Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists

Bioorganic & Medicinal Chemistry 14 (2006) 3307–3319

Identiﬁcation of 4-amino-2-cyclohexylaminoquinazolines

as metabolically stable melanin-concentrating

hormone receptor 1 antagonists

Kosuke Kanuma,^aKatsunori Omodera,^aMariko Nishiguchi,^aTakeo Funakoshi,^a

Shigeyuki Chaki,^aYasuko Nagase,^aIzumi Iida,^aJun-ichi Yamaguchi,^aGraeme Semple,^b

Thuy-Anh Tran^band Yoshinori Sekiguchi^a,*

^aMedicinal Research Laboratories, Taisho Pharmaceutical Co. Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama, Saitama 331-9530, Japan

^bArena Pharmaceuticals Inc., 6166 Nancy Ridge Drive, San Diego, CA 92121, USA

Received 21 November 2005; revised 20 December 2005; accepted 21 December 2005

Available online 24 January 2006

Abstract—The optimization of the distance between two key pharmacophore features within our ﬁrst hit compounds 1a and 2a led

to the identiﬁcation of a new class of potent non-peptidic antagonists for the MCH-R1, based around 4-amino-2-cyclohexylamino-

quinazolines. In particular, ATC0065 (2c), N²-[cis-4-({2-[4-Bromo-2-(triﬂuoromethoxy)phenyl]ethyl}amino)cyclohexyl]-N⁴,N⁴-dim-

ethylquinazoline-2,4-diamine dihydrochloride, bound with high aﬃnity to the MCH-R1 (IC₅₀value of 16 nM) and showed good

metabolic stability in liver microsomes from human and rat.

1. Introduction

non-functional MCH-R2 pseudogene.¹³Due to this spe-

cies-speciﬁc expression of MCH-R2, potential physiolog-

ical functions of the receptor have not been elucidated.

Melanin-concentrating hormone (MCH) was originally

isolated from salmon pituitary as a cyclic 17 amino

acid peptide.¹Subsequently, rat MCH (rMCH) was

identiﬁed and characterized as a cyclic 19 amino acid

peptide.²In 1999, human MCH was identiﬁed as the

endogenous ligand of the orphan G-protein-coupled

receptor (GPCR), SLC-1 (subsequently termed MCH-

R1). This pairing of the MCH ligand with its receptor,

which has signiﬁcant homology with the somatostatin

receptors,³was made independently by several

groups.^4–7Subsequently, another human orphan recep-

tor, SLT, originally identiﬁed from the human genomic

sequence and cloned from a human hippocampus

cDNA library, was identiﬁed as a second MCH recep-

tor (MCH-R2).^8–12Interestingly, MCH-R2 is found in

humans, monkeys, dogs, and ferrets, whereas several low-

er species such as rats, mice, hamsters, guinea pigs, and

rabbits either lack functional MCH-R2 or encode a

There has been signiﬁcant interest in the pharmacology

of MCH since the discovery of the MCH-R1.¹⁴Neurons

containing MCH are located in the lateral hypothalamic

area (LHA) and the rostromedial zona incerta, suggest-

ing a role for MCH in feeding behavior and energy

expenditure. Indeed, the intracerebroventricular (icv)

injection of MCH in rats increases food intake.^15,16

Moreover, MCH mRNA has been reported to be upreg-

ulated in both normal and ob/ob mice, and to be in-

creased in ob/ob mice relative to wild-type animals.¹⁵

In contrast, prepro-MCH-knockout mice were found

to be hypophagic and have reduced bodyweight and in-

creased leanness relative to their wild-type counter-

parts.¹⁷Mice in which the MCH-R1 receptor has been

knocked out have normal bodyweights but are lean

and hyperphagic.^18,19A peptide MCH-R1 antagonist

when dosed icv reduced feeding and bodyweight gains

over a period of 14 days.²⁰These data suggest that a

centrally acting MCH-R1 antagonist may reduce food

intake and bodyweight, and hence may be useful for

the treatment of obesity. This appears to be conﬁrmed

with a non-peptide antagonist (SNAP-7941) that when

Keywords: Melanin-concentrating hormone receptor 1 antagonists;

MCH-R1 antagonists; ATC0065.

*

Corresponding author. Tel.: +81 48 669 3064; fax: +81 48 652

7254; e-mail: yoshi.sekiguchi@po.rd.taisho.co.jp

doi:10.1016/j.bmc.2005.12.052

3308

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

dosed twice daily ip (20 mg/kg) reduced feeding and

bodyweight in diet induced obese rats.²¹

for the treatment of obesity. Synaptic has reported an

extensive series of substituted anilinic piperidines, show-

ing that signiﬁcant structural diversity in the left portion

of their series maintained MCH-R1 antagonist activity.

Among them, SNAP-7941 (III), featuring an N-acyl

moiety in the meta-position, has been revealed as a com-

petitive antagonist of MCH-R1 in a phosphoinositide

accumulation assay (pA₂value of 9.24).²¹By combining

MCH-R1 modeling and structural input from dopamine

D₂receptor ligands, compound IV (IC₅₀value of

28 nM) was identiﬁed as the lead compound among a

new series of MCH-R1 antagonists.²⁶Based on reported

structure–activity relationships (SAR), the antagonist

activity of the phenoxyphenylurea derivatives appeared

in general to exhibit a considerable tolerance around

the amine in the right part of the molecule, whilst it

was generally quite sensitive to structural modiﬁcations

in the left part, which is somewhat distinct from the pre-

viously known series. The last structural motif is the

substituted biaryl scaﬀold, which is similar to known

acyl-CoA cholesterol acyltransferase (ACAT) inhibi-

tors. Since the discovery of this motif, Schering has con-

tinued far-reaching investigations, claiming a number of

MCH-R1 selective antagonists in diverse patent applica-

tions. One of the most noticeable structural modiﬁca-

tions involves incorporating the trans-bicycloheptyl

moiety in place of the biaryl structure to avoid potential

mutagenicity of the biarylaniline in vivo; this modiﬁca-

In addition, MCH was reported to induce anxiogenic

eﬀects when injected into the medial preoptic area in

rats,²²while icv injections of MCH increased the

number of entries into the open arms in the elevated

plus-maze test,²³suggesting that MCH may also be

implicated in the modulation of emotional states.

This hypothesis is supported by a recent ﬁnding that

SNAP-7941, a selective antagonist for the MCH-R1,

exhibits anxiolytic and antidepressant eﬀects in rodent

models.²¹

Since the pioneering discovery of the small molecule

MCH-R1 antagonist, T-226296 ((ꢀ)-enantiomer of

compound I), which exhibits high aﬃnity for MCH-

R1 in receptor binding assays (IC₅₀value of 5.5 nM),

a plethora of structurally diverse MCH-R1 selective

antagonists have been reported (Fig. 1).²⁴An alternative

cyclization pattern and incorporation of a thieno[3,2-

d]prymidin-4(3H)-one ring that served to lock the amide

conformation led to the discovery of GW3430 (II)

(pIC₅₀value of 9.3).²⁵GW3430 is an orally active com-

pound causing signiﬁcant and dose-dependent weight

loss in obese AKR mice over a 12-day period. Glaxo-

SmithKline has also reported the initiation of ﬁrst clin-

ical evaluation of an MCH-R1 antagonist with GW3430

O

N

O

N

S

N

H

N

OMe

Cl

F

II, GW3430

I, T-226296

pIC₅₀= 9.3

IC₅₀= 5.5 nM

F

O

N

MeO₂C

MeO

N

H

N

H

N

O

N

H

N

H

O

N

H

N

H

OMe

O

IV

III, SNAP-7941

I C₅₀= 28 nM

pA₂= 9.24

F

CF₃

O

NH

O

NH

N

NC

OH

CN

VI

Ki = 2.7 nM

V

Ki = 8.9 nM

Figure 1. Non-peptidic MCH-R1 antagonists.

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

3309

tion is exempliﬁed by compounds V and VI (K_ivalues of

8.9 and 2.7 nM, respectively).²⁷

and 2a (IC₅₀values of 4.9 and 35 nM, respectively).²⁹

Although these initial hit compounds showed potent

aﬃnity for the MCH-R1, in particular compound 1a,

compound 1a displayed metabolic instability (T_1/2val-

ues of 8.9 and 5.8 min for human and rat liver micro-

somes, respectively). We considered that the metabolic

instability of compounds 1a might be caused by the ben-

zylic amine structure. Therefore, we believed that both

compounds 1a and 2a had suﬃcient potency, as well as

a pathway forward to improving metabolic stability, to

deserve further optimization of the distance between the

two key pharmacophore features. In this report, we

disclose our eﬀorts to investigate SAR at the MCH-R1

based on the 4-amino-2-cyclohexylaminoquinazoline

series and to improve metabolic stability, leading to the

identiﬁcation of ATC0065, a potent and orally eﬃcacious

antagonist of MCH-R1 for the treatment of either obesity

or, as previously reported, anxiety and depression.³⁰

As previously reported, high-throughput screening

resulted in the identiﬁcation of our lead compound

AR129330 (Table 1).²⁸During our initial exploration,

we identiﬁed that both 4-amino-substitution of the qui-

nazoline and the terminal phenyl group are critical for

MCH-R1 antagonist activity and the quinazoline and

the benzene rings form two key parts of the MCH-R1

pharmacophore. Subsequent systematic optimization

of the spacer and the linker portions of our lead series

identiﬁed a core 4-dimethylamino-2-cyclohexylamino-

quinazoline structure, exempliﬁed by compounds 1a

Table 1. Quinazoline derivatives as MCH-R1 antagonists

N

m

2. Chemistry

Br

N

H

N

Preparation of the quinazoline derivatives with the cis-4-

aminomethylcyclohexylamine is summarized in Scheme

1. Quinazoline cores 5A or 5B were synthesized in two

steps from commercially available 1H,3H-quinazoline-

2,4-dione 3. The starting material was reacted with

phosphorus oxychloride (POCl₃) under reﬂux in the

presence of N,N-dimethylaniline to provide 2,4-dichlo-

ro-quinazoline 4. Selective substitution of the chlorine

at the 4-position with 50% aqueous dimethylamine or

40% aqueous methylamine gave the corresponding

4-amino-2-chloro-quinazoline 5A or 5B.³¹The mono-

A

n

OCF

3

a

MCH-R1 IC₅₀(nM)

Compound Conformation

m

n

A

AR129330 trans

1

0

1

0

SO₂160

1a

2a

cis

CH₂

4.9

35

^aCompounds were evaluated for their ability to compete with

¹²⁵I](Phe¹³, Tyr¹⁹)MCH at a human MCH-R1 stably expressed in

[

HEK293 cells. Data represent means of 1–3 separate experiments

performed from 5 concentrations in duplicate.

