Synthesis, characterization and in vitro pharmacology of novel pregabalin derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.12.049

A series of novel pregabalin derivatives were synthesized starting from N-protected pregabalin, different amino sugars, adamantylamine, serotonin and tryptamine. New compounds were spectroscopically characterized and in vitro tested on gabapentin receptor binding assay. The serotonin-pregabalin adduct showed significant binding effect and its IC₅₀ value was determined.

Full text of DOI:10.1016/j.ejmech.2009.12.049

European Journal of Medicinal Chemistry 45 (2010) 1447–1452
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and in vitro pharmacology of novel
pregabalin derivatives
a
a
,
a
b
ˇ
*
ˇ
´
Stefica Horvat , Zdenko Hamersak , Irena Stipetic , Thierry Jolas
a
ˇ
´
Department of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, P.O. Box 180, 10002 Zagreb, Croatia
Cerep, Le Bois l’Eveque, B.P. 30001, Celle l’Evescault 86600, France
b
ˆ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pregabalin derivatives were synthesized starting from N-protected pregabalin, different
amino sugars, adamantylamine, serotonin and tryptamine. New compounds were spectroscopically
characterized and in vitro tested on gabapentin receptor binding assay. The serotonin-pregabalin adduct
showed significant binding effect and its IC50 value was determined.
Received 31 August 2009
Received in revised form
1 December 2009
Accepted 18 December 2009
Available online 4 January 2010
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Pregabalin
Gabapentin
Binding affinity
1. Introduction
Among the different strategies developed for improving proper-
ties of different drugs, the ‘‘sugar approach’’ has recently emerged as
Pregabalin (PGB; S-(þ)-3-isobutylgaba) and gabapentin (GBP) are
efficacious drugs in the treatment of epilepsy, neuropathic pain, and
anxiety states [1,2]. Both PGB and GBP are considered to bind to the
a2d subunit of voltage-gated Ca2þ channels (VGCC) with nanomolar
affinity. This interaction appears to diminish the amount of Ca2þ
entering the presynaptic terminal following depolarization, leading
to a decrease in neurotransmitter release [3]. Previous studies on
structure–activity relationships of pregabalin focused on the incor-
poration of substituents at positions along the hexanoic acid back-
bone [4] or to synthesis of the carboxylate bioisostere analogs in
which carboxyl group has been replaced by tetrazole moieties [5].
The goal of this study was to design and generate novel
pregabalin analogs and to evaluate their binding affinity for the a2d
subunit of voltage-gated Ca2þ channels. We thereby aim to
optimize the binding affinity of these analogs through structure–
activity relationships focusing mainly on C-terminal modifications
of PGB. Our manipulations involved amidation of PGB
a new attractive and versatile tool. [for review see Ref. [6]]. Due to
the poly-hydroxylated nature of sugars and the large array of sugars
that are available, attachment of sugar to drugs can (i) utilize active
transport systems, (ii) modify the physico-chemical properties of the
construct, and (iii) target the compound. The clinical use of a highly
active carbohydrate-based carbonic anhydrase inhibitor, i.e., top-
iramate, constitutes an interesting demonstration of the validity of
this approach [7]. The carbohydrate moieties can also serve as
recognition sites for carbohydrate-binding proteins – lectins. Studies
with sugar modified catanionic vesicles demonstrated the ability of
this method to control the glycoconjugate concentration in the
membrane and to explore relationship between ligand separation
distance and multivalent lectin binding at the bilayer interface [8]. In
addition, glycopolymers could be used to control interspecies
transmission and epidemic in specific host. Thus, in vitro and in vivo
infection experiments on influenza viruses using glycopolymers that
were carrying terminal 2,6-sialic acid residues on lactosamine
repeats demonstrated marked differences in inhibitory activity
against different species of viruses [9].
