Transformation of μ-opioid receptor agonists into biologically potent μ-opioid receptor antagonists

Bioorganic & Medicinal Chemistry 15 (2007) 1237–1251

Transformation of l-opioid receptor agonists into biologically

potent l-opioid receptor antagonists

Tingyou Li,^a,ꢀYunden Jinsmaa,^bMasahiro Nedachi,^cAnna Miyazaki,^cYuko Tsuda,^a,c

Akihiro Ambo,^dYusuke Sasaki,^dSharon D. Bryant,^bEwa Marczak,^bQiang Li,^e

H. Scott Swartzwelder,^fLawrence H. Lazarus^b,*and Yoshio Okada^a,c,

*

^aThe Graduate School of Food and Medicinal Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan

^bMedicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,

Research Triangle Park, NC 27709, USA

^cFaculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan

^dDepartment of Biochemistry, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan

^eDepartment of Pharmacology and Cancer Biology, and Department of Psychiatry, Duke University Medical Center,

Durham, NC 27710, USA

^fNeurobiology Research Laboratory, VA Medical Center, Durham, NC 27705, USA

Received 1 October 2006; revised 9 November 2006; accepted 10 November 2006

Available online 14 November 2006

Abstract—N-Allylation (–CH₂–CH@CH₂) of [Dmt¹]endomorphins yielded the following: (i) [N-allyl-Dmt¹]endomorphin-2

(Dmt = 2⁰,6⁰-dimethyl-L-tyrosine) (12) and [N-allyl-Dmt¹]endomorphin-1 (15) (K_il = 0.45 and 0.26 nM, respectively) became l-an-

tagonists (pA₂= 8.59 and 8.18, respectively) with weak d-antagonism (pA₂= 6.32 and 7.32, respectively); (ii) intracerebroventricu-

larly administered 12 inhibited morphine-induced CNS-mediated antinociception in mice [AD₅₀(0.148 ng/mouse) was 16-fold more

potent than naloxone], but not spinal antinociception, and (iii) 15 reversed the alcohol-elevated frequency in spontaneous inhibitory

post-synaptic currents (IPSC) in hippocampal CA1 pyramidal cells in rat brain slices (P = 0.0055). Similarly, N-allylation of the

potent l-opioidmimetic agonists, 1,6-bis-[H-Dmt-NH]-hexane and 3,6-bis-[Dmt-NH-propyl]-2(1H)-pyrazinone, converted them

into l-antagonists (pA₂= 7.23 and 7.17 for the N-allyl-derivatives 17 and 19, respectively), and exhibited weak d-antagonism. Thus,

N-allylation of Dmt containing opioid peptides or opioidmimetics continues to provide a facile means to convert selective l-opioid

agonists into potent l-opioid antagonists.

1. Introduction

these receptors, but also to serve as clinically and thera-

peutically relevant agents.¹Interestingly, the addictive

behavior associated with morphine in animal studies

when treated by some alkaloid-derived antagonists can

result in severe withdrawal symptoms due to their ability

to act as inverse agonists.¹Furthermore, the predilection

to alcoholism, in part, depends on the presence of intact

l-opioid receptors evidenced by the existence of sub-

stantial data using knock-out mice and selective opiate

antagonists which verify that these receptors are inti-

mately associated with a neural reward pathway(s) in

the CNS (central nervous system) that is aﬀected by

alcohol consumption²as well as morphine and its

cognates.

Whereas the existence of potent opioid peptide agonists

is well known for the major opioid-receptor classes

(l, d, j), the development of selective opioid antagonists

continues to be a key objective in pharmacology. In par-

ticular, l-opioid antagonists are important pharmaco-

logical tools not only to delineate critical biochemical,

pharmacological, and physiological roles played by

Keywords: Opioid; l-Opioid receptor antagonists; Endomorphin; Dmt;

Antinociception; Ethanol; Spontaneous IPSC.

*

Corresponding authors. Tel.: +81 78 974 1551; fax: +81 78 974 5689

(Y.O.), tel.: +1 919 541 3238; fax: +1 919 541 5737 (L.H.L).; e-mail

addresses: lazarus@niehs.nih.gov; okada@pharm.kobegakuin.ac.jp

Present address: College of Chemistry, Jilin University, Changchun

130012, China.

ꢀ

Opioid antagonists do not occur naturally and modiﬁca-

tion of the alkaloid architecture of morphine led to the

doi:10.1016/j.bmc.2006.11.019

1238

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

elucidation of structural requirements for their develop-

ment; however, the conversion was neither straightfor-

ward nor predictable. In particular, an allyl (–CH₂–

CH@CH₂) substitution³yielded either non-speciﬁc

antagonist for l-, d-, and j-opioid receptors (naloxone),

or a mixed agonist/antagonist (nalorphine); a compara-

ble dichotomy in pharmacological activity with

N-allylated opioid peptides occurred: for example

N,N-diallylation of the multisubstituted [Leu⁵]enkepha-

lin framework yielded two distinct d-opioid antagonists,

namely N,N-(allyl)₂-Tyr-Gly-Gly-w-(CH₂S)-Phe-Leu-

OH (ICI 154129)⁴and N,N-(allyl)₂-Tyr-Aib-Aib-Phe-

Leu-OH (ICI 174864),⁵while N-allylation produced

mixed agonist/antagonist opioids.^6,7Similarly, [N,N-(al-

lyl)₂-Tyr¹,D-Pro¹⁰]dynorphin A (1-11) imparted antago-

nism toward both j- and l-opioid receptors with weak

j-opioid receptor selectivity;⁸however, [N-allyl-Tyr¹,

D-Pro¹⁰]dynorphin A (1-11) retained the j-agonism of

the parent compound.³

other opioid peptides sporadically exhibit weak l-antag-

onism, but fail to meet the criteria for potent bio-

activity.^13–15

The l-opioid agonists endomorphin-1 (EM-1: H-Tyr-

Pro-Trp-Phe-NH₂) and endomorphin-2 (EM-2: H-Tyr-

Pro-Phe-Phe-NH₂) exhibit the highest selectivity for

the l-opioid receptor,¹⁶and thus represent a potential

opioid framework for modiﬁcation into l-antagonists.

In this communication, l-agonists containing 2⁰,6⁰-di-

methyl-^L-tyrosine (Dmt) in lieu of Tyr, such as [Dmt¹]-

endomorphin,¹⁷the bis-Dmt opioidmimetic ligands

linked by an alkyldiamine¹⁸or 3,6-bis-(aminoalkyl)-

2(1H)-pyrazinone,¹⁹were transformed by N-allylation

into potent l-opioid antagonists.

2. Chemistry

The key intermediates (Scheme 1) of N-allyl and N,N-

(allyl)₂-Xaa-OCH₃(3 and 2, Xaa = Tyr or Dmt) were

prepared in one pot from allylbromide (2.5 equiv) and

H-Xaa-OMe (1) in the presence of diisopropylethyl-

amine (DIPEA) (2.7 equiv) at 50 ꢁC for 5 h. N,N-(Al-

lyl)₂-Xaa-OCH₃(2) was hydrolyzed to give

N,N-(allyl)₂-Xaa-OH (4). N^a-H of N-allyl-Xaa-OCH₃

(3) was protected with tert-butyloxycarbonyl (Boc)

group in dioxane to produce N-allyl-N-Boc-Xaa-OCH₃

(5), and then hydrolyzed with NaOH to N-allyl-N-

Boc-Xaa-OH (6). As shown in Scheme 2, [N,N-(allyl)₂-

Inasmuch as that many speciﬁc d-antagonists exist (e.g.,

ICI 174864,⁵naltrindole,⁹the TIP(P) family,¹⁰and

Dmt-Tic pharmacophore derivatives¹¹), a majority of

the l-antagonists used pharmacologically lack a high

degree of biological speciﬁcity or potency, such as nalox-

one, naltrexone, nalmefene or CTAP (^D-Phe-Cys-Tyr-D-

Trp-Arg-Thr-Pen-Thr-NH₂).¹²On the other hand, while

the alkaloids naloxone and naltrexone are non-selective

opioid receptor antagonists, b-funaltrexamine behaves

as an irreversible l-antagonist. Nonetheless, a host of

iii

N,N-(Allyl)₂-Xaa-OH

N,N-(Allyl)₂-Xaa-OCH₃

ii

i

2

4

H-Xaa-OCH₃

H-Xaa-OH

1

iv

N-Allyl-Xaa-OCH₃

N-Allyl-N-Boc-Xaa-OCH₃

Xaa = Tyr (a) or Dmt (b)

3

5

iii

N-Allyl-N-Boc-Xaa-OH

6

Scheme 1. Synthesis of N-terminal mono- and diallyl-substituted amino acid derivatives. Reagents and conditions: (i) SOCl₂/MeOH; (ii) allyl-Br,

DIPEA, MeOH, 50 ꢁC, 5 h; (iii) 1 N NaOH; (iv) (Boc)₂O, Et₃N, dioxane.

i

N,N-(Allyl)₂-Xaa-OH

[N,N-(Allyl)₂-Xaa¹]EM

4a Xaa = Tyr

4b Xaa = Dmt

7 Xaa = Tyr (EM-2)

8 Xaa = Dmt (EM-2)

13 Xaa = Dmt (EM-1)

i

ii

[N-Allyl-N-Boc-Xaa¹]EM

[N-Allyl-Xaa¹]EM

N-Allyl-N-Boc-Xaa-OH

6a Xaa = Tyr

6b Xaa = Dmt

9 Xaa = Tyr (EM-2)

10 Xaa = Dmt (EM-2)

14 Xaa = Dmt (EM-1)

11 Xaa = Tyr (EM-2)

12 Xaa = Dmt (EM-2)

15 Xaa = Dmt (EM-1)

Scheme 2. Synthesis of N-terminal N-allyl- and N,N-(allyl)₂-substituted endomorphin-1 and endomorphin-2 analogues. Reagents: (i) H-Pro-Phe/

Trp-Phe-NH₂, DIPEA, PyBop, DMF; (ii) TFA/anisole.

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1239

ii

i

N-Allyl-N-Boc-Dmt-OH

Bis-(N-allyl-N-Boc-Dmt-NH)-hexane

6b

16

Bis-(N-allyl-Dmt-NH)-hexane

17

iii

N-Allyl-N-Boc-Dmt-OH

3,6-Bis-(N-allyl-N-Boc-Dmt-NH-propyl)-pyrazinone

6b

18

ii

3,6-Bis-(N-allyl-Dmt-NH-propyl)-pyrazinone

19

Scheme 3. Synthesis of N-terminal bis-(allyl-Dmt)-opioidmimetic ligands. Reagents and conditions: (i) 1,6-diaminohexane, DIPEA, PyBop, DMF;

(ii) TFA/anisole; (iii) 3,6-bis-(3⁰-aminopropyl)-pyrazinone, DIPEA, PyBop, DMF.

Xaa¹]- (7 and 8) and (N-allyl-Xaa¹)EM-2 analogues (11

and 12) were prepared in solution using Boc-protection;

[N,N-(allyl)₂-Dmt¹]- (13) and [N-allyl-Dmt¹]EM-1 (15)

receptor,²⁰since deletion reduces ligand eﬃcacy.¹³On

the other hand, the impairment of binding by allyl

groups^3–5,7–9might be attributed to steric hindrance

preventing the formation of an ionic bond required

for agonism, yet permitting the ligand to function as

an antagonist through interaction with a diﬀerent bind-

ing region within the receptor.^11aFurthermore, that

Dmt overcame the detrimental eﬀect of N-allylation

(Table 1) implies that hydrophobic factors also play a

dominant role in anchoring the opioid in the receptor

by aligning or stabilizing the hydroxyl group to form

a H-bond, enhancing p–p interactions or aromatic ring

stacking.^11a

were prepared identically. After deprotection of Boc-

17a

Pro-Phe-Phe-NH₂

with HCl/dioxane, the resulting

amino component was condensed with 4 or 6 using ben-

zotriazol-1-yloxytris-(pyrrolidino)phosphate (PyBop) as

the coupling reagent to give 7 and 8, and Boc-protected

9 and 10, respectively. Removal of the Boc-group (9 and

10) with triﬂuoroacetic acid (TFA) gave 11 and 12.

Compound 15 was synthesized using the same method

for 11, but using [N-allyl-N-Boc-Dmt¹]EM-1 (14). As

shown in Scheme 3, N-allyl-N-Boc-Dmt-OH (6b) was

coupled with 1,6-diaminohexane or 3,6-bis-(aminopro-

pyl)-2(1H)-pyrazinone to produce bis(N-allyl-N-Boc-

Dmt)-compounds, followed by removal of Boc group

with TFA to give 17 and 19, respectively. All ﬁnal com-

pounds were puriﬁed by semi-preparative reversed-

phase HPLC to >98% purity, and veriﬁed using mass

spectrometry, ¹H and ¹³C NMR, optical rotation,

thin-layer chromatography, analytical HPLC, and ele-

mental analysis.

3.2. Functional bioactivity

N-Allylation completely eliminated the l-agonism of

[H-Dmt¹]EM-1 (4⁰) and [H-Dmt¹]EM-2 (2⁰) to yield 15

and 12 with potent l-antagonism (pA₂= 8.18–8.59)

(Table 1). Furthermore, both compounds are 6.6- and

17-fold, respectively, more eﬀective than the published

data on CTAP;¹²even the N,N-diallyl ligand 13 has

nearly twice the potency of this molecule.¹²As shown

in Table 1, [H-Dmt¹]EM-2 (2⁰) is an agonist for both

l- and d-opioid receptors, while [H-Dmt¹]EM-1 (4⁰)

exhibited potent l-agonism/d-antagonism (GPI:

IC₅₀= 0.27 nM; MVD: pA₂= 8.60). Aromatic moiety

of Trp at position 3 in 4⁰is bulkier and more hydropho-

bic than that of Phe³in 2⁰. It can be deduced that

aromatic properties of the moiety at position 3 of

[H-Dmt¹]EM-1 or -2 are an important factor that deter-

mines whether [H-Dmt¹]EM exhibits d-agonism or d-an-

tagonism. This phenomenon is compatible with the

following results: ligand 12 exhibited high l-opioid

antagonism with an A₂ratio of 186, while ligand 15

had only an A₂ratio of 7.2. This diﬀerence in the A₂ra-

tio (26-fold) between 12 and 15 is mainly due to the

weaker d-antagonism exhibited by 12 (pA₂= 6.32) com-

pared with that of 15 (pA₂= 7.32) which can be ex-

plained by the hypothesis given above.