O

Cl

N

X

N

b: 5A

c: 5B

a

NH

N

H

3

O

Cl

4

5A: X = NMe₂

5B: X = NHMe

H₂N

BOCHN

H₂N

d

e

f

OH

NHCbz

CO₂H

6

7

8

X

N

OCF₃

n

g

N

NHCbz

N

H

N

H

N

H

Y

9A : X = NMe₂

9B : X = NHMe

1A : X = NMe₂, Y = H or Br

1B : X = NHMe, Y = H or Br

Scheme 1. Reagents and conditions: (a) POCl₃, N,N-dimethylaniline, reﬂux, 7 h, 86%; (b) aq 50% Me₂NH, THF, rt, 80 min, 94%; (c) aq 40%

MeNH₂, THF, rt, 40 min, 94%; (d) i—SOCl₂, MeOH, rt, 4.5 h, ii—(BOC)₂O, Et₃N, CHCl₃, rt, 5 h, iii—LAH, Et₂O, rt, 2 h, 77% in three steps; (e)

i—phthalimide, 40% DEAD in toluene, PPh₃, THF, rt, 2.5 days, ii—hydrazine hydrate, EtOH, reﬂux, 2.25 h, iii—CbzCl, Et₃N, CHCl₃, rt, 16 h, 91%

in three steps, iv—4 M HCl in EtOAc, EtOAc, rt, 3 h, 95%; (f) compound 5A or 5B, isopropanol, reﬂux; (g) i—H₂, 5% Pd–C or 10% Pd–C, MeOH,

50 ꢁC, ii—RCHO, NaBH(OAc)₃, AcOH, CH₂Cl₂, rt or RCHO, NaBH₃CN, AcOH, MeOH, rt, or i—H₂, 5% Pd–C or 10% Pd–C, MeOH, 50 ꢁC,

ii—RCOCl, iPr₂NEt, CH₂Cl₂, 4 ꢁC, iii—1 M BH₃-THF in THF, THF, reﬂux.

3310

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

protected cis-1,4-cyclohexylmethyldiamine 8 was pre-

pared in seven steps from commercially available

4-aminocyclohexane carboxylic acid 6. Treatment of 6

with thionyl chloride in methanol provided the corre-

sponding methyl ester. Protection of the amine as its

tert-butyl carbamate (BOC) derivative followed by

reduction of the ester group with lithium aluminum

hydride (LAH) then produced alcohol 7. Conversion of

the alcohol 7 to the corresponding amine was accom-

plished via the phthalimide derivative, and the amine

was then protected as benzyl carbamate (Cbz). Hydrogen

chloride treatment readily removed the BOC-group of

the di-protected diamine to aﬀord amine 8, which was

then condensed with 4-amino-2-chloro-quinazoline 5A

or 5B under reﬂux in isopropanol to give the coupling

product 9A or 9B. Removal of the Cbz group by

hydrogenolysis followed by reductive amination with

appropriate aldehydes aﬀorded the desired quinazoline

derivatives 1A or 1B.

in isopropanol to aﬀord coupling product 10A or 10B.

Deprotection of the BOC-group was achieved with

hydrogen chloride followed by reductive amination with

appropriate aldehydes to aﬀord the desired quinazoline

derivatives 2A or 2B.

Scheme 3 illustrates the syntheses of the aldehydes with

diﬀerent chain lengths. Formylation of commercially

available 4-bromo-1-iodo-2-(triﬂuoromethoxy)benzene

11 with N-formylmorpholine led to the corresponding

benzaldehyde 12.³²One carbon homologation of 12

was achieved by Wittig reaction followed by hydrolysis

under acidic conditions to aﬀord phenyl acetaldehyde

13. Treatment of 12 with Horner–Emmons reagent pro-

vided the a,b-unsaturated ester 14. Reduction of 14 with

LAH followed by oxidation aﬀorded the C3-aldehyde

15. C4-aldehyde 17 was synthesized from phenyl acetal-

dehyde 13 by a similar procedure.

The general synthetic approach to the cis-cyclohexane-

1,4-diamine derivatives 2A or 2B is outlined in Scheme

2. The coupling of 5A or 5B synthesized in Scheme 1

with commercially available tert-butyl (cis-4-amin-

ocyclohexyl)carbamate was accomplished upon reﬂux

3. Results and discussion

The SAR of the MCH-R1 binding of the 4-amino-2-

cyclohexylaminoquinazolines is summarized in Table

2. Our initial eﬀorts focused on ﬁnding the optimal

Y

X

N

X

N

H

N

NHBOC

a

b

N

n

OCF₃

N

H

N

H

Cl

N

5A : X = NMe₂

5B : X = NHMe

10A : X = NMe₂

10B : X = NHMe

2A : X = NMe₂, Y = H or Br

2B : X = NHMe, Y = H or Br

Scheme 2. Reagents and conditions: (a) tert-butyl (cis-4-aminocyclohexyl)carbamate, isopropanol, reﬂux; (b) i—4 M HCl in EtOAc, EtOAc, rt,

ii—RCHO, NaBH(OAc)₃, AcOH, CH₂Cl₂, rt, or RCHO, NaBH₃CN, AcOH, MeOH, rt, or 4-bromo-1-iodo-2-(triﬂuoromethoxy)benzene,

Pd₂(dba)₃, (R)-BINAP, NaOtBu, 18-C-6, THF, reﬂux.

Br

OCF₃

CHO

Br

OCF₃

I

Br

OCF₃

CHO

b

a

12

11

13

c

Br

OCF₃

Br

OCF₃

CO₂Et

16

14

d

Br

OCF₃

Br

OCF₃

CHO

15

17

Scheme 3. Reagents and conditions: (a) N-formylmorpholine, n-BuLi in hexane, THF, rt, 80 min, 77%; (b) MeOCH=P(Ph)₃, Et₂O, rt, 1 h; 10%

H₂SO₄, AcOH, rt, 30 min, 67%; (c) (EtO)₂P(O)CH₂CO₂Et, NaH, THF, 4 ꢁC or rt; (d) i—LAH, Et₂O, rt, ii—PCC, Celite, CH₂Cl₂, rt.

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

3311

Table 2. In vitro data of quinazolines with length of spacer modiﬁ-

cations

improved aﬃnity at the MCH-R1 (IC₅₀values of 16,

11, and 16 nM for compound 2c (ATC0065), 2e, and

2f, respectively) compared to the initial hit compound

2a. Compound 2d, wherein bromine was again replaced

by hydrogen in the benzene ring, increased in MCH-R1

antagonist activity by a factor of about 2 (IC₅₀value of

8.7 nM). Among the 4-methylamino-quinazoline series,

compounds 2g, 2h, and 2i, compound 2h showed opti-

mal aﬃnity at the MCH-R1 (IC₅₀value of 16 nM).

X

OCF₃

n

m

N

H

N

H

Y

a

Compound

m

n

X

Y

MCH-R1 IC₅₀(nM)

Br 4.9

5.5

Br 41

H 42

1a

1b

1

0

1

2

1

2

0

1

2

3

4

1

2

3

NMe₂

Based on the following comparisons, compounds 1b ver-

sus 1f, compounds 1d versus 1h, compounds 2a versus

2g, and compounds 2e versus 2i—we concluded that

4-dimethylamino-quinazoline derivatives were superior

to 4-methylamino-quinazoline derivatives in terms of

MCH-R1 antagonist activity. Replacing a bromine

atom with hydrogen in the benzene ring among this ser-

ies had virtually no eﬀect on aﬃnity for the MCH-R1.

Furthermore, data for compounds 1a, 1b, 1e, 1f, 2c,

2d, and 2h, which all had potent antagonist activity at

the MCH-R1, suggested that the distance of three atoms

was the optimal length from the cyclohexane ring to the

benzene ring.

H

1c

1d

1e

NHMe Br 4.8

NHMe 7.1

NHMe Br 45

1f

1g

H

1h

2b

NHMe

NMe₂

H

79

Br 520

Br 35

Br 16

2a

2c (ATC0065)

2d

2e

2f

H

8.7

Br 11

Br 16

2g

2h

2i

NHMe Br 71

NHMe Br 16

NHMe Br 58

Although compounds 1b, 1e, and 1f showed potent

aﬃnity for the MCH-R1, all of these compounds dis-

played metabolic instability in liver microsomes from

both human and rat, probably due to the benzylic amine

structure (data not shown). However, ATC0065 showed

a signiﬁcant improvement in metabolic stability in both

of the liver microsomes (T_1/2values of 73 and 30 min for

human and rat, respectively) in comparison to our initial

hit compound 1a (T_1/2values of 8.9 and 5.8 min for

human and rat, respectively). We succeeded in the

enhancement of metabolic stability with good

MCH-R1 antagonist activity only by altering the posi-

tion of a nitrogen atom, leading to the further investiga-

tion of ATC0065 in vivo pharmacology.³⁰

^aSee footnote a for Table 1.

distance between the two regions of the MCH-R1 phar-

macophore in the cis-4-aminomethylcyclohexylamine

derivatives. Compound 1c, in which a methylene group

was incorporated between the two features, was about

8-fold less potent at the MCH-R1 than our initial hit

compound 1a. Replacement of a bromine atom with

hydrogen in the benzene ring led to compound 1b, which

maintained very potent aﬃnity at the MCH-R1 (IC₅₀

value of 5.5 nM). The lengthening of compound 1b by

a methylene group gave reduced interaction with the

MCH-R1 (IC₅₀value of 42 nM for compound 1d).

