Synthesis and biological evaluations of novel endomorphin analogues containing α-hydroxy-β-phenylalanine (AHPBA) displaying mixed μ/δ opioid receptor agonist and δ opioid receptor antagonist activities

Article abstract of DOI:10.1016/j.ejmech.2014.12.049

A novel series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues was synthesized, incorporating chiral α-hydroxy-β-phenylalanine (AHPBA), and/or Dmt¹-Tic² at different positions. Pharmacological activity and metabolic stabil

Full text of DOI:10.1016/j.ejmech.2014.12.049

European Journal of Medicinal Chemistry 92 (2015) 270e281
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluations of novel endomorphin analogues
containing -hydroxy- -phenylalanine (AHPBA) displaying mixed
opioid receptor agonist and opioid receptor antagonist activities
a
b
m/
d
d
Miao Hu a, Marc A. Giulianotti b, Jay P. McLaughlin b, Jiaan Shao a, Ginamarie Debevec b,
Laura E. Maida b, Phaedra Geer b, Margaret Cazares b, Jaime Misler b, Ling Li b,
Colette Dooley b, Michelle L. Ganno b, Shainnel O. Eans b, Elisa Mizrachi b,
,
, b, *
Radleigh G. Santos b, Austin B. Yongye b, Richard A. Houghten b **, Yongping Yu a
a Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
b Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2014
Accepted 28 December 2014
Available online 29 December 2014
A novel series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues was synthesized,
incorporating chiral
Pharmacological activity and metabolic stability of the series was assessed. Consistent with earlier
studies of -amino acid substitution into endomorphins, multiple analogues incorporation AHPBA dis-
played high affinity for and opioid receptors (MOR and DOR, respectively) in radioligand competition
a-hydroxy-b
-phenylalanine (AHPBA), and/or Dmt1-Tic2 at different positions.
b
m
d
Keywords:
binding assays, and an increased stability in rat brain membrane homogenates, notably Dmt-Tic-(2R,3S)
AHPBA-Phe-NH2 (compound 26). Intracerebroventricular (i.c.v.) administration of 26 produced anti-
a-Hydroxy-b-phenylalanine
Opioid receptors
Half-lives
MOR agonist/DOR agonist
MOR agonist/DOR antagonist
nociception (ED50 value (and 95% confidence interval) ¼ 1.98 (0.79e4.15) nmol, i.c.v.) in the mouse 55 ꢀ
C
warm-water tail-withdrawal assay, equivalent to morphine (2.35 (1.13e5.03) nmol, i.c.v.), but demon-
strated DOR-selective antagonism in addition to non-selective opioid agonism. The antinociception of 26
was without locomotor activity or acute antinociceptive tolerance. This novel class of peptides adds to
the potentially therapeutically relevant collection of previously reported EM analogues.
© 2014 Published by Elsevier Masson SAS.
1. Introduction
has been used clinically as an analgesic for decades, but possesses
significant liabilities of use, notably dependence, tolerance, con-
Opioids are currently the gold standard for the clinical treat-
ment of pain [1]. Opioid receptors belong to the superfamily of G-
protein coupled receptors (GPCRs) [2], and are classified into three
stipation, nausea, respiratory depression and abuse behavior,
driving the search for safer alternatives. Endomorphin-1 (EM-1,
YPWF-NH2) and endomorphin-2 (EM-2, YPFF-NH2) are two
endogenous peptides first isolated from the bovine brain and the
human cortex in 1997 [4], displaying high affinity and selectivity for
the MOR. Of interest, both endomorphins produced potent MOR-
mediated antinociception without some of the undesirable side
effects of morphine [4]. In particular, the rewarding effect of
endomorphins are readily separated from the therapeutic doses
producing analgesia, and they are less likely than morphine to
induce respiratory depression and cardiovascular effects at effica-
cious antinociceptive doses [5]. However, like many endogenous
opioid peptides, endomorphins are susceptible to rapid proteolysis
and metabolism, low lipid solubility and limited ability to cross the
bloodebrain barrier (BBB) [6]. These effects greatly reduce the
magnitude and duration of endomorphin activity, demonstrated by
subtypes: the
m opioid receptor (MOR), the d opioid receptor (DOR),
and the opioid receptor (KOR). Agonist activation of each opioid
k
receptor produces potent antinociception, but also results in sig-
nificant side effects including tolerance and physical dependence,
as well as respiratory depression and drug abuse (MOR), seizure
activity (DOR) and psychomimetic effects (KOR) which complicate
their clinical use [3]. Morphine, a potent MOR-preferring agonist,
* Corresponding author. Zhejiang Province Key Laboratory of Anti-Cancer Drug
Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, PR China.
** Corresponding author.
E-mail addresses: houghten@tpims.org (R.A. Houghten), yyu@zju.edu.cn (Y. Yu).
http://dx.doi.org/10.1016/j.ejmech.2014.12.049
0223-5234/© 2014 Published by Elsevier Masson SAS.

M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
271
less than 30 min' antinociception after direct administration into
the CNS, and a failure to elicit antinociception following peripheral
administration in rodents [7]. Notably, several studies have
attempted to increase endomorphin activity through chemical
modification of the peptide, seeking to overcome the problems of
rapid enzymatic degradation and BBB impermeability by utilizing
the incorporation of unnatural amino acids [8,9], various types of
cyclization [10e13], change of peptide backbone [14], modification
of the pharmacophore groups [15], glycosylation [7,16,17], lip-
idation [17], and introduction of conformational constraints [18].
The resultant peptide mimics are designed to retain or improve the
biological selectivity and activity of natural endomorphins while
overcoming the undesirable poor bioavailability and rapid
metabolism.
analogues, but resulted in low affinity for MOR [21c]. Chiral
AHPBA has been found in various natural products as well as syn-
thetic biological active compounds in drug discovery efforts [29]. In
the present study EM analogues in which these b-amino acids were
employed at positions 3 or 4 were synthesized and evaluated.
Herein, we described a detailed pharmacological characterization
in vitro and in vivo, as well as enzymatic degradation studies of
these endomorphin analogues. The opioid receptor affinity for all
analogues was first determined by radioligand competition binding
experiments, and functional activity of selected lead compounds
evaluated in vitro using a label free assay to monitor cell movement
in response to receptor activation. The degradation rate of the
endomorphin analogues was examined in the presence of rat brain
homogenate. Antinociception and the opioid receptor selectivity of
agonist and antagonist effects of the most promising compounds
was characterized in vivo with the mouse 55 ꢀC warm-water tail-
withdrawal test after intracerebroventricular (i.c.v) administration.
Finally, initial liabilities of the lead compounds were evaluated in
mouse assays of coordinated locomotor activity and acute anti-
nociceptive tolerance.
In several cases, an a-amino acid has been substituted with its b-
isomer in biologically active peptides, resulting in an increased
activity and enzymatic stability [19,20]. It has been reported that
the replacement of Pro2 (in EM-1/EM-2) with alicyclic
b
-amino
-proline [21], piperidine-3-carboxylic acid [22],
azetidine-3-carboxylic acid [23], 2-aminocyclopentane-1-
acids, such as
b
carboxylic acid, 2-aminocyclohexane-1-carboxylic acid [24], 2-
aminocyclopentene-1-carboxylic acid and 2-aminocyclohexene-1-
carboxylic acid [25], leads to compounds with increased affinity
2. Chemistry
for the MOR and improved metabolic stability. Notably, b-amino
Chiral Boc-3-amino-2-(benzyloxy)-4-phenylbutanoic acids
were prepared in solution-phase (see Experimental for details).
EMs and their analogues were synthesized by standard solid-phase
methods on MBHA resin using Boc-protected amino acids and DIC/
HOBt as coupling reagents. Peptides, as well as the benzyl group of
the protected AHPBA building blocks, were cleaved from the resin
by HF in the usual manner. Peptides were purified by using pre-
parative RP-HPLC and characterized by RP-HPLC, ESI-MS, HRMS
and 1H NMR. Purities were determined to be at least 97% in all
cases. Analytical properties of EMs and their analogues are sum-
marized in tabular form (as Table S1) with accompanying NMR
spectra (as Section S2) in the Supplementary data.
acids have also been successfully utilized as substitutes for Trp3 or
Phe4 in EM-1 analogues [26]. More generally, inclusion of the
dipeptide fragment Dmt1-Tic2 is known to introduce a versatile
opioid pharmacophore, adding a wide range of activities by inter-
action with MOR and/or DOR [27]. Dmt promotes DOR/MOR agonist
activity, while Tic is reported to promote DOR-antagonist activity
when incorporated alone into position 2 of opioid peptides [28].
In the present study, we introduced several b-amino acids into
endomorphins, including the novel unnatural chiral 3-amino-2-
hydroxy-4-phenylbutanoic acid (AHPBA) and the commercially
available 3-amino-4-phenylbutyric acid (b-HoPhe-OH) (Scheme 1).
The use of AHPBA was a key interest in this study, as it places a free
hydroxyl group onto the back-bone of the peptide which could be
utilized for future modifications such as the introduction of a lipid
or sugar group, potentially improving BBB penetration and facili-
tating oral administration of drugs. The protected chiral AHPBA
building blocks used in the peptide synthesis are shown in Scheme
2. These were obtained from chiral phenylalanine (Boc-D-Phe-OH)
and N,O-Dimethyl hydroxylamine hydrochloride. The correspond-
ing coupling adducts were further reacted with vinylmagnesium
chloride to generate the elongation of the carbon chain. The sub-
sequent reduction with sodium borohydride and cerium chloride
heptahydrate generated a new chiral center at position 3. The
resulting crude product was used directly in a reaction with benzyl
bromide. The chiral products were then separated by chromatog-
raphy and oxidized to afford the desired benzyl protected AHPBA
(Scheme 3). The same procedure was repeated with Boc-L-Phe-OH.
The benzyl group of these building blocks was removed by HF
during the cleavage of the final peptides from the solid support
generating the free hydroxyl.
3. Results
3.1. Evaluation of opioid receptor affinity and selectivity for the
endomorphin analogues
Affinities of EM-1, EM-2 and their analogues for MOR, DOR, and
KOR were determined by measuring the IC50 values for the inhi-
bition of [3H]DAMGO, [3H]DPDPE and [3H]U69,593 respectively,
from their rat brain membrane binding sites. Calculated inhibitory
constants (Ki) and opioid receptor selectivity (Kid/Kim, Kik/Kim) of the
analogues are listed in Table 1. The affinity and MOR selectivity of
EM-1 and EM-2 agreed well with previously published results [30].
The Kim values ranged from 3e2083 nM for the set of analogues
tested. The EM-1 analogues, in which Phe4 was replaced by the four
isomers of AHPBA (2e5), all showed decreased (2.5e61 fold lower)
MOR affinity, and a lower MOR selectivity (as compared vs DOR).
[(3S)-AHPBA4]EM-1 (2 and 3) showed much higher affinity for MOR
as compared to the corresponding [(3R)-AHPBA4]EM-1 (4 and 5).
Among these four analogues, [(2S,3S)-AHPBA4]EM-1 (3) displayed
In previous studies, b-HoPhe-OH was incorporated into EM-1
Scheme 1. Structures of b-amino acids incorporated into endomorphins.

