Accepting the Invitation to Open Innovation in Malaria Drug Discovery: Synthesis, Biological Evaluation, and Investigation on the Structure-Activity Relationships of Benzo[b]thiophene-2-carboxamides as Antimalarial Agents

Article abstract of DOI:10.1021/acs.jmedchem.6b01685

Malaria eradication is a global health priority, but current therapies are not always suitable for providing a radical cure. Artemisinin has paved the way for the current malaria treatment, the so-called Artemisinin-based Combination Therapy (ACT). However, with the detection of resistance to ACT, innovative compounds active against multiple parasite species and at multiple life stages are needed. GlaxoSmithKline has recently disclosed the results of a phenotypic screening of an internal library, publishing a collection of 400 antimalarial chemotypes, termed the “Malaria Box”. After analysis of the data set, we have carried out a medicinal chemistry campaign in order to define the structure-activity relationships for one of the released compounds, which embodies a benzothiophene-2-carboxamide core. Thirty-five compounds were prepared, and a description of the structural features responsible for the in vitro activity against different strains of P. falciparum, the toxicity, and the metabolic stability is herein reported.

Full text of DOI:10.1021/acs.jmedchem.6b01685

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Article
Accepting the Invitation to Open Innovation in Malaria Drug Discovery:
Synthesis, Biological Evaluation and Investigation on the Structure#Activity
Relationships of Benzo[b]thiophene-2-carboxamides as Antimalarial Agents.
Marco Pieroni, Elisa Azzali, Nicoletta Basilico, Silvia Parapini, Michal Zolkiewski, Claudia
Beato, Giannamaria Annunziato, Agostino Bruno, Federica Vacondio, and Gabriele Costantino
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01685 • Publication Date (Web): 27 Jan 2017
Downloaded from http://pubs.acs.org on January 29, 2017
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Accepting the Invitation to Open Innovation in Malaria
Drug Discovery: Synthesis, Biological Evaluation and
Investigation on the Structure‒Activity Relationships of
Benzo[b]thiopheneꢀ2ꢀcarboxamides as Antimalarial
Agents.
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Marco Pieroni,1,±,* Elisa Azzali,1,± Nicoletta Basilico,2 Silvia Parapini,3 Michal Zolkiewski,4 Claudia
Beato,1 Giannamaria Annunziato,1 Agostino Bruno,1 Federica Vacondio,5 Gabriele Costantino.1,6
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1P4T group, and Department of Pharmacy, University of Parma, Parco Area delle Scienze 27/A,
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Parma, 43124; Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università di
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Milano, via Pascal 36, Milano, Italy; Dipartimento di Scienze Farmacologiche e Biomolecolari,
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Università di Milano, Milano, Italy; Department of Biochemistry and Molecular Biophysics, Kansas
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State University, Manhattan, KS, United States; Centro Interdipartimentale Misure (CIM) ’G.
Casnati’, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.
±These authors contributed equally to this work
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Abstract
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Malaria eradication is a global health priority, but current therapies are not always suitable for
providing a radical cure. Artemisinin has paved the way for the current malaria treatment, the soꢀcalled
Artemisininꢀbased Combination Therapy (ACT). However, with the detection of resistance to ACT,
innovative compounds active against multiple parasite species and at multiple life stages are needed.
GlaxoSmithKline has recently disclosed the results of a phenotypic screening of an internal library,
publishing a collection of 400 antimalarial chemotypes, termed the “Malaria Box”. After analysis of the
dataset, we have carried out a medicinal chemistry campaign in order to define the StructureꢀActivity
Relationships for one of the released compounds, which embodies a benzothiopheneꢀ2ꢀcarboxamide
core. 35 compounds were prepared, and a description of the structural features responsible for the in
vitro activity against different strains of P. falciparum, the toxicity and the metabolic stability is herein
reported.
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Introduction
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Malaria is an infectious disease caused by the unicellular parasite Plasmodium, that is transmitted by
the females of certain species of mosquitoes of the Anopheles genus.1 Among the species of plasmodia
that may infect humans, P. vivax and P. falciparum are responsible for the majority of malaria
infections that are often fatal.2 Once in the human body, the parasites rapidly multiply in the liver and
then in the red blood cells leading, after 7ꢀ10 days, to the onset of the typical malaria symptoms such as
fever, headache, chills and vomiting.3,4 If not treated, the hyperparasitemia may cause massive
intravascular hemolysis, multiꢀorgan failure and eventually death. Over the years, malaria has become a
major healthcare challenge, especially in developing countries.5 Even more disturbing is the fact that
among the hundreds of millions people infected with malaria, children under the age of 5 years and
expectant mothers account for the majority of cases.6
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Despite the recent encouraging developments, no effective vaccines are available yet, therefore
chemotherapy still remains the only means to fight the infection.7 ArtemisininꢀCombination Therapies
(ACTs) represent the current pillar of the malaria treatment.8 After isolation of artemisinin from
Artemisia annua by Chinese researchers,9,10 a discovery awarded with the 2015 Nobel Prize for
Medicine,11 ACTs have been used to cure millions of patients in the last decades. However, as for other
tropical neglected infections, for which the firstꢀline therapeutic options are relatively limited, drug
resistance represents a persistent threat and patients with resistance to artemisinin and its coꢀ
administered drugs are regularly isolated, especially in Asia.12 In light of these facts, the search for
innovative ways to target the parasite is a crucial aim at pursuing the policies of complete malaria
eradication in the coming years.13,14 A major obstacle for drug discovery in the field of many infectious
diseases is the lack of adequate resources because of the limited commercial revenues. In spite of this
fact, at least in the case of malaria and tuberculosis, some pharmaceutical companies, in collaboration
with international agencies such as Medicine for Malaria Venture (MMV), have explored new models
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of collaboration with academia, encouraging open innovation platforms and making their chemical and
biological tools available to academic institutions.15 In 2010 GlaxoSmithKline released the structures
of 13533 chemical probes that were confirmed to inhibit P.falciparum growth by at least 80% at 2 µM
concentration.16 This work was further refined by clustering the molecules according to their structural
similarities and cherryꢀpicking 47 hit compounds based on their antimalarial activity, physicochemical
properties, and dissimilarity to known antimalarial structures.17 The final “Open Access Malaria Box”
was thus assembled and delivered by MMV, with the overarching aim of catalyzing research towards
the discovery of new efficacious small molecules suitable for clinical development.18 Among the
released hits, five were subsequently explored in‒house by GSK, whereas the malaria community was
openly invited to optimize the other chemical series. Due to our interest in novel antimalarial
chemotypes,19–21 we decided to pursue a medicinal chemistry campaign on one of the compounds of the
malaria box, namely (3ꢀchlorobenzo[b]thiophenꢀ2ꢀyl)(2,6ꢀdimethylmorpholino)methanone (77, Figure
1).