3. Results and discussion

3.1. Opioid receptor aﬃnity

Compared to the standard opioids (1⁰–6⁰, Table 1), the

allylated derivatives decreased aﬃnity toward l-opioid

receptors in the following order: parent peptides > N-al-

lyl > N,N-diallyl derivatives; of these analogues, howev-

er, 12 and 15 exhibited the highest l-opioid aﬃnities

(K_il = 0.45 and 0.26 nM, respectively) (Table 1). The ef-

fect of N-allylation of EM-2 on l-opioid aﬃnity was

more sensitive than that of [H-Dmt¹]EM-2; that is, N-al-

lyl-EM-2 (11) fell 98-fold compared to only 3-fold with

[N-allyl-Dmt¹]EM-2 (12). Although the d-opioid recep-

tor aﬃnities are lower than those for the l-opioid recep-

tor, the order was similar, except 7 and 11. In all cases,

the d-opioid aﬃnities were substantially less than those

observed for the l-opioid receptor (Table 1).

In general, N-modiﬁcation profoundly aﬀects opioid

peptide activities: for example, elimination of the

N-terminal amino group of [Met⁵]enkephalin abolished

d-opioid agonism;²¹N-formylation of H-Dmt-c[-D-

Orn-2-Nal-^D-Pro-Gly] resulted in a moderately active

l-opioid antagonist;¹³N-allyl-enkephalin⁷and N-allyl-

The primary, secondary or tertiary amino function at

the N-terminus is apparently necessary for the expres-

sion of high aﬃnity and bioactivity presumably due

to the formation of an ionic bond to a group in the

Table 1. Receptor aﬃnities and functional bioactivities of opioid ligands

No. Compound K_il (nM)

GPI (l)^dIC₅₀, nM pA₂

MVD (d)^d

IC₅₀, nM

pA₂

[A₂] ratio^f

MVD/GPI

e

K_id (nM)

d/l

1⁰

7

Endomorphin-2 (EM-2)

[N,N-(Allyl)₂]EM-2

1.33 0.15 (3) 6080 1,215 (3) 4571 6.88 0.94

1045 175 (6) 5755 1224 (4) NT

—

344 93

NT

—

8.7

6

57 NT

11

2⁰

N-Allyl-EM-2

[H-Dmt¹]EM-2^a

[N,N-(Allyl)₂-Dmt¹]EM-2

[N-Allyl-Dmt¹]EM-2

130 9.3 (6)

0.15 0.004

13.1 1.3 (6)

0.45 0.08 (5)

7358 259 (3)

28.2 8.1

188 0.07 0.016

108 >10,000 (33.9%)

1244 >10,000 (49.3%)

3125 4.03 1.14

104 0.27 0.08

1.87 0.61

8

1416 50 (4)

560 49 (4)

7.07 (7.33–7.02)

8.59 (9.05–8.33)

>10,000 (13.3%) 6.13 (6.34–5.72)

12

>10,000 (42.8%) 6.32 (7.35–5.48) 186

3⁰

4⁰

Endomorphin-1 (EM-1)

[H-Dmt¹]EM-1

[N,N-(Allyl)₂-Dmt¹]EM-1

[N-Allyl-Dmt¹]EM-1

1,6-Bis-[H-Dmt-NH]-hexane^b

1,6-Bis-[N-allyl-Dmt-NH]-hexane

3,6-Bis-[H-Dmt-NH-propyl]-2(1H)-pyrazinone^c

3,6-Bis-[N-allyl-Dmt-NH-propyl]-2(1H)-pyrazinone

0.56 0.04 (4) 1750 146 (4)

0.054 0.01 (5) 5.60 0.67 (3)

—

283 39 —

>10,000 (21.6%) 8.60 (9.58–8.01)

—

13

15

5⁰

17

6⁰

19

4.60 0.32 (4)

217 23 (5)

47 >10,000 (23.4%)

40 >10,000 (49.0%)

870 3.08 0.53

7.60 (7.68—7.51) >10,000 (19.8%) 7.15 (7.40–6.90)

>10,000 (37.5%) 7.32 (7.48–7.16)

2.8

7.2

0.26 0.01 (4) 10.3 1.1 (3)

0.053 0.01 (6) 46.1 8.8 (5)

8.18 (8.28–8.16)

—

>10,000

6.1

—

12.4 1.1 (3)

0.042 0.003

6.94 1.4 (3)

51.5 4.1 (3)

13.2 1.7

77.8 14 (4)

4 >10,000 (<1%)

314 1.33 0.2

11 >10,000 (<1%)

7.23 (7.36–6.99)

—

7.17 (7.82–6.84)

>10,000 (<1%)

>10,000

>10,000 (<1%)

6.83 (7.56–5.58)

—

6.38 (6.56–6.21)

2.5

—

6.2

The aﬃnity of each compound was assayed with 5–8 graded dosages of peptide in independent repetitions (n) and data are presented as means SE. The functional bioactivity is the mean SE (n = 5–6

separate tissue preparations). NT, not tested (due to the very weak interaction with opioid receptors, K_i). A dash (ꢀ) indicates non-relevancy.

^aData from Ref. 17.

^bData from Ref. 18.

^cData from Ref. 19.

^dValues in parentheses are maximum inhibition of the tissue contraction at the concentration of 10,000 nM.

^epA₂is the negative log of the molar concentration required to double the agonist’s IC₅₀value in order to achieve the original response, and the 95% conﬁdence limits are in parentheses.

^f[A₂] ratio is the ratio of molar concentrations at the pA₂values (MVD/GPI). Antagonism was determined using endomorphin-2 and deltorphin II as l- and d-receptor agonists, respectively.

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1241

dynorphin A-(1-11)⁸had mixed biological properties;

Mor

Mor + 0.067 ng 12

Mor + 0.67 ng 12

Mor + 6.7 ng 12

Mor + 67 ng 12

a

and N,N-dimethylation of bis-Dmt-Tic-derivatives (d-

opioid antagonists) yielded potent dual l-/d-opioid

antagonists.²²On the other hand, replacement of Tyr¹

in endomorphin by Mdp [(2S)-2-methyl-3-(2⁰,6⁰-dimeth-

yl-4⁰-hydroxyphenyl)-propionic acid] or Dhp [3-(2⁰,6⁰-

100

80

60

40

20

0

dimethyl-4⁰-hydroxyphenyl)-propionic

acid]

gave

moderately weak l-opioid antagonists.²³The parent

compounds of 17 and 19 are l-opioid agonists^18,19and

the allylated derivatives exhibited l-opioid antagonism

(pA₂= 7.23 and 7.17, respectively) (Table 1) comparable

to that of CTAP (pA₂= 7.36).¹²While the d-opioid

aﬃnity of 17 remained similar to its parent ligand, the

6.6-fold improvement in d-opioid antagonism yielded a

l-/d-opioid antagonist proﬁle similar to that of 19.

Our group,^17–19as well other investigators,¹¹published

considerable data demonstrating discrepancies and

inconsistencies between receptor aﬃnity and bioactivity

without an acceptable explanation; however, tentative

answers may, in part, involve inherent diﬀerences be-

tween the assay systems, which include the artiﬁcial nat-

ure of the synaptosomes used to determine aﬃnity (K_i)

and the bioactivity in isolated tissue preparations for

estimation of functional bioactivity, and the diﬀerential

solubility of the ligand in the membrane lipid milieu that

depends on the hydrophobic properties of opioids.

Another possible reason for the inconsistencies regard-

ing endomorphin analogues may be related to their

interactions with tachykinin NK1 and NK2 receptors,²⁴

which mediate antiopioid activity.

10 20 30 40 50 60 70

Time (min)

0

1400

1200

1000

800

600

400

200

0

b

Mor (4 mg/kg)

12

0.07

0.67

6.7

67

ng/mouse

c

100

80

60

40

20

0

12

3.3. Inhibition of morphine antinociception

naloxone

The intracerebroventricular (icv) administration of a

drug is the ﬁrst analysis of its potential central role in

aﬀecting morphine-induced antinociception using the

classic hot-plate or tail-ﬂick tests. [N-Allyl-Dmt¹]EM-2

(12) alone failed to show any signiﬁcant icv eﬀect at

the same dosages used under identical conditions (hot-

plate test) for the morphine-induced antinociception;

on the other hand, 12 was a potent l-opioid antagonist

in a dose-dependent manner against morphine, such that

the observed eﬀect implies that supraspinally (CNS) l-

opioid receptors are involved (Fig. 1) (infra vide).²⁵

The %MPE (percent of maximal possible eﬀect) at

-13 -12 -11 -10 -9 -8 -7 -6

Log dose (g)

Figure 1. The eﬀect of intracerebroventricularly injected [N-allyl-

Dmt¹]EM-2 (12) on morphine (Mor)-induced antinociception (4 mg/kg

sc) in the hot-plate test in mice (a) Time course. (b) Area under the

dose–response curve (AUC). (c) Dose–response curve of 12 and

naloxone at 10 min after icv injection. Each value is the mean SE

(n = 10–17 mice per point). ANOVA and Dunnett’s test were used to

analyze the data. ***P < 0.001 and *P < 0.05 represent signiﬁcant

diﬀerences in the AUC from morphine-treated mice.

12

10 min post-injection of 12 revealed that the AD₅₀

(0.148 ng/mouse, 95% CL₉₅: 0.02–0.993 ng/mouse) was

naloxone

16 times more potent than naloxone (AD₅₀

¼

2:38 ng=mouse, 95% CL₉₅: 0.935–6.00 ng/mouse).

Although, there is little diﬀerence between the antinoci-

ception produced by the highest dose for 12 and nalox-

one in Figure 1c, a further increase in naloxone

completely blocked morphine antinociception; however,

12 failed to show this eﬀect. Moreover, in contrast to

many alkaloid opiates and peptidic l-opioid agonists,

12 only acts supraspinally (CNS) due to the absence of

l-opioid antagonism in the classic and accepted

pharmacological tail-ﬂick test which measures spinal

nociceptive mechanisms (Fig. 2a and b); that is, the

tail-ﬂick test indicates whether a spinal mechanism is in-

volved in pain perception by activating a descending

mechanism regardless of the type of injection or route

of administration.

3.4. Changes in spontaneous IPSC frequency

The inhibition of ethanol-induced frequency of sponta-

neous IPSC (a constituent component in the promotion

of GABA_Areceptor-mediated neuronal function) in

vitro (patch-clamp analysis of isolated neurons in hippo-

campal slices) veriﬁed the direct action of 15 as a l-opioid

antagonist on neuronal activity (Fig. 3). Spontaneous

IPSC is one of the most potent CNS actions initiated

by ethanol (EtOH) and constitutes an underlying mech-

anism of the sedative²⁶and anxiolytic properties²⁷of

EtOH. In the presence of glutamatergic receptor block-

ers 50 lM AVP (^D-2-amino-5-phosphonovalerate) and

1242

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

and 15 are at least two orders of magnitude more potent

(Li; Swartzwelder, et al., unpublished data, in

preparation).

a

100

90

80

70

60

50

40

30

20

10

0

Mor

Mor+ 0.07ng 12

Mor+ 0.67ng 12

Mor+ 6.7ng 12

Mor+ 67ng 12

4. Conclusions

The present study validates the conversion of l-opioid

receptor agonists to potent l-opioid antagonists by

N-allylation; and the N-allyl-derivatives of [H-Dmt¹]

endomorphins,^17abis-[H-Dmt-amino]-alkane,¹⁸and

3,6-bis-[H-Dmt-aminoalkyl]-2(1H)-pyrazinone¹⁹could

generate new drug leads. Moreover, this approach

would be applicable to producing analogues on large-

scale synthesis with considerable ease relative to the

replacement of the C-terminal phenylalanyl in endomor-

phin that only yielded weak l-opioid antagonists.¹⁵Our

data provided evidence that [N-allyl-Dmt¹]endomor-

phin-1 (15) and -2 (12) are pharmacological probes that

could be used to study the role of l-opioid receptors in

mediating physiological and pharmacological processes

in the CNS. Their potential value could be to eﬀectively

combat alkaloid drug addiction or alleviate alcohol

dependency or abuse (Figs. 1–3) due to the involvement

of the l-opioid receptor in the neural reward pathway²

and the abolishment of spontaneous IPSC (Fig. 3). Fur-

ther studies are underway to deﬁne the biochemical

mechanisms of their l-opioid antagonist action, and

the physiological abatement of morphine and alcohol-

mediated changes in vitro and in vivo.

pre 10 20 30 40 50 60 70

time after administration (min)

300

b

250

200

150

100

50

0

Mor (0.5 μg/mouse)

12

0.07 0.67

6.7

ng/mouse

67

Figure 2. The eﬀect of intracerebroventricularly injected [N-allyl-

Dmt¹]EM-2 (12) on morphine (Mor)-induced antinociception (0.5 lg/

mouse icv) in the tail-ﬂick test in mice. (a) Time course. (b) Area under

the dose–response curve (AUC). Each value is the mean SE (n = 5–6

mice per point). ANOVA and Dunnett’s test were used to analyze the

data.

5. Experimental

5.1. Chemistry general

20 lM DNOX (6,7-dinitroquinoxaline-2,3-dione) to

block excitory synaptic transmission, spontaneous IPS-

Cs were isolated from hippocampal CA1 pyramidal cells

held at ꢀ70 mV. Similar to our previous report,²⁸after

the baseline (control) spontaneous IPSCs were estab-

lished, addition of 60 mM EtOH to the infusion medium

signiﬁcantly increased average frequency of spontaneous

IPSCs of hippocampal CA1 pyramidal cells by 26% of

control (t_[5]= 4.40, P = 0.007) and was reversed with

300 nM 15 (t_[5]= 2.87, P = 0.0055) (Fig. 3). Further-

more, 15 did not reduce the baseline recordings of the

spontaneous IPSC frequency (t_[5]= 1.05, P = 0.341)

indicative that its eﬀect was only on the elevation elicited

by EtOH and complements the in vivo antagonism of

morphine antinociception that it acted as an antagonist

(Fig. 1). Sedative and anxiolytic actions are involved in

the reinforcing properties and addictive liability of etha-

nol, and drugs that attenuate these actions at the l-opi-

oid receptor²could be used therapeutically for the

treatment of EtOH dependency. Furthermore, 15 was

potent in the nanomolar range in contrast to naloxone

and naltrexone, which are well known to inhibit GABA

(c-aminobutyric acid)-mediated chloride uptake into

hippocampal synaptoneurosomes at micromolar con-

centrations.²⁹In fact, similar results were also obtained

with 12 at a comparable concentration in which naltrex-

one was active only in the 30–60 lM range (based on

data available in the literature), demonstrating that 12

Melting points were determined on a Yanagimoto

micromelting point apparatus and are uncorrected.

