Nonpeptide small molecule agonist and antagonist original leads for neuropeptide FF1 and FF2 receptors

Article abstract of DOI:10.1021/jm500989n

Neuropeptide FF1 and FF2 receptors (NPFF1-R and NPFF2-R), and their endogenous ligand NPFF, are one of only several systems responsible for mediating opioid-induced hyperalgesia, tolerance, and dependence. Currently, no small molecules displaying good affinity or selectivity for either subtype have been reported, to decipher the role of NPFF2-R as it relates to opioid-mediated analgesia, for further exploration of NPFF1-R, or for medication development for either subtype. We report the first nonpeptide small molecule scaffold for NPFF1,2-R, the guanidino-piperidines, and SAR studies resulting in the discovery of a NPFF1 agonist (7b, K_i = 487 ± 117 nM), a NPFF1 antagonist (46, K_i = 81 ± 17 nM), and a NPFF2 partial antagonist (53a, K_i = 30 ± 5 nM), which serve as leads for the development of pharmacological probes and potential therapeutic agents. Testing of 46 alone was without effect in the mouse 48 °C warm-water tail-withdrawal test, but pretreatment with 46 prevented NPFF-induced hyperalgesia.

Full text of DOI:10.1021/jm500989n

Article
pubs.acs.org/jmc
Nonpeptide Small Molecule Agonist and Antagonist Original Leads
for Neuropeptide FF1 and FF2 Receptors
V. Blair Journigan,† Christophe Mesangeau,† Neha Vyas,† Shainnel O. Eans,§ Stephen J. Cutler,†
́
Jay P. McLaughlin,§ Catherine Mollereau,‡ and Christopher R. McCurdy*,†
†Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United
States
‡Institut de Pharmacologie et Biologie Structurale, 31077 Toulouse, France
§Department of Biology, Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987, United States
ABSTRACT: Neuropeptide FF1 and FF2 receptors (NPFF1-R and NPFF2-R), and their endogenous ligand NPFF, are one of
only several systems responsible for mediating opioid-induced hyperalgesia, tolerance, and dependence. Currently, no small
molecules displaying good affinity or selectivity for either subtype have been reported, to decipher the role of NPFF2-R as it
relates to opioid-mediated analgesia, for further exploration of NPFF1-R, or for medication development for either subtype. We
report the first nonpeptide small molecule scaffold for NPFF1,2-R, the guanidino-piperidines, and SAR studies resulting in the
discovery of a NPFF1 agonist (7b, Ki = 487 117 nM), a NPFF1 antagonist (46, Ki = 81 17 nM), and a NPFF2 partial
antagonist (53a, Ki = 30 5 nM), which serve as leads for the development of pharmacological probes and potential therapeutic
agents. Testing of 46 alone was without effect in the mouse 48 °C warm-water tail-withdrawal test, but pretreatment with 46
prevented NPFF-induced hyperalgesia.
The opioid-modulating properties (among others) of the
NPFF peptide have been chronicled throughout the literature
since its discovery in 1985,6 described as producing either a
pro- or antinociceptive effect depending on the route of
administration. For example, when administered in rats by
intracerebroventricular (icv) injection alone or in combination
with morphine or other opioid agonists, NPFF or its stable
analog 1DMe (D.YL(N-Me)FQPQRFa) produces an anti-
opioid effect,6−9 however; when given intrathecally (it.), NPFF
yields an opioid-like effect and potentiates morphine-induced
analgesia in various pain models.10−13 Thus, a precise role for
NPFF1-R and NPFF2-R as anti- or pronociceptive systems in
preliminary in vivo models has not been delineated. In rats, the
lack of NPFF1-R at the spinal level has formed the basis of the
hypothesis that the antinociceptive activity of NPFF observed
upon it. administration is a result of its interaction with NPFF2-
R, while its pronociceptive activity at the supraspinal (i.e.,
brain) level is a result of its interaction with NPFF1-R.2,14
However, the pronociceptive activity of NPFF in the rat brain
cannot be exclusively attributed to its activity at the NPFF1-R,
INTRODUCTION
■
The neuropeptide FF (NPFF) system comprises two receptor
subtypes, NPFF1 and NPFF2 receptors (NPFF1-R and
NPFF2-R), which are Gi/o coupled G-protein coupled receptors
(GPCRs) with 30−35% homology to neuropeptide Y and
orexin receptors,1 and four endogenous ligands belonging to
the Arg-Phe-NH2 (RFa) peptide family.2 These peptides
i n c l u d e h N P F F ( S Q A F L F Q P Q R F a ) , h N P S F
(SLNFEELKDWGPKNVIKMSTPAVNKMPHSFANLPLRFa),
and hNPAF (AGEGLSSPFWSLAAPQRFa) (10.7-, 1.2-, and
10.1-fold selective in radioligand competition binding assays for
the hNPFF2 subtype, respectively), and hNPVF
(VPNLPQRFa) (27.8-fold selective for the hNPFF1 subtype).2
Both NPFF1-R and NPFF2-R are present in the rat brain;
however; only NPFF2-R is found in the spinal cord.3
Conversely, in humans, the distribution of NPFF1-R is not
restricted to the CNS, since NPFF1 mRNA4 is found in the
spinal cord. Talmont and colleagues noted species-related
differences in the pharmacological profiles between mouse and
human NPFF2-R, further highlighting the consideration that
must be taken in the interpretation of preliminary in vitro and
in vivo results in mouse models as they translate to human
targets.5
Received: July 1, 2014
Published: September 30, 2014
© 2014 American Chemical Society
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because the activity of the rNPFF peptide (NPAFLFQPQRFa)
at rNPFF1,2-R and hence its selectivity has not been reported.
Failing the availability of any truly selective ligands for NPFF
receptors, gene knockout studies would prove valuable in
elucidating the pharmacology of these subtypes.
A limited number of dipeptides and small molecules have
been used to try to define the pharmacology of these subtypes,
with mixed results. Although a pronociceptive/antiopioid role
has been assigned to the NPFF system as a whole based on the
in vivo activity of a nonselective dipeptide competitive
antagonist RF9 (administered subcutaneously (sc) or intra-
peritoneally (ip)) (Chart 1)15,16 in opioid-induced hyperalgesia
AC-099 (Chart 1) and partial agonist 2 (Chart 1) (both
administered ip) reported by Acadia Pharmaceuticals.14,19,20
However, it must be noted that 1DMe is not highly selective,
exhibiting full agonist activity on both NPFF1-R (EC50 = 71
14 nM) and NPFF2-R (EC50 = 2.7 0.5 nM),21 and thus both
receptors are likely to contribute to its pharmacological
response. Moreover, the modest activity and selectivity of
aryliminoguanidines AC-099 and 2 displayed in unconventional
in vitro assays limits their utility in defining NPFF2-R with
respect to opioid-mediated analgesia (Chart 1). Additionally,
no off-target opioid affinity is reported for these aryliminogua-
nidines, which calls into question the interpretation of their in
vivo analgesic activity.
A single core scaffold, on which modifications can be made to
yield a selective agonist or antagonist functional profile, would
be of use to help define the pharmacology of NPFF2-R. The
functional profiles of the compounds summarized in Chart 1
underscore the need for compounds with NPFF1 agonist or
NPFF2 agonist and antagonist efficacy to aid in the exploration
of the pharmacology of these subtypes. Other small molecule
ligands for NPFF1-R and NPFF2-R, containing a wide range of
scaffolds and thus no definitive SAR or pharmacophore, have
been reported mainly in the patent literature.23 However; to
our knowledge, no pharmacologically pure and high affinity
NPFF1 and NPFF2 agonists and antagonists have been
reported (apart from dipeptide NPFF1 antagonist 1). More-
over, the in vivo activities of these compounds as they pertain
to pain modulation, if any, have not been reported.
Chart 1. Reported Ligands Used for Pharmacological
a
Characterization of NPFF-1 and NPFF-2 Receptors
As summarized above, the majority of these compounds are
proprietary and limited in terms of binding affinity, selectivity,
and functional profiles necessary to define the pharmacology of
these subtypes. Due to the lack of availability of these
compounds to the scientific community, a tremendous design
space exists to develop novel, selective NPFF1 and NPFF2
ligands of varying functional profiles to aid in the study of these
receptors.
SAR of the residues conserved among mouse, rat, bovine and
human NPFF (FLFQPQRFa) (Ki 0.21/0.34 nM in the
membrane of rat spinal cord or CHO hNPFF2-R)24,25 indicate
that the peptide adheres to a message−address concept,26 with
the C-terminal amide, guanidine of arginine,7 and aromatic ring
of phenylalanine8 serving as the message component conferring
the majority of the peptide’s affinity and functional activity. To
date, a good number of NPFF ligands reported in the literature
contain at least phenyl and guanidine moieties, including
dipeptides RF9 and 1, and aryliminoguanidines AC-099 and 2.
Taking these functionalities into consideration, we desired a
scaffold on which to accommodate these pharmacophoric
elements while at the same time maintaining chemical
feasibility. The 4-(phenylamino)piperidine-4-carbonitrile tem-
plate (Figure 1) seemed to be a reasonable starting point,
a
The Ki and EC50 values associated with the ligands described are
included for discussion purposes only and cannot be directly
compared. Ki values were determined by radioligand displacement
b
c
assays. EC50 values were calculated from pEC50 values determined in
RSAT assay.20 EC50 values were calculated from pEC50 values
d
determined in cAMP assay.20 EC50 values were taken directly, as
e
determined in the IP accumulation assay.22 EC50 values were
f
calculated from pEC50 values determined in RSAT assay,19 using
hNPFF1-R and hNPFF2-R.
(OIH), tolerance, and dependence assays, conflicting reports
exist as to the nociceptive- or opioid-modulating properties of
each subtype. NPFF1-R has been classified as a pronociceptive/
antiopioid system using a selective (1843-fold) dipeptide
antagonist 1 (administered sc) (Chart 1)17 and a modestly
selective (19.5-fold) small molecule antagonist AC-262620
(administered ip)14 from Acadia Pharmaceuticals (hNPFF1 Ki
= 16.4 10.1 nM, similar in structure to AC-099 and 2) based
on their activities within in vivo models of pain. Further
confirmation of NPFF1-R’s pronociceptive role in terms of
opioid analgesia could be realized with NPFF1-selective
agonists. While being similarly classified as a pronociceptive/
antiopioid construct due to its antiopioid activity at the cellular
level using the stable peptide agonist 1DMe,18 NPFF2-R is also
contrarily described as a antinociceptive/pro-opioid system
using small molecules of modest affinity, putative full agonist
Figure 1. 4-(Phenylamino)piperidine-4-carbonitrile scaffold for
designed NPFF ligands.
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a
Scheme 1. Synthesis of 7a−d and 9a−c
a
Reagents and conditions: (a) aniline, TMSCN, AcOH, 0 °C, 1 h, 70−94%; (b) LiAlH4, diethyl ether (ah), 0 °C, 35−40% (for 5a,5c) or NH3/
MeOH, Raney Ni, 3 atm H2(g), 4 h, rt, 82−90% (for 5b, 5d); (c) Boc-Arg-(Boc)2-OH, EDCI, HOBt, Et3N, DCM, rt, 24 h, 50−70%; (d) HCl/
dioxane, 3 days, rt, 95%; (e) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl2, Et3N, DMF, 0 °C → rt, 10 h, 30%; (f) HCl/dioxane, 4
days, rt, quantitative; (g) 2-bromomethylnaphthalene, K2CO3, KI, 4-methyl-2-pentanone, reflux, 5 h; (h) AcOH, conc. HCl (aq), reflux, 18 h, 40%.
a
because the 4-anilido piperidine portion is known to be a
privileged scaffold for GPCRs, particularly in the field of opioid
analgesics (i.e., fentanyl). Moreover, the piperidine nitrogen
portion allows for lipophilic substitutions to be made, and the
nitrile moiety could be converted to the corresponding amine,
to allow for the introduction of guanidine-containing groups.
We report herein a series of designed guanidino-piperidines
based on this scaffold, which led to the first class of ligands to
demonstrate a tractable SAR for modulation of affinity, efficacy,
and selectivity at NPFF1-R and NPFF2-R.
Scheme 2. Synthesis of 15
RESULTS AND DISCUSSION
a
■
Reagents and conditions: (a) Boc-Arg-(Boc)2-OH, EDCI, HOBt,
Chemistry. Piperidones 3a−c and 12d (Scheme 1) bearing
various lipophilic substitutions at the nitrogen (either
commercially available or prepared via the ketal 10) were
subjected to standard Strecker conditions27 to generate N-
substituted carbonitriles 4a−d in 70−94% yield. Reduction of
the nitriles using either LiAlH4 or hydrogenation afforded the
corresponding amines 5a−d in 35−90% yield, followed by
coupling with Boc-Arg-(Boc)2-OH using EDCI/HOBt to give
Boc-protected arginine intermediates 6a−d or HgCl2-assisted
guanidation to give protected guanidine intermediates 8a−c in
30−70% yield. Deprotection with HCl/dioxane afforded
guanidines 7a−d and 9a−c in excellent yield (95%).
Des-aniline 15 (Scheme 2) was prepared starting with (1-
benzyl-4-piperidinyl)methylamine (13) and using analogous
chemistry to that used for 6a−d (Scheme 1) in 70% yield,
followed by deprotection with HCl/dioxane in excellent yield
(95−99%).
Et3N, DCM, rt, 24 h, 70%; (b) 4 M HCl/dioxane, rt, 10 h to 4 days
(95−99%).
Guanidated glycine 21 (1-carbon aliphatic linker) was
synthesized by coupling Boc-Gly-OH with 4-(aminomethyl)-
1-benzyl-N-phenylpiperidin-4-amine (5b) using EDCI/HOBt
to give intermediate 16b in 65% yield, followed by Boc removal
with TFA to yield amine 17b (92%) (Scheme 3). Boc-
protected guanidation of amine 17b with 1,3-bis(tert-butox-
ycarbonyl)-2-methyl-2-thiopseudourea afforded intermediate
20 in 84% yield, and deprotection with TFA afforded one-
carbon linked guanidine 21 in 90% yield. Two carbon
homologue 17a was prepared in a similar manner via EDCI/
HOBt coupling of cyanoacetic acid with amine 5b (Scheme 1)
to yield intermediate 16a in 50% yield, then reduction of the
cyano moiety with H2(g)/Raney Ni to amine 18a (76%). Boc-
protected guanidation of amine 18a afforded intermediate 19a
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a
Scheme 3. Synthesis of 17a−g and 21
a
Reagents and conditions: (a) carboxylic acid, EDCI, HOBt, Et3N, DCM, 0 °C → rt, 15 h, 50−84%; (b) TFA, DCM, rt, 2 h, 77−100%; (c) 1,3-
bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl2, Et3N, THF/MeOH, rt, 15 h, 16-31%; (d) NH3/MeOH, Raney Ni, 2.4 atm H2(g), 15 h,
rt, 76%; (e) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et3N, DMF(ah), rt, 18 h, 87%; (f) TFA, DCM(ah), rt, 3 h, 77%; (g) 1,3-
bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et3N, DMF(ah), rt, 2 days, 84%; (h) TFA, DCM(ah), rt, 3 h, 90%.
a
Scheme 4. Synthesis of 26
a
Reagents and conditions: (a) aniline, Ti(OiPr)4, diethylaluminum cyanide, DCE, rt, 24 h, 74%; (b) NH3/MeOH, Raney Ni, 2.7 atm H2(g), 24 h,
78%; (c) 1,3-di-Boc-2-(carboxymethyl)guanidine, HOBt, EDCI, Et3N, DCM, 0 °C → rt, 20 h, 37%; (d) TFA, DCM, rt, 6 h, 65%.
in 87% yield; subsequent deprotection with TFA yielded two-
carbon linked guanidine 17a (77%). Three and four carbon
homologues 17c,d were prepared in a similar manner by first
converting 4-aminobutanoic acid and 5-aminopentanoic acid to
their corresponding Boc-protected guanidines as described
above (17−26%), followed by EDCI/HOBt coupling with
amine 5b to yield intermediates 16c,d (77−80%). Boc-removal
from intermediates 16c,d with TFA afforded analogs 17c,d.
Phenyl linked guanidines 17e−g were prepared by converting
3-aminobenzoic acid, 4-aminobenzoic acid, and 4-aminophenyl-
acetic acid to the Boc-protected guanidines as described above
(16-31%), followed by EDCI/HOBt coupling with amine 5b to
yield intermediates 16e−g (66−84%). Boc removal from
intermediates 16e−g with TFA afforded analogs 17e−g in
quantitative yield.
One, three-substituted analog 26 was obtained (Scheme 4)
using similar chemistry as shown in Schemes 1 and 2. 1-Benzyl-
3-piperidone 22 was coupled to aniline under Strecker
conditions to give nitrile 23 in 74% yield, followed by catalytic
hydrogenation to amine 24 (78% yield). Boc-protected
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a
Scheme 5. Phenyl (Ring 1) Modification of Lead 21: Synthesis of 30a−d
a
Reagents and conditions: (a) acetic acid, substituted aniline, KCN(aq) or TMSCN, 0 °C → rt, 4 h, 55−77%; (b) NH3/MeOH, Raney Ni, 2.7 atm
H2(g), 16−24 h, 89−95%; (c) 1,3-di-Boc-2-(carboxymethyl)guanidine, HOBt, EDCI, Et3N, DCM, 0 °C → rt, 20 h, 33−67%; (d) TFA, DCM, rt, 2−
4 h, 53−83%.
a
Scheme 6. Aniline Amine Modification of Lead 21: Synthesis of 34, 42, and 43
a
Reagents and conditions: (a) Boc-gly-OH, HOBt, EDCI, Et3N, DCM, 0 °C → rt, 24 h, 78%; (b) TFA, DCM, rt, 1 h then 1,3-bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl2, Et3N, DMF, rt, 1 h, 89%; (c) TFA, DCM, rt, 2 h; (d) benzyl bromide (for 36) or (2-
bromoethyl)benzene (for 37), LDA, THF(ah), −70 °C → rt, 2−3 h, 86−89%; (e) NH3/MeOH, Raney Ni, 2.4−2.7 atm H2(g), 24 h, 61−90%; (f)
1,3-di-Boc-2-(carboxymethyl)guanidine, HOBt, EDCI, Et3N, DCM, 0 °C → rt, 22 h, 48% (for 40); for 41 (i) Boc-gly-OH, HOBt, EDCI, Et3N,
DCM, 0 °C → rt, 20 h, 91%, (ii) TFA, DCM, rt, 1 h, (iii) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl2, Et3N, DMF, rt, 2 h, 76%;
(g) TFA, DCM, rt, 2−3 h, 71−85%.
guanidated glycine 25 was accessed by EDCI/HOBt coupling
of amine 24 with 1,3-di-Boc-2-(carboxymethyl)guanidine in
37% yield; subsequent TFA deprotection afforded 1,3-
substituted piperidine 26 in 65% yield (Scheme 4).
Analog 34 was synthesized by first coupling commercially
available 1-(1-benzyl-4-phenylpiperidin-4-yl)methanamine 31
with Boc-gly-OH using EDCI/HOBt in 78% yield to give
intermediate 32, deprotection with TFA and HgCl2-assisted
guanidation (33, 89% overall yield), and finally deprotection
with TFA (Scheme 6). Methylene 42 and ethylene 43 analogs
were prepared by initially generating the carbanion at the 4-
position of commercially available 1-benzylpiperidine-4-carbon-
itrile (35) with LDA, then displacing benzyl bromide (to
intermediate 36) or (2-bromoethyl)benzene (to intermediate
37) in 86−89% yield. Catalytic hydrogenation of the cyano
moieties of intermediates 36 and 37 afforded the corresponding
amines 38 and 39 in 61−90% yield. The Boc-protected
guanidated glycines of amines 38 and 39 (intermediates 40 and
41, respectively) were prepared by either coupling with 1,3-di-
1-Naphthyl, 3,5-dimethoxyphenyl, 3-methoxyphenyl, and
3,4-dichlorophenyl analogs were prepared by reacting the
corresponding substituted aniline with N-benzyl piperidone
under Strecker conditions to give nitriles 27a−d in 55−77%
yield (Scheme 5). Subsequent reduction of the nitriles using
catalytic hydrogenation afforded amines 28a−d in excellent
yields (89−95%), followed by EDCI/HOBt coupling with 1,3-
di-Boc-2-(carboxymethyl)guanidine to give Boc protected
guanidines 29a−d in moderate yields (33−67%). Deprotection
with TFA afforded guanidines 30a−d in good yield (53−83%).
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Boc-2-(carboxymethyl)guanidine using EDCI/HOBt or em-
ploying the sequence above (coupling with Boc-gly-OH,
deprotection, HgCl2-assisted Boc guanidation) in reasonable
yields. Deprotection with TFA afforded methylene and
ethylene analogs 42−43 in 71−85% yield (Scheme 6).
Reverse amide 46 was prepared by coupling commercially
available 1-benzyl-4-phenylamino-4-carboxy-piperidine 44
(Scheme 7) with 2-(2-aminoethyl)-1,3-di-Boc-guanidine using
EDCI/HOBt to give intermediate 45 in 70% yield, followed by
TFA deprotection in 54% yield.
of Boc-protected guanidated glycines 48a−g afforded N-
substituted analogs 49a−g in 54−85% yield.
1-Naphthyl (53a), 3,4-dichlorophenyl (53b), 2-naphthyl
(53c), and biphenyl (53d) replacements were prepared using
the same synthetic sequence as that of benzyl 42 and phenethyl
43 using 1-benzylpiperidine-4-carbonitrile, 35 (Scheme 9).
Briefly, displacement of phenyl-substituted halides with the
LDA-generated carbanion of carbonitrile 35 gave intermediates
50a−d in 59−91% yield, followed by catalytic hydrogenation of
the cyano moieties to yield amines 51a−d (38−93%). The Boc-
protected guanidated glycines of amines 51a−d (intermediates
52a−d) were prepared by either coupling with 1,3-di-Boc-2-
(carboxymethyl)guanidine using EDCI/HOBt or employing
the sequence above (coupling with Boc-gly-OH, deprotection,
HgCl2-assisted Boc guanidation) (Scheme 6) in 41−77% yield.
Deprotection with TFA afforded 1-naphthyl (53a), 3,4-
dichlorophenyl (53b), 2-naphthyl (53c), and biphenyl analogs
(53c) in 54−76% yield.
Scheme 7. Methylene Amide Modification of Lead 21:
a
Synthesis of 46
Biology. Binding Affinity and Functional Efficacy at
hNPFF1-R and hNPFF2-R. The affinities of 7a−d, 9a−c, 15,
17a−g, 21, 26, 30a−d, 34, 42−43, 46, 49a−g, and 53a−d at
hNPFF1-R and hNPFF2-R were evaluated by competition
experiments using the selective NPFF1 and NPFF2 radio-
ligands [125I]YVP (YVPNLPQRFa) and [125I]EYF (EYW-
SLAAPQRFa), or [3H]-NPVF and [3H]-EYF, respectively, in
membranes of CHO cells stably expressing each receptor
(Tables 1−4). Tested compounds were compared with
previously reported high affinity peptides 1DMe and NPVF.21
Selected compounds were tested for off-target affinity at μ-, δ-,
and κ-opioid receptors (MOP, DOP, and KOP; Table 5) in
competition experiments using the selective radioligands
[3H]DAMGO, [3H]DPDPE, and [3H]U-69,593, respectively,
in membranes of HEK 293 cells stably expressing each receptor
(Table 5) and compared to the standard naloxone. The
functional activity of 7b was tested by measuring its capacity to
increase the binding of [35S]GTPγS in the membranes of CHO
cells stably expressing each receptor (Figure 2). The functional
activity of 21, 30a−c, 42, 46, and 53a was assessed by testing
each compound’s capacity to inhibit forskolin-induced intra-
a
Reagents and conditions: (a) 2-(2-aminoethyl)-1,3-di-Boc-guanidine,
HOBt, EDCI, Et3N, 1-methyl-2-pyrrolidone, rt, 24 h, 70%; (b) TFA,
DCM, rt, 4 h, 54%.
Various aromatic and nonaromatic groups were explored at
the piperidine nitrogen (49b−g, Scheme 8) by initial
debenzylation of tert-butyl 1-(1-benzyl-4-(phenylamino)-
piperidin-4-yl)-10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaun-
decan-6-ylidenecarbamate, 20 (Scheme 8), using catalytic
hydrogenation to give intermediate 47 in 68% yield and
subsequent displacement of the corresponding substituted
bromide or chloride in 27−96% yield to afford intermediates
48b−d,f,g. Intermediate 48e was prepared by coupling 4-
(aminomethyl)-1-(naphthalen-2-ylmethyl)-N-phenylpiperidin-
4-amine, 5d (Scheme 1), with 1,3-di-Boc-2-(carboxymethyl)-
guanidine using EDCI/HOBt in 48% yield. TFA deprotection
a
Scheme 8. Phenyl (Ring 2) Modification of Lead 21: Synthesis of 49a−g
a
Reagents and conditions: (a) Pd/C 10%, 4 atm H2(g), MeOH, 7 days, 68%; (b) RCH2Br(or Cl), DMF or NMP, 0 °C → rt, 33−44 h, 27−96%; (c)
TFA, DCM, rt 3−15 h, 54−85%; (d) 1,3-di-Boc-2-(carboxymethyl)guanidine, HOBt, EDCI, Et3N, DMF, 0 °C → rt, 15 h, 48%.
