Synthesis and in vitro anti-protozoal activity of a series of benzotropolone derivatives incorporating endocyclic hydrazines

Article abstract of DOI:10.1016/j.ejmech.2003.07.004

The preparation and evaluation as potential anti-protozoal agents of molecules bearing an endocyclic hydrazine moiety is presented. The synthetic route to this new series of compounds is straightforward, involving a hetero Diels-Alder reaction between dif

Full text of DOI:10.1016/j.ejmech.2003.07.004

European Journal of Medicinal Chemistry 38 (2003) 949ꢀ957
/
www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro anti-protozoal activity of a series of
benzotropolone derivatives incorporating endocyclic hydrazines
Hongyu Ren a, Shannon Grady a, Matthew Banghart a, Jason S. Moulthrop a,
Howard Kendrick b, Vanessa Yardley b, Simon L. Croft b, Guillermo Moyna a,*
a Department of Chemistry & Biochemistry, University of the Sciences in Philadelphia, 600 South 43rd Street, Philadelphia, PA 19104, USA
b Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
Received 28 April 2003; received in revised form 17 July 2003; accepted 23 July 2003
Abstract
The preparation and evaluation as potential anti-protozoal agents of molecules bearing an endocyclic hydrazine moiety is
presented. The synthetic route to this new series of compounds is straightforward, involving a hetero DielsꢀAlder reaction between
different benzotropolone esters and diethyl azodicarboxylate (DEAD). While they show limited or no in vitro activity against
/
Leishmania donovani, Plasmodium falciparum and Trypanosoma brucei rhodesiense, several of the compounds have activities against
Trypanosoma cruzi in the 15.8ꢀ41.0 mM range. These activities are comparable to that of benznidazole (IC50 6.0 mM), the main
/
chemotherapy employed in the treatment of T. cruzi infections. In addition, all but one of the new bicyclic hydrazine esters are
virtually non-toxic, one of the most important drawbacks of currently available trypanocidal drugs.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Benzotropolone derivatives; Endocyclic hydrazines; Hetero DielsꢀAlder reaction; Anti-protozoal activity
/
1. Introduction
toxicity, and present adverse side effects [5]. In view of
this, the development of new classes of readily accessible
compounds with anti-protozoal activity and improved
pharmacological properties is imperative.
In a recent report we presented the synthesis and
biological evaluation against P. falciparum and T. cruzi
of a series of novel oxazine esters (Fig. 1a) [6]. The
preparation of these compounds was straightforward,
involving the esterification of benzotropolones followed
Parasitosis such as malaria, sleeping sickness, Leish-
maniasis and Chagas disease are a threat to half of the
world population, and constitute one of the most
important public health concerns in tropical and sub-
tropical regions. Despite the efforts made in the past 50
years to control transmission vectors, parasitic diseases
are still endemic in most areas of incidence, with the
number of infected individuals growing yearly [1,2].
Furthermore, the development of effective vaccines
against them has been hampered by the complex biology
and high adaptability through antigenic variation of the
protozoan causative agents [3]. Thus, the main line of
defense against parasitic infections has been through
chemotherapy. However, and with the exception of
artemisinin and its derivatives for the treatment of
malaria [4], most of the drugs currently used for this
purpose are old and non-specific, have significant
by a high-yielding hetero Dielsꢀ
which nitrosobenzene was used to incorporate an
endocyclic ꢀNꢀOꢀ moiety into the seven-membered
tropolone ring.
/Alder cycloaddition in
/
/
/
While these compounds displayed only limited activ-
ity against P. falciparum, several showed activities
against T. cruzi comparable to that of benznidazole
(Fig. 1b), one of the current therapies against this
parasite. Unfortunately, the most active molecules had
significant cytotoxicity due to their time-dependent
thermal decomposition into starting materials with
concomitant release of nitrosobenzene [7]. These results
showed, however, that a new class of compounds with a
completely novel molecular framework and potential
* Corresponding author.
E-mail address: g.moyna@usip.edu (G. Moyna).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00180-6

950
H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ957
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Fig. 1. General structure of the oxazines mentioned in the text (a), and of the antiparasitic drug benznidazole (b).
antiparasitic activity could be obtained from easily
accessible starting materials using well established
catalytic dimethyl aminopyridine (DMAP) using
CH2C12 as solvent. The selection of the acyl chlorides
employed in the acylations was guided by several
factors. Namely, they correspond with those used in
our earlier work [6], are readily available commercially,
and provide a small but representative series of alkyl
and aryl substitutions at the C4?-position of the
benzotropolone. Ten aliphatic and aromatic esters
were prepared in yields ranging from 55 to 93%
chemical transformations [8ꢀ
One of the attractive features of our approach is that
the flexibility of the hetero DielsꢀAlder reaction can be
exploited to incorporate moieties other than ꢀNꢀOꢀ
/12].
/
/
/
/
into the final products, while at the same time main-
taining a similar molecular scaffold. Thus, compounds
bearing bridged ꢀ
ments can be obtained by simply changing the dieno-
phile used in the cycloaddition step [9ꢀ12]. This allows
/
Nꢀ
/
Nꢀ
/
, ꢀ
/
Nꢀ
/
Sꢀ
/
, or ꢀ
/
Oꢀ
/
Oꢀ
/
frag-
following this procedure (2aꢀ
The final bicyclic hydrazines were then obtained by
treatment of esters 2aꢀ2j with 2 equiv. of DEAD in
refluxing toluene. The reaction proceeded smoothly
giving exclusively products 3aꢀ3j, with no starting
materials detectable by TLC after 16ꢀ32 hours. In
addition to these compounds, the DEAD DielsꢀAlder
/
2j, Fig. 2).
/
for a large number of compounds based on common
precursors to be rapidly synthesized and tested, increas-
ing the structural diversity and efficiency of the lead
identification process. In this report we wish to present
the evaluation as anti-protozoal agents of a series of
benzotropolone derivatives bearing an endocyclic hy-
drazine moiety which were prepared through this
approach. As described below, several of the new
compounds have activities against T. cruzi approaching
that of benznidazole, and, in contrast to our original
oxazine esters and several antiparasitic drugs, display
very low cytotoxicity.
/
/
/
/
adducts of tetramethylpurpurogallin (2k) and 1, com-
pounds 3k and 3l, were also prepared following this
procedure.
In all cases, the success of the DielsꢀAlder reaction
/
was confirmed by inspection of the spectroscopic data,
particularly the 1H-NMR spectra. Apart from the
signals corresponding to two distinct ethylcarbamate
groups in the DEAD adducts, considerable differences
for the chemical shifts of protons in the tropolone ring
are observed before and after the cycloaddition. While
protons on carbons C5, C6, and C7 in the starting
materials display signals at approximately 6.20, 6.50 and
6.90 ppm, respectively, their resonances shift to roughly
5.95, 7.25 and 5.70 ppm in the products. These changes
are consistent with the conversion of the planar pseudo-
aromatic tropolone ring system into a bicyclic frame-
2. Chemistry
As stated in the Introduction, our strategy for the
preparation of the different series of exploratory com-
pounds relies on a hetero DielsꢀAlder reaction between
/
benzotropolone derivatives and diverse dienophiles. In
order to incorporate a bridged hydrazine moiety,
dienophiles bearing an electron-deficient azo functional
group are required. A compound that has these char-
acteristics is diethyl azodicarboxylate (DEAD). Apart
from being a commercially available reagent, the use of
DEAD in cycloaddition reactions is well documented
[9], and it was therefore ideal for our purposes.
work in which the C5ꢀC6 double bond is not co-planar
/
with the remaining aromatic ring.
