Enantiomerical excess determination, purification and biological evaluation of (3S) and (3R) α,β-butenolide analogues of isobenzofuranone

Article abstract of DOI:10.1016/j.bmcl.2004.11.065

The asymmetric synthesis of isobenzofurane analogues, new potential antiviral agents, is reported. High performance liquid chromatography (HPLC) was the technique chosen to separate the enantiomers. We describe this chiral separation and then determine th

Full text of DOI:10.1016/j.bmcl.2004.11.065

Bioorganic & Medicinal Chemistry Letters 15 (2005) 501–504
Enantiomerical excess determination, purification and
biological evaluation of (3S) and (3R) a,b-butenolide
analogues of isobenzofuranone
Emmanuelle Lipka,a Marie-Pierre Vaccher,a Claude Vacchera,* and Christophe Lenb,
a
´
Laboratoire de Chimie Analytique EA 1043, Universite de Lille 2, BP 83-3, rue du Pr. Laguesse, 59006 Lille, France
Laboratoire des Glucides, FRE 2779, Universite de Picardie-Jules Verne, 33 rue Saint Leu, 80039 Amiens, France
b
´
Received 16 September 2004; revised 16 November 2004; accepted 25 November 2004
Available online 23 December 2004
Abstract—The asymmetric synthesis of isobenzofurane analogues, new potential antiviral agents, is reported. High performance
liquid chromatography (HPLC) was the technique chosen to separate the enantiomers. We describe this chiral separation and then
determine the enantiomerical excess. The biological results of each tested enantiomer are given.
ꢀ 2004 Elsevier Ltd. All rights reserved.
O
N
For several years there has been an intensive search fo-
cused on the synthesis of nucleosides and analogues in
the therapy of viral diseases. A number of nucleoside
analogues have been found to possess antiviral activities
against human immunodeficiency virus (HIV)1 such as
AZT,2 ddC,3 ddI,4 d4T,5 3TC6 and ABC.7 The use of
L-nucleosides has greatly increased due to their potent
biological activity and lower toxicity compared to their
D-nucleoside analogues.8 However, the toxicity and
emergence of mutant and resistant viral strains have
been a critical problem in using these chemotherapeutic
agents. Consequently the obvious emphasis is to design
drugs with a higher therapeutic index. Among the com-
pounds approved by the US Food and Drugs Adminis-
tration for the treatment of HIV infection, d4T shows
selective anti-HIV activity comparable to that of AZT
in vitro.6 However, d4T is less toxic and less inhibitory
to mitochondrial DNA replication than AZT14 (Fig. 1).
O
N
O
N
NH
NH
HO
NH
HO
O
HO
O
O
O
O
O
N3
AZT
d4T
BcF
Figure 1. Potent anti-HIV drugs.
no activity against HIV reverse transcriptase except for
the uracil derivative having a benzoyl group in 50-posi-
tion. One reason for the inactivity of the benzo[c]furan
nucleosides in general might be due, simply, to them
not being good substrates for HIV reverse transcriptase.
However, the activity found in the one exception might
have arisen from the lactone 10S resulting from a glyco-
sidic bond cleavage and not from the parent nucleoside.
This hypothesis has prompted us to investigate the syn-
thesis of compounds related to a,b-butenolides. The
chiral derivatives such as (S)-benzoyloxymethyl-a,b-but-
enolide have served as key intermediates for the elabora-
tion into various natural products and analogues.14–16
a,b-Butenolide derivatives are widely present in second-
ary metabolites, which show interesting physio-
logical activities as exemplified by the aglycon of ranun-
culin,17,18 namely, (S)-hydroxymethyl-a,b-butenolide
(Fig. 2). Herein we describe an alternative synthesis of
the 3-benzoyloxymethylisobenzofuranone 10S and
Novel nucleoside analogues based on 1,3-dihydroben-
zo[c]furan glycone BcF have been described as thymine
analogues of d4T.9–13 Unfortunately, all of the benzo[c]-
furan derivatives synthesized in our work have shown
Keywords: Chiral HPLC; HIV; Nucleoside.