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[Insert Figure 1 here]
We focused our attention on this compound for several reasons. First, its baseline antiꢀPlasmodium
activity was significantly higher than the average (Pf3d7 XC50 = 88 nM). Second, the molecule was
amenable for chemical manipulation at various positions. The compound modifications could lead to a
modulation and, perhaps, an improvement of potency and other pharmacokinetic characteristics.
Moreover, the benzothiophene scaffold is found in many marketed drugs (raloxifene, zileuton,
sertaconazole)22 which suggests its usefulness for developing novel pharmaceuticals. Finally, when this
work was about to be finalized, additional biological information about compound 77 were disclosed
by a comprehensive work coordinated by van Voorhis et al.23 In this paper, compounds belonging to
the Malaria Box were furtherly explored with regard to their biological activity, PK characteristics, and
metabolomic and chemogenomic profile. 77 was found to have similar drugꢀdrug chemogenomic
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profile. It has been found that the drugꢀdrug chemogenomic profile of compound 77 was similar to the
artemisinin sensitivity cluster. This result provided additional rationale for further investigation of
compound 77, although the molecular mechanism of its action could not be revealed.23 In this
publication, we report the rational design, synthesis and biological antimalarial evaluation of a series of
derivatives structurally related to compound 77, leading to the description of SAR and to the
characterization of the pharmacophore for this molecule. For the most active compounds, we also
investigated their predicted metabolic properties and their putative molecular mechanism of action
through a target fishing approach.
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Results and discussion
Chemistry
The 3ꢀchlorobenzo[b]thiopheneꢀ2ꢀcarbonyl chloride core was easily prepared through the synthetic
protocol reported by Krubsack and Higa,24 and Brabander25 starting from commercially available
cinnamic acid that was refluxed with thionyl chloride and pyridine in chlorobenzene to give
intermediates 6‒10. Subsequent synthesis of amide derivatives was carried out refluxing the acyl
chloride and the suitable amine in dioxane, to give the final compounds 11‒25 (see Table 1).
[Insert Scheme 1 here]
[Insert Scheme 2 here]
The final compounds 54‒71, 73 and intermediate 75 were obtained employing a conventional amide
coupling protocol (Scheme 2 and 3), reacting the proper carboxylic acids with the suitable amines in
the presence of 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDC) and 2ꢀ(1HꢀBenzotriazoleꢀ1ꢀyl)ꢀ
1,1,3,3ꢀtetramethyluronium tetrafluoroborate (TBTU) as coupling agents and triethylamine as the base.
In the case of compounds 50, 51, 73, either the carboxylate ester 39, 40 or the carboxylic acid 72
precursors were commercially available, whereas for the majority of the derivatives reported the
synthesis of the carboxylic acid core was carried out from commercially available precursors. The
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benzofuranꢀ2ꢀcarboxylic acid intermediate 32 was obtained according a previously reported protocol,26
reacting salicylaldehyde with methylchloroacetate in DMF and K2CO3, to obtain the corresponding
methyl ester, subsequently cleaved through basic hydrolysis. Following a similar procedure, but using
methyl thiosalicylate in place of salicylaldehyde, the ethyl 3ꢀhydroxybenzo[b]thiopheneꢀ2ꢀcarboxylate
precursor 33 was obtained. However, it was not possible to hydrolyze 33, also in harsh conditions,
likely because of the formation of a pseudoꢀcycle between the 2ꢀcarboxylic moiety and the vicinal 3ꢀ
hydroxyl. Indeed, when the hydroxyl group was masked either with a methyl or with a substituted
benzyl group (intermediates 34‒38) or protected with a methoxymethyl group (intermediate 74), the
corresponding carboxylic acid derivatives (45‒49) were easily obtained in the above reported
conditions. After coupling with 2,6ꢀdimethylmorpholine, MOM derivative 75 was deprotected with
conc HCl to give the final derivative 76.
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[Insert Scheme 3 here]
The benzo[b]thiopheneꢀ2ꢀcarboxylic acid was obtained reacting oꢀnitrobenzaldehyde with ethylꢀ2ꢀ
mercaptoacetate in DMF and K2CO3 as the base to yield the ethyl ester 28, that was hydrolyzed in basic
conditions to afford the corresponding acid 42. Following a similar procedure, but using 2ꢀ
fluoropropiophenone in place of oꢀnitrobenzaldehyde, the ethyl ester intermediate 29 was obtained and
hydrolyzed to give the precursor 43. All of the ester hydrolyses were carried out reacting the suitable
ester in a mixture of THF/MeOH/H2O with LiOH at room temperature.