272
M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
Scheme 2. Chiral AHPBA building blocks in peptide synthesis.
Scheme 3. Synthesis chiral AHPBA building blocks in peptide synthesis. reagents and conditions: (a) HOBt (1.05 eq.), DIC (1.25 eq.), Et3N (2.5 eq.), DCM, r. t., 11 h; (b) Vinyl-
magnesium chloride (1.6 M, 2 eq.), THF, 0 ꢀC, 5 h; (c) NaBH4 (1.5 eq.), CeCl3$7H2O (1 eq.), THF, ꢁ78 ꢀC, 15e30 min; (d) BnBr (1.25 eq.), NaH (2 eq.), DMF, r. t., overnight; (e) NaIO4 (3
eq.), RuCl3 (3%), CH3CN/H2O, r. t., 12 h.
the strongest MOR affinity (Kim ¼ 10 nM), just 2.5-fold lower than
the parent compound (1). Similar results occurred with the EM-2
analogues (8e11). When Trp3 in EM-1 or Phe3 in EM-2 was
substituted with AHPBA (13e16), the respective [(3S)-AHPBA3]EM-
1/EM-2 analogues (13 and 14) showed greater affinity for MOR than
their corresponding [(3R)-AHPBA3]EM-1/EM-2 analogues (15 and
16). [(2R,3S) AHPBA3]EM-1/EM-2 demonstrated the greatest MOR
affinity of the analogues (13, Kim ¼ 9 nM), and retained high MOR
selectivity over DOR and KOR. Several R4 analogues of compound 13
were also investigated (17e20), but none demonstrated improved
affinity for MOR over compound 13. Additional evaluation of EM-1
parent compound 22. All the Dmt1-Tic2 analogues showed higher
DOR affinity (compounds 24e27) than the parent compounds
(22e23). Moreover, these analogues maintained the selectivity for
DOR over MOR. The corresponding
b-HoPhe analogues (28e30)
showed similar affinity profiles as their corresponding AHPBA
analogues.
3.2. Evaluation of functional activity of the endomorphin analogues
in vitro
The functional activity of EM-1, EM-2 and several EM analogues
were assessed utilizing a label free binding assay (SRU-BIND),
monitoring the responses of cells stably transfected with MOR or
DOR after addition of the endomorphin (Table 3). The label free
assay monitors the changes of cell morphology in cells expressing
opioid receptors following addition of opioid ligands, with agonists
and EM-2 analogues containing b-HoPhe-OH found relatively lower
affinity for DOR when compared to the corresponding AHPBA
structural analogues and generally more selectivity for MOR (6 to 2
through 5 and 12 to 8 through 11, except for 9).
Using a similar strategy utilized to produce the AHPBA ana-
logues (2e6, 8e11 and 13e20), those compounds with the Dmt1-
Tic2 pharmacophore were synthesized (22e30, Table 2). (2S,3S)
AHPBA was incorporated into position 4 of [Dmt1, Tic2]EM-1/EM-2
(24 and 25, respectively), and (2R,3S)AHPBA was incorporated into
position 3 of [Dmt1, Tic2]EM-1 (26) and position 4 of [Dmt1, Tic2]
EM-2 (27). As predicted, analogues incorporating the Dmt1-Tic2
pharmacophore, 22 and 23, maintained strong affinity for MOR
while dramatically increasing affinity for DOR demonstrated by
their respective endogenous counterparts (1 and 7). Three of these
Dmt1-Tic2-AHPBA analogues (24, 26 and 27) exhibited slightly less
MOR affinity than their corresponding analogues lacking AHPBA
(22e23), while compound 25 was slightly more potent than its
inducing an increase in peak wavelength values (DPWV; Fig. 1A). As
expected EM-1 and EM-2 were found to be full agonists at MOR, but
agonist activity was also observed at DOR, unlike previously pub-
lished observations collected with the guinea pig ileum assay [31].
Consistent among the present results, all the EM analogues tested
expressed agonist activity at MOR and DOR. Analogues 22, 23, 25
and 26 were also assessed for inhibition of cAMP mediated through
DOR, with activity confirming that these compounds acted as DOR
agonists (Table 3), with 25 observed to be capable of full activity in
this assay. A dose response curve was determined for compound 26
in the label free assay for DOR-mediated activity (Fig. 1B), with an
EC50 value ¼ 23 6 nM (n ¼ 6), four times greater than the effect

M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
273
Table 1
Table 2
Opioid receptor binding affinities and selectivities of endomorphin analogues con-
taining
Opioid receptor binding affinities and selectivities of analogues containing Dmt1-
a
-hydroxy-
b
-phenylalanine (AHPBA), measured in rat brain membrane
Tic2 with AHPBA or
b-HoPhe, measured in rat brain membrane preparation.
preparation.
No. Sequence
Ki values (nM) Selectivity
No. Sequence
Ki values (nM)
Selectivity
ma
db
kc
Kim/
Kik/
ma
db
kc
Kid/Kim Kik/Kim
Kid
Kid
1
2
3
4
5
6
7
8
9
Tyr-Pro-Trp-Phe-
NH2(EM-1)
Tyr-Pro-Trp-
(2R,3S)AHPBA-NH2
Tyr-Pro-Trp-
(2S,3S)AHPBA-NH2
Tyr-Pro-Trp-
(2S,3R)AHPBA-NH2
Tyr-Pro-Trp-
(2R,3R)AHPBA-NH2
4
0.4
0
729 320
2354 200
182
18
589
111
209
22
22 Dmt-Tic-Phe-Phe-NH2
23 Dmt-Tic-Trp-Phe-NH2
24 Dmt-Tic-Phe-(2S,3S)AHPBA-
5
5
12
2
1
3
2
2
1
1
1
51
289 57
6
3
3
26
145
82
25
10
453
8
2776 580
2087 90
3800 52
4054 264
>10000
0.4 82 25 12
NH2
2
508 59
287 39
441 31
4112
51
25 Dmt-Tic-Trp-(2S,3S)AHPBA-
3
8
0.2 0.8 0.3 161 11
4
201
96
NH2
176 40
243 23
1.6
1.8
26 Dmt-Tic-(2R,3S)AHPBA-Phe-
2.3 0.7 0.3 67 23 11
NH2
17
27 Dmt-Tic-Phe-(2R,3S)AHPBA-
15
1
0.5 0.2 82
3
30
164
NH2
Tyr-Pro-Trp-
b
-
10
3
3
411 1000
28 Dmt-Tic-Phe-
29 Dmt-Tic-Trp-
b
-HoPhe-NH2
15
14
6
2
5
2
1
1
1
30
170
112
15
14
6
30
170
112
HoPhe-NH2
Tyr-Pro-Phe-Phe-
NH2(EM-2)
Tyr-Pro-Phe-
(2R,3S)AHPBA-NH2
Tyr-Pro-Phe-
b-HoPhe-NH2
0.2
6804 2460 2708 446 2268
903
52
30 Dmt-Tic-b-HoPhe- Phe-NH2
a
Displacement of [3H]DAMGO (
m
-opioid receptor selective ligand).
-opioid receptor selective ligand).
k-opioid receptor selective ligand). Data are listed
33 0.2
967 215
1725 78
29
b
c
Displacement of [3H]DPDPE (
d
Displacement of [3H]U69,593 (
17
2
3843 1323 1980 139
226
116
13
as the mean Ki values from two experiments. Opioid receptor selectivity is the ratio
) or ( ) of the affinity constants (Ki).
(2S,3S)AHPBA-NH2
(
m
/d
k/d
10 Tyr-Pro-Phe-
(2S,3R)AHPBA-NH2
11 Tyr-Pro-Phe-
(2R,3R)AHPBA-NH2
12 Tyr-Pro-Phe-
HoPhe-NH2
13 Tyr-Pro-(2R,3S)
AHPBA-Phe-NH2
14 Tyr-Pro-(2S,3S)
AHPBA-Phe-NH2
15 Tyr-Pro-(2S,3R)
AHPBA-Phe-NH2
16 Tyr-Pro-(2R,3R)
AHPBA-Phe-NH2
17 Tyr-Pro-(2R,3S)
AHPBA-2-NaI-NH2
18 Tyr-Pro-(2R,3S)
AHPBA-pFPhe-NH2
19 Tyr-Pro-(2R,3S)
AHPBA-Trp-NH2
324 19
563 36
462 17
2498
4131 651
8868 3330
>10000
1.7
1.0
109
447
23
9
1
15
Table 3
In vitro functional response data for EMs and their analogues.
b
-
7
435
301
12
No. Sequence
m
Agonist %a Agonist %b
d
d
cAMP %c
73 14
0.4
3288 1049 2711 715
1788 546 5777 419
365
4
1
7
Tyr-Pro-Trp-Phe-NH2(EM-1)
Tyr-Pro-Phe-Phe-NH2(EM-2)
102 10
49
52
46
26
5
3
5
4
98
61 11
43
51 28
50 15
7
497 28
202 15
22 Dmt-Tic-Phe-Phe-NH2
23 Dmt-Tic-Trp-Phe-NH2
24 Dmt-Tic-Phe-(2S,3S)AHPBA-NH2
25 Dmt-Tic-Trp-(2S,3S)AHPBA-NH2
26 Dmt-Tic-(2R,3S)AHPBA-Phe-NH2 42
8
87
4
593 191
4820 2843
3
24
82 14
45
30
22
7
5
3
104
9
2083 284 1218 1008 2253 204
0.6
1.1
9
5
66 12
27 Dmt-Tic-Phe-(2R,3S)AHPBA-NH2
52
39
207 17
13
86 11
7
757 211
829 354
1376 257
2711 208
19
4
35
13
a
m
agonist: Using the label free binding assay (as described in Materials and
M of tested com-
Methods). HEK293 cells expressing MOR were treated with 3.3
m
pounds. Results are expressed as % response relative to 300 nM DAMGO standard
2
2316 1261 2384 599
178
6
183
20
deviation values (n ¼ 2).
b
d
agonist: Using the label free binding assay (as described in Materials and
20 Tyr-Pro-(2R,3S)
AHPBA-Arg-NH2
21 Tyr-Pro-
483 26
385
1694 162
1130
Methods). CHO cells expressing DOR were treated with 3.3 mM of tested compounds.
Results are expressed as % response relative to 10 nM DPDPE standard error values
b-HoPhe- 24
16
47
(n ¼ 4).
Phe-NH2
c
d
cAMP: CHO cells expressing DOR were treated with 500 nM of tested com-
a
b
c
Displacement of [3H]DAMGO (
m
-opioid receptor selective ligand).
-opioid receptor selective ligand).
-opioid receptor selective ligand). Data are listed
pounds. Results are expressed as % response relative to 500 nM DPDPE standard
error values (n ¼ 4).
Displacement of [3H]DPDPE (
d
Displacement of [3H]U69,593 (
k
as the mean Ki values from two experiments. Opioid receptor selectivity is the ratio
) or ( ) of the affinity constants (Ki).
(
d/
m
k/m
increase in half-life (13, 6.52 min vs. 2.30 min for 9), potentially
indicating the utility of a b-amino acid substituent at this position.
Incorporation of Dmt1-Tic2 into EM-1 and EM-2 (23 and 22,
respectively) increased metabolic stability. Dmt-Tic-Phe-Phe-NH2
(22) displayed an 8-fold increase in stability over EM-2 (7), with a
half-life of 6.11 min, while Dmt-Tic-Trp-Phe-NH2 (23) displayed a
14-fold increase over the half-life of EM-1 (1). A significant increase
in stability was also observed when the Phe4 of compound 23 was
substituted with (2S,3S)AHPBA (25), increasing the half-life of this
compound to enzymatic degradation past 2 h. Equivalent R4 sub-
stitutions to the EM-2 analogue 22 also increased metabolic sta-
bility, increasing the half-life to 48.96 min. Likewise, (2R,3S)AHPBA4
substitution (in 27) also increased the half-life significantly, to
82.6 min. The replacement of the third position of 22 and 23 with
(2R,3S)AHPBA (26) resulted in a dramatic increase in stability, with
a half-life value greater than 120 min. The analogues incorporating
generated with the DOR agonist DPDPE (EC50 value ¼ 0.8 0.1 nM).
Together, these results show that compound 26 behaved with high
potency but with non-maximal efficacy in this assay system.
3.3. Evaluation of metabolic stability
The metabolic stability of EM-1 and EM-2 and selected ana-
logues was assessed after incubation of the compounds in rat brain
homogenate (Table 4). As expected, EM-1 and EM-2 (1 and 7)
showed high susceptibility to enzymatic degradation in the rat
brain homogenate, with half-lives of 2.30 min and 0.75 min,
respectively. Chemical substitutions to EM-1 produced only minor
increases in metabolic stability when either (2S,3S)AHPBA or
b-
b
-HoPhe3 instead of AHPBA (28e30) also displayed increased half-
lives equivalent with the AHPBA analogues (Table 4), suggesting
further value of a -amino acid substituent at this position.
Together, these results confirm the utility of position-specific
HoPhe-OH were incorporated into position 4 (3 and 6), with half-
lives of 3.57 min and 4.57 min, respectively. Analogues of EM-2
where (2S,3S)AHPBA was substituted for Phe3 (13) or Phe4 (9)
showed that the Phe3 substitution produced a more substantial
b

