Synthesis and structure-activity relationships of 2-substituted D-tryptophan-containing peptidic endothelin receptor antagonists: Importance of the C-2 substituent of the D-tryptophan residue for endothelin A and B receptor subtype selectivity

J . Med. Chem. 1996, 39, 2313-2330

2313

Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of 2-Su bstitu ted

D-Tr yp top h a n -Con ta in in g P ep tid ic En d oth elin Recep tor An ta gon ists:

Im p or ta n ce of th e C-2 Su bstitu en t of th e D-Tr yp top h a n Resid u e for En d oth elin

A a n d B Recep tor Su btyp e Selectivity

Takehiro Fukami,* Takeru Yamakawa, Kenji Niiyama, Hisaki Kojima, Yuuka Amano, Fuyuko Kanda,

Satoshi Ozaki, Takahiro Fukuroda, Masaki Ihara, Mitsuo Yano, and Kiyofumi Ishikawa

Drug Discovery Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Company, Ltd.,

Tsukuba Techno-Park Oho, Okubo 3, Tsukuba, Ibaraki 300-33, J apan

Received J anuary 31, 1996^X

Continuing studies on modifications of potent cyclic pentapeptide endothelin (ET) receptor

antagonists, represented by BQ-123, and potent linear tripeptide derivative ET receptor

antagonists, represented by BQ-788, are described herein. The introduction of ^D-tryptophan

analogues with C-2 substituents in these peptidic ET antagonists resulted in potent ET receptor

antagonists with various ET_A/ET_Bsubtype selectivity. Combined ET_A/ET_Breceptor antagonists

were found in both cyclic pentapeptide and linear tripeptide series with 2-halo- and 2-methyl-

D-tryptophans. In contrast, compounds with 2-cyano-D-tryptophan were ET_Breceptor-selective

antagonists. The C-2 substituent of the ^D-tryptophanyl residue appeared to be very important

for the discrimination of ET_A/ET_Bsubtype selectivity of the antagonists. The potent ET receptor

antagonists with various ET_A/ET_Bsubtype selectivity synthesized in this study may be useful

tools for elucidating the physiological and pathophysiological roles of ET and ET receptors.

In tr od u ction

various ET_A/ET_Bsubtype selectivity will be very impor-

tant for clarifying the physiological and pathophysi-

ological roles of ET and ET receptors.

Endothelin-1 (ET-1), which was first isolated from the

culture medium of porcine aortic endothelial cells, is a

potent vasoconstrictor consisting of 21 amino acids.¹

ET-1 is some 10-fold more potent than angiotensin II

as a vasoconstrictor and has extremely long-lasting

pressor effects.²Studies including a human genomic

analysis have identified two structurally- and function-

ally-related isopeptides of ET-1 termed ET-2 and ET-

3.^3-5Several studies have characterized two ET recep-

tor subtypes, ET_Aand ET_B, in animal and mammalian

systems.^6-9The ET_Areceptor is highly specific for ET-1

and ET-2, while the ET_Breceptor has almost equal

affinity for all three isopeptides. A third ET receptor

subtype, ET_C, which is selective for ET-3, was cloned

from Xenopus dermal melanophores,¹⁰but this subtype

has not been identified yet in mammalian tissues.

Various types of ET receptor antagonists have been

reported with various ET_A/ET_Breceptor subtype selec-

tivity (for a recent review, see ref 23). We previously

discovered a selective ET_Areceptor antagonist, BQ-123

(1),^24-26which was derived from head-to-tail cyclic

pentapeptide leads BE-18257 A and B of microbial

origin.^27,28Subsequent derivation led to the discovery

of linear tripeptide ET_A-selective antagonists repre-

sented by BQ-485 (2).^29,30Further modification of the

linear tripeptide antagonists produced a selective ET_B

receptor antagonist, BQ-788 (3).³¹In the course of our

work on the discovery of the ET_Breceptor antagonist,

we noticed that the presence of a methoxycarbonyl

group on the indole nitrogen of the D-tryptophanyl

residue of 3 was very effective for both strong ET_B

antagonistic activity and ET_A/ET_Breceptor subtype

selectivity. We conjectured that further modifications

on the indole ring of the D-tryptophanyl residue of the

linear tripeptide ET antagonist might produce potent

ET receptor antagonists with various ET_A/ET_Breceptor

subtype selectivity. Of the derivatives comprising D-

tryptophan analogues with modifications on the indole

ring, potent ET_A/ET_B-nonselective and ET_B-selective

receptor antagonists were found in 2-substituted D-

tryptophan-containing tripeptide derivatives. For ex-

ample, the 2-bromo-D-tryptophan-containing linear trip-

eptide derivative BQ-928 (4) is a combined ET_A/ET_B

receptor antagonist, while the 2-cyano-D-tryptophan-

containing derivative BQ-017 (5) is an ET_B-selective

antagonist.³²Since then, we introduced the 2-substi-

tuted D-tryptophan analogues into the head-to-tail cyclic

pentapeptides that are represented by 1 and found some

potent ET receptor antagonists with various ET_A/ET_B

receptor subtype selectivity. In this paper, we describe

in detail the structure-activity relationships for these

ET_Aand ET_Breceptors are widely distributed not only

in vascular but also in nonvascular tissues and possess

different functions depending on species and location.

ET_Areceptors are predominantly found in peripheral

tissues, especially in vascular smooth muscle tissues to

mediate vasoconstriction, though they are also present

in certain regions of the brain.^6,11In contrast, ET_B

receptors have been thought to be exclusively localized

to the endothelium and nonvascular tissues such as the

liver, kidneys, and brain.¹²Endothelial ET_Breceptors

are functionally linked to vasodilation, possibly through

the release of endothelium-derived relaxing factor.¹³

However, it has been confirmed that ET_Breceptors are

also located in certain vascular and airway smooth

muscle tissues, mediating their constriction.^14-21The

ETs possess a number of important biological activities

besides potent vasoconstricting activity.²²Therefore,

the identification of potent ET receptor antagonists with

^XAbstract published in Advance ACS Abstracts, May 15, 1996.

2314 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Fukami et al.

Ch a r t 1

Sch em e 1^a

a

Reagents: (a) see ref 33; (b) Boc₂O, DMAP, CH₃CN; (c) aq NaOH, MeOH; (d) (i) Boc₂O, DMAP, CH₃CN, (ii) 3-(dimethylamino)pro-

pylamine; (e) formic acid; (f) CuCN, DMF; (g) (trimethylsilyl)acetylene, (Ph₃P)Pd, CuI, diethylamine.

potent ET antagonists with various receptor subtype

selectivity and for related analogues.

previous paper (Scheme 1).³²The protected 2-halo-D-

tryptophans 54a ,b were synthesized by radical haloge-

nation of the protected D-tryptophan 53 according to the

method reported by Phillips and Cohen.³³Treatment

of 54a ,b with an excess amount (3-5 equiv) of di-tert-

butyldicarbonate (Boc₂O) in CH₃CN in the presence of

a catalytic amount of 4-(dimethylamino)pyridine (DMAP)

Ch em istr y

The synthesis of the tripeptide derivatives and the

cyclic pentapeptides with 2-substituted D-tryptophans

described in this article involves the synthesis of D-

tryptophan analogues with C-2 substituents and their

subsequent derivation. The D-tryptophan analogues

with 2-halo, 2-cyano, and 2-ethynyl substituents were

synthesized according to the method given in our

R

afforded the N ,N ⁱⁿ-bis-Boc-protected compounds 55a ,b

in yields of 59% and 68%, respectively. Alkaline hy-

drolysis (2.2 equiv of aqueous NaOH in MeOH) of 55a ,b

R

yielded the N ,N ⁱⁿ-bis-Boc-protected 2-halo-D-tryp-

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2315

Sch em e 2^a

a

Reagents: (a) 2-methylindole, zinc triflate, CHCl₃; (b) aq NaOH, MeOH; (c) H₂, Pd/C, MeOH.

Sch em e 3. Method A^a

a

Reagents: (a) H-D-Nle-O^tBu‚HCl, EDCI, HOBT, NMM, CH₂Cl₂; (b) formic acid; (c) EDCI, HOBT, CH₂Cl₂; (d) TFA; (e) N,N ′-

carbonyldiimidazole, Et₃N, THF; (f) H₂, Pd/C, MeOH.

tophans 56a ,b in quantitative yields. Alternatively,

treatment of 54b with Boc₂O (3-5 equiv) in CH₃CN

followed by 3-(dimethylamino)propylamine in one pot

The linear tripeptide derivatives were synthesized by

five methods (methods A-E, Scheme 3-7). 1-[3-(Dim-

ethylamino)propyl]-3-ethylcarbodiimide hydrochloride

(EDCI) and 1-hydroxybenzotriazole monohydrate (HOBT)

were exclusively employed in the coupling reactions.

R

yielded the N ,N ⁱⁿ-bis-Boc-protected ester 57 in an

almost quantitative yield. Alkaline hydrolysis of 57

R

in

using an excess of NaOH (3-5 equiv) gave the N -Boc-

Method A, used for the synthesis starting from N ,N -

protected 2-bromo-D-tryptophan 58 in a quantitative

yield. Treatment of 57 with formic acid yielded the

amine 59. Reaction of the protected 2-bromo-D-tryp-

tophan analogue 57 with CuCN (2.5 equiv) in DMF (85

°C, 1-2.5 h) afforded a 2-cyano analogue (60) in a yield

of 78-99%. Compound 57 also reacted with (trimeth-

ylsilyl)acetylene (3 equiv) in the presence of (Ph₃P)₄Pd

(10 mol %) and CuI (30 mol %) in diethylamine (40 °C,

2.5-9 h) to yield a 2-(trimethylsilyl)ethynyl analogue

(62, 57-76%). Treatment of 60 and 62 with an excess

bis-Boc-protected 2-halo-D-tryptophan 56a or 56b, is

illustrated in Scheme 3. Compound 56a or 56b was

coupled with D-norleucine (D-Nle) tert-butyl ester to give

R

the dipeptide 68. N -Boc deprotection of 68 by formic

acid (room temperature, 1.5 h) afforded a mixture of 69

and 70 (55% and 21% for R ) Cl, 33% and 47% for R )

Br, respectively). Compound 69 as well as 70 was

coupled with N-cis-(2,6-dimethylpiperidino)carbonyl

amino acid 73 to give the protected tripeptide tert-butyl

ester 71. Treatment of the protected tripeptide 71 with

TFA gave the desired compounds 6, 7, and 16-22. The

N-carbamoyl amino acid 73 was prepared from an

amino acid benzyl ester p-toluenesulfonate or hydro-

chloride by two steps. Treatment of the amino acid

benzyl ester with N,N ′-carbonyldiimidazole and tri-

ethylamine in THF followed by cis-2,6-dimethylpiperi-

dine in one pot yielded the urea compound 72 (80-98%).

Catalytic hydrogenation of 72 afforded the correspond-

ing acid 73 (94-99%).

R

of NaOH (4-10 equiv) in MeOH afforded the N -Boc-

protected 2-substituted D-tryptophan analogues 61 and

63 (in a 78-100% yield), respectively.

The 2-methyl-D-tryptophan analogue was prepared by

a method analogous to that described by Sato and

Kozikowski (Scheme 2).³⁴The N-benzyloxycarbonyl-

protected aziridine-2-carboxylate 64 prepared from D-

serine was reacted with 2-methylindole in the presence

of zinc triflate to give the protected-2-methyl-D-tryp-

tophan methyl ester 65 in a 68% yield. Alkaline

hydrolysis of 65 yielded the acid 66 in a 93% yield.

Catalytic hydrogenation of 65 afforded the amine 67.

Method B was used for the synthesis starting from

R

N -Boc-protected 2-bromo- or 2-cyano-D-tryptophan 58

or 61 and is illustrated in Scheme 4. Coupling of the

2316 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Sch em e 4. Method B^a

Fukami et al.

a

Reagents: (a) H-AA²-OMe‚HCl, EDCI, HOBT, NMM, CH₂Cl₂; (b) formic acid; (c) EDCI, HOBT, CH₂Cl₂; (d) aq NaOH, MeOH.

Sch em e 5. Method C^a

a

Reagents: (a) EDCI, HOBT, CH₂Cl₂; (b) aq NaOH, MeOH; (c) H-AA²-OMe‚HCl, EDCI, HOBT, NMM, CH₂Cl₂.

Sch em e 6. Method D^a

a

Reagents: (a) Boc-Leu-OH, EDCI, HOBT, NMM, CH₂Cl₂; (b) (i) formic acid, (ii) phenyl chloroformate, pyridine; (c) R¹R²NH, Et₃N,

CHCl₃; (d) aq NaOH, MeOH.

R

N -Boc-protected D-tryptophan analogue with the C-

Method D, which was used for the synthesis of 11-

15 with N-terminal urea alteration, involves replace-

ment of a phenoxycarbonyl group with a secondary

amine (Scheme 6).²⁷The dipeptide methyl ester 75a

was transformed to the N-terminal phenoxycarbony-

lated tripeptide methyl ester 81. Reaction of 81 with a

secondary amine in the presence of triethylamine

yielded the urea 82. Subsequent alkaline hydrolysis

afforded the target compounds.

terminal amino acid methyl ester (AA²-OMe) gave the

dipeptide 74. The N -Boc protecting group in 74 was

R

deprotected by formic acid followed by coupling with the

N-terminal component 73 to afford the tripeptide methyl

ester 76. Alkaline hydrolysis of the C-terminal methyl

ester gave the target compounds 4, 5, 8, 37, and 38.

Method C employed the 2-bromo- or 2-methyl-D-

tryptophan methyl ester 59 or 67 as a starting material

(Scheme 5). The amino ester was coupled with 73,

which after alkaline hydrolysis afforded the dipeptide

78. The dipeptide was coupled with the C-terminal

amino acid methyl ester (AA²-OMe) to yield the tripep-

tide methyl ester 79. Alkaline hydrolysis of the methyl

ester gave the target compounds 9 and 23-36.

The 2-ethyl-D-tryptophan-containing tripeptide de-

rivative 10 was synthesized by the method illustrated

in Scheme 7. We first attempted to prepare a tripeptide

R

derivative with 2-ethynyl-D-tryptophan. However, N -

Boc deprotection of the dipeptide 83, derived from 63,

by formic acid did not afford the expected primary amine

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2317

Sch em e 7. Synthesis of Compound 10^a

a

Reagents: (a) H-D-Nle-OMe‚HCl, EDCI, HOBT, NMM, CH₂Cl₂; (b) formic acid; (c) H₂, Pd/BaSO₄, MeOH; (d) (i) formic acid, (ii) cis-

[(2,6-dimethylpiperidino)carbonyl]-Leu-OH (73a ), EDCI, HOBT, CH₂Cl₂; (e) aq NaOH, MeOH.

Sch em e 8. Method F^a

a

Reagents: (a) formic acid; (b) EDCI, HOBT, DMF; (c) (i) TFA, (ii) EDCI, HOBT, NMM, DMF; (d) aq NaOH, MeOH.

84, and the only isolated compound was a cyclic imine

(85) with complete racemization at C-R of the tryp-

tophanyl residue (24% yield, as a 1:1 mixture of dias-

tereoisomers). To avoid the intramolecular addition of

an R-amino group to the 2-ethynyl group of the indole

ring, a stronger acid, TFA, was employed. However,

treatment of 83 with TFA produced the same result. We,

therefore, gave up preparing a 2-ethynyl analogue and

substituted a 2-ethynyl group with a 2-ethyl group. The

2-ethyl derivative 86 was obtained by catalytic hydro-

HOBT, yielded the D-Asp side chain methyl ester of

cyclic pentapeptide 92. Alkaline hydrolysis of the D-Asp

side chain methyl ester afforded the target cyclic pen-

tapeptides 39-46, 51, and 52.

Method G was used for the synthesis of cyclic pen-

tapeptides with 2-methyl-D-tryptophan and is illus-

trated in Scheme 9. The N-terminal Boc-protected

linear tetrapeptide benzyl ester 93 was prepared using

stepwise elongation method. The Boc group was em-

ployed as an amino-protecting group, and deprotection

of the Boc was accomplished with TFA or 4 N HCl/1,4-

dioxane. EDCI and HOBT were used in coupling

reactions. Deprotection of the N-terminal Boc in the

R

genation of 83. Deprotection of the N -Boc group in

86, followed by coupling and alkaline hydrolysis in the

usual manner, yielded compound 10.

The cyclic pentapeptides were prepared by method F

or G. Method F was used for the synthesis of com-

pounds with 2-halo- or 2-cyano-D-tryptophan and is

illustrated in Scheme 8. The dipeptides 88a -d were

R

tetrapeptide 93, followed by coupling with N -Z-

protected 2-methyl-D-tryptophan 66, afforded the N-

terminal Z- and C-terminal Bzl-protected linear pen-

tapeptide 94. Deprotection of the N-terminal- and

C-terminal-protecting groups by catalytic hydrogena-

tion, followed by cyclization by EDCI and HOBT, yielded

the D-Asp side chain methyl ester of cyclic pentapeptide

95. Alkaline hydrolysis of the methyl ester afforded the

target cyclic pentapeptides 47-50.

R

prepared by coupling of N -Boc (and Nⁱⁿ-Boc)-protected

2-halo- or 2-cyano-D-tryptophan with D-Asp(OMe)-O^tBu.

