Solid phase synthesis of a redox delivery system with the aim of targeting peptides into the brain

Article abstract of DOI:10.1039/b516737j

A solid phase approach for the preparation of peptides attached to a redox chemical delivery system derived from stable annulated NADH models is reported. The synthesis starts with the grafting on a Merrifield resin of quinoline 4b, precursor of the redox

Full text of DOI:10.1039/b516737j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Solid phase synthesis of a redox delivery system with the aim of targeting
peptides into the brain
Claudine Patteux,a Le´na¨ıg Foucout,a Pierre Bohn,a Georges Dupas,a Je´roˆme Leprince,b Marie-Christine
Tonon,b Be´ne´dicte Dehouck,c Francis Marsais,a Cyril Papamicae¨l*a and Vincent Levacher*a
Received 25th November 2005, Accepted 14th December 2005
First published as an Advance Article on the web 12th January 2006
DOI: 10.1039/b516737j
A solid phase approach for the preparation of peptides attached to a redox chemical delivery system
derived from stable annulated NADH models is reported. The synthesis starts with the grafting on a
Merrifield resin of quinoline 4b, precursor of the redox carrier. From the resulting quinoline supported
resin 4d, the stepwise SPPS of both octapeptide OP (RPGLLDLK) and octadecaneuropeptide ODN
(QATVGDVNTDRPGLLDLK), two neuropeptides exhibiting anorexigenic effects, was successfully
achieved by conventional methods. Quaternization of the quinoline moiety prior to cleavage of the
modified OP and ODN peptides from the resin, led to the expected quinolinium salt 8a and 8b
respectively linked to OP or ODN peptides. Finally, the reduction with NaBH4 monitored by UV–vis,
provided the desired annulated NADH models as peptides carriers with either the OP (11a,b) or ODN
(12a,b) moiety.
Introduction
Due to their hydrophilic properties, many neuropharmaceutical
peptides cannot cross the lipoidal bilayer of the blood–brain
barrier (BBB) and consequently exhibit limited access to the
central nervous system (CNS)1 in the absence of a specific
transfer system. Moreover, peptides may also be recognized by
a variety of neuropeptide-degrading enzymes expressed in the
BBB, thus hampering their transport into the CNS.2 To overcome
these limitations, many authors3,4 have designed a brain-targeted
chemical delivery system based on the NADH/NAD+ redox
system (Fig. 1). Thus, biologically active compounds covalently
linked to a lipophilic dihydropyridine can readily penetrate the
BBB. The dihydropyridine which may be considered as a redox
“targeter” is then subjected to enzymatic oxidation in the brain.
The corresponding water-soluble and lipid-insoluble pyridinium
salt thus formed is “locked-in” in the brain promoting retention
of the targeted compound in the CNS. Subsequently, cleavage of
the pyridinium carrier induced by enzymes leads to the release of
Fig. 1 Brain targeting by means of NADH pyridinium models.
the biologically active compound reaching its site of action.
In spite of the interesting pharmacological results described
by Bodor et al.,3 this strategy remains limited due to the use of
also interested in the study of the octadecaneuropeptide (ODN,
QATVGDVNTDRPGLLDLK). ODN, which has been charac-
terized as an endogenous ligand of benzodiazepine receptors
(BZRs) and proved to be a potent inhibitor of food intake in
rodents.8 However, we have shown that the anorexigenic effect
of ODN is not mediated through BZRs9 and that intravenous
administration of 200 times the intracerebroventricular effective
dose does not affect food intake demonstrating that ODN exerts
its anorexigenic effect centrally and is unable to cross the BBB.8 We
have also demonstrated that ODN increases intracellular calcium
concentration in cultured rat astrocytes through activation of the
G protein-coupled receptor,10 and we have found that the C-
terminal octapeptide OP (RPGLLDLK) is the shorter isoactive
fragment.11
a 1,4-dihydropyridine carrier. Indeed, 1,4-dihydropyridines are
rather unstable5 and are subjected to many side reactions. In
particular a hydration reaction may occur on the 5,6-double
bond of the dihydropyridine. For several years, our research
group has been involved in the synthesis of stable annulated
NADH models in the quinoline series.6,7 In addition, we were
aLaboratoire de Chimie Fine et He´te´rocyclique UMR 6014 IRCOF, CNRS,
Universite´ et INSA de Rouen, BP 08, 76131, Mont-Saint-Aignan Ce´dex,
France. E-mail: cyril.papamicael@insa-rouen.fr, vincent.levacher@insa-
rouen.fr; Fax: +33-23-552-2962; Tel: +33-23-552-2484
bEuropean Institute for Peptide Research (IFRMP 23), Laboratory of
Cellular and Molecular Neuroendocrinology, INSERM U413, University
of Rouen, 76821, Mont-Saint-Aignan, France
cUnite´ mixte Institut Pasteur de Lille-Universite´ d’Artois, Lens, France
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In this context, we examined the possibility of developing a
strategy for targeting these peptides to the brain by means of
a redox chemical delivery system derived from a stable dihydro-
quinoline NADH model. To link the peptide and the carrier, we
next speculated that a general solid-phase strategy would be well-
suited (Fig. 2). To this end, the grafting of the chemical carrier
on a Merrifield resin was planned prior to conventional solid-
phase peptide synthesis. Quaternization of the quinoline ring and
subsequent cleavage from the resin followed by reduction of the
quinolinium salt was expected to provide the desired peptide
bearing the dihydroquinoline carrier. This solid-phase strategy
offers not only the advantage to be general and applicable to the
brain targeting of a large range of drugs, but should also be easily
exploitable by biochemists, commonly familiar with solid-phase
peptide synthesis.
Fig. 3 Design of a redox chemical delivery system by means of stable
annulated NADH model.
Synthesis of quinolines 2a,c. To prepare quinolines 2a,c
(Scheme 1), we used a Friedlander12 type condensation with a
Borsche modification.13 In the first step, nitration of commercially
available compound 1a led to 1b (81%) and subsequent reaction
with p-toluidine afforded imine 1c (88%) which was reduced by
Na2S14 to furnish amine 1d (85%). The stable amino imine 1d
was condensed with ethyl acetoacetate to produce the expected
quinoline 2a in high yield (89%). The direct conversion of ester
2a into amide 2c with (S)-phenylalaninol was rather tedious:
heating in toluene or in the presence of trimethylaluminium in
CH2Cl2 always led to low yields (ca. 10%). Alternatively, carboxylic
acid 2b was obtained by reacting 2a with lithium hydroxide
(82%) and conversion to the corresponding acid chloride was
achieved with oxalyl chloride. Finally, subsequent reaction with
(S)-phenylalaninol gave the required amide 2c (74%).
Fig. 2 General solid-phase approach for targeting peptides.
In this paper, we describe the solid-phase synthesis of ODN or
OP linked to stable annulated dihydropyridines.