It is also worth noting that when we attempted to
1
record the H- or 13C-NMR spectra of compounds 3aꢀ
/
3l at 9.4 Tesla, most signals corresponding to nuclei in
the vicinity of the bridged nitrogen atoms were severely
broadened due to the effects of the 14N quadrupoles on
1H and 13C relaxation times. Although still observable,
these effects were not as dramatic at lower magnetic
The first step towards the DielsꢀAlder adducts is the
/
synthesis of the benzotropolone precursors, which has
been described by us in previous reports [6,13]. Briefly,
the C4?-position hydroxyl group of trimethylpurpuro-
gallin (1) was acylated by treatment with the appropriate
acyl chloride in the presence of triethylamine and
fields, and all NMR spectra for compounds 3aꢀ
/3l were
recorded at 2.1 Tesla.

H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ
/957
951
Fig. 2. Synthetic route to hydrazines 3aꢀ
/
3l. (i) Rꢀ
/
COC1 (1.5 equiv.), Et3N (1.5 equiv.), DMAP (0.5 equiv.), CH2C12, RT, 8ꢀ
/
16 h. (ii) EtCO2 ꢀ
/
NÄ
/
NꢀCO2Et (2.0 equiv.), toluene (reflux), 16ꢀ
/
/32 h. PhthN represents the phthalinido group (compounds 2j and 3j).
3. Biological results and discussion
The in vitro biological activity of compounds 3aꢀ
against L. donovani, T. brucei rhodesiense, P. falciparum
and T. cruzi is presented in Table I. Our data indicate
that the compounds are ineffective against L. donovani,
with all ICs50 being at least higher than 48.2 mM.
Hydrazines 3b and 3f display only limited activity
against P. falciparum, with ICs50 more than three orders
of magnitude larger than that measured for chloroquine.
Similar low activities against T. brucei were found for
compounds 3e, 3g, 3h and 3j, their ICs50 in this case
being at least four orders of magnitude higher than that
of the control pentamidine.
The results obtained in the T. cruzi assays were more
encouraging. The ICs50 for hydrazines 3a, 3c, 3d, 3f, and
/3l
3k against this parasite were in the 15.8ꢀ41.0 mM range.
/
These ICs50 are less than one order of magnitude higher
than the one of benznidazole, found to be 6.0 mM in the
same assay, with compound 3d being only 2.5 times less
Table I
In vitro activity of hydrazines 3aꢀ/3l against protozoan parasites
Compound
IC50 (mM)
Toxicity (ED50 mg mLꢁ1
)
L. donovani
P. falciparum
T. brucei
T. cruzi
3a
3b
3c
3d
3e
3f
ꢀ
/
62.8
61.0
59.3
55.9
55.6
52.6
52.3
53.8
53.8
48.2
68.8
66.7
ꢀ
/
62.8
51.4
59.3
55.9
55.6
39.1
52.3
53.8
53.8
48.2
68.8
66.7
ꢀ
/
62.8
61.0
59.3
55.9
30.6
52.6
17.9
44.9
53.8
38.4
68.8
66.7
41.0
ꢀ
/
300
300
300
300
300
300
300
300
ꢀ
/
ꢀ
/
ꢀ
/
61.0
26.3
15.8
55.6
23.0
52.3
53.8
53.8
48.2
37.4
66.7
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
3g
3h
3i
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
B0.3
/
3j
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
300
300
300
3k
31
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
The following controls were used: Pentostam (IC50 4.0 mg SbV mLꢁ1, L. donovani); Chloroquine (IC50 3.0 nM, P. falciparum); Pentanidine (IC50
35 nM, T. brucei); Benznidazole (IC50 6.0 mM, T. Cruzi). Toxicity was assayed against KB cells using podophyllotoxin (ED50 0.0001 mg mLꢁ1) as
standard.

952
H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ957
/
active than the control. Although no clear correlation
between the structure of the substituent on the C4?
hydroxyl and activity can be established, the presence of
aliphatic chains at this position appears to be beneficial,
with p-anisoate ester 3f being the exception. From the
compounds in this subset, acetoacetate ester 3d deserves
further attention. This molecule is the most active
against T. cruzi, and, as shown above, is ineffective
against other parasites. Interestingly, the corresponding
acetoacetate ester from our earlier oxazines had no
trypanocidal activity, but proved to be the most active
compound from that series against P. falciparum [6].
Although preliminary, these data seem to indicate that
the nature of the endocyclic moiety could be involved in
specificity, and to some extent the parasite targeted by
these compounds.
dal activity of the compounds, most likely due to an
enhancement of their membrane permeability. This
would explain why compound 3l, which has a free
phenolic hydroxyl group at the C4?-position, lacks
activity against all four parasites studied. In the case
of compound 3d, the acetoacetate ester could initially
confer permeability through the cell membrane, only to
be hydrolyzed to a free hydroxyl group after absorption.
The resulting decrease in lipophilicity could prevent the
molecule from escaping the trypanosomatids, explaining
why this derivative displayed the highest activity in the
T. cruzi assay. A similar prodrug behavior has been
proposed for chloroquine and other aminoquinolone
derivatives [14], and this could be exploited to further
improve the pharmacodynamic properties and activity
of these compounds.
Cytotoxicity against KB cells is also reported in Table
I. Except for p-fluorobenzoate ester 3i, which inciden-
tally was one of the two compounds inactive in all anti-
protozoal assays performed, the molecules were non-
Although the mode of action of these molecules is
unknown at this time, we believe that their activity
against T. cruzi is linked to the presence of the
endocyclic hydrazine moiety. The single NꢀN bond in
/
toxic to cells in concentrations of up to 300 mg mLꢁ1
.
this group has a bond dissociation energy (BDE) of only
38.4 Kcal molꢁ1 [15], making it prone to undergo
homolytic cleavage. The resulting free radicals should be
capable of disrupting oxygen metabolism processes
specific to trypanosomatids, which are known to be
highly sensitive to oxidative stress [16]. This mechanism
of action has been proposed for benznidazole, nifurti-
mox, and other nitroaromatic compounds [17,18]. Due
to their low in vitro toxicity and unique structure, it is
worth investigating if the novel compounds described in
this report have a mode of action similar to that of the
aforementioned nitroaromatic drugs.
