Carnitine Conjugate of Nipecotic acid: A new example of dual prodrug

Article abstract of DOI:10.3390/molecules14093268

As a novel example of improved entry of poorly delivered drugs into the brain by means of nutrient conjugates, L-carnitine was conjugated to nipecotic acid and the capacity to antagonize PTZ-induced convulsions of this novel entity was evaluated.

Full text of DOI:10.3390/molecules14093268

Molecules 2009, 14, 3268-3274; doi:10.3390/molecules14093268
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Carnitine Conjugate of Nipecotic Acid: A New Example of Dual
Prodrug
Carmela Napolitano 1,#, Martina Scaglianti 1,†, Emanuela Scalambra 1, Stefano Manfredini 1,3,*,
Luca Ferraro 2, Sarah Beggiato 2 and Silvia Vertuani 1,3
1
Department of Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17-19,
I-44100 Ferrara, Italy
2
Department of Clinical and Experimental Medicine, Section of Pharmacology, University of
Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
3
Ambrosialab srl, Via Saragat 4a, I-44100 Ferrara, Italy
#
Current address: Specchiasol srl, Via Bruno Rizzi 37012 Bussolengo, Italy.
†
Current address: National University of Ireland, Galway, Ireland.
* Author to whom correspondence should be addressed; E-mail: stefano.manfredini@unife.it.
Received: 27 July 2009; in revised form: 25 August 2009 / Accepted: 27 August 2009 /
Published: 31 August 2009
Abstract: As a novel example of improved entry of poorly delivered drugs into the brain
by means of nutrient conjugates, L-carnitine was conjugated to nipecotic acid and the
capacity to antagonize PTZ-induced convulsions of this novel entity was evaluated.
Keywords: L-carnitine; molecular conjugation; increased brain delivery, prodrug;
nipecotic acid
Introduction
Distribution of drugs to the central nervous system (CNS) is one of the major problems of current
therapy of brain diseases. Difficulties in the crossing of the brain blood barrier (BBB) often impair the
efficacy of valuable drugs. In the past, most of the attempts to overcome this drawback have been
directed to the amelioration of the lipophilic properties through the preparation of prodrugs by
formation of reversible linkages with suitable groups [1,2]. A new approach that takes into account the

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recent progress in molecular cloning and the expression of transporters genes is being applied too. It is
now clear that membrane transporters of native compounds also take part in drug transport in various
tissues. Several specific transporters have been identified in the brain capillary endothelia, and among
them some of those involved in the active supply of nutrients (i.e., glucose, amino acids) have been
used to prepare prodrugs with improved CNS penetration [3]. We have already reported a significative
example of the latter approach [4] and here describe a new application of this prodrug concept.
We have investigated the conjugation of L-carnitine (L-C), a vital cofactor for the mitochondrial
oxidation of fatty acids [5] that posses specific carrier-mediated transport systems for its uptake [6-7],
with nipecotic acid as a tool to demonstrate the capability of L-C to behave as a drug carrier in the
brain. The choice of L-C as carrier introduces a novel concept referred to as a “double prodrug”
approach, which involves the preparation of a novel single molecular entity with therapeutic effects
resulting from two diverse, but synergistic mechanisms of action. A suitable covalent attachment of
carnitine and nipecotic acid may have a significant therapeutic value: the crossing of the BBB of the
conjugate and the controlled release of both drugs in vivo through enzymatic hydrolysis could result
not only in a good bioavailability of anticonvulsivant agent in the brain, but also in the prevention of
induced carnitine deficiency disorders.
Results and Discussion
The strategy for the conjugation of L-C to nipecotic acid is described in Scheme 1. Nipecotic acid
and L-C needed previous protection at the amino and carboxylic functions respectively. Thus nipecotic
acid was protected as its tert-butoxycarbonyl derivative and L-C as the benzyl ester 1 under standard
conditions. Several attempts were made in order to improve the reactivity of the carboxylic function of
N-Boc-nipecotic acid and thus access to conjugate 2. The most significant improvements in the
efficiency of the reaction were obtained converting the carboxylic acid into an acyl chloride employing
oxalyl chloride in the presence of a catalytic amount of a mixture of DMAP/TEA. The ester 2 was
finally deprotected by treatment with trifluoroacetic acid, followed by classical hydrogenolysis, the
molecule was purified by ion exchange solid phase extraction to afford final compound 4 in
good yield.
Scheme 1. Conjugation of L-C to nipecotic acid.
N-Boc-nipecotic acid
COOH
(R,S)
*
N
N
N
N
O
+
O
+
(R)
-
+
-
O
Br
Br
(R)
-
(R)
Cl
O
O
O
*
O
(R,S)
*
(R,S)
O
*
*
*
(R,S) *
COOBn
COOBn
COOH
N
N
H
OH
(R)
+
N
H
+
H
-
H
CF3COO
-
Cl
O
*
O
+
COOBn
N
1
-
3
2
4
Br

