, 147.8, 148.0, 154.2, 157.9. MS (ES+): m/z

347.88/349.90 (3/1) (100) [MH⁺]. HPLC: t_R= 3.3 min

(OpenLynx, polar_5 min). Elemental Analysis calcd

for C₂₀H₁₄ClN₃O: C, 69.07; H, 4.06; N, 12.08; Cl,

10.19. Found: C, 69.03; H, 4.03; N, 12.21; Cl, 10.13.

4.2.3. (3-Chloropyrazin-2-yl)-quinolin-7-yl-methanol (5b).

A solution of 2,2,6,6-tetramethylpiperidine (0.64 mL,

0.54 g, 3.8 mmol) in dry THF (10 mL), cooled to

ꢀ78 ꢁC, was charged with n-BuLi (2.5 M in hexanes;

1.6 mL, 4.0 mmol). The solution was warmed from

ꢀ78 ꢁC with an ice/water bath for 15 min, followed by

re-cooling to ꢀ78 ꢁC with a dry ice/acetone bath for

10 min, then charged with 2-chloropyrazine (0.29 mL,

0.37 g, 3.2 mmol). After stirring for 30 min, the reaction

mixture was charged by cannula with a solution of quin-

oline-7-carbaldehyde (500 mg, 3.18 mmol) in dry THF

(5 mL), cooled to ꢀ78 ꢁC, and stirred at ꢀ78 ꢁC for

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

1367

2.5 h and at 0 ꢁC for 0.5 h. The reaction was quenched

by adding 2 mL aq HCl (2 M solution) followed by aq

NH₄Cl solution. The mixture was extracted with EtOAc

(4 · 30 mL) and the combined organic extracts were

washed with water and brine, dried over MgSO₄, ﬁl-

tered, and concentrated in vacuo. The crude material

was chromatographed on silica gel [Jones Flashmaster,

20 g/70 mL cartridge], eluting with hexanes/EtOAc

2:1 ! 1: 1 ! 1:4 ! EtOAc, yielding the title compound

as an orange foam (600 mg, 69% yield). ¹H NMR

(CDCl₃) d 4.87 (d, J = 7.6 Hz, 1H), 6.26 (d, J = 7.6 Hz,

1H), 7.41 (dd, J = 8.4, 4.4 Hz, 1H), 7.60 (dd, J = 8.4,

1.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 8.02 (d,

J = 0.8 Hz, 1H), 8.14 (dd, J = 8.4, 0.8 Hz, 1H), 8.41 (d,

J = 2.4 Hz, 1H), 8.60 (d, J = 2.4 Hz, 1H), 8.91 (dd,

J = 4.4, 1.6 Hz, 1H). MS (ES+): m/z 272.1/274.1 (100/

38) [MH⁺]. Purity: 94% [HPLC (OpenLynx) at 254 nm].

C, 69.26; H, 4.36; N, 16.15; Cl, 10.22. Found: C, 69.15;

H, 4.38; N, 16.42; Cl, 10.13.

4.2.5. C-(3-Chloropyrazin-2-yl)-C-quinolin-7-yl-methyl-

amine (6b). A suspension of (3-chloropyrazin-2-yl)-quin-

olin-7-ylmethanol (600 mg, 2.21 mmol), phthalimide

(356 mg, 2.42 mmol), and PS–PPh₃(loading 2.12 mmol/

g; 1.56 g, 3.31 mmol) in dry THF (20 mL), cooled to

0 ꢁC, was charged with DIAD (480 lL, 493 mg,

2.44 mmol). The ice bath was removed and the ﬂask was

vortexed at ambient temperature for 21.5 h. Additional

PS–PPh₃(520 mg, 1.10 mmol) and DIAD (160 lL,

164 mg, 0.810 mmol) were added, and vortexing was con-

tinued for 6.5 h. The resin was removed by ﬁltration,

washed with THF and CH₂Cl₂, and the combined organ-

ics were concentrated in vacuo. The crude material was

chromatographed on silica gel [Jones Flashmaster, 20 g/

70 mL cartridge], eluting with hexanes/EtOAc 3:1 ! 2:

1 ! 1:1 ! 1:2, yielding 2-[(3-chloropyrazin-2-yl)-quino-

lin-7-ylmethyl]-isoindole-1,3-dione as a pale yellow solid

(89% yield) which was taken on directly to the next step.

¹H NMR (CDCl₃) d 7.12 (s, 1H), 7.41 (dd, J = 8.0,

4.4 Hz, 1H), 7.54 (dd, J = 8.4, 2.0 Hz, 1H), 7.72–7.78

(m, 2H), 7.81–7.89 (m, 3H), 8.01 (d, J = 0.8 Hz, 1H),

8.16 (dd, J = 8.4, 0.8 Hz, 1H), 8.39 (d, J = 2.4 Hz, 1H),

8.50 (d, J = 2.4 Hz, 1H), 8.90 (dd, J = 4.2, 1.6 Hz, 1H).

MS (ES+): m/z 401.0/402.9 (100/38) [MH⁺]. A solution

of 2-[(3-chloropyrazin-2-yl)-quinolin-7-ylmethyl]-isoin-

dole-1,3-dione (789 mg, 1.97 mmol) and anhydrous

hydrazine (63.0 lL, 2.00 mmol) in EtOH (4 mL)/CH₂Cl₂

(2 mL) was stirred at rt for 1 d. Additional hydrazine

(93.0 lL, 3.00 mmol) was added, and stirring was contin-

ued for an additional 2 d. The solid was ﬁltered, washed

with EtOH, and the combined organics were concen-

trated in vacuo to yield a red, sticky solid. This solid

was suspended in CH₂Cl₂and ﬁltered; the ﬁltrate was con-

centrated in vacuo to aﬀord the title compound as an or-

ange gum (608 mg, 100% yield). ¹H NMR (CDCl₃) d 2.40

(br s, 2H), 5.79 (s, 1H), 7.39 (dd, J = 8.2, 4.2 Hz, 1H), 7.64

(dd, J = 8.6, 1.8 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 8.01 (d,

J = 0.8 Hz, 1H), 8.13 (dd, J = 8.0, 0.8 Hz, 1H), 8.31

(d, J = 2.4 Hz, 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.90 (dd,

J = 4.4, 1.6 Hz, 1H). MS (ES+): m/z 271.0/273.0 (30/10)

[MH⁺], 254.1/256.1 (30/10) [MH⁺–NH₃]. Purity: 97%

[HPLC (OpenLynx) at 254 nm].

4.2.4. (3-Chloropyrazin-2-yl)(2-phenylquinolin-7-yl)methyl-

amine (6a). A CHCl₃(1.63 L) solution of (3-chloropyra-

zin-2-yl)-(2-phenylquinolin-7-yl)methanol (170 g, 0.490

mol), cooled to ꢀ4 ꢁC under N₂atmosphere, was

charged with thionyl chloride (42.7 mL, 0.590 mol) over

10 min. The reaction mixture was allowed to warm to rt

and stirred for 4 h. The crude reaction mixture was con-

centrated in vacuo, dissolved in CHCl₃and toluene, re-

concentrated in vacuo, and then advanced to the next

step. 7-[Chloro-(3-chloropyrazin-2-yl)methyl]-2-phenyl-

quinoline (170 g, 0.460 mol) and potassium phthalimide

(421 g, 2.27 mol) were dissolved in anhydrous DMF

(1.5 L) and heated to 60 ꢁC. After 16 h, the reaction

was concentrated in vacuo to a solid, which was sus-

pended in water and then ﬁltered. The solid was washed

with water and then re-dissolved in EtOAc and a mini-

mum amount of CH₂Cl₂, which was subsequently

washed with water and ﬁltered through a pad of Celite

and Na₂SO₄. The resulting ﬁltrate was concentrated in

vacuo to aﬀord the desired product as a tan solid

(280 g of a 3:1 mix of product/phthalimide, 97% yield),

which was advanced to the next step. 2-[(3-Chloropyra-

zin-2-yl)-(2-phenylquinolin-7-yl)methyl]isoindole-1,3-dione

(ꢁ3:1 mix with phthalimide, 200 g, 0.42 mol) was sus-

pended in anhydrous EtOH (2.1 L) and CH₂Cl₂

(300 mL), placed under an atmosphere of N₂, and

charged with anhydrous hydrazine (105 mL, 3.36 mol).