These results contrast those obtained for our earlier
oxazine derivatives, which were cytotoxic at concentra-
tions of 60.2 mg mLꢁ1 or lower [6]. As mentioned
earlier, the benzotropolone derivatives containing a
bridged ꢀ
/
Nꢀ
/
Oꢀ
/
moiety undergo a time-dependent
Alder
thermal decomposition through a retro Dielsꢀ
/
reaction. This process, driven by the return to stable
aromatic starting materials [6,7], releases highly toxic
nitrosobenzene. Thus, all of the compounds from our
original series with activity against T. cruzi were
significantly cytotoxic, and none of them had therapeu-
tic indexes (TI) above 7.5. For example, the most active
oxazine in the T. cruzi assay, with an IC50 of 4.3 mM,
was toxic to KB cells at concentrations of 0.3 mg mLꢁ1
or higher (TIB0.19). These molecules also displayed
/
considerable activity against P. falciparum, indicating
that they were non-specific. On the other hand, hydra-
zines 3a, 3c, 3d, 3f and 3k have TI values ranging from
15.3 to 35.3, and, with the exception of p-anisoate 3f
which shows limited activity in the P. falciparum assay,
are specific against T. cruzi.
The results presented above allow us to draw several
conclusions regarding the biological properties of the
compounds reported here. First, comparison of activity
4. Conclusions
The preparation and biological evaluation of a series
of benzotropolone derivatives incorporating bridged
hydrazine fragments was presented. Our results show
that the compounds are virtually non-toxic, and despite
they posses limited or no activity against L. donovani, P.
falciparum and T. brucei rhodesiense, they display
activities against T. cruzi which approach that of
benznidazole. Comparison of these results with biologi-
cal data obtained for an earlier series of structurally
related oxazine derivatives indicates that the activity of
the new compounds stems from the presence of the
endocyclic hydrazine moiety, while the substituent at the
C4?-position is likely to modulate their pharmacody-
namic properties. Experiments aimed at improving the
activity of this novel class of compounds and determin-
ing their mode of action are underway, and the results
from these investigations will be presented in due course.
and cytotoxicity data obtained for the Dielsꢀ
/Alder
adducts bearing ꢀNꢀNꢀ and ꢀNꢀOꢀ groups indicates
/
/
/
/
/
/
that the endocyclic fragment determines the specificity
of the benzotropolone derivatives towards different
parasites. Biological data for structurally related com-
pounds bearing other endocyclic moieties, which, as
mentioned earlier, can be obtained by varying the
structure of the dienophiles used in the hetero Dielsꢀ
/
Alder reaction, are required to further prove this
hypothesis. Second, aliphatic substituents at the C4?-
position were shown to improve the in vitro trypanoci-

H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ
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953
5. Experimental
5.1.1.2. 4,2?,3?-Trimethyl-4?-propionyl-purpurogallin
(2b). Obtained in 67% yield. M.p. 114ꢀ116 8C. IR (KBr
disc, cmꢁ1): 1595 (s, CÄ
O, ketone), 1764 (s, CÄO,
H). H-NMR (400 MHz): d 1.32
7.6), 2.74 (2H, q, Jꢂ7.6), 3.80 (3H, s), 3.89
(3H, s), 3.96 (3H, s), 6.23 (1H, d, Jꢂ8.8), 6.53 (1H, dd,
11.6).
/
/
/
5.1. Synthesis
1
ester), 2942 (w, Arꢀ
/
(3H, t, Jꢂ
/
/
Melting points were determined on a Thomas-Hoover
capillary melting point apparatus and are uncorrected.
When appropriate, products were purified by column
/
Jꢂ
/
8.8, Jꢂ
/
11.6), 6.88 (1H, bs), 6.91 (1H, bd, Jꢂ
/
13C-NMR (100 MHz): d 9.4, 27.8, 56.4, 56.5, 61.5,
chromatography using 60ꢀ
Science, NJ, USA). IR spectra were obtained on a
PerkinꢀElmers spectrum 1000 FT-IR instrument. NMR
/200 mesh silica gel (EM
106.9, 110.1, 124.1, 124.4, 129.9, 133.7, 142.9, 144.0,
155.5, 159.4, 173.1, 183.1. ESI-MS: 319.4 ([M]ꢃH).
/
/
spectra were recorded on Bruker Avance 400 and
Anazasi EFT-90 spectrometers at fields of 9.4 and 2.1
Tesla, respectively, using CDC13 as a solvent unless
otherwise specified. Chemical shifts (d) are in ppm
relative to tetramethylsilane (TMS), and coupling con-
stants (J) are reported in Hz. ESI-MS spectra were
recorded on a Micromass Trio-2000 single quadrupole
5.1.1.3. 4,2?,3?-Trimethyl-4?-butyryl-purpurogallin (2c).
Obtained in 67% yield. M.p. 116ꢀ119 8C. IR (KBr
disc, cmꢁ1): 1624 (s, CÄ
O, ketone), 1769 (s, CÄO,
H). H-NMR (400 MHz): d 1.09
7.6), 1.86 (2H, sex, Jꢂ7.6), 2.69 (2H, t, Jꢂ
7.6), 3.81 (3H, s), 3.89 (3H, s), 3.97 (3H, s), 6.23 (1H, d,
Jꢂ8.8), 6.54 (1H, dd, Jꢂ8.8, Jꢂ11.6), 6.88 (1H, bs),
6.93 (1H, bd, Jꢂ
11.6). 13C-NMR (100 MHz): d 14.0,
/
/
/
1
ester), 2950 (m, Arꢀ
/
(3H, t, Jꢂ
/
/
/
/
/
/
mass spectrometer. Purpurogallin (m.p. 276ꢀ
/
277 8C,
/
dec., lit. 271ꢀ272 8C [19]) was prepared in 75% yield
/
18.7, 36.3, 56.4, 56.5, 61.4, 106.8, 110.1, 124.1, 124.4,
129.9, 133.6, 142.8, 143.9, 155.5, 159.4, 172.2, 183.1.
by oxidation of pyrogallol with aqueous KIO3 following
reported procedures [19]. Trimethylpurpurogallin (1,
ESI-MS: 331.1 ([M]ꢃH).
/
m.p. 174ꢀ
/
176 8C, lit. 174ꢀ
/
177 8C [20]) and tetramethyl-
purpurogallin (2k, m.p. 90ꢀ
/
91 8C, lit. 92ꢀ93 8C [7])
/
methylethers were obtained in 42 and 82% yield,
respectively, by treatment of purpurogallin with di-
methyl sulfate in aqueous NaOH using the protocols
of Barltrop and Nicholson [20].