274
M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
Fig. 1. Label free assay results for CHO cells expressing DOR. A. Changes in peak wavelength value observed for cells exposed to 15 nM DPDPE. Points represent the mean S.E.M. of
four replicates. B. Concentration response curves for DPDPE and Compound 26 in label free assay with CHO cells expressing DOR. Points represent the mean S.E.M. for two
separate assays.
substitutions to reduce the rate of enzymatic degradation.
3.7. Liability evaluation of compound 26: locomotor activity and
acute antinociceptive tolerance
3.4. Antinociception
The locomotor effects of compounds 22 and 26 were evaluated
in the rotorod assay (Fig. 5A). At a maximal therapeutic dose
(100 nmol, i.c.v.), compound 22 significantly impaired coordinated
locomotor activity over time (F(2,20) ¼ 5.20; P ¼ 0.015, two-way
ANOVA). However, compound 26 was without significant locomo-
tor effect at any time tested with this dose.
Antinociceptive potencies of 22 and 26 were determined with
the mouse 55 ꢀC warm-water tail-withdrawal test. Compound 22 is
a previously reported parent analogue of compound 26 [32], and
was used as a comparison control along with the established opioid
receptor agonist, morphine. Morphine, 22 and 26, all demonstrated
dose-dependent antinociception, albeit with varying potencies that
proved significantly different (F(1,90) ¼ 37.7; P < 0.0001, nonlinear
regression modeling; Fig. 2). Morphine and compound 26 exhibited
statistically equivalent antinociceptive potency (i.c.v), with ED50
(and 95% confidence interval) values of 2.35 (1.13e5.03) nmol and
1.98 (0.79e4.15) nmol, respectively. In contrast, the ED50 (and 95%
confidence interval) value for the parent compound 22 was
approximately 9.2-fold higher than morphine (21.7 (10.9e52.7)
nmol, i.c.v.). Moreover, antinociception produced by compound 26
lasted up to 80 min after i.c.v. administration of the maximal dose
tested, 20 min longer than that observed with compound 22.
As an initial evaluation of the ability of compound 26 to produce
antinociceptive tolerance, an acute antinociceptive tolerance assay
[33,34] was performed separately with both centrally administered
(i.c.v.) morphine and compound 26. As expected, a single central
administration of morphine and compound 26 produced a dose-
dependent antinociception in the 55 ꢀC warm-water tail-with-
drawal assay. The antinociceptive dose response of a second
administration of morphine (10e100 nmol, i.c.v.) given 8 h after the
first dose (3 nmol, i.c.v.) was shifted significantly rightward 9.6 fold,
to 22.5 (8.48e61.9) nmol (F(1,90) ¼ 57.02, P < 0.0001, nonlinear
regression analysis), demonstrating acute antinociceptive tolerance
(Fig. 5B). In contrast, compound 26 did not demonstrate acute
antinociceptive tolerance, as the dose response of a second
administration of compound 26 given 8 h after a first dose (2 nmol,
i.c.v.) produced an ED50 value of 0.86 (0.43e2.34) nmol, a value not
significantly different from the response of naïve mice to morphine
or compound 26 (F(1,90) ¼ 0.993, P ¼ 0.322. Nonlinear regression
analysis; Fig. 5B, squares).
3.5. In vivo characterization of agonist activity of compounds 22
and 26 in the 55 ꢀC warm-water tail-withdrawal assay
The opioid receptor mediation and selectivity of compound 22
and 26 induced antinociception was determined by i.c.v. adminis-
tration to either opioid receptor knockout (MOR KO or KOR KO)
mice or after pretreatment of wild-type mice with the delta opioid
receptor selective antagonist naltrindole prior to testing in the
55 ꢀC warm water tail-withdrawal assay (Fig. 3). The anti-
nociceptive effect of compound 22 (Fig. 3A) and 26 (Fig. 3B) were
both significantly reduced in both MOR KO and KOR KO mice, and in
4. Discussion
Seeking to improve metabolic stability while retaining peptide
function, the present study determined the effect of substituting
wild-type mice pretreated with naltrindole (F(4,59)
¼
43.65,
the unnatural b-amino acids AHPBA and b-HoPhe-OH for Trp and
Phe residues in EM-1 and EM-2 and established [Dmt1-Tic2]EM
P < 0.0001 and F(4,59) ¼ 77.77, P < 0.0001, for compounds 22 and 26
respectively; one-way ANOVA and Tukey HSD post hoc test).
analogs [27,32]. We examined each of the analogues with in vitro
assays, demonstrating that
b-amino-acid substitution altered
3.6. In vivo antagonist activity of compound 26 against the
antinociceptive activity of opioid-receptor selective agonists
opioid receptor affinity, biological activity and improved stability to
enzymatic degradation. A lead compound selected from these re-
sults, 26 (Dmt-Tic-(2R,3S)AHPBA-Phe-NH2) was evaluated in
mouse assays in vivo, and found to produce dose-dependent opioid
receptor-mediated antinociception equivalent to the control com-
pounds morphine and Dmt-Tic-Phe-Phe-NH2. However, in contrast
to these control compounds, compound 26 did not demonstrate
impairment of locomotor activity in the rotorod assay or acute
antinociceptive tolerance.
The in vivo effects of compound 26 on antinociception mediated
by opioid-receptor selective agonists were next evaluated at time
points after the dissipation of agonist activity. Intra-
cerebroventricular pretreatment with 26 (30 nmol) for approxi-
mately 2 h significantly antagonized the antinociceptive effect of
the DOR selective agonist SNC-80 (Fig. 4). In contrast, pretreatment
with 26 was without effect on antinociception induced by
morphine or U50,488, suggesting the antagonist effects of com-
pound 26 were selective for DOR.
In the initial opioid radioligand receptor competition binding
assays (Table 1, compounds 1e21), the AHPBA-incorporated EM
analogues exhibited decreased MOR affinity. However all the [(3S)-

