Identification of the benzodiazepines as a new class of antileishmanial agent

Article abstract of DOI:10.1016/j.bmcl.2006.11.004

The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have ident

Full text of DOI:10.1016/j.bmcl.2006.11.004

Bioorganic & Medicinal Chemistry Letters 17 (2007) 624–627
Identification of the benzodiazepines as a new class
of antileishmanial agent
Rachel L. Clark,a Katharine C. Carter,a Alexander B. Mullen,a Geoffrey D. Coxon,a
George Owusu-Dapaah,a Emma McFarlane,a M. Dao Duong Thi,a M. Helen Grant,b
Justice N. A. Tetteya and Simon P. Mackaya,*
aStrathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK
bBioengineering Unit, University of Strathclyde, Wolfson Centre, 106 Rottenrow, Glasgow G4 0NW, UK
Received 10 October 2006; revised 31 October 2006; accepted 1 November 2006
Available online 6 November 2006
Abstract—The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the
increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have
identified the benzodiazepines as a structural class for antileishmanial hit optimisation, and demonstrated that their in vitro activity
is comparable with the clinically used drug, sodium stibogluconate, and that the compounds are not toxic to macrophages.
Ó 2006 Elsevier Ltd. All rights reserved.
Leishmaniasis, which threatens approximately 350 mil-
lion people in 88 countries, was the cause of death of
59,000 people in 2002, and of the 1.5–2 million new cases
reported, one-third were the life-threatening form of
leishmaniasis.1 The continual increase in drug resistance,
the lack of new chemotherapeutic agents and the toxic-
ity of existing agents means that new drugs are required.
A recent study by Meijer and coworkers2 demonstrated
that paullones 1, previously identified as antitumour
agents through their inhibition of cyclin-dependent ki-
nases, completely inhibit the growth of Leishmania mex-
icana promastigotes in vitro. Whilst paullones offer a
promising starting point for chemical investigation,2
we considered their non-selectivity and multi-step, low
yielding synthesis3 a disadvantage for development of
structurally related compounds with increased antipara-
sitic efficacy. Consequently, we prepared a series of syn-
the promastigote stage, so that we could test for antipar-
asitic activity against the host-relevant form, potential
cytotoxicity and ability to enter the macrophage in the
same assay.5,6
We report here the short, efficient synthetic route and
the results of the biological testing for antileishmanial
activity. Whilst modifications were made to probe the ef-
fects of structural changes, low cost economics and syn-
thetic efficiency were also important factors in selecting
compounds for synthesis.
thetically
amenable
benzodiazepines
and
pyrrolobenzodiazepines structurally related to the paul-
lone nucleus to probe for activity. Our aim was to chem-
ically modify the basic benzodiazepine skeleton and
optimise for antileishmanial activity in a macrophage
amastigote infection model,4 rather than activity against
To establish whether tricyclic or tetracyclic systems sim-
ilar to paullones exhibited antileishmanial activity, com-
pounds 3–5 were prepared in a one-step reaction7 by
condensation of isatoic anhydride 2 and the appropriate
amino acid derivative in DMSO at 100 °C (Scheme 1).
Keywords: Antileishmanial agents; Amastigote; Benzodiazepine; Pyr-
rolobenzodiazepine; Paullone.
*
Corresponding author. Tel.: +44 141 548 2866; fax: +44 141 552
6443; e-mail: simon.mackay@strath.ac.uk
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.004