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a
Scheme 9. Phenyl (Ring 1) Modification of NPFF2-Preferring Ligand 42: Synthesis of 53a−d
a
Reagents and conditions: (a) RCH2Br(or Cl), LDA, THF(ah), −70 °C → rt, 2−3 h, 59−91%; (b) NH3/MeOH, Raney Ni, 2.4 atm H2(g), 6−20 h,
38−93%; (c) 1,3-di-Boc-2-(carboxymethyl)guanidine, HOBt, EDCI, Et3N, DCM, 0 °C → rt, 15 h, 41−72% (for 52a−c), for 52d: (i) Boc-gly-OH,
HOBt, EDCI, Et3N, DCM, 0 °C → rt, 15 h, 77%, (ii) TFA, DCM, rt, 1 h, (iii) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl2, Et3N,
DMF, rt, 15 h, 80%; (d) TFA, DCM, rt, 3 h, 54−76%.
Figure 2. NPFF1-preferring ligand 7b in the [35S]GTPγS assay in
cloned NPFF1-R and NPFF2-R. Compound 7b (10 μM) increased by
36% and 184% the binding of [35S]GTPγS on membranes of NPFF1-
R and NPFF2-R expressing CHO cells, respectively, compared with a
71% and 231% increase by the agonist 1DMe (10 μM). Data
represents the mean SEM from two-four experiments performed in
duplicate.
cellular cAMP accumulation in CHO cells stably expressing
NPFF1-R and NPFF2-R (Figures 3A,B, 6A,B, 7, and 9A−D).
The antagonist properties of 21, 46, and 53a were evaluated by
measuring its ability to reverse the NPVF- and 1DMe-induced
Figure 3. Ability of the nonselective, low efficacy NPFF1 antagonist 21
to inhibit the effect of NPFF agonists in the forskolin-induced cAMP
inhibition of cAMP production in NPFF1-R and NPFF2-R,
respectively (Figures 3A,B, 6B, and 9C,D), and in the case of
53a to right-shift the dose−response curve of the aforemen-
tioned NPFF agonists in their respective subtypes (Figures
10A,B).
Among compounds bearing either an arginine or guanidine
moiety at the 4-position of the piperidine ring and various
lipophilic substitutions at the piperidine nitrogen (methyl,
benzyl, phenethyl, and 2-naphthalenylmethyl) (7a−d, 9a−c),
arginines 7b and 7d with benzyl and 2-naphthalenylmethyl
substitutions (Scheme 1) yielded affinity below 500 nM,
indicating that both arginine and compact aromatic sub-
stitutions on the 4-(phenylamino)piperidine scaffold are
detrimental for NPFF1,2 affinity (Table 1). While 2-
naphthalenylmethyl 7d gave nonselective affinity at NPFF1
assay in CHO cells expressing human NPFF1-R (A) or NPFF2-R (B).
Increasing doses of 21 were tested either alone or in the presence of
0.1 μM NPVF in CHO hNPFF1 cells or of 0.01 μM 1DMe in CHO
hNPFF2 cells. ***, p < 0.001; **, p < 0.01; *, p < 0.05; different from
NPVF alone (white bar) (one-way ANOVA followed by Dunnett’s
multiple comparison test). Data represents the mean SEM from two
to four experiments performed in duplicate.
and NPFF2, Ki = 479
39 and 304
14 nM (1.6-fold
preference for NPFF2), respectively, we chose to utilize 7b as
the lead compound due to the N-benzyl being less bulky and
for facile analog synthesis.
This benzyl analog (7b) bound to the NPFF1 subtype at Ki =
487 117 nM with 2.2-fold preference over NPFF2. Indeed,
the 2-naphthalenylmethyl substitution improved affinity at
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Table 1. Binding Affinities of Guanidino-piperidines 7a−d,
Table 2. Binding Affinities of Guanidino-piperidines 7b,
a
a
9a−c, and 15a,b at NPFF1-R and NPFF2-R
17a−g and 21 at NPFF1-R and NPFF2-R
cmpd
hNPFF1-R (Ki, nM) hNPFF2-R (Ki, nM)
ratio
cmpd
hNPFF1-R (Ki, nM) hNPFF2-R (Ki, nM)
ratio
1DMe
7a
4
0.5
0.8 0.1
>5K
NPVF
1DMe
3.66 0.95
>5K
3.85 0.42
487 117
441 14
0.47 0.08
1052 32
2907 216
3220 53
2682 570
2223 388
993 87
b
7b
487 117
2906 576
479 39
>5K
1052 32
1694 183
304 14
>5K
2.2-fold (NPFF1)
1.6-fold (NPFF2)
7b
2.2-fold (NPFF1)
6-fold (NPFF1)
7c
17a
17b
7d
3429 377
2692 121
2454 338
1623 630
1966 324
2348 413
319 19
b
9a
17c
b
9b
1880 53
2965 112
>5K
6616 377
2773 470
7579 54
17d
b
9c
17e
b
15
17f
1620 179
2398 288
405 56
b
a
Ki values were determined by using [125I]YVP and [125I]EYF for
hNPFF1-R and hNPFF2-R, respectively, and are the mean
from two to four experiments performed in duplicate.
17g
21
SEM
a
Ki values were determined by using [3H]NPVF and [3H]EYF for
hNPFF1-R and hNPFF2-R, respectively, and are the mean SEM
from two-four experiments performed in duplicate. Ki values were
determined by using [125I]YVP and [125I]EYF for hNPFF1-R and
hNPFF2-R, respectively, and are the mean SEM from two to four
experiments performed in duplicate.
b
NPFF2 and can be incorporated into future analog develop-
ment if necessary. To further investigate the moieties of
arginine 7b responsible for NPFF1,2-binding, the aniline was
removed (15, Scheme 2). This modification resulted in a
complete loss of NPFF1 affinity and a 7-fold loss of affinity at
NPFF2-R (Table 1), emphasizing the importance of this group
on our core scaffold for generating NPFF ligands, as shown in
Figure 1.
In the GTPγS assay for functional activity, 7b exhibits agonist
activity at both subtypes when tested at a high concentration of
10 μM (36% stimulation at NPFF1-R and 184% stimulation at
NPFF2-R), albeit lower than the effect of full NPFF agonist
1DMe at 10 μM (71% stimulation at NPFF1-R and 231%
stimulation at NPFF2-R; Figure 2). Although 7b has modest
affinity and preference for NPFF1-R, this analog serves as a
good starting point to develop small molecule NPFF1-selective
agonists, a pharmacological profile that is currently lacking in
the literature (as shown in Chart 1). From the limited SAR
above, we note that preference for either subtype for these
arginine-bearing analogs can be modulated in part by
substitution at the piperidine nitrogen (7b vs 7d) with 1-C
linked aromatic groups. Further substitutions, including 1-C
linked cycloalkyl, bicyclic and tricyclic aromatic rings, and
heterocyclic rings could be explored to potentially afford
NPFF1 or NPFF2 agonists.
similar affinity at both subtypes as 17d, however; two carbon
deletion (17a) yields similar affinity at NPFF1 as 7b and 6-fold
selectivity. Three carbon deletion (21) affords a 1.5- and 2.6-
fold increase in affinity at NPFF1-R and NPFF2-R, respectively,
compared with lead 7b, with Ki values of 319 19 and 405
56 nM, greatly simplifying our lead for optimization. Thus,
considering 7b vs 21, only one positively charged group is
needed on this scaffold, projected a short distance from the
core piperidine. Substituting an amine (17b) in place of the
guanidine moiety of 21 results in an 11- and 8-fold loss of
affinity at NPFF1-R and NPFF2-R, respectively, suggesting a
potential bidentate interaction. Exploring spacers between the
guanidinium group and the amide of 7b with aromatic-linked
guanidines (three to five carbons), 17e−g afford a 3.3−4.8-fold
loss of affinity at NPFF1 relative to 7b, and a 1.5−2.3-fold loss
of affinity at NPFF2-R. Such a result indicates that only one
aromatic group is tolerable at the 4-position of the piperidine
ring, in addition, as seen with the aliphatic linked analogs above,
the placement of the guanidine group relative to the core
structure is of paramount importance.
SAR of NPFF1 Agonist 7b. From the SAR above, it was
determined that disubstitution at the 4-position of the
piperidine ring with aniline and a C-terminal linked arginine
(linked via a methylene amide) were necessary for affinity at
NPFF1,2-R, in addition to compact aromatic substitution at the
1-position. To evaluate the importance of the two positively
charged moieties of 7b, the α-amino and guanidinium groups,
the α-amino group was removed in favor of retaining the
guanidine moiety present in most reported NPFF1,2 ligands
(17d) (Scheme 3, Table 2). Additionally, the linker length
between the guanidinium and amide groups was explored with
aliphatic (one to three carbons, 17a, 17c, 21) and rigidified
aromatic linkers (three to five carbons, 17e−g) (Scheme 3,
Table 2).
Removal of the α-amino group of 7b (17d) yields a 5- and 2-
fold loss of affinity at NPFF1-R and NPFF2-R, respectively,
indicating the importance of a positively charged moiety near
the core piperidine structure. The distance between the
positively charged guanidine and the core structure was then
investigated by deleting one to three aliphatic linkers (17c, 17a,
21). Shortening the linker length by one carbon (17c) affords
When evaluated alone in the forskolin-induced cAMP assay
at both subtypes, 21 displays no agonist activity at
concentrations up to 1 μM and weak partial agonist activity
at concentrations >1 μM (data not shown), in contrast to the
effect observed for peptide agonists NPVF and 1DMe at
NPFF1-R and NPFF2-R, respectively. In the same assay,
guanidated glycine 21 partially reverses the inhibitory effect of
0.1 μM NPVF at NPFF1-R, with an EC50 of 21.98 5.08 μM
and corresponding Emax of 83 2% (Figure 3A). At NPFF2-R,
this analog exhibits low agonist activity (20−25% inhibition of
cyclase at high concentrations of 10 and 30 μM) and less
potently inhibits the effect of 0.01 μM 1DMe compared with its
effect on NPVF at NPFF1-R (Figure 3B). We considered this
simplified structure (21) as our lead for optimization to
generate NPFF2 selective small molecule ligands, since
compounds of this profile are not available and would aid in
the pharmacological classification of this subtype.
Collectively, based on these compounds born from our
proposed general scaffold for designed NPFF1,2 ligands
(Figure 1), we have noted a 2D pharmacophore for both
affinity and modulation of functional activity at both subtypes
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(Figure 4). Both subtypes favor substitution at the piperidine
nitrogen with methylene-linked aromatic groups such as a
phenyl and 2-naphthyl.
Table 3. Binding Affinities of Guanidino-piperidines 26,
30a−d, 34, 42−43, 46, and 49a−g at NPFF1-R and NPFF2-
a
R
cmpd
hNPFF1-R (Ki, nM) hNPFF2-R (Ki, nM)
ratio
NPVF
1DMe
21
3.66 0.95
3.85 0.42
319 19
904 237
94 24
0.47 0.08
405 56
1893 490
309 55
987 65
884 177
409 39
3505 847
84 12
26
30a
30b
30c
30d
34
3.3-fold (NPFF1)
8.7-fold (NPFF1)
4.4-fold (NPFF1)
2.1-fold (NPFF1)
6.9-fold (NPFF1)
5-fold (NPFF2)
3-fold (NPFF1)
17.6-fold (NPFF1)
114 17
201 35
191 31
511 79
417 74
151 15
81 17
42
43
465 108
1426 49
2757 81
298 14
330 28
129 26
504 95
46
49a
49b
49c
49d
49e
49f
15085 355
1638 499
1057 150
538 40
141 34
112 11
340 91
5.5-fold (NPFF2)
3.2-fold (NPFF2)
4.2-fold (NPFF2)
3.6-fold (NPFF1)
4-fold (NPFF1)
4.5-fold (NPFF1)
Figure 4. Two-dimensional pharmacophore for affinity and modu-
lation of functional activity at NPFF1-R and NPFF2-R.
454
4
49g
1513 33
Disubstitution at the 4-position of the piperidine ring with an
aniline moiety and a methylene amide linked to a positively
charged moiety (or moieties) is also necessary for recognition
at both subtypes. The nature of the positively charged moiety is
crucial for modulation of functional efficacy: while an arginine
side chain (7b) yields a modestly preferring NPFF1 agonist, a
guanidated glycine (21) affords a nonselective compound that
weakly antagonizes NPFF1-R.
SAR of Low Efficacy NPFF1 Antagonist 21. In our quest to
identify NPFF2 selective ligands, we performed SAR analysis of
low efficacy NPFF1 antagonist 21 (Ki NPFF1-R = 319 19
nM; Ki NPFF2-R = 405 56 nM), focusing on four areas: (1)
the aniline phenyl ring (ring 1), (2) the aniline amine, (3) the
methylene amide, and (4) the benzyl ring (ring 2) at the N-1
position (Figure 5).
a
Ki values were determined by using [3H]NPVF and [3H]EYF for
hNPFF1-R and hNPFF2-R, respectively, and are the mean
from two to four experiments performed in duplicate.
SEM
groups, as well as groups containing H-bond acceptor moieties
3,5-dimethoxyphenyl 30b and 3-methoxyphenyl 30c (Scheme
5), because the aniline moiety as a whole affects NPFF1,2
binding (7b vs 15). These modifications afforded a 1.6−3.4-
fold gain of affinity for NPFF1-R compared with lead 21 (Ki =
94 24 to 201 35 nM), while showing comparable or less
affinity at NPFF2-R (Table 3). In particular, 1-naphthyl 30a
and 3,5-dimethoxyphenyl 30b substitutions were favored at
NPFF1-R (Ki = 94 24 and 114 17 nM, respectively), while
3-methoxyphenyl 30c and 3,4-dichlorophenyl 30d proved to be
half as active at this subtype (Ki = 191 31 to 201 35 nM),
indicating that additional hydrophobic interaction(s) accessed
by 30a and 30b in this region of the scaffold are beneficial for
NPFF1 binding only.
Despite showing modest (3.3−8.7-fold) preference for
NPFF1-R binding, analogs 30a−c were assayed alone for
functional activity in the forskolin-induced cAMP assay (Figure
6A). No activity was observed at concentrations up to 10 μM;
thus, a lack of agonist activity was retained on our scaffold.
Continuing to explore the aniline moiety, the aniline NH
(modification 2, Scheme 6) was removed (34) and replaced
with methylene (42) and ethylene (43) groups. Removal of the
amine (34) results in only a modest loss of affinity at NPFF1-R
compared with lead 21 (1.6-fold) but a significant loss of
affinity at NPFF2-R for this scaffold (8.7-fold) (Table 3),
suggesting the necessity of a spacer atom between the phenyl
and piperidine rings for binding at NPFF2-R only. Substitution
with a methylene moiety (42) again yields similar affinity at
NPFF1-R, and a 5-fold increase in affinity at NPFF2-R
Figure 5. Sites of modification (1−4) for low efficacy NPFF1
antagonist 21.
To first validate the 4,4-substitution pattern of 21 about the
piperidine ring before exploring modifications 1−4 above, the
corresponding 3,3-substituted analog was investigated (26)
(Scheme 4), resulting in a 2.8- and 4.7-fold loss of affinity at
NPFF1-R and NPFF2-R (Table 3). Although disubstitution of
these pharmacophoric elements at the two-position of the
piperidine ring was not explored, we felt a 4,4-substitution
pattern was optimal for our novel scaffold.
compared with lead 21 (Ki = 84
12 nM), indicating a
greater degree of lipophilicity required for NPFF2 binding.
Additionally, the spatial arrangement of the phenyl moiety
could be important for NPFF2 preference, since switching from
a trigonal pyramidal NH (21) to an sp3 C (42) yields 5-fold
preference for NPFF2-R. In the forskolin-induced cAMP assay
We next turned our attention to substituting the aniline
phenyl ring of lead 21 (modification 1, Scheme 5) with bulky,
hydrophobic 1-naphthyl 30a and 3,4-dichlorophenyl 30d
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linker length between the amide and guanidine moieties was
extended by one carbon, in order to retain the same distance
between the guanidine and piperidine moieties as lead 21. This
change allowed for a 4-fold improvement at NPFF1 relative to
lead 21 (Ki = 81 17 nM) and 17.6-fold selectivity vs NPFF2
(Table 3). Thus, selectivity for NPFF1 is controlled by
positioning of the amine and carbonyl groups; in addition,
the spatial orientation of the guanidine in relation to the core
piperidine scaffold may also contribute to selectivity (connected
via a planar carbonyl group (46) vs a flexible methylene group
(21)). In the forskolin-induced cAMP assay at NPFF1-R,
reverse amide 46 alone is inactive at concentrations up to 10
μM (Figure 6A,B) and dose-dependently antagonizes the effect
of 0.1 μM NPVF at an EC50 = 3.52 1.42 μM and Emax = 68
16% (Figure 6B). In terms of its good affinity and selectivity,
reverse amide 46 is the first small molecule, nonpeptide
NPFF1-R antagonist known, in contrast to dipeptide NPFF1-R
antagonist 1. Considering its reduced polar surface area and
number of metabolically labile amide bonds compared with
dipeptide 1, reverse amide 46 represents a small molecule lead
for drug development targeting the NPFF1-R subtype, since
dipeptide 1 was recently shown to strongly reduce OIH
induced by the opioid agonist fentanyl.17
Various methylene-linked aromatic and nonaromatic sub-
stitutions of phenyl ring 2, located off of the 1-position of the
core piperidine ring, were also explored (49b−g; modification
4, Scheme 8). As suggested by N-methyl analogs 7a and 9a
(Scheme 1, Table 1), removal of the N-substituent (49a)
resulted in a significant loss of affinity at both subtypes (Table
3). Cyclohexyl 49b, benzyl 49c, and 1-naphthyl 49d provided
modest improvement (1.2−3.1-fold) in binding to NPFF2-R
compared with lead 21 (Ki = 298 14, 330 28, and 129 26
nM, respectively), with 3.2−5.5-fold preference vs NPFF1-R.
Interestingly, replacement of the benzyl group of lead 21 with a
cyclohexyl moiety shows the importance of this aromatic ring
for NPFF1-R affinity only, since 49b yields a 5-fold drop in
NPFF1-R affinity, while retaining similar affinity at NPFF2-R.
Conversely, 2-naphthyl 49e and 4-bromophenyl 49f are more
active at NPFF1-R, providing 2.3−2.8-fold improvement in
binding to NPFF1-R compared with lead 21 (Ki = 112 11 to
141 34 nM), with 3.6−4.5-fold preference vs NPFF2-R. 4-
Methyl benzoate analog 49g yields similar affinity at NPFF1-R
as lead 21, with 4.5-fold preference. In summary, on this
scaffold, selectivity for either subtype can be controlled by N-1
substitution: both cycloalkyl (49b) and unsubstituted aromatic
moieties (49c) afford NPFF2-R-preferring compounds, where-
as a broad range of substituted aromatic groups favor the
NPFF1-R subtype, including both bromo (49f) and methyl
ester (49g) groups. Additionally, selectivity for NPFF2-R vs
NPFF1-R can be achieved with a 1- vs 2-naphthyl group (49d
vs 49e), indicating that the NPFF2-R pocket is of a more
discriminate volume.
Figure 6. (A) NPFF1-preferring ligands 30a−c and NPFF1-selective
ligand 46 in the forksolin-induced cAMP assay in cloned NPFF1-R,
compared with the NPFF1 selective agonist NPVF. (B) Ability of the
selective NPFF1 antagonist 46 to reverse the effect of 0.1 μM NPVF in
the forskolin-induced cAMP assay in CHO cells expressing hNPFF1-
R. Increasing doses of 46 were tested either alone or in the presence of
NPVF. ***, p < 0.001; **, p < 0.01; *, p < 0.05; different from NPVF
alone (white bar) (one-way ANOVA followed by Dunnett’s multiple
comparison test). Data represents the mean
SEM from two-four
experiments performed in duplicate.
at NPFF2-R, benzyl 42 alone is inactive at concentrations up to
1 μM (Figure 7), again indicating this scaffold’s lack of agonist
Figure 7. NPFF2-preferring ligand 42 in the forksolin-induced cAMP
assay in cloned NPFF2-R, compared with the NPFF agonist 1DMe.
Data represents the mean
performed in duplicate.
SEM from two to four experiments
In Vivo Testing of NPFF1-Selective Ligand 46. Mice
exposed to a 48 °C warm-water bath demonstrated a 7.85
0.51 s latency to tail-withdrawal. NPFF (30 nmol, icv)
significantly reduced tail-withdrawal latency in the mouse 48
°C warm-water tail-withdrawal assay over time (F(9,118) = 5.56,
p < 0.0001, one-way ANOVA),6,8 whereas vehicle (icv) was
without effect (F(9,74) = 0.19, p = 0.99, one-way ANOVA;
Figure 8A,B).
activity. Extending the phenyl ring by an additional carbon
(phenethyl 43) is not tolerated at NPFF2-R, resulting in a 5.5-
fold loss of affinity relative to methylene 42 (Ki = 465 108
nM), instead binding to NPFF1-R with 151 nM affinity.
Collectively, mostly NPFF2 binding is affected by substitution
of the aniline NH, while NPFF1 binding is affected by
substitution of the aniline phenyl ring.
The importance of the methylene amide at the four-position
of the piperidine ring of lead 21 was explored by simultaneous
removal of the methylene moiety and replacement with a
reverse amide (46; modification 3, Scheme 7). Additionally, the
Administration of the nonselective NPFF1,2-R antagonist
RF9 (10 nmol, icv) was without effect on the tail-withdrawal
latency (F(9,77) = 1.74, p = 0.09, one-way ANOVA), but a 20
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nM) with 5-fold preference, we considered this as our new lead
compound to generate NPFF2 ligands and explored the effect
of replacing the proximal phenyl ring 1 of this scaffold with
bulky aromatic rings as shown in Scheme 9.
Substitution of phenyl ring 1 of lead 42 with a 1-naphthyl
group (53a) afforded a 2.8-fold increase in affinity at NPFF2-R
(Ki = 30 5 nM) relative to lead 42, with similar preference
(Table 4). Substitution with a 3,4-dichlorophenyl moiety (53b)
Table 4. Binding Affinities of Guanidino-piperidines 53a−d
a
at NPFF1-R and NPFF2-R
cmpd
hNPFF1-R (Ki, nM) hNPFF2-R (Ki, nM)
ratio
NPVF
1DMe
42
3.66 0.95
3.85 0.42
417 74
112 19
432 14
316 11
296 14
0.47 0.08
84 12
5-fold (NPFF2)
3.7-fold (NPFF2)
5.3-fold (NPFF2)
1.5-fold (NPFF2)
3-fold (NPFF1)
53a
30
82
5
8
8
53b
53c
205
53d
910 107
a
Ki values were determined by using [3H]NPVF and [3H]EYF for
hNPFF1-R and hNPFF2-R, respectively, and are the mean
SEM
from two-four experiments performed in duplicate.
decreased affinity by 2.7-fold at NPFF2 relative to 1-naphthyl
53a, suggesting a potential π−π stacking interaction in the
NPFF2 pocket. Interestingly, substitution with 2-naphthyl
(53c) or biphenyl groups (53d) results in 6.8- and 30-fold
losses of affinity at NPFF2-R relative to 1-naphthyl 53a,
indicating that correct placement of the additional aromatic
group on this scaffold is important for maintaining affinity at
NPFF2-R.
We note that substitution of phenyl ring 1 on this scaffold
improves NPFF2 binding and maintains preference for this
subtype, while the same substitutions on the aniline-containing
scaffold (30a, 30d, Scheme 5, Table 3) afford compounds with
preference toward the NPFF1 subtype. This result suggests
different binding conformations for the aniline- vs benzyl-
containing scaffolds at NPFF1-R and NPFF2-R, respectively.
This observation could be further investigated by installing
NPFF2-preferring N-1 substituents (as in 49b−d) onto the
benzyl scaffold.
