Synthesis and in vitro protozoocidal activity of diazabicyclic benzotropolone derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.05.044

We describe the synthesis and protozoocidal evaluation of a series of diazabicycles based on benzotropolone ethers. Several of the compounds, which can be obtained through a high-yielding hetero Diels-Alder reaction using simple and readily available star

Full text of DOI:10.1016/j.bmcl.2007.05.044

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4183–4186
Synthesis and in vitro protozoocidal activity of diazabicyclic
benzotropolone derivatives
Alexander Khrizman,a Jason S. Moulthrop,a Susan Little,b Hayley Wharton,b
Vanessa Yardleyb and Guillermo Moynaa,*
aDepartment of Chemistry & Biochemistry, University of the Sciences in Philadelphia, 600 South 43rd Street, Philadelphia,
PA 19104-4495, USA
bLondon School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, Keppel Street,
London WC1E 7HT, UK
Received 31 March 2007; revised 11 May 2007; accepted 14 May 2007
Available online 18 May 2007
Abstract—We describe the synthesis and protozoocidal evaluation of a series of diazabicycles based on benzotropolone ethers. Sev-
eral of the compounds, which can be obtained through a high-yielding hetero Diels–Alder reaction using simple and readily avail-
able starting materials, have in vitro activities against Trypanosoma cruzi and Leishmania donovani that are comparable to, and in
some cases better than, those of currently used chemotherapies.
Ó 2007 Elsevier Ltd. All rights reserved.
Despite continued efforts from local governments, inter-
national NGOs, and philanthropic foundations, parasit-
oses such as malaria, sleeping sickness, Chagas disease,
and Leishmaniasis are still endemic in tropical and
sub-tropical underdeveloped regions. As a matter of
fact, these ailments represent a threat to nearly half of
the World population, making them a public health con-
cern of global proportions.1,2 Attempts to control trans-
mission vectors have been largely unsuccessful, and the
complex biology and remarkable adaptability of the
causative protozoa have thwarted the development of
effective vaccines.3 As a result, chemotherapy continues
to be the mainstay of treatment against these parasitic
infections. However, most of the drugs currently avail-
able for this purpose have severe side-effects and are
significantly toxic, and their intensive and extensive
use has induced selection of drug-resistant parasites.4,5
Consequently, the search for readily accessible novel
compounds with protozoocidal activity and improved
pharmacological characteristics is of primary
importance.6
In recent reports, we have described the synthesis and
antiparasitic evaluation of a series of endocyclic oxazine
and hydrazine esters (Fig. 1).7,8 The rationale behind the
design of these compounds was the incorporation of
fragments prone to homolytic cleavage into simple
molecular frameworks through the use of high-yielding
hetero Diels–Alder cycloadditions. Molecules bearing
–N–O– or –N–N– bonds, whose standard dissociation
energies (BDEs) are between 35 and 38 kcal/mol,9 could
easily lead to the generation of free radicals. Given the
high susceptibility to oxidative stress shown by the pro-
tozoa,10,11 this could make these compounds effective in
the treatment of parasitic diseases.
Keywords: Benzotropolone derivatives; Hydrazines; Parasitoses; In
vitro protozoocidal activity; Synthesis.
*
Figure 1. General structure of previously reported benzotropolone-
based oxazine (a) and hydrazine (b) esters with antiparasitic activity.7,8
Corresponding author. Tel.: +1 215 596 8526; fax: +1 215 596
8543; e-mail: g.moyna@usip.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.044

4184
A. Khrizman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4183–4186
Indeed, several of the esters showed modest to signifi-
cant activity against Plasmodium falciparum, Trypano-
soma brucei rhodesiense, and Trypanosoma cruzi, and
while most of the oxazines were somewhat cytotoxic,7
the hydrazines displayed low to negligible toxicities.8
These exploratory studies revealed that a new class of
molecules with antiparasitic potential and completely
novel structures could be obtained from commercially
available starting materials using simple and well-estab-
lished chemistries.