In this work, a total of 35 compounds were prepared and tested for their capability to inhibit the
growth of P. falciparum using the pLDH assay. The compounds were tested both against the CQ
susceptible (D10) and CQ resistant (W2) strains, in order to evaluate their susceptibility to cross
resistance. Among the 35 derivatives, 9 showed a remarkable potency in the low nM range and
therefore were further evaluated for their toxicity against a Human Microvascular Endothelial Cell line,
and their metabolic stability in human liver microsomes. Rational modifications of the main structure
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led to a variable range of activities and allowed us to construct a plausible SAR for this series of
benzo[b]thiopheneꢀ2ꢀcarboxamides, that could eventually inspire the design of improved analogues. In
addition, an attempt to identify the actual molecular target of these derivatives was made through a
“target fishing” approach.
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We identified four portions of hit compound 77 that were amenable for chemical modifications: a)
the carboxamide substituent, b) the substituent a position Cꢀ3, c) the phenyl portion of the
benzo[b]thiophene core and d) the benzo[b]thiophene ring.
[Insert Table 1 here]
Substituents at the 2-carboxamide nitrogen. Modifications at this position were carried out in
order to answer the following structural questions: a) can the morpholine be substituted by other
heteroaliphatic structures? b) Are the methyl groups important for the activity? c) Can an aromatic
group be located at this position, in order to make the nitrogen a hydrogen bond donor (HBD)? First,
we deemed relevant to establish which configuration was the most favorable for the two methyl groups
of the morpholine. Indeed, in the work that has inspired our research, it was not specified whether the
cis or trans stereoisomer is more active. Compounds 11 and 12 were then prepared and, although both
did show the activity, the cis stereoisomer was found to be about 20 times more potent than the
corresponding trans analogue (11, IC50D10= 37.1 nM vs 12, IC50D10 = 646 nM). Thus, the cis
configuration was maintained in all of the other analogues bringing a 2,6ꢀdimethylmorpholine in their
structure (see the following paragraphs). Next obvious step was to investigate the impact of the
dimethyl pattern on the overall activity. Surprisingly, removal of the dimethyl substitution on the
morpholine ring (14, IC50D10 = >15000 nM), as well as on the piperidine (15, IC50D10 = >15000 nM), led
to a complete loss of activity. The same was observed when a linear aliphatic amine such as
diethylamine (20, IC50D10 = >15000 nM) was used in place of a cycloaliphatic one. We then
investigated whether the substitution of the oxygen with a nitrogen atom, that could serve as a
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hydrogen bond donor, could play a role in the interactions with the target binding site and affect the
activity. Substituting the 2,6ꢀdimethylmorpholine core with a 3,5ꢀdimethylpiperazine (13, IC50D10
=
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4909 nM) led to a drastic reduction of the antiparasitic activity, that was restored to some extent when
3,5ꢀdimethylpiperidine was used as the substituent (21, IC50D10 = 100 nM). Finally we investigated
whether the 2ꢀcarboxamide could have a hydrogen bond donor property to better interact with the
target binding site. Therefore compounds 16‒19, bearing either cycloaliphatic or
aromatic/heteroaromatic rings as substituents, were synthesized and tested. In particular, the 4ꢀ
aminopyridyl derivative was prepared due to its recurrent presence both in some marketed antimalarial
compounds and preclinical candidates. Unfortunately, all of these modifications only led to the loss of
activity (16−19, IC50D10 = >15000 nM). Finally, some representative compounds, namely 11, 19 and 23,
were evaluated with regard to their stability in RPMI 1640, that is the medium used for the
antiplasmodial assays. Indeed, by virtue of the 3ꢀchloro substitution, these compounds might behave as
vinylogue acid chlorides, which are very reactive and may decompose or unselectively interact with
every protein and generate false positive. However, compounds resulted unmodified at the test
conditions also after 24 h (Supporting Information, S19).
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From this first round of modifications, we can conclude that the 2,6ꢀdimethylmorpholine moiety is
crucial for conferring high antimalarial activity. Attempts to substitute the ring with other structures, as
well as to remove the dimethyl group, all led to a loss/decrease of activity. The only exception,
especially considering the difference in the activity with compound 13, is represented by the 3,5ꢀ
dimethylpiperidine derivative 21, that has an activity similar to that of the hit compound 77. It can be
then speculated that, in order to maintain the activity, hydrogen bond donor groups cannot be
introduced in place of the morpholine oxygen.
Substitutions at the phenyl portion of the benzo[b]thiophene core. After exploring the best
substituent for the amide, and maintaining the chlorine atom at position Cꢀ3 as for the hit compound,
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we investigated whether adding small functional groups at the phenyl portion of the benzo[b]thiophene
scaffold might affect the activity and the pharmacokinetic parameters. Electron withdrawing groups
(EWGs) of different size, and an electron donor group (EDG) such as the methyl, were introduced at
position Cꢀ6 of the benzo[b]thiophene scaffold. In addition, a dichloro substitution pattern was
investigated as well. Smallꢀsize substituents, regardless their EDG or EWG nature, were found to
properly maintain strong antimalarial activity (23, IC50D10 = 74 nM; 24, IC50D10 = 86 nM). At the same
position, a chlorine is still well tolerated, although decreasing the activity by 5ꢀfold compared to the
fluorine atom (22, IC50D10 = 407 nM), whereas the dichloro substitution led to a considerable drop in
the activity (25, IC50D10 = 2223 nM). Therefore, this structural portion of the molecule is amenable for
various modifications, which can affect the activity and other pharmacological properties.
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Substituents at position C-3. The 3ꢀchlorobenzothiophene scaffold has been seldom used in
medicinal chemistry, possibly due to the difficulties in its preparation. The chlorine atom at Cꢀ3 might
affect the electronic characteristics of the main benzo[b]thiophene, and also those of the amide moiety.
