Discovery of potent, selective, and orally bioavailable quinoline-based dipeptidyl peptidase IV inhibitors targeting Lys554

Bioorganic & Medicinal Chemistry 19 (2011) 4482–4498

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Discovery of potent, selective, and orally bioavailable quinoline-based

dipeptidyl peptidase IV inhibitors targeting Lys554

⇑

,

Hironobu Maezaki ^a,, Yoshihiro Banno ^a,, Yasufumi Miyamoto ^a, Yuusuke Moritou ^a, Tomoko Asakawa ^a,

Osamu Kataoka ^a,, Koji Takeuchi ^a, Nobuhiro Suzuki ^a, Koji Ikedo ^a, Takuo Kosaka ^a, Masako Sasaki ^a,

Shigetoshi Tsubotani ^a, Akiyoshi Tani ^a, Miyuki Funami ^a,à, Yoshio Yamamoto ^a, Michiko Tawada ^a,

Kathleen Aertgeerts ^b, Jason Yano ^b, Satoru Oi ^a,à

^aPharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85, Jusohomachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan

^bTakeda San Diego, Inc., 10410, Science Center Drive, San Diego, CA 92121, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting

multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent

DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal

structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and eval-

uation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpro-

pyl)quinolin-6-yl]piperazine-2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor

(IC₅₀= 1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats.

A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554,

suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition.

Received 11 April 2011

Revised 8 June 2011

Accepted 10 June 2011

Available online 7 July 2011

Keywords:

Dipeptidyl peptidase IV

Quinoline-based inhibitors

Type 2 diabetes

1. Introduction

role in glucose-dependent insulin biosynthesis and secretion from

pancreatic b-cells. Furthermore, GLP-1[7-36]amide exhibits multi-

Type 2 diabetes mellitus (T2DM) is a rapidly increasing meta-

bolic disease in the human population. Prolongation of the patho-

logical conditions may cause serious and often mortal

complications, such as nephropathy, neuropathy, retinopathy and

atherosclerosis. Because T2DM currently affects more than 230

million people worldwide and is projected to affect 366 million

individuals by 2030,^1–3a variety of therapeutic options have been

proposed to treat this disorder. Recently, glucagon-like peptide-1

(GLP-1) therapy has been investigated as a potential option for

T2DM treatment.^4,5GLP-1 is a 30-amino acid peptide hormone se-

creted from the gastrointestinal tract in response to food intake.

The active form of GLP-1 (GLP-1[7-36]amide) plays an important

ple antidiabetic effects in mammals, such as inhibiting of glucagon

secretion, slowing gastric emptying, reducing appetite, and stimu-

lating b-cell regeneration and differentiation. However, the GLP-

1[7-36]amide is rapidly inactivated by dipeptidyl peptidase-IV

(DPP-4, EC 3.4.14.5) and has a half-life of less than 1 min. DPP-4,

a ubiquitously distributed serine protease that cleaves the peptide

bond at the penultimate residue of the amino terminus position,

converts GLP-1[7-36]amide to inactive GLP-1[9-36]amide.^6–8

DPP-4 inhibition prevents rapid GLP-1 inactivation and potentiates

circulating levels of active GLP-1, improving the strict glucose-

dependent secretion of insulin with little or no risk of serious

hypoglycemia. Therefore, DPP-4 inhibitors are optimal therapeutic

options to treat T2DM.^9–13Numerous studies have identiﬁed and

conﬁrmed a number of inhibitors for their clinical antidiabetic ef-

fects.^5–8Although many reported DPP-4 inhibitors, including vil-

dagliptin^14–17and saxagliptin,¹⁸are thought to covalently

interact with the Ser630 residue in the S1 pocket,^19,20sitagliptin²¹

and alogliptin²²are believed to achieve their therapeutic effects

even without a covalent ligand–enzyme interaction.

Abbreviations: DPP-4, dipeptidyl peptidase IV; T2DM, type 2 diabetes mellitus;

GLP-1, glucagon-like peptide 1; SAR, structure–activity relationship; SBDD, struc-

ture-based drug design; Boc, tert-butoxy carbonyl; TBS, tert-butyldimethylsilyl;

Cbz, benzyloxycarbonyl; ADME, absorption, distribution, metabolism and elimina-

tion; F, Bioavailability; CL_total, plasma clearance; V_dss, distribution volume.

⇑

Corresponding author. Tel.: +81 466 32 1106; fax: +81 466 32 4449.

E-mail address: Maezaki_Hironobu@takeda.co.jp (H. Maezaki).

Present address: 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555,

Japan.

Our efforts focus on the design of novel non-covalent DPP-4

inhibitors, extensively using structural information derived from

DPP-4 structures co-crystallized with small molecules originating

from different chemical classes. Notably, we previously identiﬁed

à

Present address: Pharmaceutical Production Division, Takeda Pharmaceutical

Company Limited, 17-85, Jusohomachi 2-chome Yodogawa-ku, Osaka 532-8686,

Japan.

doi:10.1016/j.bmc.2011.06.032

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4483

a structurally novel isoquinolone DPP-4 inhibitor 2 (Fig. 1).^23,24Co-

crystallographic studies of compound 2 revealed a unique binding

mode whereby the inhibitor interacts with the catalytic His740

and Ser630 mediated by water molecules (Fig. 2). This indicates

that compound 2, unlike previously published covalent inhibi-

tors,^{12,18,25–31}can make non-covalent interactions with DPP-4 cat-

alytic residues. Our studies also revealed that compound 2 utilizes

binding sites in three directions. The 2- and 3-substituents of com-

pound 2 mimic the P2-site of known ligands and are located in the

large hydrophobic S2 pocket of the enzyme. The 2-isobutyl group is

substituent only partially ﬁlls the S1 pocket, the bottom of ₀which

lies deeper with the carbon–carbon distances of 3.9–4.4 ÅA than

the meta- or para-carbon of the 4-phenyl ring (Fig. 2B). Therefore,

a meta- or para-substitution onto the 4-phenyl ring would most

likely enhance the afﬁnity to the S1 pocket. Interestingly, the 6-

substituent is positioned in the deep cleft of S1’ pocket. This cleft

is lined by hydrophobic residues Tyr547, Trp629, Gly632 and

Val546, and terminated by hydrophilic Lys554 and Asp545 resi-

dues. The 6-substituent of compound 2 forms not only a hydropho-

bic interaction with Tyr547 but also hydrogen-bonding

interactions with Lys554 and the backbone carbonyl group of

Trp629. The hydrogen-bonding interaction with Lys554 may be

applicable in the novel design of the inhibitors although none have

been reported to date. Therefore, based on the X-ray co-crystal

structure of compound 2, we developed a pharmacophore model

positioned to allow a CH–p interaction with the aromatic ring of

Phe357. The primary amino group at the 3-position interacts with

residues Glu205, Glu206 and Tyr662, the recognition site for the

charged N-terminal end of peptide substrates. The 4-pendant phe-

nyl group extends into the hydrophobic S1 pocket. However, this

2-Cyanopyrrolidines

Non-Covalent Inhibitors

F

O

N

F

HO

Me

NH₂

O

HO

H₂N

N

H

N

O

N

O

N

F

N

O

CF₃

N

NH₂

Alogliptin

Vildagliptin

Saxagliptin

Sitagliptin

Me Me

N

O

N

O

HN

NH₂

2HCl

H₂N

NH₂

N

O

HCl

O

Me

1

2

Figure 1. Structure of reported DPP-4 inhibitors (vildagliptin, saxagliptin, sitagliptin, and alogliptin), compounds 1 and 2.

Figure 2. Compound 2 in complex with human DPP-4. (A) The amino acids (black carbons, blue texts) in the binding sites of compound 2 (green carbons) are indicated. Major

interactions are depicted as dotted lines (hydrophobic, blue; hydrophilic, red). (B) The S1 binding site is indicated in cross-sectional view. Distances between carbon atoms of

the 2-phenyl group and the representative hydrophobic residues lining the S1 pocket are shown as red dotted lines. Values listed are the carbon–carbon distances in

angstroms.

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H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

pocket occupancy for enhanced potency. We focused on the opti-

mization of quinoline derivatives with an alternative scaffold to

the isoquinolone and achieved signiﬁcant improvement of inhibi-

tion, resulting in the discovery of compound 1, dihydrochloride

of 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)

quinolin-6-yl]piperazine-2,5-dione, as a lead compound of a novel

class of DPP-4 inhibitors. Results from a docking study of com-

pound 1 suggest the usefulness of our pharmacophore model

involving the interaction with Lys554.

2. Chemistry

DPP-4 inhibitors discussed in this report were derived from

quinoline-3-carbonitriles synthesized by a modiﬁed Friedlander

reaction (Scheme 1).^32,33Weinreb amide 4a was prepared using

the method described in a previous report.^34,35Anthranilic acid

3b was protected with TBS and Cbz (for OH and NH₂, respectively)

and coupled with N,O-dimethylhydroxylamine followed by depro-

tection of Cbz to yield compound 4b. Amide 4 was reacted with

substituted or unsubstituted aryl magnesium bromides, which

were commercially available or prepared from their corresponding

aryl bromides, to afford 2-aminobenzophenone 5. The methane-

sulfonic acid-catalyzed Friedlander reaction between compound

5 and 3-ketonitrile 6, which was synthesized from ester 10 accord-

ing to a previously described method,³⁶yielded quinoline-3-ni-

triles 7 and 8. Compound 8a was prepared from compound 7a by

palladium-catalyzed etheriﬁcation³⁷and acidic cleavage of the

tert-butyl moiety. The hydroxy group of 6-hydroxyquinoline 8

was alkylated, followed by hydrogenation of the 3-cyano group

using Raney cobalt or Raney nickel as a catalyst to produce 3-ami-

nomethylquinoline 9.

Figure 3. Proposed pharmacophore model based on the co-crystal structure of

compound 2. R^aand R^bare additional substituents to potentiate the afﬁnity.

Expected interactions are indicated as arrows (solid, hydrophobic; dashed,

hydrophilic).

for DPP-4 inhibitor involving its unique interaction with Lys554

(Fig. 3). In this model, the isobutyl group, the amino methyl group,

and the pendant phenyl group make similar interactions to those

of compound 2 with Phe357, the recognition site for the amino

moiety, and the S1 pocket, respectively. The scaffold orients all of

these substituents into the appropriate positions. We hypothesized

that the introduction of R^aand R^bsubstituents into this model

would improve the afﬁnity to the enzyme. R^aon the pendant phe-

nyl would provide a better ﬁt to the bottom of the S1 pocket. R^b

extending toward the hydrophobic cleft would make hydrophobic

interactions with the hydrophobic residues, such as Tyr547 and

Trp629 and make hydrogen-bonding interactions with the back-

bone atoms of the enzyme and the side chain of Lys554, which

was utilized by compound 2.

Catalytic hydrogenation of the 3-cyano group of quinoline-3-

carbonitrile 8e and subsequent Boc protection provided intermediate

11 (Scheme 2). Intermediate 11 was alkylated with 2-bromoacetate

Here, we describe a drug design strategy utilizing structure-

based drug discovery to explore the optimal DPP-4 active site

a (for 4a)

or b (for 4b)

NH₂

R¹= H

c

5a, X = Br,

5b, X = Br,

R¹= 4-Me

O

5c, X = TBSO, R¹= 3-F

5d, X = TBSO, R¹= 3-Me

R¹5e, X = TBSO, R¹= 4-F

5f, X = TBSO, R¹= 4-Me

X

CO₂H

X

Me

N

O

Me

3a, X = Br

3b, X = OH

4a, X = Br

4b, X = TBSO

d

(for 8b-e)

Me

f, g (for 9a, 9c, 9d)

or f, h (for 9b, 9e)

e

N

(for 8a)

H₂N

NH₂

O

HO

CN

R¹

Br

CN

O

R¹

9a-e

8a, 9a, R¹= H

8a-e

R¹

, R¹= H

7b, R¹= Me

7a

8b, 9b, R¹= 3-F,

8c, 9c, R¹= 3-Me

8d, 9d, R¹= 4-F

8e, 9e, R¹= 4-Me

Me

O

Me

j

Me

CN

Me

O

10

6

Scheme 1. Synthesis of compounds 9a–e. Reagents and conditions: (a) (1) bis(trichloromethy1) carbonate, THF, reﬂux, 4 h; (2) N,O-dimethylhydroxylamine hydrochloride,

Et₃N, EtOH, 75 °C, 17 h; (b) (1) Cbz-Cl, NaHCO₃, Et₂O, water, room temperature, 0.5 h; (2) N,O-dimethylhydroxylamine hydrochloride, EDC, Et₃N, DMF, room temperature,

2.5 h; (3) TBSCl, imidazole, DMF, room temperature, 14 h; (4) H₂, Pd/C, EtOH, THF, room temperature, 20 h; (c) R¹-C₆H₄-MgBr, THF, Et₂O, 0 °C–room temperature, 0.5–1 h; (d)

6, MsOH, toluene, reﬂux, 6–60 h, Dean-Stark; (e) (1) tert-BuOH, Pd(OAc)₂, rac-BINAP, tert-BuONa, toluene, 90 °C, 1 h; (2) TFA, THF, room temperature, 1 h; (f) ClCH₂CONH₂,

K₂CO₃, DMF, 60–80 °C, 4–96 h; (g) H₂, Raney-Co, 25% NH₃, MeOH, THF, 70–85 °C, 6–12 h; (h) H₂, Raney-Ni, 25% NH₃, MeOH, THF, room temperature, 7–9 h; (j) NaH, MeCN,

THF, 70 °C, 15 h.

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4485

Me

N

a

b

H

N

H

N

R²

HO

CN

HO

Boc

O

R¹

Ar

8e

Ar

11

O

Ar

12, R¹= Boc, R²= OMe

Ar = p-Tol

c

13a, R¹= H, R²= OMe (2HCl salt)

13b, R¹= H, R²= NHMe (2HCl salt)

d, c

e

Me

N

Me

N

Me

N

H

N

f, g

h, c

Tf

H

N

O

Boc

H₂N

NH₂

R¹

Ar

2HCl

O

Ar

O

Ar

14

¹= Boc

15,

13c

R

j

Me

m

13d, R¹= H

k

N

Me

N

Me

NH₂

N

HN

Me

n, c

2HCl

N

Ar

N

H

N

R²

R¹

H

N

19a

O

Ar

NC

Boc

Me

Ar

o, c

N

, R¹= Boc, R²= OH

16

H

N

18

g, c

NH₂

O

17a, R¹= H, R²= NH₂(2HCl salt)

17b, R¹= H, R²= OH (2HCl salt)

c

2HCl

N

Ar

O

19b

Scheme 2. Synthesis of compounds 13a–d, 17a, 17b, 19a and 19b. Reagents and conditions: (a) (1) H₂, Raney-Co, 25% NH₃, MeOH, 90 °C, 8 h; (2) (Boc)₂O, THF, room

temperature, 60 h; (b) BrCH₂CO₂Me, K₂CO₃, DMF, 2.5 h; (c) 4 M HCl/(1,4-dioxane or EtOAc), room temperature, 1–3 h; (d) (1) 1 M NaOH, MeOH, THF, room temperature, 1.5 h;

(2) 1 M MeNH₂/THF, HOBt, EDC, DMF, room temperature, 15 h; (e) N-Phenyl bis(triﬂuoromethanesulfonimide), NaH, DMF, 0 °C, 0.5 h; (f) (1) ethyl acrylate, PdCl₂(PPh₃)₂, Et₃N,

DMF, 70 °C, 20 h; (2) 1 M NaOH, EtOH, THF, room temperature, 17 h; (g) HOBtꢀNH₃, EDC, DMF, room temperature, 2–17 h; (h) H₂, Pd/C, EtOH, THF, room temperature, 3.5 h; (j)

(1) 4 M HCl/EtOAc, room temperature, 1.5 h; (2) 10% K₂CO₃; (k) (1) CO, Pd(OAc)₂, dppf, Et₃N, MeOH, THF, 100 °C, 3 h, sealed tube; (2) 1 M NaOH, MeOH, THF, 60 °C, 1 h; (m)

Zn(CN)₂, Pd(PPh₃)₄, NMP, 80 °C, 2 h; (n) NaN₃, NH₄Cl, DMSO, 70 °C, 48 h; (o) (1) H₂NOHꢀHCl, tert-BuOK, EtOH, 70 °C, 6 h; (2) CDI, EtOAc, THF, reﬂux, 3 h.

and converted to ester 13a and amide 13b. Intermediate 11 was

subjected to a sulfonylation using N-phenyl bis(triﬂuoromethanesulf-

onimide) and sodium hydride to give triﬂate 14, which was used for a

variety of palladium-catalyzed coupling reactions, such as the Heck

reaction, carbonylation and cyanation.^38,39The Heck reaction using

ethyl acrylate followed by two-step conversion of the ester to an

amide provided acrylamide derivative 15. The oleﬁn moiety of 15

was hydrogenated, and deprotection resulted in propionamide 13c.

