Scholarly article on (R)-tert-butyl 1-(4-ethylbenzylamino)-3-(1H-indol-3-yl)-1-thioxopropan-2-ylcarbamate 937247-96-6 from Journal of Medicinal Chemistry p. 5790

5790

J. Med. Chem. 2007, 50, 5790-5806

Toward Potent Ghrelin Receptor Ligands Based on Trisubstituted 1,2,4-Triazole Structure. 2.

Synthesis and Pharmacological in Vitro and in Vivo Evaluations

Aline Moulin,^†Luc Demange,^†Gilbert Berge´,^†Didier Gagne,^†Joanne Ryan,^†Delphine Mousseaux,^†Annie Heitz,^§

Daniel Perrissoud,^#Vittorio Locatelli,^|Antonio Torsello,^|Jean-Claude Galleyrand,^†Jean-Alain Fehrentz,*^,†,‡and

Jean Martinez^†,‡

Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247, CNRS, UniVersite´ Montpellier I et UniVersite´ Montpellier II, BP 14491, Faculte´

de Pharmacie, 15 AVenue Charles Flahault, 34093 Montpellier Cedex 5, France, CBS, UMR 5048, 29 Rue de NaVacelles, 34090 Montpellier,

France, Zentaris GmbH, Weismuellerstrasse 50, 60314 Frankfurt am Main, Germany, and Department of Experimental Medicine, School of

Medicine, UniVersity of Milano-Bicocca, Via Cadore 48, 20052 Monza (MI), Italy

ReceiVed April 18, 2007

A series of ghrelin receptor ligands based on the trisubstituted 1,2,4-triazole structure were synthesized and

evaluated for their in vitro binding and biological activity. In this study, we explored the significance of the

aminoisobutyryl (Aib) moiety, a common feature in numerous growth hormone secretagogues described in

the literature. Potent agonist and antagonist ligands of the growth hormone secretagogue receptor type 1a

(GHS-R1a) were obtained, i.e., compounds 41 (JMV2894) and 17 (JMV3031). The best compounds were

evaluated for their in vivo activity on food intake, after sc injection in rodents. Among the tested compounds,

few of them were able to stimulate food intake and some others, i.e., compounds 4 (JMV2959), 17, and 52

(JMV3021), acted as potent in vivo antagonist of hexarelin-stimulated food intake. These compounds did

not stimulate growth hormone secretion in rats and furthermore did not antagonize growth hormone secretion

induced by hexarelin, revealing that it is possible to modulate food intake without altering growth hormone

secretion.

Introduction

Growth hormone secretagogues (GHSs^a) or ghrelin receptor

agonists are compounds able to stimulate growth hormone (GH)

release in the pituitary. They were discovered by Bowers,¹and

they act in a totally independent pathway from GHRH. Their

biological target named growth hormone secretagogue receptor

type 1a (GHS-R1a)²and its natural ligand, ghrelin,³were later

identified. GHS-R1a agonists are able to substitute direct

administration of GH for treatment of various disorders caused

by GH deficiency such as growth failure. Several peptide and

non-peptide agonists of the ghrelin receptor have been reported

Figure 1. Peptide and pseudopeptide ghrelin receptor agonists.

(Figure 1). We have recently described a new family of ghrelin

receptor ligands based on trisubstituted 1,2,4-triazoles.⁴In this

study, we explore the significance of the R-aminoisobutyryl

(Aib) moiety that is a common feature in almost all small GHS

molecules described in the literature (Figure 2).^5-8

Chemistry

To study the significance of Aib, 1,2,4-triazoles D were

prepared as depicted in the general synthetic Scheme 1 and

various substitutes of Aib were introduced and deprotected when

necessary to yield final compounds E. Starting from Boc-D-

* To whom correspondance should be addressed. Phone: 33 4 67

54 86 51. Fax: 33 4 67 54 86 54. E-mail: jean-alain.fehrentz@univ-

montp1.fr.

^†Universite´ Montpellier I et Universite´ Montpellier II.

^§CBS.

^#Zentaris GmbH.

^|University of Milano-Bicocca.

^‡Equal contributors.

^aAbbreviations: GHS-R1a, growth hormone secretagogue receptor type

Figure 2. Structures of some known GHSs containing Aib residue.

1a; GH, growth hormone; GHS, growth hormone secretagogue; GHRH,

growth hormone releasing hormone; Aib, R-aminoisobutyric acid; CHO,

Chinese hamster ovary; BOP, (benzotriazol-1-yloxy)tris(dimethylamino)-

phosphonium hexafluorophosphate; DME, ethylene glycol dimethyl ether;

DCM, dichloromethane; DMF, dimethylformamide; NMM, N-methylmor-

pholine; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid.

Trp-OH, the first point of diversity was introduced by coupling

with primary amines to yield intermediates A. After treatment

with the Lawesson’s reagent, the corresponding amides A were

Published on Web 10/10/2007

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5791

Scheme 1. General Synthetic Pathway to Trisubstituted 1,2,4-Triazoles^a

^a(a) BOP, H₂N-R₁, NMM, DCM; (b) Lawesson’s reagent, DME, 85 °C; (c) H₂N-HN-COR₂, Hg(OAc)₂, room temp, THF; (d) HCl, AcOEt.

Scheme 2. Synthetic Route to Compound 11

Results and Discussion

The synthesized compounds were tested for their ability to

displace ¹²⁵I-His⁹-ghrelin from the cloned hGHS-1a receptor

transiently expressed in LLC PK-1 cells. Binding affinities of

human ghrelin and MK-0677 obtained using these cells were

in accordance with the literature. The biological in vitro activity

of the compounds (10^-5M) was then evaluated on intracellular

calcium mobilization [Ca²⁺

] using CHO cells transiently

i

expressing GHS-R1a. Results are expressed as a percent of the

maximal response induced by 10^-7M ghrelin (Table 1). The

best compounds were tested in vivo for their ability to stimulate

food intake or to inhibit hexarelin-stimulated food intake.

Compounds 1 and 2 containing the Aib residue are reported

as reference compounds.⁴Aib was first replaced by glycine. A

loss of affinity was observed (compounds 3-6) but was not

dramatic, the corresponding IC₅₀ranging from 30 to 150 nM

(see Figure 3 for compound 4). Replacement of 4-methoxy-

benzyl or 2,4-dimethoxybenzyl in the R₁group by 4-ethylbenzyl,

phenyl, 2,4-dimethoxyphenyl, or 4-ethylphenyl led to com-

pounds 7-10 having decreased affinity. Introduction of a

“reduced bond” replacing the amide bond led to compound 11

with an IC₅₀of 190 ( 10 nM. Substitution of glycine by alanine

(reintroduction of one methyl group of Aib) did not lead to better

compounds (12, 13). Replacement of glycine by a â-alanine

led to an analogue that exhibited an IC₅₀of 59 ( 16 nM

(compound 14). Replacement of glycine with phenylalanine

(compound 15) failed to increase affinity for the GHS-R1a.

Because the Aib moiety is quite a hindered molecule, we decided

to rigidify this part of the molecule by introducing a cyclic

residue. (L)-Proline and (D)-proline were introduced with

4-methoxybenzyl as the R₁group and 1H-indole-3-ylethyl or

phenethyl as the R₂group. Compounds 16-19 exhibited

affinities comparable to those of compounds 1 and 2, compound

16 being the most potent (IC₅₀) 9 ( 1 nM). Because the

L-configuration for proline seemed better for interaction with

the GHS-1a receptor, we explored in this series the R₁

substituent with 2,4-dimethoxybenzyl (compounds 20 and 21),

4-ethylbenzyl (compound 22), phenyl (compound 23), and

4-ethylphenyl groups (compound 24). As previously observed,

the 2,4-dimethoxybenzyl group was well tolerated whereas

4-ethylbenzyl and 4-ethylphenyl groups led to less potent

ligands. An inactive compound was obtained with a phenyl

substituent in the R₁position (compound 23). Introduction of a

hydroxyl group on the pyrrolidine cycle of proline (hydrox-

yproline) caused a significant decrease of binding affinity

(compound 25) when compared with compound 18. The initial

studies with the proline scaffold validated the constrained

analogue approach and suggested to us to further explore other

converted into thioamides B. The reaction of B with different

hydrazides in the presence of a thiophile metal salt yielded

trisubstituted 1,2,4-triazoles C. After deprotection of the Boc

protecting group, the generated primary amine D reacted with

acids, aldehydes, or sulfonyl chlorides to yield respectively

amides, alkylamines, or sulfonylamides E. Starting from Boc-

D-Trp-OH and within a few steps, we describe a route for the

synthesis of 1,2,4-triazole derived compounds presenting three

points of diversity, thus allowing a structure-activity relation-

ship study.

In a previous study, we have shown that compound JMV1843

(Figure 2) was a very potent compound in stimulating GH

secretion in rodents and dogs and by oral route in man.^8-10All

attempts to replace the Aib residue resulted in less potent

compounds, indicating the significance of this residue for the

stimulation of GH secretion. Recently, we reported that 1,2,4-

triazole was a good scaffold to obtain potent GHS-R1a ligands

of the ghrelin receptor, leading to agonists, partial agonists, or

antagonists.⁴In this study, we showed that a benzyl group and

particularly 4-methoxybenzyl or 2,4-dimethoxybenzyl groups

in position 4 of the triazole ring and a two-carbon chain bearing

a phenyl or an indole group in position 5 of the triazole led to

potent ligands of the GHS-R1a. Starting from these results, we

investigated the replacement of the Aib moiety in the R₃position

by natural or unnatural R-amino acids, piperidylcarboxyl, and

piperazinecarboxyl groups. All these compounds E were

obtained by coupling a carboxylic acid function on the primary

amine D except for compound 11, which was synthesized by

reductive amination of the corresponding D product with Boc-

glycinal as depicted in Scheme 2. All final compounds were

purified by reversed-phase HPLC. The purity assessed by

analytical reversed-phase C18 HPLC was found to be greater

than 95% for target compounds and greater than 98% for key

target compounds (compounds 4, 16, 17, 35, 39, 40, 41, 52),

and the structure was confirmed by MS (electrospray), ¹H NMR,

and ¹³C NMR for the most interesting compounds.

5792 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

Table 1. Binding Affinity Constants and Biological Activities of Trisubstituted 1,2,4-Triazoles

% of max

[Ca²⁺

]

i

IC₅₀

response

EC₅₀

K_b

compd

R₁

R₂

R₃

aminoisobutyryl

glycyl

(nM)^a

at 10 µM^b

(nM)^c

(nM)^d

1

2

3

4

5

benzyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

15 ( 5

73 ( 13

9 ( 11

2 ( 3

0

64 ( 14

4-methoxybenzyl

2,4-dimethoxybenzyl

4-ethylbenzyl

6 ( 3

4 ( 1

ND^f

138 ( 23

32 ( 3

38 ( 6

154 ( 44

759 ( 38

>1000

19 ( 6

27 ( 10

ND^f

6

7

glycyl

phenethyl

0

ND^f

8

phenyl

phenethyl

glycyl

7 ( 1

27 ( 11

4 ( 1

59 ( 12

13 ( 5

1 ( 1

117 ( 15

0

2 ( 2

7 ( 5

5 ( 3

3 ( 2

0

ND^f

9

2,4-dimethoxyphenyl

4-ethylphenyl

2,4-dimethoxybenzyl

4-methoxybenzyl

4-ethylbenzyl

phenethyl

1H-indole-3-ylethyl

phenethyl

glycyl

2-aminoethyl

L-alaninyl

ND^f

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

ND^f

190 ( 10

113 ( 29

275 ( 50

59 ( 16

175 ( 10

9 ( 1

279 ( 42

ND^f

L-alaninyl

ND^f

4-methoxybenzyl

2,4-dimethoxybenzyl

4-ethylbenzyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

phenyl-methyl

phenethyl

1H-indole-3-ylethyl

phenethyl

1H-indole-3-ylethyl

phenethyl

3-aminopropanoyl (â-Ala)

L-phenylalaninyl

L-prolyl

D-prolyl

L-prolyl

D-prolyl

L-prolyl

256 ( 55

24 ( 7

6 ( 2

28 ( 9

7 ( 2

9.5 ( 2

32 ( 4

13 ( 3

8 ( 2

5.6 ( 2

44 ( 6

11 ( 4

32 ( 10

ND^f

0

490 ( 140

>1000

2 ( 2

24

phenyl

4-ethylphenyl

L-prolyl

ND^f

350 ( 10

173 ( 98

6.5 ( 1

13 ( 1

5 ( 5

87 ( 23

48 ( 19

50 ( 18

15

11 ( 6

20 ( 9

89 ( 1

11 ( 1

2 ( 1

7 ( 1

0

ND^f

4-methoxybenzyl

2,4-dimethoxybenzyl

benzyl

2-methyl pyridine

4-methoxybenzyl

4-ethylbenzyl

2,4-dimethoxybenzyl

4-methoxybenzyl

2,4-dimethoxybenzyl

4-ethylbenzyl

4-methoxybenzyl

2,4-dimethoxybenzyl

benzyl

phenyl

4-ethylphenyl

4-ethylbenzyl

2,4-dimethoxyphenyl

2-methylpyridine

(thiophen-2-yl)methyl

(furan-2-yl)methyl

4-methoxybenzyl

2,4-dimethoxybenzyl

4-ethylbenzyl

(2S,4R)-hydroxyprolyl

piperidyl-3-carboxyl^e

L-piperidyl-3-carboxyl

D-piperidyl-3-carboxyl

L-piperidyl-3-carboxyl

piperidyl-2-carboxyl^e

L-piperidyl-2-carboxyl

D-piperidyl-2-carboxyl

isonipecotyl

ND^f

5.3 ( 3

9.9 ( 0.1

ND^f

>1000

10 ( 2

2.5 ( 1

16 ( 5

10 ( 1

7 ( 1

36 ( 1.4

130 ( 1

53 ( 13

11 ( 2

ND^f

18 ( 8

27 ( 8

8 ( 1

53 ( 7

ND^f

29 ( 11

23 ( 5

3 ( 2

0

134 ( 4

375 ( 6

0.3 ( 0.2

0.6 ( 0.3

0.5 ( 0.3

0.9 ( 0.7

24 ( 2

ND^f

93 ( 1

93 ( 4

77 ( 4

95 ( 14

75 ( 10

98 ( 11

100 ( 0

101 ( 13

100 ( 0

78

93 ( 13

100 ( 0

37 ( 9

42 ( 11

2

3.0 ( 1.3

1.6 ( 0.7

0.6 ( 0.0

6.1 ( 0.0

5.4 ( 2.4

ND^f

468 ( 13

70 ( 28

96 ( 18

19 ( 6

63 ( 3

>1000

2.3 ( 1.1

15 ( 1.1

7.7 ( 1.2

14 ( 4

isonipecotyl

ND^f

255 ( 66

560 ( 31

18 ( 3

ND^f

183 ( 13

8.7 ( 1.1

12 ( 1.2

piperazine-2-carboxyl^e

tetrahydro-2H-pyran-4-carboxyl

cyclohexylcarboxyl

54 ( 7

32 ( 13

35 ( 12

178 ( 47

6 ( 2

5 ( 1

31 ( 17

68 ( 20

0

19.6 ( 6

143 ( 27

4-methoxybenzyl

phenethyl

51 ( 8

18 ( 6

17 ( 7

390 ( 77

^aSpecific binding was determined by incubation of membranes from GHS-R1a transfected LLC cells with ¹²⁵I-His⁹-ghrelin in the presence of increasing

concentrations of compounds. ^bThe activation percentage for each compound (10^-5M) was assessed relative to the maximal response, obtained with ghrelin

at 10^-7M (100%). ^cThe signaling through the GHS-R1a, as determined by the accumulation of intracellular calcium and thus fluorescence output, was

measured in the presence of increasing concentrations of agonists. ^dThe ability of the antagonists to inhibit ghrelin signaling through the GHS-R1a (measurement

of flurorescence output) was assessed using Schild plots, with increasing concentrations of ghrelin, alone, or in the presence of antagonist compound at 10^-8

10^-7, or 10^-6M. ^eRacemic mixture. ^fND: not determined.

,

heterocyclic templates. We then explored the possibility of

extending the cycle size from 5 to 6 atoms, switching from

proline to piperidine moiety and we also studied the influence

of the nitrogen position in the cycle. Piperidyl-2-, piperidyl-3-,

and piperidyl-4-carboxylic acids were introduced in place of

the Aib moiety. Compounds containing the R₁optimized groups

(benzyl, 4-methoxybenzyl and 2,4-dimethoxybenzyl groups)

with piperidyl-2-carboxyl and piperidyl-3-carboxyl substituents

in the R₃position exhibited high affinity constants in the range

of 10-50 nM (compounds 26, 27, 29-31, 33-36) with the

exception of compound 37. No real influence of the piperidyl

configuration was observed (compounds 30, 31 and 34, 36).

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5793

Figure 6. Ability of the antagonists to inhibit ghrelin signaling through

the GHS-R1a (measurement of flurorescence output) was determined

using increasing concentrations of compound 4 (0), 16 (B), or 17 (∆)

in the presence of a fixed concentration of ghrelin (10^-7M).

Figure 3. Specific binding was determined by incubation of mem-

branes from GHS-R1a transfected LLC cells with ¹²⁵I-His⁹-ghrelin in

the presence of increasing concentrations of compound 4 (0), 16 (B),

or 17 (∆).

Figure 7. Ability of the antagonists to inhibit ghrelin signaling through

the GHS-R1a (measurement of flurorescence output) was assessed using

Schild plots, with increasing concentrations of ghrelin, alone (9), or

Figure 4. Specific binding was determined by incubation of mem-

branes from GHS-R1a transfected LLC cells with ¹²⁵I-His⁹-ghrelin in

the presence of increasing concentrations of compound 41 (0), 42 (B),

or 43 (∆).

in the presence of compound 4 at 10^-8M (0), 10^-7M (B), or 10^-6

(O).

M

Figure 8. Ability of the antagonists to inhibit ghrelin signaling through

the GHS-R1a (measurement of flurorescence output) was assessed using

Schild plots, with increasing concentrations of ghrelin, alone (9), or

in the presence of compound 17 at 10^-8M (0), 10^-7M (B), or 10^-6

M (O).

Figure 5. Signaling through the GHS-R1a, as determined by the

accumulation of intracellular calcium and thus fluorescence output, was

measured in the presence of increasing concentrations of agonists,

compounds 31 (0), 39 (B), and 40 (∆).

When the benzyl group in R₁position was substituted with a

4-ethyl group, a decrease in binding affinity was observed

(compounds 32 and 38). Introduction of a nitrogen in position

2 of the R₁benzyl group led to a complete loss of affinity

(compound 28). Compounds containing the isonipecotyl moiety

in the R₃substituent exhibited affinities in the subnanomolar

range (compounds 39-42, Figure 4 for compound 42). The

affinity decreased when R₁substituents were not 4-methoxy-

benzyl and 2,4-dimethoxybenzyl (compounds 43-51, Figure 4

for compound 43). We postulated that the high binding affinity

could be related to the nitrogen position on the piperidyl cycle

that can create an extra interaction with the receptor. Introduction

of a second nitrogen atom in the cycle did not improve the

binding affinity as assessed by the results obtained with

compounds 52-56. The best binding affinity constants were

obtained with compounds bearing a piperidine with a carboxylic

acid in position 4 of the piperidine ring. We replaced the

nitrogen atom by an oxygen atom (compound 57 versus

compound 40). We observed a 10-fold decrease in the affinity

value. The loss of affinity was more pronounced when the

piperidyl ring was replaced by a cyclohexyl ring (compound

58 versus compound 40) but was not so dramatic (IC₅₀) 18 (

6 nM for compound 58).

The ability of these compounds to activate the GHS-R1a and

to stimulate a biological response was determined by assessing

the increase in intracellular calcium levels in CHO cells

transiently transfected with the GHS-R1a. The activation

percentage for each compound (10^-5M) was assessed relative

to the maximal response obtained with ghrelin (10^-7M, 100%).