To explore the effect of various substituents, we decided to introduce at Cꢀ3 a wide range of functional
groups, differing in size, electronic and physicochemical properties. Removing the chlorine atom
produced a compound that was found to be 50ꢀfold less active than the parent derivative (54, IC50D10
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1823 nM). As expected, the same results were obtained when cycloaliphatic moieties other than the
2,6ꢀdimethylmorpholine core were used to modify the carboxamide portion (56, IC50D10 = 10893 nM;
55, 57, 58, IC50D10 = >15000 nM). Substitution with polar groups, having hydrogen bond
donor/acceptor nature, led to a sharp decrease, or even loss of activity (69, IC50D10 = 8761 nM; 76,
IC50D10 = >15000 nM). On the other hand, apolar substituents at the same position, such as the methyl
(71, IC50D10 = 226 nM), methoxy (62, IC50D10 = 429 nM) or ethyl (59, IC50D10 = 682 nM) were able to
maintain a remarkable activity, only 4ꢀfold lower than that of compound 71. Therefore, it might be
speculated that, at the position being object of the described modifications, the lipophilicity of the
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substituent, rather than its EWG/EDG properties, plays an important role in conferring good overall
activity. Thus, we evaluated next how the size of the substituent at Cꢀ3 could modulate the compound
activity. A benzyl moiety was used to functionalize the amino and hydroxyl group of compounds 69
and 76, to yield derivatives 70 and 63, respectively. Although derivative 69 is more active than 76, the
corresponding benzyl derivative was found to be 70ꢀfold less active than the benzyloxy counterpart
(63, IC50D10 = 38 nM vs 70, IC50D10 = 2615 nM). Indeed, compound 63 can be considered the most
active derivative of the series, being the activity toward both the chloroquine susceptible and resistant
strain on the same range of the hit compound 11. Moreover, with this compound we have moved from
the 3ꢀchlorobenzothiophene scaffold that could be unselectively reactive, as mentioned above.
Modifications of the benzyl appendage were attempted as well, in particular by adding a lipophilic
EWG such as the chlorine, a more polar EDG such as the methoxy, and a disubstitution such as the 3,4ꢀ
difluoro moiety (64, IC50D10 = 186 nM; 65, IC50D10 = 146 nM; 66, IC50D10 = 141 nM). All of these
derivatives maintained activity against both the susceptible and resistant strain of P. falciparum,
although they were 6 to 7ꢀfold less active than the unsubstituted parent compound 63. In summary at
position Cꢀ3, along with the phenyl portion of the benzo[b]thiophene above described, we have noticed
the highest freedom for substitution. Polar groups were not found suitable substituents, whereas
lipophilic functional groups, especially the bulky ones, supported the highest activity. In particular, the
presence of polar functionalities (69, 70 and 76) strongly hampers the activity of these derivatives.
Thus it can be speculated that the target binding site is predominantly hydrophobic and lacks hydrogen
bonding groups.
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Modification of the benzo[b]thiophene core. The final part of our SAR assessment was dedicated
to explore the possibility of substituting the benzo[b]thiophene core with similar heterocycles,
maintaining the same pattern of substitution at the Cꢀ2. The sulfur atom, although present in several
drugs, might be oxidized in the biological systems, and this may represent a limitation for further
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development of these derivatives. Therefore, the benzo[b]thiophene scaffold was substituted by a
benzofuran, an indole and a benzothiazole. As reported in Table 1, benzofuran and indole analogues
(60, 61, 68, 69, IC50D10 = >15000 nM) failed to show any clue of activity, when compared to the
benzo[b]thiophene counterpart; the benzothiazole 73, that still maintains a sulfur atom at the same
position as the hit compound, showed only a negligible activity (IC50D10 = >9788 nM). It can be then
hypothesized that the sulfur atom, by virtue of its size and physical characteristics, it is crucial in
conferring the desired antiparasitic activity.
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The most active derivatives (11, 12, 21, 24, 63‒66, 71) were tested against HMECꢀ1 cells to gain
information about their general toxicity. All of them proved to be reasonably safe, the selectivity index
spanning from 250 to 5000.
Target fishing experiments. Next, we attempted to find a putative target for the compound
derivatives. Finding the target could help in further optimization of analogues with improved drugꢀlike
characteristics. An extended study sponsored by Medicine for Malaria Venture reported a close
analogue of the hit compound 11 as inhibitor of P. falciparum choline kinase.27 Although this
compound inhibits Choline Kinase quite efficiently (IC50PfCK = 6.3 µM), its inhibitory activity
toward the parasite is considerably higher (EC50Pf3d7 = 0.3 µM), suggesting that choline kinase is not
the main target for this series of derivatives. In another work, Tate and colleagues have reported
molecules based on a benzo[b]thiopheneꢀ2ꢀcarboxylic scaffold as potent inhibitors of the P. falciparum
Nꢀmyristoyltransferases (PfNMT).28,29 However, the SAR for our molecules (see above) does not
match that described by Tate et al., making it unlikely that PfNMT would be the target for our
compounds. Finally, in the recent paper reported by Van Voorhis et al. above mentioned,23
a
mechanismꢀofꢀaction screening was successfully carried out for a number of compounds of the Malaria
Box, but only a generic set of information was retrieved for 77. In an attempt to find out the putative
target for this series, we have performed an in silico target fishing study.30 We performed a 2D
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similarity search (cutoff at 70%) using 11 as query in ChEMBL database31 to verify if similar
compounds have been tested against some specific targets that can be somehow related to P.
falciparum proteins. From this similarity search, we obtained 116 resulting hits. The results were
analyzed in order to cluster compounds according to chemical classes and to rule out those molecules
showing results only on whole cell assays, with no specific target annotated. For each surviving hit and
its target, we tried to find a corresponding protein in P. falciparum proteome. To this end, we queried
the proteome of P. falciparum 3d7 available in PlasmoDB32 with the protein sequences belonging to
the targets of the ChEMBL hits by using BLASTP33. The hit compounds a and b (Table S1) became
the most promising in terms of the putative target identity. Compound a was tested as a potential
antimicrobial agent targeting ClpB,34 whereas hit b was first developed as antiproliferative agent and
evaluated for DNA binding propensities and topoisomerases I/II inhibition as part of its mechanism of
action.35 Analyzing the activity of these derivatives on the annotated target, we found that only
compound a had some hint of activity, whereas compound b was inactive. It must also be noticed that
phosphatidylinositol 4ꢀkinase (PI4K) was one of the putative targets obtained from the study. Although
the benzo[b]thiophenes were known to inhibit choline kinase, we decided against evaluating our
derivatives toward this protein, due to a low sequence similarity between PI4K and choline kinase
catalytic domains (N1261ꢀ ꢀM1559, 21.5% of identity, Figure S5). This preliminary result prompted us
to investigate more in details the similarity between compounds 11 and a, also because the molecular
target under consideration plays a crucial role in the microbial life cycle.
