Synthesis and single crystal X-ray analysis of two griseofulvin metabolites

Article abstract of DOI:10.1016/j.tetlet.2010.08.095

The two phenols, 6-O-desmethyl griseofulvin and 4-O-desmethyl griseofulvin are metabolites of the antifungal drug griseofulvin. Herein, we present an improved synthesis of the 6-phenol derivative, and an unequivocal proof of both structures by single-crystal X-ray analysis.

Full text of DOI:10.1016/j.tetlet.2010.08.095

Tetrahedron Letters 51 (2010) 5881–5882
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Tetrahedron Letters
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Synthesis and single crystal X-ray analysis of two griseofulvin metabolites
Mads H. Rønnest a,b, Pernille Harris a, Charlotte H. Gotfredsen a, Thomas O. Larsen b, Mads H. Clausen a,
⇑
a Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 201 and 206, DK-2800 Kgs. Lyngby, Denmark
b Center for Microbial Biotechnology, Department of Systems Biology, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2010
Revised 28 July 2010
Accepted 31 August 2010
Available online 6 September 2010
The two phenols, 6-O-desmethyl griseofulvin and 4-O-desmethyl griseofulvin are metabolites of the anti-
fungal drug griseofulvin. Herein, we present an improved synthesis of the 6-phenol derivative, and an
unequivocal proof of both structures by single-crystal X-ray analysis.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Griseofulvin
X-ray structures
Metabolites
Synthesis
The natural product griseofulvin (1) (see Fig. 1) was first iso-
lated by Oxford et al. in 19391 and later shown to possess anti-
fungal properties.2 This antifungal agent is still in clinical use
today3 and is the only orally administered drug approved by the
Food and Drug Administration for the treatment of tinea capitis
(ringworm of the scalp).4 Recently, griseofulvin has received re-
newed attention due to reports of both antiproliferative effects in
cancer cells5–7 as well as suppression of hepatitis C replication.8
As a result of its notoriously low water solubility, griseofulvin is
furthermore, often used as a benchmark compound in formulation
studies and in the development of drug delivery systems.9
reproduced and we were actually able to isolate a small amount of
2 at step three (Scheme 1). The lengthy synthesis and poor yield of
this route prompted us to search for a more convenient method to
access 2. Thus, we were pleased to obtain the desired phenol in 29%
yield after the treatment of griseofulvin (1) with LiI in pyridine at
115 °C (Scheme 2).22 The synthesis of 4-O-desmethylgriseofulvin
MeO
4
MeO
HO
OMe
OMe
OMe
O
O
O
3a
O
O
O
O
O
O
MeO
HO
MeO
6
The metabolism of griseofulvin has been studied both in vitro10
and in vivo and reported in several publications. In addition to
studies in fungi,11 the in vivo metabolism of griseofulvin has been
investigated in rats,12 mice,13 rabbits,14 dogs,15 and man.16,17
Known important metabolites of griseofulvin include 6-O-desm-
ethylgriseofulvin (2) and 4-O-desmethylgriseofulvin (3), but their
structures have never been proven unambiguously. In the litera-
ture, it is commonly merely stated that the metabolites were com-
pared with authentic samples.11–13,15,17 Others10,14 have used
spectroscopic properties and melting points to identify the struc-
tures by comparing these data with earlier work.18–21 We present
herein, the synthesis and crystal structures of both 6-O-desmethyl-
griseofulvin (2) and 4-O-desmethylgriseofulvin (3), which provide
final verification of the structural assignments.
Cl
Cl
Cl
1
2
3
Figure 1. The structures of griseofulvin (1), 6-O-desmethylgriseofulvin (2) and 4-O-
desmethylgriseofulvin (3).
MeO
Cl
MeO
Cl
OMe
O
O
O
O
a, b, c
2
O
OMe
O
current
work
MeO
HO
1% from 1
1
d
MeO
MeO
Cl
OMe
O
O
O
O
c
e
6-O-Desmethylgriseofulvin (2) was first synthesized by Arkley
et al. in six steps with an overall yield of 14% (Scheme 1).22 To con-
firm the outcome of these transformations, the synthetic route was
2
O
OMe
O
BnO
BnO
14% from 1
Cl
Scheme 1. Reagents: (a) HOAc, 2 M H2SO4; (b) 0.5 M NaOH; (c) 2,2-dimethoxy-
propane, p-toluenesulfonic acid, MeOH; (d) K2CO3, BnBr, acetone; (e) 5% Pd/C, H2,
EtOAc.
⇑
Corresponding author. Tel.: +45 45252131; fax: +45 45933968.
E-mail address: mhc@kemi.dtu.dk (M.H. Clausen).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.08.095