Results and discussion
Design of the peptide carriers
Scheme 1 Reagents and conditions: (i) HNO3, 1 h at 0 ◦C then 2 h at 20 ◦C
(81%); (ii) p-toluidine, EtOH, 2 h, reflux (88%); (iii) Na2S, EtOH, 15 min
at reflux then 4 h at 0 ◦C (85%); (iv) Ethyl acetoacetate, piperidine, EtOH,
8 h, reflux (89%); (v) LiOH, H2O, EtOH, 3 h, reflux (82%); (vi) (COCl)2,
NEt3, DMF, CH2Cl2, 12 h, 20 ◦C then (S)-phenylalaninol, 30 min at −5 ◦C
then 24 h at 20 ◦C (74%).
To obtain a stable and lipophilic peptide carrier based on
the NADH/NAD+ redox system (Fig. 3), we planned to use
annulated NADH models developed by our research group.6,7
Annulation protects the dihydropyridine moiety against elec-
trophilic attacks on the 5,6-enamine double bond. As a result
of this protection, the reducing properties of annulated NADH
models are significantly altered. To overcome this side effect,
the electron donating alkoxy groups are expected to restore the
reducing properties obtained with simple 1,4-dihydropyridine
carriers. Besides, alkoxy groups could be advisedly used in the solid
phase synthesis to graft the peptide carrier. Finally, phenylalaninol
was envisaged as a linker between the chemical delivery system
and the active peptide. In the final step, esterases are consequently
expected to cleave the peptide from the quinoline carrier.
Synthesis of polymer-supported quinolinium salts and optimization
of the cleavage step conditions
Polymer-supported NADH models have been previously reported
by our laboratory7,15 and others.16 Our strategy is based on the
reaction of phenolic compounds with Merrifield resins prior to
quaternization and reduction steps. However, in the present study
it was also necessary to cleave the polymer-supported quinolinium
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salts prior to the final reduction step. So, to validate the whole
sequence of the solid phase approach, it was first essential to
determine the reaction conditions for the cleavage step of the
quinolinium salt from the resin.
at 20 ◦C mediated by NaH to yield the functionalized resins 4c,d
with a loading of 0.93 mmol g−1 and 0.90 mmol g−1 respectively
(estimated by nitrogen microanalysis). Resins 4c,d were treated
following the same reaction sequence previously developed with
2a and 2c (Scheme 2). Thus, quaternization reaction gave resins
4e,f (loading 0.81 mmol g−1 and 0.63 mmol g−1 respectively). The
final cleavage step with TMSOTf was accomplished affording pure
compounds 3c,d in rather fair yields. This sequence of reactions
was validated with compounds 2c and resin 4d, both bearing the
phenylalaninol linker.
Initially, we envisaged working with ester 2a and amide 2c
as models (Scheme 2), the benzyl group mimicking in both
cases the Merrifield resin. Quaternization of quinolines 2a,c
proceeded smoothly with the highly reactive methylating agent
methyl trifluoromethanesulfonate affording quinolinium salt 3a,b
in 72% and 90% yield respectively. In the literature, TMSI and
TMSOTf were successfully used to deprotect benzyl ether groups
and proved to be compatible with a pyridinium salt.17 Thus,
the quinolinium salts 3a,b were conveniently deprotected with
TMSOTf to furnish the desired compounds 3c,d in 90% yield.
It was then necessary to validate these deprotection conditions
on a Merrifield resin. To this end, benzyl ether cleavage of 2a,c
was accomplished by hydrogenation to provide 4a,b in 64% and
100% yield respectively. Phenolic derivatives 4a,b were reacted with
Merrifield resin (1% DVB, f 0 = 1.2 mmol g−1) in DMF for 4 days
Synthesis of ODN and OP on polymer-supported quinoline carrier
The next step of the synthesis was the introduction of the first
Na-protected L-amino acid. This was achieved by the coupling
reaction either with 2c or resin 4d in a customary manner by using
Fmoc-L-Lys(Boc)–OH and PyBop/DIEA activation (Scheme 3)
giving compound 5a in moderate yield (33%) and resin 5b in good
yield (loading 0.53 mmol g−1). Surprisingly, several attempts (i.e.
FEP/DIEA or MSNT/MeIm) to improve the yield of compound
5a failed.
Scheme 3 Reagents and conditions: (i) Fmoc-L-Lys(Boc)–OH, PyBop,
DIEA, DMF, 20 ◦C, 3 h from 2c (33%) and 2 h from resin 4d.
Both peptides, OP and ODN were synthesized from resin 5b
using a solid phase peptide synthesis (SPPS) with the standard
Fmoc strategy as previously described8 (Scheme 4). All the
corresponding protected (OtBu for Asp, Pmc for Arg, Trt for
Asn and Gln, tBu for Thr) Fmoc-L-amino acids were sequentially
coupled by in situ activation with HBTU/HOBt and DIEA in
NMP. Acetic anhydride was used in the capping procedure and the
N-terminal Fmoc group was successively removed by treatment
with piperidine in NMP. According to this procedure, we obtained
the functionalised resins 5c,d with the protected peptides OP and
ODN respectively.
Quaternization reaction and cleavage of resins 5c,d
We must point out that Yajima et al.18 have reported for the
chemical synthesis of proteins, an efficient deprotecting procedure
with TMSOTf. Since this reactant could be used with proteins,
we first attempted the cleavage step on resins 5c,d with TMSOTf
associated with trifluoroacetic acid in the presence of appropriate
scavengers (Scheme 5) from 0 ◦C to 20 ◦C for 2 h. Following
this procedure, the expected compounds 6a,b were obtained after
Scheme 2 Reagents and conditions: (i) MeOTf, CH2Cl2, 1 h, 20 ◦C from
2a (72%), 4 h from resins 4c,d or CHCl3, 6 h from 2c (90%); (ii) H2,
Pd/C (10%), MeOH, 2 h, 20 ◦C from 2a (64%) and 24 h from 2c (100%);
(iii) Merrifield resin (1% DVB; 1.2 mmol g−1), NaH, DMF, 4 days, 20 ◦C;
(iv) TMSOTf, CH2Cl2, 2 h, 20 ◦C from resin 4e and 4 h from 3a,b and
resin 4f.
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Scheme 4 Reagents and conditions: (i) piperidine 20%; (ii) Na-Fmoc
corresponding L-amino acid (4 equiv.), HBTU, HOBt, DIEA, NMP;
(iii) Ac2O, HOBt, DIEA.
side-chain deprotection and cleavage of the quinoline moiety from
the resin. MALDI-TOF analysis revealed the occurrence of 6a,b
as the major products. However, we notice that, in addition to the
presence of 6b, we also observed two other products corresponding
respectively to the loss of one and two molecules of water. In
the literature,19 a dehydratation reaction of the carboxamide part
of pyroglutamide to give a nitrile group was described in the
presence of TMSCl/ZnCl2. Consequently, the use of TMSOTf in
the presence of TFA could also promote such a side reaction from
Gln and Asp residues of ODN since both contain a carboxamide
group.
