Quinolylhydrazones as novel inhibitors of Plasmodium falciparum serine protease PfSUB1

Article abstract of DOI:10.1016/j.bmcl.2012.06.023

Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.

Full text of DOI:10.1016/j.bmcl.2012.06.023

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5317–5321
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinolylhydrazones as novel inhibitors of Plasmodium falciparum
serine protease PfSUB1
Sandra Gemma a,b,c, Simone Giovani a,b,c, Margherita Brindisi a,b,c, Pierangela Tripaldi a,b,c
Simone Brogi a,b,c, Luisa Savini a,b,c, Isabella Fiorini a,b,c, Ettore Novellino a,d, Stefania Butini a,b,c
,
,
e
e
Giuseppe Campiani a,b,c, , Maria Penzo , Michael J. Blackman
⇑
a European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, Via A. Moro, 53100 Siena, Italy
b Dipartimento Farmaco Chimico Tecnologico, University of Siena, Via A. Moro, 53100 Siena, Italy
c Centro Interuniversitario di Ricerche sulla Malaria (CIRM), University of Torino, Torino, Italy
d Dipartimento di Chimica Farmaceutica e Tossicologica, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy
e Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the
egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent
invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave
the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action
and consequently able to overcome the current problems of resistance to established chemotherapies.
Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone
2 as a hit compound. Here we report a preliminary investigation of the structure–activity relationships
for a class of PfSUB1 inhibitors related to our identified hit.
Received 21 May 2012
Revised 7 June 2012
Accepted 10 June 2012
Available online 23 June 2012
Keywords:
Malaria
Egress
Serine protease inhibitors
PfSUB1
Ó 2012 Elsevier Ltd. All rights reserved.
Hydrazones
Malaria is one of the major causes of morbidity and mortality in
the world.1 Malaria is endemic in many low-income countries and
despite considerable scientific advances and the intensification of
elimination campaigns, the goal of eradicating malaria is still elu-
sive.2–4 According to WHO data, 225 million malaria cases and
approximately 781,000 deaths globally were reported in 2009.1
The continued emergence of drug-resistant parasites compromises
efforts to control the disease.5 This dramatic scenario is further
worsened by the fact that currently only a single class of drugs
(artemisinins) is active against drug-resistant parasites.6,7 Indeed,
besides the recently approval of arterolane in India,8,9 no new class
of antimalarials has been introduced into clinical practise for dec-
ades, and very few prospective clinical candidates with novel
mechanisms of action are under development.10,11
Human malaria is caused by one of five species of Plasmodium
parasites, of which Plasmodium falciparum is the most deadly.
The life cycle of P. falciparum is shared between an Anopheline mos-
quito vector and the human host and involves several phases of
development. During the erythrocytic phase, occurring inside the
human body, the parasite lives inside red blood cells (RBCs),
encircled by a membrane forming the parasitophorous vacuole
(PV). The parasite matures within the PV through several develop-
mental stages until the resulting schizont eventually divides to
form 16 to 32 mature merozoites. Merozoites are the invasive
forms of the parasite and are released upon rupture of the RBC,
in a process known as egress.12 Egress and subsequent invasion
of RBCs by the released merozoites are highly regulated events,
and over the last decade it has been demonstrated that several pro-
teases are mediators of both egress and invasion.12–16
The activity of a subtilisin-like serine protease (PfSUB1) has
been demonstrated to be essential in the egress of invasive para-
sites from the host RBC.17–20 In the context of a drug discovery pro-
cess both invasion and egress appear excellent processes to be
targeted not only because they are critical for parasite survival
but also because many components of the invasion and/or egress
machinery are conserved across the phylum. Anti-invasion and/
or anti-egress drugs developed for malarial species may therefore
prove to be applicable to others. As a result, PfSUB1 represents a
highly promising potential drug target for the development of
inhibitors useful in the fight against malaria. In previous work,18
the PfSUB1 inhibitor MRT12113 (1, Fig. 1) was found to block
egress at high concentrations, whilst allowing egress of invasion-
defective merozoites at lower concentrations. Other than 1, the
⇑
Corresponding author.
E-mail address: campiani@unisi.it (G. Campiani).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.023