In the forskolin-induced cAMP assay at NPFF1-R and
NPFF2-R, 1-naphthyl 53a alone exhibits low activity at
concentrations up to 10 μM (Figures 9A−D). To evaluate
the antagonistic property of naphthyl 53a, various concen-
trations were tested in the presence of a fixed concentration of
NPVF (0.1 μM, at NPFF1-R) and the stable NPFF peptide
agonist 1DMe (0.01 μM, at NPFF2-R) in the cAMP assay
(Figures 9C,D). 1-Naphthyl 53a dose-dependently reversed
both NPVF- and 1DMe-inhibition of forskolin-induced cAMP
accumulation with EC50 values of 2.6 0.69 μM (Figure 9C)
and 645 143 nM (Figure 9D), respectively. Corresponding
Emax values at NPFF1-R and NPFF2-R were 56.2 2.6% and
22 1.5%, respectively.
Figure 8. NPFF-induced hyperalgesia is prevented by pretreatment
with NPFF-receptor antagonists. Mouse latencies to withdraw their tail
from a 48 °C warm-water stimulus were measured repeatedly over 80
min after administration of test compounds. After collection of
baseline responses (left of arrow), arrow denotes single administration
of vehicle (50% DMSO, icv; white circles), NPFF (30 nmol, icv;
purple triangles), RF9 (10 nmol icv, squares in part A) or 46 (30 nmol
icv; squares in part B). Additional mice were pretreated 20 min with
RF9 (A) or 46 (B) prior to administration of NPFF (diamonds).
Points represent n = 7−12 mice, with average % baseline response
SEM plotted. *p < 0.05 compared with baseline response with one-
way ANOVA with Tukey HSD post hoc test; †p < 0.05 compared with
NPFF response with two-way ANOVA.
min pretreatment significantly reversed NPFF-mediated hyper-
algesia (F(3,188) = 8.92, p < 0.0001, two-way ANOVA; Figure
8A). Similarly, pretreatment with the NPFF1-R selective
antagonist 46 (30 nmol, icv) also significantly prevented the
NPFF-induced hyperalgesic effects (F(3,233) = 9.58, p < 0.0001,
two-way ANOVA; Figure 8B), without demonstrating sig-
nificant differences from either baseline or vehicle-treated
responses.
In addition, a dose−response curve of NPVF and 1DMe in
the same assay was performed in the absence or the presence of
10 μM 53a (Figures 10A,B) at each subtype. 1-Naphthyl 53a
(10 μM) induces a 12-fold loss of functional activity for NPVF
at NPFF1-R (EC50 = 181
38 nM in the presence of 53a
SAR of NPFF2-Preferring Ligand 42. Since substitution at
the aniline NH (modification 2) with a methylene group
(benzyl 42) yielded a high affinity NPFF2 ligand (Ki = 84 12
compared with 15 0.6 nM alone; Figure 10A); similarly, this
compound induces a 4-fold loss of functional activity for 1DMe
at NPFF2-R (EC50 = 11.4 3.2 nM in the presence of 53a
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Figure 10. Ability of NPFF2-preferring partial antagonist 53a to right-
shift the dose−response curve of NPVF (A) or 1DMe (B) in the
forskolin-induced cAMP assay in CHO cells expressing hNPFF1-R or
hNPFF2-R, respectively. Data represents the mean SEM from two
to four experiments performed in duplicate.
develop selective small molecule NPFF2 antagonists as
pharmacological probes.
Binding Affinity of Selected Compounds at μ-, δ-, and κ-
Opioid Receptors. Because no selective, pharmacologically
pure, and high affinity NPFF1 and NPFF2 agonists and
antagonists are available to the scientific community (save
dipeptide NPFF1 antagonist 1) to investigate the opioid-
modulating properties of the NPFF1 and NPFF2 subtypes, we
tested compounds displaying reasonable affinities and a variety
of pharmacological profiles (summarized in Table 6) at both
subtypes for off-target affinity at the three opioid subtypes
(Table 5).
In general, most compounds possessed modest preference
for NPFF1 or NPFF2 vs MOP and KOP (2−12-fold), and
most ligands did not bind at DOP with any appreciable affinity
(>5K) (Table 5). Notably, the NPFF1-selective antagonist 46
afforded ≥18-fold selectivity at NPFF1 vs MOP/DOP/KOP,
while NPFF2-preferring ligands 42 and 53a yielded 2- and 12-
fold selectivity, respectively, for NPFF2-R vs MOP and KOP.
Similar to compound 46, NPFF1-preferring ligands 30a and
30b were also reasonably selective vs MOP and KOP (7−43-
fold).
Such modest preference for NPFF1,2-R vs MOP and KOP is
not surprising, given that most of these ligands contain a 4-
anilido piperidine, a known scaffold for MOP analgesia (i.e.,
fentanyl).28 Additionally, the seemingly requisite guanidine
moiety on our scaffold is reminiscent of 5′-guanidinonaltrindole
(GNTI),29 a potent and selective KOP antagonist. These
moieties will be taken into account during further SAR
exploration of our lead compounds, namely, 7b, 46, and 53a,
in order to generate selective NPFF1 and NPFF2 ligands.
Additionally, docking of 7b, 46, and 53a into the active state
homology models and crystal structures of MOP and KOP
(depending on their functional activities at each subtype) could
Figure 9. NPFF2-preferring ligand 53a in the forskolin-induced cAMP
assay in cloned NPFF1-R (A) and NPFF2-R (B) compared with the
NPFF1 and NPFF2 selective agonists NPVF and 1DMe, respectively.
Ability of NPFF2-preferring partial antagonist 53a to reverse the effect
of 0.1 μM NPVF (C) or 0.01 μM 1DMe (D) in the forskolin-induced
cAMP assay in CHO cells expressing hNPFF1-R or hNPFF2-R,
respectively. Increasing doses of 53a were tested either alone or in the
presence of NPVF or 1DMe. ***, p < 0.001; **, p < 0.01; *, p < 0.05;
different from NPVF or 1DMe alone (white bar) (one-way ANOVA
followed by Dunnett’s multiple comparison test). Data represents the
mean SEM from two to four experiments performed in duplicate.
compared with 2.8 0.3 nM alone; Figure 10B). As a result, 1-
naphthyl 53a represents the first small molecule NPFF2 partial
antagonist reported in the literature. Although exhibiting
modest preference toward the NPFF2 subtype (3.7-fold), this
compound’s unprecedented high affinity (30 nM) at NPFF2-R
bears further SAR investigation, serving as an excellent lead to
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a
Table 5. Binding Affinities of Selected Guanidino-piperidines at μ-, δ-, and κ-Opioid Receptors
cmpd
hNPFF1-R (Ki, nM)
hNPFF2-R (Ki, nM)
MOP (Ki, nM)
DOP (Ki, nM)
KOP (Ki, nM)
naloxone
RF9
7b
3.39 0.08
37.2 1.3
4.83 0.08
b
b
b
b
b
58
5
75
9
>5K
>5K
>5K
487 117
319 19
94 24
1052 32
405 56
309 55
987 65
84 12
>5K
2022 170
688 60
>5K
>5K
>5K
1233 30
705 80
21
30a
30b
42
>5K
3678 530
4247 580
224 20
114 17
417 74
81 17
>5K
201 10
>5K
2486 200
2607 650
1516 330
46
1426 49
1470 190
362 30
53a
112 19
30
5
354 90
a
Ki values were determined using radioligands [3H]DAMGO, [3H]DPDPE, and [3H]U-69,593 for MOP, DOP, and KOP receptors, respectively,
b
and are the mean SEM from three experiments. Data taken from ref 15.
Table 6. Summary of Functional Activities of Selected
Guanidino-piperidines on hNPFF1-R and hNPFF2-R
Receptors Expressed in CHO Cells
Collectively, SAR analysis of four regions of nonselective
NPFF1 antagonist 21 led to the discovery of three small
molecule, NPFF2-preferring ligands, benzyl 42, 3,4-dichlor-
ophenyl 53b, and partial antagonist 1-naphthyl 53a. All
analogues displayed good affinity at NPFF2-R, 84 12, 82
8, and 30 5 nM, respectively, and 3.7−5-fold preference vs
NPFF1-R. Additionally, SAR analysis of 21 allowed us to
generate a 2D pharmacophore for NPFF2-preferring ligands
(Figure 11).
We found that substitution of the aniline moiety of
nonselective antagonist 21 with compact aromatic groups
such as benzyl (42), 1-naphthalenylmethyl (53a), and 3,4-
dichlorophenylmethylene (53b) improved affinity and prefer-
ence for NPFF2, particularly for 53a (14-fold improvement in
binding affinity, 3.7-fold preference vs NPFF1). The methylene
amide at the four-position of the piperidine ring proved crucial
for maintaining recognition at NPFF2 (46). Additionally,
lipophilic groups substituted at N-1, including both cycloalkyl
(49b) and aromatic (49c,d) moieties, were preferred at NPFF2,
indicating that substitution at the 4- rather than 1-position of
the ring may mimic the Phe8 interaction of the endogenous
ligand NPFF. Further SAR studies of NPFF2-preferring lead
53a, to be done by substituting NPFF2-preferring N-1
substituents (as in 49b−d), are planned to potentially increase
affinity and preference for this subtype.
cAMP hNPFF1-R
cAMP hNPFF2-R
antagonist activity
antagonist activity
a
a
cmpd
EC50, nM
Emax, %
EC50, nM
Emax, %
b
c
RF9
7b
4700 1200
79
5
d
h
h
d
36
184
e
21
21980 5080
83
2
g
g
g
20−25
f
30a
30b
42
>10K
g
g
f
>10K
g
g
g
f
g
1K
g
g
46
3520 1420
2600 690
68 16
56.2
g
53a
3
645 143
22 1.5
a
EC50 is the concentration that produces a 50% reversion of the NPVF
(0.1 μM) or 1DMe (0.01 μM)-induced inhibition of forksolin-
stimulated cAMP accumulation in CHO hNPFF1 and hNPFF2 cells,
b
c
d
respectively. Data taken from ref 15. Unpublished observation.
%
e
Maximal activation in the [35S]GTPγS assay at 10 μM. % Inhibition of
forskolin-stimulated cAMP accumulation at concentrations of 10 and
f
30 μM. Highest concentration showing no agonist activity when
g
tested alone in the forskolin-induced cAMP assay. Not determined.
h
Not reported.
also shed light on further modifications to be made to this
scaffold in order to minimize off-target opioid affinity. While
the opioid affinity of these leads is of primary concern, we also
recognize the need to assay these compounds for any off-target
affinity at neuropeptide Y and orexin receptors due to 30−35%
homology with NPFF1,2-R, other receptors within the RFa
peptide family (GPR10, GPR54, and GPR103), and the
nociceptin opioid receptor (NOPr, an opioid-modulating
receptor), as previously done for RF9, the most well
characterized NPFF ligand to date.15
CONCLUSION
■
Considering the structures of a limited number of NPFF1,2
dipeptides and low affinity small molecules, as well as the
endogenous ligand NPFF, ligand-based rational design was
employed to construct the first small molecule scaffold for
NPFF1,2-R displaying significant improvements in affinity
relative to the aryliminoguanidines (Chart 1), as well as a
tractable SAR for determining functional activity and preference
Figure 11. Two-dimensional pharmacophore for NPFF2-preferring ligands from SAR analysis of low efficacy NPFF1 antagonist 21.
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40% yield. Method B: The nitrile intermediate (7.21 mmol) was
hydrogenated in a solution of methanol saturated with ammonia (210
mL) and Raney Ni (0.4 g) under pressure (2.4−3 atm) for 4−24 h.
The catalyst was removed by filtration, and the solvent was evaporated
in vacuo. The residue was chromatographed on a silica gel column
using methylene chloride/methanol as the eluent to afford the amine.
General Procedure 3: Introduction of Arginine Side Chain. A
solution of Boc-protected arginine (0.4 mmol) in 10 mL of methylene
chloride was stirred at −10 °C for 20 min. Then, triethylamine (0.9
mmol), HOBt (0.4 mmol), and EDCI (0.4 mmol) were added, and
the mixture was stirred at −10 °C for 30 min. A solution of amine (0.4
mmol) in 10 mL of methylene chloride was cooled at −10 °C and
added dropwise. After stirring overnight at room temperature, water
was added to the reaction mixture, followed by 10% NaOH solution to
pH 12, and the mixture was extracted 3 times. The organic layer was
dried using MgSO4 and evaporated affording the Boc-protected
arginine intermediate (50−60% yield).
for either subtype, as summarized in our 2D pharmacophore
models (Figures 4 and 11). Specifically, the guanidino-
piperidine class of NPFF ligands represents the first disclosure
of a small molecule scaffold whose SAR results in a wide range
of pharmacological profiles at NPFF1,2-R to serve as lead
compounds to develop pharmacological tools, namely, a
NPFF1 agonist (7b, Ki = 487 117 nM), a NPFF1 antagonist
(46, Ki = 81 17 nM), and a NPFF2 partial antagonist (53a,
Ki = 30
5 nM). In vivo testing of the novel NPFF1-R
antagonist 46 demonstrated antagonism of NPFF-mediated
hyperalgesia without significant effect alone on tail-withdrawal
latencies (Figure 8B), similar to the effects seen with dipeptide
NPFF1,2 antagonist RF9 (Figure 8A),15,16 the most established
NPFF ligand to date, and dipeptide NPFF1 antagonist 1.17 SAR
studies of reverse amide 46, including exploration of this
scaffold with NPFF1-preferring moieties (such as in those of
30a,b, 43, and 49e,f), could afford compounds of an improved
NPFF1 profile to combat opioid-induced hyperalgesia. The
SAR of arginine 7b, reverse amide 46, and 1-naphthyl 53a will
be further explored, using the approaches described herein, in
order to generate NPFF1 and NPFF2 selective agonists and
antagonists to further explore and characterize the pharmacol-
ogy of these subtypes as they relate to opioid-mediated
analgesia.
General Procedure 4: Conversion of Various Amines to Boc-
Protected Guanidines. Method A: To a stirred mixture of Boc-
protected guanidino carboxylic acid (1.11 mmol), 1-hydroxy-
benzotriazole hydrate (1.11 mmol), and triethylamine (1.20 mmol)
in methylene chloride (20 mL) at −10 °C was added N-(3-
(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (1.11
mmol). After 2 h of stirring at −10 °C, a solution of 4-
(aminomethyl)-1-benzyl-N-phenylpiperidin-4-amine (5b) (0.85
mmol) in methylene chloride (4 mL) was slowly added, and the
reaction mixture was stirred for 1 h at −10 °C and for 15 h at room
temperature. The solvent was removed in vacuo, and the residue was
chromatographed on a silica gel column using a gradient of petroleum
ether/ethyl acetate as the eluent to give the desired material. Method
B: A mixture of amine (0.57 mmol), 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea (0.68 mmol), and triethylamine (1.42 mmol)
in anhydrous DMF (4 mL) was stirred at room temperature for 2 days.
The reaction mixture was poured into water and extracted with ethyl
acetate (3 × 30 mL). The combined organic layers were washed with
brine, dried, and evaporated. The residue was purified by
chromatography on a silica gel column using methylene chloride/
methanol as the eluent to give the desired material. Method C: To a
stirred mixture of 1,3-di-Boc-2-(carboxymethyl)guanidine (1.15
mmol), 1-hydroxy-benzotriazole hydrate (1.12 mmol), and triethyl-
amine (2.87 mmol) in methylene chloride (10 mL) under argon at 0
°C was added N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (1.18 mmol). The reaction mixture was kept at 0 °C
for 2 h, and a solution of amine (1.02 mmol) in methylene chloride
(10 mL) was slowly added. The reaction mixture was stirred 1 h at 0
°C and then allowed to warm to room temperature. After 15 h of
stirring, the solvent was evaporated. Ethyl acetate (50 mL) and water
were added. The layers were separated, and the organic layer was
washed with brine. The solvent was dried and evaporated. The residue
was purified by chromatography on a silica gel column using a gradient
of methylene chloride and methanol to give the desired material.
Method D: (Step I) To a stirred mixture of amine (1.21 mmol), 1-
hydroxy-benzotriazole hydrate (1.45 mmol), Boc-gly-OH (1.33
mmol), and triethylamine (3.64 mmol) in methylene chloride (15
mL) at 0 °C under argon was added N-(3-(dimethylamino)propyl)-
N′-ethylcarbodiimide hydrochloride (1.33 mmol). The reaction
mixture was then allowed to warm to room temperature. After 20 h
of stirring, methylene (80 mL) and water (30 mL) were added. The
layers were separated, and the organic layer was extracted again with
methylene chloride (30 mL). The combined organic layers were
washed with brine. The solvent was dried and evaporated. The residue
was purified by chromatography on a silica gel column using a gradient
of methylene chloride and methanol to yield the Boc-Gly-coupled
amine. (Step II) To a solution of the Boc-Gly-coupled amine (0.558
mmol) in anhydrous methylene chloride (6 mL) was added
trifluoroacetic acid (3 mL). The reaction mixture was stirred at
room temperature under argon for 1 h and evaporated. Methylene
chloride (80 mL) and a 10% solution of NaOH (40 mL) were added.
The layers were separated, and the organic layer was washed with
water and brine. The solvent was dried and evaporated. The residue
EXPERIMENTAL SECTION
■
Chemistry. All the solvents and reagents were obtained
commercially and used as received unless noted otherwise. Anhydrous
diethyl ether was prepared by refluxing with sodium; anhydrous THF
was prepared by refluxing with sodium and benzophenone. Flash
chromatography was performed with Merck silica gel 60 (230 × 400
mesh). NMR spectra were recorded on either a Bruker AVIII 400
spectrometer or Bruker DRX500. For 7a−7e, 9a−9c and 15a,b,
HPLC/MS analyses were recorded on a Waters Alliance LC/MS
System, consisting of a Waters ZQ mass detector, photodiode array
detector, and an Alliance HPLC system, equipped with an XTerra
column (C-18, 2.1 mm × 5 mm). HPLC analysis was done using
gradient of ACN/H2O/10% HCl (aq). For all remaining final
compounds, mass spectra were obtained on a Waters micromass ZQ
detector or a Micromass Q-TOF micro hybrid quadrupole/orthogonal
high resolution time of flight MS. HPLC analysis was performed on a
reverse phase XBridge C18 column (4.6 mm × 75 mm) column, using
40% H2O/50% ACN/10% of 0.2% TFA(aq) as the mobile phase, in
gradient conditions at a flow rate of 1 mL/min. Eluted peaks were
monitored at 254 nm with a Waters 996 PDA detector. All final
compounds tested were confirmed to be of ≥95% purity by the HPLC
methods described above.
General Procedure 1: Strecker Synthesis To Generate
Carbonitriles. To a solution of N-substituted piperidone (15.9
mmol) in acetic acid (25 mL) was slowly added substituted aniline
(17.6 mmol). The mixture was cooled to 5 °C with an ice bath, and a
solution of KCN or TMSCN (17.6 mmol) in water (5 mL) was added
dropwise. The solution was stirred for 1 h at 0 °C and for 3 h at room
temperature and then poured into water. The pH was adjusted to 10
by adding NH4OH, and the aqueous layer was extracted with
methylene chloride (3 × 50 mL). The combined organic extracts were
washed with water and brine and dried. The solvent was removed in
vacuo, and the residue was purified using one of the following
methods: flash chromatography using hexanes/EtOAc, trituration with
cold ether, or recrystallization.
General Procedure 2: Nitrile Reduction. Method A: Anhydrous
ether (500 mL) was added to the nitrile intermediate (0.5 mmol),
which was then added dropwise to a solution of LiAlH4 (1.0 mmol) in
1 L of anhydrous ether under nitrogen. The solution was quenched
with a few drops of water, decanted, and evaporated. The resulting oil
was purified by silica gel chromatography (49:50:1 chloroform−
methanol−NH4OH) affording the amine as a pale yellow solid in 35−
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was dissolved in anhydrous DMF (7 mL). Triethylamine (1.40 mmol),
mercury(II) chloride (0.558 mmol), and 1,3-bis(tert-butoxycarbonyl)-
2-methyl-2-thiopseudourea (0.670 mmol) were added, and the
reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was poured into water and extracted with ethyl
acetate (3 × 30 mL). The combined organic layers were washed with
brine, dried, and evaporated. The residue was purified by
chromatography on a silica gel column using a gradient of methylene
chloride and methanol as the eluent to provide the title material.
General Procedure 5: Boc Deprotection To Give Final
Products. Method A: The Boc-protected guanidine (0.1 mmol)
was dissolved in an excess of HCl/dioxane under nitrogen and stirred
for 3−4 days after which it was concentrated under reduce pressure to
afford the hydrochloride salt of guanidine as colorless solid. Method
B: To a solution of Boc-protected guanidine (0.3 mmol) in anhydrous
methylene chloride (4 mL) was added trifluoroacetic acid (4 mL). The
reaction mixture was stirred at room temperature under argon for 3 h.
The solvent was removed in vacuo, and the residue was purified by
chromatography on a silica gel column using a gradient of methylene
chloride and methanol as the eluent to the title material as a tritriflate
salt.
General Procedure 6: Synthesis of Various Aromatic
Substituted Carbonitriles. 1-Benzylpiperidine-4-carbonitrile (5
mmol) was added to a dry three-necked flash equipped with a low
temperature thermometer. Dry THF (60 mL) was added, and the
solution was cooled to −70 °C in a dry ice−acetone bath under
positive argon pressure. Lithium diisopropylamide (2.0 M solution in
heptane/tetrahydrofuran/ethylbenzene, 6.54 mmol) was slowly added
while maintaining the temperature below −65 °C, and the resulting
mixture was stirred for 30 min at −70 °C. Substituted bromide or
chloride (6.54 mmol) was quickly added, and the resulting mixture was
then allowed to warm to room temperature, stirred for another 2 h,
poured into 40 mL of NH4Cl saturated solution, and extracted with
ethyl acetate (3 × 40 mL). The combined organic extracts were
washed with brine and dried. The solvent was removed in vacuo, and
the residue was purified by chromatography on a silica gel column
using ethyl acetate and hexanes as the eluent to afford the title
material.
(m, 4H), 7.52−7.24 (m, 3H), 7.23−7.22 (m, 3H), 6.92−6.90 (m, 2H),
3.70 (s, 2H), 3.69 (s, 1H), 2.93−2.59 (m, 4H), 2.37−2.35 (m, 4H).
MS (ESI) 342.5 [M + H]+.
4-(Aminomethyl)-1-methyl-N-phenylpiperidin-4-amine (5a). Pre-
pared according to general procedure 2, method A, to afford the title
1
material in 35−40% yield. H NMR (600 MHz, CD3OD): δ 6.72−
6.79 (m, 3H), 7.13−7.17 (m, 2H), 3.642 (s, 3H), 2.86 (s, 2H), 2.37−
2.40 (m, 4H), 1.92−1.96 (m, 4H). MS (ESI) 220.2 [M + H]+.
4-(Aminomethyl)-1-benzyl-N-phenylpiperidin-4-amine (5b). Pre-
pared according to general procedure 2, method B, to afford the title
material in 82% yield. 1H NMR (CDCl3): δ 7.34−7.16 (m, 7H), 6.80−
6.75 (m, 3H), 3.53 (s, 2H), 2.89 (s, 2H), 2.65−2.62 (m, 2H), 2.33 (t, J
= 8.1 Hz, 2H), 2.00−1.62 (m, 6H). MS (ESI) m/z 296 [M + H]+.
4-(Aminomethyl)-1-phenethyl-N-phenylpiperidin-4-amine (5c).
Prepared according to general procedure 2, method A, to afford the
title material in 35−40% yield. 1H NMR (600 MHz, CD3OD): δ
7.30−7.14 (m, 7H), 6.83−6.78 (m, 3H), 3.68−3.67 (d, J = 4, 2H),
2.95 (s, 2H), 2.88−2.42 (m, 4H), 2.05−2.02 (m, 2H), 1.82−1.76 (m,
4H). MS (ESI) 310.5 [M + H]+.
4-(Aminomethyl)-1-(naphthalen-2-ylmethyl)-N-phenylpiperidin-
4-amine (5d). Prepared according to general procedure 2, method B,
1
to afford the title material in 90% yield. H NMR (CDCl3): δ 7.81−
7.71 (m, 4H), 7.48−7.44 (m, 3H), 7.14 (t, J = 6.2 Hz, 2H), 6.76−6.71
(m, 3H), 3.64 (s, 2H), 2.86 (s, 2H), 2.64 (d, J = 9.1 Hz, 2H), 2.34 (t, J
= 8.7 Hz, 2H), 1.95 (d, J = 10.6 Hz, 2H), 1.75 (br s, 2H), 1.65−1.60
(m, 2H). MS (ESI) m/z 346 [M + H]+.