TLC was performed on precoated plates of silica gel

F254 (Merck, Darmstadt, Germany). R_fvalues refer

to the following solvent systems: (1) AcOEt/hex-

ane = 1:1, (2) AcOEt/hexane = 1:2, (3) AcOEt/hexn-

ane = 10:1, (4) AcOEt, (5) AcOEt/MeOH = 10:1, (6)

AcOEt/MeOH = 20:1, (7) AcOEt/hexane = 2:1, (8)

n-BuOH/H₂O/AcOH = 4:1:5 (upper layer), (9) CHCl₃/

MeOH/H₂O = 8:3:1 (lower layer). Optical rotations

were determined with a DIP-1000 automatic polarime-

ter (Japan Spectroscopic Co.). Analytical RP-HPLC

and semi-preparative RP-HPLC used are Waters Delta

600 with COSMOSIL C18 column (4.6 mm · 250 mm)

and COSMOSIL C18 column (20 mm · 250 mm),

respectively. The solvent for analytical HPLC was as

follows: A, 0.05% TFA in water; B, 0.05% TFA in

CH₃CN. The column was eluted at a ﬂow rate of

1 mL/min with a linear gradient of 90% A to 10% A

in 30 min; the retention time is reported as t_R(min).

Mass spectra were measured with a KRATOS MAL-

DI-TOF (matrix-assisted laser desorption ionization

time-of-ﬂight mass spectrometry). ¹H and ¹³C NMR

spectra were measured on a Bruker DPX-400 spec-

trometer at 25 ꢁC. Chemical shift values are expressed

as ppm downﬁeld from tetramethylsilane, used as an

internal standard (d value).

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1243

Control

EtOH (60 mM)

EtOH (60 mM) and 15 (300 nM)

140

120

100

80

*

60

n=6

40

20

0

Control

EtOH (60 mM)

15 (300 nM)

Figure 3. Changes in spontaneous IPSC frequency in the presence of ethanol and ethanol plus [N-allyl-Dmt¹]endomorphin-1 (15). All the recordings

in this ﬁgure were recorded from single neurons in hippocampal slices in vitro using patch-clamp methods. Upper ﬁgure: from top to bottom, the

recordings refer to baseline spontaneous IPSC recordings, plus 60 mM EtOH, and with 300 nM 15 in 60 mM EtOH. Scale bar = 500 ms/50 pA.

Bottom ﬁgure: the bars represent the percentage changes in mean spontaneous IPSC frequency (mean SE) with n = 6 tissue slices per measurement.

Left, baseline; middle, plus 60 mM EtOH (26%, t_[5]= 4.40, P = 0.007); and right, the addition of 300 nM 15 in 60 mM EtOH (t_[5]= 2.87, P = 0.0055;

denotes signiﬁcant diﬀerence).

*

5.2. General methods for peptide synthesis

The AcOEt layer was dried over Na₂SO₄and evaporat-

ed. Petroleum ether was added to the residue to give

crystals, which were collected by ﬁltration. The crude

Boc-Dmt-Pro-OMe was puriﬁed by ﬂash chromatogra-

Gram quantities of H-^L-Dmt-OH were prepared

according to Dygos et al.,³⁰and the syntheses of the

endomorphins,¹⁷1,6-bis-[H-Dmt-NH]-hexane,¹⁸and

3,6-bis-[H-Dmt-aminopropyl]-2(1H)-pyrazinone¹⁹followed

published methods. The bold numbers in the text refer

to the synthetic products (Schemes 1–3).

phy (SiO₂, AcOEt/hexane = 1:1). Yield 2.16 g (66.3%),

20

D

Anal. Calcd for C₂₂H₃₂N₂O₆: C, 62.84; H, 7.67; N, 6.66.

mp 144–145 ꢁC, R_f1= 0.45, ½aꢁ +16.5ꢁ (c 0.63, MeOH).

1

Found: C, 62.72; H, 7.68; N, 6.65. H NMR (CDCl₃)d:

6.51 (s, 2H), 5.54 (d, 0.4H, J = 8.4 Hz), 5.31 (d, 0.6H,

J = 8.9 Hz), 4.72 (q, 0.6H, J = 8.7 Hz), 4.49 (br, 0.6H),

4.40 (q, 0.4H, J = 7.0 Hz), 3.73 and 3.69 (s · 2, 3H),

3.65–3.55 (m, 1H), 3.38–3.30 (m, 0.8H), 3.13–2.89 (m,

2.6H), 2.34 and 2.31 (s · 2, 6H), 2.15–1.50 (m, 4H),

1.43 and 1.34 (s · 2, 9H).

5.3. Synthesis of [H-Dmt¹]endomorphin-1 ([H-Dmt¹]EM-

1)

5.3.1. Boc-Dmt-Pro-OCH₃. To a solution of HClÆH-Pro-

OMe (1.28 g, 7.75 mmol) in N,N-dimethylformamide

(DMF) (50 mL) were added Boc-Dmt-OH (tert-butyl-

oxycarbonyl-Dmt-OH, 2.39 g, 7.75 mmol), benztriazol-

5.3.2. Boc-Dmt-Pro-OH. Boc-Dmt-Pro-OCH₃(1.89 g,

4.5 mmol) in methanol (MeOH; 10 mL) was treated with

1.0 N NaOH (11.2 mL, 11.2 mmol) for 2 h at room tem-

perature. MeOH was removed in vacuo, the pH of the

residue was adjusted to 3 with 10% citric acid, and the

resulting precipitate was extracted with AcOEt (3·

30 mL). The combined organic phase was washed with

saturated NaCl (3· 30 mL), dried over Na₂SO₄, and

1-yloxytris-(pyrrolidino)phosphate

(PyBop) (4.4 g,

8.52 mmol), and diisopropylethylamine (DIPEA)

(3.37 mL, 19.4 mol) at 0 ꢁC. The reaction mixture was

stirred at room temperature overnight. After removal

of DMF, the residue was dissolved in ethyl acetate

(AcOEt) and washed with 10% citric acid solution, 5%

Na₂CO₃solution, and saturated NaCl aqueous solution.

1244

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

evaporated down. Petroleum ether was added to the res-

idue to produce crystals, which were ﬁltered and dried in

5.4. Synthesis of allylated derivatives

vacuo. Yield 1.79 g (98.0%), mp 84–87 ꢁC, R_f2= 0.25,

5.4.1. N,N-(Allyl)₂-Tyr-OMe (2a) and N-allyl-Tyr-OMe

(3a). To a solution of HClÆH-Tyr-OMe (2.0 g, 8.6 mmol)

in MeOH (20 mL), DIPEA (4.8 mL, 27.5 mmol) and ally-

lbromide (1.86 mL, 21.5 mmol) were added in sequence.

After stirred at room temperature for 20 min, the temper-

ature was raised to 50 ꢁC and the reaction mixture kept at

this temperature for 5 h. The solvent was removed in vac-

uo, and the residue was extracted with AcOEt (3· 30 mL).

The combined extracts were washed with saturated NaCl,

dried over Na₂SO₄, ﬁltered, and concentrated under re-

duced pressure. The residue was puriﬁed by ﬂash chroma-

tography (SiO₂, AcOEt/hexane = 1:1) to give pure 2a

20

½aꢁ

ꢀ14.8ꢁ (c 0.54, MeOH). Anal. Calcd for

D

C₂₁H₃₀N₂O₆: C, 62.05; H, 7.44; N, 6.89. Found: C,

61.99; H, 7.70; N, 6.76. ¹H NMR (CDCl₃) d: 6.52

(s · 2, 2H), 5.80 (d, 0.3H, J = 8.2 Hz), 5.45 (d, 0.7H,

J = 8.7 Hz), 4.72 (br, 0.7H), 4.56 (dd, 0.7H, J = 8.0,

3.8 Hz), 4.40 (br, 0.3H), 3.64–3.53 (m, 1H), 3.41–3.29

(m, 0.6H), 3.14–2.85 (m, 2.7H), 2.34 and 2.31 (s · 2,

6H), 2.15–1.50 (m, 4H), 1.42 and 1.37 (s · 2, 9H).

5.3.3. Boc-Dmt-Pro-Trp-Phe-NH₂. To a solution of

HClÆH-Trp-Phe-NH₂[prepared from Boc-Trp-Phe-

NH₂(200 mg, 0.44 mmol), 7.7 N HCl/dioxane

(570 lL, 4.4 mmol)] in tetrahydrofuran (THF; 30 mL)

containing DIPEA (230 lL, 1.32 mmol), Boc-Dmt-

Pro-OH (180 mg, 0.44 mmol) and PyBop (250 mg,

0.48 mmol) were added. The reaction mixture was stir-

red for 6 h at 35 ꢁC. After removal of the solvent, the

residue was extracted with AcOEt (50 mL), and the

extract was washed with 10% citric acid, 5% Na₂CO₃,

and saturated NaCl, dried over Na₂SO₄, and evapo-

rated. The residue was puriﬁed by ﬂash chromatogra-

(1.34 g, 59.3%) and 3a (740 mg, 36.4%). Compound 2a:

20

D

Anal. Calcd for C₁₆H₂₁NO₃: C, 69.79; H, 7.69; N, 5.09.

mp 127–128 ꢁC, R_f1= 0.79, ½aꢁ ꢀ198.0ꢁ (c 0.63, MeOH).

1

Found: C, 69.51; H, 7.81; N, 5.22. H NMR (400 MHz,

CDCl₃) d: 7.04 (d, 2H, J = 8.32 Hz), 6.73 (d, 2H,

J = 8.32 Hz), 5.77–5.58 (m, 2H), 5.20–5.00 (m, 4H), 4.68

(br, 1H), 3.70–3.55 (m, 4H), 3.44–3.30 (m, 2H), 3.17–

2.93 (m, 3H), 2.90–2.75 (m, 1H). Compound 3a: mp

20

D

Anal. Calcd for C₁₃H₁₇NO₃: C, 66.36; H, 7.28; N, 5.95.

117–118 ꢁC, R_f4= 0.74, ½aꢁ +27.0ꢁ (c 0.51, MeOH).

1

Found: C, 66.14; H, 7.13; N, 5.94. H NMR (400 MHz,

phy (SiO₂, AcOEt/MeOH = 10:1). Yield 223 mg

20

D

(68.6%), mp 150–152 ꢁC, R_f3= 0.57, ½aꢁ

ꢀ34.9ꢁ

CDCl₃) d: 7.02 (d, 2H, J = 8.56 Hz), 6.71 (d, 2H,

J = 8.56 Hz), 5.88–5.76 (m, 1H), 5.18–5.07 (m, 2H), 3.67

(s, 3H), 3.57 (t, 1H, J = 6.6 Hz), 3.34–3.27 (m, 1H),

3.20–3.12 (m, 1H), 2.94 (d, 2H, J = 6.6 Hz).

(c 0.49, MeOH). Anal. Calcd for C₄₁H₅₀N₆O₇Æ1.5H₂O:

C, 64.30; H, 6.97; N, 10.97. Found: C, 64.47; H, 6.89;

N, 10.75. ¹H NMR (400 MHz, DMSO-d₆)d: 9.28 (s,

0.63H), 8.85–8.47 (m, 1.2H), 8.22 (s, 0.53H), 8.00–

7.87 (m, 0.73H), 7.63–6.82 (m, 11.32H), 6.82–6.60

(m, 0.23H), 6.60–6.33 (m, 1.64H), 6.25 (s, 0.44H),

6.15–5.90 (m, 0.5H), 5.66 (d, 0.16H, J = 8.03 Hz),

5.67 (br, 0.48H), 5.26 (br, 0.27H), 4.80–4.59 (m,

1H), 4.52 (br, 1H), 4.46–4.10 (m, 1.24H), 4.10–3.84

(m, 1H), 3.60–3.24 (m, 1.41H), 3.24–2.45 (m, 5.5H),

2.45–2.00 (m, 5H), 1.90–0.90 (m, 13.27H), 0.70–0.40

(m, 1.46H).

5.4.2. N,N-(Allyl)₂-Dmt-OMe (2b) and N-allyl-Dmt-

OMe (3b). To a solution of HClÆH-Dmt-OMe (3.0 g,

11.5 mmol) in MeOH (60 mL), DIPEA (6.0 mL,

34.6 mmol) and allylbromide (2.0 mL, 23.1 mmol) were

added in sequence. After stirred at room temperature

for 20 min, the temperature was raised to 50 ꢁC and

the reaction mixture kept at this temperature for 5 h.

The solvent was removed in vacuo, the residue extracted

with AcOEt (3· 50 mL). The combined extracts were

washed with saturated NaCl, dried over Na₂SO₄, ﬁl-

tered, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography (SiO₂,

5.3.4. H-Dmt-Pro-Trp-Phe-NH₂ÆTFA (4⁰). Boc-Dmt-

Pro-Trp-Phe-NH₂(100 mg, 0.136 mmol) was treated

with 7.7 N HCl/dioxane (0.18 mL, 1.36 mmol) and

anisole (0.15 mL, 1.36 mmol) for 1 h at room temper-

ature. Et₂O was added to the reaction mixture to pre-

cipitate the product. The crude product was collected

by centrifugation, dried over KOH pellets, and puri-

ﬁed by semi-preparative reverse phase high pressure li-

AcOEt/hexane = 1:1) to give pure 2b 1.05 g (30%) and

3b 1.72 g (56.7%). Compound 2b: oil, R_f1= 0.76; ½aꢁ

20

D

+2.5ꢁ (c 0.55, MeOH). Anal. Calcd for C₁₈H₂₅NO₃: C,

71.26; H, 8.31; N, 4.62. Found: C, 71.04; H, 8.38; N,

1

4.65. H NMR (400 MHz, DMSO-d₆) d: 8.93 (s, 1H),

quid chromatography (RP-HPLC). Yield 86.8 mg

6.37 (s, 2H), 5.74-5.63 (m, 2H), 5.20-5.04 (m, 4H), 3.50

(s, 3H), 3.46 (dd, 1H, J = 5.41, 8.74 Hz), 3.93-3.43 (m,

1H), 3.39-3.36 (m, 1H), 3.04-2.95 (m, 3H), 2.70 (dd,

20

D

(85.1%), t_R16.8 min, ½aꢁ ꢀ8.8ꢁ (c 0.41, H₂O), m/z

[M+H]⁺640. Anal. Calcd for C₄₆H₅₄N₄O₈ÆTFAÆ3H₂O:

C, 56.57; H, 6.12; N, 10.42. Found: C, 56.31; H, 5.98;

1H, J = 5.40, 14.12 Hz), 2.16 (s, 6H). Compound 3b:

20

D

1

N, 10.28. H NMR (400 MHz, DMSO-d₆)d: 10.83 and

oil, R_f1= 0.53; ½aꢁ +42.1ꢁ (c 0.34, MeOH). Anal. Calcd

10.81 (s · 2, 1H), 9.15 (br, 1H), 8.36 and 8.25 (br,

s · 2, 3H), 8.07–7.97 (m, 1.6H), 7.79 (d, 0.4H,

J = 8.1 Hz), 7.58 (d, 1H, J = 7.81 Hz), 7.46 (s, 0.5H),

7.36–7.30 (m, 1H), 7.27–7.10 (m, 6.5H), 7.10–7.03

(m, 2H), 7.03–6.95 (m, 1H), 6.44 and 6.42 (s · 2,

2H), 4.53–4.32 (m, 2.4H), 4.20–4.10 (m, 0.6H), 3.60–

3.50 (m, 0.6H), 3.42–3.30 (m, 1H), 3.21–3.12 (m,

0.7H), 3.08–2.76 (m, 6.7H), 2.34–2.23 (m, 0.4H), 2.16

(s, 2.5H), 1.98 (s, 3.5H), 1.87–1.73 (m, 0.4H), 1.67–

1.52 (m, 1.6H), 1.50–1.39 (m, 0.6H), 1.39–1.20 (m,

1H).

for C₁₅H₂₁NO₃Æ0.17H₂O: C, 67.64; H, 8.07; N, 5.26.