with different amino sugars (2-amino-2-deoxy-
2-amino-2-deoxy- -galactopyranose, 2-amino-2-deoxy-
nopyranose, -glucopyranosylamine), with bioactive antiviral
D-glucopyranose,
D
D-man-
b
-D
The rationale to conjugate 1-adamantylamine to PGB originates
from our previous results that modifications of small bioactive
compounds with hydrophobic adamantane moiety may be of
particular benefit for passive cellular absorption by membrane
penetration or attachment [10,11]. Multivalent ligands can function
as inhibitors or effectors of biological processes [12,13]. We envi-
sioned that, by amidation of PGB with tryptamine or serotonin,
agent 1-adamantylamine (Symmetrel), or with neuromodulators
such as tryptamine or serotonin (Scheme 1).
* Corresponding author. Tel.: þ385 1 4571 300; fax: þ3851 4680108.
ˇ
E-mail address: hamer@irb.hr (Z. Hamersak).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.049

ˇ
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S. Horvat et al. / European Journal of Medicinal Chemistry 45 (2010) 1447–1452
O
O
O
a, b
OR
c
O
O
HOOC
O
NHCbz
1
2
3, R=cinnamyl
4, R=H
d, e
O
O
NHR1
NHR1
NH2
g
f
NHCbz
12-18
5-11
Compound
R1NH2
Compound
R1NH2
OH
H
N
O
HO
5, 12
9, 16
10, 17
11, 18
HO
OH
NH
2
NH2
H
N
OH
OH
O
6, 13
HO
HO
OH
NH
2
NH2
OH
NH2
NH
2
O
HO
HO
7, 14
8, 15
OH
OH
O
NH2
HO
HO
OH
Scheme 1. Synthesis of pregabalin derivatives 12–18. Reactions and conditions: (a) quinine, toluene, cinnamyl alc. ꢀ30 ꢁC, 24 h; (b) resol. S-phenylethylamine; (c) (PhO)2PON3, Et3N,
benzyl alc.; (d) Pd(OAc)2, PPh3, morpholine, EtOH, reflux, 3 h; (e) (1) 1-adamantylamine, MTBE/2-PrOH (2) HCl; (f) (1) N-ethylmorpholine, ClCO2iBu, THF, ꢀ15 ꢁC, 2 min; (2) RNH2,
room temperature, 24 h; (g) H2, Pd/C, EtOH/H2O/HCl, room temperature, 2 h.
potent bioactivity can arise from the high functional affinities of
multivalent ligand–receptor interactions.
Table 1
Chemical shifts of anomeric forms (C-1, H-1) and tautomeric composition of
carbohydrate–pregabalin derivatives 12–15 in DMSO-d6 solution estimated from the
NMR data.
2. Results and discussion
2.1. Chemistry
Compound
Parent amino
sugara
Tautomeric
composition
DMSO-d6
Pregabalin derivatives 12–18 were synthesized in two steps from
N-protected pregabalin (Z-PGB, 4) (Scheme 1). Z-PGB 4 of high
enantiomeric purity (>98% ee) was obtained by a modification of
procedure we have already reported earlier [14]. Instead of cinnamyl
alcohol, benzyl alcohol was used in the Curtius rearrangement.
Cinnamyl protection of 3 was cleaved by palladium acetate-triphenyl
phosphine, affording amino-protected acid 4, which was purified via
its adamantylamine salt. The coupling of 4 with the corresponding
amine was achieved by using mixed anhydride method in the
presence of isobutyl chloroformate to yield N-terminally protected
compounds 5–11. Hydrogenation of 5–11 and subsequent purifica-
tion by RP HPLC gave the desired pregabalin amides 12–18.