*
Corresponding author. Tel.: +33 3 20 96 47 01; fax: +33 3 20 95 90
09; e-mail: cvaccher@pharma.univ-lille2.fr
´ `
Present address: Universite de Poitiers, UMR CNRS 6514, Synthese
´
´
et Reactivite des Substances Naturelles, 40, avenue du Recteur
Pineau, 86022 Poitiers Cedex, France. E-mail: christophe.len@
univ-poitiers.fr
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.065

502
E. Lipka et al. / Bioorg. Med. Chem. Lett. 15 (2005) 501–504
10R,19 which are potential metabolites of the corre-
sponding 50-benzoylated nucleoside BcF and are ben-
zoylated derivatives of hydroxymethyl-a,b-butenolide
to make available a library of novel compounds for bio-
logical evaluation.
anhydrous toluene to afford the styrene derivative 3.
Asymmetric dihydroxylation (AD) using AD-mix-a
and AD-mix-b gave a mixture of two diols 4 and 5.20–23
A selective protection of the primary hydroxyl group
was required to selectively obtain only the isobenzofu-
rane derivative. Reaction of the diol 4 with benzoyl
chloride afforded the intermediate 6 with a free second-
ary hydroxyl group. Removal of the acetal from com-
pound 6 in an acidic medium (acetone, water, PTSA)
To synthesize the target compounds 3-benzoyloxy-
methylisobenzofuranone 10S and 10R, retrosynthesis
analysis was examined to be the best way for this novel
aromatic family. The lactone was obtained starting from
the phthalaldehyde (1) as depicted in Scheme 1. Com-
pound 1 was firstly protected using an achiral group
such as propane-1,3-diol to give the benzaldehyde deriv-
ative 2. Homologation of the remaining formyl group
was effected by a Wittig reaction using methyltriphenyl-
phosphonium bromide and potassium t-butoxide in
gave the desired benzo[c]furan
8 obtained using
24
RuCl3–NaIO4 to give solely 10S in 50% yield. Com-
pound 5 gave the enantiomer 10R in a similar yield. Be-
cause the AD of the styrene derivatives with AD-mix-a
and AD-mix-b were key reactions in the synthesis of the
lactone 10S and 10R, a reliable method for the determi-
nation of the ee of the products was required. The abso-
lute configuration was determined by comparison of the
samples obtained through an unambiguous stereoselec-
tive pathway.19
OH
BzO
O
O
O
HO
HO
The enantiomerical excess determination of the lactones
10S and 10R was developed in the case of the first strat-
egy. HPLC studies have been effected by modification of
different parameters such as the concentration of mobile
phase modifier, the structure of polysaccharide deriva-
tives of the chiral stationary phases (CSPs). The effect
O
O
O
OH
Ranunculin
10S
Figure 2.
O
O
O
O
O
O
O
i
ii
1
2
3
iii
iv
RO
OH O
RO
OH O
O
O
4 (R = H)
6 (R = Bz)
5 (R = H)
7 (R = Bz)
v
v
vi
vi
O
O
OH
OH
BzO
BzO
BzO
8
9
vii
vii
O
O
O
O
BzO
10S
10R
Scheme 1. Reagents: (i) PTSA, propan-1,3-diol, toluene; (ii) tert-BuOK, CH3(C6H5)3PBr, toluene; (iii) AD-mix-a, tert-BuOH, H2O; (iv) AD-mix-b,
tert-BuOH, H2O; (v) BzCl, (C2H5)3N, toluene; (vi) PTSA, acetone, H2O; (vii) NaIO4, RuCl3, acetonitrile, ethyl acetate, H2O.