Compound 1e, in which the dimethylamino group at

the 4-position of the quinazoline was replaced by a

methylamino group, provided potency comparable to

that of our initial hit compound 1a. Elongation of the

molecule showed the same tendency as that seen in the

4-dimethylamino-quinazoline series, whereby com-

pound 1g showed signiﬁcantly decreased MCH-R1 aﬃn-

ity (IC₅₀value of 45 nM). Replacement of a bromine

atom with hydrogen in the benzene ring demonstrated

the same trend as that observed in the 4-dimethylami-

no-quinazoline series, with compound 1f demonstrating

potent MCH-R1 antagonist activity (IC₅₀value of

7.1 nM). One carbon homologation of compound 1f

provided compound 1h, which displayed signiﬁcantly re-

duced aﬃnity for the MCH-R1 (IC₅₀value of 79 nM) as

shown in the comparisons, compounds 1a versus 1c,

compounds 1b versus 1d, and compounds 1e versus 1g.

4. Conclusion

The optimization of the distance between two key phar-

macophore features within our ﬁrst hit compounds 1a

and 2a led to the identiﬁcation of a new class of potent

non-peptidic antagonists for the MCH-R1, based around

4-amino-2-cyclohexylaminoquinazolines. In particular,

ATC0065 (2c), N²-[cis-4-({2-[4-Bromo-2-(triﬂuorometh-

oxy)phenyl]ethyl}amino)cyclohexyl]-N⁴,N⁴-dimethylqui-

nazoline-2,4-diamine dihydrochloride, bound with high

aﬃnity to the MCH-R1 (IC₅₀value of 16 nM) and showed

good metabolic stability in liver microsomes from human

and rat. Additionally, ATC0065 was shown to exhibit

anxiolytic and antidepressant eﬀects in various animal

models of anxiety and depression.³⁰

We then examined the eﬀects of changes in the distance

between the two extremes of the pharmacophore among

cis-1,4-cyclohexyldiamine derivatives. The aniline com-

pound 2b was essentially devoid of antagonist activity

for the MCH-R1 (IC₅₀value of 520 nM), but lengthen-

ing of the molecule by incorporating methylene groups

5. Experimental

5.1. Chemistry

All reagents and solvents were used as purchased from

commercial sources. Moisture-sensitive reactions were

3312

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

carried out under a nitrogen atmosphere. The progress

of the reactions was followed by thin-layer chromatog-

raphy (TLC) analysis (Merck, 0.2 mm silica gel 60 F₂₅₄

on glass plates or Fuji Silysia Chemical, NH-silica gel

on glass plates). Flash column chromatography and

medium-pressure liquid column chromatography were

carried out using silica gel Wako Pure Chemical C-200

or NH-silica gel Fuji Silysia chromatorex DM1020.

Melting points were measured on a Yanaco MP-500D

melting point apparatus and are uncorrected. All com-

pounds were characterized by mass spectra (MS) and

proton nuclear magnetic resonance (¹H NMR). MS

were obtained on a Shimadzu Proﬁle (EI and CI), a

JEOL JMS-SX102 (FAB), a Micromass Platform LC

5.4. 2-Chloro-N-methylquinazolin-4-amine (5B)

A solution of 4 (125 g, 628 mmol) in THF (1 L) was

cooled to 4 ꢁC and 40% aqueous MeNH₂(136 mL,

1.57 mol) was added. The mixture was stirred at room

temperature for 40 min and concentrated. Saturated

aqueous NaHCO₃was poured into the residue and the

aqueous layer was extracted with CHCl₃three times.

The combined organic layer was dried over MgSO₄, ﬁl-

tered, and concentrated. The precipitate was collected

by ﬁltration, washed with H₂O and hexane, and dried

at 80 ꢁC to give 5B (114 g, 94%) as a white solid: H

NMR (300 MHz, CDCl₃, d): 3.22 (d, J = 4.8 Hz, 3H),

6.35 (br s, 1H), 7.39–7.50 (m, 1H), 7.64–7.78 (m, 3H);

ESI MS m/z 194 (M⁺+1, 100%).

1

(ESI), or a Micromass Q-ToF2 (ESI). H NMR spectra

were recorded on a Varian Instruments Gemini 2000

(200 MHz) or a Varian Instruments INOVA 300

(300 MHz). Chemical shifts are reported in parts per

million (ppm) relative to tetramethylsilane (TMS) as

an internal standard. Coupling constants (J) are given

in hertz (Hz) and multiplicities are given as s (singlet),

d (doublet), t (triplet), q (quartet), br s (broad), or m

(multiplet). In vitro active compounds were further

characterized by elemental analyses, which were per-

formed on a Perkin-Elmer 2400 (carbon, hydrogen,

and nitrogen) and Dionex DX-500 (ﬂuorine, chlorine,

bromine, and sulfur). The results were within 0.4% of

theoretical values.

5.5. tert-Butyl [cis-4-(hydroxymethyl)cyclohexyl]carbamate

(7)

A suspension of cis-4-aminocyclohexanecarboxylic acid

6 (244 g, 1.71 mol) in MeOH (2.45 L) was cooled to

ꢀ8 ꢁC. Thionyl chloride (440 mL, 6.03 mol) was then

added dropwise. The resulting solution was stirred at

room temperature for 4.5 h and concentrated to give a

white solid. To a suspension of the above solid in CHCl₃

(3.00 L) were added triethylamine (261 mL, 1.88 mol)

and (BOC)₂O (409 g, 1.88 mol), successively. The mix-

ture was stirred at room temperature for 5 h and poured

into water. The aqueous layer was extracted with CHCl₃

three times. The combined organic layer was dried over

MgSO₄, ﬁltered, concentrated, and puriﬁed by ﬂash col-

umn chromatography (silica gel, 11% EtOAc in hexane

to 10% MeOH in CHCl₃and NH-silica gel, 33% EtOAc

in hexane to 10% MeOH in CHCl₃) to give a colorless

oil (531 g). After a suspension of lithium aluminum hy-

dride (78.3 g, 2.06 mol) in Et₂O (7.9 L) was cooled to

ꢀ4 ꢁC, a solution of the above oil (531 g) in Et₂O

(5.3 L) was added below 0 ꢁC. The resulting suspension

was stirred at room temperature for 2 h. The reaction

mixture was cooled on an ice bath, quenched with cold

water, and ﬁltered through a pad of Celite. The ﬁltrate

was dried over MgSO₄, ﬁltered, and concentrated. A

suspension of the precipitate in hexane (300 mL) was

stirred at room temperature for 1 h, ﬁltered, washed

with hexane, and dried at 70 ꢁC to give 7 (301 g, 77%)

as a white solid: ¹H NMR (300 MHz, CDCl₃, d):

4.30–4.82 (m, 1H), 3.75 (br s, 1H), 3.51 (d, J = 6.2 Hz,

2H), 1.52–1.77 (m, 8H), 1.45 (s, 9H), 1.16–1.36 (m,

2H); ESI MS m/z 252 (M⁺+23, 100%).

5.2. 2,4-Dichloroquinazoline (4)

To a suspension of quinazoline-2,4(1H,3H)-dione 3

(150 g, 925 mmol) in POCl₃(549 mL, 5.89 mol) was

added N,N-dimethylaniline (123 mL, 962 mmol). The

mixture was stirred at reﬂux for 7 h and concentrated.

The solution was then poured into ice water and the

aqueous layer was extracted with CHCl₃three times.

The combined organic layer was dried over MgSO₄, ﬁl-

tered, concentrated, and puriﬁed by ﬂash column chro-

matography (silica gel, 50% CHCl₃in hexane to 10%

EtOAc in CHCl₃) to give 4 (159 g, 86%) as a pale yellow

1

solid: H NMR (300 MHz, CDCl₃, d): 8.27 (dt, J = 8.3,

1.1 Hz, 1H), 7.95–8.04 (m, 2H), 7.71–7.81 (m, 1H); CI

MS m/z 199 (M⁺+1, 100%).

5.3. 2-Chloro-N,N-dimethylquinazolin-4-amine (5A)

A solution of 4 (102 g, 530 mmol) in THF (1.2 L) was

cooled to 4 ꢁC and aqueous 50% Me₂NH (139 mL,

1.33 mol) was added. The mixture was stirred at room

temperature for 80 min and concentrated. Saturated

aqueous NaHCO₃was poured into the residue and

the aqueous layer was extracted with CHCl₃three

times. The combined organic layer was dried over

MgSO₄, ﬁltered, and concentrated. The residue was sus-

pended in 50% Et₂O in hexane (250 mL) and stirred at

room temperature for 30 min. The precipitate was col-

lected by ﬁltration, washed with 50% Et₂O in hexane,

and dried at 80 ꢁC to give 5A (104 g, 94%) as a pale yel-

low solid: ¹H NMR (300 MHz, CDCl₃, d): 8.00 (d,

J = 8.4 Hz, 1H), 7.65–7.78 (m, 2H), 7.38 (ddd, J = 8.4,

6.9, 1.4 Hz, 1H), 3.41 (s, 6H); ESI MS m/z 207 (M⁺,

100%).