R. L. Clark et al. / Bioorg. Med. Chem. Lett. 17 (2007) 624–627
625
Scheme 1. Simple and affordable route to the pyrollobenzodiazepines and the benzodiazepine diones. Reagents and conditions: (i) 1—DMSO, 100 °C
(2) H2O, 0 °C; (ii) NaBH4, TFA, glyme, reflux; (iii) LiAlH4, THF, reflux; (iv) 1—pyridine, reflux; 2—H2O, 0 °C; (v) 1—CH2Cl2, KPhos, pH 7.0,
BrCOCH2Br, 5 °C, DBU.
The effects of chirality on activity were investigated with
both enantiomers of proline. The compounds were iso-
lated by precipitation of the cooled reaction mixture
using iced water. To establish whether the hydrogen-
bond acceptors of the amide carbonyls were necessary
for activity, the secondary amide of dione 4 was selec-
tively reduced to the corresponding secondary amine
using sodium borohydride and trifluoroacetic acid in
glyme to give 6 in a quantitative yield. Both amides in
dione 3 were reduced with lithium aluminium hydride
to give the diamine 7.8 Reductions were performed on
both the R- and S-isomers of 4 to produce enantiomer-
ically pure compounds. In order to establish whether a
tricyclic system was an activity requirement, proline
was replaced with a series of amino acid ester hydro-
chlorides,9 which in refluxing pyridine, produced benzo-
Finally, to investigate the role of substituents at N4, a
high yielding, ‘one-pot’ reaction between N-methyl isa-
toic anhydride, an appropriate primary amine and
bromoacetyl bromide in the presence of DBU was used
to produce a series of N4-substituted benzodiazepine-
2,5-diones 9.10
The antileishmanial activity of all compounds against a
clinically derived strain of Leishmania donovani (antimo-
ny-sensitive 200016)4 is shown in Table 1. In addition,
the activity of the clinically used drug sodium stiboglu-
conate is also shown for comparison.
These data indicate that whilst the tetracycle 3(S) is
inactive, the tricyclic pyrrolobenzodiazepine-2,5-diones
are more effective antileishmanial agents than sodium
stibogluconate at the concentrations tested, with no evi-
dence of toxicity against the host macrophage cells.
Activity appears to be independent of chirality in the tri-
diazepine-2,5-diones
8 with different hydrophobic
substituents (or no substituent in the case of glycine)
at C3, whilst leaving N4 free as the secondary amide.

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R. L. Clark et al. / Bioorg. Med. Chem. Lett. 17 (2007) 624–627
cyclic 2,5-diones. Replacement of the pyrrolidine with a
thiazolidine ring 5(S) not only abolished activity but
gave rise to some evidence of cellular host toxicity char-
acterised by unusual cellular growth and vacuole forma-
tion in the macrophages (data not shown).
than the common conformation that 3(S) offers between
9 and 8d(S).
When the N4-substituent is aliphatic, the antileishmanial
activity profile is inconclusive. The inactivity of the N-
methyl 9a and nor analogue 8a suggests that a degree
of bulkiness is required at N4. However, whilst the N-
propyl derivative 9b has suppressive activity equivalent
to sodium stibogluconate, the longer chain N-hexyl 9c
and methylcyclopropyl 9h derivatives are inactive. The
activity of the conformationally restricted cyclohexyl
derivative 9i is statistically not significant, yet the bulky,
but more flexible methylcyclohexyl derivative 9j was the
first compound to instigate cell death of the host
macrophages.
Reduction of the secondary amide to the correspond-
ing amine retained activity for the S-enantiomer 6(S),
whilst there was lower suppression rate of the para-
sites for the corresponding R-isomer 6(R), albeit at
a
level comparable with sodium stibogluconate.
Reduction of both amides to the diamine produced
divergent activity between the enantiomers, with sup-
pression levels maintained for 7(R), but rendered not
significant with 7(S). Given that the physicochemical
properties of the enantiomers are the same, we can
only surmise that disparities in activity are conse-
quences of differential affinities for a target or carrier
protein. It would appear therefore that chirality be-
comes important in the absence of the 5-oxo group.
The loss of H-bond acceptor capacity in this region
does not seem to be necessary for substantial antile-
ishmanial activity.
The toxicity of compound 9j prompted us to investigate
the role of an electron-withdrawing substituent in the
aromatic ring (9k), based on the development of the
anxiolytic benzodiazepines as therapeutic agents, where
a 7-chloro or nitro substituent was incorporated to
block aromatic hydroxylation by the P450 system. Aro-
matic hydroxylation can also yield toxic metabolites,11
which may be responsible for the toxicity of 9j at higher
concentrations.
The data clearly show that when the tricyclic system is
trimmed down to a benzodiazepine-2,5-dione, a bulky
substituent at position C3 is required, with the benzyl
derivatives 8d(S) and 8e(S) producing suppression simi-
lar to that of the clinically used drug, sodium stibogluco-
nate, at comparable concentrations. The results for the
two stereoisomers of 8d suggest that stereochemical fac-
tors may be important when a bulky group is present at
C3. Comparing the activity of 3(S) with 8d(S) suggests a
degree of flexibility at C3 is required when an aromatic
substituent is present.
Comparing the activity of the parent compound 9j
with its analogue 9k suggests that the introduction of
a 7-chloro aromatic substituent overcomes the debilitat-
ing effect of the N4-methylcyclohexyl group. However,
systematic metabolism studies using rat-derived liver
microsomes and hepatocytes demonstrated identical
biotransformation routes; N-demethylation, hydroxyl-
ation, and O-glucuronidation. Although both com-
pounds produced
a marked decrease in cellular
glutathione levels in macrophages, there was neither a
significant effect on macrophage viability (leakage of
lactate dehydrogenase) nor glutathione reductase activi-
ty at the concentrations used (50 and 100 lM).
For the N4-substituent series, the benzyl derivatives 9d
and 9g retain activity in common with the phenylaniline
derivative 8d(S), although a single methylene spacer be-
tween the benzodiazepine nucleus and the aromatic
group is essential, with the phenyl 9c and phenylethyl
9f analogues demonstrating no significant antileishma-
nial activity. Again, comparison with 3(S) and 8d(S) al-
ludes to flexibility in this region of the molecule, rather
The LD50 for compound 9k when determined against
L. donovani 200016 was 115 lM (37 lg/ml) and pro-
duced a 73 5% mean parasite suppression. At this con-
centration, 9j was inactive (and non-toxic, as confirmed
Table 1. A comparison of the antileishmanial activity of synthesized compounds and the antileishmanial drug sodium stibogluconate13
Compound
Mean parasite suppression (%) and standard error
Compound
Mean parasite suppression (%) and standard error
Na stibogluconate
59.4 2.8*
25.0 11NS
70.3 2.1*
85.4 0.6*
33.0 8.0a,*
70.6 1.2*
61.7 2.2*
23.0 6.0NS
67.8 16.0*
19.0 7.5NS
28.0 17.0NS
Inactive
8d(R)
8e(S)
9a
23.0 7.0NS
64.4 16.0*
Inactive
3(S)
4(S)
4(R)
5(S)
6(S)
6(R)
7(S)
7(R)
8a
9b
9c
72.0 16.0*
Inactivea
9d
9e
51.0 8.0*
Inactive
9f
9g
19.6 6.0NS
54.6 9.0*
Inactive
9h
9i
8b(S)
8c(S)
8d(S)
26.4 6.0NS
9j
9k
Toxic
85.0 5.0*
53.5 8.2*
a Some evidence of host cell toxicity.
* The statistical significance was assessed using ANOVA and Fisher’s PLSD in Statview 5.01. Results highlighted with an asterisk indicate signifi-
cance at 99% or greater (p = 0.01, NS, not significant).