E. Lipka et al. / Bioorg. Med. Chem. Lett. 15 (2005) 501–504
503
of concentration of alcohol modifier (from 5% to 30%)
on retention k0 and resolution Rs factors was investi-
gated using ethanol. It can be seen that an increase of
the polar modifier concentration in the mobile phase,
from eluent A–D, leads to a decrease of retention and
resolution factors k0 and Rs, for the four types of CSPs
(Table 1).
n-hexane/ethanol (80:20) (eluent C). The purity was
determined by the relative percentages of peaks areas.
The lactone 10S was found with 98.50% and the enan-
tiomer 10R with 97.50% (Fig. 3).
After optimization, Chiralpak AD analytical column
was chosen in order to purify each enantiomer and to
obtain an analytical sample through repetitive injections
(200 lL; 30 mM). The mobile phase was composed of a
mixture of n-hexane and ethanol (60:40) for enantiomer
10R and (70:30) for the enantiomer 10S, the eluent rate
was 2.5 mL minꢀ1 in both cases. The purity was deter-
mined on the four CSPs to be greater than 99% for each
Nowadays it is well known, that chiral recognition pro-
cess results from several interactions of: (i) different
magnitude involving hydrophobic, hydrogen, dipole–
dipole interactions between the electronegative atoms
of the solute and the –COO and the –NHCO of the CSPs;
(ii) p–p interaction, between the aromatic ring of the sol-
ute and the substituted phenyl moiety of the CSP. Chiral
recognition process results also from inclusion pheno-
menon of the solute into the chiral cavity of the CSP.
This last contribution was due to the regular higher or-
der structure of the chiral sites of the CSP: a left handed
threefold (3:2) helicoidal chain conformation for modi-
fied cellulose and a (4:1) helicoidal chain conformation
for modified amylose. This point was illustrated by the
best results obtained with Chiralpak AD towards Chi-
ralcel OD-H, though these two CSPs had the same chi-
ral selector. The results of the investigation of the effect
of alcoholic modifier on retention and resolution
showed that for a same eluent (C) cellulose OD seems
better appropriated than cellulose OJ (Table 1). Com-
paring the two types of amylose CSPs, amylose AD
was the best one to resolve those enantiomers with an
Rs of about 20 (Table 1). Here the carbamate residue ap-
pears to be the most important adsorbing site of the phe-
nylcarbamate derivatives of polysaccharide. It is notable
that the enantiomer 10R eluted first on cellulose tris-3,5-
dimethylphenylcarbamate (OD-H), cellulose tris-meth-
ylbenzoate (OJ) and on amylose tris-(S)-1-phenylethylc-
arbamate (AS) but the enantiomer 10S eluted first on
amylose tris-3,5-dimethylphenylcarbamate (AD). The
reversal of the elution order of the two enantiomers,
on this amylose-based CSP has often been reported
and was generally related to an alteration in the steric
environment of the chiral cavities (Table 1).25 After opti-
mization, the chiral purity of each enantiomer obtained
after asymmetric synthesis has been evaluated using
the four CSPs, with a mobile phase composed of
Amylose AD; eluent C
0.150
10R
0.100
0.050
10S
0.000
0.800
Amylose AS; eluent C
0.600
0.400
0.200
10R
10S
0.000
0.600
Cellulose OJ; eluent C
Cellulose OD; eluent B
10R
0.400
0.200
10S
0.000
0.600
10R
0.400
0.200
0.000
10S
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00
Minutes
Figure 3. CLHP analysis of enantiomer 10R in the presence of
enantiomer 10S as impurity after asymmetric synthesis, on various
polysaccharide CSPs and eluents.