2-Amino-5-guanidino-N-((1-methyl-4-(phenylamino)piperidin-4-
yl)methyl)pentanamide Hydrochloride (7a). Prepared according to
general procedure 3, starting with intermediate 5a, to afford the
corresponding Boc-protected arginine intermediate 6a in 50−60%
yield, which was deprotected using general procedure 5, method A, to
afford the title material in 95% yield. 1H NMR (600 MHz, CD3OD): δ
7.17−7.14 (m, 3H), 6.95−6.93 (m, 2H), 3.95−3.72 (m, 2H), 3.66−
3.55 (m, 1H), 3.41−3.33 (m, 6H), 2.85 (s, 3H), 2.31−1.97 (m, 6H),
1.66−1.64 (2H). 13C NMR (600 MHz, CD3OD): δ 170.8, 169.7,
157.4, 130, 129.5, 126, 72.3, 71.2, 67, 61, 53.2, 53.09, 42.4, 40.7, 24.5.
MS (ESI) 376.51 [M + H]+.
2-Amino-N-((1-benzyl-4-(phenylamino)piperidin-4-yl)methyl)-5-
guanidinopentanamide Hydrochloride (7b). Prepared according to
general procedure 3, starting with intermediate 5b, to afford the
corresponding Boc-protected arginine intermediate 6b in 50−60%
yield, which was deprotected using general procedure 5, method A, to
afford the title material in 95% yield. 1H NMR (600 MHz, CD3OD): δ
7.58−7.47 (m, 7H), 6.97−6.95 (m, 3H), 3.73−3.34 (m, 11 H), 2.30−
3.31 (m, 2H), 2.12−2.03 (m, 4H), 1.31−1.28 (s, 2H). 13C NMR (600
MHz, CD3OD): δ 169, 157, 149, 132.5, 132.3, 130.3, 130.2, 124.9,
120, 118.9, 73.4, 72.3, 62.0, 61.4, 54.1, 41.7, 30.7, 25.5. MS (ESI) 452.5
[M + H]+.
General Procedure 7: Synthesis of N-Substituted Analogs. A
mixture of tert-butyl 10,10-dimethyl-3,8-dioxo-1-(4-(phenylamino)-
piperidin-4-yl)-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate (47,
0.257 mmol) and potassium carbonate (0.773 mmol) in anhydrous
DMF (3 mL) was stirred at 0 °C for 30 min under argon. A solution of
substituted bromide or chloride (0.231 mmol) in DMF (1 mL) was
added dropwise, and the mixture was stirred at 0 °C for 1 h and then
allowed to warm to room temperature. After 3 h of stirring, the
reaction mixture was poured into brine (50 mL) and extracted with
ethyl acetate (3 × 30 mL). The organic layers were combined, washed
with brine, dried, and evaporated. The residue was purified by
chromatography on a silica gel column using a gradient of methylene
chloride and methanol as the eluent to give the title material.
1-Methyl-4-(phenylamino)piperidine-4-carbonitrile (4a). Pre-
pared according to general procedure 1 to afford the title material in
2-Amino-5-guanidino-N-((1-phenethyl-4-(phenylamino)-
piperidin-4-yl)methyl)pentanamide Hydrochloride (7c). Prepared
according to general procedure 3, starting with intermediate 5c, to
afford the corresponding Boc-protected arginine intermediate 6c in
50−60% yield, which was deprotected using general procedure 5,
1
1
method A, to afford the title material in 95% yield. H NMR (600
90% yield. H NMR (600 MHz, CD3OD): δ 6.72−6.79 (m, 3H),
MHz, CD3OD): δ 7.63−7.75 (m, 3H), 7.30−7.23 (m, 7H), 3.72−3.69
(m, 4H), 3.66−3.64 (m, 1H), 3.63−3.57 (m, 2H), 3.56−3.37 (4H),
2.35−2.97 (m, 2H), 2.20−2.00 (m, 4H), 2.0−1.8 (m, 2H), 1.30−1.26
(2H). 13C NMR (600 MHz, CD3OD): δ 170.9, 169.9, 157.4, 130.8,
130.2, 130.06, 129.2, 127.4, 126.6, 72.6, 71.6, 61.3, 58.2, 53.4, 43.6,
41.1, 30.8, 24.8, 24.5. MS (ESI) 466.60 [M + H]+.
7.13−7.17 (m, 2H), 3.642 (s, 3H), 2.37−2.40 (m, 4H), 1.92−1.96 (m,
4H). MS (ESI) 216.29 [M + H]+.
1-Benzyl-4-(phenylamino)piperidine-4-carbonitrile (4b). Prepared
according to general procedure 1 to afford the title material in 94%
yield. 1H NMR (CD3OD): δ 7.32−7.20 (m, 7H), 6.90−6.87 (m, 3H),
3.67 (s, 1H), 3.53 (s, 2H), 2.79−2.76 (m, 2H), 2.43 (t, J = 7.5 Hz,
2H), 2.30−2.27 (m, 2H), 1.89 (t, J = 7.8 Hz, 2H). MS (ESI) 292 [M +
H]+.
2-Amino-5-guanidino-N-((1-(naphthalen-2-ylmethyl)-4-
(phenylamino)piperidin-4-yl)methyl)pentanamide (7d). Prepared
according to general procedure 3, starting with intermediate 5d, to
afford the corresponding Boc-protected arginine intermediate 6d in
70% yield. This material was deprotected using general procedure 5,
1-Phenethyl-4-(phenylamino)piperidine-4-carbonitrile (4c). Pre-
pared according to general procedure 1 to afford the title material in
1
90% yield. H NMR (400 MHz, CD3OD): δ 7.31−7.19 (m, 7H),
1
method A, to afford the title material in 99% yield. H NMR (600
6.94−6.92 (m, 3H), 3.72 (d, 2H), 3.01−2.42 (m, 4H), 2.42−2.39 (m,
2H), 2.08−2.05 (m, 4H). MS (ESI) 306.2 [M + H]+.
MHz, CD3OD), δ 7.81−7.71 (m, 4H), 7.48−7.43 (m, 3H), 7.16−7.13
(m, 3H), 6.79−6.66 (m, 2H), 4.09−4.06 (m, 1H), 3.85−3.83 (m, 2H),
3.66 (s, 2H), 3.63−3.47 (m, 2H), 2.64−2.62 (m, 4H), 1.49 (s, 9H).
13C NMR (400 MHz, CD3OD): δ 172.5, 163.6, 160.9, 155.1, 133.5,
1-(Naphthalen-2-ylmethyl)-4-(phenylamino)piperidine-4-car-
bonitrile (4d). Prepared according to general procedure 1 to afford the
title material in 70% yield. 1H NMR (600 MHz, CDCl3): δ 7.82−7.80
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133.0, 129.5, 128.1, 127.9, 127.8, 127.5, 126.1, 125.8, 119.3, 117.5,
63.2, 54.9, 49.3, 44.3, 33.9, 33.5, 25.3.
deprotected using general procedure 5, method A, to afford the title
material in 95−99% yield. H NMR (600 MHz, CD3OD): δ 7.62−
7.61 (m, 2H), 7.40−7.48 (m, 3H), 4.34 (s, 4H), 3.66 (s, 2H), 3.50−
3.47 (m, 2H), 3.31−3.24 (m, 3H), 3.19−3.07 (m, 4H), 1.99−1.91
(6H), 1.73−1.63 (m, 3H). 13C NMR (600 MHz, CD3OD): δ 169,
157.4, 131.4, 130, 129.3, 129.1, 66.9, 60.5, 53, 52.2, 40.64, 34, 28.6, 27,
24.5. MS (ESI) 361.5 [M + H]+.
1
tert-Butyl (tert-Butoxycarbonylamino)((1-methyl-4-
(phenylamino)piperidin-4-yl)methylamino)methylenecarbamate
(8a). Prepared according to general procedure 4, method B, also using
1
HgCl2 (1 equiv) to afford the title material in 30% yield. H NMR
(400 MHz, CD3OD): δ 6.72−6.79 (m, 3H), 7.13−7.17 (m, 2H),
3.642 (s, 2H), 2.27 (s, 3H), 1.9−2.2(m, 4H), 1.657−1.8 (m, 4H),
1.45−1.50 (db, J = 20 Hz, 18H). MS (ESI) 462.80 [M + H]+.
tert-Butyl ((1-Benzyl-4-(phenylamino)piperidin-4-yl)-
methylamino)(tert-butoxycarbonylamino)methylenecarbamate
(8b). Prepared according to general procedure 4, method B, also using
N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-2-cyanoace-
tamide (16a). Prepared according to general procedure 4, method A,
using cyanoacetic acid (1.3 equiv) to afford 1.26 g (50%) of the title
1
material as a white solid. H NMR (500 MHz, DMSO-d6): δ 7.32−
7.27 (m, 5H), 7.21 (t, J = 7.7 Hz, 2H), 6.86 (t, J = 7.2 Hz, 1H), 6.75
(d, J = 7.8 Hz, 2H), 6.68−6.66 (m, 1H), 3.57−3.53 (m, 4H), 3.30 (s,
2H), 2.62−2.60 (m, 2H), 2.34−2.30 (m, 2H), 1.93−1.90 (m, 2H),
1.75−1.71 (m, 2H).
1
HgCl2 (1 equiv) to afford the title material in 30% yield. H NMR
(400 MHz, CD3OD): δ 7.33−7.16 (m, 7H), 6.83−6.78 (m, 3H), 3.68
(s, 2H), 3.52 (s, 2H), 2.64−2.61 (m, 2H), 2.32−2.30 (m, 2H), 1.99−
1.97 (m, 2H), 1.75−1.70 (m, 2H), 1.53−1.48 (d, J = 20 Hz, 18H). MS
(ESI) 538.50 [M + H]+.
tert-Butyl 2-((1-Benzyl-4-(phenylamino)piperidin-4-yl)-
methylamino)-2-oxoethylcarbamate (16b). Prepared according to
general procedure 4, method A, using Boc-gly-OH to afford 3.99 g
tert-Butyl (tert-Butoxycarbonylamino)((1-phenethyl-4-
(phenylamino)piperidin-4-yl)methylamino)methylenecarbamate
(8c). Prepared according to general procedure 4, method B, also using
1
(65%) of the title material as a white solid. H NMR (CDCl3): δ
7.30−7.23 (m, 5H), 7.16 (t, J = 7.6 Hz, 2H), 6.79 (t, J = 7.3 Hz, 1H),
6.73 (d, J = 8.0 Hz, 2H), 6.66 (br s, 1H), 5.45 (br s, 1H), 3.72 (d, J =
5.5 Hz, 2H), 3.53 (d, J = 5.4 Hz, 2H), 3.49 (s, 2H), 3.22 (br s, 1H),
2.58−2.55 (m, 2H), 2.31 (t, J = 10.1 Hz, 2H), 1.89−1.86 (m, 2H),
1.73−1.68 (m, 2H), 1.40 (s, 9H). MS (ESI) m/z 453 (M+ + 1).
tert-Butyl 1-(1-Benzyl-4-(phenylamino)piperidin-4-yl)-12,12-di-
methyl-3,10-dioxo-11-oxa-2,7,9-triazatridecan-8-ylidenecarbamate
(16c). Prepared according to general procedure 4, method A, using 4-
(aminomethyl)-1-benzyl-N-phenylpiperidin-4-amine (5b) (0.25 g,
0.85 mmol) and 4-(2,3-bis(tert-butoxycarbonyl)guanidino)butanoic
acid (0.38 g, 1.11 mmol) to afford 0.41 g (77%) of the title material as
1
HgCl2 (1 equiv) to afford the title material in 30% yield. H NMR
(600 MHz, CD3OD): δ 7.30−7.14 (m, 7H), 6.83−6.78 (m, 3H),
3.68−3.67 (d, J = 4, 2H), 2.95 (s, 2H), 2.88−2.42 (m, 4H), 2.05−2.02
(m, 2H), 1.82−1.76 (m, 4H), 1.51−1.47 (d, J = 16 Hz, 18H). MS
(ESI) 552.50 [M + H]+.
1-((1-Methyl-4-(phenylamino)piperidin-4-yl)methyl)guanidine
Hydrochloride (9a). Prepared according to general procedure 5,
method A, from intermediate 8a to afford the title material in
1
quantitative yield. H NMR (600 MHz, CD3OD): δ 6.72−6.79 (m,
3H), 7.13−7.17 (m, 2H), 3.642 (s, 2H), 2.86 (s, 3H), 2.37−2.40 (m,
4H), 1.92−1.96 (m, 4H). 13C NMR (600 MHz, CD3OD): δ 157.9,
144.5, 129.3, 119.7, 117.6, 52.6, 50.2, 48.9, 48.5, 30.7. MS (ESI) 262.5
[M + H]+.
1
a white solid. H NMR (CDCl3): δ 11.48 (s, 1H), 8.37 (br s, 1H),
7.39−7.13 (m, 7H), 6.79−6.72 (m, 3H), 6.35 (br s, 1H), 3.55−3.49
(m, 4H), 3.43 (q, J = 4.8 Hz, 2H), 2.57−2.54 (m, 2H), 2.33 (t, J = 7.2
Hz, 2H), 2.21 (t, J = 5.4 Hz, 2H), 1.92−1.34 (m, 24 H). MS (ESI) m/
z 623 (M+ + 1). 4-(2,3-Bis(tert-butoxycarbonyl)guanidino)butanoic
acid was prepared from 4-aminobutanoic acid according to general
procedure 4, method B, also using HgCl2 (1.7 equiv), and matched
reported values.30
1-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)guanidine
Hydrochloride (9b). Prepared according to general procedure 5,
method A, from intermediate 8b to afford the title material in
1
quantitative yield. H NMR (600 MHz, CD3OD): δ 7.59−7.13 (m,
7H), 6.89−6.75 (m, 3H), 3.47 (s, 2H), 3.34 (m, 2H), 2.41−2.38 (m,
4H), 2.06−2.01 (m, 4H). 13C NMR (600 MHz, CD3OD): δ 157.9,
144.5, 134, 131.4, 130.0, 129.1, 129.11, 119.3, 117.1, 60.3, 52.9, 48.4,
48.2, 30.2. MS (ESI) 338.0 [M + H]+.
tert-Butyl 1-(1-Benzyl-4-(phenylamino)piperidin-4-yl)-13,13-di-
methyl-3,11-dioxo-12-oxa-2,8,10-triazatetradecan-9-ylidenecarba-
mate (16d). Prepared according to general procedure 4, method A,
using 4-(aminomethyl)-1-benzyl-N-phenylpiperidin-4-amine (5b)
(0.25 g, 0.85 mmol) and 5-(2,3-bis(tert-butoxycarbonyl)guanidino)-
pentanoic acid (0.41 g, 1.11 mmol) to afford 0.43 g (80%) of the title
material as a white solid. 1H NMR (CDCl3): δ 11.46 (s, 1H), 8.29 (s,
1H), 7.35−7.14 (m, 7H), 6.79 (t, J = 5.7 Hz, 1H), 6.72−6.70 (m, 2H),
5.96−5.93 (m, 1H), 3.54−3.48 (m, 4H), 3.38 (q, J = 5.1 Hz, 2H),
2.58−2.55 (m, 2H), 2.30 (t, J = 6.9 Hz, 2H), 2.18 (t, J = 5.4 Hz, 2H),
1.86−1.36 (m, 26 H). MS (ESI) m/z 637 (M+ + 1). 5-(2,3-Bis(tert-
butoxycarbonyl)guanidino)pentanoic acid was prepared from 5-
aminopentanoic acid according to general procedure 4, method B,
also using HgCl2 (1.7 equiv), and matched reported values.31
tert-Butyl N-[(1Z)-{[3-({[1-Benzyl-4-(phenylamino)piperidin-4-yl]-
methyl}carbamoyl)phenyl]amino}({[(tert-butoxy)carbonyl]imino})-
methyl]carbamate (16e). Prepared according to general procedure 4,
method A, using 4-(aminomethyl)-1-benzyl-N-phenylpiperidin-4-
amine (5b) and 3-(2,3-bis(tert-butoxycarbonyl)guanidino)benzoic
acid (1.67 g, 4.41 mmol) to yield 1.7 g (75% yield) of the title
material. 1H NMR (400 MHz, CDCl3): δ 11.65 (s, 1H), 10.45 (s, 1H),
7.86 (s, 2H), 7.54−7.14 (m, 9H), 6.86−6.79 (m, 4H), 3.75 (d, J = 4.9
Hz, 2H), 3.55 (s, 2H), 3.46 (s, 1H), 2.65−2.62 (m, 2H), 2.42−2.38
(m, 2H), 1.97−1.82 (m, 4H), 1.52 (d, J = 27.2 Hz, 18H). MS(ESI)
657.7 [M + H]+. 3-(2,3-Bis(tert-butoxycarbonyl)guanidino)benzoic
acid was prepared from 3-aminobenzoic acid according to general
procedure 4, method B, also using HgCl2 (1.7 equiv), and matched
reported values.32
1-((1-Phenethyl-4-(phenylamino)piperidin-4-yl)methyl)guanidine
Hydrochloride (9c). Prepared according to general procedure 5,
method A, from intermediate 8c to afford the title material in
1
quantitative yield. H NMR (600 MHz, CD3OD): δ 7.30−7.14 (m,
7H), 6.83−6.78 (m, 3H), 3.68−3.67 (d, J = 4, 2H), 2.95 (s, 2H),
2.88−2.42 (m, 4H), 2.05−2.02 (m, 2H), 1.82−1.76 (m, 4H). 13C
NMR (600 MHz, CD3OD): δ 163.7, 145.2, 139.1, 129.35, 128.93,
128.65, 126.35, 119.8, 118.4, 60.6, 54.0, 49.3, 33.6. MS (ESI) 352.5 [M
+ H]+.
1-(Naphthalen-2-ylmethyl)piperidin-4-one (12d). A solution of 2-
bromomethylnaphthalene (5 g, 22.6 mmol), 4-piperidone ethylene
ketal (3.23 g, 22.6 mmol), K2CO3 (9.12 g, 55.25 mmol), and KI (117
mg, 0.7 mmol) in methyl isobutyl ketone (300 mL) was heated at
reflux for 5 h, cooled, and filtered. The residue was purified using silica
gel chromatography (30:70 ethyl acetate−hexane) to afford 1-
naphthalen-2-ylmethyl-piperidin-4-one ethylene ketal (11d). 1H
NMR (600 MHz, CDCl3): δ 7.81−7.75 (m, 4H), 7.52−7.43 (m,
3H), 3.93 (s, 4H), 3.72 (s, 2H), 2.81−2.61 (m, 4H), 1.79−1.77 (m,
4H). MS (ESI) 284.36 [M + H]+. This material was hydrolyzed
directly in a mixture of concentrated HCl (40 mL) and acetic acid
(210 mL) at reflux for 18 h. The mixture was poured onto ice water,
neutralized with 32% NaOH to pH 8, extracted with ethyl acetate, and
dried using MgSO4. Evaporation afforded the title material (5g, 40%).
1H NMR (600 MHz, CDCl3): δ 7.83−7.75 (m, 4H), 7.54−7.45 (m,
3H), 3.77 (s, 2H), 2.80−2.78 (m, 4H), 2.48−2.46 (m, 4H). MS (ESI)
240.31 [M + H]+.
tert-Butyl N-[(1Z)-{[4-({[1-Benzyl-4-(phenylamino)piperidin-4-yl]-
methyl}carbamoyl)phenyl]amino}({[(tert-butoxy)carbonyl]imino})-
methyl]carbamate (16f). Prepared according to general procedure 4,
method A, using 4-(aminomethyl)-1-benzyl-N-phenylpiperidin-4-
amine (5b) and 4-(2,3-bis(tert-butoxycarbonyl)guanidino)benzoic
2-Amino-N-((1-benzylpiperidin-4-yl)methyl)-5-guanidinopenta-
namide Hydrochloride (15). Prepared according to general procedure
3 starting with intermediate 13, to afford the corresponding Boc-
protected arginine intermediate 14 in 70% yield. This material was
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1
acid (1 g, 3.45 mmol) to afford 1.5 g (66%) of the title material. H
NMR (400 MHz): δ 11.61 (s, 1 H), 10.50 (s, 1 H), 7.73−7.67 (m, 4
H), 7.40−7−28 (m, 5 H), 7.21 (t, J = 7.7, 2 H), 6.88−6.82 (m, 3 H),
6.71 (s, 1 H), 3.75 (d, J = 5.1, 2 H), 3.66 (s, 2 H), 2.73 (s, 2 H), 2.56
(s, 2 H), 2.11−1.83 (m, 4 H), 1.53 (d, J = 13.6, 19 H). MS(ESI) 657.4
[M + H]+. 4-(2,3-Bis(tert-butoxycarbonyl)guanidino)benzoic acid was
prepared from was prepared from 4-aminobenzoic acid according to
general procedure 4, method B, also using HgCl2 (1.7 equiv), and
matched reported values.33
53.23, 45.04, 40.68, 34.74, 29.87, 27.99, 22.31. HRMS (ESI) calcd for
C25H37N6O [M + H]+ 437.3029, found 437.3044.
N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-3-guanidi-
nobenzamide (17e). Prepared according to general procedure 5,
method B, (no purification) using 16e (0.1 g, 0.152 mmol). 1H NMR
(500 MHz, DMSO-d6): δ 10.33 (s, 1H), 9.70 (s, 1H), 8.59 (s, 1H),
7.78−7.73 (m, 5H), 7.51−7.39 (m, 6H), 7.19−7.05 (m, 2H), 6.89 (d, J
= 7.9 Hz, 2H), 6.66 (t, J = 7.1 Hz, 1H), 4.32 (d, J = 3.6 Hz, 2H), 3.55
(d, J = 5.7 Hz, 1H), 3.38−3.08 (m, 4H), 2.20 (d, J = 14.5 Hz, 2H),
2.04−1.90 (m, 2H). 13C NMR (126 MHz, DMSO-d6): δ 166.76,
156.64, 146.39, 136.33, 136.07, 131.74, 130.17, 130.10, 129.96, 129.31,
129.22, 127.70, 125.74, 123.77, 117.78, 116.64, 59.42, 54.07, 47.65,
45.61, 29.77. HRMS (ESI) calcd for C27H33N6O 457.2716, found
457.2708 [M + H]+.
tert-Butyl N-[(1Z)-({4-[({[1-Benzyl-4-(phenylamino)piperidin-4-yl]-
methyl}carbamoyl)methyl]phenyl}amino)({[(tert-butoxy)carbonyl]-
imino})methyl]carbamate (16g). Prepared according to general
procedure 4, method A, using 4-(aminomethyl)-1-benzyl-N-phenyl-
piperidin-4-amine (5b) (1.08 g, 3.66 mmol) and 4-((2,3-bis(tert-
butoxycarbonyl)guanidino)methyl)benzoic acid (1.7 g, 4.49 mmol) to
afford the title material in 84% yield. 1H NMR (400 MHz, CDCl3): δ
11.66 (s, 1H), 10.31 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.28−7.11 (m,
9H), 6.77 (t, J = 7.0 Hz, 1H), 6.59 (d, J = 7.5 Hz, 2H), 5.88 (s, 1H),
3.48 (s, 7H), 2.54−2.51 (m, 2H), 2.30−2.28 (m, 2H), 1.79−1.76 (m,
2H), 1.68−1.63 (m, 2H), 1.53 (d, J = 9.9 Hz, 16H). MS(ESI) 671.7
[M + H]+. 4-((2,3-Bis(tert-butoxycarbonyl)guanidino)methyl)benzoic
acid was prepared from 4-aminophenylacetic acid (2.42 g, 16 mmol)
according to general procedure 4, method B, also using HgCl2 (1.7
N-{[1-Benzyl-4-(phenylamino)piperidin-4-yl]methyl}-4-carbami-
midamidobenzamide (17f). Prepared according to general procedure
1
5, method B, (no purification) using 16f (0.1 g, 0.152 mmol). H
NMR (500 MHz, DMSO-d6): δ 10.36−10.31 (m, 1H), 9.65 (s, 1H),
8.54 (s, 1H), 7.93−7.92 (m, 2H), 7.79 (s, 4H), 7.58−7.41 (m, 4H),
7.32−7.31 (m, 2H), 7.14−7.09 (m, 2H), 6.89−6.82 (m, 2H), 6.66 (t, J
= 6.3 Hz, 1H), 4.32 (s, 2H), 3.55−3.54 (m, 2H), 3.25−3.23 (m, 2H),
3.18−3.13 (m, 2 H), 2.20−2.17 (m, 2H), 1.96 (t, J = 13.4 Hz, 2H).
13C NMR (126 MHz, DMSO-d6): δ 166.64, 156.27, 146.39, 139.04,
131.89, 131.75, 130.16, 129.98, 129.31, 129.24, 123.31, 118.24, 117.71,
116.57, 115.86, 59.40, 54.01, 47.67, 45.58, 29.69. HRMS (ESI) calcd
for C27H33N6O 457.2716, found 457.2697 [M + H]+.
1
equiv), to afford 1.02 g of target compound (16% yield). H NMR
(400 MHz, CDCl3): δ 10.31 (s, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.23 (d,
J = 8.2 Hz, 2H), 3.57 (s, 2H), 1.51 (s, 18H). MS(ESI) 394.5 [M +
H]+.
