Gabapentin hybrid peptides and bioconjugates

Article abstract of DOI:10.1016/j.bmc.2013.12.017

Synthetic approaches to gabapentin bioconjugates that overcome the tendency of gabapentin to cyclize into its γ-lactam are studied. Gabapentin was converted by N-acylation at its N-terminus into di-, tri-, and tetrapeptides (L-Ala-Gbp, L-Val-Gbp, L-Ala-L-Phe-Gbp, Gly-L-Ala-β-Ala-Gbp). Carboxyl-activated Boc-protected gabapentin was used to N-, O-, and S-acylate small peptides and hormones to give conjugates that could also provide prodrugs containing conformationally constrained gabapentin units.

Full text of DOI:10.1016/j.bmc.2013.12.017

Bioorganic & Medicinal Chemistry 22 (2014) 1479–1486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Gabapentin hybrid peptides and bioconjugates
Iryna O. Lebedyeva a, David A. Ostrov b, John Neubert c, Peter J. Steel d, Kunal Patel a, Sean M. Sileno a,
Kevin Goncalves a, Mohamed A. Ibrahim a, Khalid A. Alamry e, Alan R. Katritzky a,e,
⇑
a Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
b Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32611, USA
c Department of Orthodontics, College of Dentistry, University of Florida, Gainesville, FL 32610, USA
d Chemistry Department, University of Canterbury, Christchurch 8140, New Zealand
e Chemistry Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic approaches to gabapentin bioconjugates that overcome the tendency of gabapentin to cyclize
into its -lactam are studied. Gabapentin was converted by N-acylation at its N-terminus into di-, tri-,
and tetrapeptides -Ala-Gbp, -Val-Gbp, -Ala- -Phe-Gbp, Gly- -Ala-b-Ala-Gbp). Carboxyl-activated
Boc-protected gabapentin was used to N-, O-, and S-acylate small peptides and hormones to give conju-
gates that could also provide prodrugs containing conformationally constrained gabapentin units.
Ó 2014 Published by Elsevier Ltd.
Received 11 October 2013
Revised 27 November 2013
Accepted 6 December 2013
Available online 12 December 2013
c
(
L
L
L
L
L
Keywords:
Gabapentin
Peptide
Cyclization
Benzotriazole
Coupling
1. Introduction
co-crystals with various carboxylic acids,11,34 and (v) the formation
of N-terminus bioconjugates of gabapentin, for example, with
Gabapentin 1-(aminomethyl)cyclohexane acetic acid, Gbp 1,
(trade name Neurontin), a structural analog of -aminobutyric acid
bile29,35 acid or gabapentin enacarbil
3 (Fig. 1) (Horizant,
c
XP-13512).36,37
(GABA), is utilized for the treatment of epilepsy,1 neuropathic
pain,2–4 restless legs syndrome,5 anxiety disorders,6 hot flushes7
and numerous other conditions.8,9 Anhydrous gabapentin exists
in three polymorphic forms.10,11 The mechanism of action of gaba-
pentin remains elusive.12 Apparently, it does not directly interact
with GABA receptors.13–15 The absorption of gabapentin and clear-
ance of the drug depends on the dosage and the patient.
The preparation of the Gbp bioconjugate 3 indicated that chem-
ical instability, insufficient oral absorption, rapid pre-systemic
metabolism, and toxicity of Gbp could be overcome by formulation
of Gbp prodrugs.38 The development of gabapentin peptides or
acylated at N-, O-, S-termini derivatives that could transform
in vivo to release the active drug could thus be a feasible strategy
to improve the physicochemical, biopharmaceutical and/or phar-
macokinetic, and pharmacological properties of Gbp, and thereby
increase its usefulness.39
Gabapentin 1 easily undergoes facile intramolecular cyclization
to form the five-membered cyclic lactam 2-aza-spiro[4,5]decan-3-
one 2a (Scheme 1). While gabapentin has low toxicity (DL50, tested
in mice) at more than 8000 mg/kg, lactam 2a has a much higher
O
toxicity rating of 300 mg/kg.16 The presence of
c-lactam 2a as an
H2N
NH
impurity or the potential formation of 2a during storage in any ac-
tive ingredient must therefore be minimized both in solution and
solid states.16,17
OH
O
To improve Gbp bioavailability, much attention has been given
to: (i) Gbp conformational properties in peptide sequences,18–25 (ii)
Gbp conjugated systems,26–30 (iii) intermolecular interactions of
Gbp,31 its hydrates,32 and derivatives,33 (iv) Gbp multicomponent
2a
1
Scheme 1. Intramolecular cyclization of gabapentin (Gbp) into 2-aza-
spiro[4,5]decan-3-one 2a.
⇑
Corresponding author. Tel.: +1 352 392 0554; fax: +1 352 392 9199.
E-mail address: katritzky@chem.ufl.edu (A.R. Katritzky).
0968-0896/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.12.017

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I. O. Lebedyeva et al. / Bioorg. Med. Chem. 22 (2014) 1479–1486
Prodrugs by extension at the -terminus
A
N
O
O
O
O
O
O
N
H
O
3
O
O
HN
OH
NH
OH
3
4
Gabapentin enacarbil
O
Gabapentin-Memantine N-terminus conjugate
O
N
H
HO
O
H
HO
O
OR
O
H
N
H
N
H
H
OR
H
H
O
H
H
H
OH
R = H; CH3
R = H; CH3
HO
OH
6
5
5-6 Gbp and GbpOMe chenodeoxycholic acid conjugates that target hASBT
(human apical sodium-dependent bile acid transporter)
B
Prodrugs of Gbp by extension at the C-terminus
O
O
O
O
HClH2N
NH2HCl
H2N
H2N
O
O
O
O
O
Alk
O
n
HN
n = 2, 3, 4
Gabapentin-Memantine C-terminus conjugates
7
7-8
8
9
Gabapentin mutual prodrug to relieve neuropathic pain
Figure 1. Reported Gbp bioconjugates.