After stirring for 16 h, the reaction mixture was ﬁltered

and the solid was washed with EtOH. The ﬁltrate was

concentrated in vacuo to a foam, which was puriﬁed

using silica gel chromatography (EtOAc ! 1% 7 N

NH₃in MeOH in EtOAc) to provide the desired com-

pound as a tan solid (82.9 g, 72% three-step yield). Mp

General procedure A: synthesis of amides 7a–7c. A solu-

tion of NEt(iPr)₂(150 lL, 0.861 mmol), DMAP

(5.00 mg, 0.040 mmol), and amine 6 (0.583 mmol) in

dry CH₂Cl₂(5 mL), cooled to 0 ꢁC, was charged with

the appropriate acid chloride (0.660 mmol). The bath

was removed and the reaction mixture was stirred at rt

for 3 h. Water (10 mL) was added, the layers were sep-

arated, and the aqueous layer was extracted with

CH₂Cl₂(3 · 15 mL). The combined CH₂Cl₂layers were

washed with water, satd NaHCO₃solution, and brine,

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo

to aﬀord the desired amides.

1

119–120 ꢁC. H NMR (CDCl₃) d 2.20 (br s, 2H), 5.80

(s, 1H), 7.46 (dddd, 1H, J = 6.4, 6.4, 1.6, 1.6 Hz), 7.53

(dddd, 2H, J = 8.4, 8.4, 1.2, 1.2 Hz), 7.61 (dd, 1H,

J = 8.4, 1.6 Hz), 7.81 (d, 1H, J = 8.4 Hz), 7.86 (d, 1H,

J = 8.0 Hz), 8.07 (br s, 1H), 8.13 (ddd, 2H, J = 7.6,

2.0, 2.0 Hz), 8.20 (d, 1H, J = 8.8 Hz), 8.32 (d, 1H,

J = 2.8 Hz), 8.60 (d, 1H, J = 2.4 Hz). ¹³C NMR (CDCl₃)

d 56.9, 119.1, 125.7, 126.3, 127.4 (2C), 127.6, 128.0,

128.7 (2C), 129.3, 136.4, 139.4, 142.1, 142.3, 144.2,

148.0, 148.1, 156.0, 157.7. MS (ES+): m/z 346.94/

348.89 (3/1) [MH⁺]. HPLC: t_R= 2.2 min (OpenLynx,

polar_5 min). Elemental Analysis calcd for C₂₀H₁₅ClN₄:

4.2.6. Cyclobutanecarboxylic acid [(3-chloropyrazin-2-

yl)-(2-phenylquinolin-7-yl)-methyl]-amide (7a). The title

compound was carried forward without further puriﬁca-

tion as a yellow solid (100% yield). A puriﬁed sample

1368

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

was obtained by crystallization from EtOAc/hexanes to

give a light beige solid. Mp 171–172 ꢁC. ¹H NMR

(CDCl₃) d 1.81–1.90 (m, 1H), 1.90–2.02 (m, 1H), 2.11–

2.23 (m, 2H), 2.23–2.35 (m, 2H), 3.12 (quint,

J = 8.4 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 7.22 (d,

J = 8.0 Hz, 1H), 7.43–7.48 (m, 1H), 7.48–7.54 (m, 2H),

7.73 (dd, J = 8.4, 2.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H),

7.85 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 0.8 Hz, 1H),

8.07–8.12 (m, 2H), 8.19 (d, J = 8.4 Hz, 1H), 8.38 (d,

J = 2.4 Hz, 1H), 8.58 (d, J = 2.4 Hz, 1H). ¹³C NMR

(CDCl₃, DEPT135) d 18.14 (ꢀ), 25.18 and 25.30 (rota-

mers, 2C, ꢀ), 39.80 (+), 53.78 (+), 119.42 (+), 126.66

(C_quart), 127.12 (+), 127.51 (2C, +), 128.04 (+), 128.16

(+), 128.84 (2C, +), 129.40 (+), 136.53 (+), 139.44

(C_quart), 140.81 (C_quart), 141.77 (+), 142.99 (+), 148.09

(C_quart), 148.34 (C_quart), 152.93 (C_quart), 157.88 (C_quart),

174.02 (C_quart). MS (ES+): m/z 429.0/431.0 (38/13)

[MH⁺], 469.8/471.8 (6/2) [MH⁺+ MeCN]. HPLC:

t_R= 3.6 min (ZQ3, polar_5 min). Elemental Analysis

calcd for C₂₅H₂₁ClN₄OÆ1/8EtOAc: C, 69.62; H, 5.04;

N, 12.74; Cl, 8.06. Found: C, 69.58; H, 5.01; N, 12.69;

Cl, 8.15.

as a light yellow solid (33.7 g, 88% yield). ¹H NMR

(CDCl₃) d 2.82–2.95 (m, 2H), 2.96–3.07 (m, 3H), 3.07–

3.18 (m, 1H), 4.74–4.78 (m, 2H), 6.82 (d, 1H,

J = 7.6 Hz), 7.37–7.46 (m, 1H), 7.49–7.61 (m, 3H), 7.80–

7.95 (m, 3H), 8.16 (br d, 2H, J = 6.8 Hz), 8.33 (m, 1H),

8.40 (d, 1H, J = 2.3 Hz), 8.63 (d, 1H, J = 2.3 Hz). ¹³C

NMR (DMSO-d₆) d 32.8, 34.8, 35.0, 54.3, 106.2, 119.0,

126.3, 126.7, 127.2 (2C), 127.7, 128.1, 128.8 (2C), 129.7,

137.0, 138.4, 140.4, 143.1, 143.5, 145.3, 147.3 (2C),

153.5, 156.4, 173.4. MS (ES+): m/z 441.09/443.12 (3/1)

[MH⁺]. HPLC: t_R= 3.74 min (OpenLynx, polar_5 min).

Elemental Analysis calcd for C₂₆H₂₁ClN₄O: C, 70.82;

H, 4.80; N, 12.71; Cl, 8.04. Found: C, 70.55; H, 4.77; N,

12.52; Cl, 8.26.

General procedure B: synthesis of the 8-chloroimidazopyr-

azines 8a–8d. A suspension of amide 7 (1.0 equiv) in

anhydrous acetonitrile (0.1 M suspension) was charged

with POCl₃(1.4 equiv) dropwise and the reaction mix-

ture was stirred at rt for 5 min prior to the dropwise

addition of DMF (0.1 equiv). The reaction mixture

was stirred at 55 ꢁC for 2 h. The mixture was concen-

trated in vacuo, dissolved in CH₂Cl₂, and quenched with

2 N NH₃in i-PrOH to pH 9. The mixture was concen-

trated in vacuo, dissolved in CH₂Cl₂, washed with water

and brine, dried over Na₂SO₄, ﬁltered, and concentrated

in vacuo.

4.2.7. Cyclobutanecarboxylic acid [(3-chloropyrazin-2-

yl)-quinolin-7-yl-methyl]-amide (7b). The crude material

was chromatographed on silica gel [Jones Flashmaster,

20 g/70 mL cartridge], eluting with hexanes/EtOAc

1:1 ! 1:3 ! EtOAc, yielding the title compound as an

orange foam (81% yield). ¹H NMR (CDCl₃) d 1.81–

1.91 (m, 1H), 1.91–2.03 (m, 1H), 2.11–2.23 (m, 2H),

2.23–2.35 (m, 2H), 3.12 (quint, 1H, J = 8.6 Hz), 6.80

(d, 1H, J = 8.0 Hz), 7.22 (d, 1H, J = 8.0 Hz), 7.39 (dd,

1H, J = 8.0, 4.0 Hz), 7.77 (d, 1H, J = 8.6 Hz), 7.82 (d,

1H, J = 8.6 Hz), 7.83 (s, 1H), 8.13 (d, 1H, J = 8.4 Hz),

8.37 (d, 1H, J = 2.2 Hz), 8.56 (d, 1H, J = 2.2 Hz), 8.87

(dd, 1H, J = 4.0, 1.6 Hz). MS (ES+): m/z 353.1/355.0

(100/39) [MH⁺].

4.2.10. 7-(8-Chloro-3-cyclobutylimidazo[1,5-a]pyrazin-1-

yl)-2-phenylquinoline (8a). The solid was suspended in

CHCl₃, ﬁltered, and the ﬁltrate was concentrated in va-

cuo to obtain the title compound as a yellow solid (89%

1

yield). Mp 214–215 ꢁC. H NMR (CDCl₃) d 2.04–2.15

(m, 1H), 2.15–2.28 (m, 1H), 2.50–2.60 (m, 2H), 2.64–

2.76 (m, 2H), 3.89 (quint, J = 8.4 Hz, 1H), 7.35 (d,

J = 4.8 Hz, 1H), 7.44–7.50 (m, 1H), 7.51–7.57 (m, 3H),

7.89–7.93 (m, 3H), 8.17–8.22 (m, 2H), 8.27 (dd,

J = 8.8, 0.8 Hz, 1H), 8.53 (d, J = 0.8 Hz, 1H). ¹³C

NMR (CDCl₃, DEPT135) d 18.93 (ꢀ), 26.84 (2C, ꢀ),

31.41 (+), 113.31 (+),119.26 (+), 120.03 (C_quart), 126.52

(+), 126.62 (+), 126.72 (C_quart), 127.59 (2C, +), 128.79

(2C, +), 129.14 (+), 129.28 (+), 132.08 (+), 135.06

(C_quart), 136.45 (+), 137.26 (C_quart), 139.67 (C_quart),

144.56 (C_quart), 146.08 (C_quart), 147.76 (C_quart), 157.66

(C_quart). MS (ES+): m/z 410.9/412.9 (100/39) [MH⁺].