Acrylamide 13d was obtained by N-Boc deprotection of 15. Methoxy-

carbonylation of triﬂate 14 was followed by alkali hydrolysis of the

resulting ester to afford carboxylic acid 16. Carboxylic acid 16 was

then converted to amide 17a by amidation followed by deprotection,

whereas N-Boc deprotection of 16 yielded amino acid 17b. Bioisosteric

replacement of the carboxy group of compound 16 was achieved

through nitrile 18, which was prepared by cyanation of triﬂate 14. Ni-

trile 18 was reacted with sodium azide to form a tetrazole ring⁴⁰and

then converted to compound 19a. Reacting nitrile 18 with hydroxyl-

amine yielded the corresponding amidoxime, which was treated with

1,1⁰-carbonyldiimidazole to form a 1,2,4-oxadiazolone ring.⁴¹N-Boc

deprotection of the 1,2,4-oxadiazolone derivative resulted in com-

pound 19b.

acted with 7b by using cesium carbonate as base. The resulting

6-substituted quinoline 20 was reduced by hydrogenation to com-

pound 21a. Piperazin-2,3-dione and piperazin-2,5-dione ring were

formed by amination and subsequent N-acylation as follows. Eth-

ylenediamine was coupled with compound 7b and protected with

a Boc group. Conversion of the 3-cyano group to the corresponding

Boc-aminomethyl group yielded 22, which was acylated with ethyl

chloro(oxo)acetate to give 23. Construction of the piperazin-2,3-

dione ring system could be achieved by acidic Boc deprotection

of 23 followed by neutralization to yield compound 21b. Glycine

ester was introduced to 7b, after which the 3-cyano group was

converted to a Boc-aminomethyl group to yield 24. Glycine ana-

logue 24 was acylated with the acid chloride of Cbz-glycine to pro-

duce compound 25. Cbz deprotection resulted in subsequent

cyclization to the piperazin-2,5-dione ring, and subsequent Boc

deprotection afforded compound 1.

3. Results and discussion

The quinoline derivatives described above were evaluated their

ability to inhibit human DPP-4, and the results are shown in Tables

2 and 3 as IC₅₀values with 95% conﬁdence limits (shown in paren-

theses) that were calculated from the concentration–response

curves generated by GraphPad Prism.

Introduction of a piperazinone group to the 6-position was

achieved through palladium-catalyzed amination⁴²of quinoline-

3-carbonitrile 7b (Scheme 3). Piperazin-2-one was efﬁciently re-

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H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

Me

N

a

b

O

HN

O

NH₂

Br

CN

N

CN

N

Ar

7b

Ar

HN

Ar

20

21a

21b

1

Ar = p-Tol

c, d, b, d

Me

N

O

H

N

g, b, d

N

O

NH₂

Boc

N

Boc

R²

Ar

22, R²= H

HN

Ar

Me

f

e

N

23, R²= COCO₂Et

H

N

O

Me

t-Bu

N

Boc

R¹

O

Ar

24, R¹= H

N

O

NH₂

h

j

HN

Ar

2HCl

25, R¹= COCH₂NHCbz

O

Scheme 3. Synthesis of compounds 21a, 21b and 1. Reagents and conditions: (a) piperazin-2-one, Pd(OAc)₂, rac-BINAP, Cs₂CO₃, 1,4-dioxane, 80 °C, 24 h; (b) H₂, Raney-Ni, 25%

NH₃, MeOH, THF, room temperature, 6 h; (c) ethylenediamine, Pd(OAc)₂, rac-BINAP, tert-BuONa, toluene, 80 °C, 2 h; (d) (Boc)₂O, THF, room temperature, 0.5–1 h; (e) ethyl

chloro(oxo)acetate, satd NaHCO₃, EtOAc, room temperature, 0.5 h; (f) (1) TFA, room temperature, 0.5–1 h; (2) 10% K₂CO₃; (g) (1) tert-butyl glycinate, Pd(OAc)₂, rac-BINAP, tert-

BuONa, toluene, 85 °C, 4 h; (h) Cbz-NHCH₂COCl, DMAP, pyridine, THF, room temperature, 1 h; (j) (1) H₂, Pd/C, EtOH, THF, room temperature, 24 h; (2) 4 M HCl/1,4-dioxane or

EtOAc, room temperature, 1 h.

Table 1

Designed scaffolds and results of GOLD scoring

Me Me

O

N

O

N

H₂N

NH₂

HCl

N

O

A

E

B

C

D

H

2

Me Me

N

S

N

O

S

H₂N

NH₂

O

F

G

9a

Scaffold^a

GoldScore^b

Ext_HB

Ext_vdW

Int_Torsion

Int_vdW

A

B

C

D

E

F

G

H

73.8

78.4

79.7

76.6

69.7

69.0

70.1

68.4

24.3

24.6

21.7

23.0

23.5

24.2

21.6

22.4

64.6

62.1

64.4

61.7

62.9

58.5

60.5

56.5

ꢁ11.8

ꢁ8.0

ꢁ3.3

ꢁ0.3

ꢁ1.1

ꢁ1.7

ꢁ4.1

ꢁ3.7

ꢁ2.5

ꢁ0.5

ꢁ5.3

ꢁ6.4

ꢁ12.6

ꢁ10.1

ꢁ9.4

ꢁ10.1

a

Experimental IC₅₀values of inhibitory activity against human DPP-4 of compound 2 (scaffold A) and 9a (scaffold B) were 47 (42–53) and 34 (31–37) nM, respectively,

where the IC₅₀values are means of triplicate measurements. IC₅₀values and 95% conﬁdence limits (shown in parentheses) were calculated from the concentration–response

curves generated by GraphPad Prism.

b

GoldScore consists of protein–ligand hydrogen bond energy (Ext_HB), protein–ligand van der Waals energy (Ext_vdW), ligand torsional strain energy (Int_Torsion) and

ligand internal van der Waals energy (Int_vdW).

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4487

Table 2

positions were designed. Among them, we decided to investigate

the quinoline scaffold B based on the synthetic feasibility for struc-

tural modiﬁcation. This strategy was supported by the docking

studies of designed compounds using GOLD. In these studies, the

binding afﬁnities of the compounds were estimated as a GoldScore,

which consists of four components: Ext_HB, Ext_vdW, Int_Torsion,

and Int_vdW, and the results are listed in Table 1. The GoldScore of

compounds incorporating scaffolds A–D are more than three points

higher than that of E–H. Among them, the quinoline scaffold B was

predicted to exhibit the highest level of afﬁnity for the enzyme.

Further elemental comparison between the quinoline B and isoqu-

inolone A shows that the sum of their Ext_HB and Ext_vdW scores

are comparable, assuming a similar potential to interact with the

enzyme. However, the sum of Int_Torsion and Int_vdW scores of

the quinoline was estimated to be higher than that of the isoquino-

lone, which predicts lower conformational stability of the isoqui-

nolone scaffold. This instability of the isoquinolone is probably

explained by the steric repulsion between the 1-carbonyl group

and 2-isobutyl group, and therefore, quinolines are expected to

have an advantage over corresponding isoquinolones in adopting

a preferable conformation for interacting with the enzyme. Accord-

ingly, quinoline analogue 9a was synthesized and evaluated for

DPP-4 inhibitory activity. Quinoline 9a exhibited similar inhibitory

activity to isoquinolone 2 as shown in Table 2, and we identiﬁed

SAR summary for the 4-position of quinoline derivatives

Me

N

H₂N

NH₂

O

R

Compound

R

DPP-4

IC₅₀(nM)

a

9a

9b

9c

9d

9e

H

3-F

3-CH₃

4-F

4-CH₃

34 (31–37)

49 (41–59)

57 (50–66)

26 (24–28)

4.2 (3.5–5.0)

a

IC₅₀values are means of triplicate measurements. IC₅₀values and 95% conﬁ-

dence limits (shown in parentheses) were calculated from the concentration–

response curves generated by GraphPad Prism.

Table 3

Inhibitory activities of quinoline derivatives

Me

the quinoline as

isoquinolone.

a

potential alternative scaffold to the

N

Next, the structure–activity relationships of 3-aminomethyl-4-

aryl-2-isobutylquinolines were investigated. As described in Fig-

ure 2B, the X-ray co-crystal structure of compound 2 suggests that

introduction of a small hydrophobic substituent onto the meta- or

para-position of the 4-phenyl ring should achieve a better ﬁt into

the pocket.

NH₂

R¹

n HCl

Me

We applied this hypothesis to the quinolines, and the potency-

enhancing effects of ﬂuoro or methyl substitution onto the

4-phenyl ring were evaluated and are described in Table 2. para-

Substituted analogues (9d, 9e) exhibited similar or higher potency

compared to the lead compound 9a, whereas meta-substituted

analogues (9b, 9c) showed decreased potency. Particularly, com-

pound 9e, with a p-methyl group on the pendant phenyl, exhibited

more than eightfold increase over 9a, and was found to inhibit

DPP-4 with nanomolar potency. Surprisingly, 9e exhibited sixfold

more potent inhibition than p-ﬂuoro analogue 9d, suggesting that

the ﬂuoro group was too small to reach the bottom of the S1 pock-

et. These results clearly indicate that the p-tolyl group at the 4-po-

sition of the quinoline ring should be appropriate both in size and

orientation to occupy the S1 pocket, which leads to potentiation of

the activity.

A hydrophilic substituent at the 6-position of 2-isobutyl-4-(4-

methylphenyl)quinoline analogue was investigated, as shown in

Table 3. Inhibitory activity was signiﬁcantly inﬂuenced by the nat-

ure of the 6-substituent. Compounds bearing methoxycarbonyl or

N-methylcarbamoyl groups at the terminal end of the 6-substitu-

ent (13a and 13b, respectively) exhibited a signiﬁcant decrease

in potency by more than 12-fold compared to the unsubstituted

amide 9e. Deletion of the linker moiety at the 6-position of 9e re-

sulted in 13- and 20-fold potency loss for compound 17a and its

corresponding carboxylic acid 17b, respectively. These results

clearly indicate the importance of both the approach and interac-

tion of the 6-substituent with the side chain of Lys554. Propiona-

mide 13c, which has a methylene linker showed a 1.8-fold

decrease in potency when compared to 9e which has an ether lin-

ker. This is likely due to the lack of hydrogen bonding to the en-

zyme via water molecules observed in compound 2 (Fig. 2A).

However, the potency loss was compensated in acrylamide 13d

a

Compound

R¹

n

DPP-4 IC₅₀(nM)

13a

13b

13c

13d

17a

17b

OCH₂CO₂CH₃

OCH₂CONHCH₃

CH₂CH₂CONH₂

(E)-CH@CHCONH₂

CONH₂

CO₂H

2

0

2

84 (79–90)

52 (46–59)

7.5 (6.4–8.7)

1.8 (1.7–1.9)

55 (52–57)

100 (86–120)

N

19a

2

9.2 (8.8–9.7)

NH

N

H

O

N

19b

2

11 (10–12)

O

N

O

21a

21b

0

2.2 (2.1–2.4)

2.7 (2.4–3.1)

N

O

NH

O

N

NH

O

N

O

NH

1

2

1.3 (1.1–1.5)

a

IC₅₀values are means of triplicate measurements. IC₅₀values and 95% conﬁ-

dence limits (shown in parentheses) were calculated from the concentration–

response curves generated by GraphPad Prism.

First, we explored an alternative central scaffold to isoquinolone

2 (IC₅₀= 47 (42–53) nM, n = 3) to enhance DPP-4 inhibitory activ-

ity. As shown in Table 1, 6 new core scaffolds B–H that can pre-

serve the 2-, 3-, 4-, and 6-substituents of compound 2 in similar

by conversion to a trans-oleﬁn linker that can form a

p–p stacking

interaction with the aromatic ring of Tyr547 in the hydrophobic

4488

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

Figure 4. Docking study between DPP-4 protein and compound 1. (A) The amino acids (black carbons, blue texts) in the binding sites of compound 1 (green carbons) are

indicated. Major interactions are depicted as red dotted lines. (B) The S1 binding site is indicated in cross-sectional view. Distances between carbon atoms of the 2-p-tolyl

group and the representative hydrophobic residues lining the S1 pocket are shown as red dotted lines. Values listed are the carbon–carbon distances in angstroms.

cleft. Surprisingly, bioisosteric replacement of the carboxylic acid

17b to tetrazole 19a or oxadiazole 19b resulted in more than nine-

fold potency enhancement. These results suggested a contribution

of the hydrophobic interaction between the substituents at the 6-

position and the hydrophobic cleft of the enzyme. Thus, we focused

on a variety of heterocyclic substituents at the 6-position which

could make interactions with both hydrophilic Lys554 and hydro-

phobic residues in the target cleft.

Table 4

Selected pharmacokinetic proﬁles of compound 1^a,b

Species

V_dss(mL/kg)

CL_total(mL/h/kg)

AUC_po

(lgꢀh/mL)

F (%)

Rat

Dog

10,712 (1148)

1490 (406)

2819 (390)

352 (73)

28.5 (2.3)

1409 (126)

8.0 (1.2)

49.2 (8.0)

a

1.0 mg/kg oral dose and 0.1 mg/kg intravenous.

Values are means and standard deviations of three experiments.

b

Compound 21a, which has both a hydrogen-bonding moiety

and a lipophilic moiety in the 6-substituent, showed a ﬁvefold

increase in potency relative to 19b. The effect of an additional

oxo group was dependent on the position of the oxo group on

the piperazinone substituent. Piperazin-2,3-dione derivative 21b

is similar in potency to 21a. However, the corresponding pipera-

zin-2,5-dione derivative 1 exhibited a potency-enhancing effect

and was found to be the most active inhibitor of this series, with

an IC₅₀value of 1.3 nM.

interactions and 2 hydrogen-bonding interactions, including one

with Lys554.

Enzyme selectivity of compound 1 was evaluated and resulted

in excellent selectivity for DPP-4 over closely related peptidases,

DPP-2 (IC₅₀= 20

lM), DPP-8 (IC₅₀>60 lM), and DPP-9 (IC₅₀

>60 M). The ADME proﬁles of compound 1 revealed that it is

l

highly soluble in water (>5 mg/mL at pH 6.8) and moderately or

highly stable in liver microsomes (human, dog, rat: 16, 2.0,

96 lL/min/mg, respectively). Pharmacokinetic properties are rep-

resented in Table 4. Compound 1 exhibited moderate or high bio-

availability in rats and dogs.

To gain further insight into the binding mode of the quinoline

derivatives, a docking study between compound 1 and DPP-4

was conducted (Fig. 4). The primary amino group at the 3-position

of the quinoline ring can make hydrogen bonds with the Glu-motif

(Glu205-Glu206) and Tyr662 whereas the 2-isobutyl group can

make a hydrophobic interaction with Phe357. The methyl group

of the p-tolyl group at the 4-position of the quinoline ring ﬁts well

in the small hydrophobic hole at the bottom of the S1 pocket en-

abling it to maximize hydrophobic interactions with surrounding

residues (Fig. 4B). The isoquinolone derivative with phenyl group

2 can likely not occupy this region as efﬁciently (Fig. 2B). This

result explains the signiﬁcant potency-enhancing effects of the

introduction of methyl group, as shown in Table 2. The pipera-

zin-2,5-dione ring at the 6-position of the quinoline scaffold can

be positioned to allow a hydrophobic interaction with Tyr547.