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3270
To support the possibility that L-C may work as a shuttle for drugs, and thus that conjugation with
L-C could be a means to improve the entry into the brain of drugs that do not easily penetrate the BBB,
we evaluated the effects of the systemic injection of nipecotic acid (racemic mixture), L-C and
conjugate 4 on pentylenetetrazole (PTZ)-induced convulsions in mice [8]. In the saline group, the
injection of PTZ (80 mg/kg) induced tonic convulsions with a latency of 677 ± 51 s. As shown in
Table 1, the ip injection of 4 (0.75 mmol/kg) significantly increased the latency of the appearance of
PTZ-induced tonic convulsions, while, as expected [9,10], the administration of nipecotic acid or L-C,
at the same dose (0.75 mmol/kg), were ineffective. No lethality was observed in any of the groups.
Table 1. Effect of ip injection of nipecotic acid (0.75 mmol/kg), L-C (0.75 mmol/Kg) or
conjugate 4 (0.75 mmol/kg) on pentylenetetrazole (PTZ)-induced convulsions in mice.
Treatment
Latency to convulsions (s)
677 ± 51
Control (saline + PTZ)
Nipecotic acid + PTZ
L-carnitine + PTZ
4 + PTZ
645 ± 50
664 ± 62
1021 ± 55a
aP < 0.05, significantly different from control and nipecotic acid groups, according
to ANOVA followed by the Newman–Keuls test for multiple comparisons.
Interestingly, as shown in Figure 1, the effect of conjugate 4 on PTZ-induced convulsions in mice
was dose-dependent in the range of 0.075-1 mmol/kg.
Figure 1. Dose-response effect of conjugate 4 (0.75 mmol/kg ip) on pentylenetetrazole
(PTZ)-induced convulsions in mice. Each point represents the mean ± SEM of 6-7 animals.
1400
y=141.3x + 485.9
r2= 0.9256, p<0.001
1300
1200
1100
1000
900
800
700
600
0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05
Conjugate 4 (mmol/kg)
To prove that compound 4 can cross the BBB, nipecotic acid concentration in mouse brains was
evaluated. The mice were treated with saline solution, nipecotic acid (0.75 mmol/kg) or compound 4
(0.75 mmol/kg), respectively. Thirty minutes after injection the mice were sacrificed and nipecotic
acid concentration in brain was determined by HPLC analysis. The saline treated group did not show