Scheme 5 Reagents and conditions: (i) TMSOTf, TFA, thioanisole,
ethanedithiol, m-cresol, 0 ◦C then 20 ◦C, 2 h; (ii) MeOTf, 0 ◦C, 4 h then
piperidine (20%), DMF then AcOH (5%), CH2Cl2.
Finally, we undertook the quaternization reaction of the quino-
line moiety of resins 5c,d (Scheme 5) with MeOTf. Subsequent
removal of the Fmoc group was accomplished by treatment with
20% piperidine in DMF for 20 minutes to afford the expected resins
7a,b. Cleavage of resins 7a,b was carried out with TMSOTf/TFA
in the presence of scavengers and conducted at 0 ◦C for 2 h
to lead to compounds 8a,b (Scheme 6). MALDI-TOF analysis
showed that 8a,b were the major products in the crude mixture. In
contrast to 6b, we observed in the case of 8b only a small peak on
the mass-spectrum corresponding to the loss of one molecule of
water. This is probably due to the fact that the cleavage step was
carried out at 0 ◦C with resins 7a,b instead of from 0 ◦C to 20 ◦C
as previously done with resins 5c,d. Furthermore, MALDI-TOF
analysis suggests that permethylation also occured with MeOTf
in addition to the quaternization reaction of quinoline. With
peptide OP, only monomethylation took place while mono, di
and trimethylation were observed with peptide ODN. Before the
final reduction step of the quinolinium salt, compounds 8a,b were
purified by using a preparative HPLC.
Scheme 6 Reagents and conditions: (i) TMSOTf, TFA, thioanisole,
ethanedithiol, m-cresol, 0 ◦C, 2 h.
BNAH (1-benzyl-1,4-dihydronicotinamide) also provided regios-
elective reduction of quinolinium salts to 1,4-dihydroquinolines
derivatives. But, despite our efforts, reduction of compound
3b either with sodium dithionite or BNAH failed. Speculating
that both 1,2- and 1,4-dihydroquinoline may undergo enzymatic
oxidation in the brain to give the corresponding quinolinium
salt, we turned our attention to a non-regioselective reduction
using NaBH4 (Scheme 7). Thus, reduction of quinolinium salt 3b
gave the expected compounds 9a,b. The analysis of the 1H-NMR
spectra assigned by NOE effects showed that a 11 : 9 mixture of
9a and 9b respectively was obtained. We were mainly interested in
Reduction of quinolinium salts 3b,d and 8a,b
We have previously reported the quantitative and regioselective
reduction of quinolinium salt derivatives7 by addition of a large
excess of sodium dithionite and sodium carbonate giving rise to
1,4-dihydroquinolines. In the literature,20 an alternative route using
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Scheme 7 Reagents and conditions: (i) NaBH4, EtOH, 20 ◦C, 2 h.
the reduction of compounds 8a,b. However, it was first necessary
for 8a,b, which are available only on a small scale, to find a non-
destructive and efficient analytical tool to monitor this reduction
step. Pyridinium salts are characterised by a strong UV-absorption
at 270 nm that decreases when reduced to the corresponding
dihydropyridine. Besides, a second band at 360 nm characteristic of
the pyridinium salt should be observed.21 Then, we first attempted
to investigate the reduction reaction by using UV–vis spectrometry
with 3b. The reduction of 3b was recorded after 2, 5, 10 and 20 min
of reaction (Fig. 4). It was obvious that both bands (255 nm
and 365 nm) from the quinolinium salt 3b decreased rapidly to
afford dihydroquinolines 9a,b after 20 min. Reduction was also
undertaken with 3d to obtain dihydropyridine derivatives 10a,b.
The so-obtained UV spectra were very close to those obtained with
3b. The study being successful, the next step was to perform the
same experiment with 8a,b (Scheme 8). In both cases, the evolution
of UV–vis spectrum is similar to that observed with 3b,d (Fig. 5
for 8a). Thus, the expected dihydropyridine derivatives 11a,b and
12a,b were obtained according to the disappearance of the two
characteristic bands (255 nm and 365 nm) of the quinolinium salt
8a,b and confirmed by a MALDI-TOF analysis.
Scheme 8 Reagents and conditions: (i) NaBH4, EtOH, 20 ◦C, 2 h.
Fig. 5 UV–vis spectra of quinolium salt 8a (red) and dihydropyridine
derivatives 11a,b formed by addition of NaBH4 on 8a after 15 min (violet)
and 20 min (black).
rational design of new peptide carriers led us to prepare polymer-
supported NAD+ annulated models. To this end, the grafting of
quinolines to a Merrifield resin and subsequent stepwise SPPS of
sophisticated neuropeptides such as OP and ODN was successfully
achieved. Then, quaternization reaction and subsequent cleavage
from the Merrifield resin of the so-obtained quinolinium salt
derivatives followed by a final reduction afforded the expected
compounds 11a,b and 12a,b as a mixture of 1,2- and 1,4-
dihydropyridine derivatives.
Fig. 4 UV–vis spectra of quinolium salt 3b (red) and dihydropyridine
derivatives 9a,b formed by addition of NaBH4 on 3b after 2 min (blue),
5 min (violet), 15 min (black) and 20 min (green).
Conclusion
Experimental
The fairly good stability of annulated NADH models makes
this class of potential peptide carriers of particular interest. The
Infrared spectra were recorded on a Beckmann IR 4250
spectrometer. 1H and 13C-NMR spectra were recorded on a
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200 MHz or 300 MHz Bruker apparatus and calibrated with
the residual undeuterated solvent unless specified. Spectra were
recorded in deuteriochloroform. The UV–vis spectroscopic
measurements were carried out with a Varian Cary 100
biospectrophotometer. All amino-acid residues, O-benzotriazol-
2.40 (3 H, s), 4.00 (3 H, s), 5.32 (2 H, s), 7.23 (4 H, s), 7.39–7.53
(5 H, m), 7.64 (1 H, s), 7.90 (1 H, s), 9.04 (1 H, s); dC(50 MHz,
CDCl3) 21.0, 56.4, 71.2, 107.4, 111.3, 121.1 (×2), 125.7, 127.7
(×2), 128.4, 128.6 (×2), 129.8 (×2), 135.4, 136.6, 142.5, 148.5,
150.8, 152.2, 155.0.
1-yl-N,N,Nꢀ,Nꢀ-tetramethyluronium
hexafluorophosphate
6-(p-Tolylaminomethylidene)-4-benzyloxy-3-methoxyaniline 1d.
A solution of compound 1c (3.76 g, 10 mmol) in EtOH (250 mL)
was heated to reflux and sodium sulfite nonahydrate (5.28 g,
22 mmol) was added. After a few minutes, a vigorous reaction
occurred and the heating was maintained for 10 min. After cooling
at 0 ◦C for 4 h, the precipitate was filtered. The remaining solution
was concentrated under reduced pressure. Water (100 mL) was
added to the residue and a new crop of precipitate was obtained.
