Semisynthesis and antitumoral activity of 2-acetylfuranonaphthoquinone and other naphthoquinone derivatives from lapachol

Article abstract of DOI:10.1016/j.bmcl.2008.09.053

Ozonolysis of lapachol (1), resulting in an unusual formation of a potent antitumor agent 2-acetylfuranonaphthoquinone (3) along with the expected aldehyde 6, is described. The reaction of lapachol (1) with CAN in dry acetonitrile leading to biologically active furanonaphthoquinones is also reported. The antitumoral activity of the tested compounds on human DU-145 prostate carcinoma cells was evaluated following XTT assay. The results revealed that 2-(1-methylethenyl)-2,3-dihydronaphtho[2,3-b]furan-4,9-dione (5), β-lapachone (10) and dehydro-β-lapachone diacetate (11) showed 100% inhibition at 25 μg/ml. All the tested samples showed dose-dependent activity.

Full text of DOI:10.1016/j.bmcl.2008.09.053

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5387–5390
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Semisynthesis and antitumoral activity of 2-acetylfuranonaphthoquinone
and other naphthoquinone derivatives from lapachol
Kenneth O. Eyong a,b, Ponminor S. Kumar a, Victor Kuete c, Gabriel N. Folefoc b,
,
*
Ephriam A. Nkengfack b, Sundarababu Baskaran a,
*
a Department of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India
b Department of Organic Chemistry, University of Yaounde 1, BP 812 Yaounde, Cameroon
c Department of Biochemistry, University of Dschang, BP 63 Dschang, Cameroon
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2008
Revised 10 September 2008
Accepted 12 September 2008
Available online 17 September 2008
Ozonolysis of lapachol (1), resulting in an unusual formation of a potent antitumor agent 2-acetylfurano-
naphthoquinone (3) along with the expected aldehyde 6, is described. The reaction of lapachol (1) with
CAN in dry acetonitrile leading to biologically active furanonaphthoquinones is also reported. The antitu-
moral activity of the tested compounds on human DU-145 prostate carcinoma cells was evaluated fol-
lowing XTT assay. The results revealed that 2-(1-methylethenyl)-2,3-dihydronaphtho[2,3-b]furan-4,9-
dione (5), b-lapachone (10) and dehydro-b-lapachone diacetate (11) showed 100% inhibition at 25 lg/
ml. All the tested samples showed dose-dependent activity.
Keywords:
Anticancer
Lapachol
Ó 2008 Elsevier Ltd. All rights reserved.
Furanonaphthoquinones
Ozonolysis
CAN
Radical cyclization
In addition, furanonaphthoquinones (3–5), also phytoconstituents
of Bignoniaceae family, are known to exhibit significant biological
activities. Among those, 2-acetylfuranonaphthoquinone (3) in par-
ticular shows superior antitumoral activity4 and the influence of
furan/pyran-ring in the antitumoral activity has recently been dem-
onstrated.5 As a consequence of its biological significances, 2-acet-
ylfuranonaphthoquinone (3) has become an important synthetic
target and several studies have been carried out towards the syn-
thesis of this potent antitumor agent.6
Lapachol (1), a principal constituent of Newbouldia leavis (Big-
noniaceae family)1,2 exhibits wide spectrum of biological activities
such as antibacterial, antiplasmodial, antioxidant and trypanocidal
activities.2 Lapachol has also been used as anti-microbial and anti-
protozoal agents and many of its derivatives are proved to be po-
tent antimalarial and anti-HIV agents. AtovaquoneÒ (2), a deriva-
tive of lapachol, has already been approved for the treatment of
Pneumocystis pneumonia, toxoplasmosis and malaria.3
Since 2-acetylfuranonaphthoquinone (3) is available in minute
amounts from nature, it is essential to synthesize this biologically
active molecule in large quantities for conducting structure–activ-
ity investigation as well as to make various analogs of this com-
pound for biological assay. Lapachol is abundant in nature and
hence it is of our interest to develop methods for the semisynthesis
of biologically active and pharmaceutically important furanonaph-
thoquinone molecules through efficient manipulation of lapachol.