M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
275
Table 4
Half-lives of EMs and their potent analogues in rat brain membrane homogenate.a
No.
Sequence
Half-life (min)b
1
3
6
7
Tyr-Pro-Trp-Phe-NH2(EM-1)
Tyr-Pro-Trp-(2S,3S)AHPBA-NH2
Tyr-Pro-Trp-b-HoPhe-NH2
Tyr-Pro-Phe-Phe-NH2(EM-2)
Tyr-Pro-Phe-(2S,3S)AHPBA-NH2
Tyr-Pro-(2R,3S)AHPBA-Phe-NH2
Dmt-Tic-Phe-Phe-NH2
2.30 0.04
3.57 0.00
4.57 0.03
0.75 0.02
2.30 0.02
6.52 0.08
6.11 0.21
32.19 4.39
48.96 0.58
>120
9
13
22
23
24
25
26
27
28
29
30
Dmt-Tic-Trp-Phe-NH2
Dmt-Tic-Phe-(2S,3S)AHPBA-NH2
Dmt-Tic-Trp-(2S,3S)AHPBA-NH2
Dmt-Tic-(2R,3S)AHPBA-Phe-NH2
Dmt-Tic-Phe-(2R,3S)AHPBA-NH2
>120
82.62 14.24
28.90 16.69
>120
Dmt-Tic-Phe-
Dmt-Tic-Trp-
Dmt-Tic-
b
-HoPhe-NH2
b
-HoPhe-NH2
b
-HoPhe- Phe-NH2
>120
a
Values are arithmetic mean of at least three individual experiments SEM. The
protein content of the brain homogenate was 21.2 mg/mL.
Half-lives were calculated on the basis of pseudo-first-order kinetics of the
disappearance of the peptides.
Fig. 4. DOR selective in vivo antagonism by compound 26 (30 nmol, i.c.v.). Compound
26 was administered 90 min prior to administration of the opioid-receptor selective
agonists, morphine (10 mg/kg, i.p.; MOR agonist), U50,488 (10 mg/kg, i.p.; KOR agonist)
or SNC-80 (100 nmol, i.c.v.; DOR agonist) and the determination of antinociception
40 min later in the mouse 55 ꢀC warm-water tail-withdrawal assay. Whether com-
pound 26 was administered or not is indicated by plus and minus signs under the
b
columns. All data represent mean
%
antinociception
SEM from 8 mice.
* ¼ Significantly different from response of SNC-80 administered alone (P < 0.05);
Student's t-test.
displayed the highest MOR affinity and maintained high selectivity
over DOR and KOR.
Additional analogues were made substituting chiral variants of
AHPBA or b
-HoPhe in the third and fourth position of [Dmt1-Tic2]
EM-1 and [Dmt1-Tic2]EM-2, reported previously as potent MOR
agonists [26,28,32]. The binding assays demonstrated that these
modifications resulted in a series of analogues retaining strong
affinity for MOR and DOR (Table 2, 24e30). Confirming expecta-
tions set from earlier reports [26,28,32], each of the [Dmt1-Tic2]EM
analogues (24e30) exhibited slightly stronger DOR affinity than the
parent compounds (22 and 23). While compatible with previous
studies showing that
(confirmed here as analogue 22) increases affinity for DOR and
MOR with moderate DOR selectivity [26,28,32], the current data
Fig. 2. Antinociceptive activity of morphine, compound 22 and compound 26 was
assessed in vivo following i.c.v. administration in the mouse 55 ꢀC warm-water tail-
withdrawal test. All points represent antinociception at peak response, which was
30 min for morphine, and 20 min for compounds 22 and 26. Each point represents the
mean % antinociception SEM for 8 mice.
a
Dmt1-Tic2 substitution into EM-2
Fig. 3. In vivo agonist activity of (A) compound 22 (100 nmol, i.c.v., ꢁ30 min) and (B) compound 26 (30 nmol, i.c.v., ꢁ30 min) in MOR or KOR knockout mice, or after pretreatment
with the DOR selective antagonist naltrindole (20 mg/kg, s.c., ꢁ15 min). All points represent antinociception 30 min after test compound administration. All data represent mean %
antinociception SEM from 8 mice. * ¼ Significantly different from response of compound administered alone (P < 0.05), one-way ANOVA followed by Tukey's post hoc test.
AHPBA4]EM-1/EM-2 analogues exhibited higher affinity for MOR
when compared to their corresponding (3R)-AHPBA4 analogues.
Likewise, the respective [(3S)-AHPBA3]EM-1/EM-2 all showed
better MOR affinity than their corresponding (3R)-AHPBA3 ana-
logues. Among these analogues, [(2R,3S)-AHPBA3]EM-1/EM-2 (13)
also suggests that substitution of b
-amino acids for Phe/Trp3 or
Phe4 is without consequence for opioid receptor affinity.
In view of the fact that the majority of opioid peptides undergo
rapid degradation, the metabolic stability for a number of our EM
analogues was examined (Table 4). A potential advantage of the

276
M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
Fig. 5. Liability evaluation of compounds and morphine. (A) Effects of vehicle (50% DMSO, i.c.v.; grey triangles), compound 22 (100 nmol, i.c.v., red triangles) or 26 (100 nmol, i.c.v.,
blue squares) were assessed in the rotorod assay of evoked locomotor activity. Data represent the mean latency to fall from a rotorod expressed as the % change from baseline
performance/10 min SEM, examined over 60 min. Points represent 8e18 mice. (B) Evaluation of acute antinociceptive tolerance capability of morphine and compound 26 in the
mouse 55 ꢀC warm-water tail-withdrawal assay. Morphine (circles) or compound 26 (squares) was administered at time 0 (3 and 2 nmol, i.c.v., respectively; open symbols) and
again 8 h later (filled symbols). At 8 h, morphine demonstrated significant tolerance, whereas compound 26 did not. Points represent n ¼ 8 mice each. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
incorporation of
b
-amino acids into the endomorphins was
and dopamine [37,38]. We report here our use of a similar screening
assay (BIND) for the three opioid receptors using a label free
biosensor. All analogues screened demonstrated MOR agonist ac-
tivity, although at lower efficacy than the endogenous parent
endomorphin at the concentration tested. In contrast to earlier
studies using mouse vas deferens functional assays [31], EM-1 and
EM-2 were presently observed to exhibit modest DOR agonist ac-
tivity in the label free assay. Differences in functional activity be-
tween tissue and cell lines over expressing GPCRs have been
observed previously [39], and could account for the present effects;
while new, the cell-line based label-free assay is highly sensitive to
agonist activity. However, the DOR agonist activity of both com-
pound 26 and the [Dmt1-Tic2]-EM parent compounds was
confirmed with testing in a cell-based cAMP inhibition assay.
Notably, the full concentrationeresponse curve for compound 26 in
the label free assay suggested that 26 was a partial DOR agonist, as
it did not demonstrate a maximal effect when compared to the
concentration response of the DOR full agonist, DPDPE. This may
not be entirely surprising, as Dmt-Tic-Phe-Phe-NH2 was reported to
act as a MOR agonist and DOR antagonist during testing with
separate in vitro assays [32].
Given its stability to enzymatic degradation and suggestion of
mixed MOR agonist/DOR partial agonist activity in vitro, compound
26 was selected for further biological evaluation in vivo using a
mouse tail-withdrawal assay following central (i.c.v.) administra-
tion. Surprisingly, compound 26 demonstrated equivalent anti-
nociceptive potency with morphine, but significantly greater than
that of the parent compound, [Dmt1-Tic2]EM-2. Moreover, the
antinociception effects of the parent analogue [Dmt1-Tic2]EM-2
and compound 26 were significantly reduced (but not abolished) in
animals either lacking MOR or KOR, or where DOR was antago-
nized, suggesting that compound 26 produced agonism mediated
by all three opioid receptors. The reason for this discrepancy from
the in vitro data is unclear, but might be due to the inherent dif-
ferences between in vitro and in vivo testing. While i.c.v. adminis-
tration directly into the brain eliminates some of the factors
influencing the organism's response (such absorbance or BBB
demonstrated by the significant increase in stability displayed by
analogues 25 and 26 over both their endogenous and [Dmt1-Tic2]-
EM counterparts. For compound 26, this increase can be attributed
in large part to the b
-Phe3 isomer modification, while for com-
pound 25, it is likely attributable to the Trp3 structural bulkiness as
compared to Phe3 [3]. It is known that biodegradation and enzy-
matic cleavage of EMs occurs between position 2 and 3 (ProeTrp for
EM-1 and ProePhe for EM-2) by dipeptidyl peptidase IV (DPPIV)
[35]. Alternatively, studies have also shown that EM-1 undergoes
biodegradation by a secondary minor path, cleaving the peptide at
Tyr1 and Pro2 or between Trp3 and Phe4 [6,36]. By including the
Dmt1-Tic2 analogues in the present study, and assuming they
follow similar biodegradation pathways as EM-1 and EM-2, the
present data allow a comparison to compound 24 to 25, suggesting
that the enhanced stability of 25 could be attributed to the (2S,3S)
AHPBA-conferred resistance to cleavage between Trp3 and Phe4
and the activity of the minor pathway. A comparison of the other
analogues in our data set that contain a Phe3 to Trp3 substitution (7
to 1, 9 to 3, and 22e23) supports the observation that this substi-
tution increases enzymatic stability. Taken together, two points of
substitution on the EM analogues studied provide a dramatic in-
crease in enzymatic stability; both the incorporation of Dmt1-Tic2
over Tyr1-Pro2 and the application of a
position. Encouragingly, these AHPBA-containing peptides pro-
vided half-life values on par with their non-hydroxyl containing
b-amino acid in the third
b
-
HoPhe counterparts (28e30). Overall, compound 26 incorporates
both of these stabilizing substitutions, resulting in an endomorphin
analogue with significantly increased enzymatic stability, and good
affinity for both the MOR and DOR. Moreover, the hydroxyl func-
tionality on AHPBA offers a novel functional site for future modi-
fications such as the attachment of lipids or sugars, which have
shown utility in improving BBB penetration and oral administra-
tion of drugs. The potential for these future modifications suggest
analogues containing AHPBA may offer significant pharmacological
features advantageous for therapeutic development.
Analogues selected for the favorable opioid receptor affinity and
stability (along with matching control endomorphins) were
screened for opioid agonist functional activity in vitro with a label
free assay that monitors cellular responses to activation of GPCRs.
Assays using label free assays biosensors are a newly emerging
method for the sensitive detection of GPCR signaling through the
measurement of mass redistribution [37]. They have been used
penetration), other factors including peptide modification of the b-
amino acid residue or unexpected off-target interactions remain
possible with in vivo testing. Further work is needed to determine
the impact of these effects. Of particular value would be studies of
the activity of 26 after peripheral (s.c., i.p., or even p.o.) adminis-
tration to determine if the increased metabolic stability of this
endomorphin analogue translates into measurable antinociception.
Pretreatment with compound 26 significantly reduced the
successfully with
including acetylcholine, corticotrophin releasing factor, muscarinic
a number of G-protein coupled receptors