For the most affine agonist compounds, EC₅₀values were

determined as described in the Experimental Section and are

reported in Table 1 (see Figure 5 as an example). We considered

as potential antagonist any compound unable to elicit an increase

of intracellular calcium level greater than 10% of the maximal

response elicited by 10^-7M ghrelin. For antagonist compounds

with high affinities, K_bvalues were determined as described in

the Experimental Section and are reported in Table 1 (see Figure

6-8). In a previous study,⁴we postulated that the presence of

5794 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

Table 2. Cumulative Food Intake in Rats after Subcutaneous Administration of 160 µg/kg Compound and/or 80 µg/kg Hexarelin

cumulative food intake at 2 h for

160 µg of compound (cumulative food

intake at 2 h for 80 µg of hexarelin)^a

cumulative food intake

at 2 h for 160 µg of

compound + 80 µg

of hexarelin^a

cumulative food intake at 6 h for

160 µg of compound (cumulative food

intake at 6 h for 80 µg of hexarelin)^a

cumulative food intake

at 6 h for 160 µg of

compound + 80 µg

of hexarelin^a

compd

saline

4

5

6

7

0.06 ( 0.02

0.16 ( 0.05

0.01 ( 0.0 (0.53 ( 0.17)

0.01 ( 0.0 (0.29 ( 0.11)

0.01 ( 0.0 (0.57 ( 0.17)

0.01 ( 0.0 (0.32 ( 0.13)

0.01 ( 0.0(0.46 ( 0.15)

0.05 ( 0.05 (0.55 ( 0.13)

0.01 ( 0.0 (0.32 ( 0.13)

0.01 ( 0.0 (0.53 ( 0.19)

0.01 ( 0.0 (0.28 ( 0.08)

0.01 ( 0.0 (0.70 ( 0.16)

0.52 ( 0.29 (0.26 ( 0.11)

0.01 ( 0.0 (0.27 ( 0.12)

0.02 ( 0.02 (0.70 ( 0.16)

0.01 ( 0.0 (1.01 ( 0.15)

0.04 ( 0.01 (0.41 ( 0.17)

0.01 ( 0.0 (0.26 ( 0.13)

0.43 ( 0.07 (1.01 ( 0.15)

0.01 ( 0.0 (0.44 ( 0.16)

0.01 ( 0.0 (0.41 ( 0.12)

0.01 ( 0.0 (0.44 ( 0.16)

0.03 ( 0.01 (0.41 ( 0.17)

0.02 ( 0.01 (0.70 ( 0.27)

0.01 ( 0.0 (0.70 ( 0.11)

0.01 ( 0.0 (0.43 ( 0.16)

0.01 ( 0.01

0.39 ( 0.13

1.26 ( 0.13

0.79 ( 0.32

0.16 ( 0.12

0.44 ( 0.16

0.05 ( 0.04

0.35 ( 0.20

0.02 ( 0.01

0.33 ( 0.12

0.16 ( 0.15

0.62 ( 0.21

0.84 ( 0.27

0.85 ( 0.13

1.25 ( 0.24

0.06 ( 0.03

0.40 ( 0.20

1.27 ( 0.13

0.11 ( 0.09

0.17 ( 0.16

0.38 ( 0.13

1.40 ( 0.34

0.02 ( 0.01

0.85 ( 0.23

0.63 ( 0.32

0.21 ( 0.13

0.04 ( 0.01 (0.59 ( 0.17)

0.01 ( 0.0 (0.60 ( 0.15)

0.08 ( 0.05 (0.98 ( 0.19)

0.03 ( 0.0 (0.45 ( 0.16)

0.12 ( 0.11(0.72 ( 0.17)

0.20 ( 0.09 (0.66 ( 0.14)

0.03 ( 0.0 (0.45 ( 0.16)

0.02 ( 0.0 (1.00 ( 0.19)

0.01 ( 0.0 (0.88 ( 0.26)

0.01 ( 0.0 (0.98 ( 0.16)

0.60 ( 0.30 (0.73 ( 0.11)

0.06 ( 0.02 (0.40 ( 0.12)

0.02 ( 0.02 (0.98 ( 0.16)

0.01 ( 0.0 (1.24 ( 0.06)

0.06 ( 0.01 (0.68 ( 0.29)

0.01 ( 0.0 (0.78 ( 0.24)

0.37 ( 0.21 (0.78 ( 0.24)

0.71 ( 0.15 (1.24 ( 0.06)

0.01 ( 0.0 (0.90 ( 0.18)

0.08 ( 0.06 (0.73 ( 0.21)

0.11 ( 0.10 (0.61 ( 0.18)

0.35 ( 0.18 (0.68 ( 0.29)

0.03 ( 0.01 (0.74 ( 0.27)

0.01 ( 0.0 (1.22 ( 0.06)

0.14 ( 0.12 (0.61 ( 0.18)

0.02 ( 0.01

0.74 ( 0.33

1.48 ( 0.32

0.81 ( 0.40

0.60 ( 0.20

0.48 ( 0.18

0.07 ( 0.04

0.47 ( 0.19

0.35 ( 0.21

0.65 ( 0.31

0.62 ( 0.22

0.76 ( 0.26

1.01 ( 0.27

1.07 ( 0.08

1.68 ( 0.14

0.56 ( 0.21

1.05 ( 0.26

1.40 ( 0.12

0.86 ( 0.22

0.24 ( 0.16

0.60 ( 0.23

1.66 ( 0.35

0.11 ( 0.08

1.48 ( 0.25

0.75 ( 0.31

0.49 ( 0.26

14

16

17

19

25

26

27

29

33

34

35

36

39

40

41

42

43

47

52

53

54

58

^aExpressed in g of food per 100 g of body weight.

Table 3. Dose-Response Study for Compounds 4, 17, and 52 on Food Intake Stimulated by 80 µg/kg Hexarelin^a

compound

saline

hexarelin, 80 µg

+20 µg/kg 4

+80 µg/kg 4

+160 µg/kg 4

+320 µg/kg 4

food intake

compound

saline

hexarelin 80 µg

+20 µg/kg 17

+80 µg/kg 17

+160 µg/kg 17

+320 µg/kg 17

food intake

compound

saline

hexarelin 80 µg

+20 µg/kg 52

+80 µg/kg 52

+160 µg/kg 52

+320 µg/kg 52

food intake

0.16 ( 0.05

0.81 ( 0.09

0.63 ( 0.23

0.56 ( 0.18

0.28 ( 0.12

0.36 ( 0.14

0.16 ( 0.05

0.97 ( 0.20

0.17 ( 0.12

0.15 ( 0.08

0.36 ( 0.16

0.58 ( 0.12

0.07 ( 0.04

1.12 ( 0.06

0.66 ( 0.20

0.34 ( 0.15

0.83 ( 0.20

0.11 ( 0.05

^aCumulative food intake at 6 h expressed in g of food per 100 g of body weight.

a 4-methoxybenzyl substituent in the R₁group led to antagonist

compounds, all of them containing the Aib moiety at the

N-terminus part. In this study, we have shown that when Aib

was replaced by different groups, the presence of the 4-meth-

oxybenzyl in the R₁group was not sufficient to obtain full

antagonists. Some of these compounds were able to stimulate

[Ca²⁺]_iaccumulation. This is particularly true for compound

11 with a 2-aminoethyl group replacing the Aib moiety (59%

total response of 10^-7M ghrelin), compound 14 with a

3-aminopropanoyl group instead of Aib (117% total response),

compound 25 with a hydroxyprolyl group (87% total response).

When piperidyl-3-carboxyl (compounds 26, 27, and 31) or

isonipecotyl groups (compounds 39-51) substituted Aib, com-

pounds were also able to stimulate [Ca²⁺]_iaccumulation (from

48% of the total response induced by 10^-7M ghrelin for

compound 26 to 100% of the total response for compound 48).

The EC₅₀values of the best ligands for the GHS-1a receptor

were determined (Table 1). Surprisingly, in the case of the

isonipecotyl family, the 4-methoxy benzyl in the R₁position

can be replaced by a 4-ethyl group without a dramatic loss of

activity (i.e., compound 47, EC₅₀) 7.7 ( 1.2 nM). Compounds

containing a piperazine-2-carboxyl group exhibited good to

modest biological activities (EC₅₀ranging from 8.7 ( 1.1 to

143 ( 27 nM, compounds 52-56). When suppressing the NH

moiety in the isonipecotyl group, we observed a decrease in

the agonist efficacy [17% of the ghrelin response for compound

58 (EC₅₀) 390 ( 77 nM) compared to 93% of the ghrelin

response for compound 40 (EC₅₀) 1.6 ( 0.7 nM) bearing the

isonipecotyl group]. Replacement of the Aib moiety by glycyl,

prolyl residues, or a piperidine-2-carboxyl group generally led

to antagonist compounds. They were able to antagonize the

ghrelin-induced [Ca²⁺]_iaccumulation (Figure 6). Compounds

4, (K_b) 19 ( 6 nM), 16, (K_b) 6 ( 2 nM), 19, (K_b) 8 ( 2

nM), 35 (K_b) 8 ( 1 nM), and 54 (K_b) 5 ( 1 nM) were

among the most potent ghrelin receptor antagonists. They were

able to dose-dependently antagonize ghrelin-induced [Ca²⁺

]

i

accumulation in CHO cells transiently transfected with the GHS-

R1a. Interestingly, in the presence of various increasing

concentrations of compounds 4, 16-18, and 20, the dose-

response curves of ghrelin on [Ca²⁺]_iaccumulation were shifted

to the right in a parallel manner indicating a competitive

antagonism (see Figures 7 and 8 for compounds 4 and 17 as

examples). Introduction of an oxygen atom (compound 57) in

place of the nitrogen atom of compound 40 switched the agonist

activity to antagonist activity (compound 57, 0% total response,

K_b) 51 ( 8 nM) while a good affinity was conserved (IC₅₀

)

6 ( 2 nM). A proton that can be engaged in a hydrogen bond

in the para position of the piperidine ring seems to be necessary

to maintain the agonist activity when keeping similar R₁and

R₂groups.

Selected compounds among the most active were tested in

feeding behavior in rats. They were first evaluated alone for

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5795

Figure 11. Stimulation of hexarelin (80 µg/kg) induced cumulative

food intake (g of food/100 g of body weight) at 6 h by acute

administration (sc) of GHS-R1a antagonists 6, 7, and 35 (160 µg/kg).

Figure 9. Inhibition of hexarelin (80 µg/kg) induced cumulative food

intake (g of food/100 g of body weight) at 6 h by acute administration

(sc) of compounds 4, 17, and 52 (160 µg/kg).

A dose-response study was performed for the three com-

pounds (4, 17, and 52) able to totally inhibit the hexarelin-

induced food intake at 6 h after sc administration. These results

are gathered in Table 3. Compound 4 showed a well-correlated

progressive inhibition of hexarelin stimulated cumulative food

intake with increasing doses (20, 80, and 160 µg/kg). Almost

total inhibition was observed at a dose of 80 µg/kg. The highest

doses did not improve the inhibition. Compound 17 was found

to be effective in suppressing feeding at very low doses (20

and 80 µg/kg), but surprisingly, its efficiency seemed to decrease

at higher doses. For compound 52, a dose-response curve could

be obtained.

Information on the toxicity of the compounds could be

obtained by the observation on the adult rats that received

subcutaneous doses of the compounds (20-320 µg/kg) for

assessing the effect on food intake. We did not obtain mortality

with any of the tested compounds, and the observation of the

animals during the 6 h of the food intake experiments did not

reveal altered locomotion behavior.

Compound 4 has been evaluated in a chronic model of diet-

induced obesity in mice that were treated for 2 weeks with

subcutaneous daily doses of 1.5, 6, and 24 mg/kg. No signs of

toxicity were noticed by the routine observation of the animals

during the entire experiment.

From these observations we can infer that the tested com-

pounds did not induce toxic effects at the tested doses in the

food intake experiments.

The binding affinity of a model compound, compound 40,

was tested at 10 µM versus a panel of 92 GPCRs including

cannabinoids, melanocortins, and NPY receptors (MDS Pharma

Services’ GPCR screen). Only GHS-R1a, CCKI, motilin,

tachykinin 2, and vasopressin 1a receptors showed significant

binding with this compound. However, the binding affinity to

the ghrelin receptor GHS-R1a was 1000 times higher than the

affinity to the other receptors.

Selected compounds were also evaluated for their activity

on GH secretion in infant rats after sc administration, in the

presence or not of hexarelin. Indeed, it is known that in the

baby rats the pulsatility of the GH secretion is not yet activated

at the hypothalamic level, and therefore, a constant GH blood

level is measured in these young animals.

Results are reported in Table 4. As expected, when admin-

istered alone, GHS-R1a antagonists (compounds 4, 17, 19, and

34) did not significantly stimulate GH release. Compounds 40

and 41, found to behave as full GHS-R1a in vitro agonists,

stimulated GH secretion. Compound 52, which was found to

be a partial agonist in our in vitro test (with a total response

value of 37%), gave a very low response on GH release (18.6

ng/mL vs 10.7 ng/mL for saline). However, GHS-R1a in vitro

antagonists (compounds 4, 17, 19, and 34) were unable to

antagonize hexarelin-stimulated GH secretion. The efficacy of

Figure 10. Partial inhibition of hexarelin (80 µg/kg) induced cumula-

tive food intake (g of food/100 g of body weight) at 6 h by acute

administration (sc) of GHS-R1a agonists 25, 26, and 42 (160 µg/kg).

their effect on food intake and compared to hexarelin and then

for their ability to inhibit hexarelin-stimulated food intake.

Results are gathered in Table 2. A significant increase in

cumulative food intake at 6 h was found for compounds 27,

39, 40, and 47 when compounds were administered alone. These

compounds were also characterized as agonist compounds in

vitro (EC₅₀values of 9.9, 3.0, 1.6, and 7.7 nM, respectively).

Other potent in vitro agonists did not show any increase of

cumulative food intake, suggesting either a poor in vivo

bioavailability (for example, compound 41 with a EC₅₀value

of 0.6 nM) or the presence of ghrelin receptor subtypes or a

different mechanism of action of ghrelin receptor in vivo. As

expected, when administered alone, in vitro antagonists did not

increase food intake. When inhibition of food intake induced

by hexarelin was evaluated, two compounds (4 and 17) were

found to totally suppress food intake while compound 52 was

also very potent in inhibiting hexarelin-stimulated food intake

(Figure 9). The first two were characterized as antagonists in

vitro, while compound 52 was found to be an agonist. In fact,

there was no a clear correlation between in vitro and in vivo

experiments for several compounds. Some of them characterized

as full in vitro agonists were not very potent when administrered

alone but potentiated hexarelin-stimulated food intake (com-

pounds 39, 40), compound 47 being particularly potent, both

at 2 and 6 h after injection (Table 2). Alternatively, in vitro

agonist compounds such as 25 and 42 (Figure 10) or the partial

agonists 26 and 52 were without effect on food intake when

administered alone but were able to antagonize the effect of

hexarelin on food intake both at 2 and 6 h after injection. On

the other hand, compounds 4 and 17 characterized as in vitro

antagonists did not exhibit any activity when administered alone

and decreased food intake induced by hexarelin as expected.

Compounds 33, 35, and 54, also characterized as in vitro

antagonists and with no proper activity, were unable to

antagonize the effect of hexarelin on food intake. Compounds

6, 7, and 35 were even able to potentiate this effect (Figure 11,

Table 2).

5796 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Table 4. GH Secretion in Infant Rat (sc Injection)^a

Moulin et al.

pharmacological modulation of the ghrelin effect on appetite.

To assess the medical relevance of this observation, these

compounds will be tested in several other animal models.

compd

saline

hexarelin

19

19 + hexarelin

34

34 + hexarelin

40

[rGH] (ng/mL)

compd

saline

hexarelin

4

4 + hexarelin

saline

hexarelin

17

17 + hexarelin

52

[rGH] (ng/mL)

4.01 ( 0.47

5.24 ( 0.73

170.10 ( 13.23

9.85 ( 1.04

164.46 ( 4.44

10.72 ( 2.02

253.82 ( 12.27

16.86 ( 2.15

218.84 ( 19.72

18.63 ( 2.93

216.57 ( 19.73

162.84 ( 21.09

5.66 ( 1.19

Experimental Section

160.86 ( 13.52

6.09 ( 2.09

Chemistry. Ascending TLC was performed on precoated plates

of silica gel 60 F₂₅₄(Merck). Peptide derivatives were located with

charring reagent or ninhydrin. Column chromatography was

performed with Kieselguhr Merck G silica gel, 0.04-0.063 mm.

HPLC purifications were run on a Waters 4000 preparative

apparatus on a C18 Deltapak column (100 mm × 40 mm, 15 µm,

100 Å), with a UV detection at 214 nm, at a flow rate of 50 mL/

min of a mixture of A (water with 0.1% TFA) and B (acetonitrile

with 0.1% TFA in gradient mode). Analytical HPLC chromato-

graphs were obtained on a Beckman Gold apparatus composed of

the 126 solvent module, the 168 detector, and the 32 Karat software;

runs were performed on a VWR Chromolith column (50 mm ×

3.9 mm) at a flow rate of 5 mL/min from solution A to solution B

in a 3 min gradient (conditions A) or on a Symmetry Shield C18

column (50 mm × 4.6 mm, 3.5 µm) at a flow rate of 1 mL/min

from solution A to solution B in a 15 min gradient (conditions B).

¹H and ¹³C NMR spectra were recorded in DMSO-d₆at 300 and

75 MHz or at 400 and 100 MHz, respectively, and at 300 K.

Chemical shifts were reported as δ values (ppm) indirectly

referenced to the solvent signal. Mass spectrum analyses were

recorded on a Quatromicro (Micromass, Manchester, U.K.) triple

quadrupole mass spectrometer fitted with an electrospray interface.

The L- and D-aminoacids and derivatives were from Senn Chemi-

cals, Iris Biotech GmbH, NeoMPS, or Advanced Chemtech. Human

ghrelin was purchased from NeoMPS and iodinated in our labora-

tory. All reagents were of analytical grade.

General Procedure for Hydrazide Preparation. When hy-

drazides were not commercially available, they were synthesized

in two steps via the corresponding esters as described before.⁴

General Procedure for Thioamide B Preparation. In a solution

of DCM, amine (1.0 equiv), Boc-D-Trp (1.0 equiv), NMM (2.2

equiv) and BOP (1.0 equiv) were successively added. After 1 h

stirring at room temperature, the mixture was concentrated in vacuo

and dissolved in AcOEt. The organic layer was successively washed

with aqueous solutions of 1 M KHSO₄, saturated NaHCO₃and

brine. The organic layer was then dried over Na₂SO₄, filtered and

concentrated in vacuo to yield amide A that was used without

purification. To 1.0 equiv. of amide A in DME (10 mmol/mL) was

added the Lawesson’s reagent (0.5 equiv) under argon. The reaction

was heated to 85 °C for 2 h and then concentrated in vacuo. The

residue was purified by chromatography on silica gel with a mixture

of AcOEt/hexane 3/7 as eluent. The thioamide B was obtained as

a white powder.

145.95 ( 12.16

87.52 ( 15.07

100.52 ( 12.11

119.94 ( 33.05

103.53 ( 14.09

40 + hexarelin

41

41 + hexarelin

52 + hexarelin

^aHexarelin was tested at a dose of 80 µg/kg, and compounds were tested

at 160 µg/kg.

compounds 40 and 41 was similar in the presence or absence

of hexarelin but remained lower than hexarelin itself. Compound

52 seemed to slightly inhibit hexarelin-stimulated GH secretion

(216.57 ng/mL vs 253.82 ng/mL for hexarelin alone).

For in vitro results and both GH secretion and food intake

stimulation, the situation is complex and no straightforward

correlation can be drawn between in vitro results and in vivo

experiments on GH secretion and food intake. Compounds 4

and 17 defined as in vitro GHS-R1a antagonists had no effect

on GH secretion or food intake when administered alone.

However, they were able to totally suppress hexarelin-induced

food intake, while they were unable to inhibit GH secretion

elicited by hexarelin. The same observation was made for

compound 52, which is a GHS-R1a agonist. Compounds 19

and 34, also defined as in vitro GHS-R1a antagonists, partially

inhibited food intake induced by hexarelin but not hexarelin-

stimulated GH secretion. Compound 40, a potent in vitro agonist,

was able to stimulate food intake and GH secretion when

administered alone. However, it was able to potentiate both

hexarelin-stimulated food intake but not GH secretion. Com-

pound 41, another potent GHS-R1a agonist, had no effect on

food intake when administered alone or with hexarelin but

stimulated GH secretion.

All these findings strongly suggest the existence of either

ghrelin receptor subtypes or a particular mechanism of action

of the ghrelin receptor correlated with GH secretion on one side

and food intake on the other side. This last hypothesis was

recently suggested for ghrelin receptor inverse agonists that are

biased toward a particular signaling pathway.¹¹

Conclusion

(R)-tert-Butyl 1-(2,4-Dimethoxybenzylamino)-3-(1H-indol-3-

yl)-1-oxopropan-2-ylcarbamate. 5.2 g (90%). ¹H NMR (300 MHz,

DMSO-d₆, 300 K): δ 1.31 (s, 9H, CH₃Boc), 2.94 (dd, 1H, J ) 14

and 9 Hz, CH₂âTrp), 3.11 (dd, 1H, J ) 14 and 5 Hz, CH₂âTrp),

3.71 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 4.23 (m, 3H, CH RTrp

and CH₂o,p-dimethoxybenzyl), 6.38 (dd, 1H, J_o) 8 Hz and J_m)

2 Hz, H₅o,p-dimethoxybenzyl), 6.52 (d, 1H, J_m) 2 Hz, H₃o,p-

dimethoxybenzyl); 6.78 (d, 1H, J_o) 8 Hz, H₄Trp), 6.96 (d, 1H,

J_o) 8 Hz, H₆o,p-dimethoxybenzyl), 6.97 (t, 1H, J_o) 8 Hz, H₅

Trp), 7.06 (t, 1H, J_o) 7 Hz, H₆Trp), 7.12 (s, 1H, H₂Trp), 7.34

(d, 1H, J_o) 8 Hz, H₇Trp), 7.60 (d, 1H, J ) 8 Hz, NH Boc), 8.09

(t, 1H, J ) 6 Hz, NH amide), 10.79 (1H, s, NH indole Trp). ¹³C

NMR (75 MHz, DMSO-d₆, 300 K): δ 28.3 (C âTrp), 28.5 (CH₃

Boc), 37.4 (CH₂o,p-dimethoxybenzyl), 55.3 (OCH₃), 55.5 (C RTrp

and OCH₃), 78.4 (Cq Boc), 98.5 (C₃o,p-dimethoxybenzyl), 104.6

(C₅o,p-dimethoxybenzyl), 110.7 (C₃Trp), 111.7 (C₇Trp), 118.6

(C₄Trp), 118.9 (C₅Trp), 119.2 (C₁o,p-dimethoxybenzyl), 121.2

(C₆Trp), 124.1 (C₂Trp), 127.8 (C₉Trp), 128.9 (C₆o,p-dimethoxy-

benzyl), 136.5 (C₈Trp), 155.6 (CO Boc), 158.0 (C₂o,p-dimethoxy-

benzyl), 160.0 (C₄o,p-dimethoxybenzyl), 172.3 (CO amide). MS

(ES), m/z: 454.2 [M + H]⁺, 398.2 [M + H - ^tBu]⁺, 354.2 [M +

H - Boc]⁺. HPLC t_R: 1.70 min (conditions A).