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ClpB is a member of the Hsp100/ClpB family of AAA+ ATPases that, in bacteria, yeast, and plants,
is involved in supporting survival of bacteria, yeast, and plants under stress conditions by mediating
ATPꢀdependent protein disaggregation.36,37 Two isoforms of this ATPase were identified in P.
falciparum, PfClpB1 and PfClpB2, the latter usually known as Plasmodium Hsp101.38 PfClpB1 is
located in the apicoplast and it has been suggested to have a protective role against protein misfolding
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and aggregation, whereas PfClpB2 is crucial in Plasmodium infectivity as it participates in the export
of Plasmodium proteins to the erythrocyte cytosol.39 In addition, the ClpB system is highly conserved
among bacteria, yeast and plants but it is absent in humans, therefore both PfClpB isoforms are
regarded as potential new targets for malaria treatment.40 Although none of the two PfClpB proteins
has been yet crystallized, two bacterial forms of ClpB, one from E. coli (PDB 4CIU)41 and another
from T. thermophilus (PDB 1QVR),42 have been solved bound to ADP and an ATP derivative,
respectively. The ATP binding sites are strongly conserved among different AAA+ ATPases, and in
particular among ClpBs from different species,37 so the binding site residues are well conserved
between bacterial and Plasmodium ClpB (Figure S1). In particular, the Walker A motif in the ATP
binding site is identical in E. coli ClpB and PlClpB1. Compound 11 appears to dock well in the
bacterial ClpB ATP binding site and in the binding site of a homology model of PfClpB2, that has a
lower degree of similarity with E. coli than PfClpB1. The rates of ATP hydrolysis catalyzed by E. coli
ClpB in the absence and presence of compounds 11, 63, and 71 were measured (Figure S1). The assay
reactions were initiated by adding ClpB to the assay buffer that included ATP and the compound. This
procedure mimics the in vivo conditions, where the compounds would compete with ATP for binding
to ClpB. We did not detect any inhibition of the ClpB ATPase over a broad range the compound
concentrations (Figure S1). Since ClpB uses energy from ATP to produce its biological effects, it is
unlikely that the compounds would bind to the ATP site without inhibiting the ATPase, and still have
an effect on the biological activity. To further verify the lack of inhibition, ClpB was preincubated with
63 and the reaction was started by adding ATP in order to provide the compound a competitive
advantage over the nucleotide. However, no inhibition of the ClpB ATPase was observed in this case
either, effectively ruling out the possibility that ClpB is the molecular target of this series of
compounds.
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Conclusions
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In this work, we have taken advantage of an initiative by GSK to investigate benzo[b]thiopheneꢀ2ꢀ
carboxamides as antimalarial agents. The inꢀdepth SAR investigation that we have carried out has
revealed the important structural features underlying the pharmacological activity of these derivatives.
Also, an attempt to discover the putative molecular target of this series was undertaken through a
targetꢀfishing approach.
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The backbone structure (see Table 1) allows only for a surprisingly limited number of substitutions
that preserve the antimalarial activity. Even small backbone modifications at specific sites lead to a
complete loss of antimalarial potency.
Five of the prepared compounds maintained an activity similar to and apparently slightly better than
the parent compound 77, providing useful information to drive the development of nextꢀgeneration
derivatives. The benzo[b]thiophene core is crucial for conferring a high potency, while other bicyclic
heterostructures such as indole and benzofuran were found to be detrimental for the activity. With
regard to the carboxamide at the Cꢀ2, the 2,6ꢀdimethylmorpholine appendage not only appears to be the
best substituent to provide high potency, but it appears the only one that confers a measurable activity.
Position Cꢀ3 can be adorned with various substituents, to modulate activity and other pharmacological
properties, however hydrogenꢀcontaining functional groups are detrimental for the potency. Also, small
functional groups at the benzyl portion of the benzothiophene scaffold are well tolerated, suggesting a
way to modify these derivatives in the future. In spite of our attempts to identify the mechanism of
action of these compounds, their molecular target(s) remain unknown. Recent literature has provided
information on the importance of further pursuing exploration of the Malaria Box derivatives, and this
work sets the stage for further exploration of molecules structurally related to 77.
Experimental section
Chemistry
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General information. All the reagents were purchased from SigmaꢀAldrich, AlfaꢀAesar and
Enamine at reagent purity and, unless otherwise noted, were used without any further purification. Dry
solvents used in the reactions were obtained by distillation of technical grade materials over appropriate
dehydrating agents. MCRs were performed using CEM Microwave SynthesizerꢀDiscover model.
Reactions were monitored by thin layer chromatography on silica gelꢀcoated aluminium foils (silica gel
on Al foils, SUPELCO Analytical, SigmaꢀAldrich) at both 254 and 365 nm wavelengths. When
indicated, intermediates and final products were purified through silica gel flash chromatography (silica
gel, 0.040ꢀ0.063 mm), using appropriate solvent mixtures.
