Synthesis and separation of tritiated inhibitors of aminopeptidase A and their prodrugs

Article abstract of DOI:10.1002/jlcr.888

With the aim of studying the bioavailability of two related aminopeptidase A (APA) inhibitors, we have synthesized tritiated disulfide prodrugs. These molecules, 4,4′-dithiobis-(3,3′-amino)-1,1′-butanesulfonic acid (RB 150) and its 2,2-dimethylpropyl este

Full text of DOI:10.1002/jlcr.888

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 997–1005.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.888
Research Article
Synthesis and separation of tritiated inhibitors
of aminopeptidase A and their prodrugs
1
1
1
! "
Nicolas Inguimbert , Pascale Coric , Helene Dhotel , Catherine Llorens-
Cortes2, Marie-Claude Fournie-Zaluski1 and Bernard P. Roques1,*
´
1
! ! ! !
Laboratoire de Pharmacologie Chimique et genetique, Universite Rene Descartes Paris
V, UFR des Sciences Pharmaceutiques & Biologiques, INSERM U640, CNRS FRE
2463, 4, Avenue de l’Observatoire, 75270 Paris, Cedex 06, France
2
" ! !
INSERM U36, College de France, Chaire de medecine experimentale, 11, Place
Marcelin Berthelot, 75005 Paris, France
Summary
With the aim of studying the bioavailability of two related aminopeptidase A (APA)
inhibitors, we have synthesized tritiated disulfide prodrugs. These molecules, 4,40-
dithiobis-(3,30-amino)-1,10-butanesulfonic acid (RB 150) and its 2,2-dimethylpropyl
ester(RB 151) bearing one tritium atom per monomer in position 2 were obtained with
high purity and a final specific activity of about 30 Ci/mmol. The active radiolabeled
inhibitor EC 33, Ki=270 nM for APA was obtained by reduction of the disulfide
bond of RB 150. Copyright # 2004 John Wiley & Sons, Ltd.
3
Key Words: thiol compounds; H; aminopeptidase A; inhibitors; cardiovascular
Introduction
Aminopeptidase A (APA, EC 3.4.11.7, glutamyl aminopeptidase) is a
membrane bound zinc metalloprotease that selectively cleaves the N-terminal
glutamyl or aspartyl residue of peptides. This enzyme is found in large
amounts at the periphery in the kidney and the small intestine, but is
also present in the brain.1,2 Two physiological substrates of this enzyme
have been identified in the central nervous system. The first is the sulfated
CCK 8(Asp–Tyr(SOꢀ3 )–Met–Gly–Trp–Met–Asp–Phe–NH2),3 the second is
angiotensin II (Asp–Arg–Val–Tyr–Ile–His–Pro–Phe)4,5 which is matured
by APA into angiotensin III. These two peptides exert different physio-
logical effects. In the brain CCK 8 binds to the CCK2 receptor6 and is
!
!
*Correspondence to: B. P. Roques, Universite Rene Descartes Paris V, UFR des Sciences Pharmaceutiques
& Biologiques, 4, Avenue de l’Observatoire, 75270 Paris, Cedex 06, France. E-mail: roques@pharmacie.
univ-paris5.fr
Copyright # 2004 John Wiley & Sons, Ltd.
Received 28 June 2004
Revised 28 July 2004
Accepted 16 August 2004