HPLC: t_R= 3.7 min (ZQ3, non-polar_5 min). Elemental

Analysis calcd for C₂₅H₁₉ClN₄: C, 73.08; H, 4.66; N,

13.63; Cl, 8.63. Found: C, 72.95; H, 4.63; N, 13.57; Cl,

8.80.

4.2.8. N-[(3-Chloropyrazin-2-yl)-(2-phenylquinolin-7-yl)-

methyl]-acetamide (7c). The crude product was chro-

matographed on silica gel [Jones Flashmaster, 10 g

cartridge, eluting with 2% MeOH/CH₂Cl₂] to yield a

1

dark oil (98% yield). H NMR (CDCl₃) d 2.08 (s, 3H),

6.80 (d, 1H, J = 7.9 Hz), 7.26–7.23 (m, 4H), 7.70–7.92

(m, 4H), 8.09–8.11 (m, 2H), 8.17 (d, 1H, J = 8.6 Hz),

8.37 (d, 1H, J = 2.4 Hz), 8.57 (d, 1H, J = 2.5 Hz). MS

(ES+): m/z 430.84/432.83 [MH⁺].

4.2.9. N-[(3-Chloropyrazin-2-yl)(2-phenylquinolin-7-yl)-

methyl]-3-methylenecyclobutanecarboxamide (7d).

A

CH₂Cl₂(220 mL) solution of (3-chloropyrazin-2-yl)(2-

phenylquinolin-7-yl)methylamine (30.1 g, 87.1 mmol)

was charged with EDC (25.0 g, 131 mmol), HOBt

(11.8 g, 87.1 mmol), DIPEA (23.0 mL, 130 mmol),

and 3-methylenecyclobutanecarboxylic acid (12.7 g,

113 mmol, synthesized according to the procedures de-

scribed in J. Am. Chem. Soc. 1958, 80, 5507–5513). The

solution was stirred at rt for 16 h under an atmosphere

of N₂and then diluted with CH₂Cl₂(200 mL), washed

with satd aq NaHCO₃(75 mL), brine (75 mL), dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude

product was puriﬁed via silica gel chromatography elut-

ing with 5% MTBE:CH₂Cl₂aﬀording the title compound

4.2.11. 7-(8-Chloro-3-cyclobutylimidazo[1,5-a]pyrazin-1-

yl)-quinoline (8b). The crude material was adsorbed onto

Hydromatrix and chromatographed on silica gel [Jones

Flashmaster, 20 g/ 70 mL cartridge], eluting with hex-

anes/EtOAc 1:1 ! 1:3, yielding the title compound as

1

a yellow foam (93% yield). H NMR (CDCl₃) d 2.05–

2.14 (m, 1H), 2.16–2.28 (m, 1H), 2.50–2.60 (m, 2H),

2.63–2.75 (m, 2H), 3.89 (quint, J = 8.4 Hz, 1H), 7.35

(d, J = 4.4 Hz, 1H), 7.44 (dd, J = 8.2, 4.2 Hz, 1H), 7.55

(d, J = 5.2 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 8.13 (dd,

J = 8.4, 1.6 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.46 (s,

1H), 8.98 (dd, J = 4.2, 1.6 Hz, 1H). MS (ES+): m/z

335.1/337.1 (100/44) [MH⁺].

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

1369

4.2.12. 7-(8-Chloro-3-methylimidazo[1,5-a]pyrazin-1-yl)-

2-phenylquinoline (8c). The title compound was obtained

as a light brown oil which was carried forward without

(40 mL), washed with satd aq NaHCO₃(2 · 10 mL)

and brine (2 · 10 mL), dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude material was puriﬁed

by silica gel column chromatography eluting with

EtOAc ! 98:2 EtOAc/MeOH ! 96:4 EtOAc/MeOH,

aﬀording the title compounds 10a (cis) (90 mg, 53% yield)

and 10b (trans) (25 mg, 11% yield) as light yellow solids:

1

further puriﬁcation (100% yield). H NMR (CDCl₃) d

2.77 (s, 3H), 7.41–7.59 (m, 4H), 7.70–7.72 (m, 1H),

7.88–7.93 (m, 3H), 8.19–8.28 (br m, 3H), 8.55 (br s,

1H). MS (ES+): m/z 370.96/372.97 [MH⁺].

4.2.13. 7-[8-Chloro-3-(3-methylenecyclobutyl)imidazo[1,5-

a]pyrazin-1-yl]-2-phenylquinoline (8d). The crude material

was puriﬁed via silica gel chromatography eluting with 3–

5% CH₃CN/CH₂Cl₂to aﬀord the desired compound as a

4.2.14.1. cis-Toluene-4-sulfonic acid 3-[8-amino-1-(2-

phenylquinolin-7-yl)-imidazo[1,5-a]pyrazin-3-yl]-cyclobu-

tylmethyl ester (10a). Mp 177 ꢁC (decomposed). ¹H NMR

(CDCl₃) d 2.30–2.44 (m, 2H), 2.40 (s, 3H), 2.57–2.68 (m,

2H), 2.76–2.90 (m, 1H), 3.71 (quintet, 1H, J = 8.8 Hz),

4.11 (d, 2H, J = 6.6 Hz), 5.24 (br s, 2H, NH₂), 7.10 and

7.12 (AB, 2H, J = 5.0 Hz), 7.30 (d, 2H, J = 7.8 Hz),

7.45–7.51 (m, 1H), 7.51–7.57 (m, 2H), 7.75–7.81 (m,

2H), 7.89–8.00 (m, 3H), 8.16–8.22 (m, 2H), 8.28 (d, 1H,

J = 8.6 Hz), 8.41 (dd, 1H, J = 0.8, 0.8 Hz). ¹³C NMR

(DMSO-d₆) d 21.0, 26.7, 28.7 (2C), 30.1, 73.8, 106.3,

114.1, 118.7, 126.1, 127.2 (2C), 127.5 (2C), 127.89 (2C),

127.92, 128.8 (3C), 129.6, 130.0 (2C), 132.5, 133.2,

136.2, 137.0, 138.6, 143.3, 144.8, 147.5, 151.6, 156.5. MS

(ES+): m/z 575.98 (50) [MH⁺]. HPLC: t_R= 2.80 min

(OpenLynx, polar_5 min). Elemental Analysis calcd for

C₃₃H₂₉N₅O₃S: C, 68.85; H, 5.08; N, 12.17; S, 5.57. Found:

C, 68.81; H, 5.09; N, 12.20; S, 5.47.

1

yellow solid (93% yield). Mp 211 ꢁC (decomposed). H

NMR (CDCl₃) d 3.23–3.34 (m, 2H), 3.37–3.48 (m, 2H),

3.82–3.95 (m, 1H), 4.90–4.98 (m, 2H), 7.21–7.26 (m,

1H), 7.40 (d, 1H, J = 5.1 Hz), 7.46–7.65 (m, 4H), 7.87–

8.02 (m, 3H), 8.20–8.30 (m, 2H), 8.40 (br s, 1H). MS

(ES+): m/z 423.03/423.97 (3/1) [MH⁺]. HPLC:

t_R= 3.76 min (Openlynx, non-polar_5 min). Elemental

Analysis calcd for C₂₆H₁₉ClN₄OÆ1/5H₂O: C, 73.22; H,

4.58; N, 13.14. Found: C, 73.30; H, 4.54; N, 13.05.