5.6. Benzyl ({cis-4-[(tert-butoxycarbonyl)amino]cyclohex-

yl}methyl)carbamate (7–1)

To a solution of 7 (17.7 g, 77.2 mmol) in THF (245 mL)

were added triphenylphosphine (20.2 g, 77.0 mmol) and

phthalimide (11.4 g, 77.5 mmol), successively. The

resulting suspension was cooled to 4 ꢁC and 40% diethyl

azodicarboxylate (DEAD) in toluene was added over

1 h. The mixture was stirred at room temperature for

2.5 days, concentrated, and puriﬁed by ﬂash column

chromatography (silica gel, 33% EtOAc in hexane) to

give a white solid (27.5 g). To a suspension of the above

solid in EtOH (275 mL) was added hydrazine hydrate

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

3313

(5.76 g, 115 mmol). The mixture was stirred at reﬂux for

2.25 h and concentrated. The precipitate was dissolved

in aqueous 10% sodium hydroxide (350 mL). The aque-

ous layer was extracted with CHCl₃three times. The

combined organic layer was dried over MgSO₄, ﬁltered,

and concentrated. To a solution of the residue in CHCl₃

(275 mL) was added triethylamine (8.54 g, 84.4 mmol).

The resulting solution was cooled to 0 ꢁC and CbzCl

(14.4 g, 84.4 mmol) was added below 5 ꢁC. The mixture

was stirred at room temperature for 16 h and poured

into aqueous saturated NaHCO₃. The aqueous layer

was extracted with CHCl₃three times. The combined

organic layer was dried over MgSO₄, ﬁltered, concen-

trated, and puriﬁed by ﬂash column chromatography

(silica gel, 2% MeOH in CHCl₃) to give 7–1 (25.3 g,

5.61–5.70 (m, 1H), 7.00–7.10 (m, 1H), 7.25–7.55 (m,

8H); ESI MS m/z 420 (M⁺+1, 100%).

5.10. N²-[cis-4-({[4-Bromo-2-(triﬂuoromethoxy)ben-

zyl]amino}methyl)cyclohexyl]-N⁴,N⁴-dimethylquinazo-

line-2,4-diamine dihydrochloride (1a)

To a suspension of 9A (4.57 g, 10.5 mmol) in MeOH

(46 mL) was added 5% Pd–C (460 mg). The mixture was

stirred at 50 ꢁC under a hydrogen atmosphere for 2.5

days. The reaction mixture was ﬁltered through a pad of

Celite and concentrated to give a colorless foam. To a

solution of the above foam (500 mg, 1.67 mmol) in

CH₂Cl₂(5 mL) were added 12 (449 mg, 1.67 mmol),

AcOH (100 mg, 1.67 mmol), and NaBH(OAc)₃(531 mg,

2.51 mmol), successively. The mixture was stirred at room

temperature for 9 h and poured into aqueous saturated

NaHCO₃. The aqueous layer was extracted with CHCl₃

three times. The combined organic layer was dried over

MgSO₄, ﬁltered, concentrated, and puriﬁed by medium-

pressure liquid column chromatography (NH-silica gel,

25% EtOAc in hexane) to give a colorless oil (371 mg).

After 4 M hydrogen chloride in EtOAc (5 mL) was added

to a solution of the above oil (227 mg) in EtOAc (1 mL),

the mixture was stirred at room temperature for 2 h.

The precipitate was collected by ﬁltration, washed with

Et₂O, and dried under reduced pressure to give 1a

1

91%) as a colorless oil: H NMR (300 MHz, CDCl₃,

d): 7.27–7.38 (m, 5H), 5.09 (s, 2H), 4.76–4.92 (m, 1H),

4.42–4.76 (m, 1H), 3.72 (br s, 1H), 3.10 (t, J = 6.4 Hz,

2H), 1.48–1.75 (m, 7H), 1.44 (s, 9H), 1.13–1.31 (m,

2H); ESI MS m/z 385 (M⁺+23, 100%).

5.7. Benzyl [(cis-4-aminocyclohexyl)methyl]carbamate (8)

To a solution of 7–1 (12.9 g, 35.6 mmol) in EtOAc

(129 mL) was added 4 M hydrogen chloride in EtOAc

(129 mL). The mixture was stirred at room temperature

for 3 h. The precipitate was collected by ﬁltration,

washed with EtOAc, and dried under reduced pressure.

The solid was dissolved in aqueous saturated NaHCO₃

and the aqueous layer was extracted with CHCl₃ﬁve

times. The combined organic layer was dried over

MgSO₄, ﬁltered, concentrated, and dried under reduced

1

(147 mg, 34%) as a white solid: mp 244.5–247.0 ꢁC; H

NMR (300 MHz, CDCl₃, d): 12.62 (s, 1H), 10.07 (br s,

2H), 8.66 (d, J = 7.6 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H),

7.90 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.52

(dd, J = 8.3, 1.8 Hz, 1H), 7.33–7.48 (m, 2H), 7.26 (t,

J = 7.5 Hz, 1H), 4.11–4.36 (m, 3H), 3.51 (s, 6H),

2.76–2.97 (m, 2H), 1.51–2.28 (m, 10H); ESI MS m/z 552

[M(free)⁺+1, 95%], 554 [M(free)⁺+3, 100%]; Anal. Calcd

for C₂₅H₂₉BrF₃N₅O Æ 2HCl: C, 48.02; H, 5.00; N, 11.20;

Br, 12.78; Cl, 11.34; F, 9.11. Found: C, 47.90; H, 5.03;

N, 10.99; Br, 12.59; Cl, 11.28; F, 9.12.

1

pressure to give 8 (8.88 g, 95%) as a colorless oil: H

NMR (300 MHz, CDCl₃, d): 7.36 (s, 5H), 5.12 (br s,

3H), 2.96–3.32 (m, 3H), 1.36–1.98 (m, 9H); ESI MS m/

z 263 (M⁺+1, 100%).

5.8. Benzyl [(cis-4-{[4-(dimethylamino)quinazolin-2-

yl]amino}cyclohexyl)methyl]carbamate (9A)

5.11. N⁴,N⁴-Dimethyl-N²-[cis-4-({[2-(triﬂuorometh-

oxy)benzyl]amino}methyl)cyclohexyl]quinazoline-2,4-di-

amine dihydrochloride (1b)

A mixture of 5A (2.02 g, 9.73 mmol) and 8 (3.07 g,

11.7 mmol) in isopropanol (10 mL) was stirred at reﬂux

for 7 days. The reaction mixture was poured into aque-

ous saturated NaHCO₃and the aqueous layer was

extracted with CHCl₃three times. The combined organ-

ic layer was dried over MgSO₄, ﬁltered, concentrated,

and puriﬁed by ﬂash column chromatography (NH-sili-

ca gel, 33% EtOAc in hexane) to give 9A (3.55 g, 84%) as

The title compound was prepared from 9A and 2-(triﬂu-

oromethoxy)benzaldehyde according to the experimen-

tal procedure for 1a to provide a white solid (36%

yield): mp 255.0–259.5 ꢁC; ¹H NMR (300 MHz, CDCl₃,

d): 1.41–2.28 (m, 9H), 2.70–2.94 (m, 2H), 3.50 (s, 6H),

4.15–4.30 (m, 1H), 4.31 (br s, 2H), 7.18–7.51 (m, 5H),

7.63 (t, J = 7.6 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H),

8.16–8.28 (m, 1H), 8.69 (d, J = 7.5 Hz, 1H), 9.81–10.22

(m, 2H), 12.81 (s, 1H); ESI MS m/z 474 [M(free)⁺+1,

100%]; Anal. Calcd for C₂₅H₃₀F₃N₅O Æ 2HCl: C, 54.95;

H, 5.90; N, 12.82; Cl, 12.98; F, 10.43. Found: C,

54.63; H, 5.90; N, 12.62; Cl, 12.81; F, 10.31.

1

a colorless oil: H NMR (300 MHz, CDCl₃, d): 7.81 (d,

J = 9.0 Hz, 1H), 7.26–7.52 (m, 7H), 7.01 (ddd, J = 8.2,

6.5, 1.7 Hz, 1H), 5.10 (s, 2H), 4.93–5.06 (m, 1H),

4.82–4.93 (m, 1H), 4.18–4.28 (m, 1H), 3.26 (s, 6H), 3.11

(t, J = 6.3 Hz, 2H), 1.80–1.93 (m, 2H), 1.52–1.73 (m,

5H), 1.23–1.40 (m, 2H); ESI MS m/z 434 (M⁺+1, 100%).

5.9. Benzyl [(cis-4-{[4-(methylamino)quinazolin-2-yl]ami-

no}cyclohexyl)methyl]carbamate (9B)

5.12. N²-{cis-4-[({2-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]ethyl}amino)methyl]cyclohexyl}-N⁴,N⁴-dimethylqui-

nazoline-2,4-diamine dihydrochloride (1c)

The title compound was prepared from 5B and 8 accord-

ing to the experimental procedure for 9A to provide a

white solid (80% yield): H NMR (300 MHz, CDCl₃,

1

The title compound was prepared from 9A and 13 accord-

ing to the experimental procedure for 1a to provide a

d): 1.21–1.96 (m, 9H), 3.01–3.16 (m, 5H), 4.22–4.33

(m, 1H), 4.81–4.91 (m, 1H), 4.99–5.20 (m, 3H),

1

white solid (12% yield): mp 145.5–148.5 ꢁC; H NMR

3314

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

(300 MHz, CDCl₃, d): 1.56–2.10 (m, 9H), 2.92–3.30 (m,

4H), 3.32–3.62 (m, 8H), 4.30 (br s, 1H), 7.17–7.52 (m,

5H), 7.66 (t, J = 7.6 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H),

8.70 (d, J = 7.6 Hz, 1H), 9.06–9.82 (m, 2H), 12.45 (s,

1H); ESI HRMS m/z 566.1763 [M(free)⁺+1], calcd for

C₂₆H₃₂BrF₃N₅O 566.1742.