Gabapentin enacarbil (3) was designed to improve physico-
chemical, biopharmaceutical and pharmacokinetic properties of
the pharmacologically active gabapentin molecule.40 The oral bio-
availability of gabapentin was increased from 25% to 84% by use of
the gabapentin enacarbil prodrug in monkeys, which showed dose-
proportional gabapentin exposure in humans.41 With these prom-
ising results in mind, we developed gabapentin conjugates that
might increase bioavailability of the drug in similar ways to the
gabapentin enacarbil prodrug 3. An additional search revealed re-
ports on both C- and N-terminus gabapentin conjugated systems
(4–9, Fig. 1). In the development of bile acid prodrugs of Gbp the
authors’ strategy was to couple the Gbp drug to a natural substrate
to create a molecule that mimics the three-dimensional structure
of natural human (apical) sodium-dependent bile acid trans-
porter.29 Thus, the synthesis of glu-chenodeoxycholic acid and che-
nodeoxycholic acid Gbp and its methyl ester conjugates 5 and 6
helped overcome the limitations that gabapentin faces in its zwit-
terionic form and improve gabapentin oral bioavailability. For the
purpose of treating neurological diseases, novel Gbp-adamantine
(e.g., memantine) combinatorial N-terminus (4) and C-terminus
(7 and 8) compositions were reported.30 Gabapentin hydrochloride
dimeric esters linked via a short C2–C6 aliphatic chain 9 were also
synthesized and evaluated as mutual prodrugs to relieve neuro-
pathic pain.26
2. Results and discussion
We now report (i) benzotriazole-mediated acylations of Gbp 1
with amino acids, di- and tri-peptides; together with (ii) a series
of N-protected carbonyl-activated gabapentin conjugates with
biologically important S-, O-, N-nucleophiles.
2.1. Synthesis of Gbp-conjugated at the N-terminus with amino
acids and peptides
Our first strategy to stabilize conformationally constrained
Gabapentin 1 involved the construction of small
a-,c-peptidomi-
metics by N-acylation of 1 with carbonyl activated N-protected
amino acids and small peptides. Benzotriazolyl precursors were
constructed by solution-phase step-wise coupling of intermediate
benzotriazolides with unprotected a
-and b-amino acids.45–47 This
method allowed us to build up two- and three-amino acid units
that were further activated at the C-terminus carbonyl group as
benzotriazolides. For the synthesis of 14a,b, only one CO-activation
step was required. The conjugation of benzotriazolyl amino acids
and peptides with Gbp employed known methodology (MeCN,
1.5 equiv TEA, 6 h, rt)47 to give compounds 14a–d (77–86%);
formation of 2 was not detected. Gabapentin 1 was acylated
with COBt-activated N-Cbz protected amino acid segments
The limited literature describing gabapentin N- and C-terminus
bioconjugates is a testimony to the ease of intramolecular cycliza-
(Cbz-
dipeptide (Boc-
(Cbz-Gly- -Ala-b-Ala-OH) (83%) (Scheme 2). N-Cbz-protected
aminoacids -alanine and -valine used for acylation of 1 at its
N-terminus are structurally similar to gabapentin enacarbil 3. For
the same reason—Cbz-Gly- -Ala-b-Ala-Bt 12 was employed as
acylation reagent. Such substituents should not restrict the
L
-Val-OH, Cbz-
L
-Ala-OH) to form 14a (77%), 14b (86%) N-Boc
L-Ala-
L-Phe-OH) 14c (81%), and 14d N-Cbz-tripeptide
tion of 1 to form c
-lactam 2a10 (also encountered during our work
L
with gabapentin 1, see later). Coupling Gbp 1 at its N-terminus
with other amino42,33,22,20 or bile acids was reported using DCC/
HOBt reagents and N-Boc or N-Cbz protection.43 Moderate yields
(50–73%) were reported with DCC/HOSu in THF.44
L
L
L

I. O. Lebedyeva et al. / Bioorg. Med. Chem. 22 (2014) 1479–1486
1481
O
O
Cbz-L-Ala-Bt
Cbz-L-Ala-HN
Cbz-L-Val-HN
L-Ala-HN
L-Val-HN
ii
ii
OH
O
10
OH
Cbz-L-Val-Bt
14a
O
15a
11
O
OH
OH
H2N
OH
i
14b
O
15b
O
Boc-L-Ala-L-Phe-Bt
12
1
iii
Boc-L-Ala-L-Phe-NH
HCl*DL-Ala-DL-Phe-NH
OH
OH
Cbz-Gly-L-Ala- -Ala-Bt
β
13
15c
15d
14c
14d
O
O
ii
Cbz-Gly-L-Ala-β-Ala-NH
Gly-L-Ala-β-Ala-NH
i Et3N, MeCN/H2O rt, 16 h
ii 10 wt % Pd/C, H2, MeOH, rt, 8 h
iii MeOH/HCl, rt, 2 h
OH
OH
Scheme 2. Synthesis of conformationally restricted Gbp-terminus peptidomimetics 15a–d.
rotation of
c
-amino acid 1 while providing backbone substitution
Pg
O
O
Pg
HN
O
N
to 1 analogous to gabapentin prodrug 3. Boc-
was employed to study the peculiarities of Boc-deprotection for
gabapentin N-terminus and to see if the bulky phenyl group of
L
-Ala- -Phe-Bt 12
L
HN
Nu
16b
OH BtH, Im, Nu
16a,c
L-Phe-OH would change the structural properties of gabapentin
16a: Pg = Cbz
2b
: Pg = Cbz
derivatives 14c and 15c.
2a
b
: Pg = Phth
c: Pg = Boc
c: Pg = Boc
Deprotection of both Cbz and Boc groups did not induce the
intramolecular cyclization of products 15a–d. An HPLC/MS study
of 15a confirmed the stability of the deprotected product revealing
98.6% of the positively charged ion ((+)ESI-MS [M+1]+ 243.1 m/z)
for 15a three months after deprotection and storage at room tem-
perature which confirms the long shelf-life of products 15a–d.