Notably, the docking study indicated that the 2-oxo group on the

piperazin-2,5-dione substituent can accept a hydrogen bond from

Lys554. The 5-oxo group can form an additional hydrogen bond

with the backbone NH of Tyr631. This backbone hydrogen-bonding

can explain the observed potency increase of compound 1 com-

pared to compounds 21a and 21b. Therefore, compound 1 is as-

sumed to achieve excellent activity by taking advantage of the

desirable piperazin-2,5-dione substituent by forming hydrophobic

An ex vivo study in dogs revealed that compound 1 could exhi-

bit potent and long-lasting inhibition at low dosage (Fig. 5). In this

study, administration of compound 1 in dogs at a dosage of 0.5 mg/

kg resulted in more than 50% inhibition of the plasma DPP-4 activ-

ity for 24 h. In an oral glucose tolerance test in female Wistar fatty

rats, the compound dose-dependently elevated plasma insulin lev-

els and exhibited a potent plasma glucose lowering effect with a

minimum effective dose of 1 mg/kg, po (p <0.025 vs control, Wil-

liams test). On the basis of the results of the ex vivo and the

in vivo studies, compound 1 is hypothesized to be a new type of

antidiabetic agent.

4. Conclusions

Using the structure-based drug design starting from the isoqu-

inolone 2 scaffold, a novel class of potent DPP-4 inhibitors with a

quinoline scaffold has been discovered. The representative dihy-

drochloride of 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]piperazine-2,5-dione

(1)

showed

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4489

5.1.1. 2-Amino-5-bromo-N-methoxy-N-methylbenzamide (4a)

120

100

80

5.1.1.1. Step A. To a solution of 25 g (116 mmol) of 2-amino-5-

bromobenzoic acid (3a) in 350 mL of THF was added 24.4 g

(82 mmol) of bis(trichloromethyl) carbonate was added, and the

resulting suspension was stirred at reﬂux temperature for 4 h.

The reaction mixture was poured onto ice, and the precipitate

was collected by ﬁltration, washed with methanol and dried to af-

ford 27.1 g (96%) of 6-bromo-2H-3,1-benzoxazine-2,4(1H)-dione

1 0.3 mg/kg

1 0.5 mg/kg

as

a

white powder. ¹H NMR (200 MHz, CDCl₃) d: 7.19 (d,

60

40

20

J = 8.4 Hz, 1H), 7.96–8.02 (m, 1H), 8.09 (d, J = 2.6 Hz, 1H).

5.1.1.2. Step B. A mixture of 16.4 g (168 mmol) of N,O-dim-

ethylhydroxylamine hydrochloride and 13.4 mL (168 mmol) of tri-

ethylamine in 70 mL of ethanol was stirred at room temperature

for 30 min. To the suspension was added 27.1 g (112 mmol) of 6-

bromo-2H-3,1-benzoxazine-2,4(1H)-dione, and the resulting mix-

ture was stirred at 75 °C for 17 h. The reaction mixture was cooled

and ﬁltered. The ﬁltrate was concentrated in vacuo, and the residue

was neutralized with saturated aqueous sodium bicarbonate solu-

tion and extracted with ethyl acetate. The extract was washed

sequentially with saturated aqueous sodium bicarbonate solution

and brine, dried over anhydrous magnesium sulfate and concen-

trated in vacuo. Puriﬁcation by column chromatography (silica

gel, eluting at a gradient of 50–80% ethyl acetate in hexanes) was

followed by recrystallization from diisopropyl ether/hexanes to af-

ford 21.8 g (72%) of the title compound as pale orange crystals: mp

78–81 °C. ¹H NMR (200 MHz, CDCl₃) d: 3.34 (s, 3H), 3.59 (s, 3H),

4.67 (br s, 2H), 6.64 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 8.4, 2.4 Hz,

1H), 7.50 (d, J = 2.4 Hz, 1H). MS m/z 198 (MꢁN(Me)OMe)⁺.

0

2

4

6

8

10

12

24

Timer after drug administration (h)

Figure 5. Ex vivo study of compound 1 in beagle dogs. Compound 1 was formulated

in a 0.5% suspension of methylcellulose at 0.3 mg/mL. Five dogs (28–42 months old)

were orally administered a suspension of compound at doses of 0.3 and 0.5 mg/kg.

Blood samples were collected from the cephalic vein before (0 h) and 0.5, 1, 2, 4, 6,

8, 12, and 24 h after dosing. Plasma sample was prepared from each blood sample

and the residual DPP-4 activity was measured by using a method similar to that

described in Section 5.

excellent DPP-4 inhibition in dog plasma for more than 24 h and

potent glucose lowering effects in rats. These results suggest the

potential for once-daily treatment in T2DM patients. A docking

study of compound 1 to DPP-4 suggested that an efﬁcient ﬁt into

the binding site and a hydrogen bond to Lys554 are important in

enhancing the inhibitory activity. Therefore, our results are useful

for designing novel, potent non-covalent DPP-4 inhibitors.

5.1.2. 2-Amino-5-{[tert-butyl(dimethyl)silyl]oxy}-N-methoxy-

N-methylbenzamide (4b)

5.1.2.1. Step A. A solution of 52 mL (0.36 mmol) of benzyl chloro-

formate in 200 mL of diethyl ether was added dropwise with a

vigorous stirring to a mixture of 50 g (0.33 mmol) of 2-amino-5-

hydroxybenzoic acid (3b), 109 g (1.3 mol) of sodium bicarbonate,

200 mL of diethyl ether and 200 mL of water at room temperature.

The mixture was stirred at room temperature for 30 min, and then

neutralized with concentrated hydrochloric acid. The reaction mix-

ture was extracted with ethyl acetate, and the extract was washed

with brine, dried over anhydrous magnesium sulfate. Concentration

in vacuo was followed by crystallization from diisopropyl ether–

hexanes to afford 92 g (98%) of 2-{[(benzyloxy)carbonyl]amino}-5-

hydroxybenzoic acid as a white powder, which was used in the next

step without further puriﬁcation. ¹H NMR (200 MHz, CDCl₃) d: 5.13

(s, 2H), 7.00 (dd, J = 9.0, 3.0 Hz, 1H), 7.33–7.41 (m, 6H), 8.00 (d,

J = 9.0 Hz, 1H), 9.50 (br, 1H), 10.32 (s, 1H).

5. Experimental section

5.1. Chemistry

¹H NMR spectra were recorded on

a Varian Gemini-200

(200 MHz) or a Varian Ultra-300 (300 MHz) instrument in CDCl₃,

DMSO-d₆or CD₃OD solutions. Low-resolution mass spectra (MS)

were determined using a Waters Liquid Chromatography-Mass

Spectrometer. High-resolution mass spectra (HRMS) experiments

were carried out by Takeda Analytical Laboratories Ltd. All MS

experiments were performed using electrospray ionization (ESI)

in positive or negative ion mode. Elemental analyses were also ob-

tained from Takeda Analytical Laboratories Ltd. The purity of ﬁnal

products was assessed by HPLC analyses using Shimadzu UFLC L-

5.1.2.2. Step B. To a mixture of 91 g (0.32 mol) of 2-{[(benzyl-

oxy)carbonyl]amino}-5-hydroxybenzoic acid and 36 g (0.36 mol)

of N,O-dimethyl hydroxylamine hydrochloride in 500 mL of DMF

were sequentially added 55 mL (0.36 mol) of triethylamine and

70 g (0.36 mol) of EDC at room temperature, and the resulting mix-

ture was stirred for 2.5 h at room temperature. After quenching with

1 M hydrochloric acid, the mixture was extracted with ethyl acetate.

The extract was washed with brine, dried over anhydrous magne-

sium sulfate, and concentrated in vacuo. Puriﬁcation by column

chromatography (silica gel, eluting sequentially with 1:3 ethyl ace-

tate/hexanes, 1:1 ethyl acetate/hexanes, and ethyl acetate) gave a

brown oil. A mixture of the obtained brown oil, 48 g (0.32 mol) of

tert-butyl(chloro)dimethylsilane and 22 g (0.32 mol) of imidazole

in 500 mL of DMF was stirred at room temperature for 14 h. Water

was added and the mixture was extracted with ethyl acetate. The ex-

tract was washed sequentially with 1 M hydrochloric acid and brine,

dried over anhydrous magnesium sulfate, and concentrated in va-

cuo. Puriﬁcation by column chromatography (silica gel, 1:3 ethyl

column 2 ODS (3.0 ꢂ 50 mm, 2

lm). Elution was conducted at

1.2 mL/min using a gradient of 5–90% solvent B in solvent A (sol-

vent A was 0.1% triﬂuoroacetic acid (TFA) in water, and solvent B

was 0.1% TFA in acetonitrile). Final purity (k 220 and 254 nm) is

noted with the analytical data for each compound. Melting points

were determined using a Yanagimoto melting point apparatus or a

Büchi melting point apparatus B-545 and were uncorrected. All

commercial chemicals and solvents are reagent grade and were

used without further puriﬁcation. Amounts of Raney nickel and Ra-

ney cobalt are theoretical values calculated from their weights in

water. Column chromatography was performed on Silica Gel 60

(0.063–0.200 or 0.040–0.063 mm, E. Merck) or NH silica gel (Chro-

matorex^ÒNH, 100–200 mesh, Fuji Silysia) using the solvent sys-

tems indicated. Yields are unoptimized.

4490

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

acetate/hexanes) gave 85 g (59%) of 2-{[(benzyloxy)car-

bonyl]amino}-5-{[tert-butyl(dimethyl)silyl]oxy}benzoic acid as a

white powder. ¹H NMR (200 MHz, CDCl₃) d: 0.28 (s, 6H), 1.08 (s,

9H), 3.45 (s, 3H), 3.62 (s, 3H), 5.28 (s, 2H), 7.02 (dd, J = 8.8, 2.6 Hz,

1H), 7.06 (d, J = 2.6 Hz, 1H), 7.44–7.51 (m, 5H), 8.04 (d, J = 8.8 Hz,

1H), 8.45 (br, 1H).

methylbenzamide (4b) and 4-ﬂuorophenylmagnesium bro-

mide prepared from 5.6 g (32 mmol) of p-bromoﬂuorobenzene

using a procedure similar to that described for the synthesis of

compound 5a as an orange syrup. ¹H NMR (200 MHz, CDCl₃) d:

0.10 (s, 6H), 0.93 (s, 9H), 5.63 (br s, 2H), 6.65 (d, J = 9.2 Hz, 1H),

6.80–6.95 (m, 2H), 7.05–7.20 (m, 2H), 7.60–7.75 (m, 2H).

5.1.2.3. Step C. A mixture of 85 g (0.19 mol) of 2-{[(benzyl-

oxy)carbonyl]amino}-5-{[tert-butyl(dimethyl)silyl]oxy}benzoic

acid, 2.5 g of 10% Pd/C, 250 mL of ethanol and 250 mL of THF was

stirred under atmospheric hydrogen at room temperature for

20 h. The reaction mixture was ﬁltered, and the ﬁltrate was con-

centrated in vacuo to afford 40 g (69%) of the title compound as

a white powder. ¹H NMR (200 MHz, CDCl₃) d: 0.25 (s, 6H), 1.07

(s, 9H), 3.45 (s, 3H), 3.68 (s, 3H), 6.70 (d, J = 8.8 Hz, 1H), 6.84 (dd,

J = 8.8, 3.0 Hz, 1H), 6.97 (d, J = 3.0 Hz, 1H).

5.1.8. (2-Amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)

(4-methylphenyl)methanone (5f)

The title compound (1.4 g) was prepared from 4.6 g (14.5 mmol)

of 2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}-N-methoxy-N-meth-

ylbenzamide (4b) and 44 mL (44 mmol) of 1 M p-tolylmagnesium

bromide in THF using a procedure similar to that described for the

synthesis of compound 5a as a yellow syrup. ¹H NMR (200 MHz,

CDCl₃) d: 0.10 (s, 6H), 0.93 (s, 9H), 2.43 (s, 3H), 5.58 (br s, 2H), 6.64

(d, J = 8.8 Hz, 1H), 6.70–6.75 (m, 2H), 7.00–7.10 (m, 2H), 7.25 (d,

J = 8.8 Hz, 2H), 7.55–7.65 (m, 2H). MS m/z 342 (M+H)⁺.

5.1.3. (2-Amino-5-bromophenyl)(phenyl)methanone (5a)

To a solution of 5.0 g (19 mmol) of 2-amino-5-bromo-N-meth-

oxy-N-methylbenzamide (4a) in 50 mL of diethyl ether was added

24 mL (48 mmol) of 2 M phenylmagnesium bromide in THF drop-

wise at 0 °C. The resulting mixture was stirred at 0 °C for 15 min.

After quenching with saturated aqueous ammonium chloride solu-

tion, the reaction mixture was extracted with ethyl acetate. The ex-

tract was washed with brine, dried over anhydrous magnesium

sulfate and concentrated in vacuo. Puriﬁcation by column chroma-

tography (silica gel, eluting at a gradient of 5–25% ethyl acetate in

hexanes) afforded 3.21 g (60%) of the title compound as yellow

crystals. ¹H NMR (200 MHz, CDCl₃) d: 6.08 (br s, 2H), 6.65 (d,

J = 8.8 Hz, 1H), 7.26–7.66 (m, 7H).

5.1.9. 5-Methyl-3-oxohexanenitrile (6)

To a suspension of 30.2 g (666 mmol) of sodium hydride (60% in

oil) in 300 mL of THF was added a mixture of 50 mL (333 mmol) of

methyl 3-methylbutanoate 10 and 39 mL (666 mmol) of acetoni-

trile dropwise at 70 °C, and the resulting mixture was stirred at

70 °C for 15 h. The reaction mixture was cooled and carefully

quenched with ice water. The aqueous layer was washed with hex-

anes, acidiﬁed with concentrated hydrochloric acid and extracted

with ethyl acetate. The extract was washed with brine, dried over

anhydrous magnesium sulfate and concentrated in vacuo to give

41 g (98%) of the title compound as a pale yellow oil, which was

used in the next step without further puriﬁcations. ¹H NMR

(300 MHz, CDCl₃) d: 0.96 (d, J = 6.6 Hz, 6H), 2.11–2.28 (m, 1H),

2.50 (d, J = 6.8 Hz, 2H), 3.42 (s, 2H).

5.1.4. (2-Amino-5-bromophenyl)(4-methylphenyl)methanone

(5b)

The title compound (3.5 g) was prepared from 5.0 g

(19.3 mmol) of 2-amino-5-bromo-N-methoxy-N-methylbenza-

mide (4a) and 60 mL (60 mmol) of 1 M p-tolylmagnesium bromide

in THF using a procedure similar to that described for the synthesis

of compound 5a as yellow crystals. ¹H NMR (300 MHz, CDCl₃) d:

2.44 (s, 3H), 5.98 (br s, 2H), 6.64 (d, J = 8.9 Hz, 1H), 7.28 (d,

J = 7.9 Hz, 2H), 7.35 (dd, J = 8.9, 2.3 Hz, 1H), 7.55–7.60 (m, 3H).

MS m/z 290, 292 (M+H)⁺.

5.1.10. 6-Bromo-2-(2-methylpropyl)-4-phenylquinoline-3-

carbonitrile (7a)

A

mixture of 4.2 g (15.2 mmol) of (2-amino-5-bromophe-

nyl)(phenyl)methanone (5a), 2.28 g (18.2 mmol) of 5-methyl-3-

oxohexanenitrile (6), methanesulfonic acid (1.46 g, 15.2 mmol)

and 200 mL of toluene was heated to reﬂux for 6 h using a Dean-

Stark trap. The reaction mixture was allowed to cool to room tem-

perature, washed sequentially with saturated aqueous sodium

bicarbonate solution and brine, dried over anhydrous magnesium

sulfate and concentrated in vacuo. Puriﬁcation by column chroma-

tography (silica gel, eluting at a gradient of 5–20% ethyl acetate in

hexanes) was followed by recrystallization from ethyl acetate/hex-

anes to afford 4.85 g (88%) of the title compound as colorless crys-

tals: mp 136–137 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.06 (d,

J = 6.6 Hz, 6H), 2.25–2.55 (m, 1H), 3.11 (d, J = 7.3 Hz, 2H), 7.40–

7.50 (m, 2H), 7.55–7.65 (m, 3H), 7.79 (d, J = 2.2 Hz, 1H), 7.87 (dd,

J = 8.4, 2.2 Hz, 1H), 8.80 (d, J = 8.4 Hz, 1H). MS m/z 365, 367 (M+H)⁺.