Deprotection of the N-terminal (and Nⁱⁿ) Boc by formic

acid followed by coupling with the N-terminal Boc-

protected tripeptide 90 afforded the N-terminal Boc- and

C-terminal ^tBu-protected linear pentapeptide 91. Depro-

tection of the N-terminal and C-terminal protecting

groups by TFA, followed by cyclization by EDCI and

All final compounds were analyzed for homogeneity

by HPLC and characterized for structural integrity by

2318 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Sch em e 9. Method G^a

Fukami et al.

a

Reagents: (a) (i) TFA or 4 N HCl/1,4-dioxane, (ii) Z-D-Trp(2-Me)-OH (66), EDCI, HOBT, DMF; (b) (i) H₂, Pd/C, DMF, (ii) EDCI,

HOBT, DMF; (c) aq NaOH, MeOH.

Ta ble 1. Linear Peptide Derivatives with 2-Substituted

Ta ble 2. N-Terminus Urea Modifications

D-Tryptophans

IC₅₀(nM)

compd

R¹R²N

ET_A

ET_B

a

b

IC₅₀(µM)

7

11

12

13

14

15

cis-2,6-dimethylpiperidino

1-pyrrolidinyl

piperidino

hexahydro-1H-azepinyl

octahydro-1H-azocinyl

2-methylpiperidino

0.0056^c

0.16

0.026

0.0091

0.027

0.0081

0.0025^c

2.7

0.11

0.028

0.026

0.034

a

b

compd

R

ET_A

ET_B

6

7

8

9

10

Cl

Br

CN

CH₃

C₂H₅

0.015^c

0.0056^d

0.47

0.0096

0.18

0.0056^c

0.0025^d

0.0016

0.013

a

0.0050

Binding data in porcine aortic smooth muscle membranes.

Binding data in porcine cerebellum membranes. ^cn ) 5 IC₅₀

b

a

Binding data in porcine aortic smooth muscle membranes.

determinations. All other values represent one IC₅₀determina-

Binding data in porcine cerebellum membranes. ^cn ) 3 IC₅₀

b

tion. Variation was generally (10%.

d

determinations. n ) 5 IC₅₀determinations. All other values

represent one IC₅₀determination. Variation was generally (10%.

tives (Table 2). All replacements of the cis-(2,6-dim-

ethylpiperidino)carbonyl group in 7 with other groups

led to a decrease in binding affinity for both ET_Aand

ET_Breceptors. In particular, ET_Baffinity was signifi-

cantly decreased by the replacements. These results

suggest that a cis-(2,6-dimethylpiperidino)carbonyl group

is indispensable for potent affinity for both ET_Aand ET_B

receptor subtypes. The N-terminal urea moiety was

therefore retained during the following modifications of

the linear tripeptide series.

Table 3 shows the ET_Aand ET_Breceptor binding

affinities when the N-terminal and/or C-terminal amino

acids (AA¹and AA²) were substituted in the linear

tripeptide derivatives with 2-halo-D-tryptophans. Com-

pounds 16-22 represent substitutions at the Leu posi-

tion in the 2-chloro-D-tryptophan-containing tripeptide

derivative 6. The Nle, 3-cyclopropylalanine (Cpra), and

Nva analogues 16-18 exhibited almost the same affin-

ity at both ET_Aand ET_Breceptor subtypes. The

substitution of aliphatic amino acids with â-position-

branched side chains, such as Val, Ile, and cyclopen-

tylglycine (Cpeg), afforded compounds with almost the

same or slightly enhanced ET_Baffinity as that of the

Leu analogue but led to a 2-3-fold decrease in ET_A

affinity. In contrast, the cyclopropylglycine (Cprg)

analogue 22 exhibited a 4-fold increase in ET_Baffinity

together with slightly enhanced ET_Aaffinity, although

Cprg is a â-position-branched amino acid. The same

substitution in the 2-bromo-D-tryptophan-containing

analogue 7 yielded compound 4 with IC₅₀values of 3.8

¹H NMR and high-resolution fast atom bombardment

(FAB) mass spectrometry.

Biologica l Resu lts a n d Discu ssion

The synthesized compounds were first evaluated for

their binding affinities for both ET_Aand ET_Breceptor

subtypes. The binding assay for ET_Areceptors was

performed in porcine aortic smooth muscle membranes,

and that for ET_Breceptors was in porcine cerebellum

membranes, according to the reported method.²⁷

Table 1 shows modification at the C-2 position of the

D-tryptophanyl residue of the linear tripeptide deriva-

tives. Of these compounds, the analogues with 2-halo-

D-tryptophans, 6 and 7, exhibited potent affinity for both

ET_Aand ET_Breceptors. The 2-cyano-D-tryptophan-

containing analogue 8 showed potent affinity for the

ET_Breceptor; however, its ET_Aaffinity was 300 times

weaker than its ET_Baffinity. Analogues with 2-alkyl-

D-tryptophans were obscure. The methyl analogue 9

was nonselective, while the ethyl analogue 10 was

comparatively ET_B-selective. We then optimized other

parts of the compounds to identify potent combined ET_A/

ET_Breceptor antagonists from the linear tripeptide

derivatives with 2-halo- and 2-methyl-D-tryptophans

and to find potent ET_Breceptor-selective antagonists

from the 2-cyano-D-tryptophan-containing linear trip-

eptide derivatives.

Compounds 11-15 represent modifications at the

N-terminal urea moiety of the linear tripeptide deriva-

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2319

Ta ble 3. Linear Tripeptide Derivatives with

Ta ble 4. Linear Tripeptide Derivatives with 2-Methyl- and

2-Halo-^D-tryptophans

2-Cyano-^D-tryptophans

IC₅₀(µM)

AA^{1 a}

R

AA^{2 b}

D-Nle

ET_A

ET_B

compd

AA^{1 a}

Leu

Cprg

Leu

γMeLeu

Val

R

AA²

ET_A

ET_B

c

d

b

c

compd

6

16

17

18

19

20

21

22

7

Leu

Nle

Cpra

Nva

Val

Cl

Br

0.015^e

0.0090

0.014

0.0056^e

0.0061

0.0062

0.0079

0.0046^e

0.0046^f

0.0027^f

0.0013

0.0025^g

0.000 81^h

1.9

9

32

33

34

35

36

8

37

5

38

CH₃

CN

D-Nle

D-Met

D-His

D-Nle

D-Met

D-His

D-Nle

D-Nva

0.0096

0.0080^d

0.0028

0.0057

0.0010^e

0.0048

0.47

0.013

D-Nle

D-Aoa

D-Leu

D-Nva

D-Alg

0.013^d

0.099

0.0029

0.0029^e

0.17

0.0016

0.0071

0.0023^e

0.0056^d

0.020

0.033^f

0.051^f

0.046^f

0.0093

0.0056^g

0.0038^h

0.11

Ile

Cpeg

Cprg

Leu

Cprg

Leu

0.81

2.0^f

4

Val

3.0^d

23

24

25

26

27

28

29

30

31

a

Cprg ) 2-cyclopropylglycine, γMeLeu ) γ-methylleucine, and

0.021

0.014

b

Cpeg ) 2-cyclopentylglycine. Binding data in porcine aortic

0.0092^f

0.0098

0.0022^e

0.0019^f

0.0024

0.0038^f

0.0067

0.0021^f

0.0042

0.0045^e

0.0018^f

0.0019

0.076^f

1.1

smooth muscle membranes. ^cBinding data in porcine cerebellum

d

membranes. n ) 2 IC₅₀determinations. ^en ) 3 IC₅₀determina-

D-Met

D-Cys(Et)

D-Cys(Pr)

D-His

tions. ^fn ) 4 IC₅₀determinations. All other values represent one

IC₅₀determination. Variation was generally (10%.

D-Trp

placement of D-Nle with D-Met in 9 did not affect affinity

for the ET_Aand ET_Breceptor subtypes, although the

same replacement in the 2-bromo-D-tryptophan deriva-

tives significantly increased ET_Areceptor binding af-

finity (9 vs 32 and 7 vs 27). The D-His substitution led

to a 3-fold increase in ET_Areceptor affinity and an 8-fold

decrease in ET_Breceptor affinity. With regard to the

AA¹position, the replacement of Leu with Cprg in 9

resulted in a 2-fold increase in ET_Aaffinity together

with a 4.5-fold increase in ET_Baffinity. This result

parallels findings on the activities of the 2-halo-D-

tryptophan series (6 vs 22 and 7 vs 4). The subsequent

replacement of D-Nle with D-Met in 34 increased ET_A

affinity by 6-fold, although the same replacement in 9

did not change the affinity. This compound (35) is the

most potent combined ET_A/ET_Breceptor binding inhibi-

tor among the 2-methyl-D-tryptophan-containing trip-

eptide derivatives, with IC₅₀values of 1.0 nM for ET_A

and 2.9 nM for ET_B. The replacement of D-Nle with

D-His in 34 did not increase ET_Areceptor binding

affinity, although the same replacement in 9 resulted

in a 3-fold increase in ET_Aaffinity. The effects of

replacement at the AA¹and AA²positions were not

additive in this series.

a

Cpra ) 3-cyclopropylalanine, Cpeg ) 2-cyclopentylglycine, and

b

Cprg ) 2-cyclopropylglycine. Aoa ) 2-aminooctanoic acid, Alg )

2-allylglycine, Cys(Et) ) S-ethylcysteine, and Cys(Pr) ) S-propy-

lcysteine. ^cBinding data in porcine aortic smooth muscle mem-

branes. Binding data in porcine cerebellum membranes. ^en ) 3

d

IC₅₀determinations. ^fn ) 2 IC₅₀determinations. n ) 5 IC₅₀

g

h

determinations. n ) 4 IC₅₀determinations. All other values

represent one IC₅₀determination. Variation was generally (10%.

nM for ET_Aand 0.81 nM for ET_B. This compound is a

representative combined ET_A/ET_Breceptor binding

inhibitor, in which the affinity for the ET_Breceptor is

more potent than that for the ET_Areceptor. Compounds

23-31 represent substitutions at the D-Nle position in

the 2-bromo-D-tryptophan-containing tripeptide 7. Re-

placement of D-Nle with D-2-aminooctanoic acid (D-Aoa),

which has a longer normal alkyl side chain than does

D-Nle, resulted in a marked decrease in both ET_Aand

ET_Baffinity. The incorporation of D-Leu also led to a

significant decrease in affinity for both receptor sub-

types. The D-Nva and D-allylglycine (D-Alg) substitu-

tions did not affect binding affinity at both receptors

significantly. The substitution of sulfur-containing

amino acids, such as D-Met, S-ethyl-D-cysteine (D-Cys-

(Et)), and S-propyl-D-cysteine (D-Cys(Pr)), led to a 2-3-

fold increase in ET_Aaffinity with retention of ET_B

affinity. Of these compounds, the D-Cys(Et) analogue

28 exhibited the most potent affinity for both receptor

subtypes (ET_A) 1.9 nM and ET_B) 1.8 nM). This

compound is another representative combined ET_A/ET_B

receptor binding inhibitor among the 2-halo-D-tryp-

tophan-containing tripeptide derivatives. The incorpo-

ration of aromatic amino acids such as D-His and D-Trp

was tolerated at the ET_Areceptor but led a significant

decrease in ET_Baffinity.

Compounds 8, 37, 5, and 38 are derivatives with

2-cyano-D-tryptophan. In the course of our work on the

discovery of the selective ET_Breceptor antagonist 3,

which incorporates 1-(methoxycarbonyl)-D-tryptophan,

we observed that the replacement of Leu at AA¹with

γ-methylleucine (γMeLeu) led to enhanced ET_Breceptor

binding affinity together with decreased ET_Aaffinity,

giving increased ET_Bselectivity.³¹We first replaced Leu

in 8 with γMeLeu to produce compound 37. However,

this substitution resulted in decreases in both ET_Aand

ET_Breceptor binding affinity and in the ET_A/ET_B

selectivity ratio. The structure-activity relationships

of the 2-cyano-D-tryptophan-containing tripeptide series

seemed to differ from those of the 1-(methoxycarbonyl)-

D-tryptophan-containing tripeptide series. Substitution

Table 4 shows the ET_Aand ET_Breceptor binding

affinities of linear tripeptide derivatives with 2-methyl-

and 2-cyano-D-tryptophans. Compounds 9 and 32-36

are derivatives with 2-methyl-D-tryptophan. The re-

2320 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Fukami et al.

Ta ble 5. Cyclic Pentapeptides with 2-Substituted

Ta ble 6. Binding Affinities of Selected Analogues for Human

D-Tryptophans

ET_Aand ET_BReceptors

IC₅₀(nM)

a,b

c,d

b,e

d,f

compd

pET_A

hET_A

pET_B

hET_B

4

28

35

46

50

5

3.8^g

1.9^h

1.0

13

0.81^g

1.1

3.6

4.0

29

130

5.0

29

6.4

3.0

8.6

2.9

1.8^h

2.9

4.9

15

59

2.3

22

1.8

2000^g

2400

6200

IC₅₀(µM)

52

23 000ⁱ

c

d

compd

R

AA^{3 a}

D-Val

AA^{4 b}

ET_A

ET_B

18^e

a

b

Porcine aortic smooth muscle membranes. Values represent

the average of three independent experiments unless otherwise

noted. ^cHuman neuroblastoma-derived cell line SK-N-MC mem-

1

39

40

41

42

43

44

45

46

47

48

49

50

51

52

H

Cl

Br

Leu

Cprg

Leu

Cprg

Leu

0.022^e

0.040

0.027

0.10

0.036

0.028

0.066

0.019

0.0049^f

0.051

0.0027^g

0.0081^f

0.0018^f

2.2

D-Val

0.24

D-t-Leu

D-Pen(Me)

D-Val

D-t-Leu

D-Pen(Me)

D-Cpeg

D-Val

D-Cpeg

D-Val

D-Cpeg

D-Val

0.053

0.011

0.14

d

branes. Values represent the average of two independent experi-

ments unless otherwise noted. Variation was generally (10%.

^ePorcine cerebellum membranes. ^fHuman Girardi heart cell

0.031

0.023

0.087

0.015^f

0.87

g

h

i

membranes. n ) 4. n ) 2. n ) 1.

of our structure-activity study on the cyclic pentapep-

tide ET_Areceptor antagonists, we found that the D-Asp-

Pro sequence was very important for potent activity.^25,26

We therefore retained this sequence in the following

modifications. Compounds 39-46 are cyclic pentapep-

tide analogues with 2-halo-D-tryptophans. The replace-

ment of D-Val in 39 and 42 with D-2-amino-3,3-

dimethylbutyric acid (D-tert-Leu) increased binding

affinity for both receptor subtypes. The introduction of

D-S-methylpenicillamine (D-Pen(Me)) at the same posi-

tion in 39 and 42 led to a 6-20-fold increase in ET_B

affinity but decreased ET_Abinding affinity. The D-2-

cyclopentylglycine (D-Cpeg) analogue 45 was slightly

more potent than the D-tert-Leu analogue 43 with

respect to ET_Aaffinity, although 45 is about 3-fold less

potent than 43 in ET_Baffinity. Further replacement

of Leu with Cprg in 45 led to 4- and 6-fold increases in

affinity for ET_Aand ET_Breceptors, respectively. Com-

pound 46 is one of the representative combined ET_A/

ET_Breceptor binding inhibitors among the cyclic pen-

tapeptides, with IC₅₀values of 4.9 nM for ET_Aand 15

nM for ET_B. Compounds 47-50 are 2-methyl-D-tryp-

tophan-containing cyclic pentapeptides. The replace-

ment of D-Val with D-Cpeg resulted in 20- and 7-fold

increases in affinity for ET_Aand ET_Breceptors, respec-

tively. The substitution of Cprg for Leu led to 6- and

3-fold increases in affinity for ET_Aand ET_Breceptors,

respectively. The double substitutions acted additively

to produce 50, with IC₅₀values of 1.8 nM for ET_Aand

59 nM for ET_B. The modification of the 2-cyano-D-

tryptophan-containing cyclic pentapeptides was assisted

by the structure-activity relationships of 2-halo- and

2-methyl-D-tryptophan derivatives. The replacement of

D-Val with D-Pen(Me) in 51 afforded 52 with high

selectivity for ET_Breceptors, as expected (ET_A) 23 µM

and ET_B) 22 nM).

Br

CH₃

CN

0.12^f

0.27^f

0.059^f

0.17

D-Pen(Me)

23

0.022

a

t-Leu ) 2-amino-3,3-dimethylbutyric acid, Pen(Me) ) S-

b

methylpenicillamine, and Cpeg ) 2-cyclopentylglycine. Cprg )

2-cyclopropylglycine. ^cBinding data in porcine aortic smooth

d

muscle membranes. Binding data in porcine cerebellum mem-

branes. ^eData reported in ref 25. ^fn ) 3 IC₅₀determinations.

n

g

) 2 IC₅₀determinations. All other values represent one IC₅₀

determination. Variation was generally (10%.

of Val (5) maintained potent binding affinity for the ET_B

receptor but decreased ET_Aaffinity by 4-fold, giving

increased ET_Bselectivity. The replacement of D-Nle

with D-Nva in 5 led to a decrease in both ET_Aand ET_B

binding affinity. Consequently, compound 5 is the most

potent ET_B-selective binding inhibitor among the 2-cy-

ano-D-tryptophan-containing linear tripeptide deriva-

tives, with IC₅₀values of 2.0 µM for ET_Aand 2.3 nM

for ET_B.

