Design and Synthesis of 1-((1,5-Bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)-4-methylpiperazine (BM212) and N-Adamantan-2-yl-N′-((E)-3,7-dimethylocta-2,6-dienyl)ethane-1,2-diamine (SQ109) Pyrrole Hybrid Derivatives: Discovery of Potent Antitubercular Agents Effective against Multidrug-Resistant Mycobacteria

Article abstract of DOI:10.1021/acs.jmedchem.6b00031

Novel pyrroles have been designed, synthesized, and evaluated against mycobacterial strains. The pyrroles have originally been designed as hybrids of the antitubercular drugs BM212 (1) and SQ109 (2), which showed common chemical features with very similar topological distribution. A perfect superposition of the structures of 1 and 2 revealed by computational studies suggested the introduction of bulky substituents at the terminal portion of the pyrrole C3 side chain and the removal of the C5 aryl moiety. Five compounds showed high activity toward Mycobacterium tuberculosis, while 9b and 9c were highly active also against multidrug-resistant clinical isolates. Compound 9c showed low eukaryotic cell toxicity, turning out to be an excellent lead candidate for preclinical trials. In addition, four compounds showed potent inhibition (comparable to that of verapamil) toward the whole-cell drug efflux pump activity of mycobacteria, thus turning out to be promising multidrug-resistance-reversing agents.

Full text of DOI:10.1021/acs.jmedchem.6b00031

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Article
Design and synthesis of 1-((1,5-bis(4-chlorophenyl)-2-methyl-1H-
pyrrol-3-yl)methyl)-4-methylpiperazine (BM212) and N-Adamantan-2-
yl-N'-((E)-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine (SQ109)
pyrrole hybrid derivatives: discovery of potent anti-tubercular
agents effective against multi-drug resistant mycobacteria
Sanjib Bhakta, Nicolò Scalacci, Arundhati Maitra, Alistair K. Brown, Saiprasad
Dasugari, Dimitrios Evangelopoulos, Timothy D. McHugh, Parisa N. Mortazavi,
Alexander Twist, Elena Petricci, Fabrizio Manetti, and Daniele Castagnolo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00031 • Publication Date (Web): 23 Feb 2016
Downloaded from http://pubs.acs.org on February 25, 2016
Just Accepted
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

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Design and synthesis of 1ꢀ((1,5ꢀbis(4ꢀchlorophenyl)ꢀ2ꢀmethylꢀ1Hꢀpyrrolꢀ3ꢀyl)methyl)ꢀ4ꢀ
methylpiperazine (BM212) and NꢀAdamantanꢀ2ꢀylꢀN'ꢀ((E)ꢀ3,7ꢀdimethylꢀoctaꢀ2,6ꢀdienyl)ꢀ
ethaneꢀ1,2ꢀdiamine (SQ109) pyrrole hybrid derivatives: discovery of potent antiꢀtubercular
agents effective against multiꢀdrug resistant mycobacteria
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a,¶
b,e
a
f,b
Sanjib Bhakta,
Nicolò Scalacci, Arundhati Maitra, Alistair K. Brown, Saiprasad
b
c,‡
c
a
Dasugari, Dimitrios Evangelopoulos, Timothy D. McHugh, Parisa N. Mortazavi, Alexander
b
d
d,¶,*
e, b, ¶,*
Twist, Elena Petricci, Fabrizio Manetti,
and Daniele Castagnolo.
a
Mycobacteria Research Laboratory, Department of Biological Sciences, Institute of Structural
and Molecular Biology, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK.
b
Department of Applied Sciences, Northumbria University Newcastle, Ellison Place, NE1 8ST,
c
Newcastle upon Tyne, UK. Centre for Clinical Microbiology, University College London,
d
London, NW3 2PF, UK. Dipartimento di Biotecnologie, Chimica e Farmacia, via A. Moro 2, Iꢀ
e
5
3100 Siena, Italy. Institute of Pharmaceutical Science, King’s College London, 150 Stamford
f
Street, London SE1 9NH, UK. School of Medicine, Pharmacy and Health, Durham University,
Queen’s Campus, StocktonꢀonꢀTees, TS17 6BH, UK.
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KEYWORDS. Tuberculosis, MDRꢀTB, Antimicrobial resistance, BM212, SQ109, efflux pump,
pyrrole, HTꢀSPOTi, common feature hypothesis generation approach
ABSTRACT. Novel pyrroles have been designed, synthesized and evaluated against
mycobacterial strains. The pyrroles have originally been designed as hybrids of the antiꢀ
tubercular drugs BM212 1 and SQ109 2 that showed common chemical features with very
similar topological distribution. A perfect superposition of the structures of 1 and 2 revealed by
computational studies suggested the introduction of bulky substituents at the terminal portion of
the pyrrole C3 side chain and the removal of the C5 aryl moiety. Five compounds showed high
activity toward Mycobacterium tuberculosis, while 9b and 9c were highly active also against
multiꢀdrug resistant clinical isolates. Compound 9c showed low eukaryotic cell toxicity, turning
out to be an excellent lead candidate for preclinical trials. In addition, four compounds showed
potent inhibition (comparable to that of verapamil) towards the wholeꢀcell drug efflux pump
activity of mycobacteria, and thus turning to be promising multiꢀdrug resistance reversal agents.
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INTRODUCTION
Tuberculosis (TB) was first declared as a global health emergency by the World Health
Organisation (WHO) in 1993. More than two decades on, the world is still grappling to control
the spread of this infectious disease. In 2014, around 9.3 million people were estimated to have
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1
fallen ill with TB and 1.5 million deaths were caused by it. Although, mortality due to TB has
fallen to 47% since 1990, the increasing resistance seen in the TBꢀcausative organism,
Mycobacterium tuberculosis (MTB), is responsible for new 480,000 estimated cases of
multidrugꢀresistant TB. In fact, multiꢀ, extensivelyꢀ and even totallyꢀdrug resistant tuberculosis
2ꢀ4
5ꢀ6
7
(
MDRꢀ, XDRꢀ, TDRꢀTB ) cases make the disease, originally difficult to treat, even more so
as most drugs in use today are ineffective to treat these mutant strains. Moreover, two billion
people are estimated to be latently infected with MTB, and 10% of them reactivate to active TB
with major risk relative to immigrants from endemic areas, people with HIVꢀ1 infection, and
individuals with underlying diseases (e.g. silicosis, diabetes mellitus and malignant conditions).
Treatment, if available, is lengthy, severely toxic and prone to interfere with other therapies such
as the antiretroviral therapy in the case of TBꢀHIV coꢀinfected patients.
The need for new shorter therapeutic regimens and new classes of drugs active against MDRꢀ,
XDRꢀ, and TDRꢀTB drives pharmaceutical research to accelerate in the development process of
new antiꢀTB drugs. There is an urgent need to develop novel antiꢀtubercular agents with
alternative mechanisms of action to control TB, one of the leading causes of mortality due to a
single infectious agent.
8
About fifteen years ago, the pyrrole compound 1 (BM212) was discovered to be a potent antiꢀ
mycobacterial agent. In an effort to identify new pyrrole analogues with improved solubility and
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ability to kill MDR and XDR mycobacteria, many derivatives of 1 were designed and
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ꢀ10
synthesized.
the first series of analogues (including 1 itself) suffered from poor pharmacokinetic parameters
in terms of clearance and microsomal stability) and significant cytotoxicity. Although the most
recent derivatives showed an improved pharmacokinetics and cytotoxicity profile, their
Congeners of 1 showed a micromolar activity toward MTB H37Rv. However,
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physicochemical parameters (such as lipophilicity and aromaticity) need further optimization.
Genetic analyses of spontaneous mutants resistant to 1 led to the identification of its cellular
target as the trehalose monomycolate exporter, MmpL3 protein (Rv0206c), member of MmpL
12
(
Mycobacterial membrane protein Large) family and a validated drug target in MTB. This
family has primary structure homology to the resistanceꢀnodulationꢀcell division (RND) protein
1
1
family, mainly involved in drug resistance in Gramꢀnegative bacteria. Although the MTB
genome encodes 13 members of the MmpL family, their function has not been clearly elucidated.
Despite their annotation as multiꢀdrug transporters, they do not contribute to antiꢀmycobacterial
1
3
drug resistance. MmpL3 has also been implicated in heme acquisition by MTB, although it
might not be its primary role in an endogenous environment.
1
4
Adamantaneꢀdiamine compound 2 (SQ109) was described as an antiꢀtubercular drug which is
active against susceptible and drug resistant MTB strains. Compound 2 exhibited promising
1
5
activity in drug combination during animal preclinical studies but also experienced poor
1
6
pharmacokinetic parameters. Diamine 2 is currently undergoing phase 2 clinical trials for
17
further development. Antiꢀmycobacterial activity of both 1 and 2 was demonstrated to derive
1
2
from targeting the MmpL3 protein. Given the ability of 1 and 2 to act as antiꢀmycobacterial
agents through a common molecular target, we pursued the hypothesis that they could share
common molecular portions.
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By means of an in silico molecular modelling approach, the threeꢀdimensional structure of both
compounds was studied. Although the apparent structural dissimilarity between the two
compounds, a rough comparison of the molecular structures of 1 and 2 showed an unexpected
similarity of the topological distribution of their chemical features (Figure 1). In fact, apart from
the terminal adamantyl moiety of 2 and the pꢀCl phenyl ring at the C5 position of 1, the
remaining part of the structure of 2 was superposable to that of 1.
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On the basis of this similarity, we chose to apply a molecular hybridization approach to design
new putative antiꢀmycobacterial compounds.
The molecular hybridization approach is one of the strategies included within the rational design
protocol for identification of new biologically relevant small molecules. This approach is based
on the recognition of structurally comparable or similar molecular portions of two or more
bioactive compounds. By means of merging of these molecular portions, new hybrid chemical
entities that maintain structural elements of the parent compounds could be designed. In this
study, we have chosen the more rigid scaffold of 1 as the template, and to adjust it upon
comparison with the flexible structure of 2. As a result, the Nꢀ(substituted)phenyl pyrrole core of
1 with a piperazinylꢀmethyl side chain at position C3 was maintained, while the second phenyl
substituent at position C5 of 1 was deleted from the new hybrid compounds. In addition, the
adamantyl moiety present in 2 was inserted as the terminal group bound to N4 of the piperazine
ring leading to the design of the hybrid compound A (Figure 1).
Hence, we designed, synthesised and biologically evaluated a set of novel pyrrole derivatives
with general structure A that are hybrid compounds of the antiꢀtubercular agents 1 and 2. To
explore the chemical space around the C3 piperazinoꢀmethyl moiety of the new compounds, the
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distal piperazine nitrogen is substituted with different bulky R moieties, such as adamantyl,
norbornyl, cyclohexyl, or aryl groups. Moreover the role of the substituent on C5 and its
importance for the antiꢀtubercular activity was explored. According to our superposition
hypothesis, the aryl moiety at C5 of 1 was replaced by a methyl substituent, thus leading to
design a library of 2,5ꢀdimethylpyrroles. In addition, a second series of simplified derivatives B
was also designed through molecular simplification in order to evaluate the influence of the
piperazine ring on the antiꢀtubercular activity. Within this series, also according to previous
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works,
the piperazine ring was removed from the structure and replaced with a secondary
amine (or hydroxylamine) at C3. In both cases, the methyl substituent at C2 and C5, as well as
the N1 aryl groups were left unchanged.
RESULTS
Computational design and hypothesis on the common chemical features shared by 1 and
2
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2: To confirm the superposition pattern above described for 1 and 2, the Phase software was
used to find the threeꢀdimensional arrangements of the common chemical portions shared by the
two compounds (thereafter referred to as common feature models or superposition models). As a
result, a fourꢀfeature representation was obtained, comprised of two hydrophobic groups and two
protonable atoms (Figure 2). In detail, the pꢀCl group at the N1 position of the pyrrole ring of 1
and one of the terminal methyl group of 2 matched one of the common hydrophobic regions
(H6). The central methyl group of 2 and the 2ꢀmethyl group of 1 corresponded to the second
hydrophobic portion (H4). On the other hand, the two positively ionizable features (P7 and P8)
accommodated the two basic nitrogen atoms of both compounds.
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It is important to point out that this representation of the common chemical features of 1 and 2
showed two protonable sites that are chemically unrealistic at neutral pH. However, this
qualitative model only accounts for the presence of the two piperazine nitrogens of 1 and for the
two amine nitrogens of 2, while it does not take into consideration the mutual influence of the
two amines during the protonation steps. This limitation was eliminated in a second generation,
optimized common feature model that is characterized by only one protonable group (Figure 3).
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Analysis of the superposition pattern of 1 and 2 prompted us to omit the aromatic portion at
position 5 of 1. In fact, a visual inspection of the superposed structures of 1 and 2 showed that
this moiety does not match an analogous group on 2 (Figure 2), and, consequently, it does not
represent a common feature of both compounds. Moreover, since the three aromatic rings forced
the structure of these compounds toward planarity, the removal of one or more of them could be
in principle profitable for better solubility and bioavailability. In addition, given the wellꢀ
established relationship between late stage drug development problems (i.e., the high attrition
2
1ꢀ22
rate of compounds entering the clinical phase) and lipophilicity of compounds,
modulation of
lipophilicity should be taken into account when new putative antiꢀmycobacterial compounds are
designed. In addition, since the terminal methyl group of 1 is partially superposed to the
adamantyl moiety of 2, a hydrophobic group at this position was maintained. Following these
suggestions and taking into account the similarity between 1 and 2, a new class of pyrrole
derivatives was designed on the basis of the molecular hybridization routine with the aim of
obtaining new putative antiꢀmycobacterial compounds.
The rigid scaffold of 1 was thus chosen as the template for the design of novel antitubercular
compounds, and this choice was mainly based on the fact that our research group is well
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experienced in the synthesis of pyrrole compounds. Moreover, the adamantyl group already
found in 2 was introduced on the piperazine ring, while the 5ꢀphenyl ring of 1 was removed.
Chemistry: Synthesis of derivatives with general structure A. A library of pyrrole derivatives
bearing an Nꢀsubstituted piperazinoꢀmethyl chain at C3 was first synthesized according to
classical synthetic procedures. Pyrroles 3aꢀe were synthesized through a microwaveꢀassisted
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PaalꢀKnorr reaction starting from the appropriate diones and different anilines.
Despite
computational studies suggested to keep a pꢀCl moiety fixed on the N1 aryl group, a number of
different substituents was also introduced to evaluate their steric and electronic effects on the
antiꢀmycobacterial activity and to deduce SAR considerations. Pyrroles 3aꢀe were then coupled
with differently substituted piperazines in the presence of formaldehyde to afford desired
pyrroles 7aꢀm (Table 1). Different bulky substituents were introduced on the piperazine ring to
explore the chemical space around the pyrrole nucleus. The piperazines 6cꢀe (cyclohexylꢀ
piperazine 6c, phenylꢀpiperazine 6d, 1ꢀadamantylꢀpiperazine 6e) are commercially available and
were coupled with 3aꢀe as purchased. The piperazines 6aꢀb were synthesized as described in
Scheme 1 and then reacted with the pyrroles 3aꢀe. The Bocꢀpiperazine 4 and the appropriate
ketones 5aꢀb (2ꢀadamantanone 5a, 2ꢀnorbornanone 5b) were reacted in the presence of the
reducing agent Na(OAc) BH to yield the corresponding alkylpiperazine intermediates, which
3
were immediately converted into the desired piperazines 6aꢀb by TFAꢀmediated Bocꢀcleavage.
Piperazines 6aꢀe and pyrroles 3aꢀe were then coupled via Mannich reaction to afford pyrroles
7aꢀm (Table 1).
Synthesis of derivative with general structure B. A second series of pyrrole derivatives with
general structure B was synthesised as described in Scheme 2. Pyrrole 3aꢀc were formylated
through VilsmeirꢀHaack reaction affording the aldehydes 8aꢀc. The latter compounds were
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reacted with different primary amines in the presence of Na(AcO) BH to yield pyrroles 9aꢀj.
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Moreover, aldehydes 8aꢀb were reacted with benzylhydroxylamine to afford oximes 10aꢀb
which, after treatment with NaCNBH , led to the corresponding hydroxylamines 11aꢀb in
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excellent yields.
Biology: All the compounds were tested for their biological activity by determining the
minimum inhibitory concentrations (MIC) against a panel of fastꢀgrowing environmental and
slowꢀgrowing mycobacterial species and clinical isolates (Table 3 and 4). This included the fastꢀ
growing nonꢀpathogenic strains of M. smegmatis and relatively fastꢀgrowing intracellularly
25
surviving M. aurum followed by the vaccine strain M. bovis BCG, the nonꢀpathogenic M.
2
tuberculosis mc 7000, and finally the pathogenic M. tuberculosis H37Rv. Furthermore, we also
tested the activity of these compounds against MDRꢀTB clinical isolates. In addition,
cytotoxicity analysis was carried out. The eukaryotic cell toxicity of each compound was tested
against murine macrophage RAW264.7 cells and human monocyteꢀderived THPꢀ1 cell line to
ascertain the 50% growth inhibitory concentration (GIC ). Mouse models are routinely used for
5
0
testing the efficacy of both antiꢀtubercular drugs and vaccines and are considered a highly
appropriate model. In particular, RAW264.7 are differentiated macrophage cells capable of
eliciting a whole host of inflammatory responses and been studied and characterised in great
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detail over many decades. The ratio of the MIC observed against MTB H37Rv and the GIC
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values provided the selectivity index (SI), or the therapeutic window offered by these molecules.
It must to be brought to note that there are differences in the assay methods to determine the
inhibitory concentrations of the compounds against the bacterial pathogen and the eukaryotic
cells. However, these methods have been extensively standardised so as to reduce ambiguity in
inferring the selectivity index from these assays. Finally, the effect of these compounds on the
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efflux pump activity of the model surrogate organism M. aurum was also tested (Table 5).
Efflux pumps are transport proteins involved in the extrusion of toxic substrates, including
virtually all classes of antiꢀmycobacterial drugs, from within cells into the external environment.
Pumps may be specific for one substrate or may transport a range of structurally dissimilar
compounds and can be associated with MDR in MTB. Thus, the identification of efflux pump
inhibitors can lead to new antiꢀtubercular agents able to reverse drug resistance in TB and to be
used in combination therapy together with first line drugs.
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DISCUSSION
We first analysed the data arising from the screening on nonꢀpathogenic M. tuberculosis
2
mc 7000 (Table 4). In agreement with our initial hypothesis, the insertion of a 1ꢀ and a 2ꢀ
adamantylꢀpiperazine group in place of the Nꢀmethylpiperazine of 1 led to 7l and 7k with
significantly contrasting activity. In fact, 7k, bearing the same 2ꢀadamantyl group found in 2,
was inactive (MIC value > 64 ꢀg/mL). On the contrary, the corresponding 1ꢀadamantyl analogue
2
7
l showed a strong ability to inhibit MTB mc 7000 growth, with a MIC value of about 0.5
ꢀ
g/mL. Compound 7l was then chosen as the most representative compound to be further studied
2
for deducing SAR considerations on the new class of hybrid compounds on MTB mc 7000. In
particular, according to the suggestions derived from the superposition pattern between 1 and 2,
the pꢀCl phenyl moiety at C5 of 7l was simplified to a methyl group, leading to 7i with a
significant 16ꢀfold drop in activity (8.0 ꢀg/mL). The corresponding 2ꢀadamantyl analogue 7a
showed a slight increase in activity (3.3 ꢀg/mL). Moreover, small changes in substituents and
substitution pattern at its 1ꢀphenyl ring clearly showed that small substituents (isopropyl and
fluorine) at para and ortho positions (such as in 7c and 7b) guaranteed activity retention or
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enhancement (3.3 and 1.0 ꢀg/mL, respectively). Differently, a mꢀmethyl group as in 7d caused a
significant drop in activity (32 ꢀg/mL), in agreement with that previously found for derivatives
8
of 1. Decreasing the bulkiness of the hydrophobic moiety on the piperazine ring from an
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adamantyl (such as in 7i or 7a) to a norbornane group led to a slight improvement of activity
(MIC of 7g was 2.0 ꢀg/mL). On the contrary, a further simplification to a cyclohexyl ring (7h)
and its aromatization to a phenyl ring (7e) further reduced activity to 8.0 and 32 ꢀg/mL,
respectively. Introduction of a second pꢀCl phenyl moiety at C5 of 7e restored a 1.0 ꢀg/mL
activity in 7j. SAR considerations on both 7j and 7e (1.0 and 32 ꢀg/mL, respectively) suggested
that in several cases the presence of the pꢀCl phenyl moiety at C5 could improve antiꢀ
mycobacterial activity. On the other hand, a comparison between activity of 7e and its adamantyl
analogues 7i and 7a (8.0 and 3.3 ꢀg/mL, respectively) clearly showed that the pꢀCl phenyl
moiety at C5 was not mandatory for obtaining active compounds, as suggested by the common
feature model. Also the comparison of the biological profiles of the norbornyl derivatives 7g and
7m clearly shows that the pꢀCl phenyl moiety at C5 in 7m is detrimental for the antiꢀtubercular
activity. Moreover, the comparable antiꢀmycobacterial activities of 7j and 7l (1.0 and 0.5 ꢀg/mL,
respectively) allowed us to hypothesize that the distal nitrogen atom of the piperazine ring (that
is protonatable in 7l, while has an anilino character in 7j) could be unimportant for activity. This
result was in agreement with previous pharmacophoricꢀbased calculations and in vitro activity of
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piperidine analogues of 1. To validate this hypothesis, compounds with a linear amino spacer
instead of the piperazine ring were synthesized. Among them, 9a had an overall size comparable
to that of 7e and lacked the distal nitrogen atom of the parent piperazine ring. Its activity was 8ꢀ
fold better than that of the piperazine analogue 7e (4.0 and 32 ꢀg/mL, respectively). Attempts to
modify the phenylethylamino side chain of 9a by introduction of an oxygen atom (as in 11a and
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1b) or by partial rigidification into an aryloxime (as in 10a) led to inactive compounds (>64
ꢀg/mL). Alternatively, shortening the phenylethylamino spacer led to very active compounds. As
an example, the benzylamino analogue 9b showed a 0.5 ꢀg/mL MIC value. Decoration of the
para position of the terminal phenyl ring with small substituents (such as a F, Cl, and Me) led to
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g, 9f, and 9e with comparable or slightly lower activity (0.7, 2.0, and 2.0 ꢀg/mL, respectively),
further confirming that the distal nitrogen atom of the piperazine ring was not necessarily
required for antiꢀmycobacterial activity of the new hybrid compounds. On the contrary,
replacement of the pꢀCl of 9b with a pꢀF group as in 9h caused an 8ꢀfold drop in activity (from
0.5 to 4.0 ꢀg/mL). A further reduction of the amino side chain length to a cyclohexylamino and
to the bulky 2ꢀadamantylamino moiety as in 9c and 9d, respectively, also maintained a 0.5 and
1
.0 ꢀg/mL activity. Interestingly, when the methyl group at C5 of compounds 9b and 9c was
replaced with a pꢀCl phenyl moiety leading to derivatives 9i and 9j, a significant drop in activity
was observed (16 and 8 ꢀg/mL respectively). Compounds 9bꢀc proved to be much more active
2
than 1 against MTB mc 7000. These latter data confirm the initial hypothesis that an aryl
substituent at C5, such in 1, is not mandatory to obtain derivatives active against M. tuberculosis.
Also the new compounds bearing a secondary amine show a better activity profile than 2, thus
pointing out that a diamine backbone could be not essential for antitubercular activity.
The same compounds were also assayed on additional mycobacterial strains. Activity against
the pathogenic MTB H37Rv (Table 4) followed the same trend already shown for the MTB
2
mc 7000 strain. The only exceptions were represented by 9f and 9g that were inactive or weakly
active toward MTB H37Rv (16 and >125 ꢀg/mL, respectively), while their congener compounds
9
e and 9b showed a 1.0 and 0.5 ꢀg/mL activity. The hybrid compounds 7aꢀb, 7e and 7h showed
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a good profile with MIC = 7.8 and 3.9 ꢀg/mL. The derivative 7j bearing a pꢀClꢀphenyl
substituent at C5 showed an improved biological profile (1.9 ꢀg/mL). On the other hand, the
simplified compounds 9 showed higher activity against MTB H37Rv than derivatives 7. In
particular, compounds 9b and 9c proved to be very active showing again activity (0.5 and 0.2
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ꢀ
g/mL respectively) comparable or better than 1 and 2.
Moreover, as a general trend, the hybrid compounds showed weak activity toward M.
2
smegmatis mc 155 (MIC values ranging from 4.0 to >64 ꢀg/mL, with only two compounds
showing better activity – 3.3 and 2.0 ꢀg/mL, respectively). Table 3. Similarly, M. aurum was
scarcely sensitive to the test compounds: only four compounds (namely, 9b, 9e, 9f, and 9g)
showed a MIC value of about 2.0 ꢀg/mL (Table 3). As the whole genome sequence of M. aurum
has been published recently, comparative genomic analyses of these transporter proteins should
25
elucidate why the difference in their drug susceptibilities is observed. In general, fast growing
mycobacteria (M. aurum and M. smegmatis) were inhibited at higher concentration than other
strains. A better antiꢀmycobacterial activity profile was found toward M. bovis BCG (Table 3),
with ten compounds showing activity value in the range below 4.0 ꢀg/mL and three compounds
(namely, 9b, 9c, and 9g) with a MIC value of 0.5 ꢀg/mL. Among MDRꢀTB strains (Table 4),
MDR2 was poorly sensitive to the test compounds, with the only exception of 9a and 9b which
showed a good activity at 7.8 ꢀg/mL and 3.9 ꢀg/mL. On the other hand, MDR1 growth was
blocked by at least 7 compounds with MIC values lower than 4.0 ꢀg/mL. Of particular interest
were compounds 9b and 9c that showed MIC values of about 1.0 and 0.5 ꢀg/mL, respectively,
much better than that of the parent compound 1 and isoniazid.
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An analysis of antiꢀmycobacterial data clearly showed that the best compounds (9a, 9b, and
c) were characterized by structural features significantly different from those of the parent
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compound 1. In particular, they lacked both the second aryl moiety at the position C5 of the
pyrrole nucleus and the distal nitrogen atom of the original piperazine ring. Moreover, the overall
activity underwent an improvement when the basic linear side chain was shortened from the
phenylethylamino of 9a, to the benzylamino of 9b, to the cyclohexylamino of 9c.
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The C5 aryl moiety was not a mandatory substituent for active compounds, and small groups
are allowed as decoration of the N1ꢀphenyl ring. A bulky terminal group, such as an adamantane
and a norbornane, improve the activity of piperazine analogues, while a significant gain of antiꢀ
mycobacterial activity resulted from piperazine replacement by linear aryl and alkylamino side
chains.
The high antiꢀmycobacterial activity maintained even after a significant structural
simplification of 1,5ꢀdiarylpyrroles (derivatives and analogues of 1) into the corresponding 1ꢀ
aryl hybrid analogues could be accounted by a comparison of the original pharmacophoric model
8
for antiꢀmycobacterial compounds belonging to the pyrrole class of 1 with the simplified
8
common feature model described here. In fact, the original pharmacophoric model was able to
accommodate 1 and its derivatives in two different orientations, rotated of about 180 degrees
around the pyrrole plane. Following this, the aryl moieties at positions N1 and C5 of 1 could
reciprocally change their spatial location and, consequently, match with the pharmacophore.
These results suggested that the pharmacophoric portions able to accommodate the substituents
at positions N1 and C5 are redundant and could be simplified, as also suggested by the
superposition between 1 and 2.
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In a further attempt to codify the structural features of the new hybrid pyrroles, a second
generation common feature model was built starting from a larger set of compounds, comprised
of 1, 2, and the most active pyrroles (with MIC values ≤ 1.0 ꢀg/mL). The improved model
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(
Figure 3) showed that the distal nitrogen of the piperazine ring of 1 (corresponding to P8 in the
previous common feature model) was omitted, while an additional hydrophobic region (H2),
adjacent to the P7 feature (a positively ionizable feature), accommodated the terminal
hydrophobic groups of the C3 side chain.
In order to prove the effectiveness of our compounds, the cytotoxicity of the novel pyrroles
was then evaluated on RAW264.7 cells as well as THPꢀ1 cell lines. Compounds 7j, 9c and 9h
showed an excellent cytotoxicity profile, with a selectivity index (SI) of 92, 50 and 5.6 against
RAW264.7 and 155, 37 and 28 against THPꢀ1 cells respectively. Both 7j and 9c showed a better
cytotoxicity profile than 1 against the murine and human cells (Table 4).
Finally, the effect of pyrroles on the efflux pump inhibitory (EPI) activity of the model
surrogate organism M. aurum was tested in order to identify compounds able to reverse multiꢀ
drug resistance in TB (Table 5, Figure 4). An efflux pump activity whole cellꢀbased assay has
been carried out and, as a consequence, the results account for the total activity of the whole
population of efflux pumps present in the cells. The efflux pump assay revealed that most of
pyrrole compounds (barring 7k) possess at least low to moderate inhibitory property. However, a
weak correlation between inhibition of bacterial growth and efflux pump mechanism is observed.
For example, 9aꢀc are very efficient in killing mycobacterial cells but only display moderate
modulatory effect on the pumps. On the other hand, 11b inhibits efflux inhibition at par with the
control inhibitor Verapamil, but shows almost no effect on the growth of these cells.
Interestingly, 7c, 7h, 7i, and 7l, bearing a bulky alkyl substituent on the piperazine ring, showed
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good/excellent inhibitory activity. These findings confer that specific endogenous targets for
these compounds are still elusive, however indicate that they may have a pleiotropic modes of
action and potential to reverse antiꢀmicrobial resistance. As TB treatment regimens always
comprise of a combination of complimentary drugs, this offꢀtarget effect of the compounds could
have a positive impact on the effectiveness of drug treatment regimens. Within this context it is
noteworthy that five compounds (11b, 7c, 7h, 7i, 7l) show a much higher efflux pump inhibitory
activity than 1 and 2 (Table 5), who are known inhibitors solely of the pump transporter MmpL3.
In fact, the less potent mycobacterial growth inhibitors showing higher wholeꢀcell efflux
inhibitory properties may also prove prospective leads in a multiꢀdrug therapy owing to
synergistic combinations should that arise. Thus, unlike 1 and 2 which show poor EPI activity,
11b and 7b could be used in combination with standard antitubercular drugs such isoniazid or
rifampin to reverse multiꢀdrug resistance in tuberculosis.
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CONCLUSIONS
In conclusion, a library of pyrrole derivatives was successfully synthesized and evaluated for
their antiꢀtubercular activity against MTB and MDRꢀTB clinical isolates. The new pyrroles were
designed as hybrids of the antiꢀmycobacterial agents 1 and 2. Five compounds showed antiꢀ
tubercular activity on MTB at ≤1.0 µg/mL, and two of them (9b and 9c) proved to be highly
active also against MDRꢀ1 TB strains. SAR studies revealed the key features essential for the
antiꢀmycobacterial activity. Finally, the potent antiꢀtubercular derivative 9c showed low
eukaryotic cell toxicity, turning out to be an excellent lead candidate for preclinical trials. In
general, compound 9c showed a better drug profile than 1 in terms of activity, cytotoxicity and
potency toward MDRꢀTB clinical isolates. The pharmacokinetics parameters of 9b and 9c will
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be evaluated in due course and compared with those of 1 in order to prove the efficacy of the
new compounds and to progress toward preclinical trials.
EXPERIMENTAL SECTION
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Computational details
2
8
The structures of all the molecules were sketched using Maestro and then subjected to
LigPrep program to generate highꢀquality, allꢀatom 3D structures to be used as the input for next
calculations. The OPLS_2005 force field was applied, and possible ionization states were
generated for the structures at pH 7±2 using Epik.
2
9
Conformers were generated by MacroModel with the Systematic Pseudo Monte Carlo (a
systematic torsional sampling protocol) search algorithm, OPLSꢀ2005 force field with implicit
GB/SA distanceꢀdependent dielectric solvent model (water as the solvent) at cutoff root mean
square deviation of 0.5 with 1000 steps. All the conformers were subsequently minimized using
PolakꢀRibiere Conjugate Gradient minimization with 5000 iteractions. For each molecule, a
conformer set with a maximum energy difference of 25 kcal/mol relative to the global energy
minimum conformer was stored.
Both 1 and 2 were used to build threeꢀdimensional common feature models comprised of
chemical features common to the two compounds. Considering the reduced number of
compounds and the fact that their antiꢀmycobacterial activity was assayed by different research
groups with different tests, we chose to apply a common feature hypothesis generation routine,
2
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instead of building a quantitative model. Phase software has been applied to generate the
models. Minimum and maximum number of sites for all the features were set to 4 and 5,
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respectively. Only the positively ionizable group (P) and the hydrophobic region (H) features
were used to build a series of hypotheses, while the aromatic ring (R) feature was not further
considered by the software because it was not present in the structure of 2. The resulting fourꢀ
feature hypotheses were constituted by two H and two P features.
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Biology experimental part
Bacterial strains and growth conditions: The bacterial species used in this study were M.
2
smegmatis mc 155 (ATCC 700084), M. aurum (ATCC23366), M. bovis BCG Pasteur (ATCC
2
30ꢀ32
35734), M. tuberculosis mc 7000,
M. tuberculosis H37Rv (ATTC27294), and two MDRꢀTB
clinical isolates (MDR1 and MDR2) obtained from Royal Free Hospital NHS Trust, London,
UK. The cell line utilised for cytotoxicity studies were the murine macrophage cell line
RAW264.7 (ATCC TIBꢀ71) and the human peripheral blood monocyteꢀderived cell line THPꢀ1.
Mycobacterial species were cultured in either Middlebrook 7H9 broth or Middlebrook 7H10 agar
media supplemented with albuminꢀdextroseꢀcatalase (ADC) or oleic acidꢀalbuminꢀdextroseꢀ
catalase (OADC) enrichments, respectively, purchased from BD Biosciences. All reagents were
purchased from SigmaꢀAldrich unless stated otherwise.
Bacterial growth inhibition assays.
33ꢀ35
Anti-mycobacterial activity: HTꢀSPOTi was used as described previously
to assess the
2
7
minimum inhibitory concentrations of the compounds on M. aurum, M. tuberculosis H37Rv,
and the MDR strains of the pathogen. Briefly, the assay was conducted in a semiꢀautomated 96
well plate format. The compounds were dissolved in a suitable solvent to a concentration of 50
mg/mL and serially diluted (twoꢀfold dilutions). 2 µL of each dilution were dispensed into a well
of a 96ꢀwell plate to which 200 µL of Middlebrook 7H10 agar medium kept at 55 °C
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supplemented with 0.05% (v/v) glycerol and 10% (v/v) OADC was added. The concentration
ranges of the compounds used was 500ꢀ0.5 µg/mL. A well with no compounds (DMSO only)
and isoniazid was used as experimental control. To all the plates, a drop (2 µL) of early to midꢀ
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log bacterial culture containing 2x10 colony forming units (CFUs) was spotted in the middle of
each well and the plates were incubated at 35 or 37 °C depending on the bacterial species and
incubated till distinct spots were observed in the control wells (this took 4 days in the case of M.
aurum and 7 days in the case of M. tuberculosis H37Rv and 9 days for its drug resistant clinical
isolates). The MICs were determined as the lowest concentration of the compound where
mycobacterial growth was completely inhibited.
2
The MIC of the compounds against M. smegmatis mc 155, M. bovis BCG, and M. tuberculosis
2
mc 7000 were calculated by standard MABA (Microplate Alamar Blue assay) as previously
30
described. Briefly, 200 µL of sterile deionized water was added to all outerꢀperimeter wells of a
sterile 96ꢀwell plate (Corning Incorporated, Corning, NY) to minimize evaporation of the
medium in the test wells during incubation. The wells in rows B to G in columns 3 to 11 received
1
00 µL of 7H9 medium containing 0.2% casamino acids, 24 µg/mL pantothenate and 10%
OADC (Beckton Dickinson, Sparks, MD). Compounds were added to rows B–G followed by 1:2
serial dilutions across the plate to column 10, and 100 µL of excess medium was discarded from
the wells in column 10. The bacterial culture at 0.5 McFarland standard diluted 1:25 (100 µL)
was added to the wells in rows B to G in columns 2 to 11, where the wells in column 11 served
as drugꢀfree controls. The plates were sealed with parafilm and were incubated at 37 °C. A
freshly prepared 1:1 mixture of Alamar Blue (CelltiterꢀBlue™, Promega Corp, Madison, WI)
®
reagent and 10% Tween 80 (50 µL) and reꢀincubated at 37 °C for 24 h.
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Eukaryotic cell toxicity and selectivity indices: The cell lines were grown and maintained in
RPMIꢀ1640 supplemented with 10% FBS incubated at 37°C with 5% CO in a humidified
2
incubator. For the experiment, 2 µL of the 50 mg/mL stock solution of each compound was
diluted in 200 µL of RPMIꢀ1640 medium in the first row of a 96ꢀwell microplate and two fold
dilutions were performed along the rows leaving the last one (Rowꢀ H) as a solvent control. 100
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0
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µL containing 10 cells/mL of confluent murine macrophage cells (RAW 264.7) or THPꢀ1 cells
in logarithmic growth phase were added to each well. The plates were incubated at 37 °C in a
humidified CO incubator (5% CO ) for 48 h. Each well was washed twice with 1x PBS, and 170
2
2
µL fresh RPMIꢀ1640 was added to each well followed by 30 µL of 0.01% trypan blue. AS THPꢀ
cells are suspension cultures, the plates were centrifuged between the washing steps. After
1
overnight incubation, the change in colour was observed and the fluorescence intensity was
measured at λ exc560/λ emi590 nm.
Assaying whole-cell drug efflux pump inhibition: The assay was modified from previously
36
published protocol. Early log phase cells of M. aurum (OD600 ~ 0.8) were taken and the OD600
was adjusted to 0.4 by diluting the cells with culture medium. The cells were collected by
centrifugation and resuspended in equivalent volume 1x PBS. The test samples contained: 4ꢀ
7
6
x10 bacteria/mL in PBS, 0.4% glucose (as a source of energy for efflux pumps activity), 0.5
mg/L ethidium bromide (as a substrate for efflux pumps) and the compounds being tested at
/4xꢀMIC concentrations. Blank samples contained all of the components mentioned above
1
except the bacterial suspension, which was replaced with 1x PBS. Verapamil, a known efflux
pump inhibitor was used as the positive control at a concentration of 125 µg/mL. Compounds 1
and 2 were also included in the experiment to obtain a comparison between them and the newly
synthesised compounds with regards to their efflux inhibitory properties. The experiment was
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performed in a 96ꢀwell plate which was placed in a fluorimeter (FLUOstar OPTIMA, BMG
Labtech, UK) and the instrument was programmed with the following parameters: wavelengths
of 544 nm and 590 nm for excitation and detection of fluorescence, gain 2200, a temperature of
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7 °C, and a cycle of measurement every minute for a total period of 60 min. The accumulation
or efflux of ethidium bromide was monitored for the mentioned period on a realꢀtime basis.
Chemistry ꢀ Materials and methods
1
13
H NMR and C NMR spectra were recorded on JEOL Deltaꢀ270 or JEOL ECSꢀ400
spectrometers operating at the frequencies indicated. Chemical shifts (δ) are in ppm, referenced
to tetramethylsilane. Coupling constants (J) are reported in Hertz and rounded to 0.5 Hz.
Splitting patterns are abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), broad (br) or some combination of them. Infrared spectra were obtained using a
Durascope diamond ATR system. Mass spectra (HRMS) were recorded at the EPSRC National
Mass Spectrometry Service Centre on a Thermo Scientific LTQ Orbitrap XL Mass Spectrometer
using lowꢀresolution ESI or highꢀresolution nanoESI techniques. The purity of the compounds
was assessed by reverseꢀphase liquid chromatography coupled with a mass spectrometer (Agilent
series 1100 LC/MSD) with a UV detector at k = 254 nm and an electrospray ionization source
(ESI). HPLC analyses were performed at 0.4 mL/min flow rate and using a binary solvent
system of 95:5 methyl alcohol/water. All the solvents were of HPLC grade. Mass spectra were
acquired in positive mode scanning over the mass range of 50–1500. The following ion source
parameters were used: drying gas flow, 9 mL/min; nebulize pressure, 40 psig; and drying gas
temperature, 350 ºC. All target compounds possessed a purity of ≥ 95% as verified by HPLC
analyses. TLC was performed using commercially available preꢀcoated plates and visualized
with UV light at 254 nm; K MnO4 was used to reveal the products. Flash column
2
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chromatography was carried out using Fluorochem Davisil 40ꢀ63 u 60 Å. All reactions were
conducted under a nitrogen atmosphere in ovenꢀdried glassware unless stated otherwise. THF
was distilled under nitrogen from sodium using a benzophenone indicator. Dichloromethane was
purchased from Aldrich. Acetonitrile was further dried over 4 Å ovenꢀactivated molecular sieves
for 1 h prior to use. Petrol refers to the fraction of light petroleum ether boiling between 40 and
0
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65 °C. All other solvents and commercially available reagents were used as received.
1
8
Pyrroles 3aꢀe were synthesised as described in the literature.
Synthesis of alkyl piperazines 6aꢀb. General procedure.
The Boc piperazine 4 (2 mmol) and the appropriate ketone 5 (2 mmol) were dissolved in THF
8 mL) and the resulting mixture was stirred for 5 minutes. The reaction was cooled at 0 °C and
(
then Na(AcO) BH (2.4 mmol) and AcOH (2 mmol) were carefully added. The resulting mixture
3
was stirred at rt for 16 h. The reaction was quenched with NaOH 1N (20 mL) and the product
was extracted with AcOEt (3 x 25 mL). The AcOEt extract was dried (MgSO ), and the solvent
4
was evaporated to give the crude piperazine derivatives which were filtered through a pad of
silica gel (eluent AcOEt/petroleum ether 1:1). The filtered compounds were then dissolved in
DCM (4 mL) and treated with 1 mL TFA. The resulting mixture was stirred at rt for 12 h. The
solvents were removed and the crude compound was dissolved in AcOEt (10 mL) and washed
several times with 1N NaOH (20 mL). The organic phase was dried (MgSO ), and the solvent
4
was evaporated to yield the crude piperazines 6aꢀb which were purified by column
chromatography using petroleum ether/AcOEt 1:1 as eluent.
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ꢀ(Adamantanꢀ2ꢀyl)piperazine (6a). Tan oil. H NMR (400 MHz CDCl ) δ 3.31 (m, 4H),
3
2
.81 (m, 2H), 2.31 (br s, 2H), 2.01ꢀ1.94 (m, 4H), 1.76ꢀ1.68 (m, 5H), 1.60ꢀ1.53 (m, 4H), 1.42ꢀ
+
1
.39 (m, 1H), 1.30ꢀ1.27 (m, 1H) ppm. LRMS m/z (ES+) m/z: 221 [M+H]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
ꢀ(Bicyclo[2.2.1]heptanꢀ2ꢀyl)piperazine (6b). Tan oil. H NMR (400 MHz CDCl ) δ 2.88ꢀ
3
2.82 (m, 5H), 2.28 (br s, 3H), 2.20ꢀ2.09 (m, 3H), 1.67ꢀ1.60 (m, 2H), 1.41ꢀ1.39 (m, 1H), 1.28ꢀ
+
1
.19 (m, 4H), 0.82ꢀ0.79 (m, 1H) ppm. LRMS m/z (ES+) m/z: 181 [M+H]
Synthesis of pyrrole derivatives 7aꢀm. General Procedure.
Following the Mannich reaction, to a stirred solution of an appropriate pyrrole 3aꢀe (1.5 mmol)
in acetonitrile (5 mL), a mixture of the appropriate piperazine 6 (1.5 mmol), formaldehyde (1.5
mmol) (40% in water), and 1.3 mL of acetic acid was added dropwise. After the addition was
complete, the mixture was stirred at rt for 3 h. The mixture was then treated with a solution of
sodium hydroxide (20%, w/v) (20 mL) and extracted with AcOEt (10 mL). The organic extracts
were combined, washed with water, and dried. After removal of solvent, the residue was purified
by column chromatography, using silica gel and petroleum ether/ethyl acetate (4:1 v/v) as eluent.
1
ꢀ(Adamantanꢀ2ꢀyl)ꢀ4ꢀ((1ꢀ(4ꢀchlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7a). R 0.11 (AcOEt/hexane 1:1). Tan oil. H NMR (400 MHz CDCl ) δ
f
3
7
.41 (d, 2H, J = 8.7 Hz), 7.12 (d, 2H, J = 8.7 Hz), 5.92 (s, 1H), 3.36 (s, 2H), 2.50 (br s, 4H),
.05ꢀ2.02 (m, 6H), 1.98 (s, 3H), 1.95 (s, 3H), 1.85ꢀ1.72 (m, 5H), 1.68ꢀ1.62 (m, 5H), 1.37ꢀ1.34
2
1
3
(
m, 3H) ppm. C NMR (100 MHz CDCl ) δ 137.7, 133.4, 129.7, 129.3, 127.6, 126.8, 115.2,
3
1
08.7, 67.9, 54.4, 53.5, 49.6, 37.9, 37.3, 31.6, 31.4, 29.0, 27.6, 27.4, 22.7, 14.2, 12.9, 11.0 ppm.
+
+
LRMS m/z (ES+) m/z: 439 [M+1] . HRMS (ESI): calcd for C H ClN (M + H ) 438.2671,
2
7
37
3
found 438.2664.
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2
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2
2
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1
ꢀ(Adamantanꢀ2ꢀyl)ꢀ4ꢀ((1ꢀ(2ꢀfluorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7b). R 0.15 (AcOEt/hexane 1:1). Tan oil. H NMR (400 MHz CDCl ) δ
f
3
7
.37ꢀ7.36 (m, 1H), 7.22ꢀ7.17 (m, 3H), 5.95 (s, 1H), 3.36 (s, 2H), 2.59ꢀ2.29 (br s, 4H), 2.08ꢀ2.03
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
m, 6H), 1.97 (s, 3H), 1.93 (s, 3H), 1.83ꢀ1.75 (m, 5H), 1.67ꢀ1.62 (m, 5H), 1.36ꢀ1.24 (m, 3H)
13
ppm. C NMR (100 MHz CDCl ) δ159.81, 157.31, 130.84, 129.6, 128.06, 127.2, 126.9, 124.5,
3
1
24.4, 116.7, 116.5, 115.3, 108.6, 67.9, 54.6, 53.6, 49.6, 47.0, 39.3, 37.3, 36.3, 31.9, 31.4, 29.04,
+
2
7.6, 27.5, 22.7, 14.2, 12.4, 10.5 ppm. LRMS m/z (ES+) m/z: 422 [M+1] . HRMS (ESI): calcd
+
for C H FN (M + H ) 422.2966, found 422.2889.
2
7
37
3
1
ꢀ(Adamantanꢀ2ꢀyl)ꢀ4ꢀ((1ꢀ(4ꢀisopropylphenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7c). R 0.2 (AcOEt/hexane 1:1). Tan oil. H NMR (400MHz CDCl ) δ
f
3
7
4
3
1
2
.27ꢀ7.24 (m, 2H), 7.10ꢀ7.04 (m, 2H), 5.91 (s, 1H), 3.46 (s, 2H), 2.97ꢀ2.92 (m, 1H), 2.52 (br s,
H), 2.06ꢀ2.03 (m, 6H), 1.95 (s, 3H), 1.93 (s, 3H), 1.83ꢀ1.75 (m, 5H), 1.68ꢀ1.62 (m, 5H), 1.29 (s,
1
3
H), 1.27 (s, 3H), 1.25 (m, 3H) ppm. C NMR (100MHz CDCl ) 148.2, 136.6, 128.2, 128.1,
3
27.9, 127.1, 126.9, 108.0, 67.9, 54.5, 53.4, 50.8, 49.5, 37.9, 37.3, 33.8, 31.6, 31.4, 29.0, 27.6,
+
7.4, 24.0, 22.7, 14.2, 12.9, 11.2, 11.0 ppm. LRMS m/z (ES+) m/z: 446 [M+H] . HRMS (ESI):
+
calcd for C H N (M + H ) 446.3530, found 446.3523.
3
0
44
3
1ꢀ(Adamantanꢀ2ꢀyl)ꢀ4ꢀ((1ꢀ(3ꢀmethylphenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7d). R 0.17 (AcOEt/hexane 1:1). Tan oil. H NMR (400MHz CDCl ) δ
f
3
7.32ꢀ7.29 (m, 1H), 7.18ꢀ7.16 (m, 1H), 6.99ꢀ6.95 (m, 2H), 5.91 (s, 1H), 3.39 (s, 2H), 2.53 (br s,
4
1
1
H), 2.38 (s, 3H), 2.03 (m, 6H), 1.99 (s, 3H), 1.95 (s, 3H), 1.83ꢀ1.75 (m, 5H), 1.68ꢀ1.62 (m, 5H),
1
3
.37ꢀ1.34 (m, 3H) ppm. C NMR (100MHz CDCl ) δ 139.1, 138.9, 129.0, 128.7, 128.3, 127.7,
3
25.4, 108.2, 67.9, 54.5, 53.4, 49.5, 41.0, 37.9, 37.3, 31.4, 29.03, 28.5, 27.6, 27.4, 21.3, 12.9,
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+
+
1
1.0 ppm. LRMS m/z (ES+) m/z: 418 [M+1] . HRMS (ESI): calcd for C H N (M + H )
28
40
3
4
18.3217, found 418.3234.
1
ꢀ((1ꢀ(4ꢀChlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀyl)methyl)ꢀ4ꢀphenylpiperazine (7e). Tan
10
11
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29
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49
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59
60
1
oil. R 0.43 (AcOEt/hexane 1:1). H NMR (400 MHz CDCl ) δ 7.44 (d, 2H, J = 8 Hz), 7.26 (dd,
f
3
2H, J = 8.7, 7.3 Hz), 7.15 (d, 2H, J = 7 Hz), 6.94 (d, 2H, J = 7 Hz), 6.85 (m, 1H), 5.96 (s, 1H),
1
3
3
.45 (s, 2H), 3.23 (m, 4H), 2.66 (m, 4H), 2.02 (s, 3H), 2.00 (s, 3H) ppm. C NMR (100 MHz
CDCl ) δ 151.5, 137.6, 133.5, 129.7, 129.3, 129.1, 127.8, 126.8, 119.5, 115.0, 108.6, 54.5, 52.9,
3
+
49.2, 12.9, 11.0 ppm. LRMS m/z (ES+) m/z: 380 [M+1] . HRMS (ESI): calcd for C H ClN
2
3
27
3
+
(
M + H ) 380.1888, found 380.1884.
4
,4'ꢀ((1ꢀ(4ꢀChlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrroleꢀ3,4ꢀdiyl)bis(methylene))bis(1ꢀ
1
phenylpiperazine) (7f). Tan oil. R 0.25 (AcOEt/hexane 1:1). H NMR (400 MHz CDCl ) δ
f
3
7
.30ꢀ7.28 (d, 2H, J = 8.2 Hz), 7.08 (dd, 4H, J = 8.5, 7.6 Hz), 7.01 (d, 2H, J = 8.7 Hz), 6.77 (d,
13
4H, J = 8.2 Hz), 6.66 (m, 2H), 3.34 (s, 4H), 3.03 (m, 8H), 2.47 (m, 8H), 1.85 (s, 6H) ppm. C
NMR (100 MHz CDCl ) δ 151.5, 137.7, 133.4, 129.8, 129.3, 129.1, 126.5, 119.5, 116.0, 53.0,
3
+
5
2.8, 11.0 ppm. LRMS m/z (ES+) m/z: 555 [M+1] . HRMS (ESI): calcd for C H ClN (M +
3
4
41
5
+
H ) 554.3045, found 554.3032.
1
ꢀ((1S,4R)ꢀBicyclo[2.2.1]heptanꢀ2ꢀyl)ꢀ4ꢀ((1ꢀ(4ꢀchlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7g). R 0.15 (AcOEt/hexane 1:1). Tan oil. H NMR (400 MHz CDCl ) δ
f
3
7.40 (d, 2H, J = 5 Hz), 7.11 (d, 2H, J = 5 Hz), 5.90 (s, 1H), 3.37 (s, 2H), 2.48 (br m, 5H), 2.24ꢀ
2
0
1
.12 (m, 4H), 1.97 (s, 3H), 1.94 (s, 3H), 1.72ꢀ1.65 (m, 2H), 1.46 (m, 1H), 1.33ꢀ1.22 (m, 5H),
1
3
.88ꢀ0.81 (m, 2H) ppm. C NMR (100 MHz CDCl ) δ 137.7, 133.4, 129.7, 129.3, 127.6, 126.8,
3
15.2, 108.7, 67.9, 54.4, 52.8, 52.7, 39.0, 38.1, 36.8, 35.8, 30.6, 21.1, 12.8, 11.0 ppm. LRMS
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
m/z (ES+) m/z: 398 [M+H] . HRMS (ESI): calcd for C H ClN (M + H ) 398.2358, found
24
33
3
398.2352.
1
ꢀ((1ꢀ(4ꢀChlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀyl)methyl)ꢀ4ꢀcyclohexylpiperazine (7h).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Tan oil. R 0.15 (AcOEt/hexane 1:1). H NMR (400 MHz CDCl ) δ 7.40 (d, 2H, J = 5 Hz), 7.10
f
3
(d, 2H, J = 5 Hz), 5.89 (s, 1H), 3.37 (s, 2H), 2.60 (br s, 8H), 2.20 (m, 1H), 1.96 (s, 3H), 1.93 (s,
1
3
3
H), 1.88ꢀ1.86 (m, 2H), 1.77ꢀ1.75 (m, 2H), 1.61ꢀ1.58 (m, 1H), 1.26ꢀ1.11 (m, 5H) ppm. C NMR
(
100 MHz CDCl ) δ 137.6, 133.5, 129.7, 129.6, 129.3, 127.7, 118.7, 108.7, 63.5, 56.2, 54.3,
3
+
53.0, 48.8, 29.0, 26.3, 25.9, 12.8, 11.0, 10.5 ppm. LRMS m/z (ES+) m/z: 386 [M+H] . HRMS
+
(
ESI): calcd for C H ClN (M + H ) 386.2358, found 386.2356.
2
3
33
3
1
ꢀ(Adamantanꢀ1ꢀyl)ꢀ4ꢀ((1ꢀ(4ꢀchlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7i). R 0.10 (AcOEt/hexane 1:1). Tan oil. H NMR (400 MHz CDCl ) δ
f
3
7
.41 (d, 2H, J = 8.7 Hz), 7.10 (d, 2H, J = 8.7 Hz), 5.89 (s, 1H), 3.38 (s, 2H), 2.67 (br s, 4H), 2.50
1
3
(br s, 4H), 2.05 (m, 4H), 1.96 (s, 3H), 1.93 (s, 3H), 1.69ꢀ1.59 (m, 11H) ppm. C NMR (100
MHz CDCl ) δ 137.7, 133.4, 129.7, 129.8, 129.3, 127.6, 126.8, 115.0, 108.8, 60.4, 56.1, 54.2,
3
5
3.5, 44.0, 38.5, 37.0, 31.6, 29.7, 25.3, 22.7, 20.7, 18.8, 14.1, 11.0 ppm. LRMS m/z (ES+) m/z:
+
+
439 [M+H] . HRMS (ESI): calcd for C H ClN (M + H ) 438.2671, found 438.2680.
2
7
37
3
1
ꢀ((1,5ꢀBis(4ꢀchlorophenyl)ꢀ2ꢀmethylꢀ1Hꢀpyrrolꢀ3ꢀyl)methyl)ꢀ4ꢀphenylpiperazine
(7j).
1
White solid, mp 169 ºC. R 0.2 (AcOEt 100%). H NMR (400 MHz CDCl ) δ 7.33 (d, 2H, J = 8.2
f
3
Hz), 7.23 (m, 2H), 7.07 (dd, 4H, J = 16.9, 8.2 Hz), 6.92 (m, 4H), 6.82 (m, 1H), 6.36 (s, 1H), 3.49
13
(
s, 2H), 3.21 (m, 4H), 2.67 (m, 4H), 2.07 (s, 3H) ppm. C NMR (100 MHz CDCl ) δ 151.4,
3
1
37.8, 133.5, 132.0, 131.7, 131.5, 130.3, 129.8, 129.4, 129.1, 128.8, 128.4, 119.7, 116.9, 116.1,
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11.7, 54.5, 52.9, 49.2, 11.3 ppm. LRMS m/z (ES+) m/z: 477 [M+H] . HRMS (ESI): calcd for
+
C H Cl N (M + H ) 476.1655, found 476.1648.
28 28
2
3
1
ꢀ(Adamantanꢀ2ꢀyl)ꢀ4ꢀ((1,5ꢀbis(4ꢀchlorophenyl)ꢀ2ꢀmethylꢀ1Hꢀpyrrolꢀ3ꢀ
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yl)methyl)piperazine (7k). Yellow solid, mp 197 ºC. R 0.35 (AcOEt/MeOH 4:1). H NMR (400
f
MHz CDCl ) δ 7.34ꢀ7.32 (d, 2H, J = 4 Hz), 7.10ꢀ7.04 (m, 4H), 6.93 (d, 2H, J = 4 Hz), 6.36 (s,
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H), 3.44 (s, 2H), 2.54 (br s, 4H), 2.05 (s, 3H), 2.03 (m, 4H), 1.84ꢀ1.75 (m, 4H), 1.68ꢀ1.63 (m,
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3
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H), 1.38ꢀ1.25 (m, 5H), 0.88ꢀ0.82 (m, 2H) ppm. C NMR (100 MHz CDCl ) δ 137.8, 133.4,
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131.9, 131.5, 130.2, 129.8, 129.4, 128.8, 128.3, 117.1, 111.5, 67.9, 54.4, 53.5, 49.6, 37.9, 37.3,
+
3
1.6, 31.4, 29.0, 27.6, 27.4, 22.7, 14.2, 11.2 ppm. LRMS m/z (ES+) m/z: 534 [M+H] . HRMS
+
(
ESI): calcd for C H Cl N (M + H ) 534.2437, found 534.2425.
32 38
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1ꢀ(Adamantanꢀ1ꢀyl)ꢀ4ꢀ((1,5ꢀbis(4ꢀchlorophenyl)ꢀ2ꢀmethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7l). Yellow solid, mp 148 ºC. R 0.34 (AcOEt/MeOH 4:1). H NMR (400
f
MHz CDCl ) δ 7.34ꢀ7.32 (m, 2H), 7.10ꢀ7.03 (m, 4H), 5.92 (s, 1H), 6.91 (m, 2H), 6.33 (s, 1H),
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1
3
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.48 (s, 2H), 2.72ꢀ2.70 (br d, 8H), 2.07ꢀ2.02 (m, 4H), 2.04 (s, 3H), 1.71ꢀ1.56 (m, 11H) ppm. C
NMR (100 MHz CDCl ) δ 137.8, 133.4, 131.6, 131.5, 130.3, 129.7, 129.4, 128.8, 128.3, 125.1,
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116.5, 111.9, 66.6, 54.1, 53.2, 44.0, 38.3, 36.9, 32.2, 29.7, 26.4, 23.5, 18.7, 13.2, 11.3 ppm.
+
+
LRMS m/z (ES+) m/z: 535 [M+H] . HRMS (ESI): calcd for C H Cl N (M + H ) 534.2437,
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2
38
2
3
found 534.2424.
1
ꢀ(Bicyclo[2.2.1]heptanꢀ2ꢀyl)ꢀ4ꢀ((1,5ꢀbis(4ꢀchlorophenyl)ꢀ2ꢀmethylꢀ1Hꢀpyrrolꢀ3ꢀ
1
yl)methyl)piperazine (7m). Pale yellow solid, mp 81 ºC. R 0.40 (AcOEt/MeOH 4:1). H NMR
f
(
400 MHz CDCl ) δ 7.26 (d, 2H, J = 12 Hz), 7.04 (d, 2H, J = 12 Hz), 6.99 (d, 2H, J = 12 Hz),
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6
.85 (d, 2H, J = 12 Hz), 6.30 (s, 1H), 3.44 (s, 2H), 2.51 (brs, 6H), 2.19ꢀ2.17 (m, 2H), 2.12ꢀ2.07
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m, 1H), 2.00 (s, 3H), 1.66ꢀ1.62 (m, 2H), 1.43ꢀ1.40 (m, 1H), 1.29ꢀ1.17 (m, 7H) ppm. C NMR
(
100 MHz CDCl ) δ 137.8, 133.4, 131.9,131.5, 129.7, 128.7, 128.3, 111.8, 67.9, 65.9, 54.2, 52.6,
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8.9, 38.0, 36.8, 36.8, 31.6, 30.6, 22.7, 21.0, 14.2, 11.2 ppm. LRMS m/z (ES+) m/z: 494
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0
+
+
[
M+H] . HRMS (ESI): calcd for C H Cl N (M + H ) 494.2124, found 494.2113.
29 34 2 3
Synthesis of compounds 8aꢀc. General procedure.
POCl (6 mmol) was added dropwise to a round bottom flask containing iceꢀcooled DMF (5 mL)
3
under N atmosphere. After 15 min, a solution of the appropriate pyrrole 3aꢀb and 3e (1 mmol)
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was added to the stirring solution. Then the reaction mixture was allowed to stir at 100 ºC for 3
h. The reaction was monitored through TLC. After completion, the reaction was quenched with
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0% w/v NaOH solution (20 mL). The reaction mixture was then diluted with EtOAc (10 mL),
washed two times with EtOAc (10 mL) and once with brine (20 mL). The organic extracts were
collected and then they were dried over MgSO , filtered and concentrated under reduced
4
pressure. The residue was then purified by flash chromatography (hexanesꢀAcOEt, 4:1 v/v)
affording the desired compounds 8aꢀc.
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ꢀ(4ꢀChlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrroleꢀ3ꢀcarbaldehyde (8a). Tan oil. Rf 0.67
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(
hexane/AcOEt 9:1). H NMR (400 MHz CDCl ) δ 9.71 (s, 1H), 7.41ꢀ7.39 (d, 2H, J =7.8), 7.10ꢀ
3
1
3
7.07 (d, 2H, J =7.8), 6.25 (s, 1H), 2.17 (s, 3H), 1.88 (s, 3H) ppm. C NMR (100 MHz CDCl ) δ
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1
85.3, 162.6, 138.7, 135.5, 130.9, 129.6, 129.3, 122.1, 106.15, 12.7, 11.2 ppm. Data are in
1
8
agreement with those previously reported.
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ꢀ(4ꢀFluorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrroleꢀ3ꢀcarbaldehyde (8b). Tan oil. Rf 0.71
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(
hexane/AcOEt 9:1). H NMR (400 MHz CDCl ) δ 9.86 (s, 1H), 7.20ꢀ7.17 (m, 4H), 6.36 (s, 1H),
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.26 (s, 3H), 1.96 (s, 3H) ppm. C NMR (100 MHz CDCl ) δ 187.3, 129.8, 120.4, 117.2, 116.8,
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8
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16.6, 105.9, 12.7, 11.2 ppm. Data are in agreement with those previously reported.
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,5ꢀBis(4ꢀchlorophenyl)ꢀ2ꢀmethylꢀ1Hꢀpyrroleꢀ3ꢀcarbaldehyde (8c). White solid, mp 110
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ºC. R 0.6 (hexane/AcOEt 1:1). H NMR (400 MHz CDCl ) δ 9.89 (s, 1H), 7.33 (d, 2H, J = 12
f
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Hz), 7.09 (d, 2H, J = 12 Hz), 7.01 (d, 2H, J = 12 Hz), 6.87 (d, 2H, J = 12 Hz), 6.70 (s, 1H), 2.32
1
3
(
s, 3H) ppm. C NMR (100 MHz CDCl ) δ 185.6, 140.1, 135.6, 134.5, 133.2, 132.0, 130.0,
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+
1
29.8, 129.5, 129.4, 128.6, 122.9, 108.9, 11.5 ppm. LRMS m/z (ES+) m/z: 331 [M+H] .
Synthesis of pyrrole derivatives 9aꢀj. General Procedure.
The appropriate aldehyde 8aꢀc (1 mmol) was dissolved in 5 mL of THF in a round bottom flask.
Then AcOH (1 mmol) and the appropriate amine (1 mmol) were added to the stirring solution at
room temperature. The mixture was allowed to stir at room temperature for 20 minutes before
NaB(AcO) H (3 mmol) was added. The mixture was allowed to stir at room temperature for 18h.
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Then, after completion, the reaction was quenched with NaOH 1M solution (25 mL). The
mixture was then allowed to stir for 30 minutes. The reaction mixture was then diluted with
EtOAc (10 mL), washed two times with EtOAc (10 mL) and once with brine (20 mL). The
organic extracts were collected and then dried over MgSO , filtered and concentrated under
4
reduced pressure. The residue was then purified by flash chromatography (hexaneꢀAcOEt, 6:4
v/v) affording the desired compounds 9aꢀj.
Nꢀ((1ꢀ(4ꢀChlorophenyl)ꢀ2,5ꢀdimethylꢀ1Hꢀpyrrolꢀ3ꢀyl)methyl)ꢀ2ꢀphenylethanꢀ1ꢀamine
1
(
9a). Tan oil. R 0.29 (AcOEt/hexane 1:1). H NMR (400 MHz CDCl ) δ 7.42ꢀ7.40 (d, 2H, J =
f
3
1
.4 Hz), 7.29ꢀ7.27 (m, 2H), 7.24ꢀ7.22 (m, 3H), 7.12ꢀ7.10 (d, 2H, J = 1.4 Hz), 5.90 (s, 1H), 3.63
13
(s, 2H), 2.95 (m, 2H), 2.86 (m, 2H), 1.99 (s, 3H), 1.94 (s, 3H) ppm. C NMR (100 MHz CDCl )
3
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