5.1.5. (2-Amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)

(3-ﬂuorophenyl)methanone (5c)

The title compound (1.2 g) was obtained from 2.5 g (8.1 mmol) of

2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}-N-methoxy-N-meth-

ylbenzamide (4b) and 3-ﬂuorophenylmagnesiumbromide prepared

from 5.6 g (32 mmol) of m-bromoﬂuorobenzene using a procedure

similar to that described for the synthesis of compound 5a as a yel-

low syrup. ¹H NMR (200 MHz, CDCl₃) d: 0.09 (s, 6H), 0.92 (s, 9H),

5.75 (br s, 2H), 6.66 (d, J = 8.4 Hz, 1H), 6.80–6.95 (m, 2H), 7.15–

7.25 (m, 1H), 7.30–7.50 (m, 3H). MS m/z 346 (M+H)⁺.

5.1.11. 6-Bromo-4-(4-methylphenyl)-2-(2-

methylpropyl)quinoline-3-carbonitrile (7b)

5.1.6. (2-Amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)(3-

methylphenyl)methanone (5d)

The title compound (6.6 g) was prepared from 8.7 g (30 mmol) of

(2-amino-5-bromophenyl)(4-methylphenyl)methanone (5b) and

4.5 g (36 mmol) of 5-methyl-3-oxohexanenitrile (6) using a proce-

dure similar to that described for the synthesis of compound 7a as

pale orange crystals: mp 168–169 °C. ¹H NMR (200 MHz, CDCl₃) d:

1.05 (6H, d, J = 6.8 Hz), 2.30–2.50 (1H, m), 2.50 (3H, s), 3.10 (2H, d,

J = 7.4 Hz), 7.34 (2H, d, J = 8.2 Hz), 7.42 (2H, d, J = 8.2 Hz), 7.80–

7.90 (2H, m), 7.99 (1H, d, J = 9.0 Hz). MS m/z 379, 381 (M+H)⁺.

The title compound (1.5 g) was prepared from 2.0 g (6.4 mmol) of

2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}-N-methoxy-N-meth-

ylbenzamide (4b) and 30 mL (30 mmol) of 1 M m-tolylmagnesium

bromide in THF using a procedure similar to that described for the

synthesis of compound 5a as a crude brown syrup. MS m/z 342

(M+H)⁺.

5.1.7. (2-Amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)(4-

ﬂuorophenyl)methanone (5e)

5.1.12. 6-Hydroxy-2-(2-methylpropyl)-4-phenylquinoline-3-

carbonitrile (8a)

The title compound (1.3 g) was obtained from 2.5 g (8.1 mmol)

of 2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}-N-methoxy-N-

5.1.12.1. Step A. To 20 mL of toluene under nitrogen atmosphere

were added 0.51 g (0.82 mmol) of rac-BINAP and 0.062 g

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4491

(0.27 mmol) of palladium acetate. After being stirred at 40 °C for

1 h, 2.0 g (5.5 mmol) of 6-bromo-2-(2-methylpropyl)-4-phenyl-

quinoline-3-carbonitrile (7a), 1.1 mL (11 mmol) of tert-butanol

and 0.79 g (8.2 mmol) of sodium tert-butoxide were added. The

resulting mixture was stirred at 90 °C for 1 h. The reaction mixture

was cooled to room temperature and ﬁltered through Celite. The

ﬁltrate was washed with brine, dried over anhydrous magnesium

sulfate and concentrated in vacuo. Puriﬁcation by column chroma-

tography (silica gel, eluting at a gradient of 5–20% ethyl acetate in

hexanes) was followed by recrystallization from ethyl acetate/hex-

anes to afford 1.8 g (92%) of 6-tert-butoxy-2-(2-methylpropyl)-4-

phenyl)methanone (5f) and 3.8 g (30 mmol) of 5-methyl-3-oxo-

hexanenitrile (6) using a procedure similar to that described for

the synthesis of compound 7a as pale yellow crystals: mp 247 °C

(decomp). ¹H NMR (200 MHz, CDCl₃) d: 1.03 (d, J = 6.6 Hz, 6H),

2.20–2.45 (m, 1H), 2.46 (s, 3H), 3.06 (d, J = 7.3 Hz, 2H), 6.28 (d,

J = 2.9 Hz, 1H), 7.25–7.40 (m, 4H), 7.41 (dd, J = 9.2, 2.9 Hz, 1H),

8.02 (d, J = 9.2 Hz, 1H). MS m/z 317 (M+H)⁺.

5.1.17. 2-{[3-(Aminomethyl)-2-(2-methylpropyl)-4-

phenylquinolin-6-yl]oxy}acetamide (9a)

5.1.17.1. Step A. A mixture of 0.76 g (2.5 mmol) of 6-hydroxy-2-

(2-methylpropyl)-4-phenylquinoline-3-carbonitrile (8a), 0.36 g

(3.8 mmol) of 2-chloroacetamaide and 0.53 g (3.8 mmol) of potas-

sium carbonate in 10 mL of DMF was stirred at 60 °C for 8 h. The

reaction mixture was poured into water and extracted with ethyl

acetate. The extract was washed with 1 M hydrochloric acid, dried

over anhydrous magnesium sulfate and concentrated in vacuo.

Puriﬁcation by column chromatography (silica gel, eluting at a gra-

dient of 30–100% ethyl acetate in hexanes) to give 0.71 g (79%) of

2-{[3-cyano-2-(2-methylpropyl)-4-phenylquinolin-6-yl]oxy}acet-

amide as a white powder. ¹H NMR (200 MHz, CDCl₃) d: 1.05 (d,

J = 6.6 Hz, 6H), 2.32–2.43 (m, 1H), 3.09 (d, J = 7.2 Hz, 2H), 4.40 (s,

2H), 5.75 (br s, 1H), 6.51 (br s, 1H), 6.92 (d, J = 3.0 Hz, 1H), 7.41–

7.48 (m, 2H), 7.49 (dd, J = 9.0, 3.0 Hz, 1H), 7.57–7.64 (m, 3H),

8.09 (d, J = 9.0 Hz, 1H).

phenylquinoline-3-carbonitrile as

a

white powder. ¹H NMR

(300 MHz, CDCl₃) d: 1.06 (d, J = 6.6 Hz, 6H), 1.33 (s, 9H), 2.30–

2.50 (m, 1H), 3.10 (d, J = 7.2 Hz, 2H), 7.14 (d, J = 2.5 Hz, 1H), 7.40–

7.65 (m, 6H), 8.03 (d, J = 9.1 Hz, 1H). MS m/z 369 (M+H)⁺.

5.1.12.2. Step B. A mixture of 1.0 g (2.8 mmol) of 6-tert-butoxy-

2-(2-methylpropyl)-4-phenylquinoline-3-carbonitrile, 10 mL of

TFA and 2 mL of THF was stirred at room temperature for 1 h.

The reaction mixture was poured into water and extracted with

ethyl acetate. The extract was washed with brine, dried over anhy-

drous magnesium sulfate and concentrated in vacuo. Recrystalliza-

tion from diisopropyl ether/hexanes afforded 0.81 g (95%) of the

title compound as a yellow powder. ¹H NMR (200 MHz, CDCl₃) d:

1.10 (d, J = 6.6 Hz, 6H), 2.28–2.43 (m, 1H), 3.29 (d, J = 7.5 Hz, 2H),

7.13 (d, J = 2.4 Hz, 1H), 7.45–7.51 (m, 2H), 7.62–7.70 (m, 3H),

7.73 (dd, J = 9.3, 2.4 Hz, 1H), 8.32 (br s, 1H), 8.57 (d, J = 9.3 Hz, 1H).

5.1.17.2. Step B. A mixture of 0.65 g (1.8 mmol) of 2-{[3-cyano-

2-(2-methylpropyl)-4-phenylquinolin-6-yl]oxy}acetamide, ca. 10 g

of Raney cobalt, 5 mL of 25% aqueous ammonia, 60 mL of metha-

nol and 20 mL of THF was stirred under hydrogen atmosphere at

0.5 MPa for 7 h at 70 °C. The reaction mixture was cooled to room

temperature and ﬁltered. The ﬁltrate was poured into 5% aqueous

potassium carbonate solution and extracted with ethyl acetate. The

extract was washed with brine, dried over anhydrous magnesium

sulfate and concentrated in vacuo. Puriﬁcation by column chroma-

tography (silica gel, eluting at a gradient of 0–30% methanol in

ethyl acetate) to give 0.43 g (66%) of the title compound as a white

powder: mp 139–141 °C. ¹H NMR (200 MHz, CDCl₃) d: 1.04 (d,

J = 6.6 Hz, 6H), 1.24 (br s, 2H), 2.32–2.44 (m, 1H), 3.00 (d,

J = 7.2 Hz, 2H), 3.78 (s, 2H), 4.33 (s, 2H), 5.88 (br s, 1H), 5.55 (br

s, 1H), 6.56 (d, J = 2.7 Hz, 1H), 7.25–7.28 (m, 2H), 7.30 (dd, J = 9.3,

2.7 Hz, 1H), 7.49–7.58 (m, 3H), 8.02 (d, J = 9.3 Hz, 1H). Anal. Calcd

for C₂₂H₂₅N₃O₂: C, 72.70; H, 6.93; N, 11.56. Found: C, 72.35; H,

6.80; N, 11.28. HPLC purity: 96.69% (k 220 nm), 95.60% (k 254 nm).

5.1.13. 4-(3-Fluorophenyl)-6-hydroxy-2-(2-

methylpropyl)quinoline-3-carbonitrile (8b)

The title compound (0.83 g) was prepared from 1.2 g (3.4 mmol)

of

(2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)(3-ﬂuoro-

phenyl)methanone (5c) and 0.64 g (5.1 mmol) of 5-methyl-3-oxo-

hexanenitrile (6) using a procedure similar to that described for

the synthesis of compound 7a as an off-white powder: mp 273–

276 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.05 (d, J = 6.6 Hz, 6H), 2.25–

2.45 (m, 1H), 3.08 (d, J = 7.4 Hz, 2H), 5.46 (br s, 1H), 6.68 (d,

J = 2.8 Hz, 1H), 7.10–7.35 (m, 3H), 7.48 (dd, J = 9.2, 2.8 Hz, 1H),

7.50–7.65 (m, 1H), 8.05 (d, J = 9.2 Hz, 1H). MS m/z 321 (M+H)⁺.

5.1.14. 6-Hydroxy-4-(3-methylphenyl)-2-(2-

methylpropyl)quinoline-3-carbonitrile (8c)

The title compound (0.05 g) was prepared from 1.35 g (4.0 mmol)

of (2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)(3-methyl-

phenyl)methanone (5d) and 0.64 g (5.1 mmol) of 5-methyl-3-oxo-

hexanenitrile (6) using a procedure similar to that described for the

synthesis of compound 7a as an off-white powder: mp 275 °C (de-

comp). ¹H NMR (200 MHz, CDCl₃) d: 0.99 (d, J = 6.6 Hz, 6H), 2.20–

2.35 (m, 1H), 2.43 (s, 3H), 2.96 (d, J = 7.3 Hz, 2H), 6.81 (d, J = 2.6 Hz,

1H), 7.25–7.60 (m, 5H), 7.97 (d, J = 9.2 Hz, 1H), 10.20 (s, 1H).

5.1.18. 2-{[3-(Aminomethyl)-4-(3-ﬂuorophenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}acetamide (9b)

5.1.18.1. Step A. 2-{[3-Cyano-4-(3-ﬂuorophenyl)-2-(2-methyl-

propyl)quinolin-6-yl]oxy}acetamide (0.38 g) was prepared from

0.40 g (1.25 mmol) of 4-(3-ﬂuorophenyl)-6-hydroxy-2-(2-methyl-

propyl)quinoline-3-carbonitrile (8b) using a procedure similar to

that described as step A for the synthesis of compound 9a as an

off-white powder: mp 186–193 °C. ¹H NMR (300 MHz, CDCl₃) d:

1.06 (d, J = 6.6 Hz, 6H), 2.30–2.50 (m, 1H), 3.10 (d, J = 7.3 Hz, 2H),

4.43 (s, 2H), 5.74 (br s, 1H), 6.52 (br s, 1H), 6.88 (d, J = 2.8 Hz,

1H), 7.10–7.25 (m, 2H), 7.25–7.40 (m, 1H), 7.52 (dd, J = 9.2,

2.8 Hz, 1H), 7.55–7.65 (m, 1H), 8.11 (d, J = 9.2 Hz, 1H). MS m/z

378 (M+H)⁺.

5.1.15. 4-(4-Fluorophenyl)-6-hydroxy-2-(2-

methylpropyl)quinoline-3-carbonitrile (8d)

The title compound (0.88 g) was prepared from 1.4 g (4.0 mmol)

of

(2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)(4-ﬂuoro-

phenyl)methanone (5e) and 0.76 g (6.0 mmol) of 5-methyl-3-oxo-

hexanenitrile (6) using a procedure similar to that described for the

synthesis of compound 7a as an off-white powder: mp 247–249 °C.

¹H NMR (300 MHz, CDCl₃) d: 1.04 (d, J = 6.6 Hz, 6H), 2.15–2.45 (m,

1H), 3.07 (d, J = 7.2 Hz, 2H), 5.87 (br s, 1H), 6.89 (d, J = 2.6 Hz, 1H),

7.10–7.50 (m, 5H), 8.04 (d, J = 9.2 Hz, 1H). MS m/z 321 (M+H)⁺.

5.1.18.2. Step B. A mixture of 0.35 g (0.93 mmol) of 2-{[3-cyano-

4-(3-ﬂuorophenyl)-2-(2-methylpropyl)quinolin-6-yl]oxy}acetam-

ide, ca. 4 g of Raney nickel, 4 mL of 25% aqueous ammonia, 50 mL

of methanol and 10 mL of THF was stirred under hydrogen atmo-

sphere at 0.5 MPa for 9 h at room temperature. The reaction mix-

ture was ﬁltered, and the ﬁltrate was concentrated in vacuo.

Puriﬁcation by column chromatography (silica gel, eluting at a gra-

5.1.16. 6-Hydroxy-4-(4-methylphenyl)-2-(2-

methylpropyl)quinoline-3-carbonitrile (8e)

The title compound (7.2 g) was prepared from 8.5 g (25 mmol)

of (2-amino-5-{[tert-butyl(dimethyl)silyl]oxy}phenyl)(4-methyl-

4492

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

dient of 0–20% methanol in ethyl acetate) was followed by recrys-

tallization from aqueous ethanol to give 0.27 g (76%) of the title

compound as colorless crystals: mp 106–108 °C. ¹H NMR

(300 MHz, CDCl₃) d: 1.04 (d, J = 6.6 Hz, 6H), 1.51 (br s, 2H), 2.25–

2.50 (m, 1H), 3.00 (d, J = 7.2 Hz, 2H), 3.78 (s, 2H), 4.36 (s, 2H),

5.62 (br s, 1H), 6.53 (d, J = 2.8 Hz, 1H), 6.55 (br s, 1H), 6.90–7.30

(m, 3H), 7.34 (dd, J = 9.2, 2.8 Hz, 1H), 7.45–7.60 (m, 1H), 8.03 (d,

J = 9.2 Hz, 1H). MS m/z 382 (M+H)⁺. Anal. Calcd for

5.1.21. 2-{[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}acetamide (9e)

5.1.21.1. Step A. 2-{[3-Cyano-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-6-yl]oxy}acetamide (0.37 g) was prepared from

0.40 g (1.26 mmol) of 6-hydroxy-4-(4-methylphenyl)-2-(2-meth-

ylpropyl)quinoline-3-carbonitrile (8e) using a procedure similar

to that described as step A for the synthesis of compound 9a as a

white powder: mp 184–186 °C. ¹H NMR (200 MHz, CDCl₃) d: 1.05

(d, J = 6.6 Hz, 6H), 2.30–2.50 (m, 1H), 2.51 (s, 3H), 3.04 (d,

J = 7.3 Hz, 2H), 4.42 (s, 2H), 5.71 (br s, 1H), 6.53 (br s, 1H), 6.96

(d, J = 2.9 Hz, 1H), 7.25–7.45 (m, 4H), 7.50 (dd, J = 9.2, 2.9 Hz, 1H),

8.08 (d, J = 9.2 Hz, 1H). MS m/z 374 (M+H)⁺.