Table 5 shows the ET_Aand ET_Breceptor binding

affinities of cyclic pentapeptide analogues with 2-sub-

stituted D-tryptophans. We first replaced D-Trp in 1,

which is a representative ET_Areceptor antagonist, with

2-substituted D-tryptophans to produce compounds 39,

42, 47, and 51. Substitutions of 2-halo- and 2-methyl-

D-tryptophans maintained potent binding affinity for the

ET_Areceptor. In addition, these substitutions led to

significant, 20-130-fold, increases in ET_Breceptor

binding affinity, suggesting that optimization of other

residues may produce potent combined ET_A/ET_Brecep-

tor antagonists. In contrast, the 2-cyano-D-tryptophan

substitution decreased ET_Aaffinity by 100-fold but led

to a 100-fold increase in affinity for the ET_Breceptor,

yielding a cyclic pentapeptide ET_Breceptor-selective

binding inhibitor. These results were surprising be-

cause a comparatively small structural alteration, in-

troducing 2-halo, 2-methyl, and 2-cyano substituents on

the indolyl group of the D-Trp residue in such a large

molecule, significantly changed the ET_A/ET_Breceptor

subtype selectivity of the originally ET_A-selective an-

tagonist.

It has been suggested that there are notable species

differences in the effects of some compounds. For

example, Cody et al. reported that significant species

differences in the effects of C-terminal hexapeptide

analogue ET antagonists were observed between rat and

human cloned ET_Breceptors.³⁵Selected compounds

listed in Table 6 were tested for their affinity to human

ET_Aand ET_Breceptors to determine species differences

in the effects of these compounds. Binding assays for

human receptors were carried out in membranes of the

human neuroblastoma-derived cell line SK-N-MC and

human Girardi heart cells, expressing ET_Aand ET_B

We then optimized other residues to identify potent

combined ET_A/ET_Breceptor antagonists and potent ET_B-

selective receptor antagonists among cyclic pentapep-

tides with 2-substituted D-tryptophans. In the course

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2321

receptors, respectively,³¹using a previously reported

method.²⁶Compounds 4, 28, and 35, which are linear

tripeptide combined ET_A/ET_Breceptor binding inhibitors

in porcine receptor systems, and 46 and 50, which are

cyclic pentapeptide combined ET_A/ET_Breceptor binding

inhibitors in porcine receptor systems, have a high

affinity for both human ET_Aand ET_Breceptors. Com-

pounds 5 and 52, which are ET_B-selective receptor

binding inhibitors in porcine receptor systems among

linear tripeptides and cyclic pentapeptides, respectively,

showed high affinity for human ET_Breceptors but low

affinity for human ET_Areceptors. The differences in

IC₅₀values between human and porcine ET_Aand ET_B

subtype receptors are up to 4-fold. Compounds 4, 28,

35, 46, and 50 appeared to be combined ET_A/ET_B

receptor binding inhibitors, whereas compounds 5 and

52 are ET_B-selective binding inhibitors in human as well

as porcine receptor systems.

The combined ET_A/ET_Breceptor binding inhibitors 4

and 46 were evaluated for their antagonistic activities

against ET-1-induced constriction in isolated porcine

coronary arteries, which is mediated by ET_Areceptors,

and ET_B-selective agonist BQ-3020-induced constriction

in isolated rabbit pulmonary arteries, which is mediated

by ET_Breceptors, by a reported method.³¹These

compounds strongly antagonized ET_A-mediated vaso-

constriction with pA₂values of 7.9 and 7.5, respectively,

and also antagonized ET_B-mediated vasoconstriction

with pA₂values of 9.0 and 8.2, respectively. It was

reported that neither the ET_A- nor ET_B-selective an-

tagonist alone inhibited ET-1-induced constriction in

isolated rabbit pulmonary arteries and that the combi-

nation of both antagonists inhibited the constriction.²¹

Compounds 4 and 46 inhibited the constriction with pA₂

values of 6.4 and 5.7, respectively. The compounds

showed no intrinsic agonist activities in these arteries

even at concentrations of up to 10 µM, indicating that

they are potent combined ET_A/ET_Breceptor antagonists

with no agonist activities.

subtype selectivity. The introduction of 2-halo- and

2-methyl-D-tryptophans to both linear tripeptide deriva-

tives and cyclic pentapeptides produced combined ET_A/

ET_Breceptor antagonists, while the introduction of

2-cyano-D-tryptophan afforded ET_B-selective antago-

nists. We previously described a potent ET_Areceptor-

selective antagonist, BQ-123 (1),^25,26and a potent ET_B

receptor-selective antagonist, BQ-788 (3),³¹both of

which were derived from cyclic pentapeptide leads BE-

18257 A and B of microbial origin. Here we have

synthesized additional potent ET receptor antagonists

with ET_A/ET_B-nonselective subtype selectivity, indicat-

ing that the modification of the natural products re-

sulted in potent ET antagonists encompassing all

possible ET_A/ET_Breceptor subtype selectivity: ET_A-,

combined ET_A/ET_B-, and ET_B-selective antagonists.

The effects of the antagonists reported in this paper

did not show significant species differences between

human and porcine ET receptors. Compound 4 (BQ-

928), the representative linear tripeptide ET antagonist

of both ET_Aand ET_Breceptors, showed high affinity for

porcine and human ET_Areceptors (3.8 and 13 nM,

respectively) and ET_Breceptors (0.81 and 1.1 nM,

respectively). Compound 46 (BQ-238), the representa-

tive cyclic pentapeptide combined ET_A/ET_Breceptor

antagonist, also exhibited potent binding affinity for

porcine and human ET_Areceptors (4.9 and 8.6 nM,

respectively) and ET_Breceptors (15 and 29 nM, respec-

tively). The combined ET_A/ET_Breceptor antagonistic

character of 4 and 46 was confirmed by in vitro

contractility studies using porcine coronary arteries and

rabbit pulmonary arteries. The representative linear

tripeptide derivative ET_Bantagonist 5 (BQ-017) showed

potent binding affinity for porcine and human ET_B

receptors (2.3 and 5.0 nM, respectively), with great ET_A/

ET_Bsubtype selectivity in both receptor systems. Com-

pound 5 antagonized ET_B-selective agonist BQ-3020-

induced constriction in isolated rabbit pulmonary arteries

as potently as did the previously reported ET_B-selective

antagonist BQ-788.³¹Compound 5 is much more soluble

than BQ-788 in saline, which may make 5 a useful tool

for in vivo pharmacological studies. The potent ET

receptor antagonists with various ET_A/ET_Bsubtype

selectivity reported in this paper may be useful tools

for the in vitro and in vivo pharmacological study of

endothelins and their receptors. The results of phar-

macological studies using these potent ET receptor

antagonists will be reported elsewhere.

The ET_Breceptor-selective binding inhibitors 5 and

52 were tested for their inhibitory effects on ET-1-

induced [Ca²⁺]_iincreases in human Girardi heart cells,

according to a reported method.³¹These compounds

potently inhibited the ET-1-induced [Ca²⁺]_iincrease

with IC₅₀values of 4.0 and 35 nM, respectively. The

compounds alone (each 100 µM) did not increase [Ca²⁺

]

in the cells, indicating that they are potent ET_Breceptor

antagonists with no agonist activities. Compound 5,

which was also evaluated for its antagonistic activity

against BQ-3020-induced constriction in isolated rabbit

pulmonary arteries, appeared to antagonize this vaso-

constriction with a pA₂value of 8.5, suggesting that 5

is an ET_Breceptor antagonist as potent as 3, a potent

and selective ET_Breceptor antagonist that we previously

found.³¹In addition, 5 is much more soluble than 3 in

saline (as sodium salts of both compounds, >110 mg/

mL for 5 and 0.30 mg/mL for 3). This good solubility

may make 5 a useful tool for in vivo pharmacological

studies.

Exp er im en ta l Section

Abbr evia tion s. Abbreviations follow the recommendations

of the IUPAC-IUB J oint Commission on Biochemical Nomen-

clature for amino acids and peptides; see: Eur. J . Biochem.

1984, 138, 9-37. Additional abbreviations are defined in the

text or as follows: ^D-Alg, D-2-allylglycine; D-Aoa, D-2-aminooc-

tanoic acid; ^D-Cpeg, 2-cyclopentyl-D-glycine; Cprg, 2-cyclopro-

pylglycine; ^D-Cys(Et), S-ethyl-D-cysteine; D-Cys(Pr), S-propyl-

D-cysteine; D-tert-Leu, D-2-amino-3,3-dimethylbutyric acid;

γMeLeu, γ-methyl-^L-leucine; D-Pen(Me), S-methyl-D-penicil-

lamine; ^D-Trp(2-Br), 2-bromo-D-tryptophan; D-Trp(2-Cl), 2-chloro-

D-tryptophan; D-Trp(2-CN), 2-cyano-D-tryptophan; D-Trp(2-Et),

2-ethyl-^D-tryptophan; D-Trp(2-Me), 2-methyl-D-tryptophan; Boc,

tert-butoxycarbonyl; Z, benzyloxycarbonyl; Me, methyl; Et,

Con clu sion s

t

ethyl; Bu, tert-butyl; Bzl, benzyl; TosOH, p-toluenesulfonic

This work has demonstrated the importance of the

C-2 substituent of the D-tryptophanyl residue in both

linear tripeptide derivative and cyclic pentapeptide ET

antagonists for the discrimination of ET_A/ET_Breceptor

acid; AcOH, acetic acid; EtOAc, ethyl acetate; TFA, trifluoro-

acetic acid; DMF, N,N-dimethylformamide; EDCI, 1-[3-(dim-

ethylamino)propyl]-3-ethylcarbodiimide hydrochloride; HOBT,

1-hydroxybenzotriazole monohydrate; DMAP, 4-(dimethylami-

2322 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Fukami et al.

no)pyridine; TEA, triethylamine; NMM, N-methylmorpholine;

HPLC, high-performance liquid chromatography; t_R, retention

time.

3.65 (1 H, dd, J ) 10.3, 14.8 Hz), 3.81 (3 H, s), 5.37 (1 H, dd,

J ) 5.1, 10.3 Hz), 7.18-7.43 (3H, m), 8.06 (1 H, dd, J ) 1.5,

7.5 Hz).

N

^r,N ⁱⁿ-Bis(ter t-b u t oxyca r b on yl)-2-ch lor o-D-t r yp t o-

In str u m en ts a n d Ma ter ia ls. Melting points were deter-

mined on a Yanaco MP-S3 melting point apparatus and are

uncorrected. IR spectra were obtained with a Horiba FT-IR

FT-200 spectrometer. ¹H NMR spectra were recorded on a

Varian Gemini-200, Gemini-300, or VXR-300 or a J EOL J MN-

EX400 spectrometer. Chemical shifts are reported in parts

per million (ppm) downfield from tetramethylsilane (TMS) and

coupling constants (J ) are in hertz (Hz). (Note: In the

description of the NMR spectra, the designation “brs” used

alone indicates a broad signal of undetermined multiplicity.)

Fast atom bombardment (FAB) mass spectra (MS) were

recorded on a J EOL J MS-DX-300 spectrometer in either a

glycerol or 3-nitrobenzyl alcohol matrix using xenon as a target

gas. High-resolution mass spectra (HRMS) were determined

on the same instrument. Optical rotations were recorded on

a Horiba Sepa-200 high-sensitivity polarimeter. HPLC analy-

sis was performed on a Nihon Bunkoh instrument using a

YMC-Pack ODS-AQ column (4.6 × 150 mm, 5-µm particle size;

YMC Co., Ltd.) or a Capcell Pak C18 column (4.6 × 250 mm,

5-µm particle size; Shiseido Co., Ltd.) at 40 °C. Two different

conditions were utilized for the HPLC analysis: (a) 10:90-

80:20 CH₃CN with 0.1% TFA:0.1% aqueous TFA, linear

gradient over 35 min at 1.0 mL/min (λ ) 230 nM) on a YMC-

Pack ODS-AQ column, or (b) 45:55 CH₃CN:H₂O with 0.1% H₃-

PO₄isocratic system at 1.0 mL/min (λ ) 230 nM) on a Capcell

Pak C18 column. Column chromatography was carried out

on E. Merck silica gel 60 (230-400 mesh) unless otherwise

noted. TLC analysis was performed on precoated silica gel

glass plates 60 F254 (0.25 mm) purchased from E. Merck in

the indicated solvent systems. Components were visualized

under UV light by ninhydrin spray and/or phosphomolybdic

acid reagent.

p h a n (56a ). NaOH (1 N) (1.03 mL, 1.03 mmol) was added to

a solution of 55a (255 mg, 0.465 mmol) in MeOH (5 mL) at 0

°C. The mixture was allowed to warm to room temperature

and stirred overnight. The mixture was diluted with water

(50 mL), acidified with 10% citric acid, and extracted with

EtOAc (30 mL × 3). The combined organic extracts were

washed with brine (30 mL) and dried over MgSO₄, and the

solvent was evaporated to give crude 56a (206 mg, 104%) as a

colorless amorphous solid: TLC R_f(CHCl₃:MeOH:AcOH ) 30:

1:1) 0.46; FAB-MS m/ e 438, 440 (M⁺). This material was used

without further purification.

N

^r,N ⁱⁿ-Bis(ter t-b u t oxyca r b on yl)-2-b r om o-D-t r yp t o-

p h a n (56b). Compound 55b (994 mg, 1.68 mmol) was treated

in the same manner as described for the corresponding chloro

compound, yielding crude 56b (820 mg, 101%) as a colorless

amorphous solid: TLC R_f(CHCl₃:MeOH ) 10:1) 0.39; FAB-

MS m/ e 482, 484 (M⁺).

N

^r,N ⁱⁿ-Bis(ter t-b u t oxyca r b on yl)-2-b r om o-D-t r yp t o-

p h a n Meth yl Ester (57). Boc₂O (15.1 g, 69 mmol) and DMAP

(0.28 g, 2.3 mmol) were added to a solution of N ^R-(trifluoro-

acetyl)-2-bromo-^D-tryptophan methyl ester (54b; 9.04 g, 23.0

mmol) in dry acetonitrile (50 mL) at 0 °C. After being stirred

at 0 °C for 30 min, the mixture was allowed to warm to room

temperature and stirred overnight. The mixture was cooled

to 0 °C and 3-(dimethylamino)propylamine (4.3 mL, 34.5

mmol) was added. After being stirred at 0 °C for 20 min, the

mixture was neutralized with 10% citric acid and concentrated

under reduced pressure. The residue was taken up with

EtOAc (200 mL), washed successively with 10% citric acid (100

mL), saturated NaHCO₃(100 mL), and brine (100 mL), dried

over MgSO₄, and evaporated. Trituration of the residue with

hexane gave 57 (6.70 g, 58%) as colorless crystals. The mother

liquor was purified by column chromatography on silica gel

eluted with EtOAc/hexane (1:3) to give 57 (4.72 g, 41%, total

The amino acids or amino acid derivatives ^D-Asp(OMe),³⁶

D- and L-Cpeg,³⁷Cpra,³⁸Cprg,³⁸

N

^R-(trifluoroacetyl)-D-tryp-

99%): mp 58-62 °C; TLC R_f(EtOAc:hexane ) 1:5) 0.33; [R]²⁰

tophan methyl ester,³³

N

^R-(trifluoroacetyl)-2-bromo-D-tryp-

and N ^R-(trifluoroacetyl)-2-chloro-D-

D

5.83° (c 1.0, MeOH); FAB-MS m/ e 496, 498 (M⁺); ¹H NMR

(200 MHz, CDCl₃) δ 1.40 (9 H, s), 1.70 (9 H, s), 3.10-3.40 (2

H, m), 3.68 (3 H, s), 4.52-4.71 (1 H, m), 5.15 (1 H, d, J ) 7.3

Hz), 7.15-7.40 (2 H, m), 7.50 (1 H, d, J ) 7.3 Hz), 8.05 (1 H,

d, J ) 7.3 Hz). Anal. Calcd for C₂₂H₂₉BrN₂O₆: C, 53.13; H,

5.88; N, 5.63. Found: C, 53.31; H, 5.86; N, 5.56.

33

tophan methyl ester,

tryptophan methyl ester³³were synthesized according to the

methods described in the literature, with certain modifications.

The following amino acids were commercially available: ^D-

allylglycine (Sigma), ^D-aminooctanoic acid (Aldrich), D-tert-Leu,

and γMeLeu (Daiichi Pure Chemicals Co., Ltd., Tokyo, J apan).

Other amino acids or amino acid derivatives were obtained

from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, J apan), Kokusan

Chemicals Co., Ltd. (Tokyo, J apan), or Watanabe Chemical

Industries, Ltd. (Hiroshima, J apan). Solvents and other

reagents were reagent grade and used without further puri-

fication unless otherwise noted.

N^r-(ter t-Bu toxyca r bon yl)-2-br om o-D-tr yp top h a n (58).

NaOH (4 N) (2.5 mL, 10 mmol) was added to a solution of 57

(995 mg, 2.0 mmol) in MeOH (10 mL) at 0 °C. After being

stirred at room temperature overnight, the mixture was

concentrated to remove MeOH. The residue was diluted with

water (50 mL) and extracted with ether (50 mL). The aqueous

layer was acidified with 10% citric acid, and the resulting

mixture was extracted with ether (50 mL × 2). The combined

organic extracts were washed with brine (25 mL), dried over

MgSO₄, and evaporated to give crude 58 (806 mg, 105%) as a

colorless amorphous solid: TLC R_f(CHCl₃:MeOH:AcOH ) 30:

1:1) 0.33; FAB-MS m/ e 382, 384 (M⁺). This material was used

without further purification.