4.2.14. Toluene-4-sulfonic acid 3-[8-amino-1-(2-phenyl-

quinolin-7-yl)-imidazo[1,5-a]pyrazin-3-yl]-cyclobutylmethyl

ester (10a and 10b). A dry THF (5 mL) solution of 7-[8-

chloro-3-(3-methylenecyclobutyl)-imidazo[1,5-a]pyra-

zin-1-yl]-2-phenylquinoline (338 mg, 0.800 mmol), charged

with 0.5 M 9-BBN in THF (2.40 mL, 1.20 mmol) drop-

wise, was stirred at 0 ꢁC under N₂atmosphere for

30 min and then allowed to warm to rt and stirred for

an additional 16 h. The mixture was cooled to 0 ꢁC and

charged with 1 N aq NaOH (3 mL) and 30% aq H₂O₂

(0.600 mL). Stirring was continued at 0 ꢁC for 10 min

and then at rt for 30 min. The mixture was diluted with

CH₂Cl₂(30 mL), washed with brine (2 · 20 mL), dried

over Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated

in vacuo and the crude material was puriﬁed by silica

gel column chromatography eluting with hexanes/EtOAc

(1:1 ! 0:1), aﬀording the {3-[8-chloro-1-(2-phenylquino-

lin-7-yl)-imidazo[1,5-a]pyrazin-3-yl]-cyclobutyl}-methanol

as a mixture of cis- and trans-isomers in the ratio of 5:1

(220 mg, 62% yield). MS (ES+): m/z 441/443 (3/1)

[MH⁺]. An isopropanol (5 mL) solution of {3-[8-chloro-

1-(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-3-yl]cyclo-

butyl}methanol (265 mg, 0.600 mmol) in a stainless steel

Parr apparatus was cooled to ꢀ78 ꢁC and charged with

NH₃gas for 1 min. The apparatus was sealed and heated

at 110 ꢁC for 3 d, then cooled to ꢀ78 ꢁC, vented, and al-

lowed to warm to rt. The salt was removed by ﬁltration

and the ﬁltrate was concentrated in vacuo. The crude

material was puriﬁed via silica gel chromatography elut-

ing with EtOAc ! 9:1 EtOAc/MeOH, aﬀording {3-[8-

amino-1-(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-

3-yl]cyclobutyl}methanol (130 mg, 51% yield) as a mixture

of cis- and trans-isomers in the ratio of 5:1. MS (ES+):

m/z 422.0 [MH⁺]. A suspension of {3-[8-amino-1-(2-phe-

nylquinolin-7-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutyl}

methanol (125 mg, 0.300 mmol) in dry CH₂Cl₂(5 mL)

and pyridine (2.00 mL) was charged with a CH₂Cl₂

(1 mL) solution of Ts₂O (108 mg, 0.330 mmol) at

ꢀ40 ꢁC under N₂atmosphere. The mixture was stirred

and slowly warmed to rt overnight. The reaction mixture

was quenched with water (1 mL), diluted with CH₂Cl₂

4.2.14.2. trans-Toluene-4-sulfonic acid 3-[8-amino-1-(2-

phenylquinolin-7-yl)-imidazo[1,5-a]pyrazin-3-yl]-cyclobu-

tylmethyl ester (10b). Mp 179 ꢁC (decomposed). ¹H

NMR (CDCl₃) d 2.33–2.42 (m, 2H), 2.47 (s, 3H),

2.70–2.79 (m, 2H), 2.79–2.91 (m, 1H), 3.76–3.87 (m,

1H), 4.21 (d, J = 6.0 Hz, 2H), 5.32 (br s, 2 H), 7.06

and 7.10 (AB, J = 5.1 Hz, 2H), 7.38 (d, J = 8.0 Hz,

2H), 7.45–7.50 (m, 1H), 7.51–7.57 (m, 2H), 7.83–7.87

(m, 2H), 7.89–7.98 (m, 3H), 8.17–8.21 (m, 2H), 8.27

(d, J = 8.4 Hz, 1 H), 8.42 (dd, J = 0.8, 0.8 Hz, 1H). ¹³C

NMR (CDCl₃) d 21.7, 27.7, 28.0 (2C), 30.8, 73.0,

106.4, 114.6, 119.4, 126.6, 127.6 (2C), 127.7, 127.9

(3C), 128.3, 128.9 (2C), 129.5, 129.7, 129.9 (2C), 133.2,

134.7, 136.2, 136.6, 139.4, 143.7, 144.9, 148.2, 151.5,

158.1. MS (ES+): m/z 576.08 (50) [MH⁺]. HPLC:

t_R= 2.88 min (ZQ3, polar_5 min). Elemental Analysis

calcd for C₃₃H₂₉N₅O₃S: C, 68.85; H, 5.08; N, 12.17; S,

5.57. Found: C, 68.59; H, 5.09; N, 11.95; S, 5.55.

General procedure C: synthesis of 8-aminoimidazopyra-

zines 2a–2c. Ammonia (g) was bubbled into a 0.1 M

slurry of the 8-chloroimidazopyrazine 8 in 2 M NH₃in

i-PrOH for 8 min in a stainless steel Parr apparatus

cooled to ꢀ78 ꢁC in a dry ice/acetone bath. The Parr

apparatus was secured and the cooling bath was re-

moved. The reaction mixture was heated at 110 ꢁC for

24 h, then cooled to ꢀ78 ꢁC, vented, and allowed to

warm to rt. The reaction mixture was transferred to a

round bottom ﬂask and the solvent was concentrated

in vacuo. The product was resuspended in CH₂Cl₂and

the remaining salts were removed by ﬁltration and the

ﬁltrate was concentrated in vacuo.

4.2.15. 3-Cyclobutyl-1-(2-phenylquinolin-7-yl)-imidazo[1,5-

a]pyrazin-8-ylamine (2a). The crude material was chro-

matographed on silica gel [Jones Flashmaster, 10 g/70 mL

1370

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

cartridge], eluting with CH₂Cl₂! 1% MeOH in

CH₂Cl₂! 2% MeOH in CH₂Cl₂, giving the desired

product as a yellow solid (73% yield). Mp 184–186 ꢁC.

¹H NMR (CDCl₃) d 2.01–2.12 (m, 1H), 2.13–2.27 (m,

1H), 2.47–2.58 (m, 2H), 2.62–2.73 (m, 2H), 3.85 (quint,

J = 8.0 Hz, 1H), 6.00 (br s, 2H), 7.04 (d, J = 5.4 Hz,

1H), 7.15 (d, J = 5.4 Hz, 1H), 7.46–7.51 (m, 1H), 7.52–

7.58 (m, 2H), 7.91 (dd, J = 8.4, 1.6 Hz, 1H), 7.94 (d,

J = 8.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 8.18–8.22 (m,

2H), 8.28 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 0.8 Hz, 1H).

¹³C NMR (CDCl₃, DEPT135) d 18.89 (ꢀ), 26.92

(2C, ꢀ), 31.50 (+), 106.62 (+), 114.32 (C_quart), 119.26

(+), 126.55 (C_quart), 127.56 (3C, +), 128.06 (+), 128.15

(+), 128.83 (2C, +), 129.44 (+), 129.67 (+), 134.56

(C_quart), 136.42 (C_quart), 136.53 (+), 139.44 (C_quart),

144.40 (C_quart), 148.18 (C_quart), 151.62 (C_quart), 157.94

(C_quart). MS (ES+): m/z 392.0 (50) [MH⁺]. HPLC:

t_R= 1.7 min (OpenLynx, non-polar_5 min), 2.5 min

(ZQ3, polar_5 min). Elemental Analysis calcd for

C₂₅H₂₁N₅Æ0.4H₂O: C, 75.32; H, 5.51; N, 17.57. Found:

C, 75.22; H, 5.28; N, 17.46. HRMS (ES+) for

C₂₅H₂₁N₅ÆH⁺[MH⁺]: calcd, 392.1870; found, 392.1890.

for C₂₂H₁₇N₅ÆH⁺[MH⁺]: calcd, 352.1557; found,

352.1559.

General procedure D: synthesis of aminomethylcyclobu-

tyl-derived imidazopyrazines 2d–i. A sealed tube contain-

ing a solution of toluene-4-sulfonic acid 3-[8-amino-1-

(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-3-yl]cyc-

lobutylmethyl ester 10 (0.15 mmol) in THF (3.0 mL)

was charged with amine (3.6 mmol), sealed, and heated

at 50 ꢁC for 16 h. The mixture was concentrated in va-

cuo to aﬀord the crude product.

4.2.18. cis-3-{3-[(Dimethylamino)methyl]cyclobutyl}-1-(2-

phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2d).

The crude material was puriﬁed by mass-directed HPLC

puriﬁcation to aﬀord the title compound as a light yel-

low solid (57% yield). ¹H NMR (CDCl₃) d 2.26 (s,

6H), 2.29–2.40 (m, 2H), 2.45 (d, J = 6.3 Hz, 2H), 2.61–

2.75 (m, 3H), 3.65–3.76 (m, 1H), 5.18 (br s, 2H), 7.11

(d, J = 5.1 Hz, 1H), 7.19 (d, J = 5.1 Hz, 1H), 7.46–7.52

(m, 1H), 7.52–7.59 (m, 2H), 7.91–7.99 (m, 3H), 8.18–

8.23 (m, 2H), 8.28 (d, J = 8.8 Hz, 1H), 8.43 (s, 1H).

