Synthesis of a small library of 2-phenoxy-1,4-naphthoquinone and 2-phenoxy-1,4-anthraquinone derivatives bearing anti-trypanosomal and anti-leishmanial activity

Article abstract of DOI:10.1016/j.bmcl.2008.03.009

Taking advantage of the structural features of natural products showing anti-trypanosomatid activity, we designed and synthesized a small library of 2-phenoxy-1,4-naphthoquinone and 2-phenoxy-1,4-anthraquinone derivatives. The library was obtained following a parallel approach and using readily available synthons. All the derivatives showed inhibitory activity toward either Trypanosoma or Leishmania species, with 8, 10, and 16 being the most active compounds against Trypanosoma brucei rhodesiense, Leishmania donovani, and Trypanosoma cruzi cells (IC₅₀ = 50 nM, IC₅₀ = 0.28 μM, and IC₅₀ = 1.26 μM, respectively).

Full text of DOI:10.1016/j.bmcl.2008.03.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2272–2276
Synthesis of a small library of 2-phenoxy-1,4-naphthoquinone
and 2-phenoxy-1,4-anthraquinone derivatives bearing
anti-trypanosomal and anti-leishmanial activity
Maria Laura Bolognesi,a,* Federica Lizzi,a Remo Perozzo,b
Reto Brunc and Andrea Cavallia,*
aDepartment of Pharmaceutical Sciences, Alma Mater Studiorum, Bologna University, Via Belmeloro 6, I-40126 Bologna, Italy
bPharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Geneva,
University of Lausanne, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland
cSwiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
Received 7 February 2008; revised 3 March 2008; accepted 3 March 2008
Available online 7 March 2008
Abstract—Taking advantage of the structural features of natural products showing anti-trypanosomatid activity, we designed and
synthesized a small library of 2-phenoxy-1,4-naphthoquinone and 2-phenoxy-1,4-anthraquinone derivatives. The library was
obtained following a parallel approach and using readily available synthons. All the derivatives showed inhibitory activity toward
either Trypanosoma or Leishmania species, with 8, 10, and 16 being the most active compounds against Trypanosoma brucei rhodes-
iense, Leishmania donovani, and Trypanosoma cruzi cells (IC50 = 50 nM, IC50 = 0.28 lM, and IC50 = 1.26 lM, respectively).
Ó 2008 Elsevier Ltd. All rights reserved.
Trypanosomatids are the causative agents of various
lethal parasitic diseases such as Chagas’ disease (Trypan-
osoma cruzi), African sleeping sickness (Trypanosoma
brucei with the two sub-species T. b. gambiense and
T. b. rhodesiense), and leishmaniases (Leishmania dono-
vani, L. major, and L. tropica).1 Such diseases mainly af-
fect the least developed countries and almost half a
billion people are presently at risk. Despite its epidemi-
ological importance, the therapy of trypanosomatid
infections remains an unmet challenge.2,3 No vaccines
are currently available to prevent these diseases, and
the recommended drugs have high toxicity and limited
efficacy.4 In addition, rapidly-developing drug resistance
has become a major problem. Although new anti-proto-
zoan drugs are in development for these diseases,5 the
lead identification still represents a real bottleneck,6
and the drug development pipeline is currently almost
empty.7
Combinatorial chemistry is one potential strategy for
drug discovery.8 Drug discovery for these diseases can
be tailored to academic settings where cost-effectiveness
in synthesizing new anti-parasitic agents is a major con-
sideration. Attention should therefore focus on a strat-
egy that can yield molecules with high hit rates and a
concomitant reduced library size. In this respect, the
concept of natural-product-derived compound collec-
tions is particularly attractive. This strategy recognizes
that natural product fragments have been evolutionarily
selected and biologically pre-validated. They are there-
fore appropriate starting points for the development of
compound collections.9–11 This approach may be partic-
ularly fruitful if a link already exists between a given
compound class and the desired biological activity.11
Here, we present a small library of compounds endowed
with anti-trypanosomatid activity, obtained through a
parallel approach. In the library design, we selected
the quinone unit as the core structure for combinatorial
derivatization. Naphthoquinones and other related qui-
none compounds are one of the major natural product
classes with significant activity against Leishmania and
Trypanosoma.12 For instance, lapachol (1, Fig. 1) was
reported to exhibit marked anti-trypanosomal and leish-
Keywords: Anti-parasitic; Anti-protozoan; Neglected diseases; Tropi-
cal diseases; Compound library; Parallel synthesis; Whole-cell assays.