C

₂₂H₂₄FN₃O₂ꢀH₂O: C, 66.15; H, 6.56; N, 10.52. Found: C, 65.92; H,

6.43; N, 10.31. HPLC purity: 98.31% (k 220 nm), 97.68% (k 254 nm).

5.1.19. 2-{[3-(Aminomethyl)-4-(3-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}acetamide (9c)

5.1.19.1. Step A. 2-{[3-Cyano-4-(3-methylphenyl)-2-(2-methyl-

propyl)quinolin-6-yl]oxy}acetamide (0.21 g) was prepared from

0.25 g (0.79 mmol) of 6-hydroxy-4-(3-methylphenyl)-2-(2-meth-

ylpropyl)quinoline-3-carbonitrile (8c) using a procedure similar

to that described as step A for the synthesis of compound 9a as

an off-white powder: mp 123–125 °C. ¹H NMR (300 MHz, CDCl₃)

d: 1.05 (d, J = 6.8 Hz, 6H), 2.25–2.45 (m, 1H), 2.48 (s, 3H), 3.10 (d,

J = 7.2 Hz, 2H), 4.41 (s, 2H), 5.64 (br s, 1H), 6.53 (br s, 1H), 6.93

(d, J = 2.8 Hz, 1H), 7.15–7.30 (m, 2H), 7.30–7.55 (m, 3H), 8.09 (d,

J = 9.2 Hz, 1H). MS m/z 374 (M+H)⁺.

5.1.21.2. Step B. The title compound (0.24 g) was prepared from

0.33 g (0.88 mmol) of 2-{[3-cyano-4-(4-methylphenyl)-2-(2-meth-

ylpropyl)quinolin-6-yl]oxy}acetamide using a procedure similar to

that described as step B for the synthesis of compound 9b as a

white powder: mp 175–177 °C. ¹H NMR (200 MHz, CDCl₃) d: 1.04

(d, J = 6.6 Hz, 6H), 1.60 (br s, 2H), 2.25–2.45 (m, 1H), 2.50 (s, 3H),

2.99 (d, J = 7.3 Hz, 2H), 3.78 (br s, 2H), 4.34 (s, 2H), 5.68 (br s,

1H), 6.58 (br s, 1H), 6.59 (d, J = 2.9 Hz, 1H), 7.15 (d, J = 8.1 Hz,

2H), 7.25–7.40 (m, 3H), 8.01 (d, J = 9.2 Hz, 1H). MS m/z 378 (M +

H)⁺. Anal. Calcd for C₂₃H₂₇N₃O₂ꢀ1.25H₂O: C, 69.06; H, 7.43; N,

10.51. Found: C, 69.27; H, 7.50; N, 10.11. HPLC purity: 99.10% (k

220 nm), 98.84% (k 254 nm).

5.1.19.2. Step B. The title compound (50 mg) was prepared from

0.18 g (0.48 mmol) of 2-{[3-cyano-4-(3-methylphenyl)-2-(2-meth-

ylpropyl)quinolin-6-yl]oxy}acetamide using a procedure similar to

that described as step B for the synthesis of compound 9a as a

white powder: mp 98–101 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.04

(d, J = 6.6 Hz, 6H), 1.60 (br s, 2H), 2.30–2.45 (m, 1H), 2.45 (s, 3H),

2.99 (d, J = 7.4 Hz, 2H), 3.78 (br s, 2H), 4.34 (s, 2H), 5.63 (br s,

1H), 6.57 (d, J = 2.6 Hz, 1H), 6.60 (br s, 1H), 7.05 (d, J = 7.2 Hz,

1H), 7.25–7.40 (m, 1H), 7.32 (dd, J = 9.2, 2.6 Hz, 2H), 7.40–7.50

(m, 1H), 8.01 (d, J = 9.2 Hz, 1H). MS m/z 378 (M+H)⁺; HRMS (ESI)

calcd for C₂₃H₂₇N₃O₂(M+H)⁺m/z 378.2176, found m/z 378.2151.

Anal. Calcd for C₂₃H₂₇N₃O₂ꢀ2H₂O: C, 66.81; H, 7.56; N, 10.16.

Found: C, 66.73; H, 7.05; N, 9.93. HPLC purity: 95.87% (k

220 nm), 95.11% (k 254 nm).

5.1.22. tert-Butyl {[6-hydroxy-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-3-yl]methyl}carbamate (11)

A mixture of 27.4 g (86.6 mmol) of 6-hydroxy-4-(4-methyl-

phenyl)-2-(2-methylpropyl)quinoline-3-carbonitrile (8e), ca. 101 g

of Raney cobalt, 50 mL of 25% aqueous ammonia, 300 mL of meth-

anol and 150 mL of THF was stirred under hydrogen atmosphere at

0.5 MPa for 8 h at 90 °C. The reaction mixture was cooled to room

temperature and ﬁltered. The ﬁltrate was poured into brine and ex-

tracted with ethyl acetate. The extract was dried over anhydrous

magnesium sulfate and concentrated in vacuo. The residue was

dissolved in 500 mL of THF and then 22.7 g (104 mmol) of di-

tert-butyl dicarbonate was added. The resulting mixture was stir-

red at room temperature for 96 h, and concentrated in vacuo.

The residual solid was triturated with diisopropyl ether to afford

30.2 g (83%) of the title compound as an off-white powder. ¹H

NMR (200 MHz, CDCl₃) d: 0.97 (d, J = 6.6 Hz, 6H), 1.40 (s, 9H),

2.15–2.30 (m, 1H), 2.42 (s, 3H), 2.92 (d, J = 7.3 Hz, 2H), 4.25–4.35

(m, 3H), 6.67 (d, J = 2.5 Hz, 1H), 7.08 (d, J = 7.3 Hz, 2H), 7.20–7.30

(m, 3H), 7.88 (d, J = 8.9 Hz, 1H). MS m/z 421 (M+H)⁺.

5.1.20. 2-{[3-(Aminomethyl)-4-(4-ﬂuorophenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}acetamide (9d)

5.1.20.1. Step A. 2-{[3-Cyano-4-(4-ﬂuorophenyl)-2-(2-methyl-

propyl)quinolin-6-yl]oxy}acetamide (0.93 g) was prepared from

0.84 g (2.6 mmol) of 4-(4-ﬂuorophenyl)-6-hydroxy-2-(2-methyl-

propyl)quinoline-3-carbonitrile (8d) using a procedure similar to

that described as step A for the synthesis of compound 9a as pale

yellow crystals: mp 153–154 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.05

(d, J = 6.6 Hz, 6H), 2.30–2.50 (m, 1H), 3.10 (d, J = 7.4 Hz, 2H), 4.43 (s,

2H), 5.68 (br s, 1H), 6.51 (br s, 1H), 6.90 (d, J = 2.9 Hz, 1H), 7.25–

7.40 (m, 2H), 7.40–7.50 (m, 2H), 7.52 (dd, J = 9.2, 2.9 Hz, 1H),

8.11 (d, J = 9.2 Hz, 1H). MS m/z 378 (M+H)⁺.

5.1.23. Methyl {[3-{[(tert-butoxycarbonyl)amino]methyl}-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]oxy}acetate

(12)

A mixture of 1.5 g (3.6 mmol) of tert-butyl {[6-hydroxy-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]methyl}carba-

mate (11), 0.55 g (4.0 mmol) of methyl 2-bromoacetate and 0.92 g

(6.0 mmol) of potassium carbonate in 10 mL of DMF was stirred at

room temperature for 2.5 h. After quenching with water, the reac-

tion mixture was extracted with ethyl acetate. The extract was

washed with brine, dried over anhydrous magnesium sulfate and

concentrated in vacuo. The residual solid was triturated with diiso-

propyl ether to afford 1.86 g (94%) of the title compound as a white

powder. ¹H NMR (200 MHz, DMSO-d₆) d: 0.99 (d, J = 6.6 Hz, 6H),

1.35 (s, 9H), 2.26–2.32 (m, 1H), 2.43 (s, 3H), 3.09–3.10 (m, 2H),

3.10 (s, 3H), 4.12 (s, 2H), 4.77 (s, 2H), 6.54 (s, 1H), 7.12 (s, 1H),

7.24 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.75–7.77 (m, 1H),

8.48–8.51 (m, 1H).

5.1.20.2. Step B. The title compound (0.42 g) was prepared from

0.85 g (2.2 mmol) of 2-{[3-cyano-4-(4-ﬂuorophenyl)-2-(2-methyl-

propyl)quinolin-6-yl]oxy}acetamide using a procedure similar to

that described as step B for the synthesis of compound 9a as color-

less crystals: mp 142 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.04 (d,

J = 6.6 Hz, 6H), 1.54 (br s, 2H), 2.25–2.50 (m, 1H), 2.99 (d,

J = 7.2 Hz, 2H), 3.77 (s, 2H), 4.36 (s, 2H), 5.63 (br s, 1H), 6.54 (br

s, 1H), 6.54 (d, J = 2.8 Hz, 1H), 7.20–7.30 (m, 4H), 7.33 (dd, J = 9.2,

2.8 Hz, 1H), 8.03 (d, J = 9.2 Hz, 1H). MS m/z 378 (M+H)⁺. Anal. Calcd

for C₂₂H₂₄FN₃O₂ꢀ0.75H₂O: C, 66.90; H, 6.51; N, 10.64. Found: C,

66.56; H, 6.61; N, 10.43. HPLC purity: 99.54% (k 220 nm), 99.39%

(k 254 nm).

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4493

5.1.24. Methyl {[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}acetate dihydrochloride (13a)

To a solution of 0.15 g (0.3 mmol) of methyl {[3-{[(tert-butoxy-

carbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methylpropyl)

quinolin-6-yl]oxy}acetate (12) in 5 mL of ethyl acetate was added

5 mL of 4 M hydrogen chloride in ethyl acetate at room tempera-

ture. The resulting mixture was stirred at room temperature for

1.5 h, and then concentrated in vacuo. The residue was crystallized

from diisopropyl ether to afford 0.13 g (92%) of the title compound

C

₂₄H₂₉N₃O₂(M+H)⁺m/z 392.2333, found m/z 392.2316. HPLC pur-

ity: 99.08% (k 220 nm), 99.05% (k 254 nm).

5.1.26. 3-{[(tert-Butoxycarbonyl)amino]methyl}-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-6-yl

triﬂuoromethanesulfonate (14)

To an ice-cooled mixture of 1.0 g (2.4 mmol) of tert-butyl {[6-

hydroxy-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]

methyl}carbamate (11) and 1.28 g (3.6 mmol) of N-phenyl bis(tri-

ﬂuoromethanesulfonimide) in 15 mL of DMF was added 0.14 g

(3.6 mmol) of sodium hydride (60% in oil). The resulting mixture

was stirred at 0 °C for 30 min. After quenching with water, the

reaction mixture was extracted with ethyl acetate. The extract

was washed with brine, dried over anhydrous magnesium sulfate

and concentrated in vacuo. Puriﬁcation by column chromatogra-

phy (silica gel, eluting at a gradient of 5–20% ethyl acetate in hex-

anes) to give 1.30 g (99%) of the title compound as a white powder:

mp 194–196 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.04 (d, J = 6.7 Hz,

6H), 1.42 (s, 9H), 2.30–2.45 (m, 1H), 2.49 (s, 3H), 2.99 (d,

J = 7.2 Hz, 2H), 4.33 (br s, 3H), 7.12 (d, J = 2.8 Hz, 2H), 7.22 (d,

J = 2.8 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.53 (dd, J = 9.2, 2.8 Hz,

1H), 8.14 (d, J = 9.2 Hz, 1H). MS m/z 553 (M+H)⁺.

as

a

yellow powder. ¹H NMR (200 MHz, DMSO-d₆) d: 1.00

(d, J = 7.2 Hz, 6H), 2.24–2.32 (m, 1H), 2.46 (s, 3H), 3.25 (s, 2H),

3.59 (s, 3H), 3.93–3.99 (m, 2H), 4.75 (s, 2H), 6.49 (s, 1H), 7.33 (d,

J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 7.72–7.75 (m, 1H), 8.39–

8.42 (m, 1H), 8.56 (br s, 3H). HRMS (ESI) calcd for C₂₄H₂₈N₂O₃

(M+H)⁺m/z 393.2173, found m/z 393.2155. HPLC purity: 98.26%

(k 220 nm), 98.12% (k 254 nm).

5.1.25. 2-{[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}-N-methylacetamide

dihydrochloride (13b)

5.1.25.1. Step A. A mixture of 1.6 g (3.2 mmol) of methyl {[3-

{[(tert-butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]oxy}acetate (12), 10 mL of 1 M

aqueous sodium hydroxide solution, 5 mL of methanol and 5 mL

of THF was stirred at room temperature for 1.5 h. The reaction mix-

ture was neutralized with 1 M hydrochloric acid and extracted

with ethyl acetate. The extract was washed with brine, dried over

anhydrous magnesium sulfate and concentrated in vacuo. The

residual solid was triturated with diisopropyl ether to afford

1.32 g (85%) of {[3-{[(tert-butoxycarbonyl)amino]methyl}-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]oxy}acetic acid

as a white powder. ¹H NMR (200 MHz, DMSO-d₆) d: 1.05 (d,

J = 6.6 Hz, 6H), 1.40 (s, 9H), 2.29–2.31 (m, 1H), 2.48 (s, 3H), 3.36

(s, 2H), 4.53 (s, 2H), 4.89 (br, 1H), 6.71 (s, 1H), 7.12–7.13 (m, 2H),

7.27–7.40 (m, 3H), 8.57 (d, J = 8.1 Hz, 1H).

5.1.27. tert-Butyl ({6-[(1E)-3-amino-3-oxoprop-1-en-1-yl]-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-3-

yl}methyl)carbamate (15)

5.1.27.1. Step A. A mixture of 1.48 g (2.78 mmol) of 3-{[(tert-

butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-6-yl triﬂuoromethanesulfonate (14), 0.65 mL

(6.0 mmol) of ethyl acrylate, 0.021 mg (0.03 mmol) of bis(triphen-

ylphosphine)palladium(II) dichloride and 4.2 mL (30 mmol) of tri-

ethylamine in 10 mL of DMF was stirred under nitrogen

atmosphere at 70 °C for 20 h. The reaction mixture was partitioned

between ethyl acetate and water, and the organic layer was

washed with brine, dried over anhydrous magnesium sulfate and

concentrated in vacuo. Puriﬁcation by column chromatography

(silica gel, eluting at a gradient of 0–25% ethyl acetate in hexanes)

gave 0.87 g of crude solid, which was used directly in the next step.