dC-1 (ppm)
dH-1 (ppm)
%
12
13
2-GlcN
2-GalN
a
b
-pyranose
-pyranose
90.61
95.34
4.93
4.45
70
30
a-pyranose
b-pyranose
a-furanose
b-furanose
a-pyranose
b-pyranose
90.97
95.82
93.79
99.99
90.56
95.35
4.94
4.40
5.06
4.95
4.93
4.45
38
26
6
8
2-GlcN
2-ManN
1-GlcN
13
9
14
a
b
-pyranose
-pyranose
92.59
93.19
4.85
4.71
86
14
15
b
-pyranose
79.48
4.71
100
a
The proposed structures of compounds 12–18 were confirmed
2-GlcN, 2-amino-2-deoxy-
D
-glucose; 2-GalN, 2-amino-2-deoxy-
D
-galactose;
by NMR spectroscopy (Tables 1–3). 1H and 13C resonances were
2-ManN, 2-amino-2-deoxy-
D
-mannose; 1-GlcN, b-D-glucopyranosylamine.

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S. Horvat et al. / European Journal of Medicinal Chemistry 45 (2010) 1447–1452
1449
Table 2
NMR chemical shift data (ppm) of the major tautomer in DMSO-d6 solution of glycoconjugates 12–15 derived from pregabalin.
Residue
Atom
12 (a
-pyranose)
13 (
a
-pyranose)
14 (
a
-pyranose)
15 (b-pyranose)
dH
dC
dH
dC
dH
dC
dH
dC
Sugara
1
2
3
4
5
6
NH
4.93
3.62
3.51
3.13
3.60
90.61
54.23
70.48
71.15
72.08
61.07
–
4.94
4.01
3.65
3.72
3.80
90.97
50.00
67.31
68.18
70.43
60.55
–
4.85
3.97
3.77
3.41
3.58
92.59
53.86
68.21
67.45
72.89
61.51
–
4.71
3.05
3.18
3.07
3.09
79.48
72.38
77.48
70.00
78.51
60.95
–
3.48/3.63
7.87
3.41/3.54
7.79
3.50/3.62
7.65
3.41/3.62
8.47
Pregabalin
1
2
3
4
5
6/60
7
–
171.39
37.79
31.63
40.58
24.38
–
171.57
37.85
31.68
40.53
24.39
–
171.75
37.51
31.64
40.48
24.39
–
171.98
38.23
33.01
40.85
24.53
2.22
2.06
1.16
1.64
0.84/0.86
2.78
2.22
2.07
1.17
1.64
0.82/0.88
2.78
2.30
2.05
1.17
1.63
0.86/0.87
2.77
2.11/2.19
1.97
1.08/1.16
1.61
0.84/0.85
2.62
22.15/22.75
42.77
22.74/22.80
42.60
22.29/22.80
42.59
22.59/22.68
43.47
a
2-amino-2-deoxy-
D
-glucose for 12; 2-amino-2-deoxy-
D
-galactose for 13; 2-amino-2-deoxy-
D
-mannose for 14; D-glucopyranosylamine for 15.
assigned by homonuclear COSYexperiments combined with 1H–13
C
The 1H and 13C chemical shifts data for compounds 12–18 are
correlation techniques through one bond (HMQC) and multiple
bonds (HMBC). The population of isomers in equilibrated DMSO-d6
solutions of amino sugar-derived pregabalin compounds 12–15 was
estimated by integration of the signal intensities of the anomeric
carbon atom (C-1) in the 80–96 ppm region of the 13C NMR spectra
(Table 1).
summarized in Tables 2 and 3.
2.2. Pharmacology
Pregabalin analogs 12–18 were screened for their ability to
displace [3H]gabapentin from a2d subunit-containing calcium
channels in cell membrane homogenates from rat cerebral cortex.