*
Corresponding authors. Tel.: +39 051 2099700; e-mail addresses:
marialaura.bolognesi@unibo.it; andrea.cavalli@unibo.it
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.009

M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2272–2276
2273
O
O
OH
O
O
pound 8 was just fivefold less active than the
arsenic derivative melarsoprol (21, 10 nM), which
was used as a reference compound (Fig. 2). Unfor-
tunately, 8 was also quite cytotoxic against L6
cells (IC50 = 1000 nM) with a selectivity index
(SI = IC50L6/IC50parasite) of 20. Adding substitu-
ents to 8 did not improve its activity against the
parasitic cells. Furthermore, L6 cytotoxicity was
usually increased by the introduction of halogens
on the phenyl ring to mimic triclosan structure.
However, a fairly good increase of SI was
observed with compound 3 (i.e., 2-(2,4-dichloro-
phenoxy)-1,4-anthraquinone), which retained
nanomolar activity (IC50 = 65 nM) and showed
SI > 30. Concerning the 2-phenoxy-1,4-naphtho-
quinone derivatives, 11–18 showed quite interest-
ing profiles against T. b. rhodesiense, with 16
being active in the nanomolar range, and 11–15
and 17–18 being active in the micromolar range.
Compound 16 was the most interesting compound
we discovered. It showed an IC50 value of 80 nM
and an SI of 74, which is very close to the value
considered a hit by WHO/TDR (SI more than
100).6 All other 2-phenoxy-1,4-naphthoquinone
derivatives (11–15 and 17–18) showed both lower
inhibitory activities and lower SIs when compared
to 16.
O
Cl
Cl
Cl
O
O
R1
O
O
Ar
OH
R2
3-18
O
Figure 1. Design strategy for compounds 3–18.
manicidal activity, while displaying no serious toxic ef-
fects in humans.13 Therefore, based on the 1,4-naphtho-
quinone and 1,4-anthraquinone natural scaffolds, we
synthesized 16 compounds (3–18, Table 1), which incor-
porated, at position 2, a selection of aromatic groups
that would mimic a structural element of triclosan (2,
Fig. 1). Triclosan is a general biocide which was recently
demonstrated to kill both procyclic forms and blood-
stream forms of T. brucei14 (see Fig. 1 for the design
strategy).
(ii) Concerning the amastigote form of T. cruzi, most
of our derivatives showed activity in the micromo-
lar range (see Table 2). The most interesting result
was again obtained with 16, which exhibited an
IC50 value of 1.26 lM. Compound 16 was slightly
more potent than the reference compound benzni-
dazole (22 in Fig. 2), which has an IC50 of
1.70 lM. Here too, however, the low SI (<5) was
the main drawback for 16, pointing to the need
for further investigation of the present compound
series to decrease cell cytotoxicity. The profiles of
13 (IC50 = 2.47 lM and SI = 2.1) and 15
(IC50 = 2.88 lM and SI = 1.7) were very similar
to that of the parent compound. Among the 2-
phenoxy-1,4-anthraquinone derivatives, the most
active inhibitor was 6, showing IC50 and SI values
of 2.87 lM and 0.39, respectively. Remarkably, in
contrast to their activity against T. b. rhodesiense,
the 2-phenoxy-1,4-anthraquinone derivatives were
generally less potent against T. cruzi cells than the
2-phenoxy-1,4-naphthoquinones.