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any traces of nipecotic acid in the brain, nor did the nipecotic treated group, whereas, the mice treated
with compound 4 showed 600 nmol/g concentration of nipecotic acid in the brain (Table 2).
Table 2. Nipecotic acid concentration in mice brain, 30 minute after injection of saline,
nipecotic acid (0.75 mmol/kg) or compound 4 (0.75 mmol/kg).
Treatment
Control (saline)
Nipecotic acid
Compound 4
Nipecotic acid (nmol/g)a
n.d.
n.d.
600
a Limit of quantification (LOQ) of nipecotic acid was ~58 nmol/g.
Conclusions
The conjugation of L-C to nipecotic acid is a demonstration that the approach of delivery of drugs
by means of nutrients using their endogenous transporters is of great potential value. This result further
confirms our previous discovery of a similar role exerted by ascorbic acid and opens another window
on this interesting tool. Further studies are currently on going in order to confirm and extend this
possibility to other active molecules endowed with poor or no BBB crossing properties.
Experimental Section
General
All moisture sensitive reactions were performed under an argon atmosphere. Reaction courses and
product mixtures were routinely monitored by thin-layer chromatography (TLC) on silica gel
precoated F254 Merck plates with detection under 254 nm UV lamp and/or by spraying with a diluted
potassium permanganate solution. Column chromatography was performed with Merck 60-200 mesh
silica gel. Compound 4 was obtained by purification with Supelco DSC-SAX, ion exchange (capacity
0.14 meq/g). Melting points were determined with a capillary apparatus and are uncorrected. 1H-NMR
spectra were recorded on a Bruker Advance 400 spectrometer. All drying operations were performed
over anhydrous magnesium or sodium sulphate. Nipecotic acid was protected as its tert-butoxycarbonyl
(Boc) derivative as reported in [11].
Synthesis of L-carnitine benzyl ester (1): Benzyl bromide (440 mL, 3.71 mmol) was added to a solution
of L-carnitine (500 mg, 3.1 mmol) in a 1.6:1 mixture dioxane/ DMF (25 mL). The solution was heated
to 120 °C for 4 h. After cooling to rt, volatiles were evaporated under vacuum. Purification by
chromatography over silica gel (DCM/ MeOH = 95:5) of the crude residue afforded 560 mg of the
1
pure 1 (72%). White solid; m.p. 180-183 °C. H-NMR (CD3OD) δ 2.62-2.66 (m, 2H), 3.22 (s, 9H),
3.46 (dd, J = 4.6 Hz, J = 6.0 Hz, 2H), 4.56-4.67 (m, 1H), 5.15 (d, J = 12.4 Hz, 1H), 5.19
(d, J = 12.4 Hz, 1H), 7.28-7.42 (m, 5H).
[3-Benzyloxycarbonyl-2(R)-(1-tert-butyloxycarbonyl-piperidine-3(R,S)-carbonyloxy)propyl]-trimethyl-
ammonium bromide (2): To a stirred solution of N-Boc nipecotic acid (1.146 g, 5.0 mmol) in dry THF

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(5.0 mL) were added oxalyl chloride (525 L) and dry DMF (10 L). The resulting yellow pale
solution was stirred at room temperature for 2 h. Volatiles were evaporated under vacuum and the
residue was dissolved in dry THF (10 mL). A portion of this solution (400 L was added dropwise at
0 °C to a solution of 1 (50.0 mg, 0.20 mmol), DMAP (35.0 mg, 0.29 mmol) and TEA (0.82 mL) in dry
THF (2.7 mL). The resulting mixture was stirred at room temperature for 18 h. Volatiles were
evaporated under vacuum and the residue purified by chromatography over silica gel
(DCM/ MeOH = 8:2) to afford 82 mg of the pure conjugate 2 (89%). Colorless oil; 1H-NMR (CD3OD)
 1.36-1.49 (m, 10H), 1.52-1.74 (m, 2H), 1.84-2.01 (m, 1H), 2.38-2.53 (m, 1H), 2.83-2.94 (m, 2H),
3.06-3.20 (m, 11H), 3.61-3.80 (m, 2H), 3.83-3.97 (m, 2H), 5.15 (s, 2H), 5.60-5.71 (m, 1H), 7.31-7.42
(m, 5H).
[3-Benzyloxycarbonyl-2(R)-(piperidine-3(R,S)-carbonyloxy)-propyl]-trimethyl-ammonium
bromide
trifluoroacetate (3): TFA (1.8 ml, 23.7 mmol) was added dropwise to a solution of 2 (591 mg,
1.08 mmol) in dry CH2Cl2. (2.6 mL). The mixture was stirred at room temperature for 3 h. Volatiles
1
were removed under vacuum to afford 3 as yellow pale oil (quantitative). H-NMR (CD3OD) 1.57-
2.18 (m, 5H), 2.84-3.33 (m, 16 H), 3.46 (bt, J = 13.2 Hz, 1H), 3.74 (dd, J = 2.4 Hz, J = 14.0 Hz, 1H),
3.88-3.98 (m, 1H), 5.14 (d, J =12.4 Hz, 1H), 5.18 (d, J =12.4 Hz, 1H), 5.65-5.73 (m, 1H), 7.29-7.42
(m, 5H).
[3-Carboxy-2(R)-(piperidine-3(R,S)-carbonyloxy)-propyl]-trimethyl-ammonium dichloride (4): 10%
Pd/C (200 mg) was added to a solution of 3 (703 mg, 1.33 mmol) in MeOH (20 mL) and the mixture
was stirred 5 h under hydrogen atmosphere. The mixture was filtered on a pad of Celite and the solvent
removed under vacuum to afford 550 mg of trifluoroacetic-bromide salt that was purified by
cromatogaphy over silica-bound quaternary amine a strong anion exchange (eluant MeOH = 100) to
1
get 407mg of the pure dichloride salt 4 (93%). H-NMR (DMSO) 1.55-1.77 (m, 3H), 1.93-2.05 (m,
1H), 2.72-2.93 (m, 5H), 3.11-3.33 (m, 11H), 3.64-3.68 (m, 1H), 3.85-3.99 (m, 1H), 5.46-5.47 (m, 1H),
9.0-9.5 (m, 2H), 12.5-12.9 (broad s, 1H),
Effects of the drugs on pentylenetetrazol-induced seizures
Swiss albino male mice (25-30 g body weight) were used. In any experimental session a maximum
of 15 animals were acutely injected i.p. with saline (control), nipecotic acid (0.75 mmol/kg), L-C
(0.75 mmol/Kg) or compound 4 (0.075, 0.15, 0.3, 0.75 and 1 mmol/Kg). Twenty five minutes after the
treatment, all mice were subcutaneously injected with pentylenetetrazole (80 mg/kg) and the animals
were observed for the following 30 min by an investigator who was unaware of the treatment. The
latency (in sec) to appearance of generalized tonic convulsions and lethality were measured to evaluate
the effects of the treatments on pentylenetetrazole-induced convulsions. Lethality was defined as the
percentage of the animal died within 60 min after pentylenetetrazole injection.
Determination of nipecotic acid in mouse brains
Nipecotic acid brain standard curve. Mice were anesthetized and sacrificed by decapitation, the brains
were rapidly removed, rinsed with saline solution and homogenized in ice-cold 0.1 N HCl. The