Although C-terminally modified pregabalin analogs in which the
carboxylate has been replaced by tetrazole moiety showed binding
to a2d protein [5], contrary to our expectation that conjugation with
amino sugar moieties will improve the targeting of pregabalin
molecule towards cell membrane, carbohydrate–pregabalin
conjugates 12–15 did not express ability to compete with
[3H]gabapentin for binding to the a2d subunit of voltage-dependent
The NMR spectra of 2-GlcN and 2-ManN derivatives 12 and 14
showed two sets of sugar resonances attributable to the sugar
moiety in its
the major tautomers. NMR analysis of compound 13 provided
evidence that amidation of pregabalin with 2-amino-2-deoxy-
galactose resulted in four sets of signals consistent with the pres-
ence of galacto-sugar residue in - and -pyranose as well as - and
-furanose forms in the solution. The NMR spectra of 13 also
revealed the presence of - and -pyranose forms of compound 12
(22%) in solution (Table 1), most probably formed by epimerization
of 2-amino-2-deoxy- -galactose under applied reaction conditions.
As can be seen from the data in Table 1, the glucosylamine unit of 15
a- and b-pyranose forms. The a-pyranose forms were
D-
a
b
a
b
Ca2þ channels. From the compounds 12–18 tested at 10
mM,
a
b
and besides pregabalin, only derivative 17 with a serotonin
(5-hydroxytryptamine) moiety showed significant inhibition of
specific [3H]gabapentin binding (Table 4). Noteworthy is that
derivative 16 with a tryptamine moiety showed minor inhibition at
D
resided only in b-pyranose form.
Table 3
NMR chemical shift data (ppm) of the pregabalin conjugates 16–18 in DMSO-d6 solution.
Residue
Atom
16
17
18
dH
dC
dH
dC
dH
dC
R-NHa
1-NH
2
10.86
7.15
–
2.82
3.35
7.52
6.97
7.06
7.34
–
–
10.50
7.03
–
2.75
3.29
6.81
–
6.59
7.12
–
–
7.56
–
1.92
–
122.61
111.68
25.17
39.46
118.18b
118.16b
120.87
111.37
136.24
127.16
–
123.03
110.67
25.30
39.37
102.15
150.16
111.26
111.64
130.80
127.82
–
50.89
40.88
3
30
300
4
5
6
7
8
9
2.00
1.61
28.74
35.99
–
8.24
–
8.19
300-NH
Pregabalin
1
2
3
4
5
6/60
7
–
171.16
37.94
31.97
40.60
24.46
22.32/22.69
42.87
–
–
171.07
37.04
31.45
40.55
24.43
22.23/22.80
42.70
–
–
170.63
38.43
31.61
40.44
24.38
22.22/22.78
42.72
–
2.13/2.23
2.05
1.08/1.16
1.62
0.82/0.84
2.71
NO
2.19
2.07
1.13
1.63
0.85/0.86
2.75
7.89
2.11/2.18
2.02
1.12
1.63
0.84/0.86
2.75
NH2
7.95
a
Tryptamine for 16; serotonin for 17; 1-adamantylamine for 18.
Assignments of signals can be interchangeable. NO ¼ not observed.
b

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S. Horvat et al. / European Journal of Medicinal Chemistry 45 (2010) 1447–1452
1450
Table 4
based on 2D homonuclear (COSY, NOESY) and heteronuclear
(HMQC, HMBC) experiments. High resolution mass spectrometry
(HRMS) was performed on 4800 Plus MALDI TOF/TOFÔ Analyzer.
Reversed-phase high-performance liquid chromatography (RP
HPLC) was performed on a Varian Pro Star 230 HPLC system using
Inhibition of [3H]gabapentin binding in cell membrane homogenates from rat
cerebral cortex. Each inhibition represents the mean of duplicate determinations.
Compound
Test concentration (
m
M)
% Inhibition of control specific
[3H]gabapentin bindinga
Pregabalin
10
10
10
10
10
10
10
10
92
18
9
14
17
18
51
3
a
Eurospher 100 reversed-phase C-18 semipreparative
12
13
14
15
16
17
18
(250 ꢂ 8 mm ID, 5
m
m) (flow rate: 1.0 ml/min) or analytical
(250 ꢂ 4 mm ID, 5
m
m) (flow rate: 0.5 ml/min) column under
isocratic conditions using different concentrations of MeOH in
0.1% aqueous trifluoroacetic acid (TFA). UV detection was per-
formed at 215 nm using a Varian Pro Star 335 photodiode-array
detector.
a
Compounds showing significant inhibition are indicated in bold.