To a solution of the obtained crude solid in 10 mL of ethanol and

10 mL of THF was added 10 mL of 1 M aqueous sodium hydroxide

solution at room temperature, and the resulting mixture was stir-

red at 60 °C for 4 h. The reaction mixture was cooled to room tem-

perature, neutralized with 1 M hydrochloric acid, and extracted

with ethyl acetate. The extract was washed with brine, dried over

anhydrous magnesium sulfate and concentrated in vacuo. The

residual solid was triturated with diisopropyl ether to give 0.41 g

(70%) of (2E)-3-[3-{[(tert-butoxycarbonyl)amino]methyl}-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]prop-2-enoic acid

5.1.25.2. Step B. To a mixture of 0.48 g (1.0 mmol) of {[3-{[(tert-

butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-6-yl]oxy}acetic acid, 2 mL of 1 M methylamine in

THF and 3 mL of DMF were added 0.16 g (1.2 mmol) of HOBt and

0.23 g (1.2 mmol) of EDC. The resulting mixture was stirred at

room temperature for 15 h. The reaction mixture was diluted with

ethyl acetate, washed sequentially with 1 M hydrochloric acid and

brine, dried over anhydrous magnesium sulfate and concentrated

in vacuo. Puriﬁcation by column chromatography (silica gel, 1:1

ethyl acetate/hexanes) to give 0.27 g (55%) of tert-butyl ({6-[2-

(methylamino)-2-oxoethoxy]-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-3-yl}methyl)carbamate as a pale yellow solid. ¹H

NMR (200 MHz, CDCl₃) d: 1.00 (d, J = 6.6 Hz, 6H), 1.41 (s, 9H),

2.30–2.33 (m, 1H), 2.49 (s, 3H), 2.88–2.98 (m, 5H), 4.28–4.34 (m,

4H), 6.58–6.59 (m, 2H), 6.60 (br, 1H), 7.07 (d, J = 8.1 Hz, 2H), 7.32

(d, J = 8.1 Hz, 2H), 7.33–7.34 (m, 1H), 8.02 (d, J = 9.0 Hz, 1H).

as

a

white powder. ¹H NMR (200 MHz, CDCl₃) d: 1.13 (d,

J = 6.0 Hz, 6H), 1.41 (s, 9H), 2.36–2.39 (m, 1H), 2.52 (s, 3H), 3.15

(s, 2H), 4.35 (s, 2H), 4.35 (br, 1H), 6.43 (d, J = 15.6 Hz, 1H), 7.14

(d, J = 8.1 Hz, 2H), 7.26 (s, 1H), 7.28 (d, J = 8.1 Hz, 2H), 7.45 (s,

1H), 7.70 (d, J = 15.6 Hz, 1H), 7.93 (s, 1H).

5.1.25.3. Step C. To a solution of 0.26 g (0.53 mmol) of tert-butyl

({6-[2-(methylamino)-2-oxoethoxy]-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-3-yl}methyl)carbamate in 5 mL of ethyl

acetate was added 5 mL of 4 M hydrogen chloride in ethyl acetate

at room temperature. The resulting mixture was stirred at room

temperature for 3 h, and then concentrated in vacuo. The residue

was crystallized from diisopropyl ether to afford 0.20 g (79%) of

the title compound as a pale yellow powder. ¹H NMR (200 MHz,

DMSO-d₆) d: 0.99 (d, J = 6.6 Hz, 6H), 2.26–2.30 (m, 1H), 2.46 (s,

3H), 2.58 (s, 3H), 3.18 (s, 2H), 3.97 (s, 2H), 4.42 (s, 2H), 6.64 (s,

1H), 7.32 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.72–7.73 (m,

1H), 8.07–8.08 (m, 1H), 8.41 (br s, 3H). HRMS (ESI) calcd for

5.1.27.2. Step B. A mixture of 0.30 g (0.63 mmol) of (2E)-3-[3-

{[(tert-butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]prop-2-enoic acid, 0.18 g (1.2 mmol)

of HOBt ammonium salt and 0.23 g (1.2 mmol) of EDC in 5 mL of

DMF was stirred at room temperature for 17 h. The reaction mix-

ture was partitioned between ethyl acetate and water. The organic

layer was washed with brine, dried over anhydrous magnesium

sulfate and concentrated in vacuo. Puriﬁcation by column chroma-

tography (silica gel, eluting at a gradient of 45–100% ethyl acetate

in hexanes) gave 0.25 g (84%) of the title compound as a white

powder. ¹H NMR (300 MHz, CDCl₃) d: 1.03 (d, J = 6.6 Hz, 6H), 1.41

(s, 9H), 2.35–2.39 (m, 1H), 2.50 (s, 3H), 2.97 (d, J = 7.2 Hz, 2H),

4494

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4.32 (s, 3H), 5.49 (br, 2H), 6.42 (d, J = 15.6 Hz, 1H), 7.11 (d,

J = 3.1 Hz, 2H), 7.33–7.37 (m, 3H), 7.58 (d, J = 15.6 Hz, 1H), 7.82

(dd, J = 8.7, 2.1 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H).

5.1.30. 3-{[(tert-Butoxycarbonyl)amino]methyl}-4-(4-

methylphenyl)-2-(2-methylpropyl)quinoline-6-carboxylic acid

(16)

5.1.30.1. Step A. A mixture of 12.2 g (22 mmol) of 3-{[(tert-

butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-6-yl triﬂuoromethanesulfonate (14), 30 mL of

methanol, 0.25 g (1.1 mmol) of palladium acetate, 0.61 g

(1.1 mmol) of 1,1’-bis(diphenylphosphino)ferrocene and 3.4 mL

(24 mmol) of triethylamine in 50 mL of THF was stirred under car-

bonoxide atmosphere at 0.5 MPa for 3 h at 100 °C. After being

cooled, the reaction mixture was poured into water and extracted

with ethyl acetate. The extract was washed with brine, dried over

anhydrous magnesium sulfate and concentrated in vacuo. Puriﬁca-

tion by column chromatography (silica gel, eluting at a gradient of

10–40% ethyl acetate in hexanes) gave 6.8 g (67%) of methyl 3-

{[(tert-butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-

methylpropyl)quinoline-6-carboxylate as a white powder: mp

192–194 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.04 (d, J = 6.3 Hz, 6H),

1.42 (s, 9H), 2.34–2.37 (m, 1H), 2.49 (s, 3H), 2.99 (d, J = 7.2 Hz,

2H), 3.87 (s, 3H), 4.32 (br s, 3H), 7.13 (d, J = 7.8 Hz, 2H), 7.35 (d,

J = 7.8 Hz, 2H), 8.06–8.10 (m, 2H), 8.23 (dd, J = 8.7, 2.1 Hz, 1H).

5.1.28. 3-[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]propanamide dihydrochloride

(13c)

5.1.28.1. Step A. A mixture of 0.25 g (0.53 mmol) of tert-butyl

({6-[(1E)-3-amino-3-oxoprop-1-en-1-yl]-4-(4-methylphenyl)-2-

(2-methylpropyl)quinolin-3-yl}methyl)carbamate (15), 0.25 g of

10% Pd/C, 5 mL of ethanol and 5 mL of THF was stirred under atmo-

spheric hydrogen at room temperature for 3.5 h. The reaction mix-

ture was ﬁltered, and the ﬁltrate was concentrated in vacuo to

afford 0.20 g (81%) of tert-butyl {[6-(3-amino-3-oxopropyl)-4-(4-

methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]methyl}carbamate

as a white powder. ¹H NMR (300 MHz, CDCl₃) d: 1.03 (d, J = 6.6 Hz,

6H), 1.41 (s, 9H), 2.31–2.39 (m, 1H), 2.46 (t, J = 7.8 Hz, 2H), 2.49 (s,

3H), 3.00 (t, J = 7.8 Hz, 2H), 3.00–3.03 (m, 2H), 4.31 (s, 2H), 4.34 (br,

1H), 5.28 (br s, 1H), 5.40 (br s, 1H), 7.12 (d, J = 8.1 Hz, 2H), 7.12–

7.15 (m, 1H), 7.35 (d, J = 5.1 Hz, 2H), 7.55–7.61 (m, 1H), 8.17 (br,

1H).

5.1.28.2. Step B. To a solution of 0.15 g (0.32 mmol) of tert-butyl

{[6-(3-amino-3-oxopropyl)-4-(4-methylphenyl)-2-(2-methylpro-

pyl)quinolin-3-yl]methyl}carbamate in 5 mL of ethyl acetate was

added 5 mL of 4 M hydrogen chloride in ethyl acetate at room tem-

perature. The resulting mixture was stirred at room temperature

for 1.5 h, and then concentrated in vacuo. The residue was crystal-

lized from diisopropyl ether to afford 0.14 g (96%) of the title com-

pound as a yellow powder. ¹H NMR (200 MHz, DMSO-d₆) d: 0.84 (d,

J = 6.3 Hz, 6H), 2.11–2.17 (m, 1H), 2.15 (t, J = 7.2 Hz, 2H), 2.36 (s,

3H), 2.71 (t, J = 7.2 Hz, 2H), 3.09 (s, 2H), 3.82–3.88 (m, 2H), 6.57

(br, 1H), 7.03 (s, 1H), 7.09 (br, 1H), 7.20 (d, J = 7.8 Hz, 2H), 7.29

(d, J = 7.8 Hz, 2H), 7.75–7.76 (m, 1H), 8.28–8.33 (m, 1H), 8.32 (br

s, 3H). HRMS (ESI) calcd for C₂₄H₂₉N₃O (M+H)⁺m/z 376.2383, found

m/z 376.2366. HPLC purity: 99.10% (k 220 nm), 99.50% (k 254 nm).

5.1.30.2. Step B. To a suspension of 6.5 g (14 mmol) of methyl 3-

{[(tert-butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-

methylpropyl)quinoline-6-carboxylate in 20 mL of methanol and

20 mL of THF was added 28 mL of 1 M aqueous sodium hydroxide

solution, and the resulting mixture was stirred at 60 °C for 1 h. The

reaction mixture was pured into water, acidiﬁed with 1 M hydro-

chloric acid and extracted with ethyl acetate. The extract was

washed with brine, dried over anhydrous magnesium sulfate and

concentrated in vacuo. Recrystallization of the residual solid from

THF/diisopropyl ether afforded the title compound as a white pow-

der: mp 276–277 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.05 (d,

J = 6.6 Hz, 6H), 1.41 (s, 9H), 2.30–2.49 (m, 4H), 3.18 (br s, 2H),

4.35 (d, J = 5.1 Hz, 2H), 4.49 (br s, 1H),7.15 (d, J = 7.5 Hz, 2H),

7.33–7.38 (m, 3H), 8.19 (s, 1H), 8.29–8.41 (m, 2H). Anal. Calcd for

C

₂₇H₃₂N₂O₄ꢀ0.5H₂O: C, 70.87; H, 7.27; N, 6.12. Found: C, 70.54;

5.1.29. (2E)-3-[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]prop-2-enamide (13d)

H, 7.00; N, 5.94.

5.1.29.1. Step A. To a solution of 0.126 g (0.28 mmol) of tert-

5.1.31. 3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

butyl

({6-[(1E)-3-amino-3-oxoprop-1-en-1-yl]-4-(4-methyl-

methylpropyl)quinoline-6-carboxamide dihydrochloride (17a)

5.1.31.1. Step A. A mixture of 0.22 g (0.5 mmol) of 3-{[(tert-

butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinoline-6-carboxylic acid (16), 0.16 g (1.0 mmol) of HOBt

ammonium salt and 0.20 g (1.0 mmol) of EDC in 10 mL of DMF was

stirred at room temperature for 2 h. The reaction mixture was par-

titioned between ethyl acetate and water. The organic layer was

washed with brine, dried over anhydrous magnesium sulfate and

concentrated in vacuo. The residue was crystallized from ethyl ace-

tate/diisopropyl ether to give 0.12 g (55%) of tert-butyl {[6-carbam-

oyl-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]methyl}

carbamate as a white powder: mp 206–208 °C. ¹H NMR (300 MHz,

CDCl₃) d: 1.02 (d, J = 6.3 Hz, 6H), 1.41 (s, 9H), 2.29–2.48 (m, 1H),

2.52 (s, 3H), 3.49 (d, J = 6.6 Hz, 2H), 4.37 (d, J = 5.1 Hz, 2H), 5.26

(br s, 1H), 5.84 (br s, 1H), 7.23 (d, J = 7.8 Hz, 2H), 7.39 (d,

J = 7.8 Hz, 2H), 8.08 (br s, 2H), 8.55 (d, J = 9.0 Hz, 1H), 8.79 (d,

J = 9.0 Hz, 1H).

phenyl)-2-(2-methylpropyl)quinolin-3-yl}methyl)carbamate (15)

in 5 mL of ethyl acetate was added 5 mL of 4 M hydrogen chloride

in ethyl acetate at room temperature. The resulting mixture was

stirred at room temperature for 2 h, and then concentrated in va-

cuo. The residue was crystallized from diisopropyl ether to afford

0.094 g (93%) of (2E)-3-[3-(aminomethyl)-4-(4-methylphenyl)-2-

(2-methylpropyl)quinolin-6-yl]prop-2-enamide dihydrochloride

as a pale yellow powder.

5.1.29.2. Step B. A mixture of 0.22 g (0.50 mmol) of (2E)-3-[3-

(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-

6-yl]prop-2-enamide dihydrochloride and 100 mL of 10% aqueous

potassium carbonate solution was stirred at room temperature

for 5 min. The mixture was partitioned between ethyl acetate

and water. The organic layer was washed with brine, dried over

anhydrous magnesium sulfate and concentrated in vacuo. The

residual solid was recrystallized from aqueous ethanol to afford

0.16 g (89%) of the title compound as a white powder. ¹H NMR

(200 MHz, DMSO-d₆) d: 1.04 (d, J = 6.6 Hz, 6H), 1.26 (br s, 2H),

2.33–2.44 (m, 1H), 2.50 (s, 3H), 3.02 (d, J = 6.9 Hz, 2H), 3.82 (s,

2H), 5.51 (br s, 2H), 6.42(d, J = 15.6 Hz, 1H), 7.16 (d, J = 8.1 Hz,

2H), 7.34–7.36 (m, 3H), 7.59 (d, J = 8.1 Hz, 1H), 7.81 (dd, J = 8.7,

1.8 Hz, 1H), 8.03 (d, J = 8.7 Hz, 1H). MS m/z 374 (M+H)⁺. Anal. Calcd

for C₂₄H₂₇N₃Oꢀ2H₂O: C, 70.39; H, 7.63; N, 10.26. Found: C, 70.36; H,

7.77; N, 10.25. HPLC purity: 96.28% (k 220 nm), 95.83% (k 254 nm).

5.1.31.2. Step B. A mixture of 90 mg (0.2 mmol) of tert-butyl {[6-

carbamoyl-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]

methyl}carbamate and 5 mL of 4 M hydrogen chloride in 1,4-diox-

ane was stirred at room temperature for 1 h. The reaction mixture

was concentrated in vacuo, and the residual solid was triturated

with ethyl acetate/diisopropyl ether to afford 70 mg (88%) of the ti-

tle compound as a white powder: mp 194–196 °C. ¹H NMR

(300 MHz, DMSO-d₆) d: 1.03 (d, J = 6.3 Hz, 6H), 2.32–2.49

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4495

(m, 4H), 3.17 (br s, 2H), 3.99 (d, J = 6.3 Hz, 2H), 7.36 (d, J = 7.8 Hz,

2H), 7.47 (d, J = 7.8 Hz, 2H), 7.59 (br s, 1H), 7.89 (s, 1H), 8.21–

8.33 (m, 3H), 8.42 (br s, 3H). HRMS (ESI) calcd for C₂₂H₂₅N₃O

(M+H)⁺m/z 348.2070, found m/z 348.2044. HPLC purity: 98.61%

(k 220 nm), 98.20% (k 254 nm).

1,4-dioxane was stirred at room temperature for 2 h. The reaction

mixture was concentrated in vacuo. Recrystallization from metha-

nol/diisopropyl ether gave 0.14 g (89%) of the title compound as a

pale yellow powder. ¹H NMR (300 MHz, DMSO-d₆) d: 1.05 (d,

J = 6.8 Hz, 6H), 2.30–2.50 (m, 1H), 2.49 (s, 3H), 3.08 (d, J = 7.0 Hz,

2H), 4.00 (d, J = 5.5 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.49 (d,

J = 7.8 Hz, 2H), 8.08 (d, J = 1.5 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.33

(br s, 3H), 8.43 (dd, J = 8.8, 1.5 Hz, 1H). MS m/z 373 (M+H)⁺; HRMS

(ESI) calcd for C₂₂H₂₄N₆(M+H)⁺m/z 373.2135, found m/z 373.2115.