A novel class of ghrelin receptor (GHS-R1a) ligands was

identified from substituted 1,2,4-triazoles. The Aib moiety, often

present in the described GHS structures, has been successfully

replaced to yield very potent in vitro GHS-R1a agonists

[compound 39 (JMV2952), IC₅₀) 0.3 ( 0.2 nM, EC₅₀) 3.0

( 1.3 nM; 40 (JMV2951), IC₅₀) 0.6 ( 0.3 nM, EC₅₀) 1.6

( 0.7 nM; 41 (JMV2894), IC₅₀) 0.5 ( 0.3 nM, EC₅₀) 0.6

( 0.0 nM] and GHS-R1a antagonists [compound 4 (JMV2959),

IC₅₀) 32 ( 3 nM, K_b) 19 ( 6 nM; 16 (JMV3030), IC₅₀

)

9 ( 1 nM, K_b) 6 ( 2 nM; 17 (JMV3031), IC₅₀) 28 ( 9 nM,

K_b) 7 ( 2 nM; 35 (JMV3029), IC₅₀) 29 ( 11 nM, K_b) 8

( 1 nM]. The most potent compounds in this series were tested

in vivo for their activity on hexarelin-stimulated food intake in

the rat. Compounds 4, 17, and 52 (JMV3021) were found to be

able to inhibit hexarelin-induced food intake. These compounds

had no effect on GH secretion and were unable to inhibit

hexarelin-stimulated GH secretion in the rat. Our results show

for the first time that compounds characterized as in vitro GHS-

R1a ligands can inhibit food intake without altering GH

secretion. This study supports the concept of a specific

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5797

(R)-tert-Butyl 1-(4-Methoxybenzylamino)-3-(1H-indol-3-yl)-

1-oxopropan-2-ylcarbamate. Yield, 4.3 g (82%). H NMR (300

136.5 (C₈Trp), 137.0 (C₄p-ethylbenzyl), 142.5 (C₁p-ethylbenzyl),

155.6 (CO Boc), 172.4 (CO amide). MS (ES), m/z: 421.9 [M +

1

t

MHz, DMSO-d₆, 300 K): δ 1.28 (s, 9H, CH₃Boc), 2,88 (dd, 1H,

J ) 9 and 14 Hz, CH₂âTrp), 3.04 (dd, 1H, J ) 9 and 14 Hz, CH₂

âTrp), 3.68 (s, 3H, OCH₃), 4.17 (m, 3H, CH₂p-methoxybenzyl

and CH RTrp), 6.74 (d, 1H, J_o) 8 Hz, H₄Trp), 6.79 (d, 2H, J_o)

8 Hz, H₃and H₅p-methoxybenzyl), 6.93 (t, 1H, J_o) 8 Hz, H₅

Trp), 7.02 (t, 1H, H₆Trp), 7.02 (m, 3H, H₂Trp, H₂and H₆

p-methoxybenzyl), 7.30 (d, 1H, J_o) 8 Hz, H₇Trp), 7.56 (d, 1H,

J ) 8 Hz, NH Boc), 8.27 (t, 1H, J ) 5 Hz, NH amide), 10.76 (s,

1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ

28.2 (C âTrp), 28.5 (CH₃Boc), 41.9 (CH₂-p-methoxybenzyl), 55.4

(OCH₃), 55.7 (C RTrp), 78.4 (Cq Boc), 110.6 (C₃Trp), 111.6 (C₇

Trp), 112.0 (C₃and C₅p-methoxybenzyl), 118.5 (C₄Trp), 118.9

(C₅Trp), 121.2 (C₆Trp), 124.1 (C₂Trp), 127.7 (C₉Trp), 128.7

(C₂and C₆p-methoxybenzyl), 131.7 (C₁p-methoxybenzyl), 136.5

(C₈Trp), 155.6 (CO Boc), 158.5 (C₄p-methoxybenzyl), 172.3 (CO

H]⁺, 365.9 [M + H - Bu]⁺, 322.0 [M + H - Boc]⁺. HPLC t_R:

1.93 min (conditions A).

(R)-tert-Butyl 3-(1H-Indol-3-yl)-1-oxo-1-(thiophen-2-ylmethyl-

amino)propan-2-ylcarbamate. Yield, 3.6 g (71%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.28 (s, 9H, CH₃Boc), 2.89 (dd, 1H,

J ) 14 and 9 Hz, CH₂âTrp), 3.06 (dd, 1H, J ) 14 and 4 Hz, CH₂

âTrp), 4.20 (m, 1H, CH RTrp), 4.42 (m, 2H, CH₂-o-thiophenyl),

6.74 (d, 1H, J_o) 8 Hz, H₄Trp), 6.90 (m, 2H, H₃and H₄

o-thiophenyl), 6.94 (t, 1H, J_o) 7 Hz, H₅Trp), 7.03 (t, 1H, J_o) 7

Hz, H₆Trp), 7.08 (s, 1H, H₂Trp), 7.30 (d, 1H, J_o) 8 Hz, H₇Trp),

7.33 (dd, 1H, J ) 4 and 2 Hz, H₅o-thiophenyl), 7.58 (d, 1H, J )

8 Hz, NH Boc), 8.51 (brs, 1H, NH amide), 10.76 (s, 1H, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.2 (C âTrp),

28.6 (CH₃Boc), 37.7 (CH₂-o-thiophenyl), 55.6 (C RTrp), 78.4

(Cq Boc), 110.6 (C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp), 118.9

(C₅Trp), 121.2 (C₆Trp), 124.1 (C₂Trp), 125.6 (C₅o-thiophenyl),

127.0 (C₃and C₄o-thiophenyl), 127.7 (C₉trp), 136.5 (C₈Trp),

142.8 (C₂o-thiophenyl), 155.6 (CO Boc), 172.3 (CO amide). MS

(ES), m/z: 400.2 [M + H]⁺, 344.2 [M + H - ^tBu]⁺, 300.2 [M +

H - Boc]⁺. HPLC t_R: 1.62 min (conditions A).

t

amide). MS (ES), m/z: 423.9 [M + H]⁺, 367.8 [M + H - Bu]⁺,

324.0 [M + H - Boc]⁺. HPLC t_R: 1.75 min (conditions A).

(R)-tert-Butyl 1-(Benzylamino)-3-(1H-indol-3-yl)-1-oxopro-

pan-2-ylcarbamate. Yield, 5.3 g (95%). ¹H NMR (300 MHz,

DMSO-d₆, 300 K): δ 1.31 (s, 9H, CH₃Boc), 2.95 (dd, 1H, J ) 14

and 9 Hz, CH₂âTrp), 3.11 (dd, 1H, J ) 14 and 5 Hz, CH₂âTrp),

4.28 (m, 3H, CH₂benzyl and CH RTrp), 6.79 (d, 1H, J_o) 8 Hz,

H₄Trp), 6.96 (t, 1H, J_o) 7 Hz, H₅Trp), 7.05 (t, 1H, J_o) 7 Hz,

H₆Trp), 7.12 (s, 1H, H₂Trp), 7.15-7.27 (m, 5H, CHar benzyl),

7.33 (d, 1H, J_o) 8 Hz, H₇Trp), 7.60 (d, 1H, J ) 8 Hz, NH Boc),

8.40 (t, 1H, J ) 6 Hz, NH amide), 10.82 (s, 1H, NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.2 (C âTrp), 28.6

(CH₃Boc), 42.5 (CH₂benzyl), 55.8 (C RTrp), 78.4 (Cq Boc), 110.6

(C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp), 118.9 (C₅Trp), 121.2

(C₆Trp), 124.1 (C₂Trp), 127.0 (C₄benzyl), 127.4 (C₂, C₃, C₅, and

C₆benzyl), 127.8 (C₉Trp), 136.5 (C₈Trp), 139.8 (C₁benzyl), 155.6

(CO Boc), 172.5 (CO amide). MS (ES), m/z: 394.2 [M + H]⁺,

(R)-tert-Butyl 3-(1H-Indol-3-yl)-1-oxo-1-(phenylamino)pro-

pan-2-ylcarbamate. Yield, 5.6 g (88%). ¹H NMR (300 MHz,

DMSO-d₆, 300 K): δ 1.32 (s, 9H, CH₃Boc), 3.04 (dd, 1H, J ) 14

and 9 Hz, CH₂âTrp), 3.16 (dd, 1H, J ) 14 and 5 Hz, CH₂âTrp),

4.43 (m, 1H, CH RTrp), 6.87 (d, 1H, J_o) 8 Hz, H₄Trp), 6.97 (t,

1H, J_o) 7 Hz, H₅Trp), 7.05 (m, 2H, H₄phenyl and H₆Trp), 7.18

(s, 1H, H₂Trp), 7.28 (t, 2H, J_o) 8 Hz, H₃and H₅phenyl), 7.33

(d, 1H, J_o) 8 Hz, H₇Trp), 7.61 (d, 2H, J_o) 8 Hz, H₂and H₆

phenyl), 7.66 (d, 1H, J ) 8 Hz, NH Boc), 10.02 (s, 1H, NH amide),

10.80 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300

K): δ 28.3 (C âTrp), 28.6 (CH₃Boc), 56.2 (C RTrp), 78.5 (Cq

Boc), 110.4 (C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp), 119.0 (C₅

Trp), 119.9 (C₂and C₆phenyl), 121.3 (C₆Trp), 123.7 (C₄phenyl),

124.2 (C₂Trp), 127.8 (C₉Trp), 129.0 (C₃and C₅phenyl), 136.5

(C₈Trp), 139.4 (C₁phenyl), 155.7 (CO Boc), 171.5 (CO amide).

t

338.2 [M + H - Bu]⁺, 294.2 [M + H - Boc]⁺. HPLC t_R: 1.52

min (conditions A).

(R)-tert-Butyl 3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-2-ylmethyl-

amino)propan-2-ylcarbamate. Yield, 4.3 g (100%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.30 (s, 9H, CH₃Boc), 2.93 (dd, 1H,

J ) 14 and 9 Hz, CH₂âTrp), 3.11 (dd, 1H, J ) 14 and 5 Hz, CH₂

âTrp), 4.25 (m, 1H, CH RTrp), 4.34 (d, 2H, J ) 6 Hz, CH₂-o-

pyridyl), 6.85 (d, 1H, J_o) 8 Hz, H₄Trp), 6.95 (t, 1H, J_o) 8 Hz,

H₅Trp), 7.04 (t, 1H, J_o) 8 Hz, H₆Trp), 7.12 (m, 2H, H₂Trp and

H₅o-pyridyl), 7.21 (d, 1H, J_o) 7 Hz, H₃o-pyridyl), 7.31 (d, 1H,

J_o) 8 Hz, H₇Trp), 7.59 (m, 2H, NH Boc and H₄o-pyridyl), 8.45

(m, 2H, NH amide and H₆o-pyridyl), 10.77 (s, 1H, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.1 (C âTrp),

28.5 (CH₃Boc), 44.8 (CH₂o-pyridyl), 54.8 (C RTrp), 78.5 (Cq

Boc), 110.6 (C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp), 118.9 (C₅

Trp), 121.0 (C₅o-pyridyl), 121.2 (C₆Trp), 122.4 (C₃o-pyridyl),

124.5 (C₂Trp), 127.7 (C₉Trp), 136.5 (C₈Trp), 136.9 (C₄o-pyridyl),

149.1 (C₆o-pyridyl), 155.7 (CO Boc), 158.9 (C₂o-pyridyl),

172.7 (CO amide). MS (ES), m/z: 395.3 [M + H]⁺, 339.3

t

MS (ES), m/z: 380.2 [M + H]⁺, 324.2 [M + H - Bu]⁺, 280.2

[M + H - Boc]⁺. HPLC t_R: 2.30 min (conditions A).

(R)-tert-Butyl 1-(2,4-Dimethoxybenzylamino)-3-(1H-indol-

3-yl)-1-thioxopropan-2-ylcarbamate. Yield, 4.5 g (78%). ¹H NMR

(300 MHz, DMSO-d₆, 300 K): δ 1.27 (s, 9H, CH₃Boc), 2.97 (dd,

1H, J ) 8 and 14 Hz, CH₂âTrp), 3.30 (dd, 1H, J ) 4 and 14 Hz,

CH₂âTrp), 3.71 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 4.58 (m, 3H,

CH₂-o,p-dimethoxybenzyl and CH RTrp), 6.37 (dd, 1H, J_o) 8

Hz and J_m) 2 Hz, H₅o,p-dimethoxybenzyl), 6.53 (d, 1H, J_m) 2

Hz, H₃o,p-dimethoxybenzyl), 6.86 (d, 1H, J_o) 8 Hz, H₄Trp),

6.87 (d, 1H, J_o) 8 Hz, H₆o,p-dimethoxybenzyl), 6.95 (t, 1H, J_o

) 7 Hz, H₅Trp), 7.03 (t, 1H, J_o) 7 Hz, H₆Trp), 7.11 (s, 1H, H₂

Trp), 7.30 (d, 1H, J_o) 8 Hz, H₇Trp), 7.60 (m, 1H, NH Boc), 9.97

(t, 1H, J ) 6 Hz, NH thioamide), 10.78 (s, 1H, NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.5 (CH₃Boc), 31.3

(C âTrp), 44.2 (CH₂o,p-dimethoxybenzyl), 55.6 (OCH₃), 55.9

(OCH₃), 61.9 (C RTrp), 78.6 (Cq Boc), 98.7 (C₃o,p-dimethoxy-

benzyl), 104.8 (C₅o,p-dimethoxybenzyl), 110.4 (C₃Trp), 111.7

(C₇Trp), 116.7 (C₁o,p-dimethoxybenzyl), 118.6 (C₄Trp); 118.9

(C₅Trp), 121.2 (C₆Trp), 124.4 (C₂Trp), 127.8 (C₉Trp), 130.1

(C₆o,p-dimethoxybenzyl), 136.5 (C₈Trp), 155.5 (CO Boc), 158.6

(C₂o,p-dimethoxybenzyl), 161.1 (C₄o,p-dimethoxybenzyl),

210.4 (CS thioamide). MS (ES), m/z: 470.0 [M + H]⁺, 414.0 [M

t

[M + H - Bu]⁺, 295.3 [M + H - Boc]⁺. HPLC t_R: 1.14 min

(conditions A).

(R)-tert-Butyl 1-(4-Ethylbenzylamino)-3-(1H-indol-3-yl)-1-

oxopropan-2-ylcarbamate. Yield, 5.3 g (76%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.11 (t, 3H, J ) 7 Hz, CH₃-CH₂-

phenyl), 1.29 (s, 9H, CH₃Boc), 2.53 (q, 2H, J ) 7 Hz, CH₃-

CH₂-phenyl), 2.89 (dd, 1H, J ) 14 and 9 Hz, CH₂âTrp), 3.06

(dd, 1H, J ) 14 and 5 Hz, CH₂âTrp), 4.21 (m, 3H, CH R Trp and

CH₂p-ethylbenzyl), 6.76 (d, 1H, J_o) 8 Hz, H₄Trp), 6.94 (t, 1H,

J_o) 7 Hz, H₅Trp), 7.03 (t, 1H, J_o) 8 Hz, H₆Trp), 7.07 (m, 5H,

H₂Trp and CHar p-ethylbenzyl), 7.30 (d, 1H, J_o) 8 Hz, H₇Trp),

7.57 (d, 1H, J ) 8 Hz, NH Boc), 8.30 (t, 1H, J ) 6 Hz, NH amide),

10.77 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300

K): δ 16.2 (CH₃-CH₂-phenyl), 28.2 (CH₂âTrp and CH₃-CH₂-

phenyl), 28.6 (CH₃Boc), 42.3 (CH₂p-ethylbenzyl), 55.7 (C RTrp),

78.4 (Cq Boc), 110.6 (C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp),

118.9 (C₅Trp), 121.2 (C₆Trp), 124.1 (C₂Trp), 127.5 (C₂and C₆

p-ethylbenzyl), 127.8 (C₉Trp), 127.9 (C₃and C₅p-ethylbenzyl),

t

+ H - Bu]⁺, 370.0 [M + H - Boc]⁺. HPLC t_R: 1.97 min

(conditions A).

(R)-tert-Butyl 1-(4-Methoxybenzylamino)-3-(1H-indol-3-yl)-

1-thioxopropan-2-ylcarbamate. Yield, 4.4 g (83%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.27 (s, 9H, CH₃Boc), 2.94 (dd, 1H,

J ) 9 and 14 Hz, CH₂âTrp), 3.13 (dd, 1H, J ) 5 and 14 Hz, CH₂

âTrp), 3.69 (s, 3H, OCH₃), 4.62 (m, 3H, CH₂p-methoxybenzyl

and CH RTrp), 6.76 (s, 4H, CHar p-methoxybenzyl), 6.94 (m, 2H,

H₄and H₅Trp), 7.03 (t, 1H, H₆Trp), 7.16 (s, 1H, H₂Trp), 7.30 (d,

1H, J_o) 8 Hz, H₇Trp), 7.59 (d, 1H, J ) 8 Hz, NH Boc), 10.21

(t, 1H, J ) 5 Hz, NH thioamide), 10.78 (s, 1H, NH indole Trp).

5798 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.5 (CH₃Boc), 31.3

(C âTrp), 48.2 (CH₂p-methoxybenzyl), 55.3 (OCH₃), 62.0 (C

RTrp), 78.6 (Cq Boc), 110.4 (C₃Trp), 111.7 (C₇Trp), 114.0 (C₃

and C₅p-methoxybenzyl), 118.6 (C₄Trp), 118.9 (C₅Trp), 121.2

(C₆Trp), 124.3 (C₂Trp), 127.7 (C₉Trp), 129.0 (C₁p-methoxy-

benzyl), 129.2 (C₂and C₆p-methoxybenzyl), 136.5 (C₈Trp), 155.2

(CO Boc), 158.8 (C₄p-methoxybenzyl), 204.8 (CS thioamide). MS

(ES), m/z: 439.9 [M + H]⁺, 383.9 [M + H - ^tBu]⁺, 339.9 [M +

H - Boc]⁺. HPLC t_R: 2.01 min (conditions A).

6.82 (d, 1H, J_o) 8 Hz, H₄Trp), 6.90 (m, 2H, H₃and H₄

o-thiophenyl), 6.94 (t, 1H, J_o) 7 Hz, H₅Trp), 7.03 (t, 1H, J_o) 7

Hz, H₆Trp), 7.08 (s, 1H, H₂Trp), 7.30 (d, 1H, J_o) 8 Hz, H₇Trp),

7.33 (dd, 1H, J ) 4 and 2 Hz, H₅o-thiophenyl), 7.58 (d, 1H, J )

8 Hz, NH Boc), 10.47 (brs, 1H, NH thioamide), 10.76 (s, 1H, NH

indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.2 (C

âTrp), 28.6 (CH₃Boc), 45.4 (CH₂-o-thiophenyl), 62.0 (C RTrp),

78.4 (Cq Boc), 110.6 (C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp),

118.9 (C₅Trp), 121.2 (C₆Trp), 124.1 (C₂Trp), 125.6 (C₅

o-thiophenyl), 127.0 (C₃and C₄o-thiophenyl), 127.7 (C₉Trp),

136.5 (C₈Trp), 142.8 (C₂o-thiophenyl), 155.6 (CO Boc),

207.0 (CS thioamide). MS (ES), m/z: 416.2 [M + H]⁺, 360.2 [M

(R)-tert-Butyl 1-(Benzylamino)-3-(1H-indol-3-yl)-1-thioxopro-

pan-2-ylcarbamate. Yield, 5.2 g (77%). ¹H NMR (300 MHz,

DMSO-d₆, 300 K): δ 1.30 (s, 9H, CH₃Boc), 3.03 (dd, 1H, J ) 14

and 8 Hz, CH₂âTrp), 3.20 (dd, 1H, J ) 14 and 5 Hz, CH₂âTrp),

4.65-4.85 (m, 3H, CH₂benzyl and CH RTrp), 6.79 (d, 1H, J_o)

8 Hz, H₄Trp), 6.97 (t, 1H, J_o) 7 Hz, H₅Trp), 7.06 (t, 1H, J_o)

7 Hz, H₆Trp), 7.16 (m, 3H, H₂and H₆benzyl, H₂Trp), 7.26 (m,

3H, H₃, H₄and H₅benzyl), 7.34 (d, 1H, J_o) 8 Hz, H₇Trp), 7.64

(d, 1H, J ) 7 Hz, NH Boc), 10.32 (t, 1H, J ) 5 Hz, NH thioamide),

10.81 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300

K): δ 28.5 (CH₃Boc), 31.3 (C âTrp), 48.6 (CH₂benzyl), 62.1 (C

RTrp), 78.6 (Cq Boc), 110.4 (C₃Trp), 111.7 (C₇Trp), 118.6 (C₄

Trp), 119.0 (C₅Trp), 121.3 (C₆Trp), 124.4 (C₂Trp), 127.4 (C₄

benzyl), 127.8 (C₉Trp), 128,8 (C₂and C₆benzyl), 128.6 (C₃and

C₅benzyl), 136.5 (C₈Trp), 137.3 (C₁benzyl), 155.3 (CO Boc),

205.3 (CS thioamide). MS (ES), m/z: 410.0 [M + H]⁺, 354.0 [M

t

+ H - Bu]⁺, 316.2 [M + H - Boc]⁺. HPLC t_R: 1.92 min

(conditions A).

(R)-tert-Butyl 3-(1H-Indol-3-yl)-1-(phenylamino)-1-thioxopro-

pan-2-ylcarbamate. Yield, 5.3 g (75%). ¹H NMR (300 MHz,

DMSO-d₆, 300 K): δ 1.30 (s, 9H, CH₃Boc), 3.09 (m, 1H, CH₂

âTrp), 3.17 (dd, 1H, J ) 14 and 5 Hz, CH₂âTrp), 4.74 (m, 1H,

CH RTrp), 6.80 (d, 1H, J_o) 7 Hz, H₄Trp), 6.96 (t, 1H, J_o) 7

Hz, H₅Trp), 7.04 (t, 1H, J_o) 7 Hz, H₆Trp), 7.20 (m, 2H, H₂Trp

and H₄phenyl), 7.30 (d, 1H, J_o) 8 Hz, H₇Trp), 7.35 (t, 2H, J_o)

8 Hz, H₂and H₆phenyl), 7.65 (m, 3H, H₃and H₅phenyl, NH

Boc), 10.79 (s, 1H, NH thioamide), 11.42 (s, 1H, NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 28.6 (CH₃Boc), 31.6

(C âTrp), 62.8 (C RTrp), 78.7 (Cq Boc), 110.2 (C₃Trp), 111.7 (C₇

Trp), 118.6 (C₄Trp), 119.0 (C₅Trp), 121.2 (C₆Trp), 123.9 (C₂

Trp and C₄phenyl), 126.5 (C₂and C₆phenyl), 127.8 (C₉Trp),

128.8 (C₃and C₅phenyl), 136.4 (C₈Trp), 139.6 (C₁phenyl), 155.3

(CO Boc), 204.7 (CS thioamide). MS (ES), m/z: 396.2 [M + H]⁺,

t

+ H - Bu]⁺, 310.0 [M + H - Boc]⁺. HPLC t_R: 1.99 min

(conditions A).