Enantiomeric purity of 4 was determined on Chiralpak AS using
n-hexane/EtOH/TFA (100:5:0.1) at 206 nm.
the same testing concentration indicating that the presence of an
hydroxyl on the tryptamine’s indole group improved binding of the
pregabalin analog. IC50 values obtained for derivative 17 and
4.2. Synthesis
4.2.1. (ꢀ)-(S)-3-(Benzyloxycarbonylamino-methyl)-5-methyl-
hexanoic acid (4)
gabapentin were respectively 13
and 35 nM (Fig. 1).
mM and 43 nM and Ki values 11 mM
The solution of crude carbamate 3 [14] (10.2 g, 35 mmol, 90% ee)
in abs. EtOH (50 ml) was refluxed for 10 min, and then cooled to
70 ꢁC. Morpholine (5.1 ml, 70 mmol) was added followed by tri-
phenylphosphine (80 mg, 0.3 mmol) and Pd(OAc)2 (2 mg,
0.009 mmol). The reaction mixture was refluxed for 3 h. Most of the
solvent was evaporated at reduced pressure, 200 ml of 3% Na2CO3
and 80 ml of EtOAc were added. The mixture was stirred for 15 min,
aqueous layer was separated and washed again with EtOAc. Upon
acidification to pH 1.5 with conc. HCl, extraction with CH2Cl2
afforded 7.2 g of oily benzyl-carbamate 4 which was dissolved in
130 ml of MTBE and 40 ml of 2-propanol and treated with 1-ada-
mantylamine (3.4 g, 22 mmol). Crystals were collected on filter,
dissolved in dichloromethane and the solution was treated with 2%
HCl to liberate free acid. The organic solution was washed with H2O
and dried to obtain upon evaporation 5.6 g of pure 4 as yellowish oil
3. Conclusion
Seven new pregabalin derivatives were synthesized and spec-
troscopically characterized. Gabapentin receptor binding assay
study showed the one among them, the serotonin-pregabalin
adduct 17, possesses a promising activity (Ki ¼ 11
mM). Further
manipulation of the serotonin moiety may provide analogs with
higher affinity for this target. In addition, characterization of the
activity of the serotonin-pregabalin adduct at serotonergic targets
may provide interesting information about the therapeutic interest
of such a compound.
4. Experimental
(>98% ee).
4.1. Chemistry
25
[
a
]
D
¼ ꢀ4.4 (c ¼ 25, EtOH). 1H NMR (DMSO-d6),
d/ppm:
0.81–0.85 (m, 6H, 2CH3), 1.00–1.16 (m, 2H, CH–CH2–CH), 1.55–1.66
(m, 1H, CH3–CH–CH3), 1.91–2.27 (m, 3H, HOOC–CH2, CH2–CH–CH2),
2.84–3.09 (m, 2H, NH–CH2), 5.01 (s, 2H, Ph-CH2), 7.26–7.28 (m, 5H,
Ar-H) 12.01 (brs, 1H, COOH).