Compound 1d was obtained as a yellow solid (2.94 g, 85%). Mp
142 ◦C (from H2O); (Found: C 76.31; H 6.39; N 8.13. Calc for
C22H22N2O2: C 76.28; H 6.40; N 8.09%); mmax/cm−1 3383, 1629,
1599, 1548, 1506, 1455, 1245, 1208, 1140, 1006; dH(200 MHz,
CDCl3, TMS) 2.37 (3 H, s), 3.90 (3 H, s), 5.07 (2 H, s), 6.24 (1 H,
s), 6.46 (2 H, br s), 6.85 (1 H, s), 7.06–7.20 (4 H, m), 7.31–7.47
(5 H, m), 8.34 (1 H, s); dC(50 MHz, CDCl3) 20.9, 55.7, 72.6, 99.1,
110.0, 115.2, 120.7 (×2), 127.5 (×2), 127.8, 128.4 (×2), 129.6 (×2),
134.8, 137.4, 139.4, 145.6, 149.5, 153.9, 161.1; m/z (EI) 346.
(HBTU), 1-hydroxybenzotriazole (HOBt), piperidine and N,N-
diisopropylethylamine (DIEA) were purchased from Applied
Biosystems (St Quentin en Yvelines, France). Trifluoroacetic
acid, trichloroacetic acid, phenol, thioanisol, ethanedithiol, N-
methylpyrrolidin-2-one (NMP), N-methylmorpholine (NMM),
N,N-dimethylformamide (DMF) were from Sigma-Aldrich
Chimie. Merrifield resin (f = 1.2 meq g−1, 1% DVB, 200–400
mesh) was from Novabiochem. Resin 4c was prepared by using
R
a Questꢀ 210 parallell synthesizer (Argonaut Technologies
A.G.). Flash chromatography was performed with silica gel
60 (70–230 mesh from Merck) and monitored by thin layer
chromatography (TLC) with silica plates (Merck, Kieselgel 60
F254). Peptides 8, 11, 12a,b were purified by reversed-phase
HPLC on a semipreparative Vydac C18 column (1 × 25 cm,
Touzart and Matignon, Courtaboeuf, France) using a linear
gradient (10–50% over 40 min) of CH3CN/TFA (99.9 : 0.1, v/v)
at a flow rate of 5 mL min−1. Analytical RP-HPLC (1 mL min−1)
was performed on a Vydac C18 column (0.45 × 25 cm) using a
linear gradient (10–40% over 30 min) of CH3CN/TFA (99.9 :
0.1, v/v) at a flow rate of 1 mL min−1. The purified peptides
8, 11, 12a,b were characterized by FAB-MS on a conventional
EB geometry mass spectrometer JEOL model AX-500 equipped
with a DEC data system (JEOL-Europe SA, Croissy-sur-Seine,
France). Compounds 6a,b and 8a,b were characterized by
using matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) MS (Tofspec E, Micromass, Manchester, UK) in
the reflectron mode with a-cyano-4-hydroxycinnamic acid as a
matrix.
Ethyl 6-benzyloxy-7-methoxy-2-methylquinoline-3-carboxylate
2a. To a solution of compound 1d (3.46 g, 10 mmol) and
ethylacetoacetate (1.52 g, 12 mmol) in EtOH (150 mL) were added
a few drops of piperidine. The resulting solution was refluxed for
10 h. After cooling at 0 ◦C, the precipitate was filtered, rinsed with
petroleum ether (75 mL) and dried to furnish compound 2a as a
beige powder (3.12 g, 89%). Mp 151 ◦C (from EtOH); (Found:
C 71.38; H 5.98; N 4.04. Calc: for C21H21NO4: C 71.78; H 6.02;
N 3.99%); mmax/cm−1 2968, 1702, 1496, 1270, 1214, 1188, 1164,
1069; dH(300 MHz, CDCl3, TMS) 1.37 (3 H, t, J 7.1), 2.90 (3 H,
s), 3.95 (3 H, s), 4.34 (2 H, q, J 7.1), 5.16 (2 H, s), 7.01 (1 H, s),
7.25–7.44 (6 H, m), 8.45 (1 H, s); dC(75 MHz, CDCl3) 14.1, 25.3,
55.9, 60.8, 70.5, 107.2, 120.7, 121.3, 127.0 (×2), 127.9, 128.4 (×2),
135.9, 137.8, 145.9, 148.5, 154.4, 156.4, 166.4.
5-Benzyloxy-4-methoxy-2-nitrobenzaldehyde 1b. Nitric acid
(10 mL, 68% in water) was introduced in a round bottomed
flask fitted with a mechanical stirrer and cooled with an ice bath
at 0 ◦C. Thereafter, 3 benzyloxy-4-methoxybenzaldehyde (2 g,
14 mmol) was slowly added. The reaction mixture was stirred for
30 min at 0 ◦C and 1 h at 20 ◦C. Then, the reaction mixture was
poured on ice-water to lead to a yellow solid which was filtered
and washed with water. The yellow solid was dried under high
vacuum to afford 5-benzyloxy-4-methoxy-2-nitrobenzaldehyde 1b
(1.93 g, 81%). Mp 130 ◦C (from H2O); (Found: C, 62.93; H 4.55;
N 4.72. Calc. for C15H13NO5: C 62.72; H 4.56; N 4.88%); mmax cm−1
1683, 1577, 1512, 1338, 1280 1216, 1061; dH(200 MHz, CDCl3,
TMS) 4.03 (3 H, s), 5.27 (2 H, s), 7.44 (6 H, m), 7.63 (1 H, s), 10.42
(1 H, s); dC(50 MHz, CDCl3) 56.7, 71.3, 107.3, 111.3, 125.35, 127.5
(×2), 128.6, 128.8 (×2), 135.0, 143.9, 152.3, 152.8, 187.6.
6-Benzyloxy-7-methoxy-2-methyl-quinoline-3-carboxylic acid
2b. A solution of lithium hydroxide monohydrated (252 mg,
6 mmol) in absolute ethanol (15 mL) was refluxed and ester 2a
(702 mg, 2 mmol) was added. The reflux was continued for 3 h.
Thereafter, the hot solution was filtered and cooled to 20 ◦C. The
mixture was acidified with a solution of hydrochloric acid (2N).
The acid which precipitated was filtered and dried under vacuum
to furnish compound 2b as a white solid (529 mg, 82%). (Found:
C 70.71; H 5.09; N 4.63. Calc for C19H17NO4: C 70.58; H 5.30; N
4.33%); dH(300 MHz, DMSO-d6) 2.85 (3 H, s), 4.05 (3 H, s), 5.30
(2 H, s), 7.40–7.75 (7 H, m), 8.70 (1 H, s); dC(75 MHz, DMSO-d6)
25.4, 56.2, 70.3, 107.4, 107.9, 121.1, 122.2, 128.6 (×3), 128.8 (×2),
136.7, 138.0, 145,8, 148.6, 154.4, 155.9, 168.2.