998
N. INGUIMBERT ET AL.
associated with, among other effects, the control of memory processes and
anxiety.7,8 Inhibition of APA by a thiol inhibitor EC 339 has enabled workers
to demonstrate that in the central nervous system, angiotensin III is
responsible for vasopressin release and increase in blood pressure.10,11
Furthermore, overexpression of APA was recently observed in blood vessels
of human tumors and APA inhibitors have been reported to inhibit
angiogenesis.12
Thus APA inhibitors may find therapeutic applications in the regulation
of behavior, in the treatment of cardiovascular diseases13–15 or as antiangio-
genic agents. For the two first applications, APA inhibition needs to be
achieved at the nervous central system level and at the periphery for the
regulation of angiogenesis. Cardiac failure remain one of the major health
problems and the discovery of new targets to treat related pathologies is
of major interest. We have previously demonstrated that intracerebroven-
tricular injection of the APA inhibitor EC 33 induces a decrease of blood
pressure in spontaneously hypertensive rats (SHR). In contrast, this molecule
which is unable to enter the brain, did not affect blood pressure (BP) when
given intravenously suggesting that the antihypertensive effect of EC 33 occurs
at the brain level.16
In order to improve the bioavailability of EC 33, prodrugs were developed
based on a previously demonstrated increase in brain penetration of thiol
inhibitors by formation of their disulfide forms.17 The designed prodrugs of
EC 33 (RB 151 and RB 150) were found to be able to decrease BP in SHR
after systemic or oral administration.18,19
As previously shown in the case of a mixed NEP/APN inhibitor17 endowed
with similar chemical characteristics by comparison with RB 150 and RB
151, the disulfide bridge of these latter prodrugs is reduced by a physiologi-
cally dependent process, leading to the active inhibitor (Figure 1). In our
case we thought that a similar reaction could occur by esterase hydrolysis
at the periphery or in the brain, of the neopentyl moiety protecting RB 152
and 151. Moreover, the disulfide bridge of RB 150 or 151 has been observed
to be reduced in the brain.17 Thus RB 151 is supposed to yield three
compounds: RB 150, 152 and EC 33 (Figure 1). All these molecules are devoid
of any chromophore thus precluding a quantitative estimation of their
level using HPLC detection methods. Furthermore their derivatization using
a fluorophore such as fluorescamin is ineffective for analytical purposes.20
Generally, it is acknowledged that a tritium labeled version of the drug
candidate provides the ideal standard for pharmacokinetics assays. There-
fore, we have developed two tritium labeled prodrugs of EC 33 and a
method to generate and separate the prodrugs and their metabolites,
which will be useful for performing preclinical studies using RB 151 and
RB 150.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

SYNTHESIS AND SEPARATION
999
H2N
H2N
NH2
SH
O
S
S
O
S
O
S
O
O
S
O
OH
OH
OH
Reductase
RB 150
EC 33
Esterase
H2N
Esterase
H2N
NH2
SH
O
S
S
O
S
O
O
S
O
S
O
Reductase
O
O
O
RB 151
RB 152
Figure 1. Structure of RB 151 and its metabolites
Results and discussion
The previously described synthetic pathway to obtain EC 33 was not a good
starting point for the synthesis of tritium labeled RB 151, because of the
number of reaction steps and some stringent reaction conditions.9 We have
thus developed a shorter and new synthetic approach in order to obtain this
compound (Figure 2). Esterification of commercially available (S)-N-tertio-
butoxycarbonylamino-S-trityl-cysteine with trimethylsilyldiazomethane yields
quantitatively compound 121 which was then used for the introduction of the
sulfonate side chain by a previously described one pot reaction22 involving a
DIBAl-H induced reduction of the methyl ester moiety leading to an
aluminoxyacetal intermediate. The latter was not isolated and was submitted
to a Wittig–Horner reaction23 leading to compound 2 after semi-preparative
HPLC purification. The reduction of the a–b-unsaturated sulfonate 2 (23 mg)
was carried out using 10 Ci of sodium boro[3H]hydride in ethanol affording,
after HPLC purification, 400mCi of the radiolabelled intermediate 3. This last
compound was deprotected using TFA and triisopropylsilane and air oxidized
to yield, after HPLC purification, 50mCi of tritiated RB 151 with a specific
activity of 29 Ci/mmol and a radiochemical purity >97%. Deprotection of
5 mCi of the sulfonate 3 in 1 ml ethanol with 1 ml 12N HCl at 808C for three
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

1000
N. INGUIMBERT ET AL.
3
Figure 2. Synthetic scheme for the inactive standard and the H prodrugs RB
150 and 151
days, followed by iodine oxidation, yielded, after HPLC purification, 2.1 mCi of
RB 150 with a specific activity of 29 Ci/mmol.
Furthermore the possible metabolites of RB 150 and RB 151, respectively
EC 33 and RB 152, could be generated using a 10 fold excess of dithiothreitol
as reducing agent.24 The formation of EC 33 and RB 152 was monitored by
TLC separation in isopropanol/water/acetic acid 8-2-1 as eluent followed by
four days exposure of the plate on a Bas TR2025 plate and measurement with
a Bas 5000 phosphoimager. Moreover, this separation method of RB 151 and
their by-products can be helpful when the metabolic studies of RB 151 will be
undertaken. The sensitivity of this autoradiographic method was tested using
RB 151. After 10 days exposure, 0.1 nCi of RB 151 could be easily detected
(Figure 3 right).
RB 152
RB 151
EC 33
RB 150
Figure 3. Autoradiographic plate of RB 151 metabolites (left TLC) and
sensitivity study with RB 151 (right TLC)
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