N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-3-guanidi-
nopropanamide Tritriflate (17a). Prepared according to general
procedure 5, method B, using 19a (0.3 g, 0.49 mmol) to afford 0.29 g
(77%) of the title material. 1H NMR (500 MHz, DMSO-d6): δ 9.97 (s,
1H), 8.13−7.97 (m, 1H), 7.50−6.61 (m, 15H), 5.26 (br s, 1H), 4.30
(s, 2H), 3.32−3.11 (m, 8H), 2.45−2.37 (m, 2H), 2.11−1.84 (m, 4H).
13C NMR (125 MHz, DMSO-d6): δ 170.70, 158.81 (q, J = 34.4 Hz),
N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-2-(4-
guanidinophenyl)acetamide (17g). Prepared according to general
procedure 5, method B, (no purification) using 16g (0.19 g, 0.283
1
mmol). H NMR (500 MHz, DMSO-d6): δ 10.02 (s, 1H), 9.89 (s,
1H), 8.17 (s, 1H), 7.65−7.42 (m, 9H), 7.30 (d, J = 7.9 Hz, 2H), 7.15
(d, J = 7.8 Hz, 2H), 7.09 (t, J = 7.5 Hz, 2H), 6.81 (d, J = 7.8 Hz, 2H),
6.64 (t, J = 7.2 Hz, 1H), 4.32 (d, J = 2.3 Hz, 2H), 3.47 (s, 2H), 3.33 (d,
J = 5.8 Hz, 2H), 3.25−3.22 (m, 2H), 3.15−3.09 (m, 2H), 2.15−2.12
(m, 2H), 1.87 (t, J = 13.3 Hz, 2H). 13C NMR (126 MHz, DMSO-d6):
δ 156.56, 146.34, 134.89, 134.22, 131.66, 130.74, 129.89, 129.20,
124.54, 118.96, 117.62, 116.52, 116.45, 116.00, 59.31, 55.01, 53.47,
49.01, 47.53, 41.88, 29.70. HRMS (ESI) calcd for C28H35N6O
471.2872 [M + H]+, found 471.2856.
156.99, 145.85, 131.32, 129.77, 129.55, 128.83, 128.81, 117.29, 116.25
(q, J = 292.2 Hz), 116.14, 58.91, 53.16, 47.14, 44.49, 37.19, 34.40,
29.17. MS (ESI) m/z 409 (M+ + 1).
2-Amino-N-((1-benzyl-4-(phenylamino)piperidin-4-yl)methyl)-
acetamide (17b). Prepared according to general procedure 5, method
B, using 16b (5.5 g, 12.15 mmol) using the following modified workup
and purification: Methylene chloride (150 mL) and a 10% solution of
NaOH (80 mL) were added. The layers were separated, and the
organic layer was washed with water and brine. The solvent was dried
and evaporated. The residue was purified by chromatography on a
silica gel column using a gradient of methylene chloride and methanol
saturated with ammonia (10:0 → 8:2) as the eluent to give 4.02 g
3-Amino-N-((1-benzyl-4-(phenylamino)piperidin-4-yl)methyl)-
propanamide (18a). Prepared according to general procedure 2,
method B, using 16a (0.5 g, 1.38 mmol). The residue was purified by
chromatography on a silica gel column using a gradient of methylene
chloride and methanol saturated with ammonia (9:1 → 8:2) as the
1
eluent to give 0.38 g (76%) of the title material as a white solid. H
NMR (CDCl3): δ 7.30−7.23 (m, 5H), 7.16 (t, J = 8.0 Hz, 2H), 7.07−
7.05 (m, 1H), 6.78 (t, J = 7.3 Hz, 1H), 6.73 (d, J = 7.8 Hz, 2H), 3.55
(d, J = 5.5 Hz, 2H), 3.49 (s, 2H), 2.94 (t, J = 5.7 Hz, 2H), 2.58−2.55
(m, 2H), 2.34−2.27 (m, 4H), 1.90−1.87 (m, 2H), 1.79−1.69 (m, 2H),
1.65 (br s, 2H). MS (ESI) m/z 367 (M+ + 1).
1
(92%) of the title material as a white solid. H NMR (500 MHz,
CDCl3): δ 7.48−7.46 (m, 1H), 7.27−7.21 (m, 5H), 7.14 (t, J = 7.7 Hz,
2H), 6.77 (t, J = 7.2 Hz, 1H), 6.72 (d, J = 7.7 Hz, 2H), 3.53 (d, J = 5.7
Hz, 2H), 3.47 (s, 2H), 3.28 (s, 2H), 2.57−2.55 (m, 2H), 2.30 (t, J =
9.7 Hz, 2H), 1.88−1.85 (m, 2H), 1.73−1.68 (m, 2H), 1.62 (br s, 2H).
13C NMR (125 MHz, CDCl3): δ 173.15, 145.53, 138.28, 129.37,
tert-Butyl 1-(1-Benzyl-4-(phenylamino)piperidin-4-yl)-11,11-di-
methyl-3,9-dioxo-10-oxa-2,6,8-triazadodecan-7-ylidenecarbamate
(19a). Prepared according to general procedure 4, method B, using
18a (0.32 g, 0.88 mmol) to afford 0.47 g (87%) of the title material as
129.19, 128.33, 127.18, 119.31, 117.62, 63.12, 54.51, 49.12, 45.60,
44.92, 33.79. MS (ESI) m/z 353 (M+ + 1).
1
a white solid. H NMR (500 MHz, CDCl3): δ 11.47 (s, 1H), 8.77−
N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-4-guanidi-
nobutanamide (17c). Prepared according to general procedure 5,
method B, (no purification) using 16c (0.19 g, 0.30 mmol). 1H NMR
(DMSO-d6): δ 10.12−10.10 (m, 1H), 7.99−7.89 (m, 2H), 7.50−7.07
(m, 10H), 6.81−6.63 (m, 3H), 4.37−4.30 (m, 2H), 3.32−3.05 (m,
8H), 2.23−1.66 (m, 8H). 13C NMR (DMSO-d6): δ 172.18, 158.93,
158.58, 156.99, 145.74, 131.20, 129.63, 129.42, 128.74, 128.69, 117.50,
117.47, 117.27, 116.15, 114.58, 114.56, 58.88, 53.09, 47.09, 44.48,
31.83, 29.18, 24.60. MS (ESI) m/z 423 (M+ + 1).
N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-5-guanidi-
nopentanamide (17d). Prepared according to general procedure 5,
method B, (no purification) using 16d (0.20 g, 0.31 mmol). 1H NMR
(CD3OD): δ 7.51−7.45 (m, 5H), 7.21−7.16 (m, 3H), 6.88−6.76 (m,
2H), 4.32 (s, 2H), 3.49−3.17 (m, 7H), 2.29−2.26 (m, 5H), 1.99−1.94
(m, 2H), 1.64−1.57 (m, 4H). 13C NMR (CD3OD): δ 174.79, 157.28,
145.09, 130.94, 129.81, 128.93, 128.91, 128.81, 118.57, 116.79, 60.14,
8.75 (m, 1H), 7.31−7.23 (m, 5H), 7.18 (t, J = 7.6 Hz, 2H), 6.81 (t, J =
7.2 Hz, 1H), 6.74 (d, J = 8.0 Hz, 2H), 6.15−6.13 (m, 1H), 3.74−3.70
(m, 2H), 3.57 (d, J = 5.8 Hz, 2H), 3.51 (s, 2H), 2.60−2.57 (m, 2H),
2.44 (t, J = 6.0 Hz, 2H), 2.35−2.31 (m, 2H), 1.89−1.87 (m, 2H),
1.76−1.72 (m, 2H), 1.50 (s, 9H), 1.46 (s, 9H). MS (ESI) m/z 609
(M+ + 1).
tert-Butyl 1-(1-Benzyl-4-(phenylamino)piperidin-4-yl)-10,10-di-
methyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate
(20). Prepared according to general procedure 4, method B, using 17b
(0.2 g, 0.57 mmol) to afford 0.285 g (84%) of the title material as a
1
white solid. H NMR (500 MHz, CDCl3): δ 11.36 (s, 1H), 8.87 (s,
1H), 7.30−7.25 (m, 5H), 7.16 (s, 2H), 6.79 (s, 1H), 6.72 (s, 2H), 6.54
(s, 1H), 4.00 (s, 2H), 3.56 (s, 2H), 3.51 (s, 2H), 3.50 (br s, 1H), 2.57
(s, 2H), 2.37 (s, 2H), 1.90−1.88 (m, 2H), 1.76−1.74 (m, 2H), 1.49 (s,
9H), 1.47 (s, 9H). MS (ESI) m/z 595 (M+ + 1).
8919
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N-((1-Benzyl-4-(phenylamino)piperidin-4-yl)methyl)-2-guanidi-
noacetamide tritriflate (21). Prepared according to general procedure
5, method B, using 20 (0.18 g, 0.3 mmol) to afford 0.2 g (90%) of the
title material as a white solid. 1H NMR (500 MHz, CD3OD): δ 7.49−
7.42 (m, 5H), 7.12−7.09 (m, 2H), 6.90−6.69 (m, 3H), 4.27 (s, 2H),
3.91 (s, 2H), 3.48−3.29 (m, 6H), 2.26−1.99 (m, 4H). 13C NMR (125
MHz, CD3OD): δ 170.14, 163.18, 159.37, 146.64, 132.33, 131.10,
130.34, 130.23, 130.16, 119.86, 118.25 (q, J = 282.7 Hz), 118.15,
61.47, 54.60, 49.01, 46.59, 44.74, 31.21. MS (ESI) m/z 395 (M+ + 1).
1-Benzyl-3-(phenylamino)piperidine-3-carbonitrile (23). To a
mixture of 1-benzyl-3-piperidone (2 g, 1.06 mmol) and aniline (0.96
mL, 1.06 mmol) in 1,2-dichloroethane (40 mL) was added titanium
isopropoxide (4 mL, 1.37 mmol). The reaction mixture was stirred at
room temperature for 21 h and then diethylaluminum cyanide (1 M in
toluene, 21 mL, 2.11 mmol) was added. After 3 h of stirring, ethyl
acetate (100 mL) and a solution of saturated sodium bicarbonate were
added. The mixture was stirred for 10 min and filtered through a pad
of Celite. The Celite was washed with water and ethyl acetate, and the
layers of the filtrate were separated. The organic layer was washed with
brine, dried, and evaporated. The residue was purified by
chromatography on a silica gel column using ethyl acetate and
hexanes (4:6) as the eluent to give 2.36 g (74%) of the title material as
a white solid. 1H NMR (CDCl3): δ 7.41−7.32 (m, 5H), 7.26 (t, J = 8.0
Hz, 2H), 6.96 (d, J = 7.7 Hz, 2H), 6.90 (t, J = 7.4 Hz, 1H), 4.35 (s,
1H), 3.62 (s, 2H), 2.88−2.80 (m, 2H), 2.60 (s, 1H), 2.40 (s, 1H), 2.23
(s, 1H), 1.91 (s, 1H), 1.67 (s, 2H). MS (ESI) m/z 292 (M+ + 1).
3-(Aminomethyl)-1-benzyl-N-phenylpiperidin-3-amine (24). Pre-
pared according to general procedure 2, method B, to afford 1.34 g
(78%) of the title material as a yellow oil. 1H NMR (500 MHz,
CDCl3): δ 7.38−7.21 (m, 7H), 6.80 (br s, 3H), 4.25 (br s, 1H), 3.62−
3.53 (m, 2H), 3.23 (br s, 2H), 2.91−2.68 (m, 3H), 2.02 (s, 3H), 1.58−
1.15 (m, 4H). MS (ESI) m/z 296 (M+ + 1).
7.25 (m, 5H), 7.07 (t, J = 8.0 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 6.40
(s, 1H), 6.32 (d, J = 8.0 Hz, 1H), 6.06 (s, 1H), 3.68 (s, 3H), 3.51 (s,
2H), 2.76−2.74 (m, 2H), 2.29−2.27 (m, 4H), 1.85−1.80 (m, 2H). MS
(ESI) m/z 322 (M+ + 1).
1-Benzyl-4-(3,4-dichlorophenylamino)piperidine-4-carbonitrile
(27d). Prepared according to general procedure 1, using N-
benzylpiperidone, 3,4-dichloroaniline, and TMSCN to afford 7.31 g
1
(77%) of the title material as a light brown solid. H NMR (DMSO-
d6): δ 7.39−7.24 (m, 6H), 7.01 (d, J = 2.3 Hz, 1H), 6.83 (dd, J = 8.8,
2.4 Hz, 1H), 6.56 (s, 1H), 3.50 (s, 2H), 2.76−2.73 (m, 2H), 2.32−2.29
(m, 4H), 1.85−1.82 (m, 2H). MS (ESI) m/z 360 (M+ + 1).
4-(Aminomethyl)-1-benzyl-N-(naphthalen-1-yl)piperidin-4-
amine (28a). Prepared from 27a (3 g, 8.7 mmol) according to general
procedure 2, method B, to afford 2.7 g (89%) of the title material as a
1
light brown solid. H NMR (CDCl3): δ 7.95−7.93 (m, 1H), 7.86−
7.83 (m, 1H), 7.52−7.50 (m, 2H), 7.35−7.30 (m, 7H), 6.87−6.85 (m,
1H), 4.24 (br s, 1H), 3.52 (s, 2H), 3.06 (s, 2H), 2.70−2.67 (m, 2H),
2.39 (t, J = 10.7 Hz, 2H), 2.25−2.18 (m, 2H), 1.79−1.73 (m, 2H),
1.45 (br s, 2H). MS (ESI) m/z 346 (M+ + 1).
4-(Aminomethyl)-1-benzyl-N-(3,5-dimethoxyphenyl)piperidin-4-
amine (28b). Prepared from 27b (2.89 g, 8.24 mmol) according to
general procedure 2, method B, to afford 2.8 g (97%) of the title
material as a greenish oil. 1H NMR (CDCl3, 50 °C): δ 7.31−7.22 (m,
5H), 5.97−5.93 (m, 3H), 3.73 (s, 6H), 3.51 (s, 2H), 2.92 (br s, 2H),
2.59−2.53 (m, 3H), 2.35 (br s, 2H), 2.15−1.69 (m, 6H). MS (ESI) m/
z 356 (M+ + 1).
4-(Aminomethyl)-1-benzyl-N-(3-methoxyphenyl)piperidin-4-
amine (28c). Prepared from 27c (2.3 g, 7.16 mmol) according to
general procedure 2, method B, to afford 2.2 g (95%) of the title
material as a brown oil. 1H NMR (500 MHz, DMSO-d6): δ 7.29−7.22
(m, 5H), 6.92 (t, J = 7.8 Hz, 1H), 6.34−6.31 (m, 2H), 6.14 (d, J = 6.3
Hz, 1H), 4.82 (s, 1H), 3.65 (s, 3H), 3.18 (s, 2H), 2.70−2.75 (s, 2H),
2.50−2.28 (m, 6H), 1.91−1.89 (m, 2H), 1.55−1.53 (m, 2H). MS
(ESI) m/z 326 (M+ + 1).
4-(Aminomethyl)-1-benzyl-N-(3,4-dichlorophenyl)piperidin-4-
amine (28d). Prepared from 27d (3 g, 8.3 mmol) according to general
procedure 2, method B, to afford 2.8 g (95%) of the title material as a
thick colorless oil. 1H NMR (CDCl3): δ 7.31−7.24 (m, 5H), 7.16 (d, J
= 8.7 Hz, 1H), 6.81 (d, J = 2.6 Hz, 1H), 6.55 (dd, J = 8.7, 2.6 Hz, 1H),
3.55−3.43 (m, 3H), 2.86 (s, 2H), 2.63−2.60 (m, 2H), 2.26 (t, J = 10.4
Hz, 2H), 1.94 (d, J = 13.6 Hz, 2H), 1.67−1.60 (m, 2H), 1.44 (s, 2H).
MS (ESI) m/z 364 (M+ + 1).
tert-Butyl 1-(1-Benzyl-3-(phenylamino)piperidin-3-yl)-10,10-di-
methyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate
(25). Prepared according to general procedure 4, method C, to afford
0.23 g (37%) of the title material as a white solid. 1H NMR (500 MHz,
CDCl3): δ 11.36 (s, 1H), 8.94 (s, 1H), 7.38−7.28 (m, 5H), 7.19 (t, J =
7.6 Hz, 2H), 6.82 (t, J = 7.2 Hz, 1H), 6.73 (d, J = 7.7 Hz, 2H), 6.55−
6.53 (m, 1H), 4.50 (br s, 1H), 4.05−4.04 (m, 2H), 3.59−3.42 (m,
4H), 2.79−2.78 (m, 1H), 2.70−2.68 (m, 1H), 2.06−1.98 (m, 4H),
1.63−1.40 (m, 21H). MS (ESI) m/z 595 (M+ + 1).
N-((1-Benzyl-3-(phenylamino)piperidin-3-yl)methyl)-2-guanidi-
noacetamide Tritriflate (26). Prepared according to general
procedure 5, method B, to afford 0.16 g (65%) of the title material
tert-Butyl 1-(1-Benzyl-4-(naphthalen-1-ylamino)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (29a). Prepared from 28a (0.35 g, 1.02 mmol) according to
general procedure 4, method C, to afford 0.33 g (50%) of the title
1
as a white solid. H NMR (400 MHz, DMSO-d6): δ 9.26 (br s, 1H),
1
8.26 (s, 1H), 7.90 (s, 1H), 7.57−7.17 (m, 8H), 7.00 (s, 2H), 6.68−
6.65 (m, 3H), 5.24 (br s, 1H), 4.32−4.17 (m, 2H), 3.88 (s, 2H), 3.50−
2.95 (m, 6H), 2.00−1.59 (m, 4H). 13C NMR (125 MHz, DMSO-d6):
δ 168.15, 158.65 (q, J = 31.3 Hz), 157.81, 145.12, 131.49, 129.53,
129.14, 128.73, 128.72, 118.46, 117.23, 117.15 (q, J = 297.2 Hz),
59.81, 55.93, 55.02, 52.47, 43.68, 43.38, 28.62, 18.28. MS (ESI) m/z
395 (M+ + 1).
material as a white solid. H NMR (500 MHz, CDCl3): δ 11.24 (s,
1H), 8.79 (s, 1H), 7.90−7.88 (m, 1H), 7.82−7.81 (m, 1H), 7.51−7.47
(m, 2H), 7.31−7.25 (m, 7H), 6.86−6.84 (m, 1H), 6.46−6.45 (m, 1H),
3.96 (d, J = 5.1 Hz, 2H), 3.72 (d, J = 5.6 Hz, 2H), 3.51 (s, 2H), 2.64−
2.62 (m, 2H), 2.44−2.40 (m, 2H), 2.17−2.14 (m, 2H), 1.89−1.85 (m,
2H), 1.46 (m, 18H). MS (ESI) m/z 645 (M+ + 1).
tert-Butyl 1-(1-Benzyl-4-(3,5-dimethoxyphenylamino)piperidin-4-
yl)-10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidene-
carbamate (29b). Prepared from 28b (0.39 g, 1.10 mmol) according
to general procedure 4, method C, to afford 0.34 g (48%) of the title
1-Benzyl-4-(naphthalen-1-ylamino)piperidine-4-carbonitrile
(27a). Prepared according to general procedure 1, using N-
benzylpiperidone, 1-naphthylamine, and KCN to afford 3.2 g (59%)
1
1
of the target material as a thick oil. H NMR (CDCl3): δ 7.91−7.84
material as a white solid. H NMR (500 MHz, CDCl3): δ 11.36 (s,
(m, 2H), 7.52−7.27 (m, 10H), 4.24 (s, 1H), 3.58 (s, 2H), 2.85−2.82
(m, 1H), 2.77−2.74 (m, 1H), 2.57−2.43 (m, 4H), 2.14−2.09 (m, 2H).
MS (ESI) m/z 342 (M+ + 1).
1H), 8.85 (s, 1H), 7.31−7.25 (m, 5H), 6.42 (s, 1H), 5.94−5.89 (m,
3H), 3.99 (d, J = 5.3 Hz, 2H), 3.76 (s, 6H), 3.59 (d, J = 5.6 Hz, 2H),
3.52 (s, 2H), 2.59−2.57 (m, 2H), 2.37 (t, J = 9.3 Hz, 2H), 1.92 (d, J =
13.5 Hz, 2H), 1.78−1.74 (m, 2H), 1.51 (s, 9H), 1.49 (s, 9H). MS
(ESI) m/z 655 (M+ + 1).
1-Benzyl-4-(3,5-dimethoxyphenylamino)piperidine-4-carbonitrile
(27b). Prepared according to general procedure 1, using N-
benzylpiperidone, 3,5-dimethoxyaniline, and TMSCN to afford 6.44
tert-Butyl 1-(1-Benzyl-4-(3-methoxyphenylamino)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (29c). Prepared from 28c (0.39 g, 1.10 mmol) according to
general procedure 4, method C, to afford 0.46 g (67%) of the title
1
g (69%) of the title material as a white solid. H NMR (500 MHz,
DMSO-d6): δ 7.34−7.29 (m, 5H), 6.11−6.07 (m, 3H), 3.78 (s, 6H),
3.58 (s, 2H), 2.83−2.81 (m, 2H), 2.49 (t, J = 10.6 Hz, 2H), 2.38 (d, J
= 12.7 Hz, 2H), 1.96−1.94 (m, 2H). MS (ESI) m/z 352 (M+ + 1).
1-Benzyl-4-(3-methoxyphenylamino)piperidine-4-carbonitrile
(27c). Prepared according to general procedure 1, using N-
benzylpiperidone, m-anisidine, and TMSCN to afford 4.26 g (63%)
1
material as a white solid. H NMR (500 MHz, CDCl3): δ 11.36 (s,
1H), 8.85 (s, 1H), 7.31−7.25 (m, 5H), 7.07 (t, J = 8.0 Hz, 1H), 6.48
(s, 1H), 6.36−6.32 (m, 2H), 6.29 (s, 1H), 3.99 (d, J = 5.0 Hz, 2H),
3.77 (s, 3H), 3.58 (d, J = 5.3 Hz, 2H), 3.52 (br s, 1H), 3.51 (s, 2H),
2.58−2.56 (m, 2H), 2.37 (t, J = 9.5 Hz, 2H), 1.91 (d, J = 13.0 Hz, 2H),
1
of the title material as a white solid. H NMR (DMSO-d6): δ 7.31−
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1
1.77−1.73 (m, 2H), 1.51 (s, 9H), 1.48 (s, 9H). MS (ESI) m/z 625
(M+ + 1).
material as a white solid. H NMR (CDCl3): δ 11.36 (s, 1H), 8.74−
8.72 (m, 1H), 7.36−7.22 (m, 10H), 5.74−5.73 (m, 1H), 3.89 (d, J =
5.3 Hz, 2H), 3.47−3.46 (m, 4H), 2.61−2.59 (m, 2H), 2.36 (br s, 2H),
2.14−2.12 (m, 2H), 1.94−1.91 (m, 2H), 1.50 (s, 9H), 1.47 (s, 9H).
MS (ESI) m/z 580 (M+ + 1).
tert-Butyl 1-(1-Benzyl-4-(3,4-dichlorophenylamino)piperidin-4-
yl)-10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidene-
carbamate (29d). Prepared from 28d (0.4 g, 1.10 mmol) according to
general procedure 4, method C, to afford 0.24 g (33%) of the title
material as a white solid. 1H NMR (CDCl3): δ 11.31 (s, 1H), 8.82 (s,
1H), 7.30−7.24 (m, 5H), 7.17 (d, J = 8.7 Hz, 1H), 6.80 (d, J = 2.6 Hz,
1H), 6.66−6.63 (m, 1H), 6.56 (dd, J = 8.7, 2.6 Hz, 1H), 3.97 (d, J =
5.5 Hz, 2H), 3.62 (s, 1H), 3.52 (d, J = 5.7 Hz, 2H), 3.50 (s, 2H),
2.57−2.55 (m, 2H), 2.35−2.30 (m, 2H), 1.90−1.86 (m, 2H), 1.77−
1.71 (m, 2H), 1.49 (s, 9H), 1.46 (s, 9H). MS (ESI) m/z 663 (M+ + 1).