5318
S. Gemma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5317–5321
Table 1
PfSUB1 inhibitory activity of compounds 2 and 3a–sa
Compd
Ar
% Residual hydrolytic
IC50 (lM)
activity at 50
l
M
S.D.
Figure 1. Reference compound 1, PfSUB1 inhibitor identified during the screening
(2) and site of structural modifications at the hydrazone scaffold.
3a
85.0 7.7
—
only inhibitors of PfSUB1 identified to date are a biotinylated chlo-
3b
3c
3d
75.6 6.5
77.5 7.3
92.4 3.1
—
—
—
roisocoumarin19 or very recently developed peptidyl
a-ketoam-
ides21 based on authentic substrates of the protease.
In the search for new small molecule PfSUB1 inhibitors, we per-
formed a high-throughput screening (HTS) campaign of a proprie-
tary library of compounds (ꢀ450 peptidic and non peptidic
compounds) against PfSUB1, leading to the identification of the
quinolylhydrazone 2 (Fig. 1) as a hit compound. The library was
formed by several chemotypes belonging to different classes of bio-
logically active compounds. Our hit compound is characterised by
good predicted logP and logD values, molecular weight lower than
350, and most importantly low cost of the synthetic pathway for its
preparation allowing fast exploration of the structure-activity rela-
tionships (SARs). However, we had concerns due to the presence of
the nitro group. Here we present a preliminary exploration of the
SARs for this hydrazone class of compounds as PfSUB1 inhibitors
through the synthesis of several structural analogues bearing mod-
ifications at the aromatic aryl substituent (compounds 3a–s, Table
1), at the quinoline moiety (compounds 4–12, Table 2) and at the
hydrazone linker (compounds 13a–e, 14, Table 3).
3e
3f
76.4 2.1
70.9 4.2
—
—
3g
3h
72.2 8.5
97.2 4.2
—
—
In order to explore the effect of the substituent at the arylhyd-
razone moiety on the potency of PfSUB1 inhibition, we replaced
the nitro group of 2 with several electron withdrawing and/or H-
bond donor/acceptor groups (as in 3a–s, Table 1). Compounds 2
and 3a–s were synthesized as described in Scheme 1. Accordingly,
hydrazine 15 was reacted with equimolar amounts of the suitable
carboxaldehydes 16 and 17a–p in refluxing EtOH to furnish final
compounds 2 and 3a–p, respectively (Scheme 1 and Table 1).22
Aldehydes 16, 17a–c,f,h–p were commercially available while alde-
hydes 17d,e,g were prepared following described procedures.23–25
Compound 3a, containing a methyl ester in the p-position of the
aromatic ring, was hydrolyzed with 20% aqueous NaOH in a 1:2
MeOH/THF mixture to give 3q in high yield. Aldehyde derivative
3r was obtained from 3b through acidic hydrolysis and finally, ami-
doxime 3s was obtained after exposure of the cyano-derivative 3p
to hydroxylamine hydrochloride in the presence of potassium
tert-butoxide.26 Hydrazones 4–12 and 13a,b (Tables 2 and 3) were
synthesized as described in Scheme 2. 4-Quinolinhydrazines 18a–f,
4-pyridylhydrazine 19, and 2-benzimidazolylhydrazine 20, pre-
pared following described procedures,27,28 were coupled with
p-nitrobenzaldehyde 16, p-cyanobenzaldehyde 17p or with
p-nitrophenylmethyl ketone to afford the final hydrazones 4–12
and 13a in reasonable yields. For the synthesis of derivative 13b,
hydrazine 15 was firstly reduced to the corresponding aniline
derivative 21 using a mixture of nickel chloride and sodium borohy-
dride in methanol,29 the resulting compound 21 was then coupled
with p-nitrobenzaldehyde 16 in refluxing ethanol to afford 13b. Fi-
nally, hydrazides 13c–e and 14 were synthesized as described in
Scheme 3, by coupling the appropriate carboxylic acids 22a–c with
hydrazines 15 and 20, in the presence of DCC and HOBt.
3i
38.6 21.3
40 < IC50 < 50
3j30
77.9 2.7
78.2 1.2
97.0 4.4
86.1 4.8
—
—
—
—
3k30
3l30,31
3m
3n
3o
100
6
—
—
62.1 15
3p
3q
29.2 3.3
75.5 0.6
40
—