Deprotection of the Boc group of 15c with saturated HCl/MeOH,
rt, 2 h caused racemization of the peptide unit of 15c, although
no racemization was observed for Cbz group deprotection of
15a,b, or 15d (10% wt Pd/C, H2, MeOH, rt, 8 h). Racemization during
Boc deprotection of 15c is probably being due to high polarity of
the MeOH solvent.
Scheme 3. Intramolecular cyclization of N-protected gabapentin into lactams upon
reaction with nucleophiles (16b ? 2a) and during the CO activation with benzo-
triazole (BtH) or imidazole (Im) (16a,c ? 2b,c).
attempts to activate the carboxy group of 16c (Pg = Boc) as well as
the carboxy groups of 16a,b (Pg = Cbz, Phth) by conversion into
benzotriazolide (BtH) or imidazolide (Im) failed: we invariably found
30% to 70% of N-protected intramolecular cyclization products 2b,c.
Figure 2 shows the single crystal X-ray structure for the N-Boc
cyclized tert-butyl 3-oxo-2-azaspiro[4.5]decane-2-carboxylate 2c
Scheme 3.
Although CO-activation step for the N-protected gabapentin
induced intramolecular cyclization, standard N,N0-dicyclohexylcar-
bodiimide (DCC)50 and isobutyl chloroformate couplings for
N-protected Gbp (16c) with N-, O-, S-nucleophiles allowed us to
isolate a number of Boc-N-Gbp conjugates.
The backbone substitution in the hybrid peptides 15a–d re-
stricts rotation, sets the conformational preferences of the achiral
b-disubstituted c-amino residue of Gbp and stabilizes conforma-
tionally-constrained
c-aminoacid Gbp 1 at the C-terminus of the
small peptide chain.
2.2. Preparation of N-, O-, and S-bioconjugates linked to the
C-terminus of gabapentin
2.2.1. Gabapentin C-terminus couplings with N-nucleophiles
To synthesize 17a–c we tried coupling methods such as DCC,
DCC/HOBt and isobutyl chloroformate/N-methylmorpholine. The
use of isobutyl chloroformate (i) allowed a high yield preparation
of Gbp conjugates 17a–c with no racemization or intramolecular
cyclization into 2. The yield of 17c was higher (95%) using cou-
pling method i (Scheme 4) compared to the DCC method (81%).
DCC may have caused minor intramolecular cyclization in parallel
with nucleophilic conjugation but lactam 2c was not detected.
We first examined protection of the amino group of Gbp 1. Cbz
N-protected Gbp 16a48 is conformationally constrained and exists
as two rotamers as shown by the doubling of signals in both 1H and
13C spectra (SI). N-protection of Gbp with phthalimide (Phth) 16b
failed in the next step when 16b was coupled with nucleophilic
reagents. Thus lactam 2a was isolated as the major product when
16b was reacted with benzylamine. Boc-protected Gbp 16c was free
of the disadvantages16 found with 16a and 16b. Interestingly, no
rotamers were observed for 16b and 16c. We explain this as follows:
the phenyl ring of the Cbz-group limits rotation in 16a as confirmed
by NMR data whereas phthalimide or Boc-protection does not
sterically constrain rotation of the Gabapentin moiety in 16b,c. All
Thus treatment of N-Boc-protected Gbp 16c with
ester using isobutyl chloroformate gave 17a (91%). Analogous
coupling (i, Scheme 4) with -Phe- -Trp methyl ester gave 17b
L-valine methyl
L
L
(89%) and coupling of 16c with benzylamine gave acylated 17c
(95%).

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I. O. Lebedyeva et al. / Bioorg. Med. Chem. 22 (2014) 1479–1486
2.2.4. Stability upon storage and deprotection for gabapentin
conjugates
N-protected and deprotected C-terminus gabapentin di-, tri-
and tetrapeptides 14a–d and 15a–d proved to be stable over a
period of 12 months with no traces of intramolecular cyclization
or decomposition of the gabapentin unit. Boc-N-gabapentin-L-
menthol conjugate 19a cyclized into 2c over 12 months during
storage at room temperature. When deprotecting the Boc group
from N-protected gabapentin conjugates, we managed to get
hydrochloride salts for Gbp conjugates with N-nucleophiles
18a–c, whereas deprotection of the Boc group under different
conditions (HCl/MeOH or HCl/1,4-dioxane, TFA/DCM, rt, ꢀ4 °C)
for Gbp O- and S-conjugates 19a,b, 20a,b led to decomposition of
the conjugate releasing gabapentin 1, O-, or S-nucleophile and
cyclized 2-aza-spiro[4,5]decan-3-one 2a. Therefore, N- and
C-termini Gbp conjugates with N-nucleophiles are stable under
deprotection conditions and during long-term storage.
Figure 2. Crystal structure of tert-butyl 3-oxo-2-azaspiro[4.5]decane-2-carboxylate
2c.49
Deprotection of 17a–c with HCl saturated MeOH solution gave
stable C-terminus gabapentin bioconjugates as hydrochlorides
18a–c (Scheme 4). Boc-N-deprotection of gabapentin amides
18a–c resulted in almost quantitative yields (98% for 18a, 93% for
18b, and 92% for 18c) with no formation of hydrolized or cyclized
byproducts.
2.3. Biotesting of general toxicity and pain relieving properties
for gabapentin 1 and its conjugates 15a and 18c in vivo
2.2.2. Gabapentin C-terminus couplings with O-nucleophiles
We also employed L-menthol to give 19a (91%) and cholesterol
to give 19b (80%) by isobutyl chloroformate-mediated reactions to
give optically pure products with no detectable amount of 2c. In
this way we were able to introduce two bioactive moieties into
the N-protected gabapentin through O-acylation reactions
(Scheme 5).