N-((1-Benzyl-4-(naphthalen-1-ylamino)piperidin-4-yl)methyl)-2-
guanidinoacetamide Tritriflate (30a). Prepared from 29a (0.2 g, 0.31
mmol) according to general procedure 5, method B, to afford 0.18 g
N-((1-Benzyl-4-phenylpiperidin-4-yl)methyl)-2-guanidinoaceta-
mide Ditriflate (34). Prepared according to general procedure 5,
method B, from intermediate 33 (0.12 g, 0.207 mmol) to afford the
title material as a white solid. 1H NMR (500 MHz, CD3OD): δ 7.41−
7.25 (m, 10H), 4.03 (s, 2H), 3.85 (s, 2H), 3.37−3.23 (m, 4H), 2.79
(br s, 2H), 2.39−2.37 (m, 2H), 2.15−2.11 (m, 2H). 13C NMR (125
MHz, CD3OD): δ 169.96, 163.14 (q, J = 34.2 Hz), 159.32, 142.36,
132.09, 131.93, 130.48, 130.19, 129.98, 128.11, 127.87, 118.12 (q, J =
290.9 Hz), 61.84, 50.34, 44.70, 42.28, 31.13. MS (ESI) m/z 380 (M+ +
1).
1
(74%) of the title material as a white solid. H NMR (400 MHz,
1,4-Dibenzylpiperidine-4-carbonitrile (36). Prepared according to
DMSO-d6): δ 10.35 (s, 1H), 8.29−8.16 (m, 2H), 7.91−7.22 (m, 14H),
6.90 (d, J = 7.23 Hz, 1H), 5.33 (s, 1H), 4.40−4.27 (m, 2H), 3.96−3.74
(m, 2H), 3.55−3.53 (m, 2H), 3.25−3.07 (m, 4H), 2.52−2.49 (m, 2H),
2.03−1.97 (m, 2H). 13C NMR (125 MHz, DMSO-d6): δ 167.98,
158.95 (q, J = 31.9 Hz), 157.74, 140.42, 134.39, 131.37, 129.64,
129.52, 128.72, 128.05, 126.10, 125.72, 125.34, 124.48, 118.26, 117.74,
115.89, 115.63 (q, J = 526.6 Hz), 58.80, 53.60, 47.18, 44.28, 43.36,
28.94. MS (ESI) m/z 445 (M+ + 1).
N-((1-Benzyl-4-(3,5-dimethoxyphenylamino)piperidin-4-yl)-
methyl)-2-guanidinoacetamide Tritriflate (30b). Prepared from 29b
(0.17 g, 0.26 mmol) according to general procedure 5, method B, to
afford 0.17 g (83%) of the title material as a white solid. 1H NMR (500
MHz, DMSO-d6): δ 10.10 (s, 1H), 8.24 (s, 1H), 7.75 (s, 1H), 7.50−
7.43 (m, 9H), 5.97 (s, 2H), 5.81 (s, 1H), 5.46 (s, 1H), 4.30 (s, 2H),
3.83−3.09 (m, 14H), 2.12−1.88 (m, 4H). 13C NMR (125 MHz,
DMSO-d6): δ 167.89, 160.89, 158.88 (q, J = 31.0 Hz), 157.60, 147.54,
131.29, 129.80, 129.52, 128.78, 116.11 (q, J = 296.3 Hz), 94.27, 89.50,
58.86, 54.77, 53.15, 47.09, 44.48, 43.34, 29.18. MS (ESI) m/z 455 (M+
+ 1).
N-((1-Benzyl-4-(3-methoxyphenylamino)piperidin-4-yl)methyl)-
2-guanidinoacetamide Tritriflate (30c). Prepared from 29c (0.2 g,
0.32 mmol) according to general procedure 5, method B, to afford
0.13 g (53%) of the title material as a white solid. 1H NMR (400 MHz,
DMSO-d6): δ 10.24 (s, 1H), 8.21 (s, 1H), 7.80 (s, 1H), 7.51−7.18 (m,
9H), 6.98 (t, J = 7.9 Hz, 1H), 6.40−6.37 (m, 2H), 6.21 (d, J = 7.8 Hz,
1H), 5.39 (br s, 1H), 4.30 (s, 2H), 3.85−3.83 (m, 2H), 3.67 (s, 3H),
3.36−3.08 (m, 6H), 2.14−1.88 (m, 4H). 13C NMR (125 MHz,
DMSO-d6): δ 167.93, 160.05, 158.95 (q, J = 31.9 Hz), 157.71, 147.20,
131.32, 131.10, 129.83, 129.54, 128.80, 117.09 (q, J = 297.0 Hz),
108.51, 102.65, 101.81, 58.87, 54.68, 53.18, 47.11, 44.60, 43.36, 29.20.
MS (ESI) m/z 425 (M+ + 1).
N-((1-Benzyl-4-(3,4-dichlorophenylamino)piperidin-4-yl)methyl)-
2-guanidinoacetamide Tritriflate (30d). Prepared from 29d (0.22 g,
0.33 mmol) according to general procedure 5, method B, to afford
0.19 g (72%) of the title material as a white solid. 1H NMR (500 MHz,
DMSO-d6): δ 10.13 (s, 1H), 8.25 (s, 1H), 7.70 (s, 1H), 7.49−7.13 (m,
10H), 6.98 (s, 1H), 6.77−6.75 (m, 1H), 5.90 (s, 1H), 4.29 (s, 2H),
3.83 (s, 2H), 3.59−3.04 (m, 6H), 2.14−1.88 (m, 4H). 13C NMR (125
MHz, DMSO-d6): δ 167.85, 158.57 (q, J = 31.2 Hz), 157.52, 146.16,
131.24, 130.97, 130.27, 129.74, 129.50, 128.76, 117.65, 117.13 (q, J =
297.3 Hz), 116.34, 115.10, 58.82, 53.25, 46.96, 44.00, 43.33, 28.96. MS
(ESI) m/z 463 (M+ + 1).
general procedure 6 using benzyl bromide to afford 1.29 g (89%) of
the title material as a white solid. H NMR (500 MHz, CDCl3): δ
1
7.39−7.28 (m, 10H), 3.56 (s, 2H), 2.91−2.89 (m, 4H), 2.34 (t, J =
12.1 Hz, 2H), 1.88 (d, J = 13.2 Hz, 2H), 1.73−1.67 (m, 2H). MS
(ESI) m/z 291 (M+ + 1).
1-Benzyl-4-phenethylpiperidine-4-carbonitrile (37). Prepared ac-
cording to general procedure 6 using (2-bromoethyl)benzene to afford
1.25 g (86%) of the title material as a colorless oil. 1H NMR (CDCl3):
δ 7.35−7.20 (m, 10H), 3.55 (s, 2H), 2.89−2.81 (m, 4H), 2.37 (t, J =
12 Hz, 2H), 1.96 (d, J = 13.1 Hz, 2H), 1.88−1.83 (m, 2H), 1.64−1.57
(m, 2H). MS (ESI) m/z 305 (M+ + 1).
4-(Aminomethyl)-1-benzyl-N-phenylpiperidin-4-amine (38). Pre-
pared from 36 (0.53 g, 1.82 mmol) using general procedure 2, method
1
B, to afford 0.33 g (61%) of the title material as a colorless oil. H
NMR (500 MHz, CDCl3): δ 7.35−7.17 (m, 10H), 3.56 (s, 2H), 2.69−
2.45 (m, 8H), 1.56−1.47 (m, 4H), 1.18 (br s, 2H). MS (ESI) m/z 295
(M+ + 1).
(1-Benzyl-4-phenethylpiperidin-4-yl)methanamine (39). Prepared
from 37 (0.436 g, 1.43 mmol) using general procedure 2, method B, to
1
afford 0.4 g (90%) of the title material as a colorless oil. H NMR
(CDCl3): δ 7.36−7.23 (m, 10H), 3.55 (s, 2H), 2.69 (s, 2H), 2.57−
2.43 (m, 6H), 1.70−1.66 (m, 2H), 1.56−1.54 (m, 4H), 1.18 (br s,
2H). MS (ESI) m/z 309 (M+ + 1).
tert-Butyl 1-(1-Benzyl-4-(phenylamino)piperidin-4-yl)-10,10-di-
methyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate
(40). Prepared from 38 (0.32 g, 1.08 mmol) according to general
procedure 4, method C, to afford 0.31 g (48%) of the title material as a
1
white solid. H NMR (500 MHz, CDCl3): δ 11.45 (s, 1H), 8.89 (s,
1H), 7.33−7.20 (m, 7H), 7.10 (d, J = 7.1 Hz, 2H), 6.89−6.88 (m,
1H), 4.04 (d, J = 5.5 Hz, 2H), 3.54 (s, 2H), 3.20(d, J = 5.4 Hz, 2H),
2.63−2.58 (m, 4H), 2.43−2.41 (m, 2H), 1.52−1.43 (m, 22H). MS
(ESI) m/z 594 (M+ + 1).
tert-Butyl 1-(1-Benzyl-4-phenethylpiperidin-4-yl)-10,10-dimethyl-
3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate (41). Pre-
pared from 39 (0.374 g, 1.21 mmol) according to general procedure 4,
method D, to afford 0.258 g (76%) of the title material as a white solid.
1H NMR (500 MHz, CDCl3): δ 11.41 (s, 1H), 8.93 (s, 1H), 7.34−
7.14 (m, 10H), 6.97 (s, 1H), 4.06−4.05 (m, 2H), 3.55 (s, 2H), 3.33−
3.32 (m, 2H), 2.56−2.50 (m, 6H), 1.57−1.55 (m, 6H), 1.51 (s, 9H),
1.41 (s, 9H). MS (ESI) m/z 608 (M+ + 1).
N-((1,4-Dibenzylpiperidin-4-yl)methyl)-2-guanidinoacetamide
Ditriflate (42). Prepared from 40 (0.13 g, 0.22 mmol) according to
general procedure 5, method B, to afford 0.096 g (71%) of the title
tert-Butyl 2-((1-Benzyl-4-phenylpiperidin-4-yl)methylamino)-2-
oxoethylcarbamate (32). Prepared according to general procedure
4, method D (step I), using 1-(1-benzyl-4-phenylpiperidin-4-yl)-
methanamine (31, 0.75 g, 2.66 mmol) to afford 0.9 g (78%) of the title
1
material as a white solid. H NMR (500 MHz, DMSO-d6): δ 9.89−
9.48 (m, 1H), 8.29−8.15 (m, 1H), 7.79−7.71 (m, 1H), 7.52−7.13 (m,
14H), 4.38−4.27 (m, 2H), 3.98−3.88 (m, 2H), 3.28−3.11 (m, 4H),
2.93−2.82 (m, 2H), 2.54−2.50 (m, 2H), 1.64−1.44 (m, 4H). 13C
NMR (125 MHz, DMSO-d6): δ 168.23, 168.02, 158.60 (q, J = 31.2
Hz), 137.00, 136.52, 131.17, 130.91, 130.59, 130.04, 129.83, 129.50,
128.90, 128.80, 128.15, 127.95, 126.42, 126.32, 116.16 (q, J = 297.5
Hz), 58.88, 58.63, 47.44, 47.08, 45.55, 43.52, 41.12, 36.38, 35.44,
35.31, 28.32, 28.16. MS (ESI) m/z 394 (M+ + 1).
1
material as a white solid. H NMR (CDCl3): δ 7.39−7.35 (m, 2H),
7.31−7.23 (m, 8H), 5.73 (br s, 1H), 5.11 (br s, 1H), 3.65 (d, J = 5.8
Hz, 2H), 3.45 (s, 4H), 2.61−2.59 (m, 2H), 2.34 (br s, 2H), 2.34−2.10
(m, 2H), 1.93−1.89 (m, 2H), 1.42 (s, 9H). MS (ESI) m/z 438 (M+ +
1).
tert-Butyl 1-(1-Benzyl-4-phenylpiperidin-4-yl)-10,10-dimethyl-
3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate (33). Pre-
pared according to general procedure 4, method D (step II), from
intermediate 32 (0.3 g, 0.685 mmol) to afford 0.35 g (89%) of the title
N-((1-Benzyl-4-phenethylpiperidin-4-yl)methyl)-2-guanidinoace-
tamide Ditriflate (43). Prepared from 41 (0.1 g, 0.197 mmol)
8921
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Journal of Medicinal Chemistry
Article
according to general procedure 5, method B, to afford 0.11 g (85%) of
the title material as a white solid. H NMR (500 MHz, CD3OD): δ
7.8 Hz, 2H), 6.62−6.60 (m, 1H), 3.99 (d, J = 5.1 Hz, 2H), 3.56 (d, J =
5.3 Hz, 2H), 3.56 (br s, 1H), 2.84−2.80 (m, 2H), 2.67−2.64 (m, 4H),
2.53−2.52 (m, 2H), 1.97−1.94 (m, 2H), 1.85−1.80 (m, 2H), 1.49 (s,
9H), 1.48 (s, 9H). MS (ESI) m/z 609 (M+ + 1).
1
7.54−7.52 (m, 5H), 7.29−7.18 (m, 5H), 4.37−4.36 (m, 2H), 4.01 (m,
2H), 3.52−3.18 (m, 6H), 2.67−2.63 (m, 2H), 1.81−1.60 (m, 6H). 13C
NMR (125 MHz, 45 °C, CD3OD): δ 170.25, 163.06 (q, J = 34.5 Hz),
159.49, 143.32, 132.19, 131.02, 130.37, 130.18, 129.37, 129.33, 126.79,
118.25 (q, J = 289.2 Hz), 61.29, 49.62, 44.96, 44.86, 41.94, 35.98,
30.66, 30.15. MS (ESI) m/z 408 (M+ + 1).
tert-Butyl 10,10-Dimethyl-1-(1-(naphthalen-1-ylmethyl)-4-
(phenylamino)piperidin-4-yl)-3,8-dioxo-9-oxa-2,5,7-triazaundecan-
6-ylidenecarbamate (48d). Prepared according to general procedure
7 using intermediate 47 (0.13 g, 0.257 mmol) and 1-(chloromethyl)-
naphthalene (0.041 g, 0.231 mmol) to afford 0.096 g (65%) of the title
tert-Butyl 1-(1-Benzyl-4-(phenylamino)piperidin-4-yl)-10,10-di-
methyl-1,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate
(45). To a stirred mixture of 1-benzyl-4-phenylamino-4-carboxy-
piperidine (44, 0.3 g, 0.9 mmol), 1-hydroxy-benzotriazole hydrate (0.2
g, 1.46 mmol), 2-(2-aminoethyl)-1,3-di-Boc-guanidine (0.34 g, 1.1
mmol), and triethylamine (0.6 mL, 4.5 mmol) in 1-methyl-2-
pyrrolidone (15 mL) was added N-(3-(dimethylamino)propyl)-N′-
ethylcarbodiimide hydrochloride (0.22 g, 1.1 mmol). The reaction
mixture was stirred at room temperature for 24 h. The mixture was
then poured into 80 mL of water, extracted with ethyl acetate (3 × 30
mL), washed with saturated aqueous NaCl, and dried. The solvent was
removed in vacuo, and the residue was chromatographed on a silica gel
column using a gradient of methylene chloride and methanol (10:0 →
9.5:0.5) as the eluent to give 0.37 g (70%) of tert-butyl 1-(1-benzyl-4-
(phenylamino)piperidin-4-yl)-10,10-dimethyl-1,8-dioxo-9-oxa-2,5,7-
triazaundecan-6-ylidenecarbamate as a white solid. 1H NMR (500
MHz, CDCl3): δ 11.19 (s, 1H), 8.42 (s, 1H), 7.77 (s, 1H), 7.32−7.25
(m, 5H), 7.14 (t, J = 7.4 Hz, 2H), 6.75 (t, J = 7.2 Hz, 1H), 6.54 (d, J =
7.8 Hz, 2H), 3.99 (s, 1H), 3.55−3.43 (m, 6H), 2.74 (d, J = 11.6 Hz,
2H), 2.33 (t, J = 11.2 Hz, 2H), 2.16 (t, J = 11.6 Hz, 2H), 1.90 (d, J =
13.2 Hz, 2H), 1.53 (s, 9H), 1.49 (s, 9H). MS (ESI) m/z 595 (M+ + 1).
1-Benzyl-N-(2-guanidinoethyl)-4-(phenylamino)piperidine-4-car-
boxamide Tritriflate (46). Prepared from 45 (0.12 g, 0.20 mmol)
according to general procedure 5, method B, to afford 0.08 g (54%) of
1
material as a white solid. H NMR (CDCl3): δ 11.37 (s, 1H), 8.86−
8.84 (m, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.2 Hz, 1H), 7.76
(d, J = 7.2 Hz, 1H), 7.51−7.47 (m, 2H), 7.40−7.37 (m, 2H), 7.16 (t, J
= 7.7 Hz, 2H), 6.79 (t, J = 7.3 Hz, 1H), 6.73 (d, J = 7.9 Hz, 2H),
6.56−6.53 (m, 1H), 4.00 (d, J = 5.2 Hz, 2H), 3.90 (s, 2H), 3.55 (d, J =
5.4 Hz, 2H), 3.43 (br s, 1H), 2.64−2.61 (m, 2H), 2.43 (t, J = 9.5 Hz,
2H), 1.89−1.85 (m, 2H), 1.73−1.68 (m, 2H), 1.49 (s, 9H), 1.47 (s,
9H). MS (ESI) m/z 645 (M+ + 1).
tert-Butyl 10,10-Dimethyl-1-(1-(naphthalen-2-ylmethyl)-4-
(phenylamino)piperidin-4-yl)-3,8-dioxo-9-oxa-2,5,7-triazaundecan-
6-ylidenecarbamate (48e). Prepared according to general procedure
4, method C, using 5d (0.3 g, 0.87 mmol) to afford 0.31 g (48%) of
1
the title material as a yellow solid. H NMR (500 MHz, CDCl3): δ
11.30 (s, 1H), 8.80 (s, 1H), 7.77−7.67 (m, 4H), 7.43−7.39 (m, 3H),
7.11 (t, J = 7.4 Hz, 2H), 6.74 (t, J = 7.2 Hz, 1H), 6.68 (d, J = 7.7 Hz,
2H), 6.54 (s, 1H), 3.95 (br s, 3H), 3.62−3.51 (m, 4H), 2.56−2.55 (m,
2H), 2.38−2.37 (m, 2H), 1.87−1.84 (m, 2H), 1.74−1.72 (m, 2H),
1.44 (s, 9H), 1.40 (s, 9H). MS (ESI) m/z 645 (M+ + 1).
tert-Butyl 1-(1-(4-Bromobenzyl)-4-(phenylamino)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (48f). Prepared according to general procedure 7 using
intermediate 47 (0.13 g, 0.257 mmol) and 4-bromobenzyl bromide
(0.058 g, 0.231 mmol) to afford 0.12 g (69%) of the title material as a
white solid. 1H NMR (CDCl3): δ 11.33 (s, 1H), 8.82 (s, 1H), 7.40 (d,
J = 8.2 Hz, 2H), 7.18−7.12 (m, 4H), 6.78 (t, J = 7.1 Hz, 1H), 6.70 (d,
J = 8.0 Hz, 2H), 6.51−6.50 (m, 1H), 3.98 (d, J = 5.2 Hz, 2H), 3.54 (d,
J = 5.3 Hz, 2H), 3.44 (s, 2H), 3.42 (br s, 1H), 2.53−2.51 (m, 2H),
2.37−2.33 (m, 2H), 1.88−1.85 (m, 2H), 1.75−1.70 (m, 2H), 1.48 (s,
9H), 1.45 (s, 9H). MS (ESI) m/z 673 (M+ + 1) for 79Br, 675 (M+ + 1)
for 81Br.
1
the title material as a white solid. H NMR (500 MHz, DMSO-d6): δ
10.40 (s, 1H), 8.28−7.91 (m, 2H), 7.50−7.07 (m, 12H), 6.63−6.55
(m, 3H), 6.06 (s, 1H), 4.29 (s, 2H), 3.36−3.10 (m, 8H), 2.25−1.95
(m, 4H). 13C NMR (125 MHz, DMSO-d6): δ 174.46, 158.77 (q, J =
31.4 Hz), 157.32, 144.44, 131.30, 129.53, 128.79, 128.71, 117.52,
117.12 (q, J = 296.6 Hz), 115.02, 114.52, 59.02, 55.95, 46.73, 40.19,
38.00, 28.07. MS (ESI) m/z 395 (M+ + 1).
tert-Butyl 10,10-Dimethyl-3,8-dioxo-1-(4-(phenylamino)-
piperidin-4-yl)-9-oxa-2,5,7-triazaundecan-6-ylidenecarbamate
(47). Intermediate 20 (1 g, 1.68 mmol) was dissolved in methanol (65
mL) and hydrogenated over 10% Pd/C (0.4 g) under pressure (4 bar)
for 7 days. The catalyst was removed by filtration, and the solvent was
evaporated in vacuo. The residue was purified by chromatography on a
silica gel column using a gradient of methylene chloride and methanol
saturated with ammonia (10:0 → 9:1) as the eluent to give 0.57 g
(68%) of the title material as a white solid. 1H NMR (CDCl3): δ 11.34
(br s, 1H), 8.83 (br s, 1H), 7.15 (t, J = 7.6 Hz, 2H), 6.79 (t, J = 7.4 Hz,
1H), 6.72 (d, J = 7.8 Hz, 2H), 6.53−6.55 (m, 1H), 3.98 (d, J = 5.2 Hz,
2H), 3.55 (d, J = 5.6 Hz, 2H), 3.42 (br s, 1H), 2.89−2.87 (m, 4H),
1.86−1.82 (m, 2H), 1.68−1.63 (m, 3H), 1.49 (s, 9H), 1.47 (s, 9H).
MS (ESI) m/z 505 (M+ + 1).
tert-Butyl 1-(1-(Cyclohexylmethyl)-4-(phenylamino)piperidin-4-
yl)-10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidene-
carbamate (48b). Prepared according to general procedure 7 using
intermediate 47 (0.13 g, 0.257 mmol) and cyclohexylmethyl bromide
(0.055 g, 0.308 mmol) in anhydrous NMP (5 mL) to afford 0.04 g
(27%) of the title material as a white solid. 1H NMR (CDCl3): δ 11.31
(br s, 1H), 8.84 (br s, 1H), 7.10 (t, J = 7.6 Hz, 2H), 6.76−6.69 (m,
4H), 3.97 (d, J = 4.9 Hz, 2H), 3.50 (d, J = 5.3 Hz, 2H), 2.67 (s, 2H),
2.48 (s, 2H), 2.24 (s, 2H), 1.91−1.59 (m, 10H), 1.45 (s, 9H), 1.43 (s,
9H), 1.21−1.08 (m, 4H), 0.89−0.84 (m, 2H). MS (ESI) m/z 601 (M+
+ 1).
Methyl 4-((4-(6-(tert-Butoxycarbonylamino)-10,10-dimethyl-3,8-
dioxo-9-oxa-2,5,7-triazaundec-6-enyl)-4-(phenylamino)piperidin-1-
yl)methyl)benzoate (48g). Prepared according to general procedure 7
using intermediate 47 (0.13 g, 0.257 mmol) and methyl 4-
(bromomethyl)benzoate (0.053 g, 0.231 mmol) to afford 0.14 g
1
(96%) of the title material as a white solid. H NMR (500 MHz,
CDCl3): δ 11.33 (s, 1H), 8.82 (s, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.37
(d, J = 8.1 Hz, 2H), 7.14 (t, J = 7.7 Hz, 2H), 6.78 (t, J = 7.2 Hz, 1H),
6.70 (d, J = 7.8 Hz, 2H), 6.52−6.50 (m, 1H), 3.98 (d, J = 5.3 Hz, 2H),
3.89 (s, 3H), 3.55−3.46 (m, 5H), 2.54−2.52 (m, 2H), 2.39−2.36 (m,
2H), 1.89−1.86 (m, 2H), 1.76−1.72 (m, 2H), 1.48 (s, 9H), 1.45 (s,
9H). MS (ESI) m/z 653 (M+ + 1).
2-Guanidino-N-((4-(phenylamino)piperidin-4-yl)methyl)-
acetamide Tritriflate (49a). Prepared according to general procedure
5, method B, from 47 (0.1 g, 0.198 mmol) to afford 0.11 g (85%) of
1
the title material as a brown solid. H NMR (DMSO-d6): δ 10.85 (br
s, 2H), 8.74−8.63 (m, 2H), 8.18 (s, 1H), 7.74 (s, 1H), 7.41 (br s, 3H),
7.10 (t, J = 6 Hz, 2H), 6.85 (d, J = 6.2 Hz, 2H), 6.66−6.64 (m, 1H),
3.86−3.85 (m, 2H), 3.42−3.41 (m, 2H), 3.12−3.03 (m, 4H), 2.07−
2.06 (m, 2H), 1.80−1.75 (m, 2H). 13C NMR (DMSO-d6): δ 168.01,
158.70 (q, J = 34.2 Hz), 157.60, 145.43, 128.87, 117.59, 116.33, 116.23
(q, J = 290.6 Hz), 53.58, 44.46, 43.42, 30.65, 28.66. MS (ESI) m/z 305
(M+ + 1).