50 ꢁC under a hydrogen atmosphere for 14 h. The

reaction mixture was ﬁltered through a pad of Celite

and concentrated to give a colorless foam. To a solution

of the above foam (300 mg, 1.05 mmol) in MeOH

(3 mL) were added 12 (283 mg, 1.05 mmol), AcOH

(63 mg, 1.05 mmol), and NaBH₃CN (99 mg, 1.58 mmol),

successively. The mixture was stirred at room tempera-

ture for 3.5 h and poured into aqueous 1 M sodium

hydroxide. The aqueous layer was extracted with

CHCl₃three times. The combined organic layer was

dried over MgSO₄, ﬁltered, concentrated, and puriﬁed

by medium-pressure liquid column chromatography

(NH-silica gel, 50% EtOAc in hexane; silica gel, 10%

MeOH in CHCl₃) to give a colorless oil. After 4 M

hydrogen chloride in EtOAc (5 mL) was added to a

solution of the above oil in EtOAc (1 mL), the mixture

was stirred at room temperature for 2 h. The precipitate

was collected by ﬁltration, washed with Et₂O, and

dried under reduced pressure to give 1e (39 mg, 6%) as a

5.13. N⁴,N⁴-Dimethyl-N²-{cis-4-[({2-[2-(triﬂuorometh-

oxy)phenyl]ethyl}amino)methyl]cyclohexyl}quinazoline-

2,4-diamine dihydrochloride (1d)

To a solution of [2-(triﬂuoromethoxy)phenyl]acetic acid

(350 mg, 1.59 mmol) in CH₂Cl₂(6 mL) were added

DMF (1.0 lL, 15 lmol) and thionyl chloride (165 lL,

2.26 mmol). The mixture was stirred at reﬂux for 50 min

and concentrated to give an acid chloride as a pale yellow

oil. To a solution of N²-[cis-4-(aminomethyl)cyclohexyl]-

N⁴,N⁴-dimethylquinazoline-2,4-diamine (453 mg, 1.51

mmol) obtained by the same method as compound 1a in

CH₂Cl₂(4 mL) was added iPr₂NEt (553 lL, 3.17 mmol).

After cooling to 4 ꢁC, the above acid chloride in CH₂Cl₂

(4 mL) was added and the mixture was stirred at 4 ꢁC

for 5 h and quenched with saturated aqueous NaHCO₃.

The aqueous layer was extracted with CHCl₃three times.

The combined organic layer was dried over MgSO₄,

ﬁltered, concentrated, and puriﬁed by ﬂash column chro-

matography (NH-silica gel, 33–66% EtOAc in hexane) to

give a colorless oil (396 mg). To a solution of the above oil

(246 mg) in THF (3.5 mL) was added 1 M BH₃-THF in

THF (3.95 mL, 3.95 mmol) and the mixture was stirred

at reﬂux for 2.5 h and concentrated. To a solution of the

residue in THF (3.5 mL) was added aqueous 1 M hydro-

chloric acid (4.41 mL, 4.41 mmol) and the mixture was

stirred at reﬂux for 70 min and quenched with aqueous

2 M sodium hydroxide. The aqueous layer was extracted

with CHCl₃three times. The combined organic layer was

dried over MgSO₄, ﬁltered, concentrated, and puriﬁed by

medium-pressure liquid column chromatography (NH-

silica gel, 50% EtOAc in hexane) to give a colorless oil.

After 4 M hydrogen chloride in EtOAc (1.3 mL) was add-

ed to a solution of the above oil in EtOAc (6 mL), the mix-

ture was stirred at room temperature for 1 h and

concentrated. A suspension of the above oil in Et₂O

(15 mL) was stirred at room temperature for 1 h. The pre-

cipitate was collected by ﬁltration, washed with Et₂O, and

dried under reduced pressure to give 1d (81 mg, 30%) as a

1

white solid: mp 237.0–240.5 ꢁC; H NMR (500 MHz,

CDCl₃, d): 1.38–1.84 (m, 8H), 1.89–2.00 (m, 1H),

2.88–3.05 (m, 5H), 4.19–4.30 (m, 3H), 7.07 (d,

J = 7.9 Hz, 1H), 7.14 (t, J = 7.3 Hz, 1H), 7.44–7.52 (m,

2H), 7.55 (dd, J = 8.6, 1.8 Hz, 1H), 8.08 (d, J = 8.5 Hz,

1H), 8.29 (d, J = 8.5 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H),

9.46 (br s, 1H), 9.75 (br s, 2H), 11.23 (br s, 1H); ESI MS

m/z 538 [M(free)⁺+1, 95%], 540 [M(free)⁺+3, 100%];

Anal. Calcd for C₂₄H₂₇BrF₃N₅O Æ 2HCl Æ 0.5H₂O: C,

46.47; H, 4.87; N, 11.29; Br, 12.88; Cl, 11.43; F, 9.19.

Found: C, 46.48; H, 4.68; N, 11.18; Br, 12.76; Cl, 11.58;

F, 9.19.

5.15. N⁴-Methyl-N²-[cis-4-({[2-(triﬂuoromethoxy)ben-

zyl]amino}methyl)cyclohexyl]quinazoline-2,4-diamine

dihydrochloride (1f)

The title compound was prepared from 9B and 2-(tri-

ﬂuoromethoxy)benzaldehyde according to the experi-

mental procedure for 1e to provide a white solid

1

(33% yield): mp 160.0–164.0 ꢁC; H NMR (300 MHz,

CDCl₃, d): 1.41–2.19 (m, 9H), 2.92 (d, J = 4.8 Hz,

2H), 2.99 (d, J = 4.5 Hz, 3H), 4.18–4.38 (m, 3H),

6.95–7.16 (m, 2H), 7.24–7.51 (m, 4H), 8.13 (dd,

J = 7.5, 1.8 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.43 (d,

J = 8.4 Hz, 1H), 9.22–10.0 (m, 2H), 11.49 (br s, 1H);

ESI MS m/z 460 [M(free)⁺+1, 100%]; Anal. Calcd for

C₂₄H₂₈F₃N₅O Æ 2HCl Æ 1.2H₂O: C, 52.03; H, 5.89; N,

12.64; Cl, 12.80; F, 10.29. Found: C, 51.94; H, 5.75;

N, 12.60; Cl, 13.05; F, 10.30.

1

white solid: mp 231.5–235.5 ꢁC; H NMR (300 MHz,

CDCl₃, d): 1.50–2.01 (m, 8H), 2.10–2.20 (m, 1H),

2.92–3.07 (m, 2H), 3.11–3.30 (m, 2H), 3.38–3.60 (m,

8H), 4.22–4.35 (m, 1H), 7.15–7.32 (m, 4H), 7.42–7.54

(m, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.90 (d, J = 8.2 Hz,

1H), 8.72 (d, J = 7.7 Hz, 1H), 9.72 (br s, 2H), 12.56 (s,

1H); FAB MS m/z 488 [M(free)⁺+1, 100%]; Anal Calcd

for C₂₆H₃₂F₃N₅O Æ 2HCl Æ 1.5H₂O: C, 53.15; H, 6.35; N,

11.92; Cl, 12.07; F, 9.70. Found: C, 53.17; H, 6.20; N,

11.99; Cl, 11.99; F, 9.76.

5.16. N²-{cis-4-[({2-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]ethyl}amino)methyl]cyclohexyl}-N⁴-methylquinazoline-

2,4-diamine dihydrochloride (1g)

The title compound was prepared from 9B and 13

according to the experimental procedure for 1e to

provide a white solid (10% yield): mp 170–175.5 ꢁC;

¹H NMR (300 MHz, CDCl₃, d): 1.30–2.24 (m, 9H),

2.75–3.31 (m, 7H), 3.32–3.51 (m, 2H), 4.26 (br s,

1H), 6.96–7.58 (m, 6H), 8.05–8.50 (m, 2H),

9.22–9.72 (m, 2H), 11.51–11.72 (m, 1H); ESI HRMS

m/z 552.1605 [M(free)⁺+1], calcd for C₂₅H₃₀BrF₃N₅O

552.1586.