Found: C, 67.52; H, 8.08; N, 5.27. ¹H NMR

(400 MHz, DMSO-d₆) d: 8.93 (s, 1H), 6.38 (s, 2H),

5.78–5.60 (m, 1H), 5.08–4.95 (m, 2H), 3.53 (m, 3H),

3.29 (t, 1H, J = 9.28 Hz), 3.17–3.08 (m, 1H), 2.98–2.90

(m, 1H), 2.83–2.70 (m, 2H), 2.16 (s, 6H).

5.4.3. N,N-(Allyl)₂-Tyr-OH (4a). In an ice bath, 1 N

NaOH (11 mL, 11 mmol) was added to a solution of

N,N-(allyl)₂-Tyr-OMe (2a) (1.31 g, 4.8 mmol) in MeOH

(15 mL) and the resulting solution was stirred at room

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1245

temperature overnight. The solvent was removed in vac-

uo, the pH of the residue was adjusted to 3, and the

resulting precipitate was ﬁltered, washed with cold

and saturated NaCl (2· 15 mL), dried over Na₂SO₄, ﬁl-

tered, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography (SiO₂,

water, and dried in vacuo. Yield 866 mg (69%), mp

AcOEt/hexane = 1:1) to give pure 5b. Yield 1.12 g

20

D

20

D

214–216 ꢁC, R_f8= 0.43, ½aꢁ ꢀ7.5ꢁ (c 0.48, MeOH).

(53%), oil, R_f1= 0.65, ½aꢁ ꢀ174.5ꢁ (c 0.49, MeOH).

Anal. Calcd for C₁₅H₁₉NO₃Æ0.5H₂O: C, 66.65; H, 7.46;

N, 5.18. Found: C, 66.72; H, 7.25; N, 5.13. H NMR

Anal. Calcd for C₂₀H₂₉NO₅Æ0.25H₂O: C, 65.28; H,

8.08; N, 3.81. Found: C, 65.27; H, 8.10; N, 3.80. H

1

(400 MHz, DMSO-d₆) d: 12.20 (br, 1H), 9.20 (br, 1H),

6.95 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz),

5.73–5.56 (m, 2H), 5.11 (d, 2H, J = 17.24 Hz), 5.04 (d,

2H, J = 10.24 Hz), 3.43 (t, 1H, J = 7.54 Hz), 3.28 (dd,

2H, J = 5.05, 14.8 Hz), 3.06 (dd, 2H, J = 6.92,

14.80 Hz), 2.82 (dd, 1H, J = 7.82, 13.65 Hz), 2.67 (dd,

1H, J = 7.31, 13.65 Hz).

NMR (400 MHz, DMSO-d₆) d: 8.98 (s, 1H), 6.41 (s,

2H), 5.40–5.20 (m, 1H), 5.00–4.83 (m, 2H), 3.96–3.83

(m, 1H), 3.80–3.53 (m, 4H), 3.22–2.96 (m, 3H), 2.12 (s,

6H), 1.36 (s, 9H).

5.4.7. N-Allyl-N-Boc-Tyr-OH (6a). 1 N NaOH

(9.33 mL, 9.33 mmol) was added to a solution of N-al-

lyl-N-Boc-Tyr-OMe (5a) (1.36 g, 4.05 mmol) in MeOH

(10 mL) at 0 ꢁC. After stirred at 0 ꢁC for 20 min, the

solution was stirred at room temperature overnight.

MeOH was removed in vacuo, and the pH of the residue

was adjusted to 3 with 10% citric acid. The resulting pre-

cipitate was extracted with AcOEt (2· 40 mL), and the

combined extracts were washed with saturated NaCl

solution (2· 20 mL), dried over Na₂SO₄, ﬁltered, and

5.4.4. N,N-(Allyl)₂-Dmt-OH (4b). In an ice bath, 1 N

NaOH (8 mL, 8 mmol) was added to a solution of

N,N-(allyl)₂-Dmt-OMe (2b) (967 mg, 3.19 mmol) in

MeOH (10 mL) and the resulting solution was stirred

at room temperature overnight. The solvent was re-

moved in vacuo, the pH of the residue was adjusted to

3, the resulting precipitate was extracted with AcOEt

(3· 50 mL), the combined extracts were washed with

saturated NaCl (2· 50 mL), dried over Na₂SO₄, ﬁltered,

and puriﬁed by ﬂash chromatography (SiO₂, AcOEt/

concentrated in vacuo to give 6a as a clear oil. Yield

20

D

1.09 g (84%), R_f5= 0.70, ½aꢁ ꢀ153.2ꢁ (c 0.83, MeOH).

Anal. Calcd for C₁₇H₂₃NO₅Æ0.2H₂O: C, 62.83; H, 7.26;

N, 4.31. Found: C, 63.06; H, 7.48; N, 4.06. H NMR

1

MeOH = 10:1). Yield 428 mg (46.5%), mp 205–207 ꢁC,

20

R_f5= 0.50, ½aꢁ +25.2ꢁ (c 0.68, MeOH). Anal. Calcd

(400 MHz, DMSO-d₆) d: 12.55 (s, 1H), 9.17 (s, 1H),

6.95 (d, 2H, J = 8.4 Hz), 6.83–6.58 (m, 2H), 5.54–5.35

(m, 1H), 5.01–4.87 (m, 2H), 4.35–4.24 (m, 0.36H),

4.05–3.95 (m, 0.57H), 3.79–3.60 (m, 1H), 3.35–3.25 (m,

0.5H), 3.18–2.87 (m, 2.57H), 1.35 (s, 9H).

D

for C₁₇H₂₃NO₃Æ0.1H₂O: C, 70.12; H, 8.03; N, 4.81.

Found: C, 69.91; H, 7.94; N, 4.77. ¹H NMR

(400 MHz, DMSO-d₆) d: 12.20 (br, 1H), 8.89 (s, 1H),

6.37 (s, 1H), 5.74–5.62 (m, 2H), 5.13 (dd, 2H, J = 1.29,

17.21 Hz), 5.05 (dd, 2H, J = 1.29, 10.23 Hz), 3.42–3.34

(m, 3H), 3.06–2.90 (m, 3H), 2.66 (dd, 1H, J = 5.70,

14.1 Hz).

5.4.8. N-Allyl-N-Boc-Dmt-OH (6b). 1 N NaOH

(7.15 mL, 7.15 mmol) was added to a solution of N-al-

lyl-N-Boc-Dmt-OMe (5b) (1.04 g, 2.86 mmol) in MeOH

(10 mL) at 0 ꢁC. After stirred at 0 ꢁC for 20 min, the

solution was stirred at room temperature overnight.

MeOH was removed in vacuo, and the pH of the residue

was adjusted to 3 with 10% citric acid. The resulting pre-

cipitate was extracted with AcOEt (2· 40 mL), and the

combined extracts were washed with saturated NaCl

solution (2· 20 mL), dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography (SiO₂, AcOEt/hexane = 1:1) to give the

5.4.5. N-Allyl-N-Boc-Tyr-OMe (5a). N-Allyl-Tyr-OMe

(3a) (580 mg, 2.5 mmol) was reacted with di-tert-butyl

dicarbonate (Boc₂O, 600 mg, 2.75 mmol) in dioxane

(15 mL) containing triethylamine (Et₃N) (380 lL,

2.75 mmol) at room temperature overnight. After

removal of the solvent, the residue was extracted with

AcOEt (60 mL), and the extract was washed with 10%

citric acid (3· 10 mL), 5% NaHCO₃(3· 10 mL), and sat-

urated NaCl (3· 10 mL), dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure. The residue

was puriﬁed by ﬂash chromatography (SiO₂, AcOEt/

title compound. Yield 830 mg (83%), mp 150–152 ꢁC,

20

R_f1= 0.35, ½aꢁ ꢀ185.8ꢁ (c 0.61, MeOH). Anal. Calcd

D

for C₁₉H₂₇NO₅: C, 65.31; H, 7.79; N, 4.01. Found: C,

hexane = 1:3) to give pure 5a. Yield 580 mg (69.4%),

20

D

1

65.20; H, 7.58; N, 3.98. H NMR (400 MHz, CDCl₃)

oil, R_f1= 0.82, ½aꢁ ꢀ13.5ꢁ (c 0.83, MeOH). Anal. Calcd

for C₁₈H₂₅NO₅Æ0.2H₂O: C, 63.77; H, 7.55; N, 4.13.

Found: C, 63.76; H, 7.51; N, 4.00. ¹H NMR

(400 MHz, CDCl₃) d: 7.10-6.97 (m, 2H), 6.80–6.70 (m,

2H), 5.65–5.44 (m, 1.3H), 5.01 (d, 2H, J = 14.52 Hz),

4.47–4.37 (m, 0.3H), 4.00–3.85 (m, 1.2H), 3.80-3.62

(m, 3.3H), 3.46–3.36 (m, 0.4H), 3.27–3.03 (m, 2.5H),

1.45 (s, 9H).

d: 6.52 (s, 2H), 5.56–5.35 (m, 1H), 5.00–4.85 (m, 2H),

4.10–3.80 (m, 2H), 3.50–2.80 (m, 3H), 2.24 (s, 6H),

1.48 (s, 9H).

5.4.9. [N,N-Diallyl-Tyr¹]EM-2ÆHCl (7). Boc-Pro-Phe-

Phe-NH₂(220 mg, 0.433 mmol) was treated with 6.7 N

HCl/dioxane (1.3 mL, 8.66 mmol) at room temperature

for 30 min to remove the Boc group. The product was

precipitated with ether, ﬁltered, and dried in vacuo.

The resulting hydrochloride salt was dissolved in

DMF (10 mL) containing DIPEA (190 lL 1.09 mmol),

and to this solution N,N-(allyl)₂-Tyr-OH (4a) (124 mg,

0.476 mmol) and PyBop (260 mg, 0.5 mmol) were add-

ed. The reaction mixture was stirred at 0 ꢁC for

10 min, then at room temperature for 4 h. After removal

5.4.6. N-Allyl-N-Boc-Dmt-OMe (5b). N-Allyl-Dmt-OMe

(3b) (1.53 g, 5.79 mmol) was reacted with Boc₂O (1.39 g,

6.37 mmol) in dioxane (30 mL) containing Et₃N

(890 lL, 6.37 mmol) at room temperature overnight.

After removal of the solvent, the residue was extracted

with AcOEt (100 mL), and the extract was washed with

10% citric acid (2· 15 mL), 5% NaHCO₃(2· 15 mL),

1246

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

of the solvent, the residue was diluted with AcOEt

(80 mL). The dilution was washed with cold 10% citric

acid (3· 10 mL), 5% Na₂CO₃(3· 10 mL), and saturated

NaCl (3· 10 mL), dried over Na₂SO₄, and evaporated to

dryness. The residue was puriﬁed by ﬂash chromatogra-

phy (SiO₂, AcOEt/MeOH = 10:1). The compound was

precipitated with hexane, ﬁltered, and dried in vacuo.

The solid was dissolved in MeOH, and TFA (36 lL,

0.472 mmol) was added. After concentrated to dryness,

the residue was puriﬁed by semi-preparative HPLC,

and the puriﬁed peptide was lyophilized from 1 N HCl

(m, 5.5H, CH₂@CH–CH₂–, CH₂@CH–CH₂–), 4.46–

4.35 (m, 2.0H, Phe aH), 4.35–4.28 (m, 0.27H, Pro

trans-aH), 4.17–3.70 (m, 3.5H, Dmt trans-aH,

CH₂@CH–CH₂–), 3.41–2.74 (m, 10.23H, Pro cis-aH,

Dmt cis-aH, CH₂@CH–CH₂–, Pro dH, Phe bH, Dmt

bH), 2.15 and 2.08 (s · 2, 6.0H, Dmt CH₃), 1.83–1.13

(m, 4.0H, Pro bH, Pro cH). ¹³C NMR d: 172.55,

170.66, 170.32, 170.25, 165.88, 156.03, 138.45, 138.42,

138.02, 137.62, 137.54 (11q), 129.11, 129.07, 128.96,

127.97, 127.93 (5t, Phe Ar-C), 127.87 (t, CH₂@CH–

CH₂–), 126.24, 126.10 (2t, Phe Ar-C), 125.40

(s, CH₂@CH–CH₂–), 119.69 (q), 115.18, 115.05 (2t,

Dmt Ar-C), 60.77 (t, Pro cis-aC), 59.95 (t, Por trans-

aC), 59.13 (t, Dmt trans-aC), 58.87 (t, Dmt cis-aC),

55.12 (t, Phe cis-aC), 54.20 (s, CH₂@CH–CH₂–), 54.04

(t, Phe trans-aC), 53.80 (t, Phe cis-aC), 53.62 (t, Phe

trans-aC), 53.22, 51.41 (2s, CH₂@CH–CH₂–), 46.99 (s,

Pro cis-dC), 45.99 (s, Pro trans-dC), 37.45, 37.22, 36.69

(3s, Phe bC), 31.33 (s, Pro cis-bC), 28.67 (s, Pro trans-

bC), 27.71 (s, Dmt bC), 23.88 (s, Pro trans-cC), 21.17

(s, Pro cis-cC), 20.22 (p, Dmt trans-CH₃), 19.76 (p,

Dmt cis-CH₃).

(3· 320 lL) to give an amorphous powder. Yield

20

220 mg (73.8%), R_f9= 0.75, t_R16.40, ½aꢁ ꢀ30.1ꢁ

D

(c 0.57, H₂O), m/z [M+1]⁺653. Anal. Calcd for

C₃₈H₄₅N₅O₅ÆHClÆ2H₂O: C, 63.01; H, 6.96; N, 9.67.