M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
277
antinociceptive activity of the DOR selective agonist SNC-80, indi-
cating DOR antagonist activity. This is not without precedence,
given demonstration of DOR antagonism by [Dmt1-Tic2]EM-2 in GPI
and MVD assays [32], but a mechanism for this activity is not
readily apparent. The in vitro data (both binding and functional
assays) found decent affinity and partial agonism at the DOR,
suggesting the source of the in vivo DOR antagonism may be
attributed to partial agonist activity. Alternatively, the agonist ac-
tivity of 26 may have desensitized the DOR, resulting in the
antagonist effects observed in vivo, although this seems unlikely
given that repeated treatment with 26 did not produce acute
antinociceptive tolerance, a phenomena attributed (at least in part)
to receptor desensitization [34]. A time course of DOR antagonism
was beyond the scope of this initial characterization, but might be
helpful in future studies of mechanism.
that i.c.v. administration of 26 possessed equivalent anti-
nociception to morphine in a non-selective manner and DOR-
selective antagonism. Additionally, 26 produced antinociception
without locomotor activity or acute tolerance. These results
confirm the use of b-amino acids in the third and fourth position of
endomorphins, specifically AHPBA, convey metabolic stability
while retaining opioid activity, potentially offering new opportu-
nity to further develop EM analogues as therapies for pain. More-
over, the hydroxyl functionality offers a novel functional site for
future modifications such as the attachment of lipids or sugars,
which have shown utility in improving BBB and oral administration
of drugs. These future modifications could result in a class of
compounds with advantageous pharmacological features for the
treatment of pain that could be administered orally.
The initial liability evaluation completed herein shows that
compound 26 produces no significant locomotor effect during an
hour of testing with a supra-therapeutic i.c.v. dose of 100 nmol.
Remarkably, in contrast to morphine, compound 26 did not
demonstrate acute antinociceptive tolerance. Admittedly, this is a
limited demonstration of opioid tolerance which would benefit
from further demonstration using traditional methods (such as
chronic administration), but the acute model used here is capable
of a rapid (1 day) shift in the opioid antinociceptive dose response
that is consistent with the action of opioid agonists tested under
repeated or chronic treatment conditions [33,34,40]. Moreover, the
current results are consistent with and growing number of dem-
onstrations that DOR antagonists decrease MOR agonist-induced
tolerance and dependence [41e43], either though co-
administration or in the growing number of novel opioid ligands
characterized as possessing both in vivo MOR agonist and DOR
6. Experimental
6.1. Materials and methods
All standard amino acids were obtained from commercial
sources, except the chiral Boc-3-amino-2-(benzyloxy)-4-
phenylbutanoic acids which was prepared in solution-phase. Sol-
vents were purchased from SigmaeAldrich and were used directly.
The isolated yields were based on the manufacturer's loading of
MBHA resin. LC-MS (APCI and ESI) were recorded on a Shimadzu
LCMS-2010EVat LCQ mass spectrometer (ThermoQuest Corpora-
tion) using a Phenomenex Luna 5
m
C18, 100 Å, 150 ꢂ 4.60 mm 5
micron column running a 0e50 (H2O/CH3CN; 0.1% formic acid)
gradient over 6 min or a 5e95 (H2O/CH3CN; 0.1% formic acid)
gradient over 6 min. Preparative RP-HPLC was performed on a
Shimadzu LC-8A preparative HPLC using a Phenomenex Luna 5
m
antagonist activity [44]. These include Dmt-Tic
j
[CH2NH]Phe-
C18 (2) 100 Å column (75 ꢂ 21.2 mm, 5 micron). High resolution
mass spectra were measured on an Agilent 1290 HPLC-6224 Time
of Flight Mass Spectrometer. 1H NMR and 13C NMR spectra were
recorded at 400 MHz on a Bruker Advance spectrometer with a 5-
mm inverse gradient probe. The final purity of the analogues was at
least 97% in all cases. The detailed analytical properties of the
synthetic analogues were provided in Table S1 (see Supplementary
data).
PheNH2 (DIPP-NH2[ ]), which produced potent antinociceptive
j
activity following intracerebroventricular (i.c.v.) administration
with less acute tolerance than morphine. Compounds with a dual
MOR/DOR activity may have additional beneficial pharmacological
effects, as DOR agonists have been reported to enhance the anal-
gesic potency and efficacy of MOR agonists [45]. A mechanism for
these actions is unclear. Of particular interest is evidence indicating
that MOR and DOR form functionally distinct heterodimeric or
hetero-oligomeric complexes in cell culture with resultant changes
in receptor-selective agonist efficacy and reduced desensitization
[46]. The physiological and pharmacological significance of MOR
and DOR interactions have been substantiated in vivo by work using
opioid receptor gene knockout animals [47]. On the basis of these
observations, the development of new opioid ligands possessing
mixed MOR agonist/DOR agonist profiles and mixed MOR agonist/
6.2. Characterization of representative Boc-3-amino-2-
(benzyloxy)-4-phenylbutanoic acids
Tert-butyl ((2R)-3-(benzyloxy)-1-phenylpent-4-en-2-yl)carba-
mate (5): The two stereoisomers of 5 were synthesized according to
the following procedure: To a stirred solution of Boc-D-Phe-OH
(13.27 g, 50 mmol) and N,O-Dimethyl hydroxylamine hydrochlo-
ride (6.10 g, 62.5 mmol) in 60 mL DCM was added HOBt (8.05 g,
52.5 mmol), DIC (8.21 mL, 52.5 mmol) and Et3N (17.4 mL,
125 mmol) at 0 ꢀC. The reaction was stirred for 1 h at 0 ꢀC and
another 11 h at room temperature. The resulting mixture was
filtered and the filtrate was then quenched with 1N HCl (aq)
(30 mL ꢂ 3), saturated Na2CO3 (aq) (30 mL ꢂ 3) and brine
(30 mL ꢂ 3), dried over anhydrous Na2SO4, filtered and concen-
trated under vacuum to give crude 2. Without further purification,
2 was placed under N2, dissolved in 30 mL THF, cooled to 0 ꢀC, and
treated with vinylmagnesium chloride (1.6 M in THF, 66 mL,
100 mmol). The reaction was stirred for 5 h at 0 ꢀC, then quenched
with 1N HCl (aq) (120 mL) for 15 min, and extracted with EtOAc
(200 mL). The organic layer was dried over dried over anhydrous
Na2SO4, filtered and concentrated under vacuum. Purification of the
crude product by chromatography (silica gel, cyclohexane/
EtOAc ¼ 5/1) to afford 5.8 g of pure 3 as a white solid (42%, overall
yield). 3 (5 g, 18.1 mmol) and CeCl3$7H2O (6.76 g, 18.1 mmol) were
dissolved in THF/MeOH (1/1, 50 mL), cooled to ꢁ78 ꢀC, and added
DOR antagonist profiles have emerged as
a promising new
approach to analgesic drug development [48]. While much work
remains to characterize the activity of compound 26, the present
data suggest the inclusion of in vivo DOR antagonist activity to the
mixed opioid receptor agonist effects may reduce clinical liabilities
arising from the use of this analogue.
5. Conclusion
In the current study, a novel series of EM-1 and EM-2 analogues
containing a chiral AHPBA amino acid substitution were synthe-
sized and pharmacologically profiled. The study demonstrated that
this novel series of compounds maintained many of the demon-
strated features of previously reported EM analogues that utilized
b
-amino acid substitutions. Specifically, Dmt-Tic-(2R,3S)AHPBA-
Phe-NH2 (compound 26) proved to be a potent opioid agonist and
DOR antagonist with significantly increased metabolic stability as
compared to the endogenous EMs. In vivo assays also demonstrated