HPLC purity: 99.44% (k 220 nm), 99.42% (k 254 nm).

5.1.32. 3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinoline-6-carboxylic acid dihydrochloride (17b)

A mixture of 0.22 g (0.5 mmol) of 3-{[(tert-butoxycarbonyl)

amino]methyl}-4-(4-methylphenyl)-2-(2-methylpropyl)quinoline-

6-carboxylic acid (16) and 5 mL of 4 M hydrogen chloride in 1,4-

dioxane was stirred at room temperature for 1 h. Concentration

in vacuo was followed by trituration with ethyl acetate to afford

0.20 g (95%) of the title compound as a white powder: mp 284–

286 °C. ¹H NMR (300 MHz, DMSO-d₆) d: 1.04 (d, J = 6.3 Hz, 6H),

2.28–2.44 (m, 1H), 2.49 (s, 3H), 3.27 (d, J = 6.9 Hz, 2H), 4.02 (d,

J = 5.4 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H),

8.00 (d, J = 1.5 Hz, 1H), 8.34–8.45 (m, 2H), 8.62 (br s, 3H). HRMS

(ESI) calcd for C₂₂H₂₄N₂O₂(M+H)⁺m/z 349.1911, found m/z

349.1876; calcd for C₂₂H₂₄N₂O₂(MꢁH)^ꢁm/z 347.1765, found m/z

347.1771. Anal. Calcd for C₂₂H₂₄N₂O₂ꢀ2HClꢀ0.45H₂O: C, 61.53; H,

6.31; N, 6.52. Found: C, 61.89; H, 6.48; N, 6.14. HPLC purity:

99.74% (k 220 nm), 99.57% (k 254 nm).

5.1.35. 3-[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]-1,2,4-oxadiazol-5(4H)-one

dihydrochloride (19b)

5.1.35.1. Step A. To a suspension of 3.0 g (7.0 mmol) of tert-butyl

{[6-cyano-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]

methyl}carbamate (18) and 0.73 g (11 mmol) of hydroxylamine

hydrochloride in 75 mL of ethanol was added 1.18 g (11 mmol) of

potassium tert-butoxide. The resulting mixture was stirred at

70 °C for 6 h, cooled to room temperature and ﬁltered. The ﬁltrate

was concentrated in vacuo, and the residue was partitioned be-

tween ethyl acetate and water. The organic layer was washed with

brine, dried over anhydrous magnesium sulfate and concentrated

in vacuo. The residual solid was triturated with dichloromethane/

ethyl acetate to afford 2.38 g (74%) of tert-butyl {[6-(hydroxycar-

bamimidoyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-

yl]methyl}carbamate as an off-white powder: mp 224–225 °C. ¹H

NMR (300 MHz, CDCl₃) d: 1.03 (d, J = 6.6 Hz, 6H), 1.43 (s, 9H),

2.25–2.45 (m, 1H), 2.47 (s, 3H), 2.96 (d, J = 7.2 Hz, 2H), 4.29 (d,

J = 4.7 Hz, 2H), 4.49 (t, J = 4.7 Hz, 1H), 4.75 (br s, 2H), 7.12 (d,

J = 7.5 Hz, 2H), 7.33 (d, J = 7.7 Hz, 2H), 7.40–7.50 (m, 1H), 7.80–

7.95 (m, 1H), 8.01 (d, J = 8.7 Hz, 1H). MS m/z 463 (M+H)⁺.

5.1.33. tert-Butyl {[6-cyano-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-3-yl]methyl}carbamate (18)

To

bonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methylpropyl)

quinolin-6-yl triﬂuoromethanesulfonate (14) and 0.49 g

a mixture of 3.87 g (7.0 mmol) of 3-{[(tert-butoxycar-

(4.2 mmol) of zinc cyanide in 40 mL of NMP under argon atmo-

sphere was added 0.40 g (0.35 mmol) of tetrakis(triphenylpho-

spine)palladium(0). The resulting mixture was stirred at 80 °C for

2 h. The reaction mixture was poured into water and extracted

with ethyl acetate. The extract was washed with brine, dried over

anhydrous magnesium sulfate and concentrated in vacuo. Puriﬁca-

tion by column chromatography (silica gel, eluting at a gradient of

0–25% ethyl acetate in hexanes) gave 2.18 g (73%) of the title com-

pound as a white powder. ¹H NMR (300 MHz, CDCl₃) d: 1.03 (d,

J = 6.6 Hz, 6H), 1.42 (s, 9H), 2.34–2.47 (m, 1H), 2.50 (s, 3H), 3.00

(d, J = 7.2 Hz, 2H), 4.34 (br s, 3H), 7.11 (d, J = 7.8 Hz, 2H), 7.37 (d,

J = 7.8 Hz, 2H), 7.72 (d, J = 1.8 Hz, 1H), 7.77 (dd, J = 8.7, 1.8 Hz,

1H), 8.12 (d, J = 8.7 Hz, 1H). Anal. Calcd for C₂₇H₃₁N₃O₂ꢀ0.1H₂O: C,

75.18; H, 7.29; N, 9.74. Found: C, 74.96; H, 7.45; N, 9.53.

5.1.35.2. Step B. To a solution of 0.25 g (0.54 mmol) of tert-butyl

{[6-(hydroxycarbamimidoyl)-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-3-yl]methyl}carbamate in 10 mL of THF and

10 mL of ethyl acetate was added 0.26 g (1.62 mmol) of 1,1’-car-

bonyldiimidazole, and the resulting mixture was stirred at reﬂux

for 3 h. The reaction mixture was poured into 50 mL of 0.1 M aque-

ous citric acid solution and extracted with 2:1 ethyl acetate/THF.

The extract was washed with brine, dried over anhydrous magne-

sium sulfate and concentrated in vacuo. The residual solid was

crystallized from ethyl acetate/hexanes to afford 0.24 g (90%) of

tert-butyl

{[4-(4-methylphenyl)-2-(2-methylpropyl)-6-(5-oxo-

5.1.34. 1-[4-(4-Methylphenyl)-2-(2-methylpropyl)-6-(2H-

tetrazol-5-yl)quinolin-3-yl]methanamine dihydrochloride

(19a)

4,5-dihydro-1,2,4-oxadiazol-3-yl)quinolin-3-yl]methyl}carbamate

as an white powder. ¹H NMR (300 MHz, DMSO-d₆) d: 0.99 (d,

J = 6.6 Hz, 6H), 1.38 (s, 9H), 2.30–2.50 (m, 1H), 2.45 (s, 3H), 2.85

(d, J = 6.8 Hz, 2H), 4.05 (d, J = 4.2 Hz, 2H), 7.08 (t, J = 4.2 Hz, 1H),

7.31 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 1.9 Hz,

1H), 8.03 (dd, J = 8.8, 1.9 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 13.21

(br s, 1H).

5.1.34.1. Step A. A mixture of 0.60 g (1.4 mmol) of tert-butyl {[6-

cyano-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]

methyl}carbamate (18), 0.18 g (2.8 mmol) of sodium azide and

0.30 g (5.6 mmol)of ammoniumchloride in 10 mLof DMSOwas stir-

red at 70 °C for 48 h. The reaction mixture was partitioned between

ethyl acetate and 0.1 M hydrochloric acid. The organic layer was

washed with brine, dried over anhydrous magnesium sulfate and

concentrated in vacuo. The residue was crystallized from ethyl ace-

tate/hexanes to afford 0.38 g (57%) of tert-butyl {[4-(4-methyl-

phenyl)-2-(2-methylpropyl)-6-(2H-tetrazol-5-yl)quinolin-3-yl]

methyl}carbamate as a white powder. ¹H NMR (300 MHz, CDCl₃) d:

1.00 (d, J = 6.4 Hz, 6H), 1.39 (s, 9H), 2.25–2.45 (m, 1H), 2.47 (s, 3H),

2.86 (d, J = 7.0 Hz, 2H), 4.07 (d, J = 4.3 Hz, 2H), 7.09 (t, J = 4.3 Hz,

1H), 7.35 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 8.05 (d,

J = 1.8 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.28 (dd, J = 8.7, 1.8 Hz, 1H).

5.1.35.3. Step C. A mixture of 0.20 g (0.41 mmol) of tert-butyl

{[4-(4-methylphenyl)-2-(2-methylpropyl)-6-(5-oxo-4,5-dihydro-

1,2,4-oxadiazol-3-yl)quinolin-3-yl]methyl}carbamate and 5 mL of

4 M hydrogen chloride in 1,4-dioxane was stirred at room temper-

ature for 3 h. Concentration in vivo was followed by recrystalliza-

tion from methanol/diisopropyl ether to afford 0.14 g (74%) of

the title compound as pale yellow crystals. ¹H NMR (300 MHz,

DMSO-d₆) d: 1.04 (d, J = 6.6 Hz, 6H), 2.30–2.45 (m, 1H), 2.49 (s,

3H), 3.08 (d, J = 7.2 Hz, 2H), 3.97 (d, J = 5.3 Hz, 2H), 7.34 (d,

J = 7.9 Hz, 2H), 7.46 (d, J = 7.9 Hz, 2H), 7.83 (d, J = 1.8 Hz, 1H),

8.16 (dd, J = 8.9, 1.9 Hz, 1H), 8.25 (d, J = 8.9 Hz, 1H), 8.36 (br s,

3H), 13.33 (br s, 1H). HRMS (ESI) calcd for C₂₃H₂₄N₄O₂(M+H)⁺m/z

389.1972, found m/z 389.1956. HPLC purity: 99.96% (k 220 nm),

99.92% (k 254 nm).

5.1.34.2. Step B. A mixture of 0.17 g (0.36 mmol) of tert-butyl {[4-

(4-methylphenyl)-2-(2-methylpropyl)-6-(2H-tetrazol-5-yl)quino-

lin-3-yl]methyl}carbamate and 5 mL of 4 M hydrogen chloride in

4496

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

5.1.36. 4-(4-Methylphenyl)-2-(2-methylpropyl)-6-(3-

oxopiperazin-1-yl)quinoline-3-carbonitrile (20)

ammonia, 50 mL of methanol and 10 mL of THF was stirred under

hydrogen atmosphere at 0.5 MPa for 6 h at room temperature. The

reaction mixture was ﬁltered, and the ﬁltrate was concentrated in

vacuo. The residue was dissolved in 50 mL of THF, and then 0.57 g

(2.6 mmol) of di-tert-butyl dicarbonate was added. The resulting

mixture was stirred at room temperature for 30 min and concen-

trated in vacuo. Puriﬁcation by column chromatography (silica gel,

eluting at a gradient of 20–50% ethyl acetate in hexanes) afforded

0.75 g (77%) of the title compound as a yellow powder: mp 57 °C.

¹H NMR (300 MHz, CDCl₃) d: 1.01 (d, J = 6.6 Hz, 6H), 1.41 (s, 9H),

1.43 (s, 9H), 2.20–2.40 (m, 1H), 2.46 (s, 3H), 2.89 (d, J = 7.2 Hz, 2H),

3.10 (q, J = 5.3 Hz, 2H), 3.31 (q, J = 5.3 Hz, 2H), 4.20–4.25 (m, 3H),

4.31 (t, J = 3.6 Hz, 1H), 4.72 (br s, 1H), 6.17 (d, J = 2.5 Hz, 1H), 7.05

(dd, J = 9.0, 2.5 Hz, 1H), 7.11 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz,

2H), 7.85 (d, J = 9.0 Hz, 1H). MS m/z 563 (M+H)⁺.

To 30 mL of 1,4-dioxane under nitrogen atmosphere were

added 0.12 g (0.19 mmol) of rac-BINAP and 0.014 g (0.063 mmol)

of palladium acetate. After being stirred at 40 °C for 30 min,

0.50 g (1.25 mmol) of 6-bromo-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinoline-3-carbonitrile (7b), 0.25 g (2.5 mmol) of pipera-

zin-2-one and 0.57 g (1.75 mmol) of cesium carbonate were

added. The resulting mixture was stirred at 80 °C for 24 h. The

reaction mixture was cooled to room temperature, diluted with

ethyl acetate and ﬁltered through Celite. The ﬁltrate was washed

sequentially with water and brine, dried over anhydrous magne-

sium sulfate and concentrated in vacuo. Puriﬁcation by column

chromatography (silica gel, eluting at a gradient of 0–5% methanol

in ethyl acetate) afforded 0.35 g (71%) of the title compound as a

yellow powder: mp 237–238 °C. ¹H NMR (300 MHz, CDCl₃) d:

1.04 (d, J = 6.6 Hz, 6H), 2.25–2.45 (m, 1H), 2.50 (s, 3H), 3.07 (d,

J = 7.4 Hz, 2H), 3.45–3.60 (m, 4H), 3.81 (s, 2H), 6.22 (s, 1H), 6.83

(d, J = 2.8 Hz, 1H), 7.30–7.45 (m, 4H), 7.53 (dd, J = 9.4, 2.7 Hz, 1H),

8.04 (d, J = 9.4 Hz, 1H). MS m/z 399 (M+H)⁺.

5.1.39. Ethyl ({2-[(tert-butoxycarbonyl)amino]ethyl}[3-{[(tert-

butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]amino)(oxo)acetate (23)

To a vigorously stirred mixture of 0.50 g (0.89 mmol) of tert-bu-

tyl {[6-({2-[(tert-butoxycarbonyl)amino]ethyl}amino)-4-(4-meth-

ylphenyl)-2-(2-methylpropyl)quinolin-3-yl]methyl}carbamate

(22), 50 mL of ethyl acetate and 50 mL of saturated aqueous so-

dium bicarbonate solution was added 0.30 g (2.2 mmol) of ethyl

chloro(oxo)acetate at room temperature. After being stirred vigor-

ously for 30 min, the organic layer was separated, washed with

brine, dried over anhydrous magnesium sulfate and concentrated

in vacuo. Puriﬁcation by column chromatography (silica gel, elut-

ing at a gradient of 20–40% ethyl acetate in hexanes) afforded

0.53 g (90%) of the title compound as a white powder: mp 110–

114 °C. ¹H NMR (300 MHz, CDCl₃) d: 0.93 (t, J = 7.1 Hz, 3H), 1.03

(d, J = 6.6 Hz, 6H), 1.33 (s, 9H), 1.42 (s, 9H), 2.30–2.45 (m, 1H),

2.48 (s, 3H), 2.98 (d, J = 7.4 Hz, 2H), 3.31 (q, J = 6.0 Hz, 2H), 3.85–

3.90 (m, 2H), 3.90 (q, J = 7.1 Hz, 2H), 4.30 (br s, 3H), 4.75 (t,

J = 4.7 Hz, 1H), 7.08 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 2.1 Hz, 1H),

7.34 (d, J = 7.9 Hz, 2H), 7.56 (dd, J = 8.9, 2.1 Hz, 1H), 8.09 (d,

J = 8.9 Hz, 1H). MS m/z 663 (M+H)⁺.

5.1.37. 4-[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]piperazin-2-one (21a)

The title compound (0.18 g) was prepared from 0.32 g

(0.80 mmol) of 4-(4-methylphenyl)-2-(2-methylpropyl)-6-(3-

oxopiperazin-1-yl)quinoline-3-carbonitrile (20) using a procedure

similar to that described as step B for the synthesis of compound

9b as a pale yellow powder: mp 155–157 °C. ¹H NMR (300 MHz,

CDCl₃) d: 1.03 (d, J = 6.6 Hz, 6H), 1.55 (br s, 2H), 2.25–2.40 (m,

1H), 2.49 (s, 3H), 2.97 (d, J = 7.2 Hz, 2H), 3.35–3.45 (m, 2H), 3.45–

3.55 (m, 2H), 3.72 (s, 2H), 3.75 (s, 2H), 6.21 (d, J = 10.9 Hz, 1H),

6.50 (d, J = 2.8 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz,

2H), 7.37 (dd, J = 9.2, 2.8 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H). MS m/z

403 (M+H)⁺; HRMS (ESI) calcd for C₂₅H₃₀N₄O (M+H)⁺m/z

403.2492, found m/z 403.2478. HPLC purity: 97.72% (k 220 nm),

95.11% (k 254 nm).