N

^r,N ⁱⁿ-Bis(ter t-bu toxyca r bon yl)-N ^r-(tr iflu or oa cetyl)-

2-ch lor o-^D-tr yp top h a n Meth yl Ester (55a ). Boc₂O (0.89 g,

4.1 mmol) and DMAP (20 mg, 0.16 mmol) were added to a

solution of N ^R-(trifluoroacetyl)-2-chloro-D-tryptophan methyl

ester (54a ; 283 mg, 0.81 mmol) in dry acetonitrile (5 mL) at 0

°C. The mixture was allowed to warm to room temperature

and stirred overnight. The mixture was concentrated under

reduced pressure, and the residue was purified by chroma-

tography on silica gel (E. Merck, Lobar, Lichroprep, Si 60)

eluted with EtOAc/hexane (1:5) to afford 55a (265 mg, 59%)

as a colorless oil: TLC R_f(EtOAc:hexane ) 1:3) 0.67; FAB-

2-Br om o-^D-tr yp top h a n Meth yl Ester (59). 57 (1.20 g,

2.41 mmol) was dissolved in formic acid (30 mL), and the

mixture was stirred at room temperature for 8 h. The mixture

was concentrated under reduced pressure. The residue was

taken up with EtOAc, washed with saturated NaHCO₃, dried

over MgSO₄, and evaporated to give crude 59 (762 mg, 106%)

as a pale yellow amorphous solid: TLC R_f(CHCl₃:MeOH )

10:1) 0.16. This material was used without further purifica-

tion.

1

MS m/ e 548, 550 (M⁺); H NMR (300 MHz, CDCl₃) δ 1.26 (9

H, s), 1.67 (9 H, s), 3.54 (1 H, dd, J ) 4.8, 14.5 Hz), 3.64 (1 H,

dd, J ) 10.5, 14.5 Hz), 3.80 (3 H, s), 5.34 (1 H, dd, J ) 4.8,

10.5 Hz), 7.23 (1 H, dt, J ) 1.5, 7.5 Hz), 7.29 (1 H, dt, J ) 1.5,

7.5 Hz), 7.39 (1 H, dd, J ) 1.5, 7.5 Hz), 8.05 (1 H, dd, J ) 1.5,

7.5 Hz).

N

^r,N ⁱⁿ-Bis(t er t -b u t oxyca r b on yl)-2-cya n o-D-t r yp t o-

p h a n Meth yl Ester (60). A mixture of N ^R,N ⁱⁿ-bis(tert-

butoxycarbonyl)-2-bromo-^D-tryptophan methyl ester (57; 500

mg, 1.01 mmol) and CuCN (225 mg, 2.51 mmol) in dry DMF

(1.0 mL) was stirred at 85 °C in an argon atmosphere for 1 h.

After being cooled to room temperature, the reaction mixture

was diluted with EtOAc (80 mL), washed with saturated

NaHCO₃(50 mL) and brine (50 mL), dried over MgSO₄, and

evaporated. The residue was purified by column chromatog-

N

^r,N ⁱⁿ-Bis(ter t-bu toxyca r bon yl)-N ^r-(tr iflu or oa cetyl)-

2-br om o-^D-tr yp top h a n Meth yl Ester (55b). Compound

54b (1.00 g, 2.54 mmol) was treated in the same manner as

described for the corresponding chloro compound, yielding 55b

(1.02 g, 68%) as a colorless oil: TLC R_f(EtOAc:hexane ) 1:5)

0.51; FAB-MS m/ e 592, 594 (M⁺); ¹H NMR (200 MHz, CDCl₃)

δ 1.25 (9 H, s), 1.68 (9 H, s), 3.54 (1 H, dd, J ) 5.1, 14.8 Hz),

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2323

raphy on silica gel eluted with EtOAc to give 60 (442 mg, 99%)

as a pale yellow amorphous solid: TLC R_f(EtOAc:hexane )

H, d, J ) 5.3 Hz), 3.65 (3 H, s), 4.63-4.70 (1 H, m), 5.06 and

5.12 (each 1 H, ABq, J ) 12.3 Hz), 5.29 (1 H, d, J ) 8.4 Hz),

7.03 (1 H, t, J ) 7.3 Hz), 7.10 (1 H, t, J ) 7.3 Hz), 7.24 (1 H,

d, J ) 7.3 Hz), 7.28-7.38 (5 H, m), 7.40 (1 H, d, J ) 7.3 Hz),

7.81 (1 H, s).

1:5) 0.30; [R]²⁰4.26° (c 0.99, MeOH); FAB-HRMS calcd for

D

C₂₃H₂₉N₃O₆(M + H)⁺444.2135, found 444.2136; IR (KBr, cm^-1

)

2224 (CN), 1722 (CdO); ¹H NMR (300 MHz, CDCl₃) δ 1.41 (9

H, s), 1.71 (9 H, s), 3.34 (1 H, dd, J ) 5.9, 14.2 Hz), 3.49 (1 H,

dd, J ) 5.9, 14.2 Hz), 3.79 (3 H, s), 4.67-4.74 (1 H, m), 5.20 (1

H, d, J ) 7.5 Hz, R-NH), 7.34 (1 H, t, J ) 7.8 Hz), 7.50 (1 H,

t, J ) 7.8 Hz), 7.69 (1 H, d, J ) 7.8 Hz), 8.21 (1 H, d, J ) 7.8

Hz).

N^r-(Ben zyloxyca r b on yl)-2-m et h yl-D-t r yp t op h a n (66).

NaOH (1 N) (17.2 mL, 17.2 mmol) was added to a solution of

65 (2.10 g, 5.73 mmol) in 1,4-dioxane (10 mL) at 0 °C. After

being stirred at 0 °C for 30 min, the mixture was allowed to

warm to room temperature and stirred for 4.5 h. The mixture

was concentrated under reduced pressure. The residue was

diluted with water (150 mL) and acidified with 10% citric acid,

and the resulting mixture was extracted with EtOAc (80 mL

× 3). The combined organic extracts were dried over MgSO₄

and evaporated to give 66 (1.92 g, 95%) as an off-white solid:

TLC R_f(CHCl₃:MeOH ) 10:1) 0.50; FAB-MS m/ e 353 (M +

N

^r-(ter t-Bu toxyca r bon yl)-2-cya n o-D-tr yp top h a n (61).

NaOH (4 N) (2.0 mL, 8.0 mmol) was added to a solution of 60

(420 mg, 0.947 mmol) in MeOH (6 mL) at 0 °C. The mixture

was allowed to warm to room temperature and stirred for 32

h. The mixture was concentrated under reduced pressure to

remove MeOH. The residue was acidified with 10% citric acid,

and the mixture was extracted with CHCl₃(30 mL × 3). The

combined organic extracts were washed with brine, dried over

MgSO₄, and evaporated to give 61 (243 mg, 78%) as a pale

yellow amorphous solid: TLC R_f(CHCl₃:MeOH:AcOH ) 10:

1:1) 0.50; FAB-MS m/ e 330 (M + H)⁺. This material was used

without further purification.

1

H)⁺; H NMR (300 MHz, CDCl₃) δ 2.28 (3 H, s), 3.48 (2 H, d,

J ) 7.1 Hz), 4.65-4.71 (1 H, m), 5.06 and 5.12 (each 1 H, ABq,

J ) 12.4 Hz), 5.27 (1 H, d, J ) 8.2 Hz), 7.02 (1 H, t, J ) 7.4

Hz), 7.10 (1 H, t, J ) 7.4 Hz), 7.24 (1 H, d, J ) 7.4 Hz), 7.27-

7.38 (5 H, m), 7.46 (1 H, d, J ) 7.4 Hz), 7.83 (1 H, brs).

2-Meth yl-^D-tr yp top h a n Meth yl Ester (67). A mixture

of 65 (133 mg, 0.363 mmol) and 10% Pd/C (60 mg) in MeOH

(5 mL) was stirred in a hydrogen atmosphere for 8 h. The

catalyst was removed by filtration, and the filtrate was

concentrated under reduced pressure to give 67 (96 mg, 114%)

as a pale yellow amorphous solid: TLC R_f(CHCl₃:MeOH )

10:1) 0.22; FAB-MS m/ e 233 (M + H)⁺. This material was

used without further purification.

N

^r,N ⁱⁿ-Bis(ter t-bu toxyca r bon yl)-2-[2-(tr im eth ylsilyl)-

eth yn yl]-^D-tr yp top h a n Meth yl Ester (62). (Trimethylsilyl)-

acetylene (590 mg, 6.0 mmol) was added to a mixture of

N

^R,N ⁱⁿ-bis(tert-butoxycarbonyl)-2-bromo-D-tryptophan methyl

ester (57; 994 mg, 2.00 mmol), CuI (115 mg, 0.60 mmol), and

(Ph₃P)₄Pd (232 mg, 0.20 mmol) in dry diethylamine (10 mL)

in an argon atmosphere. The mixture was stirred at 40 °C

for 9 h and then cooled. Ethyl ether (60 mL) was added to

the reaction mixture, and the resulting suspension was

filtered. The filtrate was evaporated. The residue was dis-

solved in ethyl ether (60 mL), washed with 10% citric acid (60

mL), saturated NaHCO₃(60 mL), and brine (60 mL), dried

over MgSO₄, and evaporated. The residue was purified by

column chromatography on silica gel eluted with EtOAc/

hexane (1:10) to give 62 (782 mg, 76%) as a pale yellow

cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Leu -OBzl (72a).

Triethylamine (1.53 mL, 11 mmol) was added to a suspension

of H-Leu-OBzl‚TosOH (3.94 g, 10 mmol) and N,N ′-carbonyl-

diimidazole (1.78 g, 11 mmol) in dry THF (20 mL) at 0 °C

under a nitrogen atmosphere over a period of 10 min. After a

30 min stirring at 0 °C, cis-2,6-dimethylpiperidine (1.48 mL,

11 mmol) was added to the mixture. The mixture was allowed

to warm to room temperature and stirred overnight. The

reaction was quenched by the addition of water (100 mL), and

the resulting mixture was extracted with EtOAc (50 mL × 3).

The combined organic extracts were washed with brine (50

mL), dried over MgSO₄, and evaporated. The residue was

purified by column chromatography on silica gel eluted with

EtOAc/hexane (1:2) to give cis-[(2,6-dimethylpiperidino)car-

bonyl]-Leu-OBzl (2.88 g, 80%) as a white solid: TLC R_f(EtOAc:

hexane ) 1:2) 0.49; FAB-MS m/ e 361 (M + H)⁺; ¹H NMR (200

MHz, CDCl₃) δ 0.93 (3 H, d, J ) 6.2 Hz), 0.94 (3 H, d, J ) 6.2

Hz), 1.19 (3 H, d, J ) 7.0 Hz), 1.24 (3 H, d, J ) 7.0 Hz), 1.40-

1.82 (9 H, m), 4.04-4.32 (2 H, m), 4.53-4.69 (1 H, m), 4.78 (1

H, d, J ) 7.9 Hz), 5.10 and 5.20 (each 1 H, ABq, J ) 12.3 Hz),

7.30-7.40 (5 H, m).

amorphous solid: TLC R_f(EtOAc:hexane ) 1:5) 0.49; [R]²⁰

D

22.4° (c 0.85, MeOH); FAB-HRMS calcd for C₂₇H₃₈N₂O₆Si (M⁺)

1

514.2499, found 514.2522; H NMR (300 MHz, CDCl₃) δ 0.32

(9 H, s), 1.38 (9 H, s), 1.70 (9 H, s), 3.12-3.37 (2 H, m), 3.69 (3

H, s), 4.43-4.62 (1 H, m), 5.32 (1 H, d, J ) 8.1 Hz), 7.27 (1 H,

dt, J ) 1.4, 7.8 Hz), 7.37 (1 H, dt, J ) 1.4, 7.8 Hz), 7.55 (1 H,

dd, J ) 1.4, 7.8 Hz), 8.19 (1 H, dd, J ) 1.4, 7.8 Hz).

N^r-(ter t-Bu toxyca r bon yl)-2-eth yn yl-D-tr yp top h a n (63).

NaOH (4 N) (5.0 mL, 20 mmol) was added to a solution of 62

(400 mg, 0.78 mmol) in MeOH (10 mL) at 0 °C under an argon

atmosphere. After 1 h, the mixture was allowed to warm to

room temperature and stirred overnight. The mixture was

concentrated under reduced pressure to remove MeOH. The

residue was acidified with 10% citric acid and extracted with

EtOAc (30 mL × 3). The combined organic extracts were

washed with brine, dried over MgSO₄, and evaporated to give

63 (280 mg, 110%) as a pale yellow oil: TLC R_f(CHCl₃:MeOH:

cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpr g-OBzl (72b).

This compound was prepared from H-Cprg-OBzl‚HCl in 91%

yield according to the procedure for the synthesis of 72a . 72b:

white solid; TLC R_f(EtOAc:hexane ) 1:3) 0.25; FAB-MS m/ e

1

AcOH ) 10:1:1) 0.33; FAB-MS m/ e 329 (M + H)⁺; H NMR

1

345 (M + H)⁺; H NMR (200 MHz, CDCl₃) δ 0.31-0.64 (4 H,

(200 MHz, CDCl₃) δ 1.40 (9 H, s), 3.20-3.55 (2 H, m), 3.46 (1

H, s), 4.50-4.70 (1 H, m), 5.11 (1 H, brs), 7.14 (1 H, dt, J )

2.0, 7.9 Hz), 7.20-7.38 (2 H, m), 7.60 (1 H, dd, J ) 2.0, 7.9

Hz), 8.19 (1 H, s). This material was used without further

purification.

m), 0.99-1.15 (1 H, m), 1.20 (3 H, d, J ) 7.3 Hz), 1.24 (3 H, d,

J ) 7.3 Hz), 1.40-1.89 (6 H, m), 4.02-4.31 (3 H, m), 4.96 (1

H, d, J ) 7.2 Hz), 5.12 and 5.28 (each 1 H, ABq, J ) 12.4 Hz),

7.29-7.43 (5 H, m).

N^r-(Ben zyloxyca r bon yl)-2-m eth yl-D-tr yp top h a n Meth -

yl Ester (65). A mixture of methyl (2R)-1-(benzyloxycarbo-

nyl)-2-aziridinecarboxylate (64; 540 mg, 2.30 mmol), 2-meth-

ylindole (544 mg, 4.15 mmol), and zinc triflate (1.67 g, 4.60

mmol) in dry CHCl₃was placed in a pressure bottle and stirred

at 78 °C for 19 h. After being cooled to room temperature,

the mixture was partitioned between CHCl₃(20 mL) and water

(50 mL). The aqueous layer was extracted twice with CHCl₃

(20 mL). The combined organic layers were dried over MgSO₄

and concentrated under reduced pressure. The residue was

purified by chromatography on silica gel (E. Merck, Lobar,

Lichroprep, Si 60) eluted with CHCl₃/MeOH (50:1) to afford

65 (571 mg, 68%) as off-white crystals: mp 140-141 °C; TLC

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Nle-OBzl (72c).

This compound was prepared from H-Nle-OBzl‚HCl in 98%

yield according to the procedure for the synthesis of 72a . 72c:

white solid; TLC R_f(EtOAc:hexane ) 1:2) 0.32; FAB-MS m/ e

1

361 (M + H)⁺; H NMR (200 MHz, CDCl₃) δ 0.85 (3 H, t, J )

6.7 Hz), 1.20 (3 H, d, J ) 7.0 Hz), 1.24 (3 H, d, J ) 7.0 Hz),

1.20-1.95 (12 H, m), 4.06-4.32 (2 H, m), 4.53-4.66 (1 H, m),

4.90 (1 H, d, J ) 7.5 Hz), 5.10 and 5.24 (each 1 H, ABq, J )

12.3 Hz), 7.30-7.40 (5 H, m).

cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpr a-OBzl (72d).

This compound was prepared from H-Cpra-OBzl‚HCl in 96%

yield according to the procedure for the synthesis of 72a . 72d :

colorless oil; TLC R_f(EtOAc:hexane ) 1:3) 0.29; FAB-MS m/ e

359 (M + H)⁺; ¹H NMR (200 MHz, CDCl₃) δ -0.96-0.16 (2 H,

m), 0.34-0.54 (2 H, m), 0.57-0.78 (1 H, m), 1.23 (3 H, d, J )

6.9 Hz), 1.26 (3 H, d, J ) 6.9 Hz), 1.42-1.90 (9 H, m), 4.10-

R_f(EtOAc:hexane ) 1:3) 0.15; [R]²⁰-59.2° (c 1.0, CHCl₃);

D

FAB-HRMS calcd for C₂₁H₂₂N₂O₄(M⁺) 366.1580, found

1

366.1585; H NMR (300 MHz, CDCl₃) δ 2.31 (3 H, s), 3.25 (2

2324 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Fukami et al.

4.34 (2 H, m), 4.61-4.75 (1 H, m), 5.15 and 5.23 (each 1 H,

ABq, J ) 12.4 Hz), 7.30-7.41 (5 H, m).

7.1 Hz), 1.27-1.98 (12 H, m), 4.11-4.33 (3 H, m), 5.22 (1 H,

d, J ) 6.6 Hz).

cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpr a-OH (73d).

This compound was prepared from 72d in 91% yield according

to the procedure for the synthesis of 73a . 73d : white

amorphous solid; TLC R_f(CHCl₃:MeOH:AcOH ) 50:1:1) 0.31;

FAB-MS m/ e 269 (M + H)⁺; ¹H NMR (300 MHz, CDCl₃) δ

0.07-0.16 (2 H, m), 0.41-0.51 (2 H, m), 0.71-0.84 (1 H, m),

1.22 (3 H, d, J ) 7.2 Hz), 1.25 (3 H, d, J ) 7.2 Hz), 1.30-1.90

(8 H, m), 4.15-4.27 (2 H, m), 4.31-4.40 (1 H, m), 5.42 (1 H, d,

J ) 6.6 Hz).

cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Nva-OBzl (72e).