R. L. Clark et al. / Bioorg. Med. Chem. Lett. 17 (2007) 624–627
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enhancing effect for the 7-substituent. This could possi-
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proved lipophilicity, or an improved pharmacodynamic
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In conclusion, we have identified a promising new hit for
the treatment of leishmaniasis. The 1,4-benzodiazepine-
2,5-dione 9k has an amastigote suppression efficacy com-
parable with the clinically used sodium stibogluconate
and is non-toxic in our model. 9k demonstrates efficacy
at a concentration of 37 lg/ml, whereas sodium stiboglu-
conate has plasma concentrations of 10–20 lg/ml after
clinical dosing.12 We have therefore designed a detailed
programme of further optimisation to enhance the activ-
ity of 9k. In future, we hope to report on our compound’s
progression through preclinical studies and confirm its
economical therapeutic efficacy in the treatment of this
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Acknowledgment
GO-D and MD were supported by grants from the Gov-
ernments of Ghana and Vietnam, respectively.
13. Bone marrow-derived macrophages were infected with L.
donovani antimony-sensitive strain 2000169 promastigotes,
using a 20:1 parasite: host cell ratio. Twenty-four hours
later cells were treated with medium alone (controls), a
synthesized compound (100lg/ml) or sodium stibogluco-
nate solution (70 lg/ml Sbv/ml); using four replicates/
treatment. Seventy-two hours post-treatment cells were
fixed, stained with 10% v/v aqueous Giemsa stain and the
mean percentage of cells infected determined, using 200
randomly selected cells. The mean percentage suppres-
sion SE was calculated using the relevant mean control
value. A Mann–Whitney U test using the control data and
test compound data was carried out to determine whether
a compound caused significant parasite killing, and a
p < 0.05 was considered significant.
Supplementary data
Experimental procedures and analysis data are available
on-line. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2006.11.004.
References and notes
1. World Health Organisiation (WHO), http://www.who.int/
leishmaniasis/burden/en/.