(R)-tert-Butyl 3-(1H-Indol-3-yl)-1-(pyridin-2-ylmethylamino)-

1-thioxopropan-2-ylcarbamate. Yield, 2.5 g (49%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.29 (s, 9H, CH₃Boc), 3.01 (dd, 1H,

J ) 14 and 8 Hz, CH₂âTrp), 3.23 (dd, 1H, J ) 14 and 5 Hz, CH₂

âTrp), 4.62 (m, 1H, CH RTrp), 4.89 (m, 2H, CH₂-o-pyridyl), 6.93

(m, 2H, H₄and H₅Trp), 7.01 (m, 2H, H₆Trp and H₅o-pyridyl),

7.17 (s, 1H, H₂Trp), 7.32 (d, 1H, J_o) 8 Hz, H₇Trp), 7.46 (d, 1H,

J_o) 8 Hz, H₃o-pyridyl), 7.61 (d, 1H, J ) 8 Hz, NH Boc), 7.86 (t,

1H, J_o) 8 Hz, H₄o-pyridyl), 8.60 (brs, 1H, H₆o-pyridyl), 10.53

(brs,1H, NH thioamide), 10.83 (s, 1H, NH indole Trp). ¹³C NMR

(75 MHz, DMSO-d₆, 300 K): δ 28.5 (CH₃Boc), 31.0 (C âTrp),

48.8 (CH₂-o-pyridyl), 62.4 (C RTrp), 78.8 (Cq Boc), 110.3 (C₃

Trp), 111.7 (C₇Trp), 118.7 (C₄Trp), 118.9 (C₅Trp), 121.3

(C₆Trp), 123.7 (C₅o-pyridyl), 124.5 (C₃o-pyridyl), 124.5

(C₂Trp), 127.7 (C₉Trp), 132.7 (C₄o-pyridyl), 136.5 (C₈Trp),

140.0 (C₆o-pyridyl), 155.2 (CO Boc), 155.5 (C₂o-pyridyl),

206.6 (CS thioamide). MS (ES), m/z: 411.0 [M + H]⁺, 355.0 [M

t

340.2 [M + H - Bu]⁺, 296.2 [M + H - Boc]⁺. HPLC t_R: 1.85

min (conditions A).

General Procedure for Preparation of Triazole C. To a

solution of 1.0 equiv of thioamide B in 5 mL of tetrahydrofuran (1

mmol/10 mL) were added 2.0 equiv of hydrazide and then 1.1 equiv

of mercury(II) acetate at room temperature. After 2 days, the

mixture was filtered on Celite and the filtrate was concentrated in

vacuo. The residue was purified by chromatography on silica gel

with a mixture of AcOEt/MeOH, 96/4, as eluent. The desired

compounds were obtained as a white powder (yield ranging between

48% and 65%).

(R)-tert-Butyl 1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(2,4-dimethoxy-

benzyl)-4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbam-

1

ate. Yield, 1.2 g (60%). H NMR (300 MHz, DMSO-d₆, 300 K):

δ 1.21 (s, 9H, CH₃Boc), 2.90 (m, 4H, CH₂-CH₂-indole), 3.28

(m, 2H, CH₂âTrp), 3.59 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃), 5.01

(m, 3H, CH₂-o,p-dimethoxyphenyl and CH RTrp), 6.26 (d, 1H,

J_o) 8 Hz, H₅o,p-dimethoxyphenyl), 6.49 (d, 1H, J_o) 8 Hz, H₆

o,p-dimethoxyphenyl), 6.51 (s, 1H, H₃o,p-dimethoxyphenyl), 6.89

(m, 2H, H₅Trp and H₅indole), 7.02 (m, 2H, H₆indole and H₆

Trp), 7.03 (s, 1H, H₂indole), 7.05 (s, 1H, H₂Trp), 7.26 (d, 1H, J_o

) 8 Hz, H₄Trp), 7.29 (m, 3H, H₄and H₇indole, H₇Trp), 7.57 (d,

1H, J ) 9 Hz, NH Boc), 10.79 (s, 1H, NH indole), 10.81 (1H, s,

NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300K): δ 22.4

(CH₂-CH₂-indole), 25.7 (CH₂-CH₂-indole), 28.4 (CH₃Boc),

28,9 (C âTrp), 42.6 (CH₂-o,p-dimethoxyphenyl), 46.8 (C RTrp),

55.6 (OCH₃), 55.8 (OCH₃), 78.8 (Cq Boc), 98.9 (C₃o,p-dimethox-

yphenyl), 105.1 (C₅o,p-dimethoxyphenyl), 110.0 (C₃Trp), 111.7

(C₇Trp), 111.8 (C₇indole), 112.9 (C₃indole), 114.8 (C₁o,p-

dimethoxyphenyl), 118.4 (C₄indole and C₄Trp), 118.7 (C₅indole

and C₅Trp), 121.3 (C₆Trp), 121.4 (C₆indole), 123.0 (C₂indole),

125.1 (C₂Trp), 127.1 (C₉indole), 127.5 (C₉Trp), 128.5 (C₆o,p-

dimethoxyphenyl), 136.4 (C₈Trp), 136.6 (C₈indole), 155.5 (Cq

triazole and CO Boc), 156.1 (Cq triazole), 157.8 (C₂o,p-dimethox-

yphenyl), 161.0 (C₄o,p-dimethoxyphenyl). MS (ES), m/z: 621.0

[M + H]⁺. HPLC t_R: 2.20 min (conditions A).

t

+ H - Bu]⁺, 311.0 [M + H - Boc]⁺. HPLC t_R: 1.33 min

(conditions A).

(R)-tert-Butyl 1-(4-Ethylbenzylamino)-3-(1H-indol-3-yl)-1-

thioxopropan-2-ylcarbamate. Yield, 6.3 g (95%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.11 (t, 3H, J ) 8 Hz, CH₃-CH₂-

phenyl), 1.28 (s, 9H, CH₃Boc), 2.53 (q, 2H, J ) 8 Hz, CH₃-

CH₂-phenyl), 2.97 (dd, 1H, J ) 14 and 9 Hz, CH₂âTrp), 3.15

(dd, 1H, J ) 14 and 5 Hz, CH₂âTrp), 4.68 (m, 3H, CH₂

p-ethylbenzyl and CH RTrp), 6.75 (d, 1H, J_o) 8 Hz, H₄Trp),

6.85 (t, 1H, J_o) 8 Hz, H₅Trp), 6.97 (t, 1H, J_o) 8 Hz, H₆Trp),

6.99-7.10 (m, 4H, CHar p-ethylbenzyl), 7.13 (s, 1H, H₂Trp), 7.31

(d, 1H, J_o) 8 Hz, H₇Trp), 7.60 (d, 1H, J ) 8 Hz, NH Boc),

10.24 (t, 1H, J ) 5 Hz, NH thioamide), 10.79 (s, 1H, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 16.1 (CH₃-CH₂-

phenyl), 28.2 (CH₃-CH₂-phenyl), 28.5 (CH₃Boc), 31.3 (C âTrp),

48.5 (CH₂p-ethylbenzyl), 62.1 (C RTrp), 78.6 (Cq Boc), 110.4

(C₃Trp), 111.7 (C₇Trp), 118.6 (C₄Trp), 118.9 (C₅Trp), 121.2

(C₆Trp), 124.4 (C₂Trp), 127.7 (C₉Trp), 127.9 (C₂, C₃, C₅and C₆

p-ethylbenzyl), 134.5 (C₄p-ethylbenzyl), 136.5 (C₈Trp), 143.0 (C₁

p-ethylbenzyl), 155.2 (CO Boc), 205.0 (CS thioamide). MS (ES),

t

m/z: 438.2 [M + H]⁺, 382.2 [M + H - Bu]⁺, 338.2 [M + H -

Boc]⁺. HPLC t_R: 2.17 min (conditions A).

(R)-tert-Butyl 1-(4-(2,4-Dimethoxybenzyl)-5-phenethyl-4H-

1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield, 2.2 g

(R)-tert-Butyl 3-(1H-Indol-3-yl)-1-(thiophen-2-ylmethylamino)-

1-thioxopropan-2-ylcarbamate. Yield, 4.5 g (69%). ¹H NMR (300

MHz, DMSO-d₆, 300 K): δ 1.28 (s, 9H, CH₃Boc), 2.95 (dd, 1H,

J ) 14 and 9 Hz, CH₂âTrp), 3.12 (dd, 1H, J ) 14 and 4 Hz, CH₂

âTrp), 4.55 (m, 1H, CH RTrp), 4.80 (m, 2H, CH₂-o-thiophenyl),

1

(65%). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.23 (s, 9H,

CH₃Boc), 2.81 (m, 2H, CH₂-CH₂-phenyl), 2.94 (m, 2H, CH₂-

CH₂-phenyl), 3.28 (m, 2H, CH₂âTrp), 3.58 (s, 3H, OCH₃), 3.68

(s, 3H, OCH₃), 5.05 (m, 3H, CH₂-o,p-dimethoxyphenyl and CH

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5799

RTrp), 6.30 (d, 1H, J_o) 8 Hz, H₅o,p-dimethoxyphenyl), 6.51 (s,

1H, H₃o,p-dimethoxyphenyl), 6.62 (d, 1H, J_o) 8 Hz, H₆o,p-

dimethoxyphenyl), 6.89 (t, 1H, J_o) 7 Hz, H₅Trp), 7.02 (t, 1H, J_o

) 8 Hz, H₆Trp), 7.06 (d, 2H, J_o) 8 Hz, H₂and H₆phenyl), 7.08

(s, 1H, H₂Trp), 7.17 (d, 1H, J_o) 7 Hz, H₄Trp), 7.21-7.31 (m,

4H, H₇Trp, H₃, H₄, and H₅phenyl), 7.65 (d, 1H, J ) 8 Hz, NH

Boc), 10.85 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-

d₆, 300 K): δ 26.1 (CH₂-CH₂-phenyl), 28.4 (CH₃Boc), 28.7 (C

âTrp), 32.0 (CH₂-CH₂-phenyl), 43.1 (CH₂-o,p-dimethoxyphe-

nyl), 46.8 (C RTrp), 55.7 (OCH₃), 55.8 (OCH₃), 79.0 (Cq Boc),

99.0 (C₃o,p-dimethoxyphenyl), 105.2 (C₅o,p-dimethoxyphenyl),

109.7 (C₃Trp), 111.8 (C₇Trp), 114.2 (C₁o,p-dimethoxyphenyl),

118.4 (C₄Trp), 118.8 (C₅Trp), 121.3 (C₆Trp), 124.4 (C₂Trp),

126.8 (C₆o,p-dimethoxyphenyl), 127.4 (C₉Trp), 128.6-129.1 (C₂,

C₃, C₄, C₅, and C₆phenyl), 136.4 (C₈Trp), 140.0 (C₁phenyl), 155.1

(Cq triazole), 155.5 (Cq triazole), 156.2 (CO Boc), 158.0 (C₂o,p-

dimethoxyphenyl), 161.2 (C₄o,p-dimethoxyphenyl). MS (ES),

m/z: 583.0 [M + H]⁺. HPLC t_R: 1.97 min (conditions A).

(R)-tert-Butyl 1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-

triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield, 1.7 g (65%).

¹H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.21 (s, 9H, CH₃Boc),

2.79 (m, 2H, CH₂-CH₂-phenyl), 2.88 (m, 2H, CH₂-CH₂-

phenyl), 3.33 (m, 2H, CH₂âTrp), 3.67 (s, 3H, OCH₃), 5.03 (m,

1H, CH RTrp), 5.17 (s, 2H, CH₂-p-methoxyphenyl), 6.72 (d, 2H,

J_o) 8 Hz, H₃and H₅p-methoxyphenyl), 6.81 (d, 2H, J_o) 8 Hz,

H₂and H₆p-methoxyphenyl), 6.88 (t, 1H, J_o) 8 Hz, H₅Trp),

7.02 (t, 1H, J_o) 8 Hz, H₆Trp), 7.04 (m, 2H, H₂and H₆phenyl),

7.06 (s, 1H, H₂Trp), 7.16 (d, 1H, J_o) 7 Hz, H₄Trp), 7.19-7.32

(m, 4H, H₇Trp, H₃, H₄, and H₅phenyl), 7.73 (d, 1H, J ) 8 Hz,

NH Boc), 10.84 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz,

DMSO-d₆, 300 K): δ 26.2 (CH₂-CH₂-phenyl), 28.4 (CH₃Boc),

28.7 (C âTrp), 32.0 (CH₂-CH₂-phenyl), 46.3 (CH₂-p-methox-

yphenyl), 46.8 (C RTrp), 55.5 (OCH₃), 79.0 (Cq Boc), 109.7 (C₃

Trp), 111.8 (C₇Trp), 114.5 (C₃and C₅p-methoxyphenyl), 118.4

(C₄Trp), 118.8 (C₅Trp), 121.3 (C₆Trp), 124.5 (C₂Trp), 126.7

(C₄phenyl, C₉Trp), 127.4 (C₁p-methoxyphenyl), 128.2 (C₂and

C₆p-methoxyphenyl), 128.8 (C₂, C₃, C₅, and C₆phenyl), 136.4

(C₈Trp), 140.1 (C₁phenyl), 154.9 (CO Boc), 155.6 (Cq triazole),

156.0 (Cq triazole), 159.3 (C₄p-methoxyphenyl). MS (ES), m/z:

552.1 [M + H]⁺. HPLC t_R: 1.95 min (conditions A).

indole and H₄Trp), 10.70 (s, 2H, NH indole and NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 23.3 (CH₂-CH₂-

indole), 26.2 (CH₂-CH₂-indole), 28.6 (CH₃Boc), 30.1 (C âTrp),

47.7 (C RTrp), 78.4 (Cq Boc), 110.1 (C₃Trp), 111.7 (C₇Trp), 111.8

(C₇indole), 113.2 (C₃indole), 118.2 (C₄indole and C₄Trp), 118.6

(C₅indole and C₅Trp), 121.2 (C₆indole), 121.3 (C₆Trp), 122.8

(C₂indole and C₂Trp), 127.1 (C₉indole), 127.4 (C₉Trp), 127.7

(C₄phenyl), 130.0 (C₂, C₃, C₅, and C₆phenyl), 133.5 (C₁phenyl),

136.4 (C₈trp), 136.6 (C₈indole), 154.3 (CO Boc), 155.3 (Cq

triazole), 156.1 (Cq triazole). MS (ES), m/z: 547.3 [M + H]⁺.

HPLC t_R: 1.67 min (conditions A).

(R)-tert-Butyl 1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-ethylbenzyl)-

4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield, 0.8

1

g (58%). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.08 (t, 3H,

J ) 8 Hz, CH₃-CH₂-p-ethylphenyl), 1.16 (s, 9H, CH₃Boc), 2.51

(q, 2H, J ) 7 Hz, CH₃-CH₂-p-ethylphenyl), 2.92 (m, 4H, CH₂-

CH₂-indole), 3.31 (m, 2H, CH₂âTrp), 5.01 (m, 1H, CH RTrp),

5.22 (m, 2H, CH₂p-ethylbenzyl), 6.79 (d, 2H, J_o) 8 Hz, H₃and

H₅p-ethylbenzyl), 6.88 (t, 2H, J_o) 7 Hz, H₅indole and H₅Trp),

6.89 (d, 2H, J_o) 8 Hz, H₂and H₆p-ethylbenzyl), 6.99-7.11 (m,

5H, H₂and H₆indole, H₂, H₄, and H₆Trp), 7.23 (d, 1H, J_o) 8

Hz, H₄indole), 7.29 (d, 2H, J_o) 8 Hz, H₇indole and H₇Trp),

7.69 (d, 1H, J ) 8 Hz, NH Boc), 10.79 (s, 1H, NH indole), 10.82

(s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K):

δ 15.9 (CH₃-CH₂-p-ethylphenyl), 22.2 (CH₂-CH₂-indole), 25.7

(CH₂-CH₂-indole), 28.1 (CH₃-CH₂-p-ethylphenyl), 28.3 (CH₃

Boc), 28.6 (C âTrp), 46.7 (CH₂p-ethylbenzyl), 46.8 (C RTrp), 79.0

(Cq Boc), 109.7 (C₃Trp), 111.7 (C₇Trp), 111.8 (C₇indole), 112.6

(C₃indole), 118.4 (C₄Trp and C₄indole), 118.7 (C₅Trp and C₅

indole), 121.3 (C₆indole), 121.4 (C₆Trp), 123.1 (C₂indole), 124.4

(C₂Trp), 126.5 (C₃and C₅p-ethylbenzyl), 127.0 (C₉Trp), 127.4

(C₉indole), 128.5 (C₂and C₆p-ethylbenzyl), 132.1 (C₁p-

ethylbenzyl), 136.4 (C₈Trp), 136.6 (C₈indole), 143.9 (C₄p-

ethylbenzyl), 155.5 (2 Cq triazole), 156.1 (CO Boc). MS (ES),

m/z: 589.3 [M + H]⁺. HPLC t_R: 2.00 min (conditions A).

(R)-tert-Butyl 1-(4-(4-Ethylbenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield, 0.8 g (63%). ¹H

NMR (300 MHz, DMSO-d₆, 300 K): δ 1.08 (t, 3H, J ) 8 Hz,

CH₃-CH₂-p-ethylphenyl), 1.17 (s, 9H, CH₃Boc), 2.51 (q, 2H, J

) 7 Hz, CH₃-CH₂-p-ethylphenyl), 2.86 (m, 4H, CH₂-CH₂-

phenyl), 3.31 (m, 2H, CH₂âTrp), 5.00 (m, 1H, CH RTrp), 5.20

(m, 2H, CH₂p-ethylbenzyl), 6.78 (d, 2H, J_o) 8 Hz, H₃and H₅

p-ethylbenzyl), 6.87 (t, 1H, J_o) 7 Hz, H₅Trp), 7.02 (t, 1H, J_o)

8 Hz, H₆Trp), 7.04 (m, 5H, H₂Trp, H₂and H₆p-ethylbenzyl, H₂

and H₆phenyl), 7.15 (d, 1H, J_o) 8 Hz, H₄Trp), 7.18-7.31 (m,

4H, H₇Trp, H₃, H₄, and H₆phenyl), 7.69 (d, 1H, J ) 8 Hz, NH

Boc), 10.82 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-

d₆, 300 K): δ 15.9 (CH₃-CH₂-p-ethylphenyl), 26.3 (CH₂-CH₂-

phenyl), 28.1 (CH₃-CH₂-p-ethylphenyl), 28.3 (CH₃Boc), 28.6

(C âTrp), 32.1 (CH₂-CH₂-phenyl), 46.5 (CH₂p-ethylbenzyl), 46.8

(C RTrp), 78.9 (Cq Boc), 109.8 (C₃Trp), 111.7 (C₇Trp), 118.4

(C₄Trp), 118.8 (C₅Trp), 121.3 (C₆Trp), 124.4 (C₂Trp), 126.6

(C₃and C₅p-ethylbenzyl), 126.7 (C₄phenyl), 127.4 (C₉Trp),

128.5-128.8 (C₂and C₆p-ethylbenzyl, C₂, C₃, C₅, and C₆phenyl),

132.3 (C₁p-ethylbenzyl), 136.4 (C₈Trp), 140.2 (C₁phenyl), 143.9

(C₄p-ethylbenzyl), 155.0 (CO Boc), 155,5 (Cq triazole), 156.0 (Cq

triazole). MS (ES), m/z: 549.9 [M + H]⁺. HPLC t_R: 2.03 min

(conditions A).

(R)-tert-Butyl 1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxyben-

zyl)-4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield,

1

1.4 g (62%). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.20 (s,

9H, CH₃Boc), 2.93 (m, 4H, CH₂-CH₂-indole), 3.32 (m, 2H, CH₂

âTrp), 3.66 (s, 3H, OCH₃), 5.02 (m, 1H, CH RTrp), 5.18 (m, 2H,

CH₂-p-methoxyphenyl), 6.71 (d, 2H, J_o) 8 Hz, H₃and H₅

p-methoxyphenyl), 6.81 (d, 2H, J_o) 8 Hz, H₂and H₆p-

methoxyphenyl), 6.90 (t, 3H, J_o) 7 Hz, H₅and H₆Trp, H₅indole),

7.00-7.07 (m, 4H, H₂and H₆indole, H₂and H₄Trp), 7.26-7.32

(m, 3H, H₄and H₇indole, H₇Trp), 7.72 (d, 1H, J ) 8 Hz, NH

Boc), 10.80 (s, 1H, NH indole), 10.83 (s, 1H, NH indole Trp). ¹³

C

NMR (75 MHz, DMSO-d₆, 300 K): δ 22.1 (CH₂-CH₂-indole),

25.7 (CH₂-CH₂-indole), 27.5 (CH₃Boc), 27.7 (C âTrp), 46.5

(CH₂-p-methoxyphenyl), 46.9 (C RTrp), 55.5 (OCH₃), 79.0 (Cq

Boc), 109.7 (C₃Trp), 111.8 (C₇Trp and C₇indole), 112.6 (C₃

indole), 114.3 (C₃and C₅p-methoxyphenyl), 118.1 (C₄Trp and

C₄indole), 119.1 (C₅Trp and C₅indole), 121.5 (C₆Trp and C₆

indole), 123.1 (C₂indole), 124.5 (C₂Trp), 126.6 (C₉indole), 127.0

(C₉Trp), 127.4 (C₁p-methoxyphenyl), 128.1 (C₂and C₆p-

methoxyphenyl), 136.4 (C₈Trp), 136.6 (C₈indole), 155.4 (CO Boc),

155.5 (Cq triazole), 156.0 (Cq triazole), 162.2 (C₄p-methoxyphe-

nyl). MS (ES), m/z: 561.1 [M + H]⁺. HPLC t_R: 1.98 min

(conditions A).

(R)-tert-Butyl 1-(5-(2-(1H-Indol-3-yl)ethyl)-4-phenyl-4H-1,2,4-

triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield, 0.8 g (49%).