MS (ES+): m/z 449.34 (100) [MH⁺]. HPLC:

t_R= 1.76 min (OpenLynx, polar_5 min). HRMS (ES+)

for C₂₈H₂₈N₆ÆH⁺[MH⁺]: calcd, 449.2449; found,

449.2444.

4.2.16. 3-Cyclobutyl-1-quinolin-7-ylimidazo[1,5-a]pyrazin-

8-ylamine (2b). The crude material was adsorbed onto

Hydromatrix and chromatographed on silica gel [Jones

Flashmaster, 10 g/70 mL cartridge], eluting with

CH₂Cl₂! 2% MeOH in CH₂Cl₂! 4% MeOH in

CH₂Cl₂! 5% MeOH in CH₂Cl₂! 7% MeOH in

CH₂Cl₂, yielding the title compound as a yellow solid

4.2.19. trans-3-{3-[(Dimethylamino)methyl]cyclobutyl}-1-

(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2e).

The crude material was puriﬁed by silica gel column chro-

matography [Jones Flashmaster, 5 g cartridge], eluting

with 5% 7 N NH₃in MeOH/CH₂Cl₂, to aﬀord the title

compound as a yellow solid (86% yield). ¹H NMR

(CDCl₃) d 2.27 (s, 6H), 2.28–2.36 (m, 2H), 2.55 (d,

J = 6.8 Hz, 2H), 2.73–2.84 (m, 3H), 3.80–3.90 (m, 1H),

5.20 (br s, 2H), 7.09–7.14 (m, 2H), 7.45–7.52 (m, 1H),

7.52–7.59 (m, 2H), 7.93 (d, J = 8.6 Hz, 1H), 7.97 (s, 2H),

8.18–8.23 (m, 2H), 8.28 (d, J = 8.6 Hz, 1H), 8.45 (s, 1H).

MS (ES+): m/z 449.37 (100) [MH⁺]. HPLC: t_R= 1.78 min

(OpenLynx, polar_5 min). HRMS (ES+) for C₂₈H₂₈N₆ÆH⁺

[MH⁺]: calcd, 449.2449; found, 449.2448.

1

(90% yield). Mp 94–96 ꢁC. H NMR (CDCl₃) d 2.00–

2.10 (m, 1H), 2.12–2.25 (m, 1H), 2.47–2.57 (m, 2H),

2.61–2.73 (m, 2H), 3.85 (quint, J = 8.4 Hz, 1H), 5.23 (br

s, 2H), 7.10 (d, J = 4.4 Hz, 1H), 7.16 (d, J = 4.4 Hz,

1H), 7.44 (dd, J = 8.2, 4.2 Hz, 1H), 7.95 (d, J = 8.4 Hz,

1H), 8.00 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H),

8.36 (s, 1H), 8.95–9.00 (m, 1H). ¹³C NMR (DMSO-d₆,

DEPT135) d 18.31 (ꢀ), 26.54 (2C, +), 30.57 (+), 106.41

(+), 114.03 (C_quart), 121.50 (+), 127.05 (C_quart), 127.89

(+), 128.09 (+), 128.22 (+), 128.61 (+), 133.04 (C_quart),

135.85 (+), 135.92 (C_quart), 143.85 (C_quart), 147.66

(C_quart), 151.03 (+), 151.66 (C_quart). MS (ES+): m/z

316.2 (30) [MH⁺]. HPLC: t_R= 1.9 min (ZQ3, polar_5 -

min). Elemental Analysis calcd for C₂₅H₂₁N₅Æ1/12

CH₂Cl₂Æ1/12 hexane: C, 71.36; H, 5.61; N, 21.25. Found:

C, 71.53; H, 5.69; N, 21.11.

4.2.20. cis-3-(3-Azetidin-1-ylmethylcyclobutyl)-1-(2-phe-

nylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine

(2f).

The crude material was puriﬁed by mass-directed HPLC

puriﬁcation to aﬀord the title compound as a light yel-

low solid (75% yield). Mp 161 ꢁC (decomposed). ¹H

NMR (CDCl₃) d 2.03–2.16 (m, 2H), 2.27–2.38 (m,

2H), 2.46–2.59 (m, 3H), 2.59–2.70 (m, 2H), 3.25 (t,

J = 7.1 Hz, 4H), 3.68–3.78 (m, 1H), 5.19 (br s, 2H,

NH₂), 7.11 (d, J = 5.1 Hz, 1H), 7.18 (d, J = 4.8 Hz,

1H), 7.46–7.51 (m, 1H), 7.52–7.59 (m, 2H), 7.89–7.99

(m, 3H), 8.15–8.23 (m, 2H), 8.28 (d, J = 8.5 Hz, 1H),

8.43 (s, 1H). ¹³C NMR (DMSO-d₆) d 17.6, 27.5, 30.3,

31.6 (2C), 54.9 (2C), 64.9, 106.4, 114.0, 118.7, 126.1,

127.2 (2C), 127.9, 128.0, 128.8, 128.87 (2C), 128.93,

129.7, 133.1, 136.3, 137.0, 138.6, 143.8, 147.5, 151.7,

156.5. MS (ES+): m/z 461.06 (90) [MH⁺]. HPLC:

t_R= 1.97 min (OpenLynx, polar_5 min). Elemental

Analysis calcd for C₂₉H₂₈N₆Æ1.75 H₂O: C, 70.78; H,

6.45; N, 17.08. Found: C, 70.59; H, 6.22; N, 17.06.

HRMS (ES+) for C₂₉H₂₈N₆ÆH⁺[MH⁺]: calcd,

461.2449; found, 461.2431.

4.2.17. 3-Methyl-1-(2-phenylquinolin-7-yl)-imidazo[1,5-a]-

pyrazin-8-ylamine (2c). The compound was puriﬁed by

silica gel column chromatography [Jones Flashmaster,

10 g cartridge], eluting with 1% MeOH/EtOAc, yielding

the title compound as a dark yellow solid (24% yield).

¹H NMR (CDCl₃) d 2.71 (s, 3H), 5.61 (br s, 2H), 7.13

(d, 1H, J = 5.1 Hz), 7.20 (d, 1H, J = 5.1 Hz), 7.48–7.56

(m, 3H), 7.89–7.97 (m, 3H), 8.18–8.21 (m, 2H), 8.27 (d,

1H, J = 8.6 Hz), 8.39 (s, 1H). ¹³C NMR (CDCl₃,

DEPT135) d 12.47 (+), 106.43 (+), 114.31 (C_quart),

119.34 (+), 126.58 (C_quart), 127.56 (2C, +), 127.86 (+),

127.93 (+), 128.21 (+), 128.84 (2C, +), 129.42 (+),

129.48 (+), 134.63 (C_quart), 136.15 (C_quart), 136.57 (+),

137.82 (C_quart), 139.38 (C_quart), 148.13 (C_quart), 151.52

(C_quart), 158.00 (C_quart). MS (ES+): m/z 352.06 [MH⁺].

HPLC: t_R= 2.2 min (ZQ3, polar_5 min). HRMS (ES+)

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

1371

4.2.21. trans-3-(3-Azetidin-1-ylmethylcyclobutyl)-1-(2-phe-

nylquinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (2g).

The combined organic phases were dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. The crude material

was puriﬁed via silica gel chromatography eluting with

3 ! 5% MeOH:CH₂Cl₂to aﬀord the desired compound

as a yellow solid (17.7 g, 88% yield in two steps). Mp

250 ꢁC (decomposed). ¹H NMR (CDCl₃) d 3.63–3.71

(m, 2H), 3.85–3.99 (m, 3H), 7.44–7.56 (m, 4H), 7.65

(d, J = 4.8 Hz, 1H), 7.90–7.95 (m, 3H), 8.22 (d,

J = 8.0 Hz, 2H), 8.32 (d, J = 8.8 Hz, 1H), 8.61 (s, 1H).

¹³C NMR (DMSO-d₆) d 19.9, 52.2 (2C), 115.3, 119.1,

119.8, 126.4 (2C), 126.9, 127.2 (2C), 128.8 (2C), 129.0,

129.6, 130.9, 134.9, 135.2, 137.0, 138.5, 143.4, 143.7,

146.9, 156.6, 205.5. MS (ES+): m/z 425.04/427.06 (3/1)

[MH⁺]. HPLC: t_R= 2.38 min (ZQ3, non-polar_5 min).