5.1.1.4. 4,2?,3?-Trimethyl-4?-acetoxyacetyl-purpurogallin
(2d). Obtained in 55% yield. M.p. 142ꢀ144 8C. IR (KBr
disc, cmꢁ1): 1624 (s, CÄ
O, ketone), 1750, 1786 (s, CÄO,
H). H-NMR (400 MHz): d 2.22
(3H, s), 3.84 (3H, s), 3.93 (3H, s), 4.00 (3H, s), 5.04 (2H,
bs), 6.29 (1H, d, Jꢂ8.8), 6.59 (1H, dd, Jꢂ8.8, Jꢂ
11.6), 6.94 (1H, bs), 6.98 (1H, bd, Jꢂ
11.6). 13C-NMR
/
/
/
1
ester), 2943 (w, Arꢀ
/
/
/
/
5.1.1. General procedure for the preparation of 4,2?,3?-
/
trimethylpurpurogallin derivatives (2aꢀ2j)
/
(100 MHz): d 21.0, 56.4, 56.6, 61.3, 61.7, 107.3, 110.7,
124.1, 124.3, 130.0, 133.7, 143.1, 143.2, 155.7, 159.5,
The appropriate acyl chloride (15.2 mmol) in CH2C12
(5 mL) was added gradually to a solution of trimethyl-
purpurogallin (1, 7.6 mmol), DMAP (3.8 mmol), and
triethylamine (15.2 mmol) in CH2C12 (20 mL) at 0 8C
under a nitrogen atmosphere. The resulting solution was
166.7, 170.7, 182.6. ESI-MS: 363.0 ([M]ꢃH).
/
5.1.1.5. 4,2?,3?-Trimethyl-4?-benzoyl-purpurogallin (2e).
Obtained in 63% yield. M.p. 158ꢀ160 8C. IR (KBr disc,
cmꢁ1): 1598 (s, CÄ
O, ketone), 1742 (s, CÄO, ester),
H). H-NMR (400 MHz): d 3.77 (3H, s),
3.91 (3H, s), 4.00 (3H, s), 6.25 (1H, d, Jꢂ8.8), 6.56 (1H,
dd, Jꢂ8.8, Jꢂ11.6), 6.94 (1H, bs), 6.97 (1H, bd, Jꢂ
11.6), 7.52 (2H, dd, Jꢂ7.6, Jꢂ8.3), 7.63 (1H, tt, Jꢂ
1.3, Jꢂ7.6), 8.26 (2H, dd, Jꢂ1.3, Jꢂ
8.3). 13C-NMR
stirred for a period of 8ꢀ16 h at room temperature (r.t.)
/
/
until TLC showed no starting material. The reaction
mixture was then washed with 1 N HC1, saturated
aqueous NaHCO3, and water, the organic extracts were
dried with MgSO4, and the solvent was evaporated
under vacuum. The crude solid was recrystallized from
/
/
1
2942 (w, Arꢀ
/
/
/
/
/
/
/
/
etherꢀ
tography using CH2C12ꢀ
solvent.
/
hexanes, and further purified by column chroma-
/
/
/
/
EtOAc (10:1ꢀ1:1) as eluting
/
(100 MHz): d 56.4, 56.5, 61.6, 107.1, 110.3, 124.2, 124.6,
128.9, 129.9, 130.0, 130.9, 133.6, 133.7, 143.0, 144.0,
155.6, 159.5, 165.3, 183.0. ESI-MS: 367.4 ([M]ꢃH).
/
5.1.1.1. 4,2?,3?-Trimethyl-4?-acetyl-purpurogallin (2a).
Obtained in 57% yield. M.p. 141ꢀ143 8C. IR (KBr
disc, cmꢁ1): 1625 (s, CÄ
O, ketone), 1773 (s, CÄO,
H). H-NMR (400 MHz): d 2.41
(3H, s), 3.80 (3H, s), 3.89 (3H, s), 3.95 (3H, s), 6.23 (1H,
d, Jꢂ8.8), 6.52 (1H, dd, Jꢂ8.8, Jꢂ11.6), 6.87 (1H,
bs), 6.91 (1H, bd, Jꢂ
11.6). 13C-NMR (100 MHz): d
/
5.1.1.6. 4,2?,3?-Trimethyl-4?-(p-anisoyl)-purpurogallin
(2f). Obtained in 85% yield. M.p. 126ꢀ130 8C. IR (KBr
disc, cmꢁ1): 1601 (s, CÄ
O, ketone), 1732 (s, Cꢂ0,
ester). 1H-NMR (400 MHz): d 3.76 (3H, s), 3.90 (3H, s),
3.91 (3H, s), 4.00 (3H, s), 6.24 (1H, d, Jꢂ8.8), 6.56 (1H,
/
/
/
1
ester), 2950 (w, Arꢀ
/
/
/
/
/
/
/
/
dd, Jꢂ
/
8.8, Jꢂ
/
11.6), 6.93 (1H, bs), 6.97 (1H, bd, Jꢂ
/
21.2, 56.4, 56.5, 61.5, 106.9, 110.3, 124.1, 124.2, 129.9,
133.7, 142.9, 143.9, 155.5, 159.4, 169.7, 183.0. ESI-
11.6), 6.99 (2H, AA?XX?), 8.21 (2H, AA?XX?). 13C-
NMR (100 MHz): d 55.9, 56.4, 56.5, 61.6, 107.0, 110.1,
114.2, 122.4, 124.1, 124.8, 129.8, 133.1, 133.5, 143.1,
MS:305.2 ([M]ꢃH).
/

954
H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ957
/
144.1, 155.5, 159.5, 164.2, 165.0, 183.1. ESI-MS: 397.4
([M]ꢃH).
124.3, 129.9, 132.6, 133.6, 134.5, 142.9, 143.3, 155.6,
159.5, 166.4, 167.7, 182.6. ESI-MS: 452.2 ([M]ꢃH).
/
/
5.1.2. General procedure for the preparation of the
diethyl azodicarboxylate (DEAD) DielsꢀAlder adducts
3aꢀ3l
DEAD (7.0 mmol) was added to a solution of the
appropriate trimethylpurpurogallin derivative (2aꢀ2k
5.1.1.7. 4,2?,3?-Trimethyl-4?-(p-chlorobenzoyl)-
purpurogallin (2g). Obtained in 55% yield. M.p. 150ꢀ
152 8C. IR (KBr disc, cmꢁ1): 1599 (s, CÄ
O, ketone),
O, ester). H-NMR (400 MHz): d 3.87 (3H,
s), 3.91 (3H, s), 4.01 (3H, s), 6.26 (1H, d, Jꢂ8.8), 6.58
(1H, dd, Jꢂ8.8, Jꢂ11.6), 6.95 (1H, bs), 6.98 (1H, bd,
Jꢂ
11.6), 7.49 (2H, AA?XX?), 8.19 (2H, AA?XX?). 13C-
/
/
/
/
1
1744 (s, CÄ
/
/
/
and 1, 3.5 mmol) in toluene (25 mL), and the resulting
mixture heated to reflux until no benzotropolone was
/
/
/
detectable by TLC analysis (16ꢀ
then removed under reduced pressure, and the resulting
oily solid was triturated with etherꢀhexanes to eliminate
unreacted DEAD. The crude products were further
purified by column chromatography using CH2C12ꢀ
EtOAc (10:1ꢀ1:1) as eluting solvent.
/
32 h). The solvent was
NMR (100 MHz): d 56.4, 56.5, 61.6, 107.1, 110.4, 124.3,
124.4, 128.5, 129.2, 129.9, 132.3, 133.7, 140.2, 143.0,
/
143.8, 155.6, 159.5, 164.5, 182.9. ESI-MS: 401.1 ([M]ꢃ
/
H).