SYNTHESIS AND SEPARATION
1001
In conclusion, three tritiated prodrugs and their parent APA inhibitor have
been synthesized. Furthermore, a TLC separation and a quantification method
of the prodrug and of the different metabolites was carried out. These
compounds and the separation method should provide useful tools for
pharmacokinetic studies of APA inhibitors.
Experimental part
General
All reagents and solvents are commercially available and used directly without
purification. The natural amino acid derivatives were purchased from Bachem
(Budendorf, Switzerland). Reagents were from Aldrich (Strasbourg, France).
Solvents were from SDS (Peypin, France). The tritiation was undertaken by
Amersham Biosciences (Cardiff, Walles). TLC was performed on Merck 60
F254 precoated silica plates. Analytical HPLC was performed on a LC 10
ADvp Shimadzu instrument (Touzart & Matignon, France) equipped with a
Kromasil C18 or a spherisorb S5CN column (4.6 ꢁ 150 mm) and an UV
detector (210 and 254 nm, Shimadzu SPD10Avp detector). Semi-preparative
HPLC was performed on a Waters 600 instrument (Milford, MA, USA)
equipped with a Kromasil C18 column (21 ꢁ 150 mm) and peak detection was
achieved by UV at 210 and 254 nm. Radioactivity measurements were
performed on a Wallac 1409 liquid scintillation counter (Turku, Finland)
using Beckman scintillant. A Bruker 400 MHz instrument (Wissenbourg,
France) was used for proton NMR characterization using TMS as internal
reference. TLC plates were exposed on a Bas-TR2025 Fuji imaging plate
(Raytest, Paris) and the levels of radioactive material evaluated using a Fuji
Bas 5000 phosphoimager (Tokyo, Japan). Electropray mass spectra were
recorded on a Esquier–Bruker spectrometer (Wissenbourg, France).
(S)-N-tertiobutoxycarbonylamino-S-trityl-cysteine methyl ester (1). (Trimethyl-
silyl)diazomethane (7 ml) was added dropwise at room temperature to a stirred
solution of (S)-N-Boc-S-trityl-cysteine 5 g (10.7 mmol) in a mixture of ether
80 ml and methanol 20 ml. After 6 h reaction time the solvent was removed under
reduced pressure to afford a white foam which was used in the next step without
further purification. 5.15 g (100%). TLC (Eluent cHex, EtOAc 6/4 Rf = 0.57. 1H
NMR (CDCl3) d 1.35 (s, 9H, C(CH3)3), 2.5 (d, J = 5.9 Hz, 2 H, S–CH2), 3.6 (s,
3H, O–CH3), 4.2 (td, 1H, CHNH), 4.95 (d, 1H, CHNH), 7.15 (td, 3H, CH
arom.), 7.2 (td, 6H, CH arom.), 7.35 (d, 6H, CH arom.). HPLC C18 Kromasil (5 m
(
100 A) CH3CN/H2O (TFA) 85/15, tR = 8.3min.
(3S)-3-tertiobutoxycarbonylamino-4-tritylsulfanyl-but-1-ene-1-sulfonic acid 2,2-
dimethyl-propyl ester ð2Þ. To a solution of neopentyl diethoxyphosphoryl-
methanesulfonate 3.22 g (1.2 equiv.) in 20 ml dry THF was added dropwise, at
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