This compound was prepared from H-Nva-OBzl‚TosOH in 98%

yield according to the procedure for the synthesis of 72a . 72e:

colorless oil; TLC R_f(EtOAc:hexane ) 1:3) 0.37; FAB-MS m/ e

1

347 (M + H)⁺; H NMR (200 MHz, CDCl₃) δ 0.91 (3 H, t, J )

7.2 Hz), 1.20 (3 H, d, J ) 7.7 Hz), 1.23 (3 H, d, J ) 7.7 Hz),

1.00-1.93 (10 H, m), 4.04-4.33 (2 H, m), 4.52-4.68 (1 H, m),

4.90 (1 H, d, J ) 7.5 Hz), 5.11 and 5.22 (each 1 H, ABq, J )

12.3 Hz), 7.20-7.44 (5 H, m).

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Nva -OH (73e).

This compound was prepared from 72e in 94% yield according

to the procedure for the synthesis of 73a . 73e: white

amorphous solid; TLC R_f(CHCl₃:MeOH ) 10:1) 0.44; FAB-

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Va l-OBzl (72f).

This compound was prepared from H-Val-OBzl‚TosOH in 89%

yield according to the procedure for the synthesis of 72a . 72f:

colorless oil; TLC R_f(EtOAc:hexane ) 1:2) 0.48; FAB-MS m/ e

347 (M + H)⁺; ¹H NMR (200 MHz, CDCl₃) δ 0.87 (3 H, d, J )

6.9 Hz), 0.94 (3 H, d, J ) 6.9 Hz), 1.21 (3 H, d, J ) 7.0 Hz),

1.24 (3 H, d, J ) 7.0 Hz), 1.43-1.70 (6 H, m), 2.10-2.28 (1 H,

m), 4.10-4.33 (2 H, m), 4.58 (1 H, dd, J ) 4.6, 8.3 Hz), 4.94 (1

H, d, J ) 8.2 Hz), 5.11 and 5.23 (each 1 H, ABq, J ) 12.3 Hz),

7.30-7.44 (5 H, m).

1

MS m/ e 257 (M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.93 (3

H, t, J ) 7.0 Hz), 1.21 (3 H, d, J ) 7.0 Hz), 1.24 (3 H, d, J )

7.0 Hz), 1.29-1.96 (10 H, m), 4.11-4.35 (3 H, m), 5.16 (1 H,

d, J ) 6.6 Hz).

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Va l-OH (73f).

This compound was prepared from 72f in 97% yield according

to the procedure for the synthesis of 73a . 73f: white amor-

phous solid; TLC R_f(CHCl₃:MeOH:AcOH ) 30:1:1) 0.38; FAB-

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Ile-OBzl (72g).

This compound was prepared from H-Ile-OBzl‚TosOH in 98%

yield according to the procedure for the synthesis of 72a . 72g:

colorless oil; TLC R_f(EtOAc:hexane ) 1:2) 0.48; FAB-MS m/ e

1

MS m/ e 257 (M + H)⁺; H NMR (200 MHz, CDCl₃) δ 0.97 (3

H, d, J ) 6.8 Hz), 1.02 (3 H, d, J ) 6.8 Hz), 1.23 (3 H, d, J )

7.0 Hz), 1.26 (3 H, d, J ) 7.0 Hz), 1.43-1.87 (6 H, m), 2.17-

2.39 (1 H, m), 4.09-4.29 (2 H, m), 4.32 (1 H, dd, J ) 5.7, 7.8

Hz), 5.03 (1 H, d, J ) 7.8 Hz).

1

361 (M + H)⁺; H NMR (200 MHz, CDCl₃) δ 0.88 (3 H, t, J )

7.3 Hz), 0.90 (3 H, d, J ) 7.6 Hz), 1.20 (3 H, d, J ) 7.1 Hz),

1.24 (3 H, d, J ) 7.1 Hz), 1.30-2.00 (9 H, m), 4.07-4.31 (2 H,

m), 4.61 (1 H, dd, J ) 4.6, 8.2 Hz), 4.95 (1 H, d, J ) 8.0 Hz),

5.09 and 5.23 (each 1 H, ABq, J ) 12.2 Hz), 7.30-7.40 (5 H,

m).

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Ile-OH (73g).

This compound was prepared from 72g in 94% yield according

to the procedure for the synthesis of 73a . 73g: white

amorphous solid; TLC R_f(CHCl₃:MeOH ) 10:1) 0.42; FAB-

cis-[(2,6-Dim e t h ylp ip e r id in o)ca r b on yl]-Cp e g-OBzl

(72h ). This compound was prepared from H-Cpeg-OBzl‚HCl

in 94% yield according to the procedure for the synthesis of

72a . 72h : colorless oil; TLC R_f(EtOAc:hexane ) 1:3) 0.42;

FAB-MS m/ e 347 (M + H)⁺; ¹H NMR (200 MHz, CDCl₃) δ 1.20

(3 H, d, J ) 7.1 Hz), 1.23 (3 H, d, J ) 7.1 Hz), 1.40-2.00 (14

H, m), 2.15-2.31 (1 H, m), 4.10-4.30 (2 H, m), 4.54 (1 H, dd,

J ) 7.1, 8.1 Hz), 4.90 (1 H, d, J ) 8.1 Hz), 5.10 and 5.22 (each

1 H, ABq, J ) 12.2 Hz), 7.29-7.50 (5 H, m).

cis-[(2,6-Dim et h ylp ip er id in o)ca r b on yl]-γMeLeu-OBzl

(72i). This compound was prepared from H-γMeLeu-OBzl‚-

TosOH in 91% yield according to the procedure for the

synthesis of 72a . 72i: colorless crystals; TLC R_f(EtOAc:

hexane ) 1:3) 0.33; FAB-MS m/ e 375 (M + H)⁺; ¹H NMR (300

MHz, CDCl₃) δ 0.97 (9 H, s), 1.19 (3 H, d, J ) 7.1 Hz), 1.23 (3

H, d, J ) 7.1 Hz), 1.40-1.80 (8 H, m), 4.10-4.23 (2 H, m),

4.58-4.70 (2 H, m), 5.10 and 5.19 (each 1 H, ABq, J ) 12.5

Hz), 7.30-7.36 (5 H, m).

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Leu -OH (73a ).

A mixture of cis-[(2,6-dimethylpiperidino)carbonyl]-Leu-OBzl

(2.86 g, 7.93 mmol) and 10% Pd/C (0.15 g) in MeOH (30 mL)

was stirred under an atmospheric pressure of hydrogen for 1

h. The catalyst was removed by filtration, and the filtrate was

concentrated under reduced pressure to give 73a (2.12 g, 99%)

as a white amorphous solid: TLC R_f(CHCl₃:MeOH:AcOH )

30:1:1) 0.51; FAB-MS m/ e 271 (M + H)⁺; ¹H NMR (200 MHz,

CDCl₃) δ 0.94 (3 H, d, J ) 6.4 Hz), 0.98 (3 H, d, J ) 6.4 Hz),

1.22 (3 H, d, J ) 6.8 Hz), 1.26 (3 H, d, J ) 6.8 Hz), 1.43-1.92

(9 H, m), 4.04-4.39 (3 H, m), 4.64-4.79 (1 H, m).

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Cpr g-OH (73b).

This compound was prepared from 72b in 94% yield according

to the procedure for the synthesis of 73a . 73b: white

amorphous solid; TLC R_f(CHCl₃:MeOH:AcOH ) 50:1:1) 0.19;

FAB-MS m/ e 255 (M + H)⁺; ¹H NMR (300 MHz, DMSO-d₆) δ

0.19-0.54 (4 H, m), 1.00-1.18 (1 H, m), 1.07 (3 H, d, J ) 7.0

Hz), 1.11 (3 H, d, J ) 7.0 Hz), 1.33-1.80 (6 H, m), 3.44 (1 H,

dd, J ) 7.3, 8.8 Hz), 4.06-4.25 (2 H, m), 6.30 (1 H, d, J ) 7.3

Hz).

1

MS m/ e 271 (M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.92 (3

H, t, J ) 7.3 Hz), 0.96 (3 H, d, J ) 6.9 Hz), 1.21 (3 H, d, J )

7.2 Hz), 1.24 (3 H, d, J ) 7.2 Hz), 1.29-2.02 (9 H, m), 4.12-

4.30 (2 H, m), 4.27 (1 H, dd, J ) 5.3, 7.4 Hz), 5.24 (1 H, d, J

) 7.4 Hz).

cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpeg-OH (73h ).

This compound was prepared from 72h in 97% yield according

to the procedure for the synthesis of 73a . 73h : white

amorphous solid; TLC R_f(CHCl₃:MeOH:AcOH ) 30:1:1) 0.48;

FAB-MS m/ e 283 (M + H)⁺; ¹H NMR (300 MHz, CDCl₃) δ 1.21

(3 H, d, J ) 7.4 Hz), 1.23 (3 H, d, J ) 7.4 Hz), 1.27-1.89 (14

H, m), 2.23-2.41 (1 H, m), 4.12-4.28 (2 H, m), 4.24 (1 H, t, J

) 7.3 Hz), 5.23 (1 H, d, J ) 7.3 Hz).

cis-[(2,6-Dim et h ylp ip er id in o)ca r b on yl]-γMeLeu -OH

(73i). This compound was prepared from 72i in 99% yield

according to the procedure for the synthesis of 73a . 73i: white

amorphous solid; TLC R_f(CHCl₃:MeOH:AcOH ) 30:1:1) 0.33;

FAB-MS m/ e 285 (M + H)⁺; ¹H NMR (300 MHz, CDCl₃) δ 0.98

(9 H, s), 1.23 (3 H, d, J ) 6.9 Hz), 1.25 (3 H, d, J ) 6.9 Hz),

1.42-1.88 (6 H, m), 1.47 (1 H, dd, J ) 8.7, 14.6 Hz), 2.14 (1 H,

dd, J ) 3.4, 14.6 Hz), 4.07-4.25 (2 H, m), 4.28-4.35 (1 H, m),

4.62 (1 H, d, J ) 7.6 Hz).

Met h od A. cis-[(2,6-Dim et h ylp ip er id in o)ca r b on yl]-

Leu -^D-Tr p (2-Cl)-D-Nle-OH (6). (a ) Boc-D-Tr p (1-Boc,2-Cl)-

D-Nle-O^tBu (68a ). EDCI (107 mg, 0.56 mmol) was added to

a mixture of 56a (206 mg, 0.465 mmol), ^D-Nle-O^tBu‚HCl (125

mg, 0.56 mmol), HOBT (86 mg, 0.56 mmol), and NMM (62 µL,

0.56 mmol) in CH₂Cl₂(10 mL) at 0 °C. After being stirred at

0 °C for 1 h, the mixture was allowed to warm to room

temperature and stirred for 3 h. The mixture was diluted with

CH₂Cl₂(20 mL), washed with 10 mL each of saturated

NaHCO₃, 10% citric acid, and brine, dried over MgSO₄, and

evaporated. The residue was purified by column chromatog-

raphy on silica gel eluted with EtOAc/hexane (1:5) to give 68a

(235 mg, 83%) as a white amorphous solid: TLC R_f(EtOAc:

hexane ) 1:3) 0.53; FAB-MS m/ e 608, 610 (M + H)⁺; ¹H NMR

(300 MHz, CDCl₃) δ 0.83 (3 H, t, J ) 7.1 Hz), 1.41 (9 H, s),

1.55 (9 H, s), 1.69 (9 H, s), 1.01-1.83 (6 H, m), 3.21 (2 H, d, J

) 6.3 Hz), 4.28-4.50 (2 H, m), 5.20 (1 H, brs), 6.34 (1 H, d, J

) 7.3 Hz), 7.20-7.35 (2 H, m), 7.57 (1 H, d, J ) 7.6 Hz), 8.04

(1 H, d, J ) 7.6 Hz).

cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Nle-OH (73c).

This compound was prepared from 72c in 94% yield according

to the procedure for the synthesis of 73a . 73c: white

amorphous solid; TLC R_f(CHCl₃:MeOH ) 10:1) 0.36; FAB-

(b) H-^D-Tr p (1-Boc,2-Cl)-D-Nle-O^tBu (69a ) a n d H-D-Tr p -

(2-Cl)-^D-Nle-O^tBu (70a ). 68a (252 mg, 0.415 mmol) was

dissolved in formic acid (10 mL). After being stirred at room

1

MS m/ e 271 (M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.89 (3

H, t, J ) 7.1 Hz), 1.21 (3 H, d, J ) 7.1 Hz), 1.24 (3 H, d, J )

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2325

temperature for 1.5 h, the mixture was concentrated under

reduced pressure. The residue was taken up with EtOAc (50

mL), which was washed with saturated NaHCO₃(50 mL × 2)

and brine (50 mL), dried over MgSO₄, and evaporated. The

residue was purified by preparative TLC (CHCl₃:MeOH ) 30:

1) to give 69a (116 mg, 55%) and 70a (35 mg, 21%). 69a : TLC

R_f(CHCl₃:MeOH ) 30:1) 0.53; FAB-MS m/ e 508, 510 (M +

(c) cis-[(2,6-Dim eth ylp iper idin o)ca r bon yl]-Cp r g-^D-Tr p -

(2-Br )-^D-Nle-OMe (76a ). EDCI (490 mg, 2.55 mmol) was

added to a mixture of 75a (960 mg, 2.34 mmol), 73b (540 mg,

2.12 mmol), and HOBT (390 mg, 2.55 mmol) in CH₂Cl₂(10

mL) at 0 °C. After being stirred at 0 °C for 0.5 h and at room

temperature for 1 h, the mixture was diluted with CH₂Cl₂,

washed with saturated NaHCO₃, 10% citric acid, and brine,

dried over MgSO₄, and concentrated. The residue was purified

by column chromatography on silica gel eluted with EtOAc/

hexane (1:2) to give 76a (1.25 g, 91%) as a white amorphous

solid: TLC R_f(EtOAc:hexane ) 1:2) 0.34; FAB-MS m/ e 646,

1

H)⁺; H NMR (300 MHz, CDCl₃) δ 0.87 (3 H, t, J ) 6.9 Hz),

1.46 (9 H, s), 1.55 (9 H, s), 1.12-1.82 (6 H, m), 2.95 (1 H, dd,

J ) 9.6, 14.5 Hz), 3.38 (1 H, dd, J ) 3.9, 14.5 Hz), 3.75 (1 H,

dd, J ) 3.9, 9.6 Hz), 4.38-4.49 (1 H, m), 7.20-7.34 (2 H, m),

7.59 (1 H, dd, J ) 1.5, 7.2 Hz), 8.06 (1 H, dd, J ) 1.5, 7.2 Hz),

7.80 (1 H, d, J ) 8.4 Hz).

1

648 (M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.21-0.35 (2 H,

m), 0.40-0.52 (2 H, m), 0.79 (3 H, t, J ) 7.0 Hz), 1.00-1.80

(13 H, m), 1.18 (3 H, d, J ) 6.9 Hz), 1.18 (3 H, d, J ) 6.9 Hz),

3.24 (1 H, dd, J ) 6.7, 14.6 Hz), 3.36 (1 H, dd, J ) 10.0, 14.6

Hz), 3.30-3.44 (1 H, m), 3.63 (3 H, s), 4.00-4.22 (2 H, m),

4.35-4.46 (1 H, m), 4.73-4.84 (1 H, m), 5.04 (1 H, d, J ) 5.6

Hz), 6.37 (1 H, d, J ) 8.6 Hz), 7.05 (1 H, d, J ) 9.6 Hz), 7.09

(1 H, dt, J ) 1.3, 7.1 Hz), 7.16 (1 H, dt, J ) 1.3, 7.1 Hz), 7.27

(1 H, dd, J ) 1.3, 7.1 Hz), 7.64 (1 H, dd, J ) 1.3, 7.1 Hz), 8.35

(1 H, s).

70a : TLC R_f(CHCl₃:MeOH ) 30:1) 0.22; FAB-MS m/ e 408,

1

410 (M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.87 (3 H, t, J )

7.1 Hz), 1.46 (9 H, s), 1.14-1.83 (6 H, m), 2.93 (1 H, dd, J )

9.3, 14.7 Hz), 3.36 (1 H, dd, J ) 4.0, 14.7 Hz), 3.75 (1 H, dd, J

) 4.0, 9.3 Hz), 4.41-4.51 (1 H, m), 7.13 (1 H, dt, J ) 1.6, 7.5

Hz), 7.19 (1 H, dt, J ) 1.6, 7.5 Hz), 7.28 (1 H, dd, J ) 1.6, 7.5

Hz), 7.61 (1 H, dd, J ) 1.6, 7.5 Hz), 7.77 (1 H, d, J ) 9.5 Hz),

8.11 (1 H, s).

(d) cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpr g-^D-Tr p-

(2-Br )-^D-Nle-OH (4). NaOH (1 N) (2.93 mL, 2.93 mmol) was

added to a solution of 76a (1.13 g, 1.74 mmol) in MeOH (6

mL) at 0 °C. After 10 min, the mixture was allowed to warm

to room temperature and stirred for 3.5 h. The mixture was

diluted with water (15 mL) and concentrated to remove MeOH.