278
M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
NaBH4 (1.03 g, 27.2 mmol) for three times. The reaction was stirred
for 15 min, and warmed to 0 ꢀC. 30 mL water was added and the
mixture was then extracted with 50 mL EtOAc twice. The organic
layer was dried over anhydrous Na2SO4, filtered and concentrated
under vacuum. Crude 4 was lyophilized for 4 h and used directly for
the next step. 4 and Benzyl bromide (2.7 mL, 22.6 mmol) were
dissolved in 35 mL DMF, cooled to 0 ꢀC, and treated with NaH
(0.872 g, 36.2 mmol) slowly. The reaction was stirred at room
temperature overnight. 150 mL DCM was added and the mixture
was washed with water (30 mL ꢂ 3). The organic layer was dried
over dried over anhydrous Na2SO4, filtered and concentrated under
vacuum. The diastereomers were separated by chromatography
(20:1 cyclohexane: EtOAc to 10:1 cyclohexane: EtOAc) to give 1.72 g
of pure (R,R)-5 (26% overall yield) and 2.67 g of pure (R,S)-5 (40%
overall yield). The relative configurations of compounds 5 were
according to the references reported [49e51]. NaIO4 (3.20 g,
15 mmol) and (R,R)-5 (1.84 g, 5 mmol) were dissolved in 200 mL
CH3CN/H2O (1/1), added RuCl3 (32.5 mg, 0.16 mmol) and stirred for
4 h at room temperature. Then another NaIO4 (3.20 g,15 mmol) was
added and stirred for 12 h at room temperature. The reaction
mixture was filtered and the filtrate was added 150 mL EtOAc. The
organic layer was dried over dried over anhydrous Na2SO4, filtered
and concentrated under vacuum to give 1.8 g of crude (3R,2S)-6
(93% yield) [52e55]. NaIO4 (3.20 g, 15 mmol) and (R,S)-5 (1.84 g,
5 mmol) were dissolved in 200 mL CH3CN/H2O (1/1), added RuCl3
(32.5 mg, 0.16 mmol) and stirred for 4 h at room temperature. Then
another NaIO4 (3.20 g, 15 mmol) was added and stirred for 12 h at
room temperature. The reaction mixture was filtered and the
filtrate was added 150 mL EtOAc. The organic layer was dried over
dried over anhydrous Na2SO4, filtered and concentrated under
vacuum to give 1.9 g of crude (3R,2R)-6 (98% yield), which was used
directly for the peptide synthesis. The same procedure was
repeated with Boc-L-Phe-OH to give (S,S)-5, (S,R)-5 and (3S,2R)-6
and (3S,2S)-6.
155.78, 137.52, 137.09, 129.28, 128.56, 128.50, 128.19, 126.57, 80.10,
76.65, 72.75, 54.25, 38.53, 28.28. HRMS (ESIþ) Calcd for C22H27NO5:
386.1889, found: (M þ H)þ 386.1793, (M þ Na)þ 408.1786.
tR ¼ 7.55 min (LC-MS: 5e95% buffer B in A (B: MeOH containing
0.1% formic acid, A: H2O containing 0.1% formic acid) over 6 min
with a flow rate of 0.3 mL/min and 254 nm UV detection).
(2R,3R)-2-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-
phenylbutanoic acid, (3R,2R)-6. 1H NMR (400 MHz, CDCl3)
d
7.42e7.32 (m, 5H), 7.31e7.17 (m, 5H), 4.81 (d, J ¼ 11.5 Hz, 1H), 4.76
(s, 1H), 4.51 (d, J ¼ 11.4 Hz, 1H), 4.42 (s, 1H), 4.17 (s, 1H), 2.95 (dd,
J ¼ 14.1, 5.0 Hz, 1H), 2.87 (s, 1H), 1.35 (s, 9H). 13C NMR (100 MHz,
CDCl3)
d 174.20, 155.32, 136.91, 129.42, 128.56, 128.38, 128.24,
128.16, 126.52, 79.03, 73.02, 53.04, 36.18, 29.70, 28.23. HRMS (ESIþ)
Calcd for
C22H27NO5: 386.1889, found: (M
þ
H)þ 386.2217,
(M þ Na)þ 408.2980. tR ¼ 7.50 min (LCMS: 5e95% buffer B in A (B:
MeOH containing 0.1% formic acid, A: H2O containing 0.1% formic
acid) over 6 min with a flow rate of 0.3 mL/min and 254 nm UV
detection).
6.3. Peptide synthesis
Endomorphins and their analogues were obtained by the stan-
dard tea-bag solid-phase method on MBHA resin using Boc-
protected amino acids and DIC/HOBt as coupling reagents [56].
Coupling was monitored with the ninhydrin test. The Boc group
was removed with 55% TFA/DCM at room temperature for 30 min,
followed by neutralization with 5% diisopropyl-ethylamine (DIEA)
in DCM. The peptides were removed from the solid support with
anhydrous HF in the presence of anisole at 0 ꢀC for 1.5 h. After
evaporation of the HF by N2 blowing, the resin was extracted with
95% acetic acid in water. Crude peptides were obtained in solid form
after lyophilization. Then peptides were subjected to RP-HPLC pu-
rification. Purity of peptides was determined by analytical RP-HPLC.
The molecular weights of the peptides were confirmed by ESIeMS
and HRMS. The identities of all peptides were confirmed by 1H NMR
and their purities were found to be >97% (see Supporting
Information).
Tert-butyl
carbamate, (R,R)-5. White solid. m.p. 54e56 ꢀC. 1H NMR (400 MHz,
CDCl3) 7.33e7.22 (m, 5H), 7.19e7.02 (m, 5H), 5.73 (m, 5.77e5.68,
((2R,3R)-3-(benzyloxy)-1-phenylpent-4-en-2-yl)
d
1H), 5.17 (dd, J ¼ 22.5, 13.8 Hz, 2H), 4.86 (d, J ¼ 9.2 Hz, 1H), 4.51 (d,
J ¼ 11.5 Hz, 1H), 4.21 (d, J ¼ 11.5 Hz, 1H), 3.83 (dd, J ¼ 15.3, 7.5 Hz,
6.4. Radioligand competition binding assay
1H), 3.66 (d, J ¼ 6.4 Hz, 1H), 2.80 (d, J ¼ 7.5 Hz, 2H), 1.32 (s, 9H). 13
C
NMR (100 MHz, CDCl3)
d
155.65, 138.49, 138.28, 135.65, 129.37,
MOR binding assay: rat cortices were homogenized using 50 mM
Tris, pH 7.4, and centrifuged at 16,500 rpm for 10 min. The pellets
were resuspended in fresh buffer and incubated at 37 ꢀC for 30 min.
Following incubation, the suspensions were centrifuged as before,
the resulting pellets were then resuspended in 100 volumes of
50 mM Tris, pH 7.4 plus 2 mg/mL bovine serum and the suspensions
combined. Each assay tube contained 0.5 mL of membrane sus-
pension, 2 nM [3H]-DAMGO, and 0.02 mg/mL mixture in a total
volume of 0.65 mL. DOR binding assay: rat cortices were homoge-
128.45, 128.38, 128.05, 127.76, 126.22, 118.54, 79.09, 78.96, 70.51,
55.74, 38.54, 28.40. HRMS (ESIþ) Calcd for C23H29NO3: 368.2147,
found: (M þ H)þ 368.2225. tR ¼ 8.34 min (LCMS: 5e95% buffer B in
A (B: MeOH containing 0.1% formic acid, A: H2O containing 0.1%
formic acid) over 6 min with a flow rate of 0.3 mL/min and 254 nm
UV detection).
Tert-butyl
carbamate. (R,S)-5. White solid. m.p. 80e82 ꢀC. 1H NMR (400 MHz,
CDCl3) 7.28e7.15 (m, 7H), 7.12e7.06 (m, 3H), 5.76 (m, 5.80e5.71,
((2R,3S)-3-(benzyloxy)-1-phenylpent-4-en-2-yl)
d
nized using 50 mM Tris, pH 7.4, 10 mM MgCl2e6H2O, 200 mM PMSF,
1H), 5.30e5.23 (m, 2H), 4.55 (d, J ¼ 11.8 Hz, 1H), 4.51 (s, 1H), 4.29 (d,
J ¼ 11.8 Hz, 1H), 3.92 (br, 1H), 3.81 (br, 1H), 2.89 (dd, J ¼ 14.1, 4.5 Hz,
1H), 2.75e2.66 (m, 1H), 1.24 (s, 9H). 13C NMR (100 MHz, CDCl3)
centrifuged and incubated as above. Each assay tube contained
2 nM [3H]-DPDPE, Assay tubes were incubated for 2.5 h at 25 ꢀC.
Unlabeled DPDPE was used as a competitor to generate a standard
curve and determine nonspecific binding. KOR binding assay:
Guinea pig cortices and cerebella were homogenized using 50 mM
d
155.31,138.41,135.66,129.44,128.41,128.28,127.73,127.62,126.17,
119.14, 81.84, 79.06, 70.72, 54.71, 35.69, 28.32. HRMS (ESIþ) Calcd
for C23H29NO3: 368.2147, found: (M þ H)þ 368.2224. tR ¼ 8.17 min
(LCMS: 5e95% buffer B in A (B: MeOH containing 0.1% formic acid,
A: H2O containing 0.1% formic acid) over 6 min with a flow rate of
0.3 mL/min and 254 nm UV detection).
Tris, pH 7.4, 10 mM MgCl2e6H2O, 200
mM and centrifuged and
incubated as above. Each assay tube contained 2 nM [3H]U69,593.
Assay tubes were incubated for 2 h at 25 ꢀC. Unlabeled U50,488 was
used as a competitor to generate a standard curve and determine
nonspecific binding. All three binding assays were terminated by
filtration through GF/B filters, soaked in 5 mg/mL bovine serum
albumin, 50 mM Tris, pH 7.4, on a Tomtec Mach II Harvester 96. The
filters were subsequently washed with 6 mL of assay buffer. Bound
radioactivity was counted on a Wallac Betaplate Liquid Scintillation
Counter.
(2S,3R)-2-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-
phenylbutanoic acid, (3R,2S)-6. 1H NMR (400 MHz, CDCl3)
d 8.12 (s,
1H), 7.48e7.33 (m, 5H), 7.29e7.18 (m, 3H), 7.12 (d, J ¼ 6.7 Hz, 2H),
5.22 (d, J ¼ 9.8 Hz, 1H), 4.85 (d, J ¼ 11.2 Hz, 1H), 4.37 (d, J ¼ 11.2 Hz,
2H), 3.92 (d, J ¼ 2.0 Hz, 1H), 2.94 (dd, J ¼ 13.4, 6.5 Hz, 1H), 2.84 (dd,
J ¼ 13.4, 9.0 Hz, 1H), 1.40 (s, 9H). 13C NMR (100 MHz, CDCl3)
d 174.13,