/
/
5.1.1.8. 4,2?,3?-Trimethyl-4?-(o-flurobenzoyl)-
purpurogallin (2h). Obtained in 77% yield. M.p. 174ꢀ
176 8C. IR (NaCl film, cmꢁ1): 1600 (s, CÄ
O, ketone),
H). H-NMR (400
MHz): d 3.78 (3H, s), 3.94 (3H, s), 4.00 (3H, s), 6.26
(1H, d, Jꢂ8.8), 6.57 (1H, dd, Jꢂ8.8, Jꢂ11.6), 6.94
(1H, bs), 6.97 (1H, bd, Jꢂ11.6), 7.20 (1H, dddd, Jꢂ
0.5, Jꢂ1.0, Jꢂ 10.6), 7.28 (1H, ddd, Jꢂ1.0,
8.3, 3JHF
Jꢂ7.3, Jꢂ8.0), 7.59 (1H, dddd, Jꢂ2.0, Jꢂ7.3, Jꢂ
4.8), 8.21 (1H, dddd, Jꢂ0.5, Jꢂ2.0, Jꢂ
7.3). 13C-NMR (100 MHz): d 56.4, 56.5,
22.0), 118.6 (2JCF
4.4), 129.9, 133.4,
8.8), 143.0, 143.7, 155.6, 159.5,
3.7), 162.8 (1JCF
261.3), 182.9. ESI-
MS: 385.4 ([M]ꢃH).
/
5.1.2.1. 4,2?,3?-Trimethyl-4?-acetyl-purpurogallin×
DielsꢀAlder adduct (3a). Obtained from 2a in 82%
yield. M.p. 116ꢀ
118 8C. IR (KBr disc, cmꢁ1): 1597 (s,
CÄO, ketone), 1720, 1774 (s, CÄO, ester), 2983 (m, Arꢀ
H). H-NMR (90 MHz): d 1.01 (3H, bt, Jꢂ6.8), 1.32
(3H, t, Jꢂ7.1), 2.40 (3H, s), 3.53 (3H, s), 3.81 (3H, s),
3.95 (3H, s), 4.06 (2H, bq, Jꢂ6.8), 4.30 (3H, q, Jꢂ7.1),
5.70 (1H, bdd, Jꢂ0.7, Jꢂ6.0), 5.99 (1H, dd, Jꢂ0.7,
Jꢂ 6.0, Jꢂ9.1).
/
DEAD
/
/
1
1752 (s, CÄ
/
O, ester), 2944 (w, Arꢀ
/
/
/
/
/
/
/
/
1
/
/
/
/
/
/
ꢂ
/
/
/
/
/
/
/
/
/
/
/
/
4
8.3, JHF
ꢂ
ꢂ
/
/
/
/
/
9.1), 6.88 (1H, bs), 7.26 (1H, dd, Jꢂ
/
/
4
8.0, JHF
/
13C-NMR (22.5 MHz): d 13.3, 13.8, 20.0, 51.5, 55.8,
60.5, 61.4, 62.6, 62.8, 92.4, 111.1, 117.5, 127.5, 132.2,
142.9, 144.2, 146.7, 156.0, 156.8, 157.0, 169.3, 185.7.
61.6, 107.1, 110.4, 117.4 (2JCF
ꢂ
/
ꢂ
/
9.5), 124.2, 124.4, 124.5 (3JCF
133.7, 135.2 (3JCF
162.7 (3JCF
ꢂ
/
ꢂ
/
ESI-MS: 479.4 ([M]ꢃH).
/
ꢂ
/
ꢂ
/
/
5.1.2.2. 4,2?,3?-Trimethyl-4?-propionyl-purpurogallin×
DEAD DielsꢀAlder adduct (3b). Obtained from 2b in
63% yield. M.p. 142ꢀ
143 8C. IR (KBr disc, cmꢁ1): 1596
(m, CÄO, ketone), 1708, 1720, 1762 (s, CÄO, ester),
2983 (w, ArꢀH). H-NMR (90 MHz): d 1.01 (3H, bt,
Jꢂ7.0), 1.28 (3H, t, Jꢂ7.3), 1.30 (3H, t, Jꢂ7.0), 2.72
(2H, ABX), 3.51 (3H, s), 3.76 (3H, s), 3.94 (3H, s), 4.05
(2H, bq, Jꢂ7.0), 4.28 (2H, q, Jꢂ7.3), 5.67 (1H, bdd,
Jꢂ0.8, Jꢂ5.8), 5.97 (1H, dd, Jꢂ0.8, Jꢂ9.2), 6.84
(1H, bs), 7.24 (1H, dd, Jꢂ5.8, Jꢂ
9.2). 13C-NMR (22.5
/
/
5.1.1.9. 4,2?,3?-Trimethyl-4?-(p-fluorobenzoyl)-
purpurogallin (2i). Obtained in 93% yield. M.p. 155ꢀ
157 8C. IR (NaCl film, cmꢁ1): 1600 (s, CÄ
O, ketone),
H). H-NMR (400
MHz): d 3.78 (3H, s), 3.91 (3H, s), 4.02 (3H, s), 6.26
(1H, d, Jꢂ8.8), 6.58 (1H, dd, Jꢂ8.8, Jꢂ11.6), 6.96
(1H, bs), 6.99 (1H, bd, Jꢂ
3JHF
8.8), 8.27 (2H, dd, Jꢂ
/
/
/
/
1
/
/
1
1744 (s, CÄ
/
O, ester), 2945 (w, Arꢀ
/
/
/
/
/
/
/
/
/
/
11.6), 7.19 (2H, dd, Jꢂ
/
8.8,
/
/
/
/
4
ꢂ
/
/8.8, JHF
ꢂ
/
5.1). 13C-
/
/
NMR (100 MHz): d 56.4, 56.5, 61.6, 103.3, 107.0,
110.3, 116.0 (2JCF 22.0), 124.2, 124.5, 126.3 (4JCF
9.5), 133.6, 143.0, 155.6, 159.5,
253.9), 182.9. ESI-MS: 385.4 ([M]ꢃ
MHz): d 8.3, 13.4, 13.9, 26.6, 51.6, 55.8, 60.6, 61.5, 62.7,
62.9, 92.5, 111.1, 120.9, 127.6, 132.3, 143.0, 144.2, 147.0,
ꢂ
/
ꢂ
/
2.9), 129.8, 133.5 (3JCF
ꢂ
/
156.1, 156.8, 157.2, 172.8, 185.8. ESI-MS: 493.5 ([M]ꢃ
/
164.4, 166.5 (1JCF
ꢂ
/
/
H).
H).