¹H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.24 (s, 9H, CH₃Boc),

2.73 (m, 2H, CH₂-CH₂-indole), 2.85 (m, 2H, CH₂-CH₂-indole),

3.04 (m, 2H, CH₂âTrp), 4.51 (m, 1H, CH RTrp), 6.75 (m, 2H, H₅

indole and H₅Trp), 6.83 (t, 2H, J_o) 7 Hz, H₆indole and H₆Trp),

6.92-7.04 (m, 7H, H₂indole, H₂Trp and CHar phenyl), 7.26 (d,

2H, J_o) 8 Hz, H₇indole and H₇Trp), 7.46 (m, 3H, NH Boc, H₄

(R)-tert-Butyl 1-(5-(3-(1H-Indol-3-yl)propyl)-4-benzyl-4H-

1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethylcarbamate. Yield, 0.9 g

1

(48%). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.18 (s, 9H,

CH₃Boc), 1.85 (m, 2H, CH₂-CH₂-CH₂-indole), 2.53 (t, 2H, J

) 7 Hz, CH₂-CH₂-CH₂-indole), 2.63 (t, 2H, J ) 7 Hz, CH₂-

CH₂-CH₂-indole), 3.32 (m, 2H, CH₂âTrp), 4.89 (m, 1H, CH

RTrp), 5.05 (s, 2H, CH₂benzyl), 6.74 (d, 2H, J_o) 7 Hz, H₂and

H₆benzyl), 6.86 (t, 1H, J_o) 7 Hz, H₅Trp), 6.88-7.02 (m, 5H, H₂

and H₆Trp, H₂, H₅, and H₆indole), 7.14 (m, 3H, H₃, H₄, and H₅

benzyl), 7.28 (m, 3H, H₄indole, H₄and H₇Trp), 7.39 (d, 1H, J_o)

8 Hz, H₇indole), 7.50 (d, 1H, J ) 7 Hz, NH Boc), 10.68 (s, 1H,

NH indole), 10.73 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz,

DMSO-d₆, 300 K): δ 24.3 (CH₂-CH₂-CH₂-indole), 24.5 (CH₂-

5800 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

CH₂-CH₂-indole), 27.7 (CH₂-CH₂-CH₂-indole), 28.4 (CH₃

Boc), 29.3 (C âTrp), 45.7 (CH₂benzyl), 46.8 (C RTrp), 78.5 (Cq

Boc), 110.7 (C₃Trp), 111.6 (C₇indole and C₇Trp), 114.2 (C₃

indole), 118.5 (C₄indole and C₄Trp), 118.7 (C₅indole and C₅

Trp), 121.2 (C₆indole and C₆Trp), 122.7 (C₂indole), 124.3 (C₂

Trp), 126.4 (C₄benzyl), 127.5 (C₉Trp), 127.6 (C₉indole), 127.8

(C₂and C₆benzyl), 128.9 (C₃and C₅benzyl), 136.4 (C₈indole

and C₈Trp), 136.7 (C₁benzyl), 154.8 (CO Boc), 155.5 (Cq triazole),

155.6 (Cq triazole). MS (ES), m/z: 575.4 [M + H]⁺. HPLC t_R:

1.82 min (conditions A).

General Procedure for Preparation of Compound E. The Boc

protecting group of compound C was removed at room temperature

for 1 h with a solution of AcOEt/HCl, 4 M. The mixture was then

concentrated in vacuo, diluted with MeOH, and concentrated several

times in vacuo. The residue was then coupled with the correspond-

ing acid (1.1 equiv), in the presence of BOP (1.1 equiv) and NMM

(2.2 equiv) for 2 h, in DCM. The mixture was then concentrated

in vacuo and the residue dissolved in AcOEt. The organic layer

was successively washed with aqueous solutions of 1 M KHSO₄,

saturated NaHCO₃, and brine. The organic layer was then dried

over Na₂SO₄, filtered, and concentrated in vacuo to yield the desired

compound, which was then treated with 4 M AcOEt/HCl as already

described. The final compound was purified by preparative HPLC

on a C18 column using a water/acetonitrile/TFA, 0.1% gradient

(yield around 50% for the three steps).

(R)-N-(1-(5-(2-(1H-Indol-3-yl)ethyl)-4-benzyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)-2-amino-2-methylpropanamide Tri-

fluoroacetate Salt (1). ¹H NMR (400 MHz, DMSO-d₆, 300 K): δ

1.29 (s, 3H, CH₃Aib), 1.30 (s, 3H, CH₃Aib), 2.88 (m, 2H, CH₂-

CH₂-indole), 2.97 (m, 2H, CH₂-CH₂-indole), 3.37 (m, 2H, CH₂

âTrp), 5.11 (s, 2H, CH₂benzyl), 5.21 (m, 1H, CH RTrp), 6.86 (t,

1H, J ) 7.4, H₅Trp), 6.88 (d, 2H, H₂and H₆benzyl), 6.92 (t, 1H,

J ) 7.6, H₅indole), 7.03 (t, 1H, J ) 7.6, H₆Trp), 7.05 (2H, H₆

indole, H₂indole), 7.09 (d, 1H, J ) 1.8, H₂Trp), 7.17 (d, 1H, J )

7.9, H₄Trp), 7.26 (m, 2H, H₃and H₅benzyl), 7.27 (t, 1H, H₄

benzyl), 7.30 (d, 1H, H₄indole), 7.32 (m, 2H, H₇Trp and H₇

indole), 8.03 (brs, 3H, NH₂Aib TFA salt), 8.95 (d, 1H, J ) 8.1,

NH amide), 10.77 (s, 1H, NH indole), 10.81 (s, 1H, NH indole

Trp). ¹³C NMR (100 MHz, DMSO-d₆, 300 K): δ 22.4 (CH₂-CH₂

indole), 23.1 (CH₃Aib), 23.3 (CH₃Aib), 25.4 (CH₂-CH₂indole),

28.7 (C âTrp), 45.3 (C RTrp, CH₂benzyl), 56.3 (Cq Aib), 109.5

(C₃Trp), 111.3 (C₇Trp, C₇indole), 113.0 (C₃indole), 117.8 (C₄

Trp), 118.0 (C₄indole), 118.2 (C₅indole), 118.3 (C₅Trp), 120.9

(C₆Trp, C₆indole), 122.4 (C₂indole), 124.3 (C₂Trp), 125.9 (C₂,

C₆benzyl), 126.7 (C₉Indole), 126.9 (C₉Trp), 127.6 (C₄benzyl),

128.8 (C₃, C₅benzyl), 135.7 (C₁benzyl), 136.0 (C₈Trp), 136.1

(C₈indole), 154.3 (Cq triazole), 154.5 (Cq triazole), 171.4 (CO

amide).

(R)-N-(1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-4H-

1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)-2-amino-2-methylpro-

panamide Trifluoroacetate Salt (2). ¹H NMR (400 MHz, DMSO-

d₆, 300 K): δ 1.30 (s, 3H, CH₃Aib), 1.33 (s, 3H, CH₃Aib), 2.91

(m, 2H, CH₂-CH₂-indole), 2.97 (m, 2H, CH₂-CH₂-indole), 3.37

(d, 2H, CH₂âTrp), 3.71(s, 3H, OCH₃), 5.02 (s, 2H, CH₂p-

methoxybenzyl), 5.23 (m, 1H, CH RTrp), 6.78 (s, 4H, CHar

p-methoxybenzyl), 6.87 (t, 1H, J ) 7.5, H₅Trp), 6.93 (t, 1H, J )

7.5, H₅indole), 7.03 (t, 1H, H₆Trp), 7.05 (t, 1H, H₆indole), 7.07

(s, 1H, H₂indole), 7.09 (s, 1H, H₂Trp), 7.21 (d, 1H, J ) 8, H₄

Trp), 7.32 (3H, H₄and H₇indole, H₇Trp), 8.02 (brs, 3H, NH₂Aib

TFA salt), 8.97 (d, 1H, J ) 8.1, NH amide), 10.77 (s, 1H, NH

indole), 10.80 (s, 1H, NH indole Trp). ¹³C NMR (100 MHz, DMSO-

d₆, 300 K): δ 22.4 (CH₂-CH₂indole), 23.1 (CH₃Aib), 23.4 (CH₃

Aib), 25.5 (CH₂-CH₂indole), 28.9 (C âTrp), 44.9 (CH₂p-

methoxybenzyl), 45.3 (C RTrp), 55.0 (OCH₃), 56.3 (Cq Aib), 109.5

(C₃Trp), 111.3 (C₇Trp, C₇indole), 113.0 (C₃indole), 114.1 (C₃,

C₅p-methoxybenzyl), 117.9 (C₄Trp), 118.0 (C₄indole), 118.2 (C₅

indole), 118.3 (C₅Trp), 120.9 (C₆indole, C₆Trp), 122.0 (C₂indole),

124.4 (C₂Trp), 126.7 (C₉indole), 126.9 (C₉Trp), 127.3 (C₂, C₆

p-methoxybenzyl), 127.4 (C₁p-methoxybenzyl), 135.9 (C₈Trp),

136.1 (C₈indole), 154.2 (Cq triazole), 154.5 (Cq triazole), 158.4

(C₄p-methoxybenzyl), 171.4 (CO amide).

N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-4H-

1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)-2-aminoacetamide (3).

¹H NMR (300 MHz, DMSO-d₆, 300 K): δ 2.86 (m, 4H, CH₂-

CH₂-indole), 3.25 (dd, 1H, J ) 14 Hz and 7 Hz, CH₂âTrp), 3.38

(m, 3H, CH₂âTrp and CH₂RGly), 3.64 (s, 3H, OCH₃), 4.93 (m,

2H, CH₂p-methoxybenzyl), 5.19 (m, 1H, CH RTrp), 6.63 (s, 4H,

CHar p-methoxybenzyl), 6.89 (t, 1H, J_o) 7 Hz, H₅indole), 6.86

(t, 1H, J_o) 7 Hz, H₅Trp), 6.99-7.04 (m, 5H, H₂, H₄, and H₆Trp,

H₂and H₆indole), 7.22 (d, 1H, J_o) 8 Hz, H₄indole), 7.30 (m,

2H, H₇indole and H₇Trp), 7.92 (brs, 3H, NH₂Gly TFA salt),

9.18 (d, 1H, J ) 8 Hz, NH amide), 10.74 (s, 1H, NH indole), 10.80

(d, 1H, J ) 2 Hz, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆,

300 K): δ 22.8 (CH₂-CH₂-indole), 25.8 (CH₂-CH₂-indole), 29.8

(C âTrp), 39.1 (CRGly), 45.4 (CH₂p-methoxybenzyl and C RTrp),

55.4 (OCH₃), 109.7 (C₃Trp), 111.8 (C₇indole and C₇Trp), 113.4

(C₃indole), 114.5 (C₃and C₅p-methoxybenzyl), 118.3 (C₄indole),

118.5 (C₄Trp), 118.6 (C₅indole), 118.8 (C₅Trp), 121.3 (C₆indole

and C₆Trp), 123.0 (C₂indole), 124.6 (C₂Trp), 127.2 (C₉indole),

127.4 (C₉Trp), 127.7 (C₂and C₆p-methoxybenzyl), 136.4 (C₈Trp),

136.6 (C₈indole), 154.7 (2Cq triazole), 159.0 (CO amide), 166.1

(C₄p-methoxybenzyl).

(R)-N-(1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)-2-aminoacetamide Trifluoroacetate

Salt (4). ¹H NMR (300 MHz, DMSO-d₆, 300 K): δ 2.78 (m, 4H,

CH₂-CH₂-phenyl), 3.26 (1H, dd, J ) 14 Hz and 7 Hz, CH₂âTrp),

3.39 (m, 3H, CH₂âTrp and CH₂RGly), 3.65 (s, 3H, OCH₃), 4.95

(m, 2H, CH₂-p-methoxybenzyl), 5.20 (m, 1H, CH RTrp), 6.63 (s,

4H, CHar p-methoxybenzyl), 6.86 (t, 1H, J_o) 7 Hz, H₅Trp), 6.99

(s, 1H, H₂Trp), 7.02 (t, 1H, J_o) 7 Hz, H₆Trp), 7.10 (m, 2H, H₂,

and H₆phenyl), 7.15 (d, 1H, J_o) 7 Hz, H₄Trp), 7.23 (m, 3H, H₃,

H₄, and H₅phenyl), 7.31 (d, 1H, J_o) 8 Hz, H₇Trp), 7.95 (brs,

3H, NH₂Gly, TFA salt), 9.20 (d, 1H, J ) 8 Hz, NH amide), 10.82

(s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K):

δ 26.5 (CH₂-CH₂-phenyl), 29.8 (C âTrp), 32.7 (CH₂-CH₂-

phenyl), 39.0 (C RGly), 45.3 (CH₂p-methoxybenzyl), 45.4 (C

RTrp), 55.4 (OCH₃), 109.7 (C₃Trp), 111.8 (C₇Trp), 114.5 (C₃

and C₅p-methoxybenzyl), 118.3 (C₄Trp), 118.9 (C₅Trp), 121.4

(C₆Trp), 124.6 (C₂Trp), 126.5 (C₄phenyl), 127.3 (C₉Trp), 127.7

(C₁p-methoxybenzyl), 127.8 (C₂and C₆p-methoxybenzyl), 128.7

(C₂, C₃, C₅, and C₆phenyl), 136.4 (C₈Trp), 140.9 (C₁phenyl),

154.3 (Cq triazole), 154.8 (Cq triazole), 159.0 (C₄p-methoxyben-

zyl), 166.1 (CO amide).

(R)-N-(1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(2,4-dimethoxybenzyl)-

4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)-2-aminoaceta-

1

mide Trifluoroacetate Salt (5). H NMR (300 MHz, DMSO-d₆,

300 K): δ 2.89 (m, 4H, CH₂-CH₂-indole), 3.25-3.43 (m, 4H,

CH₂âTrp and CH₂RGly), 3.60 (s, 3H, OCH₃), 3.65 (s, 3H, OCH₃),

4.80 (d, 1H, J ) 17 Hz, CH₂-o,p-dimethoxybenzyl), 4.97 (d, 1H,

J ) 17 Hz, CH₂-o,p-dimethoxybenzyl), 5.16 (m, 1H, CH RTrp),

6.15 (dd, 1H, J_o) 8 Hz and J_m) 2 Hz, H₅o,p-dimethoxybenzyl),

6.25 (d, 1H, J_o) 8 Hz, H₆o,p-dimethoxybenzyl), 6.49 (d, 1H, J

) 2 Hz, H₃o,p-dimethoxybenzyl), 6.89 (m, 2H, H₅Trp and H₅

indole), 6.98-7.05 (m, 4H, H₂and H₆Trp, H₂and H₆indole), 7.17

(d, 1H, J_o) 8 Hz, H₄Trp), 7.29 (m, 3H, H₄and H₇indole, H₇

Trp), 7.93 (brs, 3H, NH₂Gly TFA salt), 9.16 (d, 1H, J ) 8 Hz,

NH amide), 10.76 (s, 1H, NH indole), 10.80 (s, 1H, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 22.8 (CH₂-CH₂-

indole), 25.8 (CH₂-CH₂-indole), 29.8 (C âTrp), 39.1 (CRGly),

41.7 (CH₂o,p-dimethoxybenzyl), 45.5 (C RTrp), 55.6 (OCH₃), 55.9

(OCH₃), 98.9 (C₃o,p-dimethoxybenzyl), 105.0 (C₅o,p-dimethoxy-

benzyl), 109.8 (C₃Trp), 111.8 (C₇indole and C₇Trp), 113.4 (C₃

indole), 115.5 (C₁o,p-dimethoxybenzyl), 118.2 (C₄indole), 118.4

(C₄Trp), 118.6 (C₅indole), 118.8 (C₅Trp), 121.3 (C₆indole and

C₆Trp), 123.0 (C₂indole), 125.1 (C₂Trp), 127.2 (C₉indole and

C₉Trp), 127.9 (C₆o,p-dimethoxybenzyl), 136.4 (C₈indole and C₈

Trp), 154.9 (Cq triazole), 155.1 (Cq triazole), 157.6 (C₂o,p-

dimethoxybenzyl), 160.8 (C₄o,p-dimethoxybenzyl), 166.0 (CO

amide).

(R)-N-(1-(4-(2,4-Dimethoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)-2-aminoacetamide Trifluoro-

1

acetate Salt (6). H NMR (300 MHz, DMSO-d₆, 300 K): δ 2.81

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5801

(m, 4H, CH₂-CH₂-phenyl), 3.27 (t, 2H, J ) 8 Hz, CH₂RGly),

3.43 (m, 2H, CH₂âTrp), 3.60 (s, 3H, OCH₃), 3.67 (s, 3H, OCH₃),

4.79 (d, 1H, J ) 17 Hz, CH₂-o,p-dimethoxybenzyl), 4.96 (d, 1H,

J ) 17 Hz, CH₂-o,p-dimethoxybenzyl), 5.18 (m, 1H, CH RTrp),

6.18 (dd, 1H, J_o) 8 Hz and J_m) 2 Hz, H₅o,p-dimethoxybenzyl),

6.29 (d, 1H, J_o) 8 Hz, H₆o,p-dimethoxybenzyl), 6.49 (d, 1H, J_m

) 2 Hz, H₃o,p-dimethoxybenzyl), 6.85 (t, 1H, J_o) 8 Hz, H₅Trp),

6.99 (m, 2H, H₂and H₆Trp), 7.11 (m, 2H, H₂and H₆phenyl),

7.18 (d, 1H, J_o) 8 Hz, H₄Trp), 7.21 (m, 3H, H₃, H₄, and H₅

phenyl), 7.29 (d, 1H, J_o) 8 Hz, H₇Trp), 7.93 (brs, 3H, NH₂Gly

TFA salt), 9.14 (d, 1H, J ) 8 Hz, NH amide), 10.81 (s, 1H, NH

indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 26.4 (CH₂-

CH₂-phenyl), 29.8 (C âTrp), 32.7 (CH₂-CH₂-phenyl), 39.4

(CRGly), 41.7 (CH₂o,p-dimethoxybenzyl), 45.5 (CH RTrp), 55.6

(OCH₃), 55.9 (OCH₃), 99.0 (C₃o,p-dimethoxybenzyl), 105.1 (C₅

o,p-dimethoxybenzyl), 109.7 (C₃Trp), 111.8 (C₇Trp), 115.4 (C₁

o,p-dimethoxybenzyl), 118.2 (C₄Trp), 118.8 (C₅Trp), 121.4 (C₆

Trp), 124.6 (C₂Trp), 127.9 (C₆o,p-dimethoxybenzyl), 127.3 (C₉

Trp), 128.2 (C₄phenyl), 128.7 (C₂, C₃, C₅, and C₆phenyl), 136.4

(C₈Trp), 140.8 (C₁phenyl), 154.4 (Cq triazole), 155.1 (Cq triazole),

157.7 (C₂o,p-dimethoxybenzyl), 160.8 (C₄o,p-dimethoxybenzyl),

166.0 (CO amide).

(d, 1H, J ) 17 Hz, CH₂p-methoxybenzyl), 5.01 (d, 1H, J ) 17

Hz, CH₂p-methoxybenzyl), 5.21 (m, 1H, CH RTrp), 6.63 (s, 4H,

CHar p-methoxybenzyl), 6.86 (t, 1H, J_o) 8 Hz, H₅Trp), 6.97 (d,

1H, J ) 2 Hz, H₂Trp), 7.01 (t, 1H, J_o) 8 Hz, H₆Trp), 7.08 (m,

2H, H₂and H₆phenyl), 7.15 (d, 1H, J_o) 7 Hz, H₄Trp), 7.18-

7.31 (m, 4H, H₇Trp, H₃, H₄, and H₅phenyl), 7.63 (brs, 3H, NH₂

TFA salt), 8.92 (d, 1H, J ) 8 Hz, NH amide), 10.79 (s, 1H, NH

indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 26.5 (CH₂-

CH₂-phenyl), 29.4 (C âTrp), 32.2 (CH₂-CH₂-NH₂), 32.6 (CH₂-

CH₂-phenyl), 35.4 (CH₂-CH₂-NH₂), 45.0 (C RTrp), 45.3 (CH₂

p-methoxybenzyl), 55.5 (OCH₃), 110.1 (C₃Trp), 111.8 (C₇Trp),

114.4 (C₃and C₅p-methoxybenzyl), 118.4 (C₄Trp), 118.8 (C₅Trp),

121.3 (C₆Trp), 124.5 (C₂Trp), 126.5 (C₄phenyl), 127.4 (C₉Trp,

C₂and C₆p-methoxybenzyl), 127.7 (C₁p-methoxybenzyl), 128.7

(C2₃, C₃, C₅, and C₆phenyl), 136.4 (C₈Trp), 140.8 (C₁phenyl),

154.3 (Cq triazole), 155.1 (Cq triazole), 159.0 (C₄p-methoxyben-

zyl), 169.5 (CO amide).

(S)-N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-

1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)pyrrolidine-2-carboxa-

mide Trifluoroacetate Salt (16). ¹H NMR (300 MHz, DMSO-d₆,

300 K): δ 1.42 (m, 1H, H₃Pro), 1.52 (m, 1H, H₄Pro), 1.72 (m,

1H, H₄Pro), 2.07 (m, 1H, H₃Pro), 2.94 (m, 4H, CH₂-CH₂-indole),

3.05 (t, 2H, J ) 6 Hz, H₅Pro), 3.30 (m, 2H, CH₂âTrp), 3.67 (s,

3H, OCH₃), 3.96 (m, 1H, CH RPro), 5.02 (s, 2H, CH₂p-

methoxybenzyl), 5.19 (m, 1H, CH RTrp), 6.73 (s, 4H, CHar

p-methoxybenzyl), 6.84 (t, 1H, J_o) 8 Hz, H₅indole), 6.90 (t, 1H,

J_o) 8 Hz, H₅Trp), 6.93-7.06 (m, 4H, H₂and H₆indole, H₂and

H₆Trp), 7.17 (d, 1H, J_o) 8 Hz, H₄Trp), 7.29 (d, 3H, J_o) 8 Hz,

H₄and H₇indole, H₇Trp), 8.39 and 9.10 (2 m, 2H, NH Pro TFA

salt), 9.25 (d, 1H, J ) 8 Hz, NH amide), 10.76 (s, 1H, NH indole),

10.80 (d, 1H, J ) 2 Hz, NH indole Trp). ¹³C NMR (75 MHz,

DMSO-d₆, 300 K): δ 22.8 (CH₂-CH₂-indole), 23.5 (C₄Pro), 25.8

(CH₂-CH₂-indole), 29.4 (C âTrp), 29.8 (C₃Pro), 45.2 (C RTrp),

45.5 (CH₂p-methoxybenzyl), 46.0 (C₅Pro), 55.5 (OCH₃), 59.3 (C

RPro), 109.6 (C₃Trp), 111.8 (C₇indole and C₇Trp), 113.4 (C₃

indole), 114.6 (C₃and C₅p-methoxybenzyl), 118.4 (C₄indole),

118.5 (C₄Trp), 118.7 (C₅indole and C₅Trp), 121.4 (C₆indole

and C₆Trp), 123.0 (C₂Trp), 124.7 (C₂indole), 127.2 (C₉indole),

127.3 (C₉Trp), 127.7 (C₁p-methoxybenzyl), 127.8 (C₂and C₆

p-methoxybenzyl), 126.5 (C₈Trp), 136.6 (C₈indole), 154.8 (Cq

triazole), 154.9 (Cq triazole), 159.2 (C₄p-methoxybenzyl), 168.2

(CO amide). MS (ES), m/z: 588.2 [M + H]⁺. HPLC t_R: 1.60 min

(conditions A).