Found: C, 63.02; H, 6.68; N, 9.64. ¹H NMR

(400 MHz, DMSO-d₆) d: 11.55 (br, 0.3H, Dmt NH⁺),

10.50 (br, 0.2H, Dmt NH⁺), 9.63 and 9.42 (br · 2,

0.6H, Dmt OH), 8.40 (d, 0.40H, J = 7.95 Hz, Phe cis-

NH), 8.15 (d, 0.40H, J = 8.15 Hz, Phe cis-NH), 8.11

(d, 0.54H, J = 8.22 Hz, Phe trans-NH), 7.99 (d, 0.52H,

J = 7.76 Hz, Phe trans-NH), 7.38–7.04 (m, 13.1H, Phe

Ar-H, CONH₂, Tyr Ar-H), 6.94 (d, 0.90H,

J = 8.44 Hz, Tyr Ar-H), 6.76 (d, 0.90H, J = 8.44 Hz,

Tyr Ar-H), 6.69 (d, 1.1H, J = 8.42 Hz, Tyr Ar-H),

6.15–5.97 (m, 1.0H, CH₂@CH–CH₂–), 5.86–5.72 (m,

5.4.11. [N-Allyl-N-Boc-Tyr]EM-2 (9). Boc-Pro-Phe-Phe-

NH₂(220 mg, 0.433 mmol) was treated with 6.7 N HCl/

dioxane (1.3 mL, 8.66 mmol) at room temperature for

30 min to remove the Boc group. The product was pre-

cipitated with ether, ﬁltered, and dried in vacuo. The

resulting hydrochloride salt was dissolved in DMF

(10 mL) containing DIPEA (190 lL, 1.09 mmol), and

to this solution N-allyl-N-Boc-Tyr-OH (6a) (153 mg,

0.476 mmol) and PyBop (260 mg, 0.5 mmol) were add-

ed. The reaction mixture was stirred at 0 ꢁC for

10 min, then at room temperature for 4 h. After removal

of the solvent, the residue was diluted with AcOEt

(80 mL). The dilution was washed with cold 10% citric

acid (3· 10 mL), 5% Na₂CO₃(3· 10 mL), and saturated

NaCl (3· 10 mL), dried over Na₂SO₄, and evaporated to

dryness. The residue was puriﬁed by ﬂash chromatogra-

phy (SiO₂, AcOEt). The compound was precipitated

1.0H, CH₂@CH–CH₂–), 5.72–5.20

(m, 4.0H,

CH₂@CH–CH₂–), 4.50–4.35 (m, 2.0H, Phe aH), 4.35–

4.28 (m, 0.57H, Pro trans-aH), 4.15–3.57 (m, 5.0H,

Tyr aH, CH₂@CH–CH₂–), 3.50–3.20 (m, 2.43H, Pro

cis-aH, Pro trans-dH, Pro cis-dH, Tyr trans-bH, Tyr

cis-bH), 3.20–2.73 (m, 5.54H, Pro cis-dH, Phe bH, Tyr

trans-bH, Tyr cis-bH), 2.23 (br, 0.46H, Pro trans-dH),

1.92–1.78 (m, 0.54H, Pro trans-bH), 1.66–1.45

(m, 2.08H, Pro cis-bH, Pro trans-bH, Pro trans-cH),

1.45–1.26 (m, 0.92H, Pro cis-cH), 1.27–1.10 (m, 0.46H,

Pro cis-bH). ¹³C NMR d: 172.54, 172.50, 170.56,

170.46, 165.50, 156.84, 156.39, 137.83, 137.72, 137.59

(11q), 130.62, 130.41 (2t, Tyr Ar-C), 129.13, 129.09,

129.01, 128.00, 127.96, 127.94, 127.89, 126.31, 126.13

(9t, Phe Ar-C), 124.40 (s, CH₂@CH–CH₂–), 115.41,

115.26 (2t, Tyr Ar-C), 63.46 (t, Tyr trans-aC), 62.03

(t, Tyr cis-aC), 59.85 (t, Pro trans-aC), 59.20 (t, Pro

cis-aC), 54.82 (t, Phe cis-aC), 54.43 (t, Phe trans-aC),

53.84 (t, Phe cis-aC), 53.74 (t, Phe trans-aC), 53.28

(s, CH₂@CH–CH₂-), 46.80 (s, Pro trans-dC), 46.54

(s, Pro cis-dC), 37.38, 37.21, 36.88 (3s, Phe bC), 33.68

(s, Tyr trans-bC), 32.74 (s, Tyr cis-bC), 30.98 (s, Pro

cis-bC), 28.63 (s, Pro trans-bC), 23.99 (s, Pro trans-

cC), 21.14 (s, Pro cis-cC).

with hexane, ﬁltered, and dried in vacuo. Yield 247 mg

20

D

(80%), mp 121-122 ꢁC, R_f4= 0.42, ½aꢁ ꢀ75.1ꢁ (c 0.58,

MeOH). Anal. Calcd for C₄₀H₄₉N₅O₇Æ0.9H₂O: C,

65.99; H, 7.03; N, 9.62. Found: C, 65.99; H, 6.85; N,

1

9.61. H NMR (400 MHz, DMSO-d₆) d: 9.14 and 9.07

(s · 2, 1.0H, Dmt OH), 8.02–7.88 (m, 1.0H, Phe NH),

7.85–7.76 (m, 1.0H, Phe NH), 7.30–7.08 (m, 12.0H,

Phe Ar-H, CONH₂), 6.98–6.89 (m, 2.0H, Tyr Ar-H),

6.69–6.57 (m, 2.0H, Tyr Ar-H), 5.73–5.59 (m, 1.0H,

CH₂@CH–CH₂–), 5.07–4.93 (m, 2.4H, CH₂@CH–

CH₂–, Tyr cis-aH), 4.83–4.71 (m, 0.6H, Tyr trans-aH),

4.47–4.35 (m, 2.0H, Phe aH), 4.16–4.06 (m, 1.0H, Pro

aH), 3.94–3.82 (m, 0.6H, trans CH₂@CH–CH₂–),

3.82–3.71 (m, 0.4H, cis CH₂@CH–CH₂–), 3.63–3.37

(m, 2.0H, CH₂@CH–CH₂–, Pro cis-dH, Pro trans-dH),

3.35–3.18 (m, 1.0H, Pro cis-dH, Pro trans-dH), 3.14–

2.95 (m, 2.0H, Phe bH), 2.95–2.63 (m, 4.0H, Phe bH,

Tyr bH), 1.90–1.57 (m, 4.0H, Pro bH, Pro cH), 1.25

and 1.14 (s · 2, 9.0H, Boc). ¹³C NMR d: 172.48,

171.31, 170.47, 169.25, 168.72, 155.75, 155.58, 154.36,

153.45, 137.82, 137.72 (11q), 134.70 (t, cis CH₂@CH–

CH₂–), 134.15 (t, trans CH₂@CH–CH₂–), 130.28,

130.05 (t, Tyr Ar-C), 128.98, 127.99, 127.89 (3t, Phe

5.4.10. [N,N-(Allyl)₂-Dmt¹]EM-2ÆHCl (8). The title com-

pound was synthesized using the same method for 7.

20

Yield 200 mg (71.3%), R_f9= 0.73, t_R17.28, ½aꢁ ꢀ15.6ꢁ

D

(c 0.44, H₂O), m/z [M+1]⁺681. Anal. Calcd for

C₄₀H₄₉N₅O₅ÆHClÆ3H₂O: C, 62.36; H, 7.33; N, 9.09.

Found: C, 62.59; H, 7.08; N, 9.13. ¹H NMR

(400 MHz, DMSO-d₆) d: 10.60 (br, 1H, Dmt NH⁺),

9.40 and 9.16 (br · 2, 1H, Dmt OH), 8.42 (br, 0.7H,

Phe NH), 8.19 (br, 0.7H, Phe NH), 7.99 (br, 0.6H,

Phe NH), 7.44 (s, 0.7H, CONH₂), 7.37–7.00 (m,

11.3H, Phe Ar-H, CONH₂), 6.47 and 6.41 (s · 2, Dmt

Ar-H), 6.15 (br, 0.5H, CH₂@CH–CH₂–), 5.92–5.28

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1247

Ar-C), 127.64, 127.27 (2q), 126.16, 126.10 (2t, Phe Ar-

C), 116.09 (s, trans CH₂@CH–CH₂–), 115.98 (s, cis

CH₂@CH–CH₂–), 114.77, 114.69 (2t, Tyr Ar-C), 79.04,

78.83 (2q, Boc), 60.04 (t, Pro aC), 58.04 (t, Tyr trans-

aC), 56.28 (t, Tyr cis-aC), 54.08, 53.98, 53.74 (3t, Phe

aC), 46.31 (s, Pro cis-dC), 46.13 (s, Pro trans-dC),

45.42 (s, cis CH₂@CH–CH₂–), 44.67 (s, trans

CH₂@CH–CH₂–), 37.27, 36.73, 36.67 (3s, Phe bC),

33.68 (s, Tyr bC), 28.48 (s, Pro bC), 27.73 (p, cis-Boc),

27.41 (p, trans-Boc), 23.98 (s, Pro cC).

J = 8.40 Hz, Tyr Ar-H), 6.75 (d, 1.0H, J = 8.37 Hz,

Tyr Ar-H), 6.69 (d, 1.0H, J = 8.42 Hz, Tyr Ar-H),

5.95–5.71 (m, 1.0H, CH₂@CH–CH₂–), 5.41–5.22 (m,

2.0H, CH₂@CH–CH₂–), 4.53–4.32 (m, 2.40H, Pro

trans-aH, Phe aH), 4.13 (br, 0.42H, Tyr cis-aH), 3.49

(br, 0.84H, trans CH₂@CH–CH₂–), 3.43–3.09 (m,

4.30H, Pro cis-aH, Tyr cis-aH, cis CH₂@CH–CH₂–,

Pro trans-dH, Pro cis-dH, Tyr cis-bH, Tyr trans-bH),

3.09–2.65 (m, 6.14H, cis CH₂@CH–CH₂–, Pro trans-

dH, Phe bH, Tyr cis-bH), 2.65–2.53 (m, 0.46H, Pro

trans-dH), 1.97–1.84 (m, 0.44H, Pro trans-bH), 1.71–

1.53 (m, 1.88H, Pro cis-bH, Pro trans-bH, Pro trans-

cH), 1.52–1.38 (m, 0.56H, Pro cis-cH), 1.38–1.20 (m,

1.12H, Pro cis-bH, Pro cis-cH). ¹³C NMR d: 172.65,

172.57, 170.48, 170.45, 170.40, 169.81, 165.84, 165.81,

156.72, 156.47, 137.92, 137.73, 137.47 (13q), 130.92,

130.84 (2t, Tyr Ar-C), 129.14, 129.11 (2t, Phe Ar-C),

129.09, 128.76 (2t, CH₂@CH–CH₂–), 127.99, 127.97,

127.94, 127.88, 126.64, 126.20 (6t, Phe Ar-C), 124.35,

123.42 (2q), 122.39, 121.98 (2s, CH₂@CH–CH₂–),

115.41, 115.15 (2t, Tyr Ar-C), 59.59 (s, Pro trans-aC),

59.19 (s, Pro cis-aC), 58.98 (s, Tyr cis-aC), 58.38

(s, Tyr cis-aC), 54.52, 54.19 (2s, Phe trans-aC), 53.78,

53.75 (2s, Phe cis-aC), 48.06 (s, cis CH₂@CH–CH₂–),

47.84 (s, trans CH₂@CH–CH₂–), 46.63 (s, Pro trans-

dC), 46.39 (s, Pro cis-dC), 37.45, 37.39, 37.10 (3s, Phe

bC), 35.58 (s, Tyr cis-bC), 34.84 (s, Tyr trans-bC),

31.11 (s, Pro cis-bC), 28.85 (s, Pro trans-bC), 24.09 (s,

Pro trans-cC), 21.17 (s, Pro cis-cC).

5.4.12. [N-Allyl-N-Boc-Dmt¹]EM-2 (10). The title com-

pound was synthesized using the same method for 9.

20

D

Yield 225 mg (70%), mp 131–133 ꢁC, R_f5= 0.70, ½aꢁ

ꢀ82.9ꢁ (c 0.51, MeOH). Anal. Calcd for

C₄₂H₅₃N₅O₇Æ0.5H₂O: C, 67.36; H, 7.27; N, 9.35. Found:

C, 67.22; H, 7.33; N, 9.06. ¹H NMR (400 MHz, DMSO-

d₆) d: 8.90 and 8.85 (s · 2, 1.0H, Dmt OH), 8.06–7.81

(m, 2.0H, Phe NH), 7.35–7.00 (m, 12.0H, Phe Ar-H,

CONH₂), 6.40 and 6.36 (s · 2, 2.0H, Dmt Ar-H), 5.64

(br, 1.0H, CH₂@CH–CH₂–), 5.10–4.88 (m, 2.27H,

CH₂@CH–CH₂–, Dmt cis-aH), 4.88–4.73 (m, 0.73H,

Dmt trans-aH), 4.50–4.30 (m, 2.0H, Phe aH), 4.07–

3.93 (m, 0.73H, trans CH₂@CH–CH₂–), 3.93–3.82

(m, 0.27H, cis CH₂@CH–CH₂–), 3.73–3.60 (m, 0.27H,

cis CH₂@CH–CH₂–), 3.60–3.48 (m, 0.73H, trans

CH₂@CH–CH₂–), 3.48–3.18 (m, 2.0H, Pro dH), 3.15–

2.62 (m, 6.0H, Phe bH, Dmt bH), 2.16 and 2.12 (s · 2,

6.0H, Dmt CH₃), 1.90–1.55 (m, 4.0H, Pro bH, Pro

cH), 1.28 and 1.08 (s · 2, 9.0H, Boc). ¹³C NMR d:

172.48, 171.15, 170.41, 168.94, 168.29, 155.10, 153.36,

137.77, 137.62, 137.57 (10q), 134.72 (t, cis CH₂@CH–

CH₂–), 133.85 (t, trans CH₂@CH–CH₂–), 129.06,

127.95, 127.88, 126.13 (4t, Phe Ar-C), 116.10 (s, trans

CH₂@CH–CH₂–), 115.79 (s, cis CH₂@CH–CH₂–),

114.64 (t, Dmt Ar-C), 79.15, 78.84 (2q, Boc), 59.71

(t, Pro aC), 55.47 (t, Dmt trans-aC), 54.74 (t, Dmt,

cis-aC), 54.21, 53.73 (2t, Phe aC), 46.41 (s, Pro cis-

dC), 46.15 (s, Pro trans-dC), 45.36 (s, CH₂@CH–CH₂–

), 37.35, 36.96 (2s, Phe bC), 28.55 (s, Pro bC), 28.18

(s, Dmt bC), 27.70 (p, trans-Boc), 27.16 (p, cis-Boc),

24.00 (s, Pro cC), 19.85 (p, Dmt CH₃).