Table 1. Retention factors (k0) enantioselectivities (a) and resolution factors (Rs) of enantiomers 10S and 10R on different CSPs
CSP
Eluent
k10
a
Rs
First eluted isomer
Cellulose OJ
A
B
C
B
D
B
C
C
D
10.97
6.00
3.74
2.90
0.73
4.32
2.23
3.42
2.13
1.15
1.17
1.16
1.27
1.15
1.30
1.27
5.99
5.65
1.94
10R
10R
10R
10R
10R
10R
10R
10S
10S
1.55
1.53
Cellulose OD
Amylose AS
Amylose AD
2.48
1.03
3.72
2.77
22.96
20.50
Eluents A: n-hexane/ethanol: 95:5; B: n-hexane/ethanol: 90:10; C: n-hexane/ethanol: 80:20; D: n-hexane/ethanol: 70:30. The flow-rate was
1 mL minꢀ1, k = 200 and 226 nm, T = 30 ꢁC, t0 = 3.5 min, 20 lL.
Chiralpak AD column (amylose tris-3,5-dimethylphenylcarbamate), Chiralpak AS column (amylose tris-(S)-1-phenylethylcarbamate; 250 · 4.6 mm
i.d.; 10 lm), Chiralcel OD-H column (cellulose tris-3,5-dimethylphenylcarbamate; 250 · 4.6 mm i.d.; 5 lm), Chiralcel OJ column (cellulose tris-
methylbenzoate; 250 · 4.6 mm i.d.; 10 lm) were purchased from Daicel Chemical Industries, Baker France.

504
E. Lipka et al. / Bioorg. Med. Chem. Lett. 15 (2005) 501–504
0.40
Acknowledgements
10S
0.30
0.20
0.10
We thank ÔLe Conseil Regional de PicardieÕ for financial
support.
References and notes
0.00
0.40
1. De Clercq, E. J. Med. Chem. 1995, 38(14), 2491.
2. Mitsuya, H.; Weinhold, K. J.; Furman, P. A.; St Clair, M.
H.; Nusinoff-Lehrman, S.; Gallo, R. C.; Bolognesi, D. P.;
Barry, D. W.; Broder, S. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 7096.
3. Cooney, D. A.; Dalal, M.; Mitsuya, H.; McMahon, J. B.;
Nadkarni, M.; Balzarini, J.; Broder, S.; Johns, D. G.
Biochem. Pharmacol. 1986, 35, 2065.
4. Ahluwalia, G.; Cooney, D. A.; Mitsuya, H.; Fridland, A.;
Flora, K. P.; Hao, Z.; Dalal, M.; Broder, S.; Johns, D. G.
Biochem. Pharmacol. 1986, 35, 3797.
5. Coates, J. A. V.; Cammack, N. S.; Jenkinson, H. J.;
Jowett, A. J.; Jowett, M. I.; Pearson, B. A.; Pen, C. R.;
Rouse, P. L.; Viner, K. C.; Cameron, J. M. Antimicrob.
Agents Chemother. 1992, 36, 733.
10R
0.30
0.20
0.10
0.00
9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00
Minutes
6. Balzarini, J.; Kang, G. J.; Dalal, M.; Herdewijn, P.; De
Clercq, E.; Broder, S.; Johns, D. G. Mol. Pharmacol. 1987,
32, 162.
Figure 4. Extended scale chromatogram of CLHP analysis of the two
enantiomers on Chiralpak AS (eluent C) after purification through
preparative CLHP.
7. Crimmins, M. T.; King, B. W. J. Org. Chem. 1996, 61,
4192.
8. Agrofoglio, L. A.; Challand, S. R. The Chemistry of L-
Nucleosides; Kluwer Academic: Dordrecht, 1998; p 285.
9. Simpson, M. V.; Chin, C. D.; Keilbaugh, S. A.; Lin, T. S.;
Prusoff, W. H. Biochem. Pharmacol. 1989, 38, 1033.
10. Ewing, D. F.; Fahmi, N.-E.; Len, C.; Mackenzie, G.;
Ronco, G.; Villa, P.; Shaw, G. Collect. Czech. Chem.
Commun. 1996, 61, S145.
11. Ewing, D. F.; Fahmi, N.-E.; Len, C.; Mackenzie, G.;
Ronco, G.; Villa, P.; Shaw, G. Nucleos. Nucleot. 1999, 18,
2613.