Elemental Analysis calcd for C₂₅H₁₇ClN₄OÆ0.2H₂O: C,

70.08; H, 4.09; N, 13.08. Found: C, 70.10; H, 4.02; N,

12.88.

The mixture was concentrated in vacuo and the residue

was puriﬁed by mass-directed HPLC puriﬁcation to af-

ford the title compound as a light yellow solid (13%

yield). ¹H NMR (CDCl₃) d 2.11 (quintet, J = 7.0 Hz,

2H), 2.34–2.25 (m, 2H), 2.63–2.50 (m, 1H), 2.67 (d,

J = 7.4 Hz, 2H), 2.79–2.70 (m, 2H), 3.26 (t, J = 6.8 Hz,

4H), 3.86–3.77 (m, 1H), 5.20 (br s, 2H), 7.10 (s, 2H),

7.50–7.45 (m, 1H), 7.57–7.51 (m, 2H), 7.92 (d,

J = 8.6 Hz, 1H), 7.97–7.94 (m, 2H), 8.22–8.18 (m, 2H),

8.27 (dd, J = 8.6, 0.6 Hz, 1H), 8.43 (d, J = 1.0 Hz, 1H).

MS (ES+): m/z 461 (100) [MH⁺]. HPLC: t_R= 1.81 min

(OpenLynx, polar_5 min).

4.2.22.

cis-3-[3-(Morpholin-4-ylmethyl)cyclobutyl]-1-(2-

phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2h).

The crude material was puriﬁed by mass-directed HPLC

puriﬁcation to aﬀord the title compound as a light yellow

solid (69% yield). ¹H NMR (CDCl₃) d 2.31–2.41 (m, 2H),

2.43–2.50 (m, 4H), 2.54 (d, J = 6.1 Hz, 2H), 2.64–2.74 (m,

3H), 3.66–3.75 (m, 5H), 5.19 (br s, 2H), 7.12 (d,

J = 5.1 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H), 7.46–7.52 (m,

1H), 7.52–7.59 (m, 2H), 7.91–7.97 (m, 3H), 8.17–8.22

(m, 2H), 8.28 (d, J = 8.6 Hz, 1H), 8.43 (s, 1H). MS

(ES+): m/z 491 (100) [MH⁺]. HPLC: t_R= 1.78 min

General procedure E: synthesis of 3-aminocyclobutyl-de-

rived imidazopyrazines 2j–o from 3-[8-chloro-1-(2-phenyl-

quinolin-7-yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanone

(11). A solution of 3-[8-chloro-1-(2-phenylquinolin-7-

yl)imidazo[1,5-a]pyrazin-3-yl]cyclobutanone (0.234 mmol)

and amine (0.200 mmol) in DCE (5 mL) was charged with

sodium triacetoxyborohydride (0.468 mmol). The result-

ing mixture was stirred at rt for 16 h. The reaction mixture

was diluted with CH₂Cl₂, washed with NaHCO₃, brine,

dried over Na₂SO₄, ﬁltered, and then concentrated in va-

cuo, aﬀording the desired product as a yellow solid, which

was carried forward without further puriﬁcation. Ammo-

nia (g) was bubbled into a 0.1 M slurry of the 8-chloroim-

idazopyrazine in 2 M NH₃in i-PrOH for 8 min in a

stainless steel Parr apparatus cooled to ꢀ78 ꢁC in a dry

ice/acetone bath. The Parr apparatus was secured and

the cooling bath was removed. The reaction mixture

was heated at 110 ꢁC for 24 h, then cooled to ꢀ78 ꢁC,

vented, and allowed to warm to rt. The reaction mixture

was concentrated in vacuo and the residue was suspended

in CH₂Cl₂. The remaining salts were removed by ﬁltration

and the ﬁltrate was concentrated in vacuo.

(OpenLynx,

polar_5 min).

HRMS

(ES+)

for

C₃₀H₃₀N₆OÆH⁺[MH⁺]: calcd, 491.2554; found, 491.2547.

4.2.23. trans-3-[3-(Morpholin-4-ylmethyl)cyclobutyl]-1-(2-

phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2i).

The crude material was puriﬁed by preparative TLC elut-

ing with 1% MeOH:EtOAc followed by recrystallization

from EtOAc/Hexanes to aﬀord the title compound as a

1

yellow solid (66% yield). H NMR (CDCl₃) d 2.28–2.38

(m, 2H), 2.44–2.50 (m, 4H), 2.62 (d, J = 6.8 Hz, 2H),

2.72–2.87 (m, 3H), 3.69–3.75 (m, 4H), 3.78–3.89 (m,

1H), 5.24 (br s, 2H), 7.11 (s, 2H), 7.45–7.52 (m, 1H),

7.52–7.58 (m, 2H), 7.94 (d, J = 8.6 Hz, 1H), 7.95–7.98

(m, 2H), 8.17–8.23 (m, 2H), 8.29 (d, J = 8.6 Hz, 1H),

8.45 (s, 1H). MS (ES+): m/z 491.37 (100) [MH⁺]. HPLC:

t_R= 1.78 min (OpenLynx, polar_5 min). HRMS (ES+)

for C₃₀H₃₀N₆OÆH⁺[MH⁺]: calcd, 491.2554; found,

491.2553.

4.2.25. cis-3-[3-(Dimethylamino)cyclobutyl]-1-(2-phenyl-

quinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2j). The

product was submitted to mass-directed HPLC puriﬁca-

tion to aﬀord the title compound as a yellow solid (54%

1

4.2.24. 3-[8-Chloro-1-(2-phenylquinolin-7-yl)imidazo[1,5-

a]pyrazin-3-yl]cyclobutanone (11). A solution of 7-[8-

chloro-3-(3-methylenecyclobutyl)imidazo[1,5-a]pyrazin-

1-yl]-2-phenylquinoline (20.0 g, 47.3 mmol) in THF

(600 mL) was charged with NMO (6.09 g, 0.520 mol),

potassium osmate dihydrate (0.870 g, 0.002 mol), and

water (200 mL). The reaction mixture was stirred at rt

for 20 h and then quenched with excess Na₂SO₃. After

stirring for an additional 10 min, the mixture was di-

luted with EtOAc (400 mL) and washed with brine

(2 · 250 mL). The organic phase was passed through

Celite, dried over Na₂SO₄, ﬁltered, and concentrated

in vacuo to aﬀord the desired compound as a yellow so-

lid (24.5 g). The crude material (24.0 g, 47.3 mmol) in

THF (600 mL) and water (200 mL) was charged with

NaIO₄at 0 ꢁC and the mixture was allowed to warm

to rt and stirred for 4 h. The mixture was diluted with

EtOAc (400 mL) and washed with brine (2 · 150 mL).

yield). H NMR (CDCl₃) d 2.23 (s, 6H), 2.54 (m, 2H),

2.69 (m, 2H), 2.90 (m, 1H), 3.45 (m, 1H), 5.27 (br s,

2H), 7.11 (d, J = 4.8 Hz, 1H), 7.21 (d, J = 4.8 Hz, 1H),

7.47–7.56 (m, 3H), 7.91–7.94 (m, 3H), 8.18–8.20 (m,

2H), 8.27 (d, J = 8.8 Hz, 1H), 8.39 (s, 1H). MS (ES+):

m/z 435 [MH⁺]. HPLC: t_R= 2.28 min (ZQ3, polar_5 -

min). HRMS (ES+) for C₂₇H₂₆N₆ÆH⁺[MH⁺]: calcd,

435.2292; found, 435.2292.

4.2.26. cis-3-((3-Azetidin-1-yl)-cyclobutyl)-1-(2-phenyl-

quinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2k). The

crude material was puriﬁed by silica gel column chroma-

tography eluting with 5% 2 M NH₃in MeOH/CH₂Cl₂to

aﬀord the title compound as a yellow solid (71% yield).

¹H NMR (CDCl₃) d 2.05–2.09 (m, 2H), 2.50–2.60 (m,

4H), 3.25–3.29 (m, 6H), 5.20 (br s, 2H), 7.11 (d,

J = 5.2 Hz, 1H), 7.19 (d, J = 5.2 Hz, 1H), 7.48–7.57

(m, 3H), 7.91–7.95 (m, 3H), 8.18–8.20 (m, 2H), 8.27

1372

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

(d, J = 8.4 Hz, 1H), 8.41 (s, 1H). MS (ES+): m/z 447.10

[MH⁺]. HPLC: t_R= 1.76 min (Open Lynx, polar_5 -

min). HRMS (ES+) for C₂₈H₂₆N₆ÆH⁺[MH⁺]: calcd,

447.2292; found, 447.2309.