(R)-N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-

4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)pyrrolidine-2-car-

boxamide Trifluoroacetate Salt (17). ¹H NMR (300 MHz, DMSO-

d₆, 300 K): δ 1.23 (m, 1H, H₃Pro), 1.52 (m, 1H, H₄Pro), 1.74

(m, 1H, H₄Pro), 2.08 (m, 1H, H₃Pro), 2.81 (m, 2H, H₅Pro), 2.90-

3,14 (m, 4H, CH₂-CH₂-indole), 3.31 (dd, 1H, J ) 14 Hz and 7

Hz, CH₂âTrp), 3.41 (dd, 1H, J ) 14 Hz and 8 Hz, CH₂âTrp),

3.63 (s, 3H, OCH₃), 4.05 (m, 1H, CH RPro), 4.86 (s, 2H, CH₂

p-methoxybenzyl), 5.21 (m, 1H, CH RTrp), 6.63 (s, 4H, CHar

p-methoxybenzyl), 6.88 (t, 2H, J_o) 7 Hz, H₅indole and H₅Trp),

7.02 (m, 4H, H₂and H₆indole, H₂and H₆Trp), 7.26-7.34 (m,

4H, H₄and H₇indole, H₄and H₇Trp), 8.51 and 9.18 (2 m, 2H,

NH Pro, TFA salt), 9.27 (d, 1H, J ) 8 Hz, NH amide), 10.73 (s,

1H, NH indole), 10.80 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz,

DMSO-d₆, 300 K): δ 22.8 (CH₂-CH₂-indole), 23.8 (C₄Pro), 25.9

(CH₂-CH₂-indole), 29.7 (C âTrp and C₃Pro), 45.4 (CH₂

p-methoxybenzyl), 45.8 (C RTrp), 46.1 (C₅Pro), 55.5 (OCH₃), 59.2

(CH RPro), 109.7 (C₃Trp), 111.8 (C₇Trp), 111.9 (C₇indole), 113.4

(C₃indole), 114.4 (C₃and C₅p-methoxybenzyl), 118.3 (C₄indole),

118.5 (C₄Trp), 118.6 (C₅indole), 118.9 (C₅Trp), 121.4 (C₆indole

and C₆Trp), 122.9 (C₂indole and C₂Trp), 127.2 (C₉indole), 127.4

(C₉Trp), 127.6 (C₁, C₂, and C₆p-methoxybenzyl), 136.5 (C₈Trp),

136.6 (C₈indole), 154.4 (Cq triazole), 155.0 (Cq triazole), 159.1

(C₄p-methoxybenzyl), 168.3 (CO amide).

(S)-N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)-2-aminopropanamide Trifluo-

1

roacetate Salt (12). H NMR (300 MHz, DMSO-d₆, 300 K): δ

1.09 (d, 3H, J ) 7 Hz, CH₃Ala), 2.84 (m, 4H, CH₂-CH₂-phenyl),

3.29 (m, 2H, CH₂âTrp), 3.68 (s, 4H, OCH₃and CH RAla), 5.06

(s, 2H, CH₂- p-methoxybenzyl), 5.19 (m, 1H, CH RTrp), 6.73 (s,

4H, CHar p-methoxybenzyl), 6.81 (t, 1H, J_o) 7 Hz, H₅Trp), 6.99

(m, 2H, H₂and H₆Trp), 7.12-7.26 (m, 6H, H₄Trp and CHar

phenyl), 7.29 (d, 1H, J_o) 8 Hz, H₇Trp), 7.97 (brs, 3H, NH₂Ala

TFA salt), 9.14 (d, 1H, J ) 8 Hz, NH amide), 10.81 (s, 1H, NH

indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 17.3 (CH₃

Ala), 26.4 (CH₂-CH₂-phenyl), 29.5 (C âTrp), 32.7 (CH₂-CH₂-

phenyl), 45.0 (C RTrp), 45.5 (CH₂p-methoxybenzyl), 48.9 (C

RAla), 55.5 (OCH₃), 109.6 (C₃Trp), 111.7 (C₇Trp), 114.6 (C₃

and C₅p-methoxybenzyl), 118.3 (C₄Trp), 118.7 (C₅Trp), 121.3

(C₆Trp), 124.8 (C₂Trp), 126.6 (C₄phenyl), 127.3 (C₉Trp), 127.8

(C₁p-methoxybenzyl), 128.0 (C₂and C₆p-methoxybenzyl), 128.7

(C₂, C₃, C₅, and C₆phenyl), 136.5 (C₈Trp), 140.8 (C₁phenyl),

154.3 (Cq triazole), 155.0 (Cq triazole), 159.2 (C₄p-methoxyben-

zyl), 169.6 (CO amide).

(2S)-N-((R)-1-(4-(4-Ethylbenzyl)-5-phenethyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)-2-aminopropanamide Trifluoroac-

1

etate Salt (13). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.06

(d, 3H, J ) 7 Hz, CH₃Ala), 1.11 (t, 3H, J ) 8 Hz, CH₃-CH₂-

p-ethylphenyl), 2.53 (q, 2H, J ) 8 Hz, CH₃-CH₂- p-ethylphenyl),

2.83 (m, 4H, CH₂-CH₂-phenyl), 3.26 (m, 2H, CH₂âTrp), 4.65

(m, 1H, CH RAla), 5.10 (s, 2H, CH₂- p-ethylphenyl), 5.13 (m,

1H, CH RTrp), 6.74 (d, 2H, J_o) 8 Hz, H₃and H₅p-ethylphenyl),

6.80 (t, 1H, J_o) 7 Hz, H₅Trp), 6.99 (t, 1H, J_o) 7 Hz, H₆Trp),

7.0-7.17 (m, 6H, H₂and H₄Trp, H₂and H₆p-ethylphenyl, H₂

and H₆phenyl), 7.20-7.29 (m, 4H, H₇Trp, H₃, H₄, and H₅phenyl),

7.90 (brs, 3H, NH₂TFA salt), 9.09 (d, 1H, J ) 8 Hz, NH amide),

10.76 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300

K): δ 15.9 (CH₃-CH₂-p-ethylphenyl), 17.3 (CH₃Ala), 26.5

(CH₂-CH₂-phenyl), 28.1 (CH₃-CH₂-p-ethylphenyl), 29.6 (C

âTrp), 32.8 (CH₂-CH₂-phenyl), 45.0 (C RTrp), 45.6 (CH₂-p-

ethylphenyl), 48.5 (C RAla), 109.7 (C₃Trp), 111.7 (C₇Trp), 118.3

(C₄Trp), 118.7 (C₅Trp), 121.3 (C₆Trp), 122.9 (C₂Trp), 126.5

(C₃and C₅p-ethylphenyl, C₄phenyl), 127.3 (C₉Trp), 128.6 (C₂

and C₆p-ethylphenyl, C₂, C₃, C₅and C₆phenyl), 133.5 (C₁

p-ethylphenyl), 136.5 (C₈Trp), 141.0 (C₁phenyl), 143.7 (C₄

p-ethylphenyl), 154.3 (Cq triazole), 155.0 (Cq triazole), 169.5 (CO

amide).

(R)-N-(1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)-3-aminopropanamide Trifluoroac-

1

etate Salt (14). H NMR (300 MHz, DMSO-d₆, 300 K): δ 2.33

(S)-N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)pyrrolidine-2-carboxamide Tri-

(m, 2H, CH₂-CH₂-NH₂), 2.78 (m, 6H, CH₂-CH₂-phenyl and

CH₂-CH₂-NH₂), 3.26 (dd, 1H, J ) 14 and 7 Hz, CH₂âTrp), 3.40

(dd, 1H, J ) 14 and 8 Hz, CH₂âTrp), 3.64 (s, 3H, OCH₃), 4.91

1

fluoroacetate Salt (18). H NMR (400 MHz, DMSO-d₆, 300 K):

δ 1.46 (m, 1H, H₃Pro), 1.58 (m, 1H, H₄Pro), 1.76 (m, 1H, H₄

5802 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

Pro), 2.12 (m, 1H, H₃Pro), 2.88 (m, 4H, CH₂-CH₂-phenyl), 3.11

(m, 2H, H₅Pro), 3.35 (m, 2H, CH₂âTrp), 3.72 (s, 3H, OCH₃),

4.01 (m, 1H, CH RPro), 5.05 (s, 2H, CH₂p-methoxybenzyl), 5.25

(m, 1H, CH RTrp), 6.77 (s, 4H, CHar p-methoxybenzyl), 6.88 (t,

1H, J_o) 8 Hz, H₆Trp), 7.03 (t, 1H, J_o) 8 Hz, H₅Trp), 7.04 (s,

1H, H₂Trp), 7.22 (d, 1H, J_o) 8 Hz, H₄Trp), 7.25-7.29 (m, 5H,

CHar phenyl), 7.33 (d, 1H, J_o) 8 Hz, H₇Trp), 8.44 and 9.16 (2

m, 2H, NH Pro TFA salt), 9.27 (d, 1H, J ) 8 Hz, NH amide),

10.83 (s, 1H, NH indole Trp). ¹³C NMR (100 MHz, DMSO-d₆,

300 K): δ 23.1 (C₄Pro), 26.0 (CH₂-CH₂-phenyl), 29.0 (C âTrp),

29.3 (C₃Pro), 32.3 (CH₂-CH₂-phenyl), 44.7 (C RTrp), 44.9 (CH₂

p-methoxybenzyl), 45.5 (C₅Pro), 55.0 (OCH₃), 58.8 (C RPro), 109.3

(C₃Trp), 111.2 (C₇Trp), 114.1 (C₃and C₅p-methoxybenzyl), 117.9

(C₄Trp), 118.3 (C₅Trp), 120.9 (C₆Trp), 124.2 (C₂Trp), 126.0

(C₄phenyl), 126.9 (C₉Trp), 127.4 (C₁, C₂and C₆p-methoxybenzyl),

128.2 (C₂, C₃, C₅, and C₆phenyl), 136.0 (C₈Trp), 140.5 (C₁phenyl),

153.9 (Cq triazole), 154.3 (Cq triazole), 158.7 (C₄p-methoxyben-

zyl), 167.7 (CO amide).

126.6 (C₆o,p-dimethoxybenzyl), 127.3 (C₉Trp), 128.2 (C₄phenyl),

128.7 (C₂, C₃, C₅, and C₆phenyl), 136.5 (C₈Trp), 141.0 (C₁phenyl),

154.5 (Cq triazole), 155.0 (Cq triazole), 157.8 (C₂o,p-dimethoxy-

benzyl), 160.9 (C₄o,p-dimethoxybenzyl), 168.1 (CO amide).

(2S)-N-((R)-2-(1H-Indol-3-yl)-1-(5-phenethyl-4-phenyl-4H-

1,2,4-triazol-3-yl)ethyl)pyrrolidine-2-carboxamide Trifluoroac-

1

etate Salt (23). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.52

(m, 1H, H₃Pro), 1.60 (m, 1H, H₄Pro), 1.75 (m, 1H, H₄Pro), 2.17

(m, 1H, H₃Pro), 2.71 (m, 4H, CH₂-CH₂-phenyl), 3.13 (m, 4H,

CH₂âTrp and H₅Pro), 4.06 (1H, m, CH RPro), 4.79 (1H, m, CH

RTrp), 6.77 (1H, t, J_o) 7 Hz, H₅Trp), 6.84 (t, 1H, J_o) 8 Hz, H₆

Trp), 6.91 (s, 1H, H₂Trp), 6.96-7,21 (m, 10H, CHar phenyl), 7.28

(d, 1H, J_o) 8 Hz, H₄Trp), 7.47 (d, 1H, J_o) 7 Hz, H₇Trp), 8.37

and 9.15 (2 m, 2H, NH Pro TFA salt), 9.34 (d, 1H, J ) 8 Hz, NH

amide), 10.78 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-

d₆, 300 K): δ 23.7 (C₄Pro), 26.8 (CH₂-CH₂-phenyl), 29.9 (C₃

Pro), 30.6 (C âTrp), 33.9 (CH₂-CH₂-phenyl), 46.0 (C₅Pro), 46.2

(C RTrp), 59.3 (C RPro), 109.5 (C₃Trp), 111.8 (C₇Trp), 118.1

(C₄Trp), 118.7 (C₅Trp), 121.3 (C₆Trp), 124.3 (C₂Trp), 126.5

(C₄phenyl from CH₂-CH₂-phenyl), 127.2 (C₉Trp), 127.6 (C₄

phenyl), 128.6 and 130.1 (C₂, C₃, C₅, and C₆phenyl and phenyl

from CH₂-CH₂-phenyl), 133.2 (C₁phenyl), 136.4 (C₈Trp), 140.8

(C₁phenyl from CH₂-CH₂-phenyl), 153.7 (Cq triazole), 155.1

(Cq triazole), 167.9 (CO amide).

(S)-N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(2,4-dimethoxyben-

zyl)-4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)pyrrolidine-2-

1

carboxamide Trifluoroacetate Salt (20). H NMR (300 MHz,

DMSO-d₆, 300 K): δ 1.40 (m, 1H, H₃Pro), 1.53 (m, 1H, H₄Pro),

1.71 (m, 1H, H₄Pro), 2.09 (m, 1H, H₃Pro), 2.90 (m, 4H, CH₂-

CH₂-indole), 3.06 (t, 2H, J ) 6 Hz, H₅Pro), 3.28 (m, 2H, CH₂

âTrp), 3.40 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.80 (m, 1H, CH

RPro), 4.88 (d, 1H, J ) 17 Hz, CH₂o,p-dimethoxybenzyl), 5.04

(d, 1H, J ) 17 Hz, CH₂o,p-dimethoxybenzyl), 5.21 (m, 1H, CH

RTrp), 6.26 (dd, 1H, J_o) 8 Hz and J_m) 2 Hz, H₅o,p-

dimethoxybenzyl), 6.38 (d, 1H, J_o) 8 Hz, H₆o,p-dimethoxyben-

zyl), 6.53 (d, 1H, J_m) 2 Hz, H₃o,p-dimethoxybenzyl), 6.84 (t,

1H, H₅indole), 6.91 (t, 1H, J_o) 8 Hz, H₅Trp), 6.93-7.07 (m,

4H, H₂and H₆indole, H₂and H₆Trp), 7.16 (d, 1H, J_o) 8 Hz, H₄

Trp), 7.29 (m, 3H, H₄and H₇indole, H₇Trp), 8.39 and 9.10 (2 m,

2H, NH Pro TFA salt), 9.22 (d, 1H, J ) 8 Hz, NH amide), 10.76

(s, 1H, NH indole), 10.80 (s, 1H, NH indole Trp). ¹³C NMR (75

MHz, DMSO-d₆, 300 K): δ 22.9 (CH₂-CH₂-indole), 23.5 (C₄

Pro), 25.8 (CH₂-CH₂-indole), 29.6 (C âTrp), 29.8 (C₃Pro), 41.9

(CH₂-o,p-dimethoxybenzyl), 45.2 (C RTrp), 46.0 (C₅Pro), 55.7

(OCH₃), 55.9 (OCH₃), 59.3 (C RPro), 99.0 (C₃o,p-dimethoxyben-

zyl), 105.1 (C₅o,p-dimethoxybenzyl), 109.7 (C₃Trp), 111.8 (C₇

indole and C₇Trp), 113.4 (C₃indole), 115.6 (C₁o,p-dimethoxy-

benzyl), 118.4 (C₄indole and C₄Trp), 118.7 (C₅indole and C₅

Trp), 121.4 (C₆indole and C₆Trp), 123.0 (C₂indole and C₂Trp),

127.2 (C₉indole), 127.3 (C₉Trp), 128.1 (C₆o,p-dimethoxybenzyl),

136.5 (C₈Trp), 136.6 (C₈indole), 155.0 (2Cq triazole), 157.8 (C₂

o,p-dimethoxybenzyl), 160.9 (C₄o,p-dimethoxybenzyl), 168.1 (CO

amide).

(2S,4R)-N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-

triazol-3-yl)-2-(1H-indol-3-yl)ethyl)-4-hydroxypyrrolidine-2-car-

boxamide Trifluoroacetate Salt (25). ¹H NMR (300 MHz, DMSO-

d₆, 300 K): δ 1.50 (m, 1H, H₃hydroxyprolyl), 2.10 (m, 1H, H₃

hydroxyprolyl), 2.80 (m, 4H, CH₂-CH₂-phenyl), 3.0 (m, 1H, H₅

hydroxyprolyl), 3.20 (m, 1H, H₅hydroxyprolyl), 3.29 (d, 2H, J )

7 Hz, CH₂âTrp), 3.63 (m, 1H, H₂hydroxyprolyl), 3.67 (s, 3H,

OCH₃), 4.50 (brs, 3H, OH and NH hydroxyprolyl TFA salt), 5.00

(s, 2H, CH₂p-methoxybenzyl), 5.17 (m, 1H, CH RTrp), 6.72 (s,

4H, CHar p-methoxybenzyl), 6.84 (t, 1H, J_o) 8 Hz, H₅Trp), 6.99

(m, 2H, H₂and H₆Trp), 7.09 (d, 1H, J_o) 8 Hz, H₄Trp), 7.13-

7.25 (5H, CHar phenyl), 7.29 (d, 1H, J_o) 8 Hz, H₇Trp), 9.30 (d,

1H, J ) 8 Hz, NH amide), 10.79 (s, 1H, NH indole Trp). ¹³C NMR

(75 MHz, DMSO-d₆, 300 K): δ 26.5 (CH₂-CH₂-phenyl), 29.5

(C âTrp), 32.8 (CH₂-CH₂-phenyl), 40.8 (C₃hydroxyprolyl), 46.3

(C RTrp), 53.9 (CH₂p-methoxybenzyl and C₅hydroxyprolyl), 55.5

(OCH₃), 58.2 (C₂hydroxyprolyl), 69.3 (C₄hydroxyprolyl), 109.7

(C₃Trp), 111.8 (C₇Trp), 114.6 (C₃and C₅p-methoxybenzyl), 118.4

(C₄Trp), 118.8 (C₅Trp), 121.4 (C₆Trp), 124.6 (C₂Trp), 126.5

(C₄phenyl), 127.3 (C₉Trp), 127.8 (C₂and C₆p-methoxybenzyl),

128.7 (C₂, C₃, C₅, and C₆phenyl), 136.4 (C₈Trp), 141.0 (C₁phenyl),

154.4 (Cq triazole), 154.7 (Cq triazole), 159.1 (C₄p-methoxyben-

zyl), 168.2 (CO amide).

(S)-N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-

4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-3-car-

boxamide Trifluoroacetate Salt (29). ¹H NMR (300 MHz, DMSO-

d₆, 300 K): δ 1.33 (m, 1H, H₄piperidyl), 1.53 (m, 3H, H₃and H₅

piperidyl), 2.72 (m, 2H, H₂and H₆piperidyl), 2.83 (m, 4H, CH₂-

CH₂-indole), 3.03 (m, 2H, H₂and H₆piperidyl), 3.26 (dd, 1H, J

) 14 and 7 Hz, CH₂âTrp), 3.39 (dd, 1H, J ) 14 and 8 Hz, CH₂

âTrp), 3.68 (s, 3H, OCH₃), 4.92 (s, 2H, CH₂p-methoxybenzyl),

5.20 (m, 1H, CH RTrp), 6.65 (s, 4H, CHar p-methoxybenzyl), 6.87

(t, 2H, J_o) 7 Hz, H₅indole and H₅Trp), 7.00 (m, 4H, H₂and H₆

indole, H₂and H₆Trp), 7.23-7.34 (m, 4H, H₄and H₇indole, H₄

and H₇Trp), 8.53 (brs, 2H, NH piperidyl TFA salt), 8.88 (d, 1H,

J ) 8 Hz, NH amide), 10.75 (s, 1H, NH indole), 10.78 (s, 1H, NH

indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 21.2 (C₅

piperidyl), 22.7 (CH₂-CH₂-indole), 25.9 (CH₂-CH₂-indole and

C₄piperidyl), 29.3 (C âTrp), 38.7 (C₃piperidyl), 43.3 (C₆piperidyl),

44.5 (C₂piperidyl), 45.1 (C RTrp), 45.5 (CH₂p-methoxybenzyl),

55.5 (OCH₃), 110.1 (C₃Trp), 111.8 (C₇indole and C₇Trp), 113.2

(C₃indole), 114.4 (C₃and C₅p-methoxybenzyl), 118.5 (C₄indole

and C₄Trp), 118.6 (C₅indole), 118.8 (C₅Trp), 121.4 (C₆indole

and C₆Trp), 123.0 (C₂indole and C₂Trp), 127.1 (C₁p-

methoxybenzyl), 127.5 (C₉indole and C₉Trp), 127.7 (C₂and C₆

p-methoxybenzyl), 136.4 (C₈Trp), 136.6 (C₈indole), 155.0 (2Cq

triazole), 159.1 (C₄p-methoxybenzyl), 171.9 (CO amide).

(S)-N-((R)-1-(4-(2,4-Dimethoxybenzyl)-5-phenethyl-4H-1,2,4-

triazol-3-yl)-2-(1H-indol-3-yl)ethyl)pyrrolidine-2-carboxamide

Trifluoroacetate Salt (21). H NMR (300 MHz, DMSO-d₆, 300

1

K): δ 1.41 (m, 1H, H₃Pro), 1.54 (m, 1H, H₄Pro), 1.71 (m, 1H,

H₄Pro), 2.15 (m, 1H, H₃Pro), 2.84 (m, 4H, CH₂-CH₂-phenyl),

3.06 (m, 2H, H₅Pro), 3.27 (m, 2H, CH₂âTrp), 3.65 (s, 3H, OCH₃),

3.69 (s, 3H, OCH₃), 3.98 (t, 1H, J ) 7 Hz, CH RPro), 4.85 (d, 1H,

J ) 17 Hz, CH₂o,p-dimethoxybenzyl), 5.02 (d, 1H, J ) 17 Hz,

CH₂-o,p-dimethoxybenzyl), 5.22 (m, 1H, CH RTrp), 6.27 (dd, 1H,

J_o) 8 Hz and J_m) 2 Hz, H₅o,p-dimethoxybenzyl), 6.40 (d, 1H,

J_o) 8 Hz, H₆o,p-dimethoxybenzyl), 6.54 (d, 1H, J_m) 2 Hz, H₃

o,p-dimethoxybenzyl), 6.84 (t, 1H, J_o) 8 Hz, H₅Trp), 7.0 (t, 1H,

J_o) 8 Hz, H₆Trp), 7.01 (d, 1H, J ) 2 Hz, H₂Trp), 7.11-7.18

(m, 3H, H₄Trp, H₂and H₆phenyl), 7.21-7.29 (m, 4H, H₇Trp,

H₃, H₄, and H₅phenyl), 8.39 and 9.07 (2 m, 2H, NH Pro TFA

salt), 9.21 (d, 1H, J ) 8 Hz, NH amide), 10.79 (s, 1H, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 23.5 (C₄Pro),

26.5 (CH₂-CH₂-phenyl), 29.6 (C âTrp), 29.8 (C₃Pro), 32.7

(CH₂-CH₂-phenyl), 41.8 (CH₂o,p-dimethoxybenzyl), 45.2 (C

RTrp), 46.0 (C₅Pro), 55.7 (OCH₃), 55.9 (OCH₃), 59,3 (C RPro),

99.0 (C₃o,p-dimethoxybenzyl), 105.2 (C₅o,p-dimethoxybenzyl),

109.8 (C₃Trp), 111.7 (C₇Trp), 115.7 (C₁o,p-dimethoxybenzyl),

118.3 (C₄Trp), 118.7 (C₅Trp), 121.3 (C₆Trp), 124.4 (C₂Trp),

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5803

(S)-N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-3-carboxamide Tri-

fluoroacetate Salt (30). H NMR (400 MHz, DMSO-d₆, 300 K):

ethylbenzyl), 110.2 (C₃Trp), 111.7 (C₇Trp), 118.5 (C₄Trp), 118.7

(C₅Trp), 121.3 (C₆Trp), 124.4 (C₂Trp), 126.2 (C₃and C₅

p-ethylbenzyl), 126.5 (C₄phenyl), 127.5 (C₉Trp), 128.5 (C₂, C₃,

C₅, and C₆phenyl, C₂and C₆p-ethylbenzyl), 133.0 (C₁p-

ethylbenzyl), 136.4 (C₈Trp), 140.8 (C₁phenyl), 143.6 (C₄p-

ethylbenzyl), 154.5 (Cq triazole), 155.1 (Cq triazole), 171.9 (CO

amide).