The resulting aqueous solution was acidified with 1 N HCl at

0 °C to form a precipitate. The precipitate was collected by

filtration, washed with a small amount of water, and dried to

give 4 (1.02 g, 92%) as a white crystalline powder: TLC R_f

(CHCl₃:MeOH:AcOH ) 30:1:1) 0.27; ¹H NMR (400 MHz,

CDCl₃) δ -0.13-0.23 (4 H, m), 0.84 (3 H, t, J ) 7.1 Hz), 0.75-

0.92 (1 H, m), 1.03 (3 H, d, J ) 7.1 Hz), 1.05 (3 H, d, J ) 7.1

Hz), 1.18-1.78 (12 H, m), 2.87 (1 H, dd, J ) 10.3, 14.7 Hz),

3.12-3.55 (2 H, m), 4.04-4.20 (3 H, m), 4.55-4.65 (1 H, m),

6.21 (1 H, d, J ) 6.4 Hz), 6.96 (1 H, t, J ) 7.7 Hz), 7.04 (1 H,

t, J ) 7.7 Hz), 7.22 (1 H, d, J ) 7.7 Hz), 7.64 (1 H, d, J ) 7.7

Hz), 7.99 (1 H, d, J ) 9.3 Hz), 8.09 (1 H, d, J ) 7.3 Hz), 11.56

(1 H, s).

Met h od C. cis-[(2,6-Dim et h ylp ip er id in o)ca r b on yl]-

Cp r g-^D-Tr p (2-Me)-D-His-OH (36). (a ) cis-[(2,6-Dim eth -

ylpiper idin o)car bon yl]-Cpr g-^D-Tr p(2-Me)-OMe (77a). EDCI

(92 mg, 0.48 mmol) was added to a mixture of 67 (93 mg, 0.40

mmol), 73b (101 mg, 0.40 mmol), and HOBT (73 mg, 0.48

mmol) in DMF (5 mL) at 0 °C. The mixture was allowed to

warm to room temperature and stirred overnight. The mixture

was partitioned between EtOAc (50 mL) and water (50 mL).

The organic layer was washed with 50 mL each of 10% citric

acid, saturated NaHCO₃, and brine, dried over MgSO₄, and

concentrated under reduced pressure. The residue was puri-

fied by chromatography on silica gel (E. Merck, Lobar, Lichro-

prep, Si 60) eluted with EtOAc/hexane (1:1) to afford 77a (112

mg, 60%): TLC R_f(EtOAc:hexane ) 1:2) 0.30; FAB-MS m/ e

469 (M + H)⁺.

(b) cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpr g-^D-Tr p-

(2-Me)-OH (78a ). NaOH (1 N) (0.5 mL, 0.5 mmol) was added

to a solution of 77a (112 mg, 0.24 mmol) in MeOH (2 mL) at

0 °C. The mixture was stirred at 0 °C for 2.5 h and at room

temperature for 2 h; 10% citric acid (30 mL) was added to the

mixture, which was extracted with EtOAc (15 mL × 3). The

combined organic extracts were washed with brine (15 mL)

and dried over MgSO₄. The solvent was evaporated under

reduced pressure to give 78a (76 mg, 70%). This material

showed a single spot on TLC and was used in the next reaction

without further purification: TLC R_f(CHCl₃:MeOH:AcOH )

10:1:1) 0.60; FAB-MS m/ e 455 (M + H)⁺.

(c) cis-[(2,6-Dim eth ylp ip er idin o)ca r bon yl]-Cpr g-^D-Tr p -

(2-Me)-^D-His-OMe (79a ). EDCI (15.8 mg, 0.082 mmol) was

added to a mixture of 78a (24.9 mg, 0.055 mmol), ^D-His-OMe‚-

2HCl (19.9 mg, 0.082 mmol), HOBT (12.6 mg, 0.082 mmol),

and NMM (23 µL, 0.16 mmol) in DMF (1.0 mL) at 0 °C. After

being stirred at 0 °C for 3 h, the mixture was allowed to warm

to room temperature and stirred for 6 h. The mixture was

partitioned between EtOAc (50 mL) and saturated NaHCO₃

(50 mL). The organic layer was dried over MgSO₄and

(c) cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Leu -^D-Tr p -

(1-Boc,2-Cl)-^D-Nle-O^tBu (71a ). EDCI (17 mg, 0.091 mmol)

was added to a mixture of 73a (25 mg, 0.091 mmol), 69a (31

mg, 0.061 mmol), and HOBT (14 mg, 0.091 mmol) in CH₂Cl₂

(3 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and

then at room temperature for 4 h. The mixture was diluted

with CH₂Cl₂(30 mL), washed with 20 mL each of saturated

NaHCO₃, 10% citric acid, and brine, dried over MgSO₄, and

evaporated to give a crude product of 71a (51.3 mg, 111%).

This material showed a single spot on TLC and was used in

the next reaction without further purification: TLC R_f(EtOAc:

hexane ) 1:2) 0.30; FAB-MS m/ e 760, 762 (M + H)⁺.

(d ) cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Leu -^D-Tr p -

(2-Cl)-^D-Nle-OH (6). 71a (49.8 mg, 0.059 mmol) was dissolved

in TFA (1 mL), and the mixture was stirred at room temper-

ature for 1 h. The mixture was concentrated under reduced

pressure. The residue was purified by preparative TLC

(CHCl₃:MeOH:AcOH ) 30:1:1) followed by trituration with

water (10 mL) to give 6 (16.7 mg, 47%) as a pale yellow

powder: TLC R_f(CHCl₃:MeOH:AcOH ) 30:1:1) 0.29; ¹H NMR

(300 MHz, CDCl₃) δ 0.79 (3 H, t, J ) 7.2 Hz), 0.86 (3 H, d, J

) 4.9 Hz), 0.88 (3 H, d, J ) 4.9 Hz), 1.15 (3 H, d, J ) 6.9 Hz),

1.16 (3 H, d, J ) 6.9 Hz), 0.93-1.87 (15 H, m), 3.21 (1 H, dd,

J ) 6.3, 14.4 Hz), 3.38 (1 H, dd, J ) 6.3, 14.4 Hz), 3.91-4.07

(2 H, m), 4.07-4.20 (1 H, m), 4.20-4.34 (1 H, m), 4.52-4.73

(1 H, m), 4.82-5.02 (1 H, m), 6.52-6.70 (1 H, m), 7.11 (1 H, t,

J ) 7.1 Hz), 7.17 (1 H, t, J ) 7.1 Hz), 7.26 (1 H, d, J ) 7.1

Hz), 7.59 (1 H, d, J ) 7.1 Hz), 7.31-7.49 (1 H, m), 8.63 (1 H,

brs).

Met h od B. cis-[(2,6-Dim et h ylp ip er id in o)ca r b on yl]-

Cp r g-^D-Tr p (2-Br )-D-Nle-OH (4). (a ) Boc-D-Tr p (2-Br )-D-

Nle-OMe (74a ). EDCI (1.39 g, 7.24 mmol) was added to a

mixture of 58 (2.31 g, 6.03 mmol), ^D-Nle-OMe‚HCl (1.20 g, 6.63

mmol), HOBT (1.11 g, 7.24 mmol), and NMM (0.73 mL, 6.63

mmol) in CH₂Cl₂(20 mL) at 0 °C. After being stirred at 0 °C

for 0.5 h, the mixture was allowed to warm to room temper-

ature and stirred for 1.5 h. The mixture was diluted with CH₂-

Cl₂, washed with saturated NaHCO₃, 10% citric acid, and

brine, dried over MgSO₄, and evaporated. The residue was

purified by column chromatography on silica gel eluted with

EtOAc/hexane (1:3) to give 74a (2.62 g, 85%) as a white solid:

TLC R_f(EtOAc:hexane ) 1:3) 0.18; FAB-MS m/ e 510, 512 (M

+ H)⁺.

(b) H-^D-Tr p (2-Br )-D-Nle-OMe (75a ). 74a (2.08 g, 4.08

mmol) was dissolved in formic acid (30 mL), and the mixture

was stirred at room temperature for 2 h. The mixture was

concentrated under reduced pressure, and the residue was

taken up with EtOAc (60 mL), which was washed with

saturated NaHCO₃(40 mL × 2) and dried over MgSO₄. The

solvent was evaporated under reduced pressure to give 75a

(1.60 g, 97%) as a white amorphous solid. This material

showed a single spot on TLC and was used in the next reaction

without further purification: TLC R_f(CHCl₃:MeOH ) 30:1)

0.31; FAB-MS m/ e 410, 412 (M + H)⁺.

2326 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Fukami et al.

concentrated. The residue was purified by preparative TLC

(CHCl₃:MeOH ) 10:1) to give 79a (9.9 mg, 30%) as a white

amorphous solid: TLC R_f(CHCl₃:MeOH ) 10:1) 0.38; FAB-

(1 H, m), 4.69-4.77 (1 H, m), 5.47 (1 H, d, J ) 8.1 Hz), 6.32 (1

H, d, J ) 7.6 Hz), 6.58 (1 H, d, J ) 7.9 Hz), 7.07-7.22 (5 H,

m), 7.28 (1 H, d, J ) 7.5 Hz), 7.32 (2 H, t, J ) 7.5 Hz), 7.65 (1

H, d, J ) 7.6 Hz), 8.15 (1 H, s).

1

MS m/ e 606 (M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.23-

0.33 (1 H, m), 0.44-0.70 (3 H, m), 1.13 (3 H, d, J ) 7.1 Hz),

1.18 (3 H, d, J ) 7.1 Hz), 1.07-1.28 (1 H, m), 1.38-1.82 (6 H,

m), 2.11 (3 H, s), 2.89 (1 H, dd, J ) 10.6, 14.7 Hz), 2.85-2.98

(1 H, m), 3.14 (1 H, dd, J ) 6.6, 14.7 Hz), 3.23 (1 H, dd, J )

4.0, 14.7 Hz), 3.45 (1 H, dd, J ) 3.9, 14.7 Hz), 3.69 (3 H, s),

3.93-4.12 (2 H, m), 4.38-4.50 (1 H, m), 4.62-4.73 (1 H, m),

5.03 (1 H, d, J ) 5.2 Hz), 6.23 (1 H, d, J ) 7.5 Hz), 6.79 (1 H,

s), 7.03-7.19 (3 H, m), 7.23-7.40 (1 H, m), 7.33 (1 H, d, J )

7.4 Hz), 7.49 (1 H, d, J ) 7.4 Hz), 8.24 (1 H, s).

(c) [(Oct a h yd r o-1H -a zocin yl)ca r b on yl]-Leu -^D-Tr p (2-

Br )-^D-Nle-OH (14). A mixture of 81 (38 mg, 0.059 mmol),

heptamethyleneimine (75 µL, 0.59 mmol), and TEA (118 µL,

0.85 mmol) in CHCl₃(1.6 mL) and THF (1.0 mL) was stirred

at 50 °C for 4.5 h. The mixture was diluted with EtOAc (30

mL), washed with 20 mL each of 1 N HCl, saturated NaHCO₃,

and brine, and dried over MgSO₄. The solvent was evaporated,

and the residue was purified by preparative TLC (EtOAc:

hexane ) 3:2) to give the methyl ester 82a (33 mg, 85%) as a

colorless oil: TLC R_f(EtOAc:hexane ) 3:2) 0.40; FAB-MS m/ e

662, 664 (M + H)⁺.

(d) cis-[(2,6-Dim eth ylpiper idin o)car bon yl]-Cpr g-^D-Tr p-

(2-Me)-^D-His-OH (36). NaOH (1 N) (80 µL, 0.080 mmol) was

added to a solution of 79a (10.0 mg, 0.016 mmol) in MeOH

(0.30 mL) at 0 °C. After being stirred at 0 °C for 30 min, the

mixture was allowed to warm to room temperature and stirred

for 3.5 h. The mixture was cooled to 0 °C, and 1 N HCl (80

µL, 0.080 mmol) was added. The resulting mixture was

concentrated under reduced pressure. The residue was dis-

solved in water (3 mL), and the solution was passed through

a Sep-Pak C18 cartridge (Waters Chromatography Division,

Millipore Corp., MA). The cartridge was washed with water

(5 mL) and then eluted with MeOH (5 mL). The eluate was

concentrated under reduced pressure to give 36 (9.2 mg, 94%)

as a pale yellow powder: TLC R_f(CHCl₃:MeOH:AcOH ) 10:

A mixture of the methyl ester 82a (28 mg, 0.042 mmol) and

2 N NaOH (0.20 mL, 0.40 mmol) in MeOH (0.8 mL) was stirred

at 0 °C for 1 h and at room temperature for 3 h. The mixture

was concentrated under reduced pressure; 1 N HCl (0.8 mL)

was added to the residue at 0 °C to form a precipitate. The

precipitate was collected by filtration, washed with a small

amount of water, and dried to give 14 (13.1 mg, 49%) as a

1

white powder: TLC R_f(CHCl₃:MeOH ) 10:1) 0.33; H NMR

(300 MHz, CDCl₃) δ 0.68 (3 H, d, J ) 7.3 Hz), 0.71 (3 H, d, J

) 7.3 Hz), 0.82 (3 H, t, J ) 6.7 Hz), 1.01-1.68 (19 H, m), 2.87

(1 H, dd, J ) 9.4, 14.5 Hz), 3.00 (1 H, dd, J ) 4.4, 14.5 Hz),

3.14-3.47 (4 H, m), 3.80-3.86 (1 H, m), 4.03-4.09 (1 H, m),

4.43-4.52 (1 H, m), 5.88 (1 H, d, J ) 7.8 Hz), 6.94 (1 H, t, J )

7.4 Hz), 7.03 (1 H, t, J ) 7.4 Hz), 7.21 (1 H, d, J ) 7.4 Hz),

7.59 (1 H, d, J ) 7.1 Hz), 7.63 (1 H, d, J ) 7.4 Hz), 7.97 (1 H,

d, J ) 8.5 Hz), 11.58 (1 H, s).

1

1:1) 0.17; H NMR (300 MHz, CDCl₃) δ 0.00-0.28 (4 H, m),

0.80-0.95 (1 H, m), 1.06 (3 H, d, J ) 6.6 Hz), 1.07 (3 H, d, J

) 6.6 Hz), 1.31-1.77 (6 H, m), 2.30 (3 H, s), 2.70-3.60 (5 H,

m), 4.06-4.21 (2 H, m), 4.30-4.53 (2 H, m), 6.18 (1 H, d, J )

6.3 Hz), 6.80 (1 H, s), 6.86 (1 H, t, J ) 7.5 Hz), 6.94 (1 H, t, J

) 7.5 Hz), 7.17 (1 H, d, J ) 7.5 Hz), 7.51 (1 H, d, J ) 7.5 Hz),

7.53 (1 H, s), 8.02 (1 H, d, J ) 8.6 Hz), 8.27 (1 H, d, J ) 7.0

Hz), 10.62 (1 H, s).

Met h od E . cis-[(2,6-Dim et h ylp ip er id in o)ca r b on yl]-

Leu -^D-Tr p (2-Et)-D-Nle-OH (10). (a ) Boc-D-Tr p (2-eth yn yl)-

D-Nle-OMe (83). EDCI (224 mg, 1.17 mmol) was added to a

mixture of 63 (280 mg, 0.78 mmol), H-^D-Nle-OMe‚HCl (156

mg, 0.86 mmol), HOBT (179 mg, 0.17 mmol), and NMM (94

µL, 0.86 mmol) in CH₂Cl₂(10 mL) at 0 °C. After being stirred

at 0 °C for 1 h and at room temperature for 30 min, the mixture

was diluted with EtOAc (60 mL), washed with 60 mL each of

saturated NaHCO₃, 10% citric acid, and brine, and dried over

MgSO₄. The solvent was evaporated, and the residue was

purified by chromatography on silica gel (E. Merck, Lobar,

Lichroprep, Si 60) eluted with EtOAc/hexane (1:2) to give 83

(303 mg, 86%) as a pale yellow oil: TLC R_f(EtOAc:hexane )

1:2) 0.34; FAB-MS m/ e 456 (M + H)⁺; ¹H NMR (300 MHz,

CDCl₃) δ 0.80 (3 H, t, J ) 7.1 Hz), 0.92-1.11 (2 H, m), 1.11-

1.23 (2 H, m), 1.41 (9 H, s), 1.33-1.73 (2 H, m), 3.21-3.43 (2

H, m), 3.50 (1 H, s), 3.65 (3 H, s), 4.39-4.52 (2 H, m), 5.24-

5.37 (1 H, m), 6.30 (1 H, d, J ) 7.3 Hz), 7.13 (1 H, dt, J ) 1.3,

7.8 Hz), 7.24 (1 H, dt, J ) 1.3, 7.8 Hz), 7.29 (1 H, dd, J ) 1.3,

7.8 Hz), 7.66 (1 H, dd, J ) 1.3, 7.8 Hz), 8.17 (1 H, brs).

(b) Dep r otection of th e Boc Gr ou p in 83. 83 (115 mg,

0.252 mmol) was dissolved in formic acid (5 mL), and the

mixture was stirred at room temperature for 1.5 h. The

mixture was concentrated under reduced pressure, and the

residue was partitioned between EtOAc and saturated NaH-

CO₃. The organic layer was dried over MgSO₄and concen-

trated. The residue was purified by preparative TLC (CHCl₃:

MeOH ) 25:1) to give the cyclic imine 85 (21 mg, 24%) as a

1:1 mixture of two diastereoisomers presumably due to a

racemization at the C-R position of the tryptophanyl residue:

TLC R_f(CHCl₃:MeOH ) 20:1) 0.42; FAB-MS m/ e 356 (M +

Meth od D. [(Octa h yd r o-1H-a zocin yl)ca r bon yl]-Leu -^D-

Tr p (2-Br )-^D-Nle-OH (14). (a ) Boc-Leu -D-Tr p (2-Br )-D-Nle-

OMe (80). EDCI (214 mg, 1.12 mmol) was added to a mixture

of Boc-Leu-OH‚H₂O (278 mg, 1.12 mmol), 75a hydrochloride

(415 mg, 0.93 mmol), HOBT (171 mg, 1.12 mmol), and NMM

(0.12 mL, 1.12 mmol) in CH₂Cl₂(15 mL) at 0 °C. After being

stirred at 0 °C for 1.5 h and at room temperature for 2.5 h,

the mixture was diluted with CH₂Cl₂(30 mL). The mixture

was washed with saturated NaHCO₃(20 mL × 3), 10% citric

acid (20 mL × 2), and brine (20 mL), dried over MgSO₄, and

concentrated. The residue was purified by column chroma-

tography on silica gel eluted with CHCl₃/MeOH (20:1) to give

80 (564 mg, 97%) as a white amorphous solid: TLC R_f(CHCl₃:

MeOH ) 20:1) 0.28; FAB-MS m/ e 623, 625 (M + H)⁺.