In this communication, we report simple approaches for the syn-
thesis of 2-acetylfuranonaphthoquinone (3) and other furanonaph-
thoquinones from lapachol.
Intriguingly, exposure of lapachol under ozonolysis conditions re-
sulted in the formation of expected aldehyde 6 in 70% yield along with
the unusual compound, 2-acetylfuranonaphthoquinone, 3in30%yield.
The structure of compound 3 is established by spectroscopic da-
ta6g and further unambiguously confirmed by single crystal X-ray
analysis (Fig. 1).7
O
Cl
OH
O
O
O
2
O
3
O
O
O
OH
1
O
O
O
O
O
O
4
5
* Corresponding authors. Tel.: +91 44 2257 4218; fax: +91 44 2257 0545.
E-mail address: sbhaskar@iitm.ac.in (S. Baskaran).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.053

5388
K. O. Eyong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5387–5390
Thus, lapachol on treatment with DDQ in dry benzene under re-
flux conditions underwent 6-endo-trig cyclization to give the corre-
sponding dehydro-
-lapachone (8) in low yield (10%).8 On the
a
other hand, lapachol on treatment with CAN in dry acetonitrile
underwent 5-exo-trig cyclization to give 1-(2,3,4,9-tetrahydro-
4,9-dioxonaphtho[2,3-b]furany-2-yl)-1-methylethyl
nitrate
9
(55%) and 2-(1-methylethenyl)-2,3-dihydronaphtho[2,3-b]furan-
4,9-dione 5 (35%) in good yields along with 2-(10-methylethe-
nyl)-naphtho[2,3-b]furan-4,9-dione (4) in low yield (5%) (Scheme
2).9 The structure of the major compound 9 was unambiguously
confirmed by single crystal X-ray analysis (Fig. 2).7
The CAN mediated radical cyclization reaction suggests that the
antitumor agent 3 is likely to be formed via intermediate 4 during
the ozonolysis of lapachol.
Figure 1. ORTEP diagram of compound 3.
A plausible mechanism for the formation of compounds 4, 5 and
9 from lapachol (1) under radical condition is shown in Scheme 3.
After achieving the synthesis, the anticancer activity of the syn-
thetic compounds on human DU-145 prostate carcinoma cells was
evaluated following XTT assay as described10,11 and the results are
summarized in both Figure 3 and Table 1. The results in Figure 3
indicate that compounds 5, b-lapachone (10) and dehydro-b-
Further reaction of compound 6 with acetic anhydride in pyri-
dine at room temperature resulted in (E)-2-(3-hydroxy-1,4-di-
oxo-1,4-dihydronaphthalene-2-yl)vinyl acetate (7) in good yield
(80%) (Scheme 1).
Encouraged by the interesting reaction of lapachol with ozone
leading to the biologically important compound 3, we were moti-
vated to gain deeper insight into the mechanism and also to im-
prove the yield of this unusual transformation. We believed that
lapachol under ozonolysis reaction conditions forms an intermedi-
ate through radical cyclization, which on further ozonolysis results
in the formation of compound 3. In order to understand this novel
transformation leading to 3, a systematic investigation on the
mode of cyclization of lapachol under radical conditions was
initiated.
lapachone diacetate (11) showed 100% inhibition at 25 l
g/ml.12
All the tested samples showed dose-dependent activity.
However, the anticancer efficacy of the tested samples is mostly
highlighted by their IC50 values (Table 1).
From the activity obtained with a reference antineoplastic com-
pound (doxorubicin), some of the tested compounds such as 5, 6, 9,
b-lapachone (10) and dehydro-b-lapachone diacetate (11) can be
considered as promising antitumor candidates. Compounds 5 and
9 are known to exhibit significant cytotoxic activities.5a
O
O
O
O
OH
OH
O
O
O3
+
O
O
O
6
3
1
30%
Ac2O/Py,
rt
70%
O
OH
OAc
7
O
(80%)
Scheme 1. Ozonolysis reaction of lapachol.