5.14. N²-[cis-4-({[4-Bromo-2-(triﬂuoromethoxy)benzyl]ami-

no}methyl)cyclohexyl]-N⁴-methylquinazoline-2,4-diamine

dihydrochloride (1e)

To a solution of 9B (2.73 g, 6.50 mmol) in MeOH (27 mL)

was added 10% Pd–C (273 mg). The mixture was stirred at

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

3315

5.17. N⁴-Methyl-N²-{cis-4-[({2-[2-(triﬂuorometh-

oxy)phenyl]ethyl}amino)methyl]cyclohexyl}quinazoline-

2,4-diamine dihydrochloride (1h)

The combined organic layer was dried over MgSO₄,

ﬁltered, concentrated, and puriﬁed by medium-pres-

sure liquid column chromatography (NH-silica gel,

1–9% MeOH in CHCl₃) to give N²-(cis-4-amin-

ocyclohexyl)-N⁴,N⁴-dimethylquinazoline-2,4-diamine

(11.0 g, 92%) as a pale yellow solid. To a solution of

the above solid (400 mg, 1.40 mmol) in CH₂Cl₂

(4 mL) were added 12 (377 mg, 1.40 mmol), AcOH

(84 mg, 1.40 mmol), and NaBH(OAc)₃(446 mg,

2.10 mmol), successively. The mixture was stirred at

room temperature for 5 h and poured into aqueous

saturated NaHCO₃. The aqueous layer was extracted

with CHCl₃three times. The combined organic layer

was dried over MgSO₄, ﬁltered, concentrated, and

puriﬁed by medium-pressure liquid column chroma-

tography (NH-silica gel, 50% EtOAc in hexane) to

give 2a (416 mg, 55%) as a white solid: mp 63.5–

65.0 ꢁC; ¹H NMR (200 MHz, CDCl₃, d): 7.80 (dd,

J = 7.9, 0.9 Hz, 1H), 7.36–7.51 (m, 5H), 7.01 (ddd,

J = 8.3, 6.4, 1.9 Hz, 1H), 4.95–5.18 (m, 1H), 4.08–

4.22 (m, 1H), 3.81 (s, 2H), 3.25 (s, 6H), 2.55–2.70

(m, 1H), 1.65–1.90 (m, 6H), 1.29–1.65 (m, 2H); ESI

MS m/z 538 (M⁺+1, 30%), 560 (M⁺+23, 100%); Anal.

Calcd for C₂₄H₂₇BrF₃N₅O Æ 0.2H₂O; C, 53.18; H, 5.10;

N, 12.92; Br, 14.74; F, 10.52. Found: C, 52.92; H,

5.02; N, 12.56; Br, 15.03; F, 10.83.

To a solution of 1g (290 mg, 0.525 mmol) in EtOH

(5.8 mL) was added 10% Pd–C (87 mg). The mixture

was stirred at room temperature under a hydrogen

atmosphere for 18.5 h. The reaction mixture was ﬁltered

through a pad of Celite, concentrated, and puriﬁed by

medium-pressure liquid column chromatography (NH-

silica gel, 50% EtOAc in hexane to EtOAc) to give a col-

orless oil. After 4 M hydrogen chloride in EtOAc

(230 lL) was added to a solution of the above oil in

EtOAc (8 mL), the mixture was stirred at room temper-

ature for 2 h and concentrated. A suspension of the

above oil in Et₂O (10 mL) was stirred at room tempera-

ture for 2 h. The precipitate was collected by ﬁltration,

washed with Et₂O, and dried under reduced pressure

to give 1h (156 mg, 55%) as a white solid: mp 192.0–

195.5 ꢁC; ¹H NMR (200 MHz, CDCl₃, d): 1.37–2.34

(m, 9H), 2.83–3.60 (m, 9H), 4.26 (br s, 1H), 6.84–7.60

(m, 7H), 8.20–8.66 (m, 2H), 9.23–9.94 (m, 3H), 11.72

(br s, 1H); ESI MS m/z 474 [M(free)⁺+1, 100%]; Anal.

Calcd for C₂₅H₃₀F₃N₅O Æ 1.9HCl Æ 1.5H₂O: C, 52.69;

H, 6.17; N, 12.29; Cl, 11.82; F, 10.00. Found: C,

52.97; H, 5.95; N, 11.93; Cl, 11.99; F, 10.36.

5.18. tert-Butyl (cis-4-{[4-(dimethylamino)quinazolin-2-

yl]amino}cyclohexyl)carbamate (10A)

5.21. N²-(cis-4-{[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]amino}cyclohexyl)-N⁴,N⁴-dimethylquinazoline-2,4-di-

amine dihydrochloride (2b)

The title compound was prepared from 5A and tert-bu-

tyl (cis-4-aminocyclohexyl)carbamate according to the

experimental procedure for 9A to provide a pale yellow

A mixture of 18-C-6 (647 mg, 2.45 mmol), 4-bromo-1-

iodo-2-(triﬂuoromethoxy)benzene (770 mg, 2.10 mmol),

and N²-(cis-4-aminocyclohexyl)-N⁴,N⁴-dimethylquinaz-

oline-2,4-diamine (500 mg, 1.75 mmol) obtained by

the same method as compound 2a, sodium tert-butoxide

(235 mg, 2.45 mmol), tris(dibenzylideneacetone)dipalladi-

um (160 mg, 0.175 mmol), and (R)-(+)-2,2⁰-bis(diph-

enylphosphino)-1,1⁰-binaphthyl (109 mg, 0.175 mmol)

in THF (3.5 mL) was stirred at reﬂux for 18.5 h. After

cooling, Et₂O was added and the mixture was stirred

at room temperature for 30 min, ﬁltered through a

pad of Celite, washed with CHCl₃, concentrated, and

puriﬁed by medium-pressure liquid column chroma-

tography (NH-silica gel, 20–33% EtOAc in hexane; sil-

ica gel, 1–17% MeOH in CHCl₃) to give a colorless

oil. After 4 M hydrogen chloride in EtOAc (300 lL)

was added to a solution of the above oil in Et₂O

(2 mL), the mixture was stirred at room temperature

for 15 min. The precipitate was collected by ﬁltration,

washed with Et₂O, and dried under reduced pressure

to give 2b (189 mg, 18%) as a white solid: mp

254.0–258.5 ꢁC; ¹H NMR (300 MHz, CDCl₃, d):

1.64–2.08 (m, 8H), 3.30–3.78 (m, 8H), 4.20–4.38 (m,

1H), 6.65 (s, 1H), 6.79 (s, 1H), 6.94 (s, 1H), 7.22–

7.31 (m, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.61–7.70 (m,

1H), 7.90 (d, J = 8.1 Hz, 1H), 8.85 (d, J = 7.9 Hz,

1H), 13.04 (s, 1H); ESI MS m/z 524 [M(free)⁺+1,

95%], 526 [M(free)⁺+3, 100%]; Anal. Calcd for

C₂₃H₂₅BrF₃N₅O Æ 1.4HCl Æ H₂O Æ 0.3Et₂O; C, 47.21; H,

5.14; N, 11.38; Br, 12.98; Cl, 8.06; F, 9.26. Found:

C, 47.51; H, 4.78; N, 11.76; Br, 12.63; Cl, 7.92; F,

9.38.

1

foam (93% yield): H NMR (300 MHz, CDCl₃, d): 7.81

(d, J = 8.4 Hz, 1H), 7.25–7.50 (m, 2H), 6.98–7.05 (m,

1H), 4.96–5.04 (m, 1H), 4.50–4.65 (m, 1H), 4.05–4.16

(m, 1H), 3.55–3.72 (m, 1H), 3.26 (s, 6H), 1.48–1.88 (m,

8H), 1.45 (s, 9H); ESI MS m/z 386 (M⁺+1, 30%), 408

(M⁺+23, 100%).

5.19. tert-Butyl (cis-4-{[4-(methylamino)quinazolin-2-

yl]amino}cyclohexyl)carbamate (10B)

The title compound was prepared from 5B and tert-

butyl (cis-4-aminocyclohexyl)carbamate according to

the experimental procedure for 9A to provide a white

solid (65% yield): ¹H NMR (300 MHz, CDCl₃, d):

1.46 (s, 9H), 1.50–1.94 (m, 8H), 3.12 (d, J = 4.8 Hz,

3H), 3.54–3.73 (m, 1H), 4.05–4.22 (m, 1H), 4.49–4.63

(m, 1H), 5.01–5.22 (m, 1H), 5.64–5.82 (m, 1H),

7.03–7.10 (m, 1H), 7.39–7.56 (m, 3H); ESI MS m/z

372 (M⁺+1, 100%).

5.20. N²-(cis-4-{[4-Bromo-2-(triﬂuoromethoxy)ben-

zyl]amino}cyclohexyl)-N⁴,N⁴-dimethylquinazoline-2,4-di-

amine (2a)

To a solution of 10A (16.1 g, 41.7 mmol) in EtOAc

(160 mL) was added 4 M hydrogen chloride in EtOAc

(145 mL). The mixture was stirred at room tempera-

ture for 70 min and concentrated. Aqueous 1 M sodi-

um hydroxide was poured into the residue and the

aqueous layer was extracted with CHCl₃three times.

3316

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

5.22. N²-[cis-4-({2-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]ethyl}amino)cyclohexyl]-N⁴,N⁴-dimethylquinazoline-

2,4-diamine dihydrochloride (2c)

column chromatography (NH-silica gel, 1–9% MeOH

in CHCl₃) to give N²-(cis-4-aminocyclohexyl)-N⁴,

N⁴-dimethylquinazoline-2,4-diamine (11.0 g, 92%) as a

pale yellow solid. To a solution of the above solid

(240 mg, 0.841 mmol) in MeOH (3 mL) were added 17

(262 mg, 0.842 mmol), AcOH (51 mg, 0.849 mmol),

and NaBH₃CN (79 mg, 1.26 mmol), successively. The

mixture was stirred at room temperature for 8 h and

poured into aqueous 1 M sodium hydroxide. The aque-

ous layer was extracted with CHCl₃three times. The

combined organic layer was dried over MgSO₄, ﬁltered,

concentrated, and puriﬁed by medium-pressure liquid

column chromatography (NH-silica gel, 50% EtOAc in

hexane) to give a colorless oil. After 4 M hydrogen chlo-

ride in EtOAc (10 mL) was added to a solution of the

above oil in EtOAc (2 mL), the mixture was stirred at

room temperature for 1 h and concentrated. A suspen-

sion of the above oil in Et₂O (20 mL) was stirred at

room temperature for 1 h. The precipitate was collected

by ﬁltration, washed with Et₂O, and dried under re-

duced pressure to give 2f (220 mg, 40%) as a white solid:

mp 121.5–125.5 ꢁC; ¹H NMR (200 MHz, CDCl₃, d):

1.58–2.59 (m, 12H), 2.60–2.78 (m, 2H), 2.92–3.42 (m,

3H), 3.52 (s, 6H), 4.20–4.42 (m, 1H), 7.12–7.40 (m,

3H), 7.48 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.3 Hz, 1H),

7.92 (d, J = 7.9 Hz, 1H), 8.66–8.88 (m, 1H), 9.55 (br s,

2H), 12.73 (br s, 1H); ESI HRMS m/z 580.1911

[M(free)⁺+1], calcd for C₂₇H₃₄BrF₃N₅O 580.1899.