N-((1-(Cyclohexylmethyl)-4-(phenylamino)piperidin-4-yl)methyl)-
2-guanidinoacetamide Tritriflate (49b). Prepared according to
general procedure 5, method B, from 48b (0.04 g, 0.066 mmol) to
tert-Butyl 10,10-Dimethyl-3,8-dioxo-1-(1-phenethyl-4-
(phenylamino)piperidin-4-yl)-9-oxa-2,5,7-triazaundecan-6-ylidene-
carbamate (48c). Prepared according to general procedure 7 using
intermediate 47 (0.15 g, 0.297 mmol) and 2-(bromoethyl)benzene
(0.055 g, 0.297 mmol) to afford 0.13 g (72%) of the title material as a
1
afford 0.04 g (81%) of the title material as a yellow solid. H NMR
(400 MHz, CD3OD): δ 7.14 (t, J = 4 Hz, 2H), 6.90−6.88 (m, 2H),
6.73 (t, J = 4 Hz, 1H), 3.97 (s, 2H), 3.53 (s, 2H), 3.45−3.42 (m, 2H),
3.26−3.20 (m, 2H), 2.95 (d, J = 8 Hz, 2H), 2.30−2.26 (m, 2H), 2.16−
2.09 (m, 3H), 1.84−1.70 (m, 7H), 1.38−1.28 (m, 3H). MS (ESI) m/z
401 (M+ + 1).
1
white solid. H NMR (CDCl3): δ 11.35 (s, 1H), 8.85 (s, 1H), 7.29−
7.25 (m, 2H), 7.19−7.14 (m, 5H), 6.79 (t, J = 7.2 Hz, 1H), 6.73 (d, J =
8922
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2-Guanidino-N-((1-phenethyl-4-(phenylamino)piperidin-4-yl)-
methyl)acetamide Tritriflate (49c). Prepared according to general
procedure 5, method B, from 48c (0.12 g, 0.197 mmol) to afford 0.076
4-carbonitrile (35, 0.9 g, 4.49 mmol) and 3,4-dichlorobenzyl bromide
(1.4 g, 5.84 mmol) to afford 0.98 g (61%) of the title material as a
white solid. 1H NMR (DMSO-d6): δ 7.59 (d, J = 8.2 Hz, 1H), 7.55 (s,
1H), 7.32−7.23 (m, 6H), 3.46 (s, 2H), 2.91 (s, 2H), 2.78 (d, J = 11.9
Hz, 2H), 2.07 (t, J = 11.0 Hz, 2H), 1.72−1.61 (m, 4H). MS (ESI) m/z
359 [M + H]+.
1
g (51%) of the title material as a yellow solid. H NMR (400 MHz,
CD3OD): δ 7.35−7.24 (m, 5H), 7.16 (t, J = 7.6 Hz, 2H), 6.89 (d, J =
8.0 Hz, 2H), 6.75 (t, J = 7.2 Hz, 1H), 3.96 (s, 2H), 3.56−3.51 (m,
4H), 3.35−3.31 (m, 4H), 3.10−3.05 (m, 2H), 2.35−2.31 (m, 2H),
2.12−2.01 (m, 2H). 13C NMR (125 MHz, CD3OD): δ 170.15, 163.05
(q, J = 37.0 Hz), 159.37, 146.68, 137.65, 130.19, 129.93, 129.79,
128.21, 119.85, 118.26 (q, J = 290.9 Hz), 118.12, 59.02, 54.58, 46.61,
44.72, 43.00, 31.39, 30.62. MS (ESI) m/z 409 (M+ + 1).
1-Benzyl-4-(naphthalen-2-ylmethyl)piperidine-4-carbonitrile
(50c). Prepared according to general procedure 6 using 1-
benzylpiperidine-4-carbonitrile (35, 0.7 g, 3.49 mmol) and 2-
(bromomethyl)naphthalene (1 g, 4.54 mmol) to afford 0.85 g
1
(71%) of the title material as a white solid. H NMR (CDCl3): δ
7.86−7.83 (m, 3H), 7.75 (s, 1H), 7.53−7.43 (m, 3H), 7.34−7.27 (m,
5H), 3.54 (s, 2H), 3.04 (s, 2H), 2.89 (d, J = 12.3 Hz, 2H), 2.33 (t, J =
12.2 Hz, 2H), 1.90 (d, J = 12.4 Hz, 2H), 1.74 (td, J = 12.5, 2.9 Hz,
2H). MS (ESI) m/z 341 [M + H]+.
2-Guanidino-N-((1-(naphthalen-1-ylmethyl)-4-(phenylamino)-
piperidin-4-yl)methyl)acetamide Tritriflate (49d). Prepared accord-
ing to general procedure 5, method B, from 48d (0.09 g, 0.139 mmol)
1
to afford 0.07 g (65%) of the title material as a white solid. H NMR
1-Benzyl-4-(biphenyl-4-ylmethyl)piperidine-4-carbonitrile (50d).
Prepared according to general procedure 6 using 1-benzylpiperidine-
4-carbonitrile (35, 0.7 g, 3.49 mmol) and 4-(bromomethyl)biphenyl
(1.12 g, 4.54 mmol) to afford 0.76 g (59%) of the title material as a
white solid. 1H NMR (500 MHz, CDCl3): δ 7.59−7.55 (m, 4H), 7.44
(t, J = 7.5 Hz, 2H), 7.35−7.26 (m, 8H), 3.53 (s, 2H), 2.89−2.86 (m,
4H), 2.31 (d, J = 10.6 Hz, 2H), 1.88 (d, J = 12.4 Hz, 2H), 1.71−1.66
(m, 2H). MS (ESI) m/z 367 (M+ + 1).
(500 MHz, CD3OD): δ 8.15 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.2 Hz,
1H), 7.87 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.57−7.46 (m,
3H), 7.09−7.06 (m, 2H), 6.80−6.65 (m, 3H), 4.71 (s, 2H), 3.84 (s,
2H), 3.40−3.25 (m, 6H), 2.17−1.92 (m, 4H). 13C NMR (125 MHz,
CD3OD): δ 170.05, 163.07 (q, J = 34.4 Hz), 159.33, 146.63, 135.33,
133.46, 132.45, 132.13, 130.16, 130.07, 128.50, 127.53, 126.32, 124.23,
120.36, 119.83, 118.15 (q, J = 291.0 Hz), 118.09, 57.85, 54.50, 49.56,
46.47, 44.66, 31.17. MS (ESI) m/z 445 (M+ + 1).
(1-Benzyl-4-(naphthalen-1-ylmethyl)piperidin-4-yl)methanamine
(51a). Prepared according to general procedure 2, method B, from 50a
(1.3 g, 3.81 mmol) at 2.38 atm for 15 h. The catalyst was removed by
filtration, and the solvent was evaporated in vacuo. The residue was
purified by chromatography on a silica gel column using a gradient of
methylene chloride and methanol (10:0 → 8:2) as the eluent to give
0.49 g (38%) of the title material as a white solid. 1H NMR (CDCl3):
δ 8.29 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 8.0
Hz, 1H), 7.55−7.43 (m, 3H), 7.38−7.27 (m, 6H), 3.52 (s, 2H), 3.17
(s, 2H), 2.72 (br s, 2H), 2.65−2.62 (m, 2H), 2.30 (t, J = 9.8 Hz, 2H),
1.70−1.64 (m, 4H), 1.57−1.54 (m, 2H). MS (ESI) m/z 345 (M+ + 1).
(1-Benzyl-4-(3,4-dichlorobenzyl)piperidin-4-yl)methanamine
(51b). Prepared according to general procedure 2, method B, from
50b (0.9 g, 2.5 mmol) to afford 0.85 g (93%) of the title material as a
2-Guanidino-N-((1-(naphthalen-2-ylmethyl)-4-(phenylamino)-
piperidin-4-yl)methyl)acetamide Tritriflate (49e). Prepared according
to general procedure 5, method B, from 48e (0.1 g, 0.15 mmol) to
afford 0.08 g (66%) of the title material as a white solid. 1H NMR (400
MHz, DMSO-d6): δ 10.44 (s, 1H), 8.19 (s, 1H), 8.07 (s, 1H), 7.97−
7.78 (m, 4H), 7.66−7.17 (m, 8H), 7.08−7.05 (t, J = 7.4 Hz, 2H), 6.80
(d, J = 8.0 Hz, 2H), 6.61 (t, J = 7.0 Hz, 1H), 5.32 (br s, 1H), 4.46 (s,
2H), 3.84−3.83 (m, 2H), 3.58−3.16 (m, 6H), 2.14−2.11 (m, 2H),
1.98−1.92 (m, 2H). 13C NMR (125 MHz, DMSO-d6): δ 167.86,
158.65 (q, J = 31.6 Hz), 157.69, 145.90, 133.07, 132.53, 131.11,
128.81, 128.37, 128.08, 127.99, 127.71, 127.42, 127.07, 126.72, 117.32,
117.13 (q, J = 295.4 Hz), 116.19, 59.02, 53.19, 47.28, 44.74, 43.38,
29.21. MS (ESI) m/z 445 (M+ + 1).
N-((1-(4-Bromobenzyl)-4-(phenylamino)piperidin-4-yl)methyl)-2-
guanidinoacetamide Tritriflate (49f). Prepared according to general
procedure 5, method B, from 48f (0.1 g, 0.148 mmol) to afford 0.066 g
1
white solid. H NMR (DMSO-d6): δ 7.49−7.43 (m, 2H), 7.29−7.15
(m, 6H), 3.43 (s, 2H), 3.17 (s, 2H), 2.60−2.26 (m, 8H), 1.31 (s, 4H).
MS (ESI) m/z 363 (M+ + 1), 365 (M+ + 1).
1
(54%) of the title material as a white solid. H NMR (500 MHz,
(1-Benzyl-4-(naphthalen-2-ylmethyl)piperidin-4-yl)methanamine
(51c). Prepared according to general procedure 2, method B, from 50c
(0.8 g, 2.35 mmol) to afford 0.62 g (77%) of the title material as a
CD3OD): δ 7.50 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.10 (t,
J = 7.4 Hz, 2H), 6.81 (d, J = 8.1 Hz, 2H), 6.68 (t, J = 7.1 Hz, 1H), 3.92
(s, 2H), 3.84 (s, 2H), 3.51 (s, 2H), 2.89−2.81 (m, 4H), 2.11−2.09 (m,
2H), 1.86−1.84 (m, 2H). 13C NMR (125 MHz, CD3OD): δ 170.00,
163.18 (q, J = 35.8 Hz), 159.33, 146.87, 134.00, 133.36, 132.83,
130.03, 123.64, 119.60, 118.26, 118.09 (q, J = 290.7 Hz), 61.81, 54.99,
49.46, 46.62, 44.78, 32.36. MS (ESI) m/z 473 (M+ + 1) for 79Br, 475
(M+ + 1) for 81Br.
Methyl 4-((4-((2-Guanidinoacetamido)methyl)-4-(phenylamino)-
piperidin-1-yl)methyl)benzoate Tritriflate (49g). Prepared according
to general procedure 5, method B, from 48g (0.13 g, 0.199 mmol) to
afford 0.09 g (56%) of the title material as a white solid. 1H NMR (500
MHz, CD3OD): δ 8.10 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H),
7.16 (t, J = 7.8 Hz, 2H), 6.87 (d, J = 7.8 Hz, 2H), 6.76 (s, 1H), 4.42 (s,
2H), 3.94 (s, 5H), 3.54 (s, 2H), 3.36−3.33 (m, 4H), 2.30−2.06 (m,
4H). 13C NMR (125 MHz, CD3OD): δ 170.14, 167.70, 163.09 (q, J =
35.6 Hz), 159.38, 146.64, 135.40, 132.89, 132.58, 131.16, 130.18,
119.90, 118.29 (q, J = 288.8 Hz), 118.15, 60.77, 54.61, 52.89, 49.34,
46.61, 44.72, 31.22. MS (ESI) m/z 453 (M+ + 1).
1-Benzyl-4-(naphthalen-1-ylmethyl)piperidine-4-carbonitrile
(50a). Prepared according to general procedure 6 using 1-
benzylpiperidine-4-carbonitrile (35, 0.9 g, 4.49 mmol) and 1-
(chloromethyl)naphthalene (1 g, 5.84 mmol) to afford 1.4 g (91%)
of the title material as a white solid. 1H NMR (CDCl3): δ 8.11 (d, J =
8.2 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.58−
7.47 (m, 4H), 7.36−7.27 (m, 5H), 3.53 (s, 2H), 3.40 (s, 2H), 2.87 (d,
J = 12.1 Hz, 2H), 2.32 (t, J = 10.0 Hz, 2H), 1.88−1.78 (m, 4H). MS
(ESI) m/z 341 (M+ + 1).
1
colorless oil. H NMR (CDCl3): δ 7.84−7.76 (m, 3H), 7.62 (s, 1H),
7.50−7.44 (m, 2H), 7.37−7.27 (m, 6H), 3.57 (s, 2H), 2.85 (s, 2H),
2.63−2.58 (m, 4H), 2.48−2.46 (m, 2H), 1.63−1.50 (m, 4H), 1.21 (br
s, 2H). MS (ESI) m/z 345 [M + H]+.
(1-Benzyl-4-(biphenyl-4-ylmethyl)piperidin-4-yl)methanamine
(51d). Prepared according to general procedure 2, method B, from
50d (0.69 g, 1.88 mmol) to afford 0.67 g (96%) of the title material as
a colorless oil. 1H NMR (CDCl3): δ 7.60 (d, J = 7.5 Hz, 2H), 7.52 (d,
J = 8.0 Hz, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.35−7.32 (m, 4H), 7.29−
7.23 (m, 3H), 3.56 (s, 2H), 2.72 (s, 2H), 2.60−2.57 (m, 4H), 2.47−
2.46 (m, 2H), 1.59−1.52 (m, 4H), 1.32 (br s, 2H). MS (ESI) m/z 371
(M+ + 1).
tert-Butyl 1-(1-Benzyl-4-(naphthalen-1-ylmethyl)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (52a). Prepared according to general procedure 4, method C,
from 51a (0.35 g, 1.02 mmol). The residue was purified by
chromatography on a silica gel column using a gradient of methylene
chloride and methanol (10:0 → 97:3) to give 0.27 g (41%) of the title
material as a white solid. 1H NMR (CDCl3): δ 11.45 (s, 1H), 8.87 (s,
1H), 8.07 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.4 Hz, 1H), 7.73 (d, J = 8.1
Hz, 1H), 7.49−7.42 (m, 2H), 7.39 (t, J = 7.2 Hz, 1H), 7.27−7.22 (m,
6H), 6.91−6.89 (m, 1H), 3.98 (d, J = 5.4 Hz, 2H), 3.48 (s, 2H), 3.35
(d, J = 5.6 Hz, 2H), 3.09 (s, 2H), 2.62−2.59 (m, 2H), 2.31 (t, J = 10.3
Hz, 2H), 1.70−1.65 (m, 2H), 1.51−1.47 (m, 11H), 1.40 (s, 9H). MS
(ESI) m/z 644 (M+ + 1).
(tert-Butyl 1-(1-Benzyl-4-(3,4-dichlorobenzyl)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (52b). Prepared according to general procedure 4, method C,
1-Benzyl-4-(3,4-dichlorobenzyl)piperidine-4-carbonitrile (50b).
Prepared according to general procedure 6 using 1-benzylpiperidine-
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from 51b (0.37 g, 1.02 mmol) to afford 0.33 g (49%) of the title
material as a white solid. 1H NMR (CDCl3): δ 11.42 (s, 1H), 8.92 (s,
1H), 7.33−7.19 (m, 8H), 6.94 (d, J = 8.1 Hz, 1H), 4.05 (d, J = 5.8 Hz,
2H), 3.50 (s, 2H), 3.15 (d, J = 5.4 Hz, 2H), 2.57−2.52 (m, 4H), 2.39−
2.37 (m, 2H), 1.50−1.40 (m, 22H). MS (ESI) m/z 662 (M+ + 1), 664
(M+ + 1).
Biology. NPFF1 and NPFF2 Receptor Binding Studies.
Compounds 7a−d, 9a−c, and 15a,b were tested against [125I]YVP
and [125I]EYF for NPFF1 and NPFF2 affinity, respectively, as
described in Mollereau et al.21 The affinities of 17a−g, 21, 30a−d,
34, 42−43, 46, 49a−g, and 53a−c on NPFF1 and NPFF2 receptors
were evaluated by competition experiments using the selective NPFF1
and NPFF2 radioligands [3H]-NPVF and [3H]-EYF, respectively, in
membranes of CHO cells stably expressing each receptor. For
membrane preparation, CHO cells expressing human NPFF receptors
were harvested in phosphate buffer saline (PBS), frozen at least for 1 h
at −70 °C, and then homogenized in 50 mM Tris-HCl, pH 7.4, in a
Potter Elvehjem tissue grinder. The nuclear pellet was discarded by
centrifugation at 1000g for 15 min at 4 °C, and the membrane fraction
was collected upon centrifugation of the supernatant at 100000g for 30
min at 4 °C. Membranes were aliquoted and stored at −80 °C in Tris
50 mM, pH 7.4, and the protein concentration was determined by the
Lowry method. [3H]-NPVF and [3H]-EYF were custom-made by RC
TRITEC AG (Teufen, Switzerland) by hydrogenation of the
unsaturated peptide precursors with 99% tritium gas. Tritiated
products were purified (>98%) by HPLC and dissolved in ethanol
to obtain 1 mCi/mL (37 MBq/mL). Binding of [3H]-NPVF and [3H]-
EYF was measured by rapid filtration. Membranes (5−15 μg of
protein) were incubated in polypropylene tubes in a final volume of
500 μL containing 50 mM Tris-HCl, pH 7.4, 0.1% bovine serum
albumin, 60 mM NaCl (for NPFF2 receptors only), the radioligand at
0.5−1 nM, and compounds to be tested at the desired concentration.
The nonspecific binding was determined in the presence of 1 μM
YVPNLPQRFa (for hNPFF1 receptor) and 1 μM EYWSLAAPQRFa
(for hNPFF2 receptor). After 1 h incubation at 25 °C, samples were
rapidly filtered on Whatman GF/B filters preincubated in 0.3%
polyethylenimine. The filters were rinsed three times with 4 mL of ice
cold buffer containing 0.1% bovine serum albumin, and the bound
radioactivity was counted in a liquid scintillation spectrophotometric
counter (50% efficiency, Packard).
cAMP Assay at NPFF1 and NPFF2 Receptors. Recombinant
cells (200000) were seeded in glass tubes and incubated overnight as
usual. The culture medium was then removed and replaced by fresh
medium (200 μL) containing 0.1 μM adenine and 0.6 μCi of
[3H]adenine (26 Ci/mmole, Amersham, France). After 1 h incubation
at 37 °C in the incubator under 5% CO2 atmosphere, cells were rinsed
two times with 400 μL of HEPES-buffered Krebs−Ringer saline
(KRH, 124 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.5 mM CaCl2,
1.25 mM KH2PO4, 25 mM HEPES, 8 mM glucose, 0.5 mg/mL bovine
serum albumin; pH 7.4). Prewarmed KRH (150 μL) was added to
each tube, and the reaction was initiated by the addition of 50 μL of
KRH containing 20 μM forskolin (Sigma, France), 0.4 mM IBMX (3-
isobutyl-1-methylxanthine; Sigma, France), 0.4 mM Ro-20 1724 (4-(3-
butoxy-4methoxyphenyl)methyl-2-imidazolidone; Fisher, France), and
the ligand to be tested. To evaluate the antagonist activity, the
compound was tested in the presence of the NPFF agonists NPVF and
1DMe for hNPFF-1 and hNPFF-2 receptors, respectively. After 10
min at 37 °C, the reaction was stopped by addition of 20 μL of HCl
(2.2 N) with rapid mixing. The [3H]cAMP content of each tube was
isolated by chromatographic procedures on acid alumina columns as
described by Alvarez and Daniels34 and counted in a liquid scintillation
analyzer (Packard).
tert-Butyl 1-(1-Benzyl-4-(naphthalen-2-ylmethyl)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (52c). Prepared according to general procedure 4, method C,
from 51c (0.35 g, 1.02 mmol) to afford 0.38 g (72%) of the title
1
material as a white solid. H NMR (CDCl3): δ 11.47 (s, 1H), 8.97−
8.95 (m, 1H), 7.80−7.72 (m, 3H), 7.53 (s, 1H), 7.46−7.42 (m, 2H),
7.31−7.19 (m, 7H), 4.08 (d, J = 5.6 Hz, 2H), 3.52 (s, 2H), 3.21 (d, J =
5.6 Hz, 2H), 2.94 (s, 1H), 2.88 (s, 1H), 2.78 (s, 2H), 2.60−2.58 (m,
2H), 2.43−2.40 (m, 2H), 1.63−1.56 (m, 2H), 1.51 (s, 9H), 1.40 (s,
9H). MS (ESI) m/z 644 (M+ + 1).
(tert-Butyl 1-(1-Benzyl-4-(biphenyl-4-ylmethyl)piperidin-4-yl)-
10,10-dimethyl-3,8-dioxo-9-oxa-2,5,7-triazaundecan-6-ylidenecar-
bamate (52d). Prepared according to general procedure 4, method D,
from 51d (0.413 g, 1.11 mmol) to afford 0.223 g (80%) of the title
1
material as a white solid. H NMR (500 MHz, CDCl3): δ 11.47 (s,
1H), 8.95 (s, 1H), 7.59 (d, J = 7.3 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H),
7.44 (t, J = 7.5 Hz, 2H), 7.36−7.31 (m, 5H), 7.28−7.25 (m, 1H), 7.18
(d, J = 8.0 Hz, 2H), 7.12 (br s, 1H), 4.09 (d, J = 6.0 Hz, 2H), 3.57 (s,
2H), 3.22 (d, J = 5.7 Hz, 2H), 2.68 (s, 2H), 2.61 (s, 2H), 2.46 (s, 2H),
1.64−1.60 (m, 4H), 1.52 (s, 9H), 1.44 (s, 9H). MS (ESI) m/z 670
(M+ + 1).
N-((1-Benzyl-4-(naphthalen-1-ylmethyl)piperidin-4-yl)methyl)-2-
guanidinoacetamide Tritriflate (53a). Prepared according to general
procedure 5, method B, from 52a (0.12 g, 0.189 mmol). The residue
was purified by chromatography on a silica gel column using column
using a gradient of methylene chloride and methanol (95:5 → 8:2) as
the eluent to give 0.07 g (54%) of the title material as a white solid. 1H
NMR (500 MHz, CD3OD): δ 8.03 (d, J = 8.3 Hz, 1H), 7.81 (d, J = 8.0
Hz, 1H), 7.74 (t, J = 8.1 Hz, 1H), 7.48−7.27 (m, 9H), 4.27−4.13 (m,
2H), 4.08−3.84 (m, 2H), 3.59 (s, 2H), 3.35−3.02 (m, 6H), 1.88−1.61
(m, 4H). 13C NMR (125 MHz, CD3OD): δ 170.65, 163.05 (q, J =
34.2 Hz), 159.53, 135.52, 134.74, 134.49, 133.79, 132.13, 131.09,
131.00, 130.21, 129.86, 128.69, 127.06, 126.57, 126.15, 125.34, 118.24
(q, J = 292.9 Hz), 61.40, 49.47, 44.84, 44.13, 40.42, 38.22, 29.86. MS
(ESI) m/z 444 (M+ + 1).
N-((1-Benzyl-4-(3,4-dichlorobenzyl)piperidin-4-yl)methyl)-2-gua-
nidinoacetamide Ditriflate (53b). Prepared according to general
procedure 5, method B, from 52b (0.1 g, 0.151 mmol) to afford 0.07 g
1
(72%) of the title material as a white solid. H NMR (500 MHz,
CD3OD): δ 7.57−7.41 (m, 7H), 7.22−7.13 (m, 1H), 4.39−4.29 (m,
2H), 4.07−4.00 (m, 2H), 3.46−3.14 (m, 6H), 2.91−2.67 (m, 2H),
1.88−1.85 (m, 2H), 1.69−1.68 (m, 2H). 13C NMR (125 MHz, 45 °C,
CD3OD): δ 170.49, 163.06 (q, J = 34.4 Hz), 159.56, 138.37, 133.71,
133.10, 132.18, 131.82, 131.75, 131.24, 131.08, 130.33, 130.23, 118.56
(q, J = 280.0 Hz), 61.43, 49.45, 44.90, 44.14, 42.65, 36.89, 29.81. MS
(ESI) m/z 462 (M+ + 1) 464 (M+ + 1).
N-((1-Benzyl-4-(naphthalen-2-ylmethyl)piperidin-4-yl)methyl)-2-
guanidinoacetamide Ditriflate (53c). Prepared according to general
procedure 5, method B, from 52c (0.13 g, 0.202 mmol) to afford 0.10
1
g (75%) of the title material as a white solid. H NMR (500 MHz,
[35S]GTPγS Binding Assay at NPFF1 and NPFF2 Receptors.