Melting points were determined on a Tottoli (Bu¨ chi) apparatus
and are uncorrected. Optical rotations were measured at 25 ꢁC
using an Optical Activity LTD automatic AA-10 polarimeter. NMR
spectra were recorded on a Bruker AV 600 spectrometer, operating
at 150.91 MHz for 13C and 600.13 MHz for 1H nuclei. The spectra
were measured in DMSO-d6 solutions at 25 ꢁC. Chemical shifts in
parts per million were referenced to TMS. Spectra were assigned
13C NMR (DMSO-d6),
d/ppm: 22.9 (CH3), 23.2 (CH3), 25.1 (CH),
33.6 (CH), 37.6 (CH2), 41.4 (CH2), 44.3 (CH2), 65.6 (CH2), 128.1
(2 ꢂ Ar-CH), 128.2 (Ar-CH), 128.7 (2 ꢂ Ar-CH), 137.8 (Ar-C), 156.8
(NH–C]O), 174.4 (COOH). IR (film on KBr): 3340 (O]CO–H), 2956,
2929, 2871, 1708 (C]O), 1534, 1258 (CO–O), 697 cmꢀ1
.
Anal. Calcd. for C16H23NO4 (293.36): C, 65.51; H, 7.90; N, 4.77.
Found: C, 65.70; H, 8.15; N, 4.41.
100
75
50
25
0
4.2.2. Synthesis of pregabalin derivatives 5–11
To a chilled solution (ꢀ15 ꢁC) of N-benzyloxycarbonyl-pre-
gabalin (4) (147 mg, 0.5 mmol) in THF (4 ml), N-ethylmorpholine
(NEM, 63
ml, 0.5 mmol) and isobutyl chloroformate (65 ml,
0.5 mmol) were added. The resulting mixture was stirred for 2 min
at the same temperature and a precooled solution of the corre-
sponding amine (0.5 mmol) in THF–water mixture (6:4, 10 ml) was
then added. The reaction mixture was stirred for 10 min at ꢀ15 ꢁC
and then overnight at room temperature. The solvent was evapo-
rated in vacuo and the residue was purified by semipreparative RP
HPLC using different concentrations of MeOH/0.1% TFA.
Molecular structures of compounds 5–11 were confirmed by
NMR spectroscopy (see Supplementary data).
-10
-9
-8
-7
-6
-5
-4
Log [compound] (M)
Compound 5 was prepared from Z-PGB (4) and 2-amino-2-
deoxy-
D-glucopyranose hydrochloride. Purification with RP HPLC
Fig. 1. Inhibition of [3H]gabapentin binding in cell membrane homogenates from rat
cerebral cortex. The graph shows curves for gabapentin (open diamond) and 17 (filled
diamond). Each point represents the mean of duplicate determinations.
by using 62.75% MeOH/0.1% TFA afforded 132 mg (58%) of 5. M.p.
122–125 ꢁC; [
25
a]
¼ þ30 (c ¼ 1.0, MeOH).
D

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S. Horvat et al. / European Journal of Medicinal Chemistry 45 (2010) 1447–1452
1451
25
Compound 6 was prepared from Z-PGB (4) and 2-amino-2-
deoxy- -galactopyranose hydrochloride. Purification with RP HPLC
by using 62.75% MeOH/0.1% TFA afforded 136 mg (60%) of 6. M.p.
Oil; [
a
]
D
¼ þ6 (c ¼ 1.0, MeOH). HRMS calcd for C18H27N3O (Mþ)
D
302.2227, found 302.2233.
00
25
103–105 ꢁC; [
Compound 7 was prepared from Z-PGB (4) and 2-amino-2-
deoxy- -mannopyranose hydrochloride. Purification with RP HPLC
by using 62.75% MeOH/0.1% TFA afforded 129 mg (57%) of 7. M.p.
a
]
D
¼ þ34 (c ¼ 1.0, MeOH).
4.2.3.6. N3 -Pregabalyl-serotonin (17). Compound 17 was prepared
from 10. Purification with RP HPLC by using 57.5% MeOH/0.1% TFA
afforded 28 mg (80%) of 17, which was 98.0% pure by HPLC; M.p.
142–145 ꢁC (violet crystals). HRMS calcd for C18H27N3O2 (Mþ)
318.2176, found 318.2164.
D
25
76–78 ꢁC; [
Compound 8 was prepared from Z-PGB (4) and
a
]
¼ ꢀ10 (c ¼ 1.0, MeOH).