(b) (P h en oxycar bon yl)-Leu -^D-Tr p(2-Br )-D-Nle-OMe (81).

80 (530 mg, 0.85 mmol) was dissolved in formic acid (20 mL),

and the mixture was stirred at room temperature for 2 h. The

mixture was concentrated, and the residue was taken up with

EtOAc (50 mL). The mixture was washed with saturated

NaHCO₃(40 mL × 2) and brine (40 mL) and dried over MgSO₄.

The solvent was evaporated to give the corresponding amine

(434 mg, 98%): TLC R_f(CHCl₃:MeOH ) 10:1) 0.50; FAB-MS

m/ e 523, 525 (M + H)⁺.

The above-mentioned amine (422 mg, 0.806 mmol) was

dissolved in pyridine (3.0 mL), and phenyl chloroformate (0.20

mL, 1.61 mmol) was added at 0 °C. The mixture was stirred

at 0 °C for 1.5 h, and the reaction was quenched by the addition

of 1 drop of water. The mixture was concentrated under

reduced pressure, and the residue was partitioned between

EtOAc (40 mL) and water (40 mL). The aqueous layer was

extracted with EtOAc (20 mL × 2), and the combined organic

layers were washed with 10% citric acid (40 mL), saturated

NaHCO₃(40 mL), and brine (40 mL) and dried over MgSO₄.

The solvent was evaporated, and the residue was purified by

column chromatography on silica gel eluted with CHCl₃/MeOH

(50:1) to give 81 (472 mg, 91%) as a white solid: mp 193-195

°C; TLC R_f(CHCl₃:MeOH ) 30:1) 0.25; FAB-MS m/ e 643, 645

1

H)⁺; H NMR (300 MHz, CDCl₃) δ 0.90 and 0.92 (3 H, t, J )

7.0 Hz), 1.27-1.45 (4 H, m), 1.71-2.00 (2 H, m), 2.43 (3 H, s),

2.72 and 2.77 (1 H, dd, J ) 4.1, 16.5 Hz), 3.52 and 3.55 (1 H,

dd, J ) 7.6, 16.5 Hz), 3.76 and 3.78 (3 H, s), 4.13-4.29 (1 H,

m), 4.67-4.77 (1 H, m), 7.17 (1 H, t, J ) 7.4 Hz), 7.30 (1 H, t,

J ) 7.4 Hz), 7.39 (1 H, d, J ) 7.4 Hz), 7.62 (1 H, t, J ) 7.4

Hz), 8.21 (1H, brs), 8.29 and 8.32 (1 H, d, J ) 7.8 Hz).

(c) Boc-^D-Tr p (2-Et)-D-Nle-OMe (86). A mixture of 83 (180

mg, 0.395 mmol) and 5% Pd/BaSO₄(50 mg) in MeOH (4 mL)

was stirred in a hydrogen atmosphere for 3.5 h. The catalyst

was removed by filtration, and the solvent was evaporated to

give 86 (166 mg, 91%) as a pale yellow oil: TLC R_f(EtOAc:

hexane ) 1:2) 0.37; FAB-MS m/ e 459 (M⁺); ¹H NMR (300

MHz, CDCl₃) δ 0.81 (3 H, t, J ) 7.0 Hz), 0.94-1.25 (4 H, m),

1

(M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.77 (3 H, t, J ) 7.1

Hz), 0.89 (3 H, d, J ) 6.0 Hz), 0.90 (3 H, d, J ) 6.0 Hz), 0.80-

1.70 (9 H, m), 3.14 (1 H, dd, J ) 7.7, 14.6 Hz), 3.31 (1 H, dd,

J ) 6.6, 14.6 Hz), 3.64 (3 H, s), 4.13-4.19 (1 H, m), 4.36-4.43

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2327

1.29 (3 H, t, J ) 7.7 Hz), 1.43 (9 H, s), 1.36-1.73 (2 H, m),

2.79 (2 H, q, J ) 7.7 Hz), 3.08 (1 H, dd, J ) 8.6, 14.8 Hz), 3.27

(1 H, dd, J ) 2.7, 14.8 Hz), 3.58 (3 H, s), 4.29-4.42 (2 H, m),

5.15-5.30 (1 H, m), 6.08 (1 H, d, J ) 7.3 Hz), 7.06 (1 H, dt, J

) 1.5, 7.3 Hz), 7.11 (1 H, dt, J ) 1.5, 7.3 Hz), 7.28 (1 H, dd, J

) 1.5, 7.3 Hz), 7.54 (1 H, dd, J ) 1.5, 7.3 Hz), 7.88 (1 H, brs).

(d ) cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Leu -^D-Tr p -

(2-Et)-^D-Nle-OMe (87). A mixture of 86 (160 mg, 0.348 mmol)

in formic acid (5 mL) was stirred at room temperature for 1.5

h. The mixture was concentrated, and the resulting residue

was taken up with EtOAc, which was washed with saturated

NaHCO₃and dried over MgSO₄. The solvent was evaporated

to give the corresponding primary amine (123 mg, 98%) as a

pale yellow oil: TLC R_f(CHCl₃:MeOH:AcOH ) 10:1:1) 0.49;

FAB-MS m/ e 360 (M + H)⁺.

H, m), 5.18 (1 H, brs), 6.70 (1 H, d, J ) 7.2 Hz), 7.18-7.33 (2

H, m), 7.55 (1 H, dd, J ) 1.7, 7.0 Hz), 8.05 (1 H, dd, J ) 1.7,

7.0 Hz).

Boc-

D-Tr p (1-Boc,2-Br )-D-Asp (OMe)-O^tBu (88c). This

compound was prepared from 56b and H-^D-Asp(OMe)-O^tBu‚-

HCl in 94% yield according to the procedure for the synthesis

of 88a . 88c: colorless oil; FAB-MS m/ e 668, 670 (M + H)⁺;

¹H NMR (300 MHz, CDCl₃) δ 1.37 (9 H, s), 1.40 (9 H, s), 1.70

(9 H, s), 2.76 (1 H, dd, J ) 4.9, 16.8 Hz), 2.88 (1 H, dd, J )

4.1, 16.8 Hz), 3.22 (2 H, d, J ) 6.8 Hz), 3.62 (3 H, s), 4.38-

4.51 (2 H, m), 5.21 (1 H, d, J ) 5.9 Hz), 6.66 (1 H, d, J ) 7.1

Hz), 7.18-7.33 (2 H, m), 7.57 (1 H, d, J ) 7.5 Hz), 8.05 (1 H,

d, J ) 7.5 Hz).

Boc-^D-Tr p (2-CN)-D-Asp (OMe)-O^tBu (88d ). This com-

pound was prepared from 61 and H-^D-Asp(OMe)-O^tBu‚HCl in

52% yield according to the procedure for the synthesis of 88a .

The primary amine (120 mg, 0.334 mmol) was dissolved in

CH₂Cl₂(5 mL). 73a (100 mg, 0.367 mmol), HOBT (62 mg, 0.40

mmol), and EDCI (78 mg, 0.40 mmol) were added to the

mixture at 0 °C, and the resulting mixture was stirred at room

temperature for 2 h. The mixture was diluted with EtOAc,

washed with saturated NaHCO₃, 10% citric acid, and brine,

and dried over MgSO₄. The solvent was evaporated. The

residue was purified by chromatography on silica gel (E.

Merck, Lobar, Lichroprep, Si 60) eluted with EtOAc/hexane

(2:1) to give 87 (143 mg, 64%) as a pale yellow amorphous

solid: TLC R_f(EtOAc:hexane ) 2:1) 0.42; FAB-MS m/ e 612

1

88d : white amorphous solid; FAB-MS m/ e 515 (M + H)⁺; H

NMR (300 MHz, CDCl₃) δ 1.38 (9 H, s), 1.45 (9 H, s), 2.80 (1

H, dd, J ) 4.9, 16.9 Hz), 2.93 (1 H, dd, J ) 4.3, 16.9 Hz), 3.27-

3.50 (2 H, m), 3.64 (3 H, s), 4.48-4.70 (2 H, m), 5.29 (1 H, d,

J ) 6.2 Hz), 6.81 (1 H, d, J ) 6.8 Hz), 7.18 (1 H, t, J ) 8.0

Hz), 7.24-7.38 (2 H, m), 7.74 (1 H, d, J ) 8.0 Hz), 8.73 (1 H,

s).

Boc-P r o-^D-Cp eg-Cp r g-OH (90a ). EDCI (138 mg, 0.72

mmol) was added to a mixture of Boc-^D-Cpeg-OH (145 mg, 0.60

mmol), H-Cprg-OBzl‚HCl (160 mg, 0.66 mmol), HOBT (110

mg, 0.72 mmol), and NMM (0.10 mL, 0.72 mmol) in DMF (3

mL) at 0 °C. The mixture was allowed to warm to room

temperature and stirred overnight. The mixture was parti-

tioned between EtOAc (30 mL) and water (20 mL), and the

organic layer was washed with 20 mL each of 10% citric acid,

saturated NaHCO₃, and brine, dried over MgSO₄, and con-

centrated. Crystallization of the residue from EtOAc-hexane

gave Boc-^D-Cpeg-Cprg-OBzl (221 mg, 86%) as colorless crys-

tals.

Boc-^D-Cpeg-Cprg-OBzl (220 mg, 0.51 mmol) was dissolved

in 4 N HCl/EtOAc (10 mL), and the mixture was stirred at

room temperature for 1 h. The mixture was concentrated

under reduced pressure to give H-^D-Cpeg-Cprg-OBzl‚HCl (189

mg, 100%) as a white amorphous solid.

EDCI (173 mg, 0.90 mmol) was added to a mixture of H-^D-

Cpeg-Cprg-OBzl‚HCl (276 mg, 0.75 mmol), Boc-Pro-OH (194

mg, 0.90 mmol), HOBT (138 mg, 0.90 mmol), and NMM (124

µL, 0.90 mmol) in DMF (10 mL) at 0 °C. The mixture was

stirred at 0 °C for 2 h and then at room temperature overnight.

The mixture was partitioned between EtOAc (50 mL) and

water (50 mL), and the organic layer was washed with 50 mL

each of 10% citric acid, saturated NaHCO₃, and brine and dried

over MgSO₄. The solvent was evaporated to give Boc-Pro-D-

Cpeg-Cprg-OBzl (399 mg, 100%). Boc-Pro-^D-Cpeg-Cprg-OBzl

(399 mg, 0.756 mmol) was hydrogenated over 10% Pd/C (50

mg) in MeOH (10 mL) under an atmospheric pressure of

hydrogen to give 90a (332 mg, 100%) as a white amorphous

solid: FAB-MS m/ e 438 (M + H)⁺; ¹H NMR (300 MHz, CDCl₃)

δ 0.43-0.65 (4 H, m), 1.09-1.22 (1 H, m), 1.28-1.92 (10 H,

m), 1.44 (9 H, s), 1.98-2.33 (3 H, m), 3.29-3.60 (2 H, m), 4.08

(1 H, t, J ) 7.7 Hz), 4.21-4.37 (1 H, m), 4.73 (1 H, t, J ) 9.6

Hz), 7.14-7.50 (2 H, brs).

1

(M + H)⁺; H NMR (300 MHz, CDCl₃) δ 0.75-0.90 (9 H, m),

1.16 (3 H, d, J ) 7.1 Hz), 1.17 (3 H, d, J ) 7.1 Hz), 1.27 (3 H,

t, J ) 7.8 Hz), 1.02-1.82 (15 H, m), 2.70-2.91 (2 H, m), 3.15

(1 H, dd, J ) 7.5, 14.5 Hz), 3.38 (1 H, dd, J ) 5.9, 14.5 Hz),

3.60 (3 H, s), 3.91-4.20 (3 H, m), 4.29-4.40 (1 H, m), 4.67-

4.80 (2 H, m), 6.44 (1 H, d, J ) 8.5 Hz), 6.92 (1 H, d, J ) 7.5

Hz), 7.05 (1 H, dt, J ) 1.2, 7.6 Hz), 7.11 (1 H, dt, J ) 1.2, 7.6

Hz), 7.21 (1 H, dd, J ) 1.2, 7.6 Hz), 7.53 (1 H, dd, J ) 1.2, 7.6

Hz), 7.90 (1 H, brs).

(e) cis-[(2,6-Dim eth ylp ip er id in o)ca r bon yl]-Leu -^D-Tr p -

(2-Et)-^D-Nle-OH (10). NaOH (4 N) (1 mL) was added to a

solution of 87 (43.4 mg, 0.071 mmol) in MeOH (2 mL) at 0 °C,

and the resulting mixture was stirred at room temperature

for 2 h. MeOH was removed by evaporation, and 1 N HCl

was added to the aqueous residue at 0 °C. The precipitate

formed was collected by filtration, washed with a small amount

of water and dried to give 10 (36 mg, 85%) as a pale yellow

powder: TLC R_f(CHCl₃:MeOH:AcOH ) 30:1:1) 0.39; ¹H NMR

(300 MHz, CDCl₃) δ 0.71 (6 H, d, J ) 6.8 Hz), 0.83 (3 H, t, J

) 6.7 Hz), 1.03 (3 H, d, J ) 6.3 Hz), 1.05 (3 H, d, J ) 6.3 Hz),

1.19 (3 H, t, J ) 7.6 Hz), 0.95-1.76 (15 H, m), 2.59-2.89 (3 H,

m), 3.18 (1 H, dd, J ) 4.8, 14.6 Hz), 3.94-4.20 (4 H, m), 4.40-

4.48 (1 H, m), 6.07 (1 H, d, J ) 7.3 Hz), 6.87 (1 H, t, J ) 7.4

Hz), 6.94 (1 H, t, J ) 7.4 Hz), 7.18 (1 H, d, J ) 7.4 Hz), 7.53

(1 H, d, J ) 7.4 Hz), 7.89 (1 H, brs), 7.98 (1 H, d, J ) 9.8 Hz),

10.64 (1 H, s).

Boc-^D-Tr p (2-Br )-D-Asp (OMe)-O^tBu (88a ). A mixture of

58 (766 mg, 2.00 mmol), H-^D-Asp(OMe)-O^tBu‚HCl (480 mg,

2.00 mmol), NMM (0.30 mL, 2.20 mmol), HOBT (337 mg, 2.20

mmol), and EDCI (422 mg, 2.20 mmol) in CH₂Cl₂(20 mL) was

stirred at 0 °C for 1 h and at room temperature for 14 h. The

mixture was washed with saturated NaHCO₃(20 mL), 10%

citric acid (20 mL), and brine (20 mL), dried over MgSO₄, and

concentrated under reduced pressure. The residue was puri-

fied by column chromatography on silica gel eluted with

hexane/EtOAc (2:1) to give 88a (1.09 g, 96%) as a white

Boc-P r o-^D-Va l-Leu -OH (90b): white amorphous solid;

FAB-MS m/ e 428 (M + H)⁺; ¹H NMR (300 MHz, CDCl₃) δ

0.81-1.00 (12 H, m), 1.43 (9 H, s), 1.51-2.24 (8 H, m), 3.25-

3.53 (2 H, m), 4.20-4.34 (1 H, m), 4.49-4.68 (2 H, m), 7.15-

7.60 (2 H, brs).

1

amorphous solid: FAB-MS m/ e 568, 570 (M + H)⁺; H NMR

Boc-P r o-^D-ter t-Leu -Leu -OH (90c): white amorphous solid;

FAB-MS m/ e 442 (M + H)⁺; ¹H NMR (200 MHz, CDCl₃) δ

0.88-1.08 (6 H, m), 1.02 (9 H, s), 1.45 (9 H, s), 1.57-2.22 (7

H, m), 3.28-3.60 (2 H, m), 4.20-4.38 (1 H, m), 4.48-4.64 (1

H, m), 4.81 (1 H, d, J ) 9.7 Hz), 7.05-7.42 (2 H, brs).

Boc-P r o-^D-Cp eg-Leu -OH (90d ): white amorphous solid;

FAB-MS m/ e 454 (M + H)⁺; ¹H NMR (300 MHz, CDCl₃) δ

0.85-2.38 (15 H, m), 1.44 (9 H, s), 3.25-3.53 (2 H, m), 4.20-

4.30 (1 H, m), 4.49-4.65 (2 H, m), 7.05-7.35 (2 H, brs).