M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
279
6.5. Label free assay (SRU-BIND)
in accordance with the Institutional Animal Care and Use Com-
mittees at the Torrey Pines Institute for Molecular Studies as
specified by the 2008 National Institutes of Health Guide for the
Care and Use of Laboratory Animals. Consistent with these guide-
lines, ongoing statistical testing of data collected was used to
minimize the number of animals used, within the constraints of
necessary statistical power.
HEK293 cells expressing MOR or CHO cells expressing DOR were
seeded 2 ꢂ 104 per well in 384-well plates coated with 10
mg/mL
Fibronectin, 10 mg/mL Collagen and 2% Albumin (SRU Biosystems or
Resonant Sensors). Following overnight incubation, media was
replaced with 25 L assay buffer containing HBSS media and
m
0.5e1% DMSO. The plates were equilibrated at room temperature
for 30 min and read on a BIND Profiler (SRU Biosensors) to obtain
6.9. Drug administration
baseline wavelength values. For opioid testing, 25
mL of each test
compound in assay buffer was added into each well (for a final
All compounds were administered through the intraperitoneal
concentration of 3.3
control, whereas the
or the d-opioid receptor agonist DPDPE (10 nM) were used as
positive controls. Wavelength values for each well on the plate was
read over 30 min. Increased peak wavelength values (PWV) occur
when the cells spread out on the plate surface, characteristic of
opioid agonist stimulation.
m
M). Buffer alone was used for a negative
(i.p.)
or
intracerebroventricular
(i.c.v.)
routes.
Intra-
m-opioid receptor agonists DAMGO (300 nM)
cerebroventricular injections were made directly into the lateral
ventricle according to the modified method of Haley and McCor-
mick [57]. The mouse was first lightly anesthetized with isoflurane,
an incision was made in the scalp, and the injection was made
2 mm lateral and 2 mm caudal to bregma at a depth of 3 mm. The
volume of all i.c.v. injections was 5
microliter syringe.
mL, using a 10-mL Hamilton
6.6. cAMP inhibition assay
6.10. Antinociception test
CHO cells expressing DOR were plated at 20,000 cells/well and
exposed to buffer, endomorphin analogues and 4
assay buffer (1 ꢂ PBS and 1 mM IMBX) with or without opioid
agonist standards (at 10 M) for 30 min at RT. The cells were
mM forskolin in
The 55 ꢀC warm-water tail-withdrawal assay was performed as
described in McLaughlin et al. [58], although modified by the
collection of tail-withdrawal latencies over time. Briefly, each
mouse was tested for baseline tail-withdrawal latency before drug
administration. Tested compound was administered by the i.c.v.
route. Latency to withdraw the tail was subsequently measured in
10-min post-drug administration intervals as indicated in the text,
with the tail-withdrawal latency in doseeresponse lines reported
20 min after compound administration or 30 min after morphine
administration (by the i.c.v. route), as this time point corresponds
with the peak antinociceptive effect of the respective agents. A
maximum response time of 15 s was utilized to prevent tissue
damage.
m
incubated with Europium cryptate and a fluorescence acceptor dye
(d2), both diluted in a conjugate lysis buffer for one hour (Cisbio
cAMP HiRange Kit, as specified by the manufacturer's instructions).
The FRET reaction was analyzed using a Tecan Safire2 as specified
by manufacturer's instructions.
6.7. Metabolic stability
Rat brain homogenate was prepared and test compound added
to a final concentration of 10
incubated in a water bath at 37 ꢀC. At each time point tested, the
sample was vortexed and a 100 L aliquot were drawn, then added
mM (in 1.5 mL). The sample was
m
6.11. Determination of in vivo agonist and antagonist activity
to 400
mL of 19 mM internal standard acetonitrile in triplicate.
Samples were centrifuged (10,000 rpm, 10 min, 4 ꢀC) and super-
To determine the opioid receptor selectivity of the agonist ac-
tivity of compounds 22 and 26, MOR KO and KOR KO mice were
administered either compound 22 or 26. Additional wild-type
C57BL/6J mice were pretreated 20 min with a single dose of the
DOR-selective antagonist naltrindole (20 mg/kg, i.p.) before
administration of each compound. Antinociception was tested
30 min later. To determine antagonist activity, mice were pre-
treated with compound 26 90 min before administration of the
MOR-preferring agonist morphine (10 mg/kg, i.p.), KOR-selective
agonist U50,488 (10 mg/kg, i.p.) or DOR-selective agonist SNC-80
(100 nmol, i.c.v.). Antinociception produced by these established
agonists was then measured 40 min after their administration
(thereby testing the antagonist effects approximately 2 h after
analogue administration). Reference agonists and antagonists were
administered using sterile saline (0.9%) as the vehicle, except for
SNC-80 which was dissolved in 35% DMSO in sterile saline (0.9%).
natant was transferred and dried using a Thermo Scientific speed
vacuum concentrator. Samples were reconstituted in 100 mL of 10%
acetonitrile. Samples were analyzed in reverse phase MRM mode
on a Shimazdu LC-20A prominence LC system connected to Absciex
3200 Q Trap MS/MS on Phenomenex Luna C18 5
m
m 250 ꢂ 4.6 mm
column. A ratio of analyte peak area to internal standard peak area
was calculated. The average ratio for each time point (A) was
calculated and (A/A0) versus time was plotted. Weighted linear
least-squares estimation with a fixed intercept of 100% was used to
determine the decay constant
turn was used to calculate the half-life using the equation t1/2 ¼ ln
(2)/
b and its standard error, which in
b
.
6.8. Animals
Adult (8e11 weeks old), male C57BL/6J were obtained from the
Jackson Laboratory, Bar Harbor, Maine, USA. MOR gene-disrupted
(MOR KO) and KOR gene disrupted (KOR KO) mice were obtained
from colonies established at Torrey Pines Institute for Molecular
Studies from homozygous breeding pairs of mice obtained from the
Jackson Laboratory. All mice were housed four to a cage. Mice were
maintained in a temperature- and humidity-controlled room at the
Torrey Pines Institute for Molecular Studies (Port Saint Lucie, Flor-
ida, USA) vivarium on a 12:12-h light/dark cycle (lights off at
19:00 h) with free access to food and water except during experi-
mental sessions. All procedures were preapproved and carried out
6.12. Liability evaluation
Rotorod assay: Possible sedative or hyperlocomotor effects of
vehicle or compounds 22 and 26 were assessed by rotorod per-
formance, as modified from previous protocols [59]. Following
seven habituation trials (the last utilized as a baseline measure of
rotorod performance), mice were administered i.c.v. vehicle (50%
DMSO), compound 22 or 26 (100 nmol, i.c.v., each) 15 min prior to
assessment in accelerated speed trials (180 s max. latency at
0ꢁ20 rpm) over a 60 min period. Decreased latencies to fall in the

280
M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
[7] P. Varamini, F.M. Mansfeld, J.T. Blanchfield, B.D. Wyse, M.T. Smith, I. Toth,
J. Med. Chem. 55 (2012) 5859e5867.
[8] R. Perlikowska, J. Fichna, A. Wyrebska, J. Poels, J.V. Broeck, G. Toth, M. Storr,
J.C. Rego, A. Janecka, Basic Clin. Pharmacol. Toxicol. 106 (2010) 106e113.
[9] R. Staniszewska, J. Fichna, K. Gach, G. Toth, J. Poels, J.V. Broeck, A. Janecka,
Chem. Biol. Drug Des. 72 (2008) 91e94.
[10] (a) A. Mollica, F. Pinnen, A. Stefanucci, F. Feliciani, C. Campestre, L. Mannina,
A.P. Sobolev, G. Lucente, P. Davis, J. Lai, S.W. Ma, F. Porreca, V.J. Hruby, J. Med.
Chem. 55 (2012) 3027e3035;
rotorod test indicate impaired motor performance. Data are
expressed as the percent change from baseline performance.
Acute opioid antinociceptive tolerance assay: For quantitative
measurements of acute opioid tolerance, a standardized state of
tolerance was induced by administration of the opioid agonist at
the approximate ED50 dose, as shown to be effective with morphine
[33,34]. To begin this protocol (time ¼ 0 h), the first dose of
morphine (3 nmol, i.c.v.) or compound 26 (2 nmol, i.c.v.) was
administered 8 h before administration of a second dose of
morphine or compound 26 (of varying dose, but each between 0.1
and 100 nmol, i.c.v.). Morphine-induced antinociception was
assessed 30 min after the second injection of morphine, whereas
compound 26-induced antinociception was assessed 20 min after
the second injection of opioid. Previous reports have indicated that
this dosing schedule induced relatively rapid, reliable, acute
morphine antinociceptive tolerance [33,34,40].
(b) A. Mollica, F. Pinnen, A. Stefanucci, L. Mannina, A.P. Sobolev, G. Lucente,
P. Davis, J. Lai, S.W. Ma, F. Porreca, V.J. Hruby, J. Med. Chem. 55 (2012)
8477e8482.
[11] P.W. Schiller, I. Berezowska, T.M.D. Nguyen, R. Schimidt, C. Lemieux,
N.N. Chung, M.L. Falcone-Hindley, W. Yao, J. Liu, S. Iwama, A.B. Smith,
R. Hirschmann, J. Med. Chem. 43 (2000) 551e559.
[12] J.E. Burden, P. Davis, F. Porreca, A.F. Spatola, Bioorg. Med. Chem. Lett. 12
(2002) 213e216.
[13] R.D. Dolle, M. Michaut, B. Martinez-Teipel, P.R. Seida, C.W. Ajello, A.L. Muller,
R.N. DeHaven, P.J. Carroll, Bioorg. Med. Chem. Lett. 19 (2009) 3647e3650.
[14] A. Nefzi, J.M. Ostresh, J.M. Appel, J. Bidlack, C.T. Dooley, R.A. Houghten, Bioorg.
Med. Chem. Lett. 16 (2006) 4331e4338.
[15] K. Guillemyn, P. Kleczkowska, A. Novoa, B. Vandormael, I. Van den Eynde,
P. Kosson, M.F. Asim, O.W. Schiller, M. Spetea, A.W. Lipkowski, D. Tourwe,
S. Ballet, Mol. Brain 5 (2012) 4.
Acknowledgments
[16] J. Fichna, M. Mazur, D. Grzwacz, W. Kamysz, R. Perlikowska, J. Piekielna,
M. Sobczak, G. Toth, A. Janecka, C. Chen, J. Olczak, Bioorg. Med. Chem. Lett. 23
(2013), 6676e6676.
[17] P. Varamini, I. Toth, Front. Pharmacol. 4 (2013) 155.
[18] D. Torino, A. Mollica, F. Pinnen, F. Feliciani, G. Lucente, G. Fabrizi, G. Portalone,
P. Davis, J. Lai, S.W. Ma, F. Porreca, V. Hruby, J. Med. Chem. 53 (2010)
4550e4554.
[19] R.P. Cheng, S.H. Gellman, W.F. DeGrado, Chem. Rev. 101 (2001) 3219e3232.
[20] J. Frackenpohl, P.I. Arvidsson, J.V. Schreiber, D. Seebach, ChemBioChem 2
(2001) 445e455.
[21] (a) G. Giuliana, L. Gentilucci, P. Melchiorre, S. Spampinato, Bioorg. Med. Chem.
Lett. 10 (2000) 2755e2758;
This research was supported by the National Natural Science
Foundation of China (No. 81473074 and No. 81273356), Natural
Science Foundation of Zhejiang Province (No. Z2110655), Program
for Zhejiang Leading Team of S&T Innovation, NIH Grant
1R01DA031370, Arthritis & Chronic Pain Research Institute (USA),
and the State of Florida, Executive Office of the Governor's
Department of Economic Opportunity.
Abbreviations
(b) Y. Okada, Y. Tsuda, Y. Fujita, T. Yokoi, Y. Sasaki, A. Ambo, R. Konishi,
M. Nagata, S. Salvadori, Y. Jinsmaa, S. Bryant, L.H. Lazarus, J. Med. Chem. 46
(2003) 3201e3209;
(c) G. Cardillo, L. Gentilucci, A.R. Qasem, F. Sgarzi, S. Spampinat, J. Med. Chem.
45 (2002) 2571e2578.
cAMP
DCM
DIEA
DIC
Dmt
EC50
ED50
cyclic adenosine monophosphate
dichloromethane
N,N'-diisopropylethylamine
1,3-diisopropylcarbodiimide
20,6'-dimethyltyrosine
[22] R. Perlikowska, K. Gach, J. Fichna, G. Toth, B. Walkowiak, J.C. do-Rego,
A. Janecka, Bioorg. Med. Chem. 17 (2009) 3789e3794.
[23] D. Torino, A. Mollica, F. Pinnen, G. Lucente, F. Feliciani, P. Davis, J. Lai, S.W. Ma,
F. Porreca, V.J. Hruby, Bioorg. Med. Chem. Lett. 19 (2009) 4115e4118.
€
[24] A. Keresztes, M. Szucs, A. Borics, K.E. Kover, E. Forro, F. Fülop, C. Tomboly,
concentration for 50% of maximal effect
dose for 50% of maximal effect
A. Peter, A. Pahi, G. Fabian, M. Muranyi, G. Toth, J. Med. Chem. 51 (2008)
4270e4279.
[25] A. Borics, J.R. Mallareddy, I. Timari, K.E. Kover, A. Keresztes, G. Toth, J. Med.
Chem. 55 (2012) 8418e8428.
[26] (a) Y. Wang, Y. Xing, X. Liu, H. Ji, M. Kai, Z. Chen, J. Yu, D. Zhao, H. Ren, R. Wang,
J. Med. Chem. 55 (2012) 6224e6236;
DMSO dimethyl sulfoxide
EtOAc
GPI
HOBt
KO
ethyl acetate
guinea pig ileum
1-hydroxybenzotriazole
knock out
(b) X. Liu, Y. Wang, Y. Xing, J. Yu, H. Ji, M. Kai, Z. Wang, D. Wang, Y. Zhang,
D. Zhao, R. Wang, J. Med. Chem. 56 (2013) 3102e3114.
[27] G. Balboni, S. Salvadori, R. Guerrini, L. Negri, E. Giannini, Y. Jinsmaa,
S.D. Bryant, L.H. Lazarus, J. Med. Chem. 45 (2002) 5556e5563.
[28] G. Balboni, R. Guerrini, S. Salvadori, C. Bianchi, D. Rizzi, S.D. Bryant,
L.H. Lazarus, J. Med. Chem. 45 (2002) 713e720.
[29] (a) S.L. Harbeson, D.H. Rich, Biochemistry 27 (1988) 7301e7310;
(b) Y. Hamada, H. Abdel-Rahman, A. Yamani, J. Nguyen, M. Stochaj, K. Hidaka,
T. Kimura, Y. Hayashi, K. Saito, S. Ishiura, Y. Kiso, Bioorg. Med. Chem. Lett. 18
(2008) 1649e1653;
MBHA 4-methylbenzhydrylamine
MeOH methanol
MVD
PMSF
s.c.
mouse vas deferens
phenylmethanesulfonyl fluoride
subcutaneous
TFA
THF
Tic
trifluoroacetic acid
tetrahydrofuran
1,2,3,4-tetrahydroisoquinoline
(c) T. Kakizama, K. Hidaka, D. Hamada, R. Yamaguchi, T. Uemura, H. Kitamura,
H.D. Tagad, T. Hamada, Z. Ziora, Y. Hamada, T. Kimura, Y. Kiso, J. Pept. Sci. 16
(2010) 257e262;
(d) T. Mimoto, N. Hattori, Y. Nagano, S. Makoto, Y. Kiso, Eur. Pat. Appl.
0490667 B1, 1999; (e) Y. Hayashi, M. Skwaczynski, Y. Hamada, Y. Sohma,
T. Kimura, Y. Kiso, J. Med. Chem. 46 (2003) 3782e3784.
Appendix A. Supplementary data
[30] (a) C. Tomboly, K.E. Kover, A. Peter, D. Tourwe, D. Biyashev, S. Benyhe,
A. Borsodi, M. Al-Khrasani, A.Z. Ronai, G. Toth, J. Med. Chem. 47 (2004)
735e743;
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.049.
(b) T. Honda, N. Shirasu, K. Isozaki, M. Kawano, D. Shigehiro, Y. Chuman,
T. Fujita, T. Nose, Y. Shimohigashi, Bioorg. Med. Chem. 15 (2007) 3883e3888.
[31] M. Al-Khrasani, G. Orosz, L. Kocsis, V. Farkas, A. Magyar, I. Lengyel, S. Benyhe,
References
ꢀ
A. Borsodi, A.Z. Ronai, Eur. J. Pharmacol. 421 (2001) 61e67.
[32] G. Toth, E. Ioja, C. Tomboly, S. Ballet, D. Tourwe, A. Peter, T. Martinek,
N.N. Chung, P.W. Schiller, S. Benyhe, A. Borsodi, J. Med. Chem. 50 (2007)
328e333.
[33] Q. Jiang, A. Seyed-Mozaffari, A. Sebastian, S. Archer, J.M. Bidlack, J. Pharmacol.
Exp. Ther. 273 (1995) 680e688.
[34] M.R. Hoot, E.I. Sypek, K.J. Reilley, A.N. Carey, J.M. Bidlack, J.P. McLaughlin,
Behav. Pharmacol. 24 (2013) 144e152.
[35] P.L. Gelman, N.E.G. Herrera, M.E.M. Ortega, L.P. Romero, C.T. Santillan,
A.S. Juarez, B.A. Palma, Salud Ment. 33 (2010) 179e196.
[1] N. Dietis, R. Guerrini, G. Calo, S. Salvadori, D.J. Rowbothamand, D.G. Lambert,
Br. J. Anaesth. 103 (2009) 38e49.
[2] A. Gupta, F.M. Decaillot, L.A. Devi, AAPS J. 8 (2006) E153eE159.
[3] A. Janecka, J. Fichna, T. Janecki, Curr. Top. Med. Chem. 4 (2004) 1e17.
[4] (a) J.E. Zadina, L. Hackler, L.J. Ge, A.J. Kastin, Nature 386 (1997) 499e502;
(b) L. Hackler, J.E. Zadina, L.J. Ge, A.J. Kastin, Peptides 18 (1997) 1635e1639;
(c) L. Gentilucci, A. Tolomelli, Curr. Top. Med. Chem. 4 (2004) 105e121.
[5] A. Janecka, R. Staniszewska, K. Gach, J. Fichna, Peptides 29 (2008) 2066e2073.
[6] (a) A. Giannis, T. Kolter, Angew. Chem. Int. Ed. Engl. 32 (1993) 1244e1267;
(b) C. Tomboly, A. Peter, G. Toth, Peptides 23 (2002) 1573e1580.
[36] L. Wu, J. Chen, D. Zhong, C. Lin, X. Wang, Acta Neurobiol. Exp. 62 (2002) 1e5.