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1HꢀNMR and CꢀNMR spectra were recorded on a BRUKER AVANCE spectrometer at 300, 400
1
and 100 MHz respectively, with TMS as internal standard. HꢀNMR spectra are reported in this order:
multiplicity and number of protons. Standard abbreviation indicating the multiplicity was used as
follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet, m = multiplet and
br = broad signal. HPLC/MS experiments were performed with HPLC: Agilent 1100 series, equipped
with a Waters Symmetry C18, 3.5 µm, 4.6 mm x 75 mm column and MS: Applied Biosystem/MDS
SCIEX, with API 150EX ion source. HRMS experiments were performed with LTQ ORBITRAP XL
THERMO. All of the target compounds were tested as 95% purity samples or higher (by HPLC/MS).
Biology: parasite growth and drug susceptibility assay. The CQ sensitive (D10) and the CQ
resistant (W2) strains of P. falciparum were sustained in vitro as described by Trager and Jensen.43
Parasites were maintained at 5% hematocrit (human type Aꢀpositive red blood cells) in RPMI 1640
(EuroClone, Celbio) medium with the addition of 1% AlbuMax (Invitrogen, Milan, Italy), 0.01%
hypoxanthine, 20 mM HEPES, and 2 mM glutamine. All cultures were maintained at 37°C in a
standard gas mixture consisting of 1% O2, 5%CO2, and 94% N2. Compounds were dissolved in DMSO
and then diluted with medium to achieve the required concentrations (final DMSO concentration <1%,
which is nontoxic to the parasite). Drugs were placed in 96 well flatꢀbottom microplates (COSTAR)
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and serial dilutions made. Asynchronous cultures with parasitemia of 1–1.5% and 1% final hematocrit
were aliquoted into the plates and incubated for 72 h at 37°C. Parasite growth was determined
spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase
(pLDH), according to a modified version of Makler's method in control and drugꢀtreated cultures.44
Antiplasmodial activity is expressed as the 50%inhibitory concentrations (IC50). Each IC50 value is the
mean ± standard deviation of at least three separate experiments performed in duplicate.
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Cytotoxicity assay. The longꢀterm human microvascular endothelial cell line (HMECꢀ1) immortalized
by SV 40 large T antigen38 was maintained in MCDB 131 medium (Invitrogen, Milan, Italy)
supplemented with 10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/ml of epidermal growth
factor (Chemicon), 1 lg/ml of hydrocortisone, 2 mM glutamine, 100 U/ml of penicillin, 100 lg/ml of
streptomycin, and 20 mM Hepes buffer (EuroClone). Unless stated otherwise all reagents were from
Sigma Italia, Milan, Italy. For the cytotoxicity assays, cells were treated with serial dilutions of test
compounds for 72 h and cell proliferation evaluated using the MTT assay already described.33 The
results are expressed as IC50, which is the dose of compound necessary to inhibit cell growth by 50%.
Each IC50 value is the mean and standard deviation of at least three separate experiments performed in
duplicate.
Inhibition of ClpB. E. coli ClpB was produced as previously described.45 Protein concentration was
determined spectrophotometrically and reported in monomer units. To determine the rate of ATP
hydrolysis, the samples were incubated in the assay buffer (100 mM Tris/HCl pH 8, 1 mM DTT, 1 mM
EDTA, 10 mM MgCl2) at 37 °C. The compounds 11, 63, and 71 were dissolved in DMSO and then
diluted in the assay buffer to the final concentration. The corresponding amounts of DMSO added to
the buffer were used as controls. The reaction was initiated by adding ClpB to the buffer containing the
compound and 4 mM ATP or by adding ATP to the buffer containing ClpB and the compound. The
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concentration of inorganic phosphate generated from ATP by ClpB was measured as described
before.46
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Stability studies in HLM for compounds 11, 63 and 71. Stability of selected compounds in the
presence of HLM was assessed by incubation of a 1 µM concentration for 60 min in the presence of
HLM (1 mg protein mLꢀ1), at 37 °C, in the presence of a NADPHꢀregenerating system (2 mM
NADP+, 10 mM glucoseꢀ6ꢀphosphate, 0.4 U mLꢀ1 glucoseꢀ6ꢀphosphate dehydrogenase, 5 mM MgCl2)
in 100 mM PBS buffer solution pH 7.4. The reaction mixtures were preheated (37 °C) for 5 min before
adding the parent compound. At fixed time points (t = 0; 15; 30; 60; min), aliquots of samples were
withdrawn, deproteinized with two volumes of acetonitrile, centrifuged (9,000 g, 4 °C, 10 min) and the
supernatant analyzed by injection in HPLCꢀMS/MS system. The chromatographic separation was
performed employing a gradient elution starting from 70% water+0.1% formic acid (solvent A):30%
methanol (solvent B) to 90%B:10%A in 10 min; 90%B:10%A was kept for further 5 min; then back to
70%A:30%B and further 5ꢀmin of reconditioning time. HPLCꢀMS analysis employed a Thermo
Quantum Access Max TSQ triple quadrupole mass spectrometer (Thermo, USA) equipped with a Hꢀ
ESI (HeatedꢀElectroSpray Ionization) interface and coupled to an Accela UHPLC system (Thermo,
USA) constituted of a quaternary pump, a degasser and a thermostated autosampler. Compounds were
analyzed in positive ion mode using both total ion monitoring mode, over a mass range from 50 to 500
amu, and single ion monitoring mode. Data were acquired and analyzed employing Thermo Excalibur
1.4 software (Thermo, USA).