2.73 (m, 2H), 2.91–3.01 (m, 1H), 3.45–3.56 (m, 1H),

3.70–3.79 (m, 4H), 5.23 (br s, 2H), 7.12 (d, J = 4.8 Hz,

1H), 7.20 (d, J = 4.8 Hz, 1H), 7.45–7.59 (m, 3H), 7.90–

7.98 (m, 3H), 8.17–8.23 (m, 2H), 8.27 (d, J = 8.4 Hz,

1H), 8.40 (s, 1H). ¹³C NMR (DMSO-d₆, DEPT135) d

23.25 (+), 31.63 (2C, ꢀ), 49.37 (2C, ꢀ), 56.12 (+),

65.98 (2C, ꢀ), 106.41 (+), 114.04 (C_quart), 118.74 (+),

126.13 (C_quart), 127.21 (2C, +), 127.91 (2C, +), 127.97

(+), 128.78 (+), 128.86 (2C, +), 129.64 (+), 133.23

(C_quart), 136.24 (C_quart), 137.02 (+), 138.57 (C_quart),

143.46 (C_quart), 147.51 (C_quart), 151.64 (C_quart), 156.49

(C_quart). MS (ES+): m/z 477.30 [MH⁺]. HPLC:

t_R= 1.75 min (Open Lynx, polar_5 min); 1.86 min

(ZQ3, polar_5 min). HRMS (ES+) for C₂₉H₂₈N₆OÆH⁺

[MH⁺]: calcd, 477.2398; found, 477.2408.

4.2.27. cis-3-[3-(Pyrrolidine)cyclobutyl]-1-(2-phenylquino-

lin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2l). The prod-

uct was submitted to mass-directed HPLC puriﬁcation

to aﬀord the title compound as a yellow solid (18%

yield). ¹H NMR (CDCl₃) d 1.80 (m, 4H), 2.54–2.69

(m, 8H), 3.15 (m, 1H), 3.5 (m, 1H), 5.23 (br s, 2H),

7.10 (d, J = 4.4 Hz, 1H), 7.19 (d, J = 4.8 Hz, 1H),

7.47–7.55 (m, 3H), 7.90–7.94 (m, 3H), 8.19 (m, 2H),

8.26 (d, J = 8.0 Hz, 1H), 8.39 (s, 1H). MS (ES+): m/z

461.3 [MH⁺]. HPLC: t_R= 1.84 min (OpenLynx, po-

lar_5 min). HRMS (ES+) for C₂₉H₂₈N₆ÆH⁺[MH⁺]:

calcd, 461.2449; found, 461.2426.

4.2.31. 3-Cyclobutyl-1-(2-phenylquinolin-7-yl)-imidazo[1,5-

a]pyrazin-8-ol (9). A THF (2.0 mL) solution of 7-(8-

chloro-3-cyclobutyl-2H-imidazo[1,5-a]pyrazin-1-yl)-2-

phenylquinoline (50 mg, 0.12 mmol) was charged with

water (2 mL) and 37% HCl (2 mL) and heated at 60 ꢁC

for 30 min. The solution was allowed to cool to rt and stir-

red for 16 h. THF was removed in vacuo and the aqueous

solution was treated with 5 N NaOH until pH 10 was

achieved. The resulting solid was ﬁltered, washed with

water (2 · 5 mL), and dried in vacuo to aﬀord 45 mg of

the title compound as a yellow solid (94% yield). ¹H

NMR (DMSO-d₆) d 1.91–2.02 (m, 1H), 2.10 (quintet,

J = 9.2 Hz, 1H), 2.39–2.50 (m, 4H), 3.96 (quintet,

J = 8.4 Hz, 1H), 6.70 (dd, J = 5.6, 5.6 Hz, 1H), 7.22 (d,

J = 5.6 Hz, 1H), 7.49–7.62 (m, 3H), 8.00 (d, J = 8.8 Hz,

1H), 8.13 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 6.8 Hz, 2H),

8.44 (d, J = 8.4 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 9.11 (s,

1H), 10.71 (d, J = 2.8 Hz, 1H). MS (ES+): m/z 393 (100)

[MH⁺].

4.2.28. cis-1-(2-Phenylquinolin-7-yl)-3-(3-piperidin-1-yl-

cyclobutyl)-imidazo[1,5-a]pyrazin-8-ylamine (2m). The

crude material was puriﬁed by recrystallization from

CH₂Cl₂/Hexanes (4:1) to aﬀord the title compound as

1

a yellow solid (37% yield). H NMR (CDCl₃) d 1.45–

1.46 (m, 2H), 1.57–1.62 (m, 4H), 2.31–2.39 (m, 4H),

2.50–2.55 (m, 2H), 2.65–2.72 (m, 2H), 2.88 (m, 1H),

3.47 (m, 1H), 5.22 (br s, 2H), 7.10 (d, J = 4.8 Hz, 1H),

7.20 (d, J = 5.2 Hz, 1H), 7.47–7.56 (m, 3H), 7.91–7.94

(m, 3H), 8.18–8.21 (m, 2H), 8.26 (d, J = 8.4 Hz, 1H),

8.38 (s, 1H). MS (ES+): m/z 475.10 [MH⁺]. HPLC:

t_R= 1.83 min (OpenLynx, polar_5 min). HRMS (ES+)

for C₃₀H₃₀N₆ÆH⁺[MH⁺]: calcd, 475.2605; found,

475.2605.

4.2.29. cis-1-(2-Phenylquinolin-7-yl)-3-(3-thiomorpholin-

4-ylcyclobutyl)-imidazo[1,5-a]pyrazin-8-ylamine

(2n).

The crude material was puriﬁed by recrystallization

from EtOAc/Hexanes (4:1) to aﬀord the title compound

as a yellow solid (60% yield). Mp 145–150 ꢁC (decom-

4.3. Biological evaluation

1

posed). H NMR (CDCl₃) d 2.43–2.51 (m, 2H), 2.64–

4.3.1. Cell lines. 3T3/huIGFIR cells, derived from NIH

3T3 cells stably overexpressing full-length human IGF-

IR, were obtained from Dr. J. Beebe (Pﬁzer, Inc.).

GEO human cancer cells were kindly provided from

Dr. M. Brattain (Roswell Park Cancer Institute) and

were maintained in McCoy’s 5A medium supplemented

with 10% FCS and 1% ^L-glutamine or in serum-free con-

ditioned culture system. MCF-7, NCI-H292, Colo-699,

NCI-H358, HT-29, Colo-205, and HepG2 cells were

from ATCC (Manassas, VA) and cultured in the appro-

priate media according to ATCC recommendations.

2.73 (m, 10H), 2.94–2.98 (m, 1H), 3.45–3.50 (m, 1H),

5.24 (br s, 2H), 7.11 (d, J = 5.2 Hz, 1H), 7.19 (d,

J = 5.2 Hz, 1H), 7.46–7.57 (m, 3H), 7.91–7.97 (m, 3H),

8.18–8.21 (m, 2H), 8.27 (d, J = 8.8 Hz, 1H), 8.40 (s,

1H). ¹³C NMR (DMSO-d₆, DEPT135) d 23.14 (+),

26.90 (2C, ꢀ), 32.19 (2C, ꢀ), 50.52 (2C, ꢀ), 56.03 (+),

106.42 (+), 114.05 (C_quart), 118.75 (+), 126.15 (C_quart),

127.22 (2C, +), 127.93 (2C, +), 127.99 (+), 128.81 (+),

128.88 (2C, +), 129.67 (+), 133.26 (C_quart), 136.24

(C_quart), 137.04 (+), 138.59 (C_quart), 143.44 (C_quart),

147.53 (C_quart), 151.66 (C_quart), 156.51 (C_quart). MS

(ES+): m/z 493.08 [MH⁺]. HPLC: t_R= 1.92 min (ZQ3,

4.3.2. Antibodies. The following antibodies were used for

immunoprecipitation or as the capture antibody in ELI-

SAs: human IGF-IR (Ab-1, Calbiochem, EMD, CA)

and IR (Ab-2, Lab Vision Corp., CA) for capture; human

IGF-IRb (sc-713, Santa Cruz Biotechnology) for immu-

noprecipitation. The following antibodies were used for

immunoblotting analysis: human IGF-IRb (sc-713, Santa

Cruz Biotechnology), antiphosphotyrosine (X1021, Exal-

pha Biologicals, MA), antiphosphotyrosine-horseradish

peroxidase (HRP) conjugate (mouse anti-Phosphotyro-

sine-HRP, Invitrogen-ZYMED), pAKT⁴⁷³(9271, Cell

Signaling Technology), p-p70S6K (9205, Cell Signaling

Technology), and GAPDH (9482, Abcam).

polar_5 min).