M. Hu et al. / European Journal of Medicinal Chemistry 92 (2015) 270e281
281
[37] P.H. Lee, A. Gao, C. van Staden, J. Ly, J. Salon, A. Xu, Y. Fang, R. Verkleeren,
Assay. Drug Dev. Technol. 6 (2008) 83e94.
(d) L.C. Purington, I.D. Pogozheva, J.R. Traynor, H.I. Mosberg, J. Med. Chem. 52
(2009) 7724e7731.
[38] M.F. Peters, F. Vaillancourt, M. Heroux, M. Valiquette, C.W. Scott, Assay. Drug
Dev. Technol. 8 (2010) 219e227.
[46] S. Ananthan, AAPS J. 8 (2006) E118eE125.
[47] P.W. Schiller, AAPS J. 8 (2008) E560eE565.
[39] E. Kelly, Br. J. Pharmacol. 169 (2013) 1430e1446.
[48] (a) G. Balboni, V. Onnis, C. Congiu, M. Zotti, Y. Sasaki, A. Ambo, S.D. Bryant,
Y. Jinsmaa, L.H. Lazarus, C. Trapella, S. Salvadori, J. Med. Chem. 49 (2006)
5610e5617;
[40] J.L. Mathews, A.V. Smrcka, J.M. Bidlack, J. Neurosci. 28 (2008) 12183e12189.
[41] E.E. Abdelhamid, M. Sultana, P.S. Portoghese, A.E. Takemori, J. Pharmacol. Exp.
Ther. 258 (1991) 299e303.
[42] M.E. Fundytus, P.W. Schiller, M. Shapiro, G. Weltrowska, T.J. Coderre, Eur. J.
Pharmacol. 286 (1995) 105e108.
(b) B. Vandormael, D.D. Fourla, A. Gramowski-Voss, P. Kosson, D.G. Weiss,
O.H. Schroder, A. Lipkowski, Z. Georgoussi, D. Tourwe, J. Med. Chem. 54 (2011)
7848e7859.
[43] M.J. Hepburn, P.J. Little, J. Gingras, C.M. Kuhn, J. Pharmacol. Exp. Ther. 281
(1997) 1350e1356.
[49] B.A. Harrison, T.M. Gierasch, C. Neilan, G.W. Pasternak, G.L. Verdine, J. Am.
Chem. Soc. 124 (2002) 13352e13353.
[44] (a) P.W. Schiller, M.E. Fundytus, L. Merovitz, G. Weltrowska, T.M. Nguyen,
C. Lemieux, N.N. Chung, T.J. Coderre, J. Med. Chem. 42 (1999) 3520e3526;
(b) Y. Fujita, Y. Tsuda, T. Li, T. Motoyama, M. Takahashi, Y. Shimizu, T. Yokoi,
Y. Sasaki, A. Ambo, A. Kita, Y. Jinsmaa, S.D. Bryant, L.H. Lazarus, Y. Okada,
J. Med. Chem. 47 (2004) 3591e3599;
[50] R. Nishizawa, T. Saino, J. Med. Chem. 20 (1997) 510e515.
[51] A. Bartoszewicz, M. Livendahl, B. MartíneMatute, Chem. Eur. J. 14 (2008)
10547e10550.
[52] J.S. Arnold, G.T. Cizio, D.R. Heitz, H.M. Nguyen, Chem. Commun. 48 (2012)
11531e11533.
(c) L.C. Purington, I.D. Pogozheva, J.R. Traynor, H.I. Mosberg, J. Med. Chem. 52
(2009) 7724e7731.
[45] (a) E.J. Bilsky, R.D. Egleton, S.A. Mitchell, M.M. Palian, P. Davis, J.D. Huber,
H. Jones, H.I. Yamamura, J. Janders, T.P. Davis, F. Porreca, V.J. Hruby, R. Polt,
J. Med. Chem. 43 (2000) 2586e2590;
[53] G.Q. Yang, C.R. Shen, W.B. Zhang, Angew. Chem. Int. Ed. 51 (2012) 9141e9145.
[54] J.R. Rich, S.G. Withers, Angew. Chem. Int. Ed. 51 (2012) 8640e8643.
[55] Z.Y. Wang, L. Hunter, J. Fluor. Chem. 143 (2012) 143e147.
[56] R.A. Houghten, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 5131e5135.
[57] T.J. Haley, W.G. McCormick, Br. J. Pharmacol. Chemother. 12 (1957) 12e15.
[58] J.P. McLaughlin, K.P. Hill, Q. Jiang, A. Sebastian, S. Archer, J.M. Bidlack,
J. Pharmacol. Exp. Ther. 289 (1999) 304e311.
(b) S.Q. He, Z.N. Zhang, J.S. Guan, H.R. Liu, B. Zhao, H.B. Wang, Q. Li, H. Yang,
J. Luo, Z.Y. Li, Q. Wang, Y.J. Lu, L. Bao, X. Zhang, Neuron 69 (2011) 120e131;
(c) Y.S. Lee, V. Kulkarani, S.M. Cowell, S.W. Ma, P. Davis, K.E. Hanlon,
T.W. Vanderah, J. Lai, F. Porreca, R. Vardanyan, V.J. Hruby, J. Med. Chem. 54
(2011) 382e386;
[59] S.O. Eans, M.L. Ganno, K.J. Reilley, K.A. Patkar, S.N. Senadheera, J.V. Aldrich,
J.P. McLaughlin, Br. J. Pharmacol. 169 (2013) 426e436.