Boc-P r o-^D-P en (Me)-Leu -OH (90e). Iodomethane (0.68

mL, 11 mmol) was added to a mixture of ^D-penicillamine (1.49

g, 10 mmol) and 1 N NaOH (11 mL, 11 mmol) in EtOH (10

mL) at 0 °C. The mixture was allowed to warm to room

(200 MHz, CDCl₃) δ 1.38 (9 H, s), 1.41 (9 H, s), 2.74 (1 H, dd,

J ) 5.0, 16.6 Hz), 2.87 (1 H, dd, J ) 5.3, 16.6 Hz), 3.10-3.28

(2 H, m), 3.60 (3 H, s), 4.35-4.60 (2 H, m), 5.15 (1 H, brs),

6.67 (1 H, d, J ) 7.2 Hz), 7.03-7.32 (3 H, m), 7.57 (1 H, d, J

) 7.3 Hz), 8.11 (1 H, s).

Boc-^D-Tr p(1-Boc,2-Cl)-D-Asp(OMe)-O^tBu (88b). This com-

pound was prepared from 56a and H-^D-Asp(OMe)-O^tBu‚HCl

in 85% yield according to the procedure for the synthesis of

1

88a . 88b: colorless oil; FAB-MS m/ e 624, 626 (M + H)⁺; H

NMR (200 MHz, CDCl₃) δ 1.38 (9 H, s), 1.41 (9 H, s), 1.69 (9

H, s), 2.74 (1 H, dd, J ) 3.9, 16.4 Hz), 2.89 (1 H, dd, J ) 4.1,

16.4 Hz), 3.21 (2 H, d, J ) 6.8 Hz), 3.62 (3 H, s), 4.33-4.52 (2

2328 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12

Fukami et al.

temperature and stirred overnight. Di-tert-butyldicarbonate

(2.53 mL, 11 mmol) and 1 N NaOH (11 mL, 11 mmol) were

added to the mixture, and the resulting mixture was stirred

at room temperature for 3 h. The mixture was concentrated

to remove EtOH, and the resulting aqueous solution was

extracted with ethyl ether (10 mL). The aqueous layer was

acidified with 10% citric acid and extracted with EtOAc (10

mL × 3). The combined organic extracts were washed with

brine (10 mL) and dried over MgSO₄, and the solvent was

evaporated to give Boc-^D-Pen(Me)-OH (2.47 g, 94%) as a

colorless oil.

NMM (0.66 mL, 6.0 mmol), HOBT (0.92 g, 6.0 mmol), and

EDCI (1.15 g, 6.0 mmol) were added to a mixture of Boc-Pen-

(Me)-OH (1.32 g, 5.0 mmol) and H-Leu-OMe‚HCl (1.09 g, 6.0

mmol) in CH₂Cl₂(20 mL) at 0 °C. The mixture was allowed

to warm to room temperature and stirred overnight. The

mixture was washed successively with saturated NaHCO₃,

10% citric acid, and brine and dried over MgSO₄. The solvent

was evaporated to give Boc-^D-Pen(Me)-Leu-OMe (1.75 g, 90%)

as a white amorphous solid.

91a (599 mg, 0.68 mmol) was dissolved in TFA (10 mL),

and the mixture was stirred at room temperature for 1 h. The

mixture was concentrated under reduced pressure to give

H-Pro-^D-Cpeg-Cprg-D-Trp(2-Br)-D-Asp(OMe)-OH as a brown

amorphous solid (535 mg, 92%).

The above-mentioned linear pentapeptide (511 mg, 0.60

mmol) was dissolved in DMF (40 mL). NMM (83 µL, 0.60

mmol), HOBT (174 mg, 0.91 mmol), and EDCI (174 mg, 0.91

mmol) were added to the mixture at 0 °C. The mixture was

stirred at 0 °C for 1 h and then at room temperature overnight.

The mixture was concentrated, and the residue was taken up

with water (50 mL), which was extracted with CH₂Cl₂(30 mL

× 3). The combined organic extracts were washed with 10%

citric acid, saturated NaHCO₃, and brine and dried over

MgSO₄, and the solvent was evaporated. The residue was

purified by chromatography on silica gel (E. Merck, Lobar,

Lichroprep, Si 60) eluted with EtOAc/hexane (5:1) to give a

protected cyclic pentapeptide, 92a (151 mg, 35%), as a pale

yellow powder.

92a (148 mg, 0.21 mmol) was dissolved in MeOH (5 mL),

and 1 N NaOH (1.0 mL, 1.0 mmol) was added at 0 °C. After

being stirred at 0 °C for 1 h, the mixture was allowed to warm

to room temperature and stirred overnight. The mixture was

concentrated under reduced pressure to remove MeOH, and 1

N HCl was added to the resulting aqueous residue at 0 °C to

adjust the pH to 2-3. The precipitate was collected by

filtration and dried to give 46 (132 mg, 91%) as a pale yellow

powder: TLC R_f(CHCl₃:MeOH:AcOH ) 10:1:1) 0.37; ¹H NMR

(400 MHz, DMSO-d₆) δ -0.13-0.03 (3 H, m), 0.30-0.40 (1 H,

m), 0.76-0.88 (1 H, m), 1.19-1.70 (9 H, m), 1.70-1.87 (1 H,

m), 1.87-2.00 (2 H, m), 2.19-2.30 (1 H, m), 2.32 (1 H, dd, J )

3.9, 16.1 Hz), 2.76 (1 H, dd, J ) 10.7, 16.1 Hz), 3.01 (1 H, dd,

J ) 9.8, 14.7 Hz), 3.08-3.51 (4 H, m), 4.24 (1 H, t, J ) 9.8

Hz), 4.30-4.40 (1 H, m), 4.73 (1 H, d, J ) 7.3 Hz), 4.90-5.01

(1 H, m), 6.97 (1 H, t, J ) 7.6 Hz), 7.06 (1 H, t, J ) 7.6 Hz),

7.25 (1 H, d, J ) 7.6 Hz), 7.48 (1 H, d, J ) 9.8 Hz), 7.68 (1 H,

d, J ) 7.6 Hz), 7.84 (1 H, d, J ) 9.3 Hz), 8.46 (1 H, d, J ) 8.8

Hz), 9.07 (1 H, d, J ) 4.9 Hz), 11.64 (1 H, s), 12.30 (1 H, brs).

Meth od G. cyclo(-^D-Tr p (2-Me)-D-Asp -P r o-D-Cp eg-Leu -)

(48). (a ) Boc-^D-Asp (OMe)-P r o-D-Cp eg-Leu -OBzl (93a ).

EDCI (620 mg, 3.24 mmol) was added to a mixture of H-Leu-

OBzl‚TosOH (935 mg, 2.38 mmol), Boc-^D-Cpeg-OH (540 mg,

2.16 mmol), NMM (0.26 mL, 2.38 mmol), and HOBT (496 mg,

3.24 mmol) in CH₂Cl₂(10 mL) at 0 °C. The mixture was

stirred at 0 °C for 1 h and at room temperature for 3 h. The

mixture was washed with saturated NaHCO₃, 10% citric acid,

and brine, dried over MgSO₄, and concentrated in vacuo. The

residue was purified by column chromatography on silica gel

eluted with hexane/EtOAc (3:1) to give Boc-^D-Cpeg-Leu-OBzl

(0.86 g, 89%) as a white solid.

Boc-^D-Pen(Me)-Leu-OMe (1.75 g, 4.48 mmol) was dissolved

in 4 N HCl/1,4-dioxane (20 mL). After being stirred for 30

min, the mixture was concentrated under reduced pressure.

Trituration of the residue with ethyl ether gave H-^D-Pen(Me)-

Leu-OMe‚HCl (1.387 g, 95%) as white crystals.

NMM (0.24 mL, 2.2 mmol), HOBT (337 mg, 2.2 mmol), and

EDCI (422 mg, 2.2 mmol) were added to a mixture of H-^D-

Pen(Me)-Leu-OMe‚HCl (654 mg, 2.0 mmol) and Boc-Pro-OH

(474 mg, 2.2 mmol) in CH₂Cl₂(10 mL) at 0 °C. The mixture

was allowed to warm to room temperature and stirred over-

night. The mixture was washed with saturated NaHCO₃, 10%

citric acid, and brine, dried over MgSO₄, and concentrated to

give Boc-Pro-^D-Pen(Me)-Leu-OMe (0.946 g, 97%) as a white

amorphous solid.

Boc-Pro-^D-Pen(Me)-Leu-OMe (0.943 g, 1.93 mmol) was dis-

solved in MeOH (2.5 mL), and 1 N NaOH (2.5 mL, 2.5 mmol)

was added to the mixture at 0 °C. After 30 min, the mixture

was allowed to warm to room temperature and stirred for 1

h. The mixture was concentrated under reduced pressure to

remove MeOH, and the resulting aqueous solution was ex-

tracted with ethyl ether. The aqueous layer was acidified with

10% citric acid and extracted with EtOAc (10 mL × 3). The

combined organic extracts were washed with brine and dried

over MgSO₄, and the solvent was evaporated to give 90e (0.92

g, 100%) as a white amorphous solid: FAB-MS m/ e 474 (M +

1

H)⁺; H NMR (200 MHz, CDCl₃) δ 0.96 (3 H, d, J ) 6.0 Hz),

0.97 (3 H, d, J ) 6.0 Hz), 1.35 (3 H, s), 1.38 (3 H, s), 1.45 (9 H,

s), 1.56-2.22 (7 H, m), 2.09 (3 H, s), 3.28-3.60 (2 H, m), 4.22-

4.39 (1 H, m), 4.48-4.63 (1 H, m), 4.93-5.12 (1 H, m), 7.30-

7.60 (2 H, brs).

Boc-^D-Cpeg-Leu-OBzl (100 mg, 0.224 mmol) was dissolved

in 4 N HCl/1,4-dioxane (5 mL), and the solution was stirred

at room temperature for 1 h. The solvent was evaporated, and

the residue was dissolved in DMF (3 mL). Boc-Pro-OH (53

mg, 0.25 mmol), NMM (25 µL, 0.22 mmol), HOBT (41 mg, 0.27

mmol), and EDCI (52 mg, 0.27 mmol) were added to the

solution at 0 °C. The mixture was allowed to warm to room

temperature and stirred overnight. The mixture was diluted

with EtOAc (50 mL), washed with saturated NaHCO₃, 10%

citric acid, and brine, dried over MgSO₄, and concentrated. The

residue was purified by column chromatography on silica gel

eluted with hexane/EtOAc (2:1) to give Boc-Pro-^D-Cpeg-Leu-

OBzl (107 mg, 88%).

The Boc protecting group in Boc-Pro-^D-Cpeg-Leu-OBzl (103

mg, 0.19 mmol) was removed by 4 N HCl/1,4-dioxane (room

temperature, 1.5 h, 100%). The corresponding primary amine

hydrochloride and Boc-^D-Asp(OMe)-OH (71 mg, 0.28 mmol)

were dissolved in DMF (3 mL). NMM (21 µL, 0.19 mmol),

HOBT (44 mg, 0.28 mmol), and EDCI (55 mg, 0.28 mmol) were

added to the solution at 0 °C. The resulting mixture was

allowed to warm to room temperature and stirred overnight.

The mixture was diluted with EtOAc (50 mL), washed with

saturated NaHCO₃, 10% citric acid, and brine, dried over

MgSO₄, and concentrated. The residue was purified by column

chromatography on silica gel eluted with hexane/EtOAc (1:2)

Meth od F . cyclo(-^D-Tr p (2-Br )-D-Asp -P r o-D-Cp eg-Cp r g-)

(46). 88a (1.09 g, 1.92 mmol) was dissolved in formic acid (20

mL). After being stirred for 1.5 h, the mixture was concen-

trated under reduced pressure. The residue was partitioned

between EtOAc (50 mL) and saturated NaHCO₃(50 mL), and

the organic layer was dried over MgSO₄. The solvent was

evaporated, and the residue was dissolved in ethyl ether (50

mL); 4 N HCl/1,4-dioxane (0.5 mL) was added to the solution

at 0 °C, and the volatiles were evaporated to give a hydro-

chloride of the corresponding primary amine 89a (0.826 g,

85%) as a white hygroscopic powder.

An aliquot (420 mg, 0.83 mmol) of the hydrochloride of 89a

and Boc-Pro-^D-Cpeg-Cprg-OH (90a ; 332 mg, 0.76 mmol) were

dissolved in DMF (10 mL). NMM (114 µL, 0.83 mmol), HOBT

(127 mg, 0.83 mmol), and EDCI (159 mg, 0.83 mmol) were

added to the mixture at 0 °C, and the resulting mixture was

allowed to warm to room temperature and stirred overnight.

The mixture was partitioned between EtOAc (50 mL) and

water (50 mL), and the organic layer was washed with 50 mL

each of 10% citric acid, saturated NaHCO₃, and brine and dried

over MgSO₄. The solvent was evaporated. The residue was

purified by chromatography on silica gel (E. Merck, Lobar,

Lichroprep, Si 60) eluted with CHCl₃/MeOH (50:1) to give a

protected linear pentapeptide, 91a (619 mg, 92%), as a pale

yellow amorphous solid.

D-Trp-Containing ET Receptor Antagonists

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2329

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to give 93a (127 mg, 100%) as a pale yellow amorphous solid:

TLC R_f(hexane:EtOAc ) 1:2) 0.38; FAB-MS m/ e 673 (M +

H)⁺.

(b ) Z-^D-Tr p (2-Me)-D-Asp (OMe)-P r o-D-Cp eg-Leu -OBzl

(94a ). The Boc protecting group in 93a (125 mg, 0.19 mmol)

was removed by 4 N HCl/1,4-dioxane (room temperature, 1.5

h, 115 mg, 100%). An aliquot (65 mg, 0.095 mmol) of the

corresponding primary amine hydrochloride and Z-^D-Trp(2-

Me)-OH (66; 37 mg, 0.10 mmol) were dissolved in DMF (3 mL).

NMM (23 µL, 0.21 mmol), HOBT (22 mg, 0.14 mmol), and

EDCI (27 mg, 0.14 mmol) were added to the solution at 0 °C.

The resulting mixture was allowed to warm to room temper-

ature and stirred overnight. The mixture was diluted with

EtOAc (50 mL), washed with saturated NaHCO₃, 10% citric

acid, and brine, dried over MgSO₄, and concentrated. The

residue was purified by preparative TLC (hexane/EtOAc ) 1:4)

to give 94a (62 mg, 72%) as a pale yellow amorphous solid:

TLC R_f(hexane:EtOAc ) 1:5) 0.35; FAB-MS m/ e 907 (M +

H)⁺.

(c) cyclo(-^D-Tr p (2-Me)-D-Asp -P r o-D-Cp eg-Leu -) (48). A

mixture of 94a (60 mg, 0.066 mmol) and 10% Pd/C (40 mg) in

DMF (3 mL) was stirred under an atmospheric pressure of

hydrogen for 1.5 h. The catalyst was removed by filtration

and washed with DMF (10 mL). EDCI (19 mg, 0.099 mmol)

and HOBT (15 mg, 0.099 mmol) were added to the combined

filtrate and washings at 0 °C. The resulting mixture was

stirred at 0 °C for 1 h and at room temperature overnight.

The mixture was concentrated, and the residue was taken up

with EtOAc (30 mL), which was washed with saturated

NaHCO₃, 1 N HCl, and brine and dried over MgSO₄. The

solvent was evaporated, and the residue was purified by

preparative TLC (hexane/EtOAc ) 1:5) to give cyclo(-^D-Trp-

(2-Me)-^D-Asp(OMe)-Pro-D-Cpeg-Leu-) (95a ; 19 mg, 43%).

95a (15.5 mg, 0.023 mmol) was dissolved in MeOH (1 mL),

and 1 N NaOH (0.5 mL, 0.5 mmol) was added at 0 °C. After

being stirred at 0 °C for 4 h, the mixture was concentrated

under reduced pressure to remove MeOH; 1 N HCl was added

to the residue. The precipitate was collected by filtration and

dried to give 48 (12.7 mg, 84%) as a white powder: TLC R_f

(CHCl₃:MeOH:AcOH ) 30:1:1) 0.35; ¹H NMR (300 MHz,

DMSO-d₆) δ 0.61 (3 H, d, J ) 6.0 Hz), 0.69 (3 H, d, J ) 6.0

Hz), 0.98-2.02 (15 H, m), 2.17-2.50 (2 H, m), 2.32 (3 H, s),

2.80 (1 H, dd, J ) 10.5, 16.0 Hz), 2.88 (1 H, dd, J ) 10.6, 14.7

Hz), 3.05-3.50 (3 H, m), 3.92-4.05 (1 H, m), 4.19 (1 H, t, J )

10.0 Hz), 4.27-4.39 (1 H, m), 4.73 (1 H, d, J ) 7.1 Hz), 4.91-

5.03 (1 H, m), 6.87 (1 H, t, J ) 7.6 Hz), 6.94 (1 H, t, J ) 7.6

Hz), 7.18 (1 H, d, J ) 7.6 Hz), 7.48 (1 H, d, J ) 7.6 Hz), 7.51

(1 H, d, J ) 10.0 Hz), 7.76 (1 H, d, J ) 8.7 Hz), 8.67 (1 H, d,

J ) 9.0 Hz), 8.78 (1 H, d, J ) 5.0 Hz), 10.67 (1 H, s).

Ack n ow led gm en t. We gratefully acknowledge the

contributions of S. Abe (for mass spectra) and M. Saito

(for HPLC and solubility analyses). We are also grateful

to Ms. A. Thomas, Merck & Co., for her critical reading

of the manuscript.

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and HPLC data of all final compounds and ¹H NMR data of

target compounds not reported in the text (15 pages). Order-

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J M9600914

Article Doi

Synthesis and structure-activity relationships of 2-substituted D-tryptophan-containing peptidic endothelin receptor antagonists: Importance of the C-2 substituent of the D-tryptophan residue for endothelin A and B receptor subtype selectivity

DOI: 10.1021/jm9600914

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