5.1.38. tert-Butyl {[6-({2-[(tert-butoxycarbonyl)amino]ethyl}

amino)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-yl]

methyl}carbamate (22)

5.1.40. 1-[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]piperazine-2,3-dione (21b)

5.1.38.1. Step A. 6-[(2-Aminoethyl)amino]-4-(4-methylphenyl)-

2-(2-methylpropyl)quinoline-3-carbonitrile (2.2 g) was prepared

using a procedure similar to that described for the synthesis of

the synthesis of compound 8a from 1.0 g (2.6 mmol) of 6-bromo-

4-(4-methylphenyl)-2-(2-methylpropyl)quinoline-3-carbonitrile

(7b) instead of compound 7a and 0.44 mL (6.6 mmol) of ethylene-

diamine instead of tert-butanol as a crude yellow syrup, which was

used directly in the next step.

A mixture of 0.50 g (0.75 mmol) of ethyl ({2-[(tert-butoxycar-

bonyl)amino]ethyl}[3-{[(tert-butoxycarbonyl)amino]methyl}-4-

(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]amino)(oxo)

acetate (23) and 3 mL of TFA was stirred at room temperature for

30 min. The reaction mixture was concentrated in vacuo, and the

residue was partitioned between ethyl acetate and 10% aqueous

potassium carbonate solution. The organic layer was dried over

anhydrous potassium carbonate, and concentrated in vacuo. Puriﬁ-

cation by column chromatography (silica gel, eluting at a gradient

of 0–20% methanol in ethyl acetate) afforded 0.21 g (65%) of the ti-

tle compound as a pale yellow powder: mp 229–232 °C. ¹H NMR

(300 MHz, CDCl₃) d: 1.02 (d, J = 6.6 Hz, 6H), 1.66 (br s, 2H), 2.30–

2.45 (m, 1H), 2.46 (s, 3H), 3.01 (d, J = 7.2 Hz, 2H), 3.55–3.65 (m,

2H), 3.80 (s, 2H), 3.85–3.90 (m, 2H), 7.16 (d, J = 8.0 Hz, 2H), 7.18

(d, J = 2.3 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.57 (dd, J = 9.0, 2.3 Hz,

1H), 8.06 (d, J = 9.0 Hz, 1H), 8.08 (br s, 1H). MS m/z 417 (M+H)⁺;

HRMS (ESI) calcd for C₂₅H₂₈N₄O₂(M+H)⁺m/z 417.2285, found m/

z 417.2271. HPLC purity: 99.67% (k 220 nm), 99.42% (k 254 nm).

5.1.38.2. Step B. The obtained crude syrup was dissolved in

100 mL of THF, and then 0.70 g (3.2 mmol) of di-tert-butyl dicar-

bonate was added. The resulting mixture was stirred at room tem-

perature for 1 h and concentrated in vacuo. Puriﬁcation by column

chromatography (silica gel, eluting at a gradient of 20–40% ethyl

acetate in hexanes) afforded 0.85 g (70%) of tert-butyl (2-{[3-cya-

no-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]amino}

ethyl)carbamate as a yellow powder. ¹H NMR (300 MHz, CDCl₃) d:

1.03 (d, J = 6.6 Hz, 6H), 1.43 (s, 9H), 2.25–2.40 (m, 1H), 2.48 (s, 3H),

3.03 (d, J = 7.4 Hz, 2H), 3.15 (q, J = 5.5 Hz, 2H), 3.36 (q, J = 5.5 Hz,

2H), 4.57 (br, 1H), 4.74 (br, 1H), 6.49 (d, J = 2.5 Hz, 1H), 7.18 (dd,

J = 9.1, 2.5 Hz, 1H), 7.30–7.40 (m, 4H), 7.89 (d, J = 9.1 Hz, 1H). MS

m/z 459 (M+H)⁺.

5.1.41. tert-Butyl N-[3-{[(tert-butoxycarbonyl)amino]methyl}-

4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]glycinate

(24)

5.1.41.1. Step A. tert-Butyl N-[3-cyano-4-(4-methylphenyl)-2-

(2-methylpropyl)quinolin-6-yl]glycinate (2.0 g) was prepared

using a procedure similar to that described for the synthesis of

compound 8a from 2.0 g (5.3 mmol) of 6-bromo-4-(4-methyl-

5.1.38.3. Step C. A mixture of 0.80 g (1.7 mmol) of tert-butyl (2-

{[3-cyano-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]

amino}ethyl)carbamate, ca. 2 g of Raney nickel, 5 mL of 25% aqueous

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

4497

phenyl)-2-(2-methylpropyl)quinoline-3-carbonitrile (7b) instead

of compound 7a and 1.52 g (11.6 mmol) of tert-butyl glycinate in-

tead of tert-butanol as yellow crystals: mp 128–129 °C. ¹H NMR

(300 MHz, CDCl₃) d: 1.03 (d, J = 6.6 Hz, 6H), 1.44 (s, 9H), 2.25–

2.40 (m, 1H), 2.49 (s, 3H), 3.04 (d, J = 7.2 Hz, 2H), 3.71 (d,

J = 5.2 Hz, 2H), 4.81 (t, J = 5.2 Hz, 1H), 6.45 (d, J = 2.6 Hz, 1H), 7.22

(dd, J = 9.0, 2.6 Hz, 1H), 7.30–7.40 (m, 4H), 7.92 (d, J = 9.0 Hz, 1H).

MS m/z 430 (M+H)⁺.

NMR (300 MHz, CDCl₃) d: 1.02 (d, J = 6.6 Hz, 6H), 1.42 (s, 9H), 2.25–

2.45 (m, 1H), 2.48 (s, 3H), 2.98 (d, J = 7.2 Hz, 2H), 4.16 (s, 2H), 4.28

(s, 2H), 4.30 (br s, 3H), 6.13 (s, 1H), 7.12 (d, J = 7.8 Hz, 2H), 7.14 (d,

J = 2.3 Hz, 1H), 7.34 (d, J = 7.8 Hz, 2H), 7.62 (dd, J = 9.0, 2.3 Hz, 1H),

8.11 (d, J = 9.0 Hz, 1H). MS m/z 517 (M+H)⁺.

5.1.43.2. Step B. A mixture of 0.14 g (0.27 mmol) of tert-butyl

{[6-(2,5-dioxopiperazin-1-yl)-4-(4-methylphenyl)-2-(2-methyl-

propyl)quinolin-3-yl]methyl}carbamate and 5 mL of 4 M hydrogen

chloride in 1,4-dioxane was stirred at room temperature for 1 h.

The reaction mixture was concentrated in vacuo. Recrystallization

from aqueous ethanol gave 0.11 g (83%) of the title compound as a

pale yellow powder: mp 250 °C (decomp). ¹H NMR (300 MHz,

DMSO-d₆) d: 1.03 (d, J = 6.6 Hz, 6H), 2.25–2.40 (m, 1H), 2.46 (s,

3H), 3.13 (d, J = 7.0 Hz, 2H), 3.92 (s, 2H), 3.98 (d, J = 5.5 Hz, 2H),

4.21 (s, 2H), 7.23 (d, J = 1.9 Hz, 1H), 7.34 (d, J = 7.9 Hz, 2H), 7.45

(d, J = 7.9 Hz, 2H), 7.88 (d, J = 8.8 Hz, 1H), 8.24 (dd, J = 8.8, 1.9 Hz,

1H), 8.30 (br s, 1H), 8.35 (br s, 3H). MS m/z 417 (M+H)⁺; HRMS

(ESI) calcd for C₂₅H₂₈N₄O₂(M+H)⁺m/z 417.2285, found m/z

417.2272. Anal. Calcd for C₂₅H₂₈N₄O₂ꢀ2HCl: C, 61.35; H, 6.18; N,

11.45; Cl, 14.49. Found: C, 61.11; H, 6.22; N, 11.33; Cl, 14.44. HPLC

purity: 99.90% (k 220 nm), 99.93% (k 254 nm).

5.1.41.2. Step B. The title compound (0.96 g) was prepared from

1.4 g (3.3 mmol) of tert-butyl N-[3-cyano-4-(4-methylphenyl)-2-

(2-methylpropyl)quinolin-6-yl]glycinate using a procedure similar

to that described as step C for the synthesis of compound 22 as pale

yellow crystals: mp 181–185 °C. ¹H NMR (300 MHz, CDCl₃) d: 1.01

(d, J = 6.6 Hz, 6H), 1.41 (s, 9H), 1.44 (s, 9H), 2.20–2.40 (m, 1H), 2.48

(s, 3H), 2.89 (d, J = 7.4 Hz, 2H), 3.65 (d, J = 5.3 Hz, 2H), 4.22 (d,

J = 4.9 Hz, 2H), 4.30 (br, 1H), 4.42 (t, J = 4.9 Hz, 1H), 6.14 (d,

J = 2.5 Hz, 1H), 7.08 (dd, J = 9.0, 2.5 Hz, 1H), 7.11 (d, J = 8.0 Hz,

2H), 7.31 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 9.0 Hz, 1H). MS m/z 534

(M+H)⁺.

5.1.42. tert-Butyl N-[(benzyloxy)carbonyl]glycyl-N-[3-{[(tert-

butoxycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]glycinate (25)

5.2. Theoretical calculation

Docking studies were performed using GOLD software version

3.0 (Cambridge Crystallographic Data Centre, www.ccdc.cam.ac.uk),

based on the co-crystal structure model of DPP-4 in complex with

compound 2. After the automatic docking into DPP-4 using GOLD,

the conformations of the substituents in the docked compounds

were aligned to the corresponding moiety of compound 2 and then

energy-minimized at the MMFF94s force ﬁeld using MOE 2005.06

(Chemical Computing Group Inc., www.chemcomp.com).

To an ice-cooled mixture of 0.78 g (3.75 mmol) of N-[(benzyl-

oxy)carbonyl]glycine, 0.1 mL of DMF and 20 mL of THF was added

0.33 mL (3.7 mmol) of oxalyl chloride in a dropwise manner. The

resulting mixture was stirred at 0 °C for 1 h, and added dropwise

to a mixture of 0.40 g (0.75 mmol) of tert-butyl N-[3-{[(tert-butox-

ycarbonyl)amino]methyl}-4-(4-methylphenyl)-2-(2-methylpro-

pyl)quinolin-6-yl]glycinate (24) and 0.3 mL of pyridine in 100 mL

of THF at room temperature. After being stirred at room tempera-

ture for 1 h, 10 mg (0.08 mmol) of DMAP was added, and the

resulting mixture was stirred at room temperature for 60 h. The

reaction mixture was partitioned between ethyl acetate and water.

The organic layer was washed sequentially with water, saturated

aqueous sodium bicarbonate solution and brine, dried over anhy-

drous magnesium sulfate and concentrated in vacuo. Puriﬁcation

by column chromatography (silica gel, eluting at a gradient of

25–50% ethyl acetate in hexanes) afforded 0.47 g (87%) of the title

compound as a white powder: mp 81–83 °C. ¹H NMR (300 MHz,

CDCl₃) d: 1.04 (d, J = 6.8 Hz, 6H), 1.37 (s, 9H), 1.42 (s, 9H), 2.25–

2.45 (m, 1H), 2.46 (s, 3H), 3.00 (d, J = 7.4 Hz, 2H), 3.69 (d,

J = 4.3 Hz, 2H), 4.22 (s, 2H), 4.32 (br, 3H), 5.04 (s, 2H), 5.60 (t,

J = 4.3 Hz, 1H), 7.11 (d, J = 7.9 Hz, 2H), 7.25–7.40 (m, 8H), 7.60

(dd, J = 8.9, 2.1 Hz, 1H), 8.11 (d, J = 8.9 Hz, 1H). MS m/z 725 (M+H)⁺.

5.3. Biology

5.3.1. In vitro DPP-4, DPP-2, DPP-8 and DPP-9 enzyme assay

Human DPP-4 was partially puriﬁed from Caco-2 cells (ATCC

No. HTB-37). The compounds (1

were added to 79 L of assay buffer (0.25 M Tris–HCl pH 7.5, 0.25%

bovine serum albumin, 0.125% CHAPS) and mixed with 20 L of

lL in DMSO) at each concentration

l

human DPP-4 fraction. After the mixture was incubated at room

temperature for 15 min, the reaction was initiated by adding

100

l

L of 1 mM of Gly-Pro-pNAꢀTos as a substrate and run for

60 min at 37 °C.

Rat DPP-2 was partially puriﬁed from rat kidney according to

the method previously reported.⁴³Compounds (1

l

L) dissolved in

L of 1 M 3,3-

L of the DPP-2 frac-

tion. After the mixture was incubated at room temperature for

DMSO was mixed with 29

lL of distilled water, 10 l

dimethylglutamic acid buffer (pH 5.5), and 10

l

5.1.43. 1-[3-(Aminomethyl)-4-(4-methylphenyl)-2-(2-

methylpropyl)quinolin-6-yl]piperazine-2,5-dione

20 min, the reaction was initiated by adding 50 lL of 1 mM of H-

dihydrochloride (1)

Lys-Ala-pNAꢀ2HCl and run at 37 °C for 60 min.

5.1.43.1. Step A. A mixture of 0.45 g (0.62 mmol) of tert-butyl N-

[(benzyloxy)carbonyl]glycyl-N-[3-{[(tert-butoxycarbonyl)amino]

methyl}-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]

glycinate (25) and 40 mg of 10% Pd/C in 100 mL of ethanol was stir-

red under atmospheric hydrogen at room temperature for 24 h. The

reaction mixture was ﬁltered through Celite, and the ﬁltrate was

concentrated in vacuo. The residue was dissolved in 10 mL of etha-

nol, and the resulting solution was stirredat reﬂux for 17 h. The reac-

tion mixture was concentrated in vacuo, and the residue was

puriﬁed by column chromatography (silica gel, eluting at a gradient

of 0–10% methanol in ethyl acetate) followed by crystallization from

ethyl acetate to afford 0.16 g (44%) of tert-butyl {[6-(2,5-dioxopi-

perazin-1-yl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-3-

yl]methyl}carbamate as pale yellow crystals: mp 128–129 °C. ¹H

Human DPP-8 and DPP-9 were puriﬁed, respectively by afﬁn-

ity chromatography from the 293-F cells expressing each FLAG-

tagged protein.

1

l

L

of compounds dissolved in DMSO was

L of distilled water, 10 L of 1 M Tris–HCl buffer

L of the enzyme fraction. After the mixture

mixed with 29

l

(pH 7.5), and 10

l

was incubated at room temperature for 20 min, the reaction

was initiated by adding 50

l

L of 2 mM of Gly-Pro-pNAꢀTos for

DPP-8 or 4 mM of Gly-Pro-pNAꢀTos for DPP-9 and run at 37 °C

for 90 min.

Absorbance at 405 nm of each reaction mixture was measured

using a microplate reader at the initial time and the end of the

reaction. The well containing substrate alone was used as a basal

control. The well containing the substrate and the enzyme without

the compound was used as a total reaction.

4498

H. Maezaki et al. / Bioorg. Med. Chem. 19 (2011) 4482–4498

5.3.2. Effects of single administration of compound 1 on glucose

tolerance in female Wistar fatty rats

References and notes

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Acknowledgments

The authors thank Drs. Yu Momose, Junichi Sakamoto, Hiroshi

Nara, Yuji Ishihara, Kaneyoshi Kato, Shigenori Ohkawa, Takashi

Sohda and Akio Miyake for helpful discussions; Dr. Yukihiro Ikeda

for measuring solubility; and Dr. Akihiko Sumita for measuring

metabolic stabilities. We also thank ALS, which is supported by

the Director, Ofﬁce of Science, Ofﬁce of Basic Energy Sciences,

Materials Sciences Division of the U.S. Department of Energy under

contract DE-AC03-76SF00098 at Lawrence Berkeley National

Laboratory.

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