5.1.2.3. 4,2?,3?-Trimethyl-4?-butyryl-purpurogallin×
DEAD DielsꢀAlder adduct (3c). Obtained from 2c in
91% yield. M.p. 173ꢀ
175 8C. IR (KBr disc, cmꢁ1): 1598
(m, CÄO, ketone), 1717, 1735, 1768 (s, CÄO, ester),
2983 (w, ArꢀH). H-NMR (90 MHz): d 1.06 (3H, bt,
Jꢂ7.1), 1.32 (3H, t, Jꢂ6.9), 1.80 (2H, ABX5), 2.68
(2H, ABX2), 3.52 (3H, s), 3.79 (3H, s), 3.95 (3H, s), 4.05
(2H, bq, Jꢂ7.1), 4.31 (3H, t, Jꢂ6.9), 5.69 (1H, bdd,
Jꢂ0.9, Jꢂ5.9), 5.98 (1H, dd, Jꢂ0.9, Jꢂ9.1), 6.85
(1H, bs), 7.25 (1H, dd, Jꢂ5.9, Jꢂ
9.1). 13C-NMR (22.5
/
5.1.1.10. 4,2?,3?-Trimethyl-4?-phtaloylglycil-
purpurogallin (2j). Obtained in 71% yield. M.p. 98ꢀ
100 8C. IR (NaCl film, cmꢁ1): 1600 (m, CÄ
O, ketone),
1723, 1772 (s, CÄO, ester), 2945 (w, Arꢀ
H).1H-NMR
(400 MHz): d 3.81 (3H, s), 3.94 (3H, s), 3.96 (3H, s),
4.87 (2H, bs), 6.25 (1H, d, Jꢂ8.8), 6.53 (1H, dd, Jꢂ8.8,
11.6), 7.73
/
/
/
/
/
/
1
/
/
/
/
/
/
/
Jꢂ
/
11.6), 6.89 (1H, bs), 6.91 (1H, bd, Jꢂ
/
/
/
(2H, AA?MM?), 7.89 (2H, AA?MM?). 13C-NMR (100
MHz): d 39.4, 56.4, 56.6, 61.7, 107.4, 110.7, 123.9, 124.0,
/
/
/
/
/
/

H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ
/957
955
MHz): d 13.1, 13.5, 13.9, 17.6, 35.2, 51.6, 55.9, 60.6,
61.6, 62.8, 63.0, 92.6, 111.1, 125.9, 127.8, 132.4, 143.0,
144.3, 147.0, 156.1, 156.9, 157.3, 171.9, 185.7. ESI-MS:
(3H, s), 3.79 (3H, s), 3.95 (3H, s), 4.11 (2H, bq, Jꢂ
4.29 (2H, q, Jꢂ7.2), 5.72 (1H, bd, Jꢂ
bd, Jꢂ
/
7.1),
/
/
5.9), 5.96 (1H,
/
9.2), 6.90 (1H, bs), 7.25 (1H, dd, Jꢂ
/
5.9, Jꢂ
/
507.2 ([M]ꢃ
/
H).
9.2), 7.46 (2H, AA?XX?), 8.13 (2H, AA?XX?). 13C-NMR
(22.5 MHz): d 13.6, 14.0, 51.6, 56.0, 60.8, 61.6, 62.7,
63.0, 92.5, 111.4, 117.8, 127.7, 127.9, 128.8, 132.0, 132.4,
139.8, 143.2, 144.2, 146.8, 156.1, 156.9, 157.2, 164.3,
5.1.2.4. 4,2?,3?-Trimethyl-4?-acetoxyacetyl-
purpurogallin×DEAD DielsꢀAlder adduct (3d). Ob-
tained from 2d in 67% yield. M.p. 167ꢀ169 8C. IR
(NaCl film, cmꢁ1): 1598 (m, CÄ
O, ketone), 1716, 1749,
1790 (s, CÄO, ester), 2983 (w, Arꢀ
MHz): d 1.00 (3H, bt, Jꢂ6.8), 1.30 (3H, t, Jꢂ
(3H, s), 3.51 (3H, s), 3.80 (3H, s), 3.95 (3H, s), 4.00 (2H,
bq, Jꢂ6.8), 4.28 (2H, q, Jꢂ7.1), 5.00 (2H, AB), 5.67
(1H, bdd, Jꢂ0.8, Jꢂ5.9), 5.97 (1H, dd, Jꢂ0.8, Jꢂ
8.9), 6.85 (1H, bs), 7.23 (1H, dd, Jꢂ5.9, Jꢂ
8.9). 13C-
/
/
/
185.6. ESI-MS: 575.3 ([M]ꢃH).
/
/
1
/
/
H). H-NMR (90
7.1), 2.17
5.1.2.8. 4,2?,3?-Trimethyl-4?-(o-fluorobenzoyl)-
purpurogallin×DEAD DielsꢀAlder adduct (3h). Ob-
tained from 2h in 88% yield. M.p. 196ꢀ197 8C. IR
(KBr disc, cmꢁ1): 1598 (m, CÄ
O, ketone), 1719, 1730 (s,
/
/
/
/
/
/
/
/
1
/
/
/
/
CÄ
1.04 (3H, bt, Jꢂ
3.82 (3H, s), 3.96 (3H, s), 4.09 (2H, bq, Jꢂ
(2H, q, Jꢂ7.0), 5.72 (1H, bd, Jꢂ5.9), 5.97 (1H, bd,
Jꢂ9.1), 6.91 (1H, bs), 7.21 (3H, m), 7.57 (1H, m), 8.14
(1H, m). 13C-NMR (22.5 MHz): d 13.4, 13.8, 51.5, 55.9,
60.7, 61.6, 62.8, 62.9, 92.5, 111.4, 116.8 (2JCF
21.8),
117.7, 117.9 (2JCF 9.2), 124.1 (3JCF
4.1), 127.5,
132.5, 132.9, 135.0 (3JCF
9.1), 143.0, 144.3, 146.5,
156.2, 156.8, 157.2, 162.3, 162.4 (1JCF
262.4), 185.6.
ESI-MS: 559.1 ([M]ꢃH).