12. Ewing, D. F.; Fahmi, N.-E.; Len, C.; Mackenzie, G.;
Pranzo, A. J. Chem. Soc., Perkin Trans. 1 2000, 3561.
13. Ewing, D. F.; Len, C.; Mackenzie, G.; Ronco, G.; Villa, P.
Tetrahedron: Asymmetry 2000, 11, 4995.
14. Harcken, C.; Bruckner, R. Angew. Chem., Int. Ed. Engl.
1997, 36(24), 2750.
15. Kang, K.; Cha, M.; Pae, A.; Choi, K.; Cho, Y.; Koh, H.;
Chung, B. Tetrahedron Lett. 2000, 41(42), 8137.
16. Fazio, F.; Schneider, M. Tetrahedron: Asymmetry 2000,
11(9), 1869.
17. Camps, P.; Cardellach, J.; Font, J.; Ortuno, R. M.;
Ponsati, O. Tetrahedron 1982, 38(15), 2395.
18. Ng, T. B.; Liu, F.; Wang, Z. T. Life Sci. 2000, 66(8), 709.
19. Len, C.; Selouane, A.; Weiling, A.; Coicou, F.; Postel, D.
Tetrahedron Lett. 2003, 44, 663.
20. Moitessier, N.; Henry, C.; Len, C.; Postel, D.; Chapleur,
Y. J. Carbohydr. Chem. 2003, 22, 25.
enantiomer. Chromatograms of each enantiomer ob-
tained after purification are given as examples on Chir-
alpak AS (Fig. 4).
Two methods have been developed to give the lactones
10S and 10R via dihydroxylation. The first one was ef-
fected using D-xylose derivatives as chiral auxiliaries.19
Starting from the chiral styrene AD using AD-mix-a
(de 99%) and AD-mix-b (de 98%) or dihydroxylation
using OsO4 (de 0%), permitted the preparation of the
corresponding diastereoisomerically pure intermediates
easily after flash chromatography. In this case the pres-
ence of a stereogenic protecting group has achieved the
desired objective by allowing the effective resolution of
the stereocentre of the lactone 10S and 10R. The second
one was effected using achiral substrate 3. Starting from
this achiral styrene 3 AD using AD-mix-a (ee 98.5%)
and AD-mix-b (ee 97.5%), permitted the preparation
of the corresponding enantiomerically pure lactones
10S and 10R after HPLC purification using chiral sta-
tionary phase. Both methods afforded the lactone 10S
28
D
28
D
(½aꢁ +35 (c 0.8, CHCl3)) and the lactone 10R (½aꢁ
ꢀ34 (c 0.8, CHCl3)) with the same physical data.
21. Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B.
Chem. Rev. 1994, 94, 2483.
22. Vanhessche, K. P. M.; Sharpless, K. B. J. Org. Chem.
1996, 61, 7978.
23. Moitessier, N.; Henry, C.; Len, C.; Chapleur, Y. J. Org.
In conclusion the benzo[c]furanones 10S and 10R hav-
ing one asymmetric carbon atom were obtained in good
yields starting from the achiral phthalaldehyde. The key
step of this route was the use of the asymmetric dihydr-
oxylation developed by Sharpless. In anti-HIV studies,
compounds 10S and 10R were evaluated for their inhib-
itory effects on the replication of HIV-126 in human T4-
lymphoblastoid cells, CEM-SS and MT-4. Compounds
10S and 10R have been found inactive against HIV-1
replication at concentrations up to 10 lM.
Chem. 2002, 67, 7275.
´
24. Szurmai, Z.; Rako, J.; Agoston, K.; Danan, A.; Charon,
´ ´
D. Org. Lett. 2000, 13, 1839.
25. Wang, T.; Chen, Y. W. J. Chromatogr. A 1999, 855, 411.
26. Moog, C.; Wick, A.; Le Ber, P.; Kirn, A.; Aubertin, A. M.
Antiviral Res. 1994, 24, 275.