For the development of an efficient and cheap parallel
synthesis approach, we focused our attention on the dis-
placement reaction of 2-bromoquinones (19a and 19b)
with phenoxides (20a–h) (Table 1). We were able to car-
ry out a one-pot reaction at room temperature that, in
most cases, could achieve the quantitative conversion
of the starting reactant within 3 h (Scheme 1). More-
over, we developed an operationally simple and versatile
workup protocol, which involved the recovery of high-
purity final products by filtration upon addition of water
to the reaction mixture.15 As 19a is the only reagent that
is not commercially available, we carried out an efficient
bromination procedure starting from the readily avail-
able 1,4-anthraquinone (Scheme 2).16
(iii) When tested against the axenic amastigote form of
L. donovani, the derivatives were all less potent
than they were against T. b. rhodesiense. All the
compounds were active in the micromolar range.
The most potent of the present series was 10 (i.e.,
2-(2,4-difluoro-phenoxy)-1,4-anthraquinone) with
an IC50 value of 0.28 lM. Remarkably, this value
was very similar to that of the reference compound
miltefosine (23 in Fig. 2), which has an IC50 of
0.31 lM. This indicates 10 as a possible hit candi-
date for the development of new anti-leishmanial
derivatives. However, 10 also showed low selectiv-
ity with an SI of 7. Among the 2-phenoxy-1,4-
naphthoquinone derivatives, 13 was the most inter-
esting compound, endowed with an IC50 value of
Table 2 reports the activity of compounds 3–18 against
T. b. rhodesiense, T. cruzi, and L. donovani as well as
their cytotoxicity against L6 cells (rat skeletal myo-
blasts). The anti-parasitic potential of all compounds
was analyzed with regard to the WHO/TDR screening
activity criteria.6 In the following paragraphs, we discuss
the results of compounds 3–18 against (i) T. b. rhodes-
iense, (ii) T. cruzi, and (iii) L. donovani.
(i) All the derivatives showed good activity against
the blood trypomastigote form of T. b. rhodes-
iense. The unsubstituted 2-phenoxy-1,4-anthraqui-
none derivative (8) was the most potent of the
series, showing an IC50 value of 50 nM. Com-

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M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2272–2276
Table 1. The compound library obtained by a one-pot parallel
synthesis approach
Table 1 (continued)
Quinones (19)
Phenols (20)
Entries (3–18)
Quinones (19)
Phenols (20)
Entries (3–18)
O
Br
Br
O
O
O
Cl
F
HO
Cl
Br
O
O
O
O
HO
F
20d
F
14
O
O
Cl
Cl
20a
Cl
Cl
19a
3
O
O
HO
O
O
O
F
HO
20e
Br
15
16
O
O
Br
20b
4
O
HO
O
20f
O
O
HO
20c
Br
F
5
Br
O
O
O
HO
Br
F
20g
O
Br
Br
F
17
18
Br
O
O
HO
F
20d
F
F
6
O
HO
20h
O
O
HO
O
O
20e
Br
7
O
Br
O
OH
O
R
K2CO3
DMF
Br
O
Ar
R
Ar
+
O
HO
O
O
20f
19a and 19b
20a-h
3-18
8
O
Scheme 1. Synthesis of the compound library. Reagents and condi-
tions: phenol, K2CO3 in DMF, 30 min, then bromoquinone, room
temperature, 3 h.
O
Br
Br
O
HO
Br
20g
H
Br
F
9
O
H2N
N
N
N
N
S
O
F
As
S
F
O
NH2
OH
HO
21 (melarsoprol)
F
20h
O
N
10
11
12
13
—O
O
O
N+
N
H
N
H
O
O
O
O
Cl
22 (benznidazole)
Cl
Br
HO
O
P
O-
H3C(H2C)15
O
O
(CH2)2N+(CH3)3
Cl
Cl
20a
Cl
Cl
19b
O
O
23 (miltefosine)
F
Figure 2. Chemical structure of some currently marketed drugs used as
reference compounds (21–23).
F
O
O
HO
20b
O
O
0.51 lM and an SI of 10. Here too, the potency was
fairly good, while the selectivity needs to be
increased to reduce possible adverse effects.