1

δ 1.33 (m, 1H, H₄piperidyl), 1.50 (m, 1H, H₅piperidyl), 1.61 (m,

1H, H₄piperidyl), 1.64 (m, 1H, H₅piperidyl), 2.54 (m, 1H, H₃

piperidyl), 2.77 (m, 1H, H₂piperidyl), 2.80 (m, 3H, H₆piperidyl

and CH₂-CH₂-phenyl), 2.84 (m, 2H, CH₂-CH₂-phenyl), 2.96

(m, 1H, H₂piperidyl), 3.08 (m, 1H, H₆piperidyl), 3.30 (dd, 1H, J

) 14 and 7 Hz, CH₂âTrp), 3.45 (dd, 1H, J ) 14 and 8 Hz, CH₂

âTrp), 3.69 (s, 3H, OCH₃), 4.94 (m, 2H, CH₂p-methoxybenzyl),

5.25 (m, 1H, CH RTrp), 6.70 (s, 4H, CHar p-methoxybenzyl), 6.91

(t, 1H, J_o) 8 Hz, H₆Trp), 7.02 (d, 1H, J ) 2 Hz, H₂Trp), 7.05

(t, 1H, J_o) 8 Hz, H₅Trp), 7.29-7.08 (m, 5H, CHar phenyl), 7.34

(d, 1H, J_o) 8 Hz, H₄Trp), 7.39 (d, 1H, J_o) 8 Hz, H₇Trp), 8.48

(brs, 2H, NH piperidyl TFA salt), 8.90 (d, 1H, J ) 8 Hz, NH

amide), 10.81 (s, 1H, NH indole Trp). ¹³C NMR (100 MHz, DMSO-

d₆, 300 K): δ 20.7 (C₅piperidyl), 25.4 (C₄piperidyl), 26.1 (CH₂-

CH₂-phenyl), 28.9 (C âTrp), 32.2 (CH₂-CH₂-phenyl), 38.2 (C₃

piperidyl), 42.9 (C₆piperidyl), 44.0 (C₂piperidyl), 44.6 (C RTrp),

44.7 (CH₂p-methoxybenzyl), 55.0 (OCH₃), 109.8 (C₃Trp), 111.3

(C₇Trp), 114.0 (C₃and C₅p-methoxybenzyl), 118.0 (C₄Trp), 118.3

(C₅Trp), 120.8 (C₆Trp), 124.0 (C₂Trp), 126.0 (C₄phenyl), 127.1

(C₉Trp), 127.2 (C₁, C₂and C₆p-methoxybenzyl), 128.2 (C₂, C₃,

C₅, and C₆phenyl), 136.0 (C₈Trp), 140.4 (C₁phenyl), 154.0 (Cq

triazole), 154.4 (Cq triazole), 158.6 (C₄p-methoxybenzyl), 171.4

(CO amide).

(R)-N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-

4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-2-car-

boxamide Trifluoroacetate Salt (35). ¹H NMR (300 MHz, DMSO-

d₆, 300 K): δ 1.28 (m, 1H, H₃piperidyl), 1.36 (m, 1H, H₄

piperidyl), 1.45 (m, 1H, H₅piperidyl), 1.66 (m, 2H, H₄and H₅

piperidyl), 1.90 (d, 1H, J ) 13 Hz, H₃piperidyl), 2.82 (m, 4H,

CH₂-CH₂-indole), 2.89 (m, 1H, H₆piperidyl), 3.14 (d, 1H, J )

12 Hz, H₆piperidyl), 3.25 (dd, 1H, J ) 14 and 7 Hz, CH₂âTrp),

3.39 (dd, 1H, J ) 14 and 8 Hz, CH₂âTrp), 3.64 (s, 4H, OCH₃and

H₂piperidyl), 4.88 (s, 2H, CH₂-p-methoxybenzyl), 5.19 (m, 1H,

CH RTrp), 6.65 (s, 4H, CHar p-methoxybenzyl), 6.80 (m, 2H, H₅

indole and H₅Trp), 7.02 (m, 5H, H₂, H₄, and H₆Trp, H₂and H₆

indole), 7.29 (m, 3H, H₄and H₇indole, H₇Trp), 8.58 and 8.88 (2

m, 2H, NH piperidyl TFA salt), 9.22 (d, 1H, J ) 8 Hz, NH amide),

10.73 (s, 1H, NH indole), 10.81 (s, 1H, NH indole Trp). ¹³C NMR

(75 MHz, DMSO-d₆, 300 K): δ 21.6 (C₅piperidyl), 21.9 (C₄

piperidyl), 22.8 (CH₂-CH₂-indole), 25.9 (CH₂-CH₂-indole), 27.2

(C₃piperidyl), 29.7 (C âTrp), 43.6 (C₆piperidyl), 45.4 (CH₂-p-

methoxybenzyl), 45.7 (C RTrp), 55.5 (OCH₃), 57.2 (C₂piperidyl),

109.7 (C₃Trp), 111.8 (C₇Trp), 111.9 (C₇indole), 113.4 (C₃indole),

114.5 (C₃and C₅p-methoxybenzyl), 118.3 (C₄indole), 118.5 (C₄

Trp), 118.6 (C₅Trp), 118.9 (C₅indole), 121.4 (C₆indole and C₆

Trp), 122.9 (C₂indole and C₂Trp), 127.2 (C₉indole), 127.3 (C₉

Trp), 127.5 (C₁p-methoxybenzyl), 127.7 (C₂and C₆p-methoxy-

benzyl), 136.5 (C₈Trp), 136.6 (C₈indole), 154.5 (Cq triazole), 154.9

(Cq triazole), 159.1 (C₄p-methoxybenzyl), 168.8 (CO amide).

(R)-N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-2-carboxamide Tri-

(R)-N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-3-carboxamide Tri-

1

fluoroacetate Salt (31). H NMR (400 MHz, DMSO-d₆, 300 K):

δ 1.27 (m, 1H, H₄piperidyl), 1.49 (m, 2H, H₅piperidyl), 1.65 (m,

1H, H₄piperidyl), 2.53 (m, 1H, H₃piperidyl), 2.70 (m, 1H, H₂

piperidyl), 2.75 (m, 1H, H₆piperidyl), 2.79 (m, 1H, H₂piperidyl),

2.83 (m, 2H, CH₂-CH₂-phenyl), 2.84 (m, 2H, CH₂-CH₂-

phenyl), 3.01 (m, 1H, H₆piperidyl), 3.33 (dd, 1H, J ) 15 and 8

Hz, CH₂âTrp), 3.38 (dd, 1H, J ) 15 and 7 Hz, CH₂âTrp), 3.71

(s, 3H, OCH₃), 5.06 (m, 2H, CH₂p-methoxybenzyl), 5.26 (m, 1H,

CH RTrp), 6.77 (s, 4H, CHar p-methoxybenzyl), 6.89 (t, 1H, J_o)

8 Hz, H₅Trp), 7.03 (t, 1H, J_o) 8 Hz, H₆Trp), 7.06 (d, 1H, J )

2 Hz, H₂Trp), 7.29-7.08 (m, 5H, CHar phenyl), 7.29 (d, 1H, J_o

) 8 Hz, H₄Trp), 7.32 (d, 1H, J_o) 8 Hz, H₇Trp), 8.54 and 8.88

(2 m, 2H, NH piperidyl TFA salt), 8.92 (d, 1H, J ) 8 Hz, NH

amide), 10.82 (s, 1H, NH indole Trp). ¹³C NMR (100 MHz, DMSO-

d₆, 300 K): δ 20.5 (C₅piperidyl), 25.7 (C₄piperidyl), 26.0 (CH₂-

CH₂-phenyl), 28.6 (C âTrp), 32.2 (CH₂-CH₂-phenyl), 37.8 (C₃

piperidyl), 42.7 (C₆piperidyl), 43.8 (C₂piperidyl), 44.0 (C RTrp),

45.0 (CH₂p-methoxybenzyl), 55.1 (OCH₃), 109.6 (C₃Trp), 111.2

(C₇Trp), 114.0 (C₃and C₅p-methoxybenzyl), 118.0 (C₄Trp), 118.2

(C₅Trp), 120.8 (C₆Trp), 124.1 (C₂Trp), 126.1 (C₄phenyl), 127.1

(C₉Trp), 127.2 (C₁, C₂, and C₆p-methoxybenzyl), 128.2 (C₂, C₃,

C₅, and C₆phenyl), 136.0 (C₈Trp), 140.4 (C₁phenyl), 154.0 (Cq

triazole), 154.4 (Cq triazole), 158.6 (C₄p-methoxybenzyl), 171.5

(CO amide).

1

fluoroacetate Salt (36). H NMR (300 MHz, DMSO-d₆, 300 K):

δ 1.28 (m, 1H, H₃piperidyl), 1.36 (m, 1H, H₄piperidyl), 1.41 (m,

1H, H₅piperidyl), 1.66 (m, 2H, H₄and H₅piperidyl), 1.90 (d, 1H,

J ) 14 Hz, H₃piperidyl), 2.77 (m, 4H, CH₂-CH₂-phenyl), 2.86

(m, 1H, H₆piperidyl), 3.15 (d, 1H, J ) 12 Hz, H₆piperidyl), 3.25

(dd, 1H, J ) 14 and 7 Hz, CH₂âTrp), 3.41 (dd, 1H, J ) 14 and

8 Hz, CH₂âTrp), 3.64 (s, 4H, OCH₃and H₂piperidyl), 4.86 (s,

2H, CH₂p-methoxybenzyl), 5.19 (m, 1H, CH RTrp), 6.65 (s, 4H,

CHar p-methoxybenzyl), 6.87 (t, 1H, J_o) 7 Hz, H₅Trp), 7.01 (m,

2H, H₂and H₆Trp), 7.09 (d, 2H, J_o) 8 Hz, H₂and H₆phenyl),

7.14 (d, 1H, H₄Trp), 7.18-7.33 (m, 4H, H₇Trp, H₃, H₄, and H₅

phenyl), 8.60 and 8.89 (2 m, 2H, NH piperidyl TFA salt), 9.21 (d,

1H, J ) 8 Hz, NH amide), 10.81 (s, 1H, NH indole Trp). ¹³C NMR

(75 MHz, DMSO-d₆, 300 K): δ 21.6 (C₅piperidyl), 21.9 (C₄

piperidyl), 26.5 (CH₂-CH₂-phenyl), 27.3 (C₃piperidyl), 29.8 (C

âTrp), 32.7 (CH₂-CH₂-phenyl), 43.6 (C₆piperidyl), 45.2 (CH₂

p-methoxybenzyl), 45.6 (C RTrp), 55.5 (OCH₃), 57.2 (C₂piperidyl),

109.7 (C₃Trp), 111.9 (C₇Trp), 114.5 (C₃and C₅p-methoxybenzyl),

118.3 (C₄Trp), 118.9 (C₅Trp), 121.4 (C₆Trp), 124.6 (C₂Trp),

126.5 (C₄phenyl), 127.3 (C₉Trp), 127.6 (C₁p-methoxybenzyl),

127.7 (C₂and C₆p-methoxybenzyl), 128.7 (C₂, C₃, C₅, and C₆

phenyl), 136.5 (C₈Trp), 140.9 (C₁phenyl), 154.4 (Cq triazole),

154.5 (Cq triazole), 159.1 (C₄p-methoxybenzyl), 168.8 (CO amide).

(R)-N-(1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(4-methoxybenzyl)-4H-

1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-4-carboxa-

mide Trifluoroacetate Salt (39). ¹H NMR (300 MHz, DMSO-d₆,

300 K): δ 1.41 (m, 3H, H₃and H₅piperidyl), 1.54 (dd, 1H, J )

13 and 2 Hz, H₅piperidyl), 2.23 (m, 1H, H₄piperidyl), 2.72 (m,

2H, H₂and H₆piperidyl), 2.77-2.93 (m, 4H, CH₂-CH₂-indole),

3.06 (m, 2H, H₂and H₆piperidyl), 3.32 (m, 2H, CH₂âTrp), 3.65

(s, 3H, OCH₃), 4.94 (s, 2H, CH₂p-methoxybenzyl), 5.22 (m, 1H,

CH RTrp), 6.68 (s, 4H, CHar p-methoxybenzyl), 6.87 (m, 3H, H₅

and H₆Trp, H₅indole), 6.98 (m, 4H, H₂and H₆indole, H₂and H₄

Trp), 7.20-7.33 (m, 3H, H₄and H₇indole, H₇Trp), 8.15 and 8.46

(2 m, 2H, NH piperidyl TFA salt), 8.64 (d, 1H, J ) 8 Hz, NH

amide), 10.74 (s, 2H, NH indole and NH indole Trp). ¹³C NMR

(3S)-N-((R)-1-(4-(4-Ethylbenzyl)-5-phenethyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-3-carboxamide Trifluo-

1

roacetate Salt (32). H NMR (300 MHz, DMSO-d₆, 300 K): δ

1.08 (t, 3H, J ) 8 Hz, CH₃-CH₂-phenyl), 1.20 (m, 1H, H₄

piperidyl), 1.44 (m, 2H, H₅piperidyl), 1.55 (m, 1H, H₄piperidyl),

2.51 (q, 2H, J ) 8 Hz, CH₃-CH₂-phenyl), 2.53 (m, 1H, H₃

piperidyl), 2.83 (m, 7H, CH₂-CH₂-phenyl, H₂and H₆piperidyl),

3.01 (m, 1H, H₆piperidyl), 3.28 (m, 1H, CH₂âTrp), 3.36 (m, 1H,

CH₂âTrp), 4.98 (s, 2H, CH₂p-ethylbenzyl), 5.20 (m, 1H, CH

RTrp), 6.65 (d, 2H, J_o) 8 Hz, H₃and H₅p-ethylbenzyl), 6.86 (t,

1H, J_o) 7 Hz, H₅Trp), 6.97-7.07 (m, 7H, H₂, H₄, and H₆Trp,

H₂and H₆p-ethylbenzyl, H₂and H₆phenyl), 7.13-7.22 (m, 3H,

H₃, H₄, and H₅phenyl), 7.29 (d, 1H, J_o) 8 Hz, H₇Trp), 8.46 (brs,

2H, NH piperidyl TFA salt), 8.85 (d, 1H, J ) 8 Hz, NH amide),

10.78 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300

K): δ 15.9 (CH₃-CH₂-phenyl), 21.2 (C₅piperidyl), 25.7 (C₄

piperidyl), 26.5 (CH₂-CH₂-phenyl), 28.1 (CH₃-CH₂-phenyl),

29.2 (C âTrp), 32.6 (CH₂-CH₂-phenyl), 38.7 (C₃piperidyl), 43.3

(C₆piperidyl), 44.5 (C₂piperidyl), 45.0 (C RTrp), 45.6 (CH₂-p-

5804 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

(75 MHz, DMSO-d₆, 300 K): δ 22.9 (CH₂-CH₂-indole), 24.9

(C₃piperidyl), 25.4 (C₅piperidyl), 26.0 (CH₂-CH₂-indole), 29.3

(C âTrp), 39.1 (C₄piperidyl), 42.7 (C₂and C₆piperidyl), 44.7 (C

RTrp), 45.3 (CH₂p-methoxybenzyl), 55.5 (OCH₃), 109.5 (C₃Trp),

111.7 (C₇indole and C₇Trp), 113.5 (C₃indole), 114.4 (C₃and C₅

p-methoxybenzyl), 118.5 (C₄indole and C₄Trp), 118.6 (C₅indole

and C₅Trp), 121.2 (C₆indole), 121.3 (C₆Trp), 122.9 (C₂indole

and C₂Trp), 127.2 (C₉indole), 127.6 (C₉Trp, C₂and C₆

p-methoxybenzyl), 127.9 (C₁p-methoxybenzyl), 136.4 (C₈Trp),

136.6 (C₈indole), 154.9 (Cq triazole), 155.2 (Cq triazole), 159.0

(C₄p-methoxybenzyl), 173.0 (CO amide).

(R)-N-(1-(4-(2,4-Dimethoxybenzyl)-5-phenethyl-4H-1,2,4-tria-

zol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-4-carboxamide Tri-

fluoroacetate Salt (42). H NMR (300 MHz, DMSO-d₆, 300 K):

1

δ 1.43 (m, 3H, H₃and H₅piperidyl), 1.56 (dd, 1H, J ) 13 and 3

Hz, H₅piperidyl), 2.24 (m, 1H, H₄piperidyl), 2.67-2.83 (m, 6H,

CH₂-CH₂-phenyl, H₂and H₆piperidyl), 3.09 (m, 2H, H₂and H₆

piperidyl), 3.28 (m, 2H, CH₂âTrp), 3.62 (s, 3H, OCH₃), 3.67 (s,

3H, OCH₃), 4.81 (d, 1H, J ) 17 Hz, CH₂o,p-dimethoxybenzyl),

4.94 (d, 1H, J ) 17 Hz, CH₂o,p-dimethoxybenzyl), 5.24 (m, 1H,

CH RTrp), 6.24 (dd, 1H, J_o) 8 Hz and J_m) 2 Hz, H₅

o,p-dimethoxybenzyl), 6.35 (d, 1H, J_o) 8 Hz, H₆o,p-dimethoxy-

benzyl), 6.50 (d, 1H, J_m) 2 Hz, H₃o,p-dimethoxybenzyl), 6.87

(t, 1H, J_o) 8 Hz, H₅Trp), 7.0 (m, 2H, H₂and H₆Trp), 7.08 (d,

2H, J_o) 7 Hz, H₂and H₆phenyl), 7.15 (d, 1H, J_o) 7 Hz, H₄

Trp), 7.19-7.30 (m, 4H, H₇Trp, H₃, H₄, and H₅phenyl), 8.35 (m,

1H, NH piperidyl TFA salt), 8.61 (d, 1H, J ) 8 Hz, NH amide),

8.69 (m, 1H, NH piperidyl TFA salt), 10.78 (s, 1H, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 22.3 (C₃piperidyl),

25.0 (C₅piperidyl), 25.3 (CH₂-CH₂-phenyl), 29.2 (C âTrp), 32.6

(CH₂-CH₂-phenyl), 38.7 (C₄piperidyl), 41.9 (C₂and C₆pip-

eridyl), 42.6 (CH₂o,p-dimethoxybenzyl), 44.7 (C RTrp), 55.7

(OCH₃), 55.9 (OCH₃), 98.9 (C₃o,p-dimethoxybenzyl), 105.1 (C₅

o,p-dimethoxybenzyl), 110.2 (C₃Trp), 111.7 (C₇Trp), 115.5 (C₁

o,p-dimethoxybenzyl), 118.5 (C₄Trp), 118.7 (C₅Trp), 121.3 (C₆

Trp), 124.4 (C₂Trp), 126.6 (C₆o,p-dimethoxybenzyl), 127.5 (C₉

Trp), 128.1 (C₄phenyl), 128.7 (C₂, C₃, C₅and C₆phenyl), 136.4

(C₈Trp), 140.7 (C₁phenyl), 154.6 (Cq triazole), 155.6 (Cq triazole),

157.7 (C₂o,p-dimethoxybenzyl), 160.8 (C₄o,p-dimethoxybenzyl),

173.1 (CO amide).

(R)-N-(1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-4-carboxamide Trifluo-

1

roacetate Salt (40). H NMR (300 MHz, DMSO-d₆, 300 K): δ

1.42 (m, 3H, H₃and H₅piperidyl), 1.55 (m, 1H, H₅piperidyl),

2.23 (m, 1H, H₄piperidyl), 2.75 (m, 6H, H₂and H₆piperidyl and

CH₂-CH₂-phenyl), 3.04 (m, 1H, H₆piperidyl), 3.13 (m, 1H, H₂

piperidyl), 3.32 (m, 2H, CH₂âTrp), 3.66 (s, 3H, OCH₃), 4.97 (m,

2H, CH₂-p-methoxybenzyl), 5.23 (m, 1H, CH RTrp), 6.70 (s, 4H,

CHar p-methoxybenzyl), 6.87 (t, 1H, J_o) 8 Hz, H₅Trp), 7.00 (m,

2H, H₂and H₆Trp), 7.07 (d, 2H, J_o) 8 Hz, H₂and H₆phenyl),

7.14 (d, 1H, J_o) 7 Hz, H₄Trp), 7.18-7.30 (m, 4H, H₇Trp, H₃,

H₄, and H₅phenyl), 8.16 and 8.46 (2 m, 2H, NH piperidyl TFA

salt), 8.66 (d, 1H, J ) 8 Hz, NH amide), 10.75 (1H, s, NH indole

Trp). ¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 24.9 (C₃piperidyl),

25.4 (C₅piperidyl), 26.5 (CH₂-CH₂-phenyl), 29.2 (C âTrp), 32.7

(CH₂-CH₂-phenyl), 38.7 (C₄piperidyl), 42.7 (C₂and C₆pip-

eridyl), 44.7 (C RTrp), 45.3 (CH₂p-methoxybenzyl), 55.5 (OCH₃),

110.2 (C₃Trp), 111.7 (C₇Trp), 114.4 (C₃and C₅p-methoxybenzyl),

118.5 (C₄Trp), 118.7 (C₅Trp), 121.3 (C₆Trp), 124.4 (C₂Trp),

126.5 (C₄phenyl), 127.5 (C₉Trp), 127.8 (C₁, C₂, and C₆

p-methoxybenzyl), 128.7 (C₂, C₃, C₅, and C₆phenyl), 136.4 (C₈

Trp), 140.8 (C₁phenyl), 155.3 (Cq triazole), 155.4 (Cq triazole),

159.1 (C₄p-methoxybenzyl), 173.1 (CO amide).