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resultant solutions were spiked with different concentrations of aqueous nipecotic acid solution and the
homogenates were centrifuged. The supernatants were collected filtered and then derivatized with
phenylisothiocyanate (PITC) to give a phenylthiocarbamyl derivative of nipecotic acid (PTC-nipecotic
acid). The filtrates were frozen, evaporated to dryness and dissolved in coupling buffer (100 μL,
acetonitrile-pyridine-triethylamine-water, 10:5:2:3) under nitrogen, frozen again and evaporate to
dryness. The samples were dissolved in coupling buffer (100 μL) of to which was added PITC (5 μL)
and after 5 minutes at room temperature the solutions were frozen and evaporated to dryness. Three
mouse brains were used for each concentration of nipecotic acid on the standard curve and triplicate
aliquots of each brain sample were assayed by HPLC as described below. The brain standard curve
was constructed as described in literature [12] by plotting the mean AUC against the amount of
nipecotic acid. The curve was derived over a range of 20-250 pmol of nipecotic acid, the minimum
sensitivity being around ~10 pmol of nipecotic acid or ~58 nmol of nipecotic acid/g brain tissue [12].
Mouse brains to which no nipecotic acid was added were subjected to these same procedures and
served as controls [12].
HPLC-separation of PTC-Nipecotic Acid. The reverse-phase separation was performed by following
the method described by Nassereddine-Sebaei et al. [12]. Briefly, a Waters Novo-pak C18 column (4
µm particle-size, 15 cm in length and 3.9 mm in diameter), maintained at 52 °C, was used. The mobile
phase consisted of 90% solvent A (0.05 M ammonium acetate solution adjusted to pH 6.8 with
phosphoric acid) and 10% of solvent B (10% 1.0 ammonium acetate solution adjusted to pH 6.8 with
phosphoric acid, 10% methanol, 44% acetonitrile, 36% water); the flow rate was 1.0 mL/min and the
samples were analyzed a wavelength of 284 nm by using an UV/Vis variable wavelength detector.
Distribution of nipecotic acid in mouse brain. Mice were treated with saline, nipecotic acid
(0.75 mmol/kg) or compound 4 (0.75 mmol/kg), they were anesthetized and sacrificed by decapitation
30 min after injection. The brains were processed as previously described and the filtrates were
derivatized as described above. The amount of nipecotic acid in brain was calculated with the brain
standard curve, from the average AUC values for three triplicates for each brain supernatant.
Statistical evaluation
Statistical analysis was performed by ANOVA followed by the Newman-Keuls test for multiple
comparisons.
Acknowledgments
The authors wish to thank A. Casolari and E. Durini for careful assistance with the NMR
experiments and structure assignments. This work was supported by grants from the University of
Ferrara and Ambrosialab.
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