HO
As a general observation, cytotoxicity is a common
problem for many natural products2 as they are usually
20c
O

M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2272–2276
2275
a
Table 2. Anti-trypanosomatid activity of the compound library expressed as IC50 (lM)
Compound
T. b. r.
T. c.
L. d.
L6
SI
T. b. r.
SI
T. c.
SI
L. d.
3
4
0.065
0.21
0.31
0.08
0.15
0.05
0.14
0.24
0.29
0.37
0.22
0.36
0.30
0.08
0.51
0.39
0.01
13.7
21.5
22.5
2.87
6.36
4.66
16.4
7.14
5.95
4.43
2.47
3.74
2.88
1.26
34.3
5.00
0.49
0.53
0.55
0.35
0.36
0.34
0.47
0.28
1.32
0.70
0.51
1.81
0.92
1.26
2.37
2.29
2.01
31.0
12.4
13.2
14.1
17.4
20.0
15.4
8.43
17.8
12.5
24.4
13.1
16.4
74.0
55.7
14.2
0.15
0.12
0.18
0.39
0.40
0.21
0.13
0.28
0.87
1.03
2.13
1.25
1.71
4.70
0.83
1.10
4.15
5.01
7.53
3.24
7.10
2.94
4.71
7.01
3.94
6.54
10.3
2.58
5.34
4.69
12.1
2.41
2.64
4.13
1.13
2.56
1.00
2.19
1.98
5.18
4.58
5.26
4.66
4.92
5.92
5
6
7
8
9
10
11
12
13
14
15
16
17
18
21
22
23
28.5
5.51
1.70
0.31
a IC50 values for the blood trypomastigote form of Trypanosoma brucei rhodesiense (T. b. r.), the amastigote form of Trypanosoma cruzi (T. c.) in L6
cells, and the axenic amastigote form of Leishmania donovani (L. d.) are reported. The L6 cytotoxicity as well as the selectivity index (SI = IC50 for
L6/IC50 for parasite) for all parasites is also shown. Compounds 21, 22, and 23 are the standard drugs melarsoprol, benznidazole, and miltefosine,
respectively. A detailed description of the assays can be found in Refs. 17 and 18.
tion of the versatile synthetic route to enlarge the pres-
O
O
O
O
ent series of compounds, with the aim of tuning down
human cell toxicity; (2) application of biochemical and
molecular biology tools19 in order to identify the target
protein(s) of these compounds. Here, a chemical proteo-
mics approach will allow us to elucidate the SARs in
more detail and, thus, to more effectively address the de-
sign of further less toxic derivatives. It is worth noting
that, in addition to a possible target-related mechanism,
the general free-radical-generation mechanism of
quinones (probably at the basis of their general cytotox-
icity) may be exploited to prevent resistance
development.20
Br
Br2
CH3COOH
19a
Scheme 2. Synthesis of 2-bromo-anthracene-1,4-dione. Reagents and
condition: Br2 dropwise, cooling, 2.5 h.
produced to work as biological defense mechanisms.
With respect to safety, however, a greater tolerance
may be acceptable for anti-trypanosomatid candidates,
given the short course of therapy and the scarce safety
profile of the currently available drugs.7
Acknowledgments
In conclusion, we report on the design, synthesis, and
anti-parasitic screening of a small library of natural-
product-derived naphtho- and anthra-quinones against
trypanosomatid parasites. Notwithstanding the small
number of synthesized compounds, we discovered some
very potent inhibitors against T. b. rhodesiense, and
some compounds that were active against T. cruzi and
L. donovani. Although the present derivatives were over-
all quite cytotoxic toward L6 cells, we identified a prom-
ising hit compound. Indeed, 16 showed an IC50 value of
80 nM against T. b. rhodesiense cells and an SI of 74,
which is very close to the specifications required by
WHO/TDR for 16 to be considered an anti-trypanoso-
matid hit. This confirms the rationale of our design
strategy (Fig. 1). In addition, the low-cost criterion
was met by using the devised straight synthetic route,
which also made use of readily available synthons. In
the light of these results, we plan to complement the
present study with two strategies: (1) further exploita-
This research was supported by the University of Bolo-
gna. We thank C. Melchiorre and M. Recanatini for the
critical reading of the manuscript. Ms. S. Vitillo is grate-
fully acknowledged for her technical assistance.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
03.009.
References and notes
1. Available from: http://www.who.int/tdr.
2. Pink, R.; Hudson, A.; Mouries, M. A.; Bendig, M. Nat.
Rev. Drug Disc. 2005, 4, 727.