N-((R)-2-(1H-Indol-3-yl)-1-(5-phenethyl-4-phenyl-4H-1,2,4-

triazol-3-yl)ethyl)piperidine-4-carboxamide Trifluoroacetate Salt

1

(44). H NMR (300 MHz, DMSO-d₆, 300 K): δ 1.50 (m, 3H, H₃

and H₅piperidyl), 1.68 (m, 1H, H₅piperidyl), 2.37 (m, 1H, H₄

piperidyl), 2.65 (m, 2H, H₂and H₆piperidyl), 2.79 (m, 4H, CH₂-

CH₂-phenyl), 3.05-3.21 (m, 4H, CH₂âTrp, H₂and H₆piperidyl),

4.77 (m, 1H, CH RTrp), 6.76 (t, 1H, J_o) 7 Hz, H₅Trp), 6.82 (t,

1H, J_o) 8 Hz, H₆Trp), 6.90 (s, 1H, H₂Trp), 6.95-7.20 (m, 10H,

CHar phenyl), 7.26 (d, 1H, J_o) 8 Hz, H₄Trp), 7.45 (d, 1H, J_o)

8 Hz, H₇Trp), 8.20 and 8.50 (2 m, 2H, NH piperidyl TFA salt),

8.64 (d, 1H, J ) 8 Hz, NH amide), 10.72 (s, 1H, NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 25.3 (C₃and C₅

piperidyl), 26.9 (CH₂-CH₂-phenyl), 30.2 (C âTrp), 33.0 (CH₂-

CH₂-phenyl), 38.6 (C₄piperidyl), 42.8 (C₂and C₆piperidyl), 45.9

(C RTrp), 110.0 (C₃Trp), 111.7 (C₇Trp), 118.2 (C₄Trp), 118.6

(C₅Trp), 121.2 (C₆Trp), 124.3 (C₂Trp), 126.5 (C₄phenyl from

CH₂-CH₂-phenyl), 127.3 (C₉Trp), 127.6 (C₄phenyl), 128.5 (C₂,

C₃, C₅, and C₆phenyl and phenyl from CH₂-CH₂-phenyl), 133.4

(C₁phenyl), 136.4 (C₈Trp), 140.8 (C₁phenyl from CH₂-CH₂-

phenyl), 153.7 (Cq triazole), 155.6 (Cq triazole), 172.9 (CO amide).

N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-phenyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-4-carboxamide Trifluo-

(R)-N-(1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(2,4-dimethoxybenzyl)-

4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)piperidine-4-car-

boxamide Trifluoroacetate Salt (41). ¹H NMR (400 MHz, DMSO-

d₆, 300 K): δ 1.43 (m, 1H, H₅piperidyl), 1.48 (m, 2H, H₃

piperidyl), 1.59 (m, 1H, H₅piperidyl), 2.27 (m, 1H, H₄piperidyl),

2.77 (m, 2H, H₂and H₆piperidyl), 2.91 (m, 2H, CH₂-CH₂-indole),

2.94 (m, 2H, CH₂-CH₂-indole), 3.10 (m, 1H, H₆piperidyl), 3.17

(m, 1H, H₂piperidyl), 3.31 (dd, 1H, J ) 15 and 8 Hz, CH₂âTrp),

3.37 (dd, 1H, J ) 15 and 7 Hz, CH₂âTrp), 3.66 (s, 3H, OCH₃),

3.70 (s, 3H, OCH₃), 4.89 (d, 1H, J ) 17 Hz, CH₂o,p-dimethoxy-

benzyl), 5.0 (d, 1H, J ) 17 Hz, CH₂o,p-dimethoxybenzyl), 5.29

(m, 1H, CH RTrp), 6.26 (dd, 1H, J_o) 8 Hz and J_m) 2 Hz, H₅

o,p-dimethoxybenzyl), 6.39 (d, 1H, J_o) 8 Hz, H₆o,p-dimethoxy-

benzyl), 6.54 (d, 1H, J_m) 2 Hz, H₃o,p-dimethoxybenzyl), 6.92

(t, 1H, J_o) 7 Hz, H₅Trp), 6.93 (t, 1H, J_o) 7 Hz, H₅indole), 7.04

(t, 1H, J_o) 8 Hz, H₆Trp), 7.05 (t, 1H, J_o) 8 Hz, H₆indole), 7.06

(s, 1H, H₂Trp), 7.07 (s, 1H, H₂indole), 7.28 (d, 1H, J_o) 8 Hz,

H₄indole), 7.32 (m, 2H, H₇indole and H₇Trp), 7.34 (d, 1H, J_o)

8 Hz, H₄Trp), 8.29 and 8.60 (2 m, 2H, NH piperidyl TFA salt),

8.65 (d, 1H, J ) 8 Hz, NH amide), 10.80 (s, 1H, NH indole), 10.82

(s, 1H, NH indole Trp). ¹³C NMR (100 MHz, DMSO-d₆, 300 K):

δ 22.3 (CH₂-CH₂-indole), 24.5 (C₃piperidyl), 24.9 (C₅piperidyl),

25.3 (CH₂-CH₂-indole), 28.7 (C âTrp), 38.2 (C₄piperidyl), 41.5

(CH₂o,p-dimethoxybenzyl), 42.2 (C₂andC₆piperidyl), 44.3 (C

RTrp), 55.2 (OCH₃), 55.4 (OCH₃), 98.5 (C₃o,p-dimethoxybenzyl),

104.6 (C₅o,p-dimethoxybenzyl), 109.7 (C₃Trp), 111.2 (C₇indole),

111.3 (C₇Trp), 112.7 (C₃indole), 114.9 (C₁o,p-dimethoxybenzyl),

117.9 (C₄indole), 118.0 (C₄Trp), 118.2 (C₅indole and C₅Trp),

120.8 (C₆indole), 120.9 (C₆Trp), 122.5 (C₂indole), 123.9 (C₂

Trp), 126.7 (C₉indole), 127.1 (C₉Trp), 127.5 (C₆o,p-dimethoxy-

benzyl), 136.0 (C₈Trp), 136.1 (C₈indole), 154.7 (Cq triazole), 155.2

(Cq triazole), 157.2 (C₂o,p-dimethoxybenzyl), 160.4 (C₄o,p-

dimethoxybenzyl), 172.6 (CO amide).

1

roacetate Salt (45). H NMR (300 MHz, DMSO-d₆, 300 K): δ

1.54 (m, 3H, H₃and H₅piperidyl), 1.71 (m, 1H, H₅piperidyl),

2.38 (m, 1H, H₄piperidyl), 2.71 (m, 2H, H₂and H₆piperidyl),

2.83 (m, 4H, CH₂-CH₂-indole), 3.06-3.17 (m, 4H, CH₂âTrp,

H₁and H₆piperidyl), 4.77 (m, 1H, CH RTrp), 6.74-6.85 (m, 3H,

H₅indole, H₅and H₆Trp), 6.91 (d, 1H, J ) 2 Hz, H₂Trp), 6.95-

7.02 (m, 7H, CHar phenyl, H₂and H₆indole), 7.26 (d, 2H, J_o) 8

Hz, H₇indole and H₇Trp), 7.43 (m, 2H, H₄indole and H₄Trp),

8.20 and 8.52 (2 m, 2H, NH piperidyl TFA salt), 8.64 (d, 1H, J )

8 Hz, NH amide), 10.71 (s, 2H, NH indole and NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 23.4 (CH₂-CH₂-

indole), 25.3 (C₃and C₅piperidyl), 26.3 (CH₂-CH₂-indole), 30.2

(C âTrp), 38.6 (C₄piperidyl), 42.8 (C₂and C₆piperidyl), 46.0 (C

RTrp), 110.1 (C₃Trp), 111.7 (C₇indole and C₇Trp), 113.3 (C₃

indole), 118.2 (C₄indole and C₄Trp), 118.6 (C₅indole and C₅

Trp), 121.2 (C₆indole), 121.3 (C₆Trp), 122.8 (C₂Trp), 124.3 (C₂

indole), 127.1 (C₉indole), 127.4 (C₉Trp), 127.7 (C₄phenyl), 129.9

(C₂, C₃, C₅, and C₆phenyl), 133.5 (C₁phenyl), 136.6 (C₈indole),

136.4 (C₈Trp), 154.3 (Cq triazole), 155.6 (Cq triazole), 172.9 (CO

amide).

1,2,4-Triazoles as Ghrelin Receptor Ligands

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5805

(R,S)-N-((R)-1-(5-((1H-Indol-3-yl)methyl)-4-(4-methoxyben-

zyl)-4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)piperazine-2-

carboxamide Trifluoroacetate Salt (52). H NMR (300 MHz,

37 °C, 5% CO₂in Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 10% FCS (v/v), glutamine (2 mM), and

antibiotics (50 units/mL penicillin and 50 µg/mL streptomycin).

LLC PK-1 cells were transiently transfected with 1 µg of hGHS-

R1a using electroporation (Easyject Optima apparatus, Equibio)

according to the manufacturer’s protocol (Equibio). Electroporation

was carried out at room temperature according to the manufacturer’s

instructions with the following parameters: 250 V, 1500 µF, and

infinite internal resistance. Transfected cells were plated in 10 cm

culture dishes containing complete growth medium without phenol

red. Approximately 48 h after transfection, cells were washed three

times with phosphate-buffered saline, pH 6.95, once with 10 mL

of homogenization buffer (HB) containing 50 mM Tris (pH 7.3), 5

mM MgCl₂, 2.5 mM EDTA, and 30 µg/mL bacitracin and were

then collected by scrapping. The cells underwent two cycles of

freeze-thawing and were then centrifuged at 10000g for 20 min

at 4 °C. The membrane pellet was resuspended in a minimal volume

of HB, aliquoted, and stored at -80 °C until use. Membrane protein

concentrations were determined by the Bradford method using the

Bio-Rad protein assay kit.

1

DMSO-d₆, 300 K): δ 2.18 (m, 2H, NH piperazinyl), 2.96 (m, 6H,

H₂, H₅, and H₆piperazinyl), 3.34 (d, 2H, J ) 7 Hz, CH₂âTrp),

3.57 (m, 4H, CH₂-CH₂-indole), 3.61 (s, 3H, OMe), 3.64 (m, 1H,

H₃piperazinyl), 4.82 (m, 2H, CH₂p-methoxybenzyl), 5.40 (m, 1H,

CH RTrp), 6.45 (d, 2H, J_o) 8 Hz, H₃and H₅p-methoxybenzyl),

6.51 (d, 2H, J_o) 8 Hz, H₂and H₆p-methoxybenzyl), 6.65-7.47

(m, 10H, CHar, indole and indole Trp), 8.95 (m, 1H, NH amide),

10.88 (d, 1H, J ) 2 Hz, NH indole), 10.91 (s, 1H, NH indole Trp).

¹³C NMR (75 MHz, DMSO-d₆, 300 K): δ 22.5 (CH₂-CH₂-

indole), 25.6 (CH₂-CH₂-indole), 31.3 (C âTrp), 41.9 (CH₂-p-

methoxybenzyl), 47.7 (C RTrp, C₅and C₆piperazinyl), 55.5 (OCH₃

and C₂piperazinyl), 61.1 (C₃piperazinyl), 109.3 (C₃Trp), 111.7

(C₇indole and C₇Trp), 114.0 (C₃indole), 114.3 (C₃and C₅

p-methoxybenzyl), 118.6 (C₄indole), 118.7 (C₄Trp), 118.9 (C₅

indole and C₅Trp), 121.4 (C₆indole), 121.5 (C₆Trp), 123.9 (C₂

indole and C₂Trp), 127.0 (C₉indole), 127.2 (C₉Trp), 127.7 (C₁

p-methoxybenzyl), 128.1 (C₂and C₆p-methoxybenzyl), 136.3 (C₈

Trp), 136.5 (C₈indole), 155.5 (2Cq triazole), 162.2 (C₄p-

methoxybenzyl), 171.1 (CO amide).

Receptor Binding Studies. Isolated plasma membranes from

LLC PK-1 cells (10 µg of protein) were incubated in HB for 60

min at 25 °C (steady-state conditions) with 60 pM ¹²⁵I-His⁹-ghrelin

(Amersham) in the presence or absence of competing compounds.

Nonspecific binding was defined using an excess (1 µM) of ghrelin.

The binding reaction was stopped by addition of 4 mL of ice-cold

HB followed by rapid filtration over Whatman GF/C filters

presoaked with 0.5% polyethyleneimine to prevent excessive

binding of radioligand to the filters. Filters were rinsed three times

with 3 mL of ice-cold wash buffer (50 mM Tris (pH 7.3), 10 mM

MgCl₂, 2.5 mM EDTA, and 0.015% (w/v) X-100 Triton), and the

radioactivity bound to membranes was measured in a γ counter.

Intracellular Calcium Mobilization Assay. The calcium experi-

ments were performed using the benchtop scanning fluorometer

FlexStation II machine (Pharmacologie & Screening Plateform of

the Institut Fe´de´ratif de Recherche 3, Montpellier, France).

CHO cells were transiently transfected with the hGHS-1a

receptor, using electroporation, and were then plated into 96-well

black-bottom plates (80 000 cells/well). Twenty-four hours later,

the cells were washed with 150 µL of buffer A (Hank’s balanced

salt solution, 0.5% BSA, 20 mM CaCl₂, 2.5 mM probenecid, pH

7.4) and were then loaded with 1 µM fluorescent calcium indicator

Fluo-4AM prepared in buffer A, containing 0.06% pluronic acid

(a mild-ionic detergent that facilitates Fluo-4AM ester loading).

The cells were left to incubate for 1 h in the dark at 37 °C.

Following the incubation, excess Fluo-4AM was removed from

the cells by washing twice with 100 µL of buffer A, and 50 µL of

the same buffer was then added to each well. The cells were left at

room temperature for 30 min to allow complete de-esterification

of intracellular Fluo-4AM esters. The black-bottom plate containing

the cells and the plate containing the compounds to be tested were

then placed into the temperature-regulated FlexStation machine.

The machine records the fluorescence output over a period of

60 s, with the compounds being automatically distributed into the

wells containing the cells after 15 s. The Fluo-4AM exhibits a large

fluorescence intensity increase on binding of calcium, and therefore,

the fluorescence output is used directly as a measure of intracellular

calcium mobilization. The excitation and emission wavelengths

were 485 and 525 nm, respectively. The basal fluorescence intensity

of dye-loaded cells was 800-1200 arbitrary units, and the

fluorescence peak upon maximal response was 5000-7000 units.

To assess the ability of each of the compounds to induce calcium

mobilization, they were tested at 10 µM in triplicate in at least two

independent experiments. In each case, the change in fluorescence

upon addition of the compound was compared with the basal

fluorescence output measured with the control (addition of buffer

A only). The maximum fluorescent output was equivalent to that

achieved when the cells where stimulated with 0.1 µM ghrelin.

For the compounds behaving as agonists and displaying a high

affinity binding for hGHS-R1a in radiolabeled binding experiments,

the EC₅₀(the molar concentration of the agonist producing 50% of

(R,S)-N-((R)-1-(5-(2-(1H-Indol-3-yl)ethyl)-4-(2,4-dimethoxy-

benzyl)-4H-1,2,4-triazol-3-yl)-2-(1H-indol-3-yl)ethyl)piperazine-

2-carboxamide Trifluoroacetate Salt (54). H NMR (300 MHz,

1

DMSO-d₆, 300 K): δ 2.95 (m, 6H, H₂, H₅, and H₆piperazinyl),

3.32 (m, 4H, CH₂âTrp and NH piperazinyl), 3.62 (m, 10H, CH₂-

CH₂-indole and OCH₃), 4.07 (m, 1H, H₃piperazinyl), 4.84 (m,

2H, CH₂-o,p-dimethoxyphenyl), 5.16 (m, 1H, CH RTrp), 6.00-

6.53 (m, 3H, CHar o,p-dimethoxyphenyl), 6.84-7.30 (m, 11H,

CHar indole, CHar indole Trp and NH amide), 10.75 (s, 1H, NH

indole), 10.79 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-

d₆, 300 K): δ 22.9 (CH₂-CH₂-indole), 25.8 (CH₂-CH₂-indole),

29.8 (C âTrp), 41.2 (CH₂-o,p-dimethoxyphenyl), 47.7 (C RTrp,

C₅and C₆piperazinyl), 55.6 (OCH₃and C₂piperazinyl), 58.4 (C₃

piperazinyl), 99.0 (C₃o,p-dimethoxyphenyl), 105.0 (C₅o,p-

dimethoxyphenyl), 109.6 (C₃Trp), 111.8 (C₇indole and C₇Trp),

113.4 (C₃indole), 115.3 (C₁o,p-dimethoxyphenyl), 118.2 (C₄

indole), 118.4 (C₄Trp), 118.6 (C₅indole), 118.8 (C₅Trp), 121.4

(C₆indole and C₆Trp), 123.9 (C₂indole and C₂Trp), 127.2 (C₉

indole and C₉Trp), 127.9 (C₆o,p-dimethoxyphenyl), 136.5 (C₈Trp),

136.6 (C₈indole), 154.6 (Cq triazole), 154.8 (Cq triazole), 155.0

(CO amide), 157.6 (C₂o,p-dimethoxyphenyl), 160.8 (C₄o,p-

dimethoxyphenyl).

N-((R)-1-(4-(4-Methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-

3-yl)-2-(1H-indol-3-yl)ethyl)tetrahydro-2H-pyran-4-carboxam-

1

ide Trifluoroacetate Salt (57). H NMR (300 MHz, DMSO-d₆,

300 K): δ 1.20 (m, 1H, H₅tetrahydropyranyl), 1.30 (m, 3H, H₃

and H₅tetrahydropyranyl), 2.17 (m, 1H, H₄tetrahydropyranyl), 2.82

(m, 4H, CH₂-CH₂-phenyl), 3.16 (m, 2H, H₂and H₆tetrahydro-

pyranyl), 3.31 (dd, 1H, J ) 14 and 8 Hz, CH₂âTrp), 3.35 (dd, 1H,

J ) 14 and 7 Hz, CH₂âTrp), 3.66 (s, 3H, OCH₃), 3.72 (m, 2H, H₂

and H₆tetrahydropyranyl), 5.08 (m, 2H, CH₂p-methoxyphenyl),

5.26 (m, 1H, CH RTrp), 6.73 (s, 4H, CHar p-methoxyphenyl), 6.87

(t, 1H, J_o) 8 Hz, H₅Trp), 7.02 (m, 2H, H₂and H₆Trp), 7.07 (d,

2H, J_o) 7 Hz, H₂and H₆phenyl), 7.18 (m, 3H, H₃, H₄, and H₅

phenyl), 7.30 (m, 2H, H₄and H₇Trp), 8.52 (d, 1H, J ) 8 Hz, NH

amide), 10.77 (s, 1H, NH indole Trp). ¹³C NMR (75 MHz, DMSO-

d₆, 300 K): δ 26.3 (CH₂-CH₂-phenyl), 28.8 (C₃and C₅tetrahy-

dropyranyl), 29.2 (C âTrp), 32.2 (CH₂-CH₂-phenyl), 40.7 (C₄

tetrahydropyranyl), 44.8 (C RTrp), 45.9 (CH₂p-methoxyphenyl),

55.5 (OCH₃), 66.6 (C₂and C₆tetrahydropyranyl), 109.8 (C₃Trp),

111.7 (C₇Trp), 114.5 (C₃and C₅p-methoxyphenyl), 118.4 (C₄Trp),

118.7 (C₅Trp), 121.3 (C₆Trp), 124.5 (C₂Trp), 126.7 (C₄phenyl),

127.2 (C₉Trp), 127.5 (C₁p-methoxyphenyl), 128.8 (C₂and C₆

phenyl), 128.7 (C₃and C₅phenyl), 127.9 (C₂and C₆p-methox-

yphenyl), 136.4 (C₈Trp), 140.4 (C₁phenyl), 154.7 (Cq triazole),

155.7 (Cq triazole), 159.2 (C₄p-methoxyphenyl), 174.2 (CO amide).

In Vitro Determination of the Binding Affinities for the

Human GHS-R1a. Transient Transfection of LLC PK-1 Cells

and Membrane Preparation: LLC PK-1 cells were grown at

5806 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23

Moulin et al.

the maximal possible effect of that agonist) was determined using

a dose-response curve.

27, 31, 39-43, 47, 52, and 55; [Ca²⁺]_iaccumulation curves in the

presence of increasing concentration of agonists 26, 27, 29, 41-

In the case of high-affinity antagonists, the IC₅₀and K_bwere

determined using antagonist inhibition curves in the presence of

0.1 µM ghrelin (submaximal concentration). The IC₅₀was calculated

as the molar concentration of antagonist that reduced the maximal

response of ghrelin by 50%, and an estimation of the K_bwas made

using the Cheng-Prusoff Equation. Schild analysis was also used

to determine the EC₅₀of ghrelin in the presence of different

concentrations of antagonist, and from this the pA₂and the exact

K_bwere determined.

43, 47, 52, and 55; inhibition curves of ghrelin-induced [Ca²⁺

]

i

accumulation by compounds 18-20; Schild plots for compounds

16, 18, and 20. This material is available free of charge via the

Internet at http://pubs.acs.org.

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the Italian Guidelines for the Use of Animals in Medical Research.

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solvent (DMSO, final dilution 1:300), hexarelin (80 µg/kg sc), or

new compounds (160 µg/kg sc). For combined treatments (test

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min before hexarelin. Pups were killed by decapitation 15 min later.

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following 6 h. Food intake was normalized for the body weight of

the animals and expressed as grams of food eaten for 100 g of

body weight.

Acknowledgment. We gratefully acknowledge Pierre Sanchez

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Supporting Information Available: RP HPLC chromato-

gramme tracings of compounds 4, 16, 17, 35, 39-41, and 52;

displacement curves of ¹²⁵I-His⁹-ghrelin for compounds 18-20, 26,

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