One of the disadvantages of the compounds shown in
Figure 1 is the susceptibility to hydrolysis of the ester
functionality. Although this behavior could, in some
cases, confer pro-drug characteristics to the mole-
cules,7,8 premature hydrolysis would lead to lipophobic
phenols with reduced membrane permeability. A simple
approach to circumvent this potential problem, while at
the same time conserving the original molecular frame-
work, involves substituting the labile aliphatic and aro-
matic esters with alkyl or benzyl ethers. We thus decided
to explore the synthesis and in vitro protozoocidal activ-
ity of a series of endocyclic hydrazines based on benzo-
tropolone ethers (Fig. 2), and our results are discussed in
this letter.
The preparation of the ethers involved the derivatization
of the C40 phenolic position of trimethylpurpurogallin (1)
through standard Williamson etherification conditions
(Fig. 2). Briefly, 1 was dissolved in DMSO and treated
with a 3- to 5-fold excess of the appropriate alkyl or benzyl
halide in the presence of solid NaOH to give ethers 2c–2j
in yields ranging from 40% to 81%. The selection of the
halides employed in the preparation of the ethers was
guided by several factors. Namely, they are all readily
available, they provide a small but representative series
of alkyl and aryl substitutions at the C40 position of the
benzotropolone, and their structure corresponds with
that of the esters studied in our earlier work.7,8
As discussed above, the incorporation of the bridged
hydrazine moiety relies on an hetero Diels–Alder reac-
tion on the electron-rich dienes of the benzotropolone
ethers. The dienophiles employed in the reaction con-
tained electron-deficient azo functional groups, and in-
Figure 2. Synthetic route to diazabicycles 3a–j, 4a–j, and 5a–j, and
numbering scheme used in the text. Complete experimental details are
available as Supplementary data.
cluded
dimethyl,
diethyl,
and
diisopropyl
ven-membered ring change considerably after the cyclo-
addition. While protons on carbons C6 and C7 in the
starting materials display signals at approximately 6.5
and 6.9 ppm, respectively, their resonances shift to
roughly 7.3 and 5.7 ppm in the products. These changes
are consistent with the conversion of the pseudo-aro-
matic tropolone ring into a diazabicyclic framework in
which the C5–C6 double bond is not co-planar with
the remaining aromatic ring. It is also worth noting that
slow conformational equilibria of the carbamate moie-
ties leads to substantial broadening of the bridged ring
azodicarboxylates (DMAD, DEAD, and DIAD, respec-
tively). These reagents were chosen because they are
commercially available, their use in cycloaddition reac-
tions is well documented,8,12 and they allow for a rela-
tively large library of compounds based on a common
molecular framework to be rapidly prepared. Treatment
of 1, tetramethylpurpurogallin (2b), and ethers 2c–2j
with two equivalents of the appropriate dienophile in
refluxing toluene afforded exclusively products 3a–3j,
4a–4j, and 5a–5j in 55% to quantitative yields (Fig. 2).
In all cases, the reactions proceeded smoothly, and no
starting materials were detectable by TLC after 2–10 h.
The success of the Diels–Alder reactions was conve-
niently confirmed by inspection of the 1H NMR spectra.
Apart from the new signals corresponding to the methyl,
ethyl, and isopropyl carbamates observed in the spectra
of the adducts, the chemical shifts for protons in the se-
1
13
system H and C NMR signals.8 To overcome this,
NMR spectra for all products were recorded at 90 °C.