1002
N. INGUIMBERT ET AL.
ꢀ788C, 8 ml (1.2 equiv.) of a solution of n-butyllithium (1.6 M in hexane).
After the mixture was stirred for 30 min at this temperature, a solution of S-N-
Boc-S-trityl-cysteine methyl ester (5.1 g in 10 ml dry THF) was added,
immediately followed by a dropwise addition of 8 ml diisobutyl aluminum
hydride (1.5M in toluene). The resulting mixture was stirred overnight. The
solvents were removed under reduced pressure. 180 ml of diethylether and
200 ml 1M hydrochloric acid were added to the residue and the mixture stirred
for 1 h. The organic layer was separated, washed with brine, dried (Na2SO4),
filtered and concentrated in vacuo. The product was purified by semi
(
preparative HPLC C18 Kromasil (5 m 100 A, 20 ꢁ 250 mm) CH3CN/H2O
(TFA) 85/15 to afford 2.4 g (37%) of a white solid. TLC (Eluent cHex, EtOAc
1
9/1 Rf = 0.14. H NMR (CDCl3) d 0.9 (s, 9H, CH2C(CH3)3), 1.35 (s, 9H,
C(CH3)3), 2.4 (dd, 2H, S–CH2), 3.6 (s, 2H, SO2O–CH2), 4.2 (td, 1H, CHNH),
4.55 (d, 1H, CHNH), 6.1 (dd, 1H, CH ¼ CH–SO3), 6.55 (d, 1H, CH–SO3),
(
7.15ꢀ7.3 (m, 15H, CH arom.). HPLC C18 Kromasil (5 m 100 A) CH3CN/H2O
(TFA) 80/20, tR = 22.8 min. [M + Na ]+ = 618.3. [a]d = ꢀ12.13, (c = 0.52
in EtOH) l = 589 nm.
ð3S Þ-3-tertiobutoxycarbonylamino-4-tritylsulfanyl-butane-1-sulfonic acid 2,2-
dimethyl-propyl ester (3). To a stirred solution of compound 2, 1 g
(1.68 mmol) in 5.7 ml EtOH (95%) was added 28.5 mg (0.84 mmol) of sodium
borohydride in 6 ml ethanol. The resulting mixture was stirred overnight at
room temperature. The reaction was quenched by addition of 2 ml water.
After concentration of the solvent under reduced pressure the residue was
(
purified by semipreparative HPLC C18 Kromasil (5 m 100 A, 20 ꢁ 250 mm)
CH3CN/H2O (TFA) 85/15 to afford 0.85 g (85%) of a white solid. TLC
1
(Eluent cHex, EtOAc 9/1 Rf = 0.14. H NMR (CDCl3) d 0.9 (s, 9H,
CH2C(CH3)3), 1.35 (s, 9H, C(CH3)3), 1.9 (m, 2H, CH2–CH2–SO3), 2.8 (d,
J=7 Hz, 2H, S–CH2), 2.9 (t, J = 7 Hz, 2H, CH2–CH2–SO3), 3.55 (m, 1H,
CHNH), 3.7 (s, 2H, SO2O–CH2), 4.55 (d, 1H, CHNH), 7.15–7.3 (m, 15H, CH
(
arom.). HPLC C18 Kromasil (5 m 100 A) CH3CN/H2O (TFA) 80/20, tR
=
21.1 min. [M + Na ]+ = 620.3. [a]d = ꢀ2.5 (c = 0.64 in EtOH) l = 589 nm.
2-3H-(3S)-3-tertiobutoxycarbonylamino-4-tritylsulfanyl-butane-1-sulfonic acid
2,2-dimethyl-propyl ester ð3Þ. After HPLC purification 400 mCi of tritiated
compound 3 was obtained as described above using 23 mg of compound 2
dissolved in 100 ml EtOH and 10 Ci of sodium boro[3H]hydride.
ð3S Þ-3-amino-4-sulfanyl-butane-1-sulfonic acid 2,2-dimethyl-propyl ester tri-
fluoroacetate salt (RB 152). To a solution of 0.23 g (0.38 mmol) of compound
2 in 1.6 ml of dry dichloromethane were added 180 ml (0.84 mmol) of
triisopropylsilane and 1.6 ml (20 mmol) trifluoroacetic acid. The mixture was
stirred under argon for 2 h. The solvents were removed under reduced
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