(5-Benzyloxy-4-methoxy-2-nitro-benzylidene)-p-tolylamine 1c.
In a flask were introduced 5-benzyloxy-4-methoxy-2-nitroben-
zaldehyde (4.31 g, 15 mmol), p-toluidine (1.93 g, 18 mmol) and
absolute ethanol (300 mL). The mixture was refluxed for 2 h and,
after cooling at 0 ◦C, a solid was filtered. Drying under high
vacuum gave compound 1c as a yellow solid (4.97 g, 88%). Mp
N-(1-Hydroxymethyl-2-phenylethyl)-6-benzyloxy-7-methoxy-2-
methyl-quinoline-3-carboxamide 2c. In a flask, flushed with
nitrogen, was introduced compound 2b (400 mg, 1.24 mmol)
and 20 mL of CH2Cl2. Oxalyl chloride (0.44 mL, 5 mmol) and
a few drops of DMF were added and the reaction mixture was
stirred overnight. After evaporation of the solvent under reduced
pressure, the residue was placed under a nitrogen atmosphere.
◦
150 C (from EtOH); (Found: C 70.27; H 5.34; N 7.56. Calc for
C22H20N2O4: C 70.18; H 5.35; N 7.44%); mmax/cm−1 1567, 1511,
1382, 1322 1274, 1221, 1062, 986; dH(200 MHz, CDCl3, TMS)
In
a
tricol flask flushed with nitrogen, were introduced
822 | Org. Biomol. Chem., 2006, 4, 817–825
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S-phenylalaninol (755 mg, 5 mmol), NEt3 (0.7 mL, 5 mmol) and
dichloromethane (20 mL). The reaction mixture was cooled at
−10 ◦C. Dichloromethane (20 mL) was added to the intermediate
acid chloride and the suspension was added dropwise to the
ether (50 mL) was added and the precipitate was collected by
filtration, rinsed with dry diethyl ether, and dried under vacuum
to give compound 3c as a beige solid (371 mg, 90%). mmax/cm−1
3460br, 1731, 1408, 1321, 1247, 1024; dH(300 MHz, (CD3)2CO)
1.44 (3 H, t, J 7.1), 3.31 (3 H, s), 4.29 (3 H, s), 4.50 (2 H, q,
J 7.1), 4.69 (3 H, s), 7.77 (1 H, s), 7.98 (1 H, s), 9.29 (1 H, s);
dC(75 MHz, (CD3)2CO) 14.8, 20.4, 41.5, 58.4, 63.8, 100.5, 112.7,
123.5 (JC–F 337 Hz), 125.2, 125.8, 139.6, 145.3, 151.0, 157.4, 159.4,
165.6; dF(282 MHz, (CD3)2CO) −78.7.
◦
tricol flask in order to keep the temperature below 0 C. At the
◦
end of addition, the reaction medium was kept at −5 C for 1 h
and stirred at 20 ◦C for 18 h. The solution was concentrated
under reduced pressure and the residue was purified by column
chromatography on silica (CH2Cl2/EtOH: gradient from 19 : 1 to
9 : 1). Compound 2c was obtained as a white-yellow solid (418 mg,
74%). (Found: C 74.01; H 6.05; N 6.04. Calc for C28H28N2O4: C
73.66; H 6.18; N 6.14%); mmax/cm−1 3277, 3028, 2925, 1639, 1495,
1252, 1028, 739, 698; dH(300 MHz, CDCl3, TMS) 2.49 (3 H, s),
2.73–2.84 (1 H, dd, J3 8, J4 13), 2.91–2.98 (1 H, dd, J1 7 J2 = 13),
3.38–3.54 (2 H, ddd, J1 7 J3 4 J2 11), 3.95 (3 H, s), 4.32 (1 H,
m), 5.19 (2 H, s), 7.24 (1 H, s), 7.21–7.42 (12 H, m), 7.71 (1 H,
s); dC(50 MHz, CDCl3, TMS) 22.6, 37.1, 53,2, 56.3, 63.5, 71.1,
106.7, 107.6, 127.5 (×2), 128.4, 128.7 (×3), 128.9 (×3), 129.5
(×2), 134.0, 136.4, 138.3, 145.1, 149.0, 154.2, 170.1.
6-Hydroxy-3-(1-hydroxymethyl-2-phenyl-ethylcarbamoyl)-7-
methoxy-1,2-dimethyl-quinolinium trifluoromethanesulfonate 3d
was obtained as described for 3c, from compound 3b (170 mg,
0.28 mmol) in CH2Cl2 (15 mL), trimethylsilyl trifluoromethane-
sulfonate (0.22 mL, 1 mmol) and stirring for 4 h. The solution
was centrifuged at 1200 rpm after addition of diethyl ether to
lead to compound 3d in a complete conversion and as a very
hygroscopic solid. (Found: C 52.21; H 4.95; N 4.99; S 5.85. Calc for
C23H25F3N2O7S: C 52.07; H 4.75; N 5.28; S 6.04%); dH(300 MHz,
(CD3)2CO) 2.60 (3 H, s), 2.60–2.70 (2 H, m), 3.60 (2 H, m), 4.00
(3 H, s), 4.30 (4 H, m), 7.00–7.20 (5 H, m), 7.30 (1 H, s), 7.50 (1 H,
s), 7.95 (1 H, d, J 12), 8.30 (1 H, s); dC(75 MHz, (CD3)2CO) 20.4,
38.1, 41.2, 55.3, 58.1, 63.5, 100.0, 112.0, 125.2, 127.1, 129.6 (×2),
130.7 (×2), 132.2, 139.9, 141.7, 150.7, 155.1, 158.1, 170.1.
6-Benzyloxy-3-ethoxycarbonyl-7-methoxy-1,2-dimethyl-quinoli-
nium trifluoromethanesulfonate 3a. In a flask flushed with
nitrogen, methyl trifluoromethanesulfonate (136 lL, 1.2 mmol)
was added to a solution of ester 2a (351 mg, 1 mmol) in CH2Cl2
(20 mL). The solution was stirred for 1 h and then concentrated in
vacuum. Dry diethyl ether (50 mL) was added and the precipitate
collected by filtration, rinsed with 10 mL of dry diethyl ether, and
dried under vacuum. Compound 3a (371 mg, 72%) was obtained
as a a beige solid. (Found: C 53.37; H 4.79; N 3.02; S 6.35. Calc
for C23H24F3NO7S: C 53.59; H 4.69; N 2.72; S 6.22%); mmax/cm−1
3072, 3002, 1723, 1625, 1428, 1321, 1262, 1033; dH(300 MHz,
CDCl3, TMS) 1.46 (3 H, t, J = 7.1), 3.17 (3 H, s), 4.21 (3 H, s),
4.46 (2 H, q, J = 7.1), 4.51 (3 H, s), 5.25 (2 H, s), 7.37–7.50 (6 H,
m), 7.65 (1 H, s), 9.06 (1 H, s); dC(75 MHz, CDCl3, TMS) 14.1,
19.8, 41.0, 58.2, 63.1, 71.4, 99.4, 109.0, 120.6 (JC–F 319 Hz), 123.3,
123.8, 127.4 (×2), 128.7, 128.9 (×2), 134.7, 139.4, 144.1, 151.1,
155.7, 160.1, 163.8; dF(282 MHz, CDCl3) −78.7.