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Target fishing. Similarity search has been conducted using similarity search method implemented in
ChEMBL database,31 with a cutoff for minimum similarity at 70%. In the attempt to find some
analogies between the ChEMBL compounds target and plasmodium proteome, we use the blastp tool
available in PlasmoDB, restricting our research to Plasmodium Falciparum 3d7 proteome only and
applying default parameters. Multiple sequence alignment between PlClpB1, PlClpB2, E. coli ClpB
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and T. th. ClpB were performed with BLASTp. Docking studies were performed using Glide
software.47,48 E coli ClpB structure has been prepared using Protein Preparation Wizard and ligand
using LigPrep, both from Schrödinger.49 ClpB2 comparative model has been built using Prime software
from Schrödinger. The heteromultimer model methodology was chosen. For the Nꢀterminal domain we
took advantage of the available crystal structure of PfClpB2 Nꢀterminal portion 4IOD (residues 10ꢀ
156). For the remaining part of the protein we used as templates Tth1QVR and E.coli 4CIU ClpB. Final
alignment is equal to blast alignment reported in Figure S1. The final model was submitted to a long
refinement procedure to reduce the steric clashes. The ionization state of the final model has been
checked with Protein Preparation Wizard. It should be noted that one of the loop regions presents a
quite high degree of uncertainty due to very low similarity between query and template.
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General procedure for the synthesis of compounds 6‒10. Freshly distilled thionyl chloride (2 eq)
and pyridine (0.1 eq) were added to a solution of the suitable cinnamic acid (1 eq) in anhydrous
chlorobenzene (0.5 mL/mmol) and the stirred reaction mixture was heated at 130 °C for 2 hours.
Residual thionyl chloride was distilled and the crude was treated with hexane at reflux to afford the
respective acyl chloride as pale yellow needles that were collected and used for the following reaction
without further purification. Analytical data for compounds 6‒9 matched the data already
published.24,50,51
General procedure for the synthesis of compounds 11‒25. A mixture of the suitable 3ꢀ
chlorobenzothiopheneꢀ2ꢀcarbonyl chloride (1 eq) and the proper amine (1 eq) was refluxed in
anhydrous dioxane until consumption of the reacting agents according to TLC. The reaction mixture is
then poured in ice water and the organic layers were extracted with ethyl acetate (3 × 10 mL), washed
with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The oil obtained was
purified through flash column chromatography eluting with dichloromethane/methanol, to give the title
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compounds. Yields, purification methods and other analytical data are reported in the supporting
information.
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Ethyl benzo[b]thiophene-2-carboxylate (28). To a solution of 2ꢀnitrobenzaldehyde 26 (2.0 g, 3.0
mmol) in anhydrous DMF (20 mL) K2CO3 (2.2 g, 16 mmol) and ethyl thioglycolate (142 µL, 13 mmol)
were added at 0 °C. The reaction mixture was kept at the same temperature for 30 minutes, and then
was allowed to react at room temperature until consumption of the limiting reagent according to the
TLC. The mixture is then poured in ice water and the precipitate obtained is collected, dried and used
in the next reaction step without further purification. Yield 68%.
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Following a similar procedure, but using 1ꢀ(2ꢀfluorophenyl)propanꢀ1ꢀone 27 in place of 2ꢀ
nitrobenzaldehyde, ethyl 3ꢀethylbenzo[b]thiopheneꢀ2ꢀcarboxylate (29) was prepared in 80% yield.
Analytical data for compounds 28, 29 matched the data already published.52,53
Ethyl benzofuran-2-carboxylate (32). To a solution of salicylaldehyde 30 (857 µL, 8.2 mmol) in
anhydrous DMF (15 mL), ethyl bromoacetate (907 µL, 2.4 mmol) and K2CO3 (2.21 g, 160 mmol) were
added, and the mixture was stirred at 130 °C until consumption of the limiting reagent according to the
TLC. The reaction mixture is then poured in ice water and the organic layers were extracted with ethyl
acetate (3 × 10 mL), washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The oil obtained was purified through flash column chromatography eluting with
dichloromethane/methanol (99:1), to give the title compound as white solid in 70% yield. Analytical
data for compound 32 matched the data already published.54
Ethyl 3-hydroxybenzo[b]thiophene-2-carboxylate (33). Methyl 2ꢀmercaptobenzoate 31 (1.5 g,
8.92 mmol), ethyl bromoacetate (789 µL, 8.92 mmol) and sodium tertꢀbutoxide (2.68 g, 17.83 mmol)
were dissolved in anhydrous THF and reacted at room temperature for 2 hours. After completion, the
reaction mixture is poured in ice water and the organic layers were extracted with ethyl acetate (3 × 10
mL), washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The
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crude obtained was purified through flash column chromatography eluting with petroleum ether/ethyl
acetate (9:1), to give the title compound as a yellowish solid in 91% yield. Analytical data for
compounds 33 matched the data already published.55
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General procedure for the alkylation of compound 33. To a suspension of NaH (2 eq) in
anhydrous DMF, compound 33 (1 eq) is added at room temperature under nitrogen atmosphere. After
10 minutes the suitable alkyl halide (2 eq), solubilized in anhydrous DMF, is added dropwise and the
reaction mixture is stirred at 60 °C for a period spanning from 1 to 3 hours. After consumption of the
limiting reagent monitored through TLC, the mixture is poured in ice water and extracted with ethyl
acetate (3 × 5 mL), and the organic layers are washed with brine, dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The crude is purified through flash column chromatography eluting
with petroleum ether/ethyl acetate (95:5), to give compounds 34‒38 in good overall yields. Yields,
purification methods and other analytical data are reported in the supporting information. Analytical
data for compound 35 matched the data already published.56
Methyl 3-(benzylamino)benzo[b]thiophene-2-carboxylate (41). Ethyl 3ꢀaminobenzo[b]thiopheneꢀ
2ꢀcarboxylate 40 (30 mg, 0.135 mmol), benzyl bromide (7.7 mg, 0.045 mmol), K2CO3 (37 mg, 0.270
mmol) and KI (1 mg, 0.045 mmol) were dissolved in acetonitrile (3mL), put in a microwave flask and
heated in a microwave oven at 120 °C for 10 minutes for two times, then at 140 °C for 10 minutes for
once. After quenching with brine (5 mL), the mixture was extracted with ethyl acetate (3 × 5 mL), and
the combined organic layers were dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The crude material was then purified through flash chromatography eluting with petroleum
1
ether/ethyl acetate 95:5 to give the title compound 41 as a yellow solid in 83% yield. H NMR (400
MHz, CDCl3): δ 3.88 (s, 3H); 4.95 (s, 2H); 7.21ꢀ7.43 (m, 8H); 7.72 (s, 1H); 7.84 (bs, 1H); 8.00 (s, 1H).