SYNTHESIS AND SEPARATION
1003
pressure. The residue was dissolved in water (10 ml) and extracted three times
with 5 ml ether. The aqueous phase was lyophilyzed to afford a white powder
0.1 g (71%). TLC (Eluent iPrOH, H2O, Acetic Acid 8/2/1 Rf = 0.6. 1H NMR
(DMSO) d 0.85 (s, 9 H, CH2C(CH3)3), 2.0 (m, 2H, CH2–CH2–SO3), 2.7 (m,
2H, S–CH2), 3.3 (m, 1H, CHNH3), 3.4 (t, J =7 Hz, 2H, CH2–CH2–SO3), 3.8
(
(s, 2H, SO2O–CH2), 7.9 (s, 3H, CHNH3). HPLC C18 Kromasil (5 m 100 A)
CH3CN/H2O (TFA) 30/70, tR = 7.6 min. [a]d = 6.4 (c = 0.77 in H2O) l =
589 nm. [RB 152 + H]+ = 509.3.
ð3S; 30SÞ-4,40-dithiobis-3,30-amino-1,10-butanesulfonic acid 2,2-dimethylpropyl
ester trifluoroacetate salt (RB 151). 20 ml of an ethanolic iodide solution
(37 mg lꢀ1) was added to a solution of RB 152 (4 mg) in 250 ml EtOH. The
colorless solution turned orange. The solvent was removed in vacuo to yield
pure RB 151 (4 mg, yield 100%). TLC (Eluent iPrOH, H2O, Acetic Acid 8/2/1)
1
Rf = 0.5. H NMR (DMSO) d 0.85 (s, 18H, CH2C(CH3)3), 2.05 (td, 2H,
CH2–CH2–SO3), 2.9 (dd, 2H, S–CH2), 3.05 (dd, 2H, S–CH2), 3.5 (m, 6H,
CHNH3, CH2–CH2–SO3), 3.85 (s, 4H, SO2O–CH2), 8.1 (s, 6H, CHNH3).
Mass spectroscopy: [RB 151 + H ]+ = 509.1852. HPLC : HPLC C18
(
Kromasil (5 m 100 A) CH3CN/H2O (TFA) 30/70, tR = 2.6 min. Spherisorb
Cyano S5CN; 150 ꢁ 4.6 mm 5 m Solvent A: 0.05% TFA in CH3CN, solvent B
0.05% TFA in H2O. Gradient 30% B to 100% B over 10 min, tR = 6.9 min,
[a]d = +78.7 (c = 0.48 in H2O), l = 589 nm.
2; 20-3H-4,40-dithiobis-(3-amino)-1,10-butanesulfonic acid 2,2-dimethylpropyl ester
trifluoroacetate salt (RB 151). Deprotection of 400 mCi of intermediate 3 in a
mixture of 100 ml CH2Cl2, 100 ml TFA and 10 ml trisisopropylsilane yields
tritiated RB152. After removal of the solvents the crude product was subjected
to air oxidation in 100 ml EtOH. After HPLC purification 50 mCi of 3H RB151
were obtained with a specific activity (determined by mass spectrometry) of
1.07 TBq/mmol (29Ci/mmol) [(3H)RB 151 + H ]+ = 513.2029.
2; 20 3H-(3s,30s)-4,40-dithiobis-(3,30-amino)-1,10-butanesulfonic acid RB 150. To
7 mg of compound 2 as a tracer were added 5 mCi of the tritiated form in 1 ml
EtOH and 1 ml 12N HCl in a sealed vial. The reaction mixture was stirred at
808C for five days. After cooling to room temperature and 3 h speedvac
concentration, the residue was dissolved in 100 ml water. The desired
radiolabeled compound was purified by HPLC on a reverse phase kromasil
(
C18 (5 m 100 A) column (mobile phase: 0.05%TFA in water/CH3CN) eluted in
isocratic mode at 90% CH3CN at a flow rate of 1 ml/min in 10 min. Fractions
of 1 ml were collected each minute to provide 2.1 mCi of product with a
specific activity of 29 Ci/mmol.
Inactive RB 150 was used as a reference and had the following spectroscopic
1
features: TLC (Eluent iPrOH, H2O, Acetic Acid 8/2/1) Rf = 0.26. H NMR
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