5.4.14. [N-Allyl-Dmt¹]EM-2ÆHCl (12). The title com-

pound was synthesized using the same method for 11.

20

Yield 72 mg (71.6%), R_f9= 0.68, t_R15.82, ½aꢁ ꢀ5.9ꢁ

D

(c 0.42, H₂O), m/z [M+1]⁺641. Anal. Calcd for

C₃₇H₄₅N₅O₅ÆHClÆ3H₂O: C, 60.85; H, 7.18; N, 9.59.

Found: C, 61.19; H, 6.85; N, 9.65. ¹H NMR

(400 MHz, DMSO-d₆) d: 10.07 (br, 0.90H, Dmt

NH₂^þ), 9.40–9.10 (m, 1.96H, Dmt NH₂^þ, Dmt OH),

8.36 (d, 0.85H, J = 8.60 Hz, Phe cis-NH), 8.18 (d,

0.85H, J = 8.28 Hz, Phe cis-NH), 8.05 (d, 0.15H,

J = 8.12 Hz, Phe trans-NH), 8.01 (d, 0.15H,

J = 8.28 Hz, Phe trans-NH), 7.47 (s, 1.0H, CONH₂),

7.35–7.05 (m, 11.0H, Phe Ar-H, CONH₂), 6.46 and

6.42 (s · 2, 2.0H, Dmt Ar-H), 6.00–5.87 (m, 0.15H, trans

CH₂@CH–CH₂–), 5.87–5.73 (m, 0.85H, cis CH₂@CH–

CH₂–), 5.43–5.27 (m, 1.15H, trans CH₂@CH–CH₂–,

cis CH₂@CH–CH₂–), 5.23 (d, 0.85H, J = 10.39 Hz, cis

CH₂@CH–CH₂–), 4.48–4.32 (m, 2.22H, Phe aH, Pro

trans-aH), 4.13–4.04 (m, 0.15H, Dmt trans-aH), 3.64–

3.56 (m, 0.22H, trans CH₂@CH–CH₂–), 3.54–3.45 (m,

0.22H, trans CH₂@CH–CH₂–), 3.33–2.73 (m, 11.20H,

Pro cis-aH, Dmt cis-aH, cis CH₂@CH–CH₂–, Pro dH,

Phe bH, Dmt bH), 2.15 and 2.10 (s · 2, 6.0H, Dmt

CH₃), 1.86–1.73 (m, 0.14H, Pro trans-bH), 1.67–1.52

(m, 1.21H, Pro cis-bH, Pro trans-bH, Pro trans-cH),

1.52–1.36 (m, 1.0H, Pro cis-cH, Pro trans-cH), 1.28–

1.12 (m, 1.66H, Pro cis-bH, Pro cis-cH). ¹³C NMR d:

172.66, 172.50, 170.58, 170.34, 170.16, 169.91, 166.40,

166.07, 155.88, 155.63, 138.50, 138.20, 137.96, 137.65,

137.61, 137.52 (16q), 129.12, 128.90 (2t, Phe Ar-C),

128.80 (t, CH₂@CH–CH₂–), 128.05, 127.96, 127.92,

127.84, 126.44, 126.08 (6t, Phe Ar-C), 122.20 (s, trans

CH₂@CH–CH₂–), 121.87 (s, cis CH₂@CH–CH₂–),

5.4.13. [N-Allyl-Tyr¹]EM-2ÆHCl (11). [N-Allyl-N-Boc-

Tyr¹]EM-2 (9) (147 mg, 0.206 mmol) was treated with

TFA (320 lL, 4.13 mmol) and anisole (32 lL) for

30 min at room temperature. The reaction solution

was diluted with hexane, the solid was collected by ﬁltra-

tion, dried over KOH pellets, and puriﬁed by semi-pre-

parative RP-HPLC. The puriﬁed peptide was

lyophilized from 1 N HCl (3· 200 lL) to give amor-

phous powder. Yield 113 mg (84%), R_f9= 0.68,

20

D

t_R15.29, ½aꢁ ꢀ20.7ꢁ (c 0.46, H₂O), m/z [M+1]⁺613.

Anal. Calcd for C₃₅H₄₁N₅O₅ÆHClÆ2H₂O: C, 61.44; H,

6.78; N, 10.24. Found: C, 61.32; H, 6.56; N, 10.26. H

1

NMR (400 MHz, DMSO-d₆) d: 9.78 (br, 0.82H,

–NH₂^þ), 9.59 (s, 0.55H, Dmt cis-OH), 9.44 (s, 0.43H,

Dmt trans-OH), 9.12 (br, 0.83H, –NH₂^þ), 8.38 (d,

0.5H, J = 8.57 Hz, Phe cis-NH), 8.19 (d, 0.53H,

J = 8.26 Hz, Phe cis-NH), 8.15 (d, 0.42H, J = 8.30 Hz,

Phe trans-NH), 8.02 (d, 0.42H, J = 7.85 Hz, Phe trans-

NH), 7.44 (s, 0.53H, CONH₂), 7.37–7.07 (m, 12.47H,

Phe Ar-H, Tyr Ar-H, CONH₂), 6.92 (d, 1.0H,

1248

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

20

D

120.86, 120.47 (2q), 115.03 (t, Dmt Ar-C), 59.76 (t, Pro

trans-aC), 58.94 (t, Pro cis-aC), 56.26 (t, Dmt cis-aC),

55.66 (t, Dmt trans-aC), 54.84, 53.65 (2t, Phe aC),

47.65 (s, cis CH₂@CH–CH₂–), 47.56 (s, trans

CH₂@CH–CH₂–), 46.63 (s, Pro cis-dC), 45.94 (s, Pro

trans-dC), 37.54 (s, Phe cis-bC), 37.44, 37.31 (2s, Phe

trans-bC), 36.83 (s, Phe cis-bC), 31.19 (s, Pro bC),

29.64 (s, Dmt cis-bC), 28.78 (s, Dmt trans-bC), 23.87

(s, Pro trans-cC), 21.09 (s, Pro cis-cC), 20.79 (p, Dmt

trans-CH₃), 19.37 (p, Dmt cis-CH₃).

Yield 246 mg (72.9%), mp 138-140 ꢁC, R_f6= 0.69, ½aꢁ

ꢀ71.9ꢁ (c 0.36, MeOH). Anal. Calcd for

C₄₄H₅₄N₆O₇Æ1.25H₂O: C, 65.94; H, 7.11; N, 10.49.

Found: C, 66.03; H, 6.80; N, 10.50. ¹H NMR

(400 MHz, DMSO-d₆) d: 10.83 (s, 0.91H, Trp Ar-NH),

8.90 (br, 0.70H, Dmt OH), 8.03 (d, 0.65H,

J = 6.98 Hz, Trp trans-NH), 7.96 (d, 0.23H,

J = 7.46 Hz, Trp cis-NH), 7.82–7.71 (m, 0.91H, Phe

NH), 7.51 (d, 1.0H, J = 7.81 Hz, Trp Ar-H), 7.32 (d,

1.0H, J = 8.02 Hz, Trp Ar-H), 7.27–7.14 (m, 6.0H, Phe

Ar-H, CONH₂), 7.14–7.01 (m, 3.0H, Trp Ar-H,

CONH₂), 7.01–6.92 (m, 1.0H, Trp Ar-H), 6.39 and

6.35 (s · 2, 2.0H, Dmt Ar-H), 5.71–5.55 (m, 1.0H,

CH₂@CH–CH₂–), 5.07–4.87 (m, 2.30H, CH₂@CH–

CH₂–, Dmt cis-aH), 4.79 (d, 0.70H, J = 9.29 Hz, Dmt

trans-aH), 4.47–4.34 (m, 2.0H, Phe aH, Trp aH),

4.05–3.93 (m, 0.73H, trans CH₂@CH–CH₂–), 3.93–3.82

(m, 0.27H, cis CH₂@CH–CH₂–), 3.70–3.59 (m, 0.27H,

cis CH₂@CH–CH₂–), 3.53 (dd, 0.73H, J = 7.16,

15.26 Hz, trans CH₂@CH–CH₂–), 3.47–3.17 (m, 2.0H,

Pro dH), 3.13–2.74 (m, 5.27H, Dmt cis-bH, Dmt trans-

bH, Phe bH, Trp bH), 2.72–2.59 (m, 0.73H, Dmt

trans-bH), 2.15–2.11 (2s, 6.0H, Dmt CH₃), 1.98–1.58

(m, 4.0H, Pro cH, Pro bH), 1.27 and 1.07 (s · 2, 9.0H,

Boc). ¹³C NMR d: 172.50, 171.41, 170.85, 168.91,

168.27, 155.08, 154.84, 153.38, 137.70, 137.57, 135.92

(11q), 134.76 (t, cis CH₂@CH–CH₂–), 133.88 (t, trans

CH₂@CH–CH₂–), 129.08, 127.93 (2t, Phe Ar-C),

127.25 (q), 126.13 (t, Phe Ar-C), 124.98 (q), 123.32,

120.75, 118.12 (3t, Trp Ar-C), 116.06 (s, CH₂@CH–

CH₂–), 114.61 (t, Dmt Ar-C), 111.17 (t, Trp Ar-C),

109.87 (q), 79.12, 78.82 (2q, Boc), 59.74 (t, Pro aC),

55.56 (t, Dmt aC), 53.90 (t, Phe aC), 53.57 (t, Trp

aC), 46.44 (s, Pro cis-aC), 46.19 (s, Pro trans-dC),

45.37 (s, CH₂@CH–CH₂–), 37.27 (s, Phe bC), 28.73

(s, Pro bC), 28.16 (s, Dmt bC), 27.71, 27.15 (2p, Boc),

26.95 (s, Trp bC), 24.05 (s, Pro cC), 19.83 (p, Dmt CH₃).

5.4.15. [N,N-(Allyl)₂-Dmt¹]EM-1ÆHCl (13). The title

compound was synthesized using the same method for

20

D

7. Yield 115 mg (71.3%), R_f9= 0.69, t_R18.55, ½aꢁ

ꢀ33.2ꢁ (c 0.36, H₂O), m/z [M+1]⁺720. Anal. Calcd for

C₄₂H₅₀N₆O₅ÆHClÆ3H₂O: C, 62.32; H, 7.10; N, 10.38.

Found: C, 62.43; H, 6.99; N, 10.42. ¹H NMR

(400 MHz, DMSO-d₆) d: 11.66 (br, 0.16H, Dmt trans-

NH⁺), 11.00 (s, 0.68H, Trp cis Ar-NH), 10.89 (s,

0.25H, Trp trans Ar-NH), 10.52 (br, 0.56H, Dmt cis-

NH⁺), 9.39 (br, 0.58H, Dmt cis-OH), 9.16 (br, 0.15H,

Dmt trans-OH), 8.39 (d, 0.67H, J = 6.90Hz, Trp cis-

NH), 8.10–8.00 (m, 1.0H, Trp trans-NH, Phe cis-NH),

7.80 (d, 0.27H, J = 8.16 Hz, Phe trans-NH), 7.68 (d,

0.74H, J = 7.79 Hz, Trp Ar-H), 7.58 (d, 0.26H,

J = 7.75 Hz, Trp Ar-H), 7.42 (br, 0.73H, cis-CONH₂),

7.38–7.30 (m, 1.0H, Trp Ar-H), 7.30–7.13 (m, 6.0H,

Phe Ar-H, Trp Ar-H), 7.13–6.95 (m, 3.27H, CONH₂,

Trp Ar-H), 6.47 and 6.41 (s · 2, 2.0H, Dmt Ar-H),

6.16 (br, 0.48H, trans CH₂@CH–CH₂–), 5.75 (br,

0.76H, cis CH₂@CH–CH₂–), 5.66–5.36 (m, 2.54H, cis

CH₂@CH–CH₂–, CH₂@CH–CH₂–), 5.23 (d, 2.20H,

J = 10.23 Hz, CH₂@CH–CH₂–), 4.48–4.36 (m, 2.0H,

Phe aH, Trp aH), 4.35–4.28 (m, 0.29H, Pro trans-aH),

4.17–3.98 (m, 0.73H, Dmt trans-aH, CH₂@CH–CH₂–),

3.98–3.64 (m, 2.81H, CH₂@CH–CH₂–), 3.42–3.16 (m,

3.13H, Pro cis-aH, Pro cis-dH, Pro trans-dH, Dmt cis-

bH, CH₂@CH–CH₂–), 3.16–2.77 (m, 6.71H, Dmt cis-

aH, Pro cis-dH, Phe bH, Dmt cis-bH, Dmt trans-bH,

Trp bH), 2.20–1.94 (m, 6.29H, Dmt CH₃, Pro trans-

dH), 1.80–1.72 (m, 0.24H, Pro trans-bH), 1.72–1.63

(m, 0.72H, Pro cis-bH), 1.60–1.44 (m, 0.72H, Pro

trans-bH, Pro trans-cH), 1.44–1.17 (m, 2.28H, Por cis-

bH, Pro cis-cH). ¹³C NMR d: 172.54, 172.49, 171.00,

170.74, 170.23, 165.85, 156.04, 138.47, 137.64, 137.55,

136.07, 135.99 (12q), 129.15, 127.90 (2t, Phe Ar-C),

127.59 (t, CH₂@CH–CH₂–), 127.23, 126.96 (2q),

126.13 (t, Phe, Ar-C), 125.16 (t, CH₂@CH–CH₂–),

124.25, (s, CH₂@CH–CH₂–), 123.88, 123.48, 118.33,

118.11 (4t, Trp Ar-C), 115.21, 115.05 (t, Dmt Ar-C),

111.32, 115.15 (2t, Trp Ar-C), 109.89, 109.73 (2q),

60.98 (t, Pro cis-aC), 60.03 (t, Pro trans-aC), 59.34 (t,

Dmt trans-aC), 58.86 (t, Dmt cis-aC), 54.42 (t, Phe

aC), 54.19 (s, cis CH₂@CH–CH₂–), 53.64 (t, Trp cis-

aC), 53.46 (t, Trp trans-aC), 51.48 (s, trans CH₂@CH–

CH₂–), 47.01 (s, Pro cis-dC), 46.09 (s, Pro trans-dC),

37.39 (s, Phe bC), 31.38 (s, Pro cis-bC), 28.63 (s, Pro

trans-bC), 27.79 (s, Dmt cis-bC), 27.49 (s, Dmt trans-

bC), 27.11 (s, Trp bC), 23.85 (s, Pro trans-cC), 21.24

(s, Pro cis-cC), 19.78, 19.66 (2p, Dmt CH₃).