O
O
DDQ
1
O
CAN
8 (10%)
O
O
O
O
O
O
O
O
O
+
+
ONO2
5 (35%)
4 (5%)
9 (55%)
Scheme 2. Reaction of lapachol with CAN and DDQ.

K. O. Eyong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5387–5390
5389
ship of the tested compounds, it could generally be noted that acet-
ylation reduces the anticancer activity (compare 6 and 7; b-
lapachone and dehydro-b-lapachone diacetate). However, the for-
mation of the pyran ring (conversion of 1 to b-lapachone) increases
antitumoral activity. It has previously been shown that the pyran
and the furan rings do influence the antitumoral activity of naph-
thoquinones.5a In this study, it is also confirmed that a modifica-
tion in the furan ring (3 and 5) as well as on the furan ring
substituents (4 and 9) induce changes in the antitumoral activity.
In conclusion, a one step protocol was developed for the conver-
sion of lapachol into the potent antitumor agent, 2-acetylfurano-
naphthoquinone (3), through ozonolysis,13 while treatment of the
ozonolysis co-product 6 with acetic anhydride in pyridine led to
the novel vinyl acetate 7 in good yield. Since compound 4 is also
a phytoconstituent of N. leavis, the reaction of lapachol with CAN
has thrown some light on the biosynthesis of different furanonaph-
thoquinones isolated from N. leavis. The antitumoral activity of
some of the synthesized naphthoquinone derivatives gives a prom-
ising basis for their possible use in the treatment of prostate
cancer.
Figure 2. ORTEP diagram of compound 9.
The activity of b-lapachone (IC50: 7.4 nM) is only 1.9-fold lower
that of doxorubicin (IC50: 3.90 nM) while those of 6 (8.80 nM),
dehydro-b-lapachone diacetate (11.14 nM) and 5 (11.67 nM) are
less than 3-fold. Upon comparing the structure–activity relation-
O
O
O
O
CAN
5
4
+ 9
1
O
O
Scheme 3. Proposed mechanism for the formation of compounds 4, 5 and 9 from lapachol.
90
80
70
60
50
40
30
20
10
0
70
Compound 3
Compound 4
Compound 5
80
70
60
50
40
30
20
10
0
60
50
40
30
20
10
0
1.6
3.13
6.25
12.5
25
1.6
3.13
6.25 12.5
25
1.6
3.13
6.25
12.5
25
Concentration (µg/ml)
Concentration (µg/ml)
Concentration (µg/ml)
Compound 6
Compound 7
Compound 9
80
70
60
50
40
30
20
10
0
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
1.6
3.13
6.25
12.5
25
1.6
3.13
6.25
12.5
25
1.6
3.13
6.25
12.5
25
Concentration (µg/ml)
Concentration (µg/ml)
Concentration (µg/ml)
60
50
40
30
20
10
0
100
80
60
40
20
0
80
70
60
50
40
30
20
10
0
Doxorubicin
Dehydro-β-lapachone
β-Lapachone (10)
diacetate (11)
1.6
3.13
6.25
12.5
25
1.6
3.13
6.25
12.5
25
1.6
3.13
6.25
12.5
25
Concentration (µg/ml)
Concentration (µg/ml)
Concentration (µg/ml)
Figure 3. The effect of test samples on DU-145 prostate cancer cells. The values are expressed as means SEM (n = 3).

5390
K. O. Eyong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5387–5390
Wagner, H.; Lombardi, J.; Lopes, M. T. P. J. Pharm. Pharmacol. 2000, 52, 347; (e)
Table 1
Gormann, R.; Kaloga, M.; Li, X.-C.; Ferreira, D.; Bergenthal, D.; Kolodziej, H.
Phytochemistry 2003, 64, 583.
3. Baramee, A.; Coppin, A.; Mortuaire, M.; Pelinski, L.; Tomavo, S. Bioorg. Med.
Chem. 2006, 14, 1294.