1004
N. INGUIMBERT ET AL.
(D2O) d 2.15 (td, 4H, CH2–CH2–SO3), 2.85 (dd, 2H, S–CH2), 2.95 (t, 4H,
CH2–CH2–SO3), 3.15 (dd, 2H, S–CH2), 3.75 (m, 2H, CHNH3). HPLC:
Spherisorb Cyano S5CN; 150 ꢁ 4.6 mm 5 m Solvent A : 0:05% TFA in
CH3CN, solvent B 0.05% TFA in H2O. 20% B, tR = 3.1 min. [a]d
+194.5 (c = 1.33 in H2O) l = 589 nm.
=
Acknowledgements
The authors thank Dr. Armand Blommaert for assistance with the manuscript
and Glaxo-SmithKline for financial support.
References
1. Li L, Wu Q, Wang J, Bucy RP, Cooper MD. Tissue Antigens 1993; 42: 488–496.
!
2. Zini S, Masdehors P, Lenkei Z, Fournie-Zaluski MC, Roques BP, Corvol P,
Llorens-Cortes C, Neurosciences 1997; 78: 1187–1193.
!
3. Migaud M, Durieux C, Viereck J, Soroca-Lucas E, Fournie-Zaluski MC, Roques
BP. Peptides 1996; 17: 601–607.
4. Nagatsu I, Nagatsu T, Yamamoto T, Glenner GG, Mehl JW. Biochim Biophys
Acta 1970; 198: 255–270.
5. Healy DP, Wilk S. Brain Research 1993; 606: 295–303.
6. Noble F, Wank SA, Crawley JN, Bradwejn J, Seroogy KB, Hamon M, Roques
BP. Pharmacol Rev 1999; 51: 745–781.
7. Taghzouti K, Lena I, Dellu F, Roques BP, Dauge V, Simon H. Psychopharma-
cology 1999; 143: 141–149.
8. Singh L, Lewis AS, Field MJ, Hughes J, Woodruff GN. Proc Natl Acad Sci 1991;
88: 1130–1133.
!
9. Chauvel EN, Coric P, Llorens-Cortes C, Wilk S, Roques BP, Fournie-Zaluski
MC. J Med Chem 1994; 37: 1339–1346.
!
10. Reaux A, De Mota N, Zini S, Cadel S, Fournie-Zaluski MC, Roques BP, Corvol
P, Llorens-Cortes C. Neuroendocrinology 1999; 69: 370–376.
!
11. Reaux A, Iturrioz X, Vazeux G, Fournie-Zaluski MC, David C, Roques BP,
Corvol P, Llorens-Cortes C. Biochem Soc Trans 2000; 28: 435–440.
12. Marchio S, Lahdenranta J, Schlingemann RO, Valdembri D, Wesseling P, Arap
MA, Hajitou A, Ozawa MG, Trepel M, Giordano RJ, Nanus DM, Dijkman
HBPM, Oosterwijk E, Sidman RL, Cooper MD, Bussolino F, Pasqualini R, Arap
W. Cancer Cell 2004; 5: 151–162.
!
13. Roques BP, Corvol P, Fournie-Zaluski MC, Llorens-Cortes C. Patent WO
09936066.
14. David C, Bischoff L, Meudal H, Mothe A, De Mota N, DaNascimento S,
!
Llorens-Cortes C, Fournie-Zaluski MC, Roques BP. J Med Chem 1999; 42:
5197–5211.
!
15. David-Basei C, Bischoff L, Fournie-Zaluski MC, Roques BP. J Label Compd
Radiopharm 2001; 44: 89–98.
!
16. Zini S, Fournie-Zaluski MC, Chauvel E, Roques BP, Corvol P, Llorens-Cortes C.
Proc Natl Acad Sci 1996; 93: 11968–11973.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005

SYNTHESIS AND SEPARATION
1005
!
17. Fournie-Zaluski MC, Coric P, Turcaud S, Lucas E, Noble F, Moldonado R,
Roques BP. J Med Chem 1992; 35: 2473–2481.
!
18. Roques BP, Corvol P, Fournie-Zaluski MC, Llorens-Cortes C. Patent WO
2004007441A2.
!
19. Fournie-Zaluski MC, Fassot C, Valentin B, Djordjijevic D, Reaux-Le Goazigo A,
Corvol P, Roques BP, Llorens-Cortes C. Proc Natl Acad Sci USA 2004; 101:
7775–7780.
20. Shifen M, Xiaojing D, Yongjian L. Chromatog B 2002; 782: 251–267.
21. Mashimoto N, Aoyama T, Shiori T. Chem Pharm Bull 1981; 29: 1475–1478.
!
22. David C, Bischoff L, Roques BP, Fournie-Zaluski M-C. Tetrahedron 2000; 56:
209–215.
23. Carretero JC, Demillequand M, Ghosez L. Tetrahedron Lett 1987; 43: 5125–5134.
24. Kumar P, Bose NK, Gupta KC. Tetrahedron Lett 1991; 32: 967–970.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 997–1005