S. Gemma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5317–5321
5319
Table 1 (continued)
Table 2
Compd
Ar
% Residual hydrolytic
IC50 (lM)
PfSUB1 inhibitory activity of compounds 4–12a
activity at 50
lM
S.D.
Compd Structure
% Residual
hydrolytic
activity at
IC50 (lM)
3r
86.5 3.4
—
50
l
M
S.D
3s
2
73.9 1.6
27.2 2.5
—
430
12.3 1.6
32.4 4.3
80.6 4.0
74.3 12.1
85.0 4.5
55.8 10.0
81.5 20.9
84.6 12.3
20 < IC50 < 30
—
20
a
Physical and spectroscopic data for compounds 2 and 3j–l are consistent with
those reported in Ref.
30,31
.
The inhibitory activity of the synthesized hydrazones and
hydrazides against PfSUB1 was assessed using a previously de-
scribed fluorimetric assay.21 PfSUB1 inhibitory potencies are ex-
pressed as percent residual hydrolytic activity after incubation of
PfSUB1 with the tested compounds. Compounds showing levels
of inhibition greater than 50% at the starting concentration of
5
50
50 lM were further examined at a range of lower concentrations.
Results are reported in Tables 1–3.
6
—
The inhibition activity data given in Table 1 clearly highlight
that H-bond acceptor/donor groups such as 3a–c, 3e–g, and 3q–s
are not beneficial substituents at the para position. This points
out that the importance of the nitro group for the activity of 2
probably relies on its high electron withdrawing properties. This
observation is further supported by the fact that the o-nitro deriv-
ative 3i has a slightly lower inhibitory activity against the enzyme
compared with the p-nitro analogue 2, while the m-nitro derivative
3h is inactive at the tested concentration. With the aim of replacing
the potentially toxic nitro group with safer substituents, we syn-
thesized a series of derivatives bearing various electron withdraw-
ing groups such as halogens (3j–l), trifluoromethyl (3m–o) and the
cyano group 3p. Interestingly, introduction of the cyano group re-
sulted in a compound with only slightly reduced potency with re-
spect to 2. The observed potencies are in agreement with the
electron withdrawing potencies of the groups (NO2 > CN > -
SO2Me > hal). Next we examined the role of the quinoline substit-
uents on the inhibitory potency and we discovered that the
substitution pattern of the quinoline ring can sensibly modulate
the inhibitory potency (hydrazones 4–11, Table 2). From our
screening, compound 4, in which the fused dioxolane ring was re-
placed by a 6-methoxy group, was slightly less potent than 2, and
removal of the 2-methyl group (5) further reduced potency. The
low activity of compounds 6–8 is due to the lack of ether function
at the quinoline ring (5 vs 6). The pyridyl-hydrazone 11 was also
not active at the tested concentration while the 1,2,3,4-tetrahydro-
acridinyl-derivative 9 showed a minimal level of inhibition. A vi-
sual inspection of the Novartis in vitro antimalarial screening
dataset34,35 revealed that compound 12, containing a p-nitro-
phenylhydrazone moiety had antimalarial activity that might be
due to a capacity to inhibit PfSUB1. We therefore synthesized
and tested compound 12, which showed some degree of inhibition
activity against PfSUB1, although this latter activity might not be
7
—
8
—
9
>50
10
—
1132
—
related to its antiplasmodial potency (EC50 = 0.81 and 0.74 lM
against 3D7 and W2,34,35 respectively). Finally, in this preliminary
evaluation of the SARs we investigated the role of the hydrazone
linker (13a–e and 14, Table 3) on PfSUB1 inhibitory potency. Meth-
ylation of the benzylidene moiety (as in 13a), or conversion of the
hydrazone moiety to an imine (as in 13b) or to a hydrazide (as in
13c–e) always resulted in a drastic decrease in inhibitory potency.
Only in the case of the benzimidazole derivatives, both hydrazone
12 and hydrazide 14 showed similar inhibition potency. The strict
1233
43.1 11.6
27.2 2.5
>50
20
2
—
a
Physical and spectroscopic data for compounds 4, 11, and 12 are consistent
with those reported in Ref.
30,32,33
.

5320
S. Gemma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5317–5321
Table 3
PfSUB1 inhibitory activity of compounds 13a–e and 14
Compd
X
R1
% Residual hydrolytic
activity at 50
lM
S.D
13a
13b
NO2
NO2
60.3 0.5
72.2 9.8
13c
13d
13e
NO2
64.4 4.0
85.5 5.8
68.7 5.1
CN
Scheme 2. Synthetic route to the hydrazones 4–12 reported in Table 2 and 13a,b
reported in Table 3. Reagents and conditions: (a) 16, or 17p, or p-nitrophenylmethyl
ketone, EtOH, reflux, 14 h; (b) 16, EtOH, reflux, 14 h; (c) NiCl2, NaBH4, MeOH, 25 °C,
1 h.
SO2Me
14
2
—
—
—
—
46.7 11.4
27.2 2.5
Scheme 3. Synthetic route to the hydrazones 13c–e and 14 reported in Table 3.
Reagents and conditions: (a) DCC, HOBt, dry DMF, 25 °C, 12 h.
bearing a strong electron withdrawing group led us to speculate
whether a covalent mechanism of inhibition, involving a direct at-
tack of the active site serine at the benzylidene carbon, could be at
the basis of the observed SARs. Experiments designed to explore
this issue revealed that the enzyme recovered its catalytic activity
after removal of inhibitor 2, highlighting a mechanism of inhibition
that could be either competitive or covalent reversible.
In conclusion, here we presented the initial exploration of the
SARs for a class of quinoline hydrazones as novel PfSUB1 inhibitors.
The key role played by this enzyme in the life cycle of the parasite,
and the current lack of antimalarial agents endowed with innova-
tive mechanisms of action make compound 2 and its cyano-
derivative 3p important lead structures for further development.
Other than 1 and a ketoamide-based inhibitor,18,21 quinolylhydraz-
ones here described represent the first series of PfSUB1 inhibitors
with clear-cut SARs.
Scheme 1. Synthesis of hydrazones 2, 3a–s reported in Table 1. Reagents and
conditions: (a) EtOH, reflux, 14 h; (b) 20% NaOH, 1:2 MeOH/THF, 25 °C, 30 min; (c)
0.5 N HCl, acetone, 25 °C, 14 h; (d) hydroxylamine hydrochloride, potassium tert-
butoxide, dry DMSO, 25 °C, 16 h.
Acknowledgments
dependence of the inhibitory activity of this set of quinolylhydraz-
ones on an intact hydrazone moiety linked to an aromatic ring
The authors thank MIUR (PRIN) for financial support. M.P. was
funded by the European Union Framework Programme 7-funded

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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.06.023.
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