6-Hydroxy-7-methoxy-2-methyl-quinoline-3-carboxylic
acid
ethyl ester 4a. In a flask purged with nitrogen were placed
compound 2a (351 mg, 1 mmol), absolute ethanol (100 mL)
and Pd/C (10%, 125 mg, 0.12 mmol). Nitrogen was replaced by
◦
hydrogen and the reaction mixture was stirred at 20 C for 2 h.
After a filtration over celite, the solution was evaporated under
reduced pressure and left for 2 h at 20 ◦C to afford compound
4a (167 mg, 64%) as beige powder. Mp 196 ◦C; (Found: C 64.04;
H 6.05; N 5.35. Calc. for C14H15NO4: C 64.36; H 5.79; N 5.36);
mmax/cm−1 3390, 1714, 1495, 1303, 1207, 1077; dH(300 MHz,
CDCl3, TMS) 1.45 (3 H, t, J 7.1), 2.95 (3 H, s), 4.06 (3 H, s), 4.42
(2 H, q, J 7.1), 6.33 (1 H, br s), 7.24 (1 H, s), 7.39 (1 H, s), 8.57
(1 H, s); dC(75 MHz, CDCl3, TMS) 14.3, 25.2, 56.3, 61.2, 106.5,
108.9, 121.7, 122.1, 138.2, 145.4, 146.2, 152.2, 156.2, 166.7.
6-Benzyloxy-3-(1-hydroxymethyl-2-phenyl-ethylcarbamoyl)-7-
methoxy-1,2-dimethyl-quinolinium trifluoromethanesulfonate 3b
was obtained as a white solid in 90% yield (56 mg), as described
for 3a, from compound 2c (50 mg, 0.1 mmol) in CHCl3 (20 mL),
methyl trifluoromethanesulfonate (15 lL, 0,12 mmol) and stirring
for 6 h. (Found C 57.86, H 4.98, N 4.52, S 5.06. Calc for C30H31
F3N2O7S C 58.06, H 5.03, N 4.51, S 5.17%); dH(300 MHz, CDCl3,
TMS) 2.60 (3 H, s), 2.80–3.00 (2 H, m), 3.70 (3 H, m), 4.00 (3
H, s), 4.20 (3 H, s), 4.32 (1 H, m), 5.15 (2 H, s), 7.05–7.42 (12 H,
m), 7.85 (1 H, d, J 12), 8.25 (1 H, s); dC(50 MHz, CDCl3, TMS)
19.7, 37.2, 39.9, 54.1, 57.4, 64.0, 71.6, 98.3, 109.3, 120.5 (JC–F 319
Hz), 124.1, 126.8, 127.8, 128.7 (×2), 128.9 (×2), 129.1 (×2), 129.5,
129.7, 131.2, 135.2, 138.1, 138.3, 141.3, 151.0, 153.6, 158.7, 166.2;
dF(282 MHz, CDCl3) −78.9; m/z (FAB-MS) 471 (M+–OTf).
N -(1-Hydroxymethyl-2-phenylethyl)-6-hydroxy-7-methoxy-2-
methyl-quinoline-3-carboxamide 4b was obtained as a yellow solid
(310 mg, 97%) as described for 4a, from compound 2c (400 mg,
0.87 mmol) in MeOH (50 mL), Pd/C (10%, 211 mg, 0.2 mmol)
and stirring for 24 h. dH(300 MHz, CD3OD) 2.30 (3 H, s), 2.65 (1
H, dd, J 9 and 13), 2.95 (1 H, dd, J 9 and 13), 3.60 (2 H, dapp, J 11),
3.85 (3 H, s), 4.35 (1 H, m), 6.90 (1 H, s), 7.00–7.20 (6 H, m), 7.65
(1 H, s); dC(75 MHz, CD3OD) 22.9, 38.7, 55.1, 56.9, 65.2, 106.9,
110.3, 123.5, 127.9, 129.9 (×2), 130.3, 130.7 (×2), 135.0, 140.4,
•
145.3, 149.6, 154.2, 155.0, 171.8; m/z (IC+) 367 [MH]+ .
Polymer-supported compound 4a (resin 4c). Reactions were
R
carried on a Questꢀ 210. In a round-bottomed flask flushed
3-Ethoxycarbonyl-6-hydroxy-7-methoxy-1,2-dimethyl-quinoli-
nium trifluoromethanesulfonate 3c. To a solution of compound 3a
(500 mg, 0.97 mmol) in CH2Cl2 (10 mL) under a nitrogen stream,
trimethylsilyl trifluoromethanesulfonate (0.57 mL, 2.91 mmol) was
added dropwise. The solution became red and was stirred for 3 h.
A solution of MeOH (3 mL) saturated with HCl gas was added
and the solution concentrated to a volume of 2 mL. Dry diethyl
with nitrogen were introduced a Merrifield resin (400 mg, f =
1.2 meq g−1), compound 4a, sodium hydride (19 mg of a dispersion
in oil at 60%, 0.47 mmol), and DMF (10 mL). The reaction mixture
was mechanically stirred for 4 days at 20 ◦C. The resin was removed
by filtration and washed successively with CH2Cl2 (3 × 50 mL),
water/THF (1 : 1, 3 × 50 mL), MeOH (3 × 50 mL), CH2Cl2 (3 ×
50 mL) and placed in a drying oven under reduced pressure. A
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beige resin was obtained (Found: N 1.30 corresponding to f =
0.93 mmol g−1); mmax/cm−1 3059–2849, 1718, 1491, 1200, 1062.
120.3, 121.1, 125.2, 127.4 (×2), 127.6 (×4), 128.0 (×2), 128.4
(×2), 128.6, 129.1 (×4), 129.6 (×2), 133.7, 136.7, 137.6, 141.5,
143.7, 144.0, 145.3, 149.0, 154.0, 154.5, 156.7, 169.2, 173.2; m/z
•
Polymer-supported compound 4b (resin 4d) was obtained as a
beige resin as described for resin 4c, from compound 4b (400 mg,
1.1 mmol), Merrifield resin (500 mg, f = 1.2 meq g−1), sodium
hydride (42 mg of a dispersion at 60% in oil, 1.05 mmol) and DMF
(5 mL). Drying was achieved with a nitrogen stream. (Found: N
2.53 corresponding to f = 0.90 mmol g−1); mmax/cm−1 3398, 1656,
1600, 1243, 1154, 1026, 739, 695.