The compounds were assayed against P. falciparum,
T. brucei rhodesiense, T. cruzi, and Leishmania donovani.
None of the hydrazines were significantly active against

A. Khrizman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4183–4186
4185
the former two parasites, with IC50s at least three orders
of magnitude larger than those determined for the
respective controls, and only results for the latter two
are presented in Table 1. Of these, T. cruzi was the most
susceptible to this series of compounds. The IC50s of 3e,
3i, 4f–4i, and 5d–5j against this trypanosomatid ranged
from 1.9 to 83.8 lM. This makes them comparable to
the control, benznidazole, which had an IC50 of
8.7 lM in the same assay. Compounds 5f, 5h, and 5j
have virtually the same activity than the control, and
5i, the most active member of the series, is nearly five
times more active with an IC50 below 1.9 lM. Further
analysis of the data presented in Table 1 leads to a loose
correlation between the structure of the C40 substituent
and the activity against T. cruzi. With the exception of
3e, 4b, 5d, and 5e, which have IC50s in the 23.4–
57.4 lM range and contain aliphatic ethers at C40, the
majority of the active compounds bear substituted ben-
zyl groups at this position. On the other hand, the activ-
ities of our previous series of ester derivatives correlated
with the presence of aliphatic substituents at the C40
position.8 As suggested above, this could be a reflection
of the different propensity to hydrolysis, and thus bio-
the T. cruzi assay and nontoxic in concentrations of
up to 300 lg/mL. Compounds 4f–4j, 5e, 5f, and 5h–5j,
which have the highest activities against the trypanoso-
matids, are fairly toxic, with ED50s ranging from 42.0 to
215.4 lg/mL. However, the therapeutic indexes (TIs) of
the hydrazines with the highest activities against T. cruzi
and L. donovani are 37.6 (compound 5i) and 21.3 (com-
pound 4h), respectively. These are higher than the TIs
obtained for our earlier series of hydrazine esters,8 and
indicate that the compounds discussed herein have
slightly improved pharmacological properties.
In summary, 28 novel diazabicycles based on simple
benzotropolone ether frameworks were prepared, and
their in vitro antiprotozoocidal properties determined.
As detailed above, T. cruzi is the most susceptible to
these new Diels–Alder adducts, with ten of the com-
pounds being active against this parasite. From this sub-
group, three had activities comparable to that of
benznidazole, and one was nearly 5-fold more active.
Additionally, two of the compounds displayed activity
against L. donovani similar to that of pentostam. Cur-
sory analysis of our results reveals that the activity
increases with benzylic substituents at the C40 position
and bulkier aliphatic carbamates on the hydrazine. This
suggests that membrane permeability is an important
factor in the activity of these compounds, and would ex-
plain why phenols 3a, 4a, and 5a are inactive. The mode
of action of these molecules is still unknown, but it is
likely related to the presence of the bridged hydrazine
group. As mentioned earlier, the low BDE of the single
–N–N– bond in this moiety could lead to free radicals
with the potential of disrupting oxygen metabolism pro-
cesses specific to trypanosomatids in general,10 and
T. cruzi in particular.13,14 This hypothesis is supported
not only by our earlier results,7,8 but also by a recent re-
port from Gamenara and co-workers on the protozoo-
cidal activity of eucarvone-based oxazines.15
`
availability, of aromatic vis-a-vis aliphatic esters. Final-
ly, it is also evident that the number of active hydrazine
ethers increases and their activities improve as the carba-
mate groups become larger and more lipophilic. While
not presented here, these trends were observed in the
P. falciparum and T. brucei rhodesiense assays as well.
In the L. donovani assays, all but two of the compounds
had IC50s of 47.4 lM or higher, or roughly 6-fold less
active than stibogluconate (pentostam), which had an
IC50 of 7.8 lM. However, hydrazines 4h and 4j showed
activities nearly identical to that of the control.
Although the results for these two are clear exceptions,
they are consistent with the trend seen in all the assays,
and confirm that compounds bearing substituted benzyl
ethers at C40 are in general the most active.
Our efforts are now focused on evaluating the effects on
biological activity associated with structural variations
to the frameworks of the hetero Diels–Alder adducts.
In order to maximize the availability and minimize the
costs of potential drug candidates, simple and easily
Cytotoxicity against KB cells is also reported in Table 1.
In general, only inactive compounds could be consid-
ered nontoxic to cells. The exception is hydrazine 5g,
which is only 2 times less active than benznidazole in
Table 1. In vitro activity of hydrazines 3a–3j, 4a–4j, and 5a–5j against trypanosomatids
Toxicity (ED50 lg/mL)a
a
Compound
IC50 (lM)
T. cruzi
L. donovani
3
4
5
3
4
5
3
4
5
a
b
c
d
e
f
>73.5
>71.0
>68.8
>66.6
57.4
>66.7b
37.4b
>64.6
>62.7
>60.9
54.1
>64.6
>62.7
>60.9
42.3
23.4
12.1
18.0
9.5
>73.5
>71.0
>68.8
>66.6
>64.6
>60.2
>58.1
>58.1
>56.3
>56.8
>66.7b
>68.8b
>64.6
>62.7
>60.9
>57.0
>55.1
8.6
>64.6
>62.7
>60.9
>59.2
>57.6
>54.1
>52.4
>52.4
47.4
>300
>300
>300
>300
>300
>300
>300b
>300b
>300
>300
76.0
>300
>300
>300
298.8
22.1
94.0
>60.2
>58.1
>58.1
50.9
215.4
51.8
99.7
g
h
i
51.3
54.1
56.6
137.2
55.9
>300
109.0
42.0
47.9
83.8
>53.9
<1.9
9.8
>53.5
9.9
99.9
63.1
j
>56.8
>51.3
>300
a Benznidazole (IC50 8.7 lM, T. cruzi) and pentostam (IC50 7.8 lMSbV, L. donovani) were used as controls. Toxicity was assayed against KB cells
using podophyllotoxin (ED50 0.006 lg/mL) as standard. All reported values are averages of three independent repeats.