The title compound was prepared from 10A and 13

according to the experimental procedure for 2a to pro-

1

vide a white solid (26% yield): mp 170.0–174.0 ꢁC; H

NMR (300 MHz, CDCl₃, d): 1.62–1.96 (m, 2H), 2.00–

2.48 (m, 6H), 3.20–3.64 (m, 11H), 4.31 (br s, 1H),

7.19–7.77 (m, 6H), 7.93 (d, J = 6.6 Hz, 1H), 8.72 (d,

J = 7.5 Hz, 1H), 9.91 (br s, 2H), 12.66 (br s, 1H); ESI

MS m/z 552 [M(free)⁺+1, 95%], 554 [M(free)⁺+3,

100%]; Anal. Calcd for C₂₅H₂₉BrF₃N₅O Æ 2HCl Æ 1.5-

H₂O; C, 46.03; H, 5.25; N, 10.74; Br, 12.25; Cl, 10.87;

F, 8.74. Found: C, 46.06; H, 5.36; N, 10.58; Br, 12.05;

Cl, 10.87; F, 8.74.

5.23. N⁴,N⁴-Dimethyl-N²-[cis-4-({2-[2-(triﬂuorometh-

oxy)phenyl]ethyl}amino)cyclohexyl]quinazoline-2,4-di-

amine dihydrochloride (2d)

The title compound was prepared from 2c according to

the experimental procedure for 1h to provide a white

solid (48% yield): mp 169.5–173.5 ꢁC; ¹H NMR

(300 MHz, CDCl₃, d): 1.62–1.89 (m, 2H), 1.93–2.53

(m, 6H), 3.15–3.71 (m, 11H), 4.33 (br s, 1H), 7.14–7.37

(m, 4H), 7.39–7.77 (m, 3H), 7.82–8.00 (m, 1H), 8.71

(br s, 1H), 9.78 (br s, 2H), 12.62 (br s, 1H); ESI MS

m/z 474 [M(free)⁺+1, 100%]; Anal. Calcd for

C₂₅H₃₀F₃N₅O Æ 2HCl Æ 1.5H₂O: C, 52.36; H, 6.15; N,

12.21; Cl, 12.36; F, 9.94. Found: C, 52.36; H, 6.13; N,

12.21; Cl, 12.35; F, 9.73.

5.26. N²-(cis-4-{[4-Bromo-2-(triﬂuoromethoxy)ben-

zyl]amino}cyclohexyl)-N⁴-methylquinazoline-2,4-diamine

dihydrochloride (2g)

5.24. N²-[cis-4-({3-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]propyl}amino)cyclohexyl]-N⁴,N⁴-dimethylquinazoline-

2,4-diamine dihydrochloride (2e)

The title compound was prepared from 10B and 12

according to the experimental procedure for 2a to pro-

vide a white solid (20% yield): mp 197.0–202.5 ꢁC; H

1

NMR (300 MHz, CDCl₃, d): 1.41–1.94 (m, 8H), 2.57–

2.72 (m, 1H), 3.12 (d, J = 4.8 Hz, 3H), 3.82 (s, 2H),

4.09–4.27 (m, 1H), 5.52 (br s, 1H), 7.05 (ddd, J = 8.2,

6.8, 1.4 Hz, 1H), 7.34–7.57 (m, 6H); ESI MS m/z 286

[M(free)⁺ꢀ237, 100%], 522 [M(free)⁺+1, 75%], 524

[M(free)⁺+3, 75%]; Anal. Calcd for C₂₃H₂₅BrF₃N₅O Æ

2HCl Æ H₂O; C, 44.90; H, 4.75; N, 11.38; Br, 12.99; Cl,

11.52; F, 9.26. Found: C, 44.71; H, 4.77; N, 11.32; Br,

12.66; Cl, 11.42; F, 9.22.

The title compound was prepared from 10A and 15

according to the experimental procedure for 2a to pro-

vide a white solid (42% yield): mp 129.0–133.5 ꢁC; H

1

NMR (300 MHz, CDCl₃, d): 1.59–1.85 (m, 2H), 1.97–

2.36 (m, 8H), 2.78 (t, J = 7.8 Hz, 2H), 3.02–3.14 (m,

2H), 3.19–3.35 (m, 1H), 3.52 (s, 6H), 4.31 (br s, 1H),

7.12–7.52 (m, 6H), 7.60–7.70 (m, 1H), 7.91 (d,

J = 7.9 Hz, 1H), 8.81 (d, J = 7.2 Hz, 1H); ESI MS m/z

566 [M(free)⁺+1, 95%], 568 [M(free)⁺+3, 100%]; Anal.

Calcd for C₂₆H₃₁BrF₃N₅O Æ 2HCl Æ 1.6H₂O Æ 0.3EtOAc;

C, 47.03; H, 5.60; N, 10.08; Br, 11.50; Cl, 10.21; F,

8.20. Found: C, 47.18; H, 5.51; N, 10.30; Br, 11.11; Cl,

10.10; F, 8.40.

5.27. N²-[cis-4-({2-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]ethyl}amino)cyclohexyl]-N⁴-methylquinazoline-2,4-di-

amine dihydrochloride (2h)

The title compound was prepared from 10B and 13

according to the experimental procedure for 2a to

5.25. N²-[cis-4-({4-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]butyl}amino)cyclohexyl]-N⁴,N⁴-dimethylquinazoline-

2,4-diamine dihydrochloride (2f)

provide

a white solid (40% yield): mp 173.0–

177.0 ꢁC; ¹H NMR (300 MHz, CDCl₃, d): 1.64–2.42

(m, 8H), 2.94 (s, 3H), 3.08–3.33 (m, 3H), 3.35–3.58

(m, 2H), 3.94–4.26 (m, 1H), 7.21–7.45 (m, 5H), 7.60

(t, J = 7.7 Hz, 1H), 7.71–7.94 (m, 1H), 8.62–8.82 (m,

1H), 9.93 (br s, 3H), 12.18 (br s, 1H); ESI MS m/z

538 [M(free)⁺+1, 98%], 540 [M(free)⁺+3, 100%]; Anal.

Calcd for C₂₄H₂₇BrF₃N₅O Æ 1.9HCl Æ 1.5H₂O; C, 45.42;

H, 5.07; N, 11.03; Br, 12.59; Cl, 10.61; F, 8.98.

Found: C, 45.32; H, 4.93; N, 10.64; Br, 12.92; Cl,

10.68; F, 9.37.

To a solution of 10A (16.1 g, 41.7 mmol) in EtOAc

(160 mL) was added 4 M hydrogen chloride in EtOAc

(145 mL). The mixture was stirred at room temperature

for 70 min and concentrated. Aqueous 1 M sodium

hydroxide was poured into the residue and the aqueous

layer was extracted with CHCl₃three times. The com-

bined organic layer was dried over MgSO₄, ﬁltered, con-

centrated, and puriﬁed by medium-pressure liquid

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

3317

5.28. N²-[cis-4-({3-[4-Bromo-2-(triﬂuoromethoxy)phen-

yl]propyl}amino)cyclohexyl]-N⁴-methylquinazoline-2,4-

diamine dihydrochloride (2i)

NaH dispersion in mineral oil (616 mg, 15.4 mmol) un-

der a nitrogen atmosphere and the mixture was stirred

at room temperature for 50 min. After cooling to 4 ꢁC,

a solution of 12 (3.00 g, 11.2 mmol) in THF (100 mL)

was added and the mixture was stirred at room temper-

ature for 11 h and poured into water. The aqueous layer

was extracted with EtOAc three times. The combined

organic layer was dried over MgSO₄, ﬁltered, concen-

trated, and puriﬁed by ﬂash column chromatography

(silica gel, 5% EtOAc in hexane) to give 14 (2.98 g,

The title compound was prepared from 10B and 15

according to the experimental procedure for 2f to pro-

1

vide a white solid (7% yield): mp 169.5–173.0 ꢁC; H

NMR (300 MHz, CDCl₃, d): 1.60–1.98 (m, 4H), 2.09–

2.50 (m, 6H), 2.51–2.89 (m, 5H), 2.94–3.24 (m, 3H),

3.78–3.96 (m, 1H), 7.27–7.43 (m, 5H), 7.55–7.67 (m,

2H), 8.68 (d, J = 8.2 Hz, 1H), 9.59 (br s, 2H), 9.78 (br

s, 1H), 12.37 (s, 1H); ESI MS m/z 552 [M(free)⁺+1,

100%], 554 [M(free)⁺+3, 98%]; Anal. Calcd for

C₂₅H₂₉BrF₃N₅O Æ 2.1HCl Æ 1.4H₂O Æ 0.3Et₂O; C, 46.52;

H, 5.50; N, 10.35; Br, 11.81; Cl, 11.01; F, 8.43. Found:

C, 46.88; H, 5.34; N, 10.71; Br, 11.49; Cl, 10.99; F, 8.67.