(FAB-MS) 907 [M]+ .
Fmoc-Lys(Boc)–Qui–P (resin 5b) was obtained as described for
compound 5a, from resin 4d (438 mg, f = 0.90 mmol g−1), PyBop
(547 mg, 1.05 mmol), and N-a-Fmoc-N-e-Boc-L-lysine (492 mg,
1.0 mmol), DMF (5 mL) and DIEA (581 lL, 3.15 mmol) for 2 h.
The resin was washed successively with DMF (3 × 5 mL), CH2Cl2
(3 × 5 mL), MeOH (3 × 5 mL), CH2Cl2 (3 × 5 mL) and dried
with a nitrogen stream to lead to resin 5b (Found N 2.95, f =
0.53 mmol g−1); mmax/cm−1 3422, 1702, 1491, 1238, 1152, 736, 670.
Polymer-supported compound 3c (resin 4e). In a flask flushed
with nitrogen were placed resin 4c (400 mg, f = 0.93 meq g−1) and
CH2Cl2 (10 mL). Methyl triflate (110 lL, 0.97 mmol) was added
dropwise and the reaction mixture was mechanically stirred for
4 h. The resin was filtered and washed three times with CH2Cl2,
twice with MeOH, and then three times with CH2Cl2. The resin
was placed in a drying oven under reduced pressure to furnish a
beige resin (Found: N 1.13 corresponding to f = 0.81 mmol g−1);
dF(282 MHz, CDCl3) −78.5; mmax/cm−1 3024, 2919, 1728, 1621,
1505, 1253, 1151, 1029.
Peptide synthesis (resins 5c,d). Peptide synthesis was carried
out on resin 5b on a 433A peptide synthesizer (Applied Biosystems,
Saint-Quentin-en-Yvelines, France) using the standard manu-
facturer’s procedures as previously described.11 All Fmoc-L-
aminoacids (4 equiv.) were coupled by in situ activation with
HBTU/HOBt (3.6 equiv., 1 : 1, mol/mol) and DIEA (8 equiv.)
in NMP. Reactive side chains were protected as followed: Gln
and Asn, tritylamide (Trt), Thr, tert-butyl ether (t-Bu), Asp,
tert-butyl ester (Ot-Bu), Arg, pentamethylchromansulfonylamide
(Pmc), and Lys, tert-butyloxycarbonyl (Boc). After completion of
chain assembly, Na-acetylation of peptides was performed on the
resin by addition of a mixture of acetic anhydride/DIEA/HOBt
(4 equiv., 1 : 1 : 1, mol/mol/mol) in NMP for 5 min. Reactions were
monitored by the Kaiser test.22 Micro cleavages were carried out
on 5c and 5d to give respectively 6a m/z (MALDI-TOF) 1482.5
(MH+) and 6b m/z (MALDI-TOF) 2483 (MH+).
Polymer-supported compound 3d (resin 4f) was obtained as
described for resin 4e, from resin 4d (354 mg, f = 0.90 mmol g−1),
CH2Cl2 (5 mL) and methyl triflate (96 lL, 0.85 mmol) after drying
with a nitrogen stream. (Found: N 1.76 corresponding to f =
0.63 mmol g−1); dF(282 MHz, CDCl3) −79.
Cleavage of resin 4e. In a dry round-bottomed flask flushed
with nitrogen, resin 4e (300 mg, f = 0.81 mmol g−1) and CH2Cl2
(10 mL) were introduced. Thereafter, trimethylsilyle trifluoro-
methanesulfonate (210 lL, 1 mmol) was added dropwise and the
reaction mixture was mechanically stirred for 2 h. A few drops of
a solution of MeOH saturated with HCl gas was added until the
solution went colourless. The solution was filtered and the resin
was washed with CH2Cl2, then with dry acetone. The solution
was concentrated under reduced pressure and a few drops of
dichloromethane and dry diethyl ether were added. The precipitate
was filtered to afford compound 3c (71 mg, 69%). The analyses
were in agreement with those described previously in this paper
for 3c.
Compounds 6a,b. To the corresponding resin 5c,d was added
a mixture of TMSOTf/TFA/thioanisole/m-cresol/ethanedithiol
(10 mL, 1.94 mmol, 6.89 mmol, 1.2 mmol, 0.2 mmol, 0.6 mmol)
for 2 h at 0 ◦C. The solution was filtered and compounds 6a,b
were precipitated by addition of TBME, centrifuged (4500 rpm),
washed twice with TBME and lyophilised. 6a m/z (MALDI-TOF)
1482.5 (MH+) and 6b m/z (MALDI-TOF) 2483 (MH+).
Quaternarization reactions of polymer-supported quinolines
(resins 7a,b). To the corresponding resin 5c,d under an at-
mosphere of nitrogen were added CH2Cl2 (4 mL) and methyl
trifluoromethanesulfonate (2 equiv.). The reaction mixture was
stirred for 2 h at 20 ◦C. The resin was washed three times with
CH2Cl2, twice with MeOH, three times with CH2Cl2 and twice with
DMF. A solution of piperidine (20% in DMF) was introduced. The
resin became red and was stirred for 20 min. The resin was filtered,
and washed twice with DMF, twice with CH2Cl2 and twice with
a solution of acetic acid (5% in CH2Cl2). The resin lost its colour
and was finally washed twice with CH2Cl2. The resin was dried
overnight in a dessicator under vacuum. Micro cleavages were
done according to the procedure described for 6a,b with resin 7a
and resin 7b to afford respectively compounds 8a m/z (MALDI-
TOF) 1273.5 (MH+) and 8b m/z (MALDI-TOF) 2274 (MH+),
2289 (MH+ + 15), 2304(MH+ + 30).
Cleavage of resin 4f was obtained as described for the cleavage
of resin 4e, from resin 4f (150 mg, f = 0.63 mmol g−1), CH2Cl2
(5 mL) and trimethylsilyl trifluoromethanesulfonate (246 lL,
1.17 mmol) to afford compound 3d (26 mg, 52%). The analyses
were in agreement with those described previously in this paper
for 3d.
6-tert-Butyloxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarb-
onylamino)-hexanoic acid 2-[(6-benzyloxy-7-methoxy-2-methyl-
quinoline-3-carbonyl)-amino]-3-phenyl-propyl ester 5a. A solution
of compound 2c (200 mg, 0.44 mmol), PyBop (230 mg, 0.44 mmol),
CH2Cl2 (20 mL), N-a-Fmoc-N-e-Boc-L-lysine (205 mg, 0.44 mol)
and DIEA (160 lL, 0.87 mmol) was stirred for 3 h at 20 ◦C. The
solvent was removed under reduced pressure and the residue was
purified by chromatography on silica (from EtOAc/cyclohexane
to EtOAc/EtOH) to afford compound 5a as a white solid (132 mg,
33%). dC(75 MHz, CDCl3) 22.5, 23.6, 28.8 (×3), 30.0 (×2), 37.7,
39.8, 47.2, 50.7, 54.6, 56.5, 65.8, 67.1, 71.1, 79.7, 107.5, 107.8,
Compounds 8a,b were obtained according to the procedure
described for 6a,b with resin 7a and resin 7b to afford respectively
compounds 8a m/z (MALDI-TOF) 1273.5 (M+) and 8b m/z
(MALDI-TOF) 2274 (M+), 2289 (M+ + 15), 2304(M+ + 30).