All the animals behaved normally following injection of com-
pounds 1, 15a, 18c and or vehicle solutions (65% EtOH, d-H2O),
and none of the animals died following the administration of the
tested compounds. There was no significant effect of treatment
on reward licking events for compounds 1, 15a, 18c as compared
to the vehicle (65% EtOH) control group (1 way ANOVA,
F3,26 = 1.18; P = 0.34). A subgroup of animals was retested using
conjugate 15a (d-H2O vehicle) and was subjected to capsaicin-in-
duced inflammatory pain. There was no significant change in lick-
ing events for the animals treated with compound 15a on the first
session compared to their d-H2O treatment session (paired t-test,
P = 0.105). This indicates that these potential analgesics did not
significantly affect the animal from completing the operant task
and showed low toxicity in animal models. Further studies for
the pain relieving properties of conjugates 15a and 18c are cur-
rently being conducted.
2.2.3. Gabapentin C-terminus couplings with S-nucleophiles
The diversity of Gbp conjugates was enhanced by the inclusion
of S-nucleophiles. Thiophenol and cysteinyl
L-phenylalanine
methyl ester reacted with N-Boc Gbp 12c using isobutyl chlorofor-
mate (i) to afford 20a in 97% and 20b in 84% yields (Scheme 6).
During purification of 20b we also isolated cyclized byproduct 2c
(7% yield). Thus coupling of N-protected Gbp with S-nucleophilic
reagents offers another method to prevent Gbp intramolecular
cyclization.
Table 1
There were no severe adverse events noted up to 3 h following
injection of the compounds or the vehicles. Reward licking was not
significantly changed following active compound administration as
compared to vehicle treatment Table 1.
Effects of compounds 1, 15a, 18c on general behaviour and operant reward licking
events
Treatment
Mortality Reward licking events
(avg sem)
Conjugate 15a (65% EtOH)
Conjugate 18c (65% EtOH)
Gabapentin 1 (65% EtOH)
Vehicle (65% EtOH)
0/7
0/8
0/7
0/7
0/7
425 198
153 95
220 84
121 87
416 170
3. Experimental
3.1. Materials and methods
Conjugate 15a (d-
H2O) + capsaicin
Vehicle (d-H2O) + capsaicin
Melting points were determined on a capillary point apparatus
equipped with a digital thermometer and are uncorrected. 1H NMR
0/7
617 157
NH
O
NH
O
O
O
O
O
Boc
HN
O
O
O
O
O
O
i
ii
ii
i
O
O
HClH2
N
Boc-NH
NH
OH
+ N-Nu
O
N
NH
N
H
H
Boc-HN
HClH2N
N
N
H
H
16c
18a
17a
i
17b
HClH2N
18b
O
O
Boc-NH
N
H
N
i = N-methylmorpholine,
ClCO2CH2CH(CH3)2, THF, rt, 24 h
ii = MeOH/HCl, rt, 2 h
ii
H
18c
17c
Scheme 4. Synthesis of C-terminus gabapentin conjugates bearing N-nucleophilic moiety.

I. O. Lebedyeva et al. / Bioorg. Med. Chem. 22 (2014) 1479–1486
1483
Boc
HN
O
O
Boc
HN
O
O
i
i
16c
+ O-Nu
H
H
H
H
i = N-methylmorpholine,
isobutyl chloroformate, N2, rt, 24 h
19a
19b
Scheme 5. Synthesis of C-terminus gabapentin conjugates bearing an O-nucleophilic moiety.
Boc
O
O
S
Boc
O
S
Boc
HN
N
N
H
O
O
i
i
16c + S-Nu
and
HN
i = N-methylmorpholine
isobutyl chloroformate, N , rt, 24 h
O
2
2c
7%
20a
Cbz
HN
20b
84%
Scheme 6. Synthesis of C-terminus gabapentin conjugates bearing an S-nucleophilic moiety.
spectra were recorded at 300 MHz and 13C NMR spectra were re-
corded at 75 MHz on Gemini or Varian spectrometers at room tem-
perature. Chemical shifts are reported in ppm relative to TMS as
with standard food chow at the completion of testing. Mice were
tested at the same time of the day and weighed weekly to monitor
health. Water was also made available ad libitum before and after
testing sessions. Animals were anesthetized with isoflurane (2–3%,
internal standard (1H NMR) or to the residual solvent peak (13
C
NMR). The following abbreviations are used to describe spin mul-
tiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multi-
plet, br s = broad singlet, dd = doublet of doublets. Elemental
analysis was performed on a Carlo Erba-1106 instrument. High
Resolution Mass Spectra were recorded using Thermo Scientific
LCQ Ion Trap. Column chromatography was performed on silica
gel (230–400 mesh). HPLC analysis was performed on Shimadzu li-
quid chromatographic system equipped with UV-detector. Peaks
were detected at 254 nm using Phenomenex Luna C18 reversed-
phase column (250 ꢁ 4.6 mm id) 5 microns. HPLC–MS analyses
were performed on reverse phase gradient using 0.2% acetic acid
inhalation) and were injected (50 ll; ip) with gabapentin 1 and
analogs 15a, 18c or vehicle (65% EtOH or d-H2O) and then tested
30 min post-injection. Animal testing procedures and general han-
dling complied with the ethical guidelines and standards estab-
lished by the Institutional Animal Care & Use Committee at the
University of Florida and all procedures complied with the Guide
for Care and Use of Laboratory Animals.55
3.3. General procedure for the synthesis of 14a–d
To a solution of acyl benzotriazoles 10–13 (2 mmol) in MeCN
(20 mL) a solution of 2-(1-(aminomethyl)cyclohexyl)acetic acid 1
(0.342 g, 2.0 mmol) in MeCN/H2O (4:1) was added slowly followed
by the addition of Et3N (2.5 mmol, 0.41 mL). The mixture was stir-
red for 16 h, then MeCN evaporated; the crude mixture was ex-
tracted with EtOAc (30 mL) washed with 2 N HCl (3 ꢁ 25 mL),
dried over MgSO4 and EtOAc evaporated under reduced pressure
to give 14a–d.
in H2O/methanol as mobile phases; k = 254 nm. L-Amino acids
were purchased from ChemImpex Inc. All commercially available
substrates were used as received without further purification.