D
b-D-glucopyr-
anosylamine. Purification with RP HPLC by using 62.75% MeOH/
4.2.3.7. N-Pregabalyl-(1-adamantyl)amine (18). Compound 18 was
prepared from 11. Purification with RP HPLC by using 68% MeOH/
25
0.1% TFA afforded 107 mg (47%) of 8. M.p. 83–85 ꢁC; [
(c ¼ 1.0, MeOH).
a]
¼ 0
D
0.1% TFA afforded 8 mg (24%) of 18, which was 97.7% pure by HPLC.
25
Compound 9 was prepared from Z-PGB (4) and tryptamine.
M.p. 142–148 ꢁC; [
a]
¼ þ6 (c ¼ 1.0, MeOH). HRMS calcd for
D
Purification with RP HPLC by using 64.5% MeOH/0.1% TFA afforded
C18H32N2O (Mþ) 293.2587, found 293.2599.
25
135 mg (62%) of 9. Solid foam; [
a]
¼ 0 (c ¼ 1.0, MeOH).
D
Compound 10 was prepared from Z-PGB (4) and serotonin.
4.3. Pharmacology
Purification with RP HPLC by using 64.5% MeOH/0.1% TFA afforded
25
126 mg (56%) of 10. Solid foam; [
a]
¼ 0 (c ¼ 1.0, MeOH).
Cell membrane homogenates from rat cerebral cortex (50 mg
D
Compound 11 was prepared from Z-PGB (4) and 1-adamantyl-
amine. Purification with RP HPLC by using 71.5% MeOH/0.1% TFA
afforded 115 mg (54%) of 11. Solid foam; [
protein), prepared as described previously [15], were incubated
60 min at 22 ꢁC with 10 nM [3H]gabapentin in the absence or
presence of the test compounds in a buffer containing 10 mM
Hepes/Tris (pH 7.4). Nonspecific binding was determined in the
25
a
]
¼ ꢀ1 (c ¼ 1.0, MeOH).
D
4.2.3. Synthesis of pregabalin derivatives 12–18
presence of 100 mM gabapentin. Following incubation, the samples
The solution of the protected pregabalin conjugate
(0.11 mmol) in ethanol (5 ml), containing water (0.2 ml) and 1 M
HCl (0.1 ml) was hydrogenated in the presence of 10% Pd/C
(50 mg) for 2 h. The catalyst was filtered off, the filtrate was
evaporated in vacuo and the residue was then purified by semi-
preparative RP HPLC using different concentrations of MeOH/0.1%
TFA. The trifluoroacetate ion present after preparative HPLC, was
removed using an SPE cartridge. The cartridge was first eluted
with water and then with MeOH to recover the pregabalin
conjugates. The effluent was evaporated and the residue dis-
solved in water and lyophilized. Compounds 12–18 were at least
95% pure as assessed by analytical RP HPLC. Molecular structures
were confirmed by mass spectrometry and NMR spectroscopy
(see Tables 1–3).
were filtered rapidly under vacuum through glass fiber filters (GF/B,
Packard) presoaked with 0.3% PEI and rinsed several times with ice-
cold 10 m
M Hepes/Tris, 100 mM NaCl using a 96-sample cell
harvester (Unifilter, Packard). The filters were dried, then counted
for radioactivity in a scintillation counter (Topcount, Packard) using
a scintillation cocktail (Microscint 0, Packard).
Compounds were prepared as 10 m
M stock solutions in pure
DMSO. All intermediate dilutions before addition to the assay well
were made in pure DMSO. A last 100-fold dilution was made in the
assay well to reach the final testing concentration.
The specific ligand binding to the receptors was defined as the
difference between the total binding and the nonspecific binding
determined in the presence of an excess of unlabelled ligand. The
results were expressed as a percent of control specific binding
((measured specific binding/control specific binding)ꢂ100) and as
a percent inhibition of control specific binding (100ꢀ((measured
specific binding/control specific binding)ꢂ100)) obtained in the
presence of the test compounds.