/
O, ester), 2982 (w, Arꢀ
/
H). H-NMR (90 MHz): d
7.0), 3.49 (3H, s),
7.0), 4.28
/
/
/
7.0), 1.30 (3H, t, Jꢂ
/
NMR (22.5 MHz): d 13.4, 13.9, 19.9, 51.6, 56.0, 60.4,
60.9, 61.5, 62.7, 63.0, 92.4, 111.5, 117.3, 127.6, 132.4,
143.2, 144.4, 145.9, 156.3, 156.8, 157.2, 166.5, 170.4,
/
/
/
/
185.9. ESI-MS: 537.2 ([M]ꢃH).
/
ꢂ
/
5.1.2.5. 4,2?,3?-Trimethyl-4?-benzoyl-purpurogallin×
DEAD DielsꢀAlder adduct (3e). Obtained from 2e in
92% yield. M.p. 218ꢀ
220 8C. IR (KBr disc, cmꢁ1): 1597
(s, CÄO, ketone), 1716, 1730 (s, CÄO, ester), 2982 (m,
ArꢀH). H-NMR (90 MHz): d 1.05 (3H, bt, Jꢂ7.0),
1.30 (3H, t, Jꢂ7.0), 3.48 (3H, s), 3.80 (3H, s), 3.96 (3H,
s), 4.12 (2H, bq, Jꢂ7.0), 4.28 (2H, t, Jꢂ7.0), 5.72 (1H,
bd, Jꢂ5.8), 5.96 (1H, bd, Jꢂ9.0), 6.90 (1H, bs), 7.24
(1H, dd, Jꢂ5.8, Jꢂ9.0), 7.50 (3H, m), 8.19 (2H, dd,
Jꢂ1.8, Jꢂ
8.0). 13C-NMR (22.5 MHz): d 13.6, 14.0,
/
ꢂ
/
ꢂ
/
/
ꢂ
/
/
ꢂ
/
/
/
/
1
/
/
/
5.1.2.9. 4,2?,3?-Trimethyl-4?-(p-fluorobenzoyl)-
purpurogallin×DEAD DielsꢀAlder adduct (3i). Obtained
from 2i in 87% yield. M.p. 203ꢀ204 8C. IR (NaCl film,
cmꢁ1): 1599 (s, CÄ
O, ketone), 1719, 1744 (s, CÄO,
ester), 2983 (m, Arꢀ
H). 1H-NMR (90 MHz): d 1.03
(3H, bt, Jꢂ7.1), 1.31 (3H, t, Jꢂ7.1), 3.48 (3H, s), 3.80
(3H, s), 3.96 (3H, s), 4.10 (2H, bq, Jꢂ7.1), 4.29 (2H, q,
Jꢂ7.1), 5.72 (1H, bd, Jꢂ5.8), 5.97 (bd, Jꢂ9.1), 6.90
(1H, bs), 7.20 (3H, m), 8.22 (2H, dd, Jꢂ8.9, JHF
5.4). 13C-NMR (22.5 MHz): d 13.6, 13.9, 51.6, 55.9,
60.7, 61.6, 62.6, 63.0, 92.5, 111.3, 115.6 (2JCF
22.6),
117.9, 125.7 (4JCF 2.5), 127.7, 132.4, 133.2 (3JCF
9.2), 143.2, 144.2, 146.9, 156.2, 156.9, 157.2, 164.1,
166.2 (1JCF
255.7), 185.6. ESI-MS: 559.1 ([M]ꢃH).
/
/
/
/
/
/
/
/
/
/
/
/
/
/
51.6, 56.0, 60.8, 61.7, 62.8, 63.0, 92.6, 111.3, 126.4,
127.8, 128.5, 129.4, 130.6, 132.4, 133.4, 143.3, 144.2,
147.2, 156.3, 157.0, 157.4, 165.3, 185.5. ESI-MS: 541.2
/
/
/
/
/
/
([M]ꢃH).
/
4
/
ꢂ
/
5.1.2.6. 4,2?,3?-Trimethyl-4?-(p-anisoyl)-purpurogallin×
DEAD DielsꢀAlder adduct (3f). Obtained from 2f in
76% yield. M.p. 200ꢀ
202 8C. IR (KBr disc, cmꢁ1): 1604
(m, CÄO, ketone), 1721, 1736, 1750 (s, CÄO, ester),
2976 (w, ArꢀH). H-NMR (90 MHz): d 1.06 (3H, bt,
Jꢂ6.9), 1.31 (3H, t, Jꢂ7.1), 3.49 (3H, s), 3.80 (3H, s),
3.86 (3H, s), 3.96 (3H, s), 4.09 (2H, bq, Jꢂ6.9), 4.30
(2H, q, Jꢂ7.1), 5.73 (1H, bd, Jꢂ5.9), 5.76 (1H, bd,
Jꢂ9.2), 6.90 (1H, bs), 6.97 (2H, AA?XX?), 7.25 (1H, dd,
Jꢂ5.9, Jꢂ
9.2), 8.15 (2H, AA?XX?). 13C-NMR (22.5
/
ꢂ
/
/
ꢂ
/
ꢂ
/
/
/
/
1
ꢂ
/
/
/
/
/
/
5.1.2.10. 4,2?,3?-Trimethyl-4?-phtaloylglycil-
purpurogallin×DEAD DielsꢀAlder adduct (3j). Obtained
from 2j in 68% yield. M.p. 212ꢀ215 8C. IR (NaCl film,
cmꢁ1): 1598 (m, CÄ
O, ketone), 1724, 1774 (s, CÄO,
ester), 2982 (w, Arꢀ
H). 1H-NMR (90 MHz): d 1.00 (3H,
bt, Jꢂ7.0), 1.29 (3H, t, Jꢂ7.1), 3.53 (3H, s), 3.84 (3H,
s), 3.95 (3H, s), 4.03 (2H, bq, Jꢂ7.0), 4.27 (2H, q, Jꢂ
7.1), 4.66 (1H, d, Jꢂ17.5), 5.07 (1H, d, Jꢂ17.5), 5.67
(1H, bdd, Jꢂ0.8, Jꢂ5.9), 6.00 (1H, dd, Jꢂ0.8, Jꢂ
5.9, Jꢂ9.2), 7.82
/
/
/
/
/
/
/
/
/
/
MHz): d 13.5, 13.9, 51.5, 55.2, 55.9, 60.6, 61.6, 62.7,
62.9, 92.5, 111.2, 113.7, 117.9, 121.7, 127.6, 132.3, 132.6,
143.2, 144.1, 147.3, 156.1, 156.8, 157.2, 164.0, 164.7,
/
/
/
/
/
185.4. ESI-MS: 571.2 ([M]ꢃ
/
H).
/
/
/
/
/
/
5.1.2.7. 4,2?,3?-Trimethyl-4?-(p-chlorobenzoyl)-
purpurogallin×DEAD DielsꢀAlder adduct (3g). Ob-
tained from 2g in 84% yield. M.p. 224ꢀ226 8C. IR
(KBr disc, cmꢁ1): 1598 (m, CÄ
O, ketone), 1749, 1734,
1749 (s, CÄO, ester), 2983 (w, Arꢀ
MHz): d 1.03 (3H, bt, Jꢂ7.1), 1.30 (3H, t, Jꢂ
9.2), 6.87 (1H, bs), 7.23 (1H, dd, Jꢂ
/
/
/
/
(4H, AA?BB?). 13C-NMR (22.5 MHz): d 13.6, 14.0, 38.6,
51.7, 56.1, 61.6, 62.9, 63.1, 92.6, 111.6, 117.1, 123.7,
127.8, 132.2, 132.3, 134.4, 143.2, 144.1, 146.1, 156.4,
/
/
1
/
/
H). H-NMR (90
7.2), 3.48
156.9, 157.3, 166.3, 167.5, 185.7. ESI-MS: 623.5 ([M]ꢃ
H).
/
/
/

956
H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ957
/
5.1.2.11. 4,2?,3?,4?-Tetramethylpurpurogallin×
DielsꢀAlder adduct (3k). Obtained from 2k in 59%
yield. M.p. 162ꢀ
164 8C. IR (NaCl film, cmꢁ1): 1589 (m,
CÄO, ketone), 1716 (s, CÄO, ester), 2983 (m, ArꢀH).