LRMS (ESI) calcd for C17H15NO2S ([MꢀH]ꢀ) 296.08; found 296.3.
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General procedure for the ester hydrolysis: synthesis of compounds 42‒53. Esters 28, 29, 32‒41
(1 eq) and LiOH·H2O (4 eq) were dissolved in a solution of THF/MeOH/H2O (3/1/1, 1 ml/mmol) and
stirred overnight at room temperature. The reaction mixture is then evaporated in vacuo, and the crude
is taken up with H2O, acidified with HCl 1N and extracted with ethyl acetate, that is on turn washed
with brine and dried over anhydrous Na2SO4. After the evaporation of the solvent the crude materials
were used in the next reaction step without any additional purification. Yields and essential analytical
data are reported in the supporting information. Analytical data for compounds 42‒46, 50‒53 matched
the data already published.56–63
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General procedure for the synthesis of amides 54‒71, 73, 75. To a solution of the appropriate
carboxylic acid (1 equiv) in anhydrous dichloromethane or dimethylformamide (DMF) (4 mL/mmol) at
room temperature were added anhydrous hydroxybenzotriazole (HOBt, 1 equiv) and 1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 1 equiv) under nitrogen atmosphere.
After stirring for 10 min, the appropriate substituted amine (1 equiv) and triethylamine (3 equiv) were
added, and the reaction mixture was stirred at room temperature until disappearance of the starting
material (usually 2 to 6 hours). After this time water (5 mL) was added, and the mixture was extracted
with EtOAc (3 × 10 mL), the organic layers were separated, washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash
chromatography eluting petroleum ether/ethyl acetate (8:2) to obtain the title compounds in yields
ranging from 13 to 73%.
3-(Methoxymethoxy)benzo[b]thiophene-2-carboxylic acid (74). To a solution of 33 (0.15 g, 0.63
mmol) in anhydrous dichloromethane (5 mL), chloromethyl methyl ether (72 µL, 0.95 mmol) is added
dropwise through a dropping funnel at room temperature. After cooling at °C, DIPEA (165 µL, 0.95
mmol) is added, and the reaction mixture is allowed to stir at room temperature until consumption of
the starting material (2 hours). The reaction is quenched with NH4Cl, and the organic layers washed
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with water (3 × 5 mL), treated with brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The oil obtained, without further purification, was hydrolysed according to the
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procedure above reported to give the acid 74 as a white powder in 86% overall yield. H NMR (300
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MHz, CDCl3): δ 3.68 (s, 3H); 5.49 (s, 2H); 7.44ꢀ7.56 (m, 2H); 7.81 (d, J = 8 Hz, 1H); 7.98 (d, J = 8 Hz,
1H). LRMS (ESI) calcd for C11H10O4S ([MꢀH]ꢀ) 237.03; found 237.3.
(2,6-Dimethylmorpholino)(3-hydroxybenzo[b]thiophen-2-yl)methanone (76). To a solution of
compound 75 (0.028 g, 0.083 mmol) in anhydrous methanol, HCl 6N is added over a 20 minutes
period. After reacting for 1 hour at room temperature, the solvent is evaporated and the residue taken
up with saturated NaHCO3 aq. solution (8 mL), extracted with ethyl acetate (3 × 5 mL) and the organic
layers were separated, washed with brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chromatography eluting petroleum ether/ethyl
1
acetate (8:2) to obtain the title compounds in 82% yield as a white solid. H NMR (300 MHz, CDCl3):
δ 1.12ꢀ1.30 (m, 6H); 2.70ꢀ2.82 (m, 2H); 3.60ꢀ3.75 (m, 2H); 4.52ꢀ4.65 (m, 2H); 7.35ꢀ7.50 (m, 2H); 7.82
(d, J = 6 Hz, 1H); 7.99 (d, J = 8 Hz, 1H). δ 17.8, 38.8, 71.6, 122.4, 122.9, 125.7, 128.5, 132.6, 135.9,
138.4, 141.2, 162.0. HRMS (ESI) calcd for C15H17NO3S ([M+H]+) 292.0929; found 292.1002.
Supporting Information Available: A Brief description of the biological assays, some of the
1
synthetic procedures, the HꢀNMR spectra of the intermediates and title compounds and the methods
used for the target fishing experiments are available as supporting information. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Marco Pieroni +39 0521 905054; Eꢀmail marco.pieroni@unipr.it
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript. # These authors contributed equally.
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Acknowledgment
Benedetta Culiersi and Elisa Sagoe are kindly acknowledged for the contribution in the synthesis of
derivatives. The Centro Interdipartimentale Misure ‘‘G. Casnati’’ is kindly acknowledged for the
contribution in the analytical determination of the molecules synthesized.
Abbreviations a
ACT, Artemisininꢀbased combination therapy; CK, choline kinase; CQ, chloroquine; DMF, N,Nꢀ
dimethyl formamide ; EDC, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide; EDG, electronꢀdonor
group; EWG, electronꢀwithdrawing group; MOM, methoxyꢀmethyl ; PDB, protein data bank; SAR,
StructureꢀActivity
Relationships;
TBTU, 2ꢀ(1HꢀBenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium
tetrafluoroborate; THF, tetrahydrofuran.
Keywords
Malaria; Open Innovation; Drug Discovery; Benzo[b]thiophenes; Target Fishing.
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