4.2.3.1. 2-(Pregabalylamino)-2-deoxy-D-glucopyranose (12). Compound
12 was prepared from 5. Purification with RP HPLC by using 10% MeOH/
0.1% TFA afforded 28 mg (80%) of 12, which was 97.0% pure by HPLC.
M.p. 125–128 ꢁC; [a D25
]
¼ þ34 (c ¼ 1.0, MeOH). HRMS calcd for
The IC50 values (concentration causing a half-maximal inhibi-
tion of control specific binding) and Hill coefficients (nH) were
determined by non-linear regression analysis of the competition
curves generated with mean replicate values using Hill equation
C14H28N2O6 (Mþ) 321.2020, found 321.2034.
4.2.3.2. 2-(Pregabalylamino)-2-deoxy-D-galactopyranose (13). Compound
13 was prepared from 6. Purification with RP HPLC by using 10% MeOH/
0.1% TFA afforded 23 mg (65%) of 13, which was 95.3% pure by HPLC. M.p.
curve fitting (Y ¼ D þ [(A ꢀ D)/(1 þ (C/C50
)
nH)], where Y ¼ specific
binding, D ¼ minimum specific binding, A ¼ maximum specific
binding, C ¼ compound concentration, C50 ¼ IC50, and nH ¼ slope
factor). This analysis was performed using a software developed at
Cerep (Hill software) and validated by comparison with data
generated by the commercial software SigmaPlotÒ 4.0 for
WindowsÒ (Ó 1997 by SPSS Inc.). The inhibition constants (Ki) were
calculated using the Cheng Prusoff equation (Ki ¼ IC50/(1þ(L/KD)),
148–145 ꢁC; [a 2D5
]
¼ þ26 (c ¼ 1.0, MeOH). HRMS calcd for C14H28N2O6
(Mþ) 321.2020, found 321.2031.
4.2.3.3. 2-(Pregabalylamino)-2-deoxy-D-mannopyranose (14). Compound
14 was prepared from 7. Purification with RP HPLC by using 10% MeOH/
0.1% TFA afforded 26 mg (76%) of 14, which was 95.7% pure by HPLC. M.p.
115–120 ꢁC; [a 2D5
]
¼ ꢀ10 (c ¼ 1.0, MeOH). HRMS calcd for C14H28N2O6
where
L
¼
concentration of radioligand in the assay, and
(Mþ) 321.2020, found 321.2007.
KD ¼ affinity of the radioligand for the receptor). A scatchard plot
was used during assay development to determine the KD value for
gabapentin. All experiments were done in duplicate. Gabapentin
was used as a standard reference compound and tested in each
experiment at several concentrations to obtain a competition curve
from which its IC50 is calculated.
4.2.3.4. N-Pregabalyl-b-D-glucopyranosylamine (15). Compound 15
was prepared from 8. Purification with RP HPLC by using 10%
MeOH/0.1% TFA afforded 31 mg (90%) of 15, which was 98.5% pure
25
by HPLC. M.p. 142–145 ꢁC; [
a]
¼ þ12 (c ¼ 1.0, MeOH). HRMS calcd
D
for C14H28N2O6 (Mþ) 321.2020, found 321.2029.
Acknowledgment
00
4.2.3.5. N3 -Pregabalyl-tryptamine
(16). Compound 16 was
prepared from 9. Purification with RP HPLC by using 50.5% MeOH/
0.1% TFA afforded 26 mg (79%) of 16, which was 99.0% pure by HPLC.
We thank the Ministry of Science, Education and Sports of the
Republic of Croatia for financing (grants no. 098-0982933-2908,

ˇ
S. Horvat et al. / European Journal of Medicinal Chemistry 45 (2010) 1447–1452
1452
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