Elemental

Analysis

calcd

for

C₂₉H₂₈N₆SÆ0.2H₂OÆ0.2 hexane: C, 70.64; H, 6.12; N,

16.37. Found: C, 70.33; H, 5.94; N, 16.29. HRMS

(ES+) for C₂₉H₂₈N₆SÆH⁺[MH⁺]: calcd, 493.2169;

found, 493.2182.

4.2.30. cis-3-(3-Morpholin-4-ylcyclobutyl)-1-(2-phenyl-

quinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (2o). The

product was submitted to mass-directed HPLC puriﬁca-

tion to aﬀord the title compound as a yellow solid (45%

yield). Mp 230–235 ꢁC (decomposed). ¹H NMR

(CDCl₃) d 2.37–2.47 (m, 4H), 2.47–2.57 (m, 2H), 2.64–

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

1373

4.3.3. Protein kinase biochemical assays. Protein kinase

assays were either performed in-house by ELISA-based

assay methods (IGF-IR, IR, Kit, EGFR, and KDR) or

at Upstate Inc. (Charlottesville, VA) by a radiometric

method (KinaseProﬁler service). In-house ELISAs used

poly(Glu:Tyr) (Sigma, St. Louis, MO) as the substrate

bound to the surface of 96-well assay plates, and phos-

phorylation was detected using an antiphosphotyrosine

antibody conjugated to HRP. The bound antibody

was quantiﬁed using ABTS as the peroxidase substrate

by measuring absorbance at 405/490 nm. All assays used

puriﬁed recombinant kinase catalytic domains. Recom-

binant enzymes of human IGF-IR or EGFR were ex-

pressed as an NH₂-terminal glutathione S-transferase

fusion protein in insect cells and were puriﬁed in-house.

The human IR protein was purchased from Calbiochem

(Cat# 407697). IC₅₀values were determined from the

sigmoidal dose–response plot of percent inhibition ver-

sus log₁₀compound concentration (Xlﬁt 3.0, IDBS).

A minimum of three measurements, performed in dupli-

cate, were carried out with in-house assays unless other-

wise indicated.

mined by luminescent quantitation of intracellular

ATP content using CellTiterGlo (Promega, Madison,

WI).

For assay of DNA fragmentation, 5 · 10³cells per well

were seeded in a 96-well plate and treated with AQIP at

10 lM for 48 h in the presence of 10% FCS. DNA

fragments were detected using Cell Death Detection

ELISA^PLUS(Cat#11774425001), according to the

instructions provided by the manufacturer. Data were

expressed as fold of DNA induction over the DMSO

control cells.

4.3.6. Metabolic stability assay. Compound was mixed

with microsomes of human and mouse, and the reaction

was initiated by the addition of NADPH for 0 (pre-

NADPH addition), 5, 10, 20 or 40 min. The reactions

were terminated with chilled methanol. After centrifuga-

tion to precipitate protein, supernatants were analyzed

by LC–MS/MS. Various pharmacokinetic parameters

were calculated, including half-life (t_1/2), intrinsic clear-

ance (Cl_int), and scaled hepatic clearance (Cl_h), depen-

dent upon the species. An ‘Extraction Ratio’ (ER) was

calculated by the formula ER = Cl_h/hepatic blood ﬂow.

4.3.4. Protein kinase inhibition in intact cells. Quantita-

tive 96-well ELISAs were developed to study the cellular

eﬀects of AQIP. Cells were placed into 96-well plates in

media containing low serum (0.5% FCS) at 37 ꢁC for 2 h

(3T3/huIGFIR) or overnight (HepG2), and then were

treated with various concentrations of compound for

2 h before lysis (the ﬁnal DMSO concentration in the as-

say was 0.4%), and the appropriate ligand was added for

the ﬁnal 15 min of the compound treatment period (100

ng/mL IGF-I, R&D systems or 10 ng/mL insulin,

Roche). Lysates were prepared in TGH buﬀer⁴⁷(1% Tri-

ton-100, 10% glycerol, 50 mmol/L Hepes, pH 7.4) sup-

plemented with 150 mmol/L NaCl, 1.5 mmol/L MgCl,

1 mmol/L EDTA, and fresh protease and phosphatase

inhibitors (10 lg/mL leupeptin, 25 lg/mL aprotinin,

and 200 lmol/L Na₃VO₄). ELISAs of the target protein

phosphorylation were done by transferring lysates into a

second 96-well plate that was precoated with the appro-

priate capture antibody. The target proteins were then

probed with an antiphosphotyrosine antibody-HRP

conjugate using a chemiluminescent HRP substrate

(Pierce) for detection by luminometry.

4.3.7. Cytochrome P450 3A4 assay. Cytochrome P450

TM

3A4 activity was measured using P450-Glo CYP3A4

Screening Systems (Promega) according to the instruc-

tions provided by the manufacturer.

4.4. In vivo evaluation

4.4.1. Animals. Female CD-1 and athymic nude nu/nu

CD-1 mice (6–8 weeks, 25–29 g) were obtained from

Charles River Laboratories (Wilmington, MA). Ani-

mals were allowed to acclimate for a minimum of one

week prior to initiation of a study. Female Sprague–

Dawley rats with implanted jugular vein canulas were

obtained from Hilltop Lab Animals (Scottdale, PA)

and were allowed to acclimate for a minimum of 1 day

prior to a study. Throughout the studies, animals were

allowed sterile rodent chow and water ad libitum, and

immunocompromised animals were maintained under

speciﬁc pathogen-free conditions. All rodent animal

studies were conducted at OSI facilities with the ap-

proval of the Institutional Animal Care and Use Com-

mittee in an American Association for Accreditation

of Laboratory Animal Care (AAALAC)-accredited

vivarium and in accordance with the Institute of Labo-

ratory Animal Research (Guide for the Care and Use

of Laboratory Animals, NIH, Bethesda, MD). Animals

for pharmacokinetic studies on male Beagle dogs were

housed at MPI Research (Mattawan, MI).

For immunoblotting analysis, lysates were cleared of

insoluble material by centrifugation at 15,000g for

5 min at 4 ꢁC and the resultant supernatant was sub-

jected to immunoprecipitation with anti-IGF-IR anti-

body (sc-713) coupled to Protein G–Sepharose beads

(Sigma, St. Louis, MO), followed by SDS–PAGE and

immunoblotting with antiphosphotyrosine antibody-

HRP conjugate and chemiluminescent detection. Alter-

natively, for highly abundant protein targets (IGF-IR,

pErk1/2, pAkt, and p-p70S6K), lysates were analyzed

directly by SDS–PAGE and immunoblotting.

4.4.2. Pharmacokinetic analysis of AQIP. For pharma-

cokinetic analysis, the compound was formulated in sal-

ine adjusted to pH 2 with 0.01 mol/L hydrochloric acid

for intravenous injection and in 25 mmol/L tartaric acid

for oral administration. Female CD-1 mice (6–8 weeks

old) received either a single intravenous dose (5 mg/kg)

or a single oral dose (25 mg/kg) of compound. For intra-

venous dosing, compound was delivered via tail vein

injection at a dosing volume of 4 mL/kg. For oral dos-

4.3.5. Cell proliferation and DNA fragmentation. For as-

says of cell proliferation, cells were seeded into 96-well

plates in appropriate media containing 10% FCS and

incubated for 3 d in the presence of AQIP at various

concentrations. Inhibition of cell growth was deter-

1374

M. J. Mulvihill et al. / Bioorg. Med. Chem. 16 (2008) 1359–1375

ing, compound was delivered via oral gavage in a dosing

volume of 10 mL/kg. Subsequently, three animals were

sacriﬁced at each designated timepoint and blood sam-

ples were collected in EDTA. After centrifugation at

1500g for 10 min, plasma samples were prepared by pro-

tein precipitation with methanol and analyzed by

HPLC–MS/MS (PE Sciex API 3000 LC/MS/MS Sys-

tem, Applied Biosystems). Pharmacokinetic parameters

for the plasma time-concentration proﬁle, including

C_max, AUC, elimination half-life (t_1/2), volume of distri-

bution at steady state (V_ss), clearance (Cl), and oral bio-

availability, were calculated by non-compartmental

analysis.

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Article Doi

Novel 2-phenylquinolin-7-yl-derived imidazo[1,5-a]pyrazines as potent insulin-like growth factor-I receptor (IGF-IR) inhibitors

DOI: 10.1016/j.bmc.2007.10.061

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Full text of DOI:10.1016/j.bmc.2007.10.061

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