The assay buffer consisted of 20 mM HEPES, pH 7.4, 20 mM NaCl, 3
mM MgCl2, and 0.1% BSA. Aliquots (50 μL, equivalent to 1.5 μg and
to 0.7 μg protein, for NPFF1 and NPFF2, respectively) were incubated
in polypropylene tubes at 30 °C for 60 min in 500 μL of buffer with 5
μg of saponin, 0.1 μM (NPFF1) or 1 μM (NPFF2) GDP, the ligand to
be tested, and 0.066 nM [35S]GTPγS. Reaction was stopped by rapid
vacuum filtration through GF/B Whatman glass fiber filters,
preincubated in the buffer at room temperature for 1 h, and washed
three times with 4 mL of ice-cold buffer. Membrane-bound
radioactivity retained on the filters was determined by liquid
scintillation spectrophotometry (94% efficiency, Packard counter)
after overnight extraction of the filters in 4 mL of Ready Protein
scintillation fluid (Beckman).
CD3OD): δ 7.85−7.69 (m, 4H), 7.52−7.32 (m, 8H), 4.42−4.26 (m,
2H), 4.11−3.99 (m, 2H), 3.52−3.27 (m, 6H), 3.09−2.86 (m, 2H),
1.85−1.76 (m, 4H). MS (ESI) m/z 444 (M+ + 1).
N-((1-Benzyl-4-(biphenyl-4-ylmethyl)piperidin-4-yl)methyl)-2-
guanidinoacetamide Ditriflate (53d). Prepared according to general
procedure 5, method B, from 52d (0.1 g, 0.149 mmol) to afford 0.08 g
1
(76%) of the title material as a white solid. H NMR (500 MHz,
CD3OD): δ 7.62−7.57 (m, 4H), 7.51−7.43 (m, 6H), 7.36−7.34 (m,
2H), 7.28−7.27 (m, 2H), 4.40−4.29 (m, 2H), 4.09−4.00 (m, 2H),
3.50−3.20 (m, 6H), 2.96−2.73 (m, 2H), 1.78−1.70 (m, 4H). 13C
NMR (125 MHz, 45 °C, CD3OD): δ 170.41, 163.02 (q, J = 32.4 Hz),
159.53, 141.89, 140.86, 136.46, 132.34, 132.16, 131.05, 130.32, 130.20,
129.78, 128.24, 127.75, 127.74, 118.56 (q, J = 290.0 Hz), 61.38, 49.45,
44.92, 44.91, 42.81, 36.91, 29.93. MS (ESI) m/z 470 (M+ + 1).
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Opioid Receptor (μ, δ, κ) Binding Studies. Reagents. Buffer
reagents were purchased from Sigma-Aldrich (St. Louis, MO). All
radioligands and MicroScint were purchased from PerkinElmer
(Waltham, MA). Nonlabeled controls were purchased from Tocris
Bioscience (Minneapolis, MN). Membrane preparation and all assay
dilutions were made using a Tris-HCl buffer (50 mM Tris-HCl), pH
7.4.
Health Guide for the Care and Use of Laboratory Animals. Consistent
with these guidelines, ongoing statistical testing of data collected was
used to minimize the number of animals used, within the constraints of
necessary statistical power.
Injection Techniques. Intracerebroventricular (icv) injections
were made directly into the lateral ventricle according to the modified
method of Haley and McCormick.35 The volume of all icv injections
was 5 μL, using a 10-μL Hamilton microliter syringe. The mouse was
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.
Hyperalgesic Testing: The 48 °C Warm-Water Tail-With-
drawal Assay. The nociceptive stimulus was 48 °C water, with the
latency to withdraw the tail taken as the end point.36,37 The water
temperature of 48 °C was selected for this work to ensure a moderate
tail-withdrawal response, with a measurable decrease in withdrawal
time possible but also a significant temperature for hyperalgesic
testing. After determining control latencies, mice received a single icv
dose of vehicle (50% DMSO) or novel ligand pretreatment as
discussed above. The dose of RF9 used in this study (10 nmol, icv)
was selected because it was previously reported effective in
antagonizing the hyperalgesic response of NPFF.38 The dose of
compound 46 (30 nmol, icv) was selected on the basis of similar
affinity for NPFF1-R as RF9 (75 nM vs the reported 58 nM).15
Animals showing an initial baseline latency fewer than 4 s or more than
15 s were removed from the study; two mice were so excluded. Mice
administered the test compound were tested for analgesia every 10
min for 80 min postinjection. If the mouse failed to display a tail-flick
in 30 s, the tail was removed from the water to minimize tissue
damage. Experimentally induced increases in tail-withdrawal latencies
provide a measure of antinociceptive effect, whereas decreases in tail-
withdrawal latency indicate hyperalgesic effects.39−41
Cell Culture. HEK293 cells stably transfected with human opioid
receptor subtypes μ, δ, or κ were used to perform the opioid receptor
binding assays. These cells were maintained at 37 °C and 5% CO2 in a
DMEM nutrient mixture supplemented with 2 mM L-glutamine, 10%
fetal bovine serum, 1% penicillin−streptomycin, and G418 antibiotic
solutions. When the cells reached 90% confluency, the media was
removed, and the cells were washed with cold PBS, pH 7.4. The cells
were lysed and scraped in cold Tris-HCl, pH 7.4, and then centrifuged
at 5200g for 10 min at 4 °C. The supernatant was discarded ,and the
pellet was resuspended in the same buffer and homogenized via Sonic
Dismembrator model 100 (Fisher Scientific, Pittsburgh, PA) for 30 s
and then centrifuged at 1000g for 10 min at 4 °C. The supernatant was
saved, and the pellet underwent the suspension and homogenization
process two more times with the same conditions. The supernatants
were combined and centrifuged at 23300g for 40 min at 4 °C. The
pellet was resuspended in cold Tris-HCl buffer, aliquoted into 2 mL
vials, and stored at −80 °C. The total protein concentration was
determined using a Pierce BCA protein assay kit (Thermo Scientific,
Rockford, IL) using manufacturer’s instructions.
Radioligand Binding Assays. For each assay, nonspecific binding
was determined using 10 μM of a positive control [U-69,593 (κ),
DPDPE (δ), or DAMGO (μ)], and total binding was ascertained with
0.1% DMSO in Tris-HCl buffer. Each test well contained 50 μL of its
respective radioligand (U-69,593,[phenyl-3,4-3H] (κ), DAMGO,
[tyrosyl-3,5-3H(N)] (μ) or enkephalin(DPDPE),[tyrosyl-3,5-3H(N)]
(δ)), 50 μL of compound, control, or vehicle, and 100 μL of cell
membrane. The assays were incubated for 60 min at room
temperature. The reaction was terminated via rapid filtration with
cold Tris-HCl buffer through a UniFilter GF/B 96-well plate
presoaked with 0.3% BSA. When the filters were dry, 50 μL of
MicroScint-20 was applied to each filter, and the plates were read on a
TopCount NXT HTS microplate scintillation counter (PerkinElmer,
Waltham, MA) where the counts per minute (CPM) were recorded.
The Kd for ligands for each receptor was established through a
membrane evaluation and saturation binding experiment. For the
membrane evaluation experiment, 1−15 μg of membrane was
incubated with 0.6 nM of its respective radioligand. Total, specific,
and nonspecific binding were used to calculate the % binding of the
nonlabeled control to receptor. The membrane concentration
exhibiting good % binding (>90%), and total binding with high signal
(thousands of CPM) was used as the optimal membrane
concentration for the assay. For the saturation assay, the optimal
membrane concentration and 0.25−10 nM of its respective radioligand
were incubated with 10 μM of a nonlabeled positive control or 0.1%
DMSO in buffer. Data was analyzed by a nonlinear curve fit model
using GraphPad Prizm 5.04 software (GraphPad, La Jolla, CA), and
the Kd value was calculated.
Data Analysis. All tail-withdrawal latency data for hyperalgesic
testing are reported as percent baseline response to control for each
animal’s baseline latency, SEM. Percent baseline response is
calculated by the following equation: % baseline response = 100(test
latency − baseline latency). Responses to treatment over time were
analyzed by one- or two-way ANOVA followed by Tukey’s post hoc
test as appropriate, with factors of treatment and time as required.
AUTHOR INFORMATION
Corresponding Author
*Tel: 662-915-5882. Fax: 662-915-5638. E-mail: cmccurdy@
olemiss.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work described in this paper was partially supported by
Grants DA29738 (C. Mesangeau), GM104932 (S. J. Cutler),
DA034777 (C. R. McCurdy) and the State of Florida, Executive
Office of the Governor’s Dept. of Economic Opportunity (J. P.
McLaughlin). The authors are grateful to Sara Pettaway for
conducting the opioid binding experiments as part of the
COBRE in vitro research core. This investigation was
conducted in a facility constructed with support from research
facilities improvement program Grant Number RR104503 from
the National Center for Research Resources, National Institutes
of Health. Cell lines used for the opioid binding were a
generous gift from Dr. B. Roth (Department of Pharmacology,
School of Medicine, University of North Carolina, Chapel Hill).
The competitive binding assay was performed used the optimal
concentration of membrane with a radioligand concentration ≤Kd and
12 concentrations of each compound ranging from 0.00001 to 1000
μM. Each compound was tested in triplicate. The assays were
performed as stated above. The Ki values were calculated by a
nonlinear curve fit model using GraphPad Prizm 5.0 software.
In Vivo Characterization. Animals. Adult (8−11 weeks old) male
C57BL/6J were obtained from the Jackson Laboratory, Bar Harbor,
Maine, USA. All mice were housed four to a cage in a temperature-
and humidity-controlled room at the Torrey Pines Institute for
Molecular Studies (Port Saint Lucie, Florida, 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 experimental sessions. All procedures were
preapproved and carried out in accordance with the Institutional
Animal Care and Use Committees at the Torrey Pines Institute for
Molecular Studies as specified by the 2008 National Institutes of
ABBREVIATIONS
■
NPFF, neuropeptide FF; 1DMe, (D.YL(N-Me)FQPQRFa);
RF9, n-adamantanecarbonyl-Arg-Phe-NH2; AC-099, 1-(3-
bromo-4,5-dimethoxybenzylideneamino)guanidine hydrochlor-
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(13) Altier, N.; Dray, A.; Menard, D.; Henry, J. L. Neuropeptide FF
attenuates allodynia in models of chronic inflammation and neuro-
pathy following intrathecal or intracerebroventricular administration.
Eur. J. Pharmacol. 2000, 407, 245−255.
(14) Lameh, J.; Bertozzi, F.; Kelly, N.; Jacobi, P. M.; Nguyen, D.;
Bajpai, A.; Gaubert, G.; Olsson, R.; Gardell, L. R. Neuropeptide FF
receptors have opposing modulatory effects on nociception. J.
Pharmacol. Exp. Ther. 2010, 334, 244−254.
(15) Simonin, F.; Schmitt, M.; Laulin, J. P.; Laboureyras, E.;
Jhamandas, J. H.; MacTavish, D.; Matifas, A.; Mollereau, C.; Laurent,
P.; Parmentier, M.; Kieffer, B. L.; Bourguignon, J. J.; Simonnet, G.
RF9, a potent and selective neuropeptide FF receptor antagonist,
prevents opioid-induced tolerance associated with hyperalgesia. Proc.
Natl. Acad. Sci. U. S. A. 2006, 103, 466−471.
(16) Elhabazi, K.; Trigo, J. M.; Mollereau, C.; Mouledous, L.; Zajac, J.
M.; Bihel, F.; Schmitt, M.; Bourguignon, J. J.; Meziane, H.; Petit-
demouliere, B.; Bockel, F.; Maldonado, R.; Simonin, F. Involvement of
neuropeptide FF receptors in neuroadaptive responses to acute and
chronic opiate treatments. Br. J. Pharmacol. 2012, 165, 424−435.
(17) Gealageas, R.; Schneider, S.; Humbert, J. P.; Bertin, I.; Schmitt,
M.; Laboureyras, E.; Dugave, C.; Mollereau, C.; Simonnet, G.;
Bourguignon, J. J.; Simonin, F.; Bihel, F. Development of sub-
nanomolar dipeptidic ligands of neuropeptide FF receptors. Bioorg.
Med. Chem. Lett. 2012, 22, 7471−7474.
(18) Mouledous, L.; Froment, C.; Dauvillier, S.; Burlet-Schiltz, O.;
Zajac, J. M.; Mollereau, C. GRK2 protein-mediated transphosphor-
ylation contributes to loss of function of mu-opioid receptors induced
by neuropeptide FF (NPFF2) receptors. J. Biol. Chem. 2012, 287,
12736−12749.
(19) Gaubert, G.; Bertozzi, F.; Kelly, N. M.; Pawlas, J.; Scully, A. L.;
Nash, N. R.; Gardell, L. R.; Lameh, J.; Olsson, R. Discovery of selective
nonpeptidergic neuropeptide FF2 receptor agonists. J. Med. Chem.
2009, 52, 6511−6514.
(20) Scully, A. L.; Davis, R. E.; Vanover, K. E.; Gardell, L. R.; Lameh,
J.; Kelly, N. M.; Bertozzi, F. Treating neuropathic pain with
neuropeptide FF receptor 2 agonists. International Patent WO
2005031000 A2, 2005.
(21) Mollereau, C.; Mazarguil, H.; Marcus, D.; Quelven, I.; Kotani,
M.; Lannoy, V.; Dumont, Y.; Quirion, R.; Detheux, M.; Parmentier,
M.; Zajac, J. M. Pharmacological characterization of human NPFF(1)
and NPFF(2) receptors expressed in CHO cells by using NPY Y(1)
receptor antagonists. Eur. J. Pharmacol. 2002, 451, 245−256.
(22) Findeisen, M.; Wurker, C.; Rathmann, D.; Meier, R.; Meiler, J.;
Olsson, R.; Beck-Sickinger, A. G. Selective mode of action of
guanidine-containing non-peptides at human NPFF receptors. J.
Med. Chem. 2012, 55, 6124−6136.
(23) Mankus, J. V.; McCurdy, C. R. Nonpeptide ligands of
neuropeptide FF: Current status and structural insights. Future Med.
Chem. 2012, 4, 1085−1092.
(24) Gicquel, S.; Mazarguil, H.; Desprat, C.; Allard, M.; Devillers, J.
P.; Simonnet, G.; Zajac, J. M. Structure−activity study of neuropeptide
FF: Contribution of N-terminal regions to affinity and activity. J. Med.
Chem. 1994, 37, 3477−3481.
(25) Mazarguil, H.; Gouarderes, C.; Tafani, J. A.; Marcus, D.; Kotani,
M.; Mollereau, C.; Roumy, M.; Zajac, J. M. Structure-activity
relationships of neuropeptide FF: Role of C-terminal regions. Peptides
2001, 22, 1471−1478.
(26) Schwyzer, R. ACTH: A short introductory review. Ann. N.Y.
Acad. Sci. 1977, 297, 3−26.
(27) Nieto, M. J.; Philip, A. E.; Poupaert, J. H.; McCurdy, C. R.
Solution-phase parallel synthesis of spirohydantoins. J. Comb. Chem.
2005, 7, 258−263.
ide; EDCI, N-(3-(dimethylamino)propyl)-N′-ethylcarbodii-
mide hydrochloride; HOBt, 1-hydroxybenzotriazole hydrate;
[35S]GTPγS, guanosine-5′-O-(3-[35S]thio)triphosphate; cAMP,
cyclic adenosine monophosphate; YVP, YVPNLPQRFa; EYF,
EYWSLAAPQRFa (EYW-NPSF); NPVF, VPNLPQRFa;
CHO, Chinese hamster ovary; ANOVA, analysis of variance;
HEK, human embryonic kidney; DAMGO, (D -
Ala2,MePhe4,Gly-ol5)enkephalin; DPDPE, [D-Pen2,D-Pen5]-
enkephalin; U69,593, (5α,7α,8β)-(−)-N-methyl-N-[7-(1-pyrro-
lidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide; TMSCN,
trimethylsilyl cyanide; DMF, dimethylformamide; THF,
tetrahydrofuran; NMP, N-methyl-2-pyrrolidone; HEPES, 4-
(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic acid; DMEM,
Dulbecco’s Modified Eagle’s medium; Boc, tert-butyloxycar-
bonyl; TFA, trifluoroacetic acid; DCM, dichloromethane; LDA,
lithium diisopropylamide; icv, intracerebroventricular; it.,
intrathecal; OIH, opioid-induced hyperalgesia
REFERENCES
■
(1) Mollereau, C.; Roumy, M.; Zajac, J. M. Opioid-modulating
peptides: Mechanisms of action. Curr. Top. Med. Chem. 2005, 5, 341−
355.
(2) Liu, Q.; Guan, X. M.; Martin, W. J.; McDonald, T. P.; Clements,
M. K.; Jiang, Q.; Zeng, Z.; Jacobson, M.; Williams, D. L., Jr.; Yu, H.;
Bomford, D.; Figueroa, D.; Mallee, J.; Wang, R.; Evans, J.; Gould, R.;
Austin, C. P. Identification and characterization of novel mammalian
neuropeptide FF-like peptides that attenuate morphine-induced
antinociception. J. Biol. Chem. 2001, 276, 36961−36969.
(3) Gouarderes, C.; Quelven, I.; Mollereau, C.; Mazarguil, H.; Rice,
S. Q.; Zajac, J. M. Quantitative autoradiographic distribution of
NPFF1 neuropeptide FF receptor in the rat brain and comparison
with NPFF2 receptor by using [125I]YVP and [(125I]EYF as selective
radioligands. Neuroscience 2002, 115, 349−361.
(4) Bonini, J. A.; Jones, K. A.; Adham, N.; Forray, C.; Artymyshyn,
R.; Durkin, M. M.; Smith, K. E.; Tamm, J. A.; Boteju, L. W.; Lakhlani,
P. P.; Raddatz, R.; Yao, W. J.; Ogozalek, K. L.; Boyle, N.; Kouranova,
E. V.; Quan, Y.; Vaysse, P. J.; Wetzel, J. M.; Branchek, T. A.; Gerald,
C.; Borowsky, B. Identification and characterization of two G protein-
coupled receptors for neuropeptide FF. J. Biol. Chem. 2000, 275,
39324−39331.
(5) Talmont, F.; Mouledous, L.; Piedra-Garcia, L.; Schmitt, M.; Bihel,
F.; Bourguignon, J. J.; Zajac, J. M.; Mollereau, C. Pharmacological
characterization of the mouse NPFF2 receptor. Peptides 2010, 31,
215−220.
(6) Yang, H. Y.; Fratta, W.; Majane, E. A.; Costa, E. Isolation,
sequencing, synthesis, and pharmacological characterization of two
brain neuropeptides that modulate the action of morphine. Proc. Natl.
Acad. Sci. U. S. A. 1985, 82, 7757−7761.
(7) Gicquel, S.; Mazarguil, H.; Allard, M.; Simonnet, G.; Zajac, J. M.
Analogues of F8Famide resistant to degradation, with high affinity and
in vivo effects. Eur. J. Pharmacol. 1992, 222, 61−67.
(8) Oberling, P.; Stinus, L.; Le Moal, M.; Simonnet, G. Biphasic
effect on nociception and antiopiate activity of the neuropeptide FF
(FLFQPQRFamide) in the rat. Peptides 1993, 14, 919−924.
(9) Desprat, C.; Zajac, J. M. Differential modulation of mu- and
delta-opioid antinociception by neuropeptide FF receptors in young
mice. Neuropeptides 1997, 31, 1−7.
(10) Gouarderes, C.; Sutak, M.; Zajac, J. M.; Jhamandas, K.
Antinociceptive effects of intrathecally administered F8Famide and
FMRFamide in the rat. Eur. J. Pharmacol. 1993, 237, 73−81.
(11) Kontinen, V. K.; Kalso, E. A. Differential modulation of alpha 2-
adrenergic and mu-opioid spinal antinociception by neuropeptide FF.
Peptides 1995, 16, 973−977.
(12) Xu, M.; Kontinen, V. K.; Panula, P.; Kalso, E. Effects of
(1DMe)NPYF, a synthetic neuropeptide FF analogue, in different pain
models. Peptides 1999, 20, 1071−1077.
(28) McCurdy, C. R.; Prisinzano, T. E. Opioid Receptor Ligands. In
Burger’s Medicinal Chemistry, Drug Discovery and Development, 7 ed.;
Abraham, D. J., Rotella, D. P., Eds.; Wiley: Hoboken, NJ, 2010; Vol. 8,
pp 569−736.
(29) Sharma, S. K.; Jones, R. M.; Metzger, T. G.; Ferguson, D. M.;
Portoghese, P. S. Transformation of a kappa-opioid receptor
antagonist to a kappa-agonist by transfer of a guanidinium group
8926
dx.doi.org/10.1021/jm500989n | J. Med. Chem. 2014, 57, 8903−8927

Journal of Medicinal Chemistry
Article
from the 5′- to 6′-position of naltrindole. J. Med. Chem. 2001, 44,
2073−2079.
(30) Thomas, J. B.; Atkinson, R. N.; Namdev, N.; Rothman, R. B.;
Gigstad, K. M.; Fix, S. E.; Mascarella, S. W.; Burgess, J. P.; Vinson, N.
A.; Xu, H.; Dersch, C. M.; Cantrell, B. E.; Zimmerman, D. M.; Carroll,
F. I. Discovery of an opioid kappa receptor selective pure antagonist
from a library of N-substituted 4β-methyl-5-(3-hydroxyphenyl)-
morphans. J. Med. Chem. 2002, 45, 3524−3530.
(31) Costanzo, M. J.; Yabut, S. C.; Almond, H. R., Jr.; Andrade-
Gordon, P.; Corcoran, T. W.; De Garavilla, L.; Kauffman, J. A.;
Abraham, W. M.; Recacha, R.; Chattopadhyay, D.; Maryanoff, B. E.
Potent, small-molecule inhibitors of human mast cell tryptase.
Antiasthmatic action of a dipeptide-based transition-state analogue
containing a benzothiazole ketone. J. Med. Chem. 2003, 46, 3865−
3876.
(32) Riches, A. G.; Cablewski, T.; Glattauer, V.; Thissen, H.;
Meagher, L. Scalable synthesis of an integrin-binding peptide mimetic
for biomedical applications. Tetrahedron 2012, 68, 9448−9455.
(33) Kayser, K. J.; Glenn, M. P.; Sebti, S. M.; Cheng, J. Q.; Hamilton,
A. D. Modifications of the GSK3beta substrate sequence to produce
substrate-mimetic inhibitors of Akt as potential anti-cancer therapeu-
tics. Bioorg. Med. Chem. Lett. 2007, 17, 2068−2073.
(34) Alvarez, R.; Daniels, D. V. A single column method for the assay
of adenylate cyclase. Anal. Biochem. 1990, 187, 98−103.
(35) Haley, T. J.; McCormick, W. G. Pharmacological effects
produced by intracerebral injection of drugs in the conscious mouse.
Br. J. Pharmacol. Chemother. 1957, 12, 12−15.
(36) McLaughlin, J. P.; Hill, K. P.; Jiang, Q.; Sebastian, A.; Archer, S.;
Bidlack, J. M. Nitrocinnamoyl and chlorocinnamoyl derivatives of
dihydrocodeinone: in vivo and in vitro characterization of mu-selective
agonist and antagonist activity. J. Pharmacol. Exp. Ther. 1999, 289,
304−311.
(37) Vaught, J. L.; Takemori, A. E. Differential effects of leucine and
methionine enkephalin on morphine-induced analgesia, acute
tolerance and dependence. J. Pharmacol. Exp. Ther. 1979, 208, 86−90.
(38) Fang, Q.; Wang, Y. Q.; He, F.; Guo, J.; Guo, J.; Chen, Q.; Wang,
R. Inhibition of neuropeptide FF (NPFF)-induced hypothermia and
anti-morphine analgesia by RF9, a new selective NPFF receptors
antagonist. Regul. Pept. 2008, 147, 45−51.
(39) Crain, S. M.; Shen, K. F. Acute thermal hyperalgesia elicited by
low-dose morphine in normal mice is blocked by ultra-low-dose
naltrexone, unmasking potent opioid analgesia. Brain Res. 2001, 888,
75−82.
(40) Crain, S. M.; Shen, K. F. Naloxone rapidly evokes endogenous
kappa opioid receptor-mediated hyperalgesia in naive mice pretreated
briefly with GM1 ganglioside or in chronic morphine-dependent mice.
Brain Res. 2007, 1167, 31−41.
(41) Dubois, D.; Gendron, L. Delta opioid receptor-mediated
analgesia is not altered in preprotachykinin A knockout mice. Eur. J.
Neurosci. 2010, 32, 1921−1929.
8927
dx.doi.org/10.1021/jm500989n | J. Med. Chem. 2014, 57, 8903−8927