824 | Org. Biomol. Chem., 2006, 4, 817–825
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Dihydroquinolines 9a,b. To compound 3b (50 mg, 0.08 mmol)
in absolute ethanol (1 mL) was added NaBH4 (4.6 mg, 0.12 mmol).
The reaction mixture was stirred for 2 h at 20 ◦C and 1 mL
of degassed water was added. The solution was extracted with
degassed dichloromethane (3 × 5 mL). The combined organic
layers were dried over sodium sulfate and concentrated under
reduced pressure to afford 9a,b (29 mg, 76%) as a yellow oil.
kmax(EtOH) 245 and 335 nm; 9a: dH(300 MHz, CDCl3, TMS) 1.00
and 1.25 (3 H, m), 2.7–3.3 (6 H, m), 3.6–3.8 (2 H, m), 3.90 (3 H, s),
4.21 (2 H, m), 5.02 and 5.24 (2 H, 2xs), 6.08 and 6.4–6.6
(2 H, m), 7.15–7.5 (10 H, m); 9b: 1.72 and 2.20 (3 H, 2xs), 2.7–
3.3 (6 H, m), 3.6–3.8 (2 H, m), 3.87 (3 H, s), 4.34 (2 H, m),
5.07 and 5.29 (2 H, 2xs), 6.08 and 6.4–6.6 (2 H, m), 7.15–7.5
(10 H, m).
4 P. F. Torrence, J. Kinjo, K. Lesiak, J. Balzarini and E. De Clercq, FEBS
Lett., 1988, 234, 135; C. K. Chu, V. S. Bhadti, K. J. Doshi, J. T. Etse,
J. M. Gallo, F. D. Boudinot and R. F. Schinazi, J. Med. Chem., 1990,
33, 2188; P. F. Torrence, J. Kinjo, S. Khamnei and N. H. Greig, J. Med.
Chem., 1993, 36, 529.
5 J. M. Linget, G. Schlewer and C. G. Wermuth, Bioorg. Med. Chem.
Lett., 1993, 3, 1309; C. C. Johnson, J. L. Gardner, C. H. Suelter
and D. E. Metzler, Biochemistry, 1963, 2, 689; C. S. Kim and S.
Chaykin, Biochemistry, 1968, 7, 2339; P. Van Eikeren, P. Kenney and
R. Tokmakian, J. Am. Chem. Soc., 1979, 101, 7402.
6 J. Cazin, G. Dupas, J. Bourguignon and G. Que´guiner, Tetrahedron
Lett., 1986, 27, 2375; V. Levacher, N. Boussad, G. Dupas, J. Bour-
guignon and G. Que´guiner, Tetrahedron, 1992, 48, 831; P. Charpentier,
V. Lobre´gat, V. Levacher, G. Dupas, G. Que´guiner and J. Bourguignon,
Tetrahedron Lett., 1998, 39, 4013.
7 C. Vitry, J.-L. Vasse, G. Dupas, V. Levacher, G. Que´guiner and J.
Bourguignon, Tetrahedron, 2001, 57, 3087.
8 J. G. de Mateos-Verchere, J. Leprince, M.-C. Tonon, H. Vaudry and J.
Costentin, Eur. J. Pharmacol., 2001, 414, 225.
9 M.-H. Orta, J.-C. Do-Re´go, J. Leprince, M.-C. Tonon, H. Vaudry and
J. Costentin, 6th colloquium of the Neurosciences Society-Rouen, France,
13–16th May 2003.
10 P. Gandolfo, C. Patte, J. Leprince, J.-L. Thoumas, H. Vaudry and M.-C.
Tonon, Eur. J. Pharmacol., 1997, 322, 275.
Dihydroquinolines 10a,b were obtained as a yellow oil (174 mg,
81%) as described for dihydroquinolines 9a,b, from 3d (300 mg,
0.56 mmol), NaBH4 (42 mg, 1.12 mmol) and absolute ethanol
(1 mL). kmax(EtOH) 245 and 335 nm.
11 J. Leprince, P. Gandolfo, J.-L. Thoumas, C. Patte, J.-L. Fauche`re,
H. Vaudry and M.-C. Tonon, J. Med. Chem., 1998, 41, 4433; J.
Leprince, H. Oulyadi, D. Vaudry, O. Masmoudi, P. Gandolfo, C. Patte,
J. Costentin, J.-L. Fauche`re, D. Davoust, H. Vaudry and M.-C. Tonon,
Eur. J. Biochem., 2001, 268, 6045.
12 C. C. Cheng and S. J. Yan, The Friedlander synthesis of quinolines,
Organic Reactions, Wiley, New York, 1982, vol. 28, 37.
13 W. Borsche and W. Ried, Justus Liebigs Ann. Chem., 1943, 554, 269; W.
Borsche and J. Barthenheier, Justus Liebigs Ann. Chem., 1941, 548, 50.
14 H. K. Porter, The Zinin reduction of nitroarenes, Org. React., 1973, 20,
455.
Dihydroquinolines 11a,b and 12a,b. To the corresponding
quinolinium salt 8a,b dissolved in the minimum of absolute
ethanol, was added NaBH4 (2 equiv.). The reaction mixture was
stirred for 2 h at 20 ◦C and the solvent was removed under reduced
pressure. The residue was dissolved in acetonitrile, and purified on
silica grafted C18 with a gradient water/acetonitrile (water 100% to
acetonitrile 100%). The collected fractions were analysed by HPLC
and those containing the dihydropyridines were lyophilised and
stocked at −4 ◦C. 11a,b and 12a,b: kmax(EtOH) 240, 265, 315 nm.
15 D. Losset, G. Dupas, J. Bourguignon and G. Que´guiner, Polym. Bull.
(Berlin), 1989, 21, 649; G. Dupas, A. Decormeille, J. Bourguignon and
G. Que´guiner, Tetrahedron, 1989, 45, 2579.
16 S. Obika, T. Nishiyama, S. Tatematsu, M. Nishimoto, K. Miyashita
and T. Imanishi, Heterocycles, 1998, 49, 261 (and references therein).
17 H. Anan, N. Seki, O. Noshiro, K. Honda, K. Yasumuro, T. Ozasa and
N. Fusetani, Tetrahedron, 1996, 52, 10849.
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