Crystallographic data were collected at 120(2) K on an Agilent
Supernova instrument using Cu K
a radiation, k = 1.54184 Å. The
structure of 2c was solved using direct methods with SHELXS
and refined on F2 using all data by full matrix least-squares proce-
dures with SHELXL.51 All non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were in-
cluded in calculated positions with isotropic displacement param-
eters 1.2 times the isotropic equivalent of their carrier carbon
atoms. CCDC-948819 contains the Supplementary crystallographic
data for this paper. These data are presented in the Supporting
information or can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif. Synthesis of Boc-, Cbz- and phthalimide-protected gaba-
pentin, di-, tripeptide building blocks and CO-activated amino
acids is described in the Supplementary information (SI) section.
(S)-2-(1-((2-(((Benzyloxy)carbonyl)amino)propanami-
do)methyl)cyclohexyl)acetic acid 14a: White solid (77%, 0.579 g,
1.54 mmol). Mp 124.2–125.3 °C; 1H NMR (300 MHz, CDCl3, d):
7.37–7.26 (m, 5H), 7.03 (br s, 1H), 5.74 (br s, 1H), 5.10 (s, 2H),
4.30 (br s, 1H), 3.36–3.23 (m, 2H), 2.25 (s, 2H), 1.48–1.28 (m,
14H); 13C NMR (75 MHz, CD3OD, d): 176.2, 175.9, 158.3, 138.2,
129.6, 129.1, 129.0, 67.8, 52.5, 46.9, 41.9, 38.7, 35.0, 27.2, 22.7,
18.3. Anal. Calcd for C20H28N2O5: C, 63.81; H, 7.50; N, 7.44. Found:
C, 63.49; H, 7.66, N, 7.37.
(S)-2-(1-((2-(((Benzyloxy)carbonyl)amino)-3-methylbutanami-
do)methyl)cyclohexyl)acetic acid 14b: White solid (86%, 0.606 g,
1.17 mmol). Mp 121.4–123.0 °C; 1H NMR (300 MHz, CD3OD, d):
7.37–7.30 (m, 5H), 5.12 (d, J = 12.3 Hz, 1H), 5.05 (d, J = 12.6 Hz,
1H), 3.92 (d, J = 7.2 Hz, 1H), 3.34 (s, 2H), 2.30–2.00 (m, 3H), 1.58–
1.38 (m, 10H), 0.96 (t, J = 6.6 Hz, 3H), 0.94 (d, J = 6.0 Hz, 3H); 13C
NMR (75 MHz, CD3OD, d): 175.9, 174.9, 158.8, 138.3, 129.6,
129.1, 129.0, 67.8, 62.7, 46.9, 41.9, 38.5, 34.9, 31.6, 27.2, 24.2,
22.7, 20.0, 18.6. Anal. Calcd for C22H32N2O5: C, 65.32; H, 7.97; N,
6.93. Found: C, 65.49; H, 8.29, N, 6.85.
3.2. In vivo mouse models of pain relief
Hairless SKH1-Hrhr mice (male, 25–30 g, Charles River) were
tested using an operant pain system as described previously.52,53
For the assessment of general behaviour, we compared the effects
on reward licking number following administration of drugs or
vehicle. For a subset of animals, capsaicin cream (0.075%) was
placed on the face prior to drug administration and testing to pro-
duce a pain state, as described previously.54 All mice were food
fasted for 15 h prior to each facial testing session and provided
2-(1-((4S,7S)-4-Benzyl-7,11,11-trimethyl-3,6,9-trioxo-10-oxa-
2,5,8-triazadodecyl)cyclohexyl) acetic acid 14c: White solid (81%,
0.793 g, 1.62 mmol). Mp 69.8–70.9 °C; 1H NMR (300 MHz, CD3OD,

1484
I. O. Lebedyeva et al. / Bioorg. Med. Chem. 22 (2014) 1479–1486
d): 7.86 (br s, 1H), 7.52–7.47 (m, 1H), 7.30–7.15 (m, 5H), 4.59 (t,
J = 7.2 Hz, 1H), 3.97 (q, J = 7.2 Hz, 1H), 3.35–3.29 (m, 2H), 3.16–
2.97 (m, 2H), 2.11 (s, 2H), 1.45–1.16 (m, 22H); 13C NMR (75 MHz,
CD3OD, d): 175.9, 173.8, 157.9, 138.3, 130.5, 130.0, 129.1, 128.0,
80.9, 56.2, 52.0, 47.3, 41.5, 38.8, 38.3, 34.6, 30.9, 28.9, 27.1, 22.7,
18.3. HRMS (-ESI) calcd for C26H38N3O6 [MꢀH]ꢀ 488.2766, found
488.2789.
2-(1-(8-Methyl-3,6,9,13-tetraoxo-1-phenyl-2-oxa-4,7,10,14-tetra-
azapentadecan-15-yl)cyclohexyl)acetic acid 14d: White solid (83%,
0.858 g, 0.17 mmol). Mp 81.8–83.6 °C; 1H NMR (300 MHz, CD3OD,
d): 7.41–7.31 (m, 5H), 5.20–4.92 (m, 4H), 4.20–4.08 (m, 1H), 3.95–
3.80 (m, 2H), 3.55–3.40 (m, 2H), 2.58–2.43 (m, 2H), 2.31 (s, 2H),
1.60–1.30 (m, 13H); 13C NMR (75 MHz, CD3OD, d): 176.8, 176.3,
174.1, 171.7, 158.7, 138.2, 129.6, 129.1, 128.9, 128.9, 67.9, 67.7,
55.8, 52.6, 47.2, 43.7, 42.8, 38.1, 37.4, 36.9, 36.7, 35.5, 34.9, 27.2,
17.9. Anal. Calcd for C25H36N4O7: C, 59.51; H, 7.19; N, 11.10 Found:
C, 59.58; H, 7.01; N, 10.93.
magnesium sulfate and solvent was removed to give the desired
product.