1

79%) as a colorless oil: H NMR (200 MHz, CDCl₃,

d): 1.35 (t, J = 7.3 Hz, 3H), 4.29 (q, J = 7.0 Hz, 2H),

6.48 (d, J = 16.3 Hz, 1H), 7.43–7.58 (m, 3H), 7.83 (d,

J = 15.8 Hz, 1H); CI MS m/z 339 (M⁺+1, 100%), 341

(M⁺+3, 98%).

5.32. 3-[4-Bromo-3-(triﬂuoromethoxy)phenyl]propan-1-ol

(15–1)

5.29. 4-Bromo-2-(triﬂuoromethoxy)benzaldehyde (12)

A solution of 4-bromo-1-iodo-2-(triﬂuoromethoxy)ben-

zene (1.00 g, 2.72 mmol) in THF (15 mL) was cooled to

ꢀ78 ꢁC and 2.66 M n-BuLi in hexane (2.05 mL,

5.44 mmol) was added dropwise. After the mixture was

stirred at ꢀ78 ꢁC for 1.5 h, N-formylmorpholine

(648 mg, 5.63 mmol) was added. The mixture was stirred

at ꢀ78 ꢁC for 15 min and at room temperature for 80 min.

The reaction was quenched with aqueous 0.25 M citric

acid (10 mL) and the aqueous layer was extracted with

EtOAc three times. The combined organic layer was dried

over MgSO₄, ﬁltered, concentrated, and puriﬁed by ﬂash

column chromatography (silica gel, 2–5% EtOAc in hex-

To a suspension of LAH (834 mg, 22.0 mmol) in Et₂O

(20 mL) cooled to 4 ꢁC was added a solution of 14

(2.98 g, 8.79 mmol) in Et₂O (9 mL). The mixture was

stirred at 0 ꢁC for 1 h and at room temperature for

2.5 h and quenched with saturated aqueous ammonium

chloride. The aqueous layer was extracted with EtOAc

three times. The combined organic layer was washed

with aqueous 1 M hydrochloric acid, dried over MgSO₄,

ﬁltered, concentrated, and puriﬁed by ﬂash column

chromatography (silica gel, 25% EtOAc in hexane) to

give 15–1 (1.14 g, 43%) as a colorless oil: ¹H NMR

(200 MHz, CDCl₃, d): 1.58 (s, 1H), 1.75–1.96 (m, 2H),

2.65–2.82 (m, 2H), 3.60–3.76 (m, 2H), 7.11–7.44 (m,

3H); EI MS m/z 280 (M⁺-18, 100%), 282 (M⁺-16,

95%), 298 (M⁺, 20%), 300 (M⁺+2, 20%).

1

ane) to give 12 (560 mg, 77%) as a pale brown solid: H

NMR (300 MHz, CDCl₃, d): 10.33 (s, 1H), 7.85 (d,

J = 8.1 Hz, 1H), 7.50–7.67 (m, 2H); CI MS m/z 269

(M⁺+1, 95%), 271 (M⁺+3, 100%).

5.33. 3-[4-Bromo-3-(triﬂuoromethoxy)phenyl]propanal (15)

5.30. [4-Bromo-3-(triﬂuoromethoxy)phenyl]acetaldehyde

(13)

To a suspension of 15–1 (1.03 g, 3.44 mmol) and Celite

(1.40 g) in CH₂Cl₂(47 mL) cooled to 4 ꢁC was added

PCC (1.11 g, 5.16 mmol). The mixture was stirred at

room temperature for 6.5 h, ﬁltered through a pad of

Celite, washed with CHCl₃, concentrated, and puriﬁed

by ﬂash column chromatography (silica gel, 17% EtOAc

in hexane) to give 15 (659 mg, 64%) as a colorless oil: ¹H

NMR (300 MHz, CDCl₃, d): 2.71–2.80 (m, 2H), 2.91–

3.01 (m, 2H), 7.17 (d, J = 8.1 Hz, 1H), 7.33–7.42 (m,

2H), 9.80 (s, 1H); CI MS m/z 297 (M⁺+1, 100%), 299

(M⁺+3, 90%).

To a suspension of (methoxymethyl)triphenylphospho-

nium chloride (31.5 g, 91.9 mmol) in Et₂O (300 mL)

was added 1.8 M PhLi in cyclohexane–ether (7:3)

(51.0 mL, 91.9 mmol) and the mixture was stirred at

room temperature for 10 min. To the mixture was added

12 (23.8 g, 88.4 mmol) in Et₂O (100 mL) and the mixture

was stirred at room temperature for 1 h, ﬁltered, and

concentrated. To the residue was added 10% H₂SO₄

(23.8 mL) in AcOH (214 mL) and the mixture was stir-

red at room temperature for 30 min and quenched with

H₂O. The aqueous layer was extracted with Et₂O three

times. The combined organic layer was washed with sat-

urated aqueous NaHCO₃, dried over MgSO₄, ﬁltered,

concentrated, and puriﬁed by ﬂash column chromatog-

raphy (silica gel, 9% EtOAc in hexane) to give 13 (16.7

g, 67%) as a pale brown oil: ¹H NMR (300 MHz,

CDCl₃, d): 3.76 (s, 2H), 7.11–7.55 (m, 3H), 9.73 (s,

1H); ESI MS m/z 283 (M⁺+1, 100%), 285 (M⁺+3, 65%).

5.34. Ethyl (2E)-4-[4-bromo-3-(triﬂuoromethoxy)phen-

yl]but-2-enoate (16)

The title compound was prepared from 13 according to

the experimental procedure for 14 to provide a colorless

1

oil (26% yield): H NMR (300 MHz, CDCl₃, d): 1.24–

1.33 (m, 3H), 3.28 (dd, J = 7.1, 1.5 Hz, 1H), 4.18 (q,

J = 7.1 Hz, 2H), 6.28–6.46 (m, 2H), 6.65 (d, J = 16.2 Hz,

1H), 7.34–7.51 (m, 3H); ESI MS m/z 353 (M⁺+1, 100%).

5.31. Ethyl (2E)-3-[4-bromo-3-(triﬂuoromethoxy)phen-

yl]acrylate (14)

5.35. 4-[4-Bromo-3-(triﬂuoromethoxy)phenyl]butanal (17)

To a solution of ethyl (diethoxyphosphoryl)acetate

(3.45 g, 15.4 mmol) in THF (230 mL) was added 60%

The title compound was prepared from 16 according to

the experimental procedure for 15 to provide a colorless

3318

K. Kanuma et al. / Bioorg. Med. Chem. 14 (2006) 3307–3319

oil (35% yield): ¹H NMR (200 MHz, CDCl₃, d): 1.83–2.05

(m, 2H), 2.42–2.56 (m, 2H), 2.60–2.78 (m, 2H), 7.07–7.49

(m, 3H), 9.79 (s, 1H); ESI MS m/z 311 (M⁺+1, 10%).

quadrupole mass spectrometer (AB/MPS SCIEX, Thorn-

hill, Ontario, Canada) equipped with an 1100 HPLC sys-

tem (Agilent Technologies, Inc., Mississauga, ON, USA).

The separation was performed with a Zorbax SB-C18 col-

umn (5 lm, 2.1 · 50 mm or 3.5 lm, 2.1 · 15 mm, Agilent)

using linear gradient elution (0.1% aqueous acetic acid to

0.1% acetic acid containing acetonitrile) at a ﬂow rate of

250 lL/min.

6. Assay

6.1. Membrane preparations

Membranes expressing human WT-MCH-R1 were pur-

chased from Euroscreen and diluted in the assay buﬀer

(25 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, 1 lM

phosphoramidon, 0.5 mM PMSF, and 0.2% BSA,

pH7.4) for human WT-MCH-R1 binding assay.

To determine the in vitro T_1/2, the slope of the linear

regression from log concentration versus incubation

time relationships (ꢀk) was used in the conversion to

in vitro T_1/2, values by in vitro T_1/2= ꢀ0.693/k.

6.2. Receptor binding assays for WT-MCH-R1

References and notes

Binding assays were performed in 96-well plates. The

receptor binding reaction was initiated by incubating

0.2 mL of membrane preparations with 0.1 nM

1. Kawauchi, H.; Kawazoe, I.; Tsubokawa, M.; Kishida, M.;

Baker, B. I. Nature 1983, 305, 321–323.

2. Vaughan, J. M.; Fischer, W. H.; Hoeger, C.; Rivier, J.;

Vale, W. Endocrinology 1989, 125, 1660–1665.

[

¹²⁵I](Phe¹³, Tyr¹⁹)MCH (Amersham). Non-speciﬁc

binding was determined using 10 lM T-226926. The reac-

tion mixture was incubated for 2 h at room temperature.

The binding reaction was terminated by rapid ﬁltration

through a GF/C glass ﬁlter plate presoaked with 0.3%

polyethylenimine, followed by washing three times with

0.3 mL of wash buﬀer (PBS containing 0.5 M NaCl).

The radioactivity retained in the ﬁlterplates was quanti-

ﬁed with a Topcount^TMscintillation counter (Packard).

Speciﬁc binding was determined by subtracting non-spe-

ciﬁc binding from the total binding. In the competition

binding assay, the concentration of the test compound

that caused 50% inhibition of speciﬁc radioligand binding

(IC₅₀values) was determined from each concentration–

response curve. Data were analyzed by a non-linear

least-squares curve-ﬁtting procedure using Origin soft-

ware (Microcal).

3. Kolakowski, L. F., Jr.; Jung, B. P.; Nguyen, T.; Johnson,

M. P.; Lynch, K. R.; Cheng, R.; Heng, H. H. Q.; George,

S. R.; O’Dowd, B. F. FEBS Lett. 1996, 398, 253–258.

4. Chambers, J.; Ames, R. S.; Bergsma, D.; Muir, A.;

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Article Doi

Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists

DOI: 10.1016/j.bmc.2005.12.052

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