5.4.17. [N-Allyl-Dmt¹]EM-1ÆHCl (15). The title com-

pound was synthesized using the same method for 11.

20

Yield 131 mg (77.0%), R_f9= 0.63, t_R17.10, ½aꢁ ꢀ12.3ꢁ

D

(c 0.34, H₂O), m/z [M+1]⁺680. Anal. Calcd for

C₃₉H₄₆N₆O₅ÆHClÆ2.5H₂O: C, 61.61; H, 6.89; N, 11.05.

Found: C, 61.59; H, 6.59; N, 11.09. ¹H NMR

(400 MHz, DMSO-d₆) d: 11.06 (s, 0.77H, Trp cis Ar-

NH), 10.85 (s, 0.13H, Trp trans Ar-H), 10.08 and 9.88

(br · 2, 0.82H, Dmt OH), 9.27 (s, 1.60H, Dmt

cis-NH₂^þ), 9.14 (s, 0.28H, Dmt trans-NH₂^þ), 8.30

(d, 0.81H, J = 8.20 Hz, Trp cis-NH), 8.04 (d, 0.14H,

J = 8.47 Hz, trp trans-NH), 8.00 (d, 0.81H,

J = 8.09 Hz, Phe cis-NH), 7.80 (d, 0.16H, J = 8.10 Hz,

Phe trans-NH), 7.63–7.46 (m, 1.0H, Trp Ar-H), 7.43

(br, 0.84H, cis-CONH₂), 7.38–7.30 (m, 1.0H, Trp Ar-

H), 7.27–7.13 (m, 5.19H, Phe Ar-H, trans-CONH₂),

7.13–7.03 (m, 3.0H, CONH₂, Trp Ar-H), 7.03–6.94 (m,

1.0H, Trp Ar-H), 6.43, 6.42 (2s, 2.0H, Dmt Ar-H),

5.97–5.86 (m, 0.16H, trasn CH₂@CH–CH₂–), 5.81–

5.76 (m, 0.84H, cis CH₂@CH–CH₂–), 5.44–5.30 (m,

0.32H, trans CH₂@CH–CH₂–), 5.21 (d, 0.84H

J = 17.06 Hz, cis CH₂@CH–CH₂–), 5.09 (d, 0.84H,

J = 10.36 Hz,

cis

CH₂@CH–CH₂–),

4.50–4.13

5.4.16. [N-Allyl-N-Boc-Dmt¹]EM-1 (14). The title com-

pound was synthesized using the same method for 9.

(m, 2.18H, Phe aH, Trp aH, Pro trans-aH), 4.15–4.04

(m, 0.15H, Dmt trans-aH), 3.65–3.55 (m, 0.18H, trans

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1249

CH₂@CH–CH₂–), 3.55–3.45 (m, 0.18H, trans

CH₂@CH–CH₂–), 3.45–2.87 (m, 11.41H, cis

DMSO-d₆) d: 9.07 (s, 2H, Dmt OH), 8.02 (br, 2H,

NHCH₂) 6.39 (s, 4H, Dmt Ar-H), 5.95–5.84 (m, 2H,

CH₂@CH–CH₂–), 5.46–5.30 (m, 4H, CH₂@CH–CH₂–),

3.72–3.60 (m, 2H, Dmt aH), 3.48–3.35 (m, 4H,

CH₂@CH–CH₂–), 3.05–2.93 (m, 6H, Dmt bH,

NHCH₂CH₂), 2.92–2.80 (m, 2H, Dmt bH), 2.17 (s, 12H,

Dmt CH₃), 1.20–1.02 (m, 4H, NHCH₂CH₂CH₂), 0.96–

0.82 (m, 4H, NHCH₂CH₂CH₂). ¹³C NMR d: 165.40,

155.46, 140.33, 138.11 (4q), 129.50 (t, CH₂@CH–CH₂–),

122.11 (s, CH₂@CH–CH₂–), 114.76 (t, Dmt Ar-C),

58.27 (t, Dmt aC), 47.54 (s, CH₂@CH–CH₂–), 38.52

(s, NHCH₂), 28.29 (s, NHCH₂CH₂), 25.71

(s, NHCH₂CH₂CH₂), 19.93 (p, Dmt CH₃).

CH₂@CH–CH₂–, Pro cis-aH, Dmt cis-aH, Pro dH,

Phe bH, Dmt bH, Trp bH), 2.15, 2.06 (2s, Dmt CH₃),

1.87–1.76 (m, 0.16H, Pro trans-bH), 1.68–1.48 (m,

1.32H, Pro cis-bH, Pro trans-bH, Pro trans-cH), 1.48–

1.33 (m, 0.84H, Pro cis-cH), 1.33–1.77 (m, 1.68H, Pro

cis-bH, Pro cis-cH). ¹³C NMR d: 172.57, 172.42,

170.86, 170.67, 170.19, 169.96, 166.31, 155.81, 155.62,

138.52, 138.27, 137.62, 137.53, 135.98, 135.89 (15q),

129.11 (t, Phe Ar-C), 128.69 (s, CH₂@CH–CH₂–),

127.83 (t, Phe Ar-C), 127.19, 126.94 (2q), 126.08

(t, Phe Ar-C), 123.43, 122.89 (t, Trp Ar-C), 121.79

(t, CH₂@CH–CH₂–), 120.83 (t, Trp Ar-C), 120.47 (q),

118.18, 118.12 (2t, Trp Ar-C), 114.96 (t, Dmt Ar-C),

111.35 (t, Trp Ar-C), 110.18, 109.67 (2q), 59.82 (t, Pro

trans-aC), 58.88 (t, Pro trans-aC), 56.29 (t, Dmt cis-

aC), 55.78 (t, Dmt, trans-aC), 53.49 (t, Phe aC), 53.49

(t, Trp aC), 47.64 (s, Pro trans-aC), 46.67 (s, Pro cis-

aC), 45.91 (s, CH₂@CH–CH₂–), 37.45 (s, Phe bC),

31.27 (s, Pro bC), 29.73 (s, Dmt bC), 27.10 (s, Trp

bC), 24.01 (s, Pro trans-cC), 21.20 (s, Pro cis-cC),

19.34, 19.28 (2p, Dmt CH₃).

5.4.20. 3,6-Bis-(N-allyl-N-Boc-Dmt-aminopropyl)-2(1H)-

pyrazinone (18). 3,6-Bis- (Z-aminopropyl)-2(1H)-pyrazi-

none (141 mg, 0.286 mmol) was stirred in 25% HBr/

HOAc (960 lL, 4 mmol) with an ice bath for 10 min and

then at room temperature for 3 h. The resulting amine

was precipitated with ether, collected by ﬁltration, and

dried in vacuo. The amine hydrobromide was dissolved

in DMF (10 mL) containing DIPEA (240 lL,

1.37 mmol), to which N-allyl-N-Boc-Dmt-OH (6b)

(200 mg, 0.572 mmol) and PyBop (312 mg, 0.60 mmol)

were added. The solution was ﬁrst stirred in an ice bath

for 10 min, then at room temperature for 4 h. After

removal of the solvent in vacuo, the residue was extracted

with AcOEt (30 mL), which was washed with 10% citric

acid (2· 10 mL), 5% NaHCO₃(2· 10 mL), and saturated

aqueous NaCl solution (2· 10 mL), dried over Na₂SO₄,

ﬁltered, and evaporated in vacuo. The resulting residue

was puriﬁed by ﬂash chromatography (SiO₂, AcOEt/

MeOH = 10:1) and the compound was precipitated with

5.4.18. 1,6-Bis-(N-allyl-N-Boc-Dmt-NH)-hexane (16). To

a solution of 1,6-diaminohexane (33 mg, 0.286 mmol) in

DMF (10 mL), N-allyl-N-Boc-Dmt-OH (6b) (200 mg,

0.572 mmol), DIPEA (119 lL, 0.687 mmol), and PyBop

(313 mg, 0.60 mmol) were added. The reaction mixture

was stirred at 0 ꢁC for 10 min, then at room temperature

for 4 h. After removal of the solvent, the residue was

extracted with AcOEt (50 mL). The organic phase was

washed with cold 10% citric acid (3· 10 mL), 5%

Na₂CO₃(3· 10 mL), and saturated aqueous NaCl (3·

10 mL), dried over Na₂SO₄, and evaporated to dryness.

The residue was puriﬁed by ﬂash chromatography

(SiO₂, AcOEt/hexane = 2:1), precipitated with hexane,

ether. Yield 109 mg (43%), mp 122–124 ꢁC, R_f5= 0.59,

20

½aꢁ

ꢀ57.0ꢁ (c 0.31, MeOH). Anal. Calcd for

D

C₄₉H₇₀N₆O₉Æ0.5H₂O: C, 65.67; H, 7.99; N, 9.38. Found:

C, 65.49; H, 7.91; N, 9.33. ¹H NMR (400 MHz, DMSO-

d₆) d: 11.89 (br, 1H, Pyrazinone NH), 8.89 (br, 2H, Dmt

OH), 7.66 (s, 2H, NHCH₂CH₂CH₂), 6.37 (s, 4H, Dmt

Ar-H), 5.62 (br, 2H, CH₂@CH–CH₂–), 5.10–4.70 (m,

4H, CH₂@CH–CH₂–), 4.45–3.94 (m, 2H, Dmt aH),

3.94–3.20 (m, 4H, CH₂@CH–CH₂–), 3.20–2.60 (m, 8H,

Dmt bH, NHCH₂CH₂CH₂), 2.60–2.00 (m, 19H, Dmt

CH₃, pyrazinone CH₃, NHCH₂CH₂CH₂), 1.80–1.05 (m,

22H, Boc, NHCH₂CH₂CH₂). ¹³C NMR d: 170.01,

169.77, 155.54, 154.97, 153.94, 137.60 (6q), 135.72,

134.81 (2t, CH₂@CH–CH₂–), 125.51 (q), 116.45, 115.61

(2s, CH₂@CH–CH₂–), 114.71 (t, Dmt Ar-C), 79.03 (q,

Boc), 58.48 (t, Dmt aC), 48.03 (s, CH₂@CH–CH₂–),

38.52, 38.01 (2s, NHCH₂CH₂CH₂), 29.21 (s, NHCH₂CH₂

CH₂), 28.51 (s, Dmt bC), 26.09 (s, NHCH₂CH₂CH₂),

19.80 (p, Dmt CH₃), 18.02 (p, pyrazinone CH₃).

ﬁltered, and dried in vacuo. Yield 132 mg (59.2%), mp

20

90–92 ꢁC, R_f7= 0.65, ½aꢁ ꢀ57.6ꢁ (c 0.32, MeOH). Anal.

Calcd for C₄₄H₆₆N₄O₈Æ^D0.5H₂O: C, 67.06; H, 8.57; N,

7.11. Found: C, 67.09; H, 8.57; N, 7.13. ¹H NMR

(400 MHz, CDCl₃) d: 8.04 (s, 1.3H, Dmt OH), 6.53 (s,

4.0H, Dmt Ar-H), 5.96–5.52 (m, 4.0H, NH, CH₂@CH–

CH₂–), 5.26–4.95 (m, 4.0H, CH₂@CH–CH₂–), 4.55–

4.23 (m, 2.0H, Dmt aH), 4.23–3.85 (m, 4.0H,

CH₂@CH–CH₂–), 3.53–3.10 (m, 4.0H, Dmt bH), 3.10–

2.55 (m, 4.0H, NHCH₂), 2.28 (s, 12.0H, Dmt CH₃),

1.50 and 1.46 (s · 2, 18.0H, Boc), 1.40–0.98 (m, 4.0H,

NHCH₂CH₂), 0.98–0.50 (m, 4.0H, NHCH₂CH₂CH₂).

¹³C NMR d: 170.75, 156.43, 154.73, 154.10, 138.44

(5q), 135.63 (t, CH₂@CH–CH₂–), 126.34 (q), 115.64

(s, CH₂@CH–CH₂–), 115.39, 115.04 (2t, Dmt Ar-C),

81.50 (q, Boc), 59.25 (t, Dmt aC), 46.87 (s, CH₂@CH–

CH₂–), 39.15 (s, NHCH₂), 29.89 (s, Dmt bC), 28.81

(s, NHCH₂CH₂), 28.38 (p, Boc), 26.29 (s,

NHCH₂CH₂CH₂), 20.31 (p, Dmt CH₃).

5.4.21. 3,6-Bis-(N-allyl-Dmt-aminopropyl)-2(1H)-pyrazi-

noneÆ2HCl (19). The title compound was synthesized

using the same method for 11. Yield 31.4 mg (64.6%),

20

R_f9= 0.60, t_R16.8, ½aꢁ +32.1ꢁ (c 0.36, H₂O), m/z

D

5.4.19. 1,6-Bis-(N-allyl-Dmt)-hexaneÆ2HCl (17). The title

compound was synthesized using the same method for 11.

[M+1]⁺688. Anal. Calcd for C₃₉H₅₄N₆O₅Æ2HClÆ5.5H₂O:

C, 54.54; H, 7.86; N, 9.78. Found: C, 54.66; H, 7.50; N,

Yield 36.4 mg (57.3%), R_f9= 0.66, t_R15.43, ½aꢁ²⁰+55.0ꢁ (c

9.84. H NMR (400 MHz, DMSO-d₆) d: 9.99 (br, 2H,

1

D

0.36, H₂O), m/z [M+1]⁺680. Anal. Calcd for

C₃₄H₅₀N₄O₄Æ2HClÆ2.5H₂O: C, 58.21; H, 8.25; N, 8.04.

Found: C, 58.30; H, 7.90; N, 8.03. H NMR (400 MHz,

Dmt NH₂^þ), 9.33 (br, 2H, Dmt NH₂^þ), 9.07 (br, 2H,

Dmt OH), 8.23 (t, 1H, J = 4.77 Hz, NHCH₂CH₂CH₂),

8.15 (t, 1H, J = 4.52 Hz, NHCH₂CH₂CH₂), 6.38

1

1250

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

(s, 4H, Dmt Ar-H), 5.98–5.85 (m, 2H, CH₂@CH–CH₂–),

5.50–5.32 (m, 4H, CH₂@CH–CH₂–), 3.80–3.30 (m, 6H,

Dmt aH, CH₂@CH–CH₂–), 3.13–2.85 (m, 8H, Dmt

bH, NHCH₂CH₂CH₂), 2.47–2.42 (m, 2H, NHCH₂

CH₂CH₂), 2.30–2.05 (m, 17H, Dmt CH₃, Pyrazinone

tween placement of mice on the hot plate and movement

consisting ofeither licking or shaking their hind paws with

a baseline latency of 15 s and maximal cut oﬀ time of 30 s.