(S)-Methyl 2-(2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclo-
hexyl)acetamido)-3-methylbutanoate 17a: Yellow oil (91%, 0.649 g,
1.69 mmol). 1H NMR (300 MHz, CD3OD, d): 4.26 (d, J = 5.7 Hz,
1H), 3.67 (s, 3H), 3.23 (d, J = 15.0 Hz, 1H), 3.00 (d, J = 14.7 Hz,
1H), 2.22–2.10 (m, 3H), 1.48–1.20 (m, 19H), 0.96 (d, J = 6.9 Hz,
3H), 0.95 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CD3OD, d): 174.3,
173.8, 159.4, 80.3, 59.6, 52.5, 47.9, 43.0, 38.8, 35.4, 34.8, 31.4,
31.1, 28.9, 27.3, 22.7, 19.8, 18.6. Anal. Calcd for C20H36N2O5: C,
62.47; H, 9.44; N, 7.29. Found: C, 62.45; H, 9.69; N, 6.99.
(S)-Methyl 2-((S)-2-(2-(1-(((tert-butoxycarbonyl)amino)methyl)
cyclohexyl)acetamido)-3-phenylpropanamido)-3-(1H-indol-3-yl)pro-
panoate 17b: White solid (89%, 1.01 g, 1.64 mmol). Mp 89.1–
90.4 °C; 1H NMR (300 MHz, CDCl3, d): 8.67 (br s, 1H), 7.48–6.85
(m, 12H), 5.11 (d, J = 4.8 Hz, 1H), 4.83–4.64 (m, 2H), 3.53 (s, 3H),
3.17 (s, 2H), 3.05(d, J = 10.2 Hz, 1H), 2.90–2.53 (m, 2H), 1.75 (br s,
2H), 1.37 (s, 9H), 1.28–1.10 (m, 7H), 0.95 (br s, 3H), 0.74 (br s,
1H); 13C NMR (75 MHz, CD3OD, d): 172.1, 171.9, 171.4, 157.1,
137.0, 136.3, 129.4, 128.5, 127.5, 126.9, 123.6, 122.0, 119.4,
118.4, 111.6, 109.4, 79.6, 54.5, 53.1, 52.5, 46.6, 42.7, 37.4, 34.2,
33.8, 29.4, 28.6, 27.7, 26.0, 21.5, 19.1. HRMS (ESI) calcd for C35H46-
N4O6Na [M+Na]+ 641.3310, found 641.3339.
3.4. General procedure for the synthesis of 15a,b,d
Products 14a,b,d (0.10 g, 0.26 mmol for 14a, 0.12 g, 0.3 mmol
for 14b, and 0.08 g, 0.16 mmol for 14d) were dissolved in MeOH
(20 mL), and 10% wt Pd/C (20 mg) was added to the solution. The
mixture was kept under positive pressure of hydrogen for 8 h at
rt. The suspension was filtered through celite and evaporated to
give 15a,b,d.
tert-Butyl
((1-(2-(benzylamino)-2-oxoethyl)cyclohexyl)methyl)
carbamate 17c: Yellow oil (95%, 0.613 g, 1.74 mmol). 1H NMR
(300 MHz, CD3OD, d): 7.31–7.29 (m, 5H), 4.35 (s, 2H), 3.11 (s,
2H), 2.18 (s, 2H), 1.53–1.33 (m, 19H); 13C NMR (75 MHz, CD3OD,
d): 173.8, 159.0, 140.1, 129.6, 128.8, 128.3, 80.1, 48.1, 44.2, 44.0,
38.7, 35.0, 28.9, 27.3, 22.7. Anal. Calcd for C21H32N2O3: C, 69.97;
H, 8.95; N, 7.77. Found: C, 69.99; H, 9.01; N, 7.48.
(S)-2-(1-((2-Aminopropanamido)methyl)cyclohexyl)acetic
acid
15a: White solid (100%, 0.064 g, 0.026 mmol). Mp 89.1–90.2 °C;
1H NMR (300 MHz, CD3OD, d): 3.90 (q, J = 6.9 Hz, 1H), 3.38 (d,
J = 13.5 Hz, 1H), 3.24 (d, J = 13.5 Hz, 1H), 2.24 (s, 2H), 1.57–1.35
(m, 13H); 13C NMR (75 MHz, CD3OD, d): 180.1, 171.7, 50.7, 47.1,
37.8, 35.6, 35.5, 27.4, 22.9, 18.4. LC/MS m/z: 243.1 [M+H]+; HRMS
(1S,2R,5S)-2-Isopropyl-5-methylcyclohexyl 2-(1-(((tert-butoxycar-
bonyl)amino)methyl) cyclohexyl) acetate 19a: Colorless oil (91%,
0.649 g, 1.69 mmol). 1H NMR (300 MHz, CDCl3, d): 3.34 (s, 2H),
2.29–2.19 (m, 1H), 2.52 (br s, 1H), 2.22 (s, 2H), 2.12–2.01 (m,
1H), 1.86–1.77 (m, 1H), 1.55–1.20 (m, 20H), 1.02–0.72 (m, 12H),
0.66 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3, d): 173.5, 150.2,
82.6, 71.1, 57.2, 49.9, 45.3, 44.9, 36.1, 34.7, 34.5, 31.6, 28.3, 27.9,
25.6, 25.4, 23.0, 22.5, 22.2, 21.3, 21.0, 16.0. Anal. Calcd for
(-ESI) calcd for
372.1169.
C
12H20N2O3Na [Mꢀ2H+Na]ꢀ 372.1177 found
(S)-2-(1-((2-Amino-3-methylbutanamido)methyl)cyclohexyl)ace-
tic acid 15b: White solid (97%, 0.079 g, 0.29 mmol). Mp 119.5–
120.6 °C; 1H NMR (300 MHz, CD3OD, d): 3.56 (d, J = 5.1 Hz, 1H),
3.43–3.21 (m, 2H), 2.26 (s, 2H), 2.19–2.12 (m, 1H), 1.60–1.28 (m,
10H), 1.04 (t, J = 5.9 Hz, 6H); 13C NMR (75 MHz, CD3OD, d): 180.0,
170.8, 60.7, 47.5, 37.5, 35.8, 35.5, 32.0, 27.4, 22.9, 19.3, 18.2. Anal.