1H-NMR (90 MHz): d 0.95 (3H, bt, Jꢂ
6.9), 1.31 (3H,
t, Jꢂ7.1), 3.52 (3H, s), 3.84 (3H, s), 3.87 (3H, s), 3.94
(3H, s), 4.01 (2H, bq, Jꢂ6.9), 4.28 (2H, q, Jꢂ7.1), 5.63
(1H, bdd, Jꢂ0.9, Jꢂ5.9), 6.07 (1H, dd, Jꢂ0.9, Jꢂ
9.0), 6.71 (1H, bs), 7.23 (1H, dd, Jꢂ5.9, Jꢂ
9.0). 13C-
/
DEAD
tissue culture plates. Drugs were added in threefold
dilution series and cultures incubated for a total of 48 h
/
/
at 37 8C in a 5% CO2ꢀ
hypoxanthine (0.2 mCi) was added to each well [22,23].
At the end of the assay, plates were rapidly freezeꢀ
/
air mixture. After 24 h, [3H]-
/
/
/
/
/
/
thawed, harvested using a Tomtec Mach III cell
harvester (Tomtec, CT) onto a 96-well format filter-
mat and MeltilexTM solid scintillant (both Wallac, Fin-
land) added prior to reading in a Microbeta 1450
scintillation counter (Wallac, Finland) at 1 min per well.
/
/
/
/
/
/
/
/
NMR (22.5 MHz): d 12.6, 13.0, 51.2, 55.5, 60.4, 61.1,
61.5, 62.2, 62.7, 92.9, 109.4, 120.4, 129.1, 132.9, 138.0,
144.4, 146.2, 157.2, 158.6, 158.5, 188.6. ESI-MS: 451.1
5.2.3. T. brucei rhodesiense
([M]ꢃ
/
H).
STIB900 blood stream form T. brucei rhodesiense
trypomastigotes were maintained in HMI-18 medium
[24], with 15% heat-inactivated fetal calf serum (Harlan-
5.1.2.12. 4,2?,3?-Trimethylpurpurogallin×
Alder adduct (3l). Obtained from 1 in 85% yield. M.p.
178ꢀ O,
180 8C. IR (KBr disc, cmꢁ1): 1656 (m, CÄ
ketone), 1716, 1742 (s, CÄO, ester), 2988 (m, ArꢀH),
3448 (s, O-H). H-NMR (90 MHz): d 0.96 (3H, bt, Jꢂ
7.2), 1.33 (3H, t, Jꢂ7.2), 3.56 (3H, s), 3.84 (3H, s), 3.97
(3H, s), 4.06 (2H, bq, Jꢂ7.2), 4.30 (2H, q, Jꢂ7.2), 5.70
(1H, bdd, Jꢂ0.8, Jꢂ6.0), 6.07 (1H, dd, Jꢂ0.8, Jꢂ
6.0, Jꢂ9.1), 11.86
/
DEAD Dielsꢀ
/
SeraLab, UK) at 37 8C in a 5% CO2ꢀ
to drugging, trypomastigotes were washed and resus-
105
/air mixture. Prior
/
/
/
/
pended in fresh medium at a concentration of 2ꢄ
/
1
/
mLꢁ1, and 100 mL of this suspension was added to the
drug dilutions. The top concentration for the test
compounds was 30 mg mLꢁ1. Pentamidine was included
as the standard drug. Plates were incubated for 72 h at
/
/
/
/
/
/
/
9.1), 6.63 (1H, s), 7.31 (1H, dd, Jꢂ
/
/
37 8C in a 5% CO2ꢀ/air mixture. At 72 h Alamar Blue
(1H, s). 13C-NMR (22.5 MHz): d 13.2, 13.7, 51.4, 55.7,
60.1, 61.5, 62.4, 62.8, 91.3, 106.2, 108.2, 127.0, 131.8,
137.5, 144.9, 156.6, 156.7, 157.0, 158.8, 194.3. ESI-MS:
was added to the plates [25]. Plates were read after 5ꢀ
/6 h
on a Gemini fluorescent plate reader (Sofimax Pro.
3.1.1, Molecular Devices, UK) at EX/EM 530/585 nm
with a filter cut-off at 550 nm.
437.1 ([M]ꢃH).
/
5.2. Biological assays
5.2.4. T. cruzi
Peritoneal exudate macrophages were infected with T.
cruzi Tulahuen LAC-Z trypomastigotes [26], harvested
from cultured L6 myoblast feeder-cell layers, at a ratio
of 5:1. Infected cells were exposed to the drugs for 72 h.
Anti-trypanosomal activity was evaluated using a b-
galactosidase assay [26], and calculating the percent
inhibition in comparison to untreated controls. The
standard drug benznidazole was included in the assay.
Stock solutions of the test compounds, plus control
drugs, were prepared at a concentration of 20 mg mLꢁ1
in DMSO (Sigma, UK), and diluted to the appropriate
concentration prior to the assays. IC50 values were
calculated with MSXLFIT (IDBS, UK).
5.2.1. L. donovani
MHOM/ET/67/L82 L. donovani amastigotes were
harvested from an infected hamster (Mesocricetus aur-
atus) spleen and used to infect murine peritoneal
exudate macrophages (PEM) at a ratio of 7:1. In brief,
infected cells were exposed to drug for a total of 5 days
[21]. The percentage of infected cells was evaluated
microscopically and the percentage inhibition in com-
parison to untreated controls was calculated. The
standard drug, sodium stibogluconate, has an IC50 of
5.2.5. Cytotoxicity assays
96-Well plates were seeded with KB cells at a
concentration of 2ꢄ
104 mLꢁ1 (200 mL per well). Drugs
/
at 300, 30, 3 and 0.3 mg mLꢁ1 were added in fresh
overlay after 24 h, in triplicate at each concentration.
The plates were incubated for a further 72 h and then
washed three times with PBS, and 100 mL PBS and 10
mL Alamar Blue added. This was followed by incubation
of the plates for 2 h before reading EX/EM 530/585 nm
on a Gemini plate reader. IC50 values were calculated
against blanks and control samples.
around 5 mg SbV mLꢁ1
.
5.2.2. P. falciparum
Chloroquine-sensitive P. falciparum strain 3D7 was
maintained in human Aꢃ erythrocytes in RPMI1640
medium (Sigma, UK) supplemented with Albumax II at
Acknowledgements
37 8C in a 5% CO2ꢀair mixture. P. falciparum intraer-
/
ythrocytic cultures were set up as above, with 1% ring
stage parasitemia, 2.5% hematocrit, in triplicate in 100
mL of medium in 96-well, flat-bottomed Microtest III
The authors wish to thank Professors Patrick Moyna
and Horacio Heinzen (Facultad de Qu´ımica, Montevi-
deo, Uruguay) for insightful discussions and comments.

H. Ren et al. / European Journal of Medicinal Chemistry 38 (2003) 949ꢀ
/957
957
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120.
We greatly acknowledge the financial support provided
by the USP Office of the Vice-President of Academic
Affairs (G.M.), the USP Merck-AAAS Summer Re-
search Program (J.S.M.), and the UNDP/World Bank/
WHO Special Programme for Research and Training in
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