5882
M. H. Rønnest et al. / Tetrahedron Letters 51 (2010) 5881–5882
MeO
Cl
OH
confirming the position of the phenol. The UV and fluorescence
OMe
OMe
O
O
spectra of 2 and 3 were all but identical, and despite small differ-
ences in the MS–MS spectra (see Supplementary data), the reten-
tion time25 is still the most reliable and sensitive analytical
method for distinguishing the two phenols.
a
b
1
O
O
O
O
HO
MeO
Cl
2
3
Scheme 2. Reagents: (a) LiI, pyridine, 115 °C, (29%); (b) MgI2, Et2O, toluene, (98%).
Acknowledgment
The authors thank the Danish Research Council (ref. 274-07-
0561) for the financial support.
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion, and purity data, HPLC traces, NMR, UV, and MS–MS spectra
for compounds 2 and 3, and crystallographic information in cif for-
mat) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2010.08.095
References and notes
1. Oxford, A. E.; Raistrick, H.; Simonart, P. Biochem. J. 1939, 33, 240.
2. Gentles, J. C. Nature 1958, 182, 476.
3. Dauk, K. C. L.; Comrov, E.; Blumer, J. L.; O’Riordan, M. A.; Furman, L. M. Clin.
Pediatr. 2010, 49, 280.
4. Gupta, A. K.; Williams, J. V.; Zaman, M.; Singh, J. Med. Mycol. 2009, 47, 796.
5. Ho, Y. S.; Duh, J. S.; Jeng, J. H.; Wang, Y. J.; Liang, Y. C.; Lin, C. H.; Tseng, C. J.; Yu,
C. F.; Chen, R. J.; Lin, J. K. Int. J. Cancer 2001, 91, 393.
6. Panda, D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 9878.
Figure 2. ORTEP view of 6-O-desmethylgriseofulvin (2).
7. Rebacz, B.; Larsen, T. O.; Clausen, M. H.; Rønnest, M. H.; Löffler, H.; Ho, A. D.;
Krämer, A. Cancer Res. 2007, 67, 6342.
8. Jin, H.; Yamashita, A.; Maekawa, S.; Yang, P. T.; He, L. M.; Takayanagi, S.;
Wakita, T.; Sakamoto, N.; Enomoto, N.; Ito, M. Hepatol. Res. 2008, 38, 909.
9. Dalvi, S. V.; Dave, R. N. Ind. Eng. Chem. Res. 2009, 48, 7581.
10. Chang, R. L.; Zampaglione, N.; Lin, C. Drug Metab. Dispos. 1975, 3, 487.
11. Boothroyd, B.; Napier, E. J.; Somerfield, G. A. Biochem. J. 1961, 80, 34.
12. Symchowi, S.; Wong, K. K. Biochem. Pharmacol. 1966, 15, 1595.
13. Lin, C.; Magat, J.; Symchowi, S.; Chang, R. J. Pharm. Pharmacol. 1972, 24, 911.
14. Barnes, M. J.; Boothroyd, B. Biochem. J. 1961, 78, 41.
15. Harris, P. A.; Riegelma, S. J. Pharm. Pharmacol. 1969, 58, 93.
16. Chiou, W. L.; Riegelma, S. J. Pharm. Pharmacol. 1971, 60, 1376.
17. Lin, C. C.; Magat, J.; Chang, R.; Mcglotte, J.; Symchowi, S. J. Pharmacol. Exp. Ther.
1973, 187, 415.
18. Duncanson, L. A.; Grove, J. F.; Macmillan, J.; Mulholland, T. P. C. J. Chem. Soc.
1957, 3555.
19. Grove, J. F.; Macmillan, J.; Mulholland, T. P. C.; Rogers, M. A. T. J. Chem. Soc.
1952, 3949.
20. Levine, S. G.; Ronald, E. H. J. Org. Chem. 1975, 40, 2540.
21. Arison, B. H.; Wendler, N. L.; Taub, D.; Hoffsommer, R. D.; Kuo, C. H.; Slates, H.
L.; Trenner, N. R. J. Am. Chem. Soc. 1963, 85, 627.
Figure 3. ORTEP view of 4-O-desmethylgriseofulvin (3).
22. Arkley, V.; Gregory, G. I.; Attenbur, J.; Walker, T. J. Chem. Soc. 1962, 1260.
23. Synthesis of 2. Griseofulvin (10 mg, 0.03 mmol) and LiI (4.7 mg, 0.04) were
dissolved in pyridine (0.5 mL) and heated to 115 °C. After 16 h, the reaction
was allowed to reach 20 °C and satd aq NH4Cl (2 mL) was added. The mixture
was extracted with EtOAc (3 Â 3 mL), and the combined organic phases were
dried (MgSO4) and concentrated. Purification was performed on a Luna HPLC
(3) was performed by treatment of 1 with MgI2 in a mixture of
diethyl ether and toluene (Scheme 2), a slight modification of the
procedure originally published by Arkley et al.23
The structures of 2 and 3 were confirmed unequivocally by the
use of single-crystal X-ray analysis (Figs. 2 and 3).24 It is not possi-
ble to distinguish between the 4 and 6 methoxy groups of 1 by
gHMBC as no 4J correlation is observed. The 1H NMR spectrum of
2 (see Supplementary data) does not exhibit a signal for the pheno-
lic hydroxy group, due to rapid proton exchange, and thus no het-
eronuclear correlations can be used to aid in the assignment of the
spectrum. For 3, the phenolic proton is observed (see Supplemen-
tary data) and the gHMBC contains a single 3JHC correlation to C-3a,
column (250 Â 10 mm, 5
lm, C-18) using 5 mL/min H2O/CH3CN (isocratic run
at 65:35, for 15 min) as the mobile phase to yield 2 (2.8 mg, 29%) as a yellow
oil, which was crystallized from EtOAc and heptane.
24. Crystallographic data (excluding structure factors) for the structures in this
paper have been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication nos. CCDC 775177 (2) and CCDC 775176 (3). Copies
of the data can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax: +44 (0)1223 336033 or email:
deposit@ccdc.cam.ac.uk).
25. Townley, E.; Roden, P. J. Pharm. Sci. 1980, 69, 523.