Tail-ﬂick latency is the latency for removal of the tail from

the onset of radiant heat with a baseline latency of 2–3 s,

and with a cut-oﬀ time of 8 s. The duration time after

icv administration was 10 min and the test was terminated

when hot-plate and tail-ﬂick latencies were close to the

pre-response times. Statistical signiﬁcance applied one-

way analysis of variance (ANOVA) followed by Dun-

nett’s test (signiﬁcant at P < 0.05). The area under the

time–response curve (AUC) combines the response time

and time after administration.^18,19The percent of maxi-

mal possible eﬀect (%MPE) = (T₁ꢀ T₀)/(T₂ꢀ T₀) ·

100, where the T₀and T₂are pre-response and cut oﬀ

times, respectively. AD₅₀presents the dose of 12 or nalox-

one to inhibit 50% of the morphine-induced antinocicep-

tion at 10 min post-injection.

CH₃,

NHCH₂CH₂CH₂),

1.70–1.38

(m,

4H,

NHCH₂CH₂CH₂). ¹³C NMR d: 166.81, 166.70, 155.57,

155.52, 155.40, 138.20 (6q), 128.54, 128.45 (2t,

CH₂@CH–CH₂–), 122.78, 122.68 (2s, CH₂@CH–CH₂–),

121.69, 121.64 (2q), 114.82 (t, Dmt Ar-C), 57.90, 57.86

(2t, Dmt aC), 47.28 (s, CH₂@CH–CH₂–), 38.59, 38.20

(2s, NHCH₂CH₂CH₂), 29.27, 28.76 (2s, NHCH₂

CH₂CH₂), 27.17 (s, Dmt bC), 25.42 (s, NHCH₂

CH₂CH₂), 19.90, 19.87 (2p, Dmt CH₃), 17.97

(p, pyrazinone CH₃).

5.5. Competitive receptor binding

Rat brain P₂synaptosomal preparations were used in

competitive displacement assays for l- and d-opioid

receptors after removal of endogenous opioids as pub-

lished elsewhere in considerable detail,^11,17–19and using

3.5 nM [³H]DAMGO [(D-Ala²,N-Me-Phe⁴,Gly-ol⁵)en-

kephalin] (50.0 Ci/mmol, Amersham Biosciences, Buck-

inghamshire, UK) and 1.9 nM [³H]deltorphin II

(45.0 Ci/mmol, Perkin-Elmer, Boston, MA) to radiola-

bel l- and d-opioid receptors, respectively. After a 2.5-

h incubation at room temperature (22–23 ꢁC), samples

were rapidly rinsed on Whatman GF/C glass ﬁber ﬁlters,

presoaked in 0.1% polyethyleneimine to enhance the re-

sponse : background ratio, washed with ice-cold buﬀer,

dried, and the radioactivity determined using EcoLume

scintillation ﬂuid.^17–19

5.8. Brain slice preparation and whole cell recording

Vibratome-prepared hippocampal slices (300 lm) of rat

brains were incubated in a holding chamber containing

artiﬁcial cerebrospinal ﬂuid (ACSF) bubbled with

O₂/CO₂(95:5%) at room temperature for 60 min.

Spontaneous GABA_Areceptor-mediated IPSCs were

pharmacologically isolated by adding 50 lM ^D-(ꢀ)-2-

amino-5-phosphonovaleric acid and 20 lM 6,7-dinitro-

quinoxaline-2,3-dione.²⁸Patch-clamp pipettes were

prepared as detailed previously.²⁸Individual CA1 pyra-

midal cells were visualized using an infrared diﬀerential

interference contrast Zeiss Axioskop microscope (40-X

water immersion objective) and spontaneous IPSCs were

recorded at ꢀ70 mV for 10 min periods consistently of

baseline conditions, plus 60 mM EtOH, and with

300 nM 15 in EtOH. Series resistance was monitored

throughout the recording and cells were discarded if they

changed signiﬁcantly, and the data were analyzed using

paired Student’s t-tests. More comprehensive controls,

dose–response curves, and analyses were conducted

using 12 with comparable data (Li; Swartzwelder, et al.

unpublished data).

5.6. Functional opioid bioassays

Myenteric plexus longitudinal muscle from the small

intestine of male Hartley strain guinea pigs (GPI) mea-

sured l-opioid agonism and a single mouse vas deferens

(MVD) determined d-opioid agonism. The isolated tis-

sues were suspended in organ baths containing balanced

salt solutions in a physiological buﬀer, pH 7.5.^17–19Ago-

nists inhibited electrically evoked contractions; the IC₅₀

(nM) was obtained from dose–response curves

(mean SE ﬁve to six separate tissue preparations).

Antagonism was determined against the d-opioid ago-

nist deltorphin II or l-opioid agonist endomorphin-2

and expressed as the pA₂(Table 1), which was deter-

mined using the Schild Plot.³¹The Schild slopes of all

antagonists were 0.917–1.189 in the GPI and 0.883–

1.088 in the MVD assays.

Acknowledgments

These studies were supported in part by a Grant-in-Aid

for Japan Society for the Promotion of the Science

(JSPS) Fellows (1503306) to T.L., in part by the Intra-

mural Research Program of the NIH, and NIEHS,

and in part by NIAAA Grant Nos. 12478 and 14894,

and VA Merit Review and Senior Research Career Sci-

entist awards to H.S.S. The authors appreciate the pro-

fessional expertise of the library staﬀ and the

Comparative Medicine Branch at NIEHS. A U.S. Provi-

sional Patent Application No. 60/714,071 covers the

compounds in Table 1 and derivatives thereof.

5.7. Inhibition of morphine antinociception

Male mice were used according to protocols approved by

the Animal Care and Use Committee at the NIEHS.

Intracerebroventricular (icv) administration was per-

formed as described, and antinociception measured using

a hot-plate (55.0 0.1 ꢁC) and by tail-ﬂick tests.^18,19Five

minutes after subcutaneous administration of morphine

(4 mg/kg), 12 or naloxone was injected icv. As a control

to determine the eﬀect of the compound on mice, only

12was injected icv. Thehot-plate latency is the interval be-

References and notes

1. Stromberg, M. F.; Casale, M.; Volpicelli, L.; Volpicelli, J.

R.; O’Brien, C. P. Alcohol 1998, 15, 281.

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251

1251

2. (a) Matthes, H. W. D.; Maldonado, R.; Simonin, F.;

Walverde, O.; Slowe, S.; Kitchen, I.; Befort, K.; Dierich,

15. Kruszynski, R.; Fichna, J.; do-Rego, J.-C.; Chung, N. N.;

Schiller, P. W.; Kosson, P.; Costentin, J.; Janecka, A. J.

Peptide Res. 2005, 66, 125.

´

A.; Le Meur, M.; Dolle, P.; Tzavara, E.; Hanoune, J.;

Roques, B.; Kieﬀer, B. Nature 1996, 383, 819; (b) Roberts,

A. J.; McDonald, J. S.; Heyser, C. J.; Kieﬀer, B. L.;

Matthes, H. W. D.; Koob, G. F.; Gold, L. H. J.

Pharmacol. Exp. Ther. 2000, 293, 1002; (c) Hall, F. S.;

Sora, I.; Uhl, G. Psychopharmacology 2001, 154, 43; (d)

Hokkanen, A.; Vilamo, L.; Wegelius, K.; Sarviharju, M.;

Hyytia, P.; Korpi, E. R. Eur. J. Pharmacol. 1996, 304, 7;

(e) Parkes, J. H.; Sinclair, J. D. Alcohol 2000, 21, 215; (f)

Bodnar, R. J.; Hadjimarkou, M. M. Peptides 2003, 24,

1241.

16. Zadina, J. E.; Hackler, L.; Ge, L. J.; Kastin, A. J. Nature

1997, 386, 499.

17. (a) Okada, Y.; Fujita, Y.; Motoyama, T.; Tsuda, Y.;

Yokoi, T.; Li, T.; Sasaki, Y.; Ambo, A.; Jinsmaa, Y.;

Bryant, S. D.; Lazarus, L. H. Bioorg. Med. Chem. 2003,

11, 1983; (b) Fujita, Y.; Tsuda, Y.; Li, T.; Motoyama, T.;

Takahashi, M.; Shimizu, Y.; Yokoi, T.; Sasaki, Y.; Ambo,

A.; Kita, A.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H.;

Okada, Y. J. Med. Chem. 2004, 47, 3591.

18. Okada, Y.; Tsuda, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.;

Nagata, M.; Yunden, J.; Bryant, S. D.; Lazarus, L. H. J.

Med. Chem. 2003, 46, 3201.

3. Choi, H.; Murray, T. F.; Delander, G. E.; Schmidt, W. K.;

Aldrich, J. V. J. Med. Chem. 1997, 40, 2733.

4. Belton, P.; Cotton, R.; Giles, M. B.; Gormley, J. J.; Miller,

L.; Shaw, J. S.; Timms, D.; Wilkinson, A. Life Sci. 1983,

33, 443.

5. Cotton, R.; Giles, M. G.; Miller, L.; Shaw, J. S.; Timms,

D. Eur. J. Pharmacol. 1984, 97, 331.

6. Pert, C. B.; Bowie, D. L.; Pert, A.; Morell, J. L.; Gross, E.

Nature 1977, 269, 73.

7. Hahn, E. F.; Fishman, J.; Shiwaku, Y.; Foldes, F. F.;

Nagashima, H.; Duncalf, D. Res. Commun. Chem. Pathol.

Pharmacol. 1977, 18, 1.

8. Gairin, J. E.; Mazarguil, H.; Alvinerie, P.; Botanch, C.;

Cros, J.; Meunier, J.-C. Br. J. Pharmacol. 1988, 95, 1023.

9. Portoghese, P.; Sultana, M.; Takemori, A. Eur. J. Phar-

macol. 1988, 146, 185.

10. Schiller, P. W.; Weltroska, G.; Berezowska, I.; Nguyen, T.

M.-D.; Wilkes, B. C.; Lemieux, C.; Chung, N. N.

Biopolymers (Peptide Sci.) 1999, 51, 411–425.

11. (a) Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.;

Lazarus, L. H. Biopolymers (Peptide Sci.) 2003, 71, 86; (b)

Balboni, G.; Salvadori, S.; Dal Piaz, A.; Bortolotti, F.;

Argazzi, R.; Negri, L.; Lattanzi, R.; Bryant, S. D.;

Jinsmaa, Y.; Lazarus, L. H. J. Med. Chem. 2004, 47,

6541; (c) Balboni, G.; Guerrini, R.; Salvadori, S.; Negri,

L.; Giannini, E.; Bryant, S. D.; Jinsmaa, Y.; Lazarus, L.

H. J. Med. Chem. 2005, 48, 8112.

19. Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.; Fujisawa, Y.;

Shiotani, K.; Li, T.; Tsuda, Y.; Yokoi, T.; Ambo, A.;

Sasaki, Y.; Bryant, S. D.; Lazarus, L. H.; Okada, Y. J.

Med. Chem. 2004, 47, 2599.

20. Ress, D. C.; Hunter, J. C. Comprehensive Medicinal

Chemistry; Pergamon Press: New York, 1990, pp 805–846..

21. Morgan, B. A.; Smith, C. F. C.; Waterﬁeld, A. A.;

Hughes, J.; Kosterlitz, H. W. J. Pharm. Pharmacol. 1976,

28, 660.

22. Li, T.; Fujita, Y.; Shiotani, K.; Miyazaki, A.; Tsuda, Y.;

Ambo, A.; Sasaki, Y.; Jinsmaa, Y.; Marczak, E.; Bryant,

S. D.; Salvadori, S.; Lazarus, L. H.; Okada, Y. J. Med.

Chem. 2005, 48, 8035.

23. Schiller, P. W.; Weltrowska, G.; Nguyen, T. M.-D.;

Lemieux, C.; Chung, N. N.; Lu, Y. Life Sci. 2003, 73, 691.

24. (a) Kosson, P.; Bonny, I.; Carr, D. B.; Lipkowsky, A. W.

Peptides 2005, 26, 1667; (b) Botros, M.; Hallberg, M.;

Johansson, T.; Zhou, Q.; Lindeberg, G.; Frandberg, P-A.;

Tomboly, C.; Toth, G.; Greves, P. L.; Nyberg, F. Peptides

2006, 27, 753.

25. (a) Porreca, F.; Mosberg, H. I.; Hurst, R.; Hruby, V. J.;

Burks, T. F. J. Pharmacol. Exp. Ther. 1984, 230, 341; (b)

Bodnar, R. J.; Williams, C. L.; Lee, S. J.; Pasternak, G. W.

Brain Res. 1988, 447, 25; (c) Jinsmaa, Y.; Shiotani, K.;

Fujita, Y.; Miyazaki, A.; Li, T.; Tsuda, Y.; Okada, Y.;

Ambo, A.; Sasaki, Y.; Bryant, S. D.; Lazarus, L. H. Eur.

J. Pharmacol. 2005, 509, 37.

26. Liljequist, S.; Engel, J. Psychopharmacology 1982, 78, 71.

27. Kapur, J.; Macdonald, R. L. Mol. Pharmacol. 1999, 55,

444.

12. Kramer, T. H.; Shook, J. E.; Kazmierski, W.; Ayres, E.

A.; Wire, W. S.; Hruby, V. J.; Burks, T. F. J. Pharmacol.

Exp. Ther. 1989, 249, 544.

13. (a) Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Salvadori,

S. Prog. Neurobiol. 1999, 57, 377; (b) Lazarus, L. H.;

Bryant, S. D.; Cooper, P. S.; Guerrini, R.; Balboni, G.;

Salvadori, S. Drug Disc. Today 1999, 3, 284; (c) Schiller, P.

W.; Berezowska, I.; Nguyen, T. M.-D.; Schmidt, R.;

Lemieux, C.; Chung, N. N.; Falcone-Hindley, M. L.; Yao,

W.; Liu, J.; Iwama, S.; Smith, A. B., III; Hirschmann, R.

J. Med. Chem. 2000, 43, 551.

28. Li, Q.; Wilson, W. A.; Swartzwelder, H. S. Clin. Exp. Res.

2006, 30, 119.

29. Svensson, A. I.; Berntsson, A.; Eirefelt, M.; Soderpalm, B.

J. Neural Trans. 2000, 107, 261.

30. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson,

O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R.

Synthesis 1992, 741.

14. Van den Eynde, I.; Laus, G.; Schiller, P. W.; Kosson, P.;

´

Chung, N. N.; Lipkowski, A. W.; Tourwe, D. J. Med.

Chem. 2005, 48, 3644.

31. Arunlakshana, Q.; Schild, H. O. Br. J. Pharmacol. 1959,

14, 48.

Article Doi

Transformation of μ-opioid receptor agonists into biologically potent μ-opioid receptor antagonists

DOI: 10.1016/j.bmc.2006.11.019

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Full text of DOI:10.1016/j.bmc.2006.11.019

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