Calcd for C14H26N2O3: C, 62.19; H, 9.69; N, 10.36; Found: C, 62.28;
H, 10.00; N, 10.11.
(S)-2-(1-((3-(2-(2-Aminoacetamido)propanamido)propanami-
do)methyl)cyclohexyl)acetic acid 15d: White solid (100%, 0.059 g,
0.016 mmol). Mp 204.0–205.2 °C; 1H NMR (300 MHz, CD3OD, d):
3.94–3.63 (m, 4H), 3.43 (t, J = 6.3 Hz, 2H), 3.35–3.16 (m, 2H), 2.40
(t, J = 6.3 Hz, 2H), 2.17 (s, 2H), 1.45–1.27 (m, 15H); 13C NMR
(75 MHz, CD3OD, d): 180.6, 173.7, 171.4, 55.8, 50.7, 48.1, 47.4,
43.7, 37.7, 37.4, 37.0, 35.6, 27.4, 22.9, 18.5, 18.2. HRMS (ESI) calcd
for C17H30N4O5Na [M+Na]+ 393.2108, found 393.2116.
C
24H43NO4: C, 70.38; H, 10.58; N, 3.42. Found: C, 70.59; H, 10.60,
N, 3.12.
10,13-Dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,
14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-(1-
(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)acetate 19b: White
solid (80%, 0.94 g, 1.47 mmol). Mp 116.1–117.4 °C; 1H NMR
(300 MHz, CDCl3, d): 5.32 (br s, 1H), 3.44 (s, 2H), 2.33 (s, 2H),
2.29–2.19 (m, 2H), 1.96–1.90 (m, 2H), 1.82–1.70 (m, 4H), 1.50–
0.94 (m, 49H), 0.87 (d, J = 6.3 Hz, 2H), 0.82 (d, J = 6.3 Hz, 2H), 0.64
(s, 3H); 13C NMR (75 MHz, CDCl3, d): 173.8, 150.5, 140.9, 121.8,
82.9, 71.9, 57.5, 56.9, 56.3, 50.3, 45.6, 42.5, 39.9, 39.7, 37.4, 36.7,
36.5, 36.4, 36.0, 35.0, 34.8, 32.8, 32.1, 31.8, 31.2, 28.6, 28.4, 28.2,
26.2, 25.7, 25.6, 25.0, 24.5, 24.0, 23.0, 22.8, 21.8, 21.3, 19.6, 18.9,
12.0. Anal. Calcd for C41H69NO4: C, 76.94; H, 10.87; N, 2.19. Found:
C, 76.79; H, 10.78, N, 2.49.
3.5. General procedure for the synthesis of conjugates 17a–c,
19a,b, 20a,b
S-Phenyl
2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclo-
hexyl)ethanethioate 20a: Yellow oil (97%, 0.654 g, 1.80 mmol). 1H
NMR (300 MHz, CDCl3, d): 7.47–7.17 (m, 5H), 4.99 (t, J = 6.6 Hz,
1H), 3.12 (d, J = 6.9 Hz, 2H), 2.59 (s, 2H), 1.60–1.38 (m, 19H); 13C
NMR (75 MHz, CDCl3, d): 197.1, 156.4, 134.4, 129.6, 129.3, 129.1,
127.9, 127.5, 127.2, 79.0, 49.7, 47.1, 39.3, 34.3, 28.5, 26.0, 21.6.
Anal. Calcd for C20H29NO3S: C, 66.08; H, 8.04; N, 3.85. Found: C,
66.32; H, 8.07, N, 3.93.
To
a solution of 2-(1-(((tert-butoxycarbonyl)amino)methyl)
cyclohexyl)acetic acid 16c (0.50 g, 1.84 mmol) in dry THF (30 mL)
N-methylmorpholine was added (0.20 mL, 1.84 mmol) and the mix-
ture was stirred at rt for 10 min under nitrogen. After cooling to 0 °C,
isobutyl chloroformate (0.18 mL, 1.84 mmol) was added dropwise.
N-Methylmorpholine (0.20 mL, 1.84 mmol) was then added in
4 min followed by the addition of nucleophile (1.84 mmol) then stir-
red for 24 h at rt under nitrogen. After the solvent was evaporated,
ethyl acetate (30 mL) was added to the crude and solution was
washed with 2 N HCl solution (2 ꢁ 50 mL) and saturated sodium car-
bonate solution (2 ꢁ 30 mL). The organic phase was dried over anhyd
Methyl N-(((benzyloxy)carbonyl)-
L-phenylalanyl)-S-(2-(1-(((tert-
butoxycarbonyl)amino) methyl)cyclohexyl)acetyl)-
L
-cysteinate 20b:
Colorless oil (84%, 1.08 g, 1.62 mmol). 1H NMR (300 MHz, CDCl3,
d): 7.32–7.02 (m, 11H), 6.94 (d, J = 7.5 Hz, 1H), 6.78 (d,
J = 7.2 Hz, 1H), 5.59 (d, J = 7.2 Hz, 1H), 5.32 (d, J = 7.2 Hz, 1H),

I. O. Lebedyeva et al. / Bioorg. Med. Chem. 22 (2014) 1479–1486
1485
4.99 (s, 2H), 4.98–4.82 (m, 1H), 4.79–4.62 (m, 1H), 4.52–4.34
Acknowledgments
(m, 1H), 3.88 (d, J = 6.6 Hz, 1H), 3.63 (s, 3H), 3.58–2.88 (m, 6H),
2.41 (s, 1H), 1.98–1.78 (m, 1H), 1.45–1.17 (m, 13H), 0.82 (dd,
J = 6.8, 1.4 Hz, 2H); 13C NMR (75 MHz, CDCl3, d): 197.7, 172.9,
171.3, 170.7, 170.2, 156.4, 156.0, 136.6, 136.4, 129.4, 128.5,
128.1, 128.0, 127.0, 126.9, 79.3, 66.9, 56.0, 52.8, 52.5, 52.1, 48.8,
48.2, 46.9, 38.8, 38.5, 34.1, 33.7, 30.8, 28.5, 25.9, 21.5, 18.2.