Anticancer activity of the tested compounds on DU-145 prostate cancer cells
Tested compounds
IC50 in
(nM)
l
g/ml
Inhibition percentage (%) at
25 g/ml
l
4. (a) Rao, M. M.; Kingston, D. G. I. J. Nat. Prod. 1982, 45, 600; (b) Ogawa, M.;
Koyanagi, J.; Sugaya, A.; Tsuda, T.; Ohguchi, H.; Nakayama, K.; Yamamoto, K.;
Tanaka, A. Biosci. Biotechnol. Biochem. 2006, 70, 1009; (c) Itoigawa, M.; Ito, C.;
Tan, H. T.-W.; Okuda, M.; Tokuda, H.; Nishino, H.; Furukawa, H. Cancer Lett.
(Shannon, Ireland) 2001, 174, 135; (d) Solorzano, L.; Rieber, M. S.; Medina, J. D.;
Rieber, M. Cancer Biol. Ther. 2005, 4, 329; (e) Mueller, K.; Sellmer, A.; Wiegrebe,
W. J. Nat. Prod. 1999, 62, 1134.
5. (a) Perez-Sacau, E.; Diaz-Penate, R. G.; Estevez-Braun, A.; Ravelo, A. G.; Garcia-
Castellano, J. M.; Pardo, L.; Campillo, M. J. Med. Chem. 2007, 50, 696; (b) Sergio,
R.; Peraza, S.; Daniel, C.; Hee-Byung, C.; Young, G. S.; Ricardo, G.; Miliciades, M.;
Craig, R. F.; Kate, E. L.; Ana, T. M.; Norman, R. F.; Geoffrey, A. C.; John, M. P.;
Douglas, A. K. J. Nat. Prod. 2000, 63, 492.
6. (a) Ferreira, V. F.; Pinto, A. V.; Pinto, M. C. F. R.; Da Cruz, M. C.; Clarino, A. Synth.
Commun. 1989, 19, 1061; (b) Lee, Y. R.; Kim, B. S. Synth. Commun. 2001, 31, 381;
(c) Hagiwara, H.; Sato, K.; Suzuki, T.; Ando, M. Heterocycles 1999, 51, 497; (d)
Koyanagi, J.; Yamamoto, K.; Nakayama, K.; Tanaka, A. J. Heterocycl. Chem. 1995,
32, 1289; (e) Lopes, C. C.; Lopes, R. S. C.; Pinto, A. V.; Costa, P. R. R. J. Heterocycl.
Chem. 1984, 21, 621; (f) Hagiwara, H.; Sato, K.; Nishino, D.; Hoshi, T.; Suzuki, T.;
Ando, M. J. Chem. Soc., Perkin Trans. 2001, 1, 2946; (g) Lee, Y. K.; Kim, B. S.; Kim,
D. H. Tetrahedron 2000, 56, 8845; (h) Richard, H.; Paul, B. Can. J. Chem. 1974, 52,
88.
1
3
4
5
6
7
9
15.63 (64.59)
5.53 (23.04)
4.73 (19.87)
78.12 6.12
69.79 4.18
79.79 5.34
2.80 (11.67) 100.00 0.00
1.90 (8.80)
12.48 (48.37)
4.41 (15.37)
87.50 4.72
79.17 6.08
77.08 4.78
β-lapachone (10)
O
O
1.80 (7.4)
100.00 0.00
O
Dehydro-β-lapachone diacetate (11)
AcO
OAc
7. Crystallographic details have been deposited at the Cambridge Crystallographic
Data Centre (deposition number for compound 3: CCDC 669086 and for
compound 9: CCDC 669085). Please see Supplementary data for more
information.
3.63 (11.14) 100.00 0.00
O
8. (a) Matsumoto, T.; Ichihara, A.; Yanagiya, M.; Yuzawa, T.; Sannai, A.; Oikawa,
H.; Sakamura, S.; Eugster, C. H. Helv. Chim. Acta 1985, 68, 2324; (b) Crombie, L.;
Rossiter, J. T.; Bruggen, N. V.; Whiting, D. A. Phytochemistry 1992, 31, 451.
9. Under aqueous conditions, the reaction of lapachol with CAN is known to give
compounds 5 (15%) and 9 (19%) in low yields Perez-Sacau, E.; Estevez-Braun,
A.; Ravelo, A. G.; Yapu, D. G.; Turba, A. G. Chem. Biodiversity 2005, 2, 264.