b The activity and cytotoxicity of 4a and 4b was reported in a previous communication.8

4186
A. Khrizman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4183–4186
4. Buckner, F. S.; Wilson, A. J.; White, T. C.; Van Voorhis,
W. C. Antimicrob. Agents Chemother. 1998, 42, 3245.
5. Eckstein-Ludwig, U.; Webb, R. J.; Van Goethem, I. D.
A.; East, J. M.; Lee, A. G.; Kimura, M.; O’Neill, P. M.;
Bray, P. G.; Ward, S. A.; Krishna, S. Nature (London)
2003, 424, 957.
accessible scaffolds, such as tropane and tropolone
derivatives, are currently being considered. Work with
these frameworks is well underway and our results will
be reported in due course.
6. Trouiller, P.; Olliaro, P.; Torreele, E.; Orbinski, J.; Laing,
R.; Ford, R. Lancet 2002, 359, 2188.
Acknowledgments
7. Ren, H.; Grady, S.; Gamenara, D.; Heinzen, H.; Moyna,
P.; Croft, S. L.; Moyna, G. Bioorg. Med. Chem. Lett.
2001, 11, 1851.
8. Ren, H.; Grady, S.; Banghart, M.; Moulthrop, J. S.;
Kendrick, H.; Yardley, V.; Croft, S. L.; Moyna, G. Eur. J.
Med. Chem. 2003, 38, 949.
9. Pauling, L. The Nature of the Chemical Bond and the
Structure of Molecules and Crystals: An Introduction to
Modern Structural Chemistry, Third ed.; Cornell Univer-
sity Press: Ithaca, New York, 1960, Chapter 3.
10. Krieger, S.; Schwarz, W.; Ariyanayagam, M. R.; Fair-
lamb, A. H.; Krauth-Siegel, R. L.; Clayton, C. Mol.
Microbiol. 2000, 35, 542.
The authors are grateful to Ms. Rachel D. Slack for
insightful discussion and suggestions regarding the
manuscript. A.K., J.S.M., and G.M. thank the finan-
cial assistance provided by the Camille and Henry
Dreyfus Foundation and the NSF CCLI-A& I Pro-
gram. Support from the UNDP/World Bank/WHO
Special Programme for Research and Training in Trop-
ical Diseases is also acknowledged (S.L., H.W., and
V.Y.).
Supplementary data
11. Trivedi, V.; Chand, P.; Srivastava, K.; Puri, S. K.; Maulik,
P. R.; Bandyopadhyay, U. J. Biol. Chem. 2006, 280,
41129.
12. Boger, D. L.; Weinreb, S. N.. In Organic Chemistry, A
Series of Monographs; Wasserman, H. H., Ed.; Academic:
New York, 1987; Vol. 47,, Chapter 3.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.05.044.
References and notes
13. Docampo, R.; Moreno, S. N. J. Rev. Infect. Dis. 1984, 6,
223.
1. Hirst, S. I.; Stapley, L. A. Parasitol. Today 2000, 16, 1.
2. Enserink, M. Science 2000, 287, 1956.
3. Barbour, A. G.; Restrepo, B. I. Emerg. Infect. Dis. 2000, 6,
449.
14. Declerq, P. J.; Deranter, C. J. Biochem. Pharmacol. 1986,
23, 1421.
15. Gamenara, D.; Heinzen, H.; Moyna, P. Tetrahedron Lett.
2007, 48, 2505.