HRMS (ESI) calcd for C35H47N3O8SNa [M+Na]+ 692.2976, found
692.3003.
This work was supported by the Kenan Foundation (University
of Florida) and the King Abdulaziz University (Saudi Arabia) and
Science for Life HHMI Intramural Award. The authors are grateful
to Dr. C.D. Hall, Dr. Levan Khelashvili, Professor Said A. El-Feky,
Mr. Justin Juelich, Mr. Ashton Innocent, Mr. Bo Nalsen, Mrs. Galyna
Vakulenko, Mr. Khanh Ha, and Dr. Joshua Kramer for reading the
manuscript, Dr. Jodie Johnson for his help in HLPC–MS analyses,
and Dr. M.C.A. Dancel for her help with HRMS analyses.
3.6. N-Boc deprotection of conjugates 14c, 17a–c
Supplementary data
N-Boc protected peptide or bioconjugate 14c, 17a,b or c
(0.5 mmol) was stirred with HCl saturated MeOH (15 mL) for 2 h.
The solvent was evaporated, and the residue was recrystallized
from ethyl acetate/hexanes 1:4 to give the corresponding product
15c, 18a–c.
Supplementary data (experimental procedures, copies of 1H and
13C NMR for all the novel compounds and details of the X-ray
structure of 2c as a CIF) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.
017.
2-(1-(((S,R)-2-((S,R)-2-Aminopropanamido)-3-phenylpropanami-
do)methyl)cyclohexyl)acetic acid hydrochloride 15c: Light yellow oil
(96%, 0.205 g, 0.48 mmol). 1H NMR (300 MHz, CD3OD, d): 3.30–
7.20 (m, 5H), 4.70–4.64 (m, 1H), 3.94–3.83 (m, 1H), 3.67–3.62
(m, 2H), 3.25–2.82 (m, 4H), 2.28–2.11 (m, 2H), 1.61–1.18 (m,
13H); 13C NMR (75 MHz, CD3OD, d): 174.5, 170.9, 138.3, 130.4,
129.7, 129.6, 128.0, 56.8, 56.3, 52.1, 50.2, 50.0, 47.1, 41.2, 39.0,
38.5, 34.5, 28.8, 27.1, 22.7, 17.9. HRMS (-ESI) calcd for
References and notes
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21H30N3O4 [MꢀH]ꢀ 388.2242, found 388.2256.
Methyl (2-(1-(aminomethyl)cyclohexyl)acetyl)valinate hydrochlo-
ride 18a: Yellow oil (98%, 0.157 g, 0.049 mmol). 1H NMR
(300 MHz, CDCl3, d): 8.32 (br s, 2H), 7.35 (br s, 1H), 4.36 (d,
J = 3.6 Hz, 2H), 3.68 (s, 3H), 2.97 (br s, 2H), 2.56 (br s, 2H), 1.56–
1.32 (m, 10H), 0.89 (br s, 6H). 13C NMR (75 MHz, CDCl3, d): 172.8,
172.2, 57.9, 52.5, 48.0, 42.7, 35.9, 34.4, 33.7, 30.7, 25.7, 21.4,
19.2, 18.4. Anal. Calcd for C15H29ClN2O3: C, 56.15; H, 9.11; N,
8.73. Found: C, 56.21; H, 9.40; N, 9.00.
Methyl (2-(1-(aminomethyl)cyclohexyl)acetyl)-
L
-phenylalanyl-
L
-
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tryptophanate hydrochloride 18b: White solid (93%, 0.282 g, 0.
465 mmol). Mp 115.0–116.2 °C; 1H NMR (300 MHz, DMSO-d6, d):
8.73 (d, J = 7.5 Hz, 1H), 8.04 (br s, 3H), 7.53 (d, J = 9.0 Hz, 1H),
7.32–7.20 (m, 8H), 4.92 (s, 2H), 4.43 (dd, J = 12.9, 7.5 Hz, 1H),
4.33–4.26 (m, 1H), 3.64–3.49 (m, 3H), 3.16–2.68 (m, 6H), 1.50–
1.40 (m, 12H). 13C NMR (75 MHz, DMSO-d6, d): 196.8, 171.7,
170.3, 155.6, 137.8, 136.9, 129.1, 128.2, 127.9, 127.6, 127.2,
126.2, 65.1, 55.9, 52.2, 51.4, 47.9, 45.0, 37.3, 36.1, 32.1, 25.3,
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Balaram, P. J. Am. Chem. Soc. 2009, 131, 5956.
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25.0, 22.4, 20.5. HRMS (ESI) calcd for
519.2966, found 519.2981.
C
30H39N4O4 [M+H]+
2-(1-(Aminomethyl)cyclohexyl)-N-benzylacetamide hydrochloride
18c: White solid (92%, 0.136 g, 0.046 mmol). Mp 189.4–
190.8 °C; 1H NMR (300 MHz, CD3OD, d): 7.33–7.26 (m, 5H),
4.39 (d, J = 3.3 Hz, 2H), 2.97 (s, 2H), 2.55 (s, 2H), 1.53–1.37 (m,
10H). 13C NMR (75 MHz, CD3OD, d): 174.1, 139.8, 129.7, 128.8,
128.5, 66.1, 44.5, 44.1, 36.7, 34.7, 26.9, 22.4. Anal. Calcd for C16-
H25ClN2O: C, 64.74; H, 8.49; N, 9.44. Found: C, 64.60; H, 8.77; N,
9.24.
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4. Conclusions
We were able to expand the polypeptide backbone by introduc-
ing the stereochemically constrained terminus unit of gabapentin.
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