10. Cytotoxicity assay: Cells were cultured in Dulbecco’s minimum essential
medium (DMEM) supplemented with 5% fetal calf serum (FCS), gentamycin
sulfate (0.004%), glucose (0.57%) and NaHCO3 (0.12%). Cells were seeded into
96-well flat-bottomed plates at a concentration of 3.0 ꢀ 105 cells per ml. After
24 h, cells were treated with compounds, which were diluted with culture
Doxorubicin
2.12 (3.90)
92.13 1.09
Acknowledgments
Dr. Eyong thanks TWAS-UNESCO Associate Scheme and IIT-
Madras for financial Support. Authors also thank the DST-FIST
program for NMR facility and Mr. V. Ramkumar for X-ray analysis.
medium to a final concentration of 25 lg/ml. XTT labeling reagent (50:1) was
added and the absorbance (560 nm) read after 72 h.11 Experiments were
carried out three times in triplicate. Active samples (with less than 50%
survival) after an exposure time of 72 h were serially diluted in a concentration
range of 1.6–25 lg/ml and tested. The concentration of the sample that
inhibited 50% cell proliferation (IC50) was determined graphically. Doxorubicin,
a known anti-tumour agent, was used as positive control.
Supplementary data
11. (a) Gerlier, D.; Thomasset, N. J. Immunol. Methods 1986, 94, 57; (b) Itharat, A.;
Houghton, P. J.; Eno-Amooquaye, E.; Burke, P. J.; Sampson, J. H.; Raman, A. J.
Ethnopharmacol. 2004, 90, 33; (c) Cheng, R. K.-Y. Z. Drugs Future 1997, 22, 519.
12. b-Lapachone (10)5a and dehydro-b-lapachone diacetate (11) were prepared as
described in the literature Manners, G. D.; Jurd, L.; Wong, R.; Palmer, K.
Tetrahedron 1975, 31, 3019.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.053.
References and notes
13. Ozonolysis reaction of lapachol: A solution of lapachol 1 (242 mg, 1 mmol) in dry
DCM (50 ml) was cooled to ꢁ78 °C and ozonised oxygen was passed till the
completion of the reaction. Then the ozonide was quenched with
dimethylsuphide (2 ml) at ꢁ78 °C and stirred for 1 h. The reaction mixture
was washed with water and extracted with DCM (3 ꢀ 20 ml). Organic layer
was dried over anhydrous Na2SO4, concentrated under reduced pressure and
purified by column chromatography over silica gel using 30% EtOAc in hexane
1. (a) Eyong, K. O.; Krohn, K.; Hussain, H.; Folefoc, G. N.; Nkengfack, A. E.; Schulz,
B.; Hu, Q. Chem. Pharm. Bull. 2005, 53, 616; (b) Eyong, K. O.; Folefoc, G. N.;
Kuete, V.; Beng, V. P.; Krohn, K.; Hussain, H.; Nkengfack, A. E.; Saeftel, M.;
Sarite, S. R.; Hoerauf, A. Phytochemistry 2006, 67, 605.
2. (a) Kuete, V.; Eyong, K. O.; Folefoc, G. N.; Beng, V. P.; Hussain, H.; Krohn, K.;
Nkengfack, A. E. Pharmazie 2007, 62, 552; (b) Wenceslau, J. P. S.; De Souza, D. F.;
De Oliveira, M. C. F.; Lemos, T. L. G.; De Sousa, A. L.; Trevisan, M. T. S.; De
Mattos, M. C. Nat. Prod. Commun. 2006, 1, 661; (c) Schmeda-Hirschmann, G.;
Papastergiou, F. Z Naturforsch 2003, 58, 495; (d) Duarte, D. S.; Dolabela, M. F.;
Salas, C. E.; Raslan, D. S.; Oliveiras, A. B.; Nenninger, A.; Wiedemann, B.;
as an eluent to give the corresponding aldehyde
6 (70%) and 2-
acetylnaphtho[2,3-b]furan-4,9-dione 3 (30%) in good yields.