2276
M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2272–2276
3. Gelb, M. H.; Hol, W. G. Science 2002, 297, 343.
4. Linares, G. E.; Ravaschino, E. L.; Rodriguez, J. B. Curr.
Med. Chem. 2006, 13, 335.
5. Hotez, P. J.; Molyneux, D. H.; Fenwick, A.; Kumaresan,
J.; Sachs, S. E.; Sachs, J. D.; Savioli, L. N. Engl. J. Med.
2007, 357, 1018.
6. Nwaka, S.; Hudson, A. Nat. Rev. Drug Disc. 2006, 5, 941.
7. Renslo, A. R.; McKerrow, J. H. Nat. Chem. Biol. 2006, 2, 701.
8. Gutteridge, W. E. Parasitology 1997, 114, S145.
9. Koch, M. A.; Wittenberg, L. O.; Basu, S.; Jeyaraj, D. A.;
Gourzoulidou, E.; Reinecke, K.; Odermatt, A.; Wald-
mann, H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16721.
10. Breinbauer, R.; Vetter, I. R.; Waldmann, H. Angew.
Chem. Int., Ed. Engl. 2002, 41, 2879.
11. Lessmann, T.; Leuenberger, M. G.; Menninger, S.; Lopez-
Canet, M.; Muller, O.; Hummer, S.; Bormann, J.; Korn,
K.; Fava, E.; Zerial, M.; Mayer, T. U.; Waldmann, H.
Chem. Biol. 2007, 14, 443.
was stirred for 1 h at room temperature. 2-Bromo-
anthracene-1,4-dione (0.35 mmol) or 2-bromo-naphtha-
lene-1,4-dione (0.35 mmol) was added. After standing for
3 h, the mixture was treated with cold water and filtered.
The obtained solid was then washed and dried under
vacuum at 40 °C for 6 h. The above synthetic protocol
was repeated using a total of eight different phenols to
produce eight 2-phenoxy-[1,4]-naphthoquinone and eight
2-phenoxy-anthracene-1,4-dione derivatives as yellow sol-
ids. If needed, 3–18 can be crystallized by petroleum ether
(for details see Supplementary Material).
16. A solution of Br2 (0.25 mL, 4.80 mmol) in glacial acetic
acid (10 mL) was added dropwise to a suspension of 1,4-
anthraquinone (1 g, 4.80 mmol) in glacial acetic acid
(20 mL) in an ice-water bath. The reaction mixture was
stirred for 2.5 h with cooling, then treated with cold water
and filtered. The formed solid was collected by filtration
and crystallized from ethanol to give 19a as an orange
1
12. Kayser, O.; Kiderlen, A. F.; Croft, S. L. Parasitol. Res.
2003, 90, S55.
13. Krohn, K.; Hussain, H.; Viqar, U. A.; Ghulam, A. M.;
Green, I. R. Arkivoc 2007, ii, 145.
solid: 81% yield; H NMR (CDCl3, 200 MHz): d 7.65 (s,
1H), d 7.73–7.78 (m, 2H, J = 10 Hz), d 8.08–8.13 (m, 2H,
J = 10 Hz), d 8.66 (s, 1H), d 8.75 (s, 1H). ESI-MS: found
309 (M++Na+, 79Br); 311 (M++Na+, 81Br).
14. Paul, K. S.; Bacchi, C. J.; Englund, P. T. Eukaryot. Cell
2004, 3, 855.
17. Orhan, I.; Sener, B.; Atici, T.; Brun, R.; Perozzo, R.;
Tasdemir, D. Phytomedicine 2006, 13, 388.
15. General procedure for the synthesis of the library. A
carousel reaction tube was charged with the selected
phenol (0.35 mmol) and DMF (9 mL). After 1 min,
K2CO3 (1.05 mmol) was added and the reaction mixture
18. Kuettel, S.; Zambon, A.; Kaiser, M.; Brun, R.; Scapozza,
L.; Perozzo, R. J. Med. Chem. 2007, 50, 5833.
19. Lokey, R. S. Curr. Opin. Chem. Biol. 2003, 7, 91.
20. Efferth, T